Hsp27 as a negative regulator of cytochrome c release
Catherine Paul, Florence Manero, Carole Kretz-Remy, Sophie Virot, and André-Patrick Arrigo
Laboratoire Stress Oxydant, Chaperons et apoptose, Centre de Génétique Moléculaire et Cellulaire, CNRS-UMR-5534, Université Claude Bernard LYON I, 16 rue Dubois, Bat. Gregor Mendel, F-69622 Villeurbanne, France
We have reported that Hsp27 protects against apoptosis through its interaction with cytosolic cytochrome c. We have revisited this protective activity in murine cell lines expressing different levels of Hsp27. We report that Hsp27 also interferes, in a level of expression-dependent manner, with the release of cytochrome c from mitochondria. Moreover, a decreased level of endogenous Hsp27, which sensitized HeLa cells to apoptosis, reduced the delay required for cytochrome c release and pro-caspase-3 activation. The molecular mechanism regulating this function of Hsp27 is unknown. In our cell systems, Hsp27 is mainly cytosolic and only a small fraction of this protein colocalized with mitochondria. Moreover, we show that only a very small fraction of cytochrome c interacted with Hsp27, hence excluding a role for this interaction in the retention of cytochrome c in mitochondria. We also report that Bid intracellular distribution was altered by changes in Hsp27 level of expression suggesting that Hsp27 interferes with apoptotic signals upstream of mitochondria. We therefore investigated if the ability of Hsp27 to act as an expression-dependent modulator of F-actin microfilaments integrity was linked to the retention of cytochrome c in mitochondria. We show here that the F-actin depolymerizating agent cytochalasin D rapidly induced the release of cytochrome c from mitochondria and caspases activation. This phenomenon was delayed in cells pretreated with the F-actin stabilizer phalloidin and in cells expressing a high level of Hsp27. This suggests the existence of an apoptotic signaling pathway linking cytoskeleton damages to mitochondria. This pathway which induces Bid intracellular redistribution is negatively regulated by the ability of Hsp27 to protect F-actin network integrity. However, this upstream pathway is probably not the only one to be regulated by Hsp27 since, in staurosporine treated cells, phalloidin only partially inhibited cytochrome c release and caspases activation. Moreover, in etoposide treated cells, Hsp27 still delayed the release of cytochrome c from mitochondria in conditions where F-actin was not altered.
References
* Mehlen, P., Schulze-Osthoff, K., and A.-P. Arrigo. Small stress proteins as novel regulators of apoptosis: Constitutive expression of hsp27 blocks Fas- and staurosporine induced cell death. J. Biol. Chem. 271, 16510-16514 (1996).
Role of toll-like receptors in HSP70-Induced Signaling
Alexzander Asea1, Edith Kabingu1, Philip E. Auron2 and Stuart K. Calderwood1
Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115
Recent studies have initiated a paradigm shift in understanding of the function of heat shock proteins (HSP). It is now clear that HSP can and do exit mammalian cells, interact with cells of the immune system and exert immunostimulatory effects. We have recently shown that HSP70 family members can bind to the surface of human monocytes with high affinity elicit a rapid intracellular calcium flux activate nuclear NF-kB and upregulate the expression of pro-inflammatory cytokines tumor necrosis factor-a (TNF-a), interleukin 1b (IL-1b) and interleukin 6 (IL-6). Two different signal transduction cascades were activated by exogenous HSP70; one dependent on CD14 and intracellular calcium which resulted in TNF-a, IL-1b and IL-6 induction and the other dependent on calcium but not CD14 which increased TNF-a release alone. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of a novel extracellular role for HSP70. These trophic effects of HSP70, superimposed on its known role as a molecular chaperone, have led to adopt the term chaperokine for this property of HSP70.
Our current studies focus on finding the cell surface factors that interact with HSP70 and initiate signaling cascades leading to the TNF-a, IL-1b and IL-6 promoters. Our studies are informed by the findings that activation of the IL1B promoter in human monocytes by HSP70 requires upstream signals emanating from the signaling intermediate MyD88. This suggests a potential role for a member of the IL1bR family of receptors (including IL-1bR itself, IL-18R and Toll-like receptors) in CD14 dependent cytokine induction by HSP70. Our current studies focus on identifying the cell surface acceptor proteins, intracellular signaling intermediates and nuclear factors that ultimately activate CD14 dependent induction of the TNF-a, IL-1b and IL-6 promoters in cells exposed to HSP70.
c-Myc sensitizes cells to undergo apoptosis via the activation of stress-activated kinases
Kerstin Bellmann, Réna Deschesnes, Katia Desbiens, Jacques Landry
Centre de recherche en cancérologie de l'Université Laval, CHUQ-HDQ, Québec, QC, G1R 2J6, Canada kerstin.bellmann@crhdq.ulaval.ca
The expression of the oncogene c-myc is very often increased or deregulated in tumor cells leading to uncontrolled cell proliferation. But uncontrolled cell growth alone does not cause tumor growth. Cancer is thought to be the result of a deregulated cell proliferation in concert with reduced apoptotic capacity. Thus one of the central questions in cancer research is what makes cells capable of escaping from apoptosis? A cell line with deregulated expression of the oncogene c-myc allowed us to determine some of the pathways leading to apoptotic cell death induced by the chemotherapeutic agent cisplatin which is widely used in the treatment of testicular and ovarian tumors.
Exposure to cisplatin of Rat-1 cells with deregulated c-myc expression rapidly induced apoptosis as judged by their fragmented DNA and cellular blebbing whereas cells expressing a non-functional deletant of c-myc were completely resistant to apoptosis. Apoptosis was detected as early as 4 h after cisplatin exposure and peaked around 6 h. Interestingly, we found a strong correlation between c-myc dependent apoptosis and activation of the stress-activated protein kinase p38 which was not activated by cisplatin in cells expressing the mutant of c-myc. This was not due to an inability to activate p38 since heat shock and hydrogen peroxide led to p38 activation in both cell types. Since p38 activation did not depend on caspase activation we went up in the cascade of apoptosis to determine the role of p38 in cisplatin induced apoptosis. One of the principal targets of cell death is the release of cytochrome c from the mitochondria. The same cells that were found to be apoptotic indeed had cytochrome c released from the mitochondria in the cytosol. This release of cytochrome c was inhibited by the p38 kinase inhibitor SB203580, suggesting that p38 acts upstream of cytochrome c release. Mitochondrial integrity is thought to be maintained by members of the bcl2-family. In response to cisplatin bax, a proapoptotic member of the bcl2-family, underwent a change in conformation and translocated from the cytoplasm to the mitochondria. Both of these events were inhibited by the p38-inhibitor SB203580 in the same cells in which cytochrome c release was inhibited.
Hence, we proceeded in determining how c-myc makes cells permissive to p38 activation. Further analysis of the stress-activated kinase pathway revealed that the upstream kinases of p38, MAP kinase kinase 3 and MAP kinase kinase 6, were induced only in cells with deregulated c-myc expression. Upstream of MKK3/6 we find the apoptosis-signal regulating kinase 1 (Ask1) which was activated in a c-myc-dependent manner. Inhibition of Ask1 completely inhibited p38 activation and all features of apoptosis (bax translocation, nuclear fragmentation). Ask1 is regulated by thioredoxin, which binds to Ask1 in its reduced form and is released by oxidation allowing Ask1 to be activated. A potent antioxidant, N-acetylcystein that inhibits oxidation of thioredoxin was able to inhibit cisplatin induced p38 activation and apoptosis in cells with deregulated c-myc-expression suggesting that apoptosis induced by cisplatin involves oxidative stress. Studies are currently ongoing to unravel further elements upstream of p38 in the signaling cascade of cisplatin induced cell death in order to find the switch/target of c-myc.
Supported by the Canadian Intitutes of Health Research.
References
R.G. Deschesnes, J. Huot, K. Valerie and J. Landry. 2001. Involvement of p38 in apoptosis-associated membrane blebbing and nuclear condensation. Mol. Biol. Cell 12, 1569–1582.
G.I. Evan and K.H. Vousden. 2001. Proliferation, cell cycle and apoptosis in cancer. Nature 411, 342–348.
E.L. Soucie, M.G. Annis, J. Sedivy, L. Filmus, B. Leber, D.W. Andrews and L.Z. Penn. 2001. Myc potentiates apoptosis by stimulating bax activity at the mitochondria. Mol. Cell. Biol. 21, 4725–4736.
Effects of hyperthermia on the induction of cell death and Hsp70 in brain, testis and thymus of the adult and developing rat
Ian R. Brown and Vania R. Khan
Center for the Neurobiology of Stress, University of Toronto at Scarborough, 1265 Military Trail, Toronto, Ontario, Canada M1C 1A4 (ibrown@utsc.utoronto.ca)
Stressful stimuli can elicit two reactive cellular responses, the heat shock (stress) response and activation of the cell death pathway. Most in vivo studies on the effect of hyperthermia on the mammalian nervous system have focused on the heat shock response, characterized by the transient induction of heat shock proteins that play roles in repair and protective mechanisms (1, 2). Recently we have demonstrated that prior heat shock confers protection to the nervous system at the functional level of synaptic transmission and that stress-induced Hsp70 (and also Hsp27 and Hsp32) localize to synaptic elements (3,4,5). The present study examines of the effect of hyperthermia on the induction of cell death via apoptosis, assayed by TUNEL and active caspase 3 cytochemistry in the adult rat brain, testis and thymus. Results show that a fever-like increase in body temperature triggered apoptosis in dividing cell populations of the testis and thymus, but not in mature, post-mitotic cells of the adult cerebellum of the brain. These differential apoptotic responses did not appear to correlate with Hsp70 induction. We further investigated whether dividing neural cells were more sensitive to heat-induced apoptosis by examining the external granule cell layer of the cerebellum at postnatal day 7 and neuroepithelial layers of the neocortex and tectum at embryonic day 17. These proliferative neural regions were found to be highly susceptible to hyperthermia-induced apoptosis, suggesting the actively dividing neural cell populations are more prone to cell death induced by hyperthermia, than fully differentiated post-mitotic neurons that are irreplaceable.
(Supported by grants from the Canadian Institutes for Health Research)
References
1. Mayer, J. and Brown, I.R. (1994) Heat shock proteins in the nervous system. Academic Press, pp 1–297.
2. Brown, I.R and Sharp, F.R. (1999) The cellular stress gene response in brain. In ‘Stress Proteins' Latchmann, D.S. (ed), Handbook of Experimental Pharmacology, Springer-Verlag, Heidelberg, 136:243–263.
3. Karunanithi, S. et al.(1999) Neuroprotection at Drosophila synapses conferred by prior heat shock. J. Neurosci. 19:4360–4369.
4. Bechtold, D.A., Rush, S.J. and Brown, I.R. (2000) Localization of the heat shock protein Hsp70 to the synapse following hyperthermic stress in the brain. J. Neurochem. 74:641–646.
5. Bechtold, D.A. and Brown, I.R. Brown (2000) Heat shock proteins Hsp27 and Hsp32 localize to synaptic sites in the rat cerebellum following hyperthermia. Molec. Brain Res. 75:309-320.
The heat shock proteins in thyroid and cutaneous tumors
Juan J. Cabrera-Galván(1), José R. Santana-Santana(2), Pedro Pérez-Correa(2), Celia Medina-Ortega(1), Buenaventura Hernández-Machín(3), Francisco Medina-Rivero(4) and Mar.Cabrera-Cardona(1)
(1)Departamento de Morfología. ULPGC. Servicio Anatomía Patológica, Hospital Insular de Las Palmas: jcabrera@cicei.ulpgc.es. Servicios de Cirugía (2), Dermatología (3) and Oftalmología (4) del Hospital Insular de Las Palmas de Gran Canaria. Canary Island. Spain
INTRODUCTION. Hsp27 and hsp70 are expressed under diverse biological situations of stress or constitutively present according to the hormonal status, cellular cycle or differentiation grade, closely related with the cellular cytoskeleton and the intermediate filaments. In cancers, hsp27 has been involved in drug resistance in breast cancer, its expression has been studied in estrogen-dependent tumors and as a prognostic factor in breast, ovary and liver cancer. Hsp70 has been evaluated in cancer tissues in relation with hormonal dependence, with p53, and proliferation factors. The present study is aimed at monitoring the behavior of hsp27 and 70 in tumors of the thyroid gland and in cutaneous annex tumors with intermediate filaments of low molecular weight.
MATERIAL AND METHODS. We have studied a total of 194 thyroid tumors treated over a 20-year period in the Insular University Hospital of Las Palmas: adenomas (20), follicular carcinomas (60), papillary carcinomas (98), medullar (8) and undifferentiated carcinomas (8). We also studied 50 cutaneous annex tumors of sweat glands. The surgical specimens were fixed in 10% formaldehyde and embedded in paraffin. The hsp27 and 70 determinations were performed by immunohistochemical methods using monoclonal antibodies and a conventional system (ABC complex hrp kit). Immunostaining was evaluated and graded on the samples according to a score intensity-proportion. Finally, a statistical analysis was made.
RESULTS. The adenomas and follicular carcinomas showed a regular and diffuse cytoplasm hsp27 staining pattern. The mean score for adenomas was 4,2 and for follicular carcinomas was 2,57. The papillary carcinomas showed a parcel or regular hsp27 stain with a mean score of 3,20 (statistically significant in comparison with the follicular carcinomas, p>0,05). The most aggressive tumors in the thyroid, the undifferentiated or anaplastic carcinomas and the medullar carcinomas showed weak hsp27 immunostaining (1,1 and 1,3 respectively). In the follicular and papillary carcinomas hsp70 showed a granular cytoplasmic stain, with a mean score in adenomas of 4,5, in follicular carcinomas of 2,97, increasing in the tumor areas with vascular infiltration (hsp70 appeared in the cellular nuclei). The papillary carcinomas showed a mean of 3,40, not statistically significant in comparison with follicular carcinomas. The undifferentiated and medullar carcinomas showed strong positive reaction (4,2 and 4,9 respectively), both in cytoplasm and nuclei as well as in the infiltration focuses and vascular invasive areas. In the cutaneous annex tumors hsp27 showed positive reaction in Hidrocistomas (+++), Siringomas (+++), Siringoma Condroide (+++), Poroma (+++), Cilindroma (+), Espiroadenoma Ecrino (+). The tumours considered apocrines showed weak activity for the hsp27: Hidrocistoma (−), Hidradenoma Papiliphero (−), Siringocistoadenoma Papiliphero (++), Tubular Adenoma (−). Hsp70 showed scarce and irregular stain in most of the studied cutaneous tumors, not been statistically significant.
DISCUSSION AND CONCLUSIONS. Our results in the thyroid tumors showed a significant increment of hsp27 in the papillary carcinomas in comparison to follicular carcinomas, with papillary carcinomas showing a better prognosis. The undifferentiated and medullar carcinomas expressed weak and scarcely the hsp27, being those of worst prognosis. The hsp70 is related more directly with the growth and cellular dedifferentiation, being higher in the tumors of worst prognosis. It is also detected in infiltrating and angioinvasive areas, and in the nuclei. The hsp27 is related with better tumor behavior in the thyroid, in opposition to hsp70. In the tumors arising from the epithelium of the cutaneous annex, there is a characteristic hsp27 immunostaining pattern that distinguishes the ecrine from the apocrine tumors. The hsp70 is not significant given the benign component of these tumors.
References
Ciocca DR, Oesterreich S, Chammes GC, McGuire WL, Fuqua SAW. Biological and clinical implications of heat shock protein 27,000(Hsp27): a review. J.Natl Cancer Inst. 1993;85:1558–70.
Arrigo AP, Landry J. Expression and function of the low-molecular weight Heat Shock Proteins. In The Biology of heat shock proteins and Molecular Chaperones. 1994 De. Cold Sring Harbor L. Press.
Liang P. and McRae TH. Molecular chaperones and the cytoskeleton. J. Cell Science 1997; 110:1431–1440.
Vargas-Roig LM, Gango FE, Tello O, Aznar JC, Ciocca DR. Heat shock protein expression and drug resistance in breast cancer patient treated with induction chemotherapy. Int J Cancer (Pred Oncol) 1998; 79:468–75.
Regulation of the heat shock response and its conservation through malignant transformation
Stuart K. Calderwood, Dan Tang, Yiqun Wang, Yue Xie, Xiaozhe Wang, Stanislav Lepchammer, and Alexander Asea
Dana Farber Cancer Institute, Harvard Medical School, Boston, USA
Heat shock is the archetypal activator of the ancestral response to protein stress that has been preserved throughout the development of cellular organisms. In the evolution of cellular life, a high concentration of diverse proteins is required to catalyze and control metabolism. Under these highly concentrated conditions, proteins are at risk of aggregation, a cellular catastrophe. Such effects however rapidly trigger the heat shock response through the induction of heat shock transcription factor (HSF) and the expression of heat shock protein (HSP) molecular chaperones. These molecules promote cell survival through mechanisms that are still not clear, but appear to involve the deterrence of aggregation, the refolding of denatured proteins and the blockade of apoptosis. Study of the regulation of HSF and the pathways of HSP expression is therefore key to understanding the function and regulation of the heat shock response under stress (heat, UV, ischemia) and in pathological disorders (neurodegeneration, aging, inflammation, cancer, cardiovascular disease). HSF as the most proximal regulator in gene expression after heat is the primary activator of the heat shock response. Intracellular regulation of HSF involves the operation of HSPs as negative regulators, HSF modification by phosphorylation and protein-protein interaction. We have studied the regulation of HSF1, HSF2 and HSF4 in normal cells and in prostate carcinoma during malignant transformation and under conditions of exposure to cancer therapy. Expression profiling studies indicate that each of the factors is expressed at equivalent levels during malignant progression, in line with the high degree of conservation of the response. However, HSF1 activity and HSP expression play significant roles in the generation of the malignant phenotype and in the resistance to clinical cancer treatment. We are attempting to translate these basic findings on regulation of the heat shock response into new approaches to cancer therapy by targeting heat shock response in prostate cancer.
Role of HSP in anti-melanoma immune response: clinical impact and immunological results
Chiara Castelli
Unit of Immunotherapy of Human Tumor, Istituto Nazionale Tumori, Milano, Italy
The last years of research in tumor immunology have witnessed an explosion in the identification of T cell-defined human tumor antigens and several epitopes recognized by patients T cells, restricted by class I and class II MHC have been identified. These epitopes, when used in vaccination trials, revealed altogether a relatively weak immunogenicity and a limited clinical efficacy. From different lines of research, studies aimed at evaluating the immunogenicity of murine tumors lead to the discovery of HSP. This family of chaperone proteins displayed a strong immunogenic potential. Murine studies have carefully assessed the usage of chaperone molecules as cancer vaccine and their immunotherapeutic efficacy was demonstrated in murine tumors of different histology in a vaccination as well as in a therapeutic setting. The immunogenicity of HSP 70 and gp96 has been demonstrated for viral as well as tumor antigens in murine systems but no data are as yet available for the immunogenic potential of human tumor derived HSP. Therefore we focused our studies on the involvement of HSP70 and gp96 in the anti-tumor response in melanoma patients.
For human melanoma a variety of tumors associated peptides recognized by T cells have recently been described. Exploiting this knowledge and the availability of melanoma specific T cells with defined peptide specificity, we analyzed the capacity of HSP70 chaperone melanoma derived peptides to specifically activate T cells in vitro. We show that melanoma derived HSP70 are able to reconstitute the epitope for HLA-class I restricted T cell clones directed against melanoma differentiation antigens. The HSP70 mediated T cell activation occurs via recognition of MHC molecules of the matched APC pulsed with the melanoma derived HSP70 and is strictly dependent on the presence of HSP chaperoned peptides. In addition, the HSP peptide presentation to the anti-melanoma specific T cell clones is likely to occur via cross-priming since MHC matching between melanoma cell lines used as source of HSP70 and responding T cells clones is not required. These findings, together with preliminary data showing the ability of melanoma -derived HSP to elicit in vitro a specific anti-tumor response, represent a rational for HSP-based vaccination in human cancer.
Simultaneously to the in vitro studies, a clinical trail has been designed with the aim of defining the in vivo immunogenicity and the anti-tumor activity of a vaccine containing autologous tumor-derived heat shock protein peptide-complex gp96 (HSPPC-96). Metastatic melanoma patients (Stage IV, AJCC) with measurable disease or disease free after surgery, received four times, at weekly intervals, 5 or 50 ugr of HSPPC-96 either subcutaneously or intradermally. Clinical and immunological evaluation were carried out before and after vaccination. Immune monitoring of vaccinated patients included the assessment of the specific T cells response against the autologus or allogenic HLA-matched melanoma cells as well as, for the HLA-A2 positive patients, the detection of the anti-Melan-A/Mart-1 reactivity evaluated by Elispot and by specific tetramers staining. Among the patients who completed the first cycle of vaccination 2 CR and 3 SD were observed. Furthermore, immunological monitoring performed in 4 of the clinically responding patients showed that an increase in the anti-tumor specific T cells occurred after vaccination. All together these results indicate that vaccination of metastatic melanoma patients with HSPPC-96 is feasible, devoid of significant toxicity and can induce clinical responses which appear to be associated with a melanoma-specific T cell-mediated immune reaction.
The small heat shock protein family(This conference will be presented in Spanish)
Aura Chavez Zobel1, H. Lambert2 and Jacques Landry2
1Universidad Centroccidental Lisandro Alvarado, Barquisimeto, Venezuela and, 2Centre de recherche en cancérologie de l'Université Laval, CHUQ-HDQ, Québec City, Canada G1R 2J6. Jacques.landry@med.ulaval.ca
Heat Shock Proteins (HSP) were discovered by virtue of their transcriptional activation during heat shock but turned out to play an essential role in cell defense mechanisms against a wide variety of aggressions, including cellular stress associated with degenerative diseases. There are 5 major groups of HSP in mammals: HSP110, HSP90, HSP70 (or HSPA), HSP60 and the small HSP (sHSP or HSPB). The sHSP family is composed of 8 members in humans (αA- and αB-crystallin, p20, HSP27/HSPB1, MKBP/HSPB2, HSPL27/HSPB3, CVHSP/HSPB7, and HSPB8). All sHSP except perhaps HSPB3 are expressed in control cells as large oligomers. The basic unit of this ultrastructure appears to be a dimer formed by intermolecular interactions mediated by the conserved C-terminal domain. The oligomerization of the dimers is mediated by interactions in the N-terminal of the protein. The structure of the sHSP is highly dynamic. For example, upon phosphorylation of HSP27 at Ser90 (rodent sequence), the N-terminal interactions weaken and the protein then dissociates into dimers.
HSP27 is the best characterized sHSP in terms of protective activity. HSP27 overexpression confers cellular resistance not only to heat shock but also to a variety of stimuli that induce cell death with either necrotic or apoptotic features including physical and chemical stress, growth factor withdrawal and activation of death receptors. Not too surprisingly, different activities have been described for HSP27. It can modulate actin dynamics and microfilament re-organization that contribute to filament stabilization during stress. HSP27 can block apoptosis by preventing cytochrome-c dependent activation of caspases. Finally, HSP27 like several other small HSP possesses chaperone activities. In vitro, HSP27 binds denatured proteins and prevent their aggregation by keeping them in a renaturation competent state. However, an in vivo chaperone function of HSP27 remains to be demonstrated.
In human, replacement of a highly conserved arginine by a glycine (R120G) at position 120 in the C-terminal domain of αB-crystallin leads to a degenerative disease called desmin-related myopathy. A similar mutation in αA-crystallin (R116C) is associated with autosomal dominant congenital cataract. Expression of αB -R120G in PTK2 cells resulted in the formation of large amorphous masses surrounded by keratin and vimentin cages. The structures contained ubiquitin and HSP70 and their formation required an intact microtubule network, identifying them as aggresomes. Interestingly, we showed that overexpression of either HSP27, wild type αB-crystallin or HSP70 (but not HSPB8) could prevent or delay the formation of the aggresome. This result confirmed the known chaperone activity of HSP70 in vivo, but represents the first demonstration of an in vivo chaperone activity for a small HSP.
Supported by the Instituts de recherche en santé du Canada and La Fundacion Gran Mariscal de Ayacucho.
References
Arrigo, A.P., and J. Landry. 1994. Expression and function of the low-molecular-weight heat shock proteins. In The biology of heat shock proteins and molecular chaperones. R.I. Morimoto, A. Tissiéres, and C. Georgopoulos, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 335–373.
Lambert, H., S.J. Charette, A.F. Bernier, A. Guimond, and J. Landry. 1999. HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus. J Biol Chem. 274:9378–9385.
Quinlan, R., and P. Van Den Ijssel. 1999. Fatal attraction: when chaperone turns harlot [news]. Nat Med. 5:25–26.
Heat shock proteins in cancer: a brief overview
Daniel R. Ciocca
Institute of Experimental Medicine and Biology, Regional Center for Scientific and Technological Research, C. C. 855, 5500 Mendoza, Argentina. dciocca@lab.cricyt.edu.ar
Several member of the heat shock protein (Hsp) family have been involved in key processes in cancer cells and tissues: 1) with cell proliferation, 2) with cell differentiation, 3) with apoptosis, 4) with tumor cell invasion, 5) with disease prognosis, 6) with response to endocrine therapy, 6) with cytotoxic drug resistance, and 7) with the immune response. It is difficult to draw general conclusions from each of these research avenues, many times researchers have reported contradictory results. Also complicated is the intend to translate basic research data into clinical situations (trying to take advantage of the research on Hsps in cancer for the benefit of the patients). There are several examples that can be mentioned here to illustrate these points:
1) Human breast cancer tumor cells transfected with a full-length hsp27 construct have shown a higher rate of cell proliferation (1). However, in breast cancer biopsy samples, overexpression of hsp27 has been associated with lower cell proliferation and with higher tumor cell differentiation (2).
2) There are reports associating hsp27 with the invasive and metastatic potential of human breast cancer cells (hsp27-overexpressing clones were more invasive in vivo to the lung) (3). This can explain the relationship of hsp27 with worse prognosis in patients with certain tumor types (hepatocellular carcinomas, gastric carcinomas, osteosarcomas) (4). However, in another clinical studies hsp27 content has not been associated with poor prognosis (breast carcinomas), or even it has been correlated with good prognosis (neuroblastomas, squamous cell carcinomas of the esophagus, malignant fibrous histiocytomas) (4).
3) Estrogen receptor-positive human breast cancer cells express hsp27 in response to estrogen administration, and hsp27 expression is correlated with the expression of estrogen receptors. However, the expression of hsp27 (and also of hsp70, which is involved in estrogen receptor assembly and trafficking) is not useful to identify breast cancer patients who will respond to endocrine therapy with the antiestrogen tamoxifen (5).
These discrepancies may be explained by the diversity of interactions of the different Hsps within each cancer cell/tissue type. At the end, this is in fact a reflection of the dynamic molecular milieu present in the cells during the different evolving steps from the normal to the diverse cancer cell stages. Therefore, we are needing more studies to specifically address the role of each Hsp in cancer cells and tissues. In this symposium we will have very interesting data regarding the involvement of Hsps with prognosis, with cytotoxic drug resistance, and with the metastatic potential in different cancer cells and tissues.
References
1. Oesterreich S, Weng C-N, Qiu M, et al. The small heat shock protein hsp27 is correlated with growth and drug resistance in human breast cancer cell lines. Cancer Res 53:4443–4448 (1993).
2. Vargas Roig LM, Fanelli MA, Lopez LA, Gago FE, Tello O, Aznar JC, Ciocca DR: Heat shock proteins and cell proliferation in human breast cancer biopsy samples. Cancer Detect Prevent 21:441-451 (1997).
3. Lemieux P, Oesterreich S, Lawrence JA, et al. The small heat shock protein hsp27 increases invasiveness but decreases motility of breast cancer cells. Invasion Metastasis 17:113–123 (1997).
4. Ciocca DR, Vargas-Roig LM: Hsp27 as prognostic and predictive factor in cancer. In: Small Stress Proteins. Progress in Molecular and Subcellular Biology. Edited by A-P Arrigo and WEG Müller. Springer-Verlag: Heidelberg, Germany (in press).
5. Ciocca DR, Green S, Elledge RM, Clark GM, Pugh R, Ravdin P, Lew D, Martino S, Osborne CK: Heat shock proteins hsp27 and hsp70: lack of correlation with response to tamoxifen and clinical course of disease in estrogen receptor-positive metastatic breast cancer (A Southwest Oncology Group study). Clin Cancer Res 5:1263–1266 (1998).
Identification and functional analysis of oxidative stress response genes in Xylella fastidiosa
R. Costa de Oliveira & L.R. Nunes
Nucleo Integrado de Biotecnologia en Universidade de Mogi das Cruzes en Sao Paulo, Brazil. reginaco@umc.br lnunes@umc.br
One of the most important components of the plant defense response against microbial invasion is the so-called Oxidative Burst, which consists of a rapid and transient production of huge amounts of Reactive Oxygen Species (ROS), specially hydroxyl radicals, peroxides and superoxides, which damage DNA and attack the cell membrane structure, working as a very efficient mechanism for killing invading microorganisms and keeping most bacterial infections under strict control (Doke et al. 1996). Thus, phytopathogenic and endophytic microbes must be capable to counteract the deleterious effects of ROS, which is achieved through the action of specific enzyme systems that are normally induced when the cell is exposed to ROS. Genes that encode such enzymes are collectively known as Oxidative Stress Response genes (OSR genes), and have been proposed to play an important role in virulence mediated by phytopathogenic bacteria (Mongkolsuk et al. 1998). The phytopathogenic bacterium Xylella fastidiosa is capable of colonizing the xylem of several different plant species, and in certain occasions, these bacteria have been observed to grow into great densities, physically blocking the vessels and interfering with the normal flow of sap in the plant. This situation seems to be associated with the onset of disease; such as in the case of Citrus Variegated Chlorosis (CVC), that affects orange and lime trees. Due to the importance of such disease to the economy of the state, FAPESP has developed the Xylella fastidiosa Genome Project, aimed at the task of gathering a large amount of genetic information about this bacterium, hoping that, in the near future, it might provide the basis for the development of new therapeutic strategies against CVC (Simpson et al 2000). Before this gap can be filled, however, there is the need to convert the genomic data generated by the sequencing into biological information, specially concerning issues related to the mechanisms of virulence in X. fastidiosa, which, at this point, are completely unknown. Nevertheless, one should expect that a few general aspects of plant-bacteria relationship, observed in other phytopathogenic systems, should also occur in the case of CVC, making the study of such mechanisms an excellent model to better understand the phenomenon of X. fastidiosa pathogenicity. We are working in the identification of genes involved in the X. fastidiosa Oxidative Stress Response (OSR genes), and analysis of such genes as to their general mechanisms of action and regulated expression under different circumstances. Gene identification and mode of regulation are being performed using direct sequence comparison using genomic databases and microarray hybridization which may also help to provide important information about OSR gene regulation in this bacterium (Roth et al. 1998).This information might set the basis for an efficient method of CVC control based on inhibition of OSR gene expression.
References
Doke, N. et al (1996) Gene, 179 (1): 45–51. Mongkolsuk, S. et al (1998) J. Bacteriol., 180(10): 2636–2643.
Roth, F.P. et al (1998) Nature Biotech., 16: 939–945.
Simpson et al (2000) Nature.
Project funded by: FAPESP, SP, Br.
Competitive mechanisms to modify chaperone signaling: chaperone co-inducers as a novel class of pharmaceuticals to correct chaperone deficit
Peter Csermely
Biorex R&D Co., H-8201 Veszprém, P.O. Box. 348., Hungary and Semmelweis University, Department of Medical Chemistry P.O. Boksz 260. H-1444 Budapest, Hungary (peter_csermely@rex.biorex.hu)
Molecular chaperones of eukaryotic cells have several competing tasks related to their assistance in protein folding. They keep a large number of unstable signaling proteins, such as nuclear hormone receptors, protein kinases, etc. in a folding competent state, and assist in the refolding of damaged proteins after environmental stress. Besides these tasks chaperones are probably associating with numerous mutant proteins keeping the resulting phenotype hidden, until it is exposed by a stressful event (1). This “phenotypical buffering” results in genome cleansing when further life of the given species becomes impossible due to the deleterious effects of the exposed mutations. The advances of medical practice and several changes in the lifestyle in the last 200 years decreased the occurrence of the large stresses, which provided the cleansing of the genome before. Therefore the number of hidden mutations is presumed to increase in the human genome. Since chaperone action becomes compromised during the aging process together with an increase in damaged proteins, silent mutants may be exposed in aged subjects. This phenomenon may contribute to the appearance of civilization diseases such as diabetes, atherosclerosis, etc. (2). Besides guarding silent mutations eukaryotic chaperones may also participate in the organization of the cytoarchitecture (3,4). Chaperones emerge a key elements in the regulation of much more areas of cellular life as previously thought, and clearly their relative deficit in aging/sick subjects should be repaired.
In recent years Biorex R&D Co. has developed a new family of compounds acting as chaperone co-inducers. Biorex drug candidates act by inducing a variety of heat shock proteins such as Hsp47, Hsp90 and Hsp70 in a number of systems (5). The induction is most pronounced, when drug treatment is accompanied with a simultaneous/preceding stress, a phenomenon, we call chaperone co-induction. BRX drugs neither affect the half-life of Hsp70 mRNA, nor induce the typical, phosphorylation-dependent supershift of HSF1. On the contrary, they promote the nuclear translocation of HSF1, when the transcription factor has been already partially activated. Chaperone induction is highly sensitive to the fluidity of cellular membranes (6). BRX-drugs significantly increased the mobility of methylene groups of several minor lipids, such as cardiolipin and phosphatidyl-ethanolamine as judged by FT-IR spectroscopy. On the other hand, they were highly efficient inhibitors of bilayer-nonbilayer lipid phase-transitions. As a consequence of these actions BRX drug candidates fluidize the membrane without the potential damage induced by other fluidizing stimuli, such as heat-shock. The pleiotropic cytoprotective effects of Biorex drug candidates (heat shock protein induction, membrane protection, etc.) explain both their efficiency preventing various cellular damages as well as their diverse pharmacological action.
References
1. Rutherford, S.L. and Lindquist, S. (1998) Hsp90 as a capacitor for morphological evolution. Nature 396, 336–342.
2. Csermely, P. (2001) Chaperone overload as a possible contribution to “civilisation diseases”: atherosclerosis, cancer, diabetes. Trends in Genetics in press.
3. Csermely, P., Schnaider, T., Soti, C., Prohaszka, Z. and Nardai, G. (1998) The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol. Ther. 79, 129“168.
4. Csermely, P. (2001) Nonconventional role for molecular chaperones: their involvement in the organization of the cytoarchitecture. News Physiol. Sci. 15, 123–126.
5. Vígh, L., Literáti, P.N., Horváth, I., Török. Z., Balogh, G., Glatz, A., Kovács, E., Boros, I., Ferdinandy, P., Farkas, B., Jaszlits, L., Jednákovits, A., Korányi, L. and Maresca, B. 1997. Bimoclomol: A nontoxic, hydroxylamine derivative with stress protein-inducing activity and cytoprotective effects. Nature Med. 3, 1150–1154.
6. Vígh, L., Maresca, B. and Harwood, J. 1998. Does the membrane's physical state control the expression of heat shock and other genes? Trends in Biochem. Sci. 23, 369–374.
Apoptosis and heat shock proteins
Fernando Darío Cuello Carrion
Laboratory of Oncology. Institute of Experimental Medicine and Biology of Cuyo, CRICYT - CONICET, Mendoza, Argentina. dcuello@lab.cricyt.edu.ar
Apoptosis or Programmed Cell Death (PCD), is currently one of the most interesting issues in the field of the modern molecular biology. This very sophisticated system genetically controlled for cellular suicide is involved in many biological processes such as embryonic morphogenesis, metamorphosis, development and selective removal of the damaged, infected or superfluous cells. This type of cell death occurs as a counterpart of cell proliferation. Altered programmed cell death has been associated with pathological processes such as cancer, infections and chronic inflammation. Susceptibility of mammalian cells to apoptosis is determined by a balance between pro-apoptotic and anti-apoptotic molecules, that mediate or suppress the process of cell death, respectively. Among of the best studied mediators are the members of the Bcl-2 gene family. This family is constituted by both, inducers and inhibitors of the apoptotic process, many of which are localized in mitochondrial membranes and regulate the release of cytochrome c from the mitochondria to the cytosol, to form a protein complex called apoptosome constituted by cytochrome c, apoptosis-inducing factor (Apaf-1) and an initiator caspase.
Apoptosis is mediated by extrinsic (receptor-mediated) and intrinsic (mitochondria-mediated) signaling pathways that converge in the activation of proteolytic enzymes known as caspases. The cysteine protease activation is an early hallmark of apoptosis, induced by a great variety of apoptotic stimuli. The activation of the inactive pro-caspases occurs in a hierarchic cascade, in which the apoptotic signal activates an initiator caspase, which in turn activates the effector caspases. These proteases initiate the degradation phase of programmed cell death, giving rise to the morphological features of the apoptosis (cytoplasmic and nuclear condensation, cytoplasmic shrinkage, cell membrane blebbing, chromatin clumping at the periphery of the nucleus, endonuclease-mediated DNA cleavage, and formation of apoptotic bodies).
In response to stress, the cell can induce the expression of heat shock proteins (HSPs) that increase the tolerance to the harmful conditions and protect against subsequent environmental insults and cell death.
Recent evidences suggest that Hsps may block the cell death pathways at different levels. Hsps may inhibit the damage induced by denatured proteins, and the formation of reactive oxygen species; HSPs may also inhibit the activity of the final effectors in the late phases of apoptosis. In fact, most of the members of HSPs subfamilies (Hsp10, Hsp27. Hsp60, Hsp70, and Hsp90) can interact with the main components of the apoptotic machinery. Due to the cellular homeostasis is the result between survival and death then, HSPs as well as other chaperones should play a pivotal role to support this sensitive balance.
References
1. Nature insight apoptosis. Nature. 407: 769–816, 2001.
2. M. Jäättelä. Escaping cell death: Survival proteins in cancer. Exp Cell Res. 248: 30–43, 1999.
3. C. Jolly and R. Morimoto. Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J Natl Cancer Inst. 92 (19): 1564–1572, 2000.
4. M. Jäättelä. Heat shock proteins as cellular lifeguards. Ann Med. 31: 261–271, 1999.
5. A. Samali and S. Orrenius. Heat shock proteins: regulators of stress response and apoptosis. Cell Stress & Chaperones. 3(4): 228–236, 1998.
Stress dependent and cell type specific expression of heat shock proteins in the heart and brain
R.W. Currie, J.P. Leger, A.M. Krueger-Naug
Laboratory of Molecular Neurobiology, Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4H7. wcurrie@is.dal.ca
Heat shock proteins are synthesized in abundance in the heart and brain after mild injury such as heat shock or brief ischemia. In the brain ischemic injury,1 or epileptic seizure activity cause increased expression of Hsp70 in neurons and of Hsp27 in glial cells. Alternatively, cutting the vagus nerve induces the expression of Hsp27 in the axotomized neurons of the dorsal motor nucleus of he vagus nerve.2 In contrast, heat shock treatment causes expression of both Hsp70 and Hsp27 in a few neurons and mostly in glial cells.3,4 Thus cells in the make specific responses to injury that are stress dependent and cell specific.
In the heart, Hsp27 is localized normally in cardiomyocytes, in a pattern reminiscent of Z-bands, and in cardiac neuronal cell bodies and axons.5 No obvious change in Hsp27 content or distribution appears to occur after heat shock. Little or no Hsp70 is detected in control hearts. After heat shock, abundant Hsp70 is detected in small blood vessels found between the ventricular cardiomyocytes, and little or no Hsp70 is detected in cardiomyocytes or neuronal elements within the heart.5 Heat shock induces a cell-type specific expression of Hsp70 in blood vessels, but not cardiomyocytes or intrinsic cardiac neurons, suggesting that blood vessels play a primary role in myocardial protection. Myocardial protection may be due to complex interactions between myocytes and blood vessels in the heart. If blood vessels are protected from reperfusion injury, then possibly the heart is also protected.
References
1. Currie, R.W., Ellison, J.A., White, R.F., Feuerstein, G.Z., Wang, X., Barone, F.C. (2000) Benign focal ischemic preconditioning induces neuronal Hsp70 and prolonged astrogliosis with expression of Hsp27. Brain Res. 863: 169–181.
2. Hopkins, D.A., Plumier, J.-C.L., Currie, R.W. (1998) Induction of the 27-kDa heat shock protein (Hsp27) in the rat medulla oblongata after vagus nerve injury. Exp. Neurol. 153: 173–183. (with cover).
3. Krueger, A.M.R., Armstrong, J.N., Plumier, J.-C.L., Robertson, H.A., Currie, R.W. (1999) Cell specific expression of Hsp70 in neurons and glia of the rat hippocampus after hyperthermia and kainic acid-induced seizure activity. Mol. Brain Res. 71: 265–278.
4. Krueger-Naug, A.M., Hopkins, D.A., Armstrong, J.N., Plumier, J.-C.L., Currie, R.W. (2000) Hyperthermic induction of the 27-kDa heat shock protein (Hsp27) in neuroglia and neurons of the rat central nervous system. J. Comp. Neurol. 428: 495–510.
5. Leger, J., Smith, F.M., Currie, R.W. (2000) Confocal microscopic localization of constitutive and heat shock-induced Hsp70 and Hsp27 in rat heart. Circulation 102: 1703–1709.
Effect of heat shock on the inflammatory response
Antonio De Maio, Virginia L. Vega, Marcella Ferlito, Jorg Karolat, Heiko Trentzsch, and Charles N. Paidas
Division of Pediatric Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. ademaio@mail.jhmi.edu
Sepsis is a major heath problem with over 500,000 cases reported every year in the US and a mortality rate of 40–60% (1). This condition is the result of an exaggerated or uncontrolled inflammatory response (2). Inflammation is characterized by several events at the cellular and molecular levels including the orderly appearance of cytokines, such as tumor necrosis factor α (TNF-α), interleukins, etc, infiltration of polymorphonuclear leukocytes (PMN) in various organs, and a reprioritization of gene expression. In sepsis, a major factor that triggers the inflammatory response is the presence of lipopolysaccharide (LPS), a component of the outer membrane of gram negative bacteria, which is shed from the pathogen during infection. The main receptor for LPS is CD14, which is expressed primarily on myeloid cells, particularly macrophages. Binding of LPS to this receptor in association with accessory proteins, i.e. toll-like receptors, triggers a signal transduction pathway responsible for the manifestation of the inflammatory response, i.e. production of cytokines (3).
Previous studies have shown that rodents subjected to a mild thermal stress become more resistant to LPS toxicity (4). To understand the underlying protective mechanism, we have investigated the effect of heat shock on CD14 expression. Heat shock (42□C) results in a transient decrease of cell surface CD14 within 2 h of recovery at 37°C in the mouse macrophage cell line J774. This decrease of CD14 surface expression correlates with a reduction of LPS response as measured by production of TNF-α. In contrast with the change of CD14 surface level, the intracellular pool of CD14 (visualized by immunostaining of permeabilized cells) is not affected by heat shock. The expression of CD14 on the cell surface requires the presence of heat shock proteins. Thus, treatment of J744 cells with geldanamycin, an inhibitor of the Hsp90 family, results in retention of this protein within the endoplasmic reticulum (ER). It is likely that Grp94, an ER resident chaperon that belongs to the Hsp90 family, is necessary for the folding of CD14. The retention of CD14 within the ER also results in the disappearance of this glycoprotein from the cell surface. Cells that do not express CD14, such a human promonocyte cell line (THP-1), show a different effect of heat shock on the secretion of TNF-α after incubation with LPS. In fact, elevated TNF-α levels after incubation with LPS are observed during recovery at 37 (C following heat shock. This effect on TNF-α production is due to translational or post-translational mechanisms, because steady-state TNF-α mRNA levels are not affected during heat shock treatment or recovery following the stress. These results suggest that the effect of heat shock on cells involved in the inflammatory response is complex. Thus, it is likely that several mechanisms are responsible for the protection observed in heat shocked rodents from LPS toxicity.
References
1. Rangel-Frausto, M. S., Pittet, D., Costigan, M., Hwang, T., Davis, C. S., and Wenzel, R. P. (1995) The natural history of the systemic inflammatory response syndrome (SIRS). A prospective study. JAMA 273, 117–123.
2. Livingston, D. H., Mosenthal, A. C., and Deitch, E. A. (1995) Sepsis and multiple organ dysfunction syndrome: A clinical-mechanistic overview. New Horizons 3, 257–266.
3. Tobias, P. S., Tapping, R. I., and Gegner, J. A. (1999) Endotoxin interactions with lipopolysaccharide-responsive cells. Clin. Infect. Dis. 28, 476–481.
4. De Maio, A. (1999) Heat shock proteins. Facts, thoughts, and dreams. Shock 11, 1–12.
Stress signalling downstream to p38: MAPKAP kinases (MKs) as essential components
Matthias Gaestel
Institute of Biochemistry, Medical School Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany, gaestel.matthias@mh-hannover.de
MAPKAP kinase 2 (MK2) and MK5 (also known as PRAK) are two of several kinases that are regulated via direct phosphorylation through p38 MAP kinase, the central component of a stress-activated kinase cascade. Both kinases are mainly localised in the nucleus of non-stressed cells and, as a consequence of a conformational change, are rapidly exported to the cytoplasm as a result of its activation in response to stress (Engel et al., 1998; Neininger et al., 2001).
By introducing a targeted mutation into the mouse MK2 gene, we determined the physiological function of this protein kinase in vivo. Mice that lack MK2 show increased stress resistance and survive LPS-induced endotoxic shock. This is caused by an 90% reduction in the production of tumor necrosis factor (TNF) □ protein, although the level and stability of TNF□ mRNA is not reduced and TNF□ secretion is not altered (Kotlyarov et al., 1999). Interestingly, deletion of the AU-rich element (ARE) in the 3' UTR of the TNF□ gene rescues the defect in LPS-induced TNF□ biosynthesis indicating that MK2 is upstream to the ARE at the same genetically defined pathway. We conclude that MK2 is an essential component in the inflammatory response which regulates biosynthesis of TNFα at a post-transcriptional level probably by acting through an ARE-dependent mechanism within the cytoplasmic compartment. In addition, data will be presented which indicate that MK2 also plays a role in growth factor induced cellular migration.
Targeted disruption of the MK5 gene was used to analyse whether the function of this stress-activated enzyme in vivo is similar to MK2. First results to answer this question will be discussed.
References
Engel, K., Kotlyarov, A. and Gaestel, M. (1998) Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation. Embo J, 17, 3363–3371.
Kotlyarov, A., Neininger, A., Schubert, C., Eckert, R., Birchmeier, C., Volk, H.D. and Gaestel, M. (1999) MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat Cell Biol, 1, 94–97.
Neininger, A., Thielemann, H. and Gaestel, M. (2001) FRET-based detection of different conformations of MK2. EMBO Rep, 19, 19.
Structural and functional characterization of rice Hsp100 protein
Manu Agarwal1, Chandan Sahi1, Surekha Katiyar-Agarwal1, Sangeeta Agarwal1, Vishva Mitra Sharma2, Todd Young3, K. Ganesan2, Daniel R. Gallie3 and Anil Grover1,*
1Department of Plant Molecular Biology, University of Delhi South Campus, Dhaula Kuan, New Delhi-110021, India, 2Institute of Microbial Technology, Chandigarh-160036, India and 3Department of Biochemistry, University of California Riverside, USA (*author for correspondence, grover_anil@hotmail.com)
Hsp100/Clp family of proteins is ubiquitously distributed in living systems. Detailed work carried out in bacterial and yeast cells has shown that regulatory members of the Clp family (mainly ClpA, ClpB and ClpC) together with the catalytic subunit (mainly Clp P) comprise an ATP-dependent two-component proteolytic system (Agarwal et al. 2001, Katiyar-Agarwal et al. 2001). Members of the Hsp100/Clp protein family are not only involved in the regulation of energy-dependent protein hydrolysis but also function as molecular chaperones. However, the biochemical/physiological role(s) of the plant Hsp100/Clp protein family in higher plants has yet to be elucidated. Recently, this protein is shown to be involved in providing thermotolerance in Arabidopsis thaliana by mutant analysis and transgenic experiments. This protein has also been implicated in translational regulation of cellular mRNAs. Yeast Hsp104 is shown to be active in the disaggregation of heat-inactivated proteins.
Rice is a major crop for the Southeast Asian countries. High temperature causes pollen and spikelet sterility in rice. The current trend on greenhouse warming may affect rice yield drastically in years to come. Our group is interested in characterizing high temperature response of rice in molecular terms. We have earlier reported that Hsp100 is a predominant stress protein in rice cells (Singla et al. 1993, Pareek et al. 1995). While this protein is high temperature- induced in vegetative organs of rice plant, significant uninduced levels of this protein are found in developing and mature rice grains and further the levels of the uninduced Hsp104 in rice grains decline during the seed germination process (Singla et al. 1998). Full-length rice Hsp100 cDNA has been isolated and sequenced. Based on Southern blotting, it appears that there is a single Hsp100 gene per haploid genome in rice. Northern blotting analysis has shown that Hsp100 transcript level was most intense after 30 min of 42oC and became progressively less even though the stress conditions were maintained. Western blotting analysis showed that in general japonica rices appear to show early accumulation of this protein in response to heat shock and retain higher levels of this protein in the recovery phase. The transformation of (hsp100 yeast cells with rice hsp100 cDNA enabled partial recovery of the thermotolerance defect in yeast cells. Yeast cells complemented with rice hsp100 gene also showed better Cd and Ar stress tolerance. Electron micrographs of (hsp104 yeast cells showed accumulation of denatured proteins and complementation of rice hsp104 cDNA with yeast cells enabled the cells to hydrolyze the protein aggregates. We have made transgenic rice over-expressing hsp100 cDNA. There appears to be no adverse effect of the over-expression of hsp100 cDNA on growth and development process in transgenic rice plants (Grover et al. 2000).
References
1. Singla SL and A Grover. 1993. Antibodies raised against a yeast heat shock protein cross-react with a heat and abscisic acid- regulated polypeptide in rice. Plant Molecular Biology 22: 1177–1180.
2. Pareek A, SL Singla and A Grover. 1995. Immunological evidence for accumulation of two novel 104 and 90 kDa HSPs in response to diverse stresses in rice and in response to high temperature stress in diverse plant genera. Plant Molecular Biology 29: 293–301.
3. Singla SL, A Pareek and A Grover. 1998. Distribution patterns of the 104 kDa stress-associated protein of rice reveal its constitutive accumulation in seeds and disappearance from the just-emerged seedlings. Plant Molecular Biology 37: 911–919.
4. Agarwal M, Katiyar-Agarwal S, Sahi C, Gallie DR and Grover A. 2001. Arabidopsis thaliana Hsp100 protein: Kith and Kin. Cell Stress and Chaperone (in press).
5. Katiyar-Agarwal S, M Agarwal, D Gallie and A Grover. 2001. Search for the cellular functions of plant Hsp100/ Clp family proteins. Critical Reviews in Plant Sciences (in press).
6. Grover A, S Katiyar-Agarwal, M Agarwal, C Sahi, O Satya Lakshmi, H Dubey, S Agarwal and A Kapoor. 2000. Production of Abiotic stress tolerant transgenic rice plants. Proc Third International Rice Genetics Symposium. International Rice Research Institute, Manila, Philippines (in press).
Regulating the mammalian ER stress response
Linda M. Hendershot and Yanjun Ma
Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105 linda.hendershot@stjude.org
Mammalian cells respond to cellular conditions that adversely affect the environment of the ER, and thus inhibit normal maturation of secretory pathway proteins, by activating a signal transduction pathway termed the unfolded protein response (UPR). During the last several years a number of components of this signaling pathway have been identified (1). Two families of transmembrane kinases exist that are proximal sensors of ER stress. One of these, Ire1, is a homologue of the single yeast kinase that responds to ER stress. The second, PERK, is a member of the eIF-2α family of kinases and is responsible for shutting down protein synthesis during ER stress to limit the accumulation of unfolded proteins. We have examined the mechanism by which these two kinases sense ER stress and transduce the signal to downstream elements. Our experiments reveal that the lumenal domains of both kinases are bound to the ER chaperone BiP in the absence of stress. Conditions that alter normal folding in the ER cause BiP to be released from both kinases, perhaps due to the unfolded proteins competing for BiP. Release of BiP results in oligomerization and transactivation of the kinases; a situation that is reversed when stress conditions are resolved (2). Over- expression of exogenous BiP inhibits the activation of the ER kinases in response to stress conditions, further suggesting that levels of free BiP are used to monitor conditions in the ER, and if they fall below a certain threshold the UPR is initiated.
Activation of the mammalian UPR results in the transcriptional up-regulation of most ER chaperones, as well as, the induction of the CHOP transcription factor. Unlike the ER chaperones, CHOP is not constitutively expressed and is a target of many cellular stress pathways that do not affect folding in the ER. Thus, it was unclear if CHOP induction during ER stress occurred through the same pathway used to regulate ER chaperone transcription or through a pathway common to other cellular stress conditions that result in CHOP induction. Our studies reveal that CHOP induction requires the convergence of two pathways. The first is shared with other chaperones and involves an ER stress element (ERSE), which is activated through CBF and ATF-6 binding. The second one is common to other cellular stress pathways and involves the binding of C/EBP and ATF-4 to a C/EBP-ATF composite site. Activation of the ER transmembrane eIF2α kinase, PERK, induced ATF4 protein expression, nuclear localization, direct binding to its composite site, and as a consequence, CHOP protein synthesis. We propose that this eIF-2?- kinase/ATF4/C/EBP-ATF composite site pathway is conserved for CHOP regulation during various cellular stress conditions including ER stress. Our data indicate that both the ERSE and the PERK-ATF pathways converge on the CHOP promoter during ER stress and provide insights into the similarities and differences between CHOP and ER chaperone expression during normal and stress conditions (3).
References
1. Kaufman, R.J. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes & Dev 13:1211–1233, 1999.
2. Bertolotti, A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interactions between BiP and the ER stress receptors IRE1 and PERK in the unfolded protein response. Nature Cell Biology, 2:326–332, 2000.
3. Ma Y, Brewer JW, Diehl JA, Hendershot LM. Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response. Mol. Cell. Biol. in revision.
Stannous chloride, an inducer of Hsp70 and cytoprotection with clinical potential
Lawrence E. Hightower
Dept of Molecular and Cell Biology, University of Connecticut, Storrs, CT USA 06269-3044, Lawrence.Hightower@uconn.edu
The heat shock response protects cells from exposures to temperatures lethal to naïve animals, a phenomenon known as thermotolerance (e.g. Landry and Chretien 1983). Reciprocal and nonreciprocal cross-protection has been observed between pairs of a variety of stressors in addition to heat, and the altered physiological state induced by these stressors has been termed more generally cytoprotection. The existence of cross-protection has suggested a medical application of cytoprotection in which this state is induced pharmacologically prior to elective surgery to protect tissues against ischemia-reperfusion injury. Ischemic injury per se induces cytoprotection in several animal model systems (Currie and White 1981), and the heat shock response protects against ischemic as well as against other forms of metabolic injury (Currie et al 1993). Inflammation is a major component of ischemia/reperfusion injury and previous studies suggest an anti-inflammatory role for Hsp70 in blood vessels. For example, the induction and accumulation of Hsp70 in vascular endothelial cells accompanies the prevention of necrosis induced by activated human polymorphonuclear leukocytes (Wang et al 1995). Furthermore, induction of Hsp70 accompanies a reduction in the number of adherent and migrated leukocytes measured using intravital microscopy in an ischemia/reperfusion model system (Chen et al 1996). This same group showed that lipopolysaccharide-induced microvascular injury was reduced in thermotolerant rats in which leukocyte-endothelial attachment (LEA) and migration decreased (Chen et al 2001). Stannous chloride has been evaluated as a possible inducer of the cellular stress response and cytoprotection in intact rats and cultured human HT-29 cells. Evidence that stannous chloride induces a state of cytoprotection in rat mesentery venules and in cultured cells will be presented along with evidence that this is a potent anti-inflammatory state. Evidence that stannous chloride induces Hsp70 both in rats and in HT-29 cells will be presented. A cell-based assay system will be described, suitable for high throughput screening of chemical libraries, that integrates assays of stress gene expression, cell viability and cell numbers. In the process of developing this assay, a novel human Hsp70 promoter was isolated, sequenced and characterized functionally in induction assays.
Acknowledgements: I would like to acknowledge the contributions of a number of colleagues who made this study possible including: Dr. George Perdrizet and Michael Rewinski at Hartford Hospital, Professor Steven House, Dr. Peter Guidon Jr., and T. Mistry at Seton Hall University, Associate Professor Charles Giardina and Lu Li at the University of Connecticut.
This study was funded by grants from the U.S. Public Health Service and by a contract from Stressgen Biotechnologies Corp.
References:
Chen G, Kelly C, Chen H, Leaky, A, Bouchier-Hayes D. 2001. Thermotolerance protects against endotoxin-mediated microvascular injury. J Surg Res 95:79–84.
Chen G, Kelly C, Stokes K, Leahy A, Bouchier-Hayes D. 1996. Induction of heat shock protein 72 attenuates ischemia/reperfusion-induced microvascular injury. Association for Academic Surgery, 30th Annual Meeting, Chicago, IL. November 13–16, 1996. Abstract #77.
Currie RW, White FP. 1981. Trauma-induced protein in rat tissues: A physiological role for a heat shock protein? Science 214:72–73.
Currie RW, Tanguay RM, Kingma JG. 1993. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation 87:963–971.
House, S.D., Guidon, Jr., P.T , Perdrizet, G., Rewinski, M., Kyriakos, R., Bockman, R.S., Mistry T., Gallagher, P.A., and Hightower, L.E. 2001. Effects of heat shock, stannous chloride, and gallium nitrate on the rat inflammatory response. Cell Stress Chaperones 6(2): 164–171.
Landry J, Chretien P. 1983. Relationship between hyperthermia induced heat shock proteins and thermotolerance in Morris hepatoma cells. Can J Biochem 61:428–437.
Wang JH, Redmond HP, Watson RW, Condron C, Bouchier-Hayes D. 1995. Induction of heat shock protein 72 prevents neutrophil-mediated human endothelial cell necrosis. Arch Surgery 130:1260–1265.
Control of tumuor cell apoptosis by heat shock protein 70
Marja Jäättelä
Apoptosis Laboratory, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark
The major stress-inducible heat shock protein, Hsp70, is a chaperone protein abundantly and preferentially expressed in human tumours and tumour cell lines. Due to its ability to protect cells from a wide range of apoptotic and necrotic stimuli, it has been assumed that Hsp70 may confer survival advantage to tumour cells. To investigate this hypothesis in human tumour cell lines, we generated an adenovirus expressing antisense Hsp70 (Ad.asHsp70). The effective and specific Ad.asHsp70-mediated depletion of Hsp70 resulted in massive apoptosis of all tumourigenic cell lines tested (carcinomas of breast, colon, prostate and liver as well as glioblastoma), but affected neither the survival nor growth of non-tumourigenic epithelial cells or fibroblasts. Inhibitors of caspases failed to rescue tumour cells from Ad.asHsp70-induced cell death suggesting that Hsp70 silences a caspase-independent death pathway in tumour cells. In line with this, Hsp70 effectively protected tumour cells from caspase-independent apoptosis induced by death receptors and Hsp70-mediated protection from caspase-dependent apoptosis occurred downstream of caspase activation. These results indicate that the high expression of Hsp70 is a prerequisite for the survival of human cancer cells of various origins and reveal Hsp70 as an important regulator of caspase-independent apoptosis.
References
Jäättelä M, Wissing D, Kokholm K, Kallunki T, Egeblad M. Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J 17:6124–6134, 1998.
Jäättelä M. Escaping cell death; survival proteins in cancer. Exp Cell Res 248:44–57, 1999.
Nylandsted J, Rohde M, Brand K, Bastholm L, Elling F, Jäättelä M. Selective depletion of Hsp70 activates a tumor-specific death program that is independent of caspases and bypasses Bcl-2. Proc Natl Acad Sci USA 97:7871–7876, 2000.
Leist M, Jäättelä M. Four deaths and a funeral: from caspases to alternative mechanisms. Nature Rev Mol Cell Biol 2: 589–598, 2001
Hsp27 Overexpression in a human melanoma cell line affects the metastatic phenotype in vitro
I. Kindas-Mügge, S. Aldrian, I. Fröhlich, F. Trautinger.* M. Micksche
Institute of Cancer Research, * Dept of Dermatology, University of Vienna, Austria. ingela-margaret.kindas-muegge@univ.ac.at
The small heat shock protein hsp27 acts as a molecular chaperone and has a variety of functions including roles in signal transduction, regulation of cell growth (1,2,3), differentiation (4) and tumorigenesis.
Overexpression of the small heat shock protein hsp27 has been shown to modify the in vitro proliferation rate and the tumorigenicity of a human melanoma (A375) in nude mice (1). We hypothesised that hsp27 may influence the malignant phenotype. In the present study, hsp27 transfectants of this cell line were analysed for various cellular aspects associated with the metastatic process. Cell lines transfected only with the plasmid for neomycin and the wild type of A375 were used as controls.
We found that hsp27 overexpressing clones exhibited an altered morphology compared to control transfected cells. Hsp27 transfected cells tended to grow in more clusters, exhibiting a plasma membrane ruffling and a lamellipodia-like structure. Control transfected cells displayed a more elongated structure with stressfibers extended through the cytoplasm. Similarly, Lanoie et al (−93), Mairesse et al (−96), Lemieux et al (−97) found a cytoskeleton reorganization by hsp27 overexpression.
To study the intracellular distribution of actin, immunofluorescense microscopy was performed using FITC-conjugated phalloidin. Overexpression of hsp27 caused increased concentration of F-actin, found at the cell cortex. In contrast, control cells showed low cortical F-actin concentration and stained more cytoplasmic stress fibers.
The invasive potential was studied in vitro by use of a reconstituted extracellular matrix coated filter (Boyden Chambers). In this model, cells are assessed for their ability to penetrate into or through a complex biological matrix; an activity which has been shown to correlate with the ability to form metastatic tumor in vivo. Compared to control transfected cells, hsp27 overexpressing cells showed a decreased cell invasiveness, probably due to reduced cell motility and lower digestion of the extracellular matrix by proteinases.
Interaction with the extra cellular matrix is mediated primarily through the integrin class of cell adhesion molecules. These cell surface receptors are heterodimers and are made up of α and β subunits. It has been observed that (vβ3 expression is restricted to melanoma and its appearance coincides with progression to the invasive phase. FACS analysis with anti-integrin specific mAbs to (vβ3 subunit, expressed on melanoma cells revealed a significant changes in the integrin expression. Control cells expressed high levels of (vβ3 whereas hsp27 transfected cells expressed essential background levels, which support the data that hsp27 transfectants show a redused motility and invasion through a reconstituted basement membrane. However, analysis with (vβ5 anti-integrin specific mAb did not reveal any significant changes in integrin expression.
Matrix metalloproteinases are a family of neutral peptidases capable of degrading essentially extracellular matrix (ECM) components. MMP-2 and MMP-9 belongs to the gelatinase family and play an important role in the progression, invasion and metastases formation of various cancers. Secretion of MMPs was studied by traditional gelatin-substrate zymogram analysis as well as with a sensitive gelatinase activity assay, a microtiter plate based screening for measuring these MMPs. Analyses with both methods revealed that hsp27 transfected A375 melanoma cell line showed decreased secretion of MMP-2 and MMP-9 compared to control transfected cells.
Our results demonstrate that hsp27 overexpression may influence the invasive and metastatic potential of a human melanoma cell line in vitro. Local/regional application of specific agents or delivery of genes for induction of hsp27 may provide a concept for development of new strategies for treatment of solid malignances.
References
1. Modification of growth and tumorigenicity in epidermal cell lines by DNA-mediated gene transfer of 27,000 heat shock protein (hsp27): Kindas-Mügge, I., Herbacek, I., Jantschitsch, Ch., Micksche, M., Trautinger, F. (1996) Cell Growth & Diff. 7, 1167–1174.
2. Modification of growth in small heat shock (hsp27) gene transfected breast carcinoma. Kindas-Mügge, I., Micksche, M., Trautinger, F., (1998) Anticancer Research, 18, 413–418.
3. Overexpression of the small heat shock protein hsp27 confers resistance to hyperthermia, but not to oxidative stress and UV-induced cell death, in a stably transfected squamous cell carcinoma cell line. (1997) J Photochem. and Photobiol. B, Biology 39, 90–95.
4. Expression of the small heat shock protein hsp27 in developing human skin. Jantschitsch, J., Kindas-Mügge, I., Metze, D., Amann, G., Micksche, M., Trautinger, F. (1998) Brit J Dermatol, 139, 247–253.
Early heat shock signal transduction through the p38 MAP kinase pathway
Jacques Landry, Steve J. Charette, Sonia Dorion, Jimmy R. Thériault and H. Lambert
Centre de recherche en cancérologie de l'Université Laval, CHUQ-HDQ, Québec City, Canada G1R 2J6 Jacques.landry@med.ulaval.ca
The cell response to heat shock (HS) represents one of the most spectacular examples of the capacity of the cell to react to stressful environment. Not only does HS induce a new transcriptional activity that leads over a period of hours to the accumulation of the heat shock proteins (HSP) and the development of a state of extreme thermotolerance, but HS also activates within minutes several signaling pathways likely involved in generating immediate homeostatic responses. One of these pathways involves the MAP kinase p38 that activates MAPKAP kinase-2, eventually leading to the phosphorylation of HSP27 and the activation of its thermoprotective activity.
Classical desensitization experiments using HS suggest the existence of very specific HS sensing elements upstream in the signaling cascade leading to p38 activation. HS treatments induce a complete desensitization of p38 activation by HS but have no effect on the activation of the pathway by several other stressing agents tested, including oxidative stress. In response to HS as well as oxidative stress, p38 is activated/phosphorylated by the MAP kinase kinases MKK3/6, itself activated/phosphorylated by the MAP kinase kinase kinase Ask1. Ask1 represents the converging point of these two pathways being activated by different mechanisms during HS and oxidative stress. Activation of Ask1 by HS does not involve the oxidation of the Ask1-repressor thioredoxin, as it is the case during oxidative stress. Instead, it involves the release from Ask1 of its repressor GSTmu1 and the activation of an upstream activator PAK.
Downstream of the p38 pathway, it is not totally clear which function of HSP27 is activated by phosphorylation, although several pieces of evidence suggest that phosphorylation regulates a function involved in protection. Upon phosphorylation, HSP27 dissociates from large oligomers of some 24 subunits into dimers, thereby increasing several-fold its protective activity. Results suggest that phosphorylation-induced dissociation causes the unmasking of a protection domain that is located at the extreme N-terminus and normally buried inside the oligomers. We have identified a protein named Daxx, which interacts strictly with phosphorylated/dimeric HSP27, and thus could be a target of its protective activity. During HS or as a consequence of the activation of the pro-apoptotic pathway Fas, Daxx translocates from the nucleus to the cytoplasm where it can enhance caspase-dependent cell death and mediates, through an interaction with Ask1, a novel caspase-independent cell death process. By binding to Daxx, phosphorylated HSP27 block its translocation to the cytoplasm and its interaction with Ask1. Phosphorylation of HSP27 and rapid inhibition of Daxx-mediated cell death may represent an early homeostatic mechanism activated downstream of p38 during heat shock.
Supported by the Canadian Institutes of Health Research.
References
Charette, S.J., H. Lambert, and J. Landry. 2001. A kinase-independent function of Ask1 in caspase-independent cell death. J Biol Chem. In press.
Charette, S.J., J.N. Lavoie, H. Lambert, and J. Landry. 2000. Inhibition of daxx-mediated apoptosis by heat shock protein 27. Mol Cell Biol. 20:7602–7612.
Dorion, S., J. Berube, J. Huot, and J. Landry. 1999. A short lived protein involved in the heat shock sensing mechanism responsible for stress-activated protein kinase 2 (SAPK2/p38) activation. J Biol Chem. 274:37591–37597.
Lambert, H., S.J. Charette, A.F. Bernier, A. Guimond, and J. Landry. 1999. HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus. J Biol Chem. 274:9378–9385.
Mechanisms of transient thermotolerance and permanent heat-resistance in mammalian cells
Andrei Laszlo, Kenzo Ohtsuka*, Mary Stevenson and Teri Davidson
Section of Cancer Biology, Department of Radiation Oncology, Washington University School of Medicine, St. Louis MO USA, *College of Bioscience and Biotechnology, Chubu University, Aichi Japan
Exposure of mammalian cells to elevated temperatures leads to their death in a time/dose dependent fashion. The exact mechanisms that are involved in this process are yet to be elucidated. Exposure of cells to mild stresses that induce the heat shock response, including a brief heat-shock, sodium arsenite, ethanol, etc. leads to the development of transient thermotolerance, the ability to survive otherwise lethal heat treatments. There are at least two types of thermotolerance: protein synthesis dependent and protein synthesis independent thermotolerance (1). Protein synthesis independent thermotolerance can only be induced by heat-shock but not other inducers of the heat shock response. The heat shock proteins have been implicated to play a role in the protein synthesis dependent thermotolerance and several heat resistant mutants have been isolated which express elevated levels of various heat shock proteins (2).
One of the well-studied functions of heat shock proteins has been their activity as molecular chaperones. As molecular chaperones, the heat shock proteins can aid in the folding of newly synthesized proteins, can prevent heat-induced aggregation of proteins and assist in the refolding of chemically and thermally denatured proteins. We have been investigating the relationship between chaperoning activity and resistance to heat-induced cell killing using luciferase molecules that are targeted to the cytoplasm (cyt-luc) or the nucleus (nuc-luc) as a model reporter system (3).
We have examined the heat-induced inactivation of cyt-luc and nuc-luc in transiently thermotolerant cells and permanently heat-resistant cells. Protection from heat-induced inactivation of both cyt-luc and nuc-luc was observed in both types of transiently thermotolerant cells; the magnitude of such protection was greater in cells in which protein synthesis dependent thermotolerance was expressed. The protective effect against heat-induced inactivation of cyt-luc and nuc-luc decayed faster than clonogenic thermotolerance in both types of thermotolerant cells. We have also examined the heat-induced inactivation of cyt-nuc and nuc-luc in two permanently heat resistant cell lines, HR-1 and OC-14. The mechanism of permanent of heat resistance involves the overexpression of hsc70 in HR-1 cells, and is unknown in OC-14 cells. Surprisingly, neither cyt-luc nor nuc-luc was protected from heat-induced inactivation in these permanently heat-resistant cell lines. Our results indicate that there may be a complex relationship between inherent chaperoning activity and the magnitude of heat-induced cell killing.
Supported by CA-49018.
References:
1. Laszlo A (1988) Evidence for two states of thermotolerance in mammalian cells. Int. J. Hyperthermia 4:513–526.
2. Laszlo A and Venetianer A (1998) Heat resistance in mammalian cells: lessons and challenges in “Stress of Life: adaptation from Molecules to Man” (P.Chermely, ed.). Annals of the New York Acad of Sci. 851: 169–179.
3. Michels AA, NguyenV-T, Konings AWT, Kampinga HH and Bensaude O (1995) Thermostability of nuclear-targeted luciferase expressed in mammalian cells: Destabilizing influence of the intranuclear environment. Eur. J. Biochem 234:383–389.
The heat shock response, heat shock proteins and chaperones: an introduction and a brief overview
Andrei Laszlo
Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA
The field of heat shock and stress proteins is one of the most exciting areas of contemporary biological and biomedical research. The purpose of the introductory course is to offer some background information for the participants in the workshop concerning the topics that will be covered in the various symposia. The field is very complex, and therefore this introduction will only include a brief history of the heat shock/stress response and a brief overview of the function of the heat shock/stress proteins, with emphasis on chaperoning and medical aspects.
The heat shock response was discovered in 1962 by F. Ritossa in the salivary glands of the fruit fly. In 1974–75 H. Mitchell and M. Ashburner demonstrated that heat shock induced the expression of a set of new proteins under these conditions. These new proteins were called the “heat shock proteins”. The induction of this response was then confirmed in every organism testes so far, including thermophilic bacteria. Since the response can be induced by agents other than heat, the heat shock proteins are now also called “stress proteins”.
In general, there are several classes of heat shock proteins, categorized according to their apparent subunit molecular weights. There is the so called “small hsp” family, with molecular weights ranging from 14–29 kDa, the hsp 40 family, the hsp 60 family, the hsp 70 family, the hsp 90 family and the hsp110 family. In this part of the introductory course, the members of the various families will be described. Various aspects of the biology of the members of the individual heat shock protein families, including their inducers, cellular localization, functions in cellular physiology and their biochemistry in both prokaryotes and eukaryotes will be reviewed. The medical aspects of the heat shock proteins will also be briefly reviewed.
Starting in the middle 1980's, the concept of molecular chaperones evolved from the work of biochemists and cell biologists. The heat shock proteins were soon recognized as having such chaperoning functions. The details of chaperoning functions of the heat shock proteins were established in E. coli. These studies have discovered that there are two major chaperone machineries in this organism, the GroEL and DnaK systems. Similar chaperone systems have been found in eukaryotic cells. In addition, there are chaperone systems in eukaryotic cells that do not involve heat shock proteins. In the second part of the course, the details of the biochemistry and cellular biology of several chaperone systems will be reviewed briefly.
References:
The Heat Shock Response. L. Nover, CRC Press, Boca Raton, 1991.
The Biology of heat shock proteins and molecular chaperones. R. Morimoto, A. Tissières, C Georgopoulos (eds.) Plainview, N.Y.: Cold Spring Harbor Laboratory Press, 1994.
The chaperonins. R. J. Ellis (ed.) San Diego, Academic Press, 1996.
Stress-inducible cellular responses/U Feige (ed.) Boston, Birkhauser Verlag, 1996.
Stress proteins in medicine. W. van Eden and D.B.Young (eds.). New York: Marcel Dekker, 1996.
Molecular chaperones in the life cycle of proteins: structure, function, and mode of action. A.L. Fink and Y. Goto (eds.) New York, Marcel Dekker, 1998.
Bacterial stress responses. G. Storz and R. Hengge-Aronis (eds.) Washington, D.C., ASM Press, 2000.
Immunological significance of cell surface heat shock protein grp94/gp96
Zihai Li
Center for Immunotherapy of Cancer and Infectious Diseases, MC 160, Univeristy of Connecticut School of Medicine, 263 Farmington Avenue, Farmington, CT 06030-1601, U.S.A. zli@up.uchc.edu
Gp96, or grp94, is predominantly a lumenal protein of the endoplasmicreticulum (ER). It has long been observed that gp96/grp94 can be displayed on the cell surface and secreted during stress and in certain physiological conditions (1–3). However, the significance of extracellular expression of gp96/grp94 remains undefined. Recent development in immunology has witnessed that soluble grp94/gp96 binds to the surface of antigen presenting cells (APCs) in a receptor dependent manner (one of the receptors is CD91), and, in doing so, it facilitates the transfer of gp96/grp94-associated peptides from the extracellular compartment to the major histocompatibility complex (MHC) class I molecules to mediate priming of naïve CD8+ T lymphocytes (4-5). This phenomenon leads us to predict that, (1) cell surface expression of gp96/grp94 has physiological bearings; (2) surface expression of gp96/grp94 on tumor cells might enhance the interaction of tumor cells with APCs through engagement with a gp96 receptor, thereby enhancing tumor-specific T cell immunity.
By targeting gp96/grp94 to the cell surface of tumor cells via fusion to a transmembrane domain at the carboxyl terminus (6), we have indeed found that direct accessibility of gp96/grp94 to the immune system induces potent anti-tumor immunity in an otherwise poorly immunogenic murine tumor model. We further showed that gp96/grp94 surface-expressing tumor cells activate dendritic cells in vitro, and prime CD8+ T cells in vivo in a tumor-specific manner. These data supports the notion that gp96/grp94 is one of the many emerging endogenous adjuvants, and establishes a principle of bridging innate and adaptive immunity for potential cancer immunotherapy based on surface expression of a heat shock protein in tumor cells. We propose that surface expression of gp96/grp94 serve as a sensor for cellular “stress” resulted from both internal and external insult (infection, transformation, etc.) and thus act as a signal for the adaptive immunity. Since non-conventional extracellular export of HSPs is not restricted to gp96/grp94, it is prudent to study the basis of plasticity of HSPs in cellular transport, and to determine if “ectopic” expression of HSPs may contribute to immunoregulation in both physiological and pathological conditions.
References:
1. Booth, C. & Koch, G.L. Perturbation of cellular calcium induces secretion of luminal ER proteins. Cell 59, 729–37. (1989).
2. Altmeyer, A. et al. Tumor-specific cell surface expression of the KDEL-containing, endoplasmic reticular heat shock protein gp96. Int J Cancer 69, 340–9. (1996).
3. Wiest, D.L. et al. Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of “ER-resident” molecular chaperones. Proc Natl Acad Sci U S A 94, 1884–9. (1997).
4. Binder, R.J., Han, D.K. & Srivastava, P.K. CD91: a receptor for heat shock protein gp96. Nat Immunol 1, 151–5. (2000).
5. Basu, S., Binder, R.J., Ramalingam, T. & Srivastava, P.K. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 14, 303–13. (2001).
6. Zheng, H, Dai, J., Stolilova, DM & Li, Z. Cell surface targeting of an intracellular heat shock protein gp96 induces dendritic cell maturation and anti-tumor immunity. J Immunol (2001), submitted.
Heat shock response in Caulobacter crescentus: regulation and function
Suely Lopes Gomes, Antonio Carlos Almeida da Silva and Marcelo Avedissian
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, São Paulo, SP, 05513-900, Brasil sulgomes@iq.usp.br
Caulobacter crescentus is an aquatic Gram-negative bacterium, member of the (-subdivision of proteobacteria. At each cell division cycle, Caulobacter yields two different progeny : a flagellated chemotactically competent swarmer cell and sessile stalked cell. These two progeny cells differ not only in morphology but also in their genetic programmes and ability to initiate DNA replication (1).
Several heat shock-inducible genes have been characterized in Caulobacter and they all present σ32-like promoters. Interestingly, the groESL operon presents both a σ32 promoter and a CIRCE element. However, the nine-nucleotide inverted repeat and the HrcA repressor protein which binds to it are involved in cell cycle regulation of the operon, but not in heat shock induction (2). The gene encoding the Caulobacter σ32 homolog has also been characterized and one of its promoters (P2) has been shown to be σ32-dependent (3).
In this work, we have investigated the role of dnaKJ in the shutoff of the heat shock response. Caulobacter strains with different levels of DnaK and DnaJ were constructed and data obtained showed that, even in the absence of DnaK and DnaJ, the heat shock response is still transient, indicating that the turning off of the response is not dependent on DnaKJ levels. Furthermore, dnaKJ operon is essential in Caulobacter, null mutants being unable to grow at low (17°C), intermediate (30°C) or high (37°C) temperature. A dnaKJ conditional mutant was obtained in which the expression of the operon is dependent on the presence of xylose. When cells are grown in the absence of xylose, the amount of DnaK and DnaJ decreases to undetectable levels and these cells are quite sensitive to heat stress and unable to acquire thermotolerance.
References
1. Hung, D.; McAdams, H & Shapiro, L. (1999). Regulation of the Caulobacter Cell Cycle. In: Microbial Development (Y. Brun and L. Shimkets, eds.) ASM Press.
2. Baldini, R.L.; Avedissian, M. & Gomes, S.L. (1998). The CIRCE element and its putative repressor control cell cycle expression of the Caulobacter crescentus groESL operon. J. Bacteriol 180: 1632–1641.
3. Wu, J. H. & Newton, A. (1997). The Caulobacter heat shock sigma factor gene rpoH is positively autoregulated from a sigma 32-dependent promoter. J. Bacteriol. 179: 514–521
Characterization of Heat shock Factor 2-deficient mice
Yunhua Chang1, Martine Manuel1, Marko Kallio2, Murielle Rallu1, Marie-Thérèse Loones1, Valérie Mezger1, Lea Sistonen2 and Michel Morange1
1 Laboratoire de Biologie Moléculaire du Stress, Unité de Génétique Moléculaire UMR8541, Ecole Normale Supérieure, 75230 Paris cedex 05, France morange@wotan.ens.fr; 2 Turku Center for Biotechnology, University of Turku, Abo Akademi University, Tykistökatu 6B, FIN-20520 Turku, Finland
HSF2 belongs to the heat shock factor family, transcriptional regulators of heat shock genes. It has been previously shown to be expressed during spermatogenesis and ex vivo differentiation of the human erythroleukemia K562 cell line. It is also abundant during the first part of embryogenesis. Its expression becomes later restricted to the ventricular zone of the developing neural tube. However, its functions and targets have so far remained obscure: there is no obvious correlation during development between the level of HSF2, and the level of expression of heat shock proteins (HSPs). We generated a mouse line in which the Hsf2 gene was inactivated by homologous recombination. The □-geo gene was placed in phase with the 5'end of the Hsf2 gene, which allows to record the pattern of Hsf2 gene expression from □-Galactosidase activity in heterozygous or homozygous animals. Hsf2-deficient mice are viable and apparently normal. However, they suffer from brain abnormalities characterized by the enlargement of lateral and third ventricles. Immunolocalization and □-galactosidase expression profile reveal that Hsf2 is expressed in the proliferating layer of the neuroepithelium in embryos; its expression is maintained in adults in some discrete cells of the ependymal layer. Interestingly, fibroblasts from Hsf2-/- mice exhibit also decreased proliferative capacities, suggesting that HSF2 might be involved in cell proliferation. HSF2 knockout mice are also affected in spermatogenesis. Many developing spermatocytes are eliminated through apoptosis at the late pachytene phase of meiotic prophase and during meiotic divisions. Analysis of the pachytene spermatocytes revealed structural defects in the synaptonemal complexes between homologous chromosomes. The observed defects may lead to the activation of the synapsis checkpoint of spermatogenesis. HSF2-deficient females are subfertile and exhibit multiple reproduction problems: dysovulation associated with abnormally elevated levels of luteinizing hormone-receptor mRNAs, disturbance of the estrus cycle and of the corpora lutea function. HSF2-deficient prepubere females can be induced to ovulate by hormonal treatment, but mainly produce abnormal ova, revealing meiotic abnormalities. HSF2 expression in the primordial germ cells, but not in the adult ovary, suggests that this meiotic defect could occur at the first meiotic division in female fetuses.
Many questions remain unsolved:
1. What are the mechanisms by which the deficiency in HSF2 affects brain formation, ovulation in females and spermatogenesis in males? Is a modification of HSP expression involved in these alterations?
2. What is the physiological meaning of the involvement of HSF2 in brain formation and reproduction?
3. Is there a partial redundancy with the other HSF which would mask other functions of HSF2?
References
Alastalo, T.P., Lonnstrom, M., Leppa, S., Kaarniranta, K., Pelto-Huikko, M., Sistonen, L. and Parvinen, M. (1998) Stage-specific expression and cellular localization of the heat shock factor 2 isoforms in the rat seminiferous epithelium. Exp Cell Res, 240, 16–27.
Chang, Y., Manuel, M., Rallu, M., Loones, M.-T., Gitton, Y., Larney, S., Hiard, S., Morange, M. and Mezger, V. Multiple female fertility defects and brain abnormalities in heat shock factor 2-deficient mice. Submitted.
Kallio, M., Alastalo, T.-P., Manuel, M., Pirkkala, L., Morange, M., Mezger, V. and Sistonen, L. Defective synapsis of meiotic chromosomes and increased apoptosis in testis of heat shock factor 2 knockout mice. Submitted.
Manuel, M., Sage, J., Mattei, M., Morange, M. and Mezger, V. (1999) Genomic structure and chromosomal localization of the mouse Hsf2 gene and promoter sequences. Gene, 232, 115-124.
Nilson, J.H., Abbud, R.A., Keri, R.A., Quirk, C.C. 2000. Chronic hypersecretion of luteinizing hormone in transgenic mice disrupts both ovarian and pituitary function, with some effects modified by the genetic background. Recent Prog Horm Res, 55, 69–91.
Rallu, M., Loones, M., Lallemand, Y., Morimoto, R., Morange, M. and Mezger, V. (1997) Function and regulation of heat shock factor 2 during mouse embryogenesis. Proc Natl Acad Sci U S A, 94, 2392–2397.
Regulation of Nitric Oxide-mediated Apoptosis by Molecular Chaperones
Masataka Mori, Tomomi Gotoh, Seiichi Oyadomari and Kazutoyo Terada
Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan masa@gpo.kumamoto-u.ac.jp
Excess nitric oxide (NO) induces apoptosis in some cell types, including macrophages and pancreatic (-cells. Heat shock protein of 70 kDa (hsp70) has been reported to protect cells from apoptosis. Several cytosolic DnaJ homologs, partner chaperones of hsp70 family members, have been identified in mammals. They include dj1 (hsp40/hdj-1) and dj2 (HSDJ/hdj-2). We asked whether these chaperones are required to prevent NO-mediated apoptosis. When mouse macrophage-like RAW 264.7 cells were treated with an NO donor SNAP, apoptosis occurred. This apoptosis could be prevented by pretreatment of the cells with heat or a low dose of SNAP. Under these conditions, hsc70 remained unchanged, whereas hsp70 and dj1 were markedly induced and dj2 was moderately induced. In transfection experiments, hsp70, hsc70, dj1 or dj2 alone was ineffective in preventing NO-mediated apoptosis. In contrast, both dj1 and dj2, in combination with hsc70 or hsp70, prevented the cells from apoptosis. We also found that hsp70-DnaJ chaperone pairs exert anti-apoptotic effects upstream of caspase 3 activation and also upstream of cytochrome c release from mitochondria.
Cytokine-induced NO causes pancreatic (-cell dysfunction leading to type 1 diabetes. It is generally thought that NO induced apoptosis is mediated by the DNA damage-p53 pathway. However, we found that NO depletes endoplasmic reticulum (ER) Ca2+ in mouse MIN6 (-cells, causes ER stress, and leads to apoptosis through the ER stress pathway. CHOP, a C/EBP homologous protein which is induced by ER stress and plays a role in growth arrest and cell death, was induced in MIN6 cells by a NO donor S-nitroso-N-acetylpenicillamine (SNAP) and the cells underwent apoptosis. Overexpression of calreticulin increased the Ca2+ capacity of ER and afforded protection to cells against NO-mediated apoptosis. Islets from CHOP knockout mice showed resistance to NO. We conclude that the ER stress pathway is involved in early steps of NO-mediated apoptosis in pancreatic (-cells. Signal transduction from the CHOP induction to mitochondria remains to be elucidated.
References
1. Gotoh T and Mori M. 1999. Arginase II down-regulates NO production and prevents NO-mediated apoptosis in murine macrophage-derived RAW264.7 cells. J Cell Biol 144: 427–434.
2. Terada K and Mori M. 2000. Human DnaJ homologs dj2 and dj3, and bag-1 are positive cochaperones of hsc70. J Biol Chem 275: 24728–24734.
3. Gotoh T, Terada K and Mori M. 2001. hsp70-DnaJ chaperone pairs prevent nitric oxide-mediated apoptosis in RAW 264.7 macrophages. Cell Death Differ 8: 357–366.
4. Oyadomari S, Takeda K, Takiguchi M et al. 2001. Nitric oxide-induced apoptosis in pancreatic (-cells is mediated by the endoplasmic reticulum stress pathway. Proc Natl Acad Sci USA, in press.
Hsp70-peptide activated autologous NK cells in the immunotherpay of cancer - a clinical pilot study
Gerald Thonigs, Mathias Gehrmann, Catharina Groβ, Markus Hantschel, Robert Gastpar, Stefan Krause, Reinhard Andreesen, Hans-Jochem Kolb*, Torsten Haferlach*, Wolfgang Hiddemann*, and Gabriele Multhoff
Dpt. of Hematology/Oncology, University Hospital Regensburg; Dpt. of Medicine III, University Hospital Grosshadern, LMU Munich; gabriele.multhoff@klinik.uni-regensburg.de
Heat shock proteins (HSP) fulfill important functions including chaperoning proteins during synthesis, folding, assembly and degradation under pysiological conditions and follwing stress. Beside their chaperoning functions membres of the Hsp60, 70 and 90 families play key roles in cancer immunity. On the one hand they act as carrier molecules for tumor-deried peptides on the other hand they function as chaperokines and thus induce a non-specific immunostimulation. Our group demonstrated an unusual plasma membrane expression of Hsp70 on tumor cells that acts as a target recognition structure for Natural Killer cells (NK cells, 1). As previously shown an incubation of NK cells with Hsp70 protein or a 14-mer Hsp70-peptide stimulates both the proliferation and cytolytic activity against Hsp70 positive tumors, in vitro (2,3). An immunoreconstitution of tumor-bearing mice with Hsp70-peptide activated NK cells results in tumor regression (4). In total 570 different tumor biopsies and bone marrow aspirates of leukemic patients have been screened for Hsp70 membrane expression (5). Especially lung, colorectal, pancreas cancer and leukemic blasts have been defined as Hsp70 positive. Therefore, patients with these tumors were included in our first clinical trial. Peripheral blood mononuclear cells (PBMC) of patients with Hsp70 positive tumors were isolated by leukapheresis followed by Ficoll separation. Then PBMC were transferred into tissue culture bags and incubated for 4 days with Hsp70-peptide (cGMP-grade) plus low dose IL-2 (100 IU/ml) in serumfree X-Vivo 20 medium (GMP-grade). Following two washing steps the activated cells were reinfused on day 4. So far, 6 patients suffering from solid tumors and 4 leukemic patients have been treated. None of the patients showed any negative side effects. An activation of NK cells as determined by cell surface markers and in functional assays was observed in all patients.
References
1. Multhoff G, Botzler C, Jennen J, et al. Hsp cell surface expression on colon carcinoma cells correlates with the sensitivity to lysis mediated by NK cells. J Immunol 158: 4341–4350, 1997.
2. Multhoff G, Mizzen L, Winchester C, et al. Hsp70 stimulates proliferation and cytolytic activity of NK cells. Exp Hematol 27: 1627–1636, 1999.
3. Multhoff G, Pfister K, Gehrmann M, et al. A 14-mer Hsp70 peptide stimulates NK cell activity, Cell Stress & Chaperones, in press.
4. Multhoff G, Pfister K, Botzler C, et al: Adoptive transfer of human NK cells in mice with severe combined immunodeficiency inhibits growth of Hsp70 expressing tumor cells. Int J Cancer 88: 791–797, 2000.
5. Hantschel, Pfister K, Jordan A, et al. Hsp70 plasma membrane expression on primary biopsy material and bone marrow of leukemic patients. Cell Stress and Chaperones 5: 438–442, 2000
Molecular mechanisms of drug resistance in cancer
Silvina B. Nadin
Institute of Experimental Medicine and Biology of Cuyo, Regional Center for Scientific and Technological Research, C.C. 855, (5500) Mendoza, Argentina. snadin@lab.cricyt.edu.ar
Chemotherapy is one of the most common therapies used against cancer. Unfortunately, the antineoplastic drug resistance is one of the most important causes of failure in cancer treatment. Drug resistance could be defined as the absence of a tumor size reduction after the initial treatment (innate drug resistance) or a recurrence after a positive response to the antineoplastic treatment (acquired drug resistance). There are two possibilities to overcome these barriers: developing new treatments and/or applying the actuals more efficiently. In both cases, it is essential to study the molecular mechanisms of resistance. It is important to examine the drug pharmacokinetics, the drug release from the blood vessels to the tumor tissue, the interaction of the antineoplastic drugs with other chemical compounds, etc.
Here, we will see the principal molecular mechanisms involved in drug resistance: altered intracellular accumulation (P-gp, MRP, LRP, TAP, Methalotioneins), altered metabolism (glutation, glutation-S-transferase) and target molecules of the drugs (topoisomerases, dihidrofolate reductases); also, the altered repair capacity of the DNA damage (NER, BER, MMR, p53) and apoptosis (BCL-2 family, p53). Furthermore, there are genes or molecules with not well known mechanism of action like the heat shock proteins (hsps), and c-erbB-2 (HER-2/neu), that have been associated with altered drug resistance.
References
1. Links M., Brown R. Clinical Relevance of the Molecular Mechanisms of Resistance to anti-cancer Drugs. Expert Reviews in Molecular Medicine, October 1999.
2. Kerbel R.S. New targets, drugs, and approaches for the treatment of cancer: an overview. Cancer Metastasis Rev 1998 Jun 17(2):145–7.
3. Robert, J. Multidrug resistance in oncology: diagnostic and therapeutic approaches. Eur. J. Invest., 29:536–545, 1999.
4. Litman T., Druley T.E., Stein W.D., Bates S.E. From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance. Cell. Mol. Life Sci. 58(2001):931–959.
5. Ciocca D.R., Vargas Roig L.M. Heat Shock Proteins and Drug Resistance in Breast Cancer. Bernal S.D., ed. Drug Resistance in Oncology. Dekker M., New York 1997; pp. 167–190.
Cross-presentation of human shared tumor antigen by dendritic cells: dual function of HSP70 as chaperone for tumor-derived T cell epitopes and cytokine for DC maturation
E. Noessner1, R. Gastpar2,*, V. Milani2,3, M. C. Kuppner3, R.D. Issels2,3
1GSF-National Research Institute for Environment and Health, Institute of Molecular Immunology; 2Clinical Cooperation Group Hyperthermia, Ludwig-Maximilians-University and GSF; 3Klinikum Groβhadern, Medical Clinic III, Ludwig-Maximilians-University; all at 81377 Munich, Germany; *present address: Klinikum University of Regensburg/Hematology and Oncology, 93053 Regensburg, Germany. E.N. (noessner@gsf.de); R.G. (RobertGastpar@web.de); V.M. (milani@gsf.de); M.C.K. (kuppner@gsf.de); R.D.I. (issels@med3.med.uni-muenchen.de)
Pioneering work by the group of Srivastava demonstrated that heat shock protein family members, Hsp70 and gp96, isolated from murine tumors carry tumor specific peptides that instruct the immune system of immunized mice to generate tumor specific T cell response able to protect vaccinated animals from tumor growth upon rechallenge with the same tumor (1). These observations provided the basis for a new type of antigen specific vaccine against cancer that does not require the knowledge of tumor antigens that, for most tumor types, is yet in its infancy. The mechanisms involved in the stimulation of T cell responses via Hsp70 and gp96 were studied in murine systems using induced tumors and model antigens, ovalbumin and viral antigens (2). These analyses provided the principle knowledge of Hsp-mediated cross-presentation and the involvement of antigen presenting cells (APC) (3). These systems, however, differ from the situation of human cancer in that they involve highly immunogenic antigens either induced by mutagenesis or overexpressed by transfection. To obtain information relevant for the clinical application of HSP-based vaccines we established a system that most closely resembled the patient situation. HSP70-peptide complexes (HSP70-PC) were isolated from two human melanoma cell lines that express both the constitutive and the inducible form of Hsp70 under normal growth condition but differ in the natural expression of tyrosinase. Tyrosinase is an endogenously expressed non-mutated tumor associated differentiation antigen that is shared among tumors of the melanocytic lineage. Isolated HSP70-PC was tested for surface binding to different human APC. As described recently, CD14+ monocytes bound HSP70-PC; however, far stronger binding was observed for immature (CD14-, CD83-) and mature (CD14-, CD83+) dendritic cells (DC). For functional assays involving cross-presentation of HSP70-chaperoned peptides and T cell stimulation human immature DC were selected because they are highly efficient in antigen uptake and have a strong capacity to process peptide fragments to the size required for binding to MHC class I. Immature DC after incubation with HSP70-PC from tyrosinase positive (HSP70-PC/tyr+) but not from tyrosinase negative (HSP70-PC/tyr-) melanoma cells showed specific and concentration dependent activation of an HLA-A2 restricted tyrosinase-peptide specific T cell clone. Receptor-mediated binding, uptake of HSP70-PC and intracellular transport were required for efficient class I restricted cross-presentation. HSP70 chaperoned the antigenic peptide derived from the tyrosinase protein providing the antigenic signal, as well as the signal for DC maturation. The resulting T cell stimulation greatly exceeded that achieved with exogenously added synthetic tyrosinase peptide. Our results describe for the first time HSP70-PC mediated crosspresentation of a non-mutated naturally expressed human tumor antigen by human DC. These observations are of special clinical interest since hyperthermia has been found to be effective if integrated within a loco-regional treatment strategy for certain solid tumors (4). Based upon our observations that HSP70-peptide complexes chaperone antigenic peptides and deliver them to DC in an immunogenic way for efficient T cell stimulation we propose the following chain of events. During clinical hyperthermia peak temperatures of up to 42°C can be achieved that upregulates Hsp70. Within this temperature range local necrosis occurs which results in release of HSP, uptake by DC and subsequent processing and presentation of associated peptides. The proposed dual role of HSP70-PC as chaperone and cytokine then leads to efficient priming of circulating T cells. Therefore, upregulating Hsp70 expression and causing local necrosis in tumor tissue by hyperthermia potentially is a new approach to directly activate the immune system. We defined some of the mechanistic events using well defined melanoma cells, since for melanoma in particular, a randomized phase III study has been completed showing improvement of local tumor control and survival benefit in patients with multiple lesions after hyperthermia (5).
References
1. Srivastava, P., et al. (1998). Immunity 8: 657–665.
2. Breloer, M., et al. (1998). Eur. J. Immunol. 28: 1016–1021.
3. Wells, D., and Malkovsky, M. (2000). Immunol. Today 21: 129–132.
4. Falk, M.H. and Issels, R.D. (2001). Int. J. Hyperthermia 17:1–18.
5. Overgaard, J. et al. (1995). Lancet 345: 540–543.
We acknowledge the enthusiastic discussion with Drs. A. Asea and S. Calderwood. The work is supported by grants from the Deutsche Forschungsgemeinschaft (SFB455/B9) and the Deutsche Krebshilfe (7023011-1s/2).
Specific gene expression and chronological gene program during Trypanosoma cruzi cellular differentiation (metacyclogenesis)
Luiz R. Nunes
Nucleo Integrado de Biotecnologia, Universidade de Mogi das Cruzes, São Paulo, Brasil
The differentiation of epimastigotes into metacyclic trypomastigotes (metacyclogenesis) involves the transformation of a replicative, non-infectious form of Trypanosoma cruzi into a non-replicative, infectious stage. This process occurs the insect's midgut and involves several types of stress stimuli, such as quorum sensing. The functional and morphological changes occurring during this process result from important changes in the gene expression program. To improve our knowledge about T. cruzi differentiation and particularly, of the metacyclogenesis process, we have been doing a systematic analysis of differentially expressed genes using microarray technology. A T. cruzi biochip, carrying 1056 EST sequences from the parasite has been constructed and used in competitive hybridization experiments with different RNA samples, extracted from T.cruzi at different times of the differentiation process. These analyses strongly suggest that specific polysomal mobilization has an important role in T.cruzi gene expression regulation. In addition, our data show that specific sets of genes are expressed during the cellular differentiation of the parasite. In order to confirm these data, some selected genes have been further characterized in terms of mRNA stability, polysomal mobilization and protein expression. These results corroborate the importance of polysomal mobilization and the gene expression profile obtained from the microarray experiments.
These analyses are now being expanded with a more comprehensive T. cruzi biochip, currently under construction with EST sequences, obtained from the public databases. Cluster analyses and BLAST searches for the 9919 T. cruzi EST's present in EMBL (Release 62.0) have been clustered using the program cap3 (Huang and Madan 1999), resulting in 1655 clusters (6995 sequences were diverted to clusters, whereas 2924 EST's remained as singletons, with no similarity with other EST's being detected). Similarity searches using BLAST (Altschul et al. 1990) were performed for each one of the singletons as well as for the consensi of each cluster against SWIR 20.0, as an initial step towards the identification and characterization of each sequence. These data were used to synthesize PCR primers for the amplification of fragments corresponding to the T. cruzi ESTs and will be used in further microarray experiments to extend our knowledge about T. cruzi gene programming during the metacyclogenesis process.
References
Altschul SF, Gish W, Mileer W, Myers EW, Lipman DJ. Basic Local Alignment Search Tool. J. Mol. Biol. 215(3):403–10 (1990).
Huang X, Madan A. CAP3: a DNA sequence assembly program. GenomeRes. 9(9):868–877 (1999)
Financial support: FAPESP, PRONEX, PADCT, CNPq and FIOCRUZ.
The quality control of glycoprotein folding in the endoplasmic reticulum
Armando J. Parodi
Institute for Biotechnological Research, University of San Martin, CC30, 1650 San Martin, P. Buenos Aires, Argentina. aparodi@inti.gov.ar
Proteins entering the secretory pathway acquire their proper tertiary and in certain cases also quaternary structures in the ER. Incompletely folded species are prevented from transit to the Golgi apparatus and eventually degraded by the proteasome. The emerging principles by which N-glycan processing in the ER participates in the quality control process will be dealth withn in the talk. Monoglucosylated glycans formed by deglucosylation of oligosaccharides transferred from lipid (dolichol) pyrophosphate derivatives to proteins (Glc3Man9GlcNAc2) mediated glucosidases I and II (GII)- mediate the binding of glycoproteins to two ER resident lectins, calnexin (CNX), a transmembrane protein, and its soluble homolog, calreticulin (CRT). Further deglucosylation of glycans by GII liberates glycoproteins from CNX/CRT. Glycans may be then reglucosylated by the UDP-Glc:glycoprotein glucosyltransferase (GT), and thus recognized again by CNX/CRT, only when linked to incompletely folded protein moieties. The reglucosylaging enzyme behaves as a sensor of glycoprotein conformations. Deglucosylation-reglucosylation cycles catalyzed by the opposing activities of GII and GT stop when proper folding is achieved as glycoproteins become then substrates for GII but not for GT. Permanent liberation from CNX/CRT allows further glycoprotein transit through the secretory pathway. The CNX/CRT-monoglucosylated glycan interaction is one of the mechanisms by which cells retain incompletely folded glycoproteins in the ER and, in addition, it enhances folding efficiency by preventing protein aggregation and allowing intervention of additional ER chaperones and folding facilitating proteins. A still controversial protein-protein interaction between folding species and CNX/CRT might further assist the folding process. In addition, there is evidence, suggesting that Man removal, mediated by ER mannosidases might act as a timer mechanism for the disposal of incompletely folded glycoproteins bound for proteasome degradation. Synthesis of mRNAs encoding both unconventional chaperones (CNX and CRT) and the sensor of glycoprotein conformations (GT) have been shown to be induced under stress conditions that promote ER accumulation of misfolded glycoproteins. Moreover, interfering with monoglucosylated glycan formation elicits the unfolded protein response as shown by the upregulation of the main ER chaperone (BiP) mRNA synthesis.
The mechanisms described constitute a novel system, different from those of classical molecular chaperones, for retaining non-native conformers and facilitating protein folding and oligomerization. CNX and CRT are unconventional chaperones that apparently do not directly sense the folding status of the substrate proteins as classical chaperones do. This task is reserved to an enzyme (GT) that introduces a covalent modification on glycoproteins lacking their native conformations. This covalent carbohydrate modification is the element recognized by this new kind of chaperones. Although the main features of this system are increasingly clear, there are several aspects that remain obscure and that will undoubtedly be the object of future studies. More detailed biochemical and structural studies are needed to understand the recognition of non-native structures by GT, as well as CNX/CRT-ligand interaction. The controversial protein-protein interaction between CNX/CRT and folding glycoproteins and the role of glycoprotein reglucosylation in multicellular organisms needs to be established. The involvement of Man removal in the disposal of permanently misfolded species has to be further substantiated, and the interplay between the components of the CNX/CRT pathway and other folding factors in the ER should be determined to advance our understanding of the quality control mechanisms.
References
Fernández, F. et al. (1996) A new stress protein: synthesis of Schizosaccharomyces pombe UDP-Glc:glycoprotein glucosyltransferase mRNA is induced under stress conditions but the enzyme is not essential for cell viability. EMBO J. 15, 705–713.
Fanchiotti, S. et al. (1998) The UDP-Glc:glycoprotein glucosyltransferase is essential for Schizosaccharomyces pombe viability under conditions of extreme endoplasmic reticulum stress. J. Cell Biol. 143, 625–635.
D'Alessio, C. et al. (1999) Genetic evidence for the heterodimeric structure of glucosidase II. The effect of disrupting the sububit-encoding genes on glycoprotein folding. J. Biol. Chem. 274, 25899–25905.
Parodi, A. J. (2000) Protein glucosylation and its role in protein folding. Annu. Rev. Biochem. 69, 69–93.
Cell suicide systems
Andrea Balan
andmar@usp.br) and Ana Clara Schenberg (acgschen@usp.br). Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil
As a biosafety device allowing the containment of genetically modified organisms (GMOs), different suicide systems have been developed, mainly for microorganisms. The rationale, first conceived by Molin et al. (1), is to establish a genetic system whereby the GMO, after completing the task for which it has been designed, commits suicide. Suicide systems rely on the expression of genes whose products are toxic to the host cell. To ensure the necessary conditionality of the system, the lethal gene must be placed under control of a regulatable promoter. Among these promoters, the promoters of stress genes are of great value, since, in contrast to life in laboratory conditions, microbial life in the natural environment consists mostly of starvation and other types of stress, like pH, extreme temperatures, toxic compounds, etc. In an attempt to construct a suicide system for yeast, we have used the Saccharomyces cerevisiae ADH2 promoter, which is responsible for the expression of alcohol dehydrogenase II. As the ADH2 promoter is glucose-repressible, the expression of the lethal gene under its control will only occur upon glucose depletion, at the end of growth (2), thus allowing maximal mass increase before cells are killed. As the lethal gene, we used the Serratia marcescens nuc gene, which codes for a powerful nuclease, able to degrade unspecifically RNA and DNA (3). The advantage of using a nuclease as the lethal gene is to ensure the destruction of all genetic material (including the suicide plasmid), before its release from the dead cells into the environment, where it could be transmitted horizontally to other living organisms. The nuc gene was deleted for the signal-peptide coding sequence and the truncated gene was put under control of the ADH2/GAPDH hybrid promoter (2). Survival curves in glucose containing media of different S. cerevisiae strains transformed with the suicide plasmid showed the killing effect of the plasmid when cells arrived in late stationary phase (48h of growth), with a steep inactivation starting from that moment and leaving no more than 10-5 survivors. The kinetics of killing was still improved when strains carrying the rad52 mutation were used as hosts of the suicide plasmid, thus preventing repair of DNA strand breaks. In vitro activity of the nuclease produced by the yeast transformants was also demonstrated using plasmid DNA as the substrate of cell extracts and this activity was comparable to the S. marcescens purified nuclease used as a control. Furthermore, the suicide system proved to be efficient in microcosm experiments performed with the yeast transformants.
References
Molin, S.; Klemm, P.; Poulsen, L. K.; Biehl, H.; Gerdes, K.; Andersson, P., Conditional suicide systems for containment of bacteria and plasmids. Bio/Technology 5:1315–1318 (1987).
Shuster, J. R., Regulated transcriptional systems for the production of proteins in yeast: regulation by carbon source. In: Yeast Genetic Engineering, P. J. Barr; A. J. Brake; P. Valenzuela (eds), Butterworth Publishers, 1989, pp.83–108.
Ball, T.K.; Saurugger, P.N.; Benedik, M.J., The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli. Gene 57:183–192 (1987).
How does Bacillus subtilis regulate its heat shock genes?
Saskia Versteeg, Silke Reischl, Thomas Wiegert and Wolfgang Schumann
Institute of Genetics, University of Bayreuth, 95440 Bayreuth, Germany
The heat shock response is induced in all cells upon a sudden rise in temperature, and results in the transient induction of a small set of proteins called heat shock proteins [1]. This response to temperature is universal among living organ- isms, and not only the response to temperature, but also most temperature-inducible proteins are conserved. Our efforts concentrate mainly on the understanding of how expression of the heat shock genes in the gram-positive soil bacterium Bacillus subtilis are regulated. Work carried out over the last ten years revealed that the about 200 heat shock genes can be grouped into six different classes where members of each class are regulated by a different mechanism [2]. Class-I heat shock genes are under the negative control of a transcriptional repressor called HrcA which binds to an operator designated as CIRCE element. HrcA controls expression of two different operons, the heptacistronic dnaK and the bi- cistronic groESL operon. The activity of the HrcA repressor is modulated by the GroE chaperonin system, and we have suggested that de novo synthesized HrcA and HrcA dissociated from its operator are present in an inactive form unable to interact with the operator. Inactive HrcA has to interact with the GroE chaperonin system to become converted into its active form. Upon a heat shock, the molecular chaperones are titrated by the non-native proteins resulting in the accumulation of inactive HrcA which in turn leads to the induction of both the dnaK and the groE operon. The more non-native proteins have been removed from the cytoplasm, the more GroESL will become available to reactivate HrcA resulting in a gradual turn off of the two operons [3]. Class-II heat shock genes comprise the by far largest family with more than 150 members. These genes are under the positive control by the alternative sigma factor sigma-B, which, in the absence of a heat shock, is bound by an anti-sigma factor thereby preventing its interaction with the core RNA po- lymerase. The second function of the anti-sigma factor is to phosphorylate an anti-anti-sigma factor. Upon a heat shock, a phosphatase becomes activated which dephosphorylates the anti-anti-sigma factor which in turn dissociates the anti- sigma factor-sigma-B complex to release sigma-B which is now able to bind to the RNA polymerase core enzyme to initiate transcription of the more than 100 genes of the Sigma-B regulon [4]. Class-III heat shock genes are controlled by a second transcriptional repressor, CtsR, which negatively controls expression of three operons, the hexacistronic clpC and the two monocistronic clpP and clpE operons. Stability of CtsR seems to be controlled by the chaperone-like MscA protein, and destabilisation of CtsR seems to be initiated by its phosphorylation by the MscB kinase followed by its degradation through the ClpCP system [5]. Class-IV genes consist of one member so far, htpG, a protein of largely unknown function. This gene is under the control by a transcriptional activator which is part of an essential two- component signal transduction system. The binding site for this activator protein, GAAAAGG, is located downstream of the promoter and has been denoted DAS (for 
ownstream 
ctivating 
equence). We assume that, upon a heat shock, a signal is formed in the cytoplasmic membrane which will lead to the autophosphorylation of the sensor kinase YycG at its invariant histidine residue. Then, the phosphate group will be transferred to the response regulator YycF which will bind to the DAS to allow the RNA polymerase holoenzyme to initiate transcription. Shut-off of this system is assumed to occur by dephosphorylation of the response regulator. Class-V heat shock genes are also controlled by another two- component system, CssSR, and two members have been identified so far, HtrA and YvtA, both serine proteases an- chored in the cytoplasmic membrane. In addition, there a several heat-inducible genes including lonA, ftsH, ssrA, sacB, clpX and ahpC where their regulation mechanism remains elusive. They have been named Class-VI genes.
References
[1] B. Bukau and A.L. Horwich (1998) Cell 92: 351–366.
[2] W. Schumann et al. (2001) In Bacillus subtilis and Its Closest Relatives: from Genes to Cells. Edited by A.L. Sonenshein, J.A. Hoch & R. Losick. Washington DC: American Society for Microbiology, pp. 367–376.
[3] A. Mogk et al. (1997) EMBO J. 16: 4579–4590.
[4] C.W. Price (2000) In Bacterial Stress Responses. Edited by G. Storz & R. Hengge-Aronis. American Society for Microbiology, pp. 179–197.
[5] E. Krüger et al. (2001) EMBO J. 20: 852–863.
Protein acetylation in the trans regulation of human hsp90 beta gene
Yu-fei Shen
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China
Hsp90 is an abundant cytoplasmic protein with functions of specific molecular chaperone towards signaling molecules and transcriptional regulators. We reported elsewhere (Shen et al, FEBS Letters, 413, 92∼98, 1997) that sequences downstream of the transcription start site at +1 (identified by Rebbe et al in 1989) are essential for constitutive statement of hsp90β gene, while they are critical for heat shock induction. The segment consists of a small first exon and the first intron of the gene, in which novel initiation sites have been found and designated as promoter II (P-II) to differentiate it from +1 (P-I). Here we report that CRE and AP1 elements are both required in the positive regulation of hsp90β gene, in which phosphorylated CREB binds to both sites and plays a dominant role in Jurkat cells. The regulatory activity of CREB on hsp90β gene can be enhanced by co-transfection of wild type p300 but not that with HAT domain deleted, which suggests that chromatin remodeling or at least acetylation of certain protein of importance participantes in hsp90β gene regulation. Although PKA activity can be induced by heat shock, it is not involved in CREB-p300 mediate hsp90β gene statement.
We have found that wild type p53 can be induced in Jurkat cells through either transit transfection or UV treatment. Wild type p53 recognizes a putative binding site in the first exon of hsp90β gene that confers repression to the gene. The levels of statement and acetylation of wild type p53 increase and peak at four hours after UV irradiation, and then concurrently fade away within 12 hours. The addition of tricostatin A, an inhibitor for histone deacetylase, to the system further down-regulates the statement of hsp90β by p53 in the first four hours after UV irradiation, which indicates the importance of p53 acetylation is dominant over histone acetylation in the repression of hsp90β gene by p53. The statement of hsp90β gene is partially recovered after p53 degraded.
This is the first evidence showing that protein acetylation through HAT and differentially sustained by HDAC inhibitor play great parts in the positive and negative trans regulation of an abundant molecular chaperone hsp90β gene in human cells.
Heat shock proteins and stress-induced signaling
Michael Y. Sherman, Vladimir L. Gabai, Anatoli B. Meriin, Julia A. Yaglom
Department of Biochemistry, Boston University Medical School
The major heat shock protein Hsp70 when expressed in cells at high levels can suppress activation of protein kinases JNK and p38 by various stressful treatments, including heat shock, UV-irradiation, oxidative stress, cytokines and others. Hsp70 suppressed JNK activation by facilitating JNK dephosphorylation. Interestingly, the ATPase domain, and thus the chaperone function, of Hsp70 is dispensable for its effect on JNK dephosphorylation. This observation has an implication in many aspects of cell physiology that are controlled by JNK and p38; for example, in the TNF-induced apoptosis of human fibroblasts, which is depend upon activation of JNK. Expression of Hsp70 in these cells repressed JNK activation by TNF and inhibited apoptosis. Both expression of Hsp70 and inhibition of JNK by a dominant-negative JNK mutant inhibited cleavage of Bid and prevented the release of cytochrome C from mitochondria. Therefore, Hsp70 appears to regulate the mitochondrial integrity upon activation of the apoptotic program.
Surprisingly, Hsp70-mediated suppression of stress-kinases is critical for protection of cells from a caspase-independent death as well. We have found that several kinases, including JNK, ERKs and Akt determine the caspase-independent death or survival of human fibroblasts exposed to heat shock. The fibroblasts developed thermotolerance after mild heat treatment, and the Hsp70-mediated suppression of JNK was critical for acquiring the thermotolerance.
In another, morphologically distinct mode of death of cardiomyocytes after exposure to the simulated ischemia-reperfusion, Hsp70 also promoted cell survival by suppression of JNK.
It is conceivable that other important cellular processes that are regulated by the stress-kinases including cardiomyocytes' hypertrophy, production of cytokines, oxidative burst, etc., may also be controlled by Hsp70.
Interaction of heat shock proteins with antigen processing cells as the primordial immunological software
Pramod K. Srivastava
Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut Health Center, 263 Farmington Avenue, Mail Code 1601, Farmington, Connecticut 06030-1601, USA. wasik@nso2.uchc.edu
Studies from our laboratory have uncovered three novel aspects of interaction of heat shock proteins (HSPs) and antigen presenting cells (see 1 for review). These observations show a key role for HSPs in adaptive and innate immunity and form the basis of creation of a new generation of vaccines against cancers, and infectious and parasitic diseases. The principles, demonstrated in mice, rats, frogs and humans, are:
• Homogeneous preparations of HSPs gp96, calreticulin, hsp90 or hsp70 are associated with peptides derived from cellular proteins, incl. normal self proteins or mutated or foreign proteins (2–5).
• If HSPs (which are actually HSP-peptide complexes) are injected into immunocompetent hosts, the hosts develop potent antigen-specific CD8+ and CD4+ T cells (6,7). This response is directed solely at the altered or foreign peptides and not against self peptides not against the HSPs themselves. The immunogenic HSP-complexes may also be reconstituted in vitro from HSPs and synthetic peptides (5, 8–10). The mechanism of immunogenicity of HSP-peptide complexes is increasingly clear and involves the interaction of the HSPs with macrophage or dendritic cells through the CD91 HSPs receptors, followed by re-presentation of the HSP-chaperoned peptides by the MHC I and MHC II molecules of the macrophage/dendritic cells (11–13).
• HSPs stimulate macrophage and dendritic cells in a peptide-independent manner to secrete inflammatory cytokines and to express co-stimulatory molecules. HSPs also cause maturation of dendritic cells (14,15).
It is our premise that HSPs play a central role in innate and adaptive immune responses including in indirect presentation and cross-priming.
A number of Phase I and Phase II trials have been carried out where patients with cancers of the colon, pancreas, kidney and melanoma have been treated with autologous cancer-derived gp96 and hsp70. Some trials have been completed (see Nature Immunology 1:363–366, 2000 for review) while others including a Phase III trial are underway. The preliminary data from the human trials is consistent with the experience with experimental cancers of mice.
References
Srivastava, Menoret, Basu, Binder, and McQuade, Immunity, 8: 657–665, 1998.
Udono and Srivastava, Journal of Experimental Medicine, 178, 1391–1396, 1993.
Ishii, Udono, Yamano, Ohta, Uenaka, Ono, Hizuta, Tanaka, Srivastava, and Nakayama, Journal of Immunology, 162: 1303–1309, 1999.
Breloer, Fleischer, and von Bonin, Journal of Immunology, 162: 3141–3147, 1999.
Navaratnam, Deshpande, Hariharan, Zatechka, Srikumaran, Vaccine, 19(11–12):1425–34, 2001.
Udono, Levey, Srivastava, Proc. Natl. Acad. Sci., 91, 3077–3081, 1994.
Matsutake and Srivastva, II International Conference on Heat Shock Proteins in Immune Response, Abstract, 2000.
Blachere, Li, Chandawarkar, Suto, Jaikaria, Basu, Udono,and Srivastava, Journal of Experimental Medicine, 186(8): 1315–1322, 1997.
Ciupitu, Petersson, O'Donnell, Williams, Jindal, Kiessling, and Welsh, Journal of Experimental Medicine, 187(5):685–91, 1998.
Moroi, Mayhew, Trcka, Hoe, Takechi, Hartl, Rothman, Houghton. PNAS, 97(7):3485–90, 2000.
Binder, Han, and Srivastava, Nature Immunology, 1 (2), 151–155, 2000.
Basu, Binder, Ramalingam and Srivastava, Immunity, 14, 303–313, 2001.
Castelli, Ciupitu, Rini, Rivoltini, Mazzocchi Kiessling and Parmiani. Cancer Res. 2001 Jan 1;61(1):222–7.
Basu, Binder, Suto, Anderson, and Srivastava, International Immunology, 12: 1539–1546, 2000.
Binder RJ, Anderson KM, Basu S, Srivastava, Journal of Immunology, Cutting Edge, 166: 4968–4972, 2000.
Srivastava Nature Immunology, 1: 363–366, 2000.
The small Hsps of Drosophila: chaperones of cell-specific processes
Robert M. Tanguay, Sébastien Michaud, Geneviève Morrow and Julie Marchand
Laboratory of Cell and Developmental Genetics, Dept Medicine, Pav. C.E. Marchand, Université Laval, Ste-Foy, Québec, Canada, G1K 7P4. robert.tanguay@rsvs.ulaval.ca
Members of the family of small heat shock proteins (sHsp) are not only expressed under stress situation but also in a cell- and stage-specific manner during normal development in many organisms. In Drosophila melanogaster there are four main sequence-related sHsps (Hsp22, 23, 26 and 27). Previous studies have identified non-stress related expression for three members of this family during gametogenesis (1–3). Different approaches were used to reveal the regulation of expression and the function(s) of these sHsps: immunocytochemical localization, in vitro and in vivo chaperone assays, promoter analysis in transgenic flies, functional domain expression in mammalian cells and search for in vivo protein partners of sHsps.
Each sHsp localizes to a distinct intracellular compartment. Hsp27 is found in the nucleus while Hsp23 and 26 are associated with distinct structures in the cytosol. Hsp22 is targeted to the mitochondria where it is found as an oligomeric complex in the matrix fraction (4). In denaturation and renaturation assays, all four sHsps show chaperone activity albeit with different efficiencies. Cell lines and transgenic flies allowing for inducible Hsp22 expression have been constructed to test if this Hsp plays a role in protection of mitochondrial integrity during heat shock, after oxydative stress and in ageing.
Hsp23 and Hsp27 have distinct patterns of expression and induction in gonads and in the central nervous system (CNS). In testis, Hsp23 is expressed in somatic cells and Hsp27 in germ cells. Hsp23 promoter studies in transgenic flies carrying a hsp23-lacZ fusion gene reveals a dynamic pattern of activity restricted to specific cells of the developing CNS. Co-localization studies with a neuronal marker (Elav) and a midline glial enhancer-trap line (slit-lacZ) show that Hsp23 is expressed in a subset of both cell types at different stages. Neuronal expression spans from stage 11 to stage 14 while glial expression starts at late 13 to late embryogenesis. Lineage analysis demonstrates that Hsp23 expression in the CNS is confined to the MP2, VUM and surviving midline glial cells. Transactivation assays in S2 cell cultures support that the hsp23 gene is modulated in part by the Sim, Tgo and Drf transcription factors. This dynamic and highly regulated expression of Hsp23 within the developing CNS suggests that this protein may carry precise function(s) within these cells. Analysis of P-element insertion lines which result in downregulated Hs23 expression in the CNS demonstrates that the loss of this Hsp does not affect the ultrastructure of the CNS. The observation that all all of the Hsp23-expressing cell types in the CNS show an association of Hsp23 with axonal or cytoplasmic projections suggests that, similarly to its mammalian counterpart (Hsp25/27), Hsp23 could be implicated in the modulation of cytoskeleton during cellular establishment in the CNS
These data suggest that the different sHsps of Drosophila perform distinct function(s) in vivo either by acting as chaperones of specific molecules in distinct intracellular compartments and/or in distinct cell types during differentiation. (Supported by Canadian Institutes of Health Research).
References
1. Marin R, Tanguay RM (1996) Stage-specific localization of the small heat shock protein Hsp27 during oogenesis in Drosophila melanogaster. Chromosoma 105 :142–149.
2. Michaud S, Marin R, Westwoood JT, Tanguay RM (1997) Cell-specific expression and heat-shock induction of Hsps during spermatogenesis in Drosophila melanogaster. J Cell Sci 110: 1989–1997.
3. Michaud S, Marin R, Tanguay RM (1997) Regulation of heat shock gene induction and expression during Drosophila development. Cell Mol Life Sci 53 : 104–113.
4. Morrow G, Inaguma Y, Kato K, Tanguay RM (2000) The small heat shock protein Hsp22 of Drosophila melanogaster is a mitochondrial protein displaying oligomeric organization. J Biol Chem 275 : 31204–31210.
Hsp27 and Hsp70 in serial biopsies from breast cancer patients treated with doxorubicin
L. Vargas-Roig1, P. Daguerre2, M. Leuzzi2, F. Gago,3, S. Nadin,1, M.A. Lazzaro1, and D. Ciocca1
1Institute of Experimental Medicine and Biology of Cuyo, CRICYT-CONICET, Medical School, University of Cuyo, Mendoza, Argentina; 2Hospital Lagomaggiore of Mendoza, Argentina; 3Hospital Italiano of Mendoza, Argentina; vargasl@lab.cricyt.edu.ar
Heat shock proteins (Hsps) may be expressed in human tumors such as in breast cancer, where some of the Hsp family members have been studied as prognostic markers (1). There is in vitro evidence that Hsps may be involved in doxorubicin resistance (2). In a previous study, we observed that the administration of doxorubicin, 5-fluorouracil and cyclophosphamide changed the expression of Hsp27 and Hsp70 in biopsies from breast cancer patients (3). We observed increased Hsp27 and Hsp70 nuclear expression and a significant decrease in the cytoplasmic content of Hsp70. These changes were relatively late events because the post-chemotherapy biopsies were taken approximately 21 days after the last cycle of induction therapy.
In the present study, we decided to evaluate the expression of Hsp27 and Hsp70 in serial biopsies from a more homogeneous group of breast cancer patients. The study involved patients with locally advanced disease treated only with doxorubicin during 4 cycles before the surgery as the initial treatment. The serial biopsies were taken at pre-chemotherapy, at days 1, 3, 7, 21, and at surgery. Forty two patients entered into this protocol, after surgery patients received 6 cycles of cyclophosphamide, methotrexate, and 5-fluorouracil. In the invasive tumors, we found that the increased nuclear expression of Hsp27 and Hsp70 was a relatively late event, since this was significant at surgery. No significant changes were noted in the cytoplasmic content of both Hsps. It is of interest to note that some tumor cells showed membrane expression of Hsp27 and Hsp70.
We also examined the other cells that were exposed to doxorubicin, the normal mammary and connective tissue cells and the non invasive tumor cells (in situ carcinomas). In normal breast epithelium, cytoplasmic and nuclear expression of Hsp27 was lower than in the invasive tumor, specially at surgery. In in situ carcinomas, Hsp27 cytoplasmic expression was higher than in the invasive tumor cells reaching statistically significant differences up to day 7, at surgery the levels were similar to the ones found in invasive tumor cells. In normal breast epithelium, nuclear Hsp70 content increased at days 1 and 3 and decreased at days 7 and 21, but at surgery nuclear Hsp70 was increased. This dynamics was not seen in tumor cells.
We also analyzed the expression of Hsps in blood vessels, Hsp27 and Hsp70 in endothelium increased shortly after chemotherapy and then decreased.
We observed the expression of Hsps in stroma fibroblasts and in lymphoid tissue. After chemotherapy we noted an increase in the expression of Hsp27 and Hsp70 specially in the “activated” fibroblasts (large cells). We observed more expression of Hsp70 than Hsp27 in the cytoplasm of lymphocytes and plasma cells, but always in a lower percentage (<10%).
This study describes the dynamics of the Hsp27 and Hsp70 response to doxorubicin administration. Normal cells showed a different response to that noted in in situ and invasive tumor cells.
References
1. Ciocca, D.R., et al. Heat shock protein hsp70 in patients with axillary lymph node-negative breast cancer: prognostic implications. J. Natl. Cancer Inst., 85: 570–574, 1993.
2. Ciocca, D.R., and Vargas Roig, L.M. “Heat shock proteins and drug resistance in breast cancer”. In: Samuel D. Bernal (ed.), Drug Resistance in Oncology, pp. 167–190. Marcel Dekker, Inc. New York, 1997.
3. Vargas-Roig, L.M., et al. Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy. Int. J. Cancer (Pred. Oncol.)., 79: 468–475, 1998.
GroEL-assisted dehydrogenase folding mediated by coenzyme is ATP-independent
Sen Zhang, Jian LI and Chih-Chen Wang*
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, China chihwang@sun5.ibp.ac.cn
It has been commonly accepted that E. coli GroEL functions as a chaperone essentially by modulation of its affinity for folding intermediates through binding and hydrolysis of ATP [1]. The reactivation of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) assisted by GroEL occurs only when ATP and Mg2+ are both present. NAD, the tightly bound coenzyme of GAPDH, increases the spontaneous reactivation of GAPDH only slightly but unexpectedly appears also stimulates the discharge of GAPDH folding intermediate from its stable complex with GroEL formed in the absence of ATP and assists refolding with the same yield as ATP/Mg2+ does. The reactivation further increases when ATP is also present, but addition of Mg2+ has no more effect. NADP, a coenzyme of glucose-6-phosphate dehydrogenase, also releases its folding intermediates from GroEL and increases reactivation. Although NAD is also the substrate of GAPDH and has ADP in its molecule the possibilities that NAD mediates the GroEL-assisted GAPDH refolding as a substrate and/or as an ATP analog have been excluded. It has been identified that different from ATP, NAD triggers the release of GAPDH intermediates bound by GroEL via binding with GAPDH itself but not with GroEL and inducing conformational change of GAPDH so that weakening its interactions with GroEL. The collaborative effect of ATP and NAD may be ascribed to the conformational change of both GroEL and the target folding intermediates so as to release and refold GAPDH more efficiently. Unlike the intermediates released in the presence of ATP/Mg2+, which face two alternative fates, either correct folded or misfolded and further aggregated, the released intermediates mediated by NAD all fold to native molecules without the formation of aggregation, suggesting only those folding intermediates destined to correct folding can be released by NAD. The collaborative effects of coenzyme and GroEL mediate GroEL-assisted dehydrogenase folding in an ATP-independent way. The working concentrations of NAD in mediating the GroEL-assisted GAPDH refolding are not excessive as compared to the known cellular level of NAD.
References
1. Sigler, P. B., Xu, Z., Rye, H. S., Burston, S. G., Fenton, W. A. and Horwich, A., L. (1998) Structure and function in GroEL-mediated protein folding. Annu. Rev. Biochem. 67, 581–608.
2. Li, X. L., Lei, X.D., Cai, H., Li, J., Yang, S. L., Wang, C. C. and Tsou, C. L. (1998) Binding of a burst-phase intermediate formed in the folding of denatured D- Glyceraldehyde-3-phosphate dehydrogenase by chaperonin 60 and 8-anilino-1-naphthalene sulphonic acid. Biochem. J. 331, 505–511.
3. Zhang, N. X. and Wang, C. C. (1999) A stable cold folding intermediate of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase. Eur. J. Biochem. 264, 1002–1008.
4. Li, J. and Wang, C. C. (1999) “Half of the sites” binding of D-Glyceraldehyde-3-phosphate dehydrogenase folding intermediate with GroEL. J. Biol. Chem. 274, 10790–10794
Association of inducible Hsp71 and its antibodies with environmental stresses and diseases
Tangchun Wu1,2, Sheng Chen1, Chengfeng Xiao1, Yajuan Gao1, Ruibo Wang1, Robert M. Tanguay2
1Institute of Occupational Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China and 2Laboratory of Cell and Developmental Genetics, Faculty of Medicine, Université Laval, Québec, Canada, G1K 7P4.(wut@mails.timu.edu.cn)
Heat shock proteins (Hsps) and autoantibodies against Hsps may play a role in the pathogenesis and/or prognosis of some diseases (1–3). To understand the relation between environmental stresses, the stress response and diseases, we have examined Hsps or/and antibodies against Hsps in different groups of workers. Firstly, we have measured titers of antibodies against Hsps in 42 young patients with acute heat-induced illness (AHII) while training and in 57 older patients with AHII. The occurrence of antibodies to Hsp71 was significantly higher in individuals with symptoms of AHII than in controls. The data suggest that measurement of antibodies to Hsps may be useful to assess how individuals are responding to abnormal stress within their living and working environment and may be used as one biomarker to evaluate the susceptibility to heat-induced diseases (4). In a second study, we have investigated the presence of antibodies against Hsp71 in 764 steel mill workers from six work sites, and analyzed its possible association with hypertension and harsh working conditions. Analysis of the results suggest that harsh workplace conditions can increase the production of antibodies against Hsp71 and that the presence of antibodies to this stress protein may be associated with hypertension (5). Thirdly, we analyzed the basal and inducible level of Hsp71 in peripheral blood lymphocytes of patients with heat-induced illness and control individuals from north and south of China in order to explore the possible mechanism of heat induced illness. These results suggest that the basal and inducible level of HSP71 might be related to thermotolerance and heat sensitivity, and that individual differences of the hsp70 gene and its expression may play a role in the occurrence of heat-induced illness. Finally, we have started investigating the polymorphism of the Hsp 71 and Hsc73 genes in young individuals coming from different regions with different climates (hot, cold) and altitudes (low oxygen) in order to uncover their possible significance in tolerance to heat, cold and low oxygen. The precise mechanisms regulating the changes of Hsps and antibodies against Hsps and environmental and workplace stresses and its relation to diseases remain to be established.
References
1. Tangchun Wu, Yuan Ye, Wu Yang, Hanzhen He, Guogao Zhang, Robert M. Tanguay (1998). Presence of antibodies to heat stress proteins in workers exposed to benzene and in patients with benzenepoisoning. Cell Stress & Chaperones 3:161–167.
2. Tangchun Wu, Hanzen He, Yang Wu, Daigen Xu, Jiade Feng, Wuxiang Shi, Ye Yuan, Gugao Zhang, Tanguay RM (1998). Antibodies to heat stress proteins in workers exposed to high temperature, carbon monoxide and benzene. Ann NY Acad Sci, 851:520–525.
3. Tangchun Wu, Yili Xiong, Sheng Chen, Shun-tang Leng, Tao Hai, Robert M, Tanguay (1999) Biochemical changes of plasma in paratroops after parachuting: a preliminary investigation. Space Med & Medical Engineering 12:235–239.
4. Tangchun Wu, Sheng Chen, Chengfeng Xiao, Changlai Wang, Qin Pan, Zizheng Wang, Meiyun Xie, Zicheng Mao, Yang Wu, Robert M. Tanguay (2001). Presence of antibody against the inducible heat shock protein Hsp71 in patients with acute heat induced illness. Cell Stress & Chaperones 6:113–120.
5. Tangchun Wu, Jinxiang Ma, Sheng Chen, Yehuan Sun, Chengfeng Xiao, Yajuan Gao, Ruibo Wang, Jacques Poudrier, Michèle Dargis, R. William Currie, Robert M. Tanguay .Association of plasma antibodies against the inducible Hsp70 with hypertension and harsh working conditions . Cell Stress & Chaperones (In press).
Heat-shock regulation in E. coli and other gram-negative bacteria: search for thermosensors
Takashi Yura
(formerly at HSP Research Institute, Kyoto, Japan) . tayura@ip.media.kyoto-u.ac.jp
Recent work on regulation of sigma32 and its homologs required for transcription of a set of heat shock genes will be summarized. In most Gram-negative bacteria studied so far, induction of Hsp occurs primarily as the result of increased level and/or activity of sigma32 (1). However, regulatory strategies used in different bacteria vary greatly, presumably reflecting differences in ecological niche (2). Translational induction and/or stabilization of sigma32 primarily account for increased sigma32 level in E. coli and other gamma-subgroup of proteobacteria, whereas activation and transcriptional induction of sigma32 appear to be characteristic features in Agrobacterium and perhaps other members of alpha subgroup.
In E. coli, temperature upshift (30 to 42°C) provokes at least two heat shock signals. First, high temperature directly disrupts part of secondary structure of rpoH mRNA (encoding sigma32) to enhance its translation, RNA serving as a built-in thermosensor (3). The elevated synthesis of sigma32 is not a transient phenomenon as had previously been thought but continues as long as cells are kept at high temperature (4). Second, sigma32 most probably undergoes conformational change that will drastically increase susceptibility to ATP-dependent heat shock proteases. However, sigma32 is temporarily stabilized rather than destabilized presumably due to titration of free DnaKJ chaperones and proteases that serve to monitor the state of protein folding in the cytoplasm (1). Thus, the continued high rate of sigma32 synthesis is effectively counteracted by the intinsic instability of sigma32 at high temperature (5). Whereas the RNA sensor plays a protective role against heat stress before accumulation of damaged proteins, the putative protein sensor represents a dynamic and adaptive response to changing levels of protein damage accumulated in the cell.
In contrast, recent work with a plant pathogen Agrobacterium tumefaciens indicated that DnaKJ-mediated control of sigma32 activity (rather than the amount) plays a major role in the heat shock response upon shifting from 25 to 37°C (6). This conclusion came from the observations that (a) although heat shock induces rpoH transcription initiated from a sigma32-specific promoter, induction of HSP occurred almost simultaneously with that of sigma32 and preceded the increase in sigma32 level, (b) synthesis of sigma32 is sensitive to rifampicin (transcription inhibitor), unlike in E. coli, (c) heat-induced accumulation of dnaK transcript occurred even in the absence of protein synthesis, (d) virtually normal induction of HSP occurred even in a strain in which the chromosomal rpoH promoter was replaced by a non-heat shock (lac) promoter, and (e) sigma32 activity can be negatively modulated by DnaKJ chaperones. However, the mechanisms underlying control of sigma32 activity in A. tumefaciens remain open for future studies.
References
(1) Yura, T., Kanemori, M. and Morita, M. T. (2000) In Storz, G. and Hengge-Aronis, R. (eds), Bacterial Stress Responses, ASM Press, Washington, DC.
(2) Nakahigashi, K. et al. (1998) J. Bacteriol. 180, 2402–2408.
(3) Morita, M.T. et al. (1999) Genes Dev. 13, 655–665.
(4) Morita, M.T. et al. (2000) Proc. Natl. Acad. Sci. USA, 97, 5860–5865.
(5) Kanemori, M. et al. (1999) J. Biol. Chem. 274, 22002–22007.
(6) Nakahigashi, K. et al. (2001) J. Bacteriol., in press.
Footnotes
(Appearing in Cell Stress & Chaperones online.)