Apoptosis Versus Cell Differentiation

Abstract

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.

Introduction

Stress or heat shock proteins (HSPs) were first discovered in 1962¹ as a set of highly conserved proteins whose expression was induced by different kinds of stress. It has subsequently been shown that most HSPs have strong cytoprotective effects and behave as molecular chaperones for other cellular proteins. HSPs are also induced at specific stages of development, differentiation and during oncogenesis.² Mammalian HSPs have been classified into five families according to their molecular size: HSP100, HSP90, HSP70, HSP60 and the small HSPs. Each family of HSPs is composed of members expressed either constitutively or regulated inducibly, and/or targeted to different sub-cellular compartments. The most studied HSPs are HSP90, the inducible HSP70 (also called HSP72) and the small heat shock protein HSP27.

HSP90 is a constitutively abundant chaperone that makes up 1–2% of cytosolic proteins. It is an ATP-dependent chaperone that accounts for the maturation and functional stability of a plethora of proteins termed HSP90 client proteins. In mammals, HSP90 comprises 2 homologue proteins (HSP90α and HSP90β) encoded by separated but highly conserved genes that arose through duplication during evolution.³ Most studies do not differentiate between the two isoforms because for a long time they have been considered as having the same function in the cells. However, recent data and notably out-of-function experiments indicate that at least some functions of the beta isoform are not overlapped by HSP90α's functions.⁴ HSP70, like HSP90, binds ATP and undergoes a conformational change upon ATP binding, needed to facilitate the refolding of denatured proteins. The chaperone function of HSP70 is to assist the folding of newly synthesized polypeptides or misfolded proteins, the assembly of multi-protein complexes and the transport of proteins across cellular membranes.^5,6 HSP90 and HSP70 chaperone activity is regulated by co-chaperones like Hip, CHIP or Bag-1 that increase or decrease their affinity for substrates through the stabilization of the ADP or ATP bound state. In contrast to HSP90 and HSP70, HSP27 is an ATP-independent chaperone, its main chaperone function being protection against protein aggregation.⁷ HSP27 can form oligomers of more than 1000 Kda. The chaperone role of HSP27 seems modulated by its state of oligomerization, the multimer being the chaperone competent state.⁸ This oligomerization is a very dynamic process modulated by the phosphorylation of the protein that favors the formation of small oligomers. Cell-cell contact and methylglyoxal can also modulate the oligomerization of the protein.⁹

It is now well accepted that HSPs are important modulators of the apoptotic pathway. Apoptosis, or programmed cell death, is a type of death essential during embryogenesis and, latter on in the organism, to assure cell homeostasis. Apoptosis is also a very frequent type of cell death observed after treatment with cytotoxic drugs.¹⁰ Mainly, two pathways of apoptosis can be distinguished, although cross-talk between the two signal transducing cascades exists (Fig. 1). The extrinsic pathway is triggered through plasma membrane proteins of the tumor necrosis factor (TNF) receptor family known as death receptors, and leads to the direct activation of the proteases called caspases, starting with the receptor-proximal caspase-8. The intrinsic pathway involves intracellular stress signals that provoke the permeabilization of the outer mitochondrial membrane, resulting in the release of pro-apoptotic molecules normally confined to the inter-membrane space. Such proteins translocate from mitochondria to the cytosol in a reaction that is controlled by Bcl-2 and Bcl-2-related proteins.¹¹ One of them is the cytochrome c, which interacts with cytosolic apoptosis protease-activating factor-1 (Apaf-1) and pro-caspase-9 to form the apoptosome, the caspase-3 activation complex.¹² Apoptosis inducing factor (AIF) and the Dnase, EndoG, are other mitochondria intermembrane proteins released upon an apoptotic stimulus. They translocate to the nucleus and trigger caspase-independent nuclear changes.^13,14 Two additional released mitochondrial proteins, Smac/Diablo and Htra2/Omi, activate apoptosis by neutralizing the inhibitory activity of the inhibitory apoptotic proteins (IAPs) that associate with and inhibit caspases¹⁵ (Fig. 1).

Figure 1.

Modulation of apoptosis and differentiation by HSP90, HSP70 and HSP27. In apoptosis (upper part), HSP90 can inhibit caspase (casp.) activation by its interaction with Apaf1. HSP90 stabilizes proteins from the survival signaling including RIP, Akt and Bcl-2. HSP70 can block apoptosis by inhibiting JNK, Bax mitochondrial translocation, by interacting with AIF and/or Apaf-1 or by protecting essential nuclear proteins from caspase-3 cleavage. HSP27 can block caspase activation through its action on F-actin, Bid, ROS or cytochrome c. HSP27 also favors the proteasomal degradation of proteins like I-kBa. During differentiation (lower part), HSP90 stabilizes proteins involved in signaling induced by differentiating factors like Akt, RIP as well as Rb. HSP70 prevents the cleavage of GATA-1 by caspase-3 allowing erythoblast to differentiate instead of dying by apoptosis. HSP70 neutralization of AIF contributes also to the survival of the differentiating cells. HSP27 might be involved in the cell cycle arrest (→, stimulation; ⊥, inhibition; *, in erythroblasts).

Apoptosis and differentiation are two physiological processes that share different features like chromatin condensation or the need of caspase activity.¹⁶ It has been demonstrated in many differentiation models that the activation of caspases is preceded by a mitochondrial membrane depolarization and release of mitochondria apoptogenic molecules.^17,18 This suggests that the mitochondrial-caspase dependent apoptotic pathway is a common intermediate for conveying apoptosis and differentiation. Timing, intensity and cellular compartmentalization might determine whether a cell is to die or differentiate. HSPs might be essential to orchestrate this decision. This review will describe the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation.

HSP27, HSP70 and HSP90 are Anti-Apoptotic Proteins

Overexpression of HSP27, HSP70 or HSP90 prevents apoptosis triggered by various stimuli, including hyperthermia, oxidative stress, staurosporine, ligation of the Fas/Apo-1/CD95 death receptor or anticancer drugs.^2,19–21 Downregulation or inhibition of HSP27, HSP70 or HSP90 have been shown to be enough to sensitize a cell to apoptosis, proving that endogenous levels of those chaperones seem to be sufficiently high to control apoptosis.^22–24 It is now known that these chaperones can interact with key proteins of the apoptotic signaling pathways (Fig. 1).

HSP90: A survival protein through its client proteins.

HSP90 client proteins include a number of signaling proteins like ligand-dependent transcription factors and signal transducing kinases that play a role in the apoptotic process. Upon binding and hydrolysis of ATP, the conformation of HSP90 changes and the client protein, which is no longer chaperoned, is ubiquitinated and degraded by the proteasome.²⁵

A function for HSP90 in the serine/threonine protein kinase Akt pathway was first suggested by studies using an HSP90 inhibitor that promoted apoptosis in HEK293T and resulted in suppressed Akt activity.²⁶ A direct interaction between Akt and HSP90 was reported later.²⁷ Binding of HSP90 protects Akt from protein phosphatase 2A (PP2A)-mediated dephosphorylation.²⁶ Phosphorylated Akt can then phosphorylate the Bcl-2 family protein Bad and caspase-9 leading to their inactivation and to cell survival.^28,29 But Akt has been also shown to phosphorylate IkB kinase, which results in promotion of NFkB-mediated inhibition of apoptosis.³⁰ When the interaction HSP90/Akt was prevented by HSP90 inhibitors, Akt was dephosphorylated and destabilized and the likelihood of apoptosis increased.²⁷ Additional studies showed that another chaperone participates in the Akt-HSP90 complex, namely Cdc37.²⁶ Together this complex protects Akt from proteasome degradation. In human endothelial cells during high glucose exposure, apoptosis can be prevented by HSP90 through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt.³¹ HSP90 has also been shown to interact with and stabilize the receptor interacting protein (RIP). Upon ligation of TNFR-1, RIP-1 is recruited to the receptor and promotes the activation of NFκB and JNK. Degradation of RIP-1 in the absence of HSP90 precludes activation of NFκB mediated by TNFα and sensitizes cells to apoptosis.³² Another route by which HSP90 can affect NFκB survival activity is via the IKK complex.³³ The HSP90 inhibitor geldanamycin prevents TNF-induced activation of IKK, highlighting the role of HSP90 in NFκB activation. Some other HSP90 client proteins through which this chaperone could participate in cell survival are p53³⁴ and the transcription factors Her2 and Hif1α.^35,36

But the anti-apoptotic role of HSP90 can also be explained by its effect and interaction with proteins not defined as HSP90 client proteins (i.e., whose stability is not regulated by HSP90). HSP90 overexpression in human leukemic U937 cells can prevent the activation of caspases in cytosolic extracts treated with cytochrome c probably because HSP90 can bind to Apaf-1 and inhibit its oligomerization and further recruitment of procaspase-9.³⁷

Unfortunately, most studies do not differentiate between HSP90α and HSP90β. It has recently been demonstrated in multiple myeloma, in which an over expression of HSP90 is necessary for cell survival, that depletion of HSP90β by siRNA is sufficient to induce apoptosis. This effect is strongly increased when also HSP90α is also depleted,²³ suggesting different and cooperating anti-apoptotic properties for HSP90α and HSP90β. Confirming this assumption, in mast cells, HSP90β has been shown to associate with the anti-apoptotic protein Bcl-2. Depletion of HSP90β with a siRNA or inhibion of HSP90 with geldanamycin inhibits HSP90β interaction with Bcl-2 and results in cytochrome c release, caspase activation and apoptosis.³⁸

In conclusion, HSP90 anti-apoptotic functions can largely be explained by its chaperone role assuring the stability of different proteins. Recent studies suggest that the two homologue proteins, HSP90α and HSP90β, might have different survival properties. It would be interesting to determine whether HSP90α and HSP90β bind to different client proteins or bind with different affinity.

HSP70: A quintessential inhibitor of apoptosis.

HSP70 loss-of-function studies demonstrated the important role of HSP70 in apoptosis. Cells lacking hsp70.1 and hsp70.3, the two genes that code for inductive HSP70, are very sensitive to apoptosis induced by a wide range of lethal stimuli.³⁹ Further, the testis specific isoform of HSP70 (hsp70.2) when ablated, results in germ cell apoptosis.⁴⁰ In cancer cells, depletion of HSP70 results in spontaneous apoptosis.⁴¹

HSP70 has been shown to inhibit the apoptotic pathways at different levels (Fig. 1). At the pre-mitochondrial level, HSP70 binds to and blocks c-Jun N-terminal Kinase (JNK1) activity.^42,43 Confirming this result, HSP70 deficiency induces JNK activation and caspase-3 activation⁴⁴ in apoptosis induced by hyperosmolarity. HSP70 also has been shown to bind to non-phosphorylated protein kinase C (PKC) and Akt, stabilizing both proteins.⁴⁵

At the mitochondrial level, HSP70 inhibits Bax translocation and insertion into the outer mitochondrial membrane. As a consequence, HSP70 prevents mitochondrial membrane permeabilization and release of cytochrome c and AIF.⁴⁶

At the post-mitochondrial level HSP70 has been demonstrated to bind directly to Apaf-1, thereby preventing the recruitment of procaspase-9 to the apoptosome.⁴⁷ However, these results have been contradicted by a study in which the authors demonstrated that HSP70 do not have any direct effect on caspase activation. They explain these contradictory results by showing that it is a high salt concentration and not HSP70 that inhibits caspase activation.⁴⁸

HSP70 also prevents cell death in conditions in which caspase activation does not occur.⁴⁹ Indeed, HSP70 binds to AIF, inhibits AIF nuclear translocation and chromatin condensation.^39,50,51 The interaction involves a domain of AIF between aminoacids 150 and 228.⁵² AIF sequestration by HSP70 has been shown to reduce neonatal hypoxic/ischemic brain injury.⁵³ HSP70 has also been shown to associate with EndoG and to prevent DNA fragmentation⁵⁴ but since EndoG can form complexes with AIF, its association with HSP70 could involve AIF as a molecular bridge.

HSP70 can also rescue cells from a later phase of apoptosis than any known survival protein, downstream caspase-3 activation.⁵⁵ During the final phases of apoptosis, chromosomal DNA is digested by the DNase CAD (caspase activated DNase), following activation by caspase-3. The enzymatic activity and proper folding of CAD has been reported to be regulated by HSP70.⁵⁶

At the death receptors level, HSP70 binds to DR4 and DR5, thereby inhibiting TRAIL-induced assembly and activity of death inducing signaling complex (DISC).⁵⁷ Finally, HSP70 has been shown to inhibit lysosomal membrane permeabilization thereby preventing cathepsines release, proteases also implicated in apoptosis.^58,59

In conclusion, HSP70 is a quintessential regulator of apoptosis that can interfere with all main apoptotic pathways. Interestingly, the ATP binding domain of HSP70 is not always required. For instance, while the ATPase function is needed for the Apaf-1⁵⁰ and AIF binding,⁵¹ it is dispensable for JNK⁶⁰ or GATA-1⁶¹ binding/protection. In this way, in erythroblasts, in which HSP70 blocks apoptosis by protecting GATA-1 from caspase-3 cleavage, a HSP70 mutant that lacks the ATP binding domain of HSP70 is as efficient as wild type HSP70 in assuring the protection of erythroblasts.⁶¹

HSP27: An inhibitor of caspase activation.

HSP27 depletion reports demonstrate that HSP27 essentially blocks caspase-dependent apoptotic pathways. Small interefence targeting HSP27 induces apoptosis through caspase-3 activation.^62,63 This may be consequence of the association of HSP27 with cytochrome c in the cytosol, thereby inhibiting the formation of the caspase-3 activation complex as demonstrated in leukemia and colon cancer cells treated with different apoptotic stimuli.^64–66 This interaction involves amino-acids 51 and 141 of HSP27 and do not need the phosphorylation of the protein.⁶⁵ In multiple myeloma cells treated with dexamethasone, HSP27 has also been shown to interact with Smac.⁶⁷

HSP27 can also interfere with caspase activation upstream of the mitochondria.⁶⁶ This effect seems related to the ability of HSP27 to interact and regulate actin microfilaments dynamics. In L929 murine fibrosarcoma cells exposed to cytochalasin D or staurosporine, overexpressed HSP27 binds to F-actin⁶⁸ preventing the cytoskeletal disruption, Bid intracellular redistribution and cytochrome c release⁶⁶ (Fig. 1). HSP27 has also important anti-oxidant properties. This is related to its ability to uphold glutathione in its reduced form,⁶⁹ to decrease reactive oxygen species cell content,¹⁹ and to neutralize the toxic effects of oxidized proteins.⁷⁰ These anti-oxidant properties of HSP27 seem particularly relevant in HSP27 protective effect in neuronal cells.⁷¹

HSP27 has been shown to bind to the kinase Akt, an interaction that is necessary for Akt activation in stressed cells. In turn, Akt could phosphorylate HSP27, thus leading to the disruption of HSP27-Akt complexes.⁷² HSP27 also affects one downstream event elicited by Fas/CD95. The phosphorylated form of HSP27 directly interacts with Daxx.⁷³ In LNCaP tumor cells, HSP27 has been shown to induce cell protection through its interaction with the activators of transcription 3 (Stat3).⁷⁴ Finally, HSP27 protective effect can also be consequence of its effect favouring the proteasomal degradation of certain proteins under stress conditions. Two of the proteins that HSP27 targets for their ubiquitination/proteasomal degradation are the transcription factor nuclear factor κB (NFκB) inhibitor IκBα and p27^kip1. The pronounced degradation of IkBα induced by HSP27 overexpression increases NFκB dependent cell survival⁷⁵ while that of p27^kip1 facilitates the passage of cells to the proliferate phases of the cellular cycle. As a consequence HSP27 allows the cells to rapidly resume proliferation after a stress.⁷⁶

Therefore, HSP27 is able to block apoptosis at different stages because of its interaction with different partners. The capacity of HSP27 to interact with one or another partner seems to be determined by the oligomerization/phosphorylation status of the protein, which, at its turn, might depend on the cellular model/experimental conditions. We have demonstrated in vitro and in vivo that for HSP27 caspase-dependent anti-apoptotic effect, large non-phosphorylated oligomers of HSP27 were the active form of the protein.⁷⁷ Confirming these results, it has recently been demonstrated that methylglyoxal modification of HSP27 induces large oligomers formation and increases the anti-apoptotic caspase-inhibitory properties of HSP27.⁷⁸ In contrast, for HSP27 interaction with the F-actin and with Daxx, phosphorylated and small oligomers of HSP27 were necessary^73,79 and it is its phosphorylated form that protects against neurotoxicity.⁸⁰

HSP27, HSP70 and HSP90 and Cell Differentiation

Under the prescribed context of HSPs as powerful inhibitors of apoptosis, it is reasonable to assume that an increase or decrease in their expression might modulate the differentiation program. The first evidence of the role of HSPs in cell differentiation comes from their tightly regulated expression at different stages of development and cell differentiation. For instance during the process of endochondrial bone formation, they are differentially expressed in a stage-specific manner.⁸¹ In addition, during post-natal development, time at which extensive differentiation takes place, HSPs expression is regulated in neuronal and non-neuronal tissues.⁸² In hemin-induced differentiation of human K562 erythroleukemic cells, genes coding for HSPs are induced.⁸³

In leukemic cells HSP27 has been described as a pre-differentiation marker⁸⁴ because its induction occurs early during differentiation.^85–88 HSP27 expression has also been suggested as a differentiation marker for skin keratinocytes⁸⁹ and for C2C12 muscle cells.⁹⁰ This role for HSP27 in cell differentiation might be related to the fact that HSP27 expression increases as cells reach the non proliferative/quiescent phases of the cellular cycle (G₀/G₁).^19,76

Subcellular localization is another mechanism whereby HSPs can determine whether a cell is to die or to differentiate. We, and others, have recently demonstrated the essential function of nuclear HSP70 for erythroid differentiation. During red blood cells' formation, HSP70 and activated caspase-3 accumulate in the nucleus of the erythroblast.⁹¹ HSP70 directly associates with GATA-1 protecting this transcription factor required for erythropoiesis from caspase-3 cleavage. As a result, erythroblats continue their differentiation process instead of dying by apoptosis.⁶¹ HSP70, during erythropoiesis in TF-1 cells, have been shown to bind to AIF and thereby to block AIF-induced apoptosis, thus allowing the differentiation of erythroblasts to proceed.¹⁸

HSP90 has been required for erythroid differentiation of leukemia K562 cells induced by sodium butyrate⁹² and for DMSO-differentiated HL-60 cells. Regulation of HSP90 isoforms may be a critical event in the differentiation of human embryonic carcinoma cells and may be involved in differentiation into specific cell lineages.⁹³ This effect of HSP90 in cell differentiation is probably because multiple transduction proteins essential for differentiation are client proteins of HSP90 such as Akt,⁹⁴ RIP³² or Rb.⁹⁵ Loss of function studies confirm that HSP90 plays a role in cell differentiation and development. In Drosophila melanogaster, point mutations of HSP83 (the drosophila HSP90 gene) are lethal as homozygotes. Heteterozygous mutant combinations produce viable adults with the same developmental defect: sterility.⁹⁶ In Caenorhabditis elegans, DAF-21, the homologue of HSP90, is necessary for oocyte development.⁹⁷ In zebrafish, HSP90 is expressed during normal differentiation of triated muscle fibres. Disruption of the activity of the proteins or the genes give rise to failure in proper somatic muscle development.⁹⁸ In mice, loss-of-function studies demonstrate that while HSP90α loss-of-function phenotype appears to be normal, HSP90β is lethal. HSP90β is essential for trophoblasts differentiation and thereby for placenta development and this function can not be performed by HSP90α.⁴

HSP90 inhibitors have also been used to study the role of HSP90 in cell differentiation. These inhibitors such as the benzoquinone ansamycin geldanamycin or its derivative the 17-allylamino-17-demethoxygeldanamycin (17-AAG), bind to the ATP-binding “pocket” of HSP90 with higher affinity than natural nucleotides and thereby HSP90 chaperone activity is impaired and its client proteins are degraded. As could be expected by the reported role of HSP90 in cell differentiation, inhibitors of HSP90 block C2C12 myoblasts differentiation.⁹⁹ In cancer cells and human leukemic blasts, 17-AAG induces a retinoblastoma-dependent G₁ block. These G₁ arrested cells do not differentiate but instead die by apoptosis.¹⁰⁰

However, some reports describe that inhibitors of HSP90 can induce the differentiation process. In acute myeloid leukemia cells, 17-AAG induced apoptosis or differentiation depending on the dose and time of the treatment.¹⁰¹ Opposite effects on cell differentiation and apoptosis are also obtained with the HSP90 inhibitor geldanamycin: in PC12 cells it induced apoptosis while in murin neuroblastoma N2A cells it induced differentiation.¹⁰² In breast cancer cells, 17-AAG-induced G₁ block is accompanied by differentiation followed by apoptosis.¹⁰³ The HSP90 inhibitor PU3, a synthetic purine that like 17-AAG binds with high affinity to the ATP “pocket” of HSP90, caused breast cancer cells arrest in G1 phase and differentiation.¹⁰⁴

These contradictory reports concerning the inhibitors of HSP90 and cell differentiation could be explained if we consider that these drugs, depending on the experimental conditions, can have some side effects more or less independent of HSP90. Another possibility is that these studies do not differentiate between the amount of HSP90α and HSP90β inhibited. It is presently unknown whether HSP90 inhibitors equally block both isoforms, HSP90α and HSP90β. It not known neither whether post-translational modifications of HSP90 (acetylation, phosphorylation.) can affect their affinity for the inhibitors. HSP90α has been reported to be induced by lethal stimuli while the HSP90β can be induced by growth factors or cell differentiating signals.¹⁰⁵ Mouse embryos out-of-function studies clearly show the role of HSP90β in the differentiation process and, at least for HSP90β role in embryo cell differentiation, there is not an overlap with HSP90α functions. Therefore, we can hypothesized that it can be the degree of inhibition of HSP90β by the HSP90 inhibitors that would determine whether or not there is a blockade of the differentiation process. This degree of inhibition of the different HSP90 isoforms might be conditioned by their cellular localization and their post-translational modifications. It should be noted, however, that the relative relevance of HSP90β in the differentiation process might depend on the differentiation model studied.

To summarize, we can hypothesize that the role in the differentiation process of a chaperone will be determined by its transient expression, subcellular redistribution and/or post-translational modifications induced at a given stage by a differ- entiation factor. How can HSPs affect the differentiation process? First by their anti-apoptotic role interfering with caspase activity, we and other authors have shown that caspase activity was generally required for cell differentiation.^16,17 Therefore, HSPs by interfering with caspase activity at a given moment, in a specific cellular compartment, may orchestrate the decision differentiation versus apoptosis. In this way, we have recently shown that HSP70 was a key protein to orchestrate this decision in erythroblasts.⁶¹ Second, HSPs may affect the differentiation process by regulating the nuclear/cytosolic shuttling of proteins that take place during differentiation. For instance, c-IAP1 is translocated from the nucleus to the cytosol during differentiation of hematopoietic and epithelial cells, and we have demonstrated that HSP90 is needed for this c-IAP1 nuclear export.¹⁰⁶ It has also been shown that, during erythroblast differentiation, HSP70 is needed to inhibit AIF nuclear translocation.¹⁸ Third, in the case of HSP90, the role in the differentiation process could be through certain of its client proteins, like RIP or Akt, whose stability is assured by the chaperone.

Repercussions and Concluding Remarks

The ability of HSPs to modulate the fate of the cells might have important repercussions in pathological situations such as cancer. Apoptosis, differentiation and oncogenesis are very related processes. Defaults in differentiation and/or apoptosis are involved in many cancer cells' aetiology. HSPs are abnormally constitutively high in most cancer cells and, in clinical tumors, they are associated with poor prognosis. In experimental models, HSP27 and HSP70 have been shown to increase cancer cells' tumorigenicty and their depletion can induce a spontaneous regression of the tumors.^24,107 Several components of tumor cell-associated growth and survival pathways are HSP90 client proteins. These qualities have made HSPs targets for anticancer drug development. Today, although many research groups and pharmaceutical companies look for soluble specific inhibitors of HSP70 and HSP27, only specific soluble inhibitors of HSP90 are available for clinical trials. For some of them (17-AAG) phase II clinical trials are almost finished.¹⁰⁸ However, considering the new role of HSP90β in cell differentiation, it seems essential to re-evaluate the functional consequences of HSP90 blockade.