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. 2004 Sep;6(5):595–602. doi: 10.1593/neo.04214

Differential Regulation of a Fibroblast Growth Factor-Binding Protein during Skin Carcinogenesis and Wound Healing1

Andreas Kurtz *, Achim Aigner *, Rafael H Cabal-Manzano *, Robert E Butler †, Dozier R Hood †, Roy B Sessions †, Frank Czubayko *, Anton Wellstein *
PMCID: PMC1531664  PMID: 15548369

Abstract

The initiation of premalignant lesions is associated with subtle cellular and gene expression changes. Here we describe a severe combined immunodeficient mouse xenograft model with human adult skin and compare chemical carcinogenesis and wound healing. We focus on a secreted binding protein for fibroblast growth factors (FGF-BP) that enhances the activity of locally stored FGFs and is expressed at high levels in human epithelial cancers. Carcinogen treatment of murine skin induced papilloma within 6 weeks, whereas the human skin grafts displayed no obvious macroscopic alterations. Microscopic studies of the human skin, however, showed p53-positive keratinocytes in the epidermis, increased angiogenesis in the dermis of the treated skin, enhanced proliferation of keratinocytes in the basal layer, and an increase of FGF-BP protein and mRNA expression. In contrast, after surgical wounding of human skin grafts or of mouse skin, FGF-BP expression was upregulated within a few hours and returned to control levels after 2 days with wound closure. Enhanced motility of cultured keratinocytes and dermal fibroblasts by FGF-BP supports a role in wound healing. We conclude that adult human skin xenografts can be used to identify early molecular events during malignant transformation as well as transient changes during wound healing.

Keywords: Human/SCID mouse skin grafts, premaligant lesions, skin carcinogenesis, FGF-BP, wound healing

Introduction

Although the use of various animal models has provided remarkable insights into the mechanisms of skin carcinogenesis [1], differences in skin morphology, life span of the species, extent of DNA repair, and capacity to metabolize chemical carcinogens have made the extrapolation of these results to human beings rather limited [2–4]. More recently, cellular interactions and molecular events occurring during carcinogenesis of human skin have been facilitated by the use of human skin grafts in severe combined immunodeficient (SCID) mice [5–7] and the human skin/SCID mouse model proved to be appropriate for diverse studies including those on human endothelium during inflammation [5], interactions between normal and malignant human cells [7], wound healing [8], and viral effects [9–11]. Interestingly, whereas in mouse skin the application of chemical carcinogens can produce squamous atypia or carcinoma, this effect was not observed in xenografted human neonatal foreskin [12,13]. The occurrence of human squamous carcinomas on such xenografts was observed very rarely and only after application of a carcinogen (dimethyl[a]benzanthracene, DMBA) with subsequent UVB promotion [12]. However, more recent studies showed that angiogenesis is an early event during chemical carcinogenesis of murine skin [14] and we wondered to what extent this might also be true for a human model because it is commonly believed that tumor angiogenesis is a rather early event that occurs at the transition of premalignant lesions to invasive cancer [15,16] (reviewed in Ref. [17]).

Many features of solid tumors such as the release of growth factors, proliferation of stromal cells and vascular remodeling or angiogenesis are reminiscent of healing wounds [18]. Among other factors, fibroblast growth factors (FGFs) have been shown to be major players in skin wound healing and in squamous cell carcinogenesis [1,19,20]. A rate-limiting role for FGF-2 in wound healing is supported by the inhibitory effect of blocking antibodies to FGF-2 [21], and wound healing is delayed in FGF-2 knockout animals [22]. However, no significant changes of FGF-2 or FGF receptor levels were found during the wounding process [23], suggesting that modulation of the activity status of FGF signaling could be brought about independent of changes in expression levels of the growth factor.

In earlier studies, we and others observed that human squamous cell carcinomas (SCCs) express FGF-2 together with high levels of a secreted FGF-binding protein (FGF-BP) that is found at high levels during the perinatal phase of development and downregulated thereafter [24–28]. We also detected FGF-BP upregulation with high frequency early during colon carcinogenesis, i.e., in dysplastic lesions [29] and during malignant progression of breast cancer [30]. Overexpression of the FGF-BP cDNA in nontumorigenic human SW-13 cells induced these cells to form highly vascularized tumors [25] and depletion of endogenous FGF-BP from human SCC or colon cancer cells reduced their growth as xenografted tumors in mice [31]. Based on these data and studies from others as well as from our laboratory [25,27,31–34], the current working model proposes that FGF-BP functions as an extracellular chaperone for FGFs that enhances the activity of locally immobilized FGFs. FGF-BP upregulation thus could be a mechanism for FGF activation during malignant progression and also serve as an early indicator of malignant transformation of epithelial cells.

We hypothesized that changes in FGF-BP expression levels could be apparent during wound healing as well as during skin carcinogenesis and we studied FGF-BP modulation during early stages of carcinogenesis compared with wound healing in human and murine skin. We used full-thickness adult upper eyelid skin as the human xenograft, which was grafted onto SCID mice and after healing treated with chemical carcinogens either by repeated application of DMBA or DMBA plus 12-O-tetradecanoylphorbol-13-acetate (TPA). We show an upregulation of FGF-BP in the human epidermis as early as 4 weeks after start of the treatment. Concomitantly, we found p53-positive keratinocytes in the epidermis, an increased number of blood vessels in the dermis of the treated skin, and enhanced proliferation of epidermal keratinocytes in the basal layer. In parallel studies, we observed a strong upregulation of FGF-BP expression within 7 hours of skin wounding, which returned to control levels after 2 days, and we show that FGF-BP can stimulate keratinocyte and dermal fibroblast motility in cell culture, suggesting a possible role during wound closure.

Materials and Methods

Antibody Generation and Purification

Rabbit anti-FGF-BP antibodies against a glutathione-S-transferase (GST)-FGF-BP fusion protein [34] were generated by Lampire Biological Laboratories (Pipersville, PA) according to a standard protocol comprising subcutaneous and intradermal injection of 500 µg antigen in Freund's complete adjuvant. After three booster injections with the same amount of antigen in Freund's incomplete adjuvant, the rabbit was bled to obtain antiserum. Antigen-affinity purification of the antibodies was performed using eukaryotic, recombinant His-tagged FGF-BP that was produced in SF9 insect cells [34] and coupled to Reacti-Gel (Pierce, Rockford, IL) according to the manufacturer's protocol. When using 100 µg His-tagged FGF-BP and 4 ml antiserum, approximately 200 µg of antibodies was obtained. In Western blots, these antibodies as well as the antiserum recognized the recombinant FGF-BP from insect cells [34] and bacterial cell lysates. To further demonstrate the specificity of the affinity-purified anti-FGF-BP antibodies, ME-180 and SW-13 cells were stained by immunofluorescence. The FGF-BP positive ME-180 cells showed strong staining in the cytoplasm, in contrast to the FGF-BP negative SW-13 cells (data not shown).

Skin Xenografts

CB-17 SCID (scid/scid) mice (Taconic, Germantown, NY), 6 to 8 weeks old, were used for xenografting. Human upper eyelid skin discarded after plastic surgery was used for grafting at the day of removal. Grafting was performed after advice from the Herlyn laboratory (Wistar Institute, Philadelphia, PA) as published in Ref. [35]. Briefly, after shaving the hair from a 5-cm2 area on each side of the lateral abdominal region, full-thickness skin was removed, creating two circular graft beds (1.5 cm in diameter). Full-thickness human skin grafts of the same size were placed onto the wound beds. The transplants were held in place with 6-0 nonadsorbable, monofilament suture material and covered with a Band-Aid clipped to the dorsal and ventral skin of the animal with a surgical staple. An additional layer of micropore cloth tape was applied.

Wound Healing Experiments

In vivo wound healing assay Wounding experiments were performed on mouse skin and on viable human skin patches that were grafted onto SCID mice. Skin wounding was performed by full-thickness incision of the shaven skin with a scalpel. Skin from within 2 mm of the wound area was removed after different time points and control skin was obtained from an area about 0.5 to 1 cm apart from the wound and processed for paraffin embedding or immediately used for RNA isolation.

In vitro “wounding” assay SW-13 cells (FGF-BP negative, FGF-2 positive), SW-13/FGF-BP transfected cells [25], primary mouse keratinocytes (MKs) and primary mouse dermal fibroblasts (MDFs) were used. Primary cells were prepared from day 3 postnatal mice as described [36]. A total of 105 cells were plated in triplicates for each of at least three independent experiments into six-well plates to reach confluence after 16 hours when an x-shaped area was scraped from the confluent monolayer using a yellow pipet tip. Cell motility was measured in the presence or absence of FGF-BP (100 µl partially purified fraction), FGF-2 (5 ng/ml; Life Technologies, Rockville, MD), or both. Closure of the gap was photographed every 12 hours for 3 days. FGF-BP was partially purified by heparin fractionation using conditioned medium from transfected SW-13/FGF-BP cells as described [25]. An equally prepared fraction from SW-13 cells transfected with an empty vector (SW-13/Co) served as a control.

Carcinogenesis Experiments

Application of chemical carcinogens or control solvent was started 2 weeks after xenograft implantation for 6 weeks and the experiment was terminated 2 weeks after the last treatment. On the human skin xenografts and adjacent mouse skin of the SCID mice, 200 µg DMBA in 200 µl acetone was applied topically and this treatment was repeated once a week for 6 weeks (DMBA alone) (n = 9). In a parallel protocol, DMBA was only applied once followed 3 days later by twice weekly topical application of 10 µg TPA/200 µl acetone for 6 weeks (n = 9). Control grafts were treated with acetone alone (n = 5). At the end of the experiment (week 10 post grafting, 2 weeks after the last treatment), mice were sacrificed, skin pieces removed, and immediately frozen for RNA analysis, or fixed in 10% formalin and embedded in paraffin.

Histology and Immunohistochemistry

Human skin grafts were harvested 6 and 10 weeks post grafting. Paraffin-embedded sections were stained as described recently [28,37]. Briefly, sections were microwaved for 10 minutes and after 5 minutes cooling at room temperature treated with fresh xylene for 10 minutes. After a duplicate wash in 100% ethanol for 5 minutes each, the slides were rehydrated by gradient ethanol washing and then washed in double-distilled H2O and 1 x PBS for 1 minute each. Heat treatment was performed in 1 x PBS/1% (vol/vol) acetic acid for 10 minutes, reaching 80°C. The slides were then transferred to warm (80°C) 1 x PBS and incubated for 20 minutes before blocking peroxidases with 0.3% H2O2 in 1 x PBS at 4°C for 20 minutes. After two washes in 1 x PBS the tissue was treated in 1 x PBS/1% BSA and blocked in a humid chamber with 10% horse or goat normal serum, depending on the secondary antibodies' origin, in 1 x PBS/2% BSA. After washing in PBS/2% BSA for 5 minutes, primary antibodies were applied for 20 minutes in a 50°C incubator: anti-FGF-BP 100 µg/ml, anti-p53 (Oncogene Research Products, Boston, MA) 1:200, anti-proliferating cell nuclear antigen (PCNA; Signet Laboratories, Dedham, MA) 1:200, or anti-human CD31 (DAKO, Carpinteria, CA) 1:200. As a secondary antibody, biotinylated goat anti-rabbit or biotinylated rabbit anti-mouse in 1 x PBS/2% BSA was applied for 20 minutes at room temperature. For detection and visualization, the ABC system (Vector, Burlingame, CA) was used according to the manufacturer's protocol with 0.1 mg/ml DAB as substrate. Sections were stained with hematoxylin and eosin. For quantitation of blood vessels, CD31-positive vessels were counted. At least five sections per treatment were scored containing 5 to 10 fields at a magnification of 250 x in a blinded fashion.

In Situ Hybridization

Sense and antisense digoxigenin-labeled (Roche, Mannheim, Germany) riboprobes for human FGF-BP were transcribed in vitro for 2 hours at 42°C in the presence of 0.25 µM digoxigenin conjugated dUTP from 1 µg of linearized plasmid containing full-length cDNAs. Transcripts were treated with RNase-free DNase I, ethanol-precipitated, and resuspended in 100 µl DEPC-treated water. In situ hybridization (ISH) was performed as described before [26] with the modifications outlined below. Briefly, 10-µm-thick tissue sections on silanized slides were washed, acetylated, and treated overnight at 55°C with hybridization buffer containing heat-denatured sense or antisense riboprobes. Sections were washed after hybridization at high stringency, immersed in blocking buffer, and immunohistochemistry was performed using an alkaline phosphatase-conjugated sheep anti-digoxigenin antibody (1:500), following the manufacturer's instructions (Roche, Mannheim, Germany). Chromogen substrate for alkaline phosphatase (NBT/BCIP) was added and the reaction was allowed to proceed overnight at 4°C. Color development was stopped with 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, and tissue was mounted in an aqueous mounting medium.

Northern Analysis

RNA was isolated from skin samples using RNAStat (Tel- Test, Friendswood, TX). Twenty micrograms of total RNA from each tissue sample were separated in an agarose gel and transferred onto nitrocellulose. Blots were hybridized with a 32P-labeled, full-length cDNA probe for human or mouse FGF-BP, respectively. For quantitation, the blots were stripped and reprobed with a 32P-labeled, 0.5-kb cDNA fragment of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene (Clontech, Palo Alto, CA). Hybridization was carried out in a buffer containing 50% formamide at 42°C. Blots were subsequently washed in 0.1 x SSC and 0.1% SDS at 65°C before exposure to phosphorimaging plates. Plates were scanned (Phosphoimager 445S1; Molecular Dynamics, Sunnyvale, CA) and quantified using ImageQuant software (Molecular Dynamics).

Data Analysis

The Graphpad Prism 3.0 software was used for data analysis. Student t tests were applied for single comparisons and ANOVA for multiple comparisons. Correlations of nonparametric data were analyzed by chi-square analysis. P values smaller than .05 were considered statistically significant.

Results

Development of Papilloma on the Murine but Not the Human Skin

We used the SCID/human skin xenograft model to study whether upregulation of FGF-BP might be a step during squamous cell carcinogenesis and thus provide an enhancer of local FGF activity we recently described for the recombinant protein [34]. Human upper eyelid skin grafts and adjacent mouse skin were treated with either DMBA alone for 6 weeks (n = 9) or with DMBA/TPA for 6 weeks (n = 9). Six weeks after starting topical treatment of the skin grafts all of the treated mice (18/18) but none of the control mice (5/5) showed papilloma with dysplasia on the mouse skin (Figure 1a, arrows; P < .0001 treated vs. control, chi-square test). At the same time none of the human skin xenografts showed any gross macroscopic alterations (Figure 1a) except for a slight thickening of the carcinogen-treated grafts (see Figure 1a vs. Figure 3a). Histological staining of the treated human xenografts after 4 weeks of treatment and at the end of the experiment at 10 weeks post grafting revealed microscopically evident subtle morphologic changes in the epidermis and dermis that are commonly associated with incipient neoplasia, such as hyperplasia and moderate dysplasia, but none of the control-treated grafts showed similar microscopic changes (not shown). The human skin grafts were viable even 10 weeks after transplantation independent of the treatment protocols.

Figure 1.

Figure 1

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Effect of carcinogen treatment on FGF-BP, PCNA and p53. (a) SCID mouse with human skin xenograft (arrowhead) treated with DMBA/TPA at 6 weeks of treatment. Carcinogen-induced papilloma on the mouse skin are indicated by the arrows. (b–d) in situ hybridization for human FGF-BP mRNA. (b) Sense probe; (c, d) antisense probe; (e, f) immunohistochemical detection of the FGF-BP protein; (g, h) staining for PCNA and (i, j) staining for p53. Samples from control- (c, e, g, i) and DMBA-treated xenograft skin (b, d, f, h, j) were taken at the end of the experiment (i.e., 2 weeks after the last topical treatment and a total of 10 weeks after the initial grafting).

Figure 3.

Figure 3

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FGF-BP expression during wound healing and effects of FGF-BP on cell motility after “in vitro wounding” of a cell monolayer. (a) SCID mouse with sutured human skin xenograft. (b) FGF-BP Northern blot of human skin xenografts at different time points of wound healing representing one of two experiments with similar results. Quantitation after GAPDH control probing is also shown. (c) Different cell types 24 hours after “wounding” of confluent monolayers. SW-13 control or SW-13/FGF-BP transfected cells and mouse primary keratinocytes (MK) or dermal fibroblasts (MDF) with exogenously added control or FGF-BP-containing media.

Upregulation of FGF-BP in Carcinogen-Treated Murine and Human Skin

The appearance of preneoplastic histologic features in the human skin was accompanied by a dramatic increase of FGF-BP levels in the tissues. Most significantly, all of the treated grafts (18/18) showed a strong staining for FGF-BP protein in the entire epidermis at 4 weeks of treatment, which persisted for at least 2 weeks after the last treatment with DMBA (Figure 1f) or DMBA/TPA, when the experiments were discontinued at 10 weeks post grafting. Furthermore, sebaceous glands, blood vessel endothelium, sweat glands, and hair follicles expressed FGF-BP in the treated skin (not shown). In contrast, control xenografts without carcinogen treatment showed very low to undetectable FGF-BP expression (Figure 1e). In situ hybridization validated that the FGF-BP mRNA is upregulated in parallel with the protein (Figure 1, b–d). These results in the human skin xenografts were corroborated by a parallel upregulation of FGF-BP in the adjacent mouse skin (not shown). Interestingly, the induction of FGF-BP expression was similar between the two different carcinogenesis protocols (DMBA alone or DMBA plus TPA), suggesting that mutagenesis induced by DMBA is sufficient to upregulate FGF-BP gene expression and does not require the tumor promoter TPA.

Vascular Density, Basal Cell Proliferation, and p53 Expression in Preneoplastic Human Skin

Because FGF-BP was shown to mobilize dormant FGFs and enhance their activity [25,27,31,34,37], we hypothesized that upregulation of FGF-BP might be paralleled by increased blood vessel density as well as keratinocyte proliferation in skin xenografts. Indeed, concomitant with the upregulation of FGF-BP during the carcinogen treatment, we found a significant 2- to 2.5-fold increase in the number of microvessels beneath the epidermis as visualized by staining with the human endothelial cell marker CD31 (Figure 2a). In addition to increased vessel density, strong expression of the PCNA in keratinocytes in the basal and squamous cell layer of the carcinogen-treated grafts was observed. Control xenografts were either negative or only weakly positive for PCNA (Figure 1, g and h). Furthermore, immunohistochemical examination of the xenografts for nuclear p53 expression revealed strong nuclear staining for p53 in all of the carcinogen-treated xenografts but not in the controls (Figures 1, i and j, and 2b). Again, no significant difference between the DMBA alone and the DMBA plus TPA protocol was apparent. p53-positive keratinocytes appeared predominantly in the basal layer and in the first layers of the squamous stratum at this time point, i.e., 2 weeks after the last treatment and 10 weeks post grafting (Figure 1j).

Figure 2.

Figure 2

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Quantitation of angiogenesis and of p53-positive keratinocytes in control- and carcinogen-treated skin xenografts. (a) Number of CD31-positive blood vessels per field in control-, DMBA-, and DMBA plus TPA-treated human skin xenografts. (b) Number of p53 positive basal and squamous keratinocytes per field in control, DMBA- and DMBA plus TPA-treated skin xenografts. *P < .05 and **P < .01 relative to control.

FGF-BP Upregulation during Wound Healing In Vivo

Wound healing shares a number of aspects with tumorigenesis, and tumors have been described as nonhealing or chronic wounds. To determine whether FGF-BP would be regulated during wound healing and if this regulation is transient or continuous, we used the human upper eyelid skin grafts as a model (Figure 3a). After ingrowth of tissue grafts, full-thickness wounds were cut and biopsied at different time points after wounding to monitor FGF-BP mRNA levels by Northern blotting and FGF-BP protein by immunohistochemistry. Whereas FGF-BP mRNA and protein in the undamaged grafted skin were low, mRNA rapidly increased up to 17-fold 7 hours after wounding and returned to background levels within 4 days (Figure 3b). FGF-BP protein expression follows this course (not shown). A similar transitory upregulation of FGF-BP during the initial phase of wound healing was observed in parallel studies with mouse skin (not shown).

The transient appearance of FGF-BP in the healing wound suggested to us a potential stimulatory activity on cell motility during the initial healing phase and we thus tested whether FGF-BP can affect motility of dermal fibroblasts and keratinocytes in an in vitro assay. In this assay, confluent monolayers of FGF-2-expressing primary MKs and MDFs as well as human epithelial SW-13 cells were scraped with the tip of a pipette, and the filling of the scraped areas with cells was monitored. Addition of FGF-2 alone had no significant stimulatory effect on cell motility in the absence of FGF-BP (not shown). However, the presence of FGF-BP promoted the filling in of monolayers of all three cell types (Figure 3c). The most dramatic effect was observed with FGF-BP transfected SW-13 cells, where closure of the gap was completed after 24 hours compared with approximately 96 hours in the control cells that do not express FGF-BP (4-fold faster closure). The effect of FGF-BP on time to closure of the gap was 3-fold for MDF and 2.5-fold for MK cells. These studies corroborate earlier findings in which addition of FGF-BP enhanced FGF-2-induced chemotaxis of human endothelial cells [38].

Discussion

The human skin/SCID mouse model was used to elucidate early stages of skin carcinogenesis in comparison to molecular changes initiated during wound healing in human and murine skin. We show here that in the human skin derived from adults the topical application of chemical carcinogens is sufficient to induce early preneoplastic stages comparable to incipient SCC in human skin. In our experiments, no differences were observed between the application of DMBA alone and a two-stage system using DMBA initiation and promotion by TPA. Previous studies showed that in a xenograft model of human neonatal skin UVB promotion is necessary to induce human squamous carcinoma [12] and that topical application of a carcinogen alone is not sufficient [13].

Our results reveal that molecular and microscopic changes are detectable after carcinogen treatment alone, although no frank carcinoma or premalignant papilloma were seen. This was the case despite the fact that we used adult human eyelid skin that had been exposed to a lifetime of sunlight in contrast to the commonly used UV-naive neonatal foreskin used in the aforementioned experiments. In our model, immunostaining for PCNA demonstrated increased and sustained proliferation of epidermal keratinocytes in the basal layer, which led to some overall thickening of the skin grafts. Despite this marked PCNA upregulation compared to control skin, histologic changes such as papillomatosis were observed only in mouse skin but not in the human xenografts. Whereas the occurrence of murine papillomas already 5 weeks after the start of the treatment is early compared to other studies [12,13], the lack of papillomas on the adult human skin in our study is in agreement with earlier findings with newborn skin [13] and indicates that UV light-exposed human adult facial skin is more resistant to chemical carcinogens than murine skin. In general, our present findings suggest that commonly used rodent models could overestimate the carcinogenic potential of agents for humans [12].

In UV-induced skin cancers numerous characteristic “hotspots” for p53 point mutations have been described [39]. In our experiments, high numbers of p53 immunopositive keratinocytes in the human skin epidermis were observed as late as 2 weeks after the end of treatment. The cause for p53 upregulation and stabilization in the carcinogen-treated tissues is unclear. Acute induction of wild-type p53 by activated ras has been observed [40], however, the prolonged presence of p53 very likely indicates a mutational cause [41,42].

FGF-1 and FGF-2 are present at significant concentrations in most normal tissues in the adult and both factors stimulate angiogenesis, cell proliferation, and motility, and thus have the potential to play an important role during tumorigenesis. However, under physiologic conditions the activities of these FGFs are restricted by their immobilization in the extracellular matrix [43]. Our previous studies show that the FGF-BP studied here can serve as an activator of FGFs by releasing these potent growth factors from their extracellular matrix [25,34] and high levels of expression of FGF-BP mRNA were observed in tumor tissues from patients with SCC of the head and neck [25,27], and in mouse skin papillomas [26]. Interestingly, the increase in FGF-BP expression reached its maximum in early stages of skin carcinogenesis in the mouse and decreased during tumor progression, indicating that FGF-BP might play a role especially during early stages of tumorigenesis [26]. This is in agreement with our present findings in the human skin xenotransplant model where FGF-BP upregulation occurs at a very early time point of skin carcinogenesis and therefore does fulfill the requirement of an angiogenic switch molecule that is activated during the transition between premalignant lesions and invasive carcinoma [15–17]. The underlying molecular mechanisms that drive FGF-BP upregulation may be due to a number of alterations in signal transduction relevant for skin carcinogenesis and wound healing, e.g., through the EGF receptor, stress kinases, as well as virally derived oncogenes that activate the FGF-BP promoter [44–48].

In the present study, upregulation of FGF-BP was paralleled by a significant (2- to 2.5-fold) increase in the number of blood vessels in carcinogen-treated skin just beneath the basal membrane in the papillary dermis. Angiogenesis is initiated early on during skin carcinogenesis [14] and accompanied by the upregulation of vascular endothelial growth factor (VEGF) observed in mice in premalignant papillomas induced by chemical carcinogenesis and correlated with Ha-ras activation [49]. In our human skin model, carcinogen treatment induced angiogenesis at preneoplastic stages, and this was accompanied by the upregulation of FGF-BP, a protein that was also found upregulated in samples of human SCC [25,27]. In line with this, a very recent study found that FGF-BP positive human SCC samples also showed a significant increased angiogenesis compared to FGF-BP negative SCC [50]. It is likely that several independent mechanisms contribute to the progression toward malignancy and our present findings support the notion of FGF-BP as a contributor.

Interestingly, in nonmalignant diseases that include tissue repair with altered FGF activity, changes in FGF-BP expression were also observed. In a collaborative effort we found a strong upregulation of FGF-BP expression in the kidney stroma of children with renal damage due to diverse causes that include HIV infection as well as bacterial toxins causing hemolytic uremia syndrome (HUS) [37]. In support of a role of the increased FGF-BP expression during kidney repair, we also observed an enhancement of FGF-induced kidney epithelial proliferation by FGF-BP [37]. Furthermore, a very recent report indicates that FGF-BP expression is found upregulated in blood vessels of mice that succumb to premature atherogenesis [51]. In contrast to this long-term upregulation of FGF-BP, the wound healing studies in murine and human tissues showed only a transient upregulation of FGF-BP that returned to baseline on wound closure. It is tempting to speculate that enhanced cell motility required for proper wound closure could be mediated by the increased production of FGF-BP.

In conclusion, we show how FGF-BP is differentially regulated during wound healing and the onset of carcinogenesis in human and murine skin. We propose a potential role for this modulator of FGF activity and we show that the human skin xenograft model can be used to identify early molecular events during malignant transformation as well as wound healing.

Acknowledgements

We thank Meenhard Herlyn and colleagues in his laboratory (Wistar Institute, Philadelphia, PA), Stuart Yuspa and Luigi DeLuca (National Institutes of Health, Bethesda, MD), Anna T. Riegel and Anke M. Schulte (Georgetown University) for helpful discussions, suggestions, and assistance, and Ryan J. Hoefen and Peter Kranz from our laboratory for their help with the experiments.

Abbreviations

DMBA

dimethyl[a]benzanthracene

TPA

12-O-tetradecanoylphorbol-13-acetate

FGF

fibroblast growth factor

FGF-BP

FGF-binding protein

GST

glutathione-S-transferase

PCNA

proliferating cell nuclear antigen

SCC

squamous cell carcinoma

SCID

severe combined immunodeficiency

Footnotes

1

This work was supported in part by grants from the Studienstiftung des Deutschen Volkes to A.A. and the National Institutes of Health/National Cancer Institute (RO1 CA71508) to A.W.

2

Both authors contributed equally to this work.

3

Present address: Robert-Koch-Institut, Berlin, Germany.

4

Present address: Philipps-University, Marburg, Germany.

5

Present address: Beth-Israel Medical Center, New York, NY.

References

  • 1.Yuspa S. The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis—thirty-third G. H. A. Clowes Memorial Award Lecture. Cancer Res. 1994;54:1178–1189. [PubMed] [Google Scholar]
  • 2.Bohr VA, Smith CA, Okumoto DS, Hanawalt PC. DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell. 1985;40:359–369. doi: 10.1016/0092-8674(85)90150-3. [DOI] [PubMed] [Google Scholar]
  • 3.Das M, Asokan P, Don PSC, Krueger GG, Bickers DR, Mukhtar H. Carcinogen metabolism in human skin grafted onto athymic nude mice: a model system for the study of human skin carcinogenesis. Biochem Biophys Res Commun. 1986;138:33–39. doi: 10.1016/0006-291x(86)90242-1. [DOI] [PubMed] [Google Scholar]
  • 4.Reifenrath WG, Chellquist EM, Shipwash EA, Jederberg WW, Krueger GG. Percutaneous penetration in the hairless dog, weanling pig and grafted athymic nude mouse: evaluation of models for predicting skin penetration in man. Br J Dermatol. 1984;111(Suppl 27):123–135. doi: 10.1111/j.1365-2133.1984.tb15590.x. [DOI] [PubMed] [Google Scholar]
  • 5.Yan HC, Juhasz I, Pilewski J, Murphy GF, Herlyn M, Albelda SM. Human/severe combined immunodeficient mouse chimeras. An experimental in vivo model system to study the regulation of human endothelial cell-leukocyte adhesion molecules. J Clin Invest. 1993;91:986–996. doi: 10.1172/JCI116320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Juhasz I, Lazarus GS, Murphy GF, Shih IM, Herlyn M. Development of pemphigus vulgaris-like lesions in severe combined immunodeficiency disease mice reconstituted with lymphocytes from patients. J Clin Invest. 1993;92:2401–2407. doi: 10.1172/JCI116846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Juhasz I, Albelda SM, Elder DE, Murphy GF, Adachi K, Herlyn D, Valyi-Nagy IT, Herlyn M. Growth and invasion of human melanomas in human skin grafted to immunodeficient mice. Am J Pathol. 1993;143:528–537. [PMC free article] [PubMed] [Google Scholar]
  • 8.Juhasz I, Murphy GF, Yan H-C, Herlyn M, Albelda SM. Regulation of extracellular matrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneous human wound healing in vivo. Am J Pathol. 1993;143:1458–1469. [PMC free article] [PubMed] [Google Scholar]
  • 9.Randazzo BP, Kucharczuk JC, Litzki LA, Kaiser LR, Brown M, Maclean A, Albelda SM, Fraser NW. Herpes simplex 1716—an ICP 34.5 mutant—is severely replication restricted in human skin xenografts in vivo. Virology. 1996;223:392–395. doi: 10.1006/viro.1996.0493. [DOI] [PubMed] [Google Scholar]
  • 10.Moffat JF, Stein MD, Kaneshima H, Arvin AM. Tropism of varicella-zoster virus for human CD4+ and CD8+ T lymphocytes and epidermal cells in SCID-hu mice. J Virol. 1995;69:5236–5242. doi: 10.1128/jvi.69.9.5236-5242.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bonnez W, Rose RC, Rin CD, Borkhuis C, Mesy Jensen KLd, Reichman RC. Propagation of human papillomavirus type 11 in human xenografts using the severe combined immunodeficiency (SCID) mouse and comparison to the nude mouse model. Virology. 1993;197:455–458. doi: 10.1006/viro.1993.1611. [DOI] [PubMed] [Google Scholar]
  • 12.Soballe PW, Monotone KT, Satyamoorthy K, Nesbit M, Herlyn M. Carcinogenesis in human skin grafted to SCID mice. Cancer Res. 1996;56:757–764. [PubMed] [Google Scholar]
  • 13.Urano K, Katai Y, Tokuda Y, Ueyama Y, Nomura T, Yamamoto S. Failure of genotoxic carcinogens to produce tumors in human skin xenografts transplanted to SCID mice. Carcinogenesis. 1995;16:2223–2226. doi: 10.1093/carcin/16.9.2223. [DOI] [PubMed] [Google Scholar]
  • 14.Bolontrade MF, Stern MC, Binder RL, Zenklusen JC, Gimenez-Conti IB, Conti CJ. Angiogenesis is an early event in the development of chemically induced skin tumors. Carcinogenesis. 1998;19:2107–2113. doi: 10.1093/carcin/19.12.2107. [DOI] [PubMed] [Google Scholar]
  • 15.Folkman J, Watson K, Ingber D, Hanahan D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature. 1989;339:58–61. doi: 10.1038/339058a0. [DOI] [PubMed] [Google Scholar]
  • 16.Smith-McCune K, Zhu YH, Hanahan D, Arbeit J. Cross-species comparison of angiogenesis during the premalignant stages of squamous carcinogenesis in the human cervix and K14-HPV16 transgenic mice. Cancer Res. 1997;57:1294–1300. [PubMed] [Google Scholar]
  • 17.Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86:353–364. doi: 10.1016/s0092-8674(00)80108-7. [DOI] [PubMed] [Google Scholar]
  • 18.Whalen GF. Solid tumors and wounds: transformed cells misunderstood as injured tissue. Lancet. 1990;336:1489–1492. doi: 10.1016/0140-6736(90)93188-u. [DOI] [PubMed] [Google Scholar]
  • 19.Gospodarowicz D. Humoral control of cell proliferation: the role of fibroblast growth factor in regeneration, angiogenesis, wound healing, and neoplastic growth. Prog Clin Biol Res. 1976;9:1–19. [PubMed] [Google Scholar]
  • 20.Arbeit JM, Olson DC, Hanahan D. Upregulation of fibroblast growth factors and their receptors during multi-stage epidermal carcinogenesis in K14-HPV16 transgenic mice. Oncogene. 1996;13:1847–1857. [PubMed] [Google Scholar]
  • 21.Broadley KN, Aquino AM, Woodward SC, Buckley-Sturrock A, Sato Y, Rifkin DB, Davidson JM. Monospecific antibodies implicate basic fibroblast growth factor in normal wound repair. Lab Invest. 1989;61:571–575. [PubMed] [Google Scholar]
  • 22.Ortega S, Ittmann M, Tsang SH, Ehrlich M, Basilico C. Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2. Proc Natl Acad Sci USA. 1998;95:5672–5677. doi: 10.1073/pnas.95.10.5672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Werner S, Peters KG, Longaker MT, Fuller-Pace F, Banda MJ, Williams LT. Large induction of keratinocyte growth factor expression in the dermis during wound healing. Proc Natl Acad Sci USA. 1992;89:6896–6900. doi: 10.1073/pnas.89.15.6896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Wu DQ, Kan MK, Sato GH, Okamoto T, Sato JD. Characterization and molecular cloning of a putative binding protein for heparin-binding growth factors. J Biol Chem. 1991;266:16778–16785. [PubMed] [Google Scholar]
  • 25.Czubayko F, Smith RV, Chung HC, Wellstein A. Tumor growth and angiogenesis induced by a secreted binding protein for fibroblast growth factors. J Biol Chem. 1994;269:28243–28248. [PubMed] [Google Scholar]
  • 26.Kurtz A, Darwiche N, Harris V, Wang HL, Wellstein A. Expression of a binding protein for FGF is associated with epithelial development and skin carcinogenesis. Oncogene. 1997;14:2671–2681. doi: 10.1038/sj.onc.1201117. [DOI] [PubMed] [Google Scholar]
  • 27.Mongiat M, Otto J, Oldershaw R, Ferrer F, Sato JD, Iozzo RV. Fibroblast growth factor-binding protein is a novel partner for perlecan protein core. J Biol Chem. 2001;276:10263–10271. doi: 10.1074/jbc.M011493200. [DOI] [PubMed] [Google Scholar]
  • 28.Aigner A, Ray PE, Czubayko F, Wellstein A. Immunolocalization of an FGF-binding protein reveals a widespread expression pattern during different stages of mouse embryo development. Histochem Cell Biol. 2002;117:1–11. doi: 10.1007/s00418-001-0360-4. [DOI] [PubMed] [Google Scholar]
  • 29.Ray R, Cabal-Manzano R, Moser AR, Waldman T, Zipper LM, Aigner A, Byers SW, Riegel AT, Wellstein A. Up-regulation of fibroblast growth factor-binding protein, by beta-catenin during colon carcinogenesis. Cancer Res. 2003;63:8085–8089. [PubMed] [Google Scholar]
  • 30.Kagan BL, Henke RT, Cabal-Manzano R, Stoica GE, Nguyen Q, Wellstein A, Riegel AT. Complex regulation of the fibroblast growth factor-binding protein in MDA-MB-468 breast cancer cells by CCAAT/enhancer-binding protein beta. Cancer Res. 2003;63:1696–1705. [PubMed] [Google Scholar]
  • 31.Czubayko F, Liaudet-Coopman ED, Aigner A, Tuveson AT, Berchem GJ, Wellstein A. A secreted FGF-binding protein can serve as the angiogenic switch in human cancer [see editorial] Nat Med. 1997;3:1137–1140. doi: 10.1038/nm1097-1137. [DOI] [PubMed] [Google Scholar]
  • 32.Wellstein A, Czubayko F. Inhibition of fibroblast growth factors. Breast Cancer Res Treat. 1996;38:109–119. doi: 10.1007/BF01803789. [DOI] [PubMed] [Google Scholar]
  • 33.Rak J, Kerbel RS. bFGF and tumor angiogenesis—back in the limelight? Nat Med. 1997;3:1083–1084. doi: 10.1038/nm1097-1083. [news; comment] [DOI] [PubMed] [Google Scholar]
  • 34.Tassi E, Al-Attar A, Aigner A, Swift MR, McDonnell K, Karavanov A, Wellstein A. Enhancement of fibroblast growth factor (FGF) activity by an FGF-binding protein. J Biol Chem. 2001;276:40247–40253. doi: 10.1074/jbc.M104933200. [DOI] [PubMed] [Google Scholar]
  • 35.Yan H-C, Juhasz I, Pilewski J, Murphy GF, Herlyn M, Albelda SM. Human/severe combined immunodeficient mouse chimeras. J Clin Invest. 1993;91:986–996. doi: 10.1172/JCI116320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell. 1980;19:245–254. doi: 10.1016/0092-8674(80)90406-7. [DOI] [PubMed] [Google Scholar]
  • 37.Liu XH, Aigner A, Wellstein A, Ray PE. Up-regulation of a fibroblast growth factor binding protein in children with renal diseases. Kidney Int. 2001;59:1717–1728. doi: 10.1046/j.1523-1755.2001.0590051717.x. [DOI] [PubMed] [Google Scholar]
  • 38.Aigner A, Butscheid M, Kunkel P, Krause E, Lamszus K, Wellstein A, Czubayko F. An FGF-binding protein (FGF-BP) exerts its biological function by parallel paracrine stimulation of tumor cell and endothelial cell proliferation through FGF-2 release. Int J Cancer. 2001;92:510–517. doi: 10.1002/1097-0215(20010515)92:4<510::aid-ijc1227>3.0.co;2-h. [DOI] [PubMed] [Google Scholar]
  • 39.Kanjilal S, Pierceall WE, Cummings KK, Kripke ML, Ananthaswamy HN. High frequency of p53 mutations in ultraviolet radiation-induced radiationinduced murine skin tumors: evidence for strand bias and tumor heterogeneity. Cancer Res. 1993;53:2961–2964. [PubMed] [Google Scholar]
  • 40.Lin AW, Lowe SW. Oncogenic ras activates the ARF-p53 pathway to suppress epithelial cell transformation. Proc Natl Acad Sci USA. 2001;98:5025–5030. doi: 10.1073/pnas.091100298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 1994;54:4855–4878. [PubMed] [Google Scholar]
  • 42.Casey G, Lopez ME, Ramos JC, Plummer SJ, Arboleda MJ, Shaughnessy M, Karlan B, Slamon DJ. DNA sequence analysis of exons 2 through 11 and immunohistochemical staining are required to detect all known p53 alterations in human malignancies. Oncogene. 1996;13:1971–1981. [PubMed] [Google Scholar]
  • 43.Vlodavsky I, Folkman J, Sullivan R, Fridman R, Ishai-Michaeli R, Sasse J, Klagsbrun M. Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix. Proc Natl Acad Sci USA. 1987;84:2292–2296. doi: 10.1073/pnas.84.8.2292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Harris VK, Liaudet-Coopman EDE, Boyle BJ, Wellstein A, Riegel AT. Phorbol ester-induced transcription of a FGF-binding protein is modulated by a complex interplay of positive and negative regulatory promoter elements. J Biol Chem. 1998;273:19130–19139. doi: 10.1074/jbc.273.30.19130. [DOI] [PubMed] [Google Scholar]
  • 45.Harris VK, Coticchia CM, Kagan BL, Ahmad S, Wellstein A, Riegel AT. Induction of the angiogenic modulator fibroblast growth factor- binding protein by epidermal growth factor is mediated through both MEK/ERK and p38 signal transduction pathways. J Biol Chem. 2000;275:10802–10811. doi: 10.1074/jbc.275.15.10802. [DOI] [PubMed] [Google Scholar]
  • 46.Harris VK, Coticchia CM, List HJ, Wellstein A, Riegel AT. Mitogen-induced expression of the fibroblast growth factor-binding protein is transcriptionally repressed through a non-canonical E-box element. J Biol Chem. 2000;275:28539–28548. doi: 10.1074/jbc.M001677200. [DOI] [PubMed] [Google Scholar]
  • 47.Harris VK, Kagan BL, Ray R, Coticchia CM, Liaudet-Coopman ED, Wellstein A, Riegel AT. Serum induction of the fibroblast growth factor-binding protein (FGF-BP) is mediated through ERK and p38 MAP kinase activation and C/EBP-regulated transcription. Oncogene. 2001;20:1730–1738. doi: 10.1038/sj.onc.1204249. [DOI] [PubMed] [Google Scholar]
  • 48.Stoppler H, Malerczyk C, Block K, Aigner A, Czubayko F. The human papillomavirus (HPV) 16 E6 oncoprotein leads to an increase in gene expression of the angiogenic switch molecule FGF-BP in nonimmortalized human keratinocytes. Oncogene. 2001;20:7430–7436. doi: 10.1038/sj.onc.1204957. [DOI] [PubMed] [Google Scholar]
  • 49.Larcher F, Robles AI, Duran H, Murillas R, Quintanilla M, Cano A, Conti CJ, Jorcano JL. Up-regulation of vascular endothelial growth factor/vascular permeability factor in mouse skin carcinogenesis correlates with malignant progression state and activated H-ras expression levels. Cancer Res. 1996;56:5391–5396. [PubMed] [Google Scholar]
  • 50.Li WM, Chen WB. Effect of FGF-BP on angiogenesis in squamous cell carcinoma. Chin Med J (Engl) 2004;117:621–623. [PubMed] [Google Scholar]
  • 51.Napoli C, de Nigris F, Welch BS, Calara FB, Stuart RO, Glass CK, Palinski W. Maternal hypercholesterolemia during pregnancy promotes early atherogenesis in LDL receptor-deficient mice and alters aortic gene expression determined by microarray. Circulation. 2002;105:1360–1367. doi: 10.1161/hc1102.106792. [DOI] [PubMed] [Google Scholar]

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