Stathmin 1 is a potential novel oncogene in melanoma.

In previous studies, we demonstrated that miR-193b expression is reduced in melanoma relative to benign nevi, and also that miR-193b represses cyclin D1 and Mcl-1 expression. We suggested that stathmin 1 (STMN1) might be a target of miR-193b. STMN1 normally regulates microtubule dynamics either by sequestering free tubulin heterodimers or by promoting microtubule catastrophe. Increased expression of STMN1 has been observed in a variety of human malignancies, but its association with melanoma is unknown. We now report that STMN1 is upregulated during the progression of melanoma relative to benign nevi, and that STMN1 is directly regulated by miR-193b. Using an experimental cell culture approach, overexpression of miR-193b using synthetic microRNAs repressed STMN1 expression, whereas inhibition of miR-193b with anti-miR oligos increased STMN1 expression in melanoma cells. The use of a luciferase reporter assay confirmed that miR-193b directly regulates STMN1 by targeting the 3'-untranslated region of STMN1 mRNA. We further demonstrated that STMN1 is overexpressed in malignant melanoma compared with nevi in two independent melanoma cohorts, and that its level is inversely correlated with miR-193b expression. However, STMN1 expression was not significantly associated with patient survival, Breslow depth, mitotic count or patient age. STMN1 knockdown by small-interfering RNA in melanoma cells drastically repressed cell proliferation and migration potential, whereas ectopic expression of STMN1 using lentivirus increased cell proliferation and migration rates. Subsequent gene expression analysis indicated that interconnected cytoskeletal networks are directly affected following STMN1 knockdown. In addition, we identified deregulated genes associated with proliferation and migration, and revealed that p21(Cip1/Waf1) and p27(Kip) could be downstream effectors of STMN1 signaling. Taken together, our study suggests that downregulation of miR-193b may contribute to increased STMN1 expression in melanoma, which consequently promotes migration and proliferation of tumor cells.

Evidence that dysregulated DNA mismatch repair characterizes human nonmelanoma skin cancer.

BACKGROUND: In addition to an established role in the repair of postreplicative DNA errors, DNA mismatch repair (MMR) proteins also contribute to cellular responses to exogenous DNA damage. Previously, we have shown that Msh2-null mice display increased sensitivity to ultraviolet (UV) B-induced tumorigenesis, but squamous cell carcinomas (SCC) generated are microsatellite stable, suggesting a role for MMR other than postreplicative repair in UV-induced cutaneous tumour formation. OBJECTIVES: We questioned whether there was evidence of MMR dysfunction in human SCC, thus validating the mouse models of MMR-dependent UVB-induced skin cancer. METHODS: Using tissue microarrays we examined both nuclear and cytoplasmic levels of MMR proteins MSH2, MSH6, MSH3, MLH1 and PMS2 in more than 200 cases of cutaneous SCC and basal cell carcinoma (BCC). RESULTS: We found that subsets of these 10 MMR protein measures were increased in nonmelanoma skin cancer (NMSC) compared with normal epidermal samples; this was particularly true of SCC. In fact, based on post hoc tests and MMR protein distribution patterns, BCC was distinct from SCC. With the exception of nuclear MSH2, the BCC had lower levels of identified MMR protein measures than SCC. We believe this to be important because not only is SCC more aggressive than BCC, but evidence suggests that these two NMSC subtypes arise through different molecular pathways. CONCLUSIONS: In combination with previously established roles for MMR proteins in response to UVB-induced DNA damage, our data point towards an expanded perspective of the importance of MMR proteins in the suppression of UVB-induced tumorigenesis and, potentially, tumour behaviour.

Thrombospondin-1 and cutaneous melanoma.

BACKGROUND: Thrombospondins (TSPs) are recognized as important glycoproteins that regulate a wide variety of cell functions and interactions. TSPs in malignant tumors can both enhance and inhibit tumor progression, invasion, and metastasis, depending on cell type, stromal interactions, and microenvironment. These proteins are potential targets for anticancer therapy. OBJECTIVE: The aim of our article is to review the role of thrombospondin-1 (TSP1) in cutaneous melanoma. CONCLUSIONS: TSP1 expression is variable in melanoma cell lines and tumors. Similar to findings in other human cancers, expression of TSP1 by melanoma cells usually inhibits tumor progression via the antiangiogenic effect of TSP1. Conversely, stromal TSP1 overexpression in melanoma is a poor prognostic factor associated with decreased survival. Understanding the interactions of TSP1 with other melanoma- and matrix-associated proteins should provide new prognostic indices and possible therapeutic targets for melanoma treatment.

p53-dependent regulation of heat shock protein 72.

BACKGROUND: p53 is a key regulator of the cellular stress response. p53 modulates the transcription of several genes. OBJECTIVES: To examine the influence of p53 on expression of heat shock protein 72 (HSP72). METHODS: Two model systems were used. (i) HSP72 expression was studied by Western blot on extracts from p53-proficient or p53-deficient primary mouse keratinocytes, and (ii) archival human anogenital skin from fibroepithelial polyps, human papillomavirus (HPV) 16/18-associated lesions or squamous cell carcinomas (SCCs) was subjected to immunostaining for HSP72. RESULTS: Basal HSP72 expression was higher in keratinocytes from p53-deficient than from p53-proficient mice. Immunostaining for HSP72 was higher in HPV 16/18 lesions and SCCs, which have reduced p53 protein. CONCLUSIONS: p53 status may influence the basal level of HSP72.

Nucleotide excision repair genes are upregulated by low-dose artificial ultraviolet B: evidence of a photoprotective SOS response?

Nucleotide excision repair is a major mechanism of defense against the carcinogenic effects of ultraviolet light. Ultraviolet B causes sunburn and DNA damage in human skin. Nucleotide excision repair has been studied extensively and described in detail at the molecular level, including identification of many nucleotide excision repair-specific proteins and the genes encoding nucleotide excision repair proteins. In this study, normal human keratinocytes were exposed to increasing doses of ultraviolet B from fluorescent sunlamps, and the effect of this exposure on expression of nucleotide excision repair genes was examined. An RNase protection assay was performed to quantify transcripts from nucleotide excision repair genes, and a slot blot DNA repair activity assay was used to assess induction of the nucleotide excision repair pathway. The activity assay demonstrated that cyclobutane pyrimidine dimers were removed efficiently after exposure to low doses of ultraviolet B, but this activity was delayed significantly at higher doses. All nucleotide excision repair genes examined demonstrated a similar trend: ultraviolet B induces expression of nucleotide excision repair genes at low doses, but downregulates expression at higher doses. In addition, we show that pre-exposure of cells to low-dose ultraviolet protected keratinocytes from apoptosis following high-dose exposure. These data support the notion that nucleotide excision repair is induced in cells exposed to low doses of ultraviolet B, which may protect damaged keratinocytes from cell death; however, exposure to high doses of ultraviolet B downregulates nucleotide excision repair genes and is associated with cell death.

Ultraviolet light (UV) regulation of the TNF family decoy receptors DcR2 and DcR3 in human keratinocytes.

BACKGROUND: Several additional members of the tumor necrosis factor (TNF) receptor family were recently identified. The existence of such receptors, which may play distinct and unique regulatory roles, suggests that complex regulatory mechanisms are involved in apoptosis. OBJECTIVE: This study examines the expression of several members of the TNF receptor family in human keratinocytes exposed to ultraviolet B (UVB) irradiation. METHODS: Human keratinocytes were exposed to increasing doses of UVB, total RNA was harvested, and a quantitative RNase protection assay was performed. RESULTS: Decoy receptor-3 (DcR3), a nonfunctional receptor that binds to Fas ligand (FasL), was constitutively expressed at high level in keratinocytes but decreased rapidly in cells exposed to UVB. Decoy receptor-2 (DcR2), a nonfunctional receptor that binds to TNF-related apoptosis-inducing ligand (TRAIL)/APO-2L, showed the opposite expression pattern. DcR2 was undetectable in unirradiated keratinocytes and was markedly up-regulated after exposure to UVB. Although the response showed significant delays at higher UVB doses, the patterns observed for DcR3 and DcR2 were consistent in this set of experiments. CONCLUSION: We conclude that UVB regulates expression of these two TNF decoy receptors in keratinocytes. This pathway may represent a novel mechanism for regulation of apoptosis in the skin.

Effect of N-acetylcysteine on UVB-induced apoptosis and DNA repair in human and mouse keratinocytes.

The incidence of skin cancer is increasing rapidly, particularly in the Caucasian population. Epidemiological and experimental studies demonstrated that ultraviolet radiation (UVR) is the primary cause for the increasing incidence of skin cancer. It is well known that UV irradiation induces DNA damage. If the damage is not repaired or removed in time, it can lead to mutations and skin carcinogenesis. N-acetylcysteine (NAC) has been shown to be an effective protector against UVB-induced immunosuppression and to modulate the expression of some oncogenes and tumor suppressor genes. To test further the protective effect of NAC against UVR, we used both in vitro and in vivo models to investigate the effect of NAC on UVB-induced apoptosis and repair of DNA damage in human and mouse keratinocytes. Our data indicate that the intracellular glutathione level was increased after treatment with NAC at 10-20 mM but decreased with 40 mM NAC treatment due to the toxicity. At concentrations up to 20 mM NAC did not have a significant effect on UVB-induced apoptosis of cultured human keratinocytes. In addition, in an in vivo mouse model, topical application of NAC (3 mumol cm-2) that has been shown to inhibit UVB-induced immunosuppression did not have any effect on UVB-induced apoptosis and did not reduce the formation or enhance the repair of UVB-induced cyclobutane pyrimidine dimers and (6-4) photoproducts. Our results indicate that NAC is ineffective in preserving the genomic stability of keratinocytes against UVB irradiation.

Expression of cell cycle regulators in human cutaneous malignant melanoma.

We postulate that genes involved in the control of cell proliferation are important determinants of melanoma growth and/or transformation. Using Western blot analysis, we compared the expression of nine key cell cycle regulators in metastatic melanomas with that in benign acquired naevi. Among the cyclin-dependent kinases (CDKs) examined, CDK2 was consistently and significantly overexpressed (three- to eight-fold) in metastatic melanomas compared with naevi. CDK1 and CDK4 exhibited no significant difference in expression between benign naevi and metastatic melanomas. CDK6 expression was variable, with four out of 10 metastatic melanomas showing higher expression than naevi. All the cyclins examined, especially cyclins A and D, were expressed more in metastatic melanomas than in naevi. Cyclin E was not detected in benign naevi, but was easily detectable in most of the metastatic melanomas. In addition, there was significantly greater expression of CDC25A, a tyrosine phosphatase that activates CDK kinases, in the metastatic melanomas. Over-expression of CDK2, CDK6, CDC25A and cyclin A was confirmed in melanoma cell lines. These cell cycle regulators may play an important role in melanoma growth and/or transformation.

Ultraviolet radiation-induced p53 responses in the epidermis are differentiation-dependent.

BACKGROUND: The tumour suppressor, p53, is recognized as a crucial molecule in regulating cellular responses to various DNA-damaging agents. Very early on in the development of nonmelanoma cancers p53 is mutated or lost, suggesting that p53 is crucial in protecting normal keratinocytes from the harmful effects of ultraviolet (UV) radiation. OBJECTIVE: Using two mouse models, one with multiple copies of mutant p53 and the other a p53 "knockout," our laboratory has examined a role for p53 in UV-induced DNA damage and determined if these effects are differentiation dependent. CONCLUSION: We outline in this review a proposed model reflecting differentiation-dependent p53 regulation of UV-induced responses in keratinocytes. After exposure to UV, basal keratinocytes repair damaged DNA, whereas differentiating keratinocytes undergo cell death, both processes are regulated by p53.

Heat shock transcription factor-1 regulates heat shock protein-72 expression in human keratinocytes exposed to ultraviolet B light.

In response to ultraviolet radiation (UVR), skin keratinocytes increase expression of heat shock proteins that can protect cells from stress-induced damage. This heat shock response is known to be transcriptionally regulated in eukaryotic cells exposed to certain forms of environmental stress. In the skin, absorption of ultraviolet B light occurs primarily in the epidermis, and therefore, using primary cultures of normal human epidermal keratinocytes, we have examined whether transcriptional activation of the hsp72 gene occurs following UVB irradiation. Cultured keratinocytes were exposed to UVB (290-320 nm, 300 J per m2) and then incubated at 37 degrees C for various intervals before harvesting. Immediately following UV exposure, the heat shock transcription factor 1 (HSF1) dissociated from HSP72-HSF1 complexes, underwent trimerization and phosphorylation, and demonstrated DNA binding activity to the heat shock element in the promoter region of the hsp72 gene. UVB also increased hsp72 mRNA, with peak levels observed 1-3 h post-UVR. HSP72 protein was constitutively expressed in keratinocytes, and its expression was increased by UVB, with maximum levels at 6 h post-UVR. The stress response may be extremely important in the protection of human skin from UVB radiation, and modulation of heat shock protein expression and/or function offers a potential therapeutic target in the prevention of photoaging and skin cancer.

p53-regulated apoptosis is differentiation dependent in ultraviolet B-irradiated mouse keratinocytes.

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (-/-) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53-/- mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53-/- cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53-/- cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.

Expression of apoptosis regulators in cutaneous malignant melanoma.

Metastatic malignant melanoma (MM) is usually incurable and responds poorly to chemotherapy. Because many cytotoxic drugs cause cell death by inducing apoptosis, an imbalance of apoptosis regulatory proteins may contribute to MM treatment resistance. We have previously shown reduced expression of Bcl-2 protein, a negative regulator of apoptosis, in MM as compared with benign nevi. It is hypothesized that other apoptosis regulators may be involved in survival of MM cells. We examined the expression of Bax, Bcl-2, Bcl-X, and Mcl-1 in human benign nevi, primary MM, and metastatic MM using immunohistochemistry. Results were confirmed with Western blotting. The proapoptotic protein, Bax, was surprisingly overexpressed in all MM samples compared with benign nevi. Interestingly, in most MM samples there was overexpression of Mcl-1 or Bcl-XL, both negative regulators of apoptosis. Increased expression of Mcl-1 and Bcl-XL was first observed in thin primary melanomas, suggesting that up-regulation of these proteins represents a relatively early event associated with malignant transformation in MM. As published previously, the majority of primary and metastatic MM exhibited reduced Bcl-2 levels. We conclude that the apoptosis inhibitors Bcl-XL or Mcl-1, alone or in combination, may circumvent the normal cell death pathway, contributing to the pathogenesis and treatment resistance in metastatic MM.

p53-dependent regulation of nucleotide excision repair in murine epidermis in vivo.

BACKGROUND: p53 protects the integrity of the genome by inducing programed cell death or by promoting DNA repair. We have previously shown that loss or mutation of p53 leads to reduced DNA repair in keratinocytes. OBJECTIVE: The hypothesis that p53 regulates repair of ultraviolet light-induced epidermal DNA damage in vivo was tested in mice. METHODS: An immunohistochemical assay for pyrimidine dimers and 6-4 photoproducts was performed on ultraviolet-irradiated skin from p53 null (-/-) and wild type (+/+) mice. Immunostaining for photoproducts was quantified using computer-assisted imaging. The level of DNA repair was then expressed as the percentage of positive cells remaining as compared to the zero hour time point. RESULTS: p53+/+ mouse skin exposed to 1000 J/m2 retained ' 25% of epidermal cyclobutane dimers at 48 h, whereas approximately 50% remained in p53-/- cells. Using the same UV dose, p53+/+ mice retained 20% of detectable 6-4 photoproducts by 24 h, whereas about 50% remained in epidermal cells of p53-deficient mice. CONCLUSION: Using in situ labelling of UV-damaged cells, we confirm our earlier conclusion that p53 regulates DNA repair within the epidermis after exposure to UV light.

Microscopic polyangiitis after influenza vaccination.

We describe a case of microscopic polyangiitis involving skin and joints after influenza vaccination. Titers of antiinfluenza A antibody were markedly elevated in synovial fluid (SF) relative to those in serum. Antiinfluenza B antibodies were not present in SF but were present in serum, suggesting a reaction specifically involving antiinfluenza A antibodies localized to the affected joint. A review identified 16 other cases of vasculitis after influenza vaccination. The cases reclassified according to the Chapel Hill diagnostic criteria identified multiple forms of vasculitis including 7 other cases of microscopic polyangiitis. Three patients had similar illnesses after previous influenza vaccination or influenza-like illness. As in our case 11 cases resolved without recurrence. While this does not provide conclusive evidence that the vaccination caused the vasculitis, together with the serologic data we present it supports this hypothesis.

The melanoma-specific XMEL antigen is expressed in dysplastic naevi.

We have previously shown that a novel monoclonal antibody, XMEL, exhibited reactivity with deep primary melanomas while showing no reactivity with other tumours and normal tissue. XMEL was raised against a part of the extracellular domain of Xmrk, a growth factor receptor presumed to mediate melanoma formation in the Xiphophorus fish model. Here we investigate the range of XMEL immunohistochemical reactivity in paraffin sections from human common acquired and dysplastic naevi of both junctional and compound type. The strongest reactivity was observed with the compound dysplastic naevi. We conclude that the antigen recognized by XMEL acts early in the cascade of genetic alterations underlying progression into malignant melanoma. Our results also support the notion that the dysplastic naevus may play a role in progression of human malignant melanoma and may indeed represent the precursor stage.

Overexpression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) in human cutaneous malignant melanoma.

p21(WAF1/CIP1) (p21) is an inhibitor of cyclin-dependent kinases recently identified as the downstream effector of wild-type p53-mediated cell cycle arrest. The gene coding for p21 may function as a negative regulator of melanoma growth, progression, and metastasis. Using immunohistochemistry and Western blotting, we investigated the expression of p21 in human melanocytic proliferations. Immunohistochemical staining was performed on 13 common acquired nevi, 12 dysplastic nevi, 23 primary malignant melanomas, and 12 metastatic melanomas. Common acquired nevi showed minimal p21 staining (1.8+/-0.3%, mean+/-SEM). The percentage of positive nuclei was slightly elevated in dysplastic nevi (8.9+/-1.7%). Both primary malignant melanoma (29+/-3%) and metastatic melanoma (33+/-5%) demonstrated a significantly increased number of p21-positive nuclei compared to benign lesions (p<0.001). p21 was strongly expressed even in actively proliferating lesions as confirmed by MIB-1 labelling, and although the majority of p21-positive cells likely represent a non-proliferating population, staining was occasionally observed in cells undergoing mitosis, suggesting abnormal function of this cell cycle inhibitor in malignant melanoma. Overexpression of p21 in metastatic melanoma compared to common acquired nevi was confirmed by Western blot analysis of human tumor samples. These findings suggest that increased p21 expression relative to benign nevi is not sufficient to control melanoma growth in vivo.

Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes.

The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compared with p53+/+ keratinocytes. The reduced DNA repair in p53-/- mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53+/+ and p53-/- keratinocytes after the cells were exposed to UV irradiation. Our results demonstrate that wildtype p53 plays a significant role in regulating NER. Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes. Differentiation of the keratinocytes by increasing the Ca2+ concentration in the culture media resulted in a marked reduction in NER equally in both p53+/+ and p53-/- groups. This finding suggests that reduced DNA repair after differentiation is p53 independent. A similar reduction in HCR was confirmed in differentiated human keratinocytes. These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells. As nonmelanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer.

The Xiphophorus-derived antibody, XMEL, shows sensitivity and specificity for human cutaneous melanoma but is not a prognostic marker.

XMEL is a monoclonal antibody raised against part of the extracellular domain of the putative tyrosine kinase receptor protein implicated in the pathogenesis of melanoma formation in the Xiphophorus fish melanoma model. Our objective in this study was to determine the diagnostic and prognostic utility of XMEL for human melanoma. Formalin-fixed tissue from 82 melanomas, 42 carcinomas, 23 neural tumors, 12 lymphomas and 12 sarcomas were immunostained with XMEL and compared with a widely used melanoma antibody, HMB-45. The sensitivity of HMB-45 (83.1%) was similar to that of XMEL (79.8%). XMEL detected 7 melanomas that were HMB-45 negative. Specificity for detection of melanoma was greater with HMB-45 (95.5%) as compared to XMEL (80.9%). Of interest, all 4 prostatic adenocarcinomas were XMEL positive. These data suggest that XMEL is as sensitive but not as specific as HMB-45 in the detection of cutaneous melanoma but may serve as an ancillary antibody to improve diagnostic yield. The consistent positivity of XMEL in melanoma lends support to the hypothesis that the detected protein plays a role in melanoma pathogenesis. XMEL reactivity is not an independent prognosticator of death from melanoma in 37 melanomas from patients with at least 10 years' follow-up. These data and the fact that XMEL shows variable reactivity with metastatic melanomas but almost 100% reactivity with the primary melanomas suggest that the antigen recognized by the XMEL antibody may be important in the early stages of melanoma progression. This is supported by our earlier observation that XMEL is reactive with dysplastic nevi, a precursor of malignant melanoma.

Mitotic granuloma annulare: a clinicopathologic study of 20 cases.

The finding of mitotic figures in granuloma annulare (GA) has not been emphasized in the literature. We describe 20 cases of a cellular, mitotically active variant of GA; we defined this group as cases having > or = 1 mitosis per 10 hpf. Clinically, the lesions could not be distinguished from typical, localized GA: there were 9 males and 11 females with a mean patient age of 49 +/- 15 years (mean +/- SD), compared to 45 +/- 20 years in a randomly selected control group of 60 patients with GA, and no unusual sites of predilection were noted. Histologically, a classic, palisading granuloma pattern predominated (18/20 cases). Lesions were located in the mid-dermis and tended to be more cellular than typical GA. The histiocytes comprising the lesion often had enlarged nuclei and prominent nucleoli. The number of mitoses per 10 hpf was 3.0 +/- 1.5 (range 1.0-7.2), control group 0.3 +/- 0.5; occasional atypical mitotic figures were observed. The proliferative nature of these lesions was confirmed using MIB-1 staining; the percentage of MIB-1 positive cells ranged from 5%-29% (mean 15 +/- 6%). Mitotic GA must be distinguished histologically from neoplastic processes, in particular epithelioid sarcoma. We conclude that histiocytes in clinically typical GA can exhibit an increased mitotic rate. Recognition of this variant is important in order to avoid overdiagnosis of a malignant condition.

Differentiation-associated overexpression of the cyclin-dependent kinase inhibitor p21waf-1 in human cutaneous squamous cell carcinoma.

p21waf-1 negatively regulates the cell cycle by inhibiting the activity of cyclin-dependent kinases. As p21waf-1 is a probable tumor suppressor, we sought to determine whether this cyclin-dependent kinase inhibitor is abnormally regulated in human cutaneous squamous cell carcinoma (SCC). An immunohistochemical technique was employed to assay p21waf-1 protein in SCCs chosen from sun-exposed and anogenital sites. We observed that p21waf-1 was greatly overexpressed in SCC versus adjacent benign epithelium. Furthermore, expression of p21waf-1 was consistently elevated in the superficial, differentiated cells versus basal keratinocytes. p21waf-1 expression correlated with the proliferative state of the cancers as measured by MIB-1 immunostaining. In vitro, keratinocytes grown in supplemented media upregulated p21waf-1 during differentiation, supporting our in vivo observations. We conclude that p21waf-1 overexpression is associated with differentiation in proliferating SCC but is not sufficient to suppress cancer development.