Protective Vaccination against Papillomavirus-Induced Skin Tumors under Immunocompetent and Immunosuppressive Conditions: A Preclinical Study Using a Natural Outbred Animal Model

Certain cutaneous human papillomaviruses (HPVs), which are ubiquitous and acquired early during childhood, can cause a variety of skin tumors and are likely involved in the development of non-melanoma skin cancer, especially in immunosuppressed patients. Hence, the burden of these clinical manifestations demands for a prophylactic approach. To evaluate whether protective efficacy of a vaccine is potentially translatable to patients, we used the rodent Mastomys coucha that is naturally infected with Mastomys natalensis papillomavirus (MnPV). This skin type papillomavirus induces not only benign skin tumours, such as papillomas and keratoacanthomas, but also squamous cell carcinomas, thereby allowing a straightforward read-out for successful vaccination in a small immunocompetent laboratory animal. Here, we examined the efficacy of a virus-like particle (VLP)-based vaccine on either previously or newly established infections. VLPs raise a strong and long-lasting neutralizing antibody response that confers protection even under systemic long-term cyclosporine A treatment. Remarkably, the vaccine completely prevents the appearance of benign as well as malignant skin tumors. Protection involves the maintenance of a low viral load in the skin by an antibody-dependent prevention of virus spread. Our results provide first evidence that VLPs elicit an effective immune response in the skin under immunocompetent and immunosuppressed conditions in an outbred animal model, irrespective of the infection status at the time of vaccination. These findings provide the basis for the clinical development of potent vaccination strategies against cutaneous HPV infections and HPV-induced tumors, especially in patients awaiting organ transplantation.     

Skin hyperproliferation and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38

The oncoproteins E6 and E7 of human papillomavirus type 38 (HPV38) display several transforming activities in vitro, including immortalization of primary human keratinocytes. To evaluate the oncogenic activities of the viral proteins in an in vivo model, we generated transgenic mice expressing HPV38 E6 and E7 under the control of the bovine homologue of the human keratin 10 (K10) promoter. Two distinct lines of HPV38 E6/E7-expressing transgenic mice that express the viral genes at different levels were obtained. In both lines, HPV38 E6 and E7 induced cellular proliferation, hyperplasia, and dysplasia in the epidermis. The rate of occurrence of these events was proportional to the levels of HPV38 E6 and E7 expression in the two transgenic lines. Exposure of the epidermis of nontransgenic mice to UV led to p21WAF1 accumulation and cell cycle arrest. In contrast, keratinocytes from transgenic mice continued to proliferate and were not positive for p21WAF1, indicating that cell cycle checkpoints are altered in keratinocytes expressing the viral genes. Although the HPV38 E6/E7-expressing transgenic mice did not develop spontaneous tumors during their life span, two-stage carcinogen treatment led to a high incidence of papillomas, keratoacanthomas, and squamous-cell carcinomas in HPV38 mice compared with nontransgenic animals. Together, these data show that HPV38 E6 and E7 display transforming properties in vivo, providing further support for the role of HPV38 in carcinogenesis.     

Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene.

The human papillomavirus type 16 (HPV-16) genome is commonly present in human cervical carcinoma, in which a subset of the viral genes, E6 and E7, are expressed. The HPV-16 E6 and E7 gene products can associated with and inactivate the tumor suppressor proteins p53 and Rb (the retinoblastoma susceptibility gene product), and in tissue culture cells, these viral genes display oncogenic properties. These findings have led to the hypothesis that E6 and E7 contribute to cervical carcinogenesis. This hypothesis has recently been tested by using transgenic mice as an animal model. HPV-16 E6 and E7 together were found to induce cancers in multiple tissues in which they were expressed, including squamous cell carcinoma, the cancer type most commonly associated with HPV-16 in the human cervix. We have extended these studies to investigate the in vivo activities of HPV-16 E7 when expressed in squamous epithelia of transgenic mice. Grossly, E7 transgenic mice had multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair on neonates, thickened ears, and loss of hair in adults. In lines of mice expressing higher levels of E7, we observed stunted growth and mortality at an early age, potentially caused by an incapacity to feed. Histological analysis demonstrated that E7 causes epidermal hyperplasia in multiple transgenic lineages with high penetrance. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and an expansion of the keratin 10-positive layer of cells and was associated with hyperkeratosis. Hyperplasia was found at multiple sites in the animals in addition to the skin, including the mouth palate, esophagus, forestomach, and exocervix. In multiple transgenic lineages, adult animals developed skin tumors late in life with low penetrance. These tumors arose from the squamous epithelia and from sebaceous glands and were characterized histologically to be highly differentiated, locally invasive, and aggressive in their growth properties. On the basis of these phenotypes, we conclude that HPV-16 E7 can alter epithelial cell growth parameters sufficiently to potentiate tumorigenesis in mice.     

E6/E7 expression of human papillomavirus type 20 (HPV-20) and HPV-27 influences proliferation and differentiation of the skin in UV-irradiated SKH-hr1 transgenic mice

The functional role of UV irradiation, in combination with the E6 and E7 proteins of the cutaneous human papillomavirus (HPV) types in the malignant conversion of benign papillomatous lesions, has not been elucidated. Transgenic SKH-hr1 hairless mice expressing HPV-20 and HPV-27 E6 and E7 proteins in the suprabasal compartment were generated and exposed to chronic UV irradiation. Histological and immunohistochemical examination of skin samples revealed enhanced proliferation of the epidermal layers and papilloma formation in both transgenic strains in comparison to what was observed with nontransgenic mice. Squamous cell carcinoma developed in the HPV-20 E6/E7 transgenic line as well as in the HPV-27 E6/E7 transgenic line. Several weeks after cessation of UV-B exposure, enhanced proliferation, as measured by BrdU incorporation, was maintained only in HPV-20 transgenic skin. Keratin 6 expression was increased in the transgenic mice throughout all cell layers. Expression of the differentiation markers involucrin and loricrin was reduced and disturbed. p63alpha expression was differentially regulated with high levels of cytoplasmic expression in clusters of cells in the granular layer of the skin in the transgenic lines 20 weeks after cessation of UV-B exposure, in contrast to uninterrupted staining in the nontransgenic lines. p53 was expressed in clusters of cells in nontransgenic and HPV-27 transgenic mice, in contrast to an even distribution in a higher number of cells in HPV-20 transgenic animals.     

Human keratin 14 driven HPV 16 E6/E7 transgenic mice exhibit hyperkeratinosis

Human papillomavirus type 16 (HPV16) has been known as a major causative factor for the development of uterine cervical carcinomas. To investigate the in vivo activity of HPV16 expressed in squamous epithelia, transgenic mice harboring HPV16 E6/E7 with human keratin 14 (hK14) promoter were generated. Grossly, hK14 driven HPV16 E6/E7 transgenic mice exhibited multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair in neonates, thickened ears, and loss of hair in adults. Transgenic mice with phenotype exhibiting severe wrinkled skin and a lack of hair growth died at the age of 3-4 weeks. Histological analysis revealed that in transgenic mice survived beyond the initial 3-4 weeks, HPV16 E6/E7 causes epidermal hyperplasia in multiple transgenic lineages with high incidence of transgene penetration. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and keratinocytes, and was associated with hyperkeratosis. Such activities were significantly higher in the skin of transgenic mice than that of the normal mice. Thus, these transgenic mice appeared to be useful for the expression of HPV16 E6/E7 gene and subsequent analysis on hyperkeratosis.     

Induction of focal epithelial hyperplasia in tongue of young bk6-E6/E7 HPV16 transgenic mice

Squamous cell carcinoma (SCC) of the oral cavity is one of the most common neoplasms in the world. During the past 2 decades, the role of high-risk human papilloma virus (HR-HPV) has been studied and the data supporting HPV as a one of the causative agents in the development and progression of a sub-set of head and neck squamous cell carcinomas (HNSCC) has accumulated. In order to investigate the role of HR-HPV oncogene expression in early epithelial alterations in vivo, we produced transgenic mice expressing HPV16 early region genes from the promoter of the bovine keratin 6 gene (Tg[bK6-E6/E7]). In this article, we demonstrate that E6/E7 transgene was abundantly expressed and cellular proliferation was increased in the middle tongue epithelia of transgenic mice, and that in the same region young (27 weeks old) Tg[bK6-E6/E7] mice spontaneously developed histological alterations, mainly focal epithelial hyperplasia (FEH).     

The Human Papillomavirus Type 16 E6 Gene Alone Is Sufficient To Induce Carcinomas in Transgenic Animals

High-risk human papillomaviruses (HPVs) are the causative agents of certain human cancers. HPV type 16 (HPV16) is the papillomavirus most frequently associated with cervical cancer in women. The E6 and E7 genes of HPV are expressed in cells derived from these cancers and can transform cells in tissue culture. Animal experiments have demonstrated that E6 and E7 together cause tumors. We showed previously that E6 and E7 together or E7 alone could induce skin tumors in mice when these genes were expressed in the basal epithelia of the skin. In this study, we investigated the role that the E6 gene plays in carcinogenesis. We generated K14E6 transgenic mice, in which the HPV16 E6 gene was directed in its expression by the human keratin 14 promoter (hK14) to the basal layer of the epidermis. We found that E6 induced cellular hyperproliferation and epidermal hyperplasia and caused skin tumors in adult mice. Interestingly, the tumors derived from E6 were mostly malignant, as opposed to the tumors from E7 mice, which were mostly benign. This result leads us to hypothesize that E6 may contribute differently than E7 to HPV-associated carcinogenesis; whereas E7 primarily contributes to the early stages of carcinogenesis that lead to the formation of benign tumors, E6 primarily contributes to the late stages of carcinogenesis that lead to malignancy.     

Both conserved region 1 (CR1) and CR2 of the human papillomavirus type 16 E7 oncogene are required for induction of epidermal hyperplasia and tumor formation in transgenic mice.

High-risk human papillomavirus type 16 (HPV-16) and HPV-18 are associated with the majority of human cervical carcinomas, and two viral genes, HPV E6 and E7, are commonly found to be expressed in these cancers. The presence of HPV-16 E7 is sufficient to induce epidermal hyperplasia and epithelial tumors in transgenic mice. In this study, we have performed experiments in transgenic mice to determine which domains of E7 contribute to these in vivo properties. The human keratin 14 promoter was used to direct expression of mutant E7 genes to stratified squamous epithelia in mice. The E7 mutants chosen had either an in-frame deletion in the conserved region 2 (CR2) domain, which is required for binding of the retinoblastoma tumor suppressor protein (pRb) and pRb-like proteins, or an in-frame deletion in the E7 CR1 domain. The CR1 domain contributes to cellular transformation at a level other than pRb binding. Four lines of animals transgenic for an HPV-16 E7 harboring a CR1 deletion and five lines harboring a CR2 deletion were generated and were observed for overt and histological phenotypes. A detailed time course analysis was performed to monitor acute effects of wild-type versus mutant E7 on the epidermis, a site of high-level expression. In the transgenic mice with the wild-type E7 gene, age-dependent expression of HPV-16 E7 correlated with the severity of epidermal hyperplasia. Similar age-dependent patterns of expression of the mutant E7 genes failed to result in any phenotypes. In addition, the transgenic mice with a mutant E7 gene did not develop tumors. These experiments indicate that binding and inactivation of pRb and pRb-like proteins through the CR2 domain of E7 are necessary for induction of epidermal hyperplasia and carcinogenesis in mouse skin and also suggest a role for the CR1 domain in the induction of these phenotypes through as-yet-uncharacterized mechanisms.     

E6 and E7 from beta HPV38 cooperate with ultraviolet light in the development of actinic keratosis-like lesions and squamous cell carcinoma in mice

Cutaneous beta human papillomavirus (HPV) types appear to be involved in the development of non-melanoma skin cancer (NMSC); however, it is not entirely clear whether they play a direct role. We have previously shown that E6 and E7 oncoproteins from the beta HPV type 38 display transforming activities in several experimental models. To evaluate the possible contribution of HPV38 in a proliferative tissue compartment during carcinogenesis, we generated a new transgenic mouse model (Tg) where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter. Viral oncogene expression led to increased cellular proliferation in the epidermis of the Tg animals in comparison to the wild-type littermates. Although no spontaneous formation of tumours was observed during the lifespan of the K14 HPV38 E6/E7-Tg mice, they were highly susceptible to 7,12-dimethylbenz(a)anthracene (DMBA)/12-0-tetradecanoylphorbol-13-acetate (TPA) two-stage chemical carcinogenesis. In addition, when animals were exposed to ultraviolet light (UV) irradiation, we observed that accumulation of p21(WAF1) and cell-cycle arrest were significantly alleviated in the skin of Tg mice as compared to wild-type controls. Most importantly, chronic UV irradiation of Tg mice induced the development of actinic keratosis-like lesions, which are considered in humans as precursors of squamous cell carcinomas (SCC), and subsequently of SCC in a significant proportion of the animals. In contrast, wild-type animals subjected to identical treatments did not develop any type of skin lesions. Thus, the oncoproteins E6 and E7 from beta HPV38 significantly contribute to SCC development in the skin rendering keratinocytes more susceptible to UV-induced carcinogenesis.     

A Humanized Mouse Model of HPV-Associated Pathology Driven by E7 Expression

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies.     

Expression of the Ly6/uPAR-Domain Proteins C4.4A and Haldisin in Non-Invasive and Invasive Skin Lesions

C4.4A and Haldisin belong to the Ly6/uPAR/α-neurotoxin protein domain family. They exhibit highly regulated expression profiles in normal epidermis, where they are confined to early (C4.4A) and late (Haldisin) squamous differentiation. We have now explored if dysregulated expressions occur in non-invasive and invasive skin lesions. In non-invasive lesions, their expression signatures were largely maintained as defined by that of normal epidermis. The scenario was, however, markedly different in the progression towards invasive squamous cell carcinomas. In its non-invasive stage (carcinoma in situ), a pronounced attenuation of C4.4A expression was observed, but upon transition to malignant invasive squamous cell carcinomas, the invasive fronts regained high expression of C4.4A. A similar progression was observed for the early stages of benign infiltrating keratoacanthomas. Interestingly, this transition was accompanied by a shift in the predominant association of C4.4A expression with CK1/10 in the normal epidermis to CK5/14 in the invasive lesions. In contrast, Haldisin expression maintained its confinement to the most-differentiated cells and was hardly expressed in the invasive lesions. Because this altered expression of C4.4A was seen in the invasive front of benign (keratoacanthomas) and malignant (squamous cell carcinomas) neoplasms, we propose that this transition of expression is primarily related to the invasive process.     

Very high incidence of germ cell tumorigenesis (seminomagenesis) in human papillomavirus type 16 transgenic mice.

Human papillomavirus type 16 (HPV16) is frequently found in carcinomas and precancerous lesions of the uterine cervix and is thought to be closely associated with carcinogenesis in these regions. However, the transforming activity of the E6 and E7 genes in vivo has not been characterized. To investigate this function, we produced transgenic mice carrying HPV16 E6 and E7 open reading frames. We obtained five transgenic founders and established three transgenic lineages. We observed testicular tumors of germ cell origin in mice of all three lineages. Morphological studies showed that these tumors were a type of seminoma. Both testes of all tumor-bearing mice were affected with this type of tumor. Strikingly, in one lineage, all of the male mice developed this tumor. On Northern (RNA) analysis, a high level of expression of HPV mRNA was detected in these tumors. These results suggest that transforming genes of HPV16 have transforming activity in vivo and preferential effects on germ cells in the testis.     

ErbB-2 Receptor Cooperates with E6/E7 Oncoproteins of HPV Type 16 in Breast Tumorigenesis

The ErbB-2 receptor is overexpressed in roughly 30% of human breast cancers. Moreover, approximately 50% of breast cancers are positive for high-risk human papillomaviruses (HPVs), specifically types 16 and 18. Recently, we reported that ErbB-2 cooperates with E6/E7 oncoproteins of HPV type 16 to induce neoplastic transformation of human normal oral epithelial cells. We also demonstrated that E6/E7 of HPV type 16 converts non-invasive breast cancer cells to an invasive form. In order to investigate the effect of ErbB-2/E6/E7 cooperation in breast carcinogenesis, we generated double transgenic mice carrying ErbB-2 and E6/E7 of HPV type 16 under mouse mammary tumor virus (MMTV) and human keratin 14 promoters, respectively. Within six months, these double transgenic mice developed large and extensive invasive breast cancer in comparison to ErbB-2 or E6/E7 singly transgenic mice. Histological analysis of ErbB-2/E6/E7 transgenic mice tumors showed the presence of invasive breast carcinomas. However, the breast tissues from ErbB-2 and E6/E7 transgenic mice showed only in-situ cancer and normal mammary phenotype, respectively. In parallel, we examined the cooperation effect of ErbB-2 and E6/E7 in the human breast cancer cell line, BT20; in comparison to ErbB-2 and E6/E7 alone as well as wild type cells, we found that ErbB 2/E6/E7 together stimulate colony formation and cell migration in the BT20 cell line. Furthermore, we found that β-catenin is constitutively phosphorylated by c-Src and consequently trans-located to the nucleus in ErbB-2/E6/E7-breast cancer cells. These findings provide evidence that the ErbB-2 receptor cooperates with high-risk HPVs in breast tumorigenesis via β-catenin activation.     

Presence of papillomavirus genomes and amplification of the c-myc and C-Ha-ras oncogenes in invasive cancers of the uterine cervix

Invasive squamous cell carcinomas of the uterine cervix from 12 untreated patients were examined for the presence of human papillomavirus (HPV) genomes and for the state of the oncogenes c-myc and c-Ha-ras. Blot hybridization experiments have demonstrated the presence of the genome of HPV type 16 (HPV 16) in six tumors and that of the genomes of HPV types weakly related to HPV 16 or HPV 18 in five others. In the nine tumors corresponding to advanced stages of the disease (stages 3 and 4) there was a 3-30 fold amplification of c-myc and/or c-Ha-ras. A concomitant amplification of both oncogenes was found in eight cancers. In only one of the three tumors confined to the cervix (stage 1), the oncogene c-Ha-ras was weakly amplified. Neither HPV DNA sequences, nor oncogene amplification were detected in the leukocytes of five patients. Thus, it seems likely that specific HPV types play a role in the development of carcinomas of the uterine cervix, and that cellular oncogenes, activated through an amplification process, are involved in at least some steps of tumor progression.     

Oncoprotein BMI‐1 induces the malignant transformation of HaCaT cells

BMI‐1 (B‐cell‐specific Moloney murine leukemia virus integration site 1), a novel oncogene, has attracted much attention in recent years for its involvement in the initiation of a variety of tumors. Recent evidence showed that BMI‐1 was highly expressed in neoplastic skin lesions. However, whether dysregulated BMI‐1 expression is causal for the transformation of skin cells remains unknown. In this study, we stably expressed BMI‐1 in a human keratinocyte cell line, HaCaT. The expression of wild‐type BMI‐1 induced the malignant transformation of HaCaT cells in vitro. More importantly, we found that expression of BMI‐1 promoted formation of squamous cell carcinomas in vivo. Furthermore, we showed that BMI‐1 expression led to the downregulation of tumore suppressors, such as p16INK4a and p14ARF, cell adhesion molecules, such as E‐Cadherin, and differentiation related factor, such as KRT6. Therefore, our findings demonstrated that dysregulated BMI‐1 could indeed lead to keratinocytes transformation and tumorigenesis, potentially through promoting cell cycle progression and increasing cell mobility. J. Cell. Biochem. 106: 16–24, 2009. © 2008 Wiley‐Liss, Inc.     

Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) and LMP2A function cooperatively to promote carcinoma development in a mouse carcinogenesis model

The Epstein-Barr virus (EBV) proteins latent membrane proteins 1 and 2 (LMP1 and LMP2) are frequently expressed in EBV-associated lymphoid and epithelial cancers and have complex effects on cell signaling and growth. The effects of these proteins on epithelial cell growth were assessed in vivo using transgenic mice driven by the keratin 14 promoter (K14). The development of papillomas and carcinomas was determined in the tumor initiator and promoter model using dimethyl benzanthracene (DMBA), followed by repeated treatments of 12-O-tetradecanoyl phorbol 13-acetate (TPA). In these assays, LMP1 functioned as a weak tumor promoter and increased papilloma formation. In contrast, mice expressing LMP2A did not induce or promote papilloma formation. Transgenic LMP1 mice had slightly increased development of squamous cell carcinoma; however, the development of carcinoma was significantly increased in the doubly transgenic mice expressing both LMP1 and LMP2A. DMBA treatment induces an activating mutation in the Harvey-ras (H-ras(61)) oncogene, and this mutation was identified in most papillomas and carcinomas although several papillomas and carcinomas in K14-LMP1 and K14-LMP1/LMP2A mice lacked the mutation. Analysis of signaling pathways that are known to be activated by LMP1 and/or LMP2 indicated that all genotypes had high levels of activated extracellular signal-regulated kinase (ERK) and Stat3 in carcinomas with significantly higher activation in the doubly transgenic carcinomas. These findings suggest that, in combination, LMP1 and LMP2 contribute to carcinoma progression and that this may reflect the combined effects of the proteins on activation of multiple signaling pathways. This study is the first to characterize the effects of LMP2 on tumor initiation and promotion and to identify an effect of the combined expression of LMP1 and LMP2 on the increase of carcinoma development.     

Dose-dependent effects of UVB-induced skin carcinogenesis in hairless p53 knockout mice

Exposure to (solar) UVB radiation gives rise to mutations in the p53 tumor suppressor gene that appear to contribute to the earliest steps in the molecular cascade towards human and murine skin cancer. To examine in more detail the role of p53, we studied UVB-induced carcinogenesis in hairless p53 knock-out mice. The early onset of lymphomas as well as early wasting of mice interfered with the development of skin tumors in p53 null-mice. The induction of skin tumors in the hairless p53+/- mice was accomplished by daily exposure to two different UV-doses of approximately 450 J/m2 and 900 J/m2 from F40 lamps corresponding to a fraction of about 0.4 and 0.8 of the minimal edemal dose. Marked differences in skin carcinogenesis were observed between the p53+/- mice and their wild type littermates. Firstly, at 900 J/m2, tumors developed significantly faster in the heterozygotes than in wild types, whereas at 450 J/m2 there was hardly any difference, suggesting that only at higher damage levels loss of one functional p53 allele is important. Secondly, a large portion (25%) of skin tumors in the heterozygotes were of a more malignant, poorly differentiated variety of squamous cell carcinomas, i.e. spindle cell carcinomas, a tumor type that was rarely observed in daily UV exposed wild type hairless mice. Thirdly, the p53 mutation spectrum in skin tumors in heterozygotes is quite different from that in wild types. Together these results support the notion that a point mutation in the p53 gene impacts skin carcinogenesis quite differently than allelic loss: the former is generally selected for in early stages of skin tumors in wild type mice, whereas the latter enhances tumor development only at high exposure levels (where apoptosis becomes more prevalent) and appears to increase progression (to a higher grade of malignancy) of skin tumors.