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Mouse skin carcinomas arise from a small subpopulation of benign papillomas with an increased risk of malignant conversion. These papillomas arise with limited stimulation by tumor promoters, appear rapidly, and do not regress, suggesting that they differ in growth properties from the majority of benign tumors. The transforming growth factor .3 proteins are expressed in the epidermis and are growth inhibitors for mouse keratinocytes in vitro; altered TGF-18 expression could influence the growth properties of high-risk papillomas. Normal epidermis, tumor promotertreated epidermis, and skin papillomas at low risk for malignant conversion express TGF-.81 in the basal cell compartment and TGF-,82 in the suprabasal strata. In low-risk tumors, 90% ofthe proliferating cells are confined to the basal compartment.In contrast, the majority of high-risk papillomas are devoid of both TGF-,B1 and TGF-f82 as soon as they arise; these tumors have up to 40% of the proliferating cells in the suprabasal layers. Squamous cell carcinomas are also devoid of TGF-3, suggesting that they arise from the TGF--deflcient high-risk papillomas. In some high-risk papillomas, TGF-(31 loss can occur first and correlates with basal cell hyperproliferation, while TGF-,32 loss correlates with suprabasal hyperproliferation. Similarly, TGF-(31-null transgenic mice, which express wild-type levels of TGF-182 in epidernds but no TGF-131 in the basal layer, have a hyperproliferative basal cell layer without suprabasal proliferation. In tumors, loss ofTGF-I is controlled at the posttranscriptional level and is associated with expression of keratin 13, a documented marker of malignant progression. These results show that TGF-.3 expression and function are compartmentafized in epidermis and epidermal tumors and that loss of TGF-(8 is an early, biologically relevant risk factor for malignant progression.In multistage chemical carcinogenesis of the mouse epidermis, malignant conversion of a benign papilloma to a squamous cell carcinoma is a rare event (1). While most papillomas and carcinomas generated by initiation with 7,12-dimethylbenz[a]anthracene (DMBA) have an A --T mutation at codon 61 of the Ha-ras allele (2, 3), there is evidence for subpopulations of papillomas with distinct risks for malignant conversion. Standard protocols utilizing DMBA initiation and continuous promotion with phorbol 12-myristate 13-acetate (PMA) produce a large number of papillomas, the majority of which arise after 10 weeks and have a malignant conversion frequency of 3-5% (4). Many of these tumors are promoter-dependent and regress rapidly when PMA treatment is terminated (5). In contrast, DMBA initiation followed by 5 weeks of PMA promotion, or continuous promotion with the weak promoter mezerein, yields fewer papillomas, which arise within 6-8 weeks, do not regress in the absence of promotion, and have a conversion frequency of 15-25% (4). This suggests that most carcinomas arise from a small population of papillomas that have relatively autonomous growth properti...

Bryostatin 1, like the phorbol esters, activates protein kinase C. However, bryostatin 1 induces only some of the effects in cultured cells which result from phorbol ester treatment, whereas it blocks other responses to the phorbol esters. In mouse keratinocytes in particular, bryostatin 1 induces ornithine decarboxylase, a marker of proliferation, but blocks induction of markers of differentiation. Because of the postulated role of induction of differentiation in tumor promotion, we have now examined bryostatin 1 as a tumor promoter and as an inhibitor of phorbol ester tumor promotion in the initiation-promotion model of skin carcinogenesis. After initiation with 7,12-dimethylbenz[a]anthracene, weekly topical treatments of the backs of mice with 1 microgram (1.1 nmol) bryostatin 1 induced epidermal hyperplasia and inflammation, although not to the extent seen after treatment with the promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment with bryostatin 15 min before each TPA exposure reduced the phorbol ester-induced hyperplasia. Bryostatin 1 was ineffective as a complete tumor promoter and displayed very weak activity as a second stage promoter upon treatment of initiated mice for 30 weeks. Combined exposure of mice to bryostatin 1 and TPA resulted in a substantial inhibition of promotion by TPA. Our in vivo results extend earlier in vitro findings that bryostatin 1 acts as a partial inhibitor of protein kinase C function.

The widespread use of FVB/N mice for the establishment of transgenic lines containing active oncogenes suggested the importance of testing the parent FVB/N mice for sensitivity to experimental carcinogenesis. After initiation of mouse skin by a single treatment with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion by 20 weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), the skin tumor incidence was compared in FVB/N mice, TPA-sensitive (SENCAR and CD-1) and TPA-resistant mice (BALB/c and C57BL/6). Initiation by 25 micrograms DMBA followed by promotion with a low dose of TPA (2 micrograms/week) induced one or more papillomas in only 25% of FVB/N mice, compared with 100% in SENCAR, 53% in CD-1, 17% in BALB/c and 0% in C57BL/6 mice. At a more effective dose of TPA (5 micrograms/week), FVB/N mice initiated by 5, 25 or 100 micrograms DMBA developed 3.4, 6.9 and 11.8 papillomas per mouse. In contrast, the incidence of squamous cell carcinomas (SCCs) (17-18/30 mice) did not increase with DMBA dose. TPA promotion of non-initiated mice induced only six papillomas, but three progressed to SCCs, a high rate of malignant conversion. Skin tumor induction by 20 weekly treatments with 10 micrograms DMBA produced few papillomas, but 50.0% of the papillomas progressed to carcinomas in FVB/N mice, compared with 9.15% in SENCAR, 37.5% in CD-1, 23.1% in BALB/c and 15.0% in C57CL/6 mice. The first carcinomas appeared after 14 weeks in FVB/N, 24 weeks in SENCAR, 26 weeks in CD-1 and C57BL/6 and 34 weeks in BALB/c mice. Thus, FVB/N mice develop an unusually high incidence of SCCs after treatment with repeated DMBA, DMBA initiation-TPA promotion and even TPA alone.

Tripartite motif protein 32, Trim32, mRNA and protein expression was elevated in independently transformed and tumorigenic keratinocytes of a mouse epidermal carcinogenesis model, in ultraviolet B (UVB)-induced squamous cell carcinomas (SCC), and in approximately 20-25% of chemically induced mouse papillomas and human head and neck SCCs. This suggests that elevated Trim32 expression occurs frequently in experimental epidermal carcinogenesis and is relevant to human cancer. Transduced Trim32 increased colony number in an epidermal in vitro transformation assay and epidermal thickening in vivo when skin-grafted to athymic nu/nu mice. These effects were not associated with proliferation and were not sufficient for tumorigenesis, even with 12-O-tetradecanoylphorbol-13-acetate treatment or defects in the tumor suppressor p53. However, transduced Trim32 inhibited the synergistic effect of tumor necrosis factor alpha (TNFalpha) on UVB-induced apoptosis of keratinocytes in vitro and the apoptotic response of keratinocyte grafts exposed to UVB-light in vivo. Consistent with its RING domain, Trim32 exhibited characteristics of E3-ubiquitin ligases, including being ubiquitylated itself and interacting with ubiquitylated proteins, with increases in these properties following treatment of cultured keratinocytes with TNFalpha/UVB. Interestingly, missense point mutation of human TRIM32 has been reported in Limb-Girdle Muscular Dystrophy Type 2H, an autosomal recessive disease. We propose a model in which Trim32 activities as an E3-ubiquitin ligase favor initiated cell survival in carcinogenesis by blocking UVB-induced TNFalpha apoptotic signaling.

Normal keratinocytes proliferate when cultured in medium with 0.02-0.10 mM calcium and terminally differentiate when medium calcium is increased to greater than 0.1 mM. In contrast, neoplastic keratinocyte cell lines maintain the potential for continued cell renewal and survive when external calcium is increased. In order to determine whether elevation of extracellular calcium produced changes in intracellular free calcium (Cai) levels, Cai was measured in individual living keratinocytes by use of the fluorescent calcium probe fura-2. Most normal keratinocytes responded to increased extracellular calcium by a gradual 2- to 3-fold increase in Cai lasting for at least 28 min. A subpopulation displayed a sharp peak of Cai at 2 min. In contrast, the Cai level in neoplastic cells in either low or high calcium medium was 2- to 3-fold higher than that in normal cells, and all cells in the population showed a transient 4- to 9-fold elevation of Cai 2 min after external calcium was increased. Thus normal and neoplastic keratinocytes differ in the level of Cai under low calcium conditions and in their response to elevated external calcium. The regulation of Cai in keratinocytes may be important in determining their potential for terminal differentiation.

Multi-stage carcinogenesis (initiation-promotion) was first demonstrated in mouse skin. The first stage, initiation, is accomplished by a low dose of carcinogen that causes no tumours. Promotion by repeated treatment of initiated mice with certain non-carcinogenic hyperplastic agents results in the rapid production of numerous benign papillomas, a few of which progress to squamous cell carcinomas. Although this models system produces mostly benign tumours, many of the concepts concerning carcinogenesis in epithelial tissues have been derived from mouse skin studies. The permanent change in growth potential accomplished by tumour initiators is generally considered to be a mutagenic event; cell selection and clonal expansion of initiated cells may be involved in promotion. In initiation-promotion experiments, more than 90% of the squamous cell carcinomas develop from papillomas, but the conversion rate is low. The factors necessary for this conversion of benign to malignant tumours have not been defined but tumour promoters have been assumed to be involved. However, we report here that the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is ineffective in the conversion of papillomas to carcinomas whereas three initiators, urethane, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline-N-oxide (4-NQO) are effective. This suggests that malignant conversion may result from a further genetic change in papilloma cells and that the ineffectiveness of TPA may be due to its inactivity as a mutagen.