Inbred SENCARA/Pt mice are very susceptible to the induction of both benign and malignant skin tumors by standard initiation-promotion protocols. Crosses between SENCARA/Pt and resistant BALB/cAnPt mice revealed that resistance to papilloma induction is incompletely dominant, and that multiple genes determine susceptibility. A genome scan led to the identification of a major locus controlling susceptibility on chromosome 5 in a 21 cM interval between D5Mit259 and D5Mit369, and modifier loci on chromosomes 9, 11 and 12. A comparison of the susceptibility of F1 progeny produced by either SENCARA/Pt or BALB/cAnPt females showed that the mice were uniformly resistant to skin tumor induction. However, the susceptibility phenotype of backcross mice that resulted from the cross of F1 females to SENCARA/Pt males depended on the strain of the females used to produce the F1 progeny. When SENCARA/Pt females were used (compared to BALB/cAnPt females) to produce F1 mice, about 45% of the backcross mice were susceptible (compared to 15% for BALB/cAnPt). This result suggested the possibility that a susceptibility locus resides on the X chromosome. However, a determination of LOD scores for 16 polymorphic Mit markers on the X chromosome showed that the highest LOD score of 1.37 was found for DXMit234, 70 cM from the centromere. Thus, susceptibility loci on the X chromosome do not explain our result. We are currently determining the susceptibility of backcross mice bred using SENCARA females crossed to F1 males to see whether susceptibility can depend on the female parent of the male mice. We have demonstrated a marked synergism for tumor promotion between the phorbol ester 12-O-tetradecanoyphorbol-13-acetate (TPA) and agents that elevate intracellular calcium, such as thapsigargin or the calcium ionophore ionomycin, suggesting a role for calcium in tumor promotion by phorbol esters. Synergism was not found at effective promoting doses of TPA, but was seen at suboptimal or non-promoting doses of TPA. The mechanism by which elevation of calcium increases promotion by TPA may involve a conventional isoform of protein kinase C (such as protein kinase C alpha) or another calcium-activated pathway. Transgenic mice which overexpress protein kinase C alpha in the basal layer of the epidermis and hair follicles have been developed in LCCTP. These mice have been bred to study the possible role of protein kinase C alpha in the enhancement of TPA promotion by thapsigargin. A high initiating dose of DMBA is followed by low doses of thapsigargin and/or TPA. Promotion by either thapsigargin or TPA is much more effective in the PKC alpha transgenics relative to littermate controls. However, tumor formation in mice pretreated with thapsigargin prior to each treatment with TPA was not enhanced in the PKC alpha transgenics. Activating mutations in the K-ras oncogene, rarely seen in mouse skin tumors, are frequent in rapidly-arising tumors on the skin of mice initiated by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promoted by mezerein. Only a few K-ras mutations were seen in skin tumors from MNNG-initiated mice promoted with TPA. Mouse skin tumors initiated by 7,12-dimethylbenzaanthracene (DMBA) and promoted by either TPA or mezerein display mutations in H-ras but not K-ras. The mechanism of the selective action of mezerein in the promotion of MNNG-initiated skin tumors may be related to the K-ras and mezerein-activated protein kinase C pathways in skin. We have analyzed skin tumors from this experiment for methylation of the promoter for the gene encoding the DNA repair enzyme MGMT, and found methylation in 41% of papillomas and 51% of carcinomas. Methylation was found more frequently in tumors initiated by DMBA and in tumors promoted by mezerein. Methylation of MNNG-initiated carcinomas was not seen in the earliest carcinomas, but was significantly increased in the later-appearing carcinomas with promotion by either TPA (p=0.007) or mezerein p=0.048). We found no significant association between methylation and the initiating mutations in either H- or K-ras; methylation appears to be associated with the later stage of malignant conversion. MGMT promoter methylation prevented MGMT expression as determined by immunohistochemistry. Individual tumors in which MGMT was not expressed were methylated at only 1-5 CpG sites, indicating that a low level of methylation can prevent MGMT expression. Methylation at the CpG sites at -22 and -122 bases upstream from the transcription start site are critical in reducing MGMT expression. Methylation at CpG sites at -383, -413, and -415 bases did not correlate with MGMT expression. In the same set of tumor samples, p53 mutations were characterized in 132 skin tumors. Mutations, found in about half of benign and malignant tumors, were most frequent in exons 10 (47%) and 4 (22%), with fewer mutations in exons 5, 7 and 8. Mutations in p53 were more frequent 1) in tumors initiated by DMBA compared to MNNG, and 2) in early tumors compared to late tumors. Most mutations involved a base pair substitution, but no single type predominated. The unexpectedly wide spectrum of p53 mutations suggests that they are not the direct result of carcinogen damage.
| Horn, Elizabeth J; Albor, Amador; Liu, Yuangang et al. (2004) RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties. Carcinogenesis 25:157-67 |
| Kozikowski, Alan P; Nowak, Ireneusz; Petukhov, Pavel A et al. (2003) New amide-bearing benzolactam-based protein kinase C modulators induce enhanced secretion of the amyloid precursor protein metabolite sAPPalpha. J Med Chem 46:364-73 |
