Perturbation in connexin 43 and connexin 26 gap-junction expression in mouse skin hyperplasia and neoplasia.

To examine the possible role of gap junctions in mouse skin tumor progression, we generated a panel of mouse skin tissue samples exhibiting normal, hyperplastic, or neoplastic changes and characterized the expression of the gap-junction genes connexin 43 (Cx43) and connexin 26 (Cx26) by in situ hybridization and immunohistochemical analyses. In normal skin, these two gap junction genes were differentially expressed; Cx43 was found predominantly in the less differentiated lower spinous layers, whereas Cx26 was found in terminally differentiating upper spinous and granular layers. In hyperplastic epidermis exhibiting an expansion of the differentiated upper layer, i.e., epidermis with a thickened granular layer or in which the granular layer was replaced with keratinocytes exhibiting tricholemmal differentiation, expression of Cx43 and Cx26 remained segregated in the lower and upper spinous layers, respectively. However, in papillomas, Cx26 was localized in the lower but not upper spinous layer, an expression pattern identical to that of Cx43. In addition, the overall expression levels of both Cx43 and Cx26 appeared to be greatly elevated in the papillomas. It is interesting that such marked alteration in the pattern of Cx26 expression occurred within the context of hyperplastic changes histologically identical to those seen in the nonpapillomous hyperplasias. Interestingly, in neoplastic skin lesions containing a squamous cell carcinoma, Cx43 and Cx26 expression was extinguished. Moreover, expression of Cx43 was also significantly reduced in adjacent apparently nonneoplastic tissues. Overall, these observations show that perturbations in gap-junction gene expression are associated with skin hyperplasia and neoplasia. Such findings suggest a possible role for gap junctions in the malignant conversion of mouse epidermal cells.

Inhibition of x-ray induced transformation and effect on myc and fos gene expression by the nucleotide analog, 2-aminopurine.

Ionizing radiation is a carcinogen that is known to induce malignant transformation of C3H/10T1/2 fibroblasts in vitro. Radiation is also known to induce c-myc expression and protease inhibitors that suppress radiation transformation reduce myc and fos gene expression. The antiproliferative protein kinase inhibitor 2-aminopurine has been shown to selectively inhibit serum induced c-fos and c-myc expression in human hemopoetic cells. The myc and fos oncogenes are thought to play a role in the regulation of cell proliferation and may be involved in early stages of carcinogenesis. We determined the ability of 2-aminopurine to affect x-ray-induced transformation in C3H/10T1/2 cells in vitro and its effect on myc and fos gene expression in these cells. Treatment with 2-aminopurine (5 x 10(-4) M) resulted in a 50-100% reduction in transformation yield when C3H/10T1/2 cells were irradiated with 6 Gy of x-irradiation. The 2-aminopurine had to be present during the post-confluent stage of cell growth in order to exert its inhibitory effect. Treatment of cells with 2-aminopurine significantly reduced the level of myc gene expression; the inhibitory effect of 2-aminopurine on fos gene expression in these cells was not statistically significant.

Amplification of the c-myc oncogene during progression of radiation-induced rat skin tumors.

Evaluation of a large panel of radiation-induced rat skin tumors of diverse size and histological type revealed a correlation between c-myc copy number and tumor size. Both the frequency and degree of c-myc gene amplification were increased in large compared to small carcinomas, but none of the sarcomas examined showed c-myc amplification. Serial biopsies of individual tumors exhibited similar trends of increasing c-myc copy number in later biopsies. In one regressing tumor, the c-myc gene copy number paralleled the growth rate of the tumor during growth and regression. The average time required from tumor appearance to significant gene amplification was close to the average period between tumor appearance and the onset of rapid growth. The data suggest that, rather than being a target gene for the direct early effects of ionizing radiation, c-myc functions as a late-stage progression-related oncogene in this model system.

An experimental model for oncogene activation during tumor progression in vivo.

Tumor progression usually involves a complex pattern of molecular alterations. In many human tumors oncogene amplification or activation has been associated with advanced stages of cancer. Transfection studies have demonstrated the ability of several cellular oncogenes to induce a more malignant phenotype in transformed cells. We have examined the role of c-myc in tumor progression in rat tracheal cell culture, and in rat skin tumors induced by ionizing radiation. In the latter in vivo model, c-myc amplification was found to occur as a function of tumor size. Serial biopsies of growing tumors confirmed the trend toward increased gene copy number with time and stage of progression. This effect was specific for the c-myc gene, in epithelial tumors. Evidence was found for a role of tumor heterogeneity and evolution of tumor cell subpopulations in determining the oncogene activation profile of individual tumors.

Activation of c-myc and c-K-ras oncogenes in primary rat tumors induced by ionizing radiation.

An activated K-ras oncogene was detected by transfection in NIH 3T3 cells and by Southern blot analysis in 6 of 12 rat skin tumors induced by ionizing radiation. The DNA from 10 of the 12 tumors also showed c-myc gene amplification and restriction polymorphisms. Evidence for tissue specificity was observed in patterns of oncogene activation, with each of three clear cell carcinomas exhibiting activation of both c-myc and K-ras oncogenes.