Molecular Genetics of Endocrine Induced Regression of Mouse Leydig Cell Tumors

Chronic estrogen treatment results in malignant Leydig cell tumor formation in genetically susceptible strains of mice. The hypothesis that the mechanism of estrogen induced carcinogenesis includes a gene amplification event was tested using this in vivo endocrine carcinogenesis model. High molecular weight DNA isolated from liver, hyperplastic chryptorchid testes, primary Leydig cell tumors and transplanted tumor lines progressing to estrogen nondependence for growth, was studied using Southern blot analysis and the in-gel renaturation technique to identify amplification of characterized and anonymous sequences. No consistent gene amplification event could be demonstrated either in carcinogenesis or tumor progression to an estrogen nondependent state. These results suggest that estrogen mediated carcinogenesis does not depend on a gene amplification event. Estrogen dependent Leydig cell tumors regress when the trophic stimulus is withdrawn. The second objective of this work was to characterize the early molecular events associated with endocrine induced regression. Using Northern blot analysis of regressing Leydig cell tumor RNA, it was shown that c-fos, c-myc, TGFbeta and p53 are induced during regression. In contrast, the expression of the TRPM-2 gene (a marker for programmed cell death) was constitutive in both growing and regressing Leydig cell tumors; DNA isolated from growing and regressing tumors was of high molecular weight and did not exhibit the nucleosome ladder pattern typical of apoptosis (programmed cell death). These results indicate that entry into the regressing state (diapause) is an active process involving the expression of several well characterized proliferation regulatory genes, and that entry into diapause does not involve an increase in the rate of programmed cell death. Three novel genes induced during regression (rig) were identified using a subtractive cloning and hybridization approach, rigl detects a 2.1 kb transcript that is induced 2 fold at 36 hr after estrogen removal. rig2 detects a 500 bp transcript expressed in growing tumors but induced at 36 hr and reaching a 10 fold induction at 96 hr after estrogen withdrawal. rig3 detects a 125 bp transcript that is not detectable in growing tumor cells but is detected at 12 hr after estrogen removal and is progressively induced to greater than 15 fold at 96 hr after estrogen withdrawal. These results suggest that entry into diapause is an active process involving the activation of a unique pattern of gene expression