Differential regulation of c-fos expression in estrogen-induced hamster renal tumors compared with kidney not due to creation of an estrogen-response element by point mutation in the gene's flanking sequence

Abstract

The conversion of a palindromic sequence, GGTCTnnnAGACC, in the 5'-flanking region of the murine c-fos proto-oncogene into a functional estrogen-response element by a single base change into GGTC(A/G)nnnAGACC has previously been postulated [Nawaz et al., 1993] as a possible mechanism of the induction of tumors by estrogens. This attractive hypothesis has been investigated in estradiol-induced Syrian hamster kidneytumors, in H-301 kidney tumor cells (a cell line derived from the Syrian hamster tumor), and in normal kidney tissue. The c-fos gene is differentially regulated by a classical estrogen receptor-mediated process in tumors, whereas in the acutely treated kidney, estradiol induces c-fos expression independent of estrogen-receptor function. In this study, we identified in the 5'-flanking region of the hamster kidney c-fos gene the sequence AGTCCnnnAGACC, which closely resembled but did not appear to function as an estrogen-response element. No mutations were detected in this sequence or in the 5'-flanking region of c-fos genes from three different primary tumors and from H-301 tumor cells. To rule out the possibility of a low copy number of mutant alleles in a tumor sample, polymerase chain reaction-based single-strand conformation polymorphism analysis was performed on 372 base pairs of the 5'-flank of the c-fos gene (-367 to +5 base pairs relative to the transcription start point). Nine different kidney tumor DNA samples and five normal kidney tissue samples (controls) produced an identical pattern of DNA bands, suggesting a lack of natural polymorphisms and mutations in this region of the c-fos gene. Acute treatment of hamsters with 17beta-estradiol for 6 h significantly induced renal c-fos mRNA expression, whereas control levels of c-fos were restored by co-treatment with estradiol and either N-acetyl-L-cysteine or alpha-naphthoflavone. We concluded that the previously observed change in regulatory control of c-fos expression in kidney versus estradiol-induced tumors does not involve the creation of a functional estrogen-response element by single point mutation in the 5'-flanking region of the gene. Additionally, c-fos expression in estradiol-treated hamster kidneys appears to be mediated by free radicals generated by the catechol metabolites of estradiol and not by the activation of any estrogen receptor.