The transcriptional effects and DNA-binding specificities of 17beta- estradiol after dimethyldioxirane activation

Abstract

It was found recently that 17β-estradiol (E2) could be activated by the epoxide-forming oxidant dimethyldioxirane (DMDO) resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in vitro (Carcinogenesis, 17, 1957(Carcinogenesis, 17, -1961(Carcinogenesis, 17, , 1996. To further study the mechanism of this inhibition, several synthetic DNAs with different base content and sequence were used to study the transcriptional effects and binding specificities of E2 after DMDO activation in vitro. The results show: (1) E2 after activation had a strong inhibitory effect on the template function of both A-T and G-C containing double-stranded DNAs, e.g. poly[d(A-T)], polydG·polydC and poly[d(I-C)], and only a weak inhibition on the single-stranded DNA template, polydC. The inhibition was dose-dependent, and only after DMDO activation. (2) 3 H-labeled E2, after DMDO activation, was able to bind DNAs containing both A-T and G-C bases. The order of the binding preference was: calf thymus DNA > poly[d(A-T)] > poly[d(G-C)]. (3)The covalent binding nature of E2 to DNA after activation was further confirmed by 32 P-post-labeling analysis using calf thymus DNA. (4) The absorption spectrum of E2 changed, after DMDO treatment, from a peak around 280-290 nm to 260-270 nm with a shoulder appearing around 300-320 nm. These studies have not only confirmed our earlier observation that E2, after DMDO activation, can inhibit DNA-dependent RNA synthesis, but also provided new insights into the DNA-binding properties after activation. Additionally, since epoxidation is often required for the activation of chemical carcinogens to bind DNA, these studies lend further support to our proposed hypothesis that E2 epoxidation may play an initiation role in estrogen carcinogenesis.

The effect of 17β‐estradiol‐DNA adducts on the replication of exon # 5 of the human suppressor gene p53

“…Therefore, one of the important tests of our hypothesis is to determine whether E 1 and E 2 after DMDO activation are able to bind to DNA. In support to our hypothesis, we have found that 3 H‐labelled E 1 and E 2 are able to bind to both G‐C and A‐T containing DNAs but only after DMDO activation [10,11]. Additionally, the covalent binding nature of E 1 and E 2 to DNA after DMDO activation was further confirmed by 32 P‐post‐labelling analysis using calf thymus as well as several synthetic DNAs of a known base content and sequence [10,11].…”

17β‐Estradiol epoxidation as the molecular basis for breast cancer initiation and prevention

“…3). 24,25 The covalent binding nature of E 1 and E 2 to DNA was further confirmed by [ 32 P]‐post‐labelling analysis (Fig. 4).…”

“…It is clear that in order to properly prevent a disease, it is necessary to know the cause of the disease. In terms of breast cancer, our findings that E 2 could be activated by epoxidation 13 and was consequently able to bind DNA, forming DNA adducts, in vitro and in vivo 24−26,28−30 have provided a strong molecular basis for an initiatory role of E 2 in breast cancer aetiology 13 . Based on this new insight, we have developed a technique to screen potential chemo­preventive agents at the initiation step of breast cancer carcinogenesis.…”

“…Based on this new insight, we have developed a technique to screen potential chemo­preventive agents at the initiation step of breast cancer carcinogenesis. This screening test determines whether a chemical agent is able prevent the formation of E 2 epoxide (i.e., prevention at the initiation step), as measured by both the loss of the ability of E 2 to inhibit nuclear DNA‐dependent RNA synthesis 13,24 , 25 and the ability of [ 3 H]‐labelled E 2 to bind to nuclear DNA 24−26,28−30 . The following two examples are used to illustrate the utility of this screening technique.…”

“…17β‐Estradiol requires activation by epoxidation to bind to DNA and form DNA adducts, 24−26,28−30 as does TAM 13 (Tables 2,3). This raises the possibility that TAM, as an effective competitor for epoxidation, may act indirectly by preventing the formation of E 2 epoxide and, consequently, breast cancer.…”