Wanda Bender

A proposed mechanism of tamoxifen in breast cancer prevention

Recent clinical trials suggest that tamoxifen (TAM) is a preventive agent for breast cancer, however, the mechanism is unknown. Previously, we found that both 17beta-estradiol (E2) and estrone (E1) could be activated by epoxidation resulting in their ability to bind to DNA, forming DNA adducts both in vitro and in vivo, and to inhibit nuclear DNA-dependent RNA synthesis. Since epoxidation is required for the activation of many well-known chemical carcinogens including benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, aflatoxins, etc., we proposed that estrogen epoxidation is the underlying mechanism for the initiation of breast cancer (Carcinogenesis 17 (1996) 1957). Here, we report that TAM is able to dramatically inhibit the formation of E2 and E1 epoxides as measured by both the loss of their ability to inhibit nuclear DNA-dependent RNA synthesis and to bind to nuclear DNA. These findings suggest that the breast cancer preventive effect of TAM may be through a competitive epoxidation inhibition mechanism that prevents the formation of E2 and E1 epoxides and consequently, the initiation of breast cancer.

Evidence for the DNA binding and adduct formation of estrone and 17β- estradiol after dimethyldioxirane activation

Estrogens, used widely from hormone replacement therapy to cancer treatment, are themselves carcinogenic, causing uterine and breast cancers. However, the mechanism of their carcinogenic action is still not known. Recently, we found that estrone (E1) and 17beta-estradiol (E2) could be activated by the versatile epoxide-forming oxidant dimethyldioxirane (DMDO), resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in a dose-dependent manner in vitro. Since epoxidation is often required for the activation of chemical carcinogens, we proposed that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis (Carcinogenesis 17 (1996) 1957-1961). It is known that initiation requires the binding of a carcinogen to DNA with the formation of DNA adducts. One of the critical tests of our hypothesis is therefore to determine whether E1 and E2 after activation are able to bind DNA. This paper reports that after DMDO activation, [3H]E1 and [3H]E2 were able to bind to both A-T and G-C containing DNAs. Furthermore. the formation of E1-DNA and E2-DNA adducts was detected by 32P-postlabeling analysis.

Studies on the isolated transcriptionally active and inactive chromatin fractions from rat liver nuclei.

Using mild sonication, nucleoplasmic, nucleolar, and subnucleolar P-3 and S-3 chromatin fractions are isolated from rat liver nuclei. These fractions differ widely (over 80-fold) from each other in transcriptional activity as measured by the chromatin bound engaged RNA polymerases. Chemical analyses indicate that the active chromatin, e.g. P-3 and nucleolar fractions, are rich in RNA and protein as compared to the inactive chromatin, e.g. nucleoplasmic, and S-3 fractions. However, the DNA base content are all the same, showing 40% GC and 60% AT, including P-3 which is enriched in rDNA. Polyacrylamide gel electrophoresis of the 0.25 N HCl extracted proteins shows that all five histones are present in active chromatin. Additionally, the gel reveals two protein bands, one ahead of histone H2B and another ahead of histone H4, that are diminished or missing from the inactive chromatin. On the other hand, there is a fast moving protein band ahead of H4 in the inactive chromatin that is almost absent in the active chromatin. Transcriptional tests using E. coli RNA polymerase and several synthetic DNA templates of known base content and sequence indicate that the 0.25 N HCl soluble protein extracts from active chromatin contain activator proteins which are capable of countering the histone suppressors present in the extracts in a DNA base and sequence specific manner. The data show that although the histone suppressors are able to strongly inhibit the template function of poly[d(A-T)], the protein activators are able to overcome the suppressor activity and stimulate RNA synthesis several-fold when poly(dA).poly(dT) or poly(dT) is used.

Transcriptional effect of aflatoxin B1 on cytosine and/or hypoxanthine containing DNAs

SummaryThe effect of aflatoxin Bt (AFB,) on the template function for RNA synthesis of several single and double-stranded synthetic DNAs containing cytosine (C) and/or hypoxanthine (H) bases is studied in vitro. The results indicate that AFB,, after liver microsome activation, strongly inhibits the template function of poly[d(I-C)] and has little, if any, effect on polydI . polydC, polydI, or polydC. This conclusion is reached whether rat liver nuclear free RNA polymerase or E. coli RNA polymerase is used for the transcription. The mechanism of this inhibition is believed mainly due to the inhibition of elongation of RNA synthesis, because autoradiography of the [α-32 P]GTP labeled RNAs after polyacrylamide gel electrophoresis clearly shows that the size of the RNA from AFB1 treated group is dramatically reduced. The evidence that the selective inhibition of poly[d(I-C)] template function is a direct reflection of the binding of AFB1 to poly[d(I-C)] is provided by the use of radioactive [3H]AFB1 for the binding and by spectrum analysis of the appearance of a broad AFB1-DNA adduct peak between 300 nm and 400 nm right after the typical DNA peak at 260 nmn. These data, which are in direct support to our recent report (F.L. Yu, et al., Carcinogenesis, 11, 475–478, 1990), suggest that the binding of AFB1 prefers alternating, double-stranded DNA, and the binding affinity of AFB1 to DNA is greatly reduced when the bases are in either single- or double-stranded homopolymer forms. Furthermore, since AFB1 binds to and inhibits the template function of poly[d(I-C)], these results also suggest that the binding of AFB1 to DNA may not be exclusively limited to guanine as previously assumed.

Evidence for an Initiation Mechanism of 17β-Estradiol Carcinogenesis

17β-estradiol (E2), the endogenous female hormone, is carcinogenic causing uterine and breast cancers. The mechanism is unknown. We found that E2 could be activated by the epoxide-forming oxidant dimethyldioxirane (DMDO) and proposed that E2 epoxidation is the mechanism of E2 carcinogenesis. One of the critical tests of our hypothesis is to determine whether E2 -DNA adducts are formed in vivo. When female ACI rats were given intramammillary injections of E2 and DMDO-activated E2, identical DNA adducts were formed. However, the activated E2 was at least 25,000-fold more active than E2 forming DNA adducts in vivo.

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

Using a PCR technique, exon # 5 of the human tumor suppressor gene p53 was amplified and ligated into the pCRII vector and transformed into Escherichia coli INVαF’ competent cells. The cloned exon # 5 was 184 bp long. Evidence is presented to show that after dimethyldioxirane epoxidation, 17β‐estradiol was able to form 17β‐estradiol‐DNA adducts and to strongly inhibit the replication of the cloned exon # 5 producing smaller sizes of DNA fragments and introducing errors of incorporation at the 3′‐end of the terminating DNAs. The errors occurred mainly at the clusters of the complementary ‘G’ and ‘A’ bases on the template strand DNA, presumably, the major sites where the 17β‐estradiol‐DNA adducts were formed.

Errors from using 3H-labeled ribonucleoside triphosphates to monitor nuclear RNA synthesis in vitro

The [3H]XTPs are used widely to monitor RNA synthesis in vitro. Recently, we discovered that they reflected only 40-45% of the true rate of nuclear RNA synthesis. Thus, when [8-14C]GTP was used, 1466 pmol [8-14C]GMP was incorporated per mg DNA/10 min. On the other hand, when [8-3H]GTP was used, only 564 pmol [8-3H]GMP was incorporated per mg DNA/10 min. There are three obvious factors that could have contributed to this greater than 2-fold difference in the apparent incorporation rate: commercial [8-3H]GTP sample was contaminated with substances causing the assay medium to be less efficient in RNA synthesis; 3H exchange occurred during acid washing of the [3H]RNA; and there was a greater quenching effect on [3H]RNA. Experiments were designed to test each of these alternatives. We are able to conclude that none of the above three are contributing factors. Our data also show that the 3H label was removed after it was incorporated into RNA. Similar differences were observed when 3H and 14C labeled pairs of ATP, UTP and CTP were compared. Furthermore, when nuclei were fractionated into nucleolar and nucleoplasmic fractions and carried out RNA synthesis, the loss of 3H label was observed mainly from the nucleoplasmic fraction.