The expression of aromatase in human breast tumors has been studied by the reverse-transcription polymerase chain reaction (RT-PCR) method on 70 breast tissue specimens. An RT-PCR analysis using two oligonucleotide primers derived from the exon II of the human aromatase gene revealed that aromatase mRNA was detected in all but three tissue specimens. Furthermore, primer-directed RT-PCR was performed to determine the exon I usage in aromatase mRNA in these breast tumor specimens. The analysis has revealed that exons I.3 and PII are the two major exon Is present in aromatase mRNA isolated from breast tumors, suggesting that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer and surrounding adipose stromal cells. The RT-PCR analysis also detected two products, I.3A (334 bp in length) and I.3B (222 bp in length), when it was carried out using a primer derived from exon I.3 and a reverse primer derived from exon II. The nucleotide sequences of these products have been determined and indicate that I.3A contains a region which was previously thought to be an intron. In addition, RT-PCR analyses of RNA isolated from eight pairs of breast tumor and neighboring normal tissue specimens were performed to evaluate the exon I usage and the distribution of I.3A- and I.3B-containing aromatase RNA messages in breast tumor and neighboring normal tissues. The results suggest that I.3B- and I.3A-containing messages are mainly present in breast tumor and neighboring normal tissues, respectively. Finally, the exon I/promoter usage for aromatase expression in eight cell lines (skin fibroblast, MCF-7, MDA-MB-231, T-47D, SK-BR-3, JAR, OVCAR-3, and human adipose stromal cells) was examined by primer-directed RT-PCR analyses. These studies provide a basis for further evaluation of the control mechanism of aromatase expression and estrogen biosynthesis in breast tumors.

The levels of the aromatase gene and its expression in MCF-7 human breast cancer cells and seven additional cultured cells were investigated. Using normal human foreskin fibroblasts as the control, the aromatase gene appeared to be amplified in MCF-7 cells as shown by Southern and DNA slot blot analyses utilizing human placental aromatase cDNA as the probe. However, the promoter I.1 and the first exon of the aromatase gene were not amplified in MCF-7 cells based on results obtained from DNA slot blot analysis using oligonucleotide probes having sequences derived from those regions of human aromatase gene. Aromatase was expressed at a very low level in this cell line as indicated by Northern blot analysis to measure the level of aromatase mRNA, immunoprecipitation analysis to measure the level of aromatase protein, and aromatase activity measurement. Furthermore, nucleotide sequence analysis of the aromatase cDNA obtained from MCF-7 cells by PCR techniques, revealed no sequence difference from that of the enzyme expressed in placenta. These results lead us to conclude that the expression of aromatase in MCF-7 cells is under the control of an unusual promoter and aromatase gene expression is repressed at the transcriptional level in these cells.

Design, methods, and study population of a long-term multidisciplinary investigation of benign and malignant breast disease were reported. This initial report focused on the relation of menstrual, reproductive, and other factors to serum and breast fluid estrogen measures [estradiol (E2), estrone (E1), percent free estrogen, and sex hormone binding globulin] among control women. After adjustment for the factors found to be related to the various estrogen measures, estrogen levels in women with benign and malignant disease were compared to those of controls. Findings were as follows: a) little evidence of any relation of most breast cancer risk factors with the various serum estrogen parameters studied; b) differences in breast fluid estrogen levels that may be relevant to the protective effect of parity on breast cancer risk; c) markedly higher levels of E2 and E1 in breast fluid than in serum and no evidence of a correlation of serum with breast fluid measures; d) no support for the hypothesis that breast cancer patients have higher serum percent free E2 than controls or women with benign breast disease; and e) higher breast fluid E2 and E1 levels in women with biopsied benign breast disease than in controls.

We investigated estrogen (estrone and estradiof) levels in serum and in nipple aspirates of breast fluid in relation to reproductive and menopausal characteristics in 104 normal women. In general, breast fluid and serum estrogen levels were not correlated and breast fluid estrogen levels were approximately 5 to 45 times higher than serum levels. Serum estrogen levels were lower in post‐menopausal than in premenopausal women. In contrast, breast fluid estrogen levels were approximately the same in pre‐ and post‐menopausal women. Breast fluid estrogen mean levels were lower in premenopausal parous women than in nulligravidous or nulliparous women whereas serum estrogen levels did not differ in these 3 groups. Breast fluid estrogen levels were positively correlated with months since last birth or since last breastfeeding. Estrogen levels were low in nipple aspirates of breast milk but gradually increased in breast fluid of non‐lactating women over a period of several years after cessation of lactation. Serum estrogen levels did not increase with months since last breast‐feeding. We were unable to evaluate the postpartum effect of pregnancy without lactation due to the small numbers of these subjects. The high concentrations of estrogen in breast fluid and the absence of a relationship to serum estrogen levels may explain why prior serum studies have I failed to link variations in serum estrogens with breast cancer risk. The prolonged low levels of breast fluid estrogens following full‐term birth and lactation may, in part, provide a mechanism by which parity reduces breast cancer risk.

Many New World primates such as the squirrel monkey have extraordinarily high plasma levels of steroid hormones including cortisol, testosterone, progesterone and vitamin D3. While plasma estrogen levels in female squirrel monkeys apparently are approximately the same as those found in other species no information is available for males. The present results indicate that the plasma levels of estrone (E1), estradiol (E2), and E1 sulfate are approximately 10-fold higher than those found in men. Comparative in vitro studies of androgen metabolism in genital skin fibroblasts indicate that squirrel monkey cells have higher aromatase and lower 5-α-reductase activity than human cells. Estimation of aromatase activity in vivo by a radiometric assay indicates that the high plasma estrogens are derived by peripheral conversion from testicular and/or adrenal androgens.

A wealth of epidemiologic and experimental information points to the involvement of estrogens and other reproductive hormones in human endometrial and breast cancer. However, despite the many endocrine studies carried out over the past 30 years, the precise role of these hormones remains to be defined. Whether stated or not, the basic premise underlying these studies has been the notion that some abnormality of hormone production, transport, metabolism or action may be causally related to the development of cancer. As more sensitive and sophisticated methods of analysis have become available, they have been applied to this problem, but usually with the same disappointing results. Recent advances in our knowledge of the biology of cancer, such as the potential role of oncogenes, suggest that while hormones may be extremely important signals for the transformation and/or stimulation of cancer cell growth, they cannot act alone in this regard. Accordingly, one may take the view that some critical level of estrogen activates those cells that already have been primed by other mechanisms but are of no consequence to normal cells. Of course, prolonged exposure may lead to benign changes, such as those found in “normal” breast tissue. Furthermore, certain estrogens, such as diethylstilbesterol, may give rise to carcinogens or alter developmental processes, thereby actively participating in genomic events such as the activation of oncogenes. Taken as a whole, the studies of estrogen in women with cancer are in accord with this “necessary but insufficient” role of estrogens. For example, the well-established relationship between age, obesity, and endometrial cancer can be explained by increased peripheral estrogen synthesis in obese postmenopausal women‘,* together with increased availability of plasma estradiol (EJ due to depressed levels of SHBG (sex-hormone-binding globulin). Thus, chronically elevated estrogens that are unopposed by progesterone appear to promote endometrial cancer. However, most postmenopausal women with the same estrogenic status do not develop this disease, suggesting that other factors, such as environmental carcincgens, are involved. Our early studies of plasma binding of & in breast cancer patients were particularly exciting when preliminary evidence suggested that plasma E$ availability was greater than predicted from the plasma SHBG levels in some patients? This possibility was strengthened by results from a more extensive collaborative blind study! More recently, Bulbrook and his associate? have suggested that the interaction of albumin with E, may be weaker in Japanese than in British women, so that E$ availability is increased in the latter because a smaller fraction is bound to SHBG.

A non-dialyzable tRNA methylase inhibitor was isolated from normal adult rat liver. The inhibition caused by this factor was eliminated on prolonged dialysis. However, when nicotinamide was added back to the dialyzed fraction, the inhibitory activity was reconstituted.

SUMMARY Levels of nicotinamide and yV'-methylnicotinamide in serum, liver, and kidney as well as renal clearances and 24-hr urine levels of /V'-methylnicotinamide were compared in normal rats and rats bearing Walker 256 tumors. There was no significant difference between normal and tumor- bearing rats with regard to nicotinamide levels. With regard to /V'-methylnicotinamide, tumor-bearing rats had signifi cantly lower serum and liver levels and significantly higher 24-hr urine levels and renal clearances. Walker 256 tumor tissue and liver and kidney from a normal and a tumor-bearing rat were separately examined for S-adenosylmethionine:nicotinamide methyltransferase activity. The specific activity in tumor tissue extract was greater than that in each liver extract, which, in turn, was much greater than the specific activity in each kidney extract. However, the specific activity in each tissue (liver and kidney) from the tumor-bearing rat was equal to the specific activity in the corresponding tissue of the normal rat. S-Adenosylmethionine:nicotinamide methyltransferase was obtained with 18-fold purification from a tissue extract of Walker 256 tumor. The enzyme activity required activa tion by thiols, and maximal activity was observed at pH 8.6. The Km's for the substrates, S-adenosylmethionine and nicotinamide, were 7.0 x 10~3mM and 0.50 mM, respec tively. The K,'s for the products, S-adenosylhomocysteine and TV'-methylnicotinamide, were, respectively, 25 x 10~3 mMand greater than 5 mM.