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Although there is a growing body of evidence that 17 beta-hydroxysteroid oxidoreductase plays a role in the regulation of steroid levels in epithelial tumors of the endometrium and breast, our knowledge of its role in other gynecologic tumors is limited. In this investigation, the 17 beta-hydroxysteroid oxidoreductase activity of cell lines derived from two ovarian tumors (OVCAR-3, CAOV-3) and an epidermoid tumor of the vulva (A431) was assayed under conditions which differentiate between 17 beta-hydroxysteroid oxidoreductase type 1, a cytosolic isoform highly specific for estradiol, and type 2, a membrane bound isoform reactive with both estradiol and testosterone. On the basis of estradiol/testosterone activity ratios, all three cell lines appear to have type 2-like activity, with the specific activity of A431 markedly greater than that of the other cell lines. Estradiol, progesterone, or EGF, alone or in combination, were without effect on the enzymatic activity of OVCAR-3 cells. EGF decreased the activity of CAOV-3 cells slightly. In contrast, EGF stimulated A431 17 beta-hydroxysteroid oxidoreductase activity 7-8-fold over a 5-day exposure. Estradiol or progesterone, singly or in combination, also did not effect the enzymatic activity of A431 cells. However, progesterone inhibited the increase in activity seen in the presence of EGF. With EGF, estradiol, and progesterone together, the increase in enzymatic activity was comparable to that with EGF alone. The effects of estradiol and progesterone appear to result from steroid actions following binding of EGF to low-affinity receptors on A431 cells.

To elucidate the relationship between epidermal growth factor (EGF)/transforming growth factor (TGF-alpha) and estradiol-17 beta (E) in cell proliferation, we examined their effects on the breast cancer cell line, CAMA-1. While E was able to consistently induce cell proliferation under a variety of experimental conditions, EGF/TGF-alpha was without effect. Despite the presence of the receptor (EGFR) gene, mature EGFR protein and mRNA were not detected by radioreceptor assay, 35S Met-labelling, and the Intron Differential RNA/PCR method under conditions in which cells remain responsive to E. Furthermore, TGF-alpha is not an autocrine factor in CAMA-1 cells. We demonstrated unequivocally that EGF/TGF-alpha interaction with EGFR is not an obligatory event in mediating estrogen-stimulated cell proliferation.

CAMA-1 cells, isolated from malignant pleural effusion, are grown in long-term cultures as monolayers. The rate of growth is dependent upon fetal bovine serum and estradiol. These cells also exhibit a dose response to added 17 beta-estradiol with respect to the incorporation of radiolabeled thymidine into cells. The uptake is increased at low levels of estradiol and decreased at pharmacological levels of estradiol. The uptake of uridine and leucine is also stimulated by estradiol in a dose-related manner. Induction of precursor uptake is not observable until cells have been exposed to estradiol for approximately 10 hr or longer. Cells plated for different periods in steroid-stripped serum remain sensitive to estrogenic stimulation and show similar lag time in response. Estrogenic effect is not noticeable in the absence of serum. Addition of prolactin can partially restore estrogenic stimulation of thymidine uptake in serum-free medium. Like other estrogen target tissues, these cells contain cytoplasmic and nuclear estrogen receptors. These results demonstrate that the CAMA-1 cell line is estrogen dependent and that these cells may provide a promising model for the in vitro investigation of the mode of estrogen action in human breast cancer.

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.

Both serum and estrogen affect cell proliferation in CAMA-1 cells. Their effects on cell cycle kinetics are being investigated with partially synchronized cells following 48 hours of serum deprivation. By comparing the growth kinetics of synchronized (serum-deprived) cells with asynchronized (normal-fed) cells, we observed that there was a delay of cell proliferation for approximately 25 hr for synchronized cells and that estrogen only induced cell proliferation in serum-supplemented culture. A serum protein (RPF), precipitated by ammonium sulfate, dialyzed in 3,500 daltons cut off membrane and reconstituted in culture medium, was shown to stimulate cell proliferation in a dose-related manner. In the absence of RPF, estrogen had no effect on cell growth and S-phase formation in serum-free medium, but significantly induced cell growth in the presence of RPF. Experiments conducted with a synchronized cell population showed that estrogen increased the proportion of G1 phase cells to enter S phase, shortened the G1 phase duration, and ultimately increased the proportion of mitotic cells per cycle. Collectively, these results show that (a) estrogen effects of cell proliferation in vitro requires a serum component, a "G1/S-promoting factor" and (b) estrogen-induced tumor growth is a result of an accelerated rate of G1/S transition and an increased number of dividing cells per cycle. The knowledge of the interaction of estrogen with the G1/S promoting factor on cell growth is of both fundamental and therapeutic importance.

Two sublines, CAMA-1R and CAMA-1N, were derived from the original CAMA-1 cells after continuous passages in our laboratory. 1R cells were supplemented in culture with estradiol and retained the properties of the original cell line, whereas 1N cells, not supplemented with estradiol, had a partial loss of estrogenic responsiveness. The doubling times for 1R and 1N cells were 2 and 2.4, respectively. These two sublines showed different effects on thymidine uptake after estradiol stimulation. Cells of 1R had a 2-fold greater thymidine incorporation than did the control without estradiol. In contrast, no difference of thymidine uptake was demonstrated in cells of 1N in the presence or absence of estradiol. Both cell lines contained cytoplasmic progesterone receptors with a Kd of 0.56 nM and a sedimentation coefficient of 6 to 7S on a 5 to 20% sucrose gradient. The progesterone receptor level in 1N was about 8-fold higher than in 1R cells. Both sublines exhibited a dose-dependent increase in progesterone receptor levels in response to estradiol with the maximal stimulation (8-fold increase) reached by using 5 nmol of estradiol. The rate of increase for estradiol-induced progesterone receptor appears to be the same for both sublines; a liner increase was demonstrated between Days 3 and 7 following plating. Our results show that estrogen regulation of thymidine uptake and progesterone receptor induction may be mediated through two separate mechanisms. Thus, following a chronically deficient supply of estradiol to 1N cells, there is apparent loss of responsiveness to estradiol-induced thymidine uptake, but the ability of these cells to respond to estradiol induction of progesterone receptor was retained.

The presence of ER in DMBA-tumors was demonstrated by the use of dextran-charcoal assay, sephadex chromatography, sucrose gradient sedimentation, and organ culture techniques. It was found that tumors have binding sites ranging from 10-13 to 10-15 moles/mg protein, and a dissociation constant of ER 10-9 to 10-10 M. In experiments with tumor explants, prolactin-insulin significantly stimulated ER binding capacity, as compared with control without prolactin. This stimulation was tissue-specific and inhibited by progesterone. Insulin had a synergistic effect on prolactin stimulation of ER. Our results presents a plausible explanation for tumor responses to these hormones in vivo. This interaction of prolactin, estrogen, and progesterone may be a common phenomenon for all estrogen-responsive tissues.

PURPOSE One of the features of thyroid carcinoma is its predilection for women of reproductive age relative to men. An increased risk has also been documented in women who have used estrogens for gynecologic reasons. The aim of this study was to explore the mechanism by which sex hormones contribute to the development of thyroid carcinoma, which is not well understood at present. MATERIALS AND METHODS In this study, we investigated the effects of estradiol and testosterone on cell proliferation in a human thyroid papillary carcinoma cell line (KAT5) by MTT assay. We also studied the expression of estrogen receptors and the levels of anti-apoptotic Bcl-xL protein, pro-apoptotic Bax protein, and messenger RNA in the cells by Western blot and reverse transcriptase polymerase chain reaction analysis. RESULTS The results showed that estradiol promotes cell proliferation when compared with cells treated with testosterone and untreated cells, and that the growth-promoting effect of estradiol was attenuated by tamoxifen. The expression of Bcl-xL was markedly increased in a dose-dependent manner, resulting in an elevated ratio of Bcl-xL to Bax. DISCUSSION We conclude that estradiol promotes KAT5 cell proliferation and that the underlying mechanism may be associated with up-regulation of Bcl-xL expression. The data provide insight into the molecular mechanism underlying the epidemiologic data that shows a two- to threefold increased prevalence of thyroid carcinoma in women relative to men. From the therapeutic point of view, the finding that estradiol enhances anti-apoptotic signaling pathways may be significant in the search for novel prevention and treatment strategies of thyroid carcinomas. [ABSTRACT FROM AUTHOR]