GYNECOLOGIC ONCOLOGY

41,56-63 (1991)

Human Epithelial Ovarian Cancer Cell Steroid Secretion and Its Control by Gonadotropins’ JAYANTHA WIMALASENA, University of Nebraska Medical Center, Department

DANIEL MEEHAN, of Physiology,

AND CARLO CAVALLO

600 South 42nd Street, Omaha, Nebraska 68198-4575

Received September 17. 1990

To elucidate the role of gonadotropins in regulating steroid metabolism in human epithelial ovarian carcinoma (OV Ca), cells were cultured from a number of OV Ca localized to the ovary. These cells uniformly secreted 17+estradiol (E,), and cells from some OV Ca also secreted progesterone (P), as well as CA 125. Secretion rates decreased with time in culture and number of subcultures. In the original and first few subcultures, l-10 pg/ml/pg DNA/24 hr of E2 was secreted and P secretion varied from 1 to 8 ng/ml/Fg DNA/24 hr under basal conditions. Secretion rates for CA 125 were between 5 and 300 U/ml/day. Approximately 30% of the primary cultures from cystadenocarcinemas responded to hCG and hFSH and 70% of cultures responded to 8-Br-CAMP with 2- to lo-fold increases in secretion of Ez. In one primary culture, hCG produced a dose-related increase in E2 production between 1 and 5 rig/ml, but the response declined to zero at 25 rig/ml. In the same cells, exposure to hCG and CAMP for 72 hr produced cell death, whereas hFSH had no such effect. Subculturing reduced steroidogenic responsesto the hormones but the responseto CAMP was maintained to a greater degree. These results suggest that some OV Ca-derived cells are steroidogenic in vitro and that some of these cells respond with increased Ez secretion to agents which are well-known stimulators of steroidogenesis in normal ovarian cells. 0 t!w Academic PRSS,

and some epithelial OV Ca may contain stromal elements [3]. Whether OV Ca cells in vivo [7-121 secrete steroids or whether cultured OV Ca cells secrete steroids [9] is poorly defined. In fact, the majority of OV Ca permanent cell lines do not appear to secrete steroids [9]; however, evidence has been presented to indicate that cells from well-differentiated OV Ca may be growth regulated by LH/FSH [13]. The question whether membrane preparations or tissue sections derived from OV Ca tissue can bind gonadotropins remains unsettled [14-181. Our aim in this study was to determine whether cultured cells derived from fresh in situ OV Ca tissue are capable of steroid synthesis/secretion. A second aim was to determine whether OV Ca cells secreted CA 125, a well-known marker for OV Ca [19-211. The third aim was to determine whether gonadotropins are able to regulate steroidogenesis by OV Ca cells. METHODS Digestion of OV Ca Tkwes

IK.

Over 90% of the tissue was obtained from the Cooperative Human Tissue Network (CHTN) or National Disease Research Interchange (NDRI). The tissue was shipped overnight in medium at 4°C.

INTRODUCTION

The common epithelial ovarian canceers are proposed to arise from the surface epithelium covering the ovary [ 11. Epidemiologic evidence suggests that gonadotropins (FSH, LH) may have a role in the genesis of human epithelial ovarian carcinoma (OV Ca) [2-51. The primary target cells of gonadotropin action in the ovary are stromal/thecal and follicular granulosa cells which secrete 17-P-estradiol (E2) and progesterone (P) in response to LH/FSH. There is evidence that at least part of the granulosa compartment arises from the surface epithelium [6]

Preparation

The tissue was placed in a (a) Mechanical digestion. plastic petri dish and l-2 ml of sterile phosphate buffered saline (PBS) containing 200 pg/ml streptomycin and 200 U/ml penicillin was placed on the tissue. Fatty material, necrotic tissue, or hemorrhagic material was removed, and the remaining tissue was finely chopped with a No. 11 surgical blade using a hemostat to hold the tissue. The resulting suspension was transferred to a sterile 50-ml tube, and the suspension was pipetted up and down re-

’ Supported by Grant PDT-329 from the American Cancer Society.

56 OO!%-8258191 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

of OV Ca Cells

GONADOTROPINS

AND HUMAN EPITHELIAL

peatedly. An additional 15 ml of PBS was added and the remaining larger pieces of tissue were allowed to settle for 5 min. The supernatant fraction was removed and centrifuged at 800 rpm on a TJ6 centrifuge (Beckman) for 10 min. The resulting pellet was resuspended and cells were counted in hemocytometer and plated at about 1 x lo5 cells/cm’. The cells were cultured in DMEM/Hams F12 1: 1 containing 10% FCS, 1 pug/ml porcine insulin, and the antibiotics. (b) Enzymatic digestion. This was performed according to standard methods as follows: To the tissue pieces that were not disrupted by mechanical agitation, a solution containing 2 mg/ml collagenase IA (Sigma) and 50 pg/ml deoxyribonuclease 1 (Sigma) in PBS was added at an approximate ratio of 5 vol to 1 vol of tissue. The mixture was then placed in a shaking water bath at 37°C and vigorously shaken; aliquots were removed for microscopic evaluation at lo-min intervals. Usually, digestion was terminated at 30 min; any remaining tissue pieces were allowed to settle, and the supernatant was collected. The undigested tissue pieces were vigorously pipetted up and down to disperse tissue mechanically and supernatant was collected as before. If the supernatant from the first digestion was poor in cellular yield, the digestion was repeated. The supernatant fractions from digestions were diluted lo-fold with PBS containing antibiotics and 10% FCS and centrifuged for 10 min (800 rpm). The pellet was resuspended, washed in PBS/antibiotics/lo% FCS, and centrifuged. The cellular pellet was then resuspended in DMEM/Hams F12; a viable cell count was measured by exclusion of 0.4% trypan blue, and cells were plated as before. Cells were grown in Falcon Primaria plates. Subculture was performed by standard procedures using a solution containing trypsin and EDTA [13]. Routinely, plates were split 1: 3 and media were changed every 3 days when cells divided relatively rapidly and once a week when cells did not divide. Determination Secretion

of Estradiol, Progesterone, and CA 125

Media from cell cultures were stored at -20°C and P and E2 were measured in 100~~1aliquots by RIA kits obtained from Diagnostic Product Corp. (Los Angeles) according to instructions from the manufacturer. (Intraassay coefficent of variation, CV < 8.5% and interassay CV < 10% for P; intraassay CV < 7.0% and interassay CV < 8.5% for E2; sensitivity for E2 is 8 pg/ml and that for P is 0.05 rig/ml.) The slopes of the RIA plots for E2 or P were similar whether the assays were performed in culture medium +FCS or in PBS/O.l% BSA. Further, the displacement of tracer E2 or P by standard concentrations of E2 or P using our assayconditions was identical

OVARIAN

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to the RIA displacement curve provided by the manufacturer. Since some of the values in the tables and figures in the manuscript appear to be lower than the detection limit for E2 and P, the following calculation is presented to demonstrate that the measurements for the steroids were well above detection limits. For example, a progesterone secretion rate of 0.17 ng/ml/pg DNA/24 hr is calculated from an RIA value of 30.5 rig/ml, a DNA value of 60 pg DNA, and a collection period of 72 hr. Similarly, an E2 secretion rate of 0.21 pg/ml/pg DNA/24 hr is calculated from an E2 or 37.8 pg/ml measured by RIA, a DNA value of 60 pg, and a collection period of 72 hr. These values are not corrected for volume/dish; in a dish containing 5 ml of medium, the corresponding E2 secretion rate would be 1.05 pg/pg DNA/24 hr. CA 125 was measured using the CA 125 RIA kit of CENTOCOR obtained through Amersham Corp. The instructions provided by the manufacturer were followed exactly. Sensitivity of the CA 125 assay is 1 U/ml. Other Methods

Cellular DNA was determined by Burton’s method [22] and iodinated hCG binding to cells was determined as described before [23]. Tissues and Pathology

As stated above, tissues were obtained from CHTN and NDRI. All the tumors, irrespective of histology, were from the ovary itself. Pathology reports were supplied by the two agencies. The numbers of the human ovarian tumor cells refer to the sample identification numbers from CHTN and NDRI. Statistical Analysis

Differences in treatment and control groups (mean & SE) was ascertained by the Student t test. Each experiment was repeated at least twice with two to three replicate wells per treatment. Materials

Hormones, hCG (11-14 x ld IU/mg) and hFSH (AFB 4822B), were kindly provided by the National Pituitary Agency. All other chemicals except those noted above were purchased from Sigma Chemical Co. RESULTS Morphology

and Growth

After culture, cells were examined microscopically. In all of the cultures examined, cells were heterogeneous with epithelial cells of more than one shape, some cells were elongated compared to the majority, and others

58

WIMALASENA,

MEEHAN.

TABLE 1 Secretion of Estradiol by 88-10-203 Mixed Epithelial Carcinoma Treatment Control hFSH (50 rig/ml) hCG (10 rig/ml) 8-Br-CAMP (0.25 mM)

Ez secretion (pg/W~g DNA/24 W 5.85 5.93 7.15 10.26

k f 2 2

0.6 0.9 0.9 1.0

AND CAVALLO

in culture (Table 2), and the rates of secretion varied considerably between the different OV Ca cells; the range for Ez was l-10 pg/ml/pg DNA/24 hr (n = 12) and for P secretion, the range was l-8 ng/ml/pg DNA/24 hr (n = 6) under basal conditions. Effects of Androstenedione

and Serum on E2 Secretion

Since many of the cultured OV Ca cells secreted E2, it was of interest to determine whether androgens could be aromatized to estorgens by the cells. To this end, cells Note. After establishment of the OV Ca cells in culture, triplicate from six cystadenocarcinoma cultures were incubated with wells were treated with agonists in the table. Media were collected after androstenedione and the estradiol secretion was measured 72 hr, and E2 was measured. Effects of CAMP are significant at P 0.05). In these cells, hCG produced a dose-related

60

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MEEHAN,

AND CAVALLO

P and, in general, secretion of E2 and P decreased with time in culture. The rates of E2 secretion varied widely depending on the tumor, ranging from 0.08 to 6.0 pg/ml/pg DNA/24 hr under unstimulated conditions. Thompson et al. [9] recently reported that three established OV Ca cell lines secreted small quantities of Et. If the assumption that diploid cells contain 6 pg DNA/cell is made, then for the diploid line OV 166 one can calculate a production rate of 0.69 pg/pg DNA/24 hr. This production rate, as the authors state, is low in comparison 0 5 10 15 20 to the results presented herein, especially after correction 25 30 hCG for media volume (see Methods). Thompson et al. [9] also h/m0 FIG. 3. Effect of increasing concentrations of hCG on HOVT 89- provided direct evidence for significant aromatase activity in OV Ca cell lines, and the results in Table 3 suggest 8-33 cystadenocarcinoma cells’ E, secretion. Media containing indicated concentrations of hCG were placed on the cell cultures for 24 hr. Stim- that the majority of cystadenocarcinoma-derived cells ulatory effects of hCG are significant (P < 0.025 or lower) at 1, 5, and have the capacity to convert androstenedione to Ez and 10 rig/ml. that this capacity decreased with repeated subculturing. Cells cultured from other cystadenocarcinomas apparently did not have reserve capacity to convert androsincrease in E2 production when cells were treated for 24 tenedione to Ez (Fig. 1). However, in these cells, serum hr (Fig. 3). Effects of hCG were most significant at 5 regulated E2 secretion in a dose-dependent manner. rig/ml (P < O.OOl), when there was about an &fold inSerum may provide LDL-derived cholesterol and may crease in E2 secretion. At 10 rig/ml the increase in E2 also contain factors which regulate steroidogenesis in was 3.7-fold (P < O.Ol), and at 1 rig/ml the increase was these cells. There is some evidence that OV Ca may 1.6-fold (P < 0.05); at 25 rig/ml there was no change in secrete steroids; thus, Plotz et al. [ll] found that microE2 secretion. somal fractions from fresh cystadenocarcinoma tissue could convert P to androgens, but androgens were not CA 125 Secretion aromatized. Aiman et al. [lo] demonstrated that venous A number of OV Ca cultures (n = 13) secreted CA blood draining ovaries with epithelial tumors contained 125 into the culture fluid. The secretion of CA 125 also significantly elevated androstenedione, Ez, and estrone declined with subculturing (Table 2 and Fig. 4), as was also observed for steroid secretion. The production rates of CA 125 varied widely among cultures (5-300 U/ml/day) from different OV Ca established in culture. In J. 11.17 A control 5 hCG cells (Fig. 4), hCG had a significant inhibitory effect on C CAMP CA 125 secretion. The inhibitory effect of S-Br-CAMP D FSH was also significant; however, FSH had no effect on CA E First Passage F Fourth Passage 125 secretion, The effect of agonists on CA 125 secretion was tested only on 5.11.17 cells. DISCUSSION A considerable body of indirect evidence which suggests that gonadotropins may play a role in the development of epithelial OV Ca has accumulated over the last two decades. On the basis of this evidence, Cramer et al. [3] proposed that gonadotropins or estrogens, whose secretion may be controlled by gonadotropins, may induce or promote OV Ca formation. According to this theory, the stromal element of in situ OV Ca may secrete steroids. Observations presented herein are in agreement with this requirement as all of the in situ OV Ca-derived cells secreted measurable quantities of gonadal steroids, especially E2. Some of the cultures secreted both E2 and

A

B

C

D

E

F

FIG. 4. Effects of agonists on CA 125 secretion. 5.11.17 cells were established in culture as described under Methods. CA 125 was determined on Day 6 following a 48-hr stimulation with the agonists shown. CA 125 was also determined after one passage (total of 11 days in culture) and after four passages (total of 30 days in culture). On Day 6, the effect of hCG is significant at P < 0.02 (42.1 2 1.2 vs 36.3 + 1.0) and the effects of CAMP are significant at P < 0.05 (42.1 f 1.2 vs 38.4 2 0.9). Secretion on Day 11 and Day 30 is the mean (+SE) from three determinations and the rates of secretion are significantly different from those of control (P < 0.01). Secretion at fourth passage is significantly different from that at first passage (P < 0.01).

GONADOTROPINS

AND HUMAN

when compared to normal postmenopausal patients. Mahlck et al. [8,38,39] and Backstrom et al. [7] demonstrated that women with pelvic OV Ca metastasis had significantly higher 20o-OH-progesterone and P when compared to age-matched controls. Rome et al. [37] demonstrated increased Ez excretion in patients with epithelial OV Ca. These results taken together support Cramer er ai.‘s [3] proposal that OV Ca cells may secrete gonadal steroids. Whether the E2 (and P) secreting cells in our cultures are derived from the in vivo steroid secreting stromal compartment as is suggested by Cramer et al. is unknown. However, we should point out that the OV Ca samples were obtained from postmenopausal patients ranging in age from 51 to 71 years; therefore, the E2 secretion by normal stromal cells contained within the tissue would be expected to be little or none. Furthermore, we would not expect the normal cells to divide in culture, but it is certainly possible that there could be steroid-secreting abnormal stroma-derived cells which could survive subculturing. There is a substantial body of evidence to suggest that OV Ca tissues have E?., P, and androgen receptors [12,25,26], and some of these receptor-positive cells also contain aromatase, which may convert locally secreted androgens to estrogens [12]. In this study, we also measured the secretion of CA 125 antigen into the culture fluid by OV Ca cells because CA 125 is widely accepted as a clinical OV Ca marker and the control of CA 125 secretion by cultured OV Ca cells has not been previously reported. There was a wide range of CA 125 secretion and some of the cultures secreted quantities of this antigen considered to be in the range secreted by OV Ca in vivo [20]. Examples would be J. 11.17 (Fig. 4) and 87-09-262 cells (Table 2). Whereas E2 secretion by J. 11.1.7 cells did show a positive response to agonists, CA 125 secretion was inhibited by hCG and CAMP and the rate of secretion appeared to decrease with time in culture. Whether changes in CA 125 secretion relate to changes in the number of CA 125 molecules or alterations in antigenicity is unknown. The secretion of CA 125 is not limited to OV Ca since ovarian adenoma cells in culture (data not shown) and normal women secrete CA 125 in a stage-specific manner during the menstrual cycle [27]. We also have observed considerable quantities of CA 125 (66.3 + 18.5 U/ml, y1 = 7) in follicular fluid from patients undergoing hormone treatment for in vitro fertilization procedures. The localization of CA 125 in follicular fluid may suggest a follicular origin for this antigen. Others have demonstrated that hormonal stimulation during IVF treatment also increased CA 125 in blood [28]. Thus, CA 125 appears to be secreted by hormone-responsive ovarian cells in addition to OV Ca cells. Could the observations of CA 125 secretion in normal subjects and the observations of in situ OV Ca-derived cultures secreting both CA 125 and gonadal steroids

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suggest that normal ovarian cells are closely related to cells of epithelial OV Ca? Such a relationship may be suggested by the postulated origin of follicle cells from the ovarian surface epithelium [6]. Cramer et al. [3] suggested that epithelial OV Ca cells may be gonadotropin-sensitive in growth on the basis of epidemiological and animal experimentation evidence. In support of a role of gonadotropins in OV Ca is the observation by Simon et al. [13] that OV Ca cells in culture demonstrate positive growth responses to hCG and hFSH. However, the ascites-derived less-differentiated OV Ca cells did not respond to hCG or hFSH unless high levels of hCG, hFSH, and Ez were added simultaneously to cultures. Simon et al. [13] also noted that responses to gonadotropins decreased with time. Our data on steroidogenie responses to hCG and hFSH also support the view that some in situ epithelial ovarian cancers contain cells whose steroidogenesis can be modulated by hCG and/or hFSH. Thus, we found that in about 30% of cultures derived from fresh cystadenocarcinomas, hCG and hFSH at low concentrations increased E2 production severalfold (Table 3 and Figs. 2 and 3). In some other cultures, such as 5.11.17 cells, both hCG and hFSH provoked small but significant increases in E2 secretion. Where tested, these steroidogenic responses to gonadotropins appeared to wane with subculturing as seen in Table 3. This decrease in response may be partly responsible for our failure to detect steroidogenic responses to the gonadotropins in the majority of cultures since the gonadotropins were usually added at the third or fourth subculture. Similarly, hCG binding was measured at the same time and specific binding was detectable only in one of six cultures tested (data not shown). The largest steroidogenic response to hCG was observed with the 89-8-33 serous papillary cystadenocarcinoma in primary culture. The dose-response curve of Ez secretion (Fig. 3), however, showed a diminution of response at 10 rig/ml and a loss of response at 25 rig/ml. This loss of effect at higher doses may be related to the cytotoxic effect of hCG observed when the cells were exposed for 3 days to 10 rig/ml hCG. Perhaps exposure of cells to a high dose of hCG (25 rig/ml) is as toxic as exposure of cells to a moderate dose over a longer period. In contrast to hCG, hFSH did not appear to produce cytotoxic effects on the 89-8-33 cells. However, CAMP was also cytotoxic in analogy to hCG. The effects of CAMP on steroidogenesis were different from those of the gonadotropins in two respects. First, 70% of OV Ca cultures responded to a low CAMP dose (0.25 mM) with increased EZ secretion. Second, the response to CAMP was maintained to a greater degree than the responses to the gonadotropins (Table 3) with subculturing. These results suggest that the differentiated cellular elements responsive to CAMP (PK-A and steroidogenic compo-

62

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nents) may be more stable during passaging of OV Ca cells than plasma-membrane-bound hormone receptors. There are conflicting data in the literature regarding the presence of gonadotropin binding sites on OV Ca tissue. Kammerman et al. [16] found evidence for hCG binding and hCG-responsive CAMP production in freshly dissociated cells from one papillary adenocarcinoma. However, no quantitative data were available. Al-Timimi et al. [17] incubated tissue sections with lo-20 pg/ml of FSH or LH and detected bound LH or FSH with specific antihormone antibodies by using immunocytochemical methods. In a large sample (111 individual specimens of various types of ovarian epithelial cancers), FSH binding was present in 51% of malignant tumors and LH binding was present in 32% of malignant tumors. These authors found marked heterogeneity in each specimen for gonadotropin binding with strongly staining cells interspersed with cells not showing any staining. Furthermore, cells from metastases had no hormone binding and the number of binding sites/cell (qualitative) decreased with a decreasing degree of cellular differentiation in the tumor. Stouffer et al. [15] did not find evidence for hCG or FSH binding in 26 epithelial OV Ca samples. It is not clear from this report whether the samples were from in situ OV Ca or a metastatic site. Rajaniemi et al. [14] used equilibrium binding methods to detect specific hCG binding in 27% (n = 21) of in situ epithelial OV Ca specimens. These authors also pointed out the fact that receptors may be occupied in viva by the high concentrations of LH and FSH circulating in postmenopausal women. In a very recent study, Nakano et al. [18] presented quantitative and qualitative data for hCG binding to 1 of 18 epithelial tumors. Whereas 3 specimens bound hFSH, these binding sites were localized in the stroma. Again, it is not clear whether the biopsy specimens were drawn from the ovary. This evidence and our observations suggest that in some in situ epithelial OV Ca, cells may be gonadotropin-sensitive (increased steroidogenesis). The hormone responsiveness may decrease following metastasis or subsequent to a decreasing degree of differentiation as seen with subculturing. An issue which we have not addressed in this communication is the question of endogeneous hCG (or hCGlike material) secretion by cultured OV Ca cells. There is a substantial body of evidence that patients with epithelial OV Ca may secrete hCG or hCG-related material [29-321 and Matias-Guiu and Prat [33] recently demonstrated the presence of hCG in 22 (using polyclonal antihCG antibodies) of 100 ovarian neoplasm sections examined, and using monoclonal hCG antibodies, 44 of 100 were positiye for hCG. However, we are not aware of any data on the secretion of hCG by cultured OV Ca cells. We did not detect hCG (using a P-subunit-specific polyclonal antibody) in the conditioned media from

AND CAVALLO

5.11.17 cells and 89-03-023 cells, but hCG was not measured in the other cultures. If hCG was produced by the hormone-unresponsive cultures (70% of the cystadenocarcinomas), the hormone may, by occupying LH/hCG receptors, prevent a steroidogenic response to exogeneous hCG. A similar point was made by Rajaniemi et al. [14] regarding LH and FSH as stated above. In summary, we have for the first time demonstrated that cells cultured from in situ epithelial OV Ca secrete EZ, P, and CA 125. The secretion of E2 was regulated by exogenous CAMP, and in some cultures, hCG and hFSH also positively regulated E2 secretion. These facts are relevant to recent studies on the treatment of OV Ca with GnRH analogs [34-361. Evidence from limited clinical trials suggests that GnRH analogs may have an antitumor action on OV Ca. The decrease in LH and FSH concomitant with decreases in CA 125 following GnRH analog treatment led Jager et al. [36] to suggest that the decrease in the gonadotropins may correlate with the antitumor effects of GnRH. ACKNOWLEDGMENTS Carlo Cavallo wassupported by an Undergraduate Summer Fellowship (1989) from the University of Nebraska Medical School Alumni Association. We thank Glennora Flanagan for typing this manuscript. We are also grateful to Dr. Stanley Cox and Dr. Shymal Roy of the University of Nebraska Medical Center and Dr. David Puett of the University of Miami Medical School for critical review of the manuscript. REFERENCES 1. Scully, R. E. Ovarian tumors, Am. J. Pathof. 87(3), 686-720 (1977). 2. Piver, M. S. Epidemiology of ovarian cancer, in Ovarian malignancies: Diagnostic and therapeutic advances (M. S. Piver, Ed.), Livingstone, Edinburgh, pp. l-10 (1987). 3. Cramer, D. W., Hutchinson, G. B., Welch, W. R., Scully, R. E., and Ryan, K. J. Determinants of ovarian cancer risk. II. Inferences regarding pathogenesis. JNCI 71, 717-721 (1983). . 4. Bleehen, N. M. (Ed.). Ovarian cancer, Springer-Verlag, New York/Berlin, pp. l-22, 98-108 (1985). 5. Heintz, A. P., Hacker, N. F., and Lagasse, L. D. Epidemiology and etiology of ovarian cancer: A review, Obstet. Gynecol. 66(l), 127-135 (1985). 6. Speroff, L., Glass, R. H., and Kase, N. G. Clinical gynecological endocrinology and infertility, Williams & Wilkins, Baltimore, Chap. 3 (1989). 7. Backstrom, T., Mahlck, C.-G., and Kjellgren, 0. Progesterone as a possible tumor marker for “nonendocrine” ovarian malignant tumors, Gynecol. Oncol. 16, 129-138 (1983). 8. Mahlk, C.-G., Backstrom, T., Kjellgren, O., and Selstam, G. Plasma 20 cY-OH-progesterone in women with malignant epithelial “non-endocrine” ovarian tumors, Acta Obstet. Gynecol. &and. 64, 515-518 (1985). 9. Thompson, M. A., Adelson, M. D., Kaufaran, L. M., Marshall, L. D., and Cable, D. A. Aromatization of testosterone by epithelial tumor cells cultured from patients with ovarian carcinoma, Cancer Res. 48, 6491-6497 (1988).

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