Pondichery G. Satyaswaroop

Characterization of the functional progesterone receptor in an endometrial adenocarcinoma cell line (Ishikawa): Progesterone-induced expression of the α1 integrin

Endometrial progesterone receptors (PR) are regulated by both estrogen (E2) and progesterone (P) and mediate the expression of specific endometrial proteins. Ishikawa cells are a well-differentiated human endometrial adenocarcinoma cell line, with both estrogen receptors (ER) and PR, regulated in a manner similar to that of normal endometrium. Immunohistochemical and biochemical analyses demonstrate that the concentration of PR is increased by E2 priming and decreased by subsequent treatment with P. Scatchard plot analysis showed a K(d) of 1 nM. On the basis of biochemical analysis, PR concentrations reached approximately 1400 fmol/mg cytosol protein in cells after treatment with E2 (10(-8) M) for 4 days. Immunoprecipitation and Western blot studies revealed the presence of both the 116 kDa and 81 kDa proteins with multiple isoforms of the high molecular weight (MW) protein. Northern blot analysis demonstrated transcriptional control of PR by steroid treatment. These studies demonstrate the coordinate regulation of all PR mRNA species. The functionality of Ishikawa PR was demonstrated by the expression of alpha1beta1 integrin in response to E2 plus P, at the level of transcription and translation. This effect was blocked by the addition of the anti-progestin, RU-486. These studies reconfirm that the Ishikawa cell line is an excellent model for the study of hormonally regulated events in the human endometrial epithelium.

Heterogeneity and progesterone‐receptor distribution in endometrial adenocarcinoma

Objective response to progestin therapy occurs in 30% to 35% of patients with recurrent or metastatic endometrial carcinoma. Measurement of progesterone receptor (PR) in these tumors has been suggested as a method for distinguishing between responders and nonresponders. However, studies on steroid receptor measurements in endometrial carcinoma may be subject to complexities of tumor, tissue, and cellular heterogeneity. The authors provide evidence for the existence of various types of heterogeneity and address the problem of cellular heterogeneity by determination of PR concentrations in the glandular and stromal cells of the endometrium. It is concluded that until the problem of tumor and tissue heterogeneity is resolved, PR concentrations in endometrial carcinoma must be interpreted with caution. Cancer 53:113‐116, 1984.

Both 17β-estradiol and tamoxifen induce c-fos messenger ribonucleic acid expression in human endometrial carcinoma grown in nude mice*

We previously demonstrated the estrogen-like effects of tamoxifen on the acceleration of growth and increased progesterone receptor concentrations of human endometrial carcinomas grown in the nude mouse experimental model. In our current study the modulation of protooncogene expression by 17 beta-estradiol and tamoxifen in human endometrial carcinomas was investigated. The protooncogenes investigated in this study were c-fos, c-jun, c-myc, N-myc, HER-2/neu, c-erbB, c-fms, and c-Ha-ras. Among those we found that c-fos expression was induced by 17 beta-estradiol in the following 17 beta-estradiol-sensitive tumors: EnCa-101 and EnCa-X. The induction was apparent within 1 hour, reached peak level at 2 hours (16-fold), and remained constant up to 4 hours. The c-fos messenger ribonucleic acid returned to prestimulation level by 12 hours. Tamoxifen also stimulated c-fos expression, the expression pattern being similar to that of 17 beta-estradiol albeit of a lesser degree. The messenger ribonucleic acid transcripts for other protooncogenes tested did not show significant changes during hormonal manipulation. The induction of c-fos expression by tamoxifen is consistent with its estrogen-like effect on endometrial carcinoma growth.

Histologic response of normal human endometrium to steroid hormones in athymic mice

Despite the widespread use of hormonal therapy for menopausal symptoms, oral contraception, and treatment of metastatic breast carcinoma, information concerning the histologic and biologic effects of individual sex steroids on the human endometrium remains incomplete. Repetitive endometrial biopsy is impractical, and ethical constraints limit the dosage and duration of administration for some steroids. The ovariectomized athymic mouse was investigated as a host for human endometrium in which the hormonal milieu may be manipulated and the histologic response determined. Minced proliferative-phase endometrium from hysterectomy specimens of normally cycling women was inserted into the subcutis of 4- to 6-week-old mice. Proliferation of endometrial gland cells occurred in the transplanted endometrium of estradiol-treated mice, while the complete sequence of secretory-phase events, including subnuclear vacuolization, luminal secretion, and decidualization of stroma, was observed during a 14-day period of treatment with estradiol and progestin (medroxyprogesterone acetate). Progestin treatment alone also caused secretory-phase changes, but the response was delayed and appeared weaker. The transplanted endometrium in control mice appeared to be inactive. These observations provide support for the use of this model to study the histologic response of human endometrium to sex steroids.

An analysis of tamoxifen-stimulated human carcinomas for mutations in the AF-2 region of the estrogen receptor

The estrogen receptor (ER) contains two transcriptional activation domains: AF-1 and AF-2. AF-2 is dependent on a highly species-conserved region of the ER. It has been shown that site-directed point mutations of conserved hydrophobic amino acids within this region reduce estrogen-dependent transcriptional activation. In addition, when these mutated ERs are transfected into HeLa cells, both tamoxifen and ICI 164,384 become strong agonists. The implication is that mutations in this region could account for the tamoxifen-stimulated tumors seen clinically. We performed single stranded conformational polymorphism (SSCP) analysis spanning the entire ER along with DNA sequencing of the AF-2 region of the ER isolated from two different tamoxifen-stimulated breast cancers, MCF-7/TAM and MCF-7/MT2, and a tamoxifen-stimulated endometrial cancer, EnCa 101. In addition, a tamoxifen-stimulated endometrial carcinoma cell line, the Ishikawa cell line, was also studied. There were no mutations found by SSCP analysis and sequencing of all four AF-2 regions also revealed no mutations. Mutations within the AF-2 region of the human ER do not appear to account for the growth of human breast and endometrial carcinomas that are used as reproducible laboratory models of tamoxifen-stimulated growth observed clinically.

Creatine kinase activity in human endometrium: Relative distribution in isolated glands and stroma

Creatine kinase (CK) activity was measured in human endometrium as a function of the normal menstrual cycle. The specific activity of CK was consistently higher in the secretory endometrium than in the proliferative tissue (proliferative, 0.9 U/mg of protein; secretory, 3.3 U/mg of protein). The relative distribution of CK activity in isolated glands and stromal cells was determined following collagenase digestion of the endometrial specimen according to our previously described procedure. These studies show an enrichment of CK activity in the glands that is greater than fivefold that present in the stromal cells. Electrophoretic mobility of CK activity on cellulose acetate further indicates that the endometrial enzyme is a BB (brain type) isoenzyme. In the rat uterus, CK has been shown to be an estrogen-induced protein. In contrast, the activity of this enzyme may be modulated by progesterone in human endometrium.

Designing a schedule of progestin administration in the control of endometrial carcinoma growth in the nude mouse model

We previously showed that combined treatment with tamoxifen and progestin was effective in arresting the growth of human endometrial carcinomas in the nude mouse model. After a 15- to 20-week tumoristatic period, the tumors began to regrow, reminiscent of the clinical situation. Lack of progestin sensitivity during the regrowth period appeared to reflect the absence of progesterone receptor. To test the prediction that intermittent progestin administration may circumvent the regrowth phenomenon, the effect of various doses of progestin on blood progestin levels, EnCa 101 tumor progesterone receptor profiles, and rate of tumor growth were examined. Whereas 1 mg progestin was ineffective in totally down-regulating tumor progesterone receptor, 2 and 5 mg doses resulted in the total disappearance of tumor progesterone receptor by 1 week followed by its reappearance at 5 to 6 weeks and 9 to 10 weeks, respectively. On the basis of these results we predict that intermittent progestin administration may result in better control of endometrial cancer growth in the nude mouse system.

Therapeutic effect of recombinant human tumor necrosis factor in ovarian carcinoma xenograft in nude mice

Tumor necrosis factor (TNF) has been found to have in vitro and in vivo cytotoxicity against a variety of mouse and human experimental tumors. Synergistic effect with interferon-gamma (IFN) has also been reported. We examined the therapeutic effect of TNF and IFN using the human ovarian carcinoma cell line NIH:OVCAR-3 intraperitoneally transplanted in nude mice. Approximately 10 to 15 million cells were injected intraperitoneally in 80 nude mice. One week later the animals were divided into four treatment groups of 20 animals each: (1) untreated controls, (2) daily ip injections of 5 micrograms TNF, (3) daily ip injections of 5 x 10(4) U of IFN, and (4) daily ip injections of 5 micrograms TNF and 5 x 10(4) U of IFN. After 13 weeks of treatment, when 50% of the control animals were dead of disease, a survival analysis was performed. The remaining animals underwent laparotomies (comparable to second-look in humans) at Weeks 15, 18, 20, and 22 to determine the presence of tumor in various tissues by histologic examination. A statistically significant survival difference was found between the untreated control group and the groups that received TNF or TNF plus IFN (P less than 0.0001). The TNF effect was further reflected in decreased serum CA-125 levels compared with controls (P less than 0.001). Only one out of 20 animals receiving TNF or TNF in combination with IFN was found to have intraperitoneal tumor at the time of laparotomy, whereas all the untreated controls had gross intraabdominal carcinomatosis. These results, in addition to existing preclinical data, may form the basis for clinical protocols employing intraperitoneal TNF in selected ovarian carcinoma patients.

Endometrial carcinoma: An aberration of endometrial cell differentiation

A model endometrial cell differentiation is being proposed. It is in agreement with the available information on the concentrations of female sex steroid receptors in the human endometrium and the effects of estradiol and progesterone during the normal menstrual cycle. The scheme is extended to include endometrial carcinoma as a derangement of normal cell differentiation. Evidence is cited, from various studied on estradiol and progesterone receptor measurements and hormone responses of human endometrial carcinoma, in support of this hypothesis. This concept provides a rational basis for the selection of patients for hormonal (progestin) therapy.

Species crossreactivity of human progesterone receptor monoclonal antibodies: western blot analysis

The crossreactivity of monoclonal antibodies (hPRa 1, 2, 3 and 6) generated against human progesterone receptor was examined in six mammalian and an avian species using the techniques of sodium-dodecylsulfate polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected on protein blots of receptor-containing preparations from human endometrial carcinoma grown in nude mice, human T47D breast cancer cells, rabbit, cow and mouse uteri, and chick oviduct. No receptor-associated, immunoreactive bands were detected in rat, guinea pig or hamster uteri. The number and molecular weights of the receptor subunits detected varied between species, and only human progesterone receptor displayed electrophoretic microheterogeneity in its high molecular weight subunit. These data demonstrate that the human progesterone receptor antibodies recognize epitopes not common to all species.