Dita Kleinman

Regulation of endometrial cancer cell growth by insulin-like growth factors and the luteinizing hormone-releasing hormone antagonist SB-75

The involvement of IGFs in growth regulation of the Ishikawa endometrial tumor cell line and the possible interference of LH-RH analogues with a potential autocrine or paracrine loop involving IGFs was evaluated. The mitogenic effects of IGF-I, IGF-II, and insulin were compared. IGF-I was found to be 3-fold more potent than IGF-II and 30-fold more potent than insulin, suggesting that the effects of these growth factors are mediated by the IGF-I receptor. Ishikawa endometrial cancer cells secrete IGF-II, but not IGF-I, and insulin (1 microM) stimulates IGF-II release. The LH-RH antagonist [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]-GnRH (SB-75, CETRORELIX) inhibited basal and IGF-induced growth. Moreover, this antagonist almost completely inhibited IGF-II release from Ishikawa cells, while having no significant effect on the number or affinity of IGF-I binding sites. Inhibition of IGF-II release occurred at a lower SB-75 concentration than that needed for a reduction in cell number. The ED50 of SB-75 for IGF-II release was 0.3 microM as compared to 1.5 microns concentration which is required for reduction in cell number, suggesting that inhibition of growth factor release precedes cell growth inhibition. We conclude that the LH-RH antagonist SB-75 can inhibit the growth of endometrial cancer cells by interfering with the autocrine action of IGF-II and also by directly inhibiting the growth-stimulatory effects of IGFs, probably through effects on a post-receptor mechanism.

Components of the IGF system mediate the opposing effects of tamoxifen on endometrial and breast cancer cell growth.

The involvement of the IGF system in the growth regulation of hormone-dependent (e.g. endometrial and breast) cancer cells was studied. We chose two opposing effects of tamoxifen: the paradoxical stimulation of Ishikawa endometrial cancer cells growth and its inhibitory effect on MCF-7 mammary cancer cells. The results clearly confirm our working hypothesis that the IGF system is involved in growth regulation of these cancer cells irrespective of the direction of the drug effect. The following parameters of the IGFs system were studied: IGF-I receptors, IGF-I stimulated protein tyrosine phosphorylation, and membrane-associated and secreted IGF-binding proteins (IGFBPs). In Ishikawa cells, tamoxifen, similar to estradiol, increased IGF-I stimulated tyrosine phosphorylation of cellular substrates in accordance with its effect on cell growth. This effect of tamoxifen was inverted in MCF-7 cells. Tamoxifen did not affect the number or affinity of IGF-I receptors in both Ishikawa and MCF-7 cells, however, it caused a three-fold decrease in membrane-associated IGFBPs in the endometrial cells but an increase in these proteins in breast cancer cells. Similar but much less pronounced changes in soluble IGFBPs were observed. Our results indicate that the opposing growth effects of tamoxifen an endometrial and mammary cancer cells are associated with modulation of the IGF system components, mainly with reciprocal changes in membrane-associated IGFBPs.

Reactivation of cytomegalovirus in endometrial cells by estradiol

The present study uses an in vitro model to test the influence of hormones on the replication and reactivation of cytomegalovirus (CMV) in endometrial tissue culture. Infection of epithelial cells of the endometrium with CMV in the presence of a high concentration of estradiol resulted in only a slightly higher yield of infectious virus. Progesterone did not cause any effect on viral replication. Arrest of CMV replication was achieved by reduction of the pH of the incubation medium. No infectious virus was detectable after incubation of infected cultures at pH 5.8. In the above-described conditions following the arrest of CMV replication by low pH, estradiol treatment was capable of reactivating the virus. The possibility that the increase in CMV infection observed in pregnancy is caused by reactivation of latent virus provoked by hormonal factors will be discussed.

Antigenicity of sperm cells after freezing and thawing

Freezing and thawing is thought to result in removal of spermatozoal membrane antigens. We investigated the presence of sperm antigens before and after freezing and thawing by means of the immunoperoxidase assay (IPAMA), sperm immobilization test (SIT), and separation of proteins by gel electrophoresis. The results of the IPAMA and SIT assays showed no difference in the membrane antigens before and after freezing and thawing. Analysis of surface proteins by gel electrophoresis demonstrated that freezing and thawing did not remove any particular group of proteins from the surface membrane of spermatozoa. According to the evidence of the three tests performed, there is no meaningful removal of antigens from the sperm cell surface membrane by the process of freezing, preservation, and thawing when carried out by the specific methods used. This work does not support the suggestion that in cases of immunologic incompatibility between spermatozoa and cervical mucus it would be possible to overcome the couples infertility by employing the process of freezing, preservation, and thawing.

Acid phosphatase levels in follicular fluids following induction of ovulation in in vitro fertilization patients.

Considerable evidence indicates that changes in acid phosphatase (AP) activity at the ovarian level play a role in the process of ovulation. In this study the concentrations of AP were determined in follicular fluids collected from follicles of 52 women at the time of laparoscopy performed in connection with an in vitro fertilization program. In each of the 52 women at least one ovum was harvested. Of the 52 ova, 28 cleaved in vitro, while in 24 ova cleavage was not seen. Levels of AP in follicular fluids of women whose ova did not cleave were significantly lower than those found in follicular fluids of women with at least one ovum undergoing cleavage: 0.45 ±0.14 (SE) and 4.78±0.37 Bess Lowry Units (BLU), respectively. Comparison of AP levels in follicular fluids of women who conceived (5) and women whose ova cleaved, but in whom pregnancy was not achieved (23), did not reveal significant differences: 5.87 ± 1.29 and 4.55 ± 0.35 BLU, respectively. Moreover, the enzyme level was typical for a woman and not for a follicle. Fluids of two different follicles of the same woman, when one ovum cleaved and one did not, showed similar AP levels. AP levels were high in all samples follicles of women in whom at least one ovum cleaved. These findings indicate that the presence of certain levels of acid phosphatase represents an important, albeit not the sole, condition for ovum maturation. Morever, the follicular fluid levels of AP could serve as an indicator of proper timing of the follicular puncture in relation to the human chorionic gonadotropin injection (or the luteinizing hormone peak).

Infection of Endometrial Cells with Human Cytomegalovirus

In order to understand the mechanism of congenital human cytomegalovirus (CMV) infection we studied the effect of CMV on epithelial cell culture of the endometrium. Endometrial cells proved to be sensitive to CMV as indicated by morphologic alterations, success to support growth of infectious virus and positive immunoperoxidase staining. The infected cells were enlarged, multinuclear and revealed intranuclear inclusions. Electron microscopy detected the presence of the viral particles in the CMV-infected endometrial cells. The possibility that CMV infection of the endometrial cells may play a role in transmitting the virus to the embryo will be discussed.

Results of in vitro fertilization in women with antisperm antibodies in serum, cervical mucus, and follicular fluid

This study was undertaken to investigate the presence of antisperm antibodies (ASA) in serum, cervical mucus, and follicular fluid (FF) of women undergoing in vitro fertilization and embryo transfer (IVF-ET). IgG and IgA ASA directed mostly against sperm head were found at similar concentrations in serum, cervical mucus, and FF of 2 of 34 patients. Ninety-one percent fertilization and 100% cleavage rates, respectively, were observed in one of the two patients. No fertilization occurred in the second patient. In both women, in vitro sperm penetration tests revealed hostile mucus and repeated postcoital tests were poor. It is concluded that the sperm-cervical mucus penetration test and mucus ASA measurements are useful in establishing the diagnosis of immunological infertility.

The effects of contraceptive hormone of Chlamydia trachomatis in human endometrial cells

Abstract Several recent reports have documented a lower incidence of pelvic inflammatory disease (PID) among oral contraceptive users. The interpretation of this observation is difficult because the action of the contraceptive hormones may be directly by action on a specific host cellparasite relationship, or indirectly via their action on other systems. In the present study we investigated the susceptibility of cultured human epithelial cells of the endometrium to infection by chlamydia and the influence of contraceptive steroids (ethinyl estradiol, mestranol and medroxyprogesterone acetate) upon replication of chlamydia in these cells. Forty-eight hours post-infection, elementary and reticulate bodies were observed in vacuoles of the infected endometrial cells by electron microscopy. Treatment of chlamydial-infected cells with contraceptive steroids in three different concentrations (10 −5 M, 10 −6 M and 10 −7 M) resulted in no effect on chlamydial replication, as examined by one-step growth curve. These results indicate that contraceptive hormones do not prevent chlamydial infection by direct effect on the replication of the agent.