A clinico-radiological correlation of breast diseases during lactation and the significance of unilateral failure of lactation

OBJECTIVES: The Ishikawa endometrial cancer cell line is hormonally responsive, expressing estrogen and progesterone receptors (ER, PR) when grown in traditional monolayer culture. The purpose of this paper is to demonstrate a three-dimensional spheroid culture system for cancer cells. We used this system to determine the response of the Ishikawa cell line to estradiol-17 beta (E), tamoxifen (T), megestrol acetate (MA), and progesterone (P). METHODS: Ishikawa cells were incubated in polyurethane culture bags using phenol red-free media containing ethanol (0.1%, controls), E (1 mumol, or 1 nmol), T (1 mumol, or 10 nmol), MA (1 mumol, or 10 nmol), or P (1 mumol). Cellular morphology was assessed by hematoxylin and eosin staining, and expression of estrogen and progesterone receptors was determined immunohistochemically using an immunoperoxidase technique. RESULTS: Cells in control cultures demonstrated minimal organization and lacked hormone receptors. In contrast, cells exposed to either E or T displayed significant glandular formation, with multicellular, microvilli-rich, columnar epithelia exhibiting polarized nuclear arrangements. Within 4 weeks, E- and T-treated cultures showed upregulated nuclear staining for PR, with little ER present. Cells treated with MA or P showed less glandular organization but expressed ER with PR downregulation. CONCLUSIONS: These data support the use of this novel three-dimensional culture system to study the modulation of tumor cell biologic activity in response to hormonal agents. Future applications of this model include examining in vitro responsiveness of cancer cell lines to additional biologic agents and chemotherapeutic regimens.     

Fatty acid synthase expression in endometrial carcinoma: correlation with cell proliferation and hormone receptors

BACKGROUND: Fatty acid synthase (FAS), a biosynthetic enzyme, normally functions in the liver to convert dietary carbohydrate to fat, but it is minimally expressed in most other normal adult tissues. FAS is expressed at markedly elevated levels in subsets of human breast, ovarian, and prostate carcinomas that are associated with poor prognoses. During the menstrual cycle, the expression of FAS in the human endometrium is closely linked to the expression of the proliferation antigen Ki-67, estrogen receptor (ER), and progesterone receptor (PR). METHODS: This study reports the expression patterns of these antigens in 35 endometrial carcinomas as determined by immunohistochemical analysis. RESULTS: All cases demonstrated a close direct correlation between FAS and Ki-67 expression. Average FAS expression levels were correlated with tumor grade. Twenty-five carcinomas that were positive for ER and PR showed close correlation in expression of FAS, Ki-67, and hormone receptors. Individual tumors displayed varying degrees of heterogeneity of expression. A few well-differentiated carcinomas showed very low expression of all four antigens, similar to the antigenic profile of secretory endometrium. Nine high grade carcinomas that were negative for ER and PR also showed close correlation in expression of FAS and Ki-67 with uniformly high expression. CONCLUSIONS: These data suggest the following hypothesis: In hormone-dependent endometrial cells, FAS expression is part of the estrogen-driven cellular response that leads to proliferation; however, its linkage to proliferation is such that FAS expression is maintained in proliferating cells in endometrial carcinomas that acquire hormone independence. The use of these four antibodies as a panel may increase the diagnostic utility of ER and PR immunohistochemistry for tumor classification and prediction of the responsiveness of tumors to hormonal therapy.     

Identification and characterization of cytosolic sulfotransferases in normal human endometrium

Understanding the factors which alter estrogen metabolism and activity in endometrial tissue is important because unopposed estrogen stimulation is an important risk factor in the development of endometrial carcinoma. The cyclic progression of the endometrium through proliferative and secretory phases is normally under the control of the ovarian hormones beta-estradiol (E2) and progesterone. One mechanism by which progesterone inhibits the activity of E2 in secretory endometrium is by elevating the degree of E2 sulfation, thereby reducing its ability to bind to the estrogen receptor and elicit a cellular response. Our laboratories have investigated the cytosolic sulfotransferases (STs) found in biopsies of both proliferative and secretory endometrium obtained from five normal pre-menopausal women who were not taking any drugs or steroids. Two of the human cytosolic STs were detected in human endometrial tissues. The phenol-sulfating form of phenol ST (P-PST) was found at varying levels in cytosol from both proliferative and secretory endometrium in all of the women studied but with no consistent correlation to the phase of the menstrual cycle. In contrast, estrogen ST (EST) was not detected in the proliferative endometrial cytosol of any of the women studied but was consistently found in all of the secretory endometrial cytosols. The presence and levels of these STs was confirmed by ST activity studies, immunoblot analysis and Northern blot analysis. These results indicate that the expression of EST in human endometrial tissues varies with the phase of the menstrual cycle and is most likely regulated by progesterone secreted from the ovaries.     

Induction of thioredoxin, a redox-active protein, by ovarian steroid hormones during growth and differentiation of endometrial stromal cells in vitro

Human thioredoxin (hTrx) is a cellular redox-active protein that catalyzes dithiol/disulfide exchange reactions, thus controlling multiple biological functions, including cell growth-promoting activity. Here we show that the expression of hTrx protein and messenger RNA was up-regulated by incubation with 17beta-estradiol (E2) in primary culture of stromal cells isolated from human endometrium. Maximal enhancement of hTrx protein and messenger RNA was observed after 6-12 h of incubation with 10-100 nM E2, and the enhancing effect was suppressed by tamoxifen, an estrogen antagonist. Release of hTrx into the culture medium was markedly augmented after 5-day exposure of E2 plus progesterone (P) accompanied by in vitro differentiation of endometrial stromal cells (decidualization). Immunocytochemical studies showed that hTrx was localized in the nucleus, nucleolus, and cytosol in the stromal cells. Strongly enhanced immunoreactivity for hTrx was observed in the E2-treated cells, whereas there was no apparent difference in the pattern of subcellular localization among the untreated and E2- and/or P-treated cells. Although 1-50 microg/ml recombinant hTrx alone did not promote endometrial stromal cell growth, epidermal growth factor-dependent mitogenesis was additively enhanced by hTrx. Our results indicate that hTrx modulates endometrial cell growth, acting as a comitogenic factor for epidermal growth factor, which is known to be a mediator of estrogen action. It is also suggested that hTrx is deeply involved in the hormonal control of the endometrium by E2 and P, playing a regulatory role in endometrial cell growth and differentiation.     

17Beta-estradiol potentiates the stimulatory effects of progesterone on cadherin-11 expression in cultured human endometrial stromal cells

Cadherin-11 (cad-11) is a novel member of the cadherin gene superfamily of calcium-dependent cell adhesion molecules. To date, the factors capable of regulating this cell adhesion molecule remain poorly characterized. We have recently determined that cad-11 expression in the human endometrium is tightly regulated during the menstrual cycle. The spatiotemporal expression of cad-11 in the stromal cells of the human endometrium during the menstrual cycle suggests that gonadal steroids regulate the expression of this endometrial cell adhesion molecule. In view of these observations, we have examined the ability of progestins, estrogens, and androgens, alone or in combination, to regulate cad-11 expression in isolated human endometrial stromal cells using Northern and Western blot analyses. In these studies, we have determined that progesterone, but not 17beta-estradiol or dihydrotestosterone, is capable of regulating cad-11 messenger RNA and protein expression levels in isolated endometrial stromal cells. In addition, 17beta-estradiol, but not dihydrotestosterone, was capable of potentiating the stimulatory effects of progesterone in a dose-dependent manner. Taken together, these observations suggest that both 17beta-estradiol and progesterone are required for maximal cad-11 expression in human endometrial stromal cells in vitro.     

Immunocytochemical localization of progesterone receptor in the reproductive tract of adult female rats

The distribution of the progesterone receptor (PR) was investigated immunocytochemically in female reproductive tracts of rats during the estrous cycle and early pregnancy through use of an anti-PR monoclonal antibody. PR was localized predominantly in the nuclei of epithelial, stromal, and muscle cells in the uterus and vagina during the estrous cycle. In the uterus, the nuclei of epithelial cells were stained intensively at diestrus, while the PR staining of the stromal cells was more intense at proestrus than at any other stage of the cycle. PR expression during the cycle in muscle cells of the myometrium was similar to that in the endometrial stromal cells. In the vagina, however, PR expression during the cycle was approximately the same among epithelial, stromal, and muscle cells, the nuclei of which were stained deeply at proestrus. Ovariectomy at various stages of the cycle altered the PR expression appearing in the uterus and vagina during the cycle. In ovariectomized rats, estrogen increased the PR immunoreaction of various types of cells examined in the uterus and vagina except for the uterine epithelial cells. The reaction of these uterine epithelial cells was decreased by estrogen but was increased by progesterone given after estrogen; however, progesterone given alone reduced the reaction. In the epithelial and stromal cells of the uterus, intensity of the staining was increased after mating, reaching maximum on Day 3 of pregnancy, and then decreased on Day 4 (day of implantation), while in epithelial and stromal cells of the vagina the staining remained weak during early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical analysis of distribution of estrogen receptors and progesterone receptors in the postmenopausal endometrium

OBJECTIVE: To examine the expression of sex steroid receptors (ER: estrogen receptor; PR: progesterone receptor) in the postmenopausal endometrium (PMEM) and the relationship to clinical data for studying its characters. METHODS: The immunohistochemical reactivity of the PMEM was studied using monoclonal antibodies against ER and PR, in 33 postmenopausal patients. RESULTS: The endometrium was thicker in patients who were postmenopausal for 1 to 10 years (1.48 +/- 1.31 mm) than in patients who were postmenopausal for more than 10 years (0.79 +/- 0.37 mm)(p < 0.05). Among the 33 postmenopausal endometrial samples, ER positivity was found in the glands in 26 cases (78.8%) and PR positivity was detected in 18 cases (54.5%). The average age of the patients with ER positive reactivity in the glands (61.69 +/- 7.26 years) was significantly lower than that of the patients with ER negative reactivity (66.00 +/- 3.56 years)(p < 0.05). Furthermore, the endometrial thickness of the patients with ER or PR positive reactivity in the glands (1.24 +/- 1.09 mm and 1.47 +/- 1.20 mm, respectively) was significantly greater than that of the patients with ER or PR negative reactivity (0.67 +/- 0.26 mm and 0.70 +/- 0.40 mm, respectively)(p < 0.05). CONCLUSION: ER in the glands of the PMEM was determined to decrease gradually with increased aging. The presence of ER and PR in the gland cells seemed likely to determine the thickness of the PMEM.     

Accumulation of 5-aminolevulinic acid-induced protoporphyrin IX in normal and neoplastic human endometrial epithelial cells

The aim of this study was to evaluate 5-Aminolevulinic acid (ALA)-induced fluorescence of normal and neoplastic endometrial epithelial cells for diagnosis and photodynamic treatment. Fluorescence of ALA-induced PpIX in vitro was measured by flow cytometry in two different human endometrial adenocarcinoma cell lines and in normal cells cultivated from fresh endometrial tissue of three premenopausal patients. The cells were analysed after incubation with different concentrations of ALA during 3, 6, or 24 hours. Both tumor cell lines showed a statistically significant higher fluorescence of PpIX than normal epithelial cells after incubation with 1 mg ALA per ml medium during 24 hours. The well-differentiated cancer cells produced significantly more PpIX than the poorly differentiated cancer cells. Relative PpIX intensity of the two cancer cell lines correlated with cell proliferation rate as measured by the doubling times of the cells. Higher accumulation of Pp IX in neoplastic endometrium compared to normal endometrial epithelial cells may provide targeted biopsies and selective photodynamic destruction of neoplastic micro-lesions.     

Estrogenic and antiprogestagenic activities of pyrethroid insecticides

Many pesticides possess hormonal activity and have thus been classified as endocrine disruptors. Pyrethroids are commonly used insecticides worldwide, but little has been done to characterize their hormone agonist/antagonist potential. We tested four frequently encountered pyrethroids, fenvalerate, sumithrin, d-trans allethrin, and permethrin, for estrogen and progesterone agonist/antagonist activities using the Ishikawa Var-I human endometrial cancer cell line and the T47D human breast cancer cell line. Both cell lines produce alkaline phosphatase as an indicator of hormonal activity. Fenvalerate and sumithrin demonstrated significant estrogenicity; at concentrations of 10 microM, these compounds achieved maximal activities comparable to that of 10 nM 17alpha-ethynylestradiol in Ishikawa Var-I cells. None of the four compounds showed statistically significant estrogen antagonist activity or acted as progestins. However, fenvalerate and d-trans allethrin significantly antagonized the action of progesterone in T47D cells. Through these hormonal pathways, exposure to certain pyrethroids may contribute to reproductive dysfunction, developmental impairment, and cancer.     

Outbreak of viral gastroenteritis due to a contaminated well. International consequences

In order to test the hypothesis that integrin and uteroglobin (UG) expression in cultured endometrial cells are affected by hormone treatment, Ishikawa-CH endometrial cancer cells were cultured and exposed to oestradiol or oestradiol and progesterone regimens and assayed using immunohistochemistry. We evaluated the intensity of immunohistochemical staining for the integrin monomers alpha(v) and beta1, the dimers alpha(v)beta3 and alpha(v)beta6, and for the secretory protein uteroglobin under various experimental conditions. Cells grown in control media stained positively for the integrin monomers alpha(v) and beta1, the dimer alpha(v)beta3, and for UG. Oestradiol and sequential oestradiol/progesterone reversibly suppressed staining for the dimer alpha(v)beta3. Hormone treatment had no effect on the staining of the beta1 and alpha(v) monomers or UG. The alpha(v)beta6 dimer antibody did not stain under any experimental treatment conditions. These data indicate that expression of the integrin complex alpha(v)beta3 is reversibly suppressed by oestradiol in Ishikawa cells and that these cells may be a good model for studying hormone-driven molecular changes in endometrium.     

Double level fractures of the femur treated with closed intramedullary nailing

Breast cancer patients receiving tamoxifen (Tam) are at an increased risk for developing endometrial carcinomas, possibly due to the partial estrogenic effect of Tam on endometrial cells. Progestational therapy has not routinely been included in Tam regimens. It was our aim to determine the presence of estrogen receptors (ERs) and progesterone receptors (PRs) in normal and abnormal endometria from postmenopausal women with breast cancer who were treated with Tam. Standard immunohistochemical staining of ERs and PRs was performed on paraffin sections from formalin-fixed uterine curettings or hysterectomy specimens from 40 patients who had received 20-40 mg of Tam daily for a minimum of 3 months. For comparison, normal endometria from 20 women who had not received Tam (11 premenopausal, 9 postmenopausal) were also studied for ER and PR expression. Staining was evaluated using semiquantitative immunoreactivity scores (IRS) ranging from 0 (negative) to 12 (strongly positive). In the group of patients receiving Tam, ERs and PRs were detected in the nuclei of glandular cells in 24/24 cases of endometrial atrophy (ER/PR-IRS, 2-12), in 8/8 endometrial polyps (ER-IRS, 6-12; PR-IRS, 4-12), in 4/4 adenomatous endometrial hyperplasias (ER-IRS, 3-8; PR-IRS, 1-12), and in 4/4 well-differentiated endometrioid adenocarcinomas (ER-IRS, 2-12; PR-IRS, 6-8). Of the 11 endometria from premenopausal patients who had not received Tam, 8 were ER+/PR+ (ER-IRS, 1-12; PR-IRS, 1-12), 1 was ER+/PR- (ER-IRS, 3; PR-IRS, 0), 1 was ER-/PR+ (ER-IRS, 0; PR-IRS, 2), and 1 was ER-/PR- (ER/PR-IRS, 0). Among 9 atrophic endometria from women not treated with Tam, 6 were ER+/PR+ (ER-IRS, 4-12; PR-IRS, 3-6), 1 was ER+/PR- (ER-IRS, 4; PR-IRS, 0), and 2 were ER-/PR- (ER/PR-IRS, 0). The consistent finding of ER and PR expression in endometria from postmenopausal women receiving Tam further supports the suspected estrogenic effect exerted by Tam on endometrial cells. Progestational therapy could be beneficial in the prevention of Tam-induced abnormal endometrial proliferations.     

Immunohistochemical localization of estradiol and progesterone receptors in human uterus throughout pregnancy: expression in endometrial blood vessels

Although progesterone and estrogens are essential to maintain human pregnancy after implantation, the localization of their specific receptors in different uterine cell types during pregnancy has not been investigated. We studied uteri (n = 40) obtained during the first 3 months of pregnancy (n = 21) and in late pregnancy (n = 9) as well as from women 5-14 weeks pregnant (n = 10) who had received the antiprogestagen RU 38486 (Roussel-UCLAF) to induce cervical dilation. Frozen tissues were processed for indirect immunocytochemical staining with specific monoclonal antibodies against estrogen receptors (ER; Abbott Laboratories) and progesterone receptors (PR; Li 417). Specific staining for steroid receptors was only detected in the nucleus. In the endometrium, PR staining remained fairly constant throughout pregnancy, whereas ER staining was initially weak and then undetectable. PR was widely expressed in stromal cells and in spiral arterial wall cells, whereas ER was expressed in scattered stromal cells and arterial cells. Both PR and ER were absent from glandular epithelium, contrasting with the secretory activity during the first trimester. Spiral arteries of the endometrium and myometrial smooth muscle cells showed intense PR and moderate ER staining in early pregnancy. The progesterone antagonist RU 38486 (mifepristone), given in early pregnancy at a dose of 200 mg, caused a marked increase in ER staining and a smaller increase in PR staining in stromal cells, whereas the glandular epithelium remained negative for both ER and PR (except for one and two specimens, respectively). We conclude the following. 1) Stromal cells retain PR despite the high progesterone levels during pregnancy, in keeping with the role of progesterone in stromal decidualization. The absence of PR from the secretory glandular epithelium suggests a paracrine link between decidualized stromal cells and epithelial cells. 2) Significant PR down-regulation by progesterone during pregnancy occurs only in epithelial cells of the endometrium. 3) In contrast, the absence or low level of ER staining in the various cell types of the endometrium during gestation concurs with the known effect (down-regulation) of steroid hormones on ER mRNA or protein levels. The increase in ER in human decidua after RU 38486 treatment indicates that the main cause of the low ER levels is progesterone secretion. 4) The intense PR staining in smooth muscle cells of spiral arteries during early pregnancy suggests that progesterone is essential for modulating blood flow during pregnancy.     

Differential expression of uterine progesterone receptor forms A and B during the menstrual cycle

Recent studies suggest that the progesterone receptor isoforms (PR-A and PR-B) activate genes differentially and that PR-A may act as a repressor of PR-B function. Hence, the absolute and relative expression of the two isoforms will determine the response to progesterone. We have measured their relative expression in the uterus of cycling women who underwent endometrial biopsy. PR isoforms were identified on blots of SDS-PAGE gels by reaction with the AB-52 antibody after immunoprecipitation from endometrial extract. Both isoforms were highest in the peri-ovulatory phase, but levels of PR-A were always higher than those of PR-B. The ratio of PR-A to PR-B changed during the menstrual cycle. Between days 2 and 8, PR-B is almost undetectable and the A:B ratio is >10:1. From days 9 to 13, the ratio is about 5:1, and it is about 2:1 between days 14 and 16. Thereafter, PR-B dwindles rapidly and is virtually undetectable at the end of the cycle. In various hypoestrogenic environments, PR-B expression was reduced. However, exogenous estrogens in the follicular phase in the form of oral contraceptives, enhanced PR-B expression. These data support the possibility that progesterone acts through cycle-specific PR isoforms.     

Establishment and characterization of six new human endometrial adenocarcinoma cell lines

The endometrial carcinoma cell lines EC-MZ-1, 2, 3, 5, 9, and 11 were established between 1986 and 1990. Four cell cultures were started from endometrial tissue, one from ascites, and one from a lymph node metastasis. Lines have to date been subcultured up to 180 times and the doubling time varies between 26 hr and 3 weeks. Immunocytochemically the coexpression of cytokeratin (predominantly simple-epithelial cytokeratin polypeptides) and vimentin intermediate filaments was detectable in all cell lines, but three lines (EC-MZ-5, 9, 11) expressed vimentin only at low level. By transmission electron microscopy the tumor cells exhibited features of epithelial differentiation. After subcutaneous transplantation into nude mice three lines (EC-MZ-1, 2, 5) produced slow-growing tumors. The histological classification of these tumors ranged from moderately differentiated adenocarcinoma to undifferentiated carcinoma and closely corresponded to the original tumor. Even after long-term in vitro culture, without any addition of estrogens to the culture medium, the moderately differentiated receptor-positive cell line (EC-MZ-2) retained its morphological differentiation. The cells were propagated without estrogens in the culture medium. The estrogen and progesterone receptor levels of cultured cells were determined. Three lines (EC-MZ-1, 2, 3) were positive for the progesterone receptor in low passage number only, the other cell lines were negative for both receptors. The transplantable lines were investigated for hormonal receptor expression in ovariectomized nude mice. In the moderately differentiated cell line (EC-MZ-2) we observed an enhanced expression of the estrogen receptor under optimal stimulation of the nude mouse with estradiol benzoate. There was no effect on the expression of the progesterone receptor.     

Impedance of a goat eye lens

Cyclins are essential proteins in cell cycle control, and their deranged expression has been reported to be associated with malignant transformation. Involvement of cyclins in the development of endometrioid carcinomas of the endometrium was studied immunohistochemically using antibodies against both cyclin A and tumor suppressor gene product p53, and their expression was compared with that of Ki-67 antigen. Sixty-two cases of endometrial endometrioid carcinoma and 20 cases of normal endometrium (10 proliferative and 10 secretory phase) were examined. Of the 62 endometrioid carcinomas, atrophic endometrium and hyperplasia were found adjacent to the cancers in 30 and 19 cases respectively. Cyclin A was expressed in < 1% of the glandular cells of normal endometrium in the proliferative phase and in hyperplasia, but was negligible in normal secretory phase and atrophic endometrium. p53 was almost always negative in normal endometrium and hyperplasia. Of the 62 endometrioid carcinomas, 12 tumors (19.4%) overexpressed cyclin A and 21 tumors (33.8%) overexpressed p53 (positive cells > 1%). Cyclin A and p53 were more frequently expressed in poorly differentiated tumors than in well differentiated tumors (cyclin A, p = 0.002; p53, p = 0.016). In addition, cyclin A-positive cells were topographically related to those cells positive for p53 as well as Ki-67. In conclusion, the abnormal expression of cyclin A and p53 is associated with high-grade endometrial endometrioid carcinomas.