Normal Uterine Tissue Research Articles

Phytoestrogens and carcinogenesis—differential effects of genistein in experimental models of normal and malignant rat endometrium

The phytoestrogen genistein was studied in normal and malignant experimental uterine models in vivo. The action of genistein on the uterus and vagina of ovariectomized DA/Han rats after 3 day oral administration (25, 50 or 100 mg/kg/BW/d) was compared to ethinyl oestradiol (0.1 mg/kg/BW/d). Effects on uterine and vaginal morphology, uterine growth and uterine gene expression were studied. A dose dependent increase of the uterine wet weight and the uterine and vaginal epithelial height, a dose dependent up-regulation of complement C3, down-regulation of clusterin mRNA expression and a stimulation of the vaginal cornification was observed after administration of genistein. Uterine gene expression and vaginal epithelium respond to genistein at doses where no significant effects on uterine wet weight were detectable. In general the vagina was more sensitive to genistein than the uterus. To analyse the action of genistein in malignant uterine tissue, the impact of a 28 d treatment with 50 mg/kg/d of genistein on the in-vivo tumour growth of RUCA I endometrial adenocarcinoma cells, following subcutaneous inoculation into syngeneic DA/Han rats, was assessed. In contrast to ethinyl oestradiol (0.1 mg/kg/BW/d), a dose of 50 mg/kg/BW/d of genistein did not affect tumour growth. Nevertheless C3 and TRPM2 mRNA expression in the tumour were both significantly stimulated by ethinyl oestradiol and genistein. In comparison to ovariectomized animals genistein up-regulated uterine wet weight and uterine dependent gene expression in tumour bearing animals. In conclusion, four independent uterine and vaginal parameters indicate genistein is a weak oestrogen receptor agonist in the uterus and vagina of female DA/Han rats, and evidence is provided for a selective oestrogen receptor modulator (SERM)-like action of genistein in normal and malignant uterine tissue.

Altered hormonal responsiveness of proliferation and apoptosis during myometrial maturation and the development of uterine leiomyomas in the rat.

Uterine leiomyomas are responsive to the ovarian steroids, estrogen and progesterone; however, a mechanistic understanding of the role of these hormones in the development of this common gynecologic lesion remains to be elucidated. We have used the Eker rat uterine leiomyoma model to investigate how ovarian hormones regulate or promote the growth of these tumors. Proliferative and apoptotic rates were quantitated in normal uterine tissues and leiomyomas in response to endogenous ovarian steroids. In 2- to 4-mo-old animals, cell proliferation in the normal uterus corresponded with high serum levels of steroid hormones during the estrous cycle, and apoptosis occurred in the rat uterus in all cell types following sharp, cyclical declines in serum hormone levels. It is interesting that the responsiveness of uterine mesenchymal cells changed between 4 and 6 mo of age, with significant decreases in both proliferative and apoptotic rates observed in myometrial and stromal cells of cycling animals. Leiomyomas displayed much higher levels of proliferation than did age-matched myometrium; however, their apoptotic index was significantly decreased in comparison with normal myometrium. This disregulation between proliferative and apoptotic responses, which were tightly regulated during ovarian cycling in the normal myometrium, may contribute to the disruption of tissue homeostasis and underlie neoplastic growth of these tumors.

Expression of tumor-rejection antigens in gynecologic cancers.

We recently reported the four tumor‐rejection antigens (SART1259 SART2, SART3, and ART4) that possess tumor epitopes capable of inducing HLA‐A2402‐restricted cytotoxic T lymphocytes (CTLs) in cancer patients. This study investigated the expression of these tumor antigens in gynecologic cancers, including 33 ovarian cancers, 38 cervical cancers, and 40 endometrial cancers. SART1259 antigen was detected in 56%, 35%, and 30% of ovarian, cervical and endometrial cancers, while SART2 antigen was detected in 46%, 66%, and 30% of these cancers, respectively. Both SART3 and ART4 antigens were detectable in the majority of these gynecologic cancers tested. In contrast, none of these antigens was detectable in any of the normal ovarian and uterine tissues tested. Peripheral blood mononuclear cells (PBMCs) of HLA‐A24+ patients with gynecologic cancers were found to produce significant levels of interferon‐γ in response to HLA‐A24+ SART3+ gynecologic cancer cells after having been stimulated three times in vitro with either SART3109–118 or SART3315–323 peptide. These PBMCs lysed HLA‐A24+ SART3+ gynecologic cancer cells, but not HLA‐A24‐ SART3+ gynecologic cancer cells or HLA‐A24+ normal cells. Therefore, these four antigens and their peptides, including SART3 peptides, would be appropriate molecules for use in specific immunotherapy of HLA‐A24+ gynecologic cancer patients.

The oestrogen receptor (ER) in normal and abnormal uterine tissue

Glandular epithelium and stroma of the endometrium show typical behavioural patterns in the expression of oestrogen receptors (ERs) due to both endogenous and exogenous hormonal influence. Thus, the ER is increasingly expelled under the influence of oestrogen during the first half of the cycle. Under the influence of progesterone, the ER disappears during the luteal phase and is not even detectable after day 21. During menopause, the atrophic endometrium typically shows very little, if any, ER expression. In cases of oestrogen-induced hyperplasias, the receptor can again be demonstrated. The ER only disappears when nuclear irregularities occur in cases of adenomatous hyperplasia. In cases of invasive carcinoma, a heterogenous picture is seen which closely correlates with the degree of differentiation.

The expansion of the CAG repeat in exon 1 of the human androgen receptor gene is associated with uterine endometrial carcinoma

Uterine endometrial carcinoma is common among women. Several reports indicated that this cancer is influenced by androgens mediated through their receptors. The human androgen receptor gene contains a polymorphic CAG repeat in the coding region of exon 1. Other studies suggested a possible association between the CAG repeat of this gene and the development of several cancers. DNA samples isolated from 29 patients with sporadic endometrial cancer were analyzed for allelic changes in 12 highly polymorphic microsatellite loci on nine chromosomes, containing CAG repeat in the exon 1 of the androgen receptor gene. A significantly high rate of allelic change in the CAG repeat was observed (51.7%) in these patients, although the frequencies of additional loci were similar to those reported by others (0-22.2%). The changed alleles of the tumor tissue were always longer than that of normal tissue except in only one case. Since only one allele on the X chromosome is commonly active in female cells, a differential methylation assay was carried out by using genomic DNA cut with HpaII. In 14 of 15 cases, we found that the activated allele was longer in these samples from tumor tissues than those from normal tissue; in the remaining case, the length of this repeat was unchanged. The expression assays were done by using poly(A+) RNA from tumor and normal uterine tissues, revealing that an activated allele in these tumor tissues was longer than that in the normal tissues in all the cases examined. These findings suggest that expansions of the CAG repeat in the androgen receptor gene may play an important role in the carcinogenesis of uterine endometrial cells.

Estrogenic regulation of clusterin mRNA in normal and malignant endometrial tissue.

Clusterin is a heterodimeric, 80kDa, glycoprotein that is synthesized in a wide variety of tissues in response to a number of diverse stimuli, including hormone ablation. We have investigated the regulation of clusterin expression by estradiol and anti-estrogens in RUCA-I rat endometrial adenocarcinoma cells in vitro and in vivo. We have also compared clusterin expression in endometrial tumors and in normal uterine tissue. Estradiol treatment significantly increases the steady state mRNA levels of clusterin in RUCA-I cells cultured on a reconstituted basement membrane, with a maximal induction 24 hr after estradiol treatment. The inductive effects of estrogen on clusterin mRNA steady state levels in vitro are significantly more pronounced than the effects on fibronectin mRNA levels, an estrogen-repressed gene in RUCA-I. In vivo, induction of clusterin expression in primary and metastatic endometrial adenocarcinoma is also dependent on the presence of estradiol, in marked contrast to expression of clusterin in the normal endometrium of the same animals. These data suggest that clusterin mRNA expression in rat endometrial adenocarcinoma cells is tightly regulated by estrogens and anti-estrogens in vitro and in vivo, and that there is a complex mechanism of regulation of clusterin expression in the normal and cancerous endometrium.

Expression and secretion of inhibin and activin in normal and neoplastic uterine tissues. High levels of serum activin A in women with endometrial and cervical carcinoma.

Inhibins and activins are growth factors belonging to the transforming growth factor-beta) family and are known to influence cell proliferation and differentiation. Because transforming growth factor-beta is involved in physiological and tumoral changes of uterine tissues, the present study aimed to evaluate whether human normal and neoplastic endometrial and cervical epithelial cells express and secrete inhibin A, inhibin B, and activin A. To test this hypothesis, different approaches were used. By RT-PCR, the expression of specific messenger RNAs (mRNAs) for the inhibin alpha, activin betaA and betaB subunits, and activin receptor type II and type IIB was investigated: 1) in primary cultures of endometrial (stroma and epithelium) or cervical (epithelium) cells from healthy women; and 2) in specimens of endometrial or cervical carcinoma. To demonstrate a possible secretion of the proteins, dimeric inhibin A, inhibin B, and activin A were measured in culture medium of normal epithelial or stromal endometrial cells and in uterine washing fluid of healthy women or patients with endometrial adenocarcinoma. Levels of the proteins were also measured in serum of women with endometrial or cervical carcinoma. Cultured endometrial stromal or epithelial cells and epithelial cervical cells expressed inhibin alpha, activin betaA and betaB, and activin receptor type II and type IIB mRNAs. The same finding was obtained in specimens of endometrial or cervical carcinomas. Dimeric inhibin A, inhibin B, and activin A were measured in culture medium of both endometrial and cervical cells. In particular, resulting activin A levels were significantly higher in epithelial than in stromal cultured endometrial cells (P < 0.01). Dimeric proteins were also detected in the washing fluid of the uterine cavities of healthy women (controls) and with endometrial adenocarcinoma, in which higher activin A levels were found (P < 0.01 vs. controls). Women with endometrial carcinoma showed serum activin A levels significantly higher than healthy controls (P < 0.01), which significantly decreased after surgical removal of endometrial or cervical tumors (P < 0.01). The present study, for the first time, showed that inhibin A, inhibin B, and activin A, as well as activin receptors, are expressed in normal and neoplastic human uterine tissues. A secretion of activin A from tumoral cells into systemic circulation is suggested by the observation that the high levels in serum of patients with endometrial or cervical carcinoma decreased after the surgical removal of the tumor.

Expression and Secretion of Inhibin and Activin in Normal and Neoplastic Uterine Tissues. High Levels of Serum Activin A in Women with Endometrial and Cervical Carcinoma

Expression and Secretion of Inhibin and Activin in Normal and Neoplastic Uterine Tissues. High Levels of Serum Activin A in Women with Endometrial and Cervical Carcinoma

Disturbed Cell Arrangement, Increased Cell Membrane Permeability and Apoptotic Cell Death Occur in Adenomyotic Uterine Tissues in Mice

Abstract The relationship of the development of uterine adenomyosis with the changes in the cell arrangement and cell membrane permeability, and incidence of DNA fragmentation was examined in mice. In uterine areas showing the invasion of endometrial tissues into musculature, rhodamine-phalloidin staining for actin fibers revealed that the stromal cells ran parallel to the direction of the infiltration, and the muscle cells lost their regular arrangement, unlike those in the normal uteri of control mice. Inner myometrium showed positive fluorescence with Evans blue, which is known to stain only cells with increased membrane permeability. Outer myometrium also became fluorescence-positive, when the disease was advanced to severe state bearing invasion of endometrial tissues into the outer myometrium. No fluorescence with Evans blue was observed in the normal uterine tissues. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL method)-positive nuclei were exclusively found in ...

Immunohistochemical localization of p21 WAF1/CIP1 in normal, hyperplastic, and neoplastic uterine tissues

p21 WAF1/CIP1 is a nuclear protein that binds to cyclin-dependent kinase complexes (CDKs) and inhibits the activity of multiple kinases. These CDKs are involved in the regulation of cell cycle progression at several checkpoints. In this study, the authors have analyzed by immunohistochemistry the expression of p21 WAF1/CIP1 in normal uterine tissues, 12 endometrial hyperplasias, 17 endocervical adenocarcinomas, and 31 endometrial adenocarcinomas. In addition, a group of 10 leiomyomas and 10 uterine leiomyosarcomas were also stained. To evaluate cell proliferation, the monoclonal antibody Ki-67 was used in all of the available cases. Terminally differentiated epithelial endocervical and endometrial cells showed variable expression of p21 WAF1/CIP1, whereas the endometrial hyperplasias, and endocervical and endometrial adenocarcinomas showed decreased expression or were negative. All of the cases of cervical squamous dysplasia were positive. Normal smooth muscle cells and 50% of leiomyomas were negative, whereas all leiomyosarcomas showed expression of p21 WAF1/CIP1. These results indicate that p21 WAF1/CIP1 contributes to differentiation in normal endometrial and endocervical glands. The decreased expression of p21 WAF1/CIP1 in endometrial hyperplasias and carcinomas maybe important in the process of neoplastic transformation. The role of certain CDK inhibitors, such as p21 WAF1/CIP1, is different in epithelial and mesenchymal tumorigenesis in the uterus.

Enzymatic Basis for the Accumulation of Lewisb Antigen in Uterine Endometrial Cancer

In order to clarify the mechanism of the abnormal expression of Lewisb antigen, which was specific for uterine endometrial cancer tissue, the activities of α1→2fucosyltransferase, α1→3fucosyltransferase, and α1→4fucosyltransferase in normal endometrial tissues and uterine endometrial cancer tissues were determined. Further, an immunocytochemical study of the expression of blood group‐related carbohydrate antigens in 6 cultured cell lines derived from various gynecologic malignant tumors was performed and the α1→2fucosyltransferase, α1→3fucosyltransferase, and α1→4fucosyltransferase activities of these cell lines were determined. Compared with normal endometrium, uterine endometrial cancer tissues showed significantly higher values of α1→2fucosyltransferase, α1→3fucosyltransferase, and α1→4fucosyltransferase activities. The specifically strong expression of type I carbohydrate chains, particularly the Lewisb antigen, was recognized in cultured cell lines derived from uterine endometrial cancer. Compared with those cell lines derived from uterine cervical cancer and ovarian cancer, the cultured cell lines derived from uterine endometrial cancer showed higher activities of α1→2fucosyltransferase and α1→4fucosyltransferase, which are enzymes related to the synthesis of Lewisb antigen. The cell lines derived from uterine endometrial cancer showed specifically high values of α1→4fucosyltransferase activity. These results suggest that the α1→2fucosyltransferase and α1→4fucosyltransferase activities, especially the α1→4fucosyltransferase activity, contribute to the abnormal expression of the Lewisb antigen in uterine endometrial cancer.

Determination of the optical properties of the human uterus using frequency-domain photon migration and steady-state techniques

The optical properties (absorption and transport scattering coefficients) of freshly excised, bulk human uterine tissues were measured at 630 nm using frequency-domain and steady-state photon migration techniques. Measurements were made on both normal (pre- and post-menopausal) and non-neoplastic fibrotic tissues. The absorption coefficient of normal postmenopausal tissue ( approximately 0.06 mm-1) was found to be significantly greater than that of normal pre-menopausal tissue (0.02-0.03 mm-1) and pre-menopausal fibrotic tissue (0.008 mm-1). The transport scattering coefficient was similar in all three tissue types considered (0.6-0.9 mm-1). From the preliminary results presented here, the authors conclude that optical properties can be reliably calculated either from the frequency-dependent behaviour of diffusely propagating photon density waves or by combining the frequency-independent photon density wave phase velocity with steady-state light penetration depth measurements. Instrument bandwidth and tissue absorption relaxation time ultimately determine the useful frequency range necessary for frequency-domain photon migration (FDPM) measurements. Based on the optical properties measured in this study, the authors estimate that noninvasive FDPM measurements of normal uterine tissue require modulation frequencies in excess of 350 MHz.

Characterization of estrogen receptor cDNAs from human uterus: identification of a novel PvuII polymorphism

Using reverse transcription and the polymerase chain reaction, we have cloned estrogen receptor complementary DNAs from normal human uterine tissue. Restriction endonuclease analysis identified a polymorphic PvuII recognition site within half of the receptor cDNAs. Sequence analysis revealed a number of differences with the sequence previously reported for the ER cDNA isolated from MCF7 cells and confirmed that the codon for amino acid 400 was erroneously assigned as valine (GTG) rather than glycine (GGG). Sequencing also defined the nature of the PvuII polymorphism, with allele A coding for Glu 22 and allele B (with an additional PvuII site) coding for Gln 22. We demonstrate that both alleles of this receptor activate transcription of an estrogen-responsive gene to the same extent. This selective cloning method should have wide application in the investigation of naturally occurring cDNA variants from diseased tissues, such as breast cancer cell lines and primary tumor specimens.

Immunohistochemical Localization of Prolactin in Normal and Neoplastic Uterine Cervical Tissues

Immunohistochemical Localization of Prolactin in Normal and Neoplastic Uterine Cervical Tissues

Telomere reduction in endometrial adenocarcinoma

OBJECTIVE: Some of the genomic instability that is observed in solid tumors may be due to the loss of telomeric sequences. These experiments were designed to compare the number of telomeric repeat sequences in endometrial adenocarcinoma with that found in adjacent normal tissue.STUDY DESIGN: Deoxyribonucleic acid was extracted from normal and malignant uterine tissue of 11 patients undergoing hysterectomy for treatment of endometrial adenocarcinoma and also from five endometrial carcinoma cell lines. The relative number of telomeric repeat sequences in each sample was measured by hybridization of these deoxyribonucleic acids to a probe specific for the human telomeric repeat. Hybridization signals were quantified by autoradiography and a β-particle detection system.RESULTS: A reduction of telomeric repeat sequences in tumor versus normal tissue was found in 10 of 11 cases. Telomere reduction was also seen in endometrial carcinoma cell lines. CONCLUSIONS: Telomere reduction is a genetic characteristic of many endometrial tumors. Telomere reduction may contribute to the genesiS and progression of endometrial carcinoma, or it may be a secondary effect of the tumorigenesis process. (AM J OBSTET GYNECOL 1992;167:1883-7.)

Patterns of keratin subsets in normal and abnormal uterine cervical tissues. An immunohistochemical study.

We investigated the use of three monoclonal antikeratin antibodies on routinely formalin-fixed and paraffin-embedded punch and cone biopsies of the normal human uterine cervix and its metaplastic and premalignant lesions. Monoclonal antibodies used were AE8, which is specific for keratin 13; 34BE12, which reacts with keratins of the stratified squamous epithelium; and CAM5.2, which is specific for keratin 8. All these antibodies performed well in routinely processed surgical pathology material. AE8 antibody stained the suprabasal layer of the normal squamous epithelium. Squamous metaplasia and dysplasia were stained in 50% of the cases. Normal suprabasal distribution of the keratin 13, however, was lost in all positive dysplasia cases. CAM5.2 reacted with normal columnar cells in all cases, and squamous metaplasia was focally positive in 20% of the cases. Dysplasia showed a positive reaction in 30% to 40% of the cases. The 34BE12 antibody was reacting with the full thickness of the squamous epithelium. Squamous metaplasia and dysplasia were positive in 80% of the cases. In addition, 34BE12 stained reserve cell hyperplasia, making it a useful marker for this condition. Our results demonstrate that keratin immunohistochemistry with the above-listed antibodies gives pathogenetically interesting information on cervical lesions.

Calponin Developmental isoforms and a low molecular weight variant

Two-dimensional gel analysis of basic proteins in developing human smooth muscle identifies calponin as a prominent marker of the differentiated phenotype. Adult tissue (human and mouse) typically expresses up to four calponin isoforms, three of which appear sequentially during fetal development: adult myometrial cells express the same three isoforms in primary culture in vitro and these are down-regulated, in reverse order, during the subsequent modulation of phenotype. Monospecific, polyclonal antibodies against calponin identify a lower molecular weight variant of calponin (L-calponin) that is strongly and specifically expressed in adult smooth muscles of the human urogenital tract. L-Calponin is down-regulated in benign smooth muscle derived tumors (leiomyoma) and is not expressed in primary cultures of normal uterine tissue.

Growth Factors in Normal and Malignant Uterine Tissue

Growth Factors in Normal and Malignant Uterine Tissue

Expression of the macrophage colony-stimulating factor and its receptor in gynecologic malignancies

Recently, hematopoietic growth factors have been implicated in protean nonhematopoietic processes. In the current study, expression of macrophage colony-stimulating factor (M-CSF) and its receptor (the c-fms proto-oncogene) was investigated in 42 samples of gynecologic tissues. There were 15 samples of normal ovarian and uterine tissue or benign conditions of these organs; 11 samples of primary ovarian cancer tissue; seven samples of metastatic ovarian cancer tissue; and nine samples of primary endometrial cancer tissue. Steady state transcript levels were assessed by Northern Blot analysis. Macrophage colony-stimulating factor (M-CSF) expression was not observed in any of the specimens of benign abnormalities or of normal organs; c-fms expression was detected in two of 15 (13%) of these specimens, albeit at very low levels. In contrast, 14 (78%) of 18 ovarian tumor specimens, and five (55%) of nine endometrial tumor specimens expressed M-CSF. Similarly, 16 (89%) of 18 ovarian tumor specimens and six (67%) of nine endometrial tumor specimens expressed c-fms. Most positive malignant tissues (19 [86%] of 22) showed coexpression of M-CSF and c-fms. Of interest, M-CSF and c-fms mRNA were detected in tumor, but not in adjacent normal tissue. Furthermore, M-CSF and c-fms transcripts were produced by all metastatic tumors, including two cases in which the corresponding primary tumor from the same patient was negative. Because M-CSF mediates its effects by binding to its receptor, the increased levels of both these gene products in gynecologic malignancies suggest that an interaction between M-CSF and c-fms may participate in the development of ovarian and endometrial carcinomas and especially in progression to the metastatic state.

Estriol binding in uterine corpus cancer and in normal uterine tissues

1. The specific bindings of estriol (E 3) and estradiol-17β (E 2) to their specific receptors were investigated in endometrial carcinoma from 7 patients and normal tissues from their respective organs or from other patients. 2. In both cytosolic and KCl-extracted fractions from them, specific binding sites for E 3 and E 2 were detected, demonstrating the presence of their separate receptors in human uterus-associated tissues. 3. In certain cases (6 cases) of well-differentiated adenocarcinoma, the ratio of concentration of E 3 receptor to that of E 2 receptor was almost equal to or higher than in other normal tissues. 4. These findings of unique localization of E 3 receptor distribution may offer new insight into identification of endometrial carcinoma more likely to respond to hormonal influence or therapy.