Screening for cancer of the cervix with simultaneous pap smear and colposcopy. The efficacy of pap smear and colposcopy.

OBJECTIVE: Some Japanese institutes have been performing a population screening program for cervix cancer involving the simultaneous use of Pap smear and colposcopy. This program may be a good model for evaluating the efficacy of Pap smears and colposcopy. METHODS & MATERIALS: The subjects included 2,000 women who underwent primary screening at the Kanagawa Health Service Association. RESULTS: 1) The incidence of ACF (atypical colposcopic findings) was 3.6%, whereas that of abnormal Pap smears (ASC-US and above) was 1.1%; 2) Of 88 women who showed abnormal findings on Pap smear and/or colposcopy, only three cases appeared abnormal in both methods, i.e., the two methods were complementary; 3) Colposcopy was more useful for detecting mild dysplasia than the Pap smear. However, colposcopy may possibly detect benign reparatory lesions; 4) The incidence of unsatisfactory colposcopic findings (UCF) was high (24.2%), whereas no unsatisfactory cases were found by Pap smear. CONCLUSIONS: The sensitivity of the Pap smear for detecting mild dysplasia is low, whereas that of colposcopy is high. However, colposcopy may not be suitable for primary screening due to its high UCF. The low sensitivity of Pap smears may be improved by repetition or adding ancillary HPV testing.

The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas.

BACKGROUND: The phosphatidylinositol 3'-kinase (PI3K)-AKT pathway is activated in many human cancers and plays a key role in cell proliferation and survival. A mutation (E17K) in the pleckstrin homology domain of the AKT1 results in constitutive AKT1 activation by means of localisation to the plasma membrane. The AKT1 (E17K) mutation has been reported in some tumour types (breast, colorectal, ovarian and lung cancers), and it is of interest which tumour types other than those possess the E17K mutation. METHODS: We analysed the presence of the AKT1 (E17K) mutation in 89 endometrial cancer tissue specimens and in 12 endometrial cancer cell lines by PCR and direct sequencing. RESULTS: We detected two AKT1 (E17K) mutations in the tissue samples (2 out of 89) and no mutations in the cell lines. These two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras. INTERPRETATION: Our results and earlier reports suggest that AKT1 mutations might be mutually exclusive with other PI3K-AKT-activating alterations, although PIK3CA mutations frequently coexist with other alterations (such as HER2, K-Ras and PTEN) in several types of tumours.

Optimal dose for stage IIIB adenocarcinoma of the uterine cervix on the basis of biological effective dose.

PURPOSE: Prognosis of uterine cervical adenocarcinoma in locally advanced stage treated with radiation therapy has been considered to be much worse than that of squamous cell carcinoma because the optimal dose for the former one has not been determined. Thus, the current study was performed to investigate the optimal dose for Stage IIIB, locally advanced stage, adenocarcinoma of the uterine cervix on the basis of the biological effective dose (BED). METHODS: One-hundred and seventy-nine patients with Stage IIIB carcinoma of the uterine cervix were treated with curative intended therapy at Kitasato University Hospital between 1976 and 2000. Out of them, 13 patients had an adenocarcinoma component in pathological findings. Nine patients were diagnosed with adenocarcinoma and four patients were diagnosed with adenosquamous cell carcinoma. All patients were treated with external radiation therapy combined with intracavitary radiation therapy. The total BED10 (T-BED10) was caluculated from the BED of the external beam radiation therapy (E-BED10) plus the BED of the intra-cavitary radiation therapy (A-BED). RESULTS: Overall survival rate was 51%. Stratified by T-BED10 overall survival rate of the T-BED10 > or = 100 Gy group was 57% and that of the T-BED10 < 100 Gy group was 30%. There was a trend toward a better survival rate of the T-BED10 > or = 100 Gy group than the T-BED10 < 100 Gy group. CONCLUSION: The current study suggested that the optimal dose for Stage IIIB adenocarcinoma of the uterine cervix might be T-BED10 > or = 100 Gy.

C-kit overexpression in neuroendocrine small cell carcinoma of the uterine cervix.

PURPOSE OF INVESTIGATION: Neuroendocrine small cell carcinoma of the uterine cervix (NESCC) grows aggressively, and is resistant to anticancer agents and radiation, having an extremely poor prognosis. The incidence of c-kit proto-oncogene overexpression is high in gastrointestinal stromal tumors (GISTs) and small cell lung cancer, and tyrosine kinase inhibitors have been used effectively to treat GISTs. Few studies have investigated whether c-kit is overexpressed in NESCC. To investigate whether NESCC can be a target for molecular targeted therapy with tyrosine kinase inhibitors, we examined the expression of c-kit in this tumor. METHODS: Twenty-one NESCCs were examined for c-kit expression by immunohistochemical staining using the labeled streptavidin-biotin complex (LSAB) method. The expression of c-kit was regarded as positive (overexpression) and negative when the membrane and cytoplasm of more or less than 25%, respectively, of tumor cells were stained. RESULTS: Nine NESCCs (43%) were c-kit-positive (overexpression). No difference in age or clinical stage was noted. No difference in prognosis was observed between the c-kit-positive and -negative patients. CONCLUSION: The incidence of c-kit overexpression was high in NESCC; therefore, the patients with this tumor may become a future target for molecular-targeted therapy with tyrosine kinase inhibitors.

Medroxyprogesterone acetate stimulates cdk inhibitors, p21 and p27, in endometrial carcinoma cells transfected with progesterone receptor-B cDNA.

PURPOSE OF INVESTIGATION: Progestin is reported to suppress the growth of endometrial carcinomas, although its precise mechanism of action is not clear. This study aimed to transfect progesterone receptor-B (PRB) cDNA into endometrial carcinoma cells and investigate the effect of medroxyprogesterone acetate (MPA) on cell growth, and p21 and p27 expression in the transfectant. METHODS: Immunoblotting for p21 and p27 was performed at predetermined times after the administration of MPA. RESULTS: PR expression was maximally induced in Ishikawa cells at 24 hrs after the transfection. At 1 x 10(-6) M, MPA suppressed the growth of the transfectant by 34% on day 6 and stimulated p21 accumulation at 48 to 72 hrs and p27 accumulation at 48 to 96 hrs after its administration. PRB cDNA was effectively transfected and in the transfectant MPA at 1 x 10(-6) M, the dosage suppressing growth, induced p21 and p27expression. CONCLUSION: p21 and p27 may be related to progesterone-induced growth suppression in human endometrial adenocarcinoma.

Angiopoietin-1, 2 and Tie2 expressions in endometrial adenocarcinoma--the Ang2 dominant balance up-regulates tumor angiogenesis in the presence of VEGF.

We investigated Ang1, Ang2 and Tie2 expressions including balance and intratumoral vessels in the role of angiogenesis of endometrial adenocarcinoma. Immunohistochemical staining was performed on 133 patients with endometrial (endometrioid) adenocarcinoma, including 73 with G1, 34 with G2, and 26 with G3. The levels of Ang1, Ang2 and Tie2 expressions were expressed as staining score. Total vessel count (TVC), microvessel count (MVC) and mean vessel diameter (VD) in the CD34-stained tissues were measured in five hot spot areas at x 200 magnification by image cytometry. These results were compared with high and low vascular endothelial growth factor (VEGF) expressions. Ang1, Ang2, Tie2 and CD34 were expressed in the cytoplasm of tumor cells. A significant correlation was found among Ang1, Ang2 and Tie2 expressions. In high VEGF cases, Ang1 expression was correlated negatively with TVC and MVC, but positively with VD, and the Angl < Ang2 group was significantly higher in TVC and MVC and tended to be smaller in VD than the Ang1 > Ang2 group. VD was significantly larger in G3 than in G1. The Ang1 < Ang2 balance may be one of the key factors for angiogenesis of endometrial carcinoma in the presence of high VEGF expression.

Stimulatory effects of 4-hydroxytamoxifen on proliferation of human endometrial adenocarcinoma cells (Ishikawa line).

The effects of trans-4-hydroxytamoxifen (OHTam) on proliferation of cells of the Ishikawa human endometrial adenocarcinoma line were studied under serum-free, phenol red-free conditions and compared to those of estradiol. The addition of OHTam (1 microM) to basal medium (BM), consisting of equal parts of Dulbecco's modified Eagle's medium and Ham's F-12 with additional glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, resulted in significant increases in cell numbers relative to controls. These effects were even greater than those obtained with estradiol (10 nM-1 microM) or 1% charcoal-treated fetal bovine serum (ctFBS). Addition of 1% ctFBS to BM containing 1 microM OHTam further increased cell numbers whereas addition of estradiol (10 nM) did not do so. The stimulation of growth was positively correlated with OHTam concentrations in the range of 10 nM to 1 microM. Dissociation of estradiol and OHTam proliferative effects was observed in a variant of Ishikawa cells in which estradiol did not increase proliferation while OHTam had a strong stimulatory effect. The growth-promoting effects of OHTam were also observed in BM containing 5% or 15% ctFBS. In contrast, in parallel experiments in which BM was replaced by minimal essential medium (Eagle's) with Earle's salts, OHTam (1 microM) did not stimulate proliferation under these conditions and acted as an antiestrogen, inhibiting the proliferative effects of estradiol. These results illustrate marked effects of medium composition on proliferation and antiestrogenic actions of OHTam. Alkaline phosphatase activity was strongly stimulated by estradiol (10 nM) but only very weakly affected by OHTam (1 microM); at these concentrations, OHTam inhibited the effect of estradiol, both in serum-free BM and in minimal essential medium plus 15% ctFBS, demonstrating dissociation in its actions on proliferation and on enzymatic activity. These findings suggest that OHTam may stimulate the proliferation of particular clones of endometrial cancer cells in human tumors. They also suggest that OHTam can exert effects not mediated by the estrogen receptor system, or form OHTam-estrogen receptor agonistic complexes unlike those resulting from estradiol-estrogen receptor interactions. Clearly, Ishikawa cells provide a useful model to investigate mechanisms of action of antiestrogens.

Effects of steroid hormones and antisteroids on alkaline phosphatase activity in human endometrial cancer cells (Ishikawa line).

Alkaline phosphatase activity in human endometrial cancer cells of the estrogen-responsive Ishikawa line was markedly stimulated (3-20-fold in 4 days) by estrogens, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone but not by testosterone, medroxyprogesterone acetate, glucocorticoids, several peptide hormones, prostaglandins, or growth factors. Maximum responses to estradiol were obtained at concentrations between 10(-9) and 10(-7) M; at 10(-8) M estradiol, the highest activity was reached 48-72 h after addition of the hormone. A linear relationship between enzyme activity at 48 h and the length of exposure to the hormone was observed. Dibutyryl cyclic guanosine 3':5'-monophosphate, but not dibutyryl cyclic adenosine 3':5'-monophosphate enhanced alkaline phosphatase activity and acted synergistically with estradiol. trans-4-Monohydroxytamoxifen completely antagonized the stimulatory effect of estradiol and had no agonistic activity. Dihydrotestosterone and dehydroepiandrosterone appear to exert their effects, at least in part, by interacting with estrogen receptors, since the simultaneous presence in the medium of monohydroxytamoxifen abolished their influence on alkaline phosphatase activity. The specific antiandrogen monohydroxyflutamide partially antagonized the effect of these hormones, suggesting that their action involved androgenic mechanisms as well. Exposure to elevated temperature and to specific inhibitors identified alkaline phosphatase of Ishikawa cells as a placental-type isoenzyme, thus contrasting with the nonplacental type found in glandular epithelial cells of normal endometrium and in another human endometrial cancer cell line, HEC-50. This study extends our previous observations of estrogen responsiveness in the Ishikawa cell line. In addition to the previously reported stimulatory effects on growth and progesterone receptor levels, we are now describing the stimulation by estrogens and C19 steroids of an enzyme, alkaline phosphatase, which can be used as a convenient end point to examine mechanisms of hormonal action.

Proliferation and responsiveness to estrogen of human endometrial cancer cells under serum-free culture conditions.

Studies of hormonal growth regulation in cultured human endometrial cancer cells are limited by the requirement of exogenous growth factors, usually supplied by addition of serum. The present report provides evidence that estradiol can stimulate proliferation of endometrial cancer cells of the Ishikawa line in the absence of serum or added growth factors. Mitogenic effects of estrogen were demonstrated in two different experimental systems, in cells attached to the substratum of mammalian tissue culture dishes, and in cells forming colonies in soft agar under anchorage-independent conditions. Addition of estradiol to a mixture of serum-free, phenol red-free Dulbecco's minimal essential medium and Ham's F-12 medium, supplemented with L-glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [basal medium: (BM)] significantly increased the proliferation of cells attached to culture dishes. Dose-response experiments revealed maximal estradiol stimulation at 10 nM; significant responses were also observed at 1 nM and at 100 nM concentrations. The mitogenic effect of 10 nM estradiol was comparable to that of 1% charcoal-treated fetal bovine serum and the two effects were additive. The presence of estradiol in serum-free BM resulted in a shortening of the doubling time of exponentially proliferating cells from 38 to 29 h. From the labeling index, measured after exposure to a pulse of [3H]thymidine, and from the mitotic index, both determined in exponentially proliferating cells, the lengths of the S and M phases were calculated to be 11 and 1 h, respectively. From these data it was estimated that estradiol shortened the G1 phase by approximately 40%, from 22 to 13 h. Estradiol doubled the colony formation efficiency of cells plated in BM containing 0.3% agar in the absence of serum as well as in the presence of 1% charcoal-treated fetal bovine serum. The stimulation of colony formation by estradiol was influenced by medium components, since no effects were observed in minimal essential medium. The colony formation efficiency was positively related to the serum concentrations and remained significantly lower in minimal essential medium than in BM at comparable serum levels. The observed positive relationship between colony formation efficiency and cell densities at plating suggests a cooperative mitogenic effect, likely due to autocrine and paracrine action of secreted growth factors. These results define a model to evaluate hormonal growth regulation mediated by autocrine mitogens in human endometrial cancer cells in the absence of interfering exogenous growth factors.(ABSTRACT TRUNCATED AT 400 WORDS)

A human gastric choriocarcinoma cell line with human chorionic gonadotropin and placental alkaline phosphatase production.

A gastric choriocarcinoma cell line synthesizing human chorionic gonadotropin (HCG) was established in 1971 by Oboshi et al. and was found to possess human placental alkaline phosphatase. The present paper also deals with the relationship between the cell growth and HCG secretion and with cellular localization of HCG and human placental alkaline phosphatase by cytochemical and ultrastructural methods. This cell line was found to secrete HCG during cellular proliferation, with the maximum secretion in the stationary phase (about 1 muIU/cell/48 hr), and the hormone could be detected in a small proportion of mono- and/or multinuclear cells in both logarithmic and stationary phases. The organ-specific, heat-stable, L-phenylalanine-sensitive, immunoreactive human placental alkaline phosphatase was localized on the cell membrane of many cells. Ultrastructurally, the line consisted mainly of cytotrophoblastic and intermediate cells in the process of syncytial formation, with more or less squamous metaplasia. From these findings it was concluded that the cell line maintained the properties of trophoblastic cells from morphological and functional aspects, i.e., it was a cell line with two distinct marker substances.

Growth and gene expression profile analyses of endometrial cancer cells expressing exogenous PTEN.

The PTEN tumor suppressor gene encodes a multifunctional phosphatase that plays an important role in inhibiting the phosphatidylinositol-3-kinase pathway and downstream functions that include activation of Akt/protein kinase B, cell survival, and cell proliferation. Enforced expression of PTEN in various cancer cell lines decreases cell proliferation through arrest of the cell cycle, accompanied in some cases by induction of apoptosis. We used cDNA microarrays containing 4009 cDNAs to examine changes in gene-expression profiles when exogenous PTEN was induced in PTEN-defective cells. The microarrays and subsequent semi-quantitative reverse transcription-PCR analysis revealed transcriptional stimulation of 99 genes and repression of 72 genes. Some of the differentially expressed genes already had been implicated in cell proliferation, differentiation, apoptosis, or cell cycle control, e.g., overexpression of PTEN-induced transactivation of cyclin-dependent inhibitor 1B (p27Kip1) and 2B (p15INK4B), members of the TNF receptor family, tumor necrosis factor-associated genes, and members of the Notch-signaling and Mad families. To our knowledge this is the first report of transactivation of those genes by PTEN. The genes differentially expressed in our experiments also included many whose correlation with cancer development had not been recognized before. Our data should contribute to a greater understanding of the broad spectrum of ways in which PTEN affects intracellular signaling pathways. Analysis of expression profiles with microarrays appears to be a powerful approach for identifying anticancer genes and/or disease-specific targets for cancer therapy.

Regulatory expression of lipoxin A4 receptor in physiologically estrus cycle and pathologically endometriosis.

Expression of receptors for prostaglandin (PG) and leukotriene (LT) has been reported to detect in endometrium and smooth muscle of uterus, suggesting involvement of these arachidonic metabolites in endometrial pathology and reproductive biology. Lipoxin (LX), which is produced by lipoxygenases from arachidonic acid, has been characterized as an anti-inflammatory lipid mediator. Biological actions of Lipoxin A4 (LXA4) are mediated through the specific receptor. In order to know roles of LXA4 in female genitalia, expression of LXA4 receptor mRNA was quantified by real-time polymerase chain reaction. Significantly higher expression of the receptor was detected in endometrium and myometrium than ovary in normal rats. Expression of the receptor in endometrium was increased at stage of proestrus cycle under physiological condition. Exogenous administration of progesterone into female rats significantly reduced the expression, while administration of estradiol or pregnant mare serum gonadotropin (PMSG) did not. Both, endometrium in experimental endometriosis induced in rats and the tissues from patients with ectopic endometriosis showed a higher expression of LXA4 receptor compared to the normal tissues. In contrast, expressions of BLT1 and BLT2, receptors for leukotriene B4, did not change in the endometriosis. These observations suggest a possible role of LXA4 and the receptor under physiological estrus cycle and pathological condition as endometriosis.

Lymph node pathway in the spread of endometrial carcinoma.

OBJECTIVE: To elucidate the sentinel nodes of endometrial carcinoma, the spread pathway was clarified. The correlation between lymph node spread and other clinicopathological variables was also analyzed. METHODS: Dissected lymph node samples in 342 patients who underwent pelvic and selective paraaortic lymphadenectomy were reviewed. Pelvic and paraaortic node (PLN and PAN) status was compared with clinicopathological parameters. RESULTS: Lymph node metastasis was demonstrated in 52 patients, including 46 cases with PLN metastasis and six patients with independent PAN metastasis. The metastatic sites were most frequent in the obturator and internal iliac nodes. Eleven of 49 patients who underwent PAN dissection were positive for metastasis. Sixteen of 23 cases with parametrial metastasis also metastasized in the retroperitoneal lymph node. CONCLUSION: The lymph node spread pathway in endometrial carcinoma consists of a major route via the obturator node or internal iliac node with or without parametrial involvement, and rarely a direct PAN pathway.

Time course of Fos and Fras expression in the hypothalamic supraoptic neurons during chronic osmotic stimulation.

The Fos family comprises Fos and several subtypes of Fos-related proteins (Fras) such as FosB, Fra-1, Fra-2, DeltaFosB, and chronic Fras. Changes in the expression of Fos family proteins with time are not well elucidated, particularly during chronic stimulation. In the present experiments, we investigated quantitatively the time course changes in Fos, FosB and Fras immunoreactivity in the magnocellular neurons of the supraoptic nucleus (SON) during acute and chronic osmotic stimulation. A small number of Fos- and FosB-positive neurons were observed in the SON of control rats, while many Fras-positive neurons were seen in control animals. Significant increases in the numbers of Fos-, FosB-, and Fras-positive neurons were observed 2 h after acute osmotic stimulation by intraperitoneal (i.p.) injection of 3% NaCl solution. Although the number of Fos-positive neurons returned to the control level 4 h after i.p. injection, a significant number of FosB- and Fras-positive neurons were still observed 8 h after i.p. injection. During chronic osmotic stimulation by giving 2% NaCl solution for 2 and 5 days, a large number of Fos-positive neurons were observed, but the cessation of chronic osmotic stimulation by normal water drinking immediately decreased the number of Fos-positive neurons to the control level within 2 h. The number of FosB-positive neurons was increased with period of chronic osmotic stimulation, and a significant number were observed 2-8 h after the cessation of the stimulation. The number of Fras-positive neurons was also significantly higher during chronic osmotic stimulation, and this number was significantly high 2-8 h after the cessation of the stimulation. RT-PCR analysis demonstrated the persistent expression of c-fos mRNA in the SON during chronic osmotic stimulation. These results suggest that c-fos mRNA and Fos protein are constitutively elevated during chronic osmotic stimulation and the time course changes in Fos are different from those seen in FosB and Fras.

Immunohistochemical study on VEGF expression in endometrial carcinoma--comparison with p53 expression, angiogenesis, and tumor histologic grade.

OBJECTIVE: Vascular endothelial growth factor (VEGF)--that activates endothelial cell growth--has been considered to induce angiogenesis, which is indispensable to tumor-genesis and progression. In this study, an immunohistochemical analysis was carried out to clarify the correlation of VEGF expression with angiogenesis, p53 expression--of which the wild-type is considered to suppress VEGF expression--and histologic grade in endometrial carcinoma. STUDY DESIGN: Immunohistochemical staining for detecting VEGF protein, factor VIII-related antigen of endothelial cells, and p53 protein was performed by the labeled streptavidin-biotin method on the formalin-fixed and paraffin-embedded tumor tissue of 104 patients with endometrial (endometrioid) carcinoma, including 69 with well-differentiated, 25 with moderately differentiated, and ten with poorly differentiated adenocarcinoma. RESULTS: The labeling index of p53 expression was 19.9+/-28.8% in the high VEGF group, whereas in the low VEGF group it was 12.2+/-17.0%, showing that VEGF expression was significantly correlated with p53 expression (P<0.05). VEGF expression, however, was not correlated with either the number of microvessels in the tumor area or tumor histologic grade. CONCLUSION: VEGF expression was not a single specific indicator of angiogenesis in endometrial carcinoma, whereas it was significantly correlated with p53 expression.

Expression of cyclin A in endometrial adenocarcinoma and its correlation with proliferative activity and clinicopathological variables.

PURPOSE: Cyclin A is known as an S- and G2-M phase regulatory protein and its abnormal expression has been reportedly implicated in cellular proliferation. This study was designed to investigate the correlation of cyclin A expression with tumorigenesis of the endometrium and clinicopathological variables. METHODS: Immunohistochemical staining using labeled streptavidin-biotin complex was performed on formalin-fixed, paraffin-embedded tissue of normal endometrium (15 cases), endometrial hyperplasia (23 cases), and endometrial adenocarcinoma (endometrioid type) (112 cases). RESULTS: Immunohistochemistry showed that the nuclei of the cells were positive for cyclin A. In normal endometrium, only proliferative phase was focally positive for cyclin A. Cyclin A was also positive for endometrial hyperplasia. Its expression in hyperplasia was significantly more frequent than that of proliferative phase and less than that of endometrioid adenocarcinoma. The labeling index (LI) of cyclin A in endometrioid adenocarcinoma was 16.3+/-6.9 in well-differentiated, 18.3+/-8.8 in moderately differentiated, and 30.2+/-11.8 in poorly differentiated adenocarcinoma, respectively. Cyclin A expression increased significantly more in high histological grades. The area of squamous metaplasia in endometrioid adenocarcinoma was negative for cyclin A. The LI of cyclin A was positively correlated with that of Ki-67 and cyclin-dependent kinase 2. Cyclin A expression was significantly associated with carcinoma without coexisting endometrial hyperplasia and lymphovascular space involvement (LVSI), but not with FIGO stage, myometrial invasion, lymph node metastasis, estrogen receptor, progesterone receptor, and menopause as well as recurrence. CONCLUSIONS: Cyclin A expression was involved in the progression to malignancy of the endometrium and was correlated with proliferative activity and prognostic features including histological grade, without coexisting endometrial hyperplasia and LVSI.

Immunohistochemical expression of cyclin E in endometrial adenocarcinoma (endometrioid type) and its clinicopathological significance.

PURPOSE: Cyclin E is known as a G1-S phase regulatory protein and its abnormal expression has been implicated in cellular proliferation. This study aimed to investigate the correlation of cyclin E expression with tumorigenesis of the endometrium, proliferative activity, and clinicopathological features of endometrial adenocarcinoma. METHODS: Immunohistochemical staining for cyclin E in addition to cyclin-dependent kinase 2 (cdk2), Ki67, p27, and p53 was performed by the labeled streptavidin-biotin method on formalin-fixed, paraffin-embedded tissues of normal endometria (20 cases), endometrial hyperplasias (20 cases), and endometrial adenocarcinomas (endometrioid type) (127 cases). Positive staining was expressed as a labeling index (LI) based on percentages of positive nuclei in tumor cells. RESULTS: Immunohistochemistry showed that the nuclei of the cells were positive for cyclin E. Both proliferative and secretory endometria, and endometrial hyperplasia regardless of type were negligible for cyclin E expression. The expression in normal endometrium and hyperplasia was significantly less than that in endometrial adenocarcinomas (P<0.0001). LIs of cyclin E in well-differentiated, moderately differentiated, and poorly differentiated endometrial adenocarcinomas were 31.5+/-33.3%, 37.8+/-31.9%, and 51.1+/-30.8%, respectively. Cyclin E expression increased significantly more in histological grades. The LI of cyclin E in carcinoma was positively correlated with that of cdk2, Ki67, and p53 but not with p27. The cyclin E expression was correlated with myometrial invasion and lymph-vascular space involvement, but not with FIGO stage, lymph node metastasis, coexisting endometrial hyperplasia, estrogen receptor, progesterone receptor, and menopause. CONCLUSION: Cyclin E as a complex with cdk2 is associated with carcinogenesis and disease progression in endometrial adenocarcinoma, and might be a prognostic indicator of endometrial adenocarcinoma.

PTEN immunohistochemical expression is suppressed in G1 endometrioid adenocarcinoma of the uterine corpus.

PURPOSE: PTEN is a tumor suppressor gene that inhibits cell proliferation by regulating intracellular signaling pathways, and this activity can be abolished by mutations of the PTEN gene. This study was designed to examine the correlation of PTEN expression with the expression of cell cycle regulators and with clinicopathological parameters in endometrioid adenocarcinoma of the uterine corpus. METHODS: Tissue samples of 117 endometrioid adenocarcinomas in addition to those of 19 normal endometria and 20 endometrial hyperplasias were used for the study. Immunohistochemical staining for PTEN protein was performed with the labeled streptavidin-biotin method on formalin-fixed and paraffin-embedded tissue samples. PTEN expression was represented as the staining score. RESULTS: Immunohistochemistry showed that the nuclei of cells were positive for PTEN. The PTEN staining score of normal endometrium was significantly higher in the proliferative phase than in the secretory phase. The scores of various endometrial hyperplasias were not significantly different from each other, regardless of the type of hyperplasia. The PTEN staining scores of endometrioid adenocarcinomas were 7.6+/-5.2 in G1, 9.6+/-5.2 in G2, and 11.9+/-3.7 in G3, and increased significantly as the histological grade increased. PTEN staining score was not significantly correlated with clinicopathological parameters such as FIGO stage, myometrial invasion, lymph-vascular space invasion (LVSI), lymph node metastasis or group, but was significantly correlated with labeling indices (LIs) of cell cycle regulators such as Ki-67, cdk2, cyclin A, cyclin D1, cyclin E, p27, and p53. The PTEN staining score of p53-wild cases was significantly lower than that of p53-mutant ones, but there was no significant difference of the score in cases with different PTEN gene status. PTEN expression was significantly lower in cases with both high levels of estrogen receptor and progesterone receptor. CONCLUSION: PTEN protein expression was decreased in well-differentiated and less growth-aggressive endometrial carcinoma with wild-type p53 gene and high levels of ER and PR. This suggests that disturbed PTEN expression occurs in an early phase of the tumorigenesis of well-differentiated endometrial carcinoma.

Immunocytochemical determination of estrogen and progesterone receptors in human endometrial adenocarcinoma cells (Ishikawa cells).

The regulation of both estrogen and progesterone receptor levels in human endometrial adenocarcinoma cells of the Ishikawa line was investigated immunocytochemically by using monoclonal antibodies. Positive staining for estrogen and progesterone receptors was observed in the nuclei of Ishikawa cells. Intercellular heterogeneity in receptor content was evident from the presence of receptor-positive or -negative cells and from differences in staining intensity of positive cells. Quantitative analysis was performed by scoring the staining intensity and the proportion of positively stained cells. The time and dose-dependent stimulatory effect of estradiol added to culture media on progesterone receptor levels was studied by applying both immunocytochemical and biochemical methods. Estradiol at 10 nM (optimal concentration) increased the intensity score for PR from an initial value of 10.1 to 78.3 after 72 h incubation, and the proportion of the positive staining cells from 6.7 to 42.7%. Promegestone (R5020) was effective at 1 microM concentration in decreasing the intensity score for ER from 31.1 to 14.6 after 72 h exposure and the proportion of positive cells from 19.0 to 11.4%.

Effects of transforming growth factors and regulation of their mRNA levels in two human endometrial adenocarcinoma cell lines.

The effects of the transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the growth of cells from 2 endometrial cancer lines, Ishikawa and HEC-50 were evaluated by measuring rates of DNA synthesis and changes in cell numbers during culture. EGF at 17 and 1.7 nM concentrations consistently enhanced HEC-50 cell proliferation. TGF-beta 1 inhibited Ishikawa cell proliferation but, unexpectedly for epithelium-derived cells, stimulated HEC-50 cell growth. This effect is of interest as it indicates that endometrial cells can acquire an altered responsiveness to a growth inhibitor during the process of malignant transformation. Northern blot analyses showed expression of TGF-alpha, TGF-beta 1 and EGF receptors mRNA in both cell lines. Neither estradiol (E2) nor 4-hydroxytamoxifen (OHTam) affected mRNA levels for either TGF-alpha or TGF-beta in HEC-50 cells, a line unresponsive to E2 for proliferation. In Ishikawa cells, previously shown to respond to both E2 and OHTam by increasing proliferation rates, E2 increased TGF-alpha mRNA and reduced TGF-beta mRNA levels. OHTam lowered the levels of both mRNA species, although the effect was greater on TGF-beta than TGF-alpha mRNA. These data are consistent with, but do not prove, the existence of a possible autocrine regulation by TGF-alpha and TGF-beta of human cancer cell proliferation, which might be under E2 influence in Ishikawa cells.