Yuko Kamata

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.MethodsTissue 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.

High expression of skp2 correlates with poor prognosis in endometrial endometrioid adenocarcinoma

Purpose: Skp2 interacts with the degradation of cyclin-dependent kinase inhibitor p27. This study aimed to investigate the correlation of skp2 expression with the expression of p27 and other cell cycle regulators, and clinicopathological parameters in endometrial endometrioid adenocarcinoma. Methods: Tissue samples of 136 endometrioid adenocarcinomas, in addition to 20 endometrial hyperplasias and 20 normal endometria, were immunohistochemically stained for skp2. The expression was represented as a labeling index (LI), which indicates the percentage of positive nuclei. Results: Skp2 staining was localized in the nuclei of the glandular cells of the proliferative phase endometrium, and endometrial hyperplasia and carcinoma cells. Skp2 expression was increased significantly in those of higher histological grade. The high level of skp2 expression was significantly correlated with the presence of lymph node metastasis and lymph-vascular space involvement. The LI of skp2 in endometrial carcinoma was significantly correlated with that of p27, Ki-67, cdk2, cyclin A, cyclin D1, cyclin E, p53 and PTEN. The high level of skp2 expression (LI≧20%) was significantly correlated with the patients’ poor survival. Conclusions: The skp2 level might have increased due to p27 accumulation and may be a good indicator of proliferative activity and poor prognosis.

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

PurposeCyclin 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.MethodsImmunohistochemical 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.ResultsImmunohistochemistry 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.ConclusionCyclin 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.

Bcl-2 expression and allelic loss of the p53 gene in gastric carcinomas.

In order to clarify the association betweenbcl-2 protein (Bcl-2) expression and genetic alteration, we investigatedp53 andDCC (deleted in the colon carcinoma gene locus) gene abnormalities in Bcl-2-positive and-negative gastric carcinomas using a polymerase chain reaction/loss of heterozygosity (LOH) assay. Bcl-2 immunoreactivity was found in 25 of 178 (14%) gastric carcinoma cases. With these 25 positive cases, the proportion 18/87 (20.6%) of the total in early stages demonstrating invasion of the mucosa and/or submucosa was significantly greater (P=0.013) than the 7/91 (7.7%) found for advanced tumors exhibiting invasion into or through the muscularis propria. However, there was no statistically significant variation between the proportions for differentiated (17/98 cases, 17.3%) and undifferentiated (8/80 cases, 10%) lesions. Sixteen Bcl-2-positive cases (9 cases were not studied because of insufficient specimen material to allow extraction of DNA) and 31 cases randomly selected from a Bcl-2-negative group were analyzed for the presence ofp53-LOH or DCC-LOH and forp53 by immunohistochemistry. The minority of the Bcl-2-positive group hadp53-LOH and were immunopositive for p53 (P=0.033,P=0.028 respectively), while no association was found in the Bcl-2-negative category. In contrast, there was no correlation at all between Bcl-2 expression and DCC-LOH although the number of informative cases analyzed was too small to allow definite conclusions. These results indicate that Bcl-2 may be predominantly expressed at an early stage in gastric carcinomas, possibly in negative association withp53 gene abnormalities.

Stimulatory effect of estrogen on the growth of endometrial cancer cells is regulated by cell-cycle regulators.

Estrogen is known as a major risk factor in tumorigenesis of the endometrium. The aim of this study is to establish stable estrogen-responsive endometrial cancer cell lines and to investigate the mechanism of estrogen action, focusing on cell-cycle regulation. Human wild-type estrogen receptor cDNA was transfected into endometrial cancer cells (Ishikawa) and estrogen-responsive cell lines were cloned. Their estrogen responsiveness was evaluated by the effect of estrogen on cellular growth and progesterone receptor expression. It was quantitatively estimated by immunocytochemistry or immunoblotting how the expression of cell-cycle regulators such as cyclin D1, cyclin E, Cyclin A, p53, p21 and p27 was regulated by estrogen. A cell line stably responsive to estrogen was established, and cells proliferated and the glandular structure was formed by estrogen stimulation. Cyclin D1 expression increased at 6-24h and cyclin A gradually increased until 48h of estrogen treatment compared with untreated cells. On the other hand, p53 and p21 expressions decreased at 6-24h, and p27 gradually decreased until 24h by estrogen. Our results show that the stimulatory effect of estrogen on cell proliferation may be regulated by the up-regulation of cyclin D1 and cyclin A, and down-regulation of p53, p21 and p27. This cell line is useful to clarify the molecular mechanism of estrogen action on endometrial cancer.

Effect of p53 gene transfection on vascular endothelial growth factor expression in endometrial cancer cells.

It has been reported that tumor suppressor gene p53 regulates vascular endothelial growth factor (VEGF) expression, but the relation between them in endometrial carcinoma remains unclear. We investigated VEGF expression in 11 endometrial carcinoma cell lines and the effect of p53 gene transfection on VEGF expression in the p53-mutated endometrial carcinoma cell line, HEC-50B. Immunoblotting for detecting VEGF, p53, and beta-actin was performed. Wild type p53 gene was transfected using the SuperFect method. The mean VEGF value of 0.8 +/- 0.3 (n = 6) in p53 wild-type group was significantly lower than the 1.6 +/- 0.8 (n = 5) that was found in the p53 mutant group (P < 0.05). Levels of VEGF in the culture medium were measured by enzyme immunoassay (EIA). VEGF levels in the p53 gene-transfected HEC-50B cells and the conditioned medium were decreased at 48 h after p53 gene transfection. VEGF expression was downregulated by p53 in endometrial carcinoma cells.

VEGF expression and its reguration by p53 gene transfection in endometrial carcinoma cells.

Vascular endothelial growth factor (VEGF) that activates endothelial cell growth induces angiogenesis, which is indispensable to tumor igenesis and tumor progression. On the other hand, tumor suppressor gene p53 has been considered to regulate VEGF expression, but the detailed relationship between them remains unclear. In this study, we aimed to study VEGF expression in endometrial carcinoma cells and the effect of p53 gene transfection on VEGF expression using p53-mutated endometrial carcinoma cell line. HEC-50B. Immunoblotting for detecting VEGF protein, p53 protein and β-actin was performed using 11 endornetrial carcinoma cell lines. Levels of VEGF in the cultured media were measured by Enzyme immunoassay(EIA). Tmsfection of wild p53 gene was carried out by SuperFect method in HEG50B cells, which had mutant p53 gene and did not express p53 protein. The results of immunoblotting were analyzed by NIH image and expressed as values. The results of EIA were expressed as the relative value. The VEGF value was 0.8±0.3 (n=6) in p53-wild group, whereas in p53-rnutant group it was 1.6±0.8 (n=5). VEGF expression was correlated significantly with p53 status (P<0.05). VEGF levels in p53 gene-msfected cells and the conditioned medium were decreased in 48 hours after p53 gene transfection. VEGF expression was down-regulated by p53 in endometrial carcinoma cells.

HEC-1 Cells: Establishment of an In Vitro Experimental System in Endometrial Carcinoma

The HEC-1 cell line was the first in vitro cell line of a human endometrial adenocarcinoma, which enabled us to perform research work on the endometrium and endometrial carcinoma at the level of a simplified cellular system; contributing to cell and molecular biological studies on endometrial carcinoma. Once a cell line is established, it provides a stable experimental system that facilitates and progresses the study of the tissues and/or neoplasias from which the cell line is derived. In this article, we report how HEC-1 cells have been established and cleared the proposed requirements to characterize an established cell line. In addition, in order to demonstrate the usefulness of the cell lines for research work once they have been established, we illustrate these concepts by recalling results obtained with HEC-1 and the HEC family of endometrial carcinoma cells and review the literature with regard to what has been achieved by using these cells.

Expression of tumor suppressor gene product p14ARF in endometrioid adenocarcinoma of the uterine corpus.

Summary:p14ARF activates p53 by inhibiting MDM2 expression and arrests the cell cycle in G1 and G2/M. Abnormal p14ARF expression has been reported in various human cancers. This study investigated p14ARF expression in endometrioid adenocarcinoma of the uterine corpus in an attempt to clarify its correlation with other cell cycle-regulators and clinicopathologic parameters. The specimen studied consisted of 124 endometrioid adenocarcinomas, 20 normal endometria, and 20 endometrial hyperplasias. Immunohistochemical staining of formalin-fixed and paraffin-embedded tissues was performed using a Catalyzed Signal Amplification System. Cells with >5% positive staining were classified as positive for p14ARF. A staining score of 1 was adopted when the percentage of positive nuclei was <5%, a score of 2 when it was 5 to 50%, and a score of 3 when it was >50%. In normal endometrium, the frequency of positive staining in the proliferative phase and secretory phase was 50% (4/8) and 58.3% (7/12), with staining scores of 1.8±0.9 and 1.6±0.5, respectively. The frequency of staining in simple hyperplasia (SH), complex hyperplasia (CH), and complex atypical hyperplasia (CAH) was 88.9% (8/9), 25% (1/4), and 42.9% (3/7), respectively; the staining scores were 1.9±0.3, 1.3±0.5, and 1.4±0.5, respectively. Among endometrioid adenocarcinomas, the frequency of staining of well-differentiated (G1), moderately differentiated (G2), and poorly differentiated (G3) adenocarcinomas was 69% (49/71), 64% (16/25), and 42.9% (12/28) respectively, with staining scores of 2.1±0.8, 2±0.9, and 1.8±1, respectively. Thus expression levels of p14ARF were higher in G1 tumors than in normal endometria or endometrial hyperplasias, and the frequency of its staining in endometrioid carcinomas was inversely correlated with histologic grade. The staining score for endometrioid adenocarcinomas also was inversely correlated with the labeling index (LI) of Ki-67, but not with that of cyclin A, cyclin D1, cyclin E, cdk2, p27, p53, or other clinicopathologic parameters. In conclusion, p14ARF expression correlated with histologic grade and Ki-67, but not other prognostic factors in endometrioid adenocarcinoma. Long-term follow-up studies are needed to analyze the significance of p14ARF expression in these tumors.

Expression of Cell Cycle Regulators in Endometrial Adenocarcinoma

Abnormal expressions of cell cycle regulators, such as cyclin-dependent kinases (cdk), cyclins, and cyclin kinase inhibitors, are supposed to play an important role in the tumorigenesis and progression of carcinoma. The aim of the present study was to investigate the correlation of the expression of these cell cycle regulators with proliferative activity and clinicopathological parameters in endometrial adenocarcinoma, and the regulation of these proteins by sex steroid hormones in endometrial cancer cells. Tissue samples of 127 endometrial adenocarcinomas (endometrioid type) were used in the present study. Immunohistochemical staining of cycle regulators was performed according to the labeled streptavidin-biotin method. Ishikawa cells, in which estrogen receptor (ER) cDNA was transfected by the SuperFect method, and HEC-265 cells positive for the progesterone receptor were used in an in vitro study. Quantitative analysis of proteins was performed by immunoblotting. Expression of cyclin D1, cyclin E, cyclin A, p53, and p27 was positively correlated with Ki-67 expression. Expression of cdk2, cyclin Dl, cyclin E, cyclin A, p53, and p27 was positively associated with histological grade. Expression of cyclin E was significantly correlated with lymphovascular space involvement (LVSI) and myometrial invasion, and expression of cyclin A was correlated with LVSI and group (coexistence with or without hyperplasia). p53 was related with stage, LVSI, myometrial invasion, and group; whereas p27 was correlated with stage, LVSI, and lymph node metastasis. Estradiol (E2) revealed a stimulatory effect on the growth of ER-transfected Ishikawa cells, in addition to enhancing their expression of cyclin A and cyclin E. In contrast, medroxyprogesterone acetate (MPA) suppressed HEC-265 cell growth after its accumulation of p27 in the cells. In conclusion, the expression of cell cycle regulators was significantly associated with cell proliferation, histological grade, and some clinicopathological parameters in endometrial adenocarcinoma. A cell line stably sensitive to E2 was established after ER cDNA was transfected into Ishikawa cells, and cyclin A and E may be involved in the enhanced cell growth induced by E2. p27 accumulation induced by MPA may be involved in the progesterone-induced growth suppression of endometrial cancer cells.