[Deletion of OPCML gene and promoter methylation in ovarian epithelial carcinoma].

OBJECTIVE: To investigate the expression of OPCML gene in ovarian epithelial carcinoma and determine the relationship between mRNA expression and methylation of their promoters. METHOD: Twenty normal ovarian tissues and 89 ovarian epithelial tumor specimens (72 malignant, 17 benign), as well as 3 ovarian carcinoma cell lines (SKOV-3, CAOV3, and 3AO), were collected for detection of OPCML gene expression by reverse transcription-polymerase chain reaction and for detection of promoter methylation by restriction enzyme cut analysis from 7. 1999 to 7. 2003. RESULTS: Among ovarian epithelial carcinoma 19.4% expressed OPCML mRNA, while 85% of normal ovarian tissue and 76.5% of benign ovarian tumor. The ratio of expression of OPCML mRNA in ovarian epithelial carcinoma was significantly lower than those of normal (chi2 = 30.108, P = 0.0000) and benign tumors (chi2 = 21.162, P = 0.000). No OPCML mRNA expression was found in SKOV-3 and CAOV3, but was found in 3AO. Methylations were detected in 44.4% of cancer cells promoter, while 0% in normal ovarian tissue and benign ovarian tumors. The ratio of methylation of ovarian epithelial carcinoma was significantly higher than those of normal (chi2 = 13.630, P = 0.0000) and benign tumors (chi2 = 11.797, P = 0.000). Methylation was found in SKOV-3 and CAOV3, but not in 3AO. The relationship between gene expression and promoter methylation was correlated (r = 11.589, P = 0.002), especially at Hap I1 site (r = 11.640, P = 0.004). Methylation was also found in SKOV-3 and CAOV3 cell lines, but not in 3AO cell line. CONCLUSION: Deletion of OPCML gene exists in ovarian epithelial carcinoma cell. The gene promoter methylations, especially Hap II motif, may be one of pathways that contribute the inhibition of OPCML expression.

Ovarian carcinoma cells effectively inhibit differentiation and maturation of dendritic cells derived from hematopoietic progenitor cells in vitro.

Loco-regional dissemination of ovarian carcinoma is associated with immunosuppression of the peritoneal cavity. One marked characteristic of the peritoneal immunity in this disease is the defective function of dendritic cells (DCs). In this study, the affect of ovarian carcinoma cells on DCs derived from hematopoetic progenitor cells was observed. The study demonstrated that the expansion, phenotype, and function of DCs generated from CD34+ precursors were significantly altered by the supernatant secreted by ovarian carcinoma cells, and this effect could be partly explained by tumoral overproduction of Vascular Endothelial Growth Factor (VEGF). The results indicated that a role of ovarian carcinoma cells in the differentiation and function of DCs could be associated with the immunosuppression and development of ovarian carcinoma.

Proportion of CD4+CD25+ regulatory T cell is increased in the patients with ovarian carcinoma.

OBJECTIVE: To study the frequency of the CD4+CD25+ regulatory T cells (Tregs) in the patients with ovarian carcinoma and its possible mechanism. METHODS: The percentages of CD4+CD25+ Tregs in the peripheral blood lymphocytes (PBLs), tumor infiltrating lymphocytes (TILs) and tumor associated lymphocytes (TALs) from 13 patients with ovarian carcinoma and in the PBLs from 14 healthy women were determined by flow cytometry. The expression of CD69 on CD4+PBLs from the patients was detected. PBLs from healthy women were cultured in complete RPMI 1640 containing the supernatant from SKOV3 cell line with or without PHA (phytohemagglutinin) stimulation for 72 hours, then the percentage of CD4+CD25+ T cells was detected. RESULTS: CD4+CD25+ Tregs in the PBLs from patients with ovarian carcinoma were significantly increased compared with those from the control. The percentage of CD4+CD25+ Tregs in TILs was higher than that in PBLs and TALs from the patients, but not significantly. The expression of CD69 on CD4+PBLs from the patients was negative. The percentages of CD4+CD25+ T cells in PBLs cultured with SKOV3 supernatant elevated significantly compared with those without supernatant whether PHA was added or not (P=0.001 and 0.001, respectively). CONCLUSION: There is an increasing of the proportion of CD4+CD25+ Tregs in PBLs, TILs and TALs of the patients with ovarian carcinoma, which probably results from up-regulation of soluble factor secreted by ovarian carcinoma cells.

[Possibility of PTEN expression on predicting pathologic risk factors of endometrioid adenocarcinoma before operation].

BACKGROUND & OBJECTIVE: Fully estimating pathologic risk factors is important for selecting operation and predicting prognosis for endometrioid adenocarcinoma. Phosphatase and tension homology deleted on chromosome ten (PTEN), taken as the housekeeping gene of endometrium, has the highest mutation rate in endometrioid adenocarcinoma. This study was to investigate the effect of PTEN on predicting pathologic risk factors of endometrioid adenocarcinoma before operation. METHODS: Clinicopathologic data of 107 patients with endometrioid adenocarcinoma were retrospectively analyzed. The expression of PTEN was detected by SP immunohistochemistry. Correlations of PTEN to high risk factors, such as differentiation, myometrium invasion, and lymphatic metastasis, were analyzed. RESULTS: Deletion rate of PTEN was 56.1% in the 107 endometrioid adenocarcinoma patients. PTEN expression had no correlations to histological differentiation (P=0.695), myometrium invasion (P=0.921), lymphatic metastasis (P=0.682), surgical stage (P=0.750), estrogen receptor (P=0.281), and progestin receptor (P=0.260). CONCLUSION: Detection of PTEN can't predict the high risk factors of endometrioid adenocarcinoma before operation.

[Expressions of VEGF/VEGFRs and activation of STATs in ovarian carcinoma].

OBJECTIVE: To study the expressions of VEGF/VEGFRs and activation of STATs in ovarian epithelial carcinoma, and to elucidate direct effect of VEGF on ovarian carcinoma cells. METHODS: Tissue samples from 42 women with primary ovarian epithelial carcinoma (OVCA), 29 with begnin ovarian tumor (OVBT) and 11 with normal ovarian tissue (NOV) were collected. LSAB immunohistochemical staining was used to determine the expression of VEGF, VEGFR1, VEGFR2 and activated STATS (P-STAT1, P-STAT3, P-STAT5, P-STAT6) proteins. RESULTS: (1) Semi-quantitative scoring showed that VEGF expression in OVCA was significantly higher than that in OVBT and NOV (P < 0.01). Expressions of VEGFR1 and VEGFR2 were significantly elevated in OVCA, including tumor cells and stromal vascular endothelial cells (P < 0.01, compared with OVBT and NOV). There was no difference in VEGFRs expressions between OVBT and NOV. (2) In OVCA, tumor cells and endothelial cells expressed P-STAT3 and P-STAT5 at significantly higher levels than those in OVBT and NOV (P = 0.000). The staining of P-STAT1 and P-STAT6 was weak with no significant differences among OVCA, OVBT and NOV. (3) Expressions of VEGFR1 and VEGFR2 in endothelial cells were significantly correlated with P-STAT5 and P-STAT3, respectively (P = 0.006 and 0.001). In cancer cells, VEGF, VEGFR1 and VEGFR2 were all significantly correlated with P-STAT3 and P-STAT5 (P = 0.000), but not with P-STAT1 or P-STAT6. CONCLUSION: VEGF affects ovarian carcinoma cells via VEGFRs, and STATs probably participate in intracellular signaling of VEGF.

[Effects of vascular endothelial growth factor on differentiation and function of dendritic cells generated from CD34+ hematopoietic progenitor cells in vitro].

OBJECTIVE: To investigate the effects of vascular endothelial growth factor (VEGF) on differentiation and function of dendritic cells derived from CD34+ hematopoietic progenitor cells. METHODS: After isolation from umbilical cord blood with a high-gradient magnetic cell sorting system (MACS), the CD34+ cells were cultured with a cocktail cytokines for differentiating into dendritic cells (DC). The cells were stimulated by VEGF (25 ng/ml) either at the beginning or at day 9 of culture. Kinetics analysis of cell proliferation was performed during the process of cell culture, and the expression of DC differentiation antigens including CD1alpha, CD83, CD80, CD54 and HLA-DR was examined by flow cytometry. DC function was evaluated by the ability to induce proliferation of allogeneic T cells in mixed lymphocyte reaction (MLR) assay, and the production of IL-12 by ELISA. RESULTS: VEGF added at day 1 of culture induced an increase of total cell numbers by (1.51 +/- 0.23)-folds (P = 0.001). VEGF added at the initial but not the late stage of culture could dramatically down-regulate the expression of CD1a [(33.00 +/- 2.12)% vs (81.20 +/- 6.93)%], CD83 [(42.23 +/- 1.15)% vs (87.98 +/- 7.97)%], CD80 (42.93 +/- 1.32)% vs (94.53 +/- 0.87)%], and HLA-DR [(37.93 +/- 5.30)% vs (74.15 +/- 3.74)%], while obviously up-regulate the expression of CD14. Moreover, the inhibitory effect of VEGF on DC function was confirmed by a reduced ability to induce proliferation of allogeneic T cells and production of IL-12 (P < 0.01). CONCLUSIONS: VEGF could induce the expansion of hematopoietic progenitor cells and inhibit at the early stage their differentiation into mature DC.

[Development of HPV16 positive cervical cancer model in the hu-PBL-SCID mouse and its immunological features].

OBJECTIVE: To develop a HPV16 positive cervical cancer model in the hu-PBL-SCID mouse and investigate its immunological features. METHODS: Thirty-two CB17SCID mice were randomly divided into 4 groups: group A (5 mice) subcutaneously injected with phosphate-buffered saline, group B (5 mice) intraperitoneally injected with human peripheral blood lymphocyte (PBL) for immune reconstruction, group C (11 mice) subcutaneously injected with human cervical carcinoma cell line SiHa, and group D (11 mice) intraperitoneally injected with PBL and subcutaneously injected with SiHa cells after 24 hours of PBL transplantation. The tumor growth, behaviors and status of xenogeneic graft versus host disease (XGVHD) were observed. Human immunoglobulins G (IgG) in mouse serum, the percentage of human CD3(+), CD4(+) and CD8(+) T cells in peripheral blood and spleen, spleen weight, tumor infiltrating lymphocytes and human CD4(+) T cells, and cytotoxicity test of spleen cells were detected. RESULTS: The rate of successful tumor transplantation was 100%. XGVHD was not found. On the 5th day, human IgG level in the group B (0.98 microg/ml +/- 0.20 microg/ml) and group D (1.39 microg/ml +/- 0.25 microg/ml) was significantly higher than that in the group A (t = 7.655, 9.937, both P = 0.000). Human IgG level in group D was significantly higher than that in the group B (t = 3.200, P = 0.006). Only very low levels of human serumal IgG were detected in the group C and group A with no significantly difference. The level of human serumal IgG was gradually elevated in all the humanized SCID mice as the the time after PBL transplantation went on, and was significantly higher than that in non-humanized mice (P < 0.05). The percentage of human CD3(+), CD4(+) and CD8(+) T cells was significantly increased in the peripheral blood and spleen of immunoreconstituted SCID mice. The weight of spleen was markedly increased in the group D. TIL infiltrating in the tumor were remarkable and human CD4(+) T cells was detected by immunohistochemistry in the group D but not in the group C. The spleen cells in the group D displayed stronger cytotoxicity to the target cells (P < 0.05). CONCLUSION: Human immune function can be successfully reconstructed in SCID mouse via intraperitoneally injecting with human PBL, and induce anti-tumor immune response to the transplantated tumor of HPV16 positive cervical cancer.

[Phosphorylation of signal transducer and activators of transcription 3 induced by vascular endothelial growth factor in ovarian carcinoma cell line in vitro].

OBJECTIVE: To observe phosphorylation of signal transducer and activators of transcription 3 (STAT3) in Caov-3 induced by vascular endothelial growth factor (VEGF), and to investigate molecular mechanisms of the effect of VEGF on ovarian carcinoma cells. METHODS: The expressions of phosphorylated STAT3 in Caov-3 induced by VEGF were detected by immunocytochemistry and Western blot methods. Furthermore, the relationship between STAT3 phosphorylation and VEGF stimulation in ovarian carcinoma cells was investigated using a peptide which could specifically bind VEGFR2 and thus block the binding of VEGF to its receptors. RESULTS: With VEGF stimulation, the expressions of phosphorylated STAT3 were significantly higher than those without VEGF stimulation in Caov-3. And 50 ng/ml was the effective dose resulting in a significant increase of phosphorylated STAT3 (2.20 +/- 0.28/1.37 +/- 0.17) compared to 0 ng/ml (0.56 +/- 0.15/0.47 +/- 0.19) (P < 0.001). Translocation into nuclei of activated STAT3 occurred after 30 min, while STAT3 phosphorylation decreased after 60 min of stimulation (0.95 +/- 0.18/0.66 +/- 0.20). A small peptide specific for VEGFR2, blocked the increase of STAT3 phosphorylation induced by VEGF in a dose dependent manner and 80 micro mol/L was the effective dose (P < 0.001). CONCLUSIONS: VEGF could induce STAT3 phosphorylation and translocation into nuclei of activated STAT3 in Caov-3, and the small peptide could effectively inhibit these effects of VEGF. It suggests that STAT3 participates in the VEGF signal transduction mediated by VEGFR2 in ovarian carcinoma cells.

Vascular endothelial growth factor (VEGF) and ovarian carcinoma cell supernatant activate signal transducers and activators of transcription (STATs) via VEGF receptor-2 (KDR) in human hemopoietic progenitor cells.

OBJECTIVE: To investigate the STATs signaling pathway activated by VEGF in human hemopoietic progenitor cells. METHODS: CD34(+) hemopoietic progenitor cells, which isolated from umbilical cord blood, were treated with VEGF or culture supernatant of ovarian carcinoma cell line which could secrete large amount of VEGF, phosphorylation and nuclear translocation of STAT3 and STAT5 were then detected by Western Blot and immunocytochemistry. Expression of VEGFR2/KDR on CD34(+) cells was studied by immunocytochemistry. The specific VEGFR2/KDR heptapeptide antagonist ATWLPPR was used to identify whether the activation of STATs signaling pathway was specifically mediated by VEGFR2/KDR. RESULTS: The concentration of VEGF in SKOV3-supernatant was 4024.84+/- 505.59 pg/ml. CD34(+) progenitor cells could express VEGFR2/KDR. When CD34(+) cells were stimulated by VEGF and SKOV3-supernatant, STAT3 appeared tyrosine-phosphorylation and nuclear translocation, but STAT5 was only phosphorylated, and not translocated. When ATWLPPR was used to block the binding of VEGF to KDR, VEGF and the SKOV3-supernatant failed to activate the phosphorylation of STAT3 and STAT5. CONCLUSIONS: STAT3 may participate in the signal transduction pathways activated by VEGF specifically mediated by VEGFR2/KDR in human hemopoietic progenitor cells, and the aforementioned signaling pathway participated in the interaction of ovarian carcinoma cells and progenitor cells.

[Role of three-dimensional transvaginal sonography in evaluation of the depth of myometrial invasion of endometrial carcinoma].

OBJECTIVE: To investigate the value of three-dimensional transvaginal sonography (3-DTVS) in diagnosing depth of myometrial invasion (MI) and analyze factors that may influence 3-DTVS diagnosis. METHODS: Fifty-three patients with endometrial carcinoma proven by histological diagnosis postoperatively in Women's Hospital, Zhejiang University from 2002 to 2003 were included in the study. All patients underwent primary surgery and 2-DTVS and 3-DTVS examinations within 7 days before operation. The diagnosis of the depth of MI was made by multiplanar mode reconstruction and volume measurement based on 3-DTVS. Clinical-pathological parameters were simultaneously recorded. RESULTS: Eighteen patients were deep MI, 31 superficial MI and 4 no myometrial involvement. The sensitivity, specificity, positive and negative predictive values of 3-DTVS and 2-DTVS in detecting superficial MI were 92%, 100%, 100%, 67%; and 44%, 100%, 100%, 21%, respectively. The sensitivity, specificity, positive and negative predictive values of 3-DTVS and 2-DTVS in detecting deep MI were 72%, 86%, 72%, 86% and 75%, 84%, 67%, 89%, respectively. There was significant difference between 3-DTVS and 2-DTVS in detecting superficial MI (chi(2) = 13.2011, P = 0.005), but no significant difference in detecting deep MI (chi(2) = 0.0000, P > 0.05). The median tumor volume of deep and superficial MI was 1.12 cm(3) (Q(25 - 75) = 1.12 - 4.49) and 9.16 cm(3) (Q(25 - 75) = 3.35 - 23.12) respectively. There was a significant difference between the two groups (z = -3.72, P = 0.000). Among all the parameters involved in the study there was no significant factor influencing 3-DTVS diagnosis. CONCLUSIONS: Three-DTVS is superior to 2-DTVS in detecting superficial MI, but not in deep MI. The measurement of tumor volume with 3-DTVS could be used as an objective parameter for detecting deep MI.

[Analysis of the risk factors for ovarian metastasis in patients with endometrial carcinoma].

OBJECTIVE: To study the risk factors for ovarian metastasis in patients with endometrial carcinoma. METHOD: The pathological and clinical features and outcomes of endometrial carcinoma patients who were diagnosed and treated in our hospital from Jan 1996 to Dec 2002 were retrospectively reviewed and analyzed. RESULTS: Of the 321 cases reviewed, 15 (4.7%) had ovarian metastasis, among which 60% were recessive metastasis. Factors predictive of ovarian metastasis on logistic forward regression were depth of myometrial invasion, histologic grade and regional lymphatic nodes invasion. CONCLUSION: Depth of myometrial invasion, histologic grade and regional lymphatic nodes invasion are the independent predictive factors for ovarian metastasis of endometrial carcinoma.

[Relationship between interleukin-10 level in ascites of patients with primary ovarian epithelial carcinoma and immune defect in peritoneal cavity].

BACKGROUND & OBJECTIVE: As a multifunctional Th2-cytokine, interleukin-10 (IL-10)plays a major role in the immune response. It is well known that IL-10 is an immunosuppressive cytokine, and participates in the development and progression of various tumors. In this study, we investigated the relationship between the IL-10 level in the ascites of the patients with primary ovarian epithelial carcinoma (POEC) and immune defect in the peritoneal cavity. METHODS: The IL-10 levels in serum and ascites of 32 patients with POEC, in culture supernatants of 4 different ovarian carcinoma cell lines and in serum of 10 patients with ovarian epithelial benign tumor and 10 health women (control) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: (1) IL-10 level in ascites was significantly higher than that in serum of patients with POEC, (159.78+/-51.20 ng/L vs 12.01+/-4.38 ng/L, P=0.000). IL-10 level in serum of the patients with POEC (12.01+/-4.38 ng/L) was significantly higher than that of the patients with benign tumor (3.79+/-2.40 ng/L, P=0.000) and control (4.45+/-2.69 ng/L, P=0.003). There was no significant difference of IL-10 level in serum between ovarian benign tumor and control (P=0.529). (2) IL-10 level in ascites of the patients with POEC was correlated with FIGO stage but not correlated with histological grade. (3) IL-10 was detectable in culture supernatants of 4 different ovarian cancer cell lines (3ao, SKOV3, CAOV3 and OVCAR). CONCLUSION: High level of IL-10 in ascites of the patients with POEC is probably associated with immune defect in their peritoneal cavity, ovarian cancer cells may promote metastasis in peritoneal cavity by secreting IL-10.

[Activation of signal transducers and activators of transcription induced by vascular endothelial growth factor in CD34+ hematopoietic progenitor cells in vitro].

OBJECTIVE: To investigate the activation pattern of signal transducers and activators of transcription (STAT) induced by vascular endothelial growth factor (VEGF) in CD34+ hematopoietic progenitor cells, and gain an insight into the molecular mechanism and signal transduction pathway of VEGF that has an effect on CD34+ hematopoietic progenitor cells. METHODS: After isolated from umbilical cord blood by using a high-gradient magnetically activated cell sorting system (MACS), CD34+ cells were stimulated by VEGF (50 ng/ml) for different time (0, 15, 30, 45, 60, 90 min) to detect the tyrosine phosphorylation and nuclear translocation of STAT-3 and STAT-5 with Western blot and immunocytochemistry methods. The expression of VEGF receptor-2 (VEGFR2) on the membrane of CD34+ progenitor cells was examined by immunocytochemistry. ATWLPPR, an effective peptide screened from phage epitope library by affinity for membrane-expressed VEGFR2 and blocking the binding of VEGF to VEGFR2, was used to determine whether the activation of STAT pathway induced by VEGF was blocked. RESULTS: Tyrosine phosphorylation of STAT-3 and STAT-5 was undetectable in unstimulated CD34+ cells, but was evident at 15 min in response to VEGF stimulation. VEGF resulted in a rapid and transient tyrosine phosphorylation of STAT-3 and STAT-5. The maximal tyrosine phosphorylation was catched at 30 and 45 min, respectively (P = 0.0001), and returned to basal levels at 90 min. Immunocytochemistry confirmed that increased phosphorylated STAT-3 was translocated into the nuclei at 30 min (P = 0.0001), and mainly in cytoplasms again at 90 min after stimulation with VEGF. However, compared with unstimulated CD34+ cells, there was only increased phosphorylation of STAT-5 appeared mainly in cytoplasms, but no significant nuclear translocation was found after stimulation with VEGF (P > 0.05). The presence of VEGFR2 was confirmed using anti-VEGFR2 antibody staining by immunocytochemistry, moreover, the phosphorylation of STAT-3 and STAT-5 failed to be activated by the co-culture with ATWLPPR and VEGF, suggesting that activation of the STAT pathway be specifically mediated by VEGFR2 in CD34+ progenitor cells. CONCLUSIONS: STAT signaling pathway participates in the signal transduction of VEGF via VEGFR2 in CD34+ hemopoietic progenitor cells.

Effect of interleukin-7 gene transfection into ovarian carcinoma cell line SKOV3 in vitro and in vivo.

OBJECTIVE: To investigate the effect of interleukin-7 (IL-7) gene transfection into an established ovarian carcinoma cell line (SKOV3) in vitro and evaluate the tumorigenicity of SKOV3-IL-7 in severe combined immunodeficient (SCID) mice. METHODS: IL-7 gene was transfected into SKOV3 cells by liposome. IL-7 mRNA and protein of SKOV3-IL-7 and their parental control cells were detected by reverse transcriptive-polymerase chain reaction (RT-PCR) and Western blot, respectively. The levels of IL-7, IL-2, TNFalpha and TGFbeta1 in the supernatant were detected by ELISA. The cell cycle, HLA-ABC, HLA-DR and ICAM-1 expressions were assayed by flow cytometry. The sensitivities of tumor cells to lymphokine-activated killer (LAK) cells were measured by lactate dehydrogenase (LDH) release assay. Tumorigenicities of SKOV3-IL-7 and their parental cells in SCID mice were evaluated by macro- and histological examination, while IL-7 expression and secretion were detected by immunohistochemistry and ELISA. RESULTS: IL-7 mRNA and protein were detectable in SKOV3-IL-7 only. ICAM-1 expression was significantly higher and TGFbeta1 level in culture supernatants was significantly decreased in SKOV-IL-7, all other variables remained unchanged. The proliferative activity and cell cycle of SKOV3-IL-7 were unchanged. The cytotoxic sensitivity of SKOV3-IL-7 to LAK cells was significantly higher. Both gene-transfected and nontransfected SKOV3 cells were successfully inoculated into the peritoneal cavities of SCID mice. IL-7 proteins in plasmas and tumor tissues were detectable only in SCID mice inoculated with SKOV3-IL-7. The IL-7 engineered murine tumor models revealed better general aspects, reduced tumor development and dissemination. CONCLUSIONS: IL-7 gene transfection into SKOV3 cells downregulates TGFbeta1 secretion, upregulates ICAM-1 expression and enhances sensitivity to LAK cells in vitro. The tumorigenicity of IL-7 engineered cells in SCID mice is reduced. These findings may offer support to the development of cytokine gene therapy for ovarian carcinoma.

Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway.

Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-gamma and higher level of IL-10 gene expression were observed when CD8+ T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8+ T cells showed most of them were growth arrested in G0/G1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R beta and gamma chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression.

[Tumor-infiltrating dendritic cells in epithelial ovarian carcinoma and correlation with the expression of vascular endothelial growth factor].

OBJECTIVE: To investigate the density and activation status of tumor infiltrating dendritic cells (TIDC) in epithelial ovarian carcinoma (EOC) and correlation with the expression of vascular endothelial growth factor (VEGF). METHODS: Streptavidin-peroxidase (SP) and Picture two-step immunohistochemistry methods were used to detect S-100(+), CD(83)(+) TIDC and the expression of VEGF in 57 primary EOCs, 32 benign ovarian tumors (benign control) and 16 normal ovarian tissues (normal control). RESULTS: (1) Two types of heterogeneous distribution pattern of TIDC in EOC were observed under the microscope. The number of S-100(+) TIDC in EOC [median 4.3 cells/high power field (HPF)], was significantly higher than that in benign controls (median 1.8 cells/HPF) and normal controls (median 2.0 cells/HPF, P = 0.000 and 0.015). The number of S-100(+)DC in early stage was significantly higher than that in advanced stage (median 6.0 and 3.8 cells/HPF, P = 0.026). Few CD(83)(+) TIDCs infiltrated tumor stromal tissue in EOC (median 0). (2) The expression of VEGF was significantly higher in EOC than in controls (P = 0.000). (3) The number of S-100(+) DC in EOC was negatively correlated to the expression of VEGF in tumor cells (P = 0.001). CONCLUSIONS: (1) The number of S-100(+) TIDC increases significantly in EOC. Ovarian carcinoma cells may stimulate recruitment of TIDC in EOC, but TIDC can be suppressed by VEGF. (2) Maturation of TIDC in EOC is severely inhibited.

[Experimental study on apoptosis in T cells induced by ovarian carcinoma cells].

OBJECTIVE: To investigate apoptosis in T cells induced by ovarian carcinoma cells and analyze the role of nitric oxide and intracellular free calcium in this process. METHODS: Apoptosis induced by ovarian carcinoma cell lines supernatants was investigated by electron microscopy and flow cytometry. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of inducible nitric oxide synthase (iNOS) mRNA. Calcium ions ([Ca(2+)]i) and cycle guanosine monophosphate (cGMP) were determined by Fura-2 fluorescein load technique and enzyme immunoassay (EIA), respectively. RESULTS: Cluster of differentiation (CD)8(+) T cells cocultured with ovarian carcinoma cell lines supernatants expressed typical change of apoptosis, such as condensation and fragmentation of chromosomes, apoptosis peak in flow cytometry and higher apoptotic ratios[(23.8 +/- 3.5)%, (23.1 +/- 2.9)%, (24.2 +/- 4.9)% vs (4.6 +/- 0.5)%, P < 0.01]. The expression of iNOS mRNA and cGMP level in the process of CD(8)(+) T cellular apoptosis were significantly enhanced as compared to that of control group [1.14 +/- 0.03, 1.05 +/- 0.04, 1.16 +/- 0.02 vs 0.60 +/- 0.03, P < 0.01; (0.65 +/- 0.09), (0.62 +/- 0.16), (0.57 +/- 0.12) pmol/Lvs (0.29 +/- 0.04) pmol/ml, P < 0.01], and [Ca(2+)]i was significantly higher [(380 +/- 38), (366 +/- 13), (356 +/- 20) nmol/L vs (184 +/- 11) nmol/L, P < 0.01]. CONCLUSION: Nitric oxide and intracellular free calcium may play a key role in apoptotic T cell induced by ovarian carcinoma.

[Biologic characteristics of intraperitoneal transplantation model of human ovarian carcinoma in severe combined immunodeficiency mice].

OBJECTIVE: To develop a human ovarian carcinoma SKOV3 model in severe combined immunodeficiency (SCID) mouse and to study its biologic characteristics. METHODS: Human ovarian carcinoma SKOV3 cells were injected intraperitoneally into female SCID mouse to establish a transplantation model of human ovarian carcinoma. The biological characteristics, metastasis and morphology of transplanted tumors were studied. RESULT: All tumors grew progressively with no sign of regression. The tumor cells spread around the peritoneal cavity and mainly on the diaphragm, mesentery, peritoneum and around the liver, which was confirmed by histopathology. The morphology, growth pattern and CA125 secretion of primary culture of transplanted cells remained as same as those of ovarian carcinoma cell line SKOV3. CONCLUSION: An intraperitoneal transplantation model of human ovarian carcinoma SKOV3 in SCID mice has been developed successfully, which can simulate the biological behavior of peritoneal metastasis of human ovarian carcinoma.

[Single methotrexate chemotherapy for low-risk gestational trophoblastic tumor].

OBJECTIVE: To investigate the efficacy and toxicity of methotrexate (MTX) give intravenously in the primary treatment of gestational trophoblastic tumor (GTT). METHODS: A total of 37 patients with low-risk GTT was primarily treated by single MTX in Women's Hospital, School of Medicine, Zhejiang University. Data on the patients' age, clinical stage, WHO classification criteria, antecedent pregnancy, presenting level of human chorionic gonadotropin, courses of chemotherapy required to achieve complete remission, and toxicity related to chemotherapy treatments were collected. RESULTS: Thirty-seven patients with low-risk GTT totally received 137 cycles of MTX between Oct. 1999 and Sep. 2002, 34 patients (91.9%) achieved complete remission. Twenty-nine patients received multiple courses of MTX, complete remission was induced in 26 patients (89.7%). The complete response rates of I stage and III stage were 100.0% and 70.0% (P = 0.03) respectively in patients who were received multiple courses of MTX. However, eight patients received single course of chemotherapy, 7 patients achieved complete remission, and 1 achieved complete remission after another additional course of MTX was conducted. Grade III side effects (WHO criteria) only appeared in 7 courses (5.1%) during MTX treatment. Follow-up data showed that only one patient with single course of chemotherapy relapsed after 6 months. CONCLUSION: Single MTX chemotherapy may be effective and well tolerated for low-risk GTT.

[Mismatch repair gene promoter methylation and expression in hydatidiform moles and the malignant transformation].

OBJECTIVE: In this study, we assayed promoter hypermethylation and protein expression of the mismatch repair gene (MMR) hMLH1 and hMSH2 in gestational trophoblastic diseases to understand the significance of MMR promoter methylation and expression in the pathogenesis and malignant transformation of hydatidiform mole. METHODS: DNA was extracted from chorion of early pregnancies, partial hydatidiform moles, complete hydatidiform moles, and invasive moles were over digested by methylation sensitive endonuclease Hpa II. Then the promoters were amplificated by polymerase chain reaction. The protein was detected by immunohistochemistry. RESULTS: In the normal placenta, neither hMLH1 nor hMSH2 promoter methylation was detected. Expression of hMLH1 and hMSH2 in cytotrophoblasts was strongly positive, and that was negative or weakly positive in syncytiotrophobasts. In all normal chorion, expression of hMLH1 and hMSH2 in cytotrophoblasts was strongly positive. In partial hydatidiform mole and complete hydatidiform mole, the methylation of hMLH1 and hMSH2 promoters was significantly higher than that of early placenta (P < 0.05), and the protein expression in cytotrophoblasts was significantly lower (P < 0.05). In the invasive mole, hMLH1 and hMSH2 promoter methylation were not significantly different as compared with the partial hydatidiform mole and complete hydatidiform mole (P > 0.05). Expression of hMLH1 in the invasive mole (54.5%, 6/11) was not significantly different as compared with the partial hydatidiform mole and complete hydatidiform mole (P > 0.05). But expression of hMSH2 in the invasive mole (36.4%, 4/11) was weaker than that in complete hydatidiform mole (P = 0.044). Promoter methylation and less expression of hMSH2 had correlations in complete hydatidiform mole or invasive mole. CONCLUSIONS: Strong expressions of hMLH1 and hMSH2 in the cytotrophoblasts of normal placenta may keep the genome stability. Promoter methylation and down-regulation of hMLH1 and hMSH2 are probably involved in the pathogenesis of hydatidiform mole.