Hes1/Hes5 gene inhibits differentiation via down-regulating Hash1 and promotes proliferation in cervical carcinoma cells.

INTRODUCTION: Hairy and enhancer of split 1 (Hes1) and Hes5 are target genes for the mammalian Notch pathway, which are highly expressed in epithelia in the process of embryogenesis or in neural stem cells, inhibit cell differentiation via the Notch-Hes-Hash signaling, and promote the survival of stem cells. Either Hes1 or Hes5 overactivation is likely to affect cell differentiation, thereby resulting in carcinogenesis. METHODS: We transfected the diced small interference RNA into SiHa cells and detected cell differentiation and proliferation by immunocytochemistry, Western blot, and methyl thiazolyl tetrazolium assay. RESULTS: Knockdown of Hes1 and Hes5 would up-regulate the downstream gene Hash1, but not the upstream gene Notch1 in the Notch-Hes-Hash pathway. After Hes1/Hes5 RNA interference, expression of differentiation-associated proteins (including Nanog, stage-specific embryonic antigen 4, and tumor rejection antigen-1-60) was reduced, and the cell differentiation was promoted; meanwhile, the cell proliferation was inhibited, which was verified by detecting proliferation-associated proteins (eg, Ki-67, proliferating cell nuclear antigen) and methyl thiazolyl tetrazolium assay. CONCLUSIONS: Our findings suggest that Hes1/Hes5 gene would inhibit the cell differentiation via down-regulating Hash1 and promote the cell proliferation in cervical carcinoma cells; the cell differentiation and proliferation can be reversed simultaneously by Hes1/Hes5 knockdown using RNA interference.

[Study of apoptosis and Fas expression of peritoneal fluid and peripheral blood T lymphocytes in patients with epithelial ovarian cancer and their relationship with CA125].

OBJECTIVE: To investigate the apoptosis and Fas (CD95) expression of T lymphocytes from the peripheral blood and peritoneal fluid of the patients with ovarian cancer and their relationship with CA125. METHODS: Apoptosis and Fas expression of peritoneal fluid and peripheral blood T lymphocytes were assessed by flow cytometry. Peripheral blood samples were obtained from the following objects respectively: patients with stage III - IV ovarian cancer (n = 18) before and after treatment, patients with stage I - II ovarian cancer (n = 15), patients with benign ovarian tumor (n = 18), patients with Krukenberg tumor (n = 6) and normal control (n = 20). Peritoneal fluids were obtained from all the patients with ovarian cancer, Krukenberg tumor and ten patients with benign ovarian tumor. Level of serum CA125 of the patients with ovarian cancer was assessed. RESULTS: In the patients with stage III - IV ovarian cancer, the apoptosis level of the peripheral blood T lymphocytes was 5.55 (3.57 - 9.62)%, significantly higher than those from the patients with stage I - II ovarian cancer, patients with benign ovarian tumor, controls (P < 0.008 in all instances) and the patients with stage III - IV ovarian cancer after treatment (P < 0.05). The intensity of Fas expression of the peripheral blood T lymphocytes from the patients with stage III - IV ovarian cancer was 51 +/- 10, significantly higher than that from controls (P < 0.05). In peritoneal fluid, the apoptosis rates of T lymphocytes, positive rate and intensity of Fas expression on T lymphocytes from patients with stage I - II and stage III - IV ovarian cancer were 17.41 (7.06 - 24.56)%, (57 +/- 16)%, (55 +/- 11)% and 34.06 (17.03 - 44.65)%, (66 +/- 12)%, (70 +/- 24)%, respectively, increased significantly compared with those from patients with benign ovarian tumor, which were 0.78 (0.67 - 1.44)%, (37 +/- 6)%, 43 +/- 6, respectively (P < 0.01 in all instances). The apoptosis level and positive rate of Fas expression on peritoneal fluid T lymphocytes from patients with stage III - IV ovarian cancer were significantly higher than those from patients with Krukenberg tumor (P < 0.01). There was a positive correlation between the serum CA125 level and the apoptosis level of peritoneal fluid T cell in the patients with stage I - II ovarian cancer (r = 0.77, P = 0.009). For ovarian cancer, the apoptosis level of peritoneal fluid T lymphocytes from patients with the serum CA125 > 500 KU/L was higher than that from the patients with the serum CA125 < or = 500 KU/L (P = 0.009). CONCLUSIONS: (1) Extraordinarily increased apoptosis of T cells may play an important role in the development of systemic and celiac immunodeficiency in the patients with ovarian cancer. In contrast with the patients with Krukenberg tumor, the patients with advanced ovarian cancer hare higher percentage of apoptotic peritoneal fluid T lymphocytes, which shows the particularity of local immunity defect. (2) For the patients with ovarian cancer, efficient treatment can decrease the percentage of apoptotic peripheral blood T lymphocytes. (3) The increased positive rate and intensity of Fas expression on peritoneal fluid T lymphocytes stressed the significance of Fas interference in the treatment of ovarian cancer. (4) Level of serum CA125 can reflect the celiac immunity defection in patients with ovarian cancer.

Fas-mediated pathway and apoptosis in normal cervix, cervical intraepithelial neoplasia and cervical squamous cancer.

The aim of this study was to examine the expression patterns of apoptosis-related antigens, such as Fas, FasL, and cFLIP, in cervical squamous cells, and investigate the role of Fas-mediated apoptosis in the pathogenesis of cervical neoplasia. Using specific antibodies for Fas, FasL, and cFLIP, we examined protein expression in 19 specimens of normal cervix, 15 mild dysplasia (CIN I), 22 moderate dysplasia (CIN II), 23 severe dysplasia (CIN III), and 34 invasive squamous cell carcinoma (SCC) by immunohistochemistry. We detected the apoptotic indices by TUNEL in the same specimens. Fas expression levels were quite similar in CIN I, CIN II and the normal cervix. Though Fas expression tended to increase in grade 2 cancer compared to grade 1 cancer and CIN III, and a slight decline was present in grade 3 compared with grade 2 cancer, these differences did not reach statistical significance. Almost all CINs did not express FasL, while FasL expression increased with the grade of the tumor. Statistically significant differences could be observed between grade 1 and grade 2 (p<0.01) and between grade 2 plus grade 3 and grade 1 (p<0.001). All cases of normal cervix and CIN, except two cases of CIN III, did not express cFLIP. cFLIP expression tended to increase with the grade of the tumor. Apoptosis was determined in all samples by TUNEL. There was a decreasing tendency of apoptotic cells from normal cervix to cancers. A negative correlation between cFLIP and apoptosis (r=-0.499 and p=0.000) was observed. Deregulated Fas/FasL system and constitutive expression of cFLIP in SCC may be an important mechanism by which SCCs escape apoptosis during the malignant transformation and progression of these tumors.

Altered expression of cellular membrane molecules of HLA-DR, HLA-G and CD99 in cervical intraepithelial neoplasias and invasive squamous cell carcinoma.

OBJECTIVE: The aim of this study was to investigate the role of HLA-DR, HLA-G and CD99 during cervical carcinogenesis and to examine the prognostic significance of these protein expressions in invasive squamous cell carcinoma (SCC). METHODS: Using specific antibodies for HLA-DR, HLA-G and CD99, we examined protein expressions in 19 normal cervix, 15 mild dysplasia (CIN I), 22 moderate dysplasia (CIN II), 23 severe dysplasia (CIN III), and 34 invasive squamous cell carcinoma by immunohistochemistry. And we detected the expression of Ki67 in the same specimens. RESULTS: None of normal cervix and CINs except three cases of CIN III expressed HLA-DR. HLA-DR expression increased progressively with the grade of the tumor, and significant differences could be observed between grade 1 and grade 2 (P<0.01) and between grade 1 and grade 3 (P<0.05). In all normal epithelial control samples, HLA-G expression was seen in ectocervical squamous and endocervical columnar epithelium and the staining was strong and uniform. Only a small proportion of CINs and SCCs showed reduced expression of HLA-G. Compared with the results in the control samples, CINs and SCCs showed significantly reduced expression of HLA-G (P<0.001). SCCs showed significantly increased expression of CD99 when compared with normal cervix and CINs (P<0.05). Ki67 was expressed in all specimens. Significant differences were observed between CINs and normal cervix (P<0.001) and SCCs and controls (P<0.001), but no significant differences could be observed between SCCs and CINs. None of the expressions of these proteins was associated with any of clinicopathological parameters. CONCLUSIONS: These results indicate that increased expression of HLA-DR and CD99 may be related to the evolution of cervical cancer. All protein expressions were not associated with clinicopathological parameters.

[The accuracy of diagnosing cervical intraepithelial neoplasia with colposcopically directed biopsy].

OBJECTIVE: To evaluate the accuracy of colposcopically directed biopsy in the diagnosis of cervical intraepithelial neoplasia (CIN). METHODS: The clinical data of 153 patients with CIN diagnosed by colposcopically directed biopsy and treated with cervical loop electrosurgical procedure (LEEP) shortly after were retrospectively studied. The consistency of pathological examination between colposcopically directed biopsy and LEEP was evaluated. The factors of missed diagnosis of cervical invasive carcinoma were analyzed. RESULTS: The diagnosis of 106 out of the 153 patients by colposcopically directed biopsy was consistent with that by LEEP as confirmed by pathologic diagnosis with a consistence rate of 69.3%, however, the diagnoses of 47 of the 153 patients by colposcopically directed biopsy were not consistent with those by LEEP, with an inconsistent rate of 30.7%, including 22 cases of overdiagnosis (14.4%, 22/153) and 25 cases of underdiagnosis (16.3%, 25/153). Eighteen patients were confirmed as with cervical invasive carcinoma by LEEP/hysterectomy at last with a missed diagnosis rate of 11.8%. The missed diagnosis rate of invasive carcinoma by colposcopically directed biopsy was significantly higher in the patients with cytologic diagnosis of with > or = HSIL than in the patients cytologically diagnosed as with < HSIL (chi(2) = 8.208, P < 0.05). CONCLUSION: The accuracy of diagnosing CIN with colposcopically directed biopsy is still unsatisfying. Attention should be paid on the patients with cytological diagnosis of > or = HSIL so as to avoid missed diagnosis of cervical carcinoma.

[Evaluation of neoadjuvant chemotherapy for locally advanced cervical cancinoma].

OBJECTIVE: To investigate the effect of neoadjuvant chemotherapy on locally advanced cervical cancer, the factors affecting outcome of chemotherapy and the long-term survival rate in the patients. METHODS: A total of 64 patients with stageIb2-IIb of locally advanced cervical cancers treated with neoadjuvant chemotherapy between June 1999 and October 2004 in Women's Hospital, School of Medicine, Zhejiang University were retrospectively analysed. The effect of chemotherapy, as well as survival rate was evaluated. The related factors of effect, including age, histology, clinical stage, grade of differentiation, initial tumor size, chemotherapy regimens, number of courses were analysed. RESULTS: Overall effective rate of neoadjuvant chemotherapy was 80%. The effective rate was associated with histology. The patients with squamous cell cancer had significantly higher 5-year survival rate than those with adenocarcinoma (82% vs 6/9, P < 0.05). The rates of positive pelvic lymph node metastasis and parametrial invasion were significantly higher in complete or partial response patients than those in stable patients (P < 0.05). The overall 5-year survival rate was 89%. The 5-year survival rate in complete or partial response group was 100%, in stable disease group was 46% (P < 0.05). The 3, 5-year disease-free survival rate in complete or partial response group was 95% and 83%, respectively, while in stable disease group was 33% and 0, respectively. The disease-free survival rates in complete or partial response group were higher than those of stable disease group (P < 0.05). CONCLUSIONS: The clinical response to neoadjuvant chemotherapy is related to histology. The patients who have good response to chemotherapy should choose surgery. Chemotherapy can decrease the high risk factors of pathology in complete or partial response patients. Neoadjuvant chemotherapy for locally advanced cervical cancer patients seems to improve the long-term survival rate.

[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.

[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.

[Tumor lymphangiogenesis in cervical squamous cell carcinoma and its clinical significance].

OBJECTIVE: To investigate the clinical significance of lymphangiogenesis in cervical cancer. METHODS: Monoclonal podoplanin was used to immunostain the lymphatic microvessels in the paraffin sections of cervical squamous cancer tissues at the I(b) and II(a) stages from 35 cases kept in the archives. Computer-assisted morphometric analysis was used to quantitatively analyze the lymphatic vessels in intratumor and peritumor areas. The association with clinicopathologic data was analyzed. RESULTS: (1) The lymphatic microvessels were larger and denser in the peritumor areas than the intratumor areas. Cancer cells were found in the intratumoral lymphatic vessels of the lymph metastatic patients. (2) The lymphatic vessel density (LVD) of the peritumor areas was (31 +/- 10) vessels/mm(2), significantly greater than that in the intratumor areas [(20 +/- 10) vessels/mm(2), P < 0.01]. The relative lymphatic vessel area (LVA) in the peritumor areas was (0.75 +/- 0.40)%, significantly larger than that of the intratumor areas [(0.19 +/- 0.11)%, P < 0.01]. (3) The intra- and peri-tumoural LVD and LVA in the patients with lymph node metastasis were (29 +/- 7) vessels/mm(2) and (40 +/- 5) vessels/mm(2), and 0.27% +/- 0.10% and 1.23% +/- 0.36% respectively, all greater than those of the patients without lymph node metastasis [(16 +/- 8) vessels/mm(2) and (28 +/- 9) vessels/mm(2), and (0.16 +/- 0.09)%, (0.56 +/- 0.20)% respectively, P < 0.01, < 0.01, < 0.05, and < 0.01]. (4) The peri-tumoral LVA and intra-tumoral LVD and LVA in the patients with histological grade I, II, and III were (0.42 +/- 0.10)%, (9 +/- 3) vessels/mm(2), and (0.06 +/- 0.04)%; (0.77 +/- 0.37)%, (21 +/- 8) vessels/mm(2), and (0.21 +/- 0.09)%; and (0.83 +/- 0.46)%, (22 +/- 11) vessels/mm(2), and (0.21 +/- 0.12)% respectively. There were significant differences among the values of the grade II patients and grade I patients (all P < 0.05) and the grade III patients and grade I patients (all P < 0.05) and there were not significant differences between the values of the grade II patients and the grade III patients (all P > 0.05), III and grade I, no statistic significances between grade II and III. CONCLUSION: Higher lymphangiogenesis in early cervical squamous cell cancer may be associated with the risk of lymph node metastasis.

[Analysis of prognostic factors in patients with cervical squamous cell carcinoma of stage Ib and IIa].

OBJECTIVE: To analyze the prognostic factors in patients with cervical squamous cell carcinoma of stage Ib and IIa treated by surgery, and to investigate their guide roles in available post-operation adjuvant therapy. METHODS: The clinicopathologic records of 306 patients with cervical squamous cell carcinoma of stage Ib and IIa who underwent radical hysterectomy and pelvic lymphadenectomy were retrospectively analyzed, and the prognostic factors were explored by univariate and multivariate methods. Independent prognostic factors were identified by COX proportional hazards regression model. RESULTS: The overall 5-year survival rate of these 306 patients was 78.1%. In univariate survival analysis, the poor prognostic factors included poor differentiation, positive pelvic lymph nodes, deep stromal invasion, parametrial extension, tumor size > or = 4 cm, and lymph vascular space involvement (P < 0.05). While in multivariate survival analysis, the independent prognostic factors included positive pelvic lymph nodes, deep stromal invasion and parametrial extension (P < 0.05). According to the risk factors, all patients were divided into low, intermediate and high risk groups with 5-year survival rate of 90.3%, 83.9% and 43.1%, respectively. In low risk group, no risk factor or only parametrial extension was involved, pelvic recurrence rate was only 2.2%. In intermediate risk group, deep stromal invasion or together with parametrial extension was involved. Pelvic recurrence rate and extra pelvic metastasis rate were 13.5% and 1.3%, respectively. In high risk group, lymph node metastasis or together with other high risk factors was involved, pelvic recurrence rate and extra pelvic metastasis rate were 25.9% and 48.3%, respectively, and 10.3% had both pelvic recurrence and extra pelvic metastasis. CONCLUSIONS: Lymph node metastasis, deep stromal invasion and parametrial extension were independent prognostic factors of stage Ib and IIa cervical squamous cell carcinoma patients undergoing radical hysterectomy and lymphadenectomy. The establishment of prognosis-predicting system based on high risk factors may be helpful to predict the risk of recurrence and metastasis, and guide adjuvant therapy after surgery.

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.

[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.

[Ovarian carcinoma cell inhibits T cell JAK-STAT signal transduction pathway, an experimental study].

OBJECTIVE: To investigate the effect of ovarian carcinoma cell on T cell JAK-STAT signal transduction pathway and its role in the ovarian carcinoma induced immunosupression. METHODS: Human ovarian carcinoma cells of OVCAR3. CAOV3, and SKOV3 lines were cultured. CD8(+) T cells were isolated from the peripheral venous blood of healthy persons. Then the supernatants of these ovarian carcinoma cell lines and RPMI-1640 were added into the culture of CD8(+) T cells (groups I, II, III, and control). Thiazolyl blue (MTT) method was used to detect the growth of CD8(+) T cell. The cell cycle was examined by flow cytometry. The secretion of the Tc1 type cytokine interferon (IFN)-gamma mRNA and the secretion of the Tc2 type cytokine interleukin (IL)-10 mRNA were detected by RT-PCR. The expression of signaling molecules JAK and JAK3 and the phosphorylated activation of STAT3 and STAT5 in the CD8(+) T cell were analyzed by Western blotting. RESULTS: The absorbance at the wavelength 570 nm of CD8(+) T cell culture was 0.23 +/- 0.03, 0.28 +/- 0.06, and 0.29 +/- 0.05 in the group I, II, and III, all significantly lower than that in the group IV (0.79 +/- 0.07, all P < 0.01). The percentages of CD8(+) T cells at the stage S and stage G(2)/M were lower, and those in stage G(1)/G(0) were higher in groups I, II, and III than in group IV (all P < 0.01). The IFN-gamma expression was significantly lower in groups I, II, and III in comparison with that in group IV. However, the expression of IL-10 was significantly higher in groups I, II, and III in comparison with that in group IV. The expression of JAK3 protein, but not JAK1 protein, was significantly lower in groups I, II, and III in comparison with that in group IV. The phosphorylated activation of STAT5 was suppressed significantly in groups I, II, and III, whereas the phosphorylated activation of STAT3 was suppressed only in group I. CONCLUSION: Ovarian carcinoma may suppress T cell proliferation through inhibition of the JAK-STAT signal transduction pathway, which may be a mechanism of ovarian carcinoma induced immunosuppression.

[Down-regulation of vascular endothelial growth factor expression in ovarian cancer cell line SKOV3 by interferon-gamma].

OBJECTIVE: To determine whether interferon-gamma (IFN-gamma)inhibit the expression of vascular endothelial growth factor (VEGF) in ovarian cancer cell line SKOV3 with the level of mRNA and protein. METHODS: Cultured SKOV3 was treated by IFN-gamma with different concentration and time. The morphological changes of SKOV3 treated by IFN-gamma through microscope and proliferation by methyl thiazolyl tetrazolium (MTT). The expression of VEGF mRNA in SKOV3 culture was determined by relative quantitative reverse-transcription polymerase chain reaction. The VEGF protein level in supernatants was determined by enzyme-linked immunosorbent assay (ELISA) and in cytoplasma by immunocytochemical staining. RESULTS: There is a small changes in shape on SKOV3, but no alter of cell proliferation during treating with different concentration and time. Expression of VEGF mRNA and protein decreased with the INF-gamma concentration increasement, but no time-effect pattern exist. The effect peak appeared when the concentration was 1,000 U/ml. CONCLUSION: IFN-gamma can inhibit the expression of VEGF in ovarian cancer cell line SKOV3 with the level of mRNA and protein.

[Study on transfer of interleukin-7 gene into ovarian carcinoma cells in vitro].

OBJECTIVE: To investigate proliferative activity, expression of cell surface antigens and cytokines of ovarian carcinoma cells transferred by interleukin-7 (IL-7) gene, and its cytotoxic sensitivity to lymphokine activated killer (LAK) cells in vitro through gene transfection technique. METHODS: Ovarian carcinoma cell line SKOV3 were cultured and transferred by IL-7 cDNA. The proliferation activity of IL-7 gene transferred and nontransferred SKOV3 was observed with inverse phase-contrast light microscope, thiazoyl blue tetrazolium bromide assay and flow cytometry. The expressions of cell surface antigen human leucocyte antigen-ABC (HLA-ABC), human leucocyte antigen-DR (HLA-DR) and intercellular adhesion molecule-I (ICAM-I) were detected by indirect targeted FITC- flow cytometry. The secretions of IL-2, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor (TGF-beta(1)) were assayed by enzyme linked immunosorbent assay. The cytotoxic sensitivity against LAK was detected by LDH release method. RESULTS: The morphology, proliferation, cell cycle distributions and HLA-ABC, HLA-DR expression of IL-7 gene transferred SKOV3 didn't change after IL-7 gene transfer. The expression of ICAM-I in IL-7 gene transferred SKOV3 significantly increased and TGF-beta(1) secretion of IL-7 gene transferred SKOV3 significantly decreased. The cytotoxic sensitivity of IL-7 gene transferred SKOV3 to LAK significantly elevated. CONCLUSION: Ovarian carcinoma cell line SKOV3, after transferred by IL-7 gene, remains unchanged proliferation and apoptosis, upregulation of ICAM-I, downregulation of TGF-beta(1) and increases the cytotoxic sensitivity to LAK in vitro.

[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.

[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.

[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.

[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.

[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.