Expression of Cell-Cycle Mediators in Ovarian Cancer Cells after Transfection with p16, p21, and p53

Objective.The purpose of this study was to determine whether transfection of ovarian cancer cell lines with recombinant adenoviral vectors containing wild-type p16^INK4a, p21^WAF1/Cip-1, and p53 caused growth inhibition and induction of apoptosis. We also measured the expression of the cell-cycle mediators Bax, Bcl-2, pRb, and mdm-2.Methods. We introduced the wild-type p16^INK4a, p21^WAF1/Cip-1, and p53 genes into the ovarian cancer cell lines SK-OV-3 (p16^INK4a and p53 null) and OVCA-420 (p16^INK4a and p53 wild-type) by adenoviral transfection. Cell growth inhibition was measured over a 10-day period. Induction of apoptosis was tested for both cell lines 48 h after cell transfection. Expression of cell-cycle mediators was evaluated by Western blot analysis and densitometry.Results. Growth inhibition was documented after transfection with p16^INK4a, p21^WAF1/Cip-1, and p53 in both SK-OV-3 cells and OVCA-420 cells. Apoptosis was greatest in SKOV-3 cells after transfection with p53. A significant expression of Bax was only seen in the SKOV-3 cells transfected with p53. The bcl-2 protein was poorly expressed in both cell lines. Expression of pRb was suppressed in OVCA-420 cells transfected with p16^INK4a and p21^WAF1/Cip-1. Infection with Adp16^INK4a and Adp53 led to an increase in the level of mdm-2 in the SK-OV-3 cell line only.Conclusions. In the ovarian cancer cell lines studied, cell growth was inhibited after transfection with p16^INK4a, p21^WAF1/Cip-1, and p53. Cell cycle arrest was highest with p53 transfection. The expression of pro-apoptosis proteins was primarily a function of p53 expression.     

The association of PTPN22 polymorphism with endometriosis: effect of genetic and clinical factors

Objective

To investigate the possible effect of clinical and genetic variables on the association between PTPN22 and endometriosis.

Methods

PTPN22, ACP₁ and p53 codon 72 genetic polymorphisms and duration of previous pharmacological treatment were studied. The study sample consisted of 132 women hospitalized for endometriosis diagnosed by laparoscopic intervention and histologically confirmed: 359 healthy blood donors were studied as controls. PTPN22, ACP₁ and p53 codon 72 genotypes were determined by DNA analysis. Discriminant statistical analysis, logistic regression analysis, chi square of independence, power test and linear correlation were performed using SPSS programs.

Results

A significant increase of PTPN22 *T allele in endometriosis is observed in women carrying ACP₁*C allele, in women carrying p53 codon 72 *Pro allele and in women with prolonged pharmacological treatment.

Conclusions

PTPN22 may not be a primary factor in the etiology of endometriosis but may cooperate with clinical and genetic factors influencing susceptibility and clinical course of disease. These new observations point to a multifactorial origin of endometriosis and help to explain the reported differences between human populations concerning the association between PTPN22 and endometriosis.     

p53 mutations and overexpression affect prognosis of ovarian endometrioid cancer but not clear cell cancer

OBJECTIVE: Although ovarian clear cell adenocarcinoma (OCCA) and ovarian endometrioid adenocarcinoma (EC) are considered to be closely related to endometriosis, the mechanisms of carcinogenesis of these two malignancies and malignant transformation of endometriosis are unclear. In this study, we examined the biology of OCCA and EC by performing large-scale analysis of K-ras activation and p53 mutation and overexpression in these malignancies. The results were subsequently analyzed for correlation with the clinicopathologic data. METHODS: In the present study of OCCA and EC, we obtained clinicopathological data and analyzed frequency of mutations and overexpression of K-ras and p53. DNA was extracted from formalin-fixed, paraffin-embedded tissue, and target sequences were amplified in vitro by polymerase chain reaction. The DNA was analyzed for K-ras and p53 mutations by testing for single-strand conformation polymorphisms and by direct sequencing. Immunohistochemical staining was performed using p53 monoclonal antibody. Univariate analysis was performed using the Kaplan-Meier algorithm, and differences in survival were analyzed using the log rank test. The prognostic significance of the studied variables for survival was assessed using multivariate analysis with Cox regression analysis. RESULTS: K-ras mutation was detected in 16.2% (6/37) of OCCA patients and 3.7% (1/27) of EC patients. No evidence of p53 mutation was detected in OCCA patients, but p53 mutation was detected in 63.0% of EC patients; these findings are consistent with the results of p53 immunohistochemistry. No statistical significance was observed for K-ras mutation in OCCA or EC. In EC patients, the absence of endometriosis and p53 overexpression was associated with a poorer survival. In OCCA patients tubulocystic and papillary histotype as well as stage II correlated with a worse survival. CONCLUSIONS: p53 mutation, which was found in 63% of EC tumors, is an independent prognostic factor for EC patients. However, no p53 mutation was found in OCCA tumors. K-ras mutations did not affect survival of OCCA or EC patients.     

Baseline characteristics and prevalence of HPV 6, 11, 16, 18 in young German women participating in phase III clinical trials of a quadrivalent HPV (6/11/16/18) vaccine

Introduction

As limited data among German women exist about HPV, Chlamydia trachomatis (CT) and Neisseria gonorrhoeae, we report the prevalence of these genital infections and general baseline demographics of the young German women enrolled in the phase III trials of the quadrivalent HPV vaccine.

Materials and methods

German females (n = 437; 9–23 years) were recruited among 3 international phase 3 studies of an HPV-6/11/16/18 vaccine. We present baseline characteristics, prevalence of HPV-6/11/16/18 and, for women aged 16–23, abnormal cervical cytology and sexually transmitted diseases.

Results

Chlamydia trachomatis and Neisseria gonorrhoeae prevalence was 5 and 0.3%, respectively. Approximately 17% of participants had HPV-6, 11, 16, or 18 DNA or antibodies. All subjects <17 years were naïve to the four vaccine types.

Discussion

The results of the vaccine trials have demonstrated that it is worth administering prophylactic HPV vaccines before sexual debut; however, none of these sexually active German women were positive to all four types and most were positive to only one type. Thus, all women had the potential to benefit from vaccination with a quadrivalent HPV vaccine.     

PTENMutations in Endometrial Cancers with 10q LOH: Additional Evidence for the Involvement of Multiple Tumor Suppressors

Objective.The aim of this study was to assess the involvement ofPTENand other putative 10q tumor suppressors in endometrioid-type adenocarcinomas characterized by loss of 10q sequences.Methods.PCR-based single-stranded conformational variant analysis and sequencing of individualPTENexons in 34 tumor specimens and their corresponding normal DNA were used.Results.Thirteen of the 34 tumors (38%) revealed aPTENmutation: 2 frameshift, 3 nonsense, 3 missense, 3 splice site alterations, and 2 homozygous deletions.Conclusion.The observation that greater than 60% of endometrial cancers with 10q LOH lackPTENmutations, in addition to previously reported LOH data, provides evidence for the existence of other tumor suppressors on 10q. Consideration ofPTENmutation status may prove important in deletion mapping studies to locate additional tumor suppressors on the long arm of chromosome 10.     

Cell proliferation and apoptosis in BRCA-associated hereditary ovarian cancer

OBJECTIVE: The goal was to test the hypothesis that cellular growth properties differ between hereditary and sporadic ovarian cancers. METHODS: Cell proliferation and apoptosis were assessed in 67 tumors associated with deleterious germline BRCA mutations (hereditary) and 69 tumors without BRCA mutations (sporadic). Cell proliferation was evaluated by immunohistochemical analysis of Ki-67 expression, and apoptosis was assessed using a TUNEL assay. RESULTS: The mean number of Ki-67-immunopositive nuclei was significantly higher in ovarian cancers from the hereditary group compared with those from the sporadic group (P = 0.017). Cell proliferation did not differ significantly between BRCA1- and BRCA2-associated hereditary tumors, and apoptosis did not differ significantly between the hereditary and sporadic tumors. CONCLUSION: These data indicate that ovarian carcinomas associated with germline BRCA mutations have a significantly higher growth fraction than sporadic cancers. This property may contribute to an improved response to cytotoxic chemotherapy, partially accounting for the longer recurrence-free interval and overall survival observed in the hereditary group.     

Emergence of cisplatin-resistant cells from the OVCAR-3 ovarian carcinoma cell line with p53 mutations, altered tumorigenicity, and increased apoptotic sensitivity to p53 gene replacement

Abstract. Mujoo K, Zhang L, Klostergaard J, Donato NJ. Emergence of cisplatin-resistant cells from the OVCAR-3 ovarian carcinoma cell line with p53 mutations, altered tumorigenicity, and increased apoptotic sensitivity to p53 gene replacement.
Resistance to chemotherapy commonly compromises the treatment of many advanced cancers. Evidence suggests a correlation between chemoresistance and more aggressive tumor growth, possibly through accumulation of additional genetic defects in drug-treated or resistant cells. To study this process in a human ovarian cancer model, we examined OVCAR-3 cells for acute sensitivity to cisplatin (cDDP) and subsequent emergence of drug-resistant clones following chronic cDDP exposure. Clonal cells (OVCAR-3/C-1) that displayed 20-fold reduced sensitivity to cisplatin but retained equivalent sensitivity to paclitaxel, as compared with the parental population, were isolated. The cDDP-resistant clone had growth kinetics similar to those of parental population, but when transplanted into the peritoneal cavity of nude mice, they acquired the ability to grow with the development of both ascites and solid tumor masses; such growth was not detectable after transplantation of the drug-sensitive parental cell line. C-1 cells had a p53 gene mutation (codon 266) that was not detected in the parental OVCAR-3 cell line, and infection of C-1 cells with p53-adenovirus (rAd-p53) caused greater apoptosis and gene transduction than that observed in the similarly infected parental population. rAd-p53 induced high levels of p21WAF1, p27Kip1, activated caspase 3 and apoptosis in C-1 cells, without causing major changes in bax or bcl-X_L levels. Together, the results suggest that alterations in tumor growth and gene mutations characterize cDDP-resistance in OVCAR-3 cells, and viral replacement of one of these defective genes (p53) may provide an effective treatment for elimination of drug-resistant cells.     

Triaging Pap cytology negative, HPV positive cervical cancer screening results with p16/Ki-67 Dual-stained cytology

Objective

Testing for human papillomavirus (HPV) has been shown to increase the sensitivity and negative predictive value for detection of high-grade cervical intraepithelial neoplasia (CIN2+), either when used in conjunction with Pap cytology testing or alone. However, there is no satisfying clinical management algorithm for women testing Pap negative/HPV positive. We therefore evaluated the clinical utility of a novel dual biomarker-based approach (p16/Ki-67 Dual-stained cytology) for the identification of CIN2+ in women with Pap negative/HPV positive screening results, without the need to refer all women to immediate colposcopy.

Methods

All women aged ≥ 30 enrolled during 2007/2008 into a regional prospective Pap/HPV co-testing screening pilot project and tested Pap negative, but positive for HPV (n = 425) were included in the analysis. p16/Ki-67 Dual-stained cytology was performed from residual cellular material available from the liquid-based cytology vial collected during the initial Pap/HPV co-testing screening visit. Results were correlated to the presence of CIN2+ confirmed during preliminary follow-up.

Results

p16/Ki-67 Dual-stained cytology tested positive at baseline in 108 out of 425 (25.4%) Pap negative/HPV positive cases. Sensitivity of Dual-stain testing for the detection of biopsy-confirmed CIN2+ during preliminary follow-up within the group of Pap negative/HPV positive women was 91.9% for CIN2+ (34/37 cases), and 96.4% for CIN3+ (27/28 cases). Specificity was 82.1% for CIN2+ on biopsy, and 76.9% for CIN3+, respectively.

Conclusions

Triaging Pap negative/HPV positive screening test results with p16/Ki-67 Dual-stained cytology may identify women with a high probability of underlying CIN2+ and may efficiently complement HPV-based screening programs to prevent cervical cancer.     

Association of TP53 codon 72 polymorphism with susceptibility to ovarian carcinomas in Serbian women

Objectives

Finding a potential genetic factor associated with a deadly disease, such as ovarian carcinoma, is of particular importance. The aim of this study was to examine the role of the TP53 codon 72 polymorphism in ovarian carcinoma development in Serbian women.

Study design

47 wild-type TP53 gene ovarian carcinoma samples and 70 cervical smears from gynecologically healthy women were analyzed. DNA was extracted by a salting-out procedure. Codon 72 polymorphism was assessed by PCR-RFLP method. χ², Fisher exact test and odds ratio were used for statistical analysis.

Results

The distribution of Arg/Arg, Arg/Pro and Pro/Pro genotypes of codon 72 of the TP53 gene was: 46.8%, 46.8% and 6.4%, respectively in the ovarian carcinomas and 64.3%, 31.4% and 4.3%, respectively in the control group. We observed an increased risk for the development of ovarian carcinoma for Pro homozygotes in relation to heterozygotes plus Arg homozygotes (OR = 1.52; 95% CI 0.29–7.89) and a higher one for Pro/Pro plus Arg/Pro genotype in relation to Arg homozygotes (OR = 2.04; 95% CI 0.96–4.34).

Conclusion

The results showed no association between codon 72 TP53 gene polymorphism and risk for development of ovarian carcinoma in Serbian women. However, this observation requires further analysis of a larger case–control study group.     

Histology of Prophylactically Removed Ovaries from BRCA1 and BRCA2 Mutation Carriers Compared with Noncarriers in Hereditary Breast Ovarian Cancer Syndrome Kindreds

Objective. The literature reports conflicting studies claiming premalignant histological features in benign ovaries from women who may have hereditary predilections for ovarian carcinoma. To test the veracity of these claims, this investigation studied ovaries prophylactically removed from members of hereditary breast ovarian cancer (HBOC) syndrome families who carry BRCA1 and BRCA2 mutations and compared these with the ovaries of mutation-negative women from the same HBOC syndrome kindred.Methods. Sixty cases of women from HBOC syndrome families who had undergone prophylactic oophorectomies and whose BRCA1 and BRCA2 mutation status had been tested were selected from our database. Thirty had tested positive for BRCA1 mutations, 3 carried BRCA2 mutations, and 27 were negative for both BRCA1 and BRCA2 germline mutations. Histologic material from each case was examined by light microscopy blinded to the mutation status. Histologic features, previously reported to be possible precursor lesions for ovarian cancer, were quantified. Data from BRCA1 and BRCA2 mutation carriers were compared with those from mutation-negative cases in the direct line of genetic inheritance from the same HBOC syndrome families.Results. Statistical analysis found that a more frequent occurrence of ovarian surface micropapillae in 87% of mutation carriers compared with just 55% of mutation-negative cases was the only histologic feature which was significantly different between the two groups (P = 0.39). Cortical clefts tended to be deeper in the ovaries of mutation carriers, but this did not reach significance (P = 0.051). There were no other significant histologic differences between the ovaries removed from mutation carriers and those from noncarriers.Conclusions. The results of our large and prospectively controlled, blinded study contrast with those reported from smaller, unblinded investigations. Except for the possible biological significance of surface micropapillae on ovaries from BRCA1 and BRCA2 mutation carriers, we found no histologic evidence for a genetically determined ovarian carcinoma precursor lesion.     

In vivo expression and antitumor activity of p53 gene transfer with naked plasmid DNA in an ovarian cancer xenograft model in nude mice

INTRODUCTION: Abnormalities in the p53 and p16 tumor suppressor genes are one of the most common occurrences associated with human neoplasia. Consequently, restoration of wild-type p53 or p16 functions is seen as a particularly promising approach for cancer gene therapy. In vitro and in vivo data have demonstrated that virus-mediated p53 gene transfer can induce active cell death and ovarian tumor regression. AIM: To evaluate the efficiency of intratumoral injection of naked DNA in tumor growth inhibition in an ovarian xenograft model. For that purpose, plasmid vectors encoding wild-type p53 (wt-p53) or p16 alone or in combination were used. METHODS: Nude mice were injected subcutaneously with the human ovarian adenocarcinoma cell line SKOV3. Three weeks after xenograft, tumor-bearing mice were injected twice a week with plasmid vectors carrying WT-p53 and/or WT-p16 cDNA. Empty plasmids and saline buffer were used as control. Tumor growth was monitored to evaluate the inhibition potential with p53 and/or p16 restoration. RESULTS: When compared to the control, intratumoral repeated injections of naked plasmid DNA encoding wt-p53 were inhibiting tumor growth. This inhibition was not observed with p16 and no synergy could be obtained between p53 and p16. p53 expression was restored in 84% of mice injected with plasmid encoding wt-p53. p16 expression was restored in 63% of mice injected with plasmid encoding p16. CONCLUSIONS: In this report we demonstrated that: (i) naked DNA represents an efficient gene transfer in the SKOV3 xenograft model; (ii) restoration of wt-p53 gene allows tumor growth inhibition; and (iii) this inhibition could be correlated with p53 expression as seen in 84% of treated mice after repeated naked DNA injections. These results allow us to envisage naked DNA as a therapeutic adjuvant in ovarian cancer treatment, concomitantly with tumor resection and chemotherapy.     

卵巢的子宫内膜样癌临床情况及DNA倍体分析与p~(53)基因蛋白增殖细胞核抗原表达测定的研究

目的：研究卵巢的子宫内膜样癌ｐ５３基因蛋白、增殖细胞核抗原（ＰＣＮＡ）的表达与ＤＮＡ倍体分析之间的相互关系，并对其与临床分期、病理学分级与残存瘤的关系等进行分析、比较。方法：应用流式细胞技术及ｐ５３、ＰＣＮＡ单克隆抗体免疫组化技术对卵巢的子宫内膜样癌１３例进行倍体分析及基因、抗原测定。结果：ＤＮＡ异倍体率为７８％，ｐ５３基因蛋白表达率为４６％，ＰＣＮＡ表达率为９２％。ＤＮＡ倍体分析与临床Ⅱ～Ⅳ期、病理２～３级及残存瘤＞２ｃｍ者有关，异倍体阳性率分别为９０．９％、８８．８％、８７．５％，明显高于临床Ⅰ期、病理１级及无残存瘤者。ｐ５３在临床Ⅱ～Ⅳ期、病理２～３级表达率分别为６８．６％和５５．５％，均高于临床Ⅰ期及病理１级者，并对ＤＮＡ倍体水平与ｐ５３表达之间的关系进行了探讨。结论：ＤＮＡ异倍体与ｐ５３基因蛋白表达阳性可作为卵巢的子宫内膜样癌恶性程度的重要指标。     

Programmed cell death (apoptosis) as a possible pathway to metalloproteinase activation and fetal membrane degradation in premature rupture of membranes

OBJECTIVE: Increased matrix metalloproteinase 2 expression and activity are associated with premature rupture of fetal membranes. A proapoptotic protein produced in response to deoxyribonucleic acid fragmentation, p53, can bind to the matrix metalloproteinase 2 gene promoter and cause increased gene expression. It promotes apoptosis by inducing the expression of the proapoptotic bax gene and inhibiting the antiapoptotic bcl-2 gene. This study was undertaken to investigate the expression pattern of apoptotic elements in pregnancy complications that may cause increased expression of the gene for matrix metalloproteinase 2. STUDY DESIGN: Amniochorial membranes were collected from the following groups of women: (1) women with premature rupture of fetal membranes, (2) women with preterm labor and intact membranes, and (3) women with term labor after vaginal delivery. Deoxyribonucleic acid fragmentation was tested with ligation-mediated polymerase chain reaction and the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyribonucleoside triphosphate end-labeling assay. Matrix metalloproteinase 2, p53, bcl-2, and bax gene expression patterns were studied with quantitative competitive polymerase chain reaction. Statistical analysis was performed with the Tukey-Kramer multiple comparison test. RESULTS: Quantitative competitive polymerase chain reaction documented a 10-fold increase in the expression of the gene for matrix metalloproteinase 2 in premature rupture of fetal membranes with respect to term and preterm labor. This induction coincided with an increase in the expressions of the proapoptotic genes p53 and bax and a drop in the expression of the antiapoptotic gene bcl-2. Ligation-mediated polymerase chain reaction revealed deoxyribonucleic acid fragmentation in specimens from premature rupture of fetal membranes and not in those from preterm labor or labor at term. Histochemical analysis documented fragmented deoxyribonucleic acid in chorionic and amniotic cells. CONCLUSION: This study suggests that apoptosis is associated with premature rupture of fetal membranes. Deoxyribonucleic acid fragmentation, associated with elevations in the levels of the two proapoptotic gene products evaluated (p53 and bax ) and a drop in the level of the antiapoptotic bcl-2, was seen in premature rupture of the fetal membranes. Induction of matrix metalloproteinase 2 may be a function of p53 gene expression increase in premature rupture of fetal membranes.     

High Serum Levels of Soluble IL-2 Receptor, Cytokines, and C Reactive Protein Correlate with Impairment of T Cell Response in Patients with Advanced Epithelial Ovarian Cancer

The serum levels of interleukin-(IL-)1α, IL-1β, IL-2, IL-6, TNFα, and sIL-2R and the proliferative response of peripheral blood mononuclear cells (PBMC) to phytohemagglutinin (PHA), anti-CD3 monoclonal antibody (mAb), recombinant IL-2 (rIL-2), and the combination of PHA or anti-CD3 mAb with rIL-2 were studied and correlated with serum levels of C-reactive protein (CRP) in women with advanced epithelial ovarian cancer. The expression of CD25 and CD122 subunities of membrane-bound IL-2R on PHA- or anti-CD3 mAb-stimulated PBMC was also studied. In comparisons with the controls, PBMC response to PHA, anti-CD3 mAb, and rIL-2 was significantly lower in the cancer patients. The addition of exogenous rIL-2 to the PBMC cultures increased response in both controls and patients but did not modify the significance of the differences. After stimulation with PHA or anti-CD3 mAb, the percentage of PBMC CD25⁺or CD122⁺was significantly lower in patients. The serum levels of IL-1α, IL-1β, IL-6, TNFα, sIL-2R, and CRP were significantly increased in patients compared to the controls. Instead, no differences were observed for serum levels of IL-2. A strong association was found between high serum levels of the above-mentioned cytokines, sIL-2R, and CRP. The results of our study on advanced stage (IIIb–IV) ovarian cancer patients are consistent with the previously reported hypothesis that high IL-6 and/or CRP serum levels may represent an important and independent prognostic factor of the likely outcome in cancer patients.     

Absence of activating somatic mutations of PI3KCA and AKT1 genes in South Indian women with endometriosis

Objective

To investigate whether the PI3KCA and AKT1 gene influences the risk of developing endometriosis in South Indian women.

Study design

Mutations in exon 9 and 20 of PI3KCA gene and E17K mutation in exon 4 of AKT1 gene were tested for association in a case-control study between eutopic and ectopic endometrium tissue from 30 endometriosis cases and eutopic endometrium tissue from 30 controls. The genotype frequencies of these mutations were compared using polymerase chain reaction and direct sequencing analysis of tissue DNA.

Results

The analysis did not reveal any activating somatic mutations in either PI3KCA or AKT1 gene in the cases.

Conclusion

In the present study we could not observe any mutation in PI3KCA and AKT1 gene, indicating that these mutations are rarely associated with endometriosis in South Indian women.