Pregnancy and Natural Delivery Following Magnetic Resonance Imaging-Guided Focused Ultrasound Surgery of Uterine Myomas

This report discusses a pregnancy case following a series of two consecutive magnetic resonance imaging-guided focused ultrasound surgery (MRgFUS) procedures for the treatment of two different myomas in an individual patient. Both procedures were completed without adverse events, and the patient conceived naturally four months after treatment. At 39 weeks, she gave birth to a healthy baby girl, via a vaginal delivery. There were no complications in the pregnancy or during labor.

Usefulness of FDG-PET/CT as a Diagnostic Tool for Routine Post Therapy Evaluation in Endometrial Cancer / 핵의학분자영상

PURPOSE: The aim of this study was to evaluate the usefulness of FDG-PET/CT as follow up imaging tool in patients with endometrial cancer after therapy. Material and Methods: One hundred one patients with endometrial cancer who underwent FDG PET/CT after the treatment of this disease were included in this study population (25-79 yr old, Mean age 50.6 yr old) and all these patients also performed various laboratory and imaging studies such as serum tumor marker, CT or MRI. The lesions having increased focal FDG uptake were classified into benign, equivocal, and malignant one according to their pattern and activity. Tumor recurrence was confirmed by histopathological results and other clinical and imaging data. RESULTS: Among the 19 patients with 30 malignant or equivocal hot uptakes, 11 of 14 patients supposed to be malignant finding in PET/CT were proved to be tumor recurrence, while one of 5 patients with equivocal lesions were recurred malignancy. Two false negative cases were turned out to be peritoneal carcinomatosis. Estimated sensitivity, specificity and accuracy of PET/CT for diagnosis of recurrence in endometrial carcinoma after treatment were 86 %, 92 % and 91%, respectively. Positive and negative predictive values in the same issue were 63% and 98%, respectively. CONCLUSION: FDG-PET/CT is useful for regular work up of endometrial carcinoma after the treatment because of its high negative predictive value as well as high sensitivity and specificity.

Comparison of postoperative radiotherapy versus postoperative paclitaxel and platinum chemotherapy in uterine endometrial carcinoma / 대한산부인과학회지

OBJECTIVE: This study was performed to compare postoperative adjuvant paclitaxel and platinum (TC) chemotherapy and radiation therapy in women with uterine endometrial carcinoma. METHODS: Total one hundred five patients were entered into this trial. Non-endometrioid histologic subtypes such as serous, clear cell and small cell types were excluded from the study because they have different biological potentials. Of 58 assessable patients, who were needed adjuvant treatment according to surgico-pathologic reports, after surgery, 34 were received TC chemotherapy and 24 were received radiation therapy. Chemotherapy consisted of paclitaxel 175 mg/m2 and carboplatin AUC 5 (or cisplatin 50 mg/m2) every 3 weeks for 3 or 6 cycles. Irradiation dosage was 4,500~5,040 cGy in 28 fractions. RESULTS: In 58 evaluated patients, median follow-up time was 40.3 months (range 7~64 months). The 5-year overall survival and 5-year disease-free survival were 91.3% and 91.0% in 34 patients treated with TC chemotherapy, and 91.4% and 82.8% in 24 cases who treated with radiation therapy, however, there were no significant difference (P=0.646, P=0.129). The most common adverse effect of TC chemotherapy was hematologic toxicity, which was manageable conservatively. The serious gastrointestinal complication of radiotherapy was noted in 5 patients (20.8%), three of these patients were received another bowel surgery, such as ileo-cecal bypass, however, symptoms were persisted after surgery. CONCLUSIONS: These data suggest that postoperative adjuvant TC chemotherapy is a promising treatment which could be substituted for radiation therapy, with major activity and a acceptable toxicity profile for the treatment of uterine endometrial carcinoma.

Expression of Wnt 1 and beta-catenin in epithelial ovarian cancer / 대한산부인과학회지

OBJECTIVE: We were trying to identify the expression of Wnt 1 and beta-catenin in normal ovarian epithelium and epithelial ovarian tumor. METHODS: We used archival formalin-fixed and paraffin-embedded tissues from Comprehensive Gynecologic Cancer Center and the Department of Pathology at Bundang CHA Hospital from 2000 to 2005. Immunohistochemical staining for Wnt 1 and beta-catenin was performed on the ovarian epithelial tissues. Statistical analyses were performed with SPSS 10.1 for Windows and significance was defined as P<0.05. RESULTS: Of 114 cases, the cases were composed of 54 carcinomas, 40 borderline tumors, 12 benign tumors and 8 normal control ovarian tissues. Abnormal nucleocytoplasmic expression of beta-catenin was found in 4 endometrioid carcinomas. The nuclear expression of beta-catenin was found especially in the components of the endometrioid carcinoma (28.6%, P<0.05). Wnt 1 was overexpressed in all 9 clear cell carcinomas, but not frequent in the other types of malignant tumors (P<0.05). We found a statistically significant correlation between beta-catenin nuclear localization and endometrioid carcinomas. And we found a significant correlation between Wnt 1 expression and clear cell carcinomas. CONCLUSION: It does not seem that Wnt 1 over expression directly provoke the nuclear localization of beta-catenin. But, deregulation of beta-catenin and Wnt 1 may play a role in the pathogenesis of ovarian epithelial carcinogenesis of endometriod carcinoma and clear cell carcinoma. Evaluating this avenue of regulation of beta-catenin and Wnt protein in ovarian epithelial carcinoma may provide a new direction for early diagnosis and treatment in ovarian epithelial carcinoma and provide opportunities for making a certain biomarkers.

Magnetic resonance imaging-guided focused ultrasound surgery for uterine fibroids: Initial experience in Korea / 대한산부인과학회지

OBJECTIVE: The purpose of this report is to review the safety and short-term efficacy of non-invasive magnetic resonance imaging-guided focused ultrasound surgery (MRgFUS) on uterine myomas in Korean women. METHODS: A total of 29 outpatient Korean women, whose mean age was 39.1+/-5.8 years, were treated using the MRgFUS system for their symptomatic uterine myomas. Patients??symptoms were recorded using a validated symptom-specific questionnaire on treatment day, and at follow-up visits, 3 and 6 months post treatment. Data on adverse events was recorded on each follow up period. RESULTS: Symptom improvement was experienced by 83% of the patients at the three months follow-up, and 90% of the patients reported on improved quality of life by the six months follow-up. There were no serious adverse events during the treatments or the follow-up period. CONCLUSION: MRgFUS appears to be a safe and effective treatment for symptoms relief of uterine fibroids. Additional reports on longer follow up should verify long-term durability.

Alterations in Promoter Usage and Expression Levels of Insulin-like Growth Factor-II and H19 Genes in Ovarian Cancers / 대한산부인과학회잡지

OBJECTIVE: To establish the possible role of imprinting in ovarian cancer, we determined the imprinting status of both IGF-2 and H-19 genes in ovarian cacner, borderline tumors of ovary, benign ovarian tumor and normal ovarian tissues. METHODS: An allelictyping assay was performed using a PCR-RFLP-based method for identification of heterozygous informative cases. The usage of Insulin-like growth factor-II (IGF-2) promoters was examined by RT-PCR using promoter-specific primers. The mRNA expression of IGF-2 and H19 was quantified using a densitometer. RESULTS: Loss of imprinting (LOI) of IGF-2 was observed in the order of borderline tumor (77%)>cancer (71%)>benign tumor (60%)>normal ovarian tissues (50%) respectively. And the LOI of H19 gene was not detected in the normal and benign tissues but observed in the borderline tumor and cancer tissues, respectively. The usage of promoter P1, P2, P3 and P4 were observed different pattern in normal, benign tumor, borderline tumor and cancer tissues. The activity of mRNA expression of promoter P4 was higher than other promoters. The cancer tissues predominantly used promoter P1, P2 with relative silencing of the promoter P3. The ovarian cancer tissues showed the higher expression levels of the IGF-2 but a down- regulation of the H19 relative to normal tissues. CONCLUSION: These results suggest that LOI, deregulation of the IGF-2 promoters, and the altered expression levels of the IGF-2 and H19 gene might be associated with progression of ovarian cancer.

Alterations in Promoter Usage and Expression Levels of Insulin-like Growth Factor-II and H19 Genes in Cervical and Endometrial Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The biallelic expression of insulin-like growth factor-II (IGF2) and H19 has been reported to be associated with the progression of several tumors. Here, the promoter usage and expression levels of IGF2 and H19 are reported to be altered in cervical and endometrial cancers showing loss of imprinting (LOI). MATERIALS AND METHODS: The imprinting status of IGF2 and H19 was examined in 32 cervical carcinomas, their matched normal tissues, 13 endometrial cancer and 33 normal endometrial tissues. RESULTS: The LOI of IGF2 was observed in 7 of 18 (39%) and 1 of 13 (8.3%) informative cervical carcinomas and informative endometrial cancers, respectively. The LOI of the H19 gene was detected in 5 of 14 (36%) and in all 11 (100%) informative cervical carcinoma cases and informative endometrial cancer cases, respectively. The use of promoter P1 was observed in the LOI tumors of IGF2, but not in the tumors showing maintenance of IGF2 imprinting (MOI), or in cervical and endometrial cancers. Unlike MOI tumors, some LOI tumors revealed a lack of IGF2 transcription from the promoter P3. The LOI tumors of IGF2 showed increased expression of the IGF2 level, but a down-regulation of the H19, relative to normal tissues, whereas the MOI tumors revealed no significant alterations. CONCLUSION: These results suggest that the promoter P1 could be involved in the biallelic expression of IGF2, and that the altered expression of the IGF2 and H19 levels might be associated with the progression of cervical and endometrial cancers that exhibit biallelic IGF2 expression.

Expression of Chemokine Receptors in Ovarian Cancer Tissue / 대한산부인과학회잡지

OBJECTIVE: We investigated the expression of chemokine receptors in human ovarian cancer to understand the role of chemokines in ovarian cancer development and metastasis. METHODS: Twenty-two cases of epithelial ovarian cancer were studied for expression of 13 chemokine receptors such as CXCR1-CXCR5 and CCR1-CCR8 by using semi- quantitative RT-PCR and immunohistochemistry. Moreover, we studied the relationship between the chemokine receptors expression and lymph nodes metastasis of ovarian cancers. RESULTS: As compared with normal ovarian tissues, ovarian cancer tissues showed higher mean expression levels of CCR1,3,4,5,7,8 and CXCR1,3,4. Of chemokine receptors, CCR7 revealed the significantly higher levels of expression in ovarian cancer tissues relative to normal tissues. In the cases of retroperitoneal lymph nodes metastasis, increased expression of CCR2,4 and CXCR 1,3,4 was observed although there was no statistical significance. CONCLUSION: These results suggest that there is a complex chemokine/chemokine receptor network in pathogenesis and the way of lymph node metastasis of ovarian cancer rather than a specific chemokine or chemokine receptor.

Atypical Glandular Cells of Undetermind Significances (AGUS) ; Histopathologic Results and the Significance of the HPV DNA Detection / 대한산부인과학회잡지

OBJECTIVES: AGUS often reflects an immediate cervical cancer precursor such as a HSIL mimicking an endocervical glandular lesion. In this study, we attempted to assess the clinical significance of a cytologic diagnosis of atypical glandular cells of undetermined significance (AGUS) and determine the usefulness of the human papillomavirus (HPV) DNA testing as the triage strategies in evaluating AGUS. METHODS: Between 1994 and 1998, 67,730 Papanicolaou smears were evaluated at Kangnam and Uijongbu St Mary's Hospital. There were 87 (0.13%) cases of AGUS smears during that time. Colposcopy was performed on all women, and HPV DNA testing was performed on 11 persons. RESULTS: Mean age of these patients was 45.8 years. Histologic diagnosis of AGUS were kolocytosis and CIN-I in 6 (6.9%), CIS in one, endometrial hyperplasia in 2 (2.3%), endometrial adenocarcinoma in 7 (8.0%), cervical adenocarcinoma in 14 (16.1%) and cervical squamous cell carcinoma in 2 (2.3%) cases. Endometriosis was 8.9% under 46 years old and none in over 46. CIN was 8.9% and 7.2%, respectively. Cervical adenocarcinoma was 6.7% under 46 and 19.1% over 46. Endometrial cancer was 4.4% and 11.9%, respectively. The risk of cervical cancer and endometrial cancer was high in the AGUS with

Novel Molecular Biologic Mechanism of Growth Suppressive Regulation by Retinoid / Interferon in Cervical Cancer Cells / 대한산부인과학회잡지

OBJECTIVE: Retinoic acids (RAs) and interferons (IFNs) have been implicated in the growth regulation of cervical cancer cells, which was suggested by clinical trials and in vitro experiments. However, the molecular mechanisms of growth regulation are not fully defined, The purpose of this study is to assess the effect of RA and/or IFN on human cervical carcinoma cells in vitro and to analyze their action mechanisms in HPV-positive cervical carcinoma cells by molecular biologic studies. METHODS: HPV-positive (CaSki, HeLa), HPV-negative (C33A, HT-3), and non-cervical cancer Cos-1 cell lines were treated with RA and/ar IFN. Their effects on cell growth were evaluated by the cell pmliferation assay and the following BrdU DNA incorporation assay. The molecular mechanism was further investigated by a series of immunoblottings and transient cotransfection assays, which were conducted in HeLa cells and C33A cells using the CAT reporter gene assay. To observe the down regulation of HPV E6/E7 gene expression by RA/IFN, reverse transcription-polymerase chain reaction (RT-PCR) was perforned. RESULTS: The powth of RA-treated cells was less suppressed than that of IFN-treated cells. Combined treatment of RA and IFN leads to additive effect on the growth suppression of HeLa and CaSki cells. The proliferation activity was most severely reduced in Hela cells by treatment of both all-trans-RA (AtRA) and IFN-r. Combined treatment of AtRA/IFN-r causes a great increase in the level of interferon regulatory factor-1 (IRF-1) protein in HeLa cells, whereas no induction of IRF-1 was observed in C33A cells. The CAT gene expression for IRF-1 was greatly induced by IFN-r in HeLa cells. Immunoblotting assays shows the concurrent induction of p21 CDK inhibitor and dephosphorylation of Rb protein in HeLa cells. In RT-PCR, an individual treatment of either RA or IFN reduced HPV E6/E7 mRNA levels and significantly cooperative when both RA and IFN were treated. By deaeasing E6 levels, the p53 level was increased in HeLs cells treated with RA and/or IFN. Transient cotransfection of IRF-1 and p53 as the transcription factors leads to the cooperative activation of a common p21 promoter to regulate the cell cycle. CONCLUSION: RA/IFN suppressed the growth of HPV-positive cervical cancer cells. When they were both treated, additive suppressive effects were observed in cellular proliferation as well as DNA synthesis. The growth suppressive effect is likely to be related to the increased expression of IRF-1 and p21 (antitumoral effect; p53-independent). The down regulation of HPV E6 gene suppression may account for the resultant increase of p53 levels (antiviral effect; p53-dependent). Both induced IRF-1 and p53 cooperatively augument tbe suppession of p21 CDK inhibitor, which results in dephosphorylation of pRb. Although clinical effects are likely complex and may include interactions of in vitro growth inhibitory effects with immunomodulatory and antiangiogeaetic effect, tbese results suggest the optimal clinical role for the combination of RA/IFN in the treatment of cervical canccers.

Development of Gene Therapy Strategy Using Plasmid and Adenovirus in Cervical Cancer Treatment / 대한산부인과학회잡지

BACKGROUND: The basic treatment of malignant tumors is surgery, radiotherapy, chemotherapy. Even though, the object of these treatments is to kill cancer cells, they have limitations. So, in future studies of treatment of cancer, we should look into increasing human immune response using gene therapy in order to induce damage to tumor cells. OBJECTIVE: The cell growth inhibitory effect of cervical cancer cells was investigated by direct transfection using liposome(pRcCMVp53/lipofectin). and by indirect transfection using Adenovirus(AdCMVp53). METHODS: The cervical cancer cell lines we used in this study were HPV16 positive, having inhibitory gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3, LacZ gene was used as the marker gene for the transfection efficacy. Direct transfection was done by using lipofectin (pRcCMVp53/lipofectin) and indirect transfection was done by using virus, AdCMVp53. The effect of tumor cell growth inhibition was measured by cell counting assay. RESULT: Inhibition of growth of cervical cancer cells in cell counts of direct transfection was CaSki(88.5%), SiHa(59.1%), HeLa(86.0%), HeLaS3(78.0%), C33A(91.3%) and HT3(74.0%). Inhibition of growth of cervical cancer cells in cell counts of indirect transfection was CaSki(97.4%), SiHa(91.6%), HeLa(95.8%), HeLaS3(99.7%), C33A(97.3%) and HT3(87.4%). CONCLUSION: The inhibition of cell growth of cervical cancer cells by direct and indirect transfection was significantly reduced, and showed little differences depending on the type of cells. These results will have a great meaning in treating cervical cancer patients using gene therapy by direct or indirect transfection

The clinical study of 37 pregnancy women with aplastic anemia / 대한산부인과학회잡지

Objectives: To determine whether the clinical aspect of aplastic anemia is influenced by pregnancy. METHODS: We reviewed 37 cases of pregnant aplastic anemia patients during Jan. 1989 to Dec. 1998, and examined age, parity, progress of pregnancy, termination methods, obstetrics & neonatal complications, hematologic change, and treatment modality by medical records. RESULTS: According to onset of disease, patients were divided into pre-pregnant diagnosed group(n=12) and during-pregnancy diagnosed group(n=25). Mean age of diagnosis was 29.4yr, 89.2% were nulliparous, and 51.4% were severe aplastic anemic patients. All patients underwent 50 pregnancy. Mean gestational period was 37wks, birth weight was 2569gram, and, except in 7 cases of abortion, 43 cases were delivered transvaginally or transabdominally(51.2% vs. 48.8%). Preeclampsia, eclampsia, preterm labor, restricted growth, and distress were complicated and decreased hemoglobin, hematocrit, reticulocyte, platelet were reversed after termination in pregnancy associated group. Treatment modality during pregnancy included transfusion, steroid, anti-lymphocytic globulin, anti-thymocytic globulin and IVGV, and remission rate was 45.5% in pregnancy associated group. CONCLUSION: We concluded that pregnancy is associated with aplastic anemia as a high risk factor, and intensive treatment is needed.

The Effects of the Induction Chemotherapy on the Radical Radiotherapy in the Locally Advanced Cervical Cancer / 대한암학회지

PURPOSE: We evaluated the prognostic factors, survivals and patterns of failure of the patients with locally advanced cervical cancer who received radical radiotherapy alone and induction chemotherapy followed by radiotherapy respectively. MATERIALS AND METHODS: Between May 1985 to December 1992, one hundred and sixty three patients with locally advaneed cervical cancer received curative radiotherapy. Patients were divided into two groups: control group included 69 patients who received curative radiotherapy and combined group included 94 patients who received induction chemotherapy followed by curative radiotherapy. The curative radiotherapy consisted of external pelvic radiotherapy and intracavitary brachytherapy. Induction chemotherapy was delivered in VBP (vincristine, bleomycin, cisplatin) and FP (5-FU, cisplatin). Follow up period ranged from 2 months to 99 months with median of 50 months. RESULTS: The overall response rate was 94.2% in the control group and 89.4% in the combined group. The response rate by control group was 66.7% for CR (complete response), 27.5% for PR (partial response), 5.8% for NR (no response). The response rate by combined group of CR, PR, NR were 64.9%, 24.5%, 10.6%, respectively. There was no difference in response for control group and combined group (p> 0.05). The 5-year overall survival had no significant difference in between control group and combined group (54.6% vs. 57.3%). The 5-year disease free survival also had no significant difference (52.9% vs. 55.0%). In the control group, 23 patients (33.3%) had treatment failure: twelve (17.4%) at a local recurrence, 9 (13.0%) as distant metastasis, and 2 (2.9%) with both local recurrence and distant metastasis. In the combined group, Thirty patients (31.9%) failed therapy, with local recurrence in 21 patients (22.3%), distant metastasis in 7 patients (7.5%), and both in 2 patients (2.1%). The difference between the two groups was not significant in view of patterns of failure. The major toxicities were nausea/ vomiting, leukopenia, anemia, and diarrhea. The prognostic factors affecting were hemoglobin level, KPS (karnofsky performance status), and treatment response in both group by multivariate analysis. CONCLUSION: This study did not prove the efficacy of induction chemotherapy followed by radiotherapy in locally advanced cervical cancer.

Inactivation of p73 Protein by Cervical Cancer-inducing HPV E6 Protein Novel p53-independent function of E6 / 대한산부인과학회잡지

OBJECTIVE: Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 and E7 oncoproteins play major roles by inactivation of cellular p53 and pRb tumor suppressor proteins, respectively. However, it has been recently suggested that p53 and/or pRb-independent functions of E6 and E7 are involved in cervical carcinogenesis. The purpose of this study is to identify novel a cellular target, p73, of E6 and to determine how E6 inactivates p73 function, METHODS: The interaction between E6 and p73 were identified by the yeast two-hybrid assay in vivo and the GST pull-down assay in vitro. The function of the interaction was determined by transient transfections using p21 promoter-CAT reporter plasmid. The molecular mechanism underlying the functional significance of the interaction was further assessed by in vivo and in vitro protein degradation assays, and gel mobility shift assays. RESULTS: Yeast two-hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro, respectively. Transactivation domain (amino acid residues 1-49) is found to be absolutely required for this interaction. Transient co-expression of E6 significantly inhibits the p73-mediated activation of p21WAF1 promoter in a p53-defective C33A cell line. Using Ga14-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. The protein degradation assays in vivo and in vitro indicate that p73, unlike p53, is not susceptible to E6-dependent proteolysis. CONCLUSION: Throughout this study, we identified p73 as a novel cellular target of HPV-E6 protein and found that E6 binds p73 through the amino-terminal transactivation domain, and inhibits its transactivation function independent of the protein degradation and DNA binding. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV-E6 may, at least in part, contribute to E6-mediated cellular transformation.

Constructtion of the Recombinant pAAVCMVp53 for Cervical Cancer Gene therapy / 대한산부인과학회잡지

OBJECTIVE: Human papillomavirus (HPV) has been identified in the majority of invasive cervical cancer patient and has been found to contribute in a significant way to the genesis of human cervical cancer. HPV has two transforming genes that encode the oncoproteins E6 and E7, E6 can form complexes with p53 and promote p53 degradation, E7 inhibit retinoblastoma protein (RB). The p53 protein is as a phosphoprotein which co-immunoprecipitated with the SV40 T-Antigen. The wild type p53 protein is capable of suppressing the tumorigenic phenotype and regulating cell cycle. Adeno-associated virus(AAV) is a linear single stranded DNA parvovirus which is dependent upon cotransfection by a second unrelated virus to undergo productive infection. It has been well documented that AAV DNA integrates into cellular DNA as one to several tandem copies joined to cellular DNA through the termini. In order to introduce wild type p53 through AAV virus into a cervical cancer patient for gene therapy, we had constructed recombinant p53 adeno associated virus plasmid (pAAVCMVp53). METHODS: pAAVCMVp53 was created new AAV-vector system, pRc/CMVp53 including p53 cDNA and AAV-derivative vector, pASPA-AAV-CMV-polyA were made to HindIII/blunt fragments. Eluated 1.8 kb fragment of wild type p53 cDNA was ligated to pAAV-CMV-polyA, 4.9 kb fragment deprived hASPA cDNA. RESULT: Recombinant AAVCMVp53 was constructed by using pRc/CMVp53 andpASPA-AAV-CMV-polyA. This pAAVCMVp53 was confirmed by various restriction enzyme-digestions and Southern-blotting. This new vector system will be studied on expression, stability in cervical cancer cell lines and animals. CONCLUSION: This system will be one of the useful vector system for cervical cancer gene therapy.

Regulation of cell growth and HPV genes by exogenous estrogen in cervical cancer cells / 대한부인종양콜포스코피학회잡지

BACKGROUNDS: Human papillomavirus (HPV) infection is known as the major causative phenomenon in the development of cervical cancer. E6 and E7 proteins of oncogenic HPV types can play critical roles in immortalization and malignant transformation of cervical epithelial cells. From the previous epidemiologic data, long term use of oral contraceptives may be one of the risk factor for cervical cancer. PURPOSE: Investigation of estrogenic and anti-estrogenic effects on the proliferation of cervical cancer cells and gene expression of HPV under the regulation of HPV upstream regulatory region (URR) would help to explain the role of estradiol in HPV-associated pathogenesis of cervical cancer. METHODS: Cervical cancer cells (HeLa, CaSki and C33A) were cultured in vitro in the presence of 17 beta-estradiol or tamoxifen and the numbers of cells were directly counted to observe the growth stimulatory or suppressive effect of the treatment. The correlation between the growth regulatory effect and HPV E6/E7 gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR). The estrogenic effect on the promoter activity of HPV URR was further confirmed by transient co-transfection assays, which were conducted in C33A cells using the HPV-18 URR-CAT reporter plasmid. Supplemental effect of estrogen receptor on the URR promoter activity was also evaluated. To analyze the growth suppressive function at the higher concentration of estradiol or tamoxifen in HeLa cells, DNA fragmentation assay was performed. RESULTS: The proliferation of HeLa and CaSki cells was stimulated by estradiol at the concentration of physiological level (< or =1 X 10-6M), reaching maximal growth at 0.5 X 10-6M. At concentration of 0.1 X 10-6M, tamoxifen also stimulated the proliferation of HeLa and CaSki cells. In contrast to HPV-positive cervical cells, C33A cells were not influenced to cell proliferation by addition of estradiol at the physiological level, indicating that HPV might play role in growth stimulatory effect of estrogen or tamoxifen. Interestingly, the proliferation of HeLa cells was totally suppressed by estradiol and tamoxifen at the higher concentration (5 and 10 X 10-6M), whereas those of CaSki and C33A cellswere not responded and little suppressed at the concentration, respectively. The levels of HPV-18 E6 and E7 mRNA were significantly increased after treatment of 0.5 X 10-6M estradiol as determined by RT-PCR. Furthermore, transient transfection experiments using the URR-CAT reporter plasmid indicated that the increased expression of HPV E6/E7 genes was related with the growth stimulatory effect of estradiol and tamoxifen. In addition, co-transfection of estrogen receptor (ER) leads to an over 4-fold increase in CAT activity after treatment of estradiol or tamoxifen with 0.5 X 10-6M. When estradiol or tamoxifen was treated at the concentration over 5 X 10-6M for 96 hr, a typical DNA ladder, a indicative of apoptosis, was observed in HeLa cells. However, DNA ladder was not detected in C33A cells of which growth was some suppressed under same concentration of estradiol. CONCLUSION: At the physiological levels, estradiol stimulated the growth of HPV-positive cervical cancer cells and tamoxifen also did at the concentration of 0.1 X 10-6M. The increased expression of HPV E6/E7 at the physiologic levels appeared to be related with the growth stimulation of HPV-positive cervical cancer cells. Growth suppression observed at the higher concentration (5 and 10 X 10-6M) might be a indicative of apoptosis shown by DNA fragmentation assay in HeLa cells. Taken together, these data suggested that the concentration of estradiol (< or =1 X 10-6M) could be a risk-factor in HPV-mediated cerivcal carcinogenesis.

Increased transcriptional activity by mutation of HPV-16URR in cervical cancers carrying episomal HPV-16 DNA / 대한부인종양콜포스코피학회잡지

HPV E2 protein is known to act as a negative regulator of transcription and the disruption of E2 open reading frame by HPV integration can release suppression of E6 and E7 mRNA expression, resulting in uncontrolled cellular growth and malignant transformation by inactivating tumor suppressor gene products (p53, pRb). YY1 mutation of HPV URR has been suggested as one of indicator that explains development of cervical neoplasia by episomal type of HPV. To extend this hypothesis, we examined whether mutation(s) in specific sites of HPV URR is functionally related to the invasiveness of cervical neoplasia and the physical status of HPV DNA. The URR sequences were obtained by PCR amplification of HPV-16 genome from CIN and invasive cancer patients, cloned into pUC18 for sequencing, and into pBLCAT8+ for functional CAT assay. Our previous data classified HPV-infected patients into three groups: 3 cancer cases carrying episomal HPV DNA; 12 cancer cases carrying integrated HPV DNA; 12 CIN cases carrying episomal HPV DNA. The specific variants in HPV-16 URR were found in Korean women: GA transition at nt 7520 (100%, 27/27), AC transition at nt 7729 (70%; 19/27), and GA transition at nt 7841 (78%; 21/27). Selective mutations were observed at the YY1-binding sites of HPV-16 URR in the 3 patients with invasive cervical cancer, who having the episomal forms of HPV-16 DNA: AC transition at nt 7484 and GA transition at nt 7488 (YY1-binding site 2; from 7481 to 7489). Additionally, CT transition at nt 7785 (YY1-binding site 3; from 7781 to 7790) was found from 2 of 3 patients. No YY1 site mutations were detected in the 12 CIN patients and in the HPV-integrated invasive cancer patients. To determine whether these mutations have effect on the expression of HPV E6/E7 genes driven by URR, the transient transfection assay was employed using URR-CAT reporter plasmid. The relative activities of three URR mutants from episomal HPV-16 DNA of cervical cancers were 2- to 4-fold higher than that of HPV-16 URR prototype. In contrast, the URRs from integrated HPV-16 DNA in cervical cancer and from episomal HPV-16 DNA in CIN, where no mutation of the YY1-binding site was detected, showed similar levels of promoter activity to that of URR prototype.Our results support the hypothesis that the mutation at YY1 binding site is functionally related to the development of cervical neoplasia caused by episomal HPV-16 DNA in Korean cervical cancer patient. Thus, mutation in YY1 site of episomal HPV-16 URR may play a role of HPV integration in the progression of cervical cancer.

Telomerase Activity in Gestational Trophoblastic Disease / 대한산부인과학회잡지

The purpose of this study was to evaluate the significance of telomerase activity in gestational trophoblastic disease and the association of telomerase activity in complete hydatidiform mole and subsequent development of persistent gestational trophoblastic tumor. By using the standard telomerase repeat assay, we examined telomerase activity in 2 normal placentas, 31 complete hydatidiform moles, 7 invasive moles, 5 choriocarcinoma tissues and choriocarcinoma cell line (JEG-3). Telomerase activity was detected in 13 of 15 (86.7%) complete hydatidiform mole patients who eventually had chemotherapy for the treatment of persistent gestational trophoblastic tumor. All of the 9 patients with metastatic disease (FIGO Stage III) had telomerase activity in their initial molar tissue. In contrast, telomerase activity was evident in only two of 16 (12.5%) complete hydatidiform mole patients with spontaneous remission. While telomerase activity was not detected in normal placentas, high level of telomerase activity was detected in all of 7 invasive moles, 5 choriocarcinoma tissues and choriocarcinoma cell line (JEG-3). The presence of telomerase activity in a complete hydatidiform mole is associated with the development of persistent gestational trophoblastic tumor, such as invasive mole and choriocarcinoma.

Integration of HPV and the Antibody Respones to HPV Proteins in Patients with Cervical Cancer / 대한암학회지

PURPOSE: HPV (human papillomavirus) are known as the major causative agent for development of cervical cancer. High-risk HPVs, especially HPV-16 /18 DNA, are often found to be integrated into the human genome in high grade CINs as well as cervial cancer. Investigation of the relationship between the genomic states of HPV genes and their antibody response against the HPV-16 Ll/L2 virus-like particles (VLPs) and the in vitro translated E6 and E7 proteins may help to explain the mechanism of HPV-related cervical carcinogenesis and host immune responses. MATERIALS AND METHODS: Cervical cancer tissues obtained from 41 patients with cervical cancer were studied by PCR, Southern blot hybridization and the antibody response against HPV-16 Ll/L2 VLPs and HPV-16 E6, E7 proteins of serum were tested by ELISA and radioimmunoprecipitation assay (RIPA), respectively. RESULTS: Integrated forms of the HPV-16 DNA were found in 23 of the 38 patients (60.5%). The HPV-16 positive cervial cancer patients had a significantly higher prevalence (39.5%; 15/38) of antibodies to HPV-16 Ll/L2 VLPs than 8.7% (2/28) of the the control group (p0.05). CONCLUSION: Integrated forms of HPV-16 DNA were prevalent in most patients with cervical cancer. Antibodies to HPV-16 Ll/L2 VLPs, in vitro translated HPV-16 E6 and E7 proteins appeared in the significantly larger proportions of the HPV-associated cervical cancer patients than in the controls. Antibodies to HPV-16 Ll/L2 VLPs were more detectable in the cervical cancer patients with episomal form of HPV-16 DNA than those who having only integrated forms of HPV-16. Antibody responses to HPV-16 E6 and E7 proteins were not influenced by the different viral states. More numbers of studies would be necessary to determine the relationship between the genomic states of HPV and the immune responses to their proteins by the such genomic and serologic parameters.

Subsequent Reproductive Experiences after Treatments for Gestational Trophoblastic Disease / 대한산부인과학회잡지

For evaluating the reproductive performances of GTD patients, we found 115 cases of GTD patients, 77 HM and 38 GTT, who became pregnant after the completion of treatments and follow-up period. The results of this study suggest subsequent pregnancies after the completion of treatments may promise normal reproductive outcomes regardless of the chemotherapy.