Methyltransferase G9a promotes cervical cancer angiogenesis and decreases patient survival.

Research suggests that the epigenetic regulator G9a, a H3K9 histone methyltransferase, is involved in cancer invasion and metastasis. Here we show that G9a is linked to cancer angiogenesis and poor patient survival. Invasive cervical cancer has a higher G9a expression than cancer precursors or normal epithelium. Pharmacological inhibition and genetic silencing of G9a suppresses H3K9 methylation, cancer cell proliferation, angiogenesis, and cancer cell invasion/migration, but not apoptosis. Microarray and quantitative reverse transcription polymerase chain reaction analyses reveal that G9a induces a cohort of angiogenic factors that include angiogenin, interleukin-8, and C-X-C motif chemokine ligand 16. Depressing G9a by either pharmacological inhibitor or gene knock down significantly reduces angiogenic factor expression. Moreover, promoting G9a gene expression augments transcription and angiogenic function. A luciferase reporter assay suggests that knockdown of G9a inhibits transcriptional activation of interleukin-8. G9a depletion suppresses xenograft tumor growth in mouse model, which is linked to a decrease in microvessel density and proliferating cell nuclear antigen expression. Clinically, higher G9a expression correlates with poorer survival for cancer patients. For patients' primary tumors a positive correlation between G9a expression and microvessel density also exists. In addition to increasing tumor cell proliferation, G9a promotes tumor angiogenesis and reduces the patient survival rate. G9a may possess great value for targeted therapies.

Differential clinical characteristics, treatment response and prognosis of locally advanced adenocarcinoma/adenosquamous carcinoma and squamous cell carcinoma of cervix treated with definitive radiotherapy.

OBJECTIVE: To compare tumor characteristics and clinical outcome of patients with cervical locally advanced adenocarcinoma (AC)/adenosquamous carcinoma (ASC) and squamous cell carcinoma (SCC). DESIGN: Retrospective study. SETTING: National Taiwan University Hospital, Taipei, Taiwan. POPULATION: All patients with cervical SCC (n = 35), AC or ASC (n = 194) with FIGO stage ≥IIB who received definitive radiotherapy or concurrent chemoradiotherapy (CCRT) from January 1995 to December 2009. METHOD: Medical and histopathological record review. MAIN OUTCOME MEASURES: Progression-free survival (PFS), local recurrence-free survival, distant metastasis-free survival, and overall survival (OS). RESULTS: Compared with the SCC subgroup, patients with AC/ASC were significantly younger (p = 0.007), more of them without clinical symptoms were diagnosed by abnormal Pap smear findings (p = 0.043), and less responded to treatment (p = 0.018). After a median follow-up of 59.3 months, patients with AC/ASC had worse 5-year PFS (30.0% vs. 47.6%, p = 0.044), worse 5-year distant metastasis-free survival (41.5% vs. 69.9%, p = 0.005), and trends toward worse 5-year local recurrence-free survival (64.4% vs. 76.2%, p = 0.165) and worse 5-year OS (41.3% vs. 58.1%, p = 0.090) than patients with SCC. In univariate analysis, early FIGO stage and complete treatment response were significantly associated with PFS and OS. Histology of non-AC/ASC and Point A biologically equivalent doses in 2-Gy fractions >85 Gy were significantly associated with better PFS, and CCRT was significantly associated with better OS. In multivariate analysis, complete treatment response and early FIGO stage remained significant factors for predicting better PFS and OS. CONCLUSIONS: Cervical AC/ASC may be more aggressive than is SCC. For cervical AC/ASC, more comprehensively effective treatments are warranted.

MUC20 overexpression predicts poor prognosis and enhances EGF-induced malignant phenotypes via activation of the EGFR-STAT3 pathway in endometrial cancer.

OBJECTIVE: Mucins play a critical role in the malignancy of various tumors and have been identified as diagnostic markers and as attractive therapeutic targets. However, the role of mucin (MUC) 20 in endometrial cancer (EC) is still unknown. METHODS: The relationship between MUC20 expression and clinical characteristics of EC was analyzed in 97 EC tumors and 16 normal tissues by immunohistochemistry. Effects of MUC20 on EC cells, HEC-1A and RL95-2, were examined by in vitro cell growth, migration, and invasion assays, as well as in vivo tumor growth in SCID mouse model. Western blotting was performed to analyze signaling pathways modulated by MUC20. RESULTS: MUC20 expression was significantly higher in EC tumors compared with the normal tissue. High levels of MUC20 expression in EC tumors were correlated with an unfavorable histologic subtype. Furthermore, MUC20 was an independent prognostic factor for poor survival as evaluated by multivariate analyses. Overexpression of MUC20 in EC cells significantly enhanced cell growth, migration, and invasion, as well as tumor growth in vivo. The MUC20-enhanced invasive behavior was significantly blocked by erlotinib, an EGFR inhibitor. Moreover, MUC20 overexpression enhanced EGF-mediated migration and invasion, suggesting a critical role of EGFR in MUC20-mediated effects. We found that MUC20 overexpression could enhance EGF-induced phosphorylation of EGFR and STAT3. Inhibition of the STAT3 activity by its inhibitor Stattic significantly suppressed the MUC20-enhanced invasive behavior. CONCLUSIONS: MUC20 is novel prognostic factor for EC and its overexpression enhances EGF-triggered invasive behavior through activation of EGFR-STAT3 pathway.

Expression of BCL10 in cervical cancer has a role in the regulation of cell growth through the activation of NF-κB-dependent cyclin D1 signaling.

OBJECTIVE: We recently characterized the molecular linkage that directs both BCL10 overexpression and nuclear translocation in response to inflammation-related NF-κB signaling pathway. Since NF-κB activation has been shown to occur in the pathogenesis of cervical cancer, we sought to investigate whether BCL10 possesses clinical significance in relation to cervical cancer. METHODS: Four cervical cancer cell lines (C33A, SiHa, HeLa, and CaSki) were used in this study. The DNA-binding activity of NF-κB was determined by the luciferase assay. The expression of BCL10, NF-κB, and cyclin D1 in tumor cells from an array of 182 tissue samples was examined using immunohistochemical staining. RESULTS: We transfected four cervical cancer cell lines with BCL10 small interfering RNA (siRNA), and discovered that the down-regulation of BCL10 inhibited the viability of these cervical cancer cells through G1 arrest. BCL10 siRNA treatment inhibited the expression of p-IKKß and p-IκB, and also down-regulated both NF-κB activation cyclin D1, its downstream cell cycle protein. Our results reveal that cervical cancer had a higher rate of positive cytoplasmic staining (74.1%, 123/166) than either carcinoma in situ (50.0%, 3/6) or normal cervix (0.0%, 0/10); and that poorly differentiated cancer had a higher rate of cytoplasm staining (80.7%, 71/88) than moderately differentiated (75.4%, 43/57) and well differentiated (40%, 4/10) carcinoma. Furthermore, nuclear expression of BCL10 was closely associated with NF-κB activation (p<0.001) and cyclin D1 expression (p<0.001). CONCLUSIONS: Our findings indicate that BCL10 plays an important role in controlling the growth of cervical cancer cells through NF-κB dependent cyclin D1 regulation.

Lysophosphatidic acid receptor 2/3-mediated IL-8-dependent angiogenesis in cervical cancer cells.

The expression of lysophosphatidic acid (LPA)-specific receptors in cervical cancer has not been clearly defined. In this study, we identified LPA1, LPA2 and LPA3 receptors' mRNA in SiHa, HeLa and CaSki cell lines by RT-PCR. These receptors were not associated with tumor cell proliferation in vitro. We then used a xenograph animal model to evaluate the effects of these receptors on in vivo cervical cancer tumorigenicity. When SiHa cells with different receptor expression patterns were seeded on the backs of SCID mice, the resulting knockout of both LPA2 and LPA3 significantly attenuated tumor growth; this decrease in tumor growth was found to be linked with decreased angiogenesis (microvessel density), suggesting that LPA2 and LPA3 are crucial for in vivo tumor growth through an angiogenic mechanism. We further investigated this mechanism of LPA receptor 2/3-mediated angiogenic capability by analyzing angiogenic factors in protein lysates from receptor knockout tumors, by detecting interleukin (IL-8) mRNA expression after treating with siRNA, by evaluating the biological role of LPA-enhanced IL-8 via endothelial cell tube formation, monolayer permeability, migration and cell growth assays, and by IL-8 knockout xenograft mice modeling. We found that the angiogenesis is mediated through IL-8. Finally, we evaluated the regulation pathways involved in LPA-induced IL-8 expression. We found that LPA receptor 2/3-mediated IL-8 expression occurs through Gi/PI3K/AKT, Gi/PKC and IκB/NF-κB signaling. In conclusion, we propose that LPA2 and LPA3 might play an important role in cervical cancer tumor growth through IL-8-dependent angiogenesis.

Resveratrol promotes osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis.

Reports of the bone-protective effects of resveratrol, a naturally occurring phytoestrogen and agonist for the longevity gene SIRT1, have highlighted this compound as a candidate for therapy of osteoporosis. Moreover, SIRT1 antagonism enhances adipogenesis. There has been speculation that resveratrol can promote osteogenesis through SIRT1, but the mechanism remains unclear. In this study we investigated the molecular mechanism of how resveratrol can modulate the lineage commitment of human mesenchymal stem cells to osteogenesis other than adipogenesis. We found that resveratrol promoted spontaneous osteogenesis but prevented adipogenesis in human embryonic stem cell-derived mesenchymal progenitors. Resveratrol upregulated the expression of osteo-lineage genes RUNX2 and osteocalcin while suppressing adipo-lineage genes PPARγ2 and LEPTIN in adipogenic medium. Furthermore, we found that the osteogenic effect of resveratrol was mediated mainly through SIRT1/FOXO3A with a smaller contribution from the estrogenic pathway. Resveratrol activated SIRT1 activity and enhanced FOXO3A protein expression, a known target of SIRT1, in an independent manner. As a result, resveratrol increased the amount of the SIRT1-FOXO3A complex and enhanced FOXO3A-dependent transcriptional activity. Ectopic overexpression or silencing of SIRT1/FOXO3A expression regulated RUNX2 promoter activity, suggesting an important role for SIRT1-FOXO3A complex in regulating resveratrol-induced RUNX2 gene transcription. Further mutational RUNX2 promoter analysis and chromatin immunoprecipitation assay revealed that resveratrol-induced SIRT1-FOXO3A complex bound to a distal FOXO response element (-1269/-1263), an action that transactivated RUNX2 promoter activity in vivo. Taken together, our results describe a novel mechanism of resveratrol in promoting osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis.

Prevalence of telomerase activity in human cancer.

Telomerase activity has been measured in a wide variety of cancerous and non-cancerous tissue types, and the vast majority of clinical studies have shown a direct correlation between it and the presence of cancerous cells. Telomerase plays a key role in cellular immortality and tumorigenesis. Telomerase is activated in 80-90% of human carcinomas, but not in normal somatic cells, therefore, its detection holds promise as a diagnostic marker for cancer. Measurable levels of telomerase have been detected in malignant cells from various samples: tissue from gestational trophoblastic neoplasms; squamous carcinoma cells from oral rinses; lung carcinoma cells from bronchial washings; colorectal carcinoma cells from colonic luminal washings; bladder carcinoma cells from urine or bladder washings; and breast carcinoma or thyroid cancer cells from fine needle aspirations. Such clinical tests for telomerase can be useful as non-invasive and cost-effective methods for early detection and monitoring of cancer. In addition, telomerase activity has been shown to correlate with poor clinical outcome in late-stage diseases such as non-small cell lung cancer, colorectal cancer, and soft tissue sarcomas. In such cases, testing for telomerase activity can be used to identify patients with a poor prognosis and to select those who might benefit from adjuvant treatment. Our review of the latest medical advances in this field reveals that telomerase holds great promise as a biomarker for early cancer detection and monitoring, and has considerable potential as the basis for developing new anticancer therapies.

Human chorionic gonadotropin up-regulates expression of myeloid cell leukemia-1 protein in human granulosa-lutein cells: implication of corpus luteum rescue and ovarian hyperstimulation syndrome.

CONTEXT: The corpus luteum is a dynamic endocrine structure with periodic development and regression during menstrual cycles. Its lifespan can be prolonged by human chorionic gonadotropin (hCG). However, the signal mechanisms of this phenomenon remain unclear. OBJECTIVE: Our objective was to investigate the molecular mechanisms of hCG in the maintenance of the viability of granulosa-lutein cells. DESIGN: Granulosa-lutein cells were obtained from women undergoing in vitro fertilization. We examined the effects of hCG on the survival of cultured granulosa-lutein cells. The signal pathway inducing antiapoptotic protein was investigated. RESULTS: hCG enhanced viability of granulosa-lutein cells through antiapoptosis but not proliferation, because the apoptotic marker of annexin V was decreased, but the proliferative markers of Ki67 and proliferating cell nuclear antigen were not increased. Myeloid cell leukemia-1 (Mcl-1) protein, but not B-cell lymphoma protein-2 or B-cell lymphoma protein-xL, was significantly induced by hCG and LH. The granulosa-lutein cells secreted vascular endothelial growth factor that induced endothelial permeability. Mcl-1 small interfering RNA increased DNA fragmentation and diminished the antiapoptotic effect of hCG. hCG induced Mcl-1 expression through the LH/hCG receptor, adenylate cyclase, protein kinase A, and cAMP response element-binding protein signal pathway. Flavopiridol inhibited Mcl-1 production, released cytochrome c, and induced apoptosis of granulosa-lutein cells. CONCLUSIONS: We first demonstrate that hCG prevents apoptosis of granulosa-lutein cells through the induction of Mcl-1 protein via the LH/hCG receptor and a cAMP response element-binding protein-dependent pathway. We may have found the molecular mechanism for luteal rescue during early pregnancy. Mcl-1 prevents apoptosis and increases cell viability but not proliferation as mechanisms for luteal rescue. Mcl-1 is a key molecule of hCG signaling.

Prognosis and treatment of squamous cell carcinoma from a mature cystic teratoma of the ovary.

BACKGROUND/PURPOSE: Squamous cell carcinoma (SCC) arising from a mature cystic teratoma of the ovary is rare and only reported sporadically. Clinical information on the disease is limited. This study assesses the clinical characteristics, treatment, outcome and prognostic factors of reported cases. METHODS: Two hundred and twenty cases from 1976 through to 2005 in MEDLINE were analyzed for patient age, clinical and laboratory data, extent of disease, tumor markers, treatment and survival rates. Only the 188 cases with surgical staging were included in the survival analysis. RESULTS: The disease occurred most often in elderly women (mean, 55.0 +/- 14.4 years) and cysts were large (mean, 13.7 +/- 5.7 cm). Abdominal pain (71.6%) was the most common symptom. Preoperative serum SCC antigen level had a high positive rate (81.3%). Overall 5-year survival rate for all stages was 48.4%. For Stage I, the 5-year survival rate was 75.7%; stage II, 33.8%; stage III, 20.6%; and stage IV, 0% (p < 0.0001). Univariate analysis revealed that tumor stage, patient age, tumor size, preoperative SCC antigen and CA125 levels, and optimal debulking were significant prognostic factors. Further investigation into treatments for all stages revealed that postoperative adjuvant chemotherapy may produce a better survival rate for both stage III and stage IV cases. However, postoperative radiotherapy did not show a similar effect. Multivariate analysis indicated that stage and optimal debulking were significant factors that influenced survival. CONCLUSION: A mature cystic teratoma should be treated as early as possible. Tumor stage and optimal debulking are critical to survival. Unlike SCCs of the uterine cervix, postoperative adjuvant chemotherapy may produce a better result than adjuvant radiotherapy for advanced-stage cases.

Clinical significance of tumor-infiltrating lymphocytes in neoplastic progression and lymph node metastasis of human breast cancer.

To investigate the clinical significance of tumor-infiltrating lymphocytes (TILs) within the tumor milieu, we quantitatively measured and compared the subpopulations of TILs in 24 patients with stage I-III breast carcinoma. Peripheral blood mononuclear cells (PBMCs), normal breast parenchyma-infiltrating lymphocytes (NILs), and TILs were isolated from tissue specimens and quantified by flow cytometry. The results showed that increased proportion of CD8(+) T cells, with decreased proportion of CD4(+) T cells, was significant in gated CD3(+) TILs as compared to autologous NILs or PBMCs (P<0.001). The tumor-infiltrating CD8(+) T cells significantly increased with stage progression, reflected in a more strongly decreased CD4/CD8 percentage (P=0.003). The CD4/CD8 percentage of TILs was strongly correlated with lymphovascular permeation and subsequent lymph node metastasis (P<0.001). Increased percentages of tumor-infiltrating CD8(+) T cells with decreased CD4/CD8 percentages are of prognostic importance for cancer progression in human breast cancer.

Perceptions of and attitudes toward estrogen therapy among surgically menopausal women in Taiwan.

OBJECTIVE: To evaluate awareness of and attitudes toward estrogen therapy (ET) among surgically menopausal women in Taiwan. DESIGN: We performed a retrospective analysis of 188 women who underwent hysterectomy with bilateral oophorectomy before menopause. Responses to a questionnaire included women's thoughts about 20 of the most common menopausal symptoms, whether they used ET, and their attitudes about taking ET. RESULTS: Mean respondent age at surgery was 45.3+/-4.2 (SD) years; 59 women (31.4%) were either current or former users of ET. Vegetarians had a lower rate of ET use (9.5%) than omnivores (34.1%, P=0.0239, chi2 test). By multiple logistic regression, insomnia (P=0.005), palpitations (P=0.024), and cold sweats (P=0.027) were the symptoms most associated with ET use. The prevalence rates of the 20 menopausal symptoms ranged from 48.9% to 85.6%. By factor analysis, the 20 symptoms were grouped into four clusters: psychological, vasomotor, genital, and somatic. Although ET can improve all 20 symptoms, the effect was superior for the vasomotor cluster (P<0.0001, analysis of variance). Of the women, 154 (81.9%) were aware of ET, and their major sources of information about it were health professionals (48.1%) and the mass media (34.4%). Only 49.5% of the women regarded ET as necessary, and 50.4% of the women who had never used ET claimed that they would use it if their doctor could persuade them that its benefits outweighed its risks. CONCLUSIONS: Even though ET can significantly improve vasomotor symptoms, surgically menopausal women in Taiwan have a low rate of ET use.

Laparoscopic-assisted vaginal hysterectomy with uterine artery ligation through retrograde umbilical ligament tracking.

STUDY OBJECTIVE: To evaluate the clinical outcomes of uterine artery ligation through retrograde tracking of the umbilical ligament (RUL) in laparoscopic-assisted vaginal hysterectomy (LAVH). DESIGN: Prospective study (Canadian Task Force classification II-3). SETTING: University-affiliated hospital. PATIENTS: Two hundred twenty-five women with myomas or adenomyosis. INTERVENTION: Laparoscopic-assisted vaginal hysterectomy with uterine artery identification and ligation through RUL. MEASUREMENTS AND MAIN RESULTS: The median age of the patients was 46 years, and the median weight of the extirpated uteri was 340 g, with 26.2% of uteri weighing more than 500 g. The median operation time was 135 minutes, and the median blood loss was 50 mL. The median intramuscular meperidine requirements were 1 ampoule (50 mg), and the median hospital stay was 3 days. It took approximately 10 minutes from identification of the umbilical ligament to ligation of the uterine artery. Uterine weight of 500 g or more required a significantly longer operation time compared with uteri weighing less than 500 g (164 min vs 127 min median, p <.001), and there was more blood loss (100 mL vs 50 mL median, p <.001). There were no differences in the median intramuscular meperidine requirements or hospital stay between the two groups. No blood transfusion was needed in either group, even in patients with a uterine weight of more than 1000 g. By regression analysis, uterine weight was significantly related to blood loss and operation time. A linear relationship was found among uterine weight, operation time, and blood loss: Uterine weight = 87.589 + 1.881 x operation time + 0.48 x blood loss (R = 0.531, F = 35.694, degrees of freedom 184, p <.001). No complications related to RUL were observed, although two bladder injuries related to severe pelvic adhesion with endometriosis and previous cesarean section occurred. CONCLUSION: Minimal blood loss and a low complication rate were noted in LAVH by uterine artery ligation through RUL. This technique should be a valid approach, especially in patients in whom minimal blood loss must be achieved.

Integration of high-risk human papillomavirus DNA correlates with HLA genotype aberration and reduced HLA class I molecule expression in human cervical carcinoma.

In human cervical cancer (CC), local immunity against this human papilloma virus (HPV)-associated neoplasia has been signified. To stratify the possibility of HPV integration on HLA mutations, we measured the genotypic and phenotypic integrity of all available HLA class I loci in 30 cases of CC. Paired normal and cancer genomic DNA was analyzed with DNA typing trays, including 57 subtypes of HLA-A, 120 subtypes of HLA-B, and 60 subtypes of HLA-C. We demonstrated significant mutations of HLA genotype with reduced HLA molecule expression in CC. HPV coincide in > 70% cases of aberrant HLA genes. Southern blot analysis revealed the presence of HPV DNA within the mutated HLA foci. Our study reveals a plausible role of HPV integration in the contexts of aberrant HLA genotypes in CC cells. Disruptions of the HLA genes can be possible tactics of HPV to attain the potential carcinogenetic purposes, and thus the cancer immune escape.

Angiomyofibroblastoma of the vulva.

Angiomyofibroblastoma is a rare, recently described, distinctive benign mesenchymal tumor that occurs mainly in the vulvar region of premenopausal women. We report a case of angiomyofibroblastoma of the vulva. A 45-year-old woman reported a small, painless nodule on the left vulva which rapidly enlarged during the 6 months before she visited the hospital. Local examination showed a pedunculated, mobile mass on the superior aspect of the left labium majus (13 x 12 x 10 cm) with elastic texture. Left vulvectomy was performed and the excised specimen revealed a well-defined and multilobulated tumor with edematous changes on gross examination. Microscopically, the tumor had alternate hyper- and hypocellular areas characterized by benign-looking round- to spindle-shaped cells with eosinophilic cytoplasm. The tumor cells were admixed with scattered thin-walled vessels in an edematous stroma. Immunohistochemical analysis was diffusely positive for desmin, estrogen receptor and progesterone receptor; reactive in small focus for actin; and negative for S-100 and CD34. Angiomyofibroblastoma, a mesenchymal tumor, was diagnosed. The postoperative course was smooth with no recurrence during the following 8 months. Simple excision for angiomyofibroblastoma is recommended because local recurrence or metastasis has not been reported.

Effect of conjugated equine estrogen in combination with two different progestogens on the risk factors of coronary heart disease in postmenopausal Chinese women in Taiwan: a randomized one-year study.

BACKGROUND: To compare the effect of hormone replacement therapy (HRT) using estrogen plus dydrogesterone or estrogen plus medroxyprogesterone acetate (MPA) on the risk factors for coronary heart disease (CHD) in postmenopausal women. METHODS: A randomized, prospective 1-year clinical trial was designed. All of the postmenopausal women (n = 279) received sequential conjugated equine estrogen (CEE) at a dose of 0.625 mg/day for 25 days (days 1-25) of each month. These women were also randomly assigned to receive either dydrogesterone 10 mg/day (E + D group, n = 140) or MPA 5 mg/day (E + P group, n = 139) for 14 days (days 12-25) of each month. Serum biochemical markers, lipoproteins, plasma prothrombin time (PT), partial prothrombin time (PPT) and antithrombin III-antigen (ATIII-Ag) were analyzed at baseline, and after 6 and 12 months of treatment. RESULTS: Liver function, renal function, PT and PPT did not change significantly during the 12-month trial. The E + D group had a more pronounced increase in high density lipoprotein cholesterol (HDL-C) than the E + P group (10.6% vs. 2.7%) after 12 months of treatment (p < 0.05). Both groups showed reduced concentrations of total cholesterol (T-CHO), low density lipoprotein cholesterol (LDL-C) and ATIII, whereas triglyceride (TG) was increased at the end of the trial (without intergroup difference). CONCLUSIONS: Our study demonstrated a favorable effect on lipoprotein profiles with both hormone replacement therapy regimens. Dydrogesterone appears to be superior to medroxyprogesterone acetate from the perspective of modification of coronary heart disease risk factors.

Immunoaffinity capillary electrophoresis: determination of binding constant and stoichiometry for antibody-antigen interaction.

Affinity capillary electrophoresis (ACE) can provide both qualitative and quantitative information on molecular interactions and affords the advantages of very low sample consumption, high mass sensitivity, short analysis time, and the use of automated instrumentation. It has been applied clinically and biochemically to the determination of the binding constant and to the measurement of the binding stoichiometry for interactions between antibodies (Ab's) and antigens (Ag's) in free solution. In many situations, the Ag molecule has two or multiple binding sites, each of which has a similar or different intrinsic affinity for binding independently to the combining site(s) on an Ab molecule. The multivalent binding reactions between Ab and Ag molecules often occur. The objective of this review is to describe the uses of ACE in the determination of binding constants and stoichiometry of Ab-Ag interactions (immunoaffnity capillary electrophoresis), focusing especially on multivalent Ab-Ag interaction modes. Five model binding systems developed recently using ACE techniques are described with principles and examples: (i) divalent mAb-monovalent Ag interaction, (ii) divalent mAb-(homo)polyvalent Ag interaction, (iii) cooperativity of two binding sites of mAb-monovalent Ag interaction, (iv) monovalent Fab-divalent Ag interaction, and (v) polyclonal Ab-monovalent Ag interaction. Finally, the determination of binding stoichiometry of Ab-Ag interactions by ACE is described.

Telomerase activity in gestational trophoblastic disease and placental tissue from early and late human pregnancies.

BACKGROUND: The aim of this study was to evaluate telomerase activity in tissue from cases of gestational trophoblastic disease (GTD) and in placental tissue from early and late human pregnancies. METHODS: We used a telomeric repeat amplification protocol assay to measure telomerase activity in 132 tissue samples from normal early pregnancies, spontaneous abortions, normal late pregnancies, cases of late-pregnancy intrauterine fetal death, and GTD. RESULTS: Telomerase activity was detected more often in normal early pregnancies and cases of GTD than in spontaneous abortions and normal late pregnancies (P < 0.001). During early gestation, no significant difference in detection rates was found between normal pregnancies and complete hydatidiform mole. As gestational age increased, detection rates for normal pregnancies decreased significantly (P = 0.0001), while for complete hydatidiform mole no significant changes occurred. CONCLUSIONS: Our findings indicate that placental tissue from normal early pregnancies and neoplastic tissue from GTD possess similar levels of telomerase activity. Decreasing regulation of telomerase activity is present in normal pregnancies but not in complete hydatidiform mole. The fact that telomerase activity decreases in cases of fetal demise, and as pregnancy progresses, also suggests that placental senescence may play a role in the development and ageing of the placenta.