Stephanie S. Liu

E-cadherin expression is silenced by DNA methylation in cervical cancer cell lines and tumours

A previous study showed E-cadherin expression was lost in some cervical cancer cell lines and tumours. This study was designed to clarify the significance of DNA methylation in silencing E-cadherin expression. We examined promoter methylation of E-cadherin in five cervical cancer cell lines and 20 cervical cancer tissues using methylation-specific PCR (MSP) and bisulphite DNA sequencing. The correlation of E-cadherin methylation and expression together with methyltransferase (DNMT1) were further studied. We found that hypermethylation of E-cadherin was involved in five cervical cancer cell lines and 40% (8/20) of cervical cancer tissues. E-cadherin protein was lost in 6/8 (75%) samples and 3/5 (60%) cell lines with promoter methylation. E-cadherin methylation was significantly correlated with increased DNMT1. Using an antisense DNMT1 oligo to transfect into SiHa HeLa C33A cell line, E-cadherin protein was re-expressed. We concluded that loss of E-cadherin expression was in part correlated with DNA methylation and DNMT1 expression in cervical cancer.

Estrogen Receptor Subtypes in Ovarian Cancer : A Clinical Correlation

OBJECTIVE: To study the distribution of estrogen receptor (ER) subtypes in ovarian tumors and to correlate the levels of expression with clinical factors. METHODS: Estrogen receptor-&agr; (ER&agr;) and &bgr;-mRNA expressions in 58 normal, 25 borderline, and 161 malignant ovarian tissue samples were determined by quantitative real-time polymerase chain reaction. The expression levels were correlated with clinical data, including the histologic subtypes, the stage of the disease, and the disease-free and overall survival, with a median follow-up of 80 months. RESULTS: Estrogen receptor-&bgr; (ER&bgr;) expression, but not ER&agr;, was significantly higher in normal tissues compared with malignant tissues (P<.001). Estrogen receptor-&bgr; expression was also significantly higher in stage I disease compared with stage II-IV disease (P<.001). A higher ER&bgr; expression was found to be significantly associated with a longer disease-free survival (P=.007) as well as overall survival (P=.011). Estrogen receptor-&bgr; expression remained a significant predictor for disease-free survival and overall survival in multivariable analysis that took into account other factors that were shown to be associated with survival in univariate analyses, including stage of disease, type of tumor (borderline or malignant), and optimal debulking. CONCLUSION: Loss of ER&bgr; expression in ovarian tumors may be a feature of malignant transformation. Determining ER subtypes expression may improve response to hormonal therapy by tailoring the use of selective estrogen receptor modulators with different ER affinity in selected women. As a prognostic indicator, ER&bgr; levels may be useful in deciding the need for and choice of adjuvant treatment in women with early ovarian cancers. LEVEL OF EVIDENCE: II

Anti-apoptotic proteins, apoptotic and proliferative parameters and their prognostic significance in cervical carcinoma

The inhibitor of apoptosis proteins (IAP) suppress apoptosis induced by a variety of stimuli. The aims of this study were to: (a) compare the expression of X-linked IAP (Xiap) and Human IAP-2 (Hiap-2) in cervical carcinoma cells and normal cervix, (b) determine the correlation between IAP expression and tumour apoptosis or proliferation, and (c) assess their prognostic significance in cervical carcinomas. Paraffin-embedded tissue sections were retrieved from 77 patients with cervical squamous carcinomas prior to treatments and 47 normal subjects. Tumour apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuracil triphosphate (dUTP) nick-end labelling (TUNEL) and apoptotic index (AI), and the proliferative rate was measured by Ki-67 and mitotic (MI) indices. Immunoreactive Xiap and Hiap-2 were found in both cervical cancer cells and normal tissues. IAP expressions in cancers did not correlate with apoptotic and proliferative parameters, disease stage and patient survival. The lower AI and Ki-67 index were associated with a better survival. In conclusion, the basal expression levels of IAPs have no prognostic significance, but AI and Ki-67 expression are potential prognostic indicators in cervical carcinoma.

p73 expression is associated with the cellular radiosensitivity in cervical cancer after radiotherapy.

Apoptosis is one of the causes of cell death in cervical cancer following radiotherapy (S. S. Liu et al., Eur. J. Cancer, 37: 1104–1110, 2001). By studying the gene expression profile with cDNA apoptotic array, the p73 gene was found overexpressed in radiosensitive cervical cancers when compared with radioresistant ones. To investigate the role of the p73 gene in relation to clinical assessment of radiosensitivity in cervical cancer based on the findings of residual tumor cells in cervical biopsies after completion of radiotherapy, we studied the protein expression of p73 in 59 cervical cancers after radiotherapy and 68 normal cervices using immunohistochemistry. The expression of p73 was found to be significantly increased in cancer samples and, more importantly, in those samples sensitive to radiotherapy (P < 0.001). The overexpression of p73 actually predicted a better prognosis in cervical cancer patients (P < 0.001). To investigate the possible involvement of p73 downstream genes, the protein expressions of p21 and Bax were studied. The expression of p21, but not Bax, was found to be positively correlated with the expression of p73 (P = 0.001). Furthermore, the epigenetic regulation of p73 expression via DNA methylation was also investigated in 103 cervical cancers and 124 normals. Hypermethylation of p73 gene was observed in 38.8% of cervical cancers, and it was significantly associated with reduced or absent p73 expression (P < 0.001). Reactivation of p73 expression in two cervical cancer cell lines by demethylation treatment supported the role of methylation in the regulation of p73 expression. Our findings suggested that p73 expression was related to the radiosensitivity of cervical cancer cells and may play an important role in the regulation of cellular radiosensitivity.

Evaluation of a Newly Developed GenoArray Human Papillomavirus (HPV) Genotyping Assay and Comparison with the Roche Linear Array HPV Genotyping Assay

ABSTRACT Persistent infection with high-risk types of human papillomavirus (HPV) is a necessary step in the development of cervical cancer. The incorporation of HPV detection into cervical screening programs may improve the ability to identify women at risk of cervical cancer. We recently evaluated the performance characteristics of a newly developed HPV detection assay, the GenoArray (GA) genotyping assay, for the detection of HPV infections by comparing it with the commercial Roche Linear Array (LA) HPV genotyping assay. The GA assay has an analytical sensitivity for the detection of HPV types 16 (HPV-16) and HPV-18 of as few as 10 to 50 copies, and its reproducibility is adequate. The GA and LA assays showed no significant difference in the rates of detection of genotypes detected by both HPV genotyping assays and oncogenic genotypes, and the interassay agreement was excellent. The GA and LA assays revealed either concordant or compatible genotyping results for 97.5% of the samples and discordant results for only eight (2.5%) samples. Compatible results were also observed for the detection of single or multiple HPV infections and the detection of most of the genotypes. The GA assay also demonstrated good clinical performance characteristics when the comparisons were carried out with clinical subgroups of samples from patients with normal cytologies, low-grade or high-grade squamous intraepithelial lesions, and cancers. Therefore, the GA assay appears to be highly sensitive and specific for the genotyping of HPV. It has the advantage that it specifically detects HPV-52, which overcomes a limitation of the LA assay, and hence, it has potential value for use for genotyping, especially in regions where HPV-52 has a high prevalence.

Prevalence of HPV infection in esophageal squamous cell carcinoma in Chinese patients and its relationship to the p53 gene mutation

Human papillomavirus (HPV), in particular types 16 and 18, is positively associated with anogenital cancers and may be an important etiologic factor in their pathogenesis. The goal of our study was to investigate the role of HPV infection in the pathogenesis of esophageal squamous cell carcinoma (ESCC) and its relationship with the p53 mutation. We have examined ESCC collected from Sichuan, China, for the presence of HPV infection and p53 mutation. The presence of HPV DNA was detected by PCR‐Southern analysis while the p53 mutation was analyzed by PCR‐SSCP. High‐risk HPV (types 16 and 18) DNA was detected in 32 out of 152 cases of ESCC examined. In contrast, HPV DNA was not detected in normal esophageal tissues excised from the distant end (tumor free) of resected ESCC. Mutation of the p53 gene was detected in 22 out of 55 cases of ESCC. The distribution of the 22 p53 mutation was: 5 in exon 5, 1 in exon 6, 5 in exon 7, 10 in exon 8 and 1 in exon 10. The p53 mutation was detected at a significantly lower rate in ESCC with HPV infection. Our results support a role of HPV infection in the pathogenesis of ESCC from a high‐incidence area. Int. J. Cancer 72:959–964, 1997.

Clinical significance of telomerase activation and telomeric restriction fragment (TRF) in cervical cancer

Telomerase activation was examined in 50 cases of cervical cancer, 27 normal cervix and five cervical cancer cell lines using the sensitive polymerase chain reaction (PCR)-based TRAP (telomeric repeat amplification protocol) assay. Telomeric restriction fragment (TRF) length of these specimens was measured by Southern hybridisation. Telomerase activation was common in cervical cancers and was detected in 46/50 cases (92%). Telomerase activity was weak in normal cervix and was detected only in 2/27 cases (7.4%). Telomerase activity was detected in all stages of cervical cancer suggesting that it is an early event in cancer progression. The clinical significance of telomerase activation was analysed in 47 squamous cell carcinoma of the cervix. High telomerase activity was more frequently detected in advanced diseases (100% in stage III and stage IV cervical cancers combined) compared with early diseases (68.6% in stage I and stage II cancers combined). The difference was statistically significant (P < 0.02). Telomerase activity was not statistically correlated with other clinical parameters examined. This is the first report of telomeric length in human cervical cancer. Both shortening and elongation of TRF length in cervical cancers was observed. Advanced cervical cancers tended to have a wider range of variation of TRF length compared with early disease and normal cervix. There was no obvious relationship between TRF length and the clinical parameters examined including clinical staging, differentiation status of tumour, human papilloma virus (HPV) infection, recurrence rate, tumour size and invasion depth. The clinical significance of TRF length appears to be limited in cervical cancers. Our results indicate that telomerase activity is closely associated with tumour cells and may be useful as a marker for detection of tumour cells in cervical biopsies.

Abnormal expression of epidermal growth factor receptor and c-erbB2 in squamous cell carcinoma of the cervix: correlation with human papillomavirus and prognosis.

The aim of this study is to assess the expression of epidermal growth factor receptor (EGFR) and c-erbB2 and their correlation with human papillomavirus (HPV) status and prognosis in squamous cell carcinoma of the cervix. The expression of EGFR and c-erbB2 was studied at the protein level using the immunohistochemical (IHC) staining method, at the RNA level using the ribonuclease protection assay and at the DNA level using Southern blot and hybridization method. One hundred and one patients with squamous cell carcinoma of the cervix were recruited. Fifty-one patients were of stage 1B/2A and 50 patients were of stage 2B and above. Positive IHC stainings of EGFR and c-erbB2 proteins were found in 74.2 and 19.8% of cases, respectively. DNA amplifications of EGFR and c-erbB2 genes were detected in 35.4 and 17.2%, respectively. Of the patients showing positive EGFR and c-erbB2 staining, only 39.2 and 25%, respectively, showed DNA amplifications. RNA overexpression of EGFR or c-erbB2 was only detected in 2% of cervical cancers and was associated with positive staining and DNA amplifications. HPV was detected in 79.2% of the cases by HPV consensus primers L1, in 57.4% for HPV 16 and 27.7% for HPV 18. The abnormal expression of EGFR and c-erbB2 had no correlation with HPV detection and had no prognostic significance on survival.

Yolk-sac transmission and post-hatching ontogeny of serum immunoglobulins in the duck (Anas platyrhynchos)

1. To investigate the immunoglobulin (Ig) class which is active in yolk-sac transmission of maternal antibodies in ducks, sera from laying ducks, yolks from their eggs, and sera from ducklings 0-87 days of age were examined by immunoelectrophoresis (IE), and Na2SO4-precipitated Igs from these sera and yolks were run in polyacrylamide gel electrophoresis (PAGE) under reducing conditions. 2. In yolk, 7.8S IgG was greatly enriched compared to 5.7S IgG and IgM, and was the only Ig to reach the sera of newly hatched ducklings. 3. The levels of maternally derived 7.8S IgG in duckling sera decreased after 5 days of age, reaching minimum levels at about 14 days of age. 4. Increases in the serum levels of 7.8S IgG, 5.7S IgG and IgM occurred after 20 days of age, reflecting de novo synthesis by the duckling, and the adult serum profile was achieved by 71 days of age. 5. The involvement of 7.8S rather than 5.7S IgG in yolk-sac transmission was probably determined by the additional heavy chain components of this molecule.

Loss of Programmed cell death 4 (Pdcd4) associates with the progression of ovarian cancer

BackgroundProgrammed cell death 4 (Pdcd4) is a novel tumour suppressor and originally identified as a neoplastic transformation inhibitor. The aim of this study was to investigate the expression, prognostic significance and potential function of Pdcd4 in ovarian cancer.ResultsThe expression of Pdcd4 was examined in 30 normal ovarian tissues, 16 borderline and 93 malignant ovarian tissues. A continuous down regulation of Pdcd4 expression in the sequence of normal, borderline and malignant tissues was observed. The expressions of Pdcd4 in both ovarian borderline tissues and carcinomas were significantly lower than the expression in normal ovarian tissues (p < 0.001). Furthermore, patients with lower Pdcd4 expressions were found to have shorter disease-free survival (p = 0.037). The expression of Pdcd4 was also assessed by immunohistochemical analysis in 13 ovarian normal tissues and 44 carcinomas. Different subcellular localization of Pdcd4 was observed in normal compared to malignant cells. Predominant nuclear localization of Pdcd4 was found in normal ovarian tissues while ovarian carcinomas showed mainly cytoplasmic localization of Pdcd4.ConclusionOur study demonstrated that the loss of Pdcd4 was a common abnormality at molecular level in ovarian cancer and it might be a potential prognostic factor in ovarian cancer patients.