Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer.
Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the pi-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression. (+info)
Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells.
DNA-dependent protein kinase (DNA-PK) functions in double-strand break repair and immunoglobulin [V(D)J] recombination. We previously established a radiation-sensitive human cell line, M059J, derived from a malignant glioma, which lacks the catalytic subunit (DNA-PKcs) of the DNA-PK multiprotein complex. Although previous Northern blot analysis failed to detect the DNA-PKcs transcript in these cells, we show here through quantitative studies that the transcript is present, albeit at greatly reduced (approximately 20x) levels. Sequencing revealed no genetic alteration in either the promoter region, the kinase domain, or the 3' untranslated region of the DNA-PKcs gene to account for the reduced transcript levels. Nuclear run-on transcription assays indicated that the rate of DNA-PKcs transcription in M059J and DNA-PKcs proficient cell lines was similar, but the stability of the DNA-PKcs message in the M059J cell line was drastically (approximately 20x) reduced. Furthermore, M059J cells lack an alternately spliced DNA-PKcs transcript that accounts for a minor (5-20%) proportion of the DNA-PKcs message in all other cell lines tested. Thus, alterations in DNA-PKcs mRNA stability and/or the lack of the alternate mRNA may result in the loss of DNA-PKcs activity. This finding has important implications as DNA-PKcs activity is essential to cells repairing damage induced by radiation or radiomimetric agents. (+info)
Hybrid capture II, a new sensitive test for human papillomavirus detection. Comparison with hybrid capture I and PCR results in cervical lesions.
AIM: To test a new assay for the detection of human papillomavirus (HPV) DNA, hybrid capture II (HC II), compared with the previous commercialized hybrid capture I (HC I) and polymerase chain reaction (PCR) results on cervical scrapes from fresh cone excision biopsy samples. METHODS: The three methods were used on cervical scrapes from 42 fresh cone excision biopsy samples. There were nine metaplastic and inflammatory lesions, five low grade lesions, and 28 high grade lesions. PCR was performed using the general primers GP5+/GP6+. The viral load of high risk HPV DNA was estimated by the ratio of relative light units to positive control values in the samples. RESULTS: The sensitivity of HC I for the detection of high grade lesions was 71.4%, while it was 92.8% for HC II and 96.4% for the PCR. Considering only the absence of detectable cervical in situ neoplasia, the specificity was 88.9% for HC I, 66.7% for HC II, and 66.7% for PCR. With HC II, for a ratio of cervical sample to normal control of > 200, the sensitivity for the detection of high grade lesion was only 34.6% with a specificity of 66.7%. CONCLUSIONS: HPV detection with the HC II assay is more sensitive than the previous HC I and represents a more convenient and easier test than PCR for routine use. Nevertheless the viral load estimated with this test cannot be a reliable predictive indicator of high grade lesions. (+info)
Human papillomavirus DNA in adenosquamous carcinoma of the lung.
AIM: To investigate the presence of human papillomavirus (HPV) DNA in adenosquamous carcinoma of the lung--which is relatively common in Okinawa but not in mainland Japan--and examine its histological features. METHODS: Of 207 cases where primary lung cancers were surgically removed between January 1995 and June 1997 in Okinawa, 23 were adenosquamous carcinoma. HPV was detected by non-isotopic in situ hybridisation (NISH) and polymerase chain reaction (PCR) amplification with primers specific for E6 and E7 regions of the HPV genome. PCR products were analysed by Southern blotting. Immunohistochemical determination of high molecular weight cytokeratin (HMC) and involucrin was also carried out. RESULTS: 18 cases were positive for HPV DNA by PCR and NISH. HPV types 6, 11, 16, and 18 were found. Seven cases were dual positive for different types of HPV. Using NISH, HPV was also found in the squamous cell components and in neighbouring enlarged adenocarcinoma cells. The HMC and involucrin were demonstrated immunohistochemically in the same areas. CONCLUSIONS: HPV DNA was found in a high proportion (78.3%) of adenosquamous carcinomas in Okinawa, a region where HPV has previously been shown to be prevalent in squamous cell carcinoma of the lung. The adenocarcinoma cells adjacent to the squamous cell carcinoma component were enlarged and positive for HPV, HMC, and involucrin. This is thought to indicate the transition from adenocarcinoma to squamous cell carcinoma. (+info)
Immunohistochemical expression of mdm2 and p21WAF1 in invasive cervical cancer: correlation with p53 protein and high risk HPV infection.
AIM: To investigate the immunocytochemical staining pattern of mdm2 and p21WAF1 proteins in invasive cervical cancer and to determine its relation with the expression of p53 and with the high risk HPV infection. METHODS: Immunocytochemistry for p53, mdm2, and p21WAF1 was performed in 31 paraffin embedded sections of invasive cervical cancer. The results were assessed by image analysis, evaluating for each protein the optical density of the immunostained area, scored as percentage of the total nuclear area. The presence of high risk human papillomavirus (HPV) infection was detected by using the polymerase chain reaction. RESULTS: Immunostaining for both mdm2 and p21WAF1 was correlated with p53 expression; however, the correlation between p53 and mdm2 (R = 0.49; p < 0.01) was more significant than between p53 and p21WAF1 (R = 0.31; p < 0.05); the less stringent correlation between p53 and p21WAF1 might reflect the p53 independent mechanisms of p21WAF1 induction. Similar average levels of p53, mdm2, and p21WAF1 immunostaining were found in the presence or absence of high risk HPV-DNA, without significant differences between the two groups. CONCLUSIONS: These data suggest that mdm2 and p21WAF1 proteins are expressed in invasive cervical cancer and that their immunocytochemical staining pattern is not abrogated by the presence of high risk HPV genomic sequences. (+info)
The significance of cagA and vacA subtypes of Helicobacter pylori in the pathogenesis of inflammation and peptic ulceration.
AIMS: To assess the significance of cagA and vacA subtypes of Helicobacter pylori in relation to inflammation and density of bacterial colonisation in vivo within a dyspeptic UK population. METHODS: Dyspeptic patients who were Helicobacter pylori positive had antral samples taken for histology and culture. Gastroduodenal pathology was noted. The grade of bacterial density and inflammation was assessed using the Sydney system. Bacterial DNA was extracted and the vacA alleles and the cagA/gene typed using PCR. RESULTS: 120 patients were studied. There was high rate of cagA positive strains in this population. Bacterial density did not correlate with the presence of peptic ulceration. There was a significant association between cagA positive strains and increased inflammation and bacterial density. The vacA s1 type independently correlated with extensive chronic inflammation but there was no association with bacterial density. The vacA m type did not correlate with extent of inflammation or bacterial density. CONCLUSIONS: The results suggest that cagA is important in the pathogenesis of inflammation and peptic ulceration. These findings are in keeping with the hypothesis that cagA acts as a marker for a cag pathogenicity island which encodes several genes involved in inflammation. The vacA s1 allele correlates with inflammation independently of cagA, possibly through its enhanced ability to produce the vacuolating cytotoxin. (+info)
Expression of vascular endothelial growth factor in human oral squamous cell carcinoma: its association with tumour progression and p53 gene status.
AIMS: To correlate vascular endothelial growth factor (VEGF) expression in oral squamous cell carcinoma with the clinicopathological characteristics and prognosis; and to assess whether p53 gene status is associated with VEGF expression in human cancers. METHODS: Tumour specimens from 45 patients with oral squamous cell carcinomas were examined. Expression of VEGF was determined using an immunohistochemical method, and a tumour was considered positive when more than 5% of the neoplastic cells showed VEGF immunoreactivity. The p53 gene status was screened using a polymerase chain reaction--single strand conformation polymorphism analysis. RESULTS: VEGF positive staining was detected in 19 (42.2%) of the 45 cases. VEGF immunoreactivity did not correlate with the histological degree of tumour differentiation, clinical stages, or lymph node metastasis. The patients with VEGF positive tumours had a significantly worse prognosis than those with VEGF negative tumours. The five year overall survival rate of the VEGF negative patients was 76.5%, as compared with 48.8% for the VEGF positive patients. No significant association between VEGF expression and the p53 gene status of the tumours was found. CONCLUSIONS: VEGF is a good prognostic indicator of the survival of patients with oral squamous cell carcinoma. The p53 gene status does not seem to be associated with VEGF expression in these cancers. (+info)
The alphaE-catenin gene (CTNNA1) acts as an invasion-suppressor gene in human colon cancer cells.
The acquisition of invasiveness is a crucial step in the malignant progression of cancer. In cancers of the colon and of other organs the E-cadherin/catenin complex, which is implicated in homotypic cell-cell adhesion as well as in signal transduction, serves as a powerful inhibitor of invasion. We show here that one allele of the alphaE-catenin (CTNNA1) gene is mutated in the human colon cancer cell family HCT-8, which is identical to HCT-15, DLD-1 and HRT-18. Genetic instability, due to mutations in the HMSH6 (also called GTBP) mismatch repair gene, results in the spontaneous occurrence of invasive variants, all carrying either a mutation or exon skipping in the second alphaE-catenin allele. The alphaE-catenin gene is therefore, an invasion-suppressor gene in accordance with the two-hit model of Knudsen for tumour-suppressor genes. (+info)