miR-106a overexpression and pRB downregulation in sporadic colorectal cancer.

Rb1 plays an important role in cell cycle progression and therefore may be involved in malignant transformation of colonic cells. The aim of our research was to define the potential role of Rb1 as a prognostic biomarker in tumorigenesis of sporadic colorectal cancer, and to examine the role of miR-106a in Rb1 regulation as it functionally binds to 3'UTR of transcribed mRNA. We examined LOH and promoter methylation status. Real-time PCR was used for Rb1 mRNA and miR-106a, and immunohistochemistry for protein expression analysis. All the results obtained from patients' samples were correlated with the clinicopathological parameters in order to determine its influence on the sporadic colorectal carcinogenesis. LOH showed no correlation with mRNA and pRb expression. 51.5% of tumor samples were scored negative for pRb staining. Despite this finding, we detected overexpression of Rb1 mRNA in tumor samples in comparison to the adjacent normal tissue (p=0.023). mRNA overexpression was consistent with Rb1 promoter methylation analysis results, which showed no methylation in the investigated samples. Expression analysis of miR-106a in the patients samples showed its overexpression in colorectal cancer (p<10(-4)). Negative pRb score was expected according to the definition of tumor suppressor genes and their proposed role in the malignant transformation of the cells. The observed discrepancy between mRNA and protein expression can be explained by a regulatory mechanism that inhibits translation, such as microRNA silencing. Our results suggest that miR-106a might have a regulatory role for Rb1 in sporadic colorectal cancer.

A dual role of p21waf1/cip1 gene in apoptosis of HEp-2 treated with cisplatin or methotrexate.

We investigated the effects of p21(waf1/cip1) gene overexpression in human laryngeal squamous carcinoma cells HEp-2 lacking p53 protein expression on apoptosis induction upon the treatment with two commonly used chemotherapeutic agents, cisplatin and methotrexate. For that purpose, we employed cDNA arrays and qPCR to monitor gene expression upon treatment with AdCMV-p21 alone or in combination with the chemotherapeutic compounds. We found that p21(waf1/cip1) gene overexpression provoked apoptosis of HEp-2 through the induction of the TNFRSF9 gene and activation of caspase 7. In addition, we have proved that p21(waf1/cip1) can assume a dual role in apoptosis in the same cell system depending on the chemotherapeutic agent: its overexpression enhances apoptosis in cisplatin-treated cells and attenuates apoptotic signals in methotrexate-treated cells. The observed dual role of p21(waf1/cip1) was in direct correlation with the modulation of caspases 3 and 7 activation and changes in the expression of GADD45a gene. The results presented herein encourage future use of targeted p21(waf1/cip1) gene therapy in cancer treatment in a well-defined therapeutic and genetic context.

NF1 gene loss of heterozygosity and expression analysis in sporadic colon cancer.

BACKGROUND AND AIMS: Colon cancer tumorigenesis is a multistep process of mutation accumulation in a number of oncogenes and tumour suppressor genes. NF1 gene protein, neurofibromin, acts as a tumour suppressor by turning the active form of Ras into an inactive form. This molecular switch has an important role in the control of the cell cycle and differentiation, and changes in Ras activity are present in many different cancers. This is the first study to investigate the role of NF1 in sporadic colon cancer. METHODS: We investigated loss of heterozygosity (LOH) at the NF1 locus. Real time reverse transcription-polymerase chain reaction was used to determine NF1 mRNA expression in tumours and corresponding normal tissue. Expression of neurofibromin was analysed by immunohistochemistry. Relative ratio of NF1 mRNA type I and II isoform expression was also examined. RESULTS: LOH of the NF1 gene was detected in 20.7% of heterozygous samples. NF1 mRNA expression was significantly increased in tumour tissue compared with corresponding normal tissue (p = 0.04291). There was a statistically significant increase in NF1 type I isoform expression (p = 0.0005) in tumour tissue compared with corresponding normal colon tissue. NF1 isoform type II was predominantly expressed in normal tissue while the NF1 isoform type I prevailed in tumour samples. The transition from dominant expression of isoform type II in normal mucous tissue 15 cm away from the tumour to dominant expression of isoform type I in tumour tissue itself was detected. Total neurofibromin expression increased as tumours were more advanced but expression of wild-type neurofibromin remained the same. CONCLUSIONS: Our findings suggest that the NF1 gene may play a role in the development and progression of colon cancer and the NF1 gene may be a potential tumour marker and a new potential target for colon cancer therapy.

nm23-H1 expression and loss of heterozygosity in colon adenocarcinoma.

BACKGROUND: The discovery that genetic alterations in oncogenes and tumour suppressor genes accompany tumour formation in many human tumours has encouraged the search for genes that promote or suppress tumour spread and metastasis; nm23 is a promising candidate for a metastasis suppressing gene. AIMS: To evaluate whether expression of nm23-H1 protein or loss of heterozygosity (LOH) of the nm23-H1 gene is associated with colon cancer progression. MATERIALS/METHODS: Paraffin wax embedded tissue sections were analysed immunohistochemically. DNA isolated from normal and tumour tissue was used for LOH analysis using a variable nucleotide tandem repeat (VNTR) marker located in the untranslated 5' region of the nm23-H1 gene. RNA isolated from tumour and normal tissue was used for "real time" RT-PCR. RESULTS: Of 102 adenocarcinomas examined, 58.8% stained weakly for nm23-H1 protein. There was a negative correlation between nm23-H1 positivity and tumour histological grade. In VNTR analysis, 70.2% of patients were informative and 27.4% of tumours had nm23-H1 LOH. There was a positive correlation between nm23-H1 LOH and both tumour histological grade and Dukes's stage. Expression of nm23-H1 mRNA was increased in 22 of 30 colon tumours compared with normal tissue. No significant correlation was found between nm23-H1 mRNA expression and histological grade or Dukes's stage of tumours. CONCLUSIONS: These findings suggest that nm23-H1 protein expression in early stages may have a role in suppressing metastasis in sporadic colon cancer, whereas at a later stage both reduced nm23-H1 protein expression and LOH of the nm23-H1 gene may play role in colon cancer progression and metastasis.

Aberration of FHIT gene is associated with increased tumor proliferation and decreased apoptosis-clinical evidence in lung and head and neck carcinomas.

BACKGROUND: Human FHIT (fragile histidine triad) gene is highly conserved gene homologous to a group of genes identified in prokaryotes and eukaryotes. Loss of FHIT function may be important in the development and/or progression of various types of cancer. MATERIALS AND METHODS: We undertook a clinical study to analyze the relation between aberrant function of FHIT gene, tumor cell proliferation, and intensity of apoptosis as well as prognostic output in lung and squamous cell head and neck carcinoma (HNSCC). Status of FHIT gene, expression of p21waf1, intensity of apoptosis, and cell proliferation were analyzed in HNSCC and lung carcinoma tissues by molecular genetic methods, immunohistochemistry, [3H]-thymidine labeling method, and FACScan analysis in frozen and paraffin-embedded tissue sections. RESULTS: The majority of the malignant lung and HNSCC lesions displayed aberrant expression of FHIT gene, followed by low or negative expression of p21waf1, and increased intensity of cell proliferation. Similar results were obtained on synchronous combinations of normal, precancerous, and cancerous head and neck tissues. The observed changes increased with progression of these lesions. We examined tumor and corresponding normal tissue samples for microsatellite markers D3S1300 and D3S4103 to evaluate the loss of heterozygosity (LOH) at the FHIT gene loci. We found high percentage of LOH in both lung tumors and HNSCC (75% for D3S1300 and 79% for D3S4103 in lung cancer, and 87% for D3S1300 and 78% for D3S4103 in HNSCC). The median survival time of the patients suffering from lung cancer without FHIT protein expression was 22.46 months and that of the patients with FHIT expression 36.04 months. FHIT-negative cases tended to correlate with a worse prognosis, but this was not statistically significant. Median survival time of HNSCC patients without FHIT protein expression was 30.86 months and that of the patients with FHIT expression was 64.04 months (p < 0.05). CONCLUSIONS: Our results show a correlation between aberrant FHIT expression, a low rate of apoptosis, and high tumor cell proliferation. Aberrant FHIT gene could be a prognostic marker in lung cancer.

Submerged gel electrophoresis on Spreadex gels--a new method for APC gene mutation detection.

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease. Patients with FAP develop hundreds to thousands of adenomatous polyps in the colon and rectum during their 2nd or 3rd decades, and one or more of them progress to cancer if left without surgical treatment. The gene responsible for FAP was identified in 1991 and termed the APC (adenomatous polyposis coli) gene. Following identification of APC, a number of germ-line mutations responsible for the development of the disease were found. The purpose of this study was to determine the usefulness of a new method, submerged gel electrophoresis, in the detection of the most-frequent mutation of the APC gene [5-base pair (bp) deletion in codon 1309], especially in the presymptomatic diagnosis of FAP. Genomic DNAs were isolated from peripheral blood of patients and their relatives. We used two methods, electrophoresis on polyacrylamide gel and submerged gel electrophoresis, for the identification of APC gene codon 1309 mutation. After only 110 min PCR fragments of 91 bp and 86 bp (5-bp deletion) were completely resolved on a Spreadex EL300 gel. Our results showed that electrophoresis using Spreadex gels provides a simple and rapid non-radioactive method for determination of the most-frequent germ-line mutations in the APC gene.

Loss of heterozygosity of DPC4 tumor suppressor gene in human sporadic colon cancer.

We investigated the prevalence of DPC4 loss of heterozygosity in sporadic colorectal cancer. Thirty-six cases of human sporadic colon carcinoma and corresponding normal tissue samples were examined to evaluate loss of heterozygosity at the DPC4 tumor suppressor locus using variable nucleotide tandem repeat (VNTR) analysis and three polymorphic markers. From 36 analyzed samples 35 (97%) were heterozygous or informative. Loss of heterozygosity at the DPC4 locus was detected in 18 (51%) of informative tumor DNAs. The DPC4 LOH was more frequent in smaller tumors (<5 cm) than in larger ones. There was no correlation between DPC4 LOH and age or sex of patients. There was a negative correlation between DPC4 LOH and histological grade or Dukes' stage of tumors, but without statistic significance. Observed results are in agreement with the view that malignant progression is consequence of many genetic changes. It can be concluded that inactivation of the DPC4 gene plays a role in a multistep process of outgrowth and progression of colon cancer.