Changes in SNAI1 and VIM gene expression in Caco2 cells after cocultivation with bacteria from colorectal cancer biopsies.

The development of distant metastases is the final stage in the progression of solid cancer and is responsible for the majority of cancer-related deaths. Epithelial-mesenchymal transition (EMT) is involved in cancer progression and metastasis. In the present study we used different types of intracellular bacteria isolated from colorectal cancer biopsies to examine their effect on the expression of SNAI1 and VIM genes in Caco2 cell line. SNAI1 gene expression was significantly decreased after cocultivation of Caco2 cells with Pseudomonas aeruginosa, Proteus vulgaris, Proteus mirabilis, Enterococcus faecalis, Klebsiella pneumoniae and Bacillus cereus, respectively (P<0.05). We observed more than 2-fold increase in VIM gene expression within Caco2 cells after cocultivation with Proteus vulgaris. On the other hand, VIM gene expression decreased by half after cocultivation with Pseudomonas aeruginosa and Proteus mirabilis (P<0.05). Our data suggest bacteria presented in colorectal carcinoma tissues may cause changes in gene expression of EMT-associated genes. Further research is needed to find out whether bacteria are capable to support EMT and cancer progression.

The study of bacteria in biopsies from Slovak colorectal adenoma and carcinoma patients.

The development of colorectal cancer is affected by many factors, especially the intestinal microbiota. However, precise knowledge of bacterial communities associated with the mucosa in various parts of the colon is limited. Herein, we applied the gentamicin protection assay and detected the presence of intracellular bacteria in colorectal biopsies from Slovak patients with colorectal adenoma and carcinoma, and we compared this with healthy controls. The ENTEROtest 24 and MALDI-TOF mass spectrometry identified the cultivated bacteria and results revealed the presence of intracellularly localized Escherichia coli, Proteus mirabilis and Proteus vulgaris in patients with colorectal adenomas and carcinomas. In addition to these species, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis and Bacillus cereus were identified in colorectal biopsies, but these were extracellular. The marked increase in relative abundance of intracellular E. coli in patients with colorectal adenomas and carcinomas was statistically significant compared to controls, and our preliminary data supports E. coli's role as a pro-oncogenic pathogen.

Novel strategies for comprehensive mutation screening of the APC gene.

Colorectal cancer is the 4th most common cause of cancer related deaths worldwide and new possibilities in accurate diagnosis and targeted treatment are highly required. Mutations in adenomatous polyposis coli (APC) gene play a pivotal role in adenoma-carcinoma pathway of colorectal tumorigenesis. The quarter century from its´ first cloning, APC became one of the most frequently mutated, known driver genes in colorectal cancer. Intensive routine molecular testing of APC has brought the benefits for patients with family history of polyposis or colorectal cancer. Nevertheless, multiple mutational disease-causing mechanisms make the genetic testing still challenging. This minireview is focused on implementation of novel APC mutation screening diagnostic strategies for polyposis families according to the current findings. A further understanding and improved algorithms may help to increase the mutation detection rate. APC germline mutations achieve close to 100% penetrance, so more comprehensive approach followed by preventive and therapeutic strategies might reflect in decrease in burden of colorectal cancer.

Modification of microflora imbalance: future directions for prevention and treatment of colorectal cancer?

Increasing incidence and mortality of colorectal cancer brings the necessity to uncover new possibilities in the prevention, diagnosis and treatment. The microbiome as the collective genetic material of the microflora, overexceed the number of genes in the human genome and is unique for each individual. Due to the benefits providing for the host and mainly for immediate interaction with the host immune system, a gastrointestinal microflora can be considered "cardinal microbiome". Host-microbial relations includes symbiotic, pathogenic and competitive interactions. Causal role of gastrointestinal microflora in colorectal carcinogenesis is still not well determined. This minireview is focused on current evidence in understanding the role of bacteria in colorectal carcinogenesis, the impact of bacterial dysbiosis on tumor formation, and ability of probiotics and bacterial vectors to modulate the gastrointestinal microflora as prevention and therapy tool in colorectal cancer.

Intestinal flora of FAP patients containing APC-like sequences.

Colorectal cancer mortality is one of the most common cause of cancer-related mortality. A multiple risk factors are associated with colorectal cancer, including hereditary, enviromental and inflammatory syndromes affecting the gastrointestinal tract. Familial adenomatous polyposis (FAP) is characterized by the emergence of hundreds to thousands of colorectal adenomatous polyps and FAP syndrome is caused by mutations within the adenomatous polyposis coli (APC) tumor suppressor gene. We analyzed 21 rectal bacterial subclones isolated from FAP patient 41-1 with confirmed 5bp ACAAA deletion within codons 1060-1063 for the presence of APC-like sequences in longest exon 15. The studied section was defined by primers 15Efor-15Erev, what correlates with mutation cluster region (MCR) in which the 75% of all APC germline mutations were detected. More than 90% homology was showed by sequencing and subsequent software comparison. The expression of APC-like sequences was demostrated by Western blot analysis using monoclonal and polyclonal antibodies against APC protein. To study missing link between the DNA analysis (PCR, DNA sequencing) and protein expresion experiments (Western blotting) we analyzed bacterial transcripts containing the 15Efor-15Erev sequence of APC gene by reverse transcription-PCR, what indicated that an APC gene derived fragment may be produced. We observed 97-100 % homology after computer comparison of cDNA PCR products. Our results suggest that presence of APC-like sequences in intestinal/rectal bacteria is enrichment of bacterial genetic information in which horizontal gene transfer between humans and microflora play an important role.

Different phenotype manifestation of familial adenomatous polyposis in families with APC mutation at codon 1309.

Germline mutation in APC gene induced development of familial adenomatous polyposis (FAP). The risk of developing specific manifestation of FAP is often correlated with the position of the inherited APC mutation. Patients with mutations localized in the largest exon 15 between codons 1286 and 1513 (mutation cluster region, MCR) have generally a worse prognosis with early onset of the disease. We found 6 FAP families with mutation at codon 1309 (3927_3931delAAAGA) in the cohort of 39 FAP Slovak families with rapid cancer progress. In addition, mutation in codon 1309 was detected in three family members, one of them with a very different phenotype. This oldest family member, aged 81, has persisted asymptomatic without clinical manifestations.

Ashkenazi founder BRCA1/BRCA2 mutations in Slovak hereditary breast and/or ovarian cancer families.

Germline mutations in BRCA1 and BRCA2 have been predominantly associated with the breast and ovarian cancers. Two mutations in BRCA1 (185delAG and 5382insC) and one mutation in BRCA2 (6174delT) are common in Ashkenazi Jewish population. To determine the proportion of these founder mutations, we analyzed DNA samples of 120 Slovak hereditary breast and/or ovarian cancer (HBOC) suspected families. Two particular exons of BRCA1 (2, 20) and 11N segment of BRCA2 were screened by single strand conformation polymorphism (SSCP) followed by DNA sequencing of fragments showing abnormal migration pattern. Mutational analysis revealed that 7 out of 20 (35%) families with detected BRCA1/BRCA2 pathogenic alteration harbored one of three Jewish mutations: five families with 5382insC, one family with 185delAG and one family with 6174delT. Interestingly, we have noted a very rare phenotype, when 5382insC in BRCA1 co-segregated also with endometrial carcinoma. Similarly to the studies from other countries of Central and Eastern Europe, the most frequent pathogenic alteration found was 5382insC that accounted for 1/4 of all gene defects detected. Following the high proportion of Ashkenazi Jewish founder mutations in Slovak HBOC families, a pre-screening for at least 5382insC mutation in individuals at even moderate risk would be appropriate.

The novel exon 11 mutation of BRCA1 gene in a high-risk family.

Germline mutations in the BRCA1 and BRCA2 genes are required for the initiation of the development of hereditary forms of breast and ovarian cancer, which represent 10-15% of all cases. The course of the disease varies from case to case that can be due even to the possibility of multiple genetic changes including inactivation of other tumor suppressor genes--TP53 and APC genes or activation of oncogenes, especially K-ras oncogene. The combination of these changes results in an early expression of the broad variety of malignancies. The analyzed proband (II-5) comes from a high-risk family, in which various types of cancer were observed. The novel BRCA1 mutation in exon 11 (2057delCAGTGAAGAG) was detected by SSCP, HDA techniques and confirmed by automatic sequencing. The same deletion was observed in DNA sample of her first daughter (III-1), but DNA of her second one was without any mutational changes (III-2). Due to the occurrence of different types of cancer in this family, the incidental mutations in the APC; resp. TP53 tumor supressor genes and K-ras oncogene were searched as well. Any mutation was found after sequencing of SSCP interesting exons of these genes. The reasons for such strong malignant manifestation in this high risk family are discussed.

The most frequent APC mutations among Slovak familial adenomatous polyposis patients. Adenomatous polyposis coli.

We screened 46 suspected families from whole Slovakia for familial adenomatous polyposis (FAP) cancer predisposition. Individuals were enrolled to the adenomatous polyposis coli (APC) gene mutations mapping program at the base of previous clinical investigation. We have used the following techniques: heteroduplex analysis (HDA), protein truncation test (PTT), single strand conformation polymorphism (SSCP) and sequencing for the identification and detailed positional analysis of APC mutations. Around 90% of all detected mutations were found being truncated. The most frequent mutations from this collection were located within codons 1309 and 1061 of exon 15 and represented 15% and 7%, respectively of all tested families. The expressive phenotype, large amount of colorectal polyps and congenital hypertrophy of the retinal pigment epithelium (CHRPE) were associated to all mutations within codons 1309 and 1060.

Mutation screening of the BRCA1 gene in Slovak patients.

Breast cancer is the most commonly observed malignancy in women of the western world. The family history is the strongest risk factor for the disease. Two major genes, BRCA1 and BRCA2 that are involved in the familial breast and ovarian cancer have been described. Germ-line mutations of the BRCA1 gene have been linked to 85% of all hereditary breast and ovarian cancers. We performed a mutation screening ofthe entire codingregion of the BRCA1 gene in 29 Slovak families suspected of having inherited predisposition to breast cancer. For the analysis we used a combination of a single strand conformation polymorphism (SSCP), denaturing high-performance liquid chromatography (DHPLC) and sequencing. Genetic alterations were consistently indicated by SSCP and DHPLC and consequently confirmed by DNA sequencing as previously described pathogenic mutations. The patients with inherited BRCA1 mutations will undergo genetic counseling and cancer prevention health care program.

A double germline mutations in the APC and p53 genes.

Germline mutation in the APC gene is required for the initiation of the development of familial adenomatous polyposis (FAP). According to Fearon and Vogelstein model, further somatic mutations in the K-ras oncogene, DCC gene and p53 tumor suppressor gene are prerequisite for development of colon carcinoma. We have found that the germline mutations in the DNA isolated from lymphocytes of an 18 years old girl with extraordinary expressive phenotype in codons 1060-1061 of the APC gene result in truncation of the APC protein. The mutation in codons 12 and 13 of the K-ras oncogene was not detected, but another germline mutation was found in codon 210 of the p53 gene. Furthermore, no one of these germline mutations was detected in the DNA of peripheral blood lymphocytes of the patient's 21 years old healthy sister. Until now, there has been no evidence about the expressive phenotype due to mutation in codons 1060-1061 of the APC gene; the role of germline missense mutation in codon 210 of the p53 gene in the FAP malignant process remains to be elucidated too. The effect of the combination of germline mutation in two different tumor suppressor genes in the progress of disease is discussed.

Characterization of APC exon 15 germ-line mutation in FAP family with severe phenotype showing extracolonic symptoms.

The adenomatous polyposis coli (APC) gene plays a crucial role in colorectal carcinogenesis. Germ-line mutations of APC gene give rise to familial adenomatous polyposis coli (FAP) - autosomal dominant syndrome manifesting hundreds to thousands of colorectal polyps, if untreated with malignant progression. We have used the techniques of heteroduplex analysis (HDA), protein truncation test (PTT), single strand conformation polymorphism (SSCP) and DNA sequencing for the identification and detailed positional analysis of mutations in IFAP family with the expressive phenotype characterized by polyposis and extracolonic lesions. Detailed analysis revealed a 5bp deletion in a mutation cluster region (MCR) in exon 15 of APC gene in codon 1308. Two screened members of the FAP family exhibited this novel mutation.

The combination of heteroduplex analysis and protein truncation test for exact detection of the APC gene mutations.

Familial adenomatous polyposis (FAP) is usually associated with mutation in the adenomatous polyposis coli (APC) gene. To examine the occurrence of these mutations in the number of FAP suspected families from the whole Slovakia effectively, we have applied heteroduplex analysis (HDA) and protein truncation test (PTT) for the analyses of 2-5 base pair deletions and point mutations of the APC gene. In the analyzed exon 15 of the APC gene determined by the primers 15Efor-15Grev for HDA and 15ET7-15J3 for PTT more than 70% of mutations should be deletions [3, 12], which are detectable by HDA. In our collection of 5 FAP families mutations in the APC gene were found in families 10, 27 and 41 using HDA. By PTT test the formation of truncated APC protein in FAP families 2, 10, 16 and 27 were revealed. The necessity of combination of at least HDA and PTT techniques for exact detection of APC mutations in analyzed APC region is discussed.

Identification of APC exon 15 mutations in families suspected of familial adenomatous polyposis (FAP).

Patients with familial adenomatous polyposis coli (FAP) reveal numerous colorectal adenomas as well as benign and malignant extracolonic lesions. Adenomatous polyposis coli (APC) gene mutations are the crucial genetic defect in FAP. The APC mutation molecular analysis of 20 FAP families was performed using the novel and effective method of the heteroduplex analysis (HDA). All of these families were screened for mutations in APC exon 15. APC mutations were identified in 4 individuals of two families. These two families were also screened by the protein truncation test (PTT). The PTT results confirmed previous findings obtained by HDA. The results of molecular analysis were correlated with the clinical manifestations of extracolonic lesions and congenital hypertrophy of retinal pigment epithelium (CHRPE). Positive correlation of all clinical examinations and mutations of APC gene was observed in all 4 FAP patients.

gp51 of bovine leukemia virus gene expression in hamster cells.

Recombinant pMMEx-bovine leukemia virus env gene DNA fragments were produced and expressed in eukaryotic cells. Clone C4, containing an SmaI-SmaI fragment of the gene coding for gp51, was co-transfected with pSV2neo DNA into Chinese hamster cells. About 800 geneticin-resistant cell clones were isolated and then morphologically and biologically characterized. The presence of gp51 encoding env gene fragments was detected in 17 of them by Southern blotting. The expression of gp51 gene in hamster cells was confirmed by Western blotting of their lysates with monoclonal antibodies (MAbs) directed against different epitopes of gp51 of bovine leukemia virus. The immunoreactivity of the expressed peptides with MAbs directed against neutralizing epitopes of gp51 of bovine leukemia virus was confirmed.