| 1. |
- Dmitriev, Alexey A, et al.
(författare)
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Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays
- 2012
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Ingår i: Epigenetics. - : Landes Bioscience. - 1559-2294 .- 1559-2308. ; 7:5, s. 502-513
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Tidskriftsartikel (refereegranskat)abstract
- This study aimed to clarify genetic and epigenetic alterations that occur during lung carcinogenesis and to design perspective sets of newly identified biomarkers. The original method includes chromosome 3 specific NotI-microarrays containing 180 NotI clones associated with genes for hybridization with 40 paired normal/tumor DNA samples of primary lung tumors: 28 squamous cell carcinomas (SCC) and 12 adenocarcinomas (ADC). The NotI-microarray data were confirmed by qPCR and bisulfite sequencing analyses. Forty-four genes showed methylation and/or deletions in more than 15% of non-small cell lung cancer (NSCLC) samples. In general, SCC samples were more frequently methylated/deleted than ADC. Moreover, the SCC alterations were observed already at stage I of tumor development, whereas in ADC many genes showed tumor progression specific methylation/deletions. Among genes frequently methylated/deleted in NSCLC, only a few were already known tumor suppressor genes: RBSP3 (CTDSPL), VHL and THRB. The RPL32, LOC285205, FGD5 and other genes were previously not shown to be involved in lung carcinogenesis. Ten methylated genes, i.e., IQSEC1, RBSP3, ITGA9, FOXP1, LRRN1, GNAI2, VHL, FGD5, ALDH1L1 and BCL6 were tested for expression by qPCR and were found downregulated in the majority of cases. Three genes (RBSP3, FBLN2 and ITGA9) demonstrated strong cell growth inhibition activity. A comprehensive statistical analysis suggested the set of 19 gene markers, ANKRD28, BHLHE40, CGGBP1, RBSP3, EPHB1, FGD5, FOXP1, GORASP1/TTC21, IQSEC1, ITGA9, LOC285375, LRRC3B, LRRN1, MITF, NKIRAS1/RPL15, TRH, UBE2E2, VHL, WNT7A, to allow early detection, tumor progression, metastases and to discriminate between SCC and ADC with sensitivity and specificity of 80-100%.
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| 2. |
- Haraldson, Klas, et al.
(författare)
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LRRC3B gene is frequently epigenetically inactivated in several epithelial malignancies and inhibits cell growth and replication
- 2012
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Ingår i: Biochimie. - : Elsevier. - 0300-9084 .- 1638-6183. ; 94:5, s. 1151-1157
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Tidskriftsartikel (refereegranskat)abstract
- Chromosome 3 specific NotI microarrays containing 180 NotI linking clones associated with 188 genes were hybridized to NotI representation probes prepared using matched tumor/normal samples from major epithelial cancers: breast (47 pairs), lung (40 pairs) cervical (43 pairs), kidney (34 pairs of clear cell renal cell carcinoma), colon (24 pairs), ovarian (25 pairs) and prostate (18 pairs). In all tested primary tumors (compared to normal controls) methylation and/or deletions was found. For the first time we showed that the gene LRRC3B was frequently methylated and/or deleted in breast carcinoma - 32% of samples, cervical - 35%, lung - 40%, renal - 35%, ovarian - 28%, colon - 33% and prostate cancer - 44%. To check these results bisulfite sequencing using cloned PCR products with representative two breast, one cervical, two renal, two ovarian and two colon cancer samples was performed. In all cases methylation was confirmed. Expression analysis using RT-qPCR showed that LRRC3B is strongly down-regulated at the latest stages of RCC and ovarian cancers. In addition we showed that LRRC3B exhibit strong cell growth inhibiting activity (more than 95%) in colony formation experiments in vitro in KRC/Y renal cell carcinoma line. All these data suggest that LRRC3B gene could be involved in the process of carcinogenesis as a tumor suppressor gene.
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| 3. |
- Kondratov, Aleksandr G, et al.
(författare)
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Alterations of the WNT7A Gene in Clear Cell Renal Cell Carcinomas
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 7:10
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Tidskriftsartikel (refereegranskat)abstract
- WNT7A (wingless-type MMTV integration site family, member 7A) is a known tumor suppressor gene of non-small cell lung carcinomas (NSCLC) and is frequently inactivated due to CpG-island hypermethylation in human cancers. The members of WNT family are involved in cell signaling and play crucial roles in cancer development. In the present work hypermethylation of the WNT7A gene was detected in 66% (29/44) of analyzed clear cell renal cell carcinomas (RCCs) using methyl-specific PCR (MSP). Moreover, bisulfite sequencing confirmed intensive hypermethylation of the 5-CpG island of the WNT7A gene. Methylation analysis revealed positive correlations between tumor stage, Fuhrman nuclear grade and WNT7A hypermethylation. Additionally, restoration of WNT7A gene expression in the A498 cell line by 5-aza-2-deoxycytidine treatment confirmed a direct contribution of hypermethylation in silencing of the WNT7A gene. High frequency of loss of heterozygosity (LOH) was demonstrated on chromosome 3p25 in regions surrounding the WNT7A gene. The frequent down-regulation of WNT7A gene expression was detected in 88% (15/17) of clear cell RCCs. We have also shown that the WNT7A gene possesses tumor suppression function by colony-formation and cell proliferation assays in RCC cell lines. In summary, the WNT7A gene is inactivated by genetic/epigenetic alterations in clear cell RCC and demonstrates tumor suppressor properties.
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| 4. |
- Mostovich, Luydmila A, et al.
(författare)
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Integrin alpha9 (ITGA9) expression and epigenetic silencing in human breast tumors
- 2011
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Ingår i: CELL ADHESION and MIGRATION. - : Landes Bioscience. - 1933-6918 .- 1933-6926. ; 5:5, s. 395-401
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Tidskriftsartikel (refereegranskat)abstract
- Integrin alpha9 (ITGA9) is one of the less studied integrin subunits that facilitates accelerated cell migration and regulates diverse biological functions such as angiogenesis, lymphangiogenesis, cancer cell proliferation and migration. In this work, integrin alpha9 expression and its epigenetic regulation in normal human breast tissue, primary breast tumors and breast cancer cell line MCF7 were studied. It was shown that integrin alpha9 is expressed in normal human breast tissue. In breast cancer, ITGA9 expression was downregulated or lost in 44% of tumors while another 45% of tumors showed normal or increased ITGA9 expression level (possible aberrations in the ITGA9 mRNA structure were supposed in 11% of tumors). Methylation of ITGA9 CpG-island located in the first intron of the gene was shown in 90% of the breast tumors with the decreased ITGA9 expression while no methylation at 5-untranslated region of ITGA9 was observed. 5-aza-dC treatment restored integrin alpha9 expression in ITGA9-negative MCF7 breast carcinoma cells, Trichostatin A treatment did not influenced it but a combined treatment of the cells with 5-aza-dC/Trichostatin A doubled the ITGA9 activation. The obtained results suggest CpG methylation as a major mechanism of integrin alpha9 inactivation in breast cancer with a possible involvement of other yet unidentified molecular pathways.
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| 5. |
- Mostovich, Luydmila A, et al.
(författare)
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The TCF4/beta-catenin pathway and chromatin structure cooperate to regulate D-glucuronyl C5-epimerase expression in breast cancer
- 2012
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Ingår i: Epigenetics. - : Landes Bioscience. - 1559-2294 .- 1559-2308. ; 7:8, s. 930-939
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Tidskriftsartikel (refereegranskat)abstract
- D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/beta-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/beta-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/beta-catenin mRNA levels and activated TCF4/beta-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/beta-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and beta-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/beta-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/beta-catenin complex activity.
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| 6. |
- Prudnikova, Tatiana Y, et al.
(författare)
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miRNA-218 contributes to the regulation of D-glucuronyl C5-epimerase expression in normal and tumor breast tissues
- 2012
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Ingår i: Epigenetics. - : Landes Bioscience. - 1559-2294 .- 1559-2308. ; 7:10, s. 1109-1114
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Tidskriftsartikel (refereegranskat)abstract
- microRNAs (miRNAs) are key posttranscriptional regulators of gene expression. In the present study, regulation of tumor-suppressor gene D-glucuronyl C5-epimerase (GLCE) by miRNA-218 was investigated. Significant downregulation of miRNA-218 expression was shown in primary breast tumors. Exogenous miRNA-218/anti-miRNA-218 did not affect GLCE mRNA but regulated GLCE protein level in MCF7 breast carcinoma cells in vitro. Comparative analysis showed a positive correlation between miRNA-218 and GLCE mRNA, and negative correlation between miRNA-218 and GLCE protein levels in breast tissues and primary tumors in vivo, supporting a direct involvement of miRNA-218 in posttranscriptional regulation of GLCE in human breast tissue. A common scheme for the regulation of GLCE expression in normal and tumor breast tissues is suggested.
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| 7. |
- Zabarovska, Veronika, et al.
(författare)
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NotI passporting to identify species composition of complex microbial systems
- 2003
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Ingår i: Nucleic Acids Research. - Oxford, United Kingdom : Oxford University Press. - 0305-1048 .- 1362-4962. ; 31:2, s. E5-5
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Tidskriftsartikel (refereegranskat)abstract
- We describe here a new method for large-scale scanning of microbial genomes on a quantitative and qualitative basis. To achieve this aim we propose to create NotI passports: databases containing NotI tags. We demonstrated that these tags comprising 19 bp of sequence information could be successfully generated using DNA isolated from intestinal or fecal samples. Such NotI passports allow the discrimination between closely related bacterial species and even strains. This procedure for generating restriction site tagged sequences (RSTS) is called passporting and can be adapted to any other rare cutting restriction enzyme. A comparison of 1312 tags from available sequenced Escherichia coli genomes, generated with the NotI, PmeI and SbfI restriction enzymes, revealed only 219 tags that were not unique. None of these tags matched human or rodent sequences. Therefore the approach allows analysis of complex microbial mixtures such as in human gut and identification with high accuracy of a particular bacterial strain on a quantitative and qualitative basis.
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| 8. |
- Zabarovsky, Eugene R, et al.
(författare)
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Restriction site tagged (RST) microarrays : a novel technique to study the species composition of complex microbial systems
- 2003
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Ingår i: Nucleic Acids Research. - Oxford, United Kingdom : Oxford University Press. - 0305-1048 .- 1362-4962. ; 31:16, s. e95-
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a new type of microarray, restriction site tagged (RST), for example NotI, microarrays. In this approach only sequences surrounding specific restriction sites (i.e. NotI linking clones) were used for generating microarrays. DNA was labeled using a new procedure, NotI representation, where only sequences surrounding NotI sites were labeled. Due to these modifications, the sensitivity of RST microarrays increases several hundred-fold compared to that of ordinary genomic microarrays. In a pilot experiment we have produced NotI microarrays from Gram-positive and Gram-negative bacteria and have shown that even closely related Escherichia coli strains can be easily discriminated using this technique. For example, two E.coli strains, K12 and R2, differ by less than 0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily discriminate between these strains. However, these strains showed distinctly different hybridization patterns with NotI microarrays. The same technique can be adapted to other restriction enzymes as well. This type of microarray opens the possibility not only for studies of the normal flora of the gut but also for any problem where quantitative and qualitative analysis of microbial (or large viral) genomes is needed.
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