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- Dmitriev, Alexey A, et al.
(author)
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Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays
- 2012
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In: Epigenetics. - : Landes Bioscience. - 1559-2294 .- 1559-2308. ; 7:5, s. 502-513
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Journal article (peer-reviewed)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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| 3. |
- Haraldson, Klas, et al.
(author)
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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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In: Biochimie. - : Elsevier. - 0300-9084 .- 1638-6183. ; 94:5, s. 1151-1157
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Journal article (peer-reviewed)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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| 4. |
- Haraldson, Klas
(author)
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NotI genome scanning to identify unknown cancer associated genes in major human epithelial malignancies
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Epithelial cancers cause many deaths every year. Changes in the genes of human chromosome 3 are particularly common in epithelial cancers in several organs. Alterations in DNA methylation is one of the best known epigenetic changes in cancer. The abnormal epigenetic landscape of the cancer cell is characterized by a massive genomic hypomethylation and hypermethylation of CpG islands in the promoter regions of tumor suppressor genes. Microarrays is a powerful tool for studying the molecular basis of diseases that are not possible with conventional methods. Being able to predict who will develop cancer and how the disease will behave and respond to treatment after diagnosis are some uses for this technology. NotI microarrays is a novel technology that makes it possible to simultaneously detect changes in methylation, amplification and deletions in cancer. The NotI microarrays technology based on restriction enzyme NotI is methylation sensitive, and therefore makes it possible to detect if methylation causes a gene to be altered in cancer. Cancer samples are hybridized with NotI microarrays and are evaluated bioinformatically. Using NotI microarray technology, 181 NotI loci in human chromosome 3 have been analyzed for methylation, amplification and deletions in different epithelial cancers; lung cancer, renal cancer, breast cancer, cervical cancer, ovarian cancer, prostate cancer and colon cancer. The genes that were shown to be altered in cancer samples compared with normal tissue samples were analyzed further with more accurate methods; bisulfitesequencing of cloned PCR products was the method used to control and verify methylation status. NotI loci that were found to have relevant changes include genes MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, ZIC4, NKIRAS1, LRRC3B, suggesting that they probably are involved in cancer development. It was found that NPRL2/G21 gene has growth inhibitory activity for renal and lung cell lines when tested under controlled physiologic conditions of gene expression both in vitro and in vivo. Mutations were found in experimental tumors and intragenic homozygous deletions in renal, lung, and other cancer cell lines. It was also observed that NPRL2 could participate in mismatch repair. Further, it was shown that somatic hypermutations in tumor suppressor genes involved in major human malignancies is a mechanism for the development, progression and spread of cancer. RASSF1 and RBSP3 were demonstrated to have a high incidence of somatic hypermutations in several cancer types. Methylation and/or deletions of LRRC3B gene were detected in more than 50% of checked renal, cervical, ovarian, colon and breast cancer samples, and it was found to inhibit renal cell cancer cell line KRC/Y cell survival and replication in vitro. Altered genes found with the NotI microarrays technology may potentially be used as biomarkers or targets for epigenetic therapy. A set of 18 markers was suggested (BHLHB2, FBLN2, FLJ44898 (EPHB1), GATA2, GORASP1, Hmm210782 (PRICKLE2), Hmm61490, ITGA9, LOC285205, LRRC3B, MINA, MITF, MRPS17P3, NKIRAS1, PLCL2, TRH, UBE2E2, WNT7A) that allow to discriminate/diagnose different types of lung cancer.
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| 5. |
- Mostovich, Luydmila A, et al.
(author)
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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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In: Epigenetics. - : Landes Bioscience. - 1559-2294 .- 1559-2308. ; 7:8, s. 930-939
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Journal article (peer-reviewed)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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