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Overexpression of Rad51C splice variants in colorectal tumors.

Kalvala A, Gao L, Aguila B, Reese T, Otterson GA, Villalona-Calero MA, Duan W - Oncotarget (2015)

Bottom Line: Functional alterations in Rad51C are the cause of the Fanconi anemia complementation group O (FANCO) gene disorder.The alternatively spliced transcript variants are formed either without exon-7 (variant 1), without exon 6 and 7 (variant 2) or without exon 7 and 8 (variant 3).Bisulfite DNA sequencing showed promoter methylation of Rad51C in tumor cells. 5-azacytidine treatment of LS-174T cells caused a 14 fold increase in variant 1, a 4.8 fold increase for variant 3 and 3.4 fold for variant 2 compared to 2.5 fold increase in WT.

View Article: PubMed Central - PubMed

Affiliation: Comprehensive Cancer Center, The Ohio State University College of Medicine and Public Health, Columbus, Ohio, U.S.A.

ABSTRACT
Functional alterations in Rad51C are the cause of the Fanconi anemia complementation group O (FANCO) gene disorder. We have identified novel splice variants of Rad51C mRNA in colorectal tumors and cells. The alternatively spliced transcript variants are formed either without exon-7 (variant 1), without exon 6 and 7 (variant 2) or without exon 7 and 8 (variant 3). Real time PCR analysis of nine pair-matched colorectal tumors and non-tumors showed that variant 1 was overexpressed in tumors compared to matched non-tumors. Among 38 colorectal tumor RNA samples analyzed, 18 contained variant 1, 12 contained variant 2, 14 contained variant 3, and eight expressed full length Rad51C exclusively. Bisulfite DNA sequencing showed promoter methylation of Rad51C in tumor cells. 5-azacytidine treatment of LS-174T cells caused a 14 fold increase in variant 1, a 4.8 fold increase for variant 3 and 3.4 fold for variant 2 compared to 2.5 fold increase in WT. Expression of Rad51C variants is associated with FANCD2 foci positive colorectal tumors and is associated with microsatellite stability in those tumors. Further investigation is needed to elucidate differential function of the Rad51C variants to evaluate potential effects in drug resistance and DNA repair.

No MeSH data available.


Related in: MedlinePlus

Western immunoblot analysis for Rad51C variant protein expression from colorectal tumors, matched non-tumors and LS-174T cellsProtein was isolated from colorectal tumors, matched non-tumors and LS-174T cells. The protein was analyzed on 4-12% SDS page gel and blotted on nitrocellulose membrane. Rad51C wild type and variant proteins were detected using a mouse monoclonal Rad51C antibody. Western analysis showed the proteins from LS-174T cells contained a 47 KDa protein for wild type and a 41 KDa for predicted Rad51C variant 1 and 33 KDa for predicted variant 2 or 3 (A). The analysis of proteins from colorectal tumor showed similar variant expression pattern as in LS-174T tumor cells (B). The analysis of non-tumor protein showed expression of wild type and variant 2 or 3. The variant 1 was absent in non-tumor protein (C).
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Figure 6: Western immunoblot analysis for Rad51C variant protein expression from colorectal tumors, matched non-tumors and LS-174T cellsProtein was isolated from colorectal tumors, matched non-tumors and LS-174T cells. The protein was analyzed on 4-12% SDS page gel and blotted on nitrocellulose membrane. Rad51C wild type and variant proteins were detected using a mouse monoclonal Rad51C antibody. Western analysis showed the proteins from LS-174T cells contained a 47 KDa protein for wild type and a 41 KDa for predicted Rad51C variant 1 and 33 KDa for predicted variant 2 or 3 (A). The analysis of proteins from colorectal tumor showed similar variant expression pattern as in LS-174T tumor cells (B). The analysis of non-tumor protein showed expression of wild type and variant 2 or 3. The variant 1 was absent in non-tumor protein (C).

Mentions: Protein was isolated from colorectal tumors, matched non-tumors and LS-174T cells (Fig. 6). The protein was size fractionated on 4-12% SDS page gel and blotted on nitrocellulose membrane. Rad51C wild type and variant proteins were detected using a mouse monoclonal Rad51C antibody that was predicted to recognize WT and all of the putative variants. Western immunoblot analysis showed that LS-174T cells contained a 47 KDa protein (wild type) and a 41 KDa protein as predicted for Rad51C variant 1, and a 33 KDa protein as predicted for variants 2 or 3. The analysis of proteins from colorectal tumors showed similar variant expression pattern as in LS-174T tumor cells. The analysis of non-tumor protein showed expression of wild type and variant 2 or 3. Variant 1 was not detected in non-tumor samples.


Overexpression of Rad51C splice variants in colorectal tumors.

Kalvala A, Gao L, Aguila B, Reese T, Otterson GA, Villalona-Calero MA, Duan W - Oncotarget (2015)

Western immunoblot analysis for Rad51C variant protein expression from colorectal tumors, matched non-tumors and LS-174T cellsProtein was isolated from colorectal tumors, matched non-tumors and LS-174T cells. The protein was analyzed on 4-12% SDS page gel and blotted on nitrocellulose membrane. Rad51C wild type and variant proteins were detected using a mouse monoclonal Rad51C antibody. Western analysis showed the proteins from LS-174T cells contained a 47 KDa protein for wild type and a 41 KDa for predicted Rad51C variant 1 and 33 KDa for predicted variant 2 or 3 (A). The analysis of proteins from colorectal tumor showed similar variant expression pattern as in LS-174T tumor cells (B). The analysis of non-tumor protein showed expression of wild type and variant 2 or 3. The variant 1 was absent in non-tumor protein (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496183&req=5

Figure 6: Western immunoblot analysis for Rad51C variant protein expression from colorectal tumors, matched non-tumors and LS-174T cellsProtein was isolated from colorectal tumors, matched non-tumors and LS-174T cells. The protein was analyzed on 4-12% SDS page gel and blotted on nitrocellulose membrane. Rad51C wild type and variant proteins were detected using a mouse monoclonal Rad51C antibody. Western analysis showed the proteins from LS-174T cells contained a 47 KDa protein for wild type and a 41 KDa for predicted Rad51C variant 1 and 33 KDa for predicted variant 2 or 3 (A). The analysis of proteins from colorectal tumor showed similar variant expression pattern as in LS-174T tumor cells (B). The analysis of non-tumor protein showed expression of wild type and variant 2 or 3. The variant 1 was absent in non-tumor protein (C).
Mentions: Protein was isolated from colorectal tumors, matched non-tumors and LS-174T cells (Fig. 6). The protein was size fractionated on 4-12% SDS page gel and blotted on nitrocellulose membrane. Rad51C wild type and variant proteins were detected using a mouse monoclonal Rad51C antibody that was predicted to recognize WT and all of the putative variants. Western immunoblot analysis showed that LS-174T cells contained a 47 KDa protein (wild type) and a 41 KDa protein as predicted for Rad51C variant 1, and a 33 KDa protein as predicted for variants 2 or 3. The analysis of proteins from colorectal tumors showed similar variant expression pattern as in LS-174T tumor cells. The analysis of non-tumor protein showed expression of wild type and variant 2 or 3. Variant 1 was not detected in non-tumor samples.

Bottom Line: Functional alterations in Rad51C are the cause of the Fanconi anemia complementation group O (FANCO) gene disorder.The alternatively spliced transcript variants are formed either without exon-7 (variant 1), without exon 6 and 7 (variant 2) or without exon 7 and 8 (variant 3).Bisulfite DNA sequencing showed promoter methylation of Rad51C in tumor cells. 5-azacytidine treatment of LS-174T cells caused a 14 fold increase in variant 1, a 4.8 fold increase for variant 3 and 3.4 fold for variant 2 compared to 2.5 fold increase in WT.

View Article: PubMed Central - PubMed

Affiliation: Comprehensive Cancer Center, The Ohio State University College of Medicine and Public Health, Columbus, Ohio, U.S.A.

ABSTRACT
Functional alterations in Rad51C are the cause of the Fanconi anemia complementation group O (FANCO) gene disorder. We have identified novel splice variants of Rad51C mRNA in colorectal tumors and cells. The alternatively spliced transcript variants are formed either without exon-7 (variant 1), without exon 6 and 7 (variant 2) or without exon 7 and 8 (variant 3). Real time PCR analysis of nine pair-matched colorectal tumors and non-tumors showed that variant 1 was overexpressed in tumors compared to matched non-tumors. Among 38 colorectal tumor RNA samples analyzed, 18 contained variant 1, 12 contained variant 2, 14 contained variant 3, and eight expressed full length Rad51C exclusively. Bisulfite DNA sequencing showed promoter methylation of Rad51C in tumor cells. 5-azacytidine treatment of LS-174T cells caused a 14 fold increase in variant 1, a 4.8 fold increase for variant 3 and 3.4 fold for variant 2 compared to 2.5 fold increase in WT. Expression of Rad51C variants is associated with FANCD2 foci positive colorectal tumors and is associated with microsatellite stability in those tumors. Further investigation is needed to elucidate differential function of the Rad51C variants to evaluate potential effects in drug resistance and DNA repair.

No MeSH data available.


Related in: MedlinePlus