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Comparative transcriptome profiling of an SV40-transformed human fibroblast (MRC5CVI) and its untransformed counterpart (MRC-5) in response to UVB irradiation.

Chang CW, Chen CR, Huang CY, Shu WY, Chiang CS, Hong JH, Hsu IC - PLoS ONE (2013)

Bottom Line: We found that, in response to UVB irradiation, MRC-5 and MRC5CVI commonly up-regulated the expression of oxidative phosphorylation genes.MRC-5 up-regulated the expressions of chromosome condensation, DNA repair, cell cycle arrest, and apoptotic genes, but MRC5CVI did not.Further cell death assays indicated that MRC5CVI was more sensitive than MRC-5 to UVB-induced cell death with increased caspase-3 activation; combining with the transcriptomic results suggested that MRC5CVI may undergo UVB-induced cell death through mechanisms other than transcriptional regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan.

ABSTRACT
Simian virus 40 (SV40) transforms cells through the suppression of tumor-suppressive responses by large T and small t antigens; studies on the effects of these two oncoproteins have greatly improved our knowledge of tumorigenesis. Large T antigen promotes cellular transformation by binding and inactivating p53 and pRb tumor suppressor proteins. Previous studies have shown that not all of the tumor-suppressive responses were inactivated in SV40-transformed cells; however, the underlying cause is not fully studied. In this study, we investigated the UVB-responsive transcriptome of an SV40-transformed fibroblast (MRC5CVI) and that of its untransformed counterpart (MRC-5). We found that, in response to UVB irradiation, MRC-5 and MRC5CVI commonly up-regulated the expression of oxidative phosphorylation genes. MRC-5 up-regulated the expressions of chromosome condensation, DNA repair, cell cycle arrest, and apoptotic genes, but MRC5CVI did not. Further cell death assays indicated that MRC5CVI was more sensitive than MRC-5 to UVB-induced cell death with increased caspase-3 activation; combining with the transcriptomic results suggested that MRC5CVI may undergo UVB-induced cell death through mechanisms other than transcriptional regulation. Our study provides a further understanding of the effects of SV40 transformation on cellular stress responses, and emphasizes the value of SV40-transformed cells in the researches of sensitizing neoplastic cells to radiations.

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Cell cycle analysis of MRC-5 and MRC5CVI.Panel (A), results of MRC-5; panel (B), results of MRC5CVI. Pairwise statistical comparisons of the corresponding cell cycle phase between the time point and control were performed using Student’s t test (*, P<0.05; **, P<0.01). Error bars show the standard deviation (n = 3).
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pone-0073311-g004: Cell cycle analysis of MRC-5 and MRC5CVI.Panel (A), results of MRC-5; panel (B), results of MRC5CVI. Pairwise statistical comparisons of the corresponding cell cycle phase between the time point and control were performed using Student’s t test (*, P<0.05; **, P<0.01). Error bars show the standard deviation (n = 3).

Mentions: The protein encoded by the gene NCAPH plays a role in chromosome condensation [27]–[28], and is important in regulating mitotic cell death [29]. Both NCAPH and GADD45A are important in regulating DNA repair [30]–[32], thus their discrepant expressions suggest the difference in repair capability between the two cell types (Figure 3). GADD45A also plays a crucial role in regulating G2/M arrest [32]–[34]. The RT-PCR results validated the discrepant regulation of GADD45A between the two cell types, as well as the consistent down-regulation of CDKN1A in both cell types (Figure S4). In addition to the regulation by p53, the expression of MEN1[35]–[36] and TSPYL5[37]–[38] may play superior roles in regulating GADD45A and CDKN1A expressions in MRC5CVI. The discrepant regulation of GADD45A between MRC-5 and MRC5CV1 suggests their differences in regulating G2/M arrest in response to UVB irradiation. We then performed cell cycle analysis and found the increments of G2/M population for MRC-5 (Figure 4A), whereas only a gradual decrease of G2/M cells for MRC5CVI after UVB irradiation (Figure 4B). The consistent down-regulation of CDKN1A implies that both cell types may not regulate G1/S arrest. The cell cycle result confirmed that G1/S arrest was abrogated in both cell types in response to UVB irradiation.


Comparative transcriptome profiling of an SV40-transformed human fibroblast (MRC5CVI) and its untransformed counterpart (MRC-5) in response to UVB irradiation.

Chang CW, Chen CR, Huang CY, Shu WY, Chiang CS, Hong JH, Hsu IC - PLoS ONE (2013)

Cell cycle analysis of MRC-5 and MRC5CVI.Panel (A), results of MRC-5; panel (B), results of MRC5CVI. Pairwise statistical comparisons of the corresponding cell cycle phase between the time point and control were performed using Student’s t test (*, P<0.05; **, P<0.01). Error bars show the standard deviation (n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760899&req=5

pone-0073311-g004: Cell cycle analysis of MRC-5 and MRC5CVI.Panel (A), results of MRC-5; panel (B), results of MRC5CVI. Pairwise statistical comparisons of the corresponding cell cycle phase between the time point and control were performed using Student’s t test (*, P<0.05; **, P<0.01). Error bars show the standard deviation (n = 3).
Mentions: The protein encoded by the gene NCAPH plays a role in chromosome condensation [27]–[28], and is important in regulating mitotic cell death [29]. Both NCAPH and GADD45A are important in regulating DNA repair [30]–[32], thus their discrepant expressions suggest the difference in repair capability between the two cell types (Figure 3). GADD45A also plays a crucial role in regulating G2/M arrest [32]–[34]. The RT-PCR results validated the discrepant regulation of GADD45A between the two cell types, as well as the consistent down-regulation of CDKN1A in both cell types (Figure S4). In addition to the regulation by p53, the expression of MEN1[35]–[36] and TSPYL5[37]–[38] may play superior roles in regulating GADD45A and CDKN1A expressions in MRC5CVI. The discrepant regulation of GADD45A between MRC-5 and MRC5CV1 suggests their differences in regulating G2/M arrest in response to UVB irradiation. We then performed cell cycle analysis and found the increments of G2/M population for MRC-5 (Figure 4A), whereas only a gradual decrease of G2/M cells for MRC5CVI after UVB irradiation (Figure 4B). The consistent down-regulation of CDKN1A implies that both cell types may not regulate G1/S arrest. The cell cycle result confirmed that G1/S arrest was abrogated in both cell types in response to UVB irradiation.

Bottom Line: We found that, in response to UVB irradiation, MRC-5 and MRC5CVI commonly up-regulated the expression of oxidative phosphorylation genes.MRC-5 up-regulated the expressions of chromosome condensation, DNA repair, cell cycle arrest, and apoptotic genes, but MRC5CVI did not.Further cell death assays indicated that MRC5CVI was more sensitive than MRC-5 to UVB-induced cell death with increased caspase-3 activation; combining with the transcriptomic results suggested that MRC5CVI may undergo UVB-induced cell death through mechanisms other than transcriptional regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan.

ABSTRACT
Simian virus 40 (SV40) transforms cells through the suppression of tumor-suppressive responses by large T and small t antigens; studies on the effects of these two oncoproteins have greatly improved our knowledge of tumorigenesis. Large T antigen promotes cellular transformation by binding and inactivating p53 and pRb tumor suppressor proteins. Previous studies have shown that not all of the tumor-suppressive responses were inactivated in SV40-transformed cells; however, the underlying cause is not fully studied. In this study, we investigated the UVB-responsive transcriptome of an SV40-transformed fibroblast (MRC5CVI) and that of its untransformed counterpart (MRC-5). We found that, in response to UVB irradiation, MRC-5 and MRC5CVI commonly up-regulated the expression of oxidative phosphorylation genes. MRC-5 up-regulated the expressions of chromosome condensation, DNA repair, cell cycle arrest, and apoptotic genes, but MRC5CVI did not. Further cell death assays indicated that MRC5CVI was more sensitive than MRC-5 to UVB-induced cell death with increased caspase-3 activation; combining with the transcriptomic results suggested that MRC5CVI may undergo UVB-induced cell death through mechanisms other than transcriptional regulation. Our study provides a further understanding of the effects of SV40 transformation on cellular stress responses, and emphasizes the value of SV40-transformed cells in the researches of sensitizing neoplastic cells to radiations.

Show MeSH
Related in: MedlinePlus