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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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Related in: MedlinePlus

Examination of cell death after UVB irradiation by percent detachment and active caspase-3 staining.Panel (A), results of percent detachment; panel (B), results of active caspase-3 staining. Pairwise statistical comparisons between the time point of two cell types were performed using Student’s t test (*, P<0.05). Error bars show the standard deviation (n = 3).
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pone-0073311-g005: Examination of cell death after UVB irradiation by percent detachment and active caspase-3 staining.Panel (A), results of percent detachment; panel (B), results of active caspase-3 staining. Pairwise statistical comparisons between the time point of two cell types were performed using Student’s t test (*, P<0.05). Error bars show the standard deviation (n = 3).

Mentions: Among the most discrepant genes between MRC-5 and MRC5CVI, five are functionally involved in apoptotic regulation (Figure 3). According to their roles in regulating apoptosis, their expressions favor a UVB-induced apoptotic response in MRC-5 cells, whereas only the significant down-regulation of SPHK1 favors the pro-apoptotic status of MRC5CVI in response to UVB irradiation (detailed in the Discussion section). To examine the cell death response of both cell types after UVB irradiation, we performed two assays, as follows: estimating the percentage of cell detachment and active caspase-3 staining. Percent detachment was calculated as the number of cells in the medium compared to the total cells. Previous studies have used percent detachment as a quantitative estimate of cell death in fibroblasts [24], [39], and the activation of caspase-3 represents a major feature of apoptosis [40]. The results of cell death assays indicated that both cell types underwent cell death with activated caspase-3 after UVB irradiation, with MRC5CVI being more sensitive than MRC-5 (Figure 5A and 5B).


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)

Examination of cell death after UVB irradiation by percent detachment and active caspase-3 staining.Panel (A), results of percent detachment; panel (B), results of active caspase-3 staining. Pairwise statistical comparisons between the time point of two cell types were performed using Student’s t test (*, P<0.05). Error bars show the standard deviation (n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760899&req=5

pone-0073311-g005: Examination of cell death after UVB irradiation by percent detachment and active caspase-3 staining.Panel (A), results of percent detachment; panel (B), results of active caspase-3 staining. Pairwise statistical comparisons between the time point of two cell types were performed using Student’s t test (*, P<0.05). Error bars show the standard deviation (n = 3).
Mentions: Among the most discrepant genes between MRC-5 and MRC5CVI, five are functionally involved in apoptotic regulation (Figure 3). According to their roles in regulating apoptosis, their expressions favor a UVB-induced apoptotic response in MRC-5 cells, whereas only the significant down-regulation of SPHK1 favors the pro-apoptotic status of MRC5CVI in response to UVB irradiation (detailed in the Discussion section). To examine the cell death response of both cell types after UVB irradiation, we performed two assays, as follows: estimating the percentage of cell detachment and active caspase-3 staining. Percent detachment was calculated as the number of cells in the medium compared to the total cells. Previous studies have used percent detachment as a quantitative estimate of cell death in fibroblasts [24], [39], and the activation of caspase-3 represents a major feature of apoptosis [40]. The results of cell death assays indicated that both cell types underwent cell death with activated caspase-3 after UVB irradiation, with MRC5CVI being more sensitive than MRC-5 (Figure 5A and 5B).

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