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Gene expression profiles resulting from stable loss of p53 mirrors its role in tissue differentiation.

Couture O, Lombardi E, Davis K, Hays E, Chandar N - PLoS ONE (2013)

Bottom Line: In this study we compare changes in gene expression resulting after either a transient or stable reduction in p53.Accordingly we reduced p53 levels transiently and stably in C2C12 cells, which are capable of both myoblast and osteoblast differentiation, and compared the changes in gene expression of candidate genes regulated by the p53 pathway.These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression affecting normal tissue physiology more than acute loss of p53.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, Illinois, United States of America.

ABSTRACT
The tumor suppressor gene p53 is involved in a variety of cellular activities such as cellular stress responses, cell cycle regulation and differentiation. In our previous studies we have shown p53's transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression that is absent in knockout mice. Mice heterozygous for p53 also show a higher incidence of osteosarcomas than p53 knockout mice. This suggests that p53 is able to modify the environment within osteoblasts. In this study we compare changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels transiently and stably in C2C12 cells, which are capable of both myoblast and osteoblast differentiation, and compared the changes in gene expression of candidate genes regulated by the p53 pathway. Using a PCR array to assay for p53 target genes, we have found different expression profiles when comparing stable versus transient knockdown of p53. As expected, several genes with profound changes after transient p53 loss were related to apoptosis and cell cycle regulation. In contrast, stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles, suggesting that long term loss of p53 affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were validated by measuring promoter activity of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression affecting normal tissue physiology more than acute loss of p53.

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Array Confirmation.Confirmation of targets from gene array by Quantitative PCR verifies array expression values. QPCR of selected genes that showed variation in expression in arrays were selected and tested with three independently isolated single cell clones using the same shRNA as the array(S36), Transient transfections were carried out in triplicate with the same shRNA , and control cells received the scrambled control plasmid (mean + S.E.M., n = 3) for verification. For each gene, the change in expression matched that of the array in both types of transfections.
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pone-0082494-g003: Array Confirmation.Confirmation of targets from gene array by Quantitative PCR verifies array expression values. QPCR of selected genes that showed variation in expression in arrays were selected and tested with three independently isolated single cell clones using the same shRNA as the array(S36), Transient transfections were carried out in triplicate with the same shRNA , and control cells received the scrambled control plasmid (mean + S.E.M., n = 3) for verification. For each gene, the change in expression matched that of the array in both types of transfections.

Mentions: Several genes belonging to apoptosis and differentiation related pathways showing alterations in the array were chosen for validation using realtime PCR. For this analysis we created new stable lines with p53 knockdown using the same shRNA. Transient knockdown of p53 was also carried out in triplicate. RNA from these newly generated cell lines were compared to results obtained from the gene array. As shown in Figure 3, the quantitative PCR results agreed with the array results in the direction of the change in expression. As expected of independent samples there was a variation in the extent of change between samples, but most importantly the directionality of the change was maintained for all of the genes tested, thus validating the results of the array. The variation seen in the case of some of the genes may relate to differences in the level of p53 knockdown as three independent clones/transfections were compared to the array. Additionally they may represent changes in steady state levels of expression dependent on the status of the cells at the time of RNA isolation.


Gene expression profiles resulting from stable loss of p53 mirrors its role in tissue differentiation.

Couture O, Lombardi E, Davis K, Hays E, Chandar N - PLoS ONE (2013)

Array Confirmation.Confirmation of targets from gene array by Quantitative PCR verifies array expression values. QPCR of selected genes that showed variation in expression in arrays were selected and tested with three independently isolated single cell clones using the same shRNA as the array(S36), Transient transfections were carried out in triplicate with the same shRNA , and control cells received the scrambled control plasmid (mean + S.E.M., n = 3) for verification. For each gene, the change in expression matched that of the array in both types of transfections.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842970&req=5

pone-0082494-g003: Array Confirmation.Confirmation of targets from gene array by Quantitative PCR verifies array expression values. QPCR of selected genes that showed variation in expression in arrays were selected and tested with three independently isolated single cell clones using the same shRNA as the array(S36), Transient transfections were carried out in triplicate with the same shRNA , and control cells received the scrambled control plasmid (mean + S.E.M., n = 3) for verification. For each gene, the change in expression matched that of the array in both types of transfections.
Mentions: Several genes belonging to apoptosis and differentiation related pathways showing alterations in the array were chosen for validation using realtime PCR. For this analysis we created new stable lines with p53 knockdown using the same shRNA. Transient knockdown of p53 was also carried out in triplicate. RNA from these newly generated cell lines were compared to results obtained from the gene array. As shown in Figure 3, the quantitative PCR results agreed with the array results in the direction of the change in expression. As expected of independent samples there was a variation in the extent of change between samples, but most importantly the directionality of the change was maintained for all of the genes tested, thus validating the results of the array. The variation seen in the case of some of the genes may relate to differences in the level of p53 knockdown as three independent clones/transfections were compared to the array. Additionally they may represent changes in steady state levels of expression dependent on the status of the cells at the time of RNA isolation.

Bottom Line: In this study we compare changes in gene expression resulting after either a transient or stable reduction in p53.Accordingly we reduced p53 levels transiently and stably in C2C12 cells, which are capable of both myoblast and osteoblast differentiation, and compared the changes in gene expression of candidate genes regulated by the p53 pathway.These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression affecting normal tissue physiology more than acute loss of p53.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, Illinois, United States of America.

ABSTRACT
The tumor suppressor gene p53 is involved in a variety of cellular activities such as cellular stress responses, cell cycle regulation and differentiation. In our previous studies we have shown p53's transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression that is absent in knockout mice. Mice heterozygous for p53 also show a higher incidence of osteosarcomas than p53 knockout mice. This suggests that p53 is able to modify the environment within osteoblasts. In this study we compare changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels transiently and stably in C2C12 cells, which are capable of both myoblast and osteoblast differentiation, and compared the changes in gene expression of candidate genes regulated by the p53 pathway. Using a PCR array to assay for p53 target genes, we have found different expression profiles when comparing stable versus transient knockdown of p53. As expected, several genes with profound changes after transient p53 loss were related to apoptosis and cell cycle regulation. In contrast, stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles, suggesting that long term loss of p53 affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were validated by measuring promoter activity of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression affecting normal tissue physiology more than acute loss of p53.

Show MeSH
Related in: MedlinePlus