Differential Expression of p53 on Thermal Stress in Wild Type Transformed Cells and Cervical Carcinoma Cells




Shalini Datta

Introduction

p53 is often described as ‘guardian of the genome’ owing to the fact that it has a pivotal role of responding to cellular stresses like DNA damage and hypoxia, to maintain the integrity of the genome. It is one of the major tumor suppressor gene identified to date and about 50% of human neoplastic transformations have p53 gene mutations. The p53 gene has been mapped to chromosome 17, at position 17p13.1. Human p53 protein is composed of 393 amino acid and has 4 main functional domains: an amino terminal acidic transactivation domain, a sequence specific DNA binding domain, a tetramerization domain near the carboxy terminal end and a C terminal domain that interacts directly with single stranded DNA. p53 is a multifunctional protein, which has a definite role in DNA damage response, signal transduction resulting in induction of apoptosis, negative regulation of cell cycle, inhibition of angiogenesis, and maintaining genome stability. The p53 mediated growth inhibition prevents the proliferation of cells with damaged DNA or a potential for tumorigenesis. In wild type cells p53 protein is labile and is kept at a low concentration primarily by HDM2 protein. Hdm2 inhibits the transcriptional activity of p53 and promotes its degradation by proteosome (5). The status of p53 is drastically altered and activated when the cells are exposed to thermal stress, radiation induced stress, DNA damage, hypoxia and nucleotide depletion .The activation of p53 leads to cell cycle arrest, senescence or apoptosis. p53 can induce the extrinsic apoptotic pathway by activating Fas, DR5 and PERP and it can induce the intrinsic apoptotic pathway dominated by the Bcl-2 family of proteins. In the intrinsic pathway, the Bax gene contains a p53 responsive element, and it can be activated by p53 (9). The Bax gene encodes a preapoptotic protein of Bcl-2 group. In response to stress activation, BAX forms a homodimer and releases cytochrome c from mitochondria, which results in caspase-9 activation at the initiation of the apoptotic cascade (9). In p53 mutated cancerous cell the p53-mediated cell cycle arrest and apoptotic pathways are disabled. Stimulation of those nonfunctional pathways as well as activation of p53 can be a potential molecular genetic approach to treat malignancies in future.



Figures


Figure 1-Visualization of the electrophoretic pattern in 1.0% agarose gel of RT-PCR products using specific primers for beta actin and p53. The p53 forward primer was matched to nucleotides of 1058 to 1078 of exon 7 of NCBI accession number NM_000546and the p53 reverse primer was matched to nucleotides of 1349 to 1367 bases of exon 9 and 10 NCBI accession number NM_000546.The beta actin primers generated a 444 bp product and the p53 primers generated a 308 bp product. The experiment was done twice and both the results are shown in the figure 1.


Figure 2-Sequence alignment of the RT-PCR product in reference with the p53 NCBI reference sequence (#NM_000546).


Cellular p53 levels are of paramount importance as p53 plays an important role in suppressing the malignancies. WI-38 when exposed to thermal stress exhibited increased accumulation of the p53 mRNA. HeLa cells, failed to show any such response after the similar treatment.

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Acknowledgments

I express my deepest thanks to Dr. Berish Rubin for providing me with the opportunity to perform the experiments and Dr. Sylvia Anderson for synthesizing the primers. I would like to thank JinSong Qui and Brian Fox for their continuous supervision and useful suggestions and guidance throughout this project, without whose support this project could not have been completed. I express my sincere thanks to Dr. William Thornhill for his useful discussion on the subject. I deeply appreciate my classmates for supporting me throughout the experiments.


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