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Introduction of p53 induces cell-cycle arrest in p53-deficient human medullary-thyroid-carcinoma cells
J A Velasco1, D L Medina, J Romero
1Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Abstract:
The structural integrity of the p53 gene in a human thyroid-medullary-carcinoma-derived cell line has been studied. Analysis of high-molecular-weight DNA showed that the p53 locus is severely rearranged. PCR and single-strand conformation polymorphism analysis revealed that a large portion of the 5' end of the p53 gene is lost, while a region encompassing exons 8 and 9 is rearranged. As a consequence, no virtual expression of a p53-specific transcript is detected in mRNA from the medullary-carcinoma cell line. The absence of a p53 protein prompted us to analyze the biological effect of exogenous expression of this tumor-suppressor gene on cell growth and viability, introducing retroviral constructs carrying full-length human wild-type p53 cDNA. Contrary to what has been described for other cell types, including most thyroid-carcinoma cell lines of follicular origin, these experiments allowed us to establish clonal-cell populations which constitutively express p53. Cytometric analysis revealed G1-specific cell-cycle arrest, responsible for growth retardation in the transfected clones when compared with the parental cell line. However, medullary-thyroid-carcinoma cells expressing p53 are able to partially overcome the G1 block and progress through the cell cycle. In the search of the mechanism(s) involved in these processes, we describe the interaction of p53 with specific p21WAF1/Cip1 promoter sequences by gel-retardation assays.
Insights
The p53 tumor suppressor gene is severely damaged in medullary thyroid carcinoma cells, preventing its expression. Introducing functional p53 causes cell cycle arrest but is only partially effective in these cancer cells.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The p53 gene is a critical tumor suppressor.
- Medullary thyroid carcinoma (MTC) cell lines often exhibit p53 gene alterations.
- Understanding p53's role in MTC is crucial for targeted therapies.
Purpose of the Study:
- To investigate the structural integrity of the p53 gene in an MTC-derived cell line.
- To determine the functional consequences of p53 loss in MTC.
- To analyze the effects of restoring wild-type p53 expression on MTC cell behavior.
Main Methods:
- High-molecular-weight DNA analysis
- Polymerase Chain Reaction (PCR)
- Single-strand conformation polymorphism (SSCP) analysis
- Retroviral gene transfer
- Flow cytometry
- Gel-retardation assays
Main Results:
- The p53 gene locus in the MTC cell line showed severe rearrangement, with loss of the 5' end and rearrangement of exons 8 and 9.
- No p53 transcript was detected in the MTC cell line.
- Exogenous expression of wild-type p53 induced G1 cell-cycle arrest and growth retardation.
- MTC cells expressing p53 could partially overcome the G1 block.
- p53 was shown to interact with p21WAF1/Cip1 promoter sequences.
Conclusions:
- Medullary thyroid carcinoma cells possess a severely compromised p53 gene, leading to a lack of functional p53 protein.
- Restoration of p53 function in MTC cells induces cell-cycle arrest but is not fully effective in halting proliferation.
- p53's interaction with the p21WAF1/Cip1 promoter is implicated in its mechanism of action in MTC cells.