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Mutational analysis of the p21/WAF1/CIP1/SDI1 coding region in human tumor cell lines
1Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Abstract:
p21/WAF1/CIP1/SDI1 is an important cell-cycle mediator with tumor suppressor gene capabilities, and its inactivation could potentially lead to tumor progression. Because tumor suppressor genes are commonly inactivated by somatic and germline mutations, we analyzed a variety of human tumor cell lines for p21 mutations. We used single-strand conformational analysis and direct sequencing to identify possible mutations in the p21 coding region. Two base-alterations were observed in 41 immortalized human tumor cell lines. A previously reported polymorphism that results in a serine-to-arginine amino-acid substitution at codon 31 was found in 24% (10 of 41) of the tumor cell lines but was also found in 10% (six of 62) of normal parental DNAs tested and 7% (three of 43) of normal DNAs from patients with primary endometrial tumors. Another nucleotide substitution found at codon 80 resulted in the replacement of threonine with methionine. Codon 80 changes were found in 7% (three of 41) of the tumor cell lines (all endometrial) and in 2% (one of 62) of the normal parental DNAs. This change was not found in any of the primary endometrial tumors examined. The biological activity of these base changes was analyzed by using in vitro cyclin-dependent kinase 2-cyclin A kinase assays and calcium phosphate transfections. We observed that wild-type p21 and the p21 variants had similar growth-inhibitory abilities. Thus, our results suggest that mutation of the p21 gene is not prevalent in human tumor cell lines and is not a probable mechanism of inactivation of this gene.
Insights
Mutations in the p21 gene, a key cell-cycle regulator, are uncommon in human tumor cell lines. The identified p21 variants showed similar growth-inhibitory abilities to the wild-type, suggesting mutation is not a primary inactivation mechanism.
Area of Science:
- Molecular Biology
- Cancer Genetics
Background:
- The p21/WAF1/CIP1/SDI1 gene acts as a crucial cell-cycle mediator and possesses tumor suppressor functions.
- Inactivation of tumor suppressor genes, often through mutations, can drive tumor progression.
Purpose of the Study:
- To investigate the prevalence of p21 gene mutations in human tumor cell lines.
- To determine if p21 gene mutations contribute to tumor development.
Main Methods:
- Analysis of 41 immortalized human tumor cell lines for p21 coding region mutations.
- Utilized single-strand conformational analysis and direct sequencing.
- Assessed biological activity of p21 variants using in vitro kinase assays and transfections.
Main Results:
- Two base alterations were identified in the p21 gene across the analyzed tumor cell lines.
- A known polymorphism at codon 31 and a novel substitution at codon 80 were observed.
- p21 variants exhibited comparable growth-inhibitory effects to wild-type p21 in functional assays.
Conclusions:
- Mutations in the p21 gene are not frequent in human tumor cell lines.
- p21 gene mutation is unlikely to be a major mechanism for its inactivation in cancer development.