Analysis of p53 status in human cell lines using a functional assay in yeast: detection of new non-sense p53 mutation in codon 124.
p53 tumor suppressor is a sequence-specific DNA-binding protein that controls the expression of many genes in response to diverse stress stimuli. p53 gene is often mutated in human cancer and in cancer cell lines. Several methods are available for identification of p53 mutations, including functional analysis of separated alleles in yeast (FASAY). FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. We analyzed the p53 status of 26 human cell lines of different tissue origin using the functional assay in yeast. Wild-type p53 was found in six cell lines and various p53 aberrations in the remaining twenty. FASAY detected temperature-sensitive p53 mutations in breast cancer cell line, BT474, and leiomyosarcoma cell line, SK-LMS-1, and two independent p53 point mutations in SK-UT-1 and SK-LMS-1 leiomyosarcoma cell lines. In addition, the assay revealed that a recombination occurred between the two mutated p53 alleles producing a stable ratio of p53 wild-type alleles (11.7 or 2.7% respectively). In the case of acute myeloid leukemia cell line, ML-1, we detected both wild-type and heterozygous p53 status, depending on the source of the cell line. In Hs913T, HL60 and Saos-2 cell lines, FASAY failed to assess p53 status due to a large deletion/rearrangement of the p53 gene. In acute lymphoid leukemia HPB cell line, we disclosed unknown non-sense mutation in codon 124 of the p53 gene.
Smardová
Jana
J
Department of Pathology and Anatomy, University Hospital Brno, Jihlavská 20, 625 00 Brno, Czech Republic. janasmarda@seznam.cz
Pavlová
Sárka
S
Svitáková
Miluska
M
Grochová
Diana
D
Ravcuková
Barbora
B
D000483:Alleles; D045744:Cell Line, Tumor; D003062:Codon; D018389:Codon, Nonsense; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000378:metabolism; D016158:Genes, p53 / Q000235:genetics*; D005821:Genetic Techniques; D018922:HL-60 Cells; D006579:Heterozygote; D006801:Humans; D015151:Immunoblotting; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
D003062:Codon; D018389:Codon, Nonsense; D005656:Fungal Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA