Many p53 mutations found in human tumors retain partial function
based on analysis in yeast.
Francesca Storici (1),
Alberto Inga (1), Mark King (1), Janet Liu (1), Tracy Thompson (2),
Jack Taylor (3), Michael Resnick (1)
(1) Lab. of Molecular Genetics,
NIEHS, NIH, 111 Alexander Dr., RTP, NC 27709, US; (2) Epidemiology
Branch, NIEHS, NIH, Research Triangle Park, NC; (3) Epidemiology
Branch, National Institute of Environmental Health Sciences, NIH,
Research Triangle Park, NC
p53 is a transactivation factor that
targets >50 genes in human cells. Over 40% of tumors are associated with
p53 missense mutations and >70% of these occur at non-hot spot codons.
Their biological activity has not been well characterized. Some of the
mutations appear to result in partial loss of p53 transactivation
function. p53 transactivation activity can be assessed easily using an
ADE2 color assay (white/red) in yeast where transcription is
initiated by p53 binding at response elements (20-22 bp) upstream from
ADE2. The assay evaluates p53 codons 53-364, where 99% of tumor
mutations are reported. A small screen of bladder cancer cell lines and
tumors identified 6 alleles (among 10) with a novel partial
function phenotype (pink speckled). To address the importance of
partial-function p53 mutations, we mutagenized p53 and selected alleles
with reduced transactivation activity toward p21, RGC and
MDM2 response elements. 57 different mutants with single amino
acid changes were identified. 65% of these are found in the tumor p53
database and represent 2% of all p53 tumor missense mutations. A yeast
luciferase-reporter assay was developed to quantify the level of
residual activity. Since our mutagenesis screen was far from saturation,
we predict that mutations retaining partial function can be as high as
20% of all p53 tumor mutations. This approach to functional
characterization of tumor mutations can be used to better understand
tumor development and treatment.
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