Application of a Yeast Assay to Detect Functional p53 Mutations in Archival Prostate Cancer Tissue

Abstract

Detection and functional evaluation of mutant p53 alleles using a yeast assay could yield significant information for predicting the prognosis of patients with prostate cancer (CaP). Since the current version of this yeast assay is not applicable to archival tissues, we developed a modified assay for use on formalin-fixed, paraffin-embedded tissue and have applied it to the study of patient samples. Using this modified assay, we examined archival CaP samples from 10 patients for mutations in exons 5-8 of p53 gene. Mutations were detected in four samples: three resulted in the formation of red yeast colonies indicating complete loss of function, while one gave pink yeast colonies, indicating that this mutant retained partial function. In parallel, we analyzed these samples for p53 abnormalities using a single-strand conformational polymorphism (SSCP) approach. Only three of the four yeast-positive samples gave abnormal SSCP bands. In each case where abnormal p53 was found by both methods, DNA sequencing revealed the identical base change. These results suggest that the modified yeast assay may be more sensitive than SSCP for detection of p53 mutations, and demonstrate that the modified method can be used to detect and evaluate the function of p53 mutants present in archival tissue.

Evidence for an alternate molecular progression in prostate cancer

“…However, in support of our finding, our group and others have documented the occurrence of Tp53 mutations in ∼30% of localized human CaP (Chi et al, 1994; Heidenberg et al, 1995; Hughes et al, 1995; Bauer et al, 1996; Moul et al, 1996; Prendergast et al, 1996; Byrne et al, 1997; Meyers et al, 1998; Schlechte et al, 1998; Shi et al, 2002; Downing et al, 2003). The difference in rates of Tp53 mutation reported by these studies is likely to be due in part to the different methodologies used to assess the presence of p53 mutations (for a review, see Robles and Harris, 2010).…”

A modified yeast assay used on archival samples of localized prostate cancer tissue improves the detection of p53 abnormalities and increases their predictive value

“…Mutations of p53 were detected using the modified yeast functional assay adapted to archival tissue [18]. Briefly, p53 exons from genomic DNA 5, 6, 7 and 8 were separately amplified using a two‐step PCR approach, using the oligonucleotide primers described previously [18]. The first‐step PCR was a multiplex run in 100 µL containing all four outer pairs of primers (20 pmol each), 100 µmol/L of each dNTP, 2.5 units of Pfu DNA polymerase and all the genomic DNA obtained from the LCM procedure.…”

“…Mutations of p53 were detected using the modified yeast functional assay adapted to archival tissue [18]. Briefly, p53 exons from genomic DNA 5, 6, 7 and 8 were separately amplified using a two-step PCR approach, using the oligonucleotide primers described previously [18].…”

“…This study was aimed at using the yeast assay on routinely fixed, paraffin‐embedded adenocarcinomas from patients after RP. This modified yeast assay is more sensitive than single‐stranded conformational polymorphism (SSCP) for detecting p53 mutations [18]. In the present study, we determined the rate of p53 mutations in localized prostate cancer and ascertained whether they could be reliably detected by the modified yeast assay on clinical samples of prostate cancer.…”

Androgen receptor signaling and mutations in prostate cancer

“…As yeast does not have steroid receptors or most of their coregulators, it can provide the intrinsic activity of the mutated AR . This method has been successfully used to investigate steroid receptor function and for functional analyses of large numbers of AR mutations in PCa or other type of receptors in mammalian cell system [157–159]. …”