Functional Analysis Of Separated Alleles In Yeast Research Articles

Detection and Functional Analysis of TP53 Mutations in CLL.

Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).

Overexpression of yeast thioredoxin TRX2 reduces p53-mediated cell death in yeast.

We have previously shown that overexpression of the human tumor suppressor protein P53 causes cell death of the yeast Saccharomyces cerevisiae. P53 overproduction led to transcriptional downregulation of some yeast genes, such as the TRX1/2 thioredoxin system, which plays a key role in cell protection against various oxidative stresses induced by reactive oxygen species (ROS). In the present work, the impact of TRX2 overexpression on apoptosis mediated by p53 overexpression in yeast is investigated. In yeast cells expressing P53 under an inducible promoter together with TRX2 under a strong constitutive promoter, we showed that Tr2p overproduction reduced the apoptotic effect exerted by P53 and increased the viability of the P53-overproducing cells. Furthermore, measurements of ROS amounts by flow cytometry and fluorescence microscopy indicated that the TRX2 protein acted probably through its increased detoxifying activity on the P53-generated ROS. The steady-state level and activity of P53 were not affected by TRX2 overexpression, as shown by western blotting and functional analysis of separated alleles in yeast (FASAY), respectively. The growth inhibitory effect of P53 was partially reversed by the antioxidant N-acetylcysteine. Our data strengthen the idea that overexpression of a single gene (trx2) decreases the p53-mediated cell death by decreasing ROS accumulation.

Benzo[a]pyrene, Aflatoxine B1 and Acetaldehyde Mutational Patterns in TP53 Gene Using a Functional Assay: Relevance to Human Cancer Aetiology

Mutations in the TP53 gene are the most common alterations in human tumours. TP53 mutational patterns have sometimes been linked to carcinogen exposure. In hepatocellular carcinoma, a specific G>T transversion on codon 249 is classically described as a fingerprint of aflatoxin B1 exposure. Likewise G>T transversions in codons 157 and 158 have been related to tobacco exposure in human lung cancers. However, controversies remain about the interpretation of TP53 mutational pattern in tumours as the fingerprint of genotoxin exposure. By using a functional assay, the Functional Analysis of Separated Alleles in Yeast (FASAY), the present study depicts the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to well-known carcinogens: benzo[a]pyrene, aflatoxin B1 and acetaldehyde. These in vitro patterns of mutations were then compared to those found in human tumours by using the IARC database of TP53 mutations. The results show that the TP53 mutational patterns found in human tumours can be only partly ascribed to genotoxin exposure. A complex interplay between the functional impact of the mutations on p53 phenotype and the cancer natural history may affect these patterns. However, our results strongly support that genotoxins exposure plays a major role in the aetiology of the considered cancers.

Assessment of p53 functional activity in tumor cells and histologically normal mucosa from patients with head and neck squamous cell carcinoma

The purpose of this study was to investigate the value of p53 functional analysis of separated alleles in yeast (FASAY) as a witness of p53/p21 pathway alteration in head and neck squamous cell carcinoma (HNSCC). The p53 transcriptional activity was prospectively analyzed in 82 newly diagnosed patients with HNSCC. FASAY and p53 immunostaining were carried out on paired tumoral and histologically normal tissues. The predictive value of FASAY for locoregional recurrence was assessed by Cox survival analysis. Loss of p53/p21 transcriptional activity was encountered in 88% tumoral and 18% histologically normal samples, associated with mutations (79%) and insertions/deletions (21%). The p53 overexpression underestimated p53 transcriptional abnormalities. FASAY-positive histologically normal mucosa was significantly associated with locoregional recurrence. FASAY positivity indicates field cancerization in a subgroup of patients with HNSCC, in which nonfunctional p53 was significantly associated with locoregional recurrence. This prompted us to pursue the study on the p53 functional status of normal mucosa in patients with HNSCC.

A comprehensive study of TP53 mutations in chronic lymphocytic leukemia: Analysis of 1287 diagnostic and 1148 follow-up CLL samples

TP53 plays a pivotal role in the process of DNA repair and apoptosis. In 10–20% of patients with chronic lymphocytic leukemia (CLL), the TP53 pathway is affected. In this study, we analyzed the TP53 mutation status in 2435 consecutive CLL samples, including 1287 diagnostic samples and 1148 samples during follow-up, using FASAY (Functional Analysis of Separated Alleles in Yeast) and direct sequencing. In a cohort of 1287 diagnostic CLL samples, we identified 237 cases with TP53 variants, including mutations, temperature-sensitive variants, deletions, insertions and aberrant splicing variants (18.4%). In 1148 follow-up samples, no TP53 clonal evolution was observed.

Abstract 1780: p53 mutation predicts limited benefit from adjuvant chemotherapy in patients with localized completely resected high grade soft tissue sarcoma: a retrospective study on 117 patients of the French Sarcoma Group

Abstract Rationale TP 53 is frequently mutated in some sarcoma subtypes and its mutation status has been reported to be correlated to survival in some studies. We investigated whether TP53 status may play a role in determining outcome following resection of the primary tumor. Material and methods The functional status of p53 was tested in a retrospective series of 117 patients of the French Sarcoma Group with localized grade 2or 3 STS treated with surgery, followed by radiotherapy (70) and/or chemotherapy (37%) in whom frozen pre-treatment material was available. FASAY (Functional Analysis of Separated Alleles in Yeast) was used to test the functional properties of p53. Statistical analysis were performed using SPSS 16.0. Results The diagnosis and primary surgery was performed between April 1990 and March 2006. Median follow up was 10,5 years. There were 62% males 38% females, aged 16 to 92 (median 64) with 74% primary locations of the limbs, 26 trunk or H&amp;N respectively. Major histotypes were MFH/undifferentiated (32%) leiomyosarcomas (LMS, 27%), liposarcomas (LPS, 16%), while grade 2 and 3 represented 27% and 73% respectively Functional inactivation of p53 was observed in 31 tumors (29%), and was not correlated to age, gender, site, histotype, with the exception of dedifferentiated LPS which never expressed a no functional p53 (0/12 vs 31/100, p=0.01). Mutations were more frequently observed in grade 3 vs grade 2 (33% vs 13%, p=0.03). 70% patients received adjuvant radiotherapy, and 17% received adjuvant chemotherapy (AdjCT). Adjuvant RT was not correlated to RFS and OS. Adjuvant CT was associated with a better RFS (trend p=0.07) and a better OS (p=0.008). P53 mutation was correlated marginally to a worse OS (p=0.03), and a worse RFS (p=0.03) in univariate analysis. Similar results were obtained when the DDLPS subgroup of patients- with mdm2 amplification, was removed from the group (p=0.01 and 0.006 for RFS and OS). Using Cox model with gender, age, size, LPS histology, depth, R, grade, trunk sites, p53, adjCT), only the last three were independently correlated to OS, while size, p53, and adjCT were retained by the model for RFS. In the WT p53 group, adjCT improved RFS (trend p=0,07) and OS (p=0,007). RT improved significantly RFS (p=0.001) and marginally OS (0.06). In Cox model in the WT group, tumor size, adjCT for RFS, and gender, DDLPS and adjCT for OS were the only independent factors Conversely, in the MUT p53 group, OS and RFS were super-imposable with adjuvant CT and adjuvant RT trending towards a worse outcome in the CT+ and RT+ subgroups. Conclusion: Mutations of p53 using Fasay correlated to RFS and OS and to a limited or no benefit of adjuvant CT for RFS and OS. A prospective series is required to confirm this observation which may be helpful for patient selection in the future. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1780.

An unusual loss of EGFR gene copy in glioblastoma multiforme in a child: a case report and analysis of a successfully derived HGG-02 cell line

The aim of this study was to perform a detailed cytogenetic and molecular genetic analysis of a tumor taken from a 14.5-year-old boy with glioblastoma multiforme who showed an atypical clinical course. Formalin-fixed, paraffin embedded tumor tissue and the corresponding HGG-02 cell line derived from this tumor were analyzed using fluorescence in situ hybridization (FISH), G-banding, multiplex ligation-dependent probe amplification (MLPA), functional analysis of separated alleles in yeast (FASAY), immunohistochemistry (IHC), and immunocytochemistry (ICC). Mutation of the p53 gene and hypermethylation of the MLH1 gene were detected by FASAY and MLPA, respectively. Cytogenetic analysis showed a polyploid karyotype with extensive heterogeneity in chromosome number. Using FISH, we identified a very unusual genetic change - a loss of EGFR gene copy in both the tumor tissue and the HGG-02 cell line. In accordance with the cytogenetic findings, IHC and ICC did not demonstrate overexpression of EGFR in the tumor tissue or HGG-02 cells. Despite his very poor prognosis, the patient experienced 34 months of event-free survival after surgery and adjuvant radiotherapy and chemotherapy. The detected loss of the EGFR gene copy may contribute to the unusual biological features of this tumor, but the forthcoming detailed expression analysis of cancer regulatory pathways is necessary to better understand this tumor phenotype.

P53 Inactivation and Genomic Aberrations in Relation to Imatinib Resistance in Chronic Myeloid Leukemia.

Abstract 4256 BackgroundImatinib resistance in chronic myeloid leukemia (CML) patients is correlated with mutations in BCR-ABL tyrosine kinase domain in about half of the cases. Additional mechanisms related to imatinib resistance are under investigation. As TP53 gene is an important tumor suppressor that triggers apoptosis in response to DNA damage, its inactivation is responsible for chemotherapy resistance in cancer. Its role in cellular response to targeted biological treatment is currently unresolved. Inactivation of p53 by deletion and/or mutation in the TP53 gene is observed in CML patients during progression to accelerated phase (AP) and blast crisis (BC). It was suggested that BCR-ABL inhibition by imatinib induce the p53 response and therefore p53 inactivation may play role in resistance to targeted treatment (Wendel et al., 2006; Yamamoto et al., 2008). On the other hand, imatinib-induced p53 independent pro-apoptotic mechanism was described recently (Liu et al., 2009). AimWe investigated the relationship between imatinib resistance and abnormalities in the TP53 gene and additional genomic changes. MethodsRNA and genomic DNA were isolated from peripheral blood mononuclear cells of CML patients that either fail to achieve or lost the major cytogenetic response (MCyR). TP53 mutational status was examined using functional analysis of separated alleles in yeast (FASAY). In defined cases direct sequencing of genomic DNA was used. Genomic changes were detected by conventional metaphase cytogenetics and CGH microarrays 4×44K (Agilent). Copy number analyses were performed using MEV software. ResultsFASAY analysis to detect functional state of TP53 gene was performed in 16 imatinib-resistant CML patients and in one Ph+ B-ALL patient. Six of these patients were negative for BCR-ABL mutations. Four patients were examined at the time of blast crisis (two patients with myeloid BC and two patients with lymphoid BC). All examined patients carried functional TP53 gene. As FASAY is based on RNA analysis, it is not able to detect some mutations leading to nonsense-mediated RNA decay; therefore, in six patients without BCR-ABL mutation, direct sequencing of TP53 gene from genomic DNA was performed. Also by this approach no TP53 mutation was detected. Alterations of the p53 pathway were found in only 2 patients, both in lymphoid BC: one patient without BCR-ABL mutation had +8 and the deletion of TP53 locus accompanied by i(17p). Second patient with BCR-ABL mutation T315I carried heterozygous deletion of 9p with biallelic loss of 9p21. In this locus two important tumor suppressor genes CDKN2A and CDKN2B are localized. CDKN2A alternative transcript contains an alternate open reading frame (ARF) that functions as a stabilizer of the tumor suppressor protein p53. In other two patients in chronic phase that did not reach MCyR and had no BCR-ABL mutations additional genomic changes potentially connected to imatinib resistance were found: 1) +der(16) coupled with amplification of 16(p11-q12), where ABCC11 and ABCC12 genes are localized. The proteins encoded by these genes are members of the superfamily of ATP-binding cassette (ABC) transporters responsible for multidrug resistance. 2) del(3)(p13p21), involving locus 3p14 where multiple tumor suppressors are localized (e.g. FHIT, ADAMTS9, LRIG1). Chromosomal region 3p14 was shown to be often deleted in different types of human cancers. ConclusionsImatinib resistance in CML patients is probably not associated with TP53 inactivation. Alterations of the p53 pathway occur within transformation to more advanced stages, what is in concordance with previous findings. Genomic aberrations potentially influencing response to imatinib treatment may be found in some patients. Larger patients' cohorts are required to identify relevant recurrent genomic aberrations involved in imatinib resistance.This work was supported with grants NR9858-4/2008 and NR9305-3/2007 provided by IGA MH CR of Czech Republic, and MSM0021622430 provided by MEYS of Czech Republic Disclosures:No relevant conflicts of interest to declare.

Benzene-induced mutational pattern in the tumour suppressor gene TP53 analysed by use of a functional assay, the functional analysis of separated alleles in yeast, in human lung cells

Recent concern has centred on the effects of continuous exposure to low concentrations of benzene, both occupationally and environmentally. Although benzene has for a long time been recognised as a carcinogen for humans, its mechanistic pathway remains unclear. Since mutations in the tumour suppressor gene TP53 are the most common genetic alterations involved in human cancer, our objective was to establish the first mutational pattern induced by benzene on the TP53 gene in human type II-like alveolar epithelial A549 cells by using the Functional Analysis of Separated Alleles in Yeast (FASAY). Seventeen mutations linked to benzene exposure were found: 3 one- or two-base deletions, and 14 single nucleotide substitutions (1 nonsense and 13 missense mutations). A>G and G>A transitions were the most prevalent (23.5% for both). Other mutations included A>C transversions and deletions (3/17, 17.6% for both), G>T transversions (2/17, 11.8%) and A>T transversions (1/17, 5.9%). Data arising from this benzene-induced mutational pattern affecting TP53, a critical target gene in human carcinogenesis, have been compared with those reported in human acute myeloid leukaemia, the aetiology of which is clearly linked to benzene exposure, and in experimental benzene-induced carcinoma. This comparison suggests that A>G transition could be a fingerprint of benzene exposure in tumours. Furthermore, our results demonstrate that FASAY is a promising tool for the study of the carcinogenic potency of benzene in the human lung.

Delta Ex6, the Novel Transactivation-Defective Splicing Variant of p53 Gene, Is Differentially Expressed in Patients with Chronic Lymphocytic Leukemia and Confers Accented Proliferative Phenotype in Vitro

The p53 gene, metaphorically named “the Guardian of the Genome“ by David Lane in 1992, is one of the most important decision makers inside the cell. By interacting with an array of downstream genes, p53 can regulate cell fate, either by precipitating events leading to cell death by apoptosis, or by acting as a regulatory factor during G1/S phase allowing the cell to repair minor damage to DNA and proceed to the next stage of cell division. Playing such a pivotal role within the cell, p53 itself is subjected to a tight and orchestrated control, creating a network of positive and negative regulations via a number of interacting proteins (MDM-2, Wip-1, Cyclin G, p14ARF). Yet another level of p53 regulation is represented by post-translational modifications, modulating its transcriptional/transactivational ability. These modifications include phosphorylations on serines 15, 18 and 20 or acetylations at C-terminal lysines of p53. Recently, it has been found that the regulation of p53 is also substantially controlled at the transcriptional level. They identified nine different splicing variants of p53 with distinct biological characteristics, resulting from combinations of an alternative splicing of intron 2, 9 and/or aberrant transcription, starting at so-far unrecognized cryptic promoter in intron 4. Herein we present evidence of a novel splicing variant of p53 gene, termed delta ex6 that is differentially expressed in patients with chronic lymphocytic leukemia (CLL) as compared to healthy donors. The delta ex6 variant was identified in 109 out of 127 (86%) CLL patients, while in healthy individuals it was not detected. Delta ex6 variant is devoid of transactivational activity as determined in vitro by FASAY (Functional Analysis of Separated Alleles in Yeast). To test the biological properties of the delta ex6 variant we have cloned its whole coding sequence and transfected the p53-double-knock-out model cell line H1299 to produce stable integrants. Stable H1299 cell lines expressing the delta ex6 variant were distinguished by a remarkable loss of intercellular contacts and semi-suspension growth properties, in contrast to the strictly adherent growth of the parental cells and mock-transfected cells. Four stable delta ex6 producing H1299 cell lines, as well as control cell lines in doublets (parental H1299 harboring the cloning vector, and H1299 stably transfected with wild type p53) were subjected to the Affymetrix GeneChip Human Exon 1.0 ST array expression analysis. The microarray data corroborated the accented and proliferative phenotype as observed in vitro in the tissue culture: overexpression of a number of cyclins (A1, G1, G2, F, I, B2, A2, T2), matrix metalloproteinases, hyaluronidases and caspase inhibitors; and downregulation of adhesion molecules and molecules of the intercellular matrix. Our data on the presence of the delta ex6 p53 variant in CLL patients supports the recent evidence on dysregulation of p53 splicing pattern in malignancies. Moreover, as assessed in vitro, overexpression of the delta ex6 variant leads to an accented and proliferative phenotype, a finding further supporting the biological role of the novel delta ex6 p53 variant in vivo.

Aflatoxin B 1-induced TP53 mutational pattern in normal human cells using the FASAY ( Functional Analysis of Separated Alleles in Yeast)

Mutations in the TP53 gene are the most common alterations in human tumours. In hepatocellular carcinoma (HCC) related to exposure to aflatoxin B 1, a specific G > T transversion in codon 249 is classically described as a hot spot. However, AFB 1 is suspected to be a potent carcinogen in tissues other than the liver. By using the FASAY functional assay in yeast, the present study aimed at depicting the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to AFB 1. Molecular analysis of mutants revealed that codon 245 was the main hot spot, whereas no mutations were found in codon 249. The locations of mutations within GG and GC/CG sequences are well in accordance with AFB 1-adduct location data. In our assay with normal human fibroblasts, AFB 1 mainly induced G > A transitions, followed by G > T and A > G mutations. This suggests that G > T transversions at codon 249 were likely the result of a selection bias in human HCC rather than a true fingerprint of AFB 1 adducts. Indeed, a comparison of the mutation pattern with that found in human HCC excluding codon 249 reveals that the two spectra are quite similar. Furthermore, the similarity between our in vitro spectrum with that identified in AFB 1-induced lung tumours in mice suggests that AFB 1 may be a potent lung carcinogen in humans.

Acetaldehyde-induced mutational pattern in the tumour suppressor gene TP53 analysed by use of a functional assay, the FASAY (functional analysis of separated alleles in yeast)

Chronic alcohol consumption is a major risk factor for upper aero-digestive tract cancers, including cancer of the esophagus. Whereas alcohol as such is not thought to be directly carcinogenic, acetaldehyde, its first metabolite, has been proven genotoxic and mutagenic in the HPRT gene. As mutations in the tumour suppressor gene TP53 are the most common genetic alterations involved in human cancers, especially esophageal tumours, the aim of this work was to establish the mutational pattern induced by acetaldehyde in vitro on the TP53 gene, and to compare this pattern with that found in human alcohol-related tumours. For this purpose, we used a functional assay in yeast, the FASAY (functional analysis of separated alleles in yeast), after in vitro exposure of human normal fibroblasts AG1521 to acetaldehyde. We noted 35 mutations, of which 32 were single-nucleotide substitutions including 2 nonsense and 30 missense mutations. The pattern showed that the main mutations were G > A transitions ( n = 23, of which 14 in CpG sites), followed by G > T transversions ( n = 4), A > G transitions ( n = 2) and A > T transversions ( n = 2). Other mutations were one-base insertion and two deletions, leading to frameshifts. Eleven mutations (31%) were located in TP53 hot-spots in codons 245, 248, 249 and 273. Finally, we compared this pattern with that found for esophageal cancers in humans. These results support the notion that acetaldehyde plays a role in TP53 mutations in esophageal cancers. The key feature of this approach is that mutagenesis is directly studied in a key gene in human carcinogenesis, allowing direct comparison of mutational patterns with those in human tumours.

Detection of heterozygous nonsense mutations in genes of interest using an Escherichia coli‐based stop codon assay

Since nonsense mutations are closely associated with severe conditions of genetic disorders, including familial cancers, rapid and precise detection of those mutations is very important for research purposes and molecular diagnosis. Currently, screening methods such as the FASAY (functional analysis of separated alleles in yeast) and the Y-SC (stop codon assay in yeast) are used for functional detection of nonsense mutations in genes of interest. But these yeast-based approaches are time-consuming, expensive and complicated. In order to circumvent these problems, we, in the present study, devised a novel Escherichia coli-based screening method, the E-SC (E. coli stop codon assay) for the detection of heterozygous nonsense mutations in genes of interest. Our strategy was based on the fact that the plasmid replicating with a low copy number in E. coli allows an effective separation of normal and mutant alleles. Moreover, it relies on the expression vector, resulting in the formation of white and blue colonies for mutant and normal alleles through the expression of PCR-amplified fragment/lacZ fusion protein respectively. To evaluate the applicability of the E-SC method for the detection of the heterozygous truncating mutation, PCR-amplified exon 7 of the StAR [steroidogenic acute regulatory protein; causative gene of the CAH (congenital lipoid adrenal hyperplasia)] and RT (reverse transcription)-PCR-amplified full-length cDNA of MeCP2 (methyl-CpG-binding protein 2; causative gene of Rett syndrome) were used. The E-SC showed an almost 1:1 ratio of blue/white colonies in all patients examined, whereas the control samples produced blue colonies only. These results demonstrate that the E-SC system is useful for rapid and precise detection of known and unknown heterozygous truncation mutations in genes that cause genetic disorders and familial cancers.

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.

Assessment of the Transcriptional Activity of p53 Improves the Prediction of Recurrence in Superficial Transitional Cell Carcinoma of the Bladder

To investigate the value of p53 functional analysis of separated alleles in yeast (FASAY) as a witness of p53/p21 pathway alteration and as a predictor of recurrence in superficial transitional cell carcinomas. p53 transcriptional activity was prospectively analyzed in 52 newly diagnosed transitional cell carcinoma using FASAY competent for the transactivation of p21 and bax promoters. TP53 and p21 gene expression was quantified by real-time PCR, and expression of corresponding proteins was assessed by immunohistochemistry. In addition to tumor stage and grade, the predictive value of FASAY, real-time PCR, and immunohistochemistry for tumor recurrence was assessed by Cox survival analysis. A total (p21 and bax) or partial (bax only) loss of transcriptional activity was observed in 15 of 52 (29%) and 4 of 52 (7.7%) cases, respectively, a partial loss being consistently associated with R283H mutation. p53 nuclear overexpression grossly overestimated (approximately 40%) or underestimated (approximately 10%) the true incidence of p53 transcriptional abnormalities, especially in Ta-T1 grade 1 to 2 tumors. Loss of p21 transactivation significantly correlated with decreased p21 gene expression and lack of expression of p21 (P = 0.001). FASAY had a better predictive value for recurrence than p53 immunohistochemistry (Cox hazard ratio, 6.57 versus 3.95; P = 0.0002 versus 0.019, respectively), whereas neither p21 immunohistochemistry (hazard ratio, 1.9; P = 0.29) nor TP53 or p21 gene expression were significant predictors of recurrence. The prognostic difference between FASAY and p53 immunohistochemistry was maintained in the subgroup of Ta-T1 grade 3 tumors. FASAY is a valuable surrogate marker for assessing p53/p21 pathway alteration and predicts transitional cell carcinoma recurrence better than p53 immunohistochemistry.

The role of p53 in the chemotherapeutic responses to cisplatin, doxorubicin and 5-fluorouracil treatment

A panel of tumour models used extensively for in vivo evaluation of new drugs was characterised for their p53 status. Basal p53 protein levels were measured by immunodetection on both formalin-fixed tumour tissue and from protein extracts of fresh tumours. High levels of nuclear-specific staining, indicative of p53 mutation, was seen in 15/25 tumours, with the remainder showing intermittent or no staining. The functional status of p53 cDNA from these tumours was assayed within the functional analysis of separated alleles in yeast (F.A.S.A.Y.) reporter system. The cDNA from those tumours with high levels of p53 protein showed 14/15 failing to activate the reporter gene. The cDNA from tumours with low or non-detectable p53 levels showed 8/10 with wild-type p53. Tumours were grown subcutaneously in mice (n=10). Each mouse was given maximum tolerated doses for either doxorubicin, 5-fluorouracil or cisplatin. Tumour volumes were measured daily, alongside untreated controls. The specific growth delay values for each tumour were separated into two groups, those with functional p53 (wild-type) and those without (mutant and null status). The Mann-Whitney U test was performed on the groups of data, to evaluate differences in their response on the basis of p53 status. Cisplatin was moderately active against tumours with wild-type and mutant p53 genes with no significant difference seen between both groups. However, a significant difference in specific growth delay was seen between the two groups when treated with doxorubicin or 5-fluorouracil (P=0.05), indicating a role for p53 protein in modulating the in vivo efficacy of these agents.

Correlations of breast carcinoma biomarkers and p53 tested by FASAY and immunohistochemistry.

p53 status is an important predictive factor in breast cancer, but the results of many studies are ambiguous. We tested p53 by functional analysis of separated alleles in yeast (FASAY) as well as by immunohistochemistry (IHC) and evaluated correlations with main prognostic factors, proliferation, and Bcl-2. Thirty-two tumors were tested with antibodies BP53-12, DO1, DO11, DO12, and by FASAY. Spearman rank correlations were tested separately with age, tumor type, pT, grade, pN, NPI, Ki-67, S-phase, proliferation index, Bcl-2, and steroid receptor status determined by ER, PR, and pS2. FASAY showed significant correlations with ductal type, grade and proliferation, and an inverse correlation with functional estrogen receptor and Bcl-2. FASAY provided better correlations compared to p53 IHC. We conclude that FASAY shows significant correlations with main prognostic/predictive factors and provides more reliable biological information compared to p53 IHC. Apoptosis is positively linked to proliferation and is not under the control of p53, which is frequently mutated in highly proliferating carcinomas. FASAY seems to be very important in assessing the predictive significance of p53 for a specific therapy of breast cancer.

Screening patients for heterozygous p53 mutations using a functional assay in yeast

Inherited mutations of the p53 gene significantly increase the risk of developing diverse malignancies, and germline p53 mutations can be detected by assaying the transcriptional activity of the p53 protein in mammalian cells. Here we describe a method starting with lymphocytes that allows detection of germline p53 mutations by 'functional' analysis of p53 protein expressed in Saccharomyces cerevisiae. The p53 PCR products are directly cloned into yeast expression vectors in vivo and subsequently tested for transcriptional activity in a simple growth assay. This technique, functional analysis of separated alleles in yeast (FASAY), requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.