Analysis of tumor suppressor p53 status in head and neck squamous cell carcinoma.

Head and neck cancer belongs to the most common types of cancer in both males and females with a mortality rate of approximately 50%. More than 90% of head and neck cancers are squamous cell carcinoma (HNSCC). Carcinogenesis of this disease involves activation of proto-oncogenes and inactivation of tumor suppressor genes. Among them, aberrations of p53 tumor suppressor gene are common events. The aim of this study was to assess the frequency of the tumor suppressor p53 aberrations in Czech population by using a functional test in yeast (FASAY) and by two immunochemical methods. We compared results of the methods and assessed the relationship between the presence of p53 aberration and some clinico-pathological parameters. The following observations were made: i) the accumulated p53 protein was detected in 33 of 50 tested samples (66%) by immunohistochemical analysis and in 27 of 49 tested samples (55.1%) by immunoblotting; ii) the presence of p53 mutation was detected in 36 of 50 tested samples (72%); iii) 6 of 36 p53 mutations detected by FASAY were temperature sensitive (16.7%); iv) 2 independent p53 mutations were found in at least 2 of the 36 positive cases; v) no statistically significant relationship was found between p53 aberration and overall survival.

High frequency of temperature-sensitive mutations of p53 tumor suppressor in acute myeloid leukemia revealed by functional assay in yeast.

The tumor suppressor p53 is a transcription factor that participates in control of many cellular functions. All these activities are mediated by direct binding of the p53 tetramer to specific target sequences in promoters of directed genes. Lack of p53 function is often connected with development of cancer, but the frequency of p53 mutations is low in almost all types of leukemia. The aim of this study was to assess the frequency of mutations in the p53 gene in leukocytes of patients with acute myeloid leukemia (AML) using the FASAY functional analysis and to assess the relationship between the presence of p53 mutation and disease outcome. The following observations were made: i) the presence of p53 mutations was detected in 13 of 62 tested AML cases (21%) and in 1 of 4 tested myelodysplastic syndrome (MDS) cases by FASAY; ii) the presence of p53 mutation was shown to be a poor prognostic/predictive factor in AML (p=0.03/0.002); iii) although there is a statistically significant relationship between the presence of p53 mutation and p53 protein accumulation (p=0.05), not all samples having p53 mutation exhibited p53 protein accumulation; iv) five of 13 p53 mutations detected in the leukocytes of AML patients (38.5%) and the mutation detected in the leukocytes of the MDS patient (altogether 6/14-42.9%) were partially inactivating ts mutations. The high frequency of the ts p53 mutations in our study; and a novel modification in performing FASAY, are discussed; v) different ts mutations differ in the level of their temperature sensitivity and in their responsiveness to the cytoprotective drug amifostine.

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.

Determination of optimal conditions for analysis of p53 status in leukemic cells using functional analysis of separated alleles in yeast.

Tumor suppressor p53 is transcription factor that participates in control of many cellular functions. Somatic mutations of the p53 gene are frequently detected in human cancers. Several methods can be used for identification of p53 mutations, including FASAY - functional analysis of separated alleles in yeast. FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. The validity of the method depends on a low background level. There are several sources of background as PCR-induced point mutations, low quality of RNA and alternative splicing of intron 9 affecting the p53 carboxy-terminus. In the present work we show that FASAY can be successfully used for analysis of mRNA isolated from blood samples that were collected and stored for 24 hours at 0 degrees C without undesired increase of background. We also measured fidelity of several commonly used DNA polymerases and determined the most suitable kinds of Pfu DNA polymerases for FASAY. Reaction conditions described in this report allow routine analysis of p53 status in leukemic cells using FASAY.