KCA
Search
- Page Path
- HOME > Search
Original Articles
- p53 Mutation and Functional Analyses by Using Yeast Functional Assay
- Byung Joo Song, Chin Seung Kim, Il Soo Kim, Su Mi Han, Hae Jung Nam, Mi Uk Chin, Dong Hwan Kim, Dong Hwang Kim, Hyun Pil Cho, Young Ho Moon
- J Korean Cancer Assoc. 1999;31(5):876-886.
-
Abstract
PDF - PURPOSE
Mutation of the p53 tumor suppressor gene is the most common genetic defect in all human tumors. Because of the widespread mutations and polymorphism in the p53 gene, the conventional screening methods cannot distinguish between polymorphisms or functionally silent mutations and inactivating mutations. It is well known that plasmids can be generated by homologous recombination in vivo in the yeast by cotransforming the PCR product with a linearized yeast expression vector encoding part of a gene and a selectable marker gene. The aim of this study is to develop more easy and reliable method for functional assay of p53 mutation.
MATERIALS AND METHODS
We constructed a gap vector which can reliably and conveniently be used to screen p53 mutations in a simple yeast growth assay. The gap vector was constructed as follows: About 100 bp DNA fragments containing parts of N- and C- terminal portion of p53 were cloned into XbaI/SmaI and HindIII/XhoI sites of yeast expressing vector, respectively. The gap vector was obtained by double cutting with SmaI and HindIII followed by gel elution. Yeast was transformed with the reporter vector containing three tandem copies of the consensus p53 binding site by lithium acetate-mediated method. RT-PCR amplification of p53 transcripts from cell lines or tumor tissues was carried out. To investigate whether p53 gene is mutated or not, yeast containing reporter gene was cotransformed with PCR product and linearized gap vector, plated on SD medium minus histidine, and incubated for 3 days. The colonies on selective media were isolated and characterized.
RESULTS
The tumor tissues examined were one hepatocellular carcinoma, three breast cancers, two stomach cancers and two colon cancers. One hepatocellular carcinoma tissue had mutation in both alleles of the p53 gene, and 7 cancer tissues had heterozygous mutations in the p53 gene. The result of functional assay was well correlated with mutational analysis by sequencing.
CONCLUSION
p53 functional assay system might be easy and reliable method for functional screening of p53 on tumor tissues and this might be used for screening of other mutated gene. This technique, 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.
- 3,034 View
- 23 Download
- A Study on the p53 Mutations in Korean Breast Cancer Tissues
- Hong Kyu Baik, Pah Jong Jung, Youl Hee Cho, Young Hyeh Ko
- J Korean Cancer Assoc. 1998;30(5):921-934.
-
Abstract
PDF
- PURPOSE
The role of mutation of p53 gene on the carcinigenesis was studied since 1991. There were some relationships of p53 mutation and clinicopathologic factors. This sutudy was designed for the clinicopathologic and genetic factor relation in Korean breast cancer.
MATERIALS AND METHOD
A retrospective study on the clinicopathologic findings such as age, menopausal status, TNM stage, histologic grade, estrogen receptor, DNA ploidy and S-phase fraction was camed out on 47 breast cancer tissues which had been resected at the Department of Surgery, Hanyang University Hospital. Forty-seven tissues were grouped into 3 groups. Group 1 was ductal carcinoma in situ, Group 2 was invasive ductal carcinoma without axillary lymph node metastasis and Group 3 was invasive ductal carcinoma with axillary lymph node metastasis. The numbers of tissues in each groups were 14, 15 and 18, respectively. Mutation screening on the p53 tumor suppressor gene was also performed with PCR-SSCP-direct sequencing method from the genomic DNA extracted from formalin fixed and paraffin-embedded pathologic tissue blocks. The results were as followings; RESULT: p53 mutations were detected in 12 cases(25.5%) of 47. In Group 1, 4 cases(28.6%) had mutations, and in Group 2, 5 cases(33.3%), and in Group3, 3 cases(16.7%). There was no significant differences in mutation rate between three groups. In 12 mutations detected, 6 cases were transition, 5 of which were missense mutation in coding sequences, and one of which was splicing mutation at acceptor site. One case was transversion and five cases were deletions or insertions of various lengths resulting in frameshift mutations. There was no statistically significant difference between groups and clinicopathologic factors except the strong relationship between the negative estrogen receptor and p53 mutation(p< 0.001).
CONCLUSIONS
From the above findings, p53 gene could be considered to be inactivated at the all stage of multistep carcinogenesis processes. The nature of mutations and genetic background of Korean breast cancers may be somewhat different from those of Caucasians. And the p53 mutation status may be used as one of the useful prognostic factors in addition to the estrogen receptor status.
- 2,612 View
- 21 Download
- p53 Mutation of Head and Neck Squamous Cell Carcinoma Cell Lines
- Chung Hwan Baek, Ye Jeung Ko, Young Ik Sun, Sung Wha Hong, Kwang Chol Chu
- J Korean Cancer Assoc. 1998;30(1):12-19.
-
Abstract
PDF
- PURPOSE
Structural alterations of p53 and overexpression of the p53 protein are the most common genetic abnormalities in various kinds of human cancers. In this study, we examined the mutational status and the frequency of p53 mutations in head and neck squamous cell carcimona (HNSCC) cell lines.
MATERIALS AND METHODS
7 human head and neck squamous cell carcinoma cell lines were included in this analysis. Using polymerase chain reaction(PCR), single strand confonmation polymorphism(SSCP) and PCR-DNA sequencing analysis, we tested the mutational status of 7 cell lines. Exon 4~9 of the p53 gene was amplified for the direct DNA sequencing analysis.
RESULTS
Our results showed 100% nuclear p53 immunostaining and 3 electrophoretic abnomalities by PCR-SSCP in three cancer cell lines and mutations of the p53 gene including 2 base substitutions and 1 base deletion were detected in 3 cancer cell lines using PCR directed DNA sequencing analysis.
CONCLUSION
7 HNSCC cell lines examined in this study provide excellent systems for study of gene therapy using p53 gene.
- 2,851 View
- 54 Download
First
Prev
