Oncogene Division

13. Oncogene Division

A major project of the Oncogene Division is to seek a comprehensive grasp of the range of DNA aberrations that accumulate in the cancer cells of each patient to obtain a better understanding of human cancers that may in turn facilitate better prevention, diagnosis and therapy of cancers.
Detection of Aberrations of Known Genes in Human Cancers

Fluorescence-based SSCP analysis using blunt-end DNA frag ments carrying a marker for polymorphic sites of the p53 gene may potentially provide a sensitive method for the detection of loss of heterozygosity (LOH) and has been successfully applied to diagnosis of bladder cancers.⁽¹⁷⁹⁾ The in vivo mutation frequency of the hprt gene in circulating T-lymphocytes was analyzed using a newly established, accessory cell-free cloning method. The results revealed no significant differences in the mean mutation frequencies of the gene in colon cancer patients and in cancer-free individuals.⁽¹⁸⁰⁾ Genetic alterations that have accumulated in pancreatic tumors of each patient are being extensively analyzed with a focus on the many tumor-related genes using DNA analysis methods such as SSCP.⁽¹⁸¹⁾
Detection of Aberrations in Anonymous Regions of the Genome

The presence of putative tumor suppressor genes involved in genesis of human non-small cell lung cancers (NSCLCs) was suggested by LOH analysis in 3 regions on chromosome 13q. Although LOH at the RB1 locus was frequent, only a few tumors lost expression and had mutations in the remaining RB1 allele. Involvement of genes other than the RB1 gene was suggested.⁽¹⁸²⁾ Three human endogenous retrovirus-like (HERV-H) sequences were unexpectedly found to be clustered within a 300-Kb region close to the GRPR locus on the X chromosome where mutations in an Alu-repeat sequence were detected in human small cell lung cancers.⁽¹⁸³⁾ Simple comparison of arbitrarily primed (AP)-PCR fingerprints obtained from normal and cancer DNA samples of the same patient revealed gain or loss of copy numbers of chromosom al regions. For chromosome assign ment of fingerprint bands a convenient and powerful method of simultaneous hybridization of AP-PCR products (SHARP) was developed. The combined use of the SHARP and radiation hybrid methods could successfully detected DNA aberrations without the targeting of particular genes or the need for previous knowledge of nucleotide sequences.
Development of Technologies for Detection of DNA Aberrations

To clarify epigenetic changes, the comprehensive identification of CpG islands hypermethylated in human cancers was attempted by the combined use of methyl-CpG binding domain (MBD) column chromatography and the segregation of partly melted molecules (SPM) method. The SPM was applied to hunt for genes in a contiguous 400-Kb region on chromosome 11q13. A total of 12 fragments associated with CpG islands were isolated using analysis of restriction endonuclease digests of 9 P1 clones covering the region. Based on the number of NotI sites, 15 CpG islands were predicted in the region analyzed. Four fragments contained a part of the CpG islands of the four genes so far located in this region, while 6 of the other 8 fragments carried nucleotide sequences identical or highly homologous to reported expressed sequence tags (ESTs). Thus most of the CpG islands in a chromosomal region can be identified by SPM analysis. Highly methylated DNA fragments from surgical specimens of lung cancers were fractionated by the column chromatography and ligated into a lambda vector. Clones in the DNA library thus prepared were then analyzed by the SPM method to identify the presence of CpG islands. The nu cleotide sequence of one of the DNA fragments isolated was nearly identical to that of a reported EST and its CpG residues were specifically hypermethylated in a primary lung cancer, suggesting that the fragment was a part of a gene silenced by methylation in lung cancers.
Identification of putative tumor suppressor genes suggested by LOH analyses was performed by functional analyses. Introduction of YAC clones carrying a contiguous region of chromosome 11q23 that was deleted in NSCLCs into the human lung cancer cell line A549 revealed suppression of cellular tumorigenicity in nude mice in one of the YAC clones carrying a 1.6 Mb human DNA. Sequential truncation of the DNA from both termini will be used to determine the minimal functional region in the same functional assay. A similar functional approach was applied to analysis of regions deleted on chromosome 10p15-p14 in human gliomas. Suppression of colony-forming ability of the human glioblastoma cell line T98G by introduction of the chromosomal fragments carrying the corresponding regions strongly suggested the presence of tumor suppressor genes.





	13. Oncogene Division



		A major project of the Oncogene Division is to seek a comprehensive grasp of the range of DNA aberrations that accumulate in the cancer cells of each patient to obtain a better understanding of human cancers that may in turn facilitate better prevention, diagnosis and therapy of cancers. Detection of Aberrations of Known Genes in Human Cancers Fluorescence-based SSCP analysis using blunt-end DNA frag ments carrying a marker for polymorphic sites of the p53 gene may potentially provide a sensitive method for the detection of loss of heterozygosity (LOH) and has been successfully applied to diagnosis of bladder cancers.⁽¹⁷⁹⁾ The in vivo mutation frequency of the hprt gene in circulating T-lymphocytes was analyzed using a newly established, accessory cell-free cloning method. The results revealed no significant differences in the mean mutation frequencies of the gene in colon cancer patients and in cancer-free individuals.⁽¹⁸⁰⁾ Genetic alterations that have accumulated in pancreatic tumors of each patient are being extensively analyzed with a focus on the many tumor-related genes using DNA analysis methods such as SSCP.⁽¹⁸¹⁾ Detection of Aberrations in Anonymous Regions of the Genome The presence of putative tumor suppressor genes involved in genesis of human non-small cell lung cancers (NSCLCs) was suggested by LOH analysis in 3 regions on chromosome 13q. Although LOH at the RB1 locus was frequent, only a few tumors lost expression and had mutations in the remaining RB1 allele. Involvement of genes other than the RB1 gene was suggested.⁽¹⁸²⁾ Three human endogenous retrovirus-like (HERV-H) sequences were unexpectedly found to be clustered within a 300-Kb region close to the GRPR locus on the X chromosome where mutations in an Alu-repeat sequence were detected in human small cell lung cancers.⁽¹⁸³⁾ Simple comparison of arbitrarily primed (AP)-PCR fingerprints obtained from normal and cancer DNA samples of the same patient revealed gain or loss of copy numbers of chromosom al regions. For chromosome assign ment of fingerprint bands a convenient and powerful method of simultaneous hybridization of AP-PCR products (SHARP) was developed. The combined use of the SHARP and radiation hybrid methods could successfully detected DNA aberrations without the targeting of particular genes or the need for previous knowledge of nucleotide sequences. Development of Technologies for Detection of DNA Aberrations To clarify epigenetic changes, the comprehensive identification of CpG islands hypermethylated in human cancers was attempted by the combined use of methyl-CpG binding domain (MBD) column chromatography and the segregation of partly melted molecules (SPM) method. The SPM was applied to hunt for genes in a contiguous 400-Kb region on chromosome 11q13. A total of 12 fragments associated with CpG islands were isolated using analysis of restriction endonuclease digests of 9 P1 clones covering the region. Based on the number of NotI sites, 15 CpG islands were predicted in the region analyzed. Four fragments contained a part of the CpG islands of the four genes so far located in this region, while 6 of the other 8 fragments carried nucleotide sequences identical or highly homologous to reported expressed sequence tags (ESTs). Thus most of the CpG islands in a chromosomal region can be identified by SPM analysis. Highly methylated DNA fragments from surgical specimens of lung cancers were fractionated by the column chromatography and ligated into a lambda vector. Clones in the DNA library thus prepared were then analyzed by the SPM method to identify the presence of CpG islands. The nu cleotide sequence of one of the DNA fragments isolated was nearly identical to that of a reported EST and its CpG residues were specifically hypermethylated in a primary lung cancer, suggesting that the fragment was a part of a gene silenced by methylation in lung cancers. Identification of putative tumor suppressor genes suggested by LOH analyses was performed by functional analyses. Introduction of YAC clones carrying a contiguous region of chromosome 11q23 that was deleted in NSCLCs into the human lung cancer cell line A549 revealed suppression of cellular tumorigenicity in nude mice in one of the YAC clones carrying a 1.6 Mb human DNA. Sequential truncation of the DNA from both termini will be used to determine the minimal functional region in the same functional assay. A similar functional approach was applied to analysis of regions deleted on chromosome 10p15-p14 in human gliomas. Suppression of colony-forming ability of the human glioblastoma cell line T98G by introduction of the chromosomal fragments carrying the corresponding regions strongly suggested the presence of tumor suppressor genes.