13. Oncogene Division

The ultimate goal of studies in the Oncogene Division is systematic elucidation of DNA aberrations accumulated in cancer tissues in individual patients. Knowledge of the accumulated DNA aberrations may yield clues allowing better understanding of human cancers as well as enhancing the diagnosis and treatment of patients. For this purpose, three categories of research are conducted.

Detection of Aberrations of Known Genes in Human Cancers

Aberrations of tumor-related genes were detected by single-strand conformation polymorphism (SSCP) analysis⁽¹³¹⁾ of polymerase chain reaction (PCR) products labeled either with radioisotopes or by fluorescence. 1) Fluorescence-based SSCP analysis using blunt-end DNA fragments carrying HaeIII-polymorphic sites in intron 1 of the p53 gene are a potentially sensitive method of detecting loss of heterozygosity in clinical materials with a tumor cell fraction below 10%.⁽¹³²⁾ 2) The relationship between genetically determined individual predispositions and mutations of target genes in the early stage of lung carcinogenesis was investigated. Mutations of the p53 gene and germ line polymorphisms of the CYP1A1 gene detected by SSCP analysis were revealed to be associated with smoking-induced lung cancer susceptibility.⁽¹³³⁾ 3) Because the expression of a negative regulator of the cell cycle, p21^WAF1 protein, is trans-activated by p53, mutations of the p53 and WAF1 genes may be complementary. However, no mutations of the WAF1 gene were detected in 70 human primary lung and 24 pancreatic cancers, regardless of the mutational state of the p53 gene, suggesting no association of the WAF1 gene with the carcinogenetic process in these cancers.⁽¹³⁴⁾ 4) Reverse transcription (RT)-PCR with primers designed from highly homologous sequences shared by the two cDNAs and fluorescence-based SSCP analysis provided a method of quantitatively determinating the ratio of related mRNA molecules.⁽¹³⁵⁾

Detection of Aberrations in Uncharacterized Regions of the Genome

To detect aberrations in unchar-acterized regions of the genome, DNA was analyzed by several different approaches. 1) Detection of LOH revealed the presence of putative tumor suppressor genes. Three of these genes involved in the genesis of human glioma were suggested to be located at commonly deleted regions on chromosome 10, one on the short arm and two on the long arm.⁽¹³⁶⁾ 2) Restriction landmark genomic scanning (RLGS) analysis of DNA from the human pancreatic cancer cell line PANC1 detected several DNA fragments with abnormally intensified signals. All five major fragments cloned were mapped to the 19q13.1-13.2 region where the AKT2 oncogene is located. By Southern blotting using the cloned DNA fragments and a fragment of AKT2 cDNA as probes, amplification of the AKT2 gene was revealed in 3 of 12 pancreatic cancer cell lines including PANC1 and in 3 of 20 primary pancreatic cancers.⁽¹³⁷⁾ 3) Arbitrarily primed (AP)-PCR using a single primer could reproducibly and semiquantitatively amplify DNA fragments from approximately 30 regions of the genome. Fingerprints of normal and cancer DNAs from the same patient revealed gain or loss of copy numbers of chromosomal regions. For chromosome assignment of fingerprint bands, a convenient and powerful method of simultaneous hybridization of arbitrarily primed PCR products (SHARP) was developed. Radioactively labeled human AP-PCR products are hybridized to DNA fingerprints generated with the same arbitrary primer from human/rodent monochromosome cell hybrids after electroblotting to a nylon membrane.⁽¹³⁸⁾ 4) A particular arbitrary primer was found to amplify multiple DNA fragments containing repeated sequences, including the poly A tracts in the Alu repeats of the human genome. The combined use of the primer labeled with fluorescence and an automated DNA sequencing analysis of AP-PCR products provides a convenient and efficient method for detecting tumor specific changes in the number of repeating units.⁽¹³⁹⁾

Development of Technologies for Detecting DNA Aberrations

1) Segregation of partly melted molecules (SPM) developed in this division is a convenient and efficient method of detecting anonymous genes carrying CpG islands in uncharacterized DNA fragments. One of the DNA fragments detected by the SPM method was revealed to contain a putative promoter region of the prostacyclin synthase gene, suggesting that combined use of the SPM technique and the database match will allow precise characterization of the regulatory regions of many genes.⁽¹⁴⁰⁾ 2) A conventional method for directly sequencing DNA fragments obtained using single primers such as inter-Alu PCR, AP-PCR and RAPD was developed. This method involves isolation of single-stranded DNAs by electrophoretic separation of complementary DNA strands in a non-denaturing polyacrylamide gel and direct sequencing of the strands using the corresponding single primers.⁽¹⁴¹⁾

List of papers from this division

Table of Contents