GENETICS DIVISION

12.GENETICS DIVISION

    The research goals of the Genetics Division include elucidation of the molecular basis of cell transformation and carcinogenesis and the application of this basic information to the development of new strategies for cancer diagnosis, treatment and prevention. Gene amplification is a form of genomic instability, and detailed structural analysis in the Division revealed that amplification is often accompanied by other gross chromosomal abnormalities such as deletion and translocation. Genes encoding WNT signaling molecules, implicated in carcinogenesis and embryonic development, are potent cancer-associated genes. The Division is attempting to characterize the still enigmatic angioblast or endothelial precursor cells implicated in tumor vasculogenesis.
Gene amplification in human cancer

    Two new amplified genes, CAB2/ hCOS16 and Gasdermin, and on the 17q12 locus containing the c-ERBB2 gene were isolated.⁽¹⁶⁸⁾ By cloning the rearranged DNAs containing recombination sites between each amplification unit on the 17q12 locus, a site-specific end-joining between each amplification unit was found to occur in a head-to-tail manner. Furthermore, two amplification units containing the translocation junctions on marker chromosomes were identified, indicating the involvement of a translocation-mediated gene amplification mechanism in human cancer. The presence of a recombination hot spot was suggested, because end-joining between each amplification unit in two cases, interstitial deletion of GRB7 in two cases and translocation in two cases all occurred in a 13 kb DNA fragment between c-ERBB-2 and GRB7. By structural analysis of other amplicons at 11q13 and 10q26, specific DNA sequences which may play key roles in gene amplification were identified and shown to be present in the limited genomic DNA region.
Establishment of Efficient Methods for RNA and DNA Amplification

    Only a limited amount of RNA and DNA can be obtained from surgical specimens of cancer tissues, especially from biopsy or microdissected samples. An effective method of amplifying of a minute amount of RNA was developed to analyze expression profiles by cDNA microarray based on a T7 RNA polymerase-mediated RNA amplification reaction combined with an adaptor ligation-mediated PCR reaction. Whole genome amplification (WGA) of a small amount of DNA is also required for detection of SNPs and mutational analysis of a large number of target genes. A quite efficient method of achieving unbiased-WGA from both blood DNAs and clinical samples such as paraffin-embedded tissues was developed.
WNT Signaling Pathway Implicated in Carcinogenesis

    WNT-2B/WNT-13 was previously identified in the Division. A novel WNT-2B isoform (WNT-2B2), in addition to the original WNT-2B isoform (WNT- 2B1), was cloned.⁽¹⁶⁹⁾ This is the first report on WNT isoforms derived from the same gene due to alternative splicing. Among the genes encoding the WNT receptors, human FZD1, FZD2, FZD4, FZD7, and FZD10 were cloned by the Division. This year, the human FZD3 and Xenopus homologue of FZD10 (Xfz10) were cloned, and the function of the latter will be investigated using the RNA interference method.^(170,¹⁷¹⁾ The bTRCP2 gene, encoding a negative regulator of the WNT signaling pathway was cloned and mapped.⁽¹⁷²⁾ TCF-3 encoding a transcription factor implicated in the WNT signaling pathway was also cloned. TCF-3 was occasionally up-regulated in primary gastric cancer, and MKN28 transformants overexpressing TCF-3 showed approximately eight-fold resistance to mitomycin C.⁽¹⁷³⁾ In addition to the WNT signaling molecules, a novel member of the FGF gene family, FGF-20, was identified. The FGF-20 mRNA was detected only in SW480 cells (colorectal cancer), using northern blot analyses.⁽¹⁷⁴⁾ Synergism of FGF-20 and WNT signaling molecules will be investigated in the future.
Flt-1 promoter as a possible marker for endothelial precursor cells

    As a forerunner to identifying factors required for endothelial and hematopoietic differentiation and their mechanisms of programming lineage commitment, the precursor cells must be isolated. The Division developed a strategy for identifying these precursor cells by labeling them with EGFP expressed by the mouse flt-1 gene promoter in the context of hematopoietic and endothelial differentiation using the embryoid body (EB) model of mouse embryogenesis.⁽¹⁷⁵⁾
Other genes implicated in carcinogenesis

    Two related transcriptional factors, Klf4 and Klf5, implicated in gastrointestinal epithelial development, were compared for developmental expression.⁽¹⁷⁶⁾




12.GENETICS DIVISION



	The research goals of the Genetics Division include elucidation of the molecular basis of cell transformation and carcinogenesis and the application of this basic information to the development of new strategies for cancer diagnosis, treatment and prevention. Gene amplification is a form of genomic instability, and detailed structural analysis in the Division revealed that amplification is often accompanied by other gross chromosomal abnormalities such as deletion and translocation. Genes encoding WNT signaling molecules, implicated in carcinogenesis and embryonic development, are potent cancer-associated genes. The Division is attempting to characterize the still enigmatic angioblast or endothelial precursor cells implicated in tumor vasculogenesis. Gene amplification in human cancer Two new amplified genes, CAB2/ hCOS16 and Gasdermin, and on the 17q12 locus containing the c-ERBB2 gene were isolated.⁽¹⁶⁸⁾ By cloning the rearranged DNAs containing recombination sites between each amplification unit on the 17q12 locus, a site-specific end-joining between each amplification unit was found to occur in a head-to-tail manner. Furthermore, two amplification units containing the translocation junctions on marker chromosomes were identified, indicating the involvement of a translocation-mediated gene amplification mechanism in human cancer. The presence of a recombination hot spot was suggested, because end-joining between each amplification unit in two cases, interstitial deletion of GRB7 in two cases and translocation in two cases all occurred in a 13 kb DNA fragment between c-ERBB-2 and GRB7. By structural analysis of other amplicons at 11q13 and 10q26, specific DNA sequences which may play key roles in gene amplification were identified and shown to be present in the limited genomic DNA region. Establishment of Efficient Methods for RNA and DNA Amplification Only a limited amount of RNA and DNA can be obtained from surgical specimens of cancer tissues, especially from biopsy or microdissected samples. An effective method of amplifying of a minute amount of RNA was developed to analyze expression profiles by cDNA microarray based on a T7 RNA polymerase-mediated RNA amplification reaction combined with an adaptor ligation-mediated PCR reaction. Whole genome amplification (WGA) of a small amount of DNA is also required for detection of SNPs and mutational analysis of a large number of target genes. A quite efficient method of achieving unbiased-WGA from both blood DNAs and clinical samples such as paraffin-embedded tissues was developed. WNT Signaling Pathway Implicated in Carcinogenesis WNT-2B/WNT-13 was previously identified in the Division. A novel WNT-2B isoform (WNT-2B2), in addition to the original WNT-2B isoform (WNT- 2B1), was cloned.⁽¹⁶⁹⁾ This is the first report on WNT isoforms derived from the same gene due to alternative splicing. Among the genes encoding the WNT receptors, human FZD1, FZD2, FZD4, FZD7, and FZD10 were cloned by the Division. This year, the human FZD3 and Xenopus homologue of FZD10 (Xfz10) were cloned, and the function of the latter will be investigated using the RNA interference method.^(170,¹⁷¹⁾ The bTRCP2 gene, encoding a negative regulator of the WNT signaling pathway was cloned and mapped.⁽¹⁷²⁾ TCF-3 encoding a transcription factor implicated in the WNT signaling pathway was also cloned. TCF-3 was occasionally up-regulated in primary gastric cancer, and MKN28 transformants overexpressing TCF-3 showed approximately eight-fold resistance to mitomycin C.⁽¹⁷³⁾ In addition to the WNT signaling molecules, a novel member of the FGF gene family, FGF-20, was identified. The FGF-20 mRNA was detected only in SW480 cells (colorectal cancer), using northern blot analyses.⁽¹⁷⁴⁾ Synergism of FGF-20 and WNT signaling molecules will be investigated in the future. Flt-1 promoter as a possible marker for endothelial precursor cells As a forerunner to identifying factors required for endothelial and hematopoietic differentiation and their mechanisms of programming lineage commitment, the precursor cells must be isolated. The Division developed a strategy for identifying these precursor cells by labeling them with EGFP expressed by the mouse flt-1 gene promoter in the context of hematopoietic and endothelial differentiation using the embryoid body (EB) model of mouse embryogenesis.⁽¹⁷⁵⁾ Other genes implicated in carcinogenesis Two related transcriptional factors, Klf4 and Klf5, implicated in gastrointestinal epithelial development, were compared for developmental expression.⁽¹⁷⁶⁾