Radiobiology Division

8. Radiobiology Division

The project that the Radiobiology Division is currently focusing on is the physical mapping of the human genome and the positional cloning of disease genes, especially of leukemia-related genes, and tumor suppressor genes. Several tumor-associated genes, including AML1, were successfully isolated by the genome-analysis based approach. Functional analysis of these gene products is also underway.
Genome Analysis and Positional Cloning of Leukemia-associated Genes

A complete NotI restriction map of the entire long arm of human chromosome 11 was constructed using linking-clone mapping. This physical map covers 77.6 Mb, from a pericentromeric NotI site to the terminus, and provides the most accurate ordering and distance estimation to date.⁽¹³³⁾ Several human disease genes have been localized to chromosome 11q13, but few have been cloned to date. A sequencing-ready contig map encompassing 11q13.1-13.3 was constructed using P1, BAC and PAC clones. The contig map spans a 3 Mb region from D11S480 to D11S913.⁽¹³⁴⁾ The map encompasses all the candidate loci for the BBS1 and SCA5 genes, one-third of the distal region for the hereditary paraganglioma 2 gene (PGL2), and one-third of the central region for insulin-dependent diabetes mellitus 4 locus (IDDM4). This contig map will facilitate identification of candidate genes by direct cDNA selection, as well as prediction of gene function from sequence information of these clones. The 2.5 Mb region between D21S17 and ERG is reportedly responsible for the main features of Down syndrome. A distal 1.6 Mb region within this 2.5 Mb region was previously focused on in an analysis of Japanese Down syndrome patients with partial trisomy 21. Nearly full-length cDNAs of four genes were isolated by screening several cDNA libraries and using RACE PCR. The gene distribution and direction of transcription were determined by mapping both ends of the cDNA sequences.⁽¹³⁵⁾
The inv(11)(p15q22) is a recurrent chromosomal abnormarily associated with de novo and therapy-related myeloid malignancies. Positional cloning of this chromosomal aberration using the NotI restriction map of 11q showed the consistent rearrangement of the DDX10 gene on chromosome 11q22, which encodes a putative RNA helicase. The translocation targets the NUP98 gene on 11p15, a member of the FG peptide repeat nucleoporin family. In DDX10 and NUP98, the inv(11) breakpoints occurred within two introns of each gene and the two genes merged in-frame to produce the chimeric transcripts characteristic of this translocation.^{(136, 137)} The 16;21 translocation results in the formation of the TLS/FUS-ERG fusion gene. The clinical chracteristics of 19 patients with t(16;21)-AML were analyzed. This indicated that the patients with t(16;21) are characterized by a relatively younger age (median age, 22 years old), involvement of various subtypes of French-American-British classification and a poor prognosis: 18 of the 19 patients died of the disease (median survival was 16 months).⁽¹³⁸⁾ Molecular analysis of cytogenetically identified t(8;12)(q22;q24) disclosed a masked type of the translocation, t(8;12;21) (p22.1;q24.1;q22.1) which contains the AML1-MTG8 fusion gene as t(8;21).⁽¹³⁹⁾ THE AML1-MTG8 fusion product is thought to play a critical role in the abnormal proliferation and differentiation of myeloid leukemia cells. The effects of various differentiation inducers of myeloid leukemia cells on growth and differentiation of Kasumi-1 and SKNO-1 cells, AML cell lines with t(8;21) were examined. This study showed that dexamethasone can induce apoptosis in these cells at low concentrations, whereas other myelomonocytic leukemia cell lines tested were resistant to glucocorticoid-induced apoptosis. The levels of glucocorticoid receptor gene expression were high in Kasumi-1 and SKNO-1 cells.⁽¹⁴⁰⁾ Glucocorticoids might induce the apoptosis of some types of AML cells, just like that of some lymphoid leukemia cells.
Mutation Induction by Extreme Dryness

When Bacillus subtilis spores are exposed to high vacuum (less than 10^-3 Pa) for several hours, the frequencies of mutations resistant to nalidixic acid or rifampicin increased several fold over the spontaneous rate of mutations. Sequence analysis of nalidixic acid-resistant mutations revealed a majority of the mutants belonged to one particular allele, gyrA12, carrying a tandem double base change from CA to TT.⁽¹⁴¹⁾ Thus, the exposure to extreme dryness is the most specific and unique mutagen for this organism.
Development and Elongation of Neurite-like Outgrowth on SCLC Cell Lines

The SCLC cell line Lu-134A differentiated morphologically into cells with remarkably long neuronal-like processes on the substrates (polyethelenimine or extracellular matrix of PC-9 cells).⁽¹⁴²⁾ This result shows the retention of neuroendocrine characteristics in SCLC cells and raises the possibility of differentiation therapy for SCLC.




	8. Radiobiology Division



		The project that the Radiobiology Division is currently focusing on is the physical mapping of the human genome and the positional cloning of disease genes, especially of leukemia-related genes, and tumor suppressor genes. Several tumor-associated genes, including AML1, were successfully isolated by the genome-analysis based approach. Functional analysis of these gene products is also underway. Genome Analysis and Positional Cloning of Leukemia-associated Genes A complete NotI restriction map of the entire long arm of human chromosome 11 was constructed using linking-clone mapping. This physical map covers 77.6 Mb, from a pericentromeric NotI site to the terminus, and provides the most accurate ordering and distance estimation to date.⁽¹³³⁾ Several human disease genes have been localized to chromosome 11q13, but few have been cloned to date. A sequencing-ready contig map encompassing 11q13.1-13.3 was constructed using P1, BAC and PAC clones. The contig map spans a 3 Mb region from D11S480 to D11S913.⁽¹³⁴⁾ The map encompasses all the candidate loci for the BBS1 and SCA5 genes, one-third of the distal region for the hereditary paraganglioma 2 gene (PGL2), and one-third of the central region for insulin-dependent diabetes mellitus 4 locus (IDDM4). This contig map will facilitate identification of candidate genes by direct cDNA selection, as well as prediction of gene function from sequence information of these clones. The 2.5 Mb region between D21S17 and ERG is reportedly responsible for the main features of Down syndrome. A distal 1.6 Mb region within this 2.5 Mb region was previously focused on in an analysis of Japanese Down syndrome patients with partial trisomy 21. Nearly full-length cDNAs of four genes were isolated by screening several cDNA libraries and using RACE PCR. The gene distribution and direction of transcription were determined by mapping both ends of the cDNA sequences.⁽¹³⁵⁾ The inv(11)(p15q22) is a recurrent chromosomal abnormarily associated with de novo and therapy-related myeloid malignancies. Positional cloning of this chromosomal aberration using the NotI restriction map of 11q showed the consistent rearrangement of the DDX10 gene on chromosome 11q22, which encodes a putative RNA helicase. The translocation targets the NUP98 gene on 11p15, a member of the FG peptide repeat nucleoporin family. In DDX10 and NUP98, the inv(11) breakpoints occurred within two introns of each gene and the two genes merged in-frame to produce the chimeric transcripts characteristic of this translocation.^{(136, 137)} The 16;21 translocation results in the formation of the TLS/FUS-ERG fusion gene. The clinical chracteristics of 19 patients with t(16;21)-AML were analyzed. This indicated that the patients with t(16;21) are characterized by a relatively younger age (median age, 22 years old), involvement of various subtypes of French-American-British classification and a poor prognosis: 18 of the 19 patients died of the disease (median survival was 16 months).⁽¹³⁸⁾ Molecular analysis of cytogenetically identified t(8;12)(q22;q24) disclosed a masked type of the translocation, t(8;12;21) (p22.1;q24.1;q22.1) which contains the AML1-MTG8 fusion gene as t(8;21).⁽¹³⁹⁾ THE AML1-MTG8 fusion product is thought to play a critical role in the abnormal proliferation and differentiation of myeloid leukemia cells. The effects of various differentiation inducers of myeloid leukemia cells on growth and differentiation of Kasumi-1 and SKNO-1 cells, AML cell lines with t(8;21) were examined. This study showed that dexamethasone can induce apoptosis in these cells at low concentrations, whereas other myelomonocytic leukemia cell lines tested were resistant to glucocorticoid-induced apoptosis. The levels of glucocorticoid receptor gene expression were high in Kasumi-1 and SKNO-1 cells.⁽¹⁴⁰⁾ Glucocorticoids might induce the apoptosis of some types of AML cells, just like that of some lymphoid leukemia cells. Mutation Induction by Extreme Dryness When Bacillus subtilis spores are exposed to high vacuum (less than 10^-3 Pa) for several hours, the frequencies of mutations resistant to nalidixic acid or rifampicin increased several fold over the spontaneous rate of mutations. Sequence analysis of nalidixic acid-resistant mutations revealed a majority of the mutants belonged to one particular allele, gyrA12, carrying a tandem double base change from CA to TT.⁽¹⁴¹⁾ Thus, the exposure to extreme dryness is the most specific and unique mutagen for this organism. Development and Elongation of Neurite-like Outgrowth on SCLC Cell Lines The SCLC cell line Lu-134A differentiated morphologically into cells with remarkably long neuronal-like processes on the substrates (polyethelenimine or extracellular matrix of PC-9 cells).⁽¹⁴²⁾ This result shows the retention of neuroendocrine characteristics in SCLC cells and raises the possibility of differentiation therapy for SCLC.