Tsutomu Nobori

The methylthioadenosine phosphorylase gene is frequently co-deleted with the p16INK4a gene in acute type adult T-cell leukemia.

Adult T‐cell leukemia (ATL) is a retrovirus‐associated leukemia with poor prognosis and often has deletions of the p16INK4a and p15INK4b genes on chromosome 9p21. The gene for methylthioadenosine phosphorylase (MTAP), a purine and methionine metabolic enzyme, resides approximately 100 Kb telomeric to the p16INK4a gene and is frequently co‐deleted with the tumor suppressor gene in a variety of cancers. This enzyme deficiency can be exploited for selective chemotherapy with de novo purine synthesis inhibitors and/or methionine depletion. To determine whether ATL can be a candidate for selective chemotherapy based on genetic alterations on chromosome 9p21, we analyzed the MTAP gene in 41 samples from ATL patients (27 acute type and 14 chronic type ATL) and 3 cell lines established from ATL patients. Five samples from the acute type had deletions of the MTAP gene (4 total deletions and 1 partial deletion of exons 6–8). The MTAP gene was always co‐deleted with p16INK4a. No deletion of the MTAP gene was detected in samples from the chronic type. Of 3 cell lines, 2 showed partial deletions of exons 5–8 of the MTAP gene, and 1 lost all exons. The p16INK4a gene was deleted in all cell lines. In conclusion, deletions of the MTAP gene were found in 5 of 27 acute type ATL samples. Acute type ATL with MTAP deficiency can be a good candidate for selective chemotherapy by depleting purines and/or methionine. Int. J. Cancer 75:51–56, 1998.© 1998 Wiley‐Liss, Inc.

Detection of p16 gene deletions in gliomas: A comparison of fluorescence in situ hybridization (FISH) versus quantitative PCR

The p16 protein plays a key role in cell cycle control by preventing CDK4 from inactivating the retinoblastoma protein (pRb). The corresponding tumor suppressor gene (p16/MTS1/CDKN2) has recently been implicated in malignant progression of astrocytomas and could potentially serve as an important marker for patient prognosis and for guiding specific therapeutic strategies. We have undertaken a study to evaluate 2 methods of detecting p16 deletion. Thirty diffuse gliomas were analyzed for p16 gene dosage. Dual color fluorescence in situ hybridization (FISH) was performed on cytologic preparations using paired centromeric (CEN) and locus-specific probes for CEN9/p16. CEN8/RB, and CEN12/CDK4. Quantitative PCR was performed using primers for p16, MTAP, and reference genes. Eleven cases were also studied using comparative genomic hybridization (CGH). Abnormalities of the p16-CDK4-RB pathway were identified in 21 (70%) cases by FISH and/or PCR. These included 15 (50%) with p16 deletion, 9 of which were detected by both techniques, 3 by FISH alone, and 3 by PCR alone (concordance rate = 81%). FISH analysis further revealed tetraploidy/aneuploidy in 14 (47%), RB deletion in 11 (37%), and CDK4 amplification in 1 (3.3%). There were 94% and 100% concordance rates between CGH and FISH or PCR, respectively. Quantitative PCR was noninformative in 4 cases. Although FISH and quantitative PCR are both reliable techniques, each has limitations. PCR is likely to miss p16 deletions when there is significant normal cell contamination or clonal heterogeneity, whereas the p16 YAC probe used for FISH analysis may miss small deletions. Replacement of the latter with a cosmid probe may improve the sensitivity of FISH in future experiments.

Chromosome 9 related aberrations and deletions of the CDKN2 and MTS2 putative tumor suppressor genes in human chondrosareomas

Deletions on the short arm of chromosome 9 (9p21 region) have been reported in a number of hematopoietic and solid tumors. These aberrations on 9p have been previously associated with the loss of the interferon gene cluster and the gene for methylthioadenosine phosphorylase (MTAP), localized to the 9p21-22 region. Recently, two putative tumor suppressor gene(s) CDKN2 and MTS2 have been mapped to the 9p21 region, and shown to be deleted in a large number of tumors including leukemias, melanomas, bladder cancers and brain tumors. We have previously reported a similar 9p21 abnormality and deletions of the CDKN2 and MTS2 genes in a myxoid chondrosarcoma cell line and its subclones. In this study we report consistent abnormalities of chromosome 9 in additional chondrosarcomas examined by a detailed cytogenetic and molecular analysis. Seven chondrosarcoma cell lines, one primary chondrosarcoma, and a benign chondroma were examined. Four of the seven tumor cell lines examined showed grossly visible aberrations of chromosome 9. Molecular analysis of these chondrosarcoma cell lines revealed hemizygous deletions of the interferon genes, and the absence of the MTAP gene, protein or activity. In addition, four of the seven chondrosarcoma cell lines also showed deletions of the CDKN2 and/or MTS2 putative tumor suppressor genes, or the absence of the CDKN2 protein product. No such chromosome 9 related aberrations were detected in the benign chondroma. These data suggest that chromosome 9p21 abnormality, and deletions of the CDKN2 and MTS2 tumor suppressor genes may be a significant event in the development of chondrosarcomas.

Genomic organization and chromosomal localization of the human casein gene family

Abstract Five yeast artificial chromosome (YAC) clones containing the human casein gene family were isolated and characterized to study the control mechanisms for the expression of these genes. Partial restriction analysis in conjunction with the chromosomal fragmentation method and fluorescence in situ hybridization (FISH) analysis were performed to construct a detailed physical map of the casein gene family and to determine the chromosomal localization of these genes. The isolated YAC clones 748F3, 750D11, 882G11, 886B3 and 960D2 were 1.2 Mb, 860 kb, 800 kb 1.5 Mb and 1.5 Mb in size, respectively. The clones 748F3, 882G11, 886B3 and 960D2 contained the entire casein gene family, while the κ-casein gene was absent in 750D11. The human αS1-, β- and κ-casein genes were found to be closely linked and arranged in the order αS1-β-κ. The distance between αS1 and β, and between αS1 and κ was approximately 10 and 300 kb, respectively. The β-casein gene was oriented in the opposite direction to the αS1- and κ-casein genes. The casein gene family was localized to chromosome 4q21.1 by FISH analysis.

Partial deletions of the CDKN2 and MTS2 putative tumor suppressor genes in a myxoid chondrosarcoma.

Cytogenetic abnormalities of chromosome 9 (9p21) have been reported in a large number of tumors that include malignant melanomas, gliomas, lung cancers and leukemias. These aberrations on 9p have been previously shown to involve the loss of the interferon gene cluster and the gene for methylthioadenosine phosphorylase (MTAP), both of which have been mapped to the 9p21 region. Recently, two putative tumor suppressor gene(s) CDKN2 and MTS2, have been mapped to the 9p21 region, and have been shown to be deleted in a large number of hematopoietic and solid malignancies. In this study we report a cytogenetic and a detailed molecular analysis of a myxoid chondrosarcoma cell line 105KC and its clonal derivatives 105AJ, 105AJ1.1, 105AJ3.1, and 105AJ5.1. Specifically, we have demonstrated chromosome 9p21 related abnormalities by cytogenetic analysis, the associated loss of the interferon gene cluster, and the loss of the immunoreactive MTAP protein and activity. In addition, we have also shown the presence of deletions involving the CDKN2 and the MTS2 putative tumor suppressor genes in these chondrosarcoma cell lines. The above studies were extended to other chondrosarcoma cell lines and primary tumors, where similar deletions of the CDKN2 and MTS2 genes were found to be present (unpublished data). This suggests a potential role for the involvement of the CDKN2 and MTS2 putative tumor suppressor genes in the development of chondrosarcomas.

A polymerase chain reaction-based method for isolation of gene-specific sequences from the interferon-α gene cluster

Abstract The interferon-α gene is a gene family of over 20 distinct genes having 80–95% homology with one another at a nucleotide level. Because of the high homology in the gene cluster, the available interferon-α gene probes can hybridize to multiple bands of different size on Southern blot analysis of restricted human genomic DNA. We used the polymerase chain reaction with the primers synthesized from Alu repetitive sequence and the conserved sequences of the interferon-α gene cluster to generate specific probes for individual interferon-α genes. The amplification products were subcloned into a plasmid vector and analyzed by DNA sequencing and Southern blotting of the restricted human placental DNA. One clone, which derived from interferon-α14 gene, produced a single 5.2-kb band in Southern blots of the HindIII-restricted human placental DNA. This stands in contrast to the 10 bands of different size that were detected with a cDNA for the interferon-αI′ gene. Our results indicate that a polymerase chain reaction-based method can be used to isolate gene-specific sequences from the interferon-α gene cluster. Since a variety of human cancers has been found to have the complete or partial deletion of the interferon-α gene cluster, the gene-specific probe generated by this method may aid in determining the breakpoints in the vicinity of the gene cluster.

Molecular Genetic Analysis of Chromosome 9p in Methylthioadenosine Phosphorylase Deficient Glioma Cell Lines

Methylthioadenosine (MTA) phosphorylase is the enzyme involved in the metabolism of polyamines and purines.1 MTA, the substrate for this enzyme, is produced during the synthesis of spermine and spermidine and is cleaved to methylthioribose 1-phosphate and adenine, which are recycled to methionine and adenine nucleotide, respectively.1 This enzyme has been known to be deficient in human leukemias, lymphomas, and solid tumors.2,3,4 The gene locus for this enzyme (designated MTAP) has been mapped to human chromosome 9p.5 Enzyme deficient malignant cell lines are frequently found to have chromosome 9p abnormalities.6 Although cytogenetic analysis of human gliomas has demonstrated a very prevalent chromosomal abnormality involving deletions or translocations of chromosome 9p,7 these tumors have not been tested for MTAP deficiency. In this report, we screened eight human glioma cell lines and six primary brain tumors for MTA phosphorylase activities using the radiochemical method and immunoblot analysis. Of 14 cell lines and primary tumors, five cell lines and five primary tumors (70%) were found to be enzyme-deficient. To elucidate the molecular mechanism of this enzyme deficiency and its relevance to the genesis of human brain tumors, DNA analysis and pulsed field gel analysis were carried out.