Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p2l. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One-and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangements is discussed.Several chromosomal mechanisms leading to the loss of function of putative tumor suppressor genes and the subsequent abnormal growth and proliferation of cancer cells have been described previously (16). Such mechanisms include point mutations, somatic crossing over, deletions and unbalanced translocations, and chromosome nondisjunction. The molecular cloning and characterization of several tumor suppressor genes, such as TP53 on chromosome 17pl3 (19, 43), RB1 on chromosome 13q14 (13), WTI on chromosome 11p13 (5), and APC on chromosome 5 (25), have greatly expanded our understanding of the role of tumor suppressor genes in the development of cancer. However, the molecular mechanisms underlying the interstitial deletions and unbalanced translocations associated with tumor suppressor genes have not been well studied.Recent data indicate that unbalanced translocations or interstitial deletions of the short arm of chromosome 9 [del(9p)] are recurring chromosomal abnormalities in a variety of tumor types, including acute lymphoblastic leukemia, glioma, melanoma, lung cancer, head and neck cancer, mesothelioma, ovarian cancer, and bladder cancer (1,2,7,8,23,28,29,35,40,42). Through molecular analysis, homozygous deletions of DNA sequences or losses of heterozygosity on 9p in a significant proportion of these tumors have been described (4, 10-12, 20, 24, 29, 31-33). Although the lengths and locations of these deletions vary, there is a common region of deletion at band 9p2l. This suggests the presence of a tumor suppressor gene in this region, whose inactivation contributes to the malignant process in all these different tumor types. The molecular studies with the different tumor types have demonstrated that the deletions involving 9p are sometimes interstitial and often include homozygous deletions of all or part of the interferon (IFN)...

Translocations involving chromosome band Ilq23, found in acute lymphoid and myeloid leukemias, disrupt the MLL gene. This gene encodes a putative transcription factor with regions of homology to several other proteins including the zinc fingers and other domains of the Drosophila trithorax gene product, and the "AT-hook" DNA-binding motif of high mobility group proteins. We have previously demonstrated that MLL contains transcriptional activation and repression domains using a GAL4 fusion protein system (21). The repression domain, which is capable of repressing transcription 3-5-fold, is located centromeric to the breakpoint region of MLL. The activation domain, located telomeric to the breakpoint region, activated transcription from a variety of promoters including ones containing only basal promoter elements. The level of activation was very high, ranging from IO-fold to more than 300-fold, depending on the promoter and cell line used for transient transfection.In translocations involving MLL, the protein produced from the der(ll) chromosome which contains the critical junction for leukemogenesis includes the AT-hook domain and the repression domain. We assessed the DNA binding capability of the MLL AT-hook domain using bacterially expressed and purified AT-hook protein. In a gel mobility shift assay, the MLL AT-hook domain could bind cruciform DNA, recognizing structure rather than sequence of the target DNA. This binding could be specifically competed with Hoechst 33258 dye and with distamycin. In a nitrocellulose protein-DNA binding assay, the MLL AT-hook domain could bind to AT-rich SARs, but not to non-SAR DNA fragments. The role that the AT-hook binding to DNA may play in vivo is unclear, but it is likely that DNA binding could affect downstream gene regulation. The AT-hook domain retained on the der(ll) would potentially recognize a different DNA target than the one normally recognized by the intact MLL protein. Furthermore, loss of an activation domain while retaining a repression domain on the der( 11) chromosome could alter the expression of various downstream target genes, suggesting potential mechanisms of action for MLL in leukemia.

By using a shuttle vector system developed in our laboratory, we have carried out studies on the molecular mechanism by which 5-bromodeoxyuridine (BrdUrd) induces mutations in mammalian cells. The target for mutagenesis in these studies was the Escherichia coli gpt gene that was contained within a retroviral shuttle vector and integrated into chromosomal DNA in mouse A9 cells. Shuttle vector-transformed cells expressing the gpt gene were mutagenized with BrdUrd and cells with mutations in the gpt gene were selected.Shuttle vector sequences were recovered from the mutant cells, and the base sequence of the mutant gpt genes was determined.The great majority of the BrdUrd-induced mutations involving single-base changes were found to be GC -> A-T transitions. We have shown that mutagenesis by BrdUrd depends upon perturbation of deoxycytidine metabolism. Thus, the current results suggest that BrdUrd mutagenesis involves mispairing and misincorporation of BrdUrd opposite guanine in DNA, driven by nucleotide pool perturbation caused by BrdUrd and the resulting imbalanced supply of triphosphates available for DNA synthesis. The results also revealed a very high degree of sequence specificity for the BrdUrd mutagenesis. BrdUrdinduced G-C -> APT transitions occurred almost exclusively in sequences with two adjacent guanine residues. Furthermore, in ,9O% of the cases, the guanine residue involved in mutation was the one in the more 3' position.Our laboratory has shown that mutagenesis by the thymidine (dThd) analog 5-bromodeoxyuridine (BrdUrd) transitions (4-6).We have studied the molecular mechanisms of BrdUrd mutagenesis in mammalian cells, by using a retroviral shuttle vector system developed in our laboratory (7). As the target for mutations, the shuttle vector contains the Escherichia coli gpt gene, coding for the enzyme xanthine guanine phosphoribosyltransferase (5-phospho-a-D-ribose-l-diphosphate: xanthine phosphoribosyltransferase; EC 2.4.2.22). The shuttle vector was introduced into mouse A9 cells and a transformed line (A9I-2) was isolated that contains a single copy of the vector integrated into chromosomal DNA. Since A9 cells are deficient in the enzyme hypoxanthine guanine phosphoribosyltransferase (IMP-pyrophosphate phosphoribosyl-transferase; EC 2.4.2.8) (8), transformants with mutations in the gpt gene can be selected with 6-thioguanine (sGua). For sequencing, the gpt genes from mutant A9I-2 cells can be recovered by fusion with monkey COS cells (9).In the present study, the gpt genes were recovered and sequenced from a large number of BrdUrd-induced mutants of A91-2 cells. We have used this shuttle vector system to analyze mutations that occurred spontaneously or were induced by ethyl methanesulfonate (EtMes) (10, 11). Mutational studies with various shuttle vector systems also have been carried out by other laboratories (12-17). Our system differs from those used in the other studies in that the vector in our system is integrated into chromosomal DNA and clonal selection for cells with mutant ge...

A major unresolved question for 11q23 translocations involving MLL is the chromosomal mechanism(s) leading to these translocations. We have mapped breakpoints within the 8.3-kb BamHI breakpoint cluster region in 31 patients with acute lymphoblastic leukemia and acute myeloid leukemia (AML) de novo and in 8 t-AML patients. In 23 of 31 leukemia de novo patients, MLL breakpoints mapped to the centromeric half (4.57 kb) of the breakpoint cluster region, whereas those in eight de novo patients mapped to the telomeric half (3.87 kb). In contrast, only two t-AML breakpoints mapped in the centromeric half, whereas six mapped in the telomeric half. The difference in distribution of the leukemia de novo breakpoints is statistically significant (P = .02). A similar difference in distribution of breakpoints between de novo patients and t-AML patients has been reported by others. We identified a low- or weak-affinity scaffold attachment region (SAR) mapping just centromeric to the breakpoint cluster region, and a high-affinity SAR mapping within the telomeric half of the breakpoint cluster region. Using high stringency criteria to define in vitro vertebrate topoisomerase II (topo II) consensus sites, one topo II site mapped adjacent to the telomeric SAR, whereas six mapped within the SAR. Therefore, 74% of leukemia de novo and 25% of t-AML breakpoints map to the centromeric half of the breakpoint cluster region map between the two SARs; in contrast, 26% of the leukemia de novo and 75% of the t-AML patient breakpoints map to the telomeric half of the breakpoint cluster region that contains both the telomeric SAR and the topo II sites. Thus, the chromatin structure of the MLL breakpoint cluster region may be important in determining the distribution of the breakpoints. The data suggest that the mechanism(s) leading to translocations may differ in leukemia de novo and in t-AML.

Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.