A putative tumor suppressor locus on the short arm of human chromosome 9 has been localized to a region of less than 40 kilobases by means of homozygous deletions in melanoma cell lines. This region contained a gene, Multiple Tumor Suppressor 1 (MTS1), that encodes a previously identified inhibitor (p16) of cyclin-dependent kinase 4. MTS1 was homozygously deleted at high frequency in cell lines derived from tumors of lung, breast, brain, bone, skin, bladder, kidney, ovary, and lymphocyte. Melanoma cell lines that carried at least one copy of MTS1 frequently carried nonsense, missense, or frameshift mutations in the gene. These findings suggest that MTS1 mutations are involved in tumor formation in a wide range of tissues.
The radiosensitive rodent mutant cell line xrs-5 is defective in DNA double-strand break repair and lacks the Ku component of the DNA-activated protein kinase, DNA-PK. Here radiosensitive human cell lines were analyzed for DNA-PK activity and for the presence of related proteins. The radiosensitive human malignant glioma M059J cell line was found to be defective in DNA double-strand break repair, but fails to express the p350 subunit of DNA-PK. These results suggest that DNA-PK kinase activity is involved in DNA double-strand break repair.
We have identified a group of 8 (among 39) human tumour cell strains deficient in the ability to support the growth of adenovirus 5 preparations treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but able to support the growth of non-treated adenovirus normally. This deficient behaviour defines the Mer- phenotype. Strains having the Mer- phenotype were found to arise from tumours originating in four different organs. Relative to Mer+ strains, Mer- tumour strains showed greater sensitivity to MNNG-produced killing, greater MNNG-stimulated "DNA repair synthesis and a more rapid MNNG-produced decrease in semi-conservative DNA synthesis. Here we report that (1) Mer- strains are deficient in removing O6-methylguanine (O6-MeG) from their DNA after [Me-14C]MMNG treatment (Table 1); (2) Mer- tumour strains originate from tumours arising in patients having Mer+ normal fibroblasts (Fig. 1a, b); (3) SV40 transformation of (Mer+) human fibroblasts often converts them to Mer- strains (Fig. 1c, d); (4) MNNG produces more sister chromatid exchanges (SCEs) in Mer- than in Mer+ cell strains (Fig. 2).
Two aneuploid cell lines which differ in their inherent sensitivity to ionizing radiation and chemotherapeutic agents were established concurrently from a single tumor specimen obtained from a patient with glioblastoma. M059J cells are approximately 30-fold more sensitive to radiation than are M059K cells (surviving fractions at 2 Gy were 0.02 and 0.64, respectively). This relative difference in radiation sensitivity has remained a stable feature of the cell lines during 2 years in continuous culture. In addition, cells of the M059J line are more sensitive than those of the M059K line to the cytotoxic effects of bleomycin, N,N-bis(2-chloroethyl)-N-nitrosourea, and nitrogen mustard. These cell lines may prove to provide a useful model system for evaluating the cellular and molecular processes which confer resistance or sensitivity in cancer treatment.
The biological consequences of O 6 -methylguanine (m6G) in DNA are well recognized. When template m6G is encountered by DNA polymerases, replication is hindered and trans-lesion replication results in the preferential incorporation of dTMP opposite template m6G. Thus, unrepaired m6G in DNA is both cytotoxic and mutagenic. Yet, cell lines tolerant to m6G in DNA have been isolated, which indicates that some cellular DNA polymerases may replicate m6G-containing DNA with reasonable efficiency. Previous reports suggested that mammalian pol  could not replicate m6G-containing DNA, but we find that pol  can catalyze trans-lesion replication; however, the lesion must reside in the optimal context for pol  activity, single-or short nucleotide gapped substrates. Primed single-stranded DNA templates, with or without template m6G, were poor substrates for pol  as reported in earlier studies. In contrast, trans-lesion replication by bacteriophage T4 DNA polymerase was observed for primed single-stranded DNA templates. Replication of m6G-containing DNA by T4 DNA polymerase required the gp45 accessory protein that clamps the polymerase to the DNA template. The rate-limiting step in replicating m6G-containing DNAs by both DNA polymerases tested was incorporation of dTMP across from the lesion. -DNA methyltransferase repairs m6G residues in DNA; however, 20 -30% of human solid tumor cell lines do not express this repair activity (1). Exposure of cells lacking m6G-DNA methyltransferase to alkylating agents such as MNNG results in high levels of mutations, sister chromatid exchanges, and cell death (reviewed in Ref. 2). Yet, cells unable to repair the m6G damage but tolerant to the killing activities of MNNG have been isolated (reviewed in Refs. 2, 3). As these tolerant cells remain sensitive to the mutagenic effects of alkylating agents, questions about the replication of m6G-containing DNA arise.DNA polymerases are predicted to encounter m6G in three DNA environments: 1) in single-or short nucleotide gaps formed during short patch DNA repair, 2) in lengthy singlestranded regions formed by long patch repair, and 3) at replication forks. Single-nucleotide gaps may be produced by short patch mismatch repair activity that is normally directed to the repair of G:T mispairs that are the result of 5-methylcytosine (5mC) deamination (4). The enzyme that initiates this repair process is a DNA G:T mismatch-specific thymine-DNA glycosylase that removes the mispaired thymine from G:T DNA to generate an apyrimidinic site (5). A G:T thymine-DNA glycosylase also initiates the removal of thymine from m6G:T base pairs (6). The abasic site is further processed to generate a single-nucleotide gap across from guanine for G:T mismatches and across from m6G for m6G:T mismatches. While pol  can efficiently fill in single-nucleotide and small gaps across from undamaged DNA templates (7), pol  activity on single-nucleotide gaps across from m6G has not been reported. Pol  replication is blocked, however, by template m6G in long singlestranded DNA ...
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