Relative rates of insertion and deletion mutations in dinucleotide repeats of various lengths in mismatch repair proficient mouse and mismatch repair deficient human cells

Microsatellites are DNA elements composed of short tandem repeats of 1-5bp. These sequences are particularly prone to frameshift mutation by insertion-deletion loop formation during replication. The mismatch repair system is responsible for correcting these replication errors, and microsatellite mutation rates are significantly elevated in the absence of mismatch repair. We have investigated the effect of varying the number of repeats in a (CA)n microsatellite on mutation rates in cultured mammalian cells proficient or deficient in mismatch repair. We have also compared the relative rates of single-repeat insertions and deletions in these cells. Two plasmid vectors were constructed for each repeat unit number (n=8, 17, and 30), such that the microsatellites, placed upstream of a bacterial neomycin resistance gene (neo), disrupted the reading frame of the gene in the (-1) or (+1) direction. Plasmids were introduced separately into the cells, where they integrated into the cellular genome. Mutation rates were determined by selection of clones with frameshift mutations in the microsatellite that restored the reading frame of the neo gene. We found that mutation rates were significantly higher for (CA)17 and (CA)30 tracts than for (CA)8 tracts in both mismatch repair proficient (mouse) and deficient (human) cells. A mutational bias favoring insertions was generally observed. In both (CA)17 and (CA)30 tracts, single-repeat insertion rates were higher than single-repeat deletion rates with or without mismatch repair; deletions of multiple repeat units (> or =8bp) were observed in these tracts, where as deletions this large were not found in the (CA)8 tract. Single-repeat mutations of both types were made at similar rates in (CA)8 tracts in human mismatch repair deficient (MMR-) cells, but single-repeat insertion rates were higher than single-repeat deletion rates in mouse mismatch repair proficient (MMR+) cells. Results of these direct studies on microsatellite mutations in cultured cells should be useful for refinement of mathematical models for microsatellite evolution.     

Expression of HLA-DR antigen in human class II mutant B-cell lines by double infection with retrovirus vectors.

A new retrovirus vector containing the gene for hygromycin B resistance (hyg) as a selectable marker under the control of an internal simian virus 40 promoter was constructed. It was used, together with an analogous previously described vector, DO1, which contains the gene for G418 resistance, to introduce and express the genes for the two chains of a human class II major histocompatibility complex antigen in NIH 3T3 cells. In addition, these vectors were used to express DR antigens in two human mutant B-lymphoblastoid cell lines, one of which was deleted for both alleles of the DR alpha gene and the other of which expressed no class II antigens because of a genetic defect in a putative trans-acting regulatory factor.     

Escherichia coli DNA polymerase IV contributes to spontaneous mutagenesis at coding sequences but not microsatellite alleles

Slipped strand mispairing during DNA synthesis is one proposed mechanism for microsatellite or short tandem repeat (STR) mutation. However, the DNA polymerase(s) responsible for STR mutagenesis have not been determined. In this study, we investigated the effect of the Escherichia colidinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability, using an HSV-tk gene episomal reporter system for microsatellite mutations. For the control vector (HSV-tk gene only) we observed a statistically significant 3.5-fold lower median mutation frequency in dinB(-) than dinB(+) cells (p<0.001, Wilcoxon Mann Whitney Test). For vectors containing an in-frame mononucleotide allele ([G/C](10)) or either of two dinucleotide alleles ([GT/CA](10) and [TC/AG](11)) we observed no statistically significant difference in the overall HSV-tk mutation frequency observed between dinB(+) and dinB(-) strains. To determine if a mutational bias exists for mutations made by Pol IV, mutational spectra were generated for each STR vector and strain. No statistically significant differences between strains were observed for either the proportion of mutational events at the STR or STR specificity among the three vectors. However, the specificity of mutational events at the STR alleles in each strain varied in a statistically significant manner as a consequence of microsatellite sequence. Our results indicate that while Pol IV contributes to spontaneous mutations within the HSV-tk coding sequence, Pol IV does not play a significant role in spontaneous mutagenesis at [G/C](10), [GT/CA](10), or [TC/AG](11) microsatellite alleles. Our data demonstrate that in a wild type genetic background, the major factor influencing microsatellite mutagenesis is the allelic sequence composition.     

Sequence-dependent effect of interruptions on microsatellite mutation rate in mismatch repair-deficient human cells

Although microsatellite mutation rates generally increase with increasing length of the repeat tract, interruptions in a microsatellite may stabilize it. We have performed a direct analysis of the effect of microsatellite interruptions on mutation rate and spectrum in cultured mammalian cells. Two mononucleotide sequences (G₁₇ and A₁₇) and a dinucleotide [(CA)₁₇] were compared with interrupted repeats of the same size and with sequences of 8 repeat units. MMR-deficient (MMR⁻) cells were used for these studies to eliminate effects of this repair process. Mutation rates were determined by fluctuation analysis on cells containing a microsatellite sequence at the 5′ end of an antibiotic-resistance gene; the vector carrying this sequence was integrated in the genome of the cells. In general, interrupted sequences had lower mutation rates than perfect ones of the same size, but the magnitude of the difference was dependent upon the sequence of the interrupting base(s). Some interrupted repeats had mutation rates that were lower than those of perfect sequences of the same length but similar to those of half the length. This suggests that interrupting bases effectively divide microsatellites into smaller repeat runs with mutational characteristics different from those of the corresponding full-length microsatellite. We conclude that interruptions decrease microsatellite mutation rate and influence the spectrum of frameshift mutations. The sequence of the interrupting base(s) determines the magnitude of the effect on mutation rate.     

Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.     

A host-vector system for molecular study of the intracellular growth of Mycobacterium tuberculosis in phagocytic cells

The mechanisms by which Mycobacterium tuberculosis survives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host-vector system is indispensable. In this study it has been shown that, in contrast with M . smegmatis strain mc²155 which has been widely used for molecular analysis, M. smegmatis strain J15cs is able to survive even at day 6 post-infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from an Escherichia coli - mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp-, hyg- and km-resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence on in vitro growth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria-Bertani broth and at day 5 post-infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes of M. tuberculosis and intracellular growth was demonstrated.
In this study, M. smegmatis strain J15cs and pYT923hyg were found to be capable of serving as an appropriate host-vector system for molecular study of the intracellular growth of M . tuberculosis in phagocytic cells; this system may be useful as a screening tool for M . tuberculosis genes.     

The human homolog of the rodent immediate early response genes, PC4 and TIS7, resides in the lung cancer tumor suppressor gene region on chromosome 3p21

Recently, human chromosome band 3p21.3 was shown to undergo overlapping homozygous deletions in several small cell lung cancer lines further defining a putative tumor suppressor gene(s) region. We report the cloning and mutational analysis of a novel human gene, SKMc15, from the commonly homozygously deleted region in three small cell lung cancer lines (NCI-H1450, NCI-H740, GLC20). It has 11 exons ranging in size from 50 to 541 bp with an open reading frame of 442 amino acids. The gene covers 7 to 10 kb of genomic DNA; the message of 1.8 to 2 kb is expressed in all analyzed fetal and adult human and mouse tissues including heart, brain, placenta, lung liver, skeletal muscle, kidney, testis and pancreas and in small cell and non-small cell cancer lines. The intron/exon boundaries were used to analyze the gene for mutations by exon PCR-SSCP sequencing in 60 small cell lung cancer cell lines. No loss-of-function mutations were detected. The cDNA sequence has high homology, 75% at the protein level, to the rat early response gene PC4 and its murine homolog TIS7. In addition, the known partial sequence of the putative mouse interferon β2 (64 amino acids) gene is highly conserved in PC4/TIS7 (94%) and in SKMc15 (83%) at the amino acid level. The sequence TAAAT, which is thought to be involved in mRNA degradation, is present in the 3′ UTR of SKMc15 and in the 3′ UTR of PC4 and TIS7 genes. Received: 28 August 1996 / Revised: 18 October 1996     

Establishment and characterization of four human pancreatic carcinoma cell lines. Genetic alterations in the TGFBR2 gene but not in the MADH4 gene

We characterized four pancreatic carcinoma cell lines (designated SNU-213, SNU-324, SNU-410, and SNU-494) established from histopathologically varied primary or liver metastatic tumor samples of Korean patients. Three cell lines grew as adherent monolayers and one as adherent and floating cell clumps. All lines had: (1) relatively high viability; (2) an absence of mycoplasma or bacterial contamination; (3) genetic heterogeneity as assessed by DNA-fingerprinting analysis; (4) an absence of MADH4 mutation. Among the lines, three lines had mutations in codon 12 in K- ras, two lines harbored p53 mutations within the DNA-binding domain; two lines had homozygous deletions in both p16 and p15 genes; and one line had a missense mutation. Two lines (SNU-324 and SNU-410) had genetic alterations in the TGFBR2 gene: the SNU-324 line had a -1-bp or +1-bp mutation in 10-bp polydeoxyadenine repeat tracts; the SNU-410 line had a genomic deletion in this gene. Mutation analysis of mismatch repair genes demonstrated that SNU-324 has two heterozygous missense mutations in different exons of the hMLH1 gene. In addition, this line showed microsatellite instability and harbored frameshift mutations in simple repeated sequences of the coding regions of the TGFBR2, BAX, and hMSH3 genes. These defects of microsatellite instability and mismatch repair genes suggest the possibility of a new mutator phenotype for pancreatic carcinogenesis. These cell lines should be very useful for studying the biology of pancreatic carcinoma, particularly those related to mutator phenotype and genetic alterations in the TGFBR2 gene.     

Antitumor activity via apoptotic cell death pathway of water soluble copper(II) complexes: effect of the diamino unit on selectivity against lung cancer NCI-H460 cell line

BioMetals - The cytotoxicity against five human tumor cell lines (THP-1, U937, Molt-4, Colo-205 and NCI-H460) of three water soluble copper(II) coordination compounds containing the ligands...     

Polarity of mutations in tumor-associated microsatellite instability

Many factors have been implicated in influencing the rate of microsatellite mutations, including the length and base composition of the repeat motif, number of repeats, base composition of flanking sequences and, perhaps most importantly, degree of perfection of the repeats. The latter is of clinical relevance, since in both spino-cerebellar ataxia and fragile X syndrome, alleles with imperfect repeats appear to be much more stable than perfect ones. As yet, the relative importance of increased replication slippage and decreased mismatch repair efficiency in the preference of mutations to occur within perfect repeats has not been fully determined. D13S308E is an asymmetric trinucleotide repeat microsatellite with the sequence (CAT)₃CAC(CAT)CAC(CAT)₂CAC(CAT)CAC(CAT) ₁₅ , thus containing two parts: an 11-repeat imperfect portion (underlined above) and a 15-repeat perfect one (bold). We sequenced eight new mutant alleles of D13S308E from three human gastric tumors with instability in this and other microsatellites. In all mutations the size variation occurred exclusively in the perfect part of the microsatellite. These results constitute direct evidence that the molecular basis of microsatellite alterations seen in normal cells is similar to those that occur in human tumors with extensive microsatellite instability. The investigation of mechanisms involved in microsatellite mutations has been handicapped by the fact that they are rare events. The microsatellite instability observed in malignant tumors provides us with a useful general system to study these mechanisms. Received: 24 June 1997 / Accepted 7 October 1997     

The influence of interstitial telomeric sequences on chromosome instability in human cells

Although most telomere repeat sequences are found at the ends of chromosomes, some telomeric repeat sequences are also found at intrachromosomal locations in mammalian cells. Several studies have found that these interstitial telomeric repeat sequences can promote chromosome instability in rodent cells, either spontaneously or following ionizing radiation. In the present study we describe the extensive cytogenetic analysis of three different human cell lines with plasmids containing telomeric repeat sequences integrated at interstitial sites. In two of these cell lines, Q18 and P8SX, instability has been detected in the chromosome containing the integrated plasmid, involving breakage/fusion/bridge cycles or amplification of the plasmid DNA, respectively. However, the data suggest that the instability observed is characteristic of the general instability in these cell lines and that the telomeric repeat sequences themselves are not responsible. Consistent with this interpretation, the chromosome containing an integrated plasmid with 500 bp of telomeric repeat sequences is highly stable in the third cell line, SNG28, which has a relatively stable genome. The stability of the chromosome containing the integrated plasmid sequences in SNG28 makes this an excellent cell line to study the effect of ionizing radiation on the stability of interstitial telomeric sequences in human cells.     

Variable stability of a selectable provirus after retroviral vector gene transfer into human cells.

Human lymphoblasts deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) were infected with an amphotropic helper-free retroviral vector expressing human HPRT cDNA. The stability and expression of the HPRT provirus in five cell lines with different proviral integration sites were examined by determining HPRT mutation and reversion frequencies and by blot hybridization studies. Mutation to the HPRT-negative phenotype occurred at frequencies of approximately 4 X 10(-5) to 3 X 10(-6) per generation. Most mutations in each of the five cell lines were associated with partial or complete deletions or rearrangements of the provirus. Several mutants retained a grossly intact HPRT provirus, and in one such mutant HPRT shutdown resulted from a revertible epigenetic mechanism that was not associated with global changes in proviral methylation. Therefore, mutation and shutdown of the HPRT provirus in human lymphoblasts result from mechanisms similar to those reported for several other avian and mammalian replication-competent retroviruses.     

Effects of oxidative and alkylating damage on microsatellite instability in nontumorigenic human cells

Microsatellite instability is a phenomenon that is well characterized in mismatch repair-deficient tumor cell lines, including the potential etiological role of endogenous DNA damage. However, our understanding of microsatellite mutational mechanisms in repair-proficient, nontumorigenic cells is limited. We determined microsatellite mutation frequencies for human lymphoblastoid cells using an episomal DNA shuttle vector in which a (TTCC/AAGG)(9) microsatellite is inserted in-frame within the herpes simplex virus thymidine kinase (HSV-tk) gene. The responses of plasmid-bearing cells to reactive oxygen species or alkylating agents were compared after treatment with hydrogen peroxide (H(2)O(2)) and N-ethyl-N-nitrosourea (ENU). H(2)O(2) treatment induced a statistically significant increase in overall HSV-tk mutation frequency relative to controls, with catalase reducing the effect. H(2)O(2) treatment increased the mutation frequency within the microsatellite and the HSV-tk coding region to a similar extent (five and six-fold, respectively, relative to the control). Mutational specificity analyses demonstrated that the proportion of mutations within the microsatellite is not statistically different among the H(2)O(2), catalase, and PBS treatment groups. In contrast, treatment of cells bearing the microsatellite vector with ENU altered the mutational spectrum, relative to solvent control. ENU induced the expected base substitutions within the HSV-tk coding region, but did not increase the microsatellite mutation frequency. The low level of microsatellite mutagenesis observed after reactive oxygen species (ROS) insult likely reflects the normal repair processes of these nontumorigenic, repair-competent cells. Our ex vivo experiments demonstrate the manner in which repetitive DNA in normal human cells might respond to endogenous mutagens.     

Shortening of microsatellite deoxy(CA) repeats involved in GL331-induced down-regulation of matrix metalloproteinase-9 gene expression

Matrix metalloproteinase-9 (MMP-9) associates with cancer cell invasion and metastasis. CL1-5 cells, a human lung adenocarcinoma cell line, expressed an elevated level of MMP-9 and exhibited a highly invasive and metastatic ability. By Matrigel assay and gelatinase zymography, the topoisomerase II poison GL331 was found to dose-dependently inhibit the invasiveness and the level of secreted MMP-9 of CL1-5 cells. Northern blot analysis indicated that cellular MMP-9 mRNA level was decreased after GL331 treatment. Furthermore, GL331-induced down-regulation of mmp-9 gene promoter was demonstrated by using a luciferase reporter gene driven by the -216 to -13 region of the mmp-9 gene promoter cloned from CL1-5 cells. By PCR amplification and gel electrophoresis, we found that GL331 caused shortening of the -216 to -13 region of the mmp-9 promoter. Direct sequencing analysis revealed that the number of d(CA) was reduced from 24 to 18 at the microsatellite d(CA) repeat region of the mmp-9 promoter. The CL1-5 cells transfected with the luciferase reporter containing 18 d(CA)s expressed only 53% of those when the reporter contained 24 d(CA)s. The promoter region of mmp-9 gene contains other positive regulatory elements, such as TRE and kappaB. We found that GL331 did not significantly influence the luciferase activity driven by TRE or kappaB. Taken together, these data suggested that GL331 inhibited MMP-9 mRNA expression at least partly through the selective induction of shortening of microsatellite d(CA) repeats. This is the first report that an anti-cancer agent can inhibit mmp-9 gene expression by inducing microsatellite DNA shortening.     

Detection of amplified genomic sequences in human small-cell lung carcinoma cells by arbitrarily primed-PCR genomic fingerprinting

The arbitrarily primed-PCR (AP-PCR) genomic fingerprinting method was applied to evaluate its effectiveness in detecting and characterizing amplified DNA fragments in two small-cell lung carcinoma (SCLC) cell lines, NCI-H69 and NCI-H82. Of the 2428 DNA fragments detected by AP-PCR using 62 arbitrary primers, 2 (0.08%) DNA fragments were amplified in NCI-H69 and 6 (0.25%) DNA fragments were amplified in NCI-H82. Based on these results, we estimate the total size of the amplified genomic regions in these cell lines to be 3000 megabase pairs (Mb) × 0.0008 = 2.4 Mb in NCI-H69 and 3000 Mb × 0.0025 = 7.5 Mb in NCI-H82. The 2 amplified fragments in NCI-H69 were mapped to chromosome 2, and all 6 amplified fragments in NCI-H82 were mapped to chromosome 8. This strongly suggests that restricted chromosomal regions are specifically amplified in these SCLC cell lines. Since the N-myc gene at 2p24 is amplified in NCI-H69 and the c-myc gene at 8q24 is amplified in NCI-H82, it is possible that these DNA fragments are co-amplified with N-myc or c-myc in these cell lines. However, since the 2 amplified fragments in NCI-H69 were not amplified in 42 other human cancer cell lines including 11 cell lines carrying amplified N-myc genes, it is also possible that there are amplified regions on chromosome 2 other than the N-myc locus at 2p24 in NCI-H69. In contrast, all 6 amplified fragments in NCI-H82 were amplified in several other human cancer cell lines carrying amplified c-myc genes. This result further indicates that these fragments were derived from an amplification unit that includes the c-myc gene. Our results show the ability of the AP-PCR method to analyze the fraction of the genome with amplification in human cancer cells. Received: 10 April 1995 / Revised: 18 December 1995, 15 April 1996     

An Inducible,Isogenic Cancer Cell Line System for Targeting the State of Mismatch Repair Deficiency

The DNA mismatch repair system (MMR) maintains genome stability through recognition and repair of single-base mismatches and small insertion-deletion loops. Inactivation of the MMR pathway causes microsatellite instability and the accumulation of genomic mutations that can cause or contribute to cancer. In fact, 10-20% of certain solid and hematologic cancers are MMR-deficient. MMR-deficient cancers do not respond to some standard of care chemotherapeutics because of presumed increased tolerance of DNA damage, highlighting the need for novel therapeutic drugs. Toward this goal, we generated isogenic cancer cell lines for direct comparison of MMR-proficient and MMR-deficient cells. We engineered NCI-H23 lung adenocarcinoma cells to contain a doxycycline-inducible shRNA designed to suppress the expression of the mismatch repair gene MLH1, and compared single cell subclones that were uninduced (MLH1-proficient) versus induced for the MLH1 shRNA (MLH1-deficient). Here we present the characterization of these MMR-inducible cell lines and validate a novel class of rhodium metalloinsertor compounds that differentially inhibit the proliferation of MMR-deficient cancer cells.