Frameshift mutator mutations.
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Base Sequence↗
Biomedical subjects
M Perucho
Publications and source records attributed to M Perucho.
Chromosomal assignment of human DNA fingerprint sequences by simultaneous hybridization to arbitrarily primed PCR products from human/rodent monochromosome cell hybrids.
We have developed a technique for the simultaneous chromosomal assignment of multiple human DNA sequences from DNA fingerprints obtained by the arbitrarily primed polymerase chain reaction (AP-PCR). Radioactively labeled human AP-PCR products are hybridized to DNA fingerprints generated with the same arbitrary primer from human/rodent monochromosome cell hybrids after electroblotting to a nylon membrane. Human-specific hybridization bands in the human/rodent fingerprints unambiguously determine their chromosome of origin. We named this method simultaneous hybridization of arbitrarily primed PCR DNA fingerprinting products (SHARP). Using this approach, we determined the chromosomal origins of most major bands of human AP-PCR fingerprints obtained with two arbitrary primers. Altogether, the chromosomal localization of near 50 DNA fragments, comprehensive of all human chromosomes except chromosomes 21 and Y, was achieved in this simple manner. Chromosome assignment of fingerprint bands is essential for molecular karyotyping of cancer by AP-PCR DNA fingerprinting. The SHARP method provides a convenient and powerful tool for this purpose.
Animals↗
Clinical implications of microsatellite instability in colorectal cancers.
BACKGROUND: Microsatellite instability (MI) has been reported in some sporadic colon tumors and in cases of hereditary nonpolyposis colorectal cancer (HNPCC). The criteria for HNPCC have not been fully defined, and clinical criteria are used to identify as many HNPCC patients as possible. To clarify the conformity of these criteria with the identification of eligible HNPCC cases, we analyzed MI in HNPCC patients diagnosed using clinical criteria. METHODS: Genomic DNA was extracted from surgical specimens of 56 colorectal cancers, including 36 from patients diagnosed with HNPCC using the clinical criteria. We analyzed four microsatellite loci using 32P-labeled primers. RESULTS: Among HNPCC patients diagnosed using clinical criteria, patients who were positive for MI accounted for 62% of Group A (a confirmed group) and 35% of Group B (a high risk group); only 5% of randomly selected colorectal cancer patients (Group C), were positive for MI. Furthermore, MI-positive tumors were found in patients who had a tendency for tumors to involve the right side of the colon, an association with cancers in other organs, a lower incidence of p53 protein positivity, and a higher proportion of poorly differentiated cancers. CONCLUSIONS: The presence of MI, in concert with modified clinical criteria, may identify legitimate cases of HNPCC in patients who might otherwise be excluded by the minimum criteria.
Age of Onset↗
Differences in the spectrum of spontaneous mutations in the hprt gene between tumor cells of the microsatellite mutator phenotype.
We have determined the frequency and spectrum of spontaneous mutations at the hprt locus in LoVo, HCT116, LS180 and DLD-1 colon carcinoma cell lines exhibiting microsatellite genetic instability. Each cell line has a different mutator gene. LoVo and HCT116 cells have mutated hMSH2 and hMLH1 genes, respectively, which account for the majority of hereditary non-polyposis colorectal cancer (HNPCC). LS180 cells are wild type for these genes and also for hPMS1 and hPMS2 mismatch repair genes. DLD-1 cells harbor a mutated GTBP mismatch binding factor and a mutated DNA Polymerase delta. The mutation rate at the hprt locus was several hundred fold higher in these cell lines relative to control cell lines without microsatellite instability. The mutations were frameshifts (deletions and insertions of a single nucleotide in short repeats) and single base substitutions (transversions and transitions). Some mutations were shared by these four cell lines. However, every cell line also exhibited a distinctive spectrum of mutations suggesting that each mutator gene induces a particular mutator phenotype. These results also suggest that the frequency and spectrum of somatic mutations in tumor cells of the microsatellite mutator phenotype may have diagnostic applications to discriminate among the diverse underlying mutator genes.
Base Sequence↗
Microsatellite instability: the mutator that mutates the other mutator.
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Adenoma↗
Ras oncogene mutations in thyroid tumors: polymerase chain reaction-restriction-fragment-length polymorphism analysis from paraffin-embedded tissues.
Ras mutations have been found in thyroid lesions. Different studies have shown different frequencies of mutations among benign and malignant lesions. The presence of point mutations in codons 12 and 13 of the c-K-ras, c-H-ras, and N-ras genes was studied in 58 thyroid lesions (10 nodular goiters, 10 follicular adenomas, and 15 papillary, 10 follicular, and 13 anaplastic carcinomas). DNA was extracted from formalin-fixed paraffin-embedded tissue, and target sequences were amplified in vitro by the polymerase chain reaction. Mutations were detected by the presence of restriction-fragment-length polymorphisms either occurring naturally or introduced artificially by the use of mutant primers. No characterization of the mutations was performed. Results were correlated with clinicopathologic features and patient follow-up. One goiter showed a mutation at codon 13, c-K-ras. All follicular adenomas, including three hyalinizing trabecular adenomas, were negative. Four papillary carcinomas presented mutations (one at codon 13, c-K-ras; three at codon 12, N-ras). Two follicular carcinomas showed mutations at codon 12, N-ras. Five anaplastic carcinomas showed mutations (two at codon 12 and two at codon 13, c-K-ras; one at codon 12, N-ras). In summary, the results confirm that ras oncogenes play a role in thyroid tumorigenesis, probably at an early step. Ras mutations appear not to be related to prognosis.
Adenoma↗
Detection of microsatellite instability in cancers by arbitrarily primed-PCR fingerprinting using a fluorescently labeled primer (FAP-PCR).
The microsatellite mutator phenotype (MMP), detected as a change in the number of repeating units in hundreds of thousands of microsatellite sequences in the tumor cell genome, underlies the carcinogenesis of a variety of tumors including sporadic and hereditary nonpolyposis colon cancers. This enhanced microsatellite instability was discovered using arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting of DNA from colon cancers. In this study, we found an arbitrary primer that can amplify multiple DNA fragments containing repeated sequences, including the poly A tracts found in the Alu repeats of the human genome. The combined use of primer labeling with fluorescence and an automated DNA sequencing analysis of AP-PCR products (FAP-PCR) detected alterations in fingerprint bands in all DNA samples previously determined to belong to the MMP. Fluorescent AP-PCR fingerprinting using this single arbitrary primer provides a convenient and efficient method for detecting tumor specific fingerprint alterations that are usually undetectable by conventional fingerprinting.
Blotting, Southern↗
Cancer of the microsatellite mutator phenotype.
This review focuses on the genomic instability underlying the microsatellite mutator phenotype (MMP) pathway for cancer. MMP was discovered by the application of DNA fingerprinting by Arbitrarily Primed PCR (AP-PCR) to the analysis of somatic genetic alterations in colon tumors. The unbiased nature of AP-PCR permitted to infer, from the mobility shifts observed in some fingerprint bands, the accumulation by a subset of colon tumors of hundreds of thousands of somatic mutations in simple repeated sequences or microsatellites. We deduced that this enormous agglomeration of clonal mutations was due to the previous occurrence of 'mutator mutations' in DNA replication or repair factors leading to a decreased fidelity of replication. These mutator mutations appeared to be the remote cause for the development of these MMP tumors, whose existence unmistakably validated the hypothesis of 'cancer as a mutator phenotype'. Since these original observations, rapid progress has occurred in the field. The mutator mutations were identified as those occurring in members of the DNA mismatch repair gene family, which are also associated with hereditary non-polyposis colorectal cancer (HN-PCC). In this review I discuss the experimental approach that allowed the discovery of MMP and the features of the genomic instability of these tumors. I also review recent developments that affect the understanding of the role of the mismatch repair mutator mutations in the unfolding of MMP during carcinogenesis.
Colorectal Neoplasms, Hereditary Nonpolyposis↗
The microsatellite mutator phenotype of colon cancer cells is often recessive.
A new mutator mechanism for tumorigenesis, characterized by somatic genomic instability (SGI) at simple repeated sequences (SRS) or microsatellites, underlies hereditary nonpolyposis colorectal cancer (HNPCC) and some sporadic tumors of the colon and other types. To determine whether the microsatellite mutator phenotype (MMP) is dominant or recessive, we generate somatic cell hybrids between a tumor cell line without SGI at SRS (D98OR) and colon carcinoma cell lines with relative low (HCT-15) and high (LS174-T) SGI at SRS. The normal fidelity of replication of these unstable sequences was observed in each of these cell hybrids. Fusion of HCT-15/DLD-1 low instability cells, with LS174-T, HCT116 and LoVo cell lines, all exhibiting relative high instability, also restored the replication fidelity of SRS in all of the hybrids. Hybrids between the high instability cell lines did not grow possibly because of senescence or apoptosis. These results indicate that, in the cell lines analysed, the characterized mutator phenotype of the mismatch repair system resulting in high SGI at SRS, and the uncharacterized mutator phenotype underlying low SGI at SRS, are both recessive. The results also suggest that different tumor cells of the MMP harbor distinct altered growth-related genes.
Colonic Neoplasms↗
Fingerprinting of DNA and RNA by arbitrarily primed polymerase chain reaction: applications in cancer research.
RNA fingerprinting by RAP-PCR is a powerful tool for the temporal and spatial analysis of differential gene expression. Many biological situations exist where differential gene expression results in distinguishable phenotypes, including, for example, tissue and cell types, responses to hormones, growth factors, stress, and the heterologous expression of certain genes. There are several methods for detecting differential gene expression and cloning differentially expressed genes that do not rely on a biological assay of phenotype. Most of these methods fall into two general categories: subtractive hybridization and differential screening. RAP-PCR offers numerous advantages over these methods, including its simplicity and its ability to compare the fluctuations in gene expression between multiple samples simultaneously using minute amounts of RNA. In addition, RAP-PCR can yield information on the overall patterns of gene expression between different cell types or between different physiological conditions of the same cell type. Comparison of the RAP-PCR fingerprints from these different experimental groups permits one to draw inferences regarding the overall cellular states of gene expression and the interrelation between gene transcripts belonging to the same or different regulatory pathways. Hypotheses regarding signal transduction pathways can be obtained using this information. RAP-PCR offers applications in cancer research in the detection of tumor-specific alterations in gene expression, providing a bountiful source of tumor markers. The pleiotropic impact of oncogene activation, tumor suppressor gene inactivation, and mutator mutations, in gene regulation, can be readily assessed by RAP-PCR in model systems both in vitro and in vivo.
Base Sequence↗
Development of a rat cell line containing stably integrated copies of a lambda/lacI shuttle vector.
A rat embryo cultured cell line was generated that carries stably integrated copies of a lambda/lacI shuttle vector, containing the lacI gene as a mutational target. After the desired treatment of the cells, this vector can be rapidly and efficiently recovered from the cell DNA by in vitro packaging and then screened for mutations in the lacI gene, using bacterial detection systems. The vector is identical to that integrated into the Big Blue transgenic mouse, which was developed for in vivo mutation analysis. Characterization of the cell line by fluorescence in situ hybridization showed that the phage DNA is integrated at two distinct sites on separate chromosomes at approximately 50-70 copies per cell and the cell line is polyploid. The rescue efficiency is approximately 100,000 pfu/micrograms of genomic DNA. To examine the ability of the cell line to detect mutations in the lacI gene, the cells were treated with 100 micrograms/ml of the direct-acting alkylating agent N-methyl-N-nitrosourea (MNU) for 30 min at 37 degrees C and grown to confluence. The shuttle vector was rescued from untreated and mutagen treated cells, and spontaneous and induced mutant frequencies were determined to be 4.0 x 10(-5) and 92.7 x 10(-5), respectively. The cell line can be used to detect mutations in the lacI gene, followed by recovery of mutants for sequence analysis. The cell line may be valuable for short-term in vitro mutagenesis studies, oncogene and tumor suppressor studies, and DNA repair studies.
Animals↗
Nucleic acid fingerprinting by PCR-based methods: applications to problems in aging and mutagenesis.
There are many methods of inference in common use in biology that are based on population sampling, including such diverse areas as sampling organisms to determine the population structure of an ecosystem, sampling a set of DNA sequences to infer evolutionary history, sampling genetic loci to build a genetic map, sampling differentially expressed genes to find phenotypic markers, and many others. Recently developed PCR-based methods for nucleic acid fingerprinting can be used as sampling tools with general applicability in molecular biology, evolution and genetics. These methods include arbitrarily primed PCR (AP-PCR; Welsh and McClelland, 1990) and random amplified polymorphic DNA (RAPD; Williams et al., 1990) for the fingerprinting of DNA, and RNA arbitrarily primed PCR (RAP-PCR; Welsh et al., 1992a) and differential display (DD; Liang and Pardee, 1992) for the fingerprinting of RNA. Novel ways of looking at genetic control are facilitated by the high data-acquisition capabilities of the fingerprinting methods. In this article, we review some of the applications of DNA fingerprinting to the study of mutagenesis, and of RNA fingerprinting to the study of normal and abnormal signal transduction. We propose that these fingerprinting approaches may also have applications in the study of senescence and aging.
Aging↗
Diagnostic detection of mutant ras genes in minor cell populations.
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Base Sequence↗
Design of a selectable reporter for the detection of mutations in mammalian simple repeat sequences.
To study the mutator phenotype characteristic of tumors showing widespread replication errors at simple DNA repeat sequences (RER+), we designed a selectable reporter system for the detection of such mutations in mammalian cells. A hygromycin B phosphotransferase gene was rendered out-of-frame by the insertion of a (CA)13 dinucleotide repeat tract immediately following the ATG start codon, and subcloned into a retroviral expression vector containing a G418 (neo) selectable marker. Following transduction of this construct into cultured cells, clonal neo+ cell lines were established and then tested for their ability to form colonies in hygromycin B-containing medium. Using this system, we found that the HCT116, LS174T and LS180 human colon carcinoma cell lines acquire hygromycin resistance (hygr) at a 100-fold higher frequency than the HT29, SW480, DLD-1 and HCT15 human colon carcinoma and NIH3T3 fibroblast cell lines, and at a 25-fold higher rate than the Rat 6 embyro fibroblast cell line. DNA sequence analysis indicated that frameshift mutations had occurred within the CA dinucleotide repeat tract in HCT116 cells that became hygr. Thus, the mutation rates at simple repeated sequences in mammalian cell lines can be readily determined and studied using this system.
3T3 Cells↗
Comparative analysis of mutations in the p53 and K-ras genes in pancreatic cancer.
Mutations in codon 12 of K-ras occur in a high proportion of pancreatic cancer cases. Although there is evidence that p53 mutations also occur in this tumor, few studies have been reported to date and no comparison has been made of K-ras and p53 mutations in the same tissues. Single-strand conformation polymorphism and sequencing of the PCR products were used to determine mutations in p53 gene; to detect mutations in K-ras genes, the artificial restriction fragment length polymorphism (RFLP) approach was used. Eight out of 30 tissues from primary pancreas cancer and 3 of 4 samples from metastases showed p53 mutations. Fifteen out of 17 pancreatic cancer cell lines had p53 mutations. In 2 cases, the same p53 mutation was identified in the original tumor and in a tumor-derived cell line. The majority of p53 mutations were present in exons 5-9 of the gene. Mutations at codon 12 of the K-ras gene were identified in 23/32 pancreas cancer tissues and in 14/17 cell lines. There was no relationship between the types of mutation observed in the 2 genes. In conclusion, mutations in K-ras and p53 genes are common in pancreatic cancer. p53 mutations may occur more frequently in metastatic lesions than in primary tumors, although further work is necessary to investigate this point.
Base Sequence↗
Defective mismatch repair in extracts of colorectal and endometrial cancer cell lines exhibiting microsatellite instability.
A replication error (RER+) phenotype, characterized by somatic instability in simple repeated sequences, is associated with several types of cancer. To determine if a defect in DNA replication fidelity or repair of replication errors might explain this instability, we compared both processes in cell-free extracts from RER+ endometrial and colorectal cancer cell lines to RER- cell lines. SV40 origin-dependent replication of a microsatellite sequence is highly accurate in cell extracts regardless of their RER phenotype. However, extracts from RER+ cell lines are defective in mismatch repair, while extracts of RER- cell lines are not. Lack of repair was observed when the signal (a nick) for strand-specific repair was either 3' or 5' to the mispair. One colorectal cancer cell line contained deletions in both alleles of the putative mismatch repair gene hMSH2, and one endometrial cancer cell line contained a 4-base pair duplication in one hMSH2 allele. No hMSH2 mutation was detected in the other allele or in the other five RER+ cell lines. Repair was readily detected when each of the defective extracts was mixed with a repair-proficient extract, demonstrating that no trans-acting inhibitor is present. Attempts to complement the repair deficiencies by mixing two different defective extracts identified three combinations that restored repair. The data suggest that: (i) defective repair is associated with colorectal and endometrial cancer and, by extrapolation, with other types of cancer; (ii) mutations in the hMSH2 gene, and possibly other genes, result in defective mismatch repair; (iii) the defect(s) in these lines likely involves pre-incision events or the excision step, but not the incision, polymerization, or ligation steps; and (iv) at least four functional complementation groups for mismatch repair may be involved in human cancer.
Cell Line↗
Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation.
Genomic instability at simple repeated sequences (SRS) is a landmark for some sporadic and hereditary cancers of the colon. We have identified several human tumour cell lines with up to 1,000-fold increases in mutation rates for endogenous microsatellite sequences, relative to normal cells or tumour cells without the mutator phenotype and show that they are very early events in tumorigenesis. Our in vivo and in vitro results show that the genomic instability persists after transformation and that microsatellite mutations accumulate as consecutive somatic slippage events of a single or a few repeated units. This mechanism may account for the repeat expansions in triplet hereditary diseases and the same defect in replication fidelity in non-polyposis colon cancer could also contribute to the non-mendelian anticipation in these diseases.
Cell Transformation, Neoplastic↗
Natural occurrence of drug resistance mutations in the reverse transcriptase of human immunodeficiency virus type 1 isolates.
Reverse transcriptase-associated amino acid substitutions related to ddC, d4T, and nevirapine resistance have been found in isolates of human immunodeficiency virus type 1 (HIV-1) from patients treated with AZT only. Sequence analysis of 23 isolates documented the presence of 4 unexpected mutations at amino acid residues related to drug resistance. Two isolates contained an aspartic residue in codon 69 associated with ddC resistance, and another a change in codon 75 associated with resistance to d4T. The Y-to-C alteration in codon 181 associated with nevirapine resistance was observed in another isolate after serial passage in cell culture in the absence of drug. Changes in substitution patterns were also noted after serial passage of four AZT resistant isolates in cell culture without inhibitors. One of the strains showed changes in codons 67 and 70 to wild-type residues. Clonal analysis showed that this alteration occurred by the selection during cell culture passage of the wild-type genotype, which was present as a minority subpopulation in the initially resistant virus stock, rather than to genetic reversion. In summary, we present evidence documenting the presence of mutations associated with drug resistance in the absence of drug treatment and supporting the role played by gentic variability in the emergence of HIV-1 antiviral resistance.
Amino Acid Sequence↗