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Biomedical subjects

M Meuth

Publications and source records attributed to M Meuth.

At least 19 recordsLinked to original sources
PubMed2006

Methylational urinalysis: a prospective study of bladder cancer patients and age stratified benign controls.

Tumour suppressor gene (TSG) methylation has been proposed as a diagnostic marker for urothelial cancer (UC). Here, we compare the frequency of urinary TSG methylation in young and elderly patients, with and without UC. Urine samples were obtained prospectively from 35 UC patients, 35 benign controls over the age of 70 years and 34 healthy volunteers under the age of 40 years. Methylation analysis was performed for eight gene promoters using quantitative methylation-specific PCR. Methylation was detected in urine DNA from all three patient groups. The highest frequencies were seen in UC patients. Significantly less methylation was present in control samples than UC cases for RASSF1a and APC (P < 0.034). The 'methylation index' and level of methylation was highest in the UC group and lowest in the young control group. A marker panel of RASSF1a, E-cad and APC generated a sensitivity of 69%, a specificity of 60% and a diagnostic accuracy of 86%. TSG methylation is detectable in urine DNA from patients with and without bladder cancer. The frequency and extent of methylation appears to increase with age and malignancy. The lack of tumour specificity suggests that further investigation is required before this test is introduced into clinical practice.

Adult↗
PubMed2006

Evidence for the early onset of aberrant promoter methylation in urothelial carcinoma.

There is evidence that carcinoma in situ (CIS) is the precursor of invasive urothelial carcinoma, a tumour characterized by frequent gene promoter methylation. The timing of altered DNA methylation is unknown in this pathway. Here we investigate gene methylation in 196 consecutive samples of normal urothelium, CIS, and tumours from 104 patients with both CIS and invasive urothelial carcinoma using quantitative methyl-sensitive polymerase chain reaction for six genes (p16, p14, E-cadherin, RARbeta2, RASSF1a, and GSTP1). Control normal urothelial samples from 15 patients with no history of urothelial carcinoma were also analysed. Immunohistochemistry established the expression of well-characterized CIS markers p53 and cytokeratin 20. Promoter methylation occurred frequently in both normal urothelium and CIS samples from patients with urothelial carcinoma, and increased with progression from normal to invasive urothelial carcinoma, at both specific loci (chi2 test: E-cadherin, p=0.0001; RASSF1a, p=0.003, RARbeta2, p=0.007, p16, p=0.024) and in general (methylation indices [t-test, p<0.0001]). Methylation was associated with cytokeratin 20 expression (t-test, p=0.004) and poor prognosis, and with increased progression to tumour death in patients whose CIS samples showed methylation, in comparison with those without methylation (log rank p<0.03). Promoter methylation occurs early in the urothelial carcinogenic pathway and appears to be a good biomarker of the invasive urothelial carcinoma phenotype.

Adult↗
PubMed2004

Proteomic analysis of voided urine after prostatic massage from patients with prostate cancer: a pilot study.

OBJECTIVES: Serum prostate-specific antigen measurements are widely used for the early detection of prostate cancer but lack specificity, thus warranting the search for additional biomarkers. METHODS: Two-dimensional gel electrophoresis followed by matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis was used to investigate the protein profiles of voided urine after prostatic massage from 6 patients with histologically confirmed prostate cancer and 6 age-matched patients with benign prostatic hyperplasia. RESULTS: The median number of protein spots per gel was lower in the urine from the patients with cancer (median 143 spots, range 118 to 163) than in the urine from those with benign prostatic hyperplasia (median 154 spots, range 142 to 209), although the difference was not statistically significant. MALDI-TOF-MS analysis identified six commonly expressed proteins: alpha-enolase, isocitrate dehydrogenase, beta-2-microglobulin, alpha-1-microglobulin, complex-forming glycoprotein HC, and PRO2044. Of the five protein spots seen in a subset of patients with cancer, one was identified as calgranulin B/MRP-14. Immunohistochemical staining of prostatic tissue showed greater expression of calgranulin B/MRP-14 in 2 of 7 well-differentiated, 1 of 12 moderately differentiated, and 0 of 8 poorly differentiated tumors relative to adjacent benign tissue; expression of calgranulin A/MRP-8, a heterodermic binding partner of calgranulin B/MRP-14, was absent. CONCLUSIONS: The role of urinary calgranulin B/MRP-14 as a potential novel marker for prostate cancer needs additional investigation.

Aged↗
PubMed2004

Genetic instability and transitional cell carcinoma of the bladder.

The development of cancer occurs in a stepwise fashion, with each step representing the mutation in one of several key genes. However, the mutation rate of somatic cells is too low to account for the number of mutations required for a cell to undergo carcinogenesis. Thus, the development of genetic instability is a vital early step towards carcinogenesis. We review the evidence for genetic instability, with particular reference to transitional cell carcinoma of the bladder. Both microsatellite instability and chromosomal instability are present in this tumour, and we discuss their incidence and clinical implications.

Carcinoma, Transitional Cell↗
PubMed2001

Suppression of the radiation-sensitive phenotype of hamster irs1 and irs2 strains selected for resistance to 3-aminobenzamide.

PURPOSE: The radiosensitive hamster cell lines irs1 and irs2 have phenotypic similarities to cells defective in the early response to DNA-damaging agents as a result of mutations of the genes encoding poly(ADP-ribose)polymerase (PARP) or ataxia-telangiectasia mutated (ATM). Whether modification of PARP activity through selection of strains resistant to 3-aminobenzamide (3-AB) would affect the radiosensitive phenotype of irs1 and irs2 was examined. MATERIALS AND METHODS: 3-AB-resistant strains of irs1, irs2 and their parent line V79 were established and their sensitivity to DNA-damaging agents was measured. In some 3-AB-resistant strains, the radiation resistance of DNA synthesis and the induction of apoptosis were also assayed. Additionally, a number of aspects of PARP function were measured. RESULTS: Independently selected 3-AB-resistant strains of irs2 showed nearly complete suppression of radiation sensitivity, sensitivity to topoisomerase inhibitors, and radioresistant DNA synthesis. 3-AB-resistant strains of irs1 showed partial suppression of phenotype while 3-AB-resistant strains of V79 had no sensitivity changes. The induction of apoptosis in 3-AB-resistant strains of irs2 required substantially higher radiation doses than for irs2 itself. 3-AB-resistant strains had no detectable alteration of PARP level or cleavage following ionizing irradiation and there were no mutations in the PARP gene. CONCLUSIONS: Suppression of radiosensitivity associated with 3-AB resistance has important implications for mechanisms of tolerance to damage because it is able to override responses associated with specific genetic defects.

Animals↗
PubMed2000

Microsatellite instability and the PTEN1 gene mutation in a subset of early onset gliomas carrying germline mutation or promoter methylation of the hMLH1 gene.

High-frequent microsatellite instability (MSI-H) was detected in two of the 80 gliomas examined, whlie the other 78 gliomas showed microsatellite stable (MSS) phenotype. Both of the two MSI-H tumors were glioblastomas which developed in teenage patients. One of the patient was diagnosed as having Turcot's syndrome and had a germline mutation in the hMLH1 gene. Loss of expression due to promoter methylation was selectively observed in the wild type allele of the hMLH1 gene in the tumor of this patient. The other patient had neither a family history nor a past personal history of malignancy. Although no mutation in the mismatch repair genes was detected in the tumor of this patient, the level of expression of the hMLH1 gene was markedly decreased and the promoter sequence of the gene was highly methylated. In the tumor of this patient, the PTEN1 gene, one of the genes carrying microsatellite sequences in their coding regions, was altered by a slippage mutation within five adenine repeat sequences. These findings indicate that the genetic or epigenentic inactivation of the hMLH1 gene is involved in a subset of early-onset gliomas and the PTEN1 gene could be a downstream target for mutation as observed in glioblastoma without MSI.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Apoptosis induced by overexpression of hMSH2 or hMLH1.

Mutations of the mismatch repair genes hMSH2 and hMLH1 have been found in a high proportion of individuals with hereditary nonpolyposis colon cancer (HNPCC), establishing the link between mismatch repair and cancer. Tumor cell lines that are deficient in mismatch repair develop a mutator phenotype that appears to drive the accumulation of mutations required for tumor development. However, mutations of other mismatch repair genes such as hPMS2 can lead to a mutator phenotype, although inherited mutations of these genes are rare in HNPCC families. Here, we show that overexpression of hMSH2 or hMLH1 but not of hMSH3, hMSH6, or hPMS2 induces apoptosis in either repair-proficient or -deficient cells. Furthermore, primary mouse embryo fibroblasts derived from Msh2-deficient mice lose their ability to undergo apoptosis after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. These results suggest that the mismatch repair proteins hMSH2 and hMLH1 may be components of a pathway that influences apoptosis. We consider the possibility that loss of apoptosis as a result of hMSH2 or hMLH1 deficiency may be an additional factor in cancer predisposition in HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed1999

MSH3 deficiency is not sufficient for a mutator phenotype in Chinese hamster ovary cells.

In the yeast Saccharomyces cerevisiae, the mutS homolog protein products MSH3 and MSH6, each in cooperation with MSH2, play well-defined and specific roles in the repair of DNA mismatches and nucleotide loops. The discrete functions of the human homologs hMSH3 and hMSH6 are less clear and current evidence suggests that the substrate specificity of these proteins may be less strict. To determine the role of MSH3 in mammalian mismatch repair, we employed MSH3-deficient Chinese hamster ovary (CHO) cell lines. No significant changes in mutation rate were detected in the MSH3-deficient strain and there were no differences in sensitivity to DNA-damaging agents. Further analysis of hprt mutants did not show a MSH3-dependent shift in the mutant spectrum. Interestingly, thorough examination of four dinucleotide microsatellite regions revealed instability at only one locus in one of the MSH3-deficient cell lines. These data support the idea of a high degree of redundancy in the function of the MutS homologs MSH3 and MSH6, at least with respect to the control of microsatellite instability.

Animals↗
PubMed1997

Conditional mutator phenotypes in hMSH2-deficient tumor cell lines.

Two human tumor cell lines that are deficient in the mismatch repair protein hMSH2 show little or no increase in mutation rate relative to that of a mismatch repair-proficient cell line when the cells are maintained in culture conditions allowing rapid growth. However, mutations accumulate at a high rate in these cells when they are maintained at high density. Thus the mutator phenotype of some mismatch repair-deficient cell lines is conditional and strongly depends on growth conditions. These observations have implications for tumor development because they suggest that mutations may accumulate in tumor cells when growth is limited.

Cell Count↗
PubMed1997

Mutator phenotype in Msh2-deficient murine embryonic fibroblasts.

Embryonic fibroblast cell lines were established from mice deficient, heterozygous, or proficient for Msh2, one of the three known DNA mismatch repair genes involved in hereditary nonpolyposis colon cancer (HNPCC). Cell lines were established by transfection of primary mouse embryo fibroblasts with E7 and Ras oncogenes or mutant p53. Spontaneously immortalized cells derived from the primary cultures were also studied. To determine whether these cells developed a mutator phenotype similar to that found in colon cancer cells deficient in mismatch repair, we measured mutation rates, microsatellite instability, and sensitivities to a range of DNA-damaging agents. The mutator phenotype detected in the E7 and Ras or mutant p53-immortalized Msh2-/- mouse cells was similar to that found in human mismatch repair-deficient colorectal carcinoma cell lines. Mutation rates to ouabain resistance were increased 8-12-fold relative to lines from Msh2+/+ mice, and microsatellite instability was detectable in 12-18% of subclones derived from the Msh2-/- line but was undetectable in subclones developed from the Msh2+/+ line. Furthermore, E7 and Ras or spontaneously immortalized Msh2-/- cells were significantly more resistant to the cytotoxic effects of 6-thioguanine relative to Msh2+/+ cells. In contrast, these lines showed various responses to UV light and cis-platinum, suggesting that mismatch repair deficiency was not the sole determinant for sensitivity to these DNA-damaging agents. Particular attention was paid to the properties of cells heterozygous for the Msh2 mutant gene, which would mimic the situation of an HNPCC carrier. However, our studies failed to reveal any properties of these cells that might provide a growth advantage or predispose them for the acquisition of further mutations. This observation is consistent with the model that inactivation of the wild-type Msh2 allele is a critical step for tumorigenesis in HNPCC patients.

Animals↗
PubMed1996

The influence of a (GT)29 microsatellite sequence on homologous recombination in the hamster adenine phosphoribosyltransferase gene.

Several DNA sequence elements are thought to stimulate homologous recombination, illegitimate recombination, or both in mammalian cells. Some are implicated by their recurrence around rearrangement breakpoints, others by their effects on recombination of extrachromosomal plasmids. None of these sequences, however, has been tested on the chromosome in a defined context. In this paper we show how the adenine phosphoribosyltransferase locus in CHO cells can be used to study the recombinogenic potential of defined DNA sequences. As an example we have measured the effect on homologous recombination of a dinucleotide repeat, (GT)29, which has been shown to stimulate homologous recombination in extrachromosomal vectors 3-20 fold. On the chromosome at the adenine phosphoribosyltransferase locus, however, this sequence shows no capacity to stimulate recombination or to influence the distribution of recombination events.

Adenine Phosphoribosyltransferase↗
PubMed1996

Loss of heterozygosity and base substitution at the APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines.

We determined the nature of mutations occurring at the autosomal APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines. The analysis of mutations that result in APRT deficiency in a mismatch-repair-deficient strain of DLD-1 heterozygous for this locus enabled us to measure the rate of loss of the wild-type gene through deletion, recombination, or gene conversion as well as the rate of point mutation. The overall rate of mutation at the APRT locus in DLD-1 was elevated 100-fold compared with the mismatch-repair-proficient colorectal carcinoma cell line SW620. Loss of heterozygosity (LOH) at APRT accounted for only 4 to 9% of mutant strains derived from DLD-1, indicating a rate for these types of events of 4 x 10(-7) to 9 x 10(-7). In SW620 the rate of LOH at APRT was about 10-fold higher. LOH was not found at polymorphic markers within the same chromosome subband as APRT, indicating that only a limited portion of the chromosome was affected by these alterations. Chromosome painting of SWS620 mutants revealed that the loss of APRT occurred together with a substantial portion of the long arm of chromosome 16. Differences in the nature of base substitutions at APRT (e.g., the proportion of mutations resulting from transitions or transversions) in these tumor cell lines were also detected. There was also an important similarity---the presence of a mutant APRT gene with multiple base substitutions that may be the result of some sort of error-prone DNA synthesis.

Adenine Phosphoribosyltransferase↗
PubMed1996

Patterns of mutation in cancer cells.

The discovery of powerful mutator phenotypes in a subset of colon cancers provides direct support for the hypothesis that destabilization of replication fidelity and repair drive the accumulation of mutations in tumour suppressor or proto-oncogenes. Nevertheless, many important questions remain. The tumour cell lines in which these mutator genes were characterized have many other mutations that may contribute to the mutator phenotype and the characteristic pattern of mutations found in these cells. Thus, mismatch repair deficiency may be necessary for the mutator phenotype, but is it sufficient? Certainly, changes in DNA replication fidelity or cell cycle checkpoint controls may contribute to the mutator phenotype. This question also has important implications for the effect of mismatch repair deficiency on tumour development. Does the mutator phenotype in HNPCC patients arise as a very early event resulting from the loss of the wild type allele or does it arise in later stages only after alterations of cell cycle controls or replication fidelity? Given that eukaryotic cells have numerous homologues of the mismatch repair genes, what are the roles of all these genes? Are these involved in the repair of very specific types of replication errors or do they have other roles in cells? Finally, what mechanisms underlie the accumulation of mutations in other types of tumours? Given the rapid progress made since the isolation of the human homologues of the E coli mismatch repair genes less than 3 years ago, we can look forward to the answers to many of these questions in the near future.

Animals↗
PubMed1995

Response of colon cancer cell lines to the introduction of APC, a colon-specific tumor suppressor gene.

The APC gene, mutations in which are responsible for the inherited colon cancer syndrome adenomatous polyposis coli (APC), is described as a tumor suppressor gene. A full-length, wild-type APC gene was introduced by transfection into three human colon carcinoma cell lines, each characterized for mutations at loci involved in colon tumor formation. The response of each cell line to the introduction of APC differed with the genotype of the cell line. Some of the cell clones derived from these transfections displayed altered morphologies; some showed suppression of tumorigenicity based on growth in soft agar and tumor formation in nude mice. One cell line, SW480, could not be stably transfected with the APC gene. These results provide the first direct evidence that the APC gene can alter the transformation properties of colon carcinoma cells.

Animals↗
PubMed1995

Deletion mapping of highly conserved transcribed sequence downstream from APRT locus.

To investigate the nature of DNA sequence rearrangements occurring in a highly malignant human colorectal carcinoma cell line (SW620) exhibiting a high level of chromosome instability, we characterized the molecular basis of deletions eliminating APRT. Deletions in SW620 resembled those in a variety of cell lines. They were joined at regions of little similarity through mono-, di-, or trinucleotide repeats. Breakpoint regions were rich in di- and trinucleotide repeats that might constitute pause sites for the replication complex. Deletions ranged in size from 1.8 to approximately 70 kb and were "directional" in that they eliminated sequences upstream of APRT but not downstream. Analysis of downstream sequences suggested that this pattern of deletion was due to the presence of another gene. Transcripts from these two genes converged but did not overlap. Given that this gene was not deleted in any hamster or human mutants, it appears essential for cell viability. This organization has important consequences for the pattern of mutation and repair of this region.

Adenine Phosphoribosyltransferase↗
PubMed1995

Molecular analysis of mutations in mutator colorectal carcinoma cell lines.

The nature of mutations occurring in two colorectal carcinoma cell lines deficient in mismatch repair and displaying mutator phenotypes was determined. One of the lines (HCT116) exhibited a higher level of microsatellite instability than the second (DLD-1), although the rate of mutation at the selectable locus encoding the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase (HPRT) was equally elevated (about 350-450-fold relative to mismatch repair proficient cell lines). Transitions were the major class of mutations in the two mutator lines. In DLD-1 these mutations recurred at several sites that appeared to be hotspots. Frameshifts at a run of six guanine residues in the coding sequence for HPRT constituted 35% of mutations in HCT116. These frameshifts were highly unstable and reverted to wild type at high frequency. Larger deletions were also detected in HCT116. Although these deletions constituted a small proportion of mutations compared with the other types, our data suggest that the rate of deletion is elevated relative to mismatch repair proficient (hMLH1+) cell lines. These observations suggest that the gene(s) altered in DLD-1 may preferentially affect the repair of base mismatches while the alteration(s) in HCT116 may affect the repair of both mismatches and frameshifts.

Base Sequence↗