In vitro transcription/translation assay for the screening of hMLH1 and hMSH2 mutations in familial colon cancer

Luce, Michael C.; Marra, Giancarlo; Chauhan, D; Laghi, Luigi; Carethers, John M.; Cherian, Sajeev; Hawn, Mary T.; Binnie, Cameron G.; Kam-Morgan, Lauren; Cayouette, Matthew C.; Koi, Minoru; Boland, C. Richard

doi:10.1016/0016-5085(95)90600-2

Cited by 60 publications

(37 citation statements)

References 30 publications

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“…The lymphoblastoid cell lines were either established as described (18) or obtained from Bert Vogelstein (Johns Hopkins Oncology Center, Baltimore) or from the Finnish HNPCC databank. The cells were grown in RPMI medium 1640 supplemented with 20% FCS (GIBCO) and 2 mM glutamine (Sigma), in 5% CO 2 at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide

Marra

D’Atri

Corti

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Members of hereditary nonpolyposis colon cancer (HNPCC) familiesharboring heterozygous germline mutations in the DNA mismatch repair genes hMSH2 or hMLH1 present with tumors generally two to three decades earlier than individuals with nonfamilial sporadic colon cancer. We searched for phenotypic features that might predispose heterozygous cells from HNPCC kindreds to malignant transformation. hMSH2 ؉/؊ lymphoblastoid cell lines were found to be on average about 4-fold more tolerant than wild-type cells to killing by the methylating agent temozolomide, a phenotype that is invariably linked with impairment of the mismatch repair system. This finding was associated with an average 2-fold decrease of the steady-state level of hMSH2 protein in hMSH2 ؉/؊ cell lines. In contrast, hMLH1 ؉/؊ heterozygous cells were indistinguishable from normal controls in these assays. Thus, despite the fact that HNPCC families harboring mutations in hMSH2 or hMLH1 cannot be distinguished clinically, the early stages of the carcinogenic process in hMSH2 and hMLH1 mutation carriers may be different. Should hMSH2 ؉/؊ colonocytes and lymphoblasts harbor a similar phenotype, the increased tolerance of the former to DNA-damaging agents present in the human colon may play a key role in the initiation of the carcinogenic process.

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Section: Methodsmentioning

confidence: 99%

Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide

Marra

D’Atri

Corti

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…The methods for IVTT has been previously described (34). Briefly, total cellular RNA was purified from the cell lines by guanidinium isothiocyanate-phenol chloroform extraction (35), and reverse transcribed.…”

Section: Methodsmentioning

confidence: 99%

Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

Carethers¹,

Hawn²,

Chauhan³

et al. 1996

J. Clin. Invest.

125

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The phenomenon of alkylation tolerance has been observed in cells that are deficient in some component of the DNA mismatch repair (MMR) system. An alkylation-induced cell cycle arrest had been reported previously in one MMR-proficient cell line, whereas a MMR-defective clone derived from this line escapes from this arrest. We examined human cancer cell lines to determine if the cell cycle arrest were dependent upon the MMR system. Growth characteristics and cell cycle analysis after MNNG treatment were ascertained in seven MMR-deficient and proficient cell lines, with and without confirmed mutations in hMLH1 or hMSH2 by an in vitro transcription/translation assay. MMR-proficient cells underwent growth arrest in the G2 phase of the cell cycle after the first S phase, whereas MMR-deficient cells escaped an initial G2 delay and resumed a normal growth pattern. The agent N -methyl-N Ј -nitro-N -nitrosoguanidine (MNNG) 1 is an alkylator of DNA capable of producing the methylated purines 7-methylguanine (7-meG), 3-methyladenine (3-meA), and O 6

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“…Oligo(dT)-primed cDNAs were synthesised using the SuperScriptt Preamplification System for First Strand cDNA Synthesis kit (Invitrogen). The complete open reading frame of MSH2 was then amplified as two overlapping segments from 2 ml of the cDNA reaction using primers previously described by Luce et al (1995).…”

Section: Dna and Rna Extraction And Rt -Pcrmentioning

confidence: 99%

Germline truncating mutations in both MSH2 and BRCA2 in a single kindred

et al. 2004

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There has been interest in the literature in the possible existence of a gene that predisposes to both breast cancer (BC) and colorectal cancer (CRC). We describe the detailed characterisation of one kindred, MON1080, with 10 cases of BC or CRC invasive cancer among 26 first-, second-or third-degree relatives. Linkage analysis suggested that a mutation was present in BRCA2. DNA sequencing from III: 22 (diagnosed with lobular BC) identified a BRCA2 exon 3 542G4T (L105X) mutation. Her sister (III: 25) had BC and endometrial cancer and carries the same mutation. Following immunohistochemical and microsatellite instability studies, mutation analysis by protein truncation test, cDNA sequencing and quantitative real-time PCR revealed a deletion of MSH2 exon 8 in III: 25, confirming her as a double heterozygote for truncating mutations in both BRCA2 and MSH2. The exon 8 deletion was identified as a 14.9 kb deletion occurring between two Alu sequences. The breakpoint lies within a sequence of 45 bp that is identical in both Alu sequences. In this large BC/CRC kindred, MON1080, disease-causing truncating mutations are present in both MSH2 and BRCA2. There appeared to be no increased susceptibility to the development of colorectal tumours in BRCA2 mutation carriers or to the development of breast tumours in MSH2 mutation carriers. Additionally, two double heterozygotes did not appear to have a different phenotype than would be expected from the presence of a mutation in each gene alone. Keywords: MSH2; BRCA2; microsatellite instability; colorectal cancer; breast cancer; astrocytoma; multiple primary cancers BRCA1 and BRCA2 are the most important susceptibility genes for breast and ovarian cancer, and mutations in these two genes account for 480% of all kindreds with hereditary breast/ovarian cancer and for about 2 -3% of breast cancer (BC) cases overall. Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common form of hereditary colorectal cancer (CRC) (Lynch and de la Chapelle, 1999) and is responsible for 0.5 -3% of all cases of CRC. Mutations in the DNA repair genes MLH1 and MSH2 segregate in up to 90% of HNPCC pedigrees (Peltomaki, 2001).Several groups have studied the genetic relationship between breast and colorectal cancer, with varying findings. In one large Dutch family with a segregating BRCA1 mutation, there are several mutation carriers who have developed CRC. However, detailed analysis suggested that the BRCA1 mutation was not contributing to the CRCs observed. By contrast, in a large international study, Thompson et al (2002) observed a relative risk of 2.03 (Po0.001) for CRC in BRCA1 mutation carriers, compared with general population cancer incidence. Studies in HNPCC kindreds have shown no excess of BC, or have shown that when BC does occur, microsatellite instability (MSI), a hallmark of HNPCC-related cancer, is usually absent (Aarnio et al, 1995;Caluseriu et al, 2001). Interestingly, Borg et al reported a family with two missense mutations in MLH1 and a single truncating mutation in BRCA1. ...

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In vitro transcription/translation assay for the screening of hMLH1 and hMSH2 mutations in familial colon cancer

Cited by 60 publications

References 30 publications

Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide

Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide

Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

Germline truncating mutations in both MSH2 and BRCA2 in a single kindred

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In vitro transcription/translation assay for the screening of hMLH1 and hMSH2 mutations in familial colon cancer

Cited by 60 publications

References 30 publications

Tolerance of human MSH2 +/− lymphoblastoid cells to the methylating agent temozolomide

Tolerance of human MSH2 +/− lymphoblastoid cells to the methylating agent temozolomide

Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

Germline truncating mutations in both MSH2 and BRCA2 in a single kindred

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Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide

Tolerance of human MSH2 ^+/− lymphoblastoid cells to the methylating agent temozolomide