A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene

Monica Francesca Blasi, Ilenia Ventura, Gabriele Aquilina, Paolo Degan, Lucio Bertario, Chiara Bassi, Paolo Radice, Margherita Bignami

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

We describe a new approach to investigate alterations in the human MLH1 mismatch repair (MMR) gene. This is based on complementation of the phenotype of a MLH1-defective subclone of the ovarian carcinoma A2780 cells by transfection of vectors encoding altered MLH1 proteins. Measurements of resistance (tolerance) to methylating agents, mutation rate at HPRT, microsatellite instability (MSI), and steady-state levels of DNA 8-oxoguanine were used to define the MMR status of transfected clones. The approach was validated by transfecting cDNA of wild-type (WT) MLH1, cDNAs bearing two previously identified polymorphisms (I219V and I219L) and two with confirmed hereditary nonpolyposis colorectal cancer (HNPCC) syndrome mutations (G224D and G67R). A low-level expression of two MLH1 polymorphisms partially reversed methylation tolerance and the mutator phenotype, including MSI. Higher levels of I219V resulted in full restoration of these properties to WT. Increased expression of I129L did not fully complement the MLH1 defect, because there was a simultaneous escalation in the level of oxidative DNA damage. The findings confirmed the important relationship between deficient MMR and increased levels of oxidative DNA damage. Mutations from Italian HNPCC families (G224D, G67R, N635S, and K618A) were all ineffective at reversing the phenotype of the MLH1-defective A2780 cells. One (K618A) was identified as a low penetrance mutation based on clinical and genetic observations.

Original languageEnglish
Pages (from-to)9036-9044
Number of pages9
JournalCancer Research
Volume66
Issue number18
DOIs
Publication statusPublished - Sep 15 2006

Fingerprint

DNA Mismatch Repair
Hereditary Nonpolyposis Colorectal Neoplasms
Microsatellite Instability
Phenotype
Mutation
DNA Damage
Complementary DNA
Genes
Hypoxanthine Phosphoribosyltransferase
Penetrance
Mutation Rate
Methylation
Transfection
Clone Cells
Carcinoma
DNA

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Blasi, M. F., Ventura, I., Aquilina, G., Degan, P., Bertario, L., Bassi, C., ... Bignami, M. (2006). A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene. Cancer Research, 66(18), 9036-9044. https://doi.org/10.1158/0008-5472.CAN-06-1896

A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene. / Blasi, Monica Francesca; Ventura, Ilenia; Aquilina, Gabriele; Degan, Paolo; Bertario, Lucio; Bassi, Chiara; Radice, Paolo; Bignami, Margherita.

In: Cancer Research, Vol. 66, No. 18, 15.09.2006, p. 9036-9044.

Research output: Contribution to journalArticle

Blasi, Monica Francesca ; Ventura, Ilenia ; Aquilina, Gabriele ; Degan, Paolo ; Bertario, Lucio ; Bassi, Chiara ; Radice, Paolo ; Bignami, Margherita. / A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene. In: Cancer Research. 2006 ; Vol. 66, No. 18. pp. 9036-9044.
@article{13cc7dc43a0340aa951940aedd843f34,
title = "A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene",
abstract = "We describe a new approach to investigate alterations in the human MLH1 mismatch repair (MMR) gene. This is based on complementation of the phenotype of a MLH1-defective subclone of the ovarian carcinoma A2780 cells by transfection of vectors encoding altered MLH1 proteins. Measurements of resistance (tolerance) to methylating agents, mutation rate at HPRT, microsatellite instability (MSI), and steady-state levels of DNA 8-oxoguanine were used to define the MMR status of transfected clones. The approach was validated by transfecting cDNA of wild-type (WT) MLH1, cDNAs bearing two previously identified polymorphisms (I219V and I219L) and two with confirmed hereditary nonpolyposis colorectal cancer (HNPCC) syndrome mutations (G224D and G67R). A low-level expression of two MLH1 polymorphisms partially reversed methylation tolerance and the mutator phenotype, including MSI. Higher levels of I219V resulted in full restoration of these properties to WT. Increased expression of I129L did not fully complement the MLH1 defect, because there was a simultaneous escalation in the level of oxidative DNA damage. The findings confirmed the important relationship between deficient MMR and increased levels of oxidative DNA damage. Mutations from Italian HNPCC families (G224D, G67R, N635S, and K618A) were all ineffective at reversing the phenotype of the MLH1-defective A2780 cells. One (K618A) was identified as a low penetrance mutation based on clinical and genetic observations.",
author = "Blasi, {Monica Francesca} and Ilenia Ventura and Gabriele Aquilina and Paolo Degan and Lucio Bertario and Chiara Bassi and Paolo Radice and Margherita Bignami",
year = "2006",
month = "9",
day = "15",
doi = "10.1158/0008-5472.CAN-06-1896",
language = "English",
volume = "66",
pages = "9036--9044",
journal = "Journal of Cancer Research",
issn = "0008-5472",
publisher = "American Association for Cancer Research Inc.",
number = "18",

}

TY - JOUR

T1 - A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene

AU - Blasi, Monica Francesca

AU - Ventura, Ilenia

AU - Aquilina, Gabriele

AU - Degan, Paolo

AU - Bertario, Lucio

AU - Bassi, Chiara

AU - Radice, Paolo

AU - Bignami, Margherita

PY - 2006/9/15

Y1 - 2006/9/15

N2 - We describe a new approach to investigate alterations in the human MLH1 mismatch repair (MMR) gene. This is based on complementation of the phenotype of a MLH1-defective subclone of the ovarian carcinoma A2780 cells by transfection of vectors encoding altered MLH1 proteins. Measurements of resistance (tolerance) to methylating agents, mutation rate at HPRT, microsatellite instability (MSI), and steady-state levels of DNA 8-oxoguanine were used to define the MMR status of transfected clones. The approach was validated by transfecting cDNA of wild-type (WT) MLH1, cDNAs bearing two previously identified polymorphisms (I219V and I219L) and two with confirmed hereditary nonpolyposis colorectal cancer (HNPCC) syndrome mutations (G224D and G67R). A low-level expression of two MLH1 polymorphisms partially reversed methylation tolerance and the mutator phenotype, including MSI. Higher levels of I219V resulted in full restoration of these properties to WT. Increased expression of I129L did not fully complement the MLH1 defect, because there was a simultaneous escalation in the level of oxidative DNA damage. The findings confirmed the important relationship between deficient MMR and increased levels of oxidative DNA damage. Mutations from Italian HNPCC families (G224D, G67R, N635S, and K618A) were all ineffective at reversing the phenotype of the MLH1-defective A2780 cells. One (K618A) was identified as a low penetrance mutation based on clinical and genetic observations.

AB - We describe a new approach to investigate alterations in the human MLH1 mismatch repair (MMR) gene. This is based on complementation of the phenotype of a MLH1-defective subclone of the ovarian carcinoma A2780 cells by transfection of vectors encoding altered MLH1 proteins. Measurements of resistance (tolerance) to methylating agents, mutation rate at HPRT, microsatellite instability (MSI), and steady-state levels of DNA 8-oxoguanine were used to define the MMR status of transfected clones. The approach was validated by transfecting cDNA of wild-type (WT) MLH1, cDNAs bearing two previously identified polymorphisms (I219V and I219L) and two with confirmed hereditary nonpolyposis colorectal cancer (HNPCC) syndrome mutations (G224D and G67R). A low-level expression of two MLH1 polymorphisms partially reversed methylation tolerance and the mutator phenotype, including MSI. Higher levels of I219V resulted in full restoration of these properties to WT. Increased expression of I129L did not fully complement the MLH1 defect, because there was a simultaneous escalation in the level of oxidative DNA damage. The findings confirmed the important relationship between deficient MMR and increased levels of oxidative DNA damage. Mutations from Italian HNPCC families (G224D, G67R, N635S, and K618A) were all ineffective at reversing the phenotype of the MLH1-defective A2780 cells. One (K618A) was identified as a low penetrance mutation based on clinical and genetic observations.

UR - http://www.scopus.com/inward/record.url?scp=33749483796&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33749483796&partnerID=8YFLogxK

U2 - 10.1158/0008-5472.CAN-06-1896

DO - 10.1158/0008-5472.CAN-06-1896

M3 - Article

VL - 66

SP - 9036

EP - 9044

JO - Journal of Cancer Research

JF - Journal of Cancer Research

SN - 0008-5472

IS - 18

ER -