A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene.
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.
Blasi
Monica Francesca
MF
Unit of Experimental Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.
Ventura
Ilenia
I
Aquilina
Gabriele
G
Degan
Paolo
P
Bertario
Lucio
L
Bassi
Chiara
C
Radice
Paolo
P
Bignami
Margherita
M
D048868:Adaptor Proteins, Signal Transducing; D020137:Base Pair Mismatch; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D045744:Cell Line, Tumor; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D000070957:MutL Protein Homolog 1; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000235:genetics*; D011110:Polymorphism, Genetic; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D000070957:MutL Protein Homolog 1