- Email: [email protected]
P III A.6 Analysis of the background hprt mutant frequency and microsatellite mutations in healthy young adults
S·IlI : Mismatch repair. replication fidelity and cancer On the other hand, the mutagen ic process occuring in E. coli C600su+ host cells, perm issive for Asus08 phage DNA replicat ion, is different from that occunng in 594su- cells. It may occur in the IlmllDC deletion mutant , C600SIl+t1llmIlDC. but requires induction of other SOS function(s). To learn more about the mutagenic pathways occuring under permissive and nonperm issive conditions for phage DNA replication , the effect of mismatch repair was studied . Results on deficien cy of mismatch repair in UV- and MMS- induced mutagenesis in ),sus08 in SIl- and su+ host cells will be presented, Keyword(s): Mutagenesis; ), phage ; Mismatch repair
S19
These findings suggest that resistance to the cytotox ic effects of 6-TG is directly associated with an increase in mutations in MMR-defective cells. Keyword(s): mismatch repair; HPRT; 6-thioguanine
Ip III A.81
Senslllvity to CCNU In methylallon toleranl mlsmatch repair defective human cells
Gabriele Aquilina, Sabrina Cecconi, Simone Martinelli , Richard Hampson' , Margherita Bignami. Istituto Superiore di Sanita', 00161 Rome. Italy;
tImperial Cancer Research Fund. South Mimms. UK
Ip III A.61
Analysis oflhe background hprl mutanl frequency and microsatelllte mutallons In healthy young adull.
Margaret Davies. Joanne Turner, Paul Rurnsby. BIBRAInternational. Wood-
mansteme Road. Carsbalton, Surrey, UK Inter-ind ividual variation in the hprt mutant frequencies obta ined in the Tcell clonal assay using human populations often makes the results difficult to interpret. To investigate some of the factors that may influence the background mutant frequency we have examined a group of fifty healthy, non-smoking, young adults, aged 18 to 25 years. Using both hpn mutant and wild-type clones from the same population we are also analysing a series of microsatellite sequences. at least one of the which is representative of each chromosome of the human genome. This will indicate inter-individual variation in the ability to repair mismatch damage and determine the likelihood of mutations occurring at multiple sites within a subpopulation of cells . A life-style questionnaire was used to assess factors such as occupation, dietary habits and alcohol intake which may influence the mutant frequency. In addition, polymorphisms in the CYPIAI, GSTMI and NAn genes were detennined to assess their role on the mutation rate. 6 The hprt mutant frequenc ies ranged from 0.25 to 9.64 per 10 cells and this distribution could not be attributed to differences in life-style factors . The higher frequencies were not related to the heterozygous Msp I polymorphism of CYPIAI (18% of subjects) , the null genotype of GST (SOO!o of subjects), nor the NAn polymorphism (50'10 of subjects) resulting in the slow acetylator phenotype. Analysis of the microsatellite sequences is in progress but again does not appear to be related to the factors described here. Keyword(s) : hprt ; microsatellites; background frequency
Ip III A.71
Resistance to 6-thloguanlne In mismatch repairdenclent human cancer cell lines correlates wllh an Inerease In mulatlons al Ihe IIPRT locus
Warren E. Glaab 1.' , K.enneth R. Tindall l ,2. /Curriculurn in Toxicology; Uni-
versityof North Carolina; Chapel Hill, NC. USA; J Laboratory of EIWironmentalCarcinogenesis and Mutagenesis. NationalInstitute ofEnoiranmental Health Sciences. Research 1l'iangle Parle. NC, USA The cytotox ic and mutagen ic response at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus to 6-thioguanine (6-TG) exposure were determined in human cancer cell lines defective in mismatch repair (MMR) . Additionally, the defects in these cell lines have been complemented by chromosome transfer, resulting in isogenic matched Jines. Differences in the cellular response to 6-TG between the MMR-defic ient cell line and the cells which complement the specific MMR-gene defects by chromosome transfer help to illustrate the role that specific MMR genes play in the observe phenotypes. Our findings support the not ion that cytotoxicity to 6-TG is mediate through functional MMR, as indicated by resistance to 6-TG in MMR-deficient cells an sensitivity 10 6-TG in cells for which the MMR defect bas been complemented by chromosome transfer. Furthermore, the induced mutant frequency following exposure to 6-TG is significantly increased in the MMR-deficient Jines as compared to the induced mutant frequencies observed in the MMR-proficient chromosome transfer cell lines.
Resistance to N-methylnitrosourea (MNU) was analyzed in several mismatch repair defect ive (MMR-) human cell lines. The majority of these lines express 06-methylguanine-DNA-methyltransferase (MGMT) activity (Mex+ phenotye) which confers resistance to MNU independently from MMR proficiency. Depletion ofMGMT by growth in the presence of06-benzylguanine (0 6-BG) sensitize MMR+ HeLaS3 and HT29 cells IOO-fold to MNU. In contrast 7 out of 7 MMR- and Mex+ human tumor cell lines (HCTlI6. LoVo, DLDI, AN3CA. HEC-IA. LSI74T. DUl4S) were not sensitized by MGMT depletion and were thus all tolerant to MNU damage. Methylation tolerant MMR- HeLa, Raji and CHO cell liines exhibited a concomitant hypersensitivity to CCNU. To determine whether CCNU sensitivity is a general feature of MMR- cells, we compared a number of MMR+ and MMR- human tumor cell lines. Maximal 06-BG sensitization to CCNU was 4-fold in the MMR+ HeLaS3 and HT29 cells and 8-fold in 5 MMRtumor cell Jines (HCTI16, LoVo, AN3CA, LS 174T and HEC·IA). Hypersens itivity to CCNU was not seen in the MMR- lines OLDI and DUl45. These were the only representative, among the 7 MMR- cell Jines, which had mutant p53 . Although mismatch repair defects are involved in conferring sensitivity to CCNU, a possible role of the p53 status of the cells is also suggested . The sensitivity to CCNU in MMR- cells suggests a possible involvement in repairing interstrand crosslinks and may have implications for clinical treatment of MMR- tumors. Keyword(s): mismatch repair; CCNU; methylation tolerance
S19
These findings suggest that resistance to the cytotox ic effects of 6-TG is directly associated with an increase in mutations in MMR-defective cells. Keyword(s): mismatch repair; HPRT; 6-thioguanine
Ip III A.81
Senslllvity to CCNU In methylallon toleranl mlsmatch repair defective human cells
Gabriele Aquilina, Sabrina Cecconi, Simone Martinelli , Richard Hampson' , Margherita Bignami. Istituto Superiore di Sanita', 00161 Rome. Italy;
tImperial Cancer Research Fund. South Mimms. UK
Ip III A.61
Analysis oflhe background hprl mutanl frequency and microsatelllte mutallons In healthy young adull.
Margaret Davies. Joanne Turner, Paul Rurnsby. BIBRAInternational. Wood-
mansteme Road. Carsbalton, Surrey, UK Inter-ind ividual variation in the hprt mutant frequencies obta ined in the Tcell clonal assay using human populations often makes the results difficult to interpret. To investigate some of the factors that may influence the background mutant frequency we have examined a group of fifty healthy, non-smoking, young adults, aged 18 to 25 years. Using both hpn mutant and wild-type clones from the same population we are also analysing a series of microsatellite sequences. at least one of the which is representative of each chromosome of the human genome. This will indicate inter-individual variation in the ability to repair mismatch damage and determine the likelihood of mutations occurring at multiple sites within a subpopulation of cells . A life-style questionnaire was used to assess factors such as occupation, dietary habits and alcohol intake which may influence the mutant frequency. In addition, polymorphisms in the CYPIAI, GSTMI and NAn genes were detennined to assess their role on the mutation rate. 6 The hprt mutant frequenc ies ranged from 0.25 to 9.64 per 10 cells and this distribution could not be attributed to differences in life-style factors . The higher frequencies were not related to the heterozygous Msp I polymorphism of CYPIAI (18% of subjects) , the null genotype of GST (SOO!o of subjects), nor the NAn polymorphism (50'10 of subjects) resulting in the slow acetylator phenotype. Analysis of the microsatellite sequences is in progress but again does not appear to be related to the factors described here. Keyword(s) : hprt ; microsatellites; background frequency
Ip III A.71
Resistance to 6-thloguanlne In mismatch repairdenclent human cancer cell lines correlates wllh an Inerease In mulatlons al Ihe IIPRT locus
Warren E. Glaab 1.' , K.enneth R. Tindall l ,2. /Curriculurn in Toxicology; Uni-
versityof North Carolina; Chapel Hill, NC. USA; J Laboratory of EIWironmentalCarcinogenesis and Mutagenesis. NationalInstitute ofEnoiranmental Health Sciences. Research 1l'iangle Parle. NC, USA The cytotox ic and mutagen ic response at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus to 6-thioguanine (6-TG) exposure were determined in human cancer cell lines defective in mismatch repair (MMR) . Additionally, the defects in these cell lines have been complemented by chromosome transfer, resulting in isogenic matched Jines. Differences in the cellular response to 6-TG between the MMR-defic ient cell line and the cells which complement the specific MMR-gene defects by chromosome transfer help to illustrate the role that specific MMR genes play in the observe phenotypes. Our findings support the not ion that cytotoxicity to 6-TG is mediate through functional MMR, as indicated by resistance to 6-TG in MMR-deficient cells an sensitivity 10 6-TG in cells for which the MMR defect bas been complemented by chromosome transfer. Furthermore, the induced mutant frequency following exposure to 6-TG is significantly increased in the MMR-deficient Jines as compared to the induced mutant frequencies observed in the MMR-proficient chromosome transfer cell lines.
Resistance to N-methylnitrosourea (MNU) was analyzed in several mismatch repair defect ive (MMR-) human cell lines. The majority of these lines express 06-methylguanine-DNA-methyltransferase (MGMT) activity (Mex+ phenotye) which confers resistance to MNU independently from MMR proficiency. Depletion ofMGMT by growth in the presence of06-benzylguanine (0 6-BG) sensitize MMR+ HeLaS3 and HT29 cells IOO-fold to MNU. In contrast 7 out of 7 MMR- and Mex+ human tumor cell lines (HCTlI6. LoVo, DLDI, AN3CA. HEC-IA. LSI74T. DUl4S) were not sensitized by MGMT depletion and were thus all tolerant to MNU damage. Methylation tolerant MMR- HeLa, Raji and CHO cell liines exhibited a concomitant hypersensitivity to CCNU. To determine whether CCNU sensitivity is a general feature of MMR- cells, we compared a number of MMR+ and MMR- human tumor cell lines. Maximal 06-BG sensitization to CCNU was 4-fold in the MMR+ HeLaS3 and HT29 cells and 8-fold in 5 MMRtumor cell Jines (HCTI16, LoVo, AN3CA, LS 174T and HEC·IA). Hypersens itivity to CCNU was not seen in the MMR- lines OLDI and DUl45. These were the only representative, among the 7 MMR- cell Jines, which had mutant p53 . Although mismatch repair defects are involved in conferring sensitivity to CCNU, a possible role of the p53 status of the cells is also suggested . The sensitivity to CCNU in MMR- cells suggests a possible involvement in repairing interstrand crosslinks and may have implications for clinical treatment of MMR- tumors. Keyword(s): mismatch repair; CCNU; methylation tolerance