Molecular changes in UV-induced and γ-ray-induced mutations in human lymphoblastoid cells

We have characterized the structural changes in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of 14 UV-induced, 15 γ-ray-induced and 17 spontaneous mutants of human lymphoblastoid cells selected for 6-thioguanine (6TG) resistance. Southern blot analysis using the full-length HPRT cDNA as a probe revealed that 29% (5/17) of the spontaneous mutants contained detectable alterations in their restriction fragment patterns. Among the 15 mutants induced by γ rays, 7 (47%) had such alterations indicative of large deletions in the HPRT gene. In contrast, all 14 UV-induced mutants exhibited hybridization patterns indistinguishable from those of the wild-type cells. These results suggest that UV is likely to induce point mutations at the HPRT locus on the human chromosome and that the molecular mechanism of UV-induced mutation is quite different from that of ionizing radiation-induced mutation or spontaneous mutation in human cells.     

Ionizing radiation and genetic risks. III. Nature of spontaneous and radiation-induced mutations in mammalian in vitro systems and mechanisms of induction of mutations by radiation

This paper (1) presents an analysis of published data on the molecular nature of spontaneously arising and radiation-induced mutations in mammalian somatic cell systems and (2) examines whether the molecular nature and mechanisms of origin of radiation-induced mutations, in mammalian in vivo and in vitro systems, as currently understood, are consistent with expectations based on the biophysical and microdosimetric properties of ionizing radiation. Depending on the test system (CHO cells, human T lymphocytes and human lymphoid cell line TK6), 80-97% of spontaneous HPRT mutations show normal Southern patterns; the remainder is due to gross changes, predominantly partial (intragenic) deletions. Total gene deletions at the HPRT locus are rare except in the TK6 cell line. At the APRT locus in CHO cells, 80-97% of spontaneous mutations are due to base-pair changes, the remainder being, mostly, partial deletions. The latter can extend upstream in the 5' direction but not beyond the APRT gene in the 3' direction. At the human HLA-A locus (T lymphocytes), the percentage of mutations with normal Southern patterns is lower than that for HPRT, and in the range of 50-60%. At the HLA-A locus, mitotic recombination contributes substantially to the mutation spectrum (approximately 30% of mutations recovered) and this is likely to be true of the TK locus in the TK6 cell line as well. With a few exceptions, most of the radiation-induced mutations show altered Southern patterns and are consistent with their being deletions and/or other gross changes (HPRT, 70-90% (CHO); 50-85% (TK6); 50-75% (T lymphocytes); TK, 60-80% (TK6); HLA-A, 80% (T lymphocytes); DHFR, 100% (CHO]. The exceptions are APRT mutations in CHO cells (16-20% of mutants with deletions or other changes) and HPRT mutations in T lymphocytes from A-bomb survivors (15-25%); the latter finding is consistent with the occurrence of in vivo selection against HPRT mutant cells. In cases of HPRT intragenic deletions analyzed (CHO cells and V79 Chinese hamster cells), there is evidence for a non-random distribution of breakpoints. The spontaneous mutation frequencies vary widely, from about 0.04/10(6) cells (sickle cell mutations at the human HBB locus) to 30.8/10(6) cells (HLA-A mutations in T lymphocytes) and are dependent on the locus, the system employed and a number of other factors. Those for the other loci fall between these limits.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular basis of X-ray-induced mutation at the HPRT locus in human lymphocytes

Human lymphocytes lacking functional HPRT enzyme after a dose of 300 rad X-radiation were cloned and the monoclonal populations expanded so that sufficient genomic DNA was obtained for Southern analysis. A total of 33 mutant clones were analysed. Wild-type clones showed no evidence of changes to the HPRT gene resolvable by Southern banding patterns whereas 17 of 33 mutant clones showed changes. The alterations observed included total gene deletions (3 clones) and partial gene deletions with or without the appearance of novel bands (12 clones). Two clones showed the appearance of novel bands only. There were no changes observed in 16 of the 33 mutant clones. Three clones showed changes inconsistent with deletion of portions of the gene. In these clones inversion seems to have been the most likely cause of the mutation. The spectrum of gene alterations following ionizing radiation appears different to that previously observed for spontaneous mutations. Consequently, ionizing radiation or radiomimetic agents would appear to be aetiologic, at the most, for only a minor proportion of in vivo somatic mutations.     

Mutational spectral analysis at the HPRT locus in healthy children

There is growing evidence linking somatic mutational events during fetal development and childhood to an increasing number of multifactorial human diseases. Despite this, little is known about the relationship between endogenous and environmentally induced exogenous mutations during human development. Here we describe a comparative spectral analysis of somatic mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene locus in healthy children. We observed an age-specific decrease in the proportion of large alterations and a corresponding increase in the proportion of small alterations with increasing age following birth (P<0.001). The age specific decrease in the proportion of large alterations (67-30%) was mainly due to a decrease in the proportion of aberrant variable (V), diversity (D) and joining (J) (V(D)J) recombinase mediated HPRT deletions (P<0.001). The increase in the proportion of small alterations with age (28-64%) was associated with an increase in transversions from 8% in children at the late stages of fetal development to 31% in children 12-16 years old (P=0.003). Transitions decreased with age, especially at CpG dinucleotides (P=0.010), as transversions increased (P=0.009). These patterns of mutations provide insight into important spontaneous, genotoxic, and site-specific recombinational somatic mutational events associated with the age-specific development of human disease in children as well as adults.     

The Chinese hamster HPRT gene: restriction map, sequence analysis, and multiplex PCR deletion screen

The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has been determined; the gene has nine exons and is dispersed over 36 kb DNA. Exons 2-9 are contained within overlapping lambda bacteriophage clones and exon 1 was obtained by an inverse polymerase chain reaction (PCR). All the exons have been sequenced, together with their immediate flanking regions, and these sequences compared to those of the mouse and human HPRT genes. Sequences immediately flanking all exons but the first show considerable homology between the different species but the region around exon 1 is less conserved, apart from the preserved location of putative functional elements. Oligonucleotide primers derived from sequences flanking the HPRT gene exons were used to amplify simultaneously seven exon-containing fragments in a multiplex PCR. This simple procedure was used to identify total and partial gene deletions among Chinese hamster HPRT-deficient mutants. The multiplex PCR is quicker to perform than Southern analysis, traditionally used to study such mutants, and also provides specific exon-containing fragments for further analysis. The Chinese hamster HPRT gene is often used as a target for mutation studies in vitro because of the ease of selection of forward and reverse mutants; the information presented here will enhance the means of investigating molecular defects within this gene.     

Mutations induced by X-rays at the HPRT locus in cultured Chinese hamster cells are mostly large deletions

We investigated the molecular basis of 19 X-ray-induced HPRT-deficient mutants of V79 Chinese hamster cells with Southern hybridisation techniques. 12 of those mutants suffer from a big deletion (greater than 10 kb) of HPRT DNA sequences. Cytological studies of chromosome preparations of those 12 deletion mutants showed that in at least 3 of these mutants part of the long arm of the X-chromosome was lost. After correction for spontaneous arising mutations we estimate that at least 70-80% of X-ray-induced mutations are caused by large deletions.     

Molecular analysis of spontaneous and ethyl methanesulphonate-induced mutations of the hprt gene in hamster cells

Independent spontaneous or ethyl methanesulphonate (EMS)-induced mutants lacking HPRT enzyme activity were analysed for changes in hprt gene structure. Of 21 spontaneous mutants, 6 had total gene deletions, 2 had partial gene deletions, and 13 were indistinguishable from wild-type by Southern analysis. In contrast a sample of 23 EMS-induced mutants, each of which showed potentially interesting characteristics (e.g. high reversion frequency, X-chromosome rearrangement), showed no detectable hprt gene changes. RNA isolated from 59 mutants with presumptive point mutations (13 spontaneous, 46 EMS-induced) was analysed on dot blots for changes in the amount of hprt mRNA. A wide range of mRNA levels was found, from mutants with undetectable amounts to those with more than wild-type amounts. However, Northern blots of all these mutant RNAs revealed only one (EMS-induced) mutation with a change in hprt mRNA size. Taken with our previously-published data on these mutants, it is argued that they represent a broad range of mutational types, and that the hprt gene mutation system provides a sensitive means of distinguishing mutational spectra of different DNA-damaging agents.     

Molecular analysis of X-ray-induced mutants at the HPRT locus in V79 Chinese hamster cells

Spontaneous and X-ray-induced mutants at the hypoxanthine phosphoribosyl transferase (HPRT) locus have been isolated from V79 Chinese hamster cells and characterized at the biochemical and cytogenetic levels. Fourteen spontaneous and 24 X-ray-induced clones were azaguanine and thioguanine resistant, did not grow in HAT medium (AZRTGRHATS) and failed to incorporate significant levels of [14C]hypoxyanthine. Cytogenetic analysis of two spontaneous and eight X-ray-induced mutants revealed no major X chromosome rearrangements. In two induced mutants, one of which was hypotetraploid (mode 35-39) with 2 X chromosomes, the short arm of the chromosome (Xp) was slightly shorter than normal. A third mutant was hyperdiploid (mode 22-23) compared with the parental clone (mode 21). When compared with wild-type clones, no other cytogenetic changes were evident in the remaining mutants. Analysis at the DNA level using a Chinese hamster HPRT cDNA probe showed major deletion of HPRT sequences in two and partial deletion in another two induced mutants. In two of the mutants with deletions of HPRT sequences there was a visible shortening of the Xp arm. In the other six mutants two spontaneous and four induced) no karyotypic changes or alterations in restriction fragment patterns were detected suggesting that they carry small deletions or point mutations at the HPRT locus.     

Southern-blot analyses of human T-lymphocyte mutants induced in vitro by gamma-irradiation

G0 phase cultures of human peripheral blood T-lymphocytes from a single individual were exposed to 300 rad of gamma-irradiation from a 137Cs source and cultured in vitro for 8 days to allow phenotypic expression. Thioguanine-resistant (TGr) mutants were isolated by a cell cloning assay in microtiter plates. These mutants were studied by Southern blot analysis to define the gross structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene by use of an hprt cDNA probe. A similar analysis of the T-cell receptor (TCR) gene rearrangement patterns was employed to define the independent nature of each mutant colony by use of TCR beta and gamma cDNA probes. 74 mutants were isolated in 5 separate experiments. TCR gene rearrangement analysis showed these to represent 24 independent mutations, of which 18 contained hprt structural alterations. These alterations included simple deletions (10/18) as well as more complex rearrangements resulting in molecular weight changes of restriction fragments representing both the 5' and 3' regions of the hprt gene (4/18 and 4/18, respectively). These results demonstrate that gamma-irradiation primarily induces TGr mutations through gross structural alterations in the hprt gene and that these alterations are randomly distributed across the gene. This approach to mutation analysis will provide information on the types of alterations induced by this irradiation, especially the extent of deletions involving the hprt gene. These results also demonstrate the feasibility of employing in vitro exposure of human T-lymphocytes to a single mutagenic agent as an aid to understanding the mechanisms of mutations occurring in vivo in humans.     

Quantitative and molecular analyses of ethyl methanesulfonate- and ICR 191-induced mutation in AS52 cells

A pSV2gpt-transformed Chinese hamster ovary (CHO) cell line has been used to study mutation at the molecular level. This cell line, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line, and has been previously shown to contain a single, functional copy of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. In this study, conditions for its use in the study of mammalian cell mutagenesis have been stringently defined. The spontaneous mutation rate (2 X 10(-6)/cell division) and phenotypic expression time (7 days) of the gpt locus compare favorably with those of the hprt locus in wild-type CHO-K1-BH4 cells. While both cell lines exhibit similar cytotoxic responses to ethyl methanesulfonate (EMSO and ICR 191, significant differences in mutation induction were observed. Ratios of XPRT to HPRT mutants induced per unit dose of EMS and ICR 191 are 0.70 and 1.6, respectively. Southern blot hybridization analyses revealed that most XPRT mutant cell lines which arose following treatment with EMS (20/22) or ICR 191 (20/24) exhibited no alterations of the gpt locus detectable by this technique. Similar observations were made for the hprt locus in EMS-(21/21) and ICR 191-induced (22/22) HPRT mutants. In contrast, most spontaneous gpt mutants (14/23) contained deletions, while most spontaneous hprt mutants (18/23) exhibited no detectable alterations. Results of this study indicate that the AS52 cell line promises to be useful for future study of mutation in mammalian cells at the DNA sequence level.     

UV radiation induces delayed hyperrecombination associated with hypermutation in human cells

Ionizing radiation induces delayed genomic instability in human cells, including chromosomal abnormalities and hyperrecombination. Here, we investigate delayed genome instability of cells exposed to UV radiation. We examined homologous recombination-mediated reactivation of a green fluorescent protein (GFP) gene in p53-proficient human cells. We observed an approximately 5-fold enhancement of delayed hyperrecombination (DHR) among cells surviving a low dose of UV-C (5 J/m2), revealed as mixed GFP+/- colonies. UV-B did not induce DHR at an equitoxic (75 J/m2) dose or a higher dose (150 J/m2). UV is known to induce delayed hypermutation associated with increased oxidative stress. We found that hypoxanthine phosphoribosyltransferase (HPRT) mutation frequencies were approximately 5-fold higher in strains derived from GFP+/- (DHR) colonies than in strains in which recombination was directly induced by UV (GFP+ colonies). To determine whether hypermutation was directly caused by hyperrecombination, we analyzed hprt mutation spectra. Large-scale alterations reflecting large deletions and insertions were observed in 25% of GFP+ strains, and most mutants had a single change in HPRT. In striking contrast, all mutations arising in the hypermutable GFP+/- strains were small (1- to 2-base) changes, including substitutions, deletions, and insertions (reminiscent of mutagenesis from oxidative damage), and the majority were compound, with an average of four hprt mutations per mutant. The absence of large hprt deletions in DHR strains indicates that DHR does not cause hypermutation. We propose that UV-induced DHR and hypermutation result from a common source, namely, increased oxidative stress. These two forms of delayed genome instability may collaborate in skin cancer initiation and progression.     

Easy detection of GFP-positive mutants following forward mutations at specific gene locus in cultured human cells

We have generated a new mutation assay system using HT1080 human fibrosarcoma cells, which consists of a combination of tetracycline-operator dependent GFP gene (TetO-EGFP) and tetracycline repressor (TetR) genes, where the expression of GFP gene is under strict control of TetR protein, and the TetR gene is located within the endogenous HPRT gene. In this system, any inactivating mutation at the TetR gene or large deletions including the gene itself results in high expression of GFP gene (>200-fold increase) in the cells, which can be readily scored not only by a flow cytometer but also under a fluorescent microscope. With this new cell line, we show that the spontaneous mutation rate at the TetR locus was 2.8-3.4×10(-6)/cell division, slightly lower than the rate at the endogenous HPRT gene of HT1080 cells, and has a dose response to X rays as a mutagen. We also isolated variant clones with elevated spontaneous mutation rate (i.e., genetically unstable cells) following X irradiation. Spontaneous GFP-positive mutants were predominantly base-change mutations at the TetR gene while those obtained after X irradiation often contained large deletions which spanned up to 6Mb. The results indicate that the bacterial TetR/TetO regulatory units work extremely well as a mutation detection system in human cells, and any part of the human genome may be tested for mutation sensitivity following targeted insertion of the TetR gene in a stably expressing gene.     

Real-time PCR and linkage studies to identify carriers presenting HPRT deleted gene

Lesch-Nyhan syndrome (LNS) is an X-linked genetic disorder resulting in hyperuricemia, choreoathetosis, mental retardation, and self-injurious behavior. It is caused by loss of activity of the ubiquitous enzyme hypoxanthine-guanine-phosphoribosyltransferase (HPRT). The biochemical analysis of residual HPRT activity in patients' red blood cells is the first step in LNS diagnosis, and it precedes molecular study to discover the specific mutation. Unfortunately, biochemical diagnosis of healthy carriers is difficult because HPRT enzymatic activity in blood cells is similar in LNS carriers and in healthy people; genetic tests can help reveal mutations at the genomic or cDNA level, whereas gross deletions involving the first or last exons of HPRT gene are not detectable. Until now, a test based on 6-thioguanine-resistant phenotype of HPRT mutant cells from LNS patients is the only method accepted for the diagnosis of any kind of mutation in carriers. In this work, we introduce a new approach to identify carriers of large deletions in HPRT gene using real-time PCR. Results were validated in a blinded manner with a linkage study and with results obtained in Italian families previously analyzed with selective medium test. Real-time PCR analysis clearly confirmed the results obtained by selective medium; linkage data strengthened real time results, allowing us to follow the allele with the mutated HPRT through the family pedigree. We hope that the real-time PCR approach will provide a useful and reliable method to diagnose LNS carriers of large deletions in HPRT gene.     

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.     

Molecular characterization of hprt mutants induced by low- and hgh-LET radiations in human cells

Southern blotting techniques were employed to examine the spectrum of molecular alterations in DNA induced by internally emitting iodine isotopes and X-rays at and around the hprt locus in a human lymphoblastoid cell line. We analyzed 165 mutant clones using a cDNA probe for the human hprt locus, and 3 anonymous sequenceprobes for regions of the X-chromosome which are linked to hrpt. The results were compared with those for 35 spontaneously arising mutant clones. The majority of ionizing radiation-induced mutants showed changes in the normal restriction patterns at the hprt locus, whereas very few alterations were seen at linked markers along the X chromosome. Total hprt coding sequence deletions comprised 30–48% of the changes observed at this locus, while partial deletions and rearrangements comprised 14–54% of the observed changes. In the case of mutants induced by [^125I]dUrd, a densely ionizing radiation, the spectrum of alterations was dose-dependent; at low doses it was not significantly different from that seen after sparsely ionizing X-ray exposure, whereas a higher proportion of gene deletions and rearrangements occurred after high doses of this incorporated isotope. Changes were rarely observed in the 3 linked markers examined. Overall, these results indicate that the distribution of mutational events at the hprt locus in irradiated human cells may not only be LET-dependent but dose-dependent, and that deletions involving large regions of the X chromosome surrounding the hprt locus are rae events.     

Characterization of HAT- and HAsT-resistant HPRT mutant clones of V79 Chinese hamster cells.

HPRT mutant clones of V79 Chinese hamster cells, isolated after 6-thioguanine (6TG) selection, normally exhibit sensitivity to growth in medium containing the folic acid inhibitor aminopterin or the glutamine analogue L-azaserine (e.g., HAT or HAsT medium). However, it has been shown that some HPRT- clones are resistant to both HAT and HAsT medium. The present study was undertaken to investigate whether any common structural gene alteration exists for such 6TGr-HATr-HAsTr clones. Four clones were studied, 1 of spontaneous origin, 2 induced by a low dose of MNU and 1 EMS-induced. In contrast to wild-type cells and a mutant clone carrying a complete deletion of the HPRT gene, these 4 investigated 6TGr-HATr-HAsTr clones all showed an enhanced incorporation of exogenous 3H-hypoxanthine in the presence of aminopterin and L-azaserine suggesting that these clones carry mutations in the structural part of the HPRT gene. Sequence analysis of PCR-amplified HPRT cDNA from these mutants showed that the spontaneous and the 2 MNU-induced mutant clones lacked exon 4, while the EMS-induced mutant had a GC to AT transition in exon 6. Southern blot analysis of genomic DNA after digestion with BglII, EcoRI and PstI showed no changes in fragment patterns as compared to the wild type. Further sequence analysis of PCR-amplified genomic DNA using exon 4-specific primers showed that all these 3 mutants had an AT to GC or GC to AT transition in exon 4, but had no alterations in the splice sites of exon 4. Based on their characteristics of hypoxanthine incorporation, the present mutant clones fit the model for the proposed functional domains of the HPRT protein.     

Restriction fragment pattern analysis of HPRT mutations induced in rat-liver epithelial cells by alkylating and arylating agents.

Structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in genomic DNA of adult rat-liver (ARL) epithelial cells that were mutated by alkylating and arylating mutagens were studied by restriction enzyme fragment pattern (RFP) analysis. ARL cells were mutated with the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the activation-dependent arylating agents 7,12-dimethylbenz[a]anthracene (DMBA) and N-2-acetylaminofluorene (AAF). Alterations in the HPRT gene of at least 10 independent 6-thioguanine-resistant (TGr) clones mutated by each chemical were analyzed using 8 different restriction endonucleases; Hind III, EcoRI, BamHI, XbaI, Hae III, XhoI, MspI and PstI, and a full-length HPRT cDNA as a probe in molecular hybridization. Among the 10 MNNG-induced mutants, the RFPs obtained with most endonucleases displayed no changes, while an altered RFP was found in only one mutant using XbaI. None of the 10 DMBA-induced mutants displayed altered RFPs. Restriction analysis of the 10 AAF-induced mutants showed no abnormality in HPRT gene structure in most restriction digests, while altered RFPs were detected in one mutant using MspI and in two mutants with XbaI digestion. Overall, the studies reveal an absence of major DNA sequence changes in 26 of 30 induced mutants although the mutant phenotype of 4 of the TGr clones can be attributed to gross chromosomal changes or a point mutation at the restriction site. The absence of detectable alterations in the RFPs of the majority of the mutants is strongly suggestive of base substitution as the major molecular alteration underlying the mutant phenotype. The HPRT activity of 14 of 30 mutants was at least 5% of the wild-type level, which is consistent with a structural alteration in the gene product expressed as partial activity of the enzyme. Therefore, the data are interpreted as indicating that in the ARL cells, all 3 mutagens induced primarily localized alterations in base sequences in the HPRT gene together with a few mutations involving large sequence changes.     

Radiation-induced HPRT mutations resulting from misrejoined DNA double-strand breaks

DNA double-strand breaks (DSBs) are the most severe lesions induced by ionizing radiation, and unrejoined or misrejoined DSBs can lead to cell lethality, mutations and the initiation of tumorigenesis. We have investigated X-ray- and alpha-particle-induced mutations that inactivate the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in human bladder carcinoma cells and in hTERT-immortalized human fibroblasts. Fifty to 80% of the mutants analyzed exhibited partial or total deletions of the 9 exons of the HPRT locus. The remaining mutants retained unaltered PCR products of all 9 exons but often displayed a failure to amplify the HPRT cDNA. Hybridization analysis of a 2-Mbp NotI fragment spanning the HPRT gene with a probe 200 kbp distal to the HPRT locus indicated altered fragment sizes in most of the mutants with a wild-type PCR pattern. These mutants likely contain breakpoints for genomic rearrangements in the intronic sequences of the HPRT gene that allow the amplification of the exons but prevent HPRT cDNA amplification. Additionally, mutants exhibiting partial and total deletions of the HPRT exons also frequently displayed altered NotI fragments. Interestingly, all mutations were very rarely associated with interchromosomal exchanges analyzed by FISH. Collectively, our data suggest that intrachromosomal genomic rearrangements on the Mbp scale represent the prevailing type of radiation-induced HPRT mutations.     

Mutations induced by benzo[a]pyrene diolepoxide at the hprt locus in human T-lymphocytes in vitro

Human T-lymphocytes have been treated with benzo[a]pyrene diolepoxide (BPDE) in vitro and T-cell clones mutated in the hprt gene have been isolated. The mutant frequencies in BPDE-treated T-cell cultures were on average 24-fold higher than those of untreated cultures. Thus, BPDE is a potent inducer of gene mutation in this system. In order to examine which types of mutations are induced by BPDE in human cells, 41 spontaneous and 44 BPDE-induced mutant clones have been characterized using the Southern blot technique. In addition, rearrangements of the T-cell-receptor beta and gamma loci have been used to determine the proportion of isolated clones that are unique, and thus likely to represent independent mutational events. Out of 23 independent spontaneous mutants 4 had large hprt alterations that could be detected on Southern blots. Two of these alterations, deletions of exons 2-6, have been confirmed using PCR of hprt cDNA and direct sequencing of the PCR product. All 33 independent BPDE-induced mutants had normal hprt restriction patterns which indicates that BPDE is mainly a point mutagen in this system.