Spectrum of somatic mutation at the hypoxanthine phosphoribosyltransferase (hprt) gene of healthy people

Understanding the significance of somatic mutations requiresknowledge of the mutations that occur in vivo in healthy people.The molecular characterization of mutations in the hypoxanthinephosphoribosyltransferase (hprt) gene in 217 independent T-lymphocytemutants from 172 donors, including smoking and non-smoking malesand females, reveals a broad spectrum of in vivo somatic mutationoccurring in a population of healthy people. Identificationof the DNA alteration in individual mutant clones was accomplishedusing either one or a combination of multiplex polymerase chainreaction analysis of genomic DNA, sequencing of cDNA, and genomicDNA sequencing. The total spectrum consists of 59% (128/217)base substitutions: 126 simple and two tandem CC>TT basesubstitutions; 39% (85/217) deletion/insertion type mutations:30 frameshifts, 26 small (3–200 basepairs) and 27 largedeletions, and two duplications; and the remaining 2% (4/217)complex mutations involving the deletion of one to 11 basepairswhich are replaced by 1 to 10 basepairs. No significant differencewas detected between the base substitution spectra for the smokersand the non-smokers. Analysis of the number of mutations occurringat any one base position led to the identification of threehotspots for mutations at basepairs 197, 508 and 617, in thehprt gene coding region. Spontaneous deamination of CpG maybe implicated in the creation of basepair 508 as a hotspot sinceall mutations detected are C>T transitions resulting in thenonsense mutation, TAG. At basepairs 197 and 617 both G>Ttransversions and G>A transitions were found indicating thatat least two mechanisms were involved in creating mutationsat these positions. Comparison of the mutation spectra fromtwo populations can provide insight into the origin of the mutations.This study provides an excellent base for comparison of mutationspectra in other human populations.     

Deletion mutations in the hprt gene of T-lymphocytes as a biomarker for genomic rearrangements important in human cancers

The DNA sequence of 11 in vivo-arising intragenic deletion junctionsoccurring in the hypoxanthine-guanine phospho-ribosyltransferase(hprt) gene of human T-lymphocytes was determined. These deletionsranged in size from 16 bp to 4057 bp. Extensive homology wasnot found at the deletion breaksites, indicating that non-homologousrecombination was responsible for these deletions. Short regionsof homology (1–3 nucleotides) at the deletiontermini,which may direct the recombination event, were found in sevenof the mutations. Only one mutation had an unaccounted for nucleotideat the junction. V(D)J recombinase recognition sequences, previouslyidentified at other hprt deletion breaksites, were not present.Such features are also found at the deletion and translocationjunctionsof rearranged oncogenes and suppressor oncogenes. Theability to isolate and molecularly analyze deletion mutationsoccurring in vivo in peripheral human T-lymphocytes allows theassay of DNA breakage/rejoining events. Such a system may serveas a biomarker of exposure to environmental and occupationalagents which may be important in the etiology of cancer.     

In vitro mutational specificity of cisplatin in the human hypoxanthine guanine phosphoribosyltransferase gene.

The in vitro mutational spectra of cisplatin [cis-diamminedichloroplatinum(II)] in exon 3 of the human hypoxanthine guanine phosphoribosyltransferase gene in B-lymphoblasts was examined by a combination of polymerase chain reaction and denaturing gradient gel electrophoresis. Several thousand independent mutants were induced at the hypoxanthine guanine phosphoribosyltransferase locus by cisplatin and were selected en masse by addition of 6-thioguanine to the bulk culture. Polymerase chain reaction was used to amplify exon 3 from the complex mutant population, and denaturing gradient gel electrophoresis was used to separate wild-type DNA sequences from mutant sequences. Mutational hotspots were visible as discrete bands on the denaturing gradient gel. Scanning densitometry was used to determine the fraction of the complex population represented by the novel bands. The mutant bands were excised from the denaturing gradient gel and sequenced. In this way, the nature and frequency of mutational hotspots in a population of several thousand mutants were determined. Cisplatin produced several mutational hotspots in exon 3. About 9-10% of the cisplatin-induced mutants had mutations in a GGGGGG sequence (base pairs 207-212). GC----AT substitutions at the second and third guanines in the 5'-GGGGGG-3' run made up about 2 and 4% of the induced mutants, respectively. About 4% of the induced mutants contained a GC----TA substitution at the sixth guanine. About 1% of the cisplatin-induced mutants had an AT----TA transversion in a TAGA sequence (base pair 271; mutated base is underlined). Our results are consistent with mutations occurring at GpG and ApG sites. These nucleotide sequences have been identified as the primary sites of cisplatin adduction.     

Site specificity of N-methyl-N-nitrosourea-induced transition mutations in the hprt gene

The reaction product of N-methyl-N-nitrosourea (MNU) with DNA, O6-methylguanine (O6-MeG), is responsible for the mutagenic and carcinogenic effects of this carcinogen. These involve activation of the H-ras proto-oncogene in rat mammary tumors by MNU, with a high frequency of GC to AT transitions in codon 12 of this gene. The present study aimed to investigate the types and position specificities of mutations induced by MNU in another gene, the hprt gene of V79 Chinese hamster cells. Furthermore, since processes involved in the expression of genetic damage, e.g. the state of the DNA precursor pool, have been suggested to be important factors in carcinogenesis, the mutagenic specificity of MNU was also studied in the presence of an imbalanced nucleotide pool. Isolation of independent hprt mutant clones from three groups treated in different manners was performed. Two different doses of MNU and a low dose of MNU in combination with hydroxyurea (HU) were employed. Comparison of the results with the two doses of MNU did not indicate any shift in mutation specificity. The majority of the mutations induced by MNU were base substitutions, mostly transitions of GC to AT showing high affinity for the middle base in 5'-purine-G-N-3' sequences (15/18) in the nontranscribing strand, suggesting a difference in repair capacity for the two strands. The relatively high frequency of the base substitutions resulting in splicing defects is explained by the presence of a consensus sequence (5'-purine-g-N-3') in the splice sites of the hprt gene. The results from the HU/MNU group showed a few more GC to TA transversions, though not statistically significant, which may be caused by a shift from miscoding to non-coding recognition of the O6-MeG lesion. The same reactive decomposition products formed from MNU are also formed from a variety of other carcinogenic compounds, e.g. N-methyl-N'-nitro-N-nitrosoguanidine, dimethylnitrosamine, nitrosocimetidine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, suggesting that our findings concerning the mutagenic specificity of MNU in mammalian cells are valid also for these other compounds as well.     

Hypoxanthine:guanine phosphoribosyltransferase activity in xenografts of human osteosarcoma

It has recently been reported that human osteosarcomas may lack the purine salvage pathway enzyme, hypoxanthine:guanine phosphoribosyltransferase (EC 2.4.2.8). We have established a quantitative assay for measurement of this enzyme in human osteosarcoma xenografts with analysis of products by thin-layer chromatography. Nucleotidase or phosphatase activity was readily detected and could be abolished by preheating cytosol at 60 degrees C for 10 min and performing the assay at pH 10. Alternatively, the use of 25 mM NaF at pH 7.4 also inhibited this activity. The pH optimum for this enzyme in red blood cell sonicates and tumor cytosols was pH 10. All six human osteosarcoma xenografts contained hypoxanthine:guanine phosphoribosyltransferase activity ranging from 0.97 to 4.06 nmol/min/mg of protein at pH 7.4. Control human red blood cell sonicates demonstrated activity of 0.83 nmol/min/mg of protein. These data demonstrate that human osteosarcoma xenografts contain substantial activities of this purine salvage pathway enzyme.     

Variation in the protein coding region of the human p53 gene

This report describes a restriction fragment length polymorphism for the enzyme BglII caused by a conserved C to T change at residue 21 of the human p53 gene. This RFLP could potentially be misinterpreted as a rearrangement or a point mutation if paired constitutional tissue is not simultaneously analyzed.     

Induction of hprt gene mutations in splenic T-lymphocytes from the rat exposed in vivo to DNA methylating agents is correlated with formation of O6-methylguanine in bone marrow and not in the spleen

The suitability of splenic T-lymphocytes as a substitute tissuefor detection of genotoxic effects induced in vivo by chemicalagents that are organ-specifically activated was tested in ratsexposed to single doses at the potent lung-carcinogen 4-(methylnitrosamino)-1-(3-pyri-dyl)-1-butanone(NNK), acetoxymethylmethylnitrosamine (AMMN) or N-methyl-N-nitrosourea(MNU). NNK, AMMN and MNU methylate DNA most likely via the formationof a methanediazohydroxide ion that decomposes to a methyl diazoniumion.For all three agents, an increase in the levels of O⁶-methylguanineand 7-methylguanine in DNA of rat liver and lung was detectedby reverse phase HPLC and electrochemical detection. Treatmentwith NNK did not result in the formation of O⁶-methylguanineand 7-methylguanine in DNA of bone marrow and spleen, correspondingwith the absence of metabolic activation pathways for this compoundin these tissues. For AMMN formation of both O⁶-methylguanineand 7-methylguanine was detectable in DNA of the spleen, whereasin DNA of bone marrow only very low frequencies of 7-methylguaninewere found at a toxic dose. MNU induced O⁶-methylguanine and7-methylguanine in both spleen and bone marrow. Using splenicT-lymphocytes from the rat no increase above control levelsof the hprt mutant frequencies was found for NNK and AMMN forall exposure levels tested, 32 days after chemical exposure.For MNU a dose-dependent increase in hprt mutant frequency wasfound at exposure levels of 0.097 mmol/kg up to 0.582 mmol/kg.DNA sequence analysis was performed on PCR products of hprtcDNA from 39 MNU-induced 6-thioguanine-resistant T-lymphocyteclones. Single base pair substitutions were found in 25 of thesemutants (64%), GC     

Specificity of mutations induced by N-methyl-N-nitrosourea in a cDNA of the hprt gene

N-Methyl-N-nitrosourea (MNU) reacts with DNA to produce a varietyof lesions, of which O⁶-methylguanine (O⁶-MeG) has been implicatedin the mutagenic and carcinogenic effects of this agent. Thepresent study aimed to investigate the types and position specificitiesof mutations induced by MNU. Mutational sequence alterationswere determined for 53 independent mutations induced by MNUin a cDNA of the human hprt gene, which is stably integratedinto chromosomes of the mouse cell line VH12. The majority ofthe mutations induced by MNU were base substitutions (85%),mostly GC to AT transitions (41/43), and the remainder (15%)were frameshift or deletion mutations resulting from loss oraddition of a few bases. The transition mutations occurred preferentiallyat the middle G in 5'-purine-G-N-3' sequences in the non-transcribedstrand, and were distributed non-randomly over the coding regionof the gene. Analysis of the results suggests that, when interpretingmutational specificity and distribution, not only the natureof the mutational target sequence, but also the functional domainsof the protein should be considered.     

Biomarkers of styrene exposure in lamination workers: levels of 06-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes

Occupational exposure to styrene was studied in nine workersof a hand lamination plant in Bohemia. Personal dosimeters wereused to monitor the styrene workplace exposure, and the levelsof styrene in blood and mandelic acid in urine were measured.Blood samples were taken at four occasions during a 7 monthperiod to determine styrene-specific 0⁶-guanine DNA adductsin lymphocytes and granulocytes, DNA strand breaks and hypoxanthineguanine phosphoribosyltransferase (HPRT) mutant frequency inT-lymphocytes. Seven administrative employees in the same factory(factory controls) and eight persons in a research laboratory(laboratory controls) were used as referents. DNA adduct levelsdetermined by the ³²P-postlabelling method in lymphocytes oflamina-tors were remarkably constant and significantly higher(P < 0.0001) than in factory controls at all four samplingtimes. HPRT mutant frequencies (MF) measured by the T-cell cloningassay were higher in the laminators (17.5 x10^–6, groupmean) than in the factory controls (15.7x10^–6, group mean)at three of the four sampling times, but the differences werenot statistically significant. However, a statistically significant(P = 0.021) difference between MF in the laminators (18.0 x10^–6,group mean) and laboratory controls (11.8 xl0^–6, groupmean) was observed at sampling time 4 (the only sampling timewhen this latter group was studied). This result indicates thatstyrene exposure may induce gene mutation in T-cells in vivo.DNA strand breaks were studied by the ‘Comet assay’at the fourth sampling time. The laminators were found to havesignificantly higher levels of DNA strand breaks than the factorycontrols (P = 0.032 for tail length, TL; P = 0.007 for percentageof DNA in tail, T%; and P = 0.020 for tail moment, TM). A statisticallysignificant correlation was also found between the levels oflymphocyte DNA adducts and all three DNA strand break parameters(TL P = 0.046; T% P = 0.026 and TM P = 0.034). On the contrary,no significant correlations were found between DNA adduct levelsand the HPRT mutant frequencies or between the mutant frequenciesand DNA strand breaks. Taken together, these results add furthersupport to the genotoxic and possibly mutagenic effects of styreneexposure in vivo. However, no simple quantitative relationshipseems to exist between the levels of styrene-induced DNA damageand frequency of HPRT mutation in T-lymphocytes.     

Spectrum of cis-diamminedichloroplatinum(II)-induced mutations in a shuttle vector propagated in human cells

The supF gene of the shuttle vector pZ189 was used as a target for the study of mutations induced by cis-diamminedichloroplatinum(II) (cis-DDP). Normal human repair-proficient fibroblasts and cis-DDP repair-deficient xeroderma pigmentosum (XP) cells were used as host cells to study the effect of cis-DDP on the inhibition of shuttle vector replication and mutagenesis. Transfection of cis-DDP-treated pZ189 into normal and XP cell lines resulted in a marked increase in the mutation frequency and a decrease in the replication efficiency of the vector. However, these effects were much greater for the plasmid propagated in XP cells. Atomic absorption spectroscopy showed that six to eight Pt-DNA adducts per plasmid were necessary to inhibit plasmid replication by 50% in normal cells. In contrast, only one to two Pt-DNA adducts were necessary to inhibit replication of the plasmid by 50% in XP cells. Analysis of mutation sites demonstrated that cis-DDP treatment resulted primarily in single and double mutations separated by one base and limited to a few locations within the 85-bp mature tRNA. Propagation of the cis-DDP-treated vector in either normal or XP cells led to predominantly transversion mutations at AGA, AGG, and GAG sites and a cis-DDP-associated deletion of 174 bp. Although mutations occurred at target sites for cis-DDP adduct formation, there was no correlation between sites of mutation and the most frequent sites of adduct formation.     

DNA sequence analysis of 1-nitrosopyrene-induced mutations in the hprt gene of Chinese hamster ovary cells.

DNA sequence was determined in 21 mutants induced at the hprt locus of Chinese hamster ovary (CHO) cells by 1-nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1-nitropyrene. Following cDNA synthesis using RNA from each of the mutants, the hprt protein-coding region was amplified by the polymerase chain reaction (PCR) and subjected to direct DNA sequence analysis. Sixteen primary mutations were found: seven were G:C----T:A transversions, five were G:C----A:T transitions, two were single basepair insertions, one was a single basepair deletion, and one was a complex mutation involving substitutions at two A:T basepairs. The simple basepair substitution mutations preferentially occurred with one or two purines 3' to the mutated dG, and mutations in exons 1-4 disproportionately occurred with the mutated dG on the nontranscribed DNA strand. In addition, 12 of the mutants produced one or more cDNA PCR products with partial or complete exon deletions. Seven mutants with multiple PCR products had point mutations in one of the products; exon deletions in the other product(s) removed these point mutations. A group of solvent control mutants had a different distribution of basepair substitution mutations and a lower proportion of cDNAs with exon deletions than that found for the 1-nitrosopyrene-induced mutants. The results indicate a specificity for the induction of mutations in the hprt gene of CHO cells by 1-nitrosopyrene with respect to both the types of mutations produced and their location in the hprt gene. Also, the elimination of point mutations in many of the cDNA PCR products with exon deletions suggests that mutations in the protein-coding sequence affect hprt mRNA processing.     

Kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the hprt gene of Chinese hamster ovary cells

The kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone(MX) in the protein coding region of the hprt gene of Chinesehamster ovary (CHO) cells were determined by direct sequencingof polymerase chain reaction (PCR)-amplified cDNA. Primary mutationswere found in 15 of 19 of the mutants: 11 were G:CT:A transversions,two were A:TT:A transversions and two were deletions of singleG:C base pairs (-1 frameshifts). The remaining four mutantshad large alterations in the cDNA that were explained by mRNAsplicing errors. A group of control mutants had more diversehprt cDNA alterations than MX-induced mutants. Transversionsyielding an A:T base pair were the predominant type of MX-inducedmutations, in agreement with previous findings in bacteria.This specificity may be explained by the ‘A rule’,that DNA polymerases preferentially insert, adenine nucleotidesopposite non-instructional lesions.     

Hprt mutations in human T-lymphocytes reflect radioprotective effects of the aminothiol, WR-1065

Aminothiols such as WR-2721 and its active free thiol WR-1065have previously been shown to reduce mutations resulting fromionizing radiation in exponentially growing cells. In this study,non-dividing human G_o T-lymphocytes were exposed to the aminothiolradioprotective agent, WR-1065, 30 min before or 3 h after externalbeam -irradiation and subsequently assessed for survival andmutation induction at the hprt locus. Cytotoxicity due to -irradiationwas reduced only when the WR-1065 was present during irradiation.The frequency of hprt mutations, however, was reduced regardlessof time of administration, although the reduction was statisticallysignificant only when WR-1065 was added 30 min before irradiation(P < 0.01). This is the first study to demonstrate the protectiveeffects of WR-1065 against radiation-induced mutation in a reportergene using a human non-cycling cell. Hprt mutations arisingin vivo in these cells may be useful for monitoring the radioprotectiveeffect of aminothiols in human populations.     

Spectrum of mutations induced by methyl and ethyl methanesulfonate at the hprt locus of normal and tag expressing Chinese hamster fibroblasts

This work describes the isolation and characterization of methylmethanesulfonate (MMS) and ethyl methane-sulfonate (EMS) induced6-thioguanine-resistant mutants in normal and Escherichia colitag gene expressing Chinese hamster fibroblast, RJKO, cells.It was previously shown that increased removal of 3-alkylatedadenine, effected by 3-methyladenine DNA glycosylase I (Tag),reduces the frequencies of hprt mutations induced by alkylatingagents which produce mostly N-alkylation (MMS and EMS) to halfthe normal rate (12). In order to identify which type of mutationis suppressed by increased 3-alkyladenine repair we have determinedthe DNA base sequence changes of the hprt cDNA in 61 independentMMS- and EMS-induced mutant clones. For both cell types andirrespective of the agent used, the majority of mutations wereGC to AT transitions originating in the non-transcribed strand.Only 6/55 base substitutions occurred at AT base pairs: fiveAT to GC transitions and one AT to CG transversion. Six mutationswere found to be deletions. These results indicate that 3-alkylatedadenines in DNA are not directly premutagenic. The fact thatthe mutation frequency is reduced by increased 3-alkyladenineremoval might be explained by postulating the existence in mammaliancells of an SOS-like response turned on by cytotoxic lesionslike 3-alkyladenine, or, alternatively, that increased removalof 3-alkyladenine increases the number of single-strand breaksin DNA, which stalls DNA replication and allows a prolongedtune for DNA repair by the alkyltransferase.     

Molecular analysis of spontaneous hypoxanthine phosphoribosyltransferase mutations in thioguanine-resistant HL-60 human leukemia cells

We have measured the forward mutation rate at the hypoxanthine phosphoribosyltransferase (HPRT) gene of the human promyelocytic leukemia cell line HL-60 and have determined the molecular spectrum of spontaneous HPRT mutations in 45 independent 6-thioguanine-resistant HL-60 sublines. Four fluctuation tests using a total of 132 replicate HL-60 cultures revealed a mean forward mutation rate of HL-60 cells to thioguanine resistance of 1.7-6 x 10(-7)/cell/generation. Blot hybridization analysis of the X-linked HPRT gene using a human HPRT complementary DNA probe revealed abnormalities in HPRT gene structure and/or HPRT mRNA expression in 24 of 45 (53%) independent thioguanine-resistant HL-60 sublines. Six different classes of mutation were identified. The most prevalent (47%; 21 of 45 mutations) consists of mutations that are not detected by blot hybridization analyses and that do not disrupt HPRT mRNA production. These results suggest that a comparatively low forward mutation rate may be found in malignant human cells that exhibit both karyotypic and molecular evidence of genomic instability and that several different molecular classes of mutation may contribute to thioguanine resistance in HL-60, and perhaps in other, malignant human cells. The forward mutation assay system we have developed using the X-linked HPRT gene of HL-60 cells may be useful for analyses of the mutagenic potential and molecular spectrum of mutations produced by chemotherapeutic agents, suspected human mutagens and carcinogens, and phagocyte respiratory burst oxidants in human cells.     

Activating point mutations of the gsp oncogene in human thyroid adenomas

The gene for the alpha polypeptide chain (alpha s) of the heterotrimeric G protein Gs can be activated to the putative oncogene gsp by specific point mutations at codons 201 and 227. Such mutations have been reported in 40% of human growth hormone-secreting pituitary adenomas and in a single autonomously functioning thyroid adenoma. We examined an archival series of 45 differentiated human thyroid tumors by polymerase chain reaction amplification and oligonucleotide hybridization to identify point mutations at each of the affected codons. Successful amplification was achieved in 38 cases, and activating mutations were identified in 5 of 13 (38%) autonomously functioning adenomas, but in none of 16 nonfunctioning adenomas, six papillary carcinomas, or three follicular carcinomas. Our results confirm that the gsp oncogene is involved in the pathogenesis of autonomously functioning tumors but do not support a role in other thyroid tumors.