T cells responsive to myelin basic protein in patients with multiple sclerosis.

Gene mutation in vivo in human T lymphocytes appears to occur preferentially in dividing cells. Individuals with multiple sclerosis (MS) are assumed to have one or more populations of diving T cells that are being stimulated by autoantigens. Mutant T cell clones from MS patients were isolated and tested for reactivity to myelin basic protein, an antigen that is thought to participate in the induction of the disease. The hypoxanthine guanine phosphoribosyltransferase (hprt) clonal assay was used to determine mutant frequency values in MS patients with chronic progressive disease. Eleven of 258 thioguanine-resistant (hprt-) T cell clones from five of the six MS patients who were tested proliferated in response to human myelin basic protein without prior in vitro exposure to this antigen. No wild-type clones from these patients, nor any hprt- or wild-type clones from three healthy individuals responded to myelin basic protein. Thus, T cell clones that react with myelin basic protein can be isolated from the peripheral blood of MS patients.

Effect of folate deficiency on mutations at the hprt locus in Chinese hamster ovary cells exposed to monofunctional alkylating agents.

Multiplex PCR amplification of hprt exons from 113 Chinese hamster ovary cell clones selected for resistance to 6-thioguanine was performed to investigate the molecular basis for the synergistic mutagenic effects of nutritional folic acid deficiency and alkylating agents. In cells treated with ethyl methanesulfonate, intragenic deletions were detected in 9 of 46 (19.6%) clones derived from folate-deficient cells, but in none of 16 mutants grown in folate-replete medium. The number of deletions found in mutants generated by N-nitroso-N-ethylurea was low in both folate-deficient (1 of 25; 4%) and folate-replete (1 of 26; 3.8%) cells. Correction of folate deficiency may decrease the frequency of intragenic deletions caused by some alkylating agents.

Increased frequency of specific genomic deletions resulting from in vitro malathion exposure.

Malathion is a widely used pesticide with high potential for human exposure. Epidemiological studies suggest that individuals with chronic environmental exposures to pesticides have increased risks of various hematological malignancies. The genotoxic data to date have been somewhat inconclusive with regard to malathion exposure. We have used a cell cloning assay to study the genotoxicity of in vitro exposure of human T lymphocytes to malathion. We exposed cells in G0 to doses of malathion ranging from 10 to 600 microg/ml. Mutant frequencies of treated samples showed both intra- and interindividual variability and, in some cases, slight significant increases over the controls. Molecular analysis of hprt mutants resulting from both in vitro and an in vivo malathion exposure was performed by genomic multiplex PCR. In seven in vitro experiments (using cells from four different individuals) and one experiment on an individual exposed in vivo, one or more independent mutant(s) containing a partial deletion of exon 3 have been isolated from each individual. In five of the seven mutants, the deleted regions overlap extensively, revealing an area within exon 3 exceptionally prone to deletions upon exposure to malathion, This work provides the first evidence of an association between malathion exposure and specific mutations in human T lymphocytes. Additional work is necessary to determine the underlying molecular mechanism for these deletions and how this may relate to agricultural workers' increased risk of cancer.

V(D)J recombinase-like activity mediates hprt gene deletion in human fetal T-lymphocytes.

Studies from several laboratories worldwide have developed a large database for in vivo hypoxanthine-guanine phosphoribosyltransferase gene mutations in human T-lymphocytes. Sufficient differences have been found thus far between the spectrum for spontaneous mutations in adults and that observed in the fetus to suggest fundamental differences in in vivo mutagenic mechanisms at these two life stages. In adults, only approximately 15% of hypoxanthine-guanine phosphoribosyltransferase mutations have structural alterations on Southern blots, while in the fetus 75% of mutations show alterations of which one-half are deletions of exons 2 and 3. We have now sequenced the breakpoint sites for these specific deletions in 18 mutant lymphocyte clones isolated from 13 normal newborns. Three classes of deletions were found. Each class had the same intron 1 breakpoint but a different intron 3 breakpoint. These mutations have all the signatures of a V(D)J recombinase-mediated event (a 5' consensus heptamer, 3' consensus heptamer and nonamer, nibbling, non-germline-encoded nucleotides, P-nucleotides). At the 3' breakpoint of the most common class (comprising 83% of the mutants) a perfect heptamer can be created by postulating a hairpin loop which could attain a Z-DNA configuration. This feature may indicate recombinase preference for certain DNA structures. These results implicate the V(D)J recombinase in illegitimate events causing mutation in this housekeeping gene during T-cell development. Inactivation of genes involved in the control of growth and differentiation (e.g., tumor suppressor genes) by this mechanism may have important implications for cancer development.

Mitochondrial genome damage associated with cigarette smoking.

We have investigated the level of mitochondrial DNA (mtDNA) damage and deletions in bronchoalveolar lavage tissues from smokers and nonsmokers using quantitative, extra-long PCR and a "common" mtDNA deletion assay. Smokers had 5.6 times the level of mtDNA damage, 2.6 times the damage at a nuclear locus (beta-globin gene cluster), and almost 7 times the level of a 4.9-kb mtDNA deletion compared to nonsmokers, although the latter increase was not significant. Although both genomes (mitochondrial and nuclear) showed significantly increased levels of DNA damage in smokers (mtDNA P = 0.00072; beta-globin P = 0.0056), the relative differences were greatest in the mtDNA. Damage to the mtDNA may inhibit oxidative phosphorylation and, therefore, potentially cause or contribute to chronic lung disease and cancer. Consequently, the mtDNA may be a sensitive biomarker for environmentally induced genetic damage and mutation.

Vital Genes That Flank Sex-Lethal, an X-Linked Sex-Determining Gene of DROSOPHILA MELANOGASTER.

The X-chromosome:autosome balance in D. melanogaster appears to control both sex determination and dosage compensation through effects on a maternally influenced sex-linked gene called Sex-lethal (Sxl; 1-19.2). To facilitate molecular and genetic analysis of Sxl, we attempted to determine the locations of all ethyl methanesulfonate (EMS)-mutable genes vital to both sexes in the region between 6E1 and 7B1. This area includes approximately 1 cM of the genetic map on each side of Sxl and was reported by C. B. Bridges to contain 26 salivary gland polytene chromosome bands. The region appears rather sparsely populated with genes vital to both sexes, since the 122 recessive lethal mutations we recovered fell into only nine complementation groups. From one to 38 alleles of each gene were recovered. There was a preponderance of embryonic lethals in this area, although the lethal periods of loss-of-function mutations included larval, pupal and adult stages as well. Since the screen required that mutations be recessive and lethal to males, our failure to recover new Sxl alleles was the result expected for a gene with a female-specific function. An attempt was made to identify recessive male-specific lethals in this region, but none were found. Precise map positions were determined for eight of the nine vital genes. An interesting feature of the map is the location of Sxl in the middle of a 0.6- to 0.7-cM interval that appears to be devoid of genes vital to both sexes. The genetic location was determined of breakpoints near Sxl for all available chromosome rearrangements. Sxl is most likely located just to the left of band 7A1. We determined the relationship of our EMS-induced mutations in these nine genes to alleles induced by others. From this we conclude that the various genes appear to differ significantly from each other in their relative sensitivity to mutation by EMS vs. X rays.

The aminothiol WR-1065 protects T lymphocytes from ionizing radiation-induced deletions of the HPRT gene.

Aminothiols, such as WR-2721 and its active free thiol, WR-1065, reduce mutations from ionizing radiation in exponentially growing cells. In this study, human noncycling G0 T lymphocytes were exposed in vitro to gamma-irradiation in the presence or absence of WR-1065. The five treatment groups were: (a) control; (b) treatment with 4 mM WR-1065; (c) treatment with 3 Gy of gamma-radiation, from a 137Cs source; and (d) and (e) treatment with WR-1065 30 min prior to or 3 h after 3 Gy of gamma-irradiaiton, respectively. A total of 224 cloned HPRT mutants representing 179 independent mutations were analyzed for genetic alterations using multiplex PCR. Ionizing radiation alone significantly increased the percentage of mutations with gross structural alterations compared to controls (P = 0.02). Although the frequency of such large structural mutations was not different from control cells treated with WR-1065 alone, this aminothiol significantly reduced their frequency among irradiated mutants (P = 0.01) when the radioprotector was present during the irradiation. Addition of WR-1065 3 h postirradiation also greatly reduced the percentage of gross structural alterations; however, due to small numbers, this was not statistically significant. This is the first demonstration that the antimutagenicity of WR-1065 in human cells specifically protects against these kinds of large-scale DNA alterations induced by ionizing radiation. WR-1065 and similar aminothiol compounds may afford protection against radiation-induced mutations through polyamine-like processes, e.g., stabilization of chromatin structure, inhibition of cell proliferation, and influences on DNA repair systems.

Southern analysis of DNA polymorphism among Dw subtypes of DR4.

DNA from individuals of four Dw subtypes of DR4 (Dw4, Dw10, Dw14, Dw15) were studied using Southern blotting to determine if subtype-specific DR beta or DQ beta restriction fragment polymorphism could be found. Although very little polymorphism was found among ten DR4 homozygous individuals (4 Dw4, 2 Dw10, 3 Dw14, 1 Dw15) using a Dr beta or a DQ beta probe, restriction fragment polymorphism was easily detected between different DR types (DR1-DRw8). The possible evolutionary significance of the lack of Dw-associated polymorphism relative to DR-associated polymorphism is discussed.

HLA loss variants of a B27+ lymphoblastoid cell line: genetic and cellular characterization.

Variants of a lymphoblastoid cell line, LCL 526 (SB3 MB1 DR1 B44 C5 A2/SB4 MT4 DR4 B27 C2 A24), which lost various HLA specificities were selected with monoclonal antibodies and complement using a method developed by Kavathas et al. (PNAS 77:4251, 1980). Using alpha B27 monoclonals, 8 B27 only loss mutants and 4 B27 haplotype multiple loss mutants were generated. The parental LCL 526 and two of the B27- mutants were used to select alpha B27 CTLs. The selection of six A2 loss, one A2-C5 loss, and 14 A2 haplotype multiple loss variants as well as secondary selection on haplotype loss variants to obtain A null, B null, DR null, and total A, B, C, null variants is also described. The usefulness of these mutants for the study of the relationship between B27 and disease and as two new haplotypes for immunologic, genetic, and molecular research is discussed. These mutants are available to other researchers.

Somatic cell gene mutations in humans: biomarkers for genotoxicity.

Somatic cell gene mutations arising in vivo in humans provide biomarkers for genotoxicity. Four assays, each measuring changes in a different "recorder" gene, are available for detecting mutations of the hemoglobin (Hb) and glycophorin A (gpa) genes in red blood cells and the hypoxanthine-guanine phosphoribosyltransferase (hprt) and HLA genes in T-lymphocytes. Mean adult background mutant frequencies have been established; i.e., approximately 4 x 10(-8) (Hb), 5-10 x 10(-6) (hprt), 10-20 x 10(-6) (gpa) and 30 x 10(-6) (HLA). All the assays have now been used in studies of individuals exposed to physical and/or chemical genotoxic agents, and all have shown elevated values following exposures; examples are presented. In addition to quantitation, the lymphocyte assays allow molecular analyses of in vivo mutations, the definition of background and induced mutational spectra, and the search for unique changes for characterizing specific mutagens. The HPRT system currently has the largest database in this regard. Approximately 15% of adult background hprt mutations are due to gross structural alterations (primarily deletions) having random breakpoints; 85% result from "point" changes detected only by sequencing. In contrast, a specific intragenic deletion due to DNA cleavage at specific sites characterizes fetal hprt mutations, implicating a developmental mistake in their genesis. (This kind of developmental mistake in other genes is frequently observed in lymphoid malignancies.) Mutational spectra are just beginning to be defined for induced hprt mutations, e.g., ionizing radiation produces large deletions.(ABSTRACT TRUNCATED AT 250 WORDS)

Future research directions for evaluating human genetic and cancer risk from environmental exposures.

The utility of biomarkers for evaluating the genotoxicity of environmental exposures is well documented. Biomarkers of both exposure and effect provide bases for assessing human-genotoxicant interactions and may be indicative of future disease risk. At present, there is little information on the predictive value of these assays for either a population or the individuals tested. This paper describes some aspects of biomarker assays, the possible use of susceptibility measures in biomonitoring protocols, and the need for evaluation of disease relevance. A population study involving epidemiologists, geneticists, toxicologists, statisticians, and physicians is proposed to determine the disease relevance of these biomarkers.

In vivo mutations in human blood cells: biomarkers for molecular epidemiology.

Mutations arising in vivo in recorder genes of human blood cells provide biomarkers for molecular epidemiology by serving as surrogates for cancer-causing genetic changes. Current markers include mutations of the glycophorin-A (GPA) or hemoglobin (Hb) genes, measured in red blood cells, or mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) or HLA genes, measured in T-lymphocytes. Mean mutant frequencies (variant frequencies) for normal young adults are approximately: Hb (4 x 10(-8)) < hprt (5 x 10(-6)) = GPA (10 x 10(-6)) < HLA (30 x 10(-6)). Mutagen-exposed individuals show decided elevations. Molecular mutational spectra are also being defined. For the hprt marker system, about 15% of background mutations are gross structural alterations of the hprt gene (e.g., deletions); the remainder are point mutations (e.g., base substitutions or frameshifts). Ionizing radiations result in dose-related increases in total gene deletions. Large deletions may encompass several megabases as shown by co-deletions of linked markers. Possible hprt spectra for defining radiation and chemical exposures are being sought. In addition to their responsiveness to environmental mutagens/carcinogens, three additional findings suggest that the in vivo recorder mutations are relevant in vivo surrogates for cancer mutations. First, a large fraction of GPA and HLA mutations show exchanges due to homologous recombination, an important mutational event in cancer. Second, hprt mutations arise preferentially in dividing T-cells, which can accumulate additional mutations in the same clone, reminiscent of the multiple hits required in the evolution of malignancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of chromosomal aberrations, sister chromatid exchange, hprt mutations, and DNA adducts in peripheral lymphocytes of human populations at increased risk for cancer.

Using a multidisciplinary approach, we have measured various indicators of DNA damage in peripheral lymphocytes of human populations potentially at increased risk for cancer. Sister chromatid exchanges (SCE) and polycyclic aromatic hydrocarbon (PAH)-DNA adducts were evaluated in a group of firefighters; chromosomal aberrations and hprt mutations were evaluated in a group of cancer patients undergoing radioimmunoglobulin therapy (RIT); SCE and acrolein-modified DNA were measured in cancer chemotherapy patients and in pharmacists preparing chemotherapy prescriptions; and SCE and PAH-DNA adducts are being measured in U.S. army troops stationed in Kuwait. Our results indicate that both SCE and PAH-DNA adduct levels were not elevated in firefighters, but that other factors such as smoking status and race were risk factors for increased SCE and PAH-DNA adducts. RIT was found to increase background rates of chromosome-type aberrations and frequencies of hprt mutations and there was a strong correlation between levels of therapy-induced chromosome damage sustained in vivo and in vitro sensitivity to radiation-induced chromosome damage. Peripheral blood lymphocytes of cancer patients treated with cyclophosphamide showed higher levels of SCE and had a higher incidence of acrolein adducts in DNA. Lymphocytes from pharmacists preparing antineoplastic drugs were found to acquire increased in vitro sensitivity to SCE induction by phosphoramide mustard with increased lifetime duration of drug handling. A prospective, longitudinal study was performed to identify environmental factors that modulate genetic damage in breast cancer patients. Women with benign breast masses and no apparent disease served as controls. Mutant frequency, cloning efficiency, and chromosomal aberration frequency did not differ significantly among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiation quality affects the efficiency of induction and the molecular spectrum of HPRT mutations in human T cells.

Human T lymphocytes can be used to determine the frequency and molecular spectrum of somatic cell gene mutations induced by ionizing radiations both in vivo and in vitro. In vitro exposure of these G0 cells to low-LET 137Cs gamma rays results in the induction of HPRT mutations and a predominant molecular spectrum of DNA deletions and rearrangements, particularly total gene deletions (11-12%). Similar results are found in samples from humans exposed to low-LET radiation from 131I. The doubling dose for mutation induction is calculated to be 0.8 and 1.0 Gy from these exposures performed in vitro and in vivo, respectively. In vitro studies of the effects of high-LET radiation from exposure to 222Rn also showed an induction of HPRT mutations, with a doubling dose of approximately 0.2 Gy. With this radiation, the predominant mutations were small partial deletions, with less than 2% total gene deletions. Studies of humans exposed to high-LET radiation from 239Pu showed an increased HPRT mutant frequency for the group, although no significant dosimetry could be defined. In contrast to the humans exposed to 131I, no increase in the frequency of total gene deletions was found. This is consistent with the results for 222Rn in vitro. The available data show that radiation quality affects both the efficiency of induction and the molecular spectrum of HPRT mutations in human T lymphocytes both in vitro and in vivo. The mutational spectrum may be relatively specific for radiations of different quality and thus allow a more precise measurement of the induction of somatic gene mutations resulting from individual exposures to radiation, and thereby provide more sensitive assessments of health risks.

TCR beta PCR from crude preparations for restriction digest or sequencing.

T cell specificity is determined by the combinatorial association of specific variable (V), diversity (D), and junctional (J) regions. Clones of T cells (clonality) can occur, in the blood or in tissue, after proliferation of activated T cells. Determining clonality in mutation assays is necessary to distinguish between mutants and mutational events. We have developed a novel approach to determine clonality among T cell isolates, using restriction digests of PCR-amplified cDNA of the T cell receptor beta gene. The T cell receptor beta gene was PCR-amplified by use of a consensus primer, beginning from a cell pellet of 2,000-5,000 cells or from extracted RNA. This TCR (T cell receptor) beta chain PCR product can also be directly sequenced, allowing simple and easy identification of Vbeta and CDR3 sequence from a small number of cells. The utility of this method is demonstrated by PCR, restriction digest, and sequencing of the TCR beta cDNA from eight T cell clones isolated from 2 individuals. A clone of three identical isolates (one 3-mer) and a clone of two identical isolates (one 2-mer) were determined from restriction digests using two different enzymes. This new method is an easier and more rapid way of determining clonality than traditional methods, e.g., Southern blotting.

Use of inverse PCR to amplify and sequence breakpoints of HPRT deletion and translocation mutations.

Deletion and translocation mutations have been shown to play a significant role in the genesis of many cancers. The hprt gene located at Xq26 is a frequently used marker gene in human mutational studies. In an attempt to better understand potential mutational mechanisms involved in deletions and translocations, inverse PCR (IPCR) methods to amplify and sequence the breakpoints of hprt mutants classified as translocations and large deletions were developed. IPCR involves the digestion of DNA with a restriction enzyme, circularization of the fragments produced, and PCR amplification around the circle with primers oriented in a direction opposite to that of conventional PCR. The use of this technique allows amplification into an unknown region, in this case through the hprt breakpoint into the unknown joined sequence. Through the use of this procedure, two translocation, one inversion, and two external deletion hprt breakpoint sequences were isolated and sequenced. The isolated IPCR products range in size from 0.4 to 1.8 kb, and were amplified from circles ranging in size from 0.6 to 7.7 kb. We have shown that inverse PCR is useful to sequence translocation and large deletion mutant breakpoints in the hprt gene.

Molecular analyses of in vivo hprt mutations in human T-lymphocytes: IV. Studies in newborns.

In order to characterize in vivo gene mutations that occur during fetal development, molecular analyses were undertaken of mutant 6-thioguanine resistant T-lymphocytes isolated from placental cord blood samples of 13 normal male newborns. These mutant T-cells were studied to define hypoxanthine-guanine phosphoribosyltransferase (hprt) gene structural alterations and to determine T-cell receptor (TCR) gene rearrangement patterns. Structural hprt alterations, as shown by Southern blot analyses, occurred in 85% of these mutant clones. These alterations consisted mostly of deletion of exons 2 and 3. These findings contrast with the 10-20% of gross structural alterations (i.e., those visible on Southern blots) occurring randomly across the entire gene previously reported for T-cell mutants isolated from normal young adults. Iterative analyses of hprt structural alterations and TCR gene rearrangement patterns show that approximately one-third of the newborn derived mutants may have originated as pre- or intrathymic hprt mutations. This too contrasts with previous findings in adults where the background in vivo hprt mutations appeared to originate in postthymic T-lymphocytes.

Cytosine arabinoside enhancement of gamma irradiation induced mutations in human T-lymphocytes.

The frequency of 6-thioguanine resistant (TGr) mutants induced in human G0 phase T-lymphocytes by 200 cGy of gamma irradiation is greatly enhanced by incubation with cytosine arabinoside (ara-C) after irradiation. The mutant frequency increased with increasing incubation time in ara-C for up to 2 hr. This mutation induction required a phenotypic expression time of 5-8 days mass culture growth, similar to that found with mutants induced by 300 cGy of irradiation alone. Southern blot analysis of 40 isolated mutant clones revealed 8 independent mutations by T-cell receptor (TCR) gene rearrangement patterns. Four of these eight showed hprt gene structural alterations (0.50). An alternative method to allow phenotypic expression was developed to minimize the isolation of hprt/TCR sibling mutants. The use of in situ expression in the microtiter dish wells resulted in the isolation of 17 independent mutations in 19 mutant clones. Ten of these 17 mutations showed hprt structural alterations (0.59). The high fraction of mutations involving structural alterations detected by Southern blot analysis is consistent with the known induction of chromosome aberrations by irradiation plus ara-C treatment. We propose that both the increase in Mf and the increase in the incidence of hprt gene structural alterations are due to the accumulation of strand breaks in repairing regions of DNA under these conditions of ara-C induced inhibition of repair. We further propose that upon release of the ara-C inhibition, these repairing regions can interact to yield both gene mutations and chromosome aberrations.

Analysis of human HPRT deletion mutations with X-linked probes and pulsed field gel electrophoresis.

Because the human hprt gene is used in numerous mutation studies, it is important to fully characterize this gene. Therefore, our laboratory has undertaken to map the region around the hprt gene at band q26 of the human X chromosome. Utilizing hprt mutant T-cell clones isolated using the hprt clonal assay, which have deletions of all or part of the hprt gene, we have ordered 5 anonymous probes previously known to map in Xq26. Results suggest that this region includes between 460 kb and 18 Mb of DNA, which is at least 10 times the size of the hprt gene itself (43 kb). Pulsed field gel analysis of the region is underway to determine the exact distances between each of the anonymous probes and hprt and to determine deletion sizes in the mutant T-cell clones.

Molecular analyses of in vivo hypoxanthine-guanine phosphoribosyltransferase mutations in human T-lymphocytes: II. Demonstration of a clonal amplification of hprt mutant T-lymphocytes in vivo.

Recent molecular analysis of in vivo-derived hprt mutant T-lymphocytes cloned from human blood show that mutants occurring at the normal frequency (approximately 5 X 10(-6) in healthy young individuals generally represent independent hprt mutations. Here we report that in an individual with a high mutant frequency (86-620 X 10(-6],92% (61/66) of the mutant clones are descendents of an original mature T-cell precursor that has undergone in vivo clonal expansion. Therefore, these mutants could represent as few as one original hprt mutation. If so, correcting for the clonal expansion yields a revised calculated mutant frequency (Mf) value for this individual that is near the normal range. These hprt mutant clones all showed identically rearranged T-cell receptor (TCR) beta and gamma gene patterns by Southern blot analysis. All the clones were surface marker CD4+, showed no obvious chromosomal aberration, and had no detectable hprt gene structural alteration. This TCR-defined T-cell clone appears to have expanded in the blood of the individual over a 6-month period and persists at high levels after nearly 4 years. This finding illustrates the need to analyze mutants from individuals with high mutant frequencies at the molecular level in order to estimate hprt mutation frequency from the calculated hprt mutant frequency. The possibility that spontaneous hprt mutants might arise in vivo preferentially in dividing cells, and implications of this, are discussed.