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H L Liber

Publications and source records attributed to H L Liber.

At least 37 records · Page 2Linked to original sources
PubMed1993

The effects of hypoxia and cysteamine on X-ray mutagenesis in human cells. I. Dose response and Southern blot analysis of induced mutants.

The effects of cysteamine and hypoxia on X-ray-induced mutation in human lymphoblast cells have been investigated. Irradiation of these cells under these two sets of radioprotective conditions resulted in a fourfold lower mutant fraction at the hprt locus, compared to irradiation in the absence of any protection. Using Southern blot analysis, the molecular nature of mutants induced in these cells by X rays delivered under hypoxic conditions, or in the presence of 25 mM cysteamine, has been compared with that of mutants induced by an equally mutagenic treatment of X rays alone. Of 60 mutants from cultures treated with X rays alone, 16 exhibited no change in the restriction fragment pattern and were defined as point mutations, 27 were total gene deletions, and 17 were partial deletions or rearrangements. Of 46 mutants from cultures treated with X rays in the presence of 25 mM cysteamine, 22 were point mutations, 18 were total gene deletions, and only 6 were partial deletions or rearrangements. Of 45 mutants from cultures treated with X rays delivered under hypoxic conditions, 17 were point mutations, 14 were total gene deletions, and 14 were partial deletions or rearrangements. Both radioprotective conditions reduced the level of mutation in each mutational class, when compared to the maximum expected in the absence of protection. The spectrum of mutations induced by X rays in cysteamine was significantly different from X rays alone. However, the mutational spectrum of X rays under hypoxic conditions was not different from X rays alone or from X rays in cysteamine. The extents of the deletions induced at the hprt locus were also examined by hybridizing the Southern blots to X-chromosome markers that have been mapped to within 1200 kb of hprt. There were no significant differences among the three groups; however, many mutants were missing one or more of these markers, indicating that deletions of several hundred kilobases are not uncommon.

Base Sequence↗
PubMed1993

Mutagenesis after therapy for Hodgkin's disease.

Therapy for Hodgkin's disease has been associated with a significant increase in risk for second cancers. To begin an investigation of the association of therapy-induced genetic damage with this risk, somatic mutations at the hypoxanthine phosphoribosyltransferase locus were measured in lymphocytes from patients previously treated for Hodgkin's disease. The results demonstrate that a subset of patients have persistently elevated mutation frequencies, perhaps suggesting that these individuals are among those at significant risk of second cancer development.

Adolescent↗
PubMed1992

Heritable alterations at the adenine phosphoribosyltransferase (APRT) locus in human lymphoblastoid cell lines.

Human lymphoblastoid cell lines derived from WI-L2 exhibit unexpected frequencies of diaminopurine (DAP) resistant mutants. The background mutant fractions of 10(-7) to 10(-8) in untreated cultures are much lower than the frequencies expected for loss of a heterozygous autosomal locus (10(-5) to 10(-6), yet much higher than expected for a homozygous locus (10(-10) to 10(-12). We used aminopterin, adenine and thymidine (AAT) to select DAP-sensitive (DAPS) revertants from one resistant line. The background frequency of DAPR in these revertant cell lines ranged from 3.5 to 6.5 x 10(-4), approximately the square root of 10(-7). Thus these data suggest that both alleles of aprt are inactivated at similarly high frequencies. They also indicate that the DAPS revertants were heterozygotes (aprt +/-) or hemizygotes (aprt +/0) and that WI-L2 was homozygous (aprt+/+). Mutational dose-response studies with X-rays, ethyl methanesulfonate (EMS), and ICR-191 were conducted in 4 of these revertant cell lines. EMS and ICR-191, which induce mainly point mutations, did not induce an increase in mutant fraction. A dose of 200 cGy X-rays, however, induced a frequency of 10(-3). Treatment of DAPR cells with 5-azacytidine induced a significant increase in reversion to DAPS. Southern blot analysis of the aprt gene after digestion with MspI or HpaII also suggests that differential methylation changes may play a major role in the generation of DAP sensitivity and resistance.

2-Aminopurine↗
PubMed1992

A comparison of induced mutation at homologous alleles of the tk locus in human cells. II. Molecular analysis of mutants.

Previously we described the dose-response relationship for X-ray-induced mutation of the two homologous alleles of the thymidine kinase (tk) gene in a human lymphoblastoid cell line (Amundson and Liber, 1991). The two alleles were differentially mutable by X-rays, with one allele 6-10 times more mutable than the other. This difference was shown to be due to the virtual absence of the class of slow growth mutants from one allele. In the present report, restriction fragment length polymorphism (RFLP) analyses of informative markers along chromosome 17 have been used to delineate a region of chromosome 17 in which heterozygosity is lost with relatively high frequency among slow growth TK- mutants from the more mutable allele. However, loss of heterozygosity of this region has never been observed in normal growth mutants obtained from the more mutable allele, or in TK- mutants from the other, less mutable, allele. This may indicate the presence of a heterozygous essential gene on chromosome 17 distal to TK1.

Alleles↗
PubMed1992

Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.

Alpha Particles↗
PubMed1991

In vivo somatic mutation in the lymphocytes of Hodgkin's disease patients.

While current medical therapies for Hodgkin's disease are usually quite effective, it has become increasingly clear that some of the therapies utilized carry an inherent risk for the induction of secondary malignancies. In order to examine the cellular and genetic responses to therapy for Hodgkin's disease among individuals, we have determined the mutant frequency of T-lymphocytes in 3 cohorts of patients (N = 86) and in controls (N = 71) using a T-cell cloning assay selecting for 6-thioguanine resistance. The Hodgkin's disease cohorts studied include 1) new and untreated, 2) radiotherapy, and 3) combined modality therapy patients. Additionally, two patients receiving chemotherapy alone were studied. In untreated patients, 3 of 18 (17%) mutant frequencies were above the upper 95% confidence limit for mutant frequency in controls (12.6 x 10(-6]. After therapy, 14 out of 45 (31%) of those treated with X-rays only and 10 of 23 (44%) patients treated with both X-rays and chemotherapy had mutant frequencies greater than 12.6 x 10(-6). Overall, the results indicated that the individual response to Hodgkin's disease therapy was a heterogeneous one with a sub-population of persons having elevated mutant frequencies even many years after their last treatment. The larger frequency of elevated MFs in those patients who received intensive therapy (chemotherapy and radiotherapy) is consistent with their increased risk for second cancer induction.

Adult↗
PubMed1991

A comparison of induced mutation at homologous alleles of the tk locus in human cells.

Using a restriction fragment length polymorphism which can distinguish the two copies of the thymidine kinase (tk) gene in the TK6 human lymphoblastoid cell line, we have identified heterozygous subclones with alternate active alleles. Quantitative mutagenesis studies with X-rays revealed a markedly different response, depending on which homolog carried the active allele. The slopes of the dose-response curves differed by approximately 10-fold for mutation of the two alleles and this relationship held true for several independently isolated cell lines. Only one of the cell lines showed a different response to ethyl methanesulfonate. There were no differences among any of the cell lines at the X-linked hprt locus. Analyses of TK- mutants recovered from these cell lines indicated that the reduced yield of mutants from the one allele may be due, at least in part, to a lack of a specific class of TK- mutant, that is, the slow-growing mutants which have been associated with large-scale mutagenic events.

Alleles↗
PubMed1991

Mutagenicity of 2-amino-N6-hydroxyadenine to TK6 human lymphoblast cells.

TK6 human lymphoblast cells (tk +/-; hprt+) were treated with various concentrations of 2-amino-N6-hydroxyadenine (AHA) for 24 h. AHA was quite toxic to TK6 cells in the dose range 0-0.05 micrograms/ml, but additional toxicity was not observed between 0.05 and 0.10 micrograms/ml. AHA induced mutations at 2 distinct genetic loci: the autosomal thymidine kinase (tk) and the X-linked hypoxanthine-guanine phosphoribosyl transferase (hprt). Significant levels of both tk-NG mutants (normal growth rate of 16-18 h, colonies visible after 10-11 days incubation) and tk-SG mutants (slow growth rate of greater than 24 h, colonies visible after 18 days incubation) were induced. 15 hprt- mutants were isolated and analyzed by Southern blot. 8 of these had normal restriction fragment patterns after digestion with PstI, EcoRI, and HindIII, and were defined as 'point' mutations; the remaining 7 had partial deletions of the hprt gene. 32 tk- mutants were also isolated. 3 of 22 normal growth mutants and 6 of 10 slow growth mutants had lost the active tk allele. These data suggest that both point mutations and larger-scale alterations are induced by AHA.

Adenine↗
PubMed1991

Genotoxic and mutagenic effects of the diagnostic use of thallium-201 in nuclear medicine.

In order to investigate possible mutagenic effects of in vivo exposure to low levels of ionizing radiation used in nuclear medicine, we have examined the hypoxanthine guanine phosphoribosyl transferase (hprt) mutant fraction (MF) and chromosome aberration (CA) frequency in 24 nuclear medicine patients before and after injection of thallium-201. The mean MF of the thallium-201-exposed cohort was 5.2 +/- 4.4 x 10(-6) before injection exposure. No significant difference in MF was observed 24 h later. In 11 patients who were studied on a third occasion, 30 days after thallium-201 exposure, there was again no significant difference in post-exposure as compared with the pre-exposure MF. The frequency of CA in peripheral blood lymphocytes was not significantly different, comparing pre- and 24 h to 1 month post-radionuclide exposure. Thus, thallium-201 exposure was not associated with significant elevations in MF or CA frequency in lymphocytes of exposed individuals.

Chromosome Aberrations↗
PubMed1991

Human lymphocytes exposed to low doses of X-rays are less susceptible to radiation-induced mutagenesis.

Human lymphocytes exposed to low doses of X-rays become refractory to the subsequent induction of chromosomal damage by high doses of radiation (Shadley and Wolff, 1987). The current study was designed to test the effect of pretreatment of human T-lymphocytes with a low dose of X-rays on the induction of mutations at the hprt locus by a subsequent challenge dose. When cells were exposed to 1 cGy X-rays 24 h after phytohemagglutinin stimulation, the yield of mutations induced by a 300 cGy X-ray dose given 16 h later was reduced by approximately 70% from the control level of X-ray-induced mutations. This indicates that this previously described adaptive response to low dose X-rays also results in lymphocytes becoming refractory to the induction of gene mutations.

Adaptation, Physiological↗
PubMed1991

In vivo exposure of human lymphocytes to technetium-99m in nuclear medicine patients does not induce detectable genetic effects.

Recent reports have demonstrated that exposure of nuclear medicine patients to thallium-201 does not result in a detectable increase in mutation at the hprt locus in human lymphocytes. In an effort to study further the potential genetic effects of medical exposures to low dose radiation, we have examined chromosome aberrations and mutations in peripheral blood lymphocytes from nuclear medicine patients exposed to clinical doses of technetium-99m. Our results show that there is no exposure-related increase in chromosomal damage; furthermore, the data do not confirm earlier reports of exposure-related increases in mutations induced by technetium-99m.

Analysis of Variance↗
PubMed1991

Single-strand conformation polymorphisms can be used to detect T cell receptor gene rearrangements: an application to the in vivo hprt mutation assay.

Epidemiologic application of the human in vivo hypoxanthine-guanine phosphoribosyltransferase (hprt) mutation assay requires screening of mutant colonies to differentiate independent from clonal origin. Previously, sibship was defined by Southern blot analysis of T cell receptor gene rearrangements. We report here a more expedient method to determine these rearrangements utilizing the polymerase chain reaction (PCR) and a DNA single-strand conformation polymorphism technique. The results are consistent with those obtained by Southern blotting in that sibship can be defined easily. A major advantage is that cells may be taken directly from the microtiter plate, eliminating the necessity to expand the clones and isolate genomic DNA. Cell lines which have not undergone receptor gene rearrangements cannot serve as PCR templates and do not interfere with this analysis. Furthermore, background from the large number of nonmutant lymphocytes present in the well does not hinder the analysis of the T cell receptor pattern of a mutant. This technique facilitates rapid screening of a large number of clones in a shorter time than Southern blotting, and is useful for the study of in vivo mutation and the clonal expansion of mutants in populations of T cells.

Base Sequence↗
PubMed1989

Long-term, low-dose benzo[a]pyrene-induced mutation in human lymphoblasts competent in xenobiotic metabolism.

We have studied the mutagenic effects of benzo[a]pyrene (BP) administered in a long-term, low-dose fashion to metabolically competent human lymphoblastoid cells. A continuous dose as low as 0.02 microM for 20 days produced a significant increase in mutant fraction at the 6TG-resistance (HGPRT) locus. The slope of the mutant fraction over time in the 0.02 microM BP-treated culture was twice that observed in the untreated concurrent control; 0.02 microM therefore represents the doubling dose of BP for gene mutation in this cell line. For higher doses of 0.1, 0.5 or 1 microM BP, the rate (or efficiency) of induced mutation was considerably higher for the first 5 or 6 days of exposure than for the last 14-15. This did not appear to be due to a growth disadvantage against early-arising mutants. Comparison to previously published data in the same cell system (Crespi and Thilly, 1984) revealed that the long-term , low-dose protocol (0-1 microM for up to 20 days) was significantly more efficient at inducing mutations than a short-term, high-dose protocol (0-10 microM for 1 day).

Benzo(a)pyrene↗
PubMed1989

A comparison of mutation induction at the tk and hprt loci in human lymphoblastoid cells; quantitative differences are due to an additional class of mutations at the autosomal tk locus.

X-Rays, ethyl methanesulfonate and ICR-191 induced 2 classes of trifluorothymidine-resistant mutants at the autosomal tk locus in human lymphoblastoid cells. These classes were differentiated by their growth rates; some mutants grew with a normal doubling time of 14-18 h (tk-NG), while others grew much more slowly, with doubling times of 21-44 h (tk-SG). Only mutants with normal growth rates were observed at the X-linked hprt locus; the frequencies of mutations induced at hprt were equal to those induced for tk-NG mutants. Thus, more mutations overall (by up to a factor of 6) were induced at tk than at hprt. These results are discussed in relation to recent studies in rodent cells, in which much greater mutation frequencies were observed at autosomal loci.

Aminacrine↗
PubMed1989

Formaldehyde-induced and spontaneous alterations in human hprt DNA sequence and mRNA expression.

Human lymphoblast mutants at the X-linked hprt locus have been examined by Southern blot, Northern blot and DNA sequence analysis. A previous study had shown that approximately a third of the spontaneously-arising mutants and half those induced by formaldehyde showed no alteration in restriction fragment pattern and thus were classified as point mutations. In this report, Northern blot analysis was used to show that these point mutants fall into 4 categories: normal size and amount of RNA, normal size but reduced amounts, reduced size of RNA or no RNA. Sequence analyses of cDNAs prepared from hprt mRNAs were performed on 1 spontaneous and 7 formaldehyde-induced mutants with normal Northern blots. The spontaneous mutant was caused by an AT----GC transition. 6 of the formaldehyde-induced mutants were base substitutions, all of which occurred at AT base-pairs. There was an apparent hot spot, in that 4/6 independent mutants were AT----CG transversions at one specific site. The remaining mutant had lost exon 8.

Base Sequence↗
PubMed1989

The molecular nature of mutants induced by X rays is altered by the presence of the radioprotector cysteamine.

The molecular structure of mutants induced in human lymphoblast cells by 500 cGy X rays in the presence of the radioprotector cysteamine (25 mM) has been compared with that induced by an equally mutagenic treatment of 150 cGy X rays alone. Sets of mutants at the hypoxanthine-guanine phosphoribosyl transferase locus were analyzed by Southern blot. Of 24 mutants induced by X rays in the presence of cysteamine, 67% exhibited no change in the restriction fragment pattern and thus were defined as point mutations; 8% appeared to be total gene deletions and 25% were partial deletions or rearrangements. In contrast, among 28 mutants induced by X rays alone (Liber et al., Mutat. Res. 178, 143-153 (1987)), 46% were point mutations, while 50% were total gene deletions and only 1 mutant (4%) was a partial deletion or rearrangement. Thus mutants isolated in the presence of cysteamine consisted of more point mutations and partial deletions/rearrangements, and considerably fewer total gene deletions. These results suggest that cysteamine may protect selectively against processes which lead to large-scale molecular changes.

Blotting, Southern↗
PubMed1988

Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E. coli.

The molecular nature of formaldehyde (HCHO)-induced mutations was studied in both human lymphoblasts and E. coli. Thirty HPRT- human lymphoblast colonies induced by eight repetitive 150 microM HCHO treatments were characterized by Southern blot analysis. Fourteen of these mutants (47%) had visible deletions of some or all of the X-linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts. In E. coli, DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target. Exposure of E. coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%). Dideoxy DNA sequencing revealed that most of the point mutations were transversions at GC base pairs. In contrast, exposure of E. coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene. Therefore, HCHO is capable of producing different genetic alterations in E. coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used. Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E. coli. Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism.

Chromosome Deletion↗