Transcriptional responses to ionizing radiation reveal that p53R2 protects against radiation-induced mutagenesis in human lymphoblastoid cells.

The p53 protein has been implicated in multiple cellular responses related to DNA damage. Alterations in any of these cellular responses could be related to increased genomic instability. Our previous study has shown that mutations in p53 lead to hypermutability to ionizing radiation. To investigate further how p53 is involved in regulating mutational processes, we used 8K cDNA microarrays to compare the patterns of gene expression among three closely related human cell lines with different p53 status including TK6 (wild-type p53), NH32 (p53-null), and WTK1 (mutant p53). Total RNA samples were collected at 1, 3, 6, 9, and 24 h after 10 Gy gamma-irradiation. Template-based clustering analysis of the gene expression over the time course showed that 464 genes are either up or downregulated by at least twofold following radiation treatment. In addition, cluster analyses of gene expression profiles among these three cell lines revealed distinct patterns. In TK6, 165 genes were upregulated, while 36 genes were downregulated. In contrast, in WTK1 75 genes were upregulated and 12 genes were downregulated. In NH32, only 54 genes were upregulated. Furthermore, we found several genes associated with DNA repair namely p53R2, DDB2, XPC, PCNA, BTG2, and MSH2 that were highly induced in TK6 compared to WTK1 and NH32. p53R2, which is regulated by the tumor suppressor p53, is a small subunit of ribonucleotide reductase. To determine whether it is involved in radiation-induced mutagenesis, p53R2 protein was inhibited by siRNA in TK6 cells and followed by 2 Gy radiation. The background mutation frequencies at the TK locus of siRNA-transfected TK6 cells were about three times higher than those seen in TK6 cells. The mutation frequencies of siRNA-transfected TK6 cells after 2 Gy radiation were significantly higher than the irradiated TK6 cells without p53R2 knock down. These results indicate that p53R2 was induced by p53 protein and is involved in protecting against radiation-induced mutagenesis.

Homologous and nonhomologous recombination resulting in deletion: effects of p53 status, microhomology, and repetitive DNA length and orientation.

Repetitive DNA elements frequently are precursors to chromosomal deletions in prokaryotes and lower eukaryotes. However, little is known about the relationship between repeated sequences and deletion formation in mammalian cells. We have created a novel integrated plasmid-based recombination assay to investigate repeated sequence instability in human cells. In a control cell line, the presence of direct or inverted repeats did not appreciably influence the very low deletion frequencies (2 x 10(-7) to 9 x 10(-7)) in the region containing the repeat. Similar to what has been observed in lower eukaryotes, the majority of deletions resulted from the loss of the largest direct repeat present in the system along with the intervening sequence. Interestingly, in closely related cell lines that possess a mutant p53 gene, deletion frequencies in the control and direct-repeat plasmids were 40 to 300 times higher than in their wild-type counterparts. However, mutant p53 cells did not preferentially utilize the largest available homology in the formation of the deletion. Surprisingly, inverted repeats were approximately 10,000 times more unstable in all mutant p53 cells than in wild-type cells. Finally, several deletion junctions were marked by the addition of novel bases that were homologous to one of the preexisting DNA ends. Contrary to our expectations, only 6% of deletions in all cell lines could be classified as arising from nonhomologous recombination.

Radiation-induced mutations at the autosomal thymidine kinase locus are not elevated in p53-null cells.

To explore further the possibility that some forms of mutated p53 may increase mutagenesis in a positive manner, a double p53 knockout cell line was created, using a promoterless gene targeting approach. The identity of these p53-null cells was confirmed by Southern blot and Western blot analyses. Radiation-induced toxicity and mutagenicity was then compared among p53-null cells, TK6 cells with wild-type p53, and WTK1 cells with a p53 point mutation in codon 237. At the autosomal, heterozygous thymidine kinase locus, p53-null cells had equivalent background mutation frequencies and were approximately equally mutable as TK6, whereas WTK1 was much more sensitive to spontaneously arising and X-ray-induced mutation. Thus, these results indicate that the lack of wild-type p53 did not lead to increased mutagenesis.

Interrelationships between radiation-induced mutations and modifications in gene expression linked to cancer.

Ionizing radiation induces cancer in humans, but the mechanisms are not established. One simple way by which radiation could induce cancer is by inducing a genetic mutation in one or more of the multiple steps in the oncogenic pathway. However, many investigators favor the hypothesis that a mutator phenotype is necessary for a single cell to acquire the multiple genetic changes found in cancer. The alternative idea that ionizing radiation might induce an epigenetic heritable mutator phenotype has gained momentum in the last decade or so. A number of laboratories have observed a phenomenon termed 'delayed reproductive death', which is manifested as a reduced cloning efficiency, and also increased frequencies of gene locus mutations or chromosome aberrations that persist for many generations after radiation exposure. It is theorized that these endpoints arise due to a long-lived alteration in gene expression induced by radiation. Ionizing radiation is well known to induce gene expression, as it triggers the p53 pathway, as well as a series of early response genes, including c-jun and egr-I. However, there is no model from the gene expression field that could account for an epigenetic mutator effect that persists for tens or even hundreds of generations.

Increased transcription decreases the spontaneous mutation rate at the thymidine kinase locus in human cells.

Transcription increases DNA repair efficiency and modulates the distribution of certain types of DNA damage. Furthermore, increased transcription level stimulates spontaneous mutation rate in yeast. We explored whether transcription level affects spontaneous mutation rate in human cells. We first developed two thymidine kinase (tk) inducible human cell lines using the Gal4-Estrogen receptor system. In our TK6i-G3 and G9 tk heterozygous cell lines, the active tk allele is linked to an inducible promoter element. Tk mRNA is induced following treatment with estrogen. Spontaneous mutation rate was significantly decreased in human cell lines after induction in contrast to the report in yeast. Thus, humans may have evolved different or additional mechanisms to deal with transcription related spontaneous mutagenesis.

Spectra of X-ray-induced and spontaneous intragenic HPRT mutations in closely related human cells differentially expressing the p53 tumor suppressor gene.

Previous work with two closely related human lymphoblast cell lines demonstrated that WTK1 cells are more resistant than TK6 cells to X-ray-induced cytotoxicity, but more mutable at both the TK and HPRT loci. It was subsequently determined that WTK1 cells overexpress a mutant form of the tumor suppressor gene p53, while TK6 cells correctly express wild-type p53. Thus these two cell lines allowed us to examine the mutational spectra at the HPRT locus in related human lymphoblast cell lines differentially expressing p53. Previously, we isolated sets of X-ray-induced and spontaneous WTK1 mutants and spontaneous TK6 mutants and analyzed the mutational spectra by the combination of multiplex polymerase chain reaction (PCR) and Southern blot analysis. Somewhat unexpectedly, we found that even though there were approximately four times as many mutants induced by X rays in WTK1 cells as in TK6 cells, there was very little difference in the mutational spectra. In the present study, to determine if there was a higher frequency of intragenic deletions among the X-ray-induced WTK1 mutants, we further examined the subsets of mutants that contained HPRT point mutations. cDNA sequence analysis was used to define the mutation precisely in 19 X-ray-induced and 25 spontaneous WTK1 mutants and 25 spontaneous TK6 mutants. While subtle differences exist in the spectra of HPRT mutations between these two cell lines, the data again suggest that p53 is associated with an increase in the frequency of mutations at HPRT without an obvious effect on the types of mutations recovered.

The tumor suppressor p53 modifies mutational processes in a human lymphoblastoid cell line.

Abnormalities in the p53 gene play an important role in genomic instability and tumorigenesis. Our previous work showed that p53 status is correlated with differential mutability in two closely related human lymphoblastoid cell lines, TK6 and WTK1. WTK1 cells, which contain a mutation in p53 (p53Ile237) show a remarkably increased mutability, larger genetic alterations at the thymidine kinase locus (tk), an increased ability to catalyze recombination, and a delay in the onset of apoptosis after X-irradiation, compared to TK6 (p53 +/+). In the present study, we demonstrate that after transfection and subsequent overexpression of the known dominant negative mutant p53 Ala143 allele (mp53Ala143) in TK6, there were significantly enhanced spontaneous and X-ray-induced mutant frequencies at the tk locus, and delayed onset of X-ray-induced apoptosis, to a similar extent as in WTK1. In addition, high protein expression of mp53Ala143 in transfectants was correlated with both increased mutation frequency and altered apoptosis kinetics. Similar results were obtained with p53 Ile237 transfection into TK6. Our observations indicate that the product of the p53 gene affects mutational processes. We hypothesize that p53 dysfunction can lead to increased mutagenicity at the endogenous tk gene in human lymphoblastoid cell lines either through delayed apoptosis in response to DNA damage or by mediating increased recombination.

Effects of cell cycle position on ionizing radiation mutagenesis. I. Quantitative assays of two genetic loci in a human lymphoblastoid cell line.

Relatively little work has been done on the influence of the position of the cell in the cell cycle on ionizing radiation-induced mutagenesis. We synchronized WTK1 human lymphoblastoid cells with 200 microM lovastatin for 48 h; under these conditions more than 80% of the cells were arrested in G1 phase. Upon release, there was a 12-15-h lag followed by movement of a large fraction into S phase. We irradiated cells with either 1.5 Gy X rays at 1, 15, 18, 21 or 24 h or 1.5 Gy gamma rays at 1, 5, 10, 15 or 24 h after release from lovastatin. We showed that WTK1 cells were most sensitive to ionizing radiation-induced toxicity in G1 and into S phase, and more resistant in mid to late S and G2/M phase. Somewhat surprisingly, we found that the two different gene loci had different sensitivities to radiation-induced mutation through the cell cycle. Cells in late G1 through mid-S phase were most sensitive to radiation-induced mutations at the autosomal thymidine kinase (TK) locus, whereas G1 phase was the most sensitive phase at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus.

Mutant frequency at the hprt locus in human lymphocytes in a case-control study of lung cancer.

A clonal assay to determine the mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human lymphocytes has been used by a number of investigators to study exposure to mutagens and carcinogens in a variety of populations. We have studied hprt MF in 106 subjects (40 controls and 66 cases) enrolled in a case-control investigation of lung cancer. Epidemiological data collected included smoking history, intake of dietary micronutrients, and occupational and environmental exposures as well as medical history, all obtained from an interviewer-administered questionnaire. All subjects were also genotyped for the known polymorphism in glutathione S-transferase class mu (GST-mu). In analysis of cases and controls, hprt MF was not associated with age, smoking, the polymorphism in GST mu, dietary intake, occupational exposures, family history of cancer or usage of medications. Since MF and cloning efficiency (CE) are not independent when CE is low, further analysis in cases and controls with a CE greater than or equal to 30% (27 cases and 22 controls) was also conducted. In analysis of controls, hprt MF increased with age and was inversely associated with intake of folate and vitamins A and C. The presence of lung cancer was not associated with hprt MF. Thus, our study supports the previous observation that dietary components may affect the MF at the hprt locus.

Spectra of spontaneous and X-ray-induced mutations at the hprt locus in related human lymphoblast cell lines that express wild-type or mutant p53.

Previous work showed that WTK1 human lymphoblastoid cells are radioresistant but more sensitive to X-ray-induced mutation than the closely related line TK6. In addition, WTK1 cells contain a mutant p53 while in TK6 cells p53 is wild-type. In this work, we examined the spectra of 68 X-ray-induced and 56 spontaneous mutants at the hemizygous hprt locus in WTK1 cells. The induced spectra were classified by Southern blot and multiplex polymerase chain reaction (PCR); there were 19 point mutations (28%) with an unaltered Southern blot or PCR pattern, 26 (38%) partial deletions or rearrangements and 23 (34%) complete gene deletions. The spontaneous spectrum included 25 (45%) point mutations, 22 (39%) partial deletions and 9 (16%) complete gene deletions. These spectra of mutations were compared to those of TK6 cells. Although distinct differences in the spectra of mutations at the tk locus were reported previously, overall there is no significant difference in the spectra of X-ray-induced or spontaneous mutations at the hprt locus in these two cell lines. While there was an increase in the proportion of large-scale changes that occurred at tk after X irradiation, the spectrum of mutations at the hprt locus shows all classes of mutations increasing proportionately in WTK1 cells. However, the proportion of internal partial deletion mutations at the hprt locus was about 2 times more frequent in WTK1 than in TK6 cells.

Altered p53 status correlates with differences in sensitivity to radiation-induced mutation and apoptosis in two closely related human lymphoblast lines.

Previous work identified TK6 and WTK1 as human lymphoblast cell lines from one donor that have different capacities to catalyze recombination and that vary significantly in their response to ionizing radiation. WTK1 cells are more resistant to the toxic effects of X-rays yet more sensitive to induced mutation. We demonstrate here that although both cell lines contain equal levels of p53 mRNA, baseline protein levels are 4 times higher in WTK1. Irradiation leads to higher levels of p53 protein in both lines but to a greater extent in TK6. TK6 contains a wild-type p53 sequence, while WTK1 has a homozygous mutation in codon 237 of exon 7. We also observed a significant difference in the kinetics but not the overall degree of apoptosis induced by X-rays in these cells; apoptotic death is delayed for 3 days in WTK1. We hypothesize that this p53 mutation is responsible for the difference in apoptosis as well as for the differences in mutability and mutational spectra reported previously.

Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

WIL2-NS and TK6 are two distinct human lymphoblast cell lines derived from a single male donor. WIL2-NS cells are significantly more resistant to the cytotoxic effects of X-irradiation but considerably more sensitive to induced mutation. In an effort to determine the mechanistic basis for these differences, we analyzed the physical structures of thymidine kinase (tk)-deficient mutants isolated after X-ray treatment of tk heterozygotes derived from TK6 and the more mutable WIL2-NS. Southern analysis showed that while 84% of TK6-derived mutants had arisen by loss of heterozygosity (LOH), all 106 mutants from WIL2-NS derivatives arose with LOH at tk and all but one showed LOH at other linked loci on chromosome 17. We adapted a fluorescence in situ hybridization technique to distinguish between LOH due to deletion, which results in retention of only one tk allele, and LOH due to a mechanism involving the homologous chromosome (e.g., recombination), which results in the retention of two alleles. Among the LOH mutants derived that were analyzed in this way, 9 of 26 from WIL2-NS and 11 of 17 from TK6 cell lines arose by deletion. The remaining mutants retained two copies of the tk gene and thus arose by a mechanism involving the homologous allele. Since many of these mutants arising by a homologous mechanism retained partial heterozygosity of chromosome 17, they must have arisen by recombination or gene conversion, and not chromosome loss and reduplication. Finally, the recombinational capacities of WIL2-NS and TK6 were compared in transfection assays with plasmid recombination substrates. Intermolecular recombination frequencies were greater in WIL2-NS than in TK6. These data are consistent with a model suggesting that a recombinational repair system is functioning at a higher level in WIL2-NS than in TK6; the greater mutability of the tk locus in WIL2-NS results from more frequent inter- and intramolecular recombination events.

Mutational spectrum of ICR-191 at the hprt locus in human lymphoblastoid cells.

Human TK6 lymphoblasts were treated with the acridine derivative ICR-191, and mutants at the hprt locus were isolated. Mutant hprt cDNA was reverse-transcribed from mRNA, amplified by polymerase chain reaction (PCR), and sequenced. Additions of single G:C base pairs (+1 frameshift mutations) in repetitive G:C sequences were found in 82% (32/39) of the mutants. Sixteen of the +1 frameshifts analyzed were located in a single sequence of six consecutive guanine bases in exon 3. The remaining +1 frameshifts occurred at six different GGG sequences (14 mutants) and a single GGGG sequence (2 mutants) in other hprt exons. The repetitive guanine sequences that underwent frameshift mutagenesis were located in both the transcribed and nontranscribed strands of hprt. No single base deletions (-1 frameshift mutations) were observed. Base substitutions were observed in 13% (5/39) of the clones analyzed and occurred at both G:C and A:T bases. Loss of exon 4 from the cDNA was also observed in 5% (2/39) of the mutants. Hprt mutants containing seven consecutive guanines (produced from a +1 frameshift in a GGGGGG sequence) were treated with ICR-191 and wild-type revertants selected in CHAT medium. Revertants were recovered at a frequency of approximately 10(-7) and contained the wild-type sequence (GGGGGG) in all clones analyzed. The observed frequency of ICR-191-induced-1 frameshift reversion in the GGGGGGG sequence was approximately 500-fold lower than the estimated frequency of +1 frameshifts observed in the wild-type GGGGGG sequence following the same ICR-191 treatment. These results suggest that ICR-191 produces predominantly +1 frameshift mutations at the hprt locus in human cells.

Genotoxicity to human cells induced by air particulates isolated during the Kuwait oil fires.

In an effort to examine the potential of exposure to soot from the 1991 oil fires in the Kuwait desert for inducing genetic effects we studied the in vitro genotoxicity of this material. Air particulates isolated near the Kuwait oil fires were studied using three assays. Dose-dependent increases were observed for both sister chromatid exchanges in human peripheral blood lymphocytes and mutation at the hprt locus in the metabolically competent human lymphoblast cell line AHH-1. Similar magnitudes of response were seen using these two assays when testing a standard air particulate sample which had been isolated from the Washington, DC, area. Using the 32P-postlabeling assay, no increase in DNA adduct formation was observed in AHH-1 cells treated with particulates isolated from sampling in Kuwait.

The effects of hypoxia and cysteamine on X-ray mutagenesis in human cells. II. hprt mRNA expression and cDNA sequence analysis of induced mutants.

Mutants at the hprt locus isolated after treatment of cells of the human lymphoblastoid cell line TK6 with X rays were examined by Northern blot and cDNA sequence analysis. In previous work, Southern blot analysis showed that approximately 25% of the mutants isolated from cultures treated with X rays displayed restriction fragment patterns indistinguishable from wild type. In addition, 38 and 48% of the mutants isolated from cultures treated with X rays under two radioprotective conditions, hypoxia or 25 mM cysteamine, respectively, had normal restriction fragment patterns. In the work presented here, Northern blot and DNA sequence analyses were used to characterize these mutants further. Mutants were classified as having normal size and amount of hprt mRNA, reduced amount or undetectable mRNA, or abnormal size message. Mutants that expressed hprt mRNA were sequenced after reverse transcription and PCR amplification. Sequence analysis is reported for 7 mutants from cultures treated in the absence of protection, 11 from cultures treated under hypoxic conditions, and 11 from cultures irradiated in cysteamine. Striking differences among the three treatment groups were not apparent. All types of mutations at both AT and GC base pairs were observed, including transitions, transversions, small deletions, and insertions. Several mutations affected RNA splicing, leading to exon skipping or inclusion of intron sequences in the final message. Approximately half (14/29) of the mutants sequenced had additions or deletions of one to several nucleotides. Also, 3/29 involved tandem DNA base changes (GG-->TT, C-->AA, AA-->CG). These observations are consistent with a mechanism involving the induction of noncoding or synthesis-blocking lesions that result in polymerase slippage or error-prone bypass synthesis. In addition, potential hotspot sites for mutation by X rays were discovered. At one site in exon 3, the same complex mutation, consisting of a G-->T transversion and a nearby six-base deletion, was detected in three independent mutants. Another mutant had a G-->C transversion at the same base, but without the deletion. At another site in exon 8, three mutations occurred at a run of three consecutive cytosines; these included a -C, a -CC, and a C-->AA. Also in exon 8, two mutations (+T, T-->C) occurred at two consecutive thymines.

Mutagenesis after cancer therapy.

A subset of Hodgkin's disease (HD) and breast cancer patients have been reported to have elevated hprt mutant frequencies in peripheral blood lymphocytes after cessation of therapy. A subset of these patients are also known to develop second therapy-related malignancies. Therefore, it is clearly important to determine if these elevations in mutant frequency represent true, persistently elevated mutation frequencies. As a follow-up to our study of patients previously treated for HD, we recruited for a prospective study six previously treated HD patients and five patients who had been treated for squamous cell carcinoma of the head and neck. These individuals were studied several times over a 6-7 months. The results confirmed that a subset of patients have persistently high mutant frequencies when compared to 71 previously studied controls. The study was designed to determine if the elevated mutant frequencies of treated patients represented independent mutations or resulted from the in vivo expansion of single mutant cells. We used the polymerase chain reaction to examine DNA single strand conformation polymorphisms at the T-cell receptor-gamma locus of individual mutant clones. This analysis showed that 20.1% of the mutants from Hodgkin's disease patients and 17.5% of the mutants from squamous cell carcinoma patients were siblings. The sibling mutants generally did not persist over time. However, one patient had one mutant clone that persisted, but slowly decreased in prevalence over a 7 month sampling period. The data demonstrate that treatments for cancer result in persistently elevated mutation frequencies at the hprt locus in some, but not all, patients.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Different cytotoxic and mutagenic responses induced by X-rays in two human lymphoblastoid cell lines derived from a single donor.

Two human lymphoblastoid cell lineages derived from the same parental line exhibit markedly different survival and mutational responses to X-irradiation, but not to chemical point mutagens. WI-L2-NS (ATCC CRL 8155) and TK6 (ATCC CRL 8015) both are derived from the original WI-L2 isolate described by Levy et al. (1968). Both lines are near diploid with stable and indistinguishable karyotypes (47, X, Y 13 +). However, differences in the extent of heterozygosity of chromosome 17 RFLP markers have been detected in these lines. Relative to TK6, WI-L2-NS and several cell lines subsequently derived from it exhibit enhanced survival after X-ray treatment. This is due partly to a more pronounced shoulder in the dose response curve for WI-L2-NS and partly to a higher D0 than is observed in TK6. X-ray-induced mutant frequencies also are markedly different. At the hprt locus, the overall magnitude of the response is similar in the two cell lines. However, in TK6, a linear equation appears to be the best fit to the data, as compared to a linear quadratic curve for WI-L2-NS. Induced mutant frequencies at the tk locus in heterozygotes derived from WI-L2-NS are 20-50-fold higher than those seen in TK6 and tk heterozygous derivatives of TK6. Analysis of the mutability of the two tk alleles in various tk heterozygotes of WI-L2-NS reveals a similar pattern to that described previously in heterozygotes derived from TK6; 3 times as many mutants were recovered from one tk allele than the other. A possible explanation for the higher survival and induced mutant frequencies seen in WI-L2-NS and its derivatives is the presence in these lines of an error prone repair system not functioning in TK6.