Hprt mutant frequency and molecular analysis of Hprt mutations in Fischer 344 rats treated with thiotepa

Tao Chen1, Anane Aidoo, Roberta A. Mittelstaedt, Daniel A. Casciano and Robert H. Heflich2

Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Thiotepa is a bifunctional alkylating anticancer drug that is a rodent carcinogen and a suspected human carcinogen. In order to determine the sensitivity of mutant induction in the Hprt lymphocyte assay for detecting tumorigenic doses of thiotepa, Fischer 344 rats were treated for 4 weeks with thiotepa using a procedure adapted from a carcinogenesis protocol. At various times after beginning the treatment regimen, rats were killed and the lymphocyte Hprt assay was performed on splenic lymphocytes isolated from the animals. The 6-thioguanine-resistant T lymphocyte mutant frequency increased with time during the period of thiotepa exposure and declined slightly thereafter. Significant dose-dependent increases in mutant frequency were found using concentrations of thiotepa that eventually result in lymphoproliferative tumors. Hprt mRNA from mutant lymphocytes was reverse transcribed to cDNA, amplified by PCR and examined for mutations by DNA sequencing. This analysis indicated that the major type of point mutation was G:C->T:A transversion and that 33% of the mutants contained simple or complex frameshifts. Also, a multiplex PCR performed on DNA from mutant clones that were expanded in vitro indicated that 34% of the clones had deletions in the Hprt gene. These results indicate that the induction of lymphocyte Hprt mutants is a sensitive biomarker for the carcinogenicity of thiotepa and that the types of mutations found in the lymphocyte Hprt gene reflect the kinds of DNA damage produced by thiotepa.

Abbreviations: CE, cloning efficiency; CM, conditioned medium; ConA, concanavalin A; DGGE, denaturing gradient gel electrophoresis; DMSO, dimethyl sulfoxide; dNTPs, deoxynucleotide triphosphates; EM, expansion medium; FBS, fetal bovine serum; GM, growth medium; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; RT, reverse transcriptase; SPBS, supplemented PBS; TGr, 6-thioguanine-resistant.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Thiotepa (N,N',N''-triethylenethiophosphoramide) is an alkylating anticancer drug with activity against a variety of human tumors, including those of the breast, ovary and brain; it has also been used to treat cases of malignant lymphoma, urinary bladder malignancies, meningeal carcinomatosis and various soft tissue tumors (17). Like other therapeutic alkylating agents, thiotepa is a carcinogen in laboratory animals (811). Also, an association has been demonstrated between risk for leukemia and treatment of patients with thiotepa (1216) and thiotepa has been classified as a group I human carcinogen by the International Agency for Research on Cancer (5).

In addition to the strong evidence of its carcinogenicity, the clastogenicity of thiotepa has been demonstrated in a number of experimental systems and in man. It induces chromosomal aberrations in animal and human cell lines (1719), chromosomal aberrations, micronuclei and dominant lethal mutations in laboratory animals (2025) and chromosomal aberrations in humans (26). A limited number of reports also indicate that thiotepa can induce gene mutations in vitro (22,2729). Thiotepa has been in use since the early 1950s (8) and there has been a recent resurgence of clinical interest in this drug, primarily because of its use at high doses in conjunction with autologous bone marrow transplantation (7,9,10).

The lymphocyte Hprt assay has been used to measure the genotoxic effects of treating cancer patients with cytotoxic chemotherapeutic agents (33,34). Somatic cell mutations have been implicated as critical events in carcinogenesis through the activation of protooncogenes and the inactivation of tumor suppressor genes (3537). Therefore, an increased mutant frequency in the Hprt reporter gene may be useful in evaluating the potential of a chemotherapeutic treatment for inducing secondary tumors. Also, the pattern of mutations produced in the Hprt gene could provide evidence associating the chemotherapeutic agent exposure with the increased mutant frequency. Little, however, is known about the quantitative relationship between mutant induction in the lymphocyte Hprt gene and the subsequent formation of tumors. In addition, the patterns of mutations produced by cancer chemotherapeutic agents in the lymphocyte Hprt assay are largely unknown.

In the present study, we examined the relationship between lymphocyte Hprt mutant frequency and tumor formation after thiotepa treatment. For this purpose, we determined the frequency of mutations induced by thiotepa in the model rat lymphocyte Hprt assay (3840) using treatment conditions adapted from a procedure that results in the formation of tumors (11). This analysis seems suitable for estimating the relationship between mutant induction and tumor formation in humans, since thiotepa causes mainly lymphoproliferative malignancies in both the male rat (11) and man (5). We measured mutations during and after only 1 month of thiotepa treatment, rather than the 52 week treatment used in the carcinogenesis protocol. We hypothesized that the mutations induced at the beginning of the treatment were likely to reflect the frequency of initiating lesions and we wanted to avoid the effects of the clonal expansion of previously initiated cells during the later stages of the treatment.

We previously determined the types of mutations produced in the rat lymphocyte Hprt assay by PCR/denaturing gradient gel electrophoresis (DGGE) analysis, a procedure that is useful for identifying point mutations and frameshifts (4144). Because the available evidence on the clastogenicity and DNA binding of thiotepa suggests that a major fraction of the mutations induced by this drug involves large disruptions of genomic DNA, we devised and applied methods designed to provide a more comprehensive analysis of mutations than is available from PCR/DGGE. We recently described a procedure for expanding 6-thioguanine-resistant (TGr) rat lymphocyte clones (45) and, in the present study, we used DNA from expanded clones to screen for the induction of deletions in the rat Hprt gene by multiplex PCR. We also used a reverse transcriptase (RT)–PCR/cDNA sequencing analysis of Hprt mRNA from mutant clones in order to provide a more comprehensive characterization of point mutations. We find that Hprt lymphocyte mutation induction can be detected with doses of thiotepa that eventually result in tumorigenicity and that the types of mutations induced by thiotepa in the Hprt gene are consistent with the nature of the DNA damage produced by the compound.


    Materials and methods
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 Introduction
 Materials and methods
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Animals and thiotepa exposure
Male Fischer 344 rats were from our institute's breeding colony. We followed the recommendations set forth by our Institutional Animal Care and Use Committee for the handling, maintenance, treatment and killing of the animals. Seven-week-old rats were administered thiotepa (Thioplex; a gift of Immunex, Seattle, WA) using a treatment procedure adapted from a carcinogenesis protocol (11). Groups of 12 rats received i.p. injections of 0, 0.7, 1.4 or 2.8 mg/kg body wt thiotepa 3 times/week (Monday, Wednesday and Friday) for 4 weeks. The agent was dissolved in phosphate-buffered saline (PBS; 0.9% NaCl, 0.14% KH2PO4, 0.08% Na2HPO4·H2O, pH 7.4) and administered in a volume of 2.5 ml/kg. At 1, 2, 4 and 8 weeks after the treatment was started, 3 rats/treatment group were killed by CO2 anesthesia and the rat lymphocyte Hprt assay was performed. For the 1, 2 and 4 week time points the rats were killed 3 days after the third dose for that week was administered.

Lymphocyte Hprt assay
Reagents and cell culture media.
Ionomycin, phorbol 12-myristate 13-acetate (PMA), concanavalin A (ConA), {alpha}-methylmannoside, 6-thioguanine, amphotericin B, dimethyl sulfoxide (DMSO) and 2-mercaptoethanol were obtained from Sigma (St Louis, MO). RPMI medium 1640 with 25 mM HEPES buffer, PBS, L-glutamine, penicillin and streptomycin and MEM sodium pyruvate were purchased from Gibco BRL (Gaithersburg, MD). Fetal bovine serum (FBS) was from Gemini Bio-Products (Calabasas, CA) and heat-inactivated before use. Lympho-paque was purchased from Accurate Chemical and Scientific (Westbury, NY) and HL-1 medium was from BioWhittaker (Walkersville, MD).

Supplemented PBS (SPBS) was PBS containing 2% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Basal growth medium (GM) was 25 mM HEPES-buffered RPMI medium 1640 supplemented with 10% FBS, 20% HL-1 medium, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 100 U/ml penicillin and 100 µg/ml streptomycin. Conditioned medium (CM) was prepared by incubating a culture of 4–5x106/ml rat spleen cells in GM containing 10 µg/ml ConA at 37°C in a humidified atmosphere containing 5% CO2 for 40 h. The cells were removed from the culture medium by centrifugation and the biological activity of ConA was inactivated by the addition of 30 mM {alpha}-methylmannoside. The resultant CM was stored at –20°C and sterilized through a 0.2 µm filter just before use. Expansion medium (EM) was 25 mM HEPES-buffered RPMI medium 1640 supplemented with 15% CM, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, 1 mM MEM sodium pyruvate, 50 µM 2-mercaptoethanol, 2.5 µg/ml 6-thioguanine, 10 ng/ml PMA and 0.25 µg/ml ionomycin.

Isolation of spleen lymphocytes.
Spleens were removed aseptically from the rats, teased apart with 25–26 gauge needles and washed with cold SPBS to release the spleen cells. The lymphocytes were isolated by Lympho-paque density gradient centrifugation as described previously (38,39). After centrifugation, the interface mononuclear cells were transferred into 15 ml sterile tubes and washed with SPBS. The isolated cells were suspended in GM and enumerated.

Mutant selection and quantification.
The Hprt mutant assay used in this study was modified from our previous protocol (38,39). In particular, the mitogens ionomycin and PMA were used for lymphocyte cloning in place of phytohemagglutinin priming and TK6 feeder cells. For each sample, two sets of lymphocyte cultures were established in three 96-well microtiter plates. Each well of the plates contained 200 µl of a mixture of 20% CM and 80% GM supplemented with 0.25 µg/ml ionomycin and 10 ng/ml PMA. In one set of plates used for determining cloning efficiency (CE) under non-selective conditions, each well was seeded with four target cells and 5x104 lethally irradiated autologous target cells. The other set of plates was used for selecting mutants: each well contained 5x104 target cells and the culture medium was supplemented with 2.5 µg/ml 6-thioguanine. Both sets of plates were incubated in a humidified atmosphere of 5% CO2 in air. After 12–14 days, the wells were examined microscopically in order to score the plates for clone formation. The CE for each set of cultures was calculated as previously described (38) and the frequency of TGr lymphocytes was determined by dividing the CE of the selection plates by the CE of the non-selection plates. Hprt mutant frequencies were analyzed for significance by a two-way analysis of variance that included the time after treatment and the dose as independent variables. Contrasts between individual data points were conducted using Holm's modification (48) of the Bonferroni procedure to correct for multiple comparisons.

Propagation of TGr lymphocyte clones.
The expansion of mutant clones was performed as recently described (45). Briefly, mutant clones obtained from the 96-well 6-thioguanine selection plates were resuspended in the medium contained in their wells. Individual clones were then transferred into 12-well culture plates containing 2 ml EM/well. Cell growth was monitored by visual inspection for up to 9 days. Lymphocytes from clones judged to have successfully expanded were counted using a hemocytometer and washed with PBS. Cell pellets from these expanded clones were frozen quickly and stored at –70°C for subsequent DNA extraction and use in multiplex PCR.

Mutation analysis
Genomic DNA isolation.
DNA was extracted from 80 successfully expanded clones using the QIAamp Blood Kit (Qiagen, Chatsworth, CA) and following the manufacturer's protocol for blood and body fluids.

Multiplex PCR analysis of the Hprt gene.
The primers used for multiplex PCR were specific to sequences flanking the protein coding regions of the rat Hprt gene (GenBank accession nos AF001278AF001282, AF009655, AF009656, U06049, U31249 and U48799) (46) and were chosen using the software program OLIGO (Molecular Biology Insights, Cascade, CO). All nine Hprt exons were amplified simultaneously from rat genomic DNA, exons 1–6 and 9 in individual fragments and exons 7 and 8 in a single fragment. In addition, the unlinked exon 2 of K-ras on chromosome 12 was amplified in the reaction as an internal positive control. The sequences of the oligodeoxynucleotide primers and the predicted sizes of the corresponding amplification products are listed in Table IGo.


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Table I. Oligodeoxynucleotide primers for multiplex PCR amplification of the protein coding regions of the rat Hprt gene
 
The amplification reactions were carried out in a total volume of 30 µl in 500 µl microcentrifuge tubes using reagents purchased from the Applied Biosystems Division of Perkin-Elmer (Foster City, CA). Each reaction mixture consisted of 0.5–1 µg genomic DNA purified from expanded lymphocyte clones, 5 U AmpliTaq DNA polymerase, 125 mM KCl, 25 mM Tris–HCl (pH 8.3), 3.75 mM MgCl2, 250 µM each of the four deoxynucleotide triphosphates (dNTPs), 5% DMSO and the nine pairs of primers (final concentrations listed in Table IGo). The reaction mixtures without the polymerase were covered with 50 µl mineral oil and incubated in a Perkin-Elmer Thermal Cycler 480 for 5 min at 100°C. The reactions were then cooled to 85°C for 5 min for the addition of the polymerase, followed by 33 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 1 min, with a 10 min incubation at 72°C after the last cycle. The reaction products (5 µl) were separated by electrophoresis through a 2% agarose gel that contained 0.5 µg/ml ethidium bromide and visualized on a UV light box. In initial experiments, difficulty was encountered in amplifying the exon 1 product, presumably because of the high GC content of the untranslated portion of exon 1. The addition of 5% DMSO to the amplification mix, however, resulted in the nine clearly distinguishable PCR products, including the product amplified from exon 1. Several examples of the multiplex PCR products amplified from the DNA of TGr clones are shown in Figure 1Go.



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Fig. 1. Multiplex PCR products amplified from DNA extracted from several TGr lymphocyte clones isolated from an untreated rat. The amplification products were separated on a 2% agarose gel containing ethidium bromide and visualized on a UV transilluminator. Lane 1, 100 bp ladder; lanes 2 and 3, wild-type pattern of products; lane 4, exon 1 product missing; lanes 5 and 6, exons 7 and 8 product missing. Labels on the left indicate fragment sizes in bp; labels on the right identify the products by exon.

 
Preparation of total RNA.
One to five thousand lymphocytes were taken directly from positive clones in the 96-well 6-thioguanine selection plates and transferred to 500 µl reaction tubes. The cells were washed once with PBS and the pellets were frozen at –70°C or used directly for preparation of total RNA. To each pellet was added 10 µl RNase-free H2O (Promega, Madison, WI) containing 0.4% RNasin (Promega) and 2.5% Nonidet P40 (Gibco BRL). The cells were mixed with a pipette tip to assist in lysis and incubated for 20 min on ice. The lysate was the source of total RNA for Hprt RT–PCR reactions. Initial experiments indicated that lysates prepared from >5x103 cells could inhibit the RT–PCR reaction (unpublished data).

RT–PCR of Hprt mRNA and sequence analysis of mutations.
The sequences of the primers used for RT–PCR and their positions relative to the rat Hprt cDNA protein coding region are shown in Figure 2Go. The primer designs were based on the rat cDNA sequence described by Jansen et al. (47). RT–PCR was performed using the Access RT–PCR System (Promega) and 20 µl RT–PCR reactions contained 2 µl cell lysate (~0.2–1x103 cell equivalents), 1x AMV/Tfl reaction buffer, 200 µM each dNTP, 1 mM MgSO4, 0.1 µg oligo(dT) (Gibco BRL), 0.1 µM each primers R1F and R2R, 2 U AMV reverse transcriptase and 2 U Tfl DNA polymerase. The reactions were overlaid with mineral oil and thermal cycled for one cycle of 45 min at 48°C and 2 min at 94°C, for 40 cycles of 30 s at 94°C, 1 min at 60°C and 2 min at 68°C, followed by an incubation of 7 min at 68°C. The products from this reaction were diluted 1:100 in H2O and 1 µl of this dilution was used in a 30 µl PCR using nested PCR primers. The nested PCR also contained 1x AmpliTaq PCR buffer II (Perkin-Elmer), 1.5 mM MgCl2, 100 µM each dNTP, 1.5 U AmpliTaq DNA polymerase and 0.15 µM each primers R3F and R4R and was carried out in 96-well plates using a GeneAmp PCR System 9600 thermocycler (Perkin-Elmer). The thermal cycling conditions consisted of an incubation of 94°C for 3 min, 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, followed by 72°C for 7 min after the last cycle. A 5 µl portion of the PCR products was evaluated by agarose gel electrophoresis. Reactions that produced a clearly defined band of PCR product were purified using a QIAquick Spin PCR Purification Kit (Qiagen). The purified cDNA products were used as the template in DNA sequencing reactions that examined the entire Hprt protein coding region for mutations. Sequencing reactions were conducted with an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction FS Kit and a 373 Stretch DNA Sequencing System (Applied Biosystems). We began sequencing with the reverse primer R6R (Figure 2Go) and usually obtained adequate sequence data for the entire coding region. Primers R3F, R5F and R7R (Figure 2Go) were used if necessary for completing and confirming the sequencing results.



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Fig. 2. Relative positions and sequences of primers used for the RT–PCR amplification of rat Hprt mRNA and for the sequencing of the protein coding region. Base positions of the rat Hprt cDNA sequence from Jansen et al. (47).

 

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 Abstract
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 Materials and methods
 Results
 Discussion
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Hprt mutant frequencies in spleen lymphocytes from thiotepa-treated rats
Male Fischer 344 rats were treated with multiple doses of thiotepa and killed 1, 2, 4 and 8 weeks after the initial treatment. Spleen lymphocytes were isolated from these rats and the frequencies of TGr T lymphocytes were determined by the modified rat lymphocyte Hprt assay (Table IIGo). Because half of the rats treated with the 2.8 mg/kg dose of thiotepa died after 2 weeks of exposure, we did not obtain any data for the high dose group at the 4 and 8 week time points.


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Table II. CE and frequency of TGr lymphocytes from Fischer 344 rats treated with thiotepa three times per week for 4 weeks
 
Both the treated and control rats displayed very low mutant frequencies at the 1 week time point. At the 2, 4 and 8 week time points, the frequencies of TGr T lymphocytes in all the treated groups were significantly higher than the concurrent control frequencies, with the increases ranging from 3.0- to 11.9-fold above the controls (P < 0.05). Also, significant dose-dependent increases in the mutant frequencies were found when the mutant frequency data from the different time points were pooled by dose (P < 0.05). The mutant frequencies in all the thiotepa exposure groups increased with time (P < 0.05) and reached their highest values by 4 weeks after the initial exposure. Thereafter, there was a significant decrease in mutant frequency at week 8 (P < 0.05).

Molecular analysis of mutations in TGr spleen lymphocyte clones from thiotepa-treated rats
A total of 130 TGr lymphocyte clones were collected from all 21 rats that were treated with multiple doses of 0.7, 1.4 and 2.8 mg/kg thiotepa and were assayed for mutations at the 2, 4 and 8 week time points. Out of these 130 clones, 80 were successfully expanded to at least 5x105 cells/clone and were examined by multiplex PCR for deletions. Twenty-seven of the 80 were missing one or more Hprt PCR products and contained potential deletion mutations; 19 of these 27 were clearly independent mutations (Table IIIGo). (In our analyses, mutations found more than once in clones from a single rat were assumed to be siblings.) Most of these putative deletions affected either the 5'- or 3'-end of the gene. Among the independent putative deletions, one was a partial exon 1 deletion, three affected multiple exons and 15 involved a single exon, the most common being exon 1.


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Table III. Multiplex PCR analysis of genomic DNA from TGr lymphocyte clones cultured from thiotepa-treated Fischer 344 rats
 
The 130 TGr clones from thiotepa-treated rats were also analyzed by direct lysis of ~1x103 cells from each clone to release cytoplasmic RNA, followed by RT–PCR/cDNA sequencing. Of the 27 clones that revealed potential genomic deletions by multiplex PCR, only one mutation was found by RT–PCR (described below); the other 26 clones either produced no RT–PCR product (15 clones) or a product with no obvious sequence change (11). In contrast, the 53 clones that were examined by multiplex PCR and had a wild-type pattern of PCR products included 43 clones that contained mutations identified by RT–PCR/cDNA sequencing; the other 10 clones produced either wild-type PCR product (six clones) or no product (four). Only eight of the remaining 50 clones, the clones that could not be expanded for multiplex PCR analysis, produced a mutation by RT–PCR/cDNA sequencing; 31 of these clones produced no PCR product and the remaining 11 amplified a product with no obvious sequence alteration.

The thiotepa-induced mutations that were identified by RT–PCR/cDNA sequencing of mutant lymphocyte RNA are listed in Table IVGo and summarized in terms of the sequence alteration in the non-transcribed DNA strand in Table VGo. Forty-six of the 52 mutations that were recovered were clearly independent. Single base pair substitutions were found in 20 out of the 46 independent mutations (44%) leading to 18 missense, one nonsense and one silent mutation. Substitutions at G:C accounted for 65% of point mutations (13 of 20), with G:C->T:A transversion being the predominant type (8/20, 40%). Only 20% (4/20) of independent point mutations were transition mutations whereas 80% (16/20) were transversions. Ninety percent (18/20) of independent point mutations occurred with the mutated purine positioned in the non-transcribed DNA strand, 11 at dG and seven at dA, while only two G:C->A:T mutations were recovered that had the mutated dG on the transcribed strand. One G:C->T:A transversion at base 151 was observed seven times in four animals. An A:T->T:A transversion at base 271 was observed in three different animals.


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Table IV. RT–PCR/cDNA sequencing analysis of mutations in TGr lymphocyte clones cultured from thiotepa-treated Fischer 344 rats
 

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Table V. Summary of Hprt mutation data from RT–PCR/cDNA sequencing analysis in terms of sequence alteration on the non-transcribed DNA strand
 
The mutation profile derived from RT–PCR analysis also exhibited a high incidence of frameshifts and complex mutations (33%, 15/46 independent mutations; Table IVGo). Among these, three were –1 or +1 frameshifts, two were complex mutations (deletions accompanied by insertions) and the other 10 were deletions or insertions of >2 bp. Two mutations resulted in the inclusion of portions of intron 1 in the cDNA, presumably due to the effects the mutations had on Hprt mRNA splicing. One of these mutations had a deletion of 10 bp in exon 1 and 2 bp in the intron, removing the consensus splice donor site. As a result, 17 bases from intron 1 were included in the mRNA, causing a frameshift. The other included the first 19 bases of intron 1 in the mRNA, without a mutation at the splice donor site.

Twenty-four percent of the independent mutations identified by RT–PCR (11/46) contained deletions of entire exons. All exons but 1, 8 and 9 were deleted in one or more of the mutants in this category. Most of the samples with exon deletions in their cDNA sequence were also analyzed by multiplex PCR and one with a cDNA exon 2 deletion was also found to have an exon 2 deletion in genomic DNA. The remaining mutants having exon deletions in their cDNA had no apparent genomic DNA deletions. Cariello and Skopek (49) indicate that point mutations occurring at splice sites cause most of the incorrect mRNA splicing in the human Hprt gene. The data from the multiplex PCR analysis are consistent with this conclusion.


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 Abstract
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 Results
 Discussion
 Conclusions
 References
 
Mutation in the rat lymphocyte Hprt gene is detected at doses of thiotepa that eventually result in tumors
It is widely accepted that the transition from a normal somatic cell to a cancer cell is due to mutations in protooncogenes and tumor suppressor genes. Experiments have shown that these mutations, which could arise through base pair substitution, deletion, insertion or rearrangement of DNA sequences, play a critical role in the initiation and progression phases of carcinogenesis (50). In the present study, the Hprt gene is used as a reporter of these mutations and, if it is an adequate reporter, its sensitivity to mutation induction should be comparable with those genes important to carcinogenesis, especially when Hprt frequencies are measured in the target tissue for tumorigenesis.

Evidence indicating that thiotepa treatment causes lymphoproliferative malignancies in both humans and male rats (1116) prompted us to use the rat model to explore the relationship between the carcinogenicity of thiotepa and its ability to induce mutations in the lymphocyte Hprt assay. In the carcinogenesis study (11), groups of 35–39 male rats, aged 35, 42 or 58 days, received i.p. injections of 0.7, 1.4 or 2.8 mg/kg thiotepa 3 times/week for up to 52 weeks and the animals were observed for up to 87 weeks. One group of 10 males served as controls. All of the high dose animals died before week 19. Malignant lymphomas, lymphocytic leukemia and granulocytic leukemia were observed in the remaining animals treated with thiotepa. The incidence of tumors [(no. of tumors/no. of rats)x100] increased from 0% in the control rats to 18% in rats given 0.7 mg/kg thiotepa and to 38% in rats treated with the 1.4 mg/kg dose. At 4 weeks after beginning the treatment, the most sensitive time point for measuring mutations in our study, the induced Hprt mutant frequencies were 15.6x10–6 for the 0.7 mg/kg dose and 34.2x10–6 for the 1.4 mg/kg dose, as compared with a control frequency of 3.1x10–6. It is clear that at this initial stage of carcinogenesis the lymphocyte Hprt gene is acting as a sensitive reporter for the mutagenicity and potential carcinogenicity of thiotepa.

Types of mutations induced by thiotepa in the rat lymphocyte Hprt gene reflect the types of DNA adducts formed by thiotepa
Several observations indicate that most of the mutations that were identified in lymphocytes from thiotepa-treated rats were due to thiotepa-induced DNA damage. First, the 21 rats from which the 130 mutants were collected for mutational analysis had an average 7.4-fold higher mutant frequency than those of the nine concurrent control animals (P = 0.0015), suggesting that most of the mutations were not of spontaneous origin. Consistent with this conclusion, the pattern of point mutations produced by thiotepa was different from our small collection of 35 independent spontaneous Hprt lymphocyte mutations from F344 and Big Blue rats. These spontaneous mutations were identified by a combination of PCR/DGGE (seven of the mutations) and RT–PCR/cDNA sequencing of TGr lymphocyte clones from this and other studies (unpublished data). Mutations involving deletions and insertions were similar between the mutant sets (23% for spontaneous versus 33% for thiotepa mutations; Table VGo), but G:C->A:T transitions accounted for 60% of spontaneous point mutations (compared with 14% of thiotepa mutations), while only 7% of spontaneous point mutations were G:C->T:A transversions (versus 40% for thiotepa mutations). In addition, 90% of all base pair substitution mutations induced by thiotepa had the mutated purine situated in the non-transcribed DNA strand (versus 44% for the spontaneous mutations). This strand bias could have resulted from transcription-coupled DNA repair preferentially removing thiotepa-induced dG and dA adducts from the transcribed DNA strand.

The mutations detected in thiotepa-treated rats were also consistent with what is known of the DNA damage induced by this drug. Thiotepa contains a four-coordinated phosphorus atom and three aziridine rings. Bifunctional alkylating agents, such as thiotepa, are thought to be effective therapeutically because they have multiple reactive groups that can produce DNA–DNA and DNA–protein cross-links in addition to DNA monoadducts (51). Thiotepa produces reactive species through direct nucleophilic ring-opening of two of its aziridinyl groups (52,53) or through hydrolysis of thiotepa to aziridine (ethylene imine), a highly reactive monofunctional alkylating species (54). In addition to cross-links, the first pathway could result in DNA strand breaks and the formation of DNA adducts (53,55); the second pathway can form either monoadducts through reaction with aziridine or cross-links through a diaziridinyl partial hydrolysis product (54). An N7-dG adduct, 7-(2-aminoethyl)-dG, and a small amount of an aminoethyl adduct with dA (possibly attached to N3) have been identified, while adducts with dC or dT have not been detected (5456). Since 7-(2-aminoethyl)-dG is not stable, the adducts produced from the reaction of thiotepa with DNA probably result in the formation of apurinic sites or imidazole ring-opened products (5658).

Among the independent Hprt mutations recovered from thiotepa-treated rats, G:C->T:A transversion was the major type of base pair substitution, accompanied by high percentages of frameshifts and large deletions. N-alkylation of DNA, as is caused by thiotepa, can give rise to both gene mutations and structural chromosome aberrations (59). N-alkyl monoadducts could also cause mutations through spontaneous hydrolysis or enzymatic removal of the alkylated base to produce an apurinic site (57). When DNA containing apurinic sites is replicated in bacteria, base pair substitutions occur due to the substitution of dA residues opposite the non-informational apurinic site, causing G:C->T:A and A:T->T:A transversions (60). If rat polymerases behave similarly, the 35% G:C->T:A and 20% A:T->T:A frequencies for thiotepa-induced base pair substitutions could be caused by the depurination of 7-(2-aminoethyl)-dG and 3-(2-aminoethyl)-dA adducts.

The induction of a high percentage of DNA insertions and deletions by thiotepa was not unexpected in view of the known clastogenic activity of this agent. DNA cross-linking and depurination of the 7-(2-aminoethyl)-dG lesion can block DNA replication and induce DNA strand breakage (6163). Blocking DNA replication and DNA strand breakage are presumably involved in the cytotoxicity of this anticancer drug, but they can also potentially produce mutations, especially deletions and insertions. Gill et al. (58) found that Chinese hamster ovary cells transfected with formamidopyrimidine-DNA glycosylase, which can repair ring-opened lesions, displayed increased resistance to the lethal and mutagenic effects of thiotepa. Therefore, imidazole ring-opening of 7-(2-aminoethyl)-dG may also play an important role in the mutagenesis induced by thiotepa in rats.

Methodological improvements to the rat lymphocyte Hprt assay and analysis of rat lymphocyte Hprt mutations
In this study, we utilized a medium containing a combination of the ionophore ionomycin and the tumor promoter PMA, rather than using lectin priming and TK6 feeder cells to promote the clonal growth of rat lymphocytes. The main advantage of the modified method was that the lymphocyte clones were much larger than those of the previous assay so that scoring of positive clones was easier and cells from the clones could be used without further expansion for mutation analysis by RT–PCR/cDNA sequencing. Initial experiments indicated that the CEs for both non-selection and selection cultures were similar using the modified method and using lectin/feeder cell stimulation (unpublished data). The low concentrations of ionomycin and PMA included in the GM obviated the need for antigenic stimulation by bypassing the antigen receptor and activating the lymphocytes directly without the toxicity associated with lectin priming (45). In addition, ionomycin and PMA are likely to provide extra requirements for T cell proliferation that are present in bulk culture (45,64), so that feeder cells are not necessary in the modified method.

An advantage of using the Hprt gene as a reporter for in vivo mutation is the large range of mutational events that the locus can recover. The lymphocyte Hprt assay has recovered base pair substitutions, frameshifts, splicing mutations and large deletions and insertions (40,51,65,66). Previous analyses of mutations produced in the rat lymphocyte Hprt assay, however, have been limited to determining point mutations by PCR/DGGE or cDNA sequencing (41,67). It is estimated that ~80% of the different mutations recovered in the human lymphocyte Hprt assay are detected by the RT–PCR/cDNA sequencing approach (68). Unfortunately, large DNA alterations cannot be characterized by RT–PCR/cDNA sequencing and even some relatively small deletion mutations could not be detected by this approach in an analysis of human Hprt mutations (69).

Recently, we elucidated the DNA sequences flanking all nine exons of the rat Hprt gene (46). Using this sequence to design primers, a multiplex PCR analysis was developed to screen for deletion mutations. The method recovered a number of different potential genomic deletions in lymphocyte mutants from thiotepa-treated rats. Although we have not confirmed that the multiplex PCR is specifically detecting deletions, other, similar multiplex PCR screens for Hprt mutations have been highly efficient and accurate procedures for detecting deletion mutations (68,70,71). Also, multiplex PCR screens on small sets of mutants from rats treated with ethylnitrosourea or N-hydroxy-2-acetylaminofluorene, compounds that might be expected to produce a high frequency of point mutations (41,66,72), detected few genomic deletions (73; unpublished data). Thus, RT–PCR/cDNA sequencing plus multiplex PCR deletion screening should give a relatively complete analysis of the types of mutations recovered by the Hprt gene. In the present study, mutations were identified in 70 of the 80 clones that could be expanded, and thus analyzed by both techniques.

Several of the RT–PCR/cDNA sequencing analyses resulted in the amplification of wild-type sequence or in no amplification product, even when multiplex PCR indicated that the Hprt gene contained no major deletions. Reasons for a lack of amplification include no Hprt mRNA expression due to a nonsense mutation (74) or a major deletion or simply a reaction failure. The apparent amplification of wild-type sequence could occur if a positive well contained more than one mutant with different mutations, a possibility when mutants are selected by cloning in 96-well dishes, or if residual Hprt mRNA was amplified from the inactivated wild-type cells that make up the mutant clones. This latter possibility appears to have occurred with several of the mutant clones we examined that were suspected to contain large genomic deletions by multiplex PCR (see Results) and were not expected to express mutant mRNA. Finally, we suspect that the low recovery of mutations by RT–PCR/cDNA sequencing in the 50 clones that could not be expanded for multiplex PCR analysis (16% of these clones produced mutations versus 55% of the clones that were capable of further expansion) was due to the poor condition of mutant cells in these clones. Besides being incapable of further replication, the mutant cells in these clones may be expressing little Hprt mRNA. It may be that processing clones for expansion and RT–PCR earlier during the cloning procedure may increase the fraction of clones that can be expanded and the number of mutations that can be detected.


    Conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Treatment of rats with doses of thiotepa that eventually result in tumors produced significant increases in the frequency of lymphocyte Hprt mutants. The types of Hprt mutations recovered in thiotepa-treated rats were consistent with what is known of the kinds of DNA damage produced by thiotepa and demonstrate that the rat lymphocyte Hprt gene is capable of recovering mutations ranging from base pair substitutions to large deletions. The results of this study suggest that the Hprt lymphocyte assay is a useful biomarker for assessing the potential carcinogenicity of cytotoxic cancer chemotherapeutic agents.


    Notes
 
1 Present address: Environmental Carcinogenesis Division, MD-68, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA Back

2 To whom correspondence should be addressed Email: rheflich{at}nctr.fda.gov Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 

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Received July 6, 1998; revised September 16, 1998; accepted October 2, 1998.