Tk Mutants Research Articles

The Mouse Lymphoma Assay Detects Recombination, Deletion, and Aneuploidy

The mouse lymphoma assay (MLA) uses the thymidine kinase (Tk) gene of the L5178Y/Tk(+/-)-3.7.2C mouse lymphoma cell line as a reporter gene to evaluate the mutagenicity of chemical and physical agents. The MLA is recommended by both the United States Food and Drug Administration and the United States Environmental Protection Agency as the preferred in vitro mammalian cell mutation assay for genetic toxicology screening because it detects a wide range of genetic alterations, including both point mutations and chromosomal mutations. However, the specific types of chromosomal mutations that can be detected by the MLA need further clarification. For this purpose, three chemicals, including two clastogens and an aneugen (3'-azido-3'-deoxythymidine, mitomycin C, and taxol), were used to induce Tk mutants. Loss of heterozygosity (LOH) analysis was used to select mutants that could be informative as to whether they resulted from deletion, mitotic recombination, or aneuploidy. A combination of additional methods, G-banding analysis, chromosome painting, and a real-time PCR method to detect the copy number (CN) of the Tk gene was then used to provide a detailed analysis. LOH involving at least 25% of chromosome 11, a normal karyotype, and a Tk CN of 2 would indicate that the mutant resulted from recombination, whereas LOH combined with a karyotypically visible deletion of chromosome 11 and a Tk CN of 1 would indicate a deletion. Aneuploidy was confirmed using G-banding combined with chromosome painting analysis for mutants showing LOH at every microsatellite marker on chromosome 11. From this analysis, it is clear that mouse lymphoma Tk mutants can result from recombination, deletion, and aneuploidy.

Multi-endpoint genotoxic assay using L5178Y (Tk+/- -3.7.2c) cells

When the mouse lymphoma Tk assay (MLA) provides a positive result, its cause can be roughly estimated by examining colony sizes. An increase in the number of large colonies means that the compound tested has point mutational potential, while an increase in small colonies indicates the potential for chromosome aberration. However, it was found to be difficult to clearly judge this in the case of caffeine known as a clastogen lacking the potential of point mutation. In our study, caffeine significantly increased the thymidine kinase (Tk) mutation frequencies derived from large colonies as well as those from small colonies in the standard protocol, although the frequencies derived from a small colony were higher than those from large colonies at higher doses. Therefore, we prolonged the expression period from 2 days, a standard period, to 6 days after treatment and then examined the Tk and Hprt mutations simultaneously. The result showed that caffeine gave a completely negative result on a mutation test for both Tk and Hprt. On the other hand, ethyl methanesulfonate (EMS), a genotoxic carcinogen, showed a positive result for both. Moreover, caffeine and EMS significantly increased the frequencies of micronucleated cells. In conclusion, when MLA gives a positive result and the cause is ambiguous, in order to identify the exact cause of the positive response, it is helpful to perform a confirmatory test investigating the potential of Tk and Hprt gene mutation simultaneously after 6-day expression and to perform an in vitro micronucleus assay during the expression period.

Biologic and Clinical Markers for Outcome after Fludarabine (F) or F Plus Cyclophosphamide (FC) - Comprehensive Analysis of the CLL4 Trial of the GCLLSG.

The CLL4 trial evaluated 1st line treatment with F or FC among 375 CLL patients up to 65 years (Eichhorst et al., Blood, 2006). Updated follow-up (median 52.8 months (mo) for alive patients) revealed 50.4% events for progression-free survival (PFS) and 24.5% for overall survival (OS). Comparing the treatment arms, significant differences were observed in favor of FC for CR (19.9% vs 5.9%, p<.001), CR+PR (94.7% vs 82.4%, p<.001), and PFS (median 67.8 vs 43.7 mo, p=.002) but not for OS (median n.r. vs 84.6 mo, p=.191).The incidences of genomic aberrations by FISH (n=321) were 13q- 50.5%, 13q- single 36.0%, 11q- 21.1%, +12 13.6%, and 17p- 5.0%. No aberration was found in 25.9%. TP53 mutations (n=340) were observed in 8.8%. VH status (n=340) was unmutated in 64.3% and V3–21 was rearranged in 5.9%. ZAP70 (n=236) was >20% in 37.7% and CD38 (n=259) was >7% in 47.9%. β2-MG and TK (both n=261) were >5 mg/L in 13.8% and > 10U/L in 73.0%, respectively. The distributions of these biologic markers and the major clinical characteristics (age: median 59 (42–65) years, sex: 73.1% male, stage: Binet A 9.2%, B 55.4%, C 35.4%, LDH > 250U/L: 39.2%, lymphocytes > 50/nl: 57.1%, comorbidities >1: 21.4%, and creatinine clearance were not significantly different between treatment arms. Outcome was analyzed for subgroups defined by the clinical and biologic parameters in univariate analyses for the entire population (i.e. both treatment arms combined). PFS was significantly shorter for 11q-, 17p- (Fig.1), TP53 mutation, elevated TK and LDH. OS was significantly shorter for the same parameters and unmutated VH, elevated β2-MG, and >1 comorbidities.All except 2 patients with 17p- had TP53 mutations. There were 13 additional cases with TP53 mutation without 17p-. OS was similarly poor for TP53 mutation without 17p- as compared to 17p- and all other cases (OS at 48 mo: 25.9%, 21.4%, and 84.6%, respectively, p<.001) (Fig. 2). To investigate specific treatment effects, the efficacy of F and FC was compared in subgroups defined by biologic markers. PFS was longer after FC only for the subgroups with unmutated VH (p=.005), no aberration (p=.010), 11q- (p=.017), unmutated TP53 (p=.001), CD38 >7% (p=.011), and β2MG <5mg/L (p=.009). OS was significantly longer after FC only in the subgroups with 11q- (p=.037), +12 (p=.044), and unmutated TP53 (p=.029).Multivariate analysis was performed by Cox regression with backward selection including all clinical and biologic parameters in addition to the treatment arms. Regarding PFS, independent prognostic factors were FC (HR 0.68, p=.047), TK (HR 1.62, p=.051), 11q- (HR 1.66, p=.015), and TP53 mutation (HR 4.16, p<.001). Similarly, for OS independent factors were FC (HR 0.56, p=.033), TK (HR 2.79, p=.009), 11q- (HR 1.98, p=.015), and TP53 mutation (HR 11.61, p<.001). If TP53 mutation was not included in the analyses, 17p- entered the model as significant factor (PFS: HR 3.60, p=.003, OS: HR 12.64, p<.001).In summary, biologic parameters are the major determinants of outcome after 1st line treatment with F and FC in CLL. The overall improvement after FC as compared to F appears to result from specific treatment effects in distinct biologic subgroups. However, in comprehensive multivariate analysis, TP53 mutation, 11q-, and TK > 10 remain predictors for PFS and OS independently of the improvement by FC. TP53 mutation and 17p-, although largely overlapping, deserve further evaluation for their independent impact. [Display omitted] [Display omitted]

Subcutaneous Alemtuzumab (Campath) in Fludarabine-Refractory CLL: Final Results of the CLL2H Trial of the GCLLSG and Comprehensive Analysis of Prognostic Markers

The multicenter CLL2H trial of the GCLLSG evaluated subcutaneous alemtuzumab 3 × 30 mg weekly in fludarabine refractory CLL. From September 2002 to February 2006, 103 patients were enrolled and received at least one dose of alemtuzumab. Median age was 63 (35.1–81.8) years, 72% were male, 74% were Binet C, and a median of 3 (1–10) prior lines had been given. Unfavorable genetics were frequent (17p deletion: 29%, 11q deletion: 19%, unmutated IgVH: 68%, TP53 mutation 34%).Subcutaneous treatment was performed on an outpatient basis in 96% and had to be temporarily interrupted in 65 patients due to neutropenia (27%), anemia (3%), thrombocytopenia (8%), infections (36%), and was stopped early in 65 cases due to insufficient response (43%), hematotoxicity (14%) and infections (29%). The median alemtuzumab dose given was 722 (3–2203) mg.Toxicity during treatment period was mostly grade I/II apart from hematotoxicity. Grade 3/4 neutropenia, thrombocytopenia, anemia occurred in 56%, 57%, and 50% of patients, respectively. Grade 3/4 non-cytomegalovirus infection occurred in 29%. CMV reactivation was observed in 15 % total, Grade 3/4 occurred in 8% of patients. All CMV episodes were successfully treated with anti-CMV therapy, and there was no CMV-related death. Injection site reaction occurred in 34% and was grade 1 or 2 except in 1 patient who had grade 3 reaction.Pegfilgrastim prophylaxis was scheduled for the second half of the trial. Grade 3/4 neutropenia occurred in 70% vs 46% and non-CMV infections occurred in 32% vs 24% in the first and second half, respectively.Development of anti-alemtuzumab antibody was assessed in samples from 21 patients. Plasma anti-alemtuzumab antibody was detectable in only 1 patient, who had a concentration marginally above the detection threshold and was found to be negative in a re-test 5 months later. Stable disease was achieved in this patient.After a median follow-up time of 37.9 months, there were 75 (73%) deaths, 56% due to disease progression, 31% due to infection, and 13% not related to CLL. Overall response rate was 34% (CR 4%, PR 30%), median progression free survival time was 7.7 months, and median overall survival time was 19.1 months.Clinical and biologic parameters (age, sex, B-symptoms, stage, ECOG, number of prior lines, node size, hepato-spenomegaly, WBC, LDH, β2-MG, TK, VH status, genomic aberrations and TP53 mutation) were evaluated for their prognostic role. In univariate analyses, OS was significantly inferior for age > 65 y (12.2 vs 29.0 mo, p<.001), ECOG > 1 (10.8 vs 21.5 mo, p=.011), TK > median (26U/L) (14.9 vs 29.0 mo, p=.001), and β2- MG > 5 (13.6 vs 27.2 mo, p=.004). Median PFS and OS were not different for 17p-, 11q-, other cytogenetic and TP53 mutation subgroups. Multivariate analysis by Cox regression revealed only age (HR 1.6, p<.001) as significant prognostic factor, while TK (p=.11), β2- MG (p=.089), and 17p- (p=.528) showed no significant impact.The choice of next therapy significantly affected survival. Seventy-four patients received subsequent salvage treatment or allogeneic stem cell transplantation (SCT). The median OS since next therapy in these patients was 11.5 months. The 2-year OS rate for allogeneic SCT as compared to other subsequent treatments (chemo-, immuno-, or chemoimmunotherapies) was 86% and 27% (p=0.009).

Chemoenzymatic synthesis of l-tyrosine derivative for a transketolase assay

We have prepared an l-tyrosine derivative bearing a d- threo ketose moiety by a convenient chemoenzymatic route. This compound is of potential interest for developing stereospecific assays for enzymes catalyzing C–C bond cleavage such as transketolase. We showed in vitro by analytical studies (LC/MS and 31P NMR) that this compound can release l-tyrosine in the presence of wild type TK extract and bovine serum albumin. This assay is the first step towards a mutant TK selection test that could be developed for yeast cells auxotrophic for l-tyrosine.

Flavouring Group Evaluation 5, Revision 1 (FGE.05Rev1):Esters of branched‐ and straight‐chain aliphatic saturated primary alcohols and of one secondary alcohol, and branched‐ and straight‐chain unsaturated carboxylic acids from chemical groups 1, 2, and 5 (Commission Regulation (EC) No 1565/2000 of 18 July 2000) ‐ Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with

Flavouring Group Evaluation 5, Revision 1 (FGE.05Rev1):Esters of branched‐ and straight‐chain aliphatic saturated primary alcohols and of one secondary alcohol, and branched‐ and straight‐chain unsaturated carboxylic acids from chemical groups 1, 2, and 5 (Commission Regulation (EC) No 1565/2000 of 18 July 2000) ‐ Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with

Mucoepidermoid carcinoma of lung: Potential target of EGFR-directed treatment

Mucoepidermoid carcinoma (MEC) of lung is a rare malignancy of lung which originates from minor salivary glands of tracheobronchial tree. EGFR targeted therapy by inhibition of EGFR activation with the specific tyrosine kinase inhibitors (TKIs) has shown meaningful anti-tumor activity in patients with EGFR TK mutation and/or amplification, or in patients with adenocarcinoma. In the present study, we find that MEC has EGFR mutation in 40% (2 out of 5) of cases, and all mutations are L858R mutation. In addition, we also observed that a MEC patient well-responded to EGFR TKI in the absence of EGFR mutation or amplification. These data indicate for the first time that MEC of lung is another potential target of EGFR inhibitor, and more extended clinical investigation is warranted.

Characterization and susceptibility to antiviral agents of herpes simplex virus type 1 containing a unique thymidine kinase gene with an amber codon between the first and the second initiation codons

A herpes simplex virus type 1 (HSV-1) containing a thymidine (TK) gene with an amber mutation at the 8th position counted from the first AUG codon was isolated from a child with acute gingivostomatitis. The virus was predicted to express a mutant viral translated from the 2nd AUG codon at the 46th amino acid position and consisting of 331 amino acids. The virus was as sensitive to acyclovir (ACV), 5-bromovinyl-2'-deoxyuridine (BVdU), 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), and 1-beta-D-arabinofuranosylthymine (araT) as a wild-type HSV-1. The mutant TK showed the same level of TK activity as the wild-type TK at reaction temperatures of 34 degrees C, 37 degrees C and 39 degrees C. ACV, BVdU, BVaraU, and araT inhibited the replication of the TK-deficient and drug-resistant HSV-1 and HSV-2 in 293T cells in which the mutant TK was expressed to the same extent as in cells in which intact HSV-1-TK was expressed, whereas BVdU and BVaraU inhibited the replication of these viruses less strongly in cells in which HSV-2-TK was expressed. It can be concluded that the mutant HSV-1 exists in nature as a variant and possesses the necessary phosphorylation activities to form ACV-monophosphate from ACV, to form BVdU-diphosphate through BVdU-monophosphate from BVdU, and to form BVaraU-diphosphate through BVaraU-monophosphate from BVaraU. These results indicate that the mutant HSV-1-TK with a deletion of the first 45 amino acid residues is phenotypically the same as that of wild-type HSV-1-TK in terms of the phosphorylation activity of TK-associated anti-herpes virus drugs.

The spectra of large second-step mutations are similar for two different mouse autosomes

Loss of tumor suppressor gene expression via mutations plays a critical role in cancer development, particularly when occurring in heterozygous cells. These so-called “second-step” mutational events are often large in size and arise most often from chromosome loss, mitotic recombination, or interstitial deletion. An open question in cancer research is whether different chromosomes are equally susceptible to formation of large mutations, or alternatively if the unique sequence of each chromosome will lead to chromosome-specific mutational spectra. To address this question, the spectra of second-step mutations were determined for chromosomes 8 and 11 in Aprt and Tk mutants, respectively, isolated from primary kidney clones heterozygous for both loci. The results showed that the spectra of large mutational events were essentially the same. This observation suggests that internal and external cellular environments provide the driving force for large autosomal mutational events, and that chromosome structure per se is the substrate upon which these forces act.

Role of EGFR but not HER2 or HER3 gene copy number in predicting sensitivity of head and neck squamous cell carcinoma (SCCHN) cell lines to EGFR tyrosine kinase inhibitors

6063 Background: EGFR gene copy number has previously been reported to predict for improved overall survival in NSCLC patients treated with gefitinib (IRESSA) or erlotinib compared with placebo [JCO 2006;24:5034–42 &amp; N Engl J Med 2005;353:133–44]. The utility of EGFR gene copy number as a predictive biomarker in other tumour types such as squamous cell carcinoma of the head and neck (SCCHN) is currently under clinical investigation. The present study examined a panel of 20 SCCHN cell lines to identify potential biomarkers predicting in vitro sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Methods: A panel of 20 SCCHN cell lines was screened for sensitivity to gefitinib, vandetanib or erlotinib using a viable cell number endpoint, with G150 values determined for each cell line (inhibitor concentration required to give 50% growth inhibition). Cell lines were blinded and assessed for EGFR, HER2 and HER3 protein expression by ELISA, mutation status by dye-terminator sequencing, and gene copy number by fluorescence in situ hybridisation (FISH). Results: A broad range in sensitivity was observed for all compounds across the panel of 20 SCCHN cell lines (G150 ranging from 0.001uM to =10uM). 12 cell lines were positive for EGFR genomic gain. Sensitivity (GI50 &lt;1uM) to all EGFR TKIs was seen in 11 lines and resistance (GI50 &gt;8uM) in 5 lines. Of the sensitive cell lines, 9 were positive for EGFR genomic gain compared with only 1 of the resistant lines. Furthermore, EGFR protein expression also had a direct association with EGFR TKI sensitivity. In contrast, only 4 cell lines were positive for HER2 or HER3 genomic gain and there was no correlation with sensitivity. The most sensitive cell line was positive for EGFR genomic gain and was the only line to have an EGFR TK mutation (S768I in exon 20). Conclusions: EGFR gene copy number and protein expression appeared to have predictive value in identifying SCCHN cell lines sensitive to EGFR TKIs. No significant financial relationships to disclose.

Transplacental drug transfer and frequency of Tk and Hprt lymphocyte mutants and peripheral blood micronuclei in mice treated transplacentally with zidovudine and lamivudine

In previous studies, we have shown that zidovudine (3'-azido-3'-deoxythymidine; AZT), but not lamivudine [(-)2',3'-dideoxy-3'-thiacytidine; 3TC], is genotoxic when administered to neonatal mice, and that 3TC when coadministered with AZT does not alter the responses observed with AZT alone (Von Tungeln et al. [2002] Carcinogenesis 23:1427-1432). We now have investigated the transplacental transfer of these drugs and the induction of mutants and micronuclei in the neonatal offspring. From gestational day 12 until parturition, female C57BL/6N and C57BL/6N/Tk(+/-) mice, which had been mated to male C3H/HeNMTV mice, were treated daily by gavage with AZT, 3TC, or a combination of AZT and 3TC. In both dams and fetuses, AZT was found at much higher levels than its metabolites, AZT 5'-glucuronide and 3'-azido-3'-deoxythymidine. In the neonates, AZT and the mixture of AZT and 3TC caused a decrease in the percentage of reticulocytes (RETs) and an increase in the percentage of micronucleated RETs and micronucleated normochromatic erythrocytes. When assessed 3 weeks after birth, AZT and the combination of AZT and 3TC increased the thymidine kinase (Tk) mutant frequency in male mice; at 5 weeks, 3TC increased the Tk mutant frequency in female mice. The increase in Tk mutants in mice treated with AZT and the mixture of AZT and 3TC was associated with loss of the wild-type (Tk(+)) allele (loss of heterozygosity; LOH) and a pattern of discontinuous LOH. These data indicate that AZT, 3TC, and the combination of AZT and 3TC are transplacental mutagens and that the increase in mutants resulting from AZT is due mainly to large-scale genetic alterations.

3′‐azido‐3′‐deoxythymidine induces deletions in L5178Y mouse lymphoma cells

3'-Azido-3'-deoxythymidine (AZT), a nucleoside analogue used for the treatment of acquired immunodeficiency syndrome (AIDS), induced a significant dose-related increase in the thymidine kinase (Tk) mutant frequency (MF) in L5178Y/Tk(+/-) 3.7.2C mouse lymphoma cells. Treatment with 1 mg/ml (3,742 muM) AZT for 24 hr resulted in a MF of 407 x 10(-6) compared to a control MF of 84 x 10(-6). The MFs of the large and small colony mutants resulting from AZT exposure were 142 x 10(-6) and 265 x 10(-6), respectively. One hundred and fifty mutants from the 1 mg/ml (3,742 muM) AZT-treated culture and sixty-nine mutants from independent untreated cultures were isolated and analyzed. LOH analysis using a heteromorphic microsatellite locus located in the Tk gene was performed to determine the presence or absence of the Tk(+) allele. Eight other microsatellite markers spanning the entire mouse chromosome 11 also were examined for heterozygosity to determine the extent of LOH. In addition, Tk gene dosage analysis was conducted using Real-Time PCR in those mutants showing LOH at the Tk locus. The presence of only one Tk allele based on Real-Time PCR indicated that the mutant resulted from deletion while the presence of two alleles was consistent with a recombination event. More mutants from the AZT-treated culture showed Tk LOH than did independent mutants from the untreated cultures (91% vs. 64%) and the induced mutants also showed distinct chromosome 11 LOH patterns. The mutation spectrum of mutants from AZT-treated cells was also significantly different from that of spontaneous mutants. More deletions and fewer intragenic mutations were observed in the mutants from the AZT-treated culture than independent mutants from the untreated control. Our data indicate that AZT primarily induced LOH mutations in L5178Y mouse lymphoma cells and a large number of LOH mutations resulted from deletions.

Evaluation of in vitro Genotoxic Potential of Kojic Acid in Human Lymphoblastoid Cells

Although kojic acid is used as a cosmetic agent for skin whitening, information of its genotoxicity in in vitro assay is much complicated. In order to evaluate its genotoxic potentials in vitro, we conducted comet assay in regular and acellular versions, chromosome aberration assay, and TK mutation assay in human lymphoblastoid cells. Positive results were obtained in all of the comet, chromosome aberration, and TK mutation assays at almost identical concentration in both TK6 and WTK1 cells. In the acellular comet assay, kojic acid led to positive responses at pH12 and pH13, suggesting that it induced DNA single strand breaks (SSBs). In the TK mutation assay, kojic acid increased the fraction of normal growing but not slowly growing mutants, suggesting that observed gene mutations are due to point mutations within the TK locus but not gross structural changes that can form chromosome aberrations observable by a microscopy.

A method to distinguish between the de novo induction of thymidine kinase mutants and the selection of pre-existing thymidine kinase mutants in the mouse lymphoma assay

The mouse lymphoma assay (MLA) is the most widely used in vitro mammalian gene mutation assay. It detects various mutation events involving the thymidine kinase ( Tk) gene in L5178Y/ Tk +/− −3.7.2C mouse lymphoma cells. Mutants are detected using a thymidine analogue that arrests the growth of cells containing a functional Tk gene. However, there are a number of potential test chemicals that are thymidine analogues, and there is a problem when using the MLA to evaluate the mutagenicity of these chemicals. Thymidine analogues are activated by Tk before eliciting their toxicity. Therefore, any pre-existing Tk −/− mutants may avoid the toxicity of the test chemical and obtain a growth advantage over the Tk +/− cells, increasing the Tk mutant frequency (MF) in the culture via a selection mechanism. This potential mutant selection effect needs to be distinguished from de novo mutant induction in order to properly evaluate the mutagenicity of these chemicals. Here we describe a simple MLA study design that can differentiate between the selection of pre-existing mutants and de novo mutant induction. Trifluorothymidine (TFT), a thymidine analogue and the selection agent normally used in the MLA, and 4-nitroquinoline-1-oxide (4-NQO), a potent mutagen, were used to treat cells from two different Tk +/− mouse lymphoma cell cultures with different background MFs (approximately 112 and 305 × 10 −6). Both agents significantly increased the Tk MFs in both the normal and high background cultures ( p < 0.01). In 4-NQO-treated cultures, the induced MFs (MF of treated culture − MF of control) for the cultures with different background MFs were about the same ( p > 0.1), while in TFT-treated cultures, they were significantly different ( p < 0.01). In TFT-treated cultures, the fold-increases of MF (MF of treated culture/MF of control) for the cultures with different background MFs were about the same ( p > 0.1), while in 4-NQO-treated cultures, they were significantly different ( p < 0.01). This study confirms that, when de novo mutations are induced, the induced MF is the same for cultures with normal and artificially high background MFs. In situations where the increase in MF is due solely to selection of pre-existing mutants, the “induced” MF will be a multiple of the background MF and the magnitude of the increase of the induced MF will depend upon the magnitude of the background MF. Our results demonstrate that it is possible, using this experimental design, to distinguish between chemicals acting primarily via the selection of pre-existing Tk mutants and those inducing de novo mutants in the MLA.

Microarray analysis distinguishes differential gene expression patterns from large and small colony Thymidine kinase mutants of L5178Y mouse lymphoma cells

BackgroundThe Thymidine kinase (Tk) mutants generated from the widely used L5178Y mouse lymphoma assay fall into two categories, small colony and large colony. Cells from the large colonies grow at a normal rate while cells from the small colonies grow slower than normal. The relative proportion of large and small colonies after mutagen treatment is associated with a mutagen's ability to induce point mutations and/or chromosomal mutations. The molecular distinction between large and small colony mutants, however, is not clear.ResultsTo gain insights into the underlying mechanisms responsible for the mutant colony phenotype, microarray gene expression analysis was carried out on 4 small and 4 large colony Tk mutant samples. NCTR-fabricated long-oligonucleotide microarrays of 20,000 mouse genes were used in a two-color reference design experiment. The data were analyzed within ArrayTrack software that was developed at the NCTR. Principal component analysis and hierarchical clustering of the gene expression profiles showed that the samples were clearly separated into two groups based on their colony size phenotypes. The Welch T-test was used for determining significant changes in gene expression between the large and small colony groups and 90 genes whose expression was significantly altered were identified (p < 0.01; fold change > 1.5). Using Ingenuity Pathways Analysis (IPA), 50 out of the 90 significant genes were found in the IPA database and mapped to four networks associated with cell growth. Eleven percent of the 90 significant genes were located on chromosome 11 where the Tk gene resides while only 5.6% of the genes on the microarrays mapped to chromosome 11. All of the chromosome 11 significant genes were expressed at a higher level in the small colony mutants compared to the large colony mutants. Also, most of the significant genes located on chromosome 11 were disproportionally concentrated on the distal end of chromosome 11 where the Tk mutations occurred.ConclusionThe results indicate that microarray analysis can define cellular phenotypes and identify genes that are related to the colony size phenotypes. The findings suggest that genes in the DNA segment altered by the Tk mutations were significantly up-regulated in the small colony mutants, but not in the large colony mutants, leading to differential expression of a set of growth regulation genes that are related to cell apoptosis and other cellular functions related to the restriction of cell growth.

Characterization of Three Essential Residues in the Conserved ATP-Binding Region of Epstein-Barr Virus Thymidine Kinase

The thymidine kinase encoded by Epstein-Barr virus (EBV TK) is an important target for antiviral therapy and the treatment of EBV-associated malignancies. Through computer-assisted alignment with other human herpesviral TK proteins, EBV TK was shown to contain a conserved ATP-binding motif as for the other TK enzymes. To investigate functional roles of three highly conserved residues (G294, K297, T298) within this region, site-directed mutagenesis was employed to generate various mutants. The TK enzyme activity and ATP-binding ability of these mutant TK enzymes were determined and compared with EBV wild-type TK (wtTK). Mutant G294V lost its ATP-binding ability and was inactive in enzyme activity assay. As the enzyme activity of G294A was reduced to 20% of that of wtTK, the K(m) for ATP binding of G294A was 48.7 microM as compared with 30.0 microM of EBV wtTK. These results suggested that G294 participates in ATP binding and contributes to maintenance of structure. EBV TK mutants K297E, K297Q, and K297R lost their ATP-binding ability and enzyme activity. However, K297R was shown to have a preference for usage of GTP (K(m): 43.0 microM) instead of ATP (K(m): 87.6 microM) as the phosphate donor. This implies that, in addition to nucleotide binding, K297 was involved in the selection of phosphate donor. While EBV TK mutant T298S retained approximately 80% of wtTK enzyme activity, T298A lost its enzyme activity, suggesting that a hydroxyl group at this position is important for the enzyme activity. Interestingly, T298A retained its ATP-binding ability, suggesting a role of T298 in the catalytic process but not in the coordination of ATP. This study demonstrated that amino acid residues G294, K297, and T298 in the ATP-binding motif of EBV TK enzyme are essential for the enzymatic activity but are involved in different aspects of its action.

Frequency of Tk and Hprt lymphocyte mutants and bone marrow micronuclei in mice treated neonatally with zidovudine and didanosine.

The nucleoside analog zidovudine (3'-azido-3'-deoxythymidine, AZT), by itself or in combination with other anti- retroviral drugs, is used perinatally to prevent mother to child transmission of human immunodeficiency virus type 1. AZT is mutagenic in vitro and mutagenic and carcinogenic when administered to neonatal mice. A previous study indicated that the anti-retroviral agent didanosine (2',3'-dideoxyinosine, ddI) potentiated the mutagenicity of AZT in the thymidine kinase (TK) gene of cultured human TK6 lymphoblastoid cells. We have evaluated whether or not ddI affects the in vivo genotoxicity of AZT by breeding C57Bl/6N/Tk+/- female mice with C3H/HeNMTV male mice and treating the offspring daily on postnatal days 1-8 with 200 mg/kg ddI alone or in combination with 200 mg/kg AZT. One day after the last dose, bone marrow polychromatic erythrocytes (PCEs) were obtained to assess the induction of micronuclei; 3 weeks following treatment, the induction of mutants was determined in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) and Tk genes of splenic T lymphocytes from B6C3F1/Tk+/- mice. The mixture of AZT and ddI, but not ddI alone, caused a significant increase in micronucleated PCEs. When assessed 3 weeks after dosing, ddI did not induce mutations in the Hprt or Tk genes. The mixture of AZT and ddI also did not induce mutations in the Hprt gene, but did induce a significant increase in Tk mutants, similar to that observed previously with AZT alone. The induction of mutations in the Tk gene by the mixture of AZT and ddI was associated with loss of the wild-type Tk+ allele. These data indicate that, under the conditions of this experiment, ddI is not mutagenic in neonatal B6C3F1/Tk+/- mice and that it does not potentiate the mutagenicity of AZT.

Loss of P53 heterozygosity is not responsible for the small colony thymidine kinase mutant phenotype in L5178Y mouse lymphoma cells.

The mouse lymphoma L5178Y Tk+/- 3.7.2C assay is a well-characterized in vitro system used for the study of somatic cell mutation. It was determined that this cell line has a heterozygous mutation in exon 5 of Trp53. Based on this assumption that the cell line is heterozygous for the Trp53 gene, it was postulated that the small colony thymidine kinase (Tk) mutant phenotype may be due to a newly induced mutation/deletion in both the Trp53 and Tk1 alleles. The resultant Tk-/- mutants would also be Trp53+/0 or Trp53+/+ and would lose their ability to grow at normal rates. Subsequently, we published our evaluation of the Trp53 status in L5178Y cells. This analysis included sequencing of Trp53 exon 4 and determined that the mouse lymphoma cell line has a mutation in both of the Trp53 alleles and, therefore, no wild-type Trp53 allele in either Tk+/- cells or Tk-/- mutants. Because the cells have no wild-type Trp53, it is not possible that the small colony phenotype results from a newly induced loss of both functional Trp53 and Tk. To determine whether small colonies might, however, include the deletion of both Trp53 and Tk we evaluated, using microsatellite marker analysis, a series of small colony mutants. We also utilized in situ hybridization to determine that the Trp53 alleles are, in fact, in their normal chromosome 11 location in Tk+/- 3.7.2C mouse lymphoma cells. From all of these analyses we can conclude that the small colony mutant phenotype is not caused by deletion of both Trp53 and Tk1.

Analysis of mutations in the Tk gene of Tk+/− mice treated as neonates with 3′-azido-3′-deoxythymidine (AZT)

Mother-to-child transmission of the human immunodeficiency virus (HIV) is reduced by perinatal treatment with the antiretroviral nucleoside analogue 3′-azido-3′-deoxythymidine (AZT, zidovudine). AZT, however, is genotoxic and carcinogenic in mice when administered either transplacentally or neonatally, suggesting a possible cancer risk for children later in life. In a previous study [Carcinogenesis 23 (2002) 1427–1432] we found that treating B6C3F1/ Tk +/− mice on postnatal days 1 through 8 with intraperitoneal injections of 200 mg AZT per kg body weight per day significantly increased spleen lymphocyte mutant frequencies in the autosomal Tk gene. Allele-specific PCR of Tk mutants from treated mice indicated that 61% had lost the Tk + allele (loss of heterozygosity; LOH), compared with 35% of Tk mutants from control mice, a difference that was significant. In the present study, Tk mutant lymphocyte clones were analyzed further using polymorphic microsatellite markers that flank the Tk gene along the length of mouse chromosome 11. The analysis indicated that allele-loss mutations in control mice were due to either total chromosome loss, mitotic recombination, or both. The pattern of marker loss in mutants from AZT-treated mice differed significantly from the control mice and was consistent with chromosome loss, mitotic recombination, interstitial deletion, gene conversion, and an unusual discontinuous LOH. The results indicate that AZT induced a unique pattern of mutations in the Tk gene of mice and that the major mechanisms of mutation by AZT involved deletion and recombination.

Comparison of gene expression changes induced in mouse and human cells treated with direct‐acting mutagens

Exposure to DNA-damaging agents can elicit a variety of stress-related responses that may alter the gene expression of numerous biological pathways. We used Affymetrix microarrays to detect gene expression changes in mouse lymphoma (L5178Y) and human lymphoblastoid (TK6) cells in response to methyl methanesulfonate (MMS; a prototypical alkylating agent) and bleomycin (a prototypical oxidative mutagen). Cells were treated for 4 hr, and RNA was isolated either at the end of the treatment or after a 20-hr recovery period. Two concentrations of each agent were used based on cytotoxicity levels and Tk mutant frequencies. Our microarray data analysis indicated that MMS and bleomycin gene expression responses were considerably different in mouse cells versus human cells. The results also suggested that more comprehensive cellular responses to MMS and bleomycin occurred in TK6 cells than in L5178Y cells. In contrast to L5178Y cells, the response of TK6 cells to MMS and bleomycin was characterized by the induction of p53-dependent genes that are involved in DNA repair, cell cycle regulation, and apoptosis. It appears that the induction of DNA damage by MMS in human TK6 cells mediated cytotoxicity and led to decreased cell survival. This may explain the greater sensitivity of TK6 cells to cytotoxic effects of MMS compared to L5178Y cells. Bleomycin exerted comparable cytotoxic effects in the two cell lines. Overall, these studies were unable to identify distinctive gene expression changes that differentiated bleomycin from MMS in either TK6 cells or mouse lymphoma cells.