Radiation-Induced Chromosome Instability in WTK1 and TK6 Human Lymphoblastoid Cells


The effects of ionizing radiation on single nucleotide polymorphism (SNP) copy number variations between TK6 and WTK1 cell lines are described herein. Specifically, the integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) was analyzed in the presence and absence of ionizing radiation. WTK1 cells contain a p53 mutation, whereas the TK6 cell line has the native p53 tumor-suppressor gene. Each cell line was isolated post-irradiation for SNP analysis, which showed significant, genome-wide impacts on both cell lines; for the mutant WTK1 sample, there were a total of 48 gene deletions and no gene amplifications, whereas for the wild-type TK6 sample, there were 217 gene deletions and 9 gene amplifications. It appears that both cell lines are affected in the areas of cell-cycle control, but that other affected areas differ significantly between the two.

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Chant, A. , Driscoll, H. , Weiss, J. , Chaudary, A. and Kraemer-Chant, C. (2023) Radiation-Induced Chromosome Instability in WTK1 and TK6 Human Lymphoblastoid Cells. Advances in Biological Chemistry, 13, 57-70. doi: 10.4236/abc.2023.132005.

1. Introduction

p53 is a critical cell-cycle check-point protein that regulates the G1 phase of the cell cycle and is directly responsible for maintaining genomic DNA stability during the DNA damage repair stage. It is known to coordinate the repair of damaged DNA and the removal of DNA lesions before the cell enters S-phase of the cell cycle. Therefore, it is easy to imagine that any problems that might occur during the G1 cell-cycle arrest would impact normal repair to the damaged DNA that occurs prior to entering S-phase. The p53 gene has been studied extensively and has been identified as one of the most frequently mutated genes in human cancers [1] [2] [3] [4] . The question of how far-reaching the effects of these mutations are on cell integrity is still being studied. The p53 regulatory protein is crucial in the cell’s response to DNA damage and plays a direct role in the DNA repair pathways, affecting the activities of a number of diverse regulatory proteins that collectively control the early stages of the cell cycle [5] - [19] . This would further lead to genomic instability, resulting in abnormal numbers of chromosomes, gene amplifications, chromosomal rearrangement, deletions, insertions, accumulation of double-strand breaks, and gene amplifications [20] - [26] . Controlled cell death (apoptosis) has also been reported to be affected by p53 mutated proteins, as cell lines with these mutations have been shown to have a reduced frequency of apoptotic death, which could in turn lead to the accumulation of gene mutations and progression of tumorigenesis [27] [28] [29] [30] .

For this study, we analyzed the effects of ionizing radiation on two closely related WIL2-derived lymphoblastoid cell lines (TK6, or thymidine kinase 6, and WTK1). The WTK1 cell line is a TP53-knockout mutant derived from the WIL2 parent cell line. Specifically, the p53 gene in the WTK1 cell line has a substitution at codon 237, which leads to a mutation in the p53 protein from methionine to isoleucine. This cell line will only overexpress mutant p53 protein; no wild type p53 will be present. This cell line has been used so frequently in previous research on the effects of ionizing radiation that it can be considered the “gold standard” for these types of studies. The TK6 cell line, in contrast, is also derived from the WIL2 parent cell line but is a thymidine kinase heterozygote cell line that is wild-type for p53 [31] [32] [33] [34] . Previous studies on the effects of ionizing radiation on the two cells lines show that the WTK1 cell line is more resistant to radiation-induced killing and that there is significantly less apoptosis in WTK1 when compared to TK6. Mutability was also shown to be drastically different, with TK6 exhibiting a 10-fold decrease when compared to WTK1 [35] [36] .

2. Materials and Methods

2.1. Cell Lines and Cell Culturing

Cells used were the thymidine kinase heterozygote cell line TK6 and the TP53 mutant cell line WTK1. Both cell lines were each cultured in a 75 cm2 tissue culture flask at 37˚C with 8% CO2 and maintained at a cell concentration between 2 × 105 and 1 × 106 cells/mL. The culture medium consisted of RPMI 1640 and 10% v/v heat-inactivated horse serum (Gibco®, a ThermoFisher Scientific Company, Waltham, MA, USA).

2.2. Irradiation of Cell Lines and Preparation for SNP Analysis

The two lymphoblastoid cell lines (TK6 and WTK1) were treated with ionizing radiation (3 Gy vs. 0 Gy control) using a 137Cs biological irradiator (Gammacell-1000 Unit). Each cell line was irradiated 0.3 cm from the surface with a dose rate of 6.41 Gy/min for 28 seconds to provide a total of 3 Gy exposure. After irradiation, cells were incubated for 48 hours at 37˚C under 8% CO2, followed by isolation for SNP analysis. Whole genomic DNA was isolated from both cell lines using a DNeasy Blood and Tissue Kit (Qiagen). Approximately 5 million cultured cells (WTK1 or TK) were centrifuged separately for 5 minutes at 300 ×g. After centrifugation, 200 μL of PBS (50 mM potassium phosphate, 150 mM NaCl, pH 7.2) was used to resuspend the pellet. Once all cells were in free suspension, 20 μL of Proteinase K (600 mAU/mL) and 4 μL of RNase A (100 mg/mL) were added, and the cells were incubated at room temperature for 2 minutes. After the incubation period, 200 μL of AL buffer (Qiagen) was added, the suspension was mixed by vortexing, and the cells were incubated again for 10 minutes at 56˚C. The sample tube was left to cool to room temperature before 200 μL 95% molecular grade ethanol was added, and the sample was vortexed until it was thoroughly mixed. The sample was then placed in a DNeasy spin column and centrifuged at 6000 ×g for 1 minute. The flowthrough and collection tube were discarded and the DNeasy spin column was placed in a new tube. 500 μL of AW1 buffer (Qiagen) was added to the tube and centrifuged at 6000 ×g for 1 minute. The DNeasy spin column containing the DNA was placed in a new collection tube and 500 μL of AW2 was added and centrifuged at 20,000 ×g for 1 minute. The DNeasy column was then put into a clean tube and 200 μL of AE buffer (Qiagen) was added to the membrane and left to incubate at room temperature for 1 minute. The sample was then eluted by centrifuging at 6000 ×g for 1 minute.

2.3. Analysis of SNPs

50 μg of whole genomic DNA from each cell type was then analyzed using a 256 k STY microarray chip, and analysis of single nucleotide polymorphisms (SNPs) was carried out using an Affymetrix GeneChip Human Mapping 250 K Sty. Partek Genomic Suite’s Copy Number analysis (CN) and Allele-specific copy number (ASCN) workflows were utilized for the two samples using the paired analysis option.

3. Results

3.1. SNP Analysis—Integration of ASCN and CN Results and P-Values

To confirm allele deletions and detect copy neutral genomic events, ASCN results were integrated with CN results. Table 1 and Table 2 show the raw data set from the SNP analysis. Statistical analysis shows the P-values for all the probes listed to be of a high degree of confidence.

3.2. SNP Analysis—Specifics of TK6 and WTK1 Results

Interestingly, no two genes from the TK6 and WTK1 samples were affected by

Table 1. Integrated ASCN results with CN results for TK6.

UV irradiation in the same way. Specifically, no one deletion or amplification was seen on the same gene of a chromosome in both samples. The genes of some chromosomes did have deletions in both samples, but for separate, distinct genes on each chromosome. In addition, most of the modifications caused by UV irradiation were gene deletions, whereas amplifications are lesser in frequency. These deletions and amplifications in specific chromosomes are uniquely linked to each cell type. The only chromosomes that showed no change were chromosomes 8, 14, 18 and 19.

When comparing the data for TK6 (Table 1) and WTK1 (Table 2) cell samples, there were 13 deletions in chromosome 1 for WTK1 and a single amplification of one gene (DAB1) in the TK6 sample. Both chromosomes 2 and 3 showed 4 deletions each, all of which were located in WTK1; TK6 showed no changes for either chromosome. Chromosome 4 showed 5 deletions for WTK1 and 4 deletions for TK6, but again, these were for different genes. TK6 showed a high incidence of deletions in chromosome 5 - 9 deletions were identified on this chromosome, as compared to 4 in the WTK1 sample. Chromosome 6 showed 2 deletions for TK6 and 4 deletions for WTK1. TK6 showed a high number of deletions again in chromosome 7 - 13 deletions were identified on this chromosome, with a single amplification for one gene (specifically SNTNAP2). WTK6 showed only 1 deletion and no amplifications for this chromosome (chromosome 7).

Only 1 gene was affected on chromosome 9 for WTK1 (on gene GBBR2), which was a deletion. There was no effect seen on chromosome 9 for the TK6 sample. For chromosome 10, there were 16 total deletions, 3 in the WTK1 sample and 13 in the TK6 sample. Chromosome 11 showed 5 deletions; 3 were in the TK6 sample, and 2 were in the WTK1 sample. Again, there were no overlaps between the two samples. Chromosome 12 had 2 deletions only, and both were in the WTK1 sample.

Chromosome 13 had 2 deletions, 1 in each sample. Chromosome 16 had a significant number of deletions (168 in all); interestingly, all these deletions were in the TK6 sample. WTK1 remained unchanged. Chromosome 17 had 2 deletions (all in WTK1) and 4 amplifications (all in TK6), and chromosome 20 had 1

Table 2. Integrated ASCN results with CN results for WTK1.

deletion for WTK1 (on gene PTPN1) but 3 amplifications for TK6. Finally, chromosome 21 had 5 gene deletions, 2 for WTK1 and 3 for TK6.

3.3. Identification of Genes Impacted by Irradiation in TK6 and WTK1

As shown in Table 3, out of the 18 genes listed for TK6, the most genes impacted are involved in gene regulation (PURB, E4F, POLRK, ZNF276, ZNF778, ERG) and cell cycle control (GFER, NME3, NME4, SOX8, CDK10, CDT1, TCF25). It is also significant to note that there are four genes involved in the DNA repair process. Finally, GAS8, which codes for a tumor suppressor protein, was also impacted.

Table 4 shows that, out of the 12 genes listed for WTK1, the ones most impacted are involved in tumor suppression (PPM1J, ST7L, CISH, CCDC6) and cell-cycle control (MAPAPK3, R3CC1L, PPP2R2C, ENDOU). The group of genes next most affected is involved in gene regulation (MOV10, CAMTA1, ZBTB 21). Only one gene was noted to be involved in the DNA repair process (GTF 2H4).

3.4. Summary

For the mutant WTK1 sample, there were a total of 48 gene deletions and no gene amplifications, whereas for the wild-type TK6 sample, there were 217 gene deletions and 9 gene amplifications. Besides the fact that none of these gene modifications overlapped between the two samples, these results are interesting in that the only sample showing amplifications is the wild-type sample, which also showed the largest number of gene deletions. To appreciate the differences between these two sets of data, we generated a shared segment karyogram, which shows a direct comparison of regions for deletions and amplification in the TK6 and WTK1 cells after irradiation (Figure 1).

Table 3. TK6 genes that were affected by ionizing radiation, their function/role, and pathology associated with aberrant gene expression.

Table 4. WTK1 genes that were affected by ionizing radiation, their function/role, and pathology associated with aberrant gene expression.

Figure 1. WTK1 genes that were affected by ionizing radiation, their function/role, and pathology associated with aberrant gene expression.

4. Discussion

In this study, we set out to understand the effects of ionizing radiation on two closely related cell lines: TK 6 (the wild type) and WTK 1 (the p53 mutant). Specifically, we wanted to investigate the copy-number variations between these two cell types to determine the level of impact that ionizing radiation has on genomic stability.

WTK1 contains the mutant p53 (M237I) at the thymidine kinase (tk) locus [37] . The p53 gene has been shown to be one of the most mutated genes as it relates to cancer. When comparing the level of mutability with the wild type p53 TK6 cells, it was shown that there is a 10-fold rate of hypermutability at the tk locus in the WTK1 cell line. p53 has also been shown to play a major role in maintaining genetic stability. p53 is a transcription factor that functions as a tumor suppressor, and p53 mutants have been shown to have loss of DNA binding function that prevents them from carrying out their regulatory role [38] . In addition, disruption of the interactions between the oncoprotein Mdm2, which promotes the rapid degradation of p53, with certain p53 mutants disrupts the p53 degradation pathway. This would indicate that mutant p53 is able to engage in aberrant interactions with other cellular factors. In fact, this has been shown to be the case and typically results in gain-of-function phenotypes [39] [40] [41] .

The application of the Affymetrix mapping arrays on our cell lines has provided a wealth of information into the global impact on chromosomal instability after being subjected to ionizing radiation. Our results show a number of notable gene deletions and amplifications that are involved in general gene regulation, DNA damage repair, direct tumor suppression, regulation of the cell cycle and posttranslational modifications. The impact of abnormal gene expression, or, conversely, loss of expression, could potentially lead to a unique set of pathologies (see for example Table 3 and Table 4).

Finally, we have demonstrated that WTK1 and TK6 do not share any genes that have been impacted in the same way by the irradiation. Instead, each cell line presents its own unique response profile. However, both cell types share changes in important cellular functions that can lead to cancer and disease. The most notable differences are that TK6 is mostly affected in the areas of gene regulation and cell-cycle control, whereas WTK1 is mainly affected in tumor suppression and cell-cycle control. These results hold significant importance in the study of the effects of ionizing radiation in human cells, and how different cell lines can be affected in significantly different ways depending on the presence of wild type p53.


Research reported in this (publication, release) was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103449. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

Conflicts of Interest

The authors declare no commercial or financial conflict of interest.


[1] De Benedetti, V., Bennett, W.P., Greenblatt, M.S. and Harris, C.C. (1996) p53 Tumor Suppressor Gene: Implications for Iatrogenic Cancer and Cancer Therapy. Medical and Pediatric Oncology, 1, 2-11.
[2] Greenblatt, M.S., Bennett, W.P., Hollstein, M. and Harris, C.C. (1994) Mutations in the p53 Tumor Suppressor Gene: Clues to Cancer Etiology and Molecular Pathogenesis. Cancer Research, 54, 4855-4878.
[3] Olivier, M., Eeles, R., Hollstein, M., Khan, M.A., Harris, C.C. and Hainaut, P. (2002) The IARC TP53 Database: New Online Mutation Analysis and Recommendations to Users. Human Mutation, 19, 607-614.
[4] Duffy, M.J., Synnott, N.C., O’Grady, S. and Crown, J. (2022) Targeting p53 for the Treatment of Cancer. Seminars in Cancer Biology, 79, 58-67.
[5] Williams, A.B. and Schumacher, B. (2016) p53 in the DNA-Damage-Repair Process. Cold Spring Harbor Perspectives in Medicine, 6, ea026070.
[6] Sancar, A., Lindsey-Boltz, L.A., Unsal-Kaçmaz, K. and Linn, S. (2004) Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints. Annual Review of Biochemistry, 73, 39-85.
[7] Albrechtsen, N., Dornreiter, I., Grosse, F., Kim, E., Wiesmüller, L. and Deppert, W. (1999) Maintenance of Genomic Integrity by p53: Complementary Roles for Activated and Non-Activated p53. Oncogene, 18, 7706-7717.
[8] Bertrand, P., Rouillard, D., Boulet, A., Levalois, C., Soussi, T. and Lopez, B.S. (1997) Increase of Spontaneous Intrachromosomal Homologous Recombination in Mammalian Cells Expressing a Mutant p53 Protein. Oncogene, 14, 1117-1122.
[9] Bill, C.A., Yu, Y., Miselis, N.R., Little, J.B. and Nickoloff, J.A. (1997) A Role for p53 in DNA End Rejoining by Human Cell Extracts. Mutation Research, 385, 21-29.
[10] Blander, G., Kipnis, J., Leal, J.F., Yu, C.E., Schellenberg, G.D. and Oren, M. (1999) Physical and Functional Interaction between p53 and the Werner’s Syndrome Protein. Journal of Biological Chemistry, 274, 29463-29469.
[11] Dudenhöffer, C., Kurth, M., Janus, F., Deppert, W. and Wiesmüller, L. (1999) Dissociation of the Recombination Control and the Sequence-Specific Transactivation Function of P53. Oncogene, 18, 5773-5784.
[12] Mathonnet, G., Leger, C., Desnoyers, J., Drouin, R., Therrien, J.-P. and Drobetsky, E.A. (2003) UV Wavelength-Dependent Regulation of Transcription-Coupled Nucleotide Excision Repair in p53-Deficient Human Cells. Proceedings of the National Academy of Sciences of the United States of America, 100, 7219-7224.
[13] Mekeel, K.L., Tang, W., Kachnic, L.A., Luo, C.M., De Frank, J.S. and Powell, S.N. (1997) Inactivation of p53 Results in High Rates of Homologous Recombination. Oncogene, 14, 1847-1857.
[14] Romanova, L.Y., Willers, H., Blagosklonny, M.V. and Powell, S.N. (2004) The Interaction of p53 with Replication Protein A Mediates Suppression of Homologous Recombination. Oncogene, 23, 9025-9033,
[15] Saintigny, Y., Rouillard, D., Chaput, B., Soussi, T. and Lopez, B.S. (1999) Mutant p53 Proteins Stimulate Spontaneous and Radiation-Induced Intrachromosomal Homologous Recombination Independently of the Alteration of the Transactivation Activity and of the G1 Checkpoint. Oncogene, 18, 3553-3563.
[16] Stürzbecher, H.W., Donzelmann, B., Henning, W., Knippschild, U. and Buchhop, S. (1996) p53 Is Linked Directly to Homologous Recombination Processes via RAD51/RecA Protein Interaction. The EMBO Journal, 15, 1992-2002.
[17] Tang, W., Willers, H. and Powell, S.N. (1999) p53 Directly Enhances Rejoining of DNA Double-Strand Breaks with Cohesive Ends in Gamma-Irradiated Mouse Fibroblasts. Cancer Research, 59, 2562-2565.
[18] Willers, H., et al. (2000) Dissociation of p53-Mediated Suppression of Homologous Recombination from G1/S Cell Cycle Checkpoint Control. Oncogene, 19, 632-639.
[19] Zurer, I., et al. (2004) The Role of p53 in Base Excision Repair Following Genotoxic Stress. Carcinogenesis, 25, 11-19.
[20] Lengauer, C., Kinzler, K.W. and Vogelstein, B. (1997) Genetic Instability in Colorectal Cancers. Nature, 386, 623-627.
[21] Lengauer, C., Kinzler, K.W. and Vogelstein, B. (1998) Genetic Instabilities in Human Cancers. Nature, 396, 643-649.
[22] Negrini, S., Gorgoulis, V.G. and Halazonetis, T.D. (2010) Genomic Instability—An Evolving Hallmark of Cancer. Nature Reviews Molecular Cell Biology, 11, 220-228.
[23] Matsuno, Y., et al. (2019) Replication Stress Triggers Microsatellite Destabilization and Hypermutation Leading to Clonal Expansion in Vitro. Nature Communications, 10, Article No. 3925.
[24] Matsuno, Y., et al. (2021) Replication-Stress-Associated DSBs Induced by Ionizing Radiation Risk Genomic Destabilization and Associated Clonal Evolution. iScience, 24, Article ID: 102313.
[25] Rajagopalan, H. and Lengauer, C. (2004) Aneuploidy and Cancer. Nature, 432, 338-341.
[26] Korbel, J.O. and Campbell, P.J. (2013) Criteria for Inference of Chromothripsis in Cancer Genomes. Cell, 152, 1226-1236.
[27] Bishop, A.J.R. and Schiestl, R.H. (2002) Homologous Recombination and Its Role in Carcinogenesis. Journal of Biomedicine and Biotechnology, 2, 75-85.
[28] Hoeijmakers, J.H.J. (2009) DNA Damage, Aging and Cancer. The New English Journal of Medicine, 361, 1475-1485.
[29] Ciccia, A. and Elledge, S.J. (2010) The DNA Damage Response: Making It Safe to Play with Knives. Molecular Cell, 40, 179-204.
[30] Sebastian, R. and Raghavan, S.C. (2016) Induction of DNA Damage and Erroneous Repair Can Explain Genomic Instability Caused by Endosulfan. Carcinogenesis, 37, 929-940.
[31] Levy, J.A., Virolainen, M. and Defendi, V. (1968) Human Lymphoblastoid Lines from Lymph Node and Spleen. Cancer, 22, 517-524.
[32] Chuang, Y.Y., Chen, Q., Brown, J.P., Sedivy, J.M. and Liber, H.L. (1999) Radiation-Induced Mutations at the Autosomal Thymidine Kinase Locus Are Not Elevated in p53-Null Cells. Cancer Research, 59, 3073-3076.
[33] Little, J.B., Nagasawa, H., Keng, P.C., Yu, Y. and Li, C.Y. (1995) Absence of Radiation-Induced G1 Arrest in Two Closely Related Human Lymphoblast Cell Lines That Differ in p53 Status. Journal of Biological Chemistry, 270, 11033-11036.
[34] Xia, F., et al. (1995) Altered p53 Status Correlates with Differences in Sensitivity to Radiation-Induced Mutation and Apoptosis in Two Closely Related Human Lymphoblast Lines. Cancer Research, 55, 12-15.
[35] Peng, Y., Zhang, Q., Nagasawa, H., Okayasu, R., Liber, H.L. and Bedford, J.S. (2002) Silencing Expression of the Catalytic Subunit of DNA-Dependent Protein Kinase by Small Interfering RNA Sensitizes Human Cells for Radiation-Induced Chromosome Damage, Cell Killing and Mutation. Cancer Research, 62, 6400-6404.
[36] Amundson, S.A., Xia, F., Wolfson, K. and Liber, H.L. (1993) Different Cytotoxic and Mutagenic Responses Induced by X-Rays in Two Human Lymphoblastoid Cell Lines Derived from a Single Donor. Mutation Research, 286, 233-241.
[37] Zhang, Q., Liu, Y., Zhou, J., Chen, W., Zhang, Y. and Liber, H.L. (2007) Wild-Type p53 Reduces Radiation Hypermutability in p53-Mutated Human Lymphoblast Cells. Mutagenesis, 22, 329-334.
[38] Kato, S., et al. (2003) Understanding the Function-Structure and Function-Mutation Relationships of p53 Tumor Suppressor Protein by High-Resolution Missense Mutation Analysis. Proceedings of the National Academy of Sciences of the United States of America, 100, 8424-8429.
[39] Haupt, Y., Maya, R., Kazaz, A. and Oren, M. (1997) Mdm2 Promotes the Rapid Degradation of p53. Nature, 387, 296-299.
[40] Midgley, C.A. and Lane, D.P. (1997) p53 Protein Stability in Tumour Cells Is Not Determined by Mutation but Is Dependent on Mdm2 Binding. Oncogene, 15, 1179-1189.
[41] Wiech, M., Olszewski, M.B., Tracz-Gaszewska, Z., Wawrzynow, B., Zylicz, M. and Zylicz A. (2012) Molecular Mechanism of mutant p53 Stabilization: The Role of HSP70 and MDM2. PLOS ONE, 7, e51426,

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