Comparison of the Occurrence of Genomic Instability in TK6 and WTK1 Human Lymphoblasts Surviving Exposure to ⁵⁶Fe Ions

Helen H. Evans1, Min-Fen Horng1, Marlene Ricanati1, Mireya Diaz-Insua1, Rob Jordan2, and Jeffrey L. Schwartz2
1Case Western Reserve University, Cleveland, OH and 2University of Washington, Seattle, WA

The occurrence of genomic instability has been compared in clones of WTK1 and TK6 cells surviving exposure to ⁵⁶Fe ions and respective control clones. The two cell lines are related (1) but differ in several respects. TK6 cells are more sensitive to the cytotoxic effects of ionizing radiation (e.g. 1), and are relatively deficient in recombination and in the repair of DNA double-strand breaks (reviewed in 2). TK6 cells express wild-type TP53 protein, while WTK1 cells express a protein with a mutation in its DNA binding region (3). However, the presence of wild-type TP53 in TK6 cells has been found not to be related to its radiation sensitivity relative to WTK1 cells (4).

WTK1 and TK6 clones surviving exposure to 56Fe and their respective controls were analyzed for 21 individual characteristics involving chromosome aberrations; growth defects; an altered response to a second radiation (or first in the case of the controls); the frequency of ouabain-resistant mutants (OUA-R); and the frequency of thymidine kinase negative mutants. For exposure to Fe-56, aliquots were placed in T25 flasks and irradiated with ⁵⁶Fe ions (1.09 GeV/ amu) (LET = 148 keV/mm) generated by the alternating gradient synchrotron at Brookhaven National Laboratory. Survival of TK6 cells was 3.2% when plated immediately after an exposure of 1.67 Gy, while survival of WTK1 cells was 3.4% after an exposure of 2.5 Gy . For radiation administered to measure the response of isolated clones, cells at a density of 2 x 10⁵ cells/ml, were placed in T25 flasks and exposed to 2 Gy 137Cs for TK6 clonal cells and 3 Gy for WTK1 clonal cells. Survival averaged 5.7% for TK6 clonal cells and 11% for WTK1 clonal cells. For isolation of clones, cells were plated 48 h after exposure to ⁵⁶Fe, and after two weeks incubation, clones were picked up, expanded in culture, and frozen at -135° until analysis. Approximately 36 generations post exposure, cells were analyzed for chromosome aberrations as described previously (5), for plating efficiency and survival after radiation by plating in soft agar, for cell cycle distribution and level apoptosis by flow cytometry , and for mutant frequency at the Na/K ATPase and thymidine kinase loci by plating in the presence of ouabain and trifluorothymidine, respectively. From 26 to 41 control and exposed TK6 and WTK1 clones were analyzed for each characteristic. The mean values were determined and outlying characteristics defined as those values that differed from the mean by three standard deviations for true outliers or two standard deviations for suspected outliers. For values that did not present a normal distribution, box plots were used to determine outlying values. Clones exhibiting an outlier value in any one of the 21 characteristics were designated as putative unstable clones

Using this definition, we found that exposure to ⁵⁶Fe ions increased the percentage of unstable clones isolated for both cell lines. The percentage of unstable clones isolated was much higher in the case of the TK6 cell line than for the WTK1 cell line for both control clones and clones surviving exposure to ⁵⁶Fe ions (Table 1). However, the extent of the instability, as determined by the average value of a specific characteristic in the outlying clones) was much less for TK6 unstable clones than in the case of the unstable clones of WTK1 (Fig. 1). Further, the phenotype of the TK6 unstable clones differed from the WTK1 unstable clones. Thus, while the phenotype of the 25 WTK1 unstable clones varied markedly from one to another, the phenotype of a majority of the 47 TK6 unstable cells was quite similar, with 94% exhibiting chromosome aberrations as an outlying characteristic vs. 52% for WTK1. Chromosome aberrations were associated with growth defects in 69% of the TK6 unstable clones and 30% of the WTK1 unstable clones.

Fig. 1 Extent of Instability in Outliers

The figure shows the average of % cells with the specific chromosome aberration per clone in the clones with outlying values in each characteristic. The values for the clones surviving exposure to ⁵⁶Fe are shown. The error bars show the standard deviation from the mean.

We suggest that the increased percentage of unstable clones in the case of TK6 cells is caused by its deficiency in DNA double strand break repair that in turn may be related to the presence of chromatid breaks and gaps as well as a deficiency in telomere maintenance. This deficiency may also explain the similarity in phenotype of the TK6 unstable cells. We also suggest that the decrease in the extent of the instability in the case of TK6 cells is related to the presence of an active TP-53 protein that eliminates heavily aberrant cells through apoptosis or permanent cell cycle delays.

1. S. Amundson, F. Xia, K. Wolfson, H. Liber, Mutat. Res. 286, 233-241, 1994.

2. H. Evans, M. Horng. M. Ricanati, J. Deahl, N. Oleinick, Photochem.Photobiol. 66, 690-696, 1997.

3. F. Xia, Y. Wang, H-H. Wang, N. Tsang, S. Yandell, K. Kelsey, H. Liber, Cancer Res. 55, 12-15, 1995

4. Y. Chuang, Q. Chen, H. Liber, Cancer Res. 59, 3073-3076, 1999.

5. J. Schwartz, R. Hordan, H. Liber, J. Murnane, H.Evans, Genes, Chromosomes & Cancer 30, 236-244. 2001.

This research was supprorted by the National Cancer Institute and tha National Aeronautics Space Administration through NIH grant CA-73931.