British Journal of Cancer (1999) 79(7/8), 1037–1041 © 1999 Cancer Research Campaign Article no. bjoc.1998.0166
U Kasten1, N Plottner1, J Johansen2, J Overgaard2 and E Dikomey1
1Institute of Biophysics and Radiobiology, University of Hamburg, Martinistrasse 52, 20246 Hamburg, Germany; 2Department of Experimental Clinical Oncology, Aarhus University Hospital, Nørrebrogade 44, 8000 Aarhus C, Denmark
Summary The expression of the Ku70 and Ku80 genes as well as the activity of the DNA-dependent protein kinase (DNA-PK) were studied in 11 normal human fibroblast lines. The proteins studied are known to be part of a double-strand break (dsb) repair complex involved in non- homologous recombination, as was demonstrated for the radiosensitive rodent mutant cell lines of the complementation groups 5–7. The 11 fibroblast lines used in this study represent a typical spectrum of normal human radiosensitivity with the surviving fraction measured for a dose of 3.5 Gy, SF3.5 Gy, ranging from 0.03 to 0.28. These differences in cell survival were previously shown to correlate with the number of non-repaired dsbs. We found that the mRNA signal intensities of both Ku70 and Ku80 genes were fairly similar for the 11 cell lines investigated. In addition, the DNA-PK activity determined by the pulldown assay was fairly constant in these fibroblast lines. Despite the correlation between cell survival and dsb repair capacity, there was no correlation between dsb repair capacity and DNA-PK activity in the tested normal human fibroblast lines. Obviously, in this respect, other proteins/pathways appear to be more relevant.
Keywords: DNA-dependent protein kinase; Ku70/80; double-strand break repair; radiosensitivity; normal human fibroblasts
XRCC7
Ku70 Ku80 XRCC6/XRCC5 Received 20 April 1998 Revised 15 July 1998 Cell culture Accepted 16 July 1998 Correspondence to: E Dikomey
1037 1038 U Kasten et al
DNA-PK pulldown assay α ° µ µ Northern blot hybridization µ µ µ ° ° ° × ° β° ° ° × × × × β ° ° ° ° µ ° µ µ µ µ µ µ µ ′ Ku70 Ku80 µ EcoPst PstSma Gene expression of Ku70 and Ku80 Ku70 Ku80 β Ku70 Ku80 ′ β-actin
British Journal of Cancer (1999) 79(7/8), 1037–1041 © Cancer Research Campaign 1999 DNA-PK activity and dsb repair in normal human fibroblasts 1039
A Cell line 1234567891011 kb 50 7.46 4.40 40
2.37 1000)
Ku70 × 1.35 30
20 B 1234567891011 kb 7.46 10 4.40
Ku80 DNA-PK activity (c.p.m. 2.37 0 1.35
1234567891011 Cell line C 1234567891011 kb 7.46 Figure 3 Activity of DNA-PK determined for 11 normal human fibroblast 4.40 lines. Protein (150 µg) of the appropriate whole cell extract was used. All 2.37 data were corrected for the background, i.e. c.p.m. in the absence of peptide. β-actin The error bars represent s.e.m. of two independent experiments, with 1.35 triplicate determination
A B Figure 1 Gene expression of Ku70, Ku80 and β-actin of 11 different normal human fibroblast lines. Total RNA (5 µg) was analysed by Northern blot, 0.3 hybridization with the appropriate DIG-labelled DNA probe and subsequent 0.08 chemiluminescence detection via exposure of a light-sensitive film ) 0.2 0.06 3.5 Gy . ... 1.4 1.4 .. Ku70 Ku80 0.04 1.2 1.2 0.1 0.02 Non-repaired dsbs 1.0 1.0 Survival (SF 0.8 0.8 0 0.00 0.6 0.6 0.02 0.04 0.06 0.08 0 1020304050
0.4 0.4 Non-repaired dsbs released 24 h after 100 Gy) (Fraction of DNA (Fraction of DNA released DNA-PK activity (c.p.m.×1000) 0.2 0.2 24 h after 100 Gy)
Gene expression (relative units) 0.0 0.0 1234567891011 1234567891011 Figure 4 Relation between cellular survival, double-strand break repair capacity and DNA-PK activity in 11 normal human fibroblast lines. (A) Cell line Cell line Correlation between cell survival and dsb repair capacity. Cell survival after 3.5 Gy was determined for plateau phase cells by colony-forming ability, with Figure 2 Relative gene expression of Ku70 and Ku80 of 11 different normal delayed plating (14 h) after irradiation; the repair capacity was expressed by human fibroblast lines. Data were obtained from blots like that one shown in the number of non-repaired dsbs detected by constant field gel Figure 1, however the hybridization signals were determined directly via the electrophoresis as a fraction of DNA released from the plug 24 h after light emission recorded by a highly sensitive camera. The data were irradiation with 100 Gy; data were taken from Dikomey et al (1999). (B) Lack normalized to the β-actin signal to correct for gel loading. The Ku80 value of correlation between dsb repair capacity and DNA-PK activity. The number represents the sum of both transcripts. For better comparison, all values of non-repaired dsbs were determined as described above; data of DNA-PK were normalized to the mean value. Error bars represent s.e.m. of three activity were taken from Figure 3 independent RNA preparations
Ku70 Ku80 β-actin Ku80 ±Ku70 ±Ku80 µ ± DNA-PK activity
© Cancer Research Campaign 1999 British Journal of Cancer (1999) 79(7/8), 1037–1041 1040 U Kasten et al
Relation between DNA double-strand break repair capacity and DNA-PK activity XRCC1–XRCC8 XRCC5XRCC7 XRCC1 XRCC3 XRCC8 XRCC2 XRCC4 ATM Ku70 Ku80
British Journal of Cancer (1999) 79(7/8), 1037–1041 © Cancer Research Campaign 1999 DNA-PK activity and dsb repair in normal human fibroblasts 1041
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© Cancer Research Campaign 1999 British Journal of Cancer (1999) 79(7/8), 1037–1041