Further characterization of loss of heterozygosity enhanced by p53 abrogation in human lymphoblastoid TK6 cells: disappearance of endpoint hotspots.
Loss of heterozygosity (LOH) is the predominant mechanism of spontaneous mutagenesis at the heterozygous thymindine kinase locus (tk) in TK6 cells. LOH events detected in spontaneous TK(-) mutants (110 clones from p53 wild-type cells TK6-20C and 117 clones from p53-abrogated cells TK6-E6) were analyzed using 13 microsatellite markers spanning the whole of chromosome 17. Our analysis indicated an approximately 60-fold higher frequency of terminal deletions in p53-abrogated cells TK6-E6 compared to p53 wild-type cells TK6-20C whereas frequencies of point mutations (non-LOH events), interstitial deletions, and crossing over events were found to increase only less than twofold by such p53 abrogation. We then made use of an additional 17 microsatellite markers which provided an average map-interval of 1.6Mb to map various LOH endpoints on the 45Mb portion of chromosome 17q corresponding to the maximum length of LOH tracts (i.e. from the distal marker D17S932 to the terminal end). There appeared to be four prominent peaks (I-IV) in the distribution of LOH endpoints/Mb of Tk6-20C cells that were not evident in p53-abrogated cells TK6-E6, where they appeared to be rather broadly distributed along the 15-20Mb length (D17S1807 to D17S1607) surrounding two of the peaks that we detected in TK6-20C cells (peaks II and III). We suggest that the chromosomal instability that is so evident in TK6-E6 cells may be due to DNA double-strand break repair occurring through non homologous end-joining rather than allelic recombination.
Yatagai
Fumio
F
Division of Radioisotope Technology, The Institute of Physical and Chemical Research, Saitama 351-0198, Japan. yatagai@postman.riken.go.jp
Morimoto
Shigeko
S
Kato
Takesi
T
Honma
Masamitsu
M
D002460:Cell Line; D002886:Chromosomes, Human, Pair 17; D006801:Humans; D019656:Loss of Heterozygosity; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D016159:Tumor Suppressor Protein p53