Published in Cancer Detection and Prevention 2000; 24(Supplement 1).

Aprt as a target for in vivo detection of loss of heterozygosity in splenocytes

SW Wijnhoven MD, PP Van Sloun PhD, H Vrieling PhD

Dept Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, Leiden, The Netherlands,

AIM: Analysis of the frequency of spontaneous and mutagen-induced LOH mutations to establish their contribution to tumour initiation. METHODS: Splenocytes of Aprt heterozygous mice were isolated and Aprt and Hprt mutant lymphocyte clones were selected using 8-azaadenine or 6-thioguanine, respectively. The type of mutation was determined by PCR allelotyping and FISH analysis. RESULTS: The mutation frequencies at both the Aprt and Hprt locus were determined in one-year old Aprt heterozygous mice. Whereas the mutation frequency at the Hprt locus remained low with increasing age, a dramatic increase in the Aprt mutant frequency was observed. This rise in mutant frequency resulted predominantly from LOH mutations, rising from 68% at 4 months to up to 90% in one-year old animals. Treatment with various carcinogens revealed that the selectivity of the induction of LOH events at the Aprt locus depends strongly on the type of agent tested and can be ranked as follows. X-rays < ENU < MNU < BNU < DMBA < MMC < etoposide. CONCLUSIONS: LOH mutations occur frequently both spontaneous and induced. The mutagenic events determined at Aprt involve predominantly mitotic recombination, gene conversion, or mitotic non-disjunction but not deletion. In principle all interactions of agents with DNA that give rise to single strand regions, either as a result from stalling of the replication machinery or via repair processes, lead to the induction of LOH mutations. References: Van Sloun et al. Nucleic Acids Res 1998. Wijnhoven et al. Proc Natl Acad Sci USA 1998. Vrieling et al. Environ Mol Mutagen 1999.

KEY WORDS: tumor suppressor genes, apoptosis, DNA repair.

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Paper presented at the International Symposium on Impact of Biotechnology on Cancer Diagnostic & Prognostic Indicators; Geneva, Switzerland; October 28 - 31, 2000; in the section on genetic instability.