AN ASSAY FOR LOSS OF HETEROZYGOSITY IN-VIVO AT THE DLB-1 LOCUS

Loss of heterozygosity (LOH) is a frequent event in many tumours, resu lting in the loss of tumour suppressor genes and ultimately magnifying the number of cells with a mutant phenotype, We have used the Dlb-1 l ocus as a simple quantitative assay for LOH in vivo, Mutations of the dominant Dlb-1(b) allele are readily detected in heterozygous (Dlb-1(a )/Dlb-1(b)) mice by the loss of histochemical staining, which results in unstained, white (DEb1(a)/Dlb-1(-)) ribbons on a stained background , Such ribbons are extremely rare in untreated C57BL mice which are ho mozygous for the dominant allele, as would be expected when two indepe ndent mutations are required, To test for LOH, we first treated the an imals with a high dose of ethylnitrosourea (ENU) which induces many mu tations and thus many heterozygous cells, and allowed 2 weeks for gene expression, Then the animals were treated with the test agent to dete rmine if it could cause LOH and thus convert heterozygous mutant cells , which would not produce detectable ribbons, into homozygotes that wo uld produce white, non-staining ribbons, Treatment with ENU alone prod uced a low but detectable, frequency of mutant ribbons, Treatment with X-rays alone produced no detectable increase in the frequency of muta nt ribbons, Combinations of these treatments produced additive effects , thus showing that no significant LOH was induced, The additivity of the two equal ENU treatments was unexpected, since double mutants shou ld increase as the square of the mutant frequency, This can be explain ed in two ways: (i) stem cells are immutable except when cycling, whic h is rare, so few stem cells are hit twice or (ii) there is a constant rate of LOH which is not significantly increased by either X-rays or ENU, The assay can be used to determine if other factors can induce si gnificant frequencies of LOH.