4-nitroquinoline-1-oxide induced Ha-ras point mutations are associated with loss of heterozygosity, gene amplification and genomic instability

Tumorigenesis is a multistep genetic process. The most current view of tumorigenesis presumes that multiple independent mutations accumulate in the cell eventually leading to neoplastic transformation. Our hypothesis is that tumorigenesis can occur by an alternative route, that is, it may occur via a process involving linked molecular events.;Using 4-nitroquinoline-1-oxide (4NQO) induced murine oral squamous cell tumorigenesis as model, we have observed a series of non-random genomic changes involving the Ha-ras locus and adjacent genes on mouse chromosome 7. These changes occurred long after exposure to the carcinogen 4NQO had ceased. To determine if multiple mutations detected in these tumors developed by linked events, we have studied the association of Ha-ras point mutations in preneoplastic lesions with loss of heterozygosity (LOH) on chr 7, gene amplification at Int-2 amplicon, and genomic instability in neoplastic lesions.;In approximately 70% of the tumors analyzed (13/19), 4NQO caused a G to A point mutation at Ha-ras codon 12. In addition, loss of the wild type Ha-ras allele was observed in half of the tumors with Ha-ras point mutations when this occurred, LOH was always detected at loci flanking the Ha-ras gene (7/7). Since only half of the tumors develop LOH, LOH was not caused by the initial Ha-ras point mutation, but was a highly selected event during the progression of tumorigenesis. However, our data suggests that gene amplification and genomic instability are non-randomly associated with LOH at chr 7. Of the tumors with LOH, 5/6 showed both gene amplification and genomic instability. Furthermore, in tumors that did not develop LOH at chr 7, gene amplification or genomic instability was never observed. Statistical analysis strongly indicates that the occurrence of these gross chromosomal changes may result from linked events (p ;Thus, our data suggests a novel mechanism for tumorigenesis. It implies that certain primary events could influence the selection and the site of secondary mutations. In one third of the tumors analyzed, the events do not appear to be independent. This observation is contrary to the current multi-hit hypothesis.