GENE AMPLIFICATION, GENOME PLASTICITY AND ONCOGENESIS

The nature of mechanism(s) of mammalian gene amplification has been ho tly discussed for over ten years and many models have been proposed. R ecently, fluorescent in situ hybridization (FISH) has provided a power ful tool to study the early stages of mammalian gene amplification. Th e results obtained in three different model systems established that e arly amplification relies on unequal segregation of gene copies at mit osis, rather than on local over-replication, and that the early amplif ied structures are extremely unstable. The latter property focuses att ention on the relationship between the molecular processes leading to amplification and those generating major genomic rearrangements. Genom ic instability and oncogene amplification are indeed two frequent prop erties of cancer cells, which adds considerable interest to further ex ploration of amplification in model systems of cultured cells where th e dynamic aspects of genome remodeling can be more conveniently analyz ed. The recent demonstration, in two models of cultured mammalian cell s, that gene amplification is prevented by the expression of the p53 g ene - a major tumor suppressor gene that appears to exert a general co ntrol on genomic stability - is of particular interest because it link s amplification to oncogenesis, and stresses the likely relevance of r esults obtained on model systems of cultured cells to the mechanisms r esponsible for genomic instability in cancer cells. At least two diffe rent mechanisms are responsible for the initial steps of the amplifica tion process in mammalian cells. Early stages of chromosomal gene ampl ification are characterized by a striking heterogeneity from cell to c ell within a mutant clone. The extracopies are generally located on a chromosome arm where one copy maps in unamplified cells, and are organ ized as megabase-long inverted repeats with one or several orders of s ymmetry. These properties are consistent with an amplification mechani sm involving chromatid breakage followed by the fusion - after replica tion - of the telomereless sister chromatids. This phenomenon is cycli c because the bridge formed between the sister chromatids leads to fur ther breaks at mitosis. However, metaphase amplified dicentric and rin g chromosomes, which are frequently observed in all model systems of m ammalian amplification, are not intermediates of this mechanism. It wa s shown that these rearranged chromosomes are created when secondary e vents occur during expansion of populations of cells that undergo the cycles of chromatid breakage. Extrachromosomal copies of the amplified genes are also observed at early stages of gene amplification. FISH a nalysis of the cells that contain double-minute (DM) chromosomes revea ls that looping out of a circular molecule appears to generate at leas t a significant part of DMs and thereby to initiate - through unequal segregation at mitosis - an independant pathway of gene amplification, alternative to the operation of the intrachromosomal mechanism.