Transient excess of MYC activity can elicit genomic instability and tumorigenesis

Abstract

Overexpression of the MYC protooncogene has been implicated in the genesis of diverse human tumors. Tumorigenesis induced by MYC has been attributed to sustained effects on proliferation and differentiation. Here we report that MYC may also contribute to tumorigenesis by destabilizing the cellular genome. A transient excess of MYC activity increased tumorigenicity of Rat1A cells by at least 50-fold. The increase persisted for >30 days after the return of MYC activity to normal levels. The brief surfeit of MYC activity was accompanied by evidence of genomic instability, including karyotypic abnormalities, gene amplification, and hypersensitivity to DNA-damaging agents. MYC also induced genomic destabilization in normal human fibroblasts, although these cells did not become tumorigenic. Stimulation of Rat1A cells with MYC accelerated their passage through G1/S. Moreover, MYC could force normal human fibroblasts to transit G1 and S after treatment with N-(phosphonoacetyl)-L-aspartate (PALA) at concentrations that normally lead to arrest in S phase by checkpoint mechanisms. Instead, the cells subsequently appeared to arrest in G2. We suggest that the accelerated passage through G1 was mutagenic but that the effect of MYC permitted a checkpoint response only after G2 had been reached. Thus, MYC may contribute to tumorigenesis through a dominant mutator effect.

Figure 1

Brief excess of MYC activity increases the frequency of tumorigenic cells. Rat1A(MYCER) untreated (□), treated with E2 (10 μM) for 2 days (■), treated with E2 for 2 days followed by 14 days (●) or 30 days (▴) in the absence of hormone treatment. Tumorigenicity assays were performed as described in Table 1.

Figure 2

Excess of MYC activity is associated with karyotypic abnormalities. Cells were treated with E2 (10 μM) for two days, followed by analysis of metaphase spreads (15). Untreated Rat1A(MYCER) cells displayed a normal karyotype (A). Rat1A(MYCER) cells treated with E2 displayed aneuploidy (B), double minute chromosomes (D), and dicentric chromosomes (E). In a separate experiment, Rat1A(MYCER) cells were treated with E2 for 48 hours and then directly injected into nude mice to induce tumors. Explanted tumors were propagated in vitro and found to be euploid, but when treated with E2 exhibited marked aneuploidy (C) and multicentric chromosomes (F).

Figure 3

Excess MYC activity accelerates transit through the cell cycle. Serum-starved Rat1A(MYCER) cells were treated with serum (10%, ■) or serum and E2 (1 μM, □). (A) S Phase was determined by measuring the frequency of cells that incorporated BrdUrd. (B) Mitotic index was measured by examining the frequency of mitotic nuclei in metaphase spreads.

Figure 4

Excess MYC activity causes inappropriate cell cycle entry. NHF(BABE) or NHF(MYCER) cells were treated with PALA (50 μM) for two days and then analyzed for DNA content by fluorescence-ativated cell sorter (FACS) analysis of propidium iodide stained cells.