C57BL/6 Mouse Primary Embryonic Fibroblasts

Early-Stage Centrosome Amplification in Primary Mouse Embryonic Fibroblasts

The centrosome is involved in cytoplasmic microtubule organization during interphase and in mitotic spindle assembly during cell division. Centrosome amplification (abnormal proliferation of centrosome number) has been observed in several types of cancer and in precancerous conditions. Therefore, it is important to elucidate the mechanism of centrosome amplification in order to understand the early stage of carcinogenesis.

Primary cells could be used to better understand the early stage of carcinogenesis rather than immortalized cells, which tend to have various genetic and epigenetic changes. When primary mouse embryonic fibroblasts (MEF) were incubated with 5 µM AG14361 (a PARP inhibitor known to induce centrosome amplification and chromosomal aneuploidy) for 72 h, there was a significant increase in the number of cells containing three or more spots of γ-tubulin, a marker of the centrosome (Fig. 1A). Moreover, the majority of spots of γ-tubulin colocalized with the spots of centriole, thus confirming that the actual number of centrosomes in primary MEF was increased via AG14361 treatment (Fig. 1B). The proliferation of cells was significantly reduced at 72 h after incubation with 5 µM AG14361 without significant changes in the flow cytometric patterns (Fig. 1C).

In addition to the numerical amplification of centrosomes (centrosome amplification), cancer cells often exhibit changes in the number of chromosomes (aneuploidy) and multipolar spindle formation. In primary MEF, the normal chromosome number was 2n = 40 ± 4 and 80 ± 4 (Fig. 1D). However, aneuploidy was not observed with 5 µM AG14361 treatment in primary MEF (Fig. 1D).