Single cell analysis of checkpoints in G1

Abstract

It is becoming evident that defective cell cycle regulation is an important property of the cancer cell. The restriction point (R), a G1 checkpoint after which cell cycle progression becomes independent of extracellular growth factors, is often aberrant in cancer cells. The molecular mechanism governing R is unknown, but hyperphosphorylation and inactivation of the tumour suppressor retinoblastoma (pRb) has been suggested as a potential candidate. We examined the temporal relationship between R and pRb phosphorylation using methods for detailed single cell analysis. We found that that phosphorylation of pRb occurs after passage through R and therefore is not the mechanism regulating passage through R. Instead, phosphorylation of pRb seems to constitute a separate checkpoint in late G1 which possibly regulates S phase entry.

Cyclin D-cyclin dependent kinase (cdk) 4/6 phosphorylates pRb in response to growth factor stimulation and we studied the timing of cyclin D and pRb colocalisation in relation to the R point and pRb phosphorylation. We found that cyclin D and pRb only colocalise in late G1, after passage through R, with kinetics similar to pRb phosphorylation. This strongly suggests that intra-nuclear translocation of cyclin D to pRb is involved in regulation of pRb phosphorylation and not in passage through R. This further supports our finding that pRb phosphorylation is not the mechanism behind the restriction point.

R occurs at a set time after mitosis, implying that the R point mechanism might be related to events that occur during mitosis. In support of this notion, time-lapse videomicroscopy analysis revealed that early post mitotic cells, prior to passage through R, undergo a transient change in cell shape associated with cell cycle exit when subjected to serum starvation. However, addition of growth factors was shown to counteract both the change in cell shape and cell cycle exit induced by serum withdrawal. In conclusion, our data suggest that reorganisation of the cytoskeleton and/or cell adhesion to ECM after exit from mitosis is involved in R point passage.

To study cell growth in relation to pRb phosphorylation we inhibited rRNA synthesis through treatment with actinomycin D. This induces cell cycle arrest in G1 cells, but not in S phase cells. This G1 arrest was shown to be associated with inhibited pRb phosphorylation. We also observed that protein synthesis was not inhibited in actinomycin D treated, G1 arrested, cells. This indicates that ribosome biogenesis is involved in the regulation of a late G1 checkpoint that is separate from the R point and involves pRb phosphorylation, but not mass accumulation.