Cyclin E-induced replicative stress drives p53-dependent whole-genome duplication.

Whole-genome duplication (WGD) is a frequent event in cancer evolution and an important driver of aneuploidy. The role of the p53 tumor suppressor in WGD has been enigmatic: p53 can block the proliferation of tetraploid cells, acting as a barrier to WGD, but can also promote mitotic bypass, a key step in WGD via endoreduplication. In wild-type (WT) p53 tumors, WGD is frequently associated with activation of the E2F pathway, especially amplification of CCNE1, encoding cyclin E1. Here, we show that elevated cyclin E1 expression causes replicative stress, which activates ATR- and Chk1-dependent G2 phase arrest. p53, via its downstream target p21, together with Wee1, then inhibits mitotic cyclin-dependent kinase activity sufficiently to activate APC/CCdh1 and promote mitotic bypass. Cyclin E expression suppresses p53-dependent senescence after mitotic bypass, allowing cells to complete endoreduplication. Our results indicate that p53 can contribute to cancer evolution through the promotion of WGD.

Multiplex Cell Fate Tracking by Flow Cytometry.

Measuring differences in cell cycle progression is often essential to understand cell behavior under different conditions, treatments and environmental changes. Cell synchronization is widely used for this purpose, but unfortunately, there are many cases where synchronization is not an option. Many cell lines, patient samples or primary cells cannot be synchronized, and most synchronization methods involve exposing the cells to stress, which makes the method incompatible with the study of stress responses such as DNA damage. The use of dual-pulse labelling using EdU and BrdU can potentially overcome these problems, but the need for individual sample processing may introduce a great variability in the results and their interpretation. Here, we describe a method to analyze cell proliferation and cell cycle progression by double staining with thymidine analogues in combination with fluorescent cell barcoding, which allows one to multiplex the study and reduces the variability due to individual sample staining, reducing also the cost of the experiment.

DNA replication and oncogene-induced replicative stress.

DNA replication must be tightly regulated to ensure that the genome is accurately duplicated during each cell cycle. When these regulatory mechanisms fail, replicative stress and DNA damage ensue. Activated oncogenes promote replicative stress, inducing a DNA damage response (DDR) early in tumorigenesis. Senescence or apoptosis result, forming a barrier against tumour progression. This may provide a selective pressure for acquisition of mutations in the DDR pathway during tumorigenesis. Despite its potential importance in early cancer development, the precise nature of oncogene-induced replicative stress remains poorly understood. Here, we review our current understanding of replication initiation and its regulation, describe mechanisms by which activated oncogenes might interfere with these processes and discuss how replicative stress might contribute to the genomic instability seen in cancers.