Failure of cell cleavage induces senescence in tetraploid primary cellsAndreas Panopoulosa, Cristina Pacios-Brasb, Justin Choia, Mythili Yenjerlaa, Mark A. Sussmanc, Rati Fotedara, and Robert L. Margolisa

aTumor Initiation and Maintenance Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; bDepartment of Immunology and Oncology, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Científicas, E-28049 Madrid, Spain; cSan Diego Heart Research Institute and Department of Biology; San Diego State University, San Diego, CA 92182

Tetraploidy is the result of several cleavage defects in mammalian cells undergoing mitosis. Tetraploid cells that continue to cycle proceed to aneuploidy due to inheritance of multiple centrosomes. Aneuploidy and chromosomal instability have been reported to be the major cause of human cancers and invasive tumors. Capacity of cells to arrest in G1 after cleavage failure and mitotic defects is critical to controlling tumor growth. Tetraploidy arrest then is a

potentially critical cellular control.

Tetraploidy is the inheritance of twice the number of complement chromosomes. It arises from several erratic cellular processes such as chromosomal nondisjunction, dysfunction of telomeres and abnormal cell fusion. Tetraploid cells may rapidly proceed to aneuploidy due to production of multipolar spindles at the next mitosis or due to random segregation of chromosomes.

Tetraploidy arrest occurs through induction of senescence. The induction of senescence requires both p53 and pRb pathways and is strictly dependent on the role of p16INK4a. p16INK4a is required for onset of senescence and is induced by drug-induced cleavage failure or siRNA transfection. Suppression of this senescence marker allows escape of

tetraploid arrest.

Several other laboratories have found that nontransformed mammalian cells stop cycling immediately after becoming tetraploid and that transformed cells continue cycling and

proceed to aneuploidy.

Primary rat embryo fibroblasts (REF52), human foreskin fibroblasts (HFF) and T-antigen-transformed REF52 were used to in this study to demonstrate the effects of drug- or small-interfering RNA induced failure of cleavage. Cells were exposed to dihydroxycytochalasin B (DCB), which inhibits actin assembly and blebbistatin, a myosin II inhibitor. Both inhibitors suppress cytokinesis in mitotic cells. Unsynchronized cells were used in this study to prevent possible ocntirbution to tetraploidy by DNA damage. DNA damage may be induced by synchronization before induction of tetraploid arrest.

To assess whether cells exposed to DCB have become tetraploid, they were subjected to flow cytometry. Cells were harvested with trypsin-EDTA and fixed for cytometry. Half the initial cell population was 4N after exposure to DCB or blebbistatin for 24h. Videos of the treated cells show that binucleate cells were abundant. Consequently, tetraploid (4N) cells were unable to proceed to 8N and thus avoided aneuploidy. REF52 cells also incorporated little 5-bromo-2’-deoxyuridine (BDU), indicating lack of DNA synthesis. Cells were also subjected to flourescent ubiquitination-based cell cycle indication (FUCCI) assay using green and orange–red fluorescent markers. Green fluorescent markers are used for geminin

expression while the orange-red is for Cdt1 expression and are exclusive for G2/M and G0/G1 phases of the cell cycle respectively. Binucleate tetraplod HFF cells were uniformly found to be in G0/G1 phase. These results show that REF52 and HFF cells arrest at the G1 cycle after cleavage failure.

T-antigen-transformed (TAG) REF52 cells, however, respond to the treatments differently. Primary REF52 cells arrest in tetraploid stage while TAG cells continue cycling. This results further re-establishes the findings of other laboratories that transformed cells do not arrest at tetraploid stage and that they proceed to become aneuploid.

The failure to reenter cell cycle is stable in REF52 and HFF cells. These cells then have lost their capacity to proliferate and thus have become senescent. To confirm continuing senescence, cells were assayed to detect primary cilia, a marker of cell quiescence. Mononucleate cells exposed to treatments were found ciliated during their exposure to DCB and reverted back to cycling after release from DCB. Binucleate cells ciliated during the treatment remained positive 72h after DCB release. Expression of senescence-associated beta-galactosidase was found to be increasing in binucleate HFF cells 3d after release from treatment. These results indicate induction of senescence and that senescence is permanent in tetraploid cells.

Also, no evidence of DNA damage brought about by cleavage failure was found via DNA damage marker, phosphorylated histone 2 variant H2AX. Ser-15 residue was not

phosphorylated in DCB-treated HFF cells. Tetraploidy does not induce another DNA damage response which is the phosphorylation of checkpoint kinase 2 on Thr-98. These results prove that failure in cytokinesis does not provoke DNA damage.

Tetraploidy is incompatible with mammalian embryonic development. In developing

embryos, tetraploid cells are reported to be restricted to extraembryonic tissue. This

indicates that tetraploidy is limited to extraembryonic cells.