Link Between DNA Damage and Centriole Disengagement/Reduplication in Untransformed Human Cells

Abstract

The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 hours, though 14% of the cells eventually go through mitosis in that time. By 72 hours after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53 dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations.

Opposing post-translational modifications regulate Cep76 function to suppress centriole amplification

“…In these cells, both centriole disengagement and centrosomal Plk1-pT210 staining were observed (Figures 7F–G). As a further proof that acetylation of Cep76 in G2 phase leads to a loss of protein function, we found that Cep76 could not prevent centriole amplification induced by doxorubicin (DOX) (Figure 7H), a radiomimetic drug that induces G2 arrest (52). Our findings suggest that acetylation of Cep76 in G2 phase renders the protein unable to function.…”

“…For analysis of protein acetylation, we expressed Flag-Cep76 in synchronized cells and showed that the protein is more highly acetylated in G2 compared to other cell cycle phases (Figure 7B). Fittingly, Cep76 failed to inhibit centriole amplification induced by RO 3306 (Figures 7C–E), a small molecule inhibitor that triggers prolonged arrest in G2 phase and repeated rounds of centriole duplication (48, 51, 52). In these cells, both centriole disengagement and centrosomal Plk1-pT210 staining were observed (Figures 7F–G).…”

“…Centriole disengagement is known to require the activities of Plk1 and the anaphase-promoting complex (APC) (52, 60, 61). Although either activity alone is sufficient to disengage centrioles, the extent to which they contribute to this intricate process remains unclear.…”

Once and only once: mechanisms of centriole duplication and their deregulation in disease

“…The clearest example is that of a prolonged arrest in G2 phase, which leads to Plk1 activation, centriole disengagement and premature centriole reduplication 160 . Consequently, DNA damage can induce centrosome amplification by increasing the time cells spend in G2 phase 161,162 .…”

Kinetic analysis of de novo centriole assembly in heat‐shocked mammalian cells

“…Splitting up of centriole pairs and reduplication of the separated centrioles have been previously observed in DNA damage‐induced (Saladino et al, ; Inanç et al, ; Douthwright and Sluder, ) and G2 arrest‐induced centrosome amplification (Lončarek et al, ; Prosser et al, ). Therefore, the observed centrosome amplification in heat‐shocked MEFs could be due to the reduplication of the separated centrioles.…”