Reduced expression of GINS complex members induces hallmarks of pre-malignancy in primary untransformed human cells.
In cancer cells ablation of the GINS complex member Psf2 elicits chromosome mis-segregation yet the precise role of Psf2 in mitosis is unknown. We investigated the putative mitotic role of the GINS complex using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF). Metaphase spreads from Psf1/Psf2-depleted HDF were normal and mitotic exit of Psf1/Psf2-depleted cells was only slightly delayed, suggesting no direct role for the GINS complex in mitosis of untransformed cells. Because the GINS complex is required for initiation and elongation events during DNA replication we hypothesized that the mitotic delay of Psf1/Psf2-deficient cells resulted indirectly from defective DNA synthesis during a prior S-phase. Therefore, we investigated the effects of Psf1/Psf2-depletion on DNA replication. Recruitment of Mcm7 to chromatin during G(1) was unaffected by Psf1/Psf2-ablation, indicating that replication licensing does not require GINS. However, chromatin-binding of Cdc45 and PCNA, onset of DNA synthesis and accumulation of G(2)/M markers were delayed in Psf1/Psf2-ablated cells. The cell cycle delay of Psf1/Psf2-depleted HDF was associated with several hallmarks of pre-malignancy including gammaH2AX, Thr 68-phosphorylated Chk2, and increased numbers of aberrant fragmented nuclei. Ectopic expression of catalytically-inactive Chk2 promoted S-phase and G(2)/M progression in Psf1/Psf2-depleted cells, as evidenced by modestly-increased rates of DNA synthesis and increased dephosphorylation of Cdc2. Therefore, S-phase progression of untransformed cells containing sub-optimal levels of Psf1/2 is associated with replication stress and acquisition of DNA damage. The ensuing Chk2-mediated DNA damage signaling likely contributes to maintenance of chromosomal integrity.
Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA, USA.
Laura R
LR
Barkley
Ihn Young
IY
Song
Ying
Y
Zou
Cyrus
C
Vaziri
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D010445:Peptide Elongation Factors / Q000378:metabolism; D011230:Precancerous Conditions / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D016196:S Phase; D015398:Signal Transduction
D018528:ATP-Binding Cassette Transporters; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D010445:Peptide Elongation Factors; D034741:RNA, Small Interfering; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases