Cdt1 and geminin are down‐regulated upon cell cycle exit and are over‐expressed in cancer‐derived cell lines

Abstract

Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells. Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin. Over-expression of Cdt1 was shown to predispose cells for malignant transformation. We show here that Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset. Cdt1's inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3-6 h delay over Cdt1, following serum re-addition. Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved. Our data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation. In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium. Cdt1 and geminin levels were compared in primary cells vs. cancer-derived human cell lines. We show that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and that the Cdt1 protein accumulates to higher levels in individual cancer cells. Geminin is similarly over-expressed in the majority of cancer cell lines tested. The relative ratios of Cdt1 and geminin differ significantly amongst cell lines. Our data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells.

Idas, a Novel Phylogenetically Conserved Geminin-related Protein, Binds to Geminin and Is Required for Cell Cycle Progression

“…A second vector for in situ hybridization was generated by subcloning the mIdas open reading frame to pBluescript between SacI and KpnI sites. The following plasmids have been described elsewhere: GemininGFP (29), Geminin⌬90 -120GFP (30,31), Cdt1dhcRed (31), GemininHA (29), plasmids for mouse Geminin in situ probes (32,33).…”

“…For control sense probe generation, the pBluescript vector containing the mIdas open reading frame was linearized with KpnI and transcribed with T7 RNA polymerase. Geminin sense and antisense probes were as described previously (32,33). Sense probes were used as the control in all experiments.…”

“…For immunofluorescence: rabbit antihIdas (hIdasAb2) (1:500), mouse monoclonal anti-Geminin (32), mouse monoclonal anti-GFP (3E6, 1181460001, Roche Applied Science), mouse monoclonal anti-HA (12CA5, sc-57592 Santa Cruz), and total mouse IgGs (Sigma). For Western blotting: rabbit anti-hIdas (hIdasAb1) (1:1000), rabbit anti-hIdas (hIdasAb2) (1:500) mouse monoclonal anti-actin (C4, Millipore, 1:3000), rabbit anti-Cdt1 (1:1000) (32), rabbit anti-Geminin (1:500) (32), rabbit anti-GFP (Santa Cruz, 1:500), and monoclonal anti-HA (12ca5, sc-57592 Santa Cruz, 1:500).…”

Geminin: A good prognostic factor in high‐grade astrocytic brain tumors

“…From these observations, it was believed that geminin likely would have tumorsuppressive functions. However, previous reports have demonstrated that the expression of geminin rises with increasing tumor grade, [11][12][13][14][15][16] leading to a poor prognosis in a variety of cancer patients, eg, breast cancer and renal cell carcinoma. 17,18 Thus there has been a discrepancy between normal molecular function and the outcomes of clinical studies of geminin in many cancers.…”

Role of geminin: from normal control of DNA replication to cancer formation and progression?

“…Immunohistochemistry and immunoblotting for geminin revealed that, except in testis, where its expression is high, this protein is absent in organs with minimal proliferation activity (heart, nerves, prostate, kidney, lung and scheletric muscle) and it is specifically expressed in proliferating lymphocytes, male germ cells and epithelial cells. [55][56][57] Interestingly, geminin is frequently overexpressed in human primary colon, rectal and breast tumors, in a subset of non-small-cell lung carcinomas, in B-cell lymphoma and in cancer cell lines of different histogenic origin and its upregulation correlated with high proliferative activity of tumor cells, [58][59][60][61][62] suggesting that this protein could exhibit oncogenic activity. Although being initially proposed as a tumor suppressor gene, geminin does not appear to exert an antiproliferative effect in these tumors but its role is supportive to consider geminin as a marker of proliferation.…”