Overexpression of the replication licensing regulators hCdt1 and hCdc6 characterizes a subset of non-small-cell lung carcinomas: synergistic effect with mutant p53 on tumor growth and chromosomal instability--evidence of E2F-1 transcriptional control over hCdt1.

Replication licensing ensures once per cell cycle replication and is essential for genome stability. Overexpression of two key licensing factors, Cdc6 and Cdt1, leads to overreplication and chromosomal instability (CIN) in lower eukaryotes and recently in human cell lines. In this report, we analyzed hCdt1, hCdc6, and hGeminin, the hCdt1 inhibitor expression, in a series of non-small-cell lung carcinomas, and investigated for putative relations with G(1)/S phase regulators, tumor kinetics, and ploidy. This is the first study of these fundamental licensing elements in primary human lung carcinomas. We herein demonstrate elevated levels (more than fourfold) of hCdt1 and hCdc6 in 43% and 50% of neoplasms, respectively, whereas aberrant expression of hGeminin was observed in 49% of cases (underexpression, 12%; overexpression, 37%). hCdt1 expression positively correlated with hCdc6 and E2F-1 levels (P = 0.001 and P = 0.048, respectively). Supportive of the observed link between E2F-1 and hCdt1, we provide evidence that E2F-1 up-regulates the hCdt1 promoter in cultured mammalian cells. Interestingly, hGeminin overexpression was statistically related to increased hCdt1 levels (P = 0.025). Regarding the kinetic and ploidy status of hCdt1- and/or hCdc6-overexpressing tumors, p53-mutant cases exhibited significantly increased tumor growth values (Growth Index; GI) and aneuploidy/CIN compared to those bearing intact p53 (P = 0.008 for GI, P = 0.001 for CIN). The significance of these results was underscored by the fact that the latter parameters were independent of p53 within the hCdt1-hCdc6 normally expressing cases. Cumulatively, the above suggest a synergistic effect between hCdt1-hCdc6 overexpression and mutant-p53 over tumor growth and CIN in non-small-cell lung carcinomas.

[1]  V. Gorgoulis,et al.  Expression of p16INK4A and alterations of the 9p21‐23 chromosome region in non‐small‐cell lung carcinomas: Relationship with tumor growth parameters and ploidy status , 2000, International journal of cancer.

[2]  J. Moreau,et al.  XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis , 2000, Nature.

[3]  T. Volkert,et al.  E2F integrates cell cycle progression with DNA repair, replication, and G(2)/M checkpoints. , 2002, Genes & development.

[4]  J. Nevins,et al.  Interaction of YY1 with E2Fs, mediated by RYBP, provides a mechanism for specificity of E2F function , 2002, The EMBO journal.

[5]  J. Kutok,et al.  Expression of geminin as a marker of cell proliferation in normal tissues and malignancies. , 2002, The American journal of pathology.

[6]  Zoi Lygerou,et al.  The Human Licensing Factor for DNA Replication Cdt1 Accumulates in G1 and Is Destabilized after Initiation of S-phase* , 2001, The Journal of Biological Chemistry.

[7]  M. Inagaki,et al.  Cell Cycle Regulation of Human CDC6 Protein , 1999, The Journal of Biological Chemistry.

[8]  Anindya Dutta,et al.  A p53-dependent checkpoint pathway prevents rereplication. , 2003, Molecular cell.

[9]  S. Shreeram,et al.  The role of the replication licensing system in cell proliferation and cancer. , 2003, Progress in cell cycle research.

[10]  L. Cooley,et al.  Control of DNA Replication and Chromosome Ploidy by Geminin and Cyclin A , 2002, Molecular and Cellular Biology.

[11]  I. Royzman,et al.  Drosophila double parked: a conserved, essential replication protein that colocalizes with the origin recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts. , 2000, Genes & development.

[12]  K. Nasmyth,et al.  Loading of an Mcm Protein onto DNA Replication Origins Is Regulated by Cdc6p and CDKs , 1997, Cell.

[13]  D. Lane,et al.  Cell type-specific responses of human cells to inhibition of replication licensing , 2002, Oncogene.

[14]  Zoi Lygerou,et al.  The Cdt1 protein is required to license DNA for replication in fission yeast , 2000, Nature.

[15]  K. Nasmyth,et al.  An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast , 1996, Nature.

[16]  J. Diffley.  DNA replication: Building the perfect switch , 2001, Current Biology.

[17]  J. Nevins,et al.  Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Trimarchi,et al.  E2f3 is critical for normal cellular proliferation. , 2000, Genes & development.

[19]  Rodrigo Bermejo,et al.  Regulation of CDC6, geminin, and CDT1 in human cells that undergo polyploidization. , 2002, Molecular biology of the cell.

[20]  H. Nishitani,et al.  Control of DNA replication licensing in a cell cycle , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[21]  J. Parvin,et al.  Human CDC6/Cdc18 Associates with Orc1 and Cyclin-cdk and Is Selectively Eliminated from the Nucleus at the Onset of S Phase , 1998, Molecular and Cellular Biology.

[22]  Kristian Helin,et al.  Cell Cycle-Regulated Expression of MammalianCDC6 Is Dependent on E2F , 1998, Molecular and Cellular Biology.

[23]  S. Taraviras,et al.  Generation of inhibitory mutants of hepatocyte nuclear factor 4. , 1997, European journal of biochemistry.

[24]  M. Lei,et al.  Initiating DNA synthesis: from recruiting to activating the MCM complex. , 2001, Journal of cell science.

[25]  T. Kelly,et al.  cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[26]  V. Gorgoulis,et al.  Proliferation, but not apoptosis, is associated with distinct beta-catenin expression patterns in non-small-cell lung carcinomas: relationship with adenomatous polyposis coli and G(1)-to S-phase cell-cycle regulators. , 2002, The American journal of pathology.

[27]  J A Wohlschlegel,et al.  Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. , 2000, Science.

[28]  C. Prives,et al.  The p53 pathway , 1999, The Journal of pathology.

[29]  V. Zoumpourlis,et al.  Altered Expression of the Cell Cycle Regulatory Molecules pRb, p53 and MDM2 Exert a Synergetic Effect on Tumor Growth and Chromosomal Instability in Non-small Cell Lung Carcinomas (NSCLCs) , 2000, Molecular medicine.

[30]  J. Blow,et al.  A role for the nuclear envelope in controlling DNA replication within the cell cycle , 1988, Nature.

[31]  J. Peters,et al.  Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1. , 2000, Genes & development.

[32]  Amanda G Paulovich,et al.  When Checkpoints Fail , 1997, Cell.

[33]  M. Méchali,et al.  Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin , 2001, Nature Cell Biology.

[34]  Xiaohua Wu,et al.  The SCFSkp2 Ubiquitin Ligase Complex Interacts with the Human Replication Licensing Factor Cdt1 and Regulates Cdt1 Degradation* , 2003, Journal of Biological Chemistry.

[35]  P. Nurse,et al.  Expression of Cdc18/Cdc6 and Cdt1 during G2 phase induces initiation of DNA replication , 2001, The EMBO journal.

[36]  C. Harris,et al.  Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. , 1994, Cancer research.

[37]  K. Shroyer,et al.  Immunohistochemical localization of cdc6 in squamous and glandular neoplasia of the uterine cervix. , 2002, Archives of pathology & laboratory medicine.

[38]  M. Kirschner,et al.  Geminin, an Inhibitor of DNA Replication, Is Degraded during Mitosis , 1998, Cell.

[39]  T. Coleman,et al.  The Xenopus Cdc6 Protein Is Essential for the Initiation of a Single Round of DNA Replication in Cell-Free Extracts , 1996, Cell.

[40]  Ituro Inoue,et al.  Regulation of Geminin and Cdt1 expression by E2F transcription factors , 2004, Oncogene.

[41]  P. Nurse,et al.  p65 cdc18 Plays a major role controlling the initiation of DNA replication in fission yeast , 1995, Cell.

[42]  D. Fremont,et al.  Oncogenic potential of the DNA replication licensing protein CDT1 , 2002, Oncogene.

[43]  R. Chakrabarti,et al.  Down-regulation of Cdc6, a Cell Cycle Regulatory Gene, in Prostate Cancer* , 2002, The Journal of Biological Chemistry.

[44]  A. Papavassiliou,et al.  Transcription factor E2F‐1 acts as a growth‐promoting factor and is associated with adverse prognosis in non‐small cell lung carcinomas , 2002, The Journal of pathology.

[45]  N. Dyson,et al.  A revised picture of the E2F transcriptional network and RB function. , 2002, Current opinion in cell biology.

[46]  G. Williams,et al.  Improved cervical smear assessment using antibodies against proteins that regulate DNA replication. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[47]  Anindya Dutta,et al.  DNA replication in eukaryotic cells. , 2002, Annual review of biochemistry.

[48]  K. Kinzler,et al.  Genetic instabilities in human cancers , 1998, Nature.

[49]  R. Laskey,et al.  Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process. , 2000, Journal of cell science.

[50]  Jiri Bartek,et al.  Phosphorylation of mammalian CDC6 by Cyclin A/CDK2 regulates its subcellular localization , 1999, The EMBO journal.

[51]  T. Weinert,et al.  Toward maintaining the genome: DNA damage and replication checkpoints. , 2002, Annual review of genetics.

[52]  T. Halazonetis,et al.  Alterations of the p16-pRb pathway and the chromosome locus 9p21-22 in non-small-cell lung carcinomas: relationship with p53 and MDM2 protein expression. , 1998, The American journal of pathology.

[53]  A. Papavassiliou,et al.  Low Levels of p27 in Association With Deregulated p53-pRb Protein Status Enhance Tumor Proliferation and Chromosomal Instability in Non-Small Cell Lung Carcinomas , 2001, Molecular medicine.

[54]  R V Shohet,et al.  A human protein related to yeast Cdc6p. , 1997, Proceedings of the National Academy of Sciences of the United States of America.