Stable low-level expression of p21WAF1/CIP1 in A549 human bronchogenic carcinoma cell line-derived clones down-regulates E2F1 mRNA and restores cell proliferation control

BackgroundDeregulated cell cycle progression and loss of proliferation control are key properties of malignant cells. In previous studies, an interactive transcript abundance index (ITAI) comprising three cell cycle control genes, [MYC × E2F1]/p21 accurately distinguished normal from malignant bronchial epithelial cells (BEC), using a cut-off threshold of 7,000. This cut-off is represented by a line with a slope of 7,000 on a bivariate plot of p21 versus [MYC × E2F1], with malignant BEC above the line and normal BEC below the line. This study was an effort to better quantify, at the transcript abundance level, the difference between normal and malignant BEC. The hypothesis was tested that experimental elevation of p21 in a malignant BEC line would decrease the value of the [MYC × E2F1]/p21 ITAI to a level below this line, resulting in loss of immortality and limited cell population doubling capacity. In order to test the hypothesis, a p21 expression vector was transfected into the A549 human bronchogenic carcinoma cell line, which has low constitutive p21 TA expression relative to normal BEC.ResultsFollowing transfection of p21, four A549/p21 clones with stable two-fold up-regulated p21 expression were isolated and expanded. For each clone, the increase in p21 transcript abundance (TA) was associated with increased total p21 protein level, more than 5-fold reduction in E2F1 TA, and 10-fold reduction in the [MYC × E2F1]/p21 ITAI to a value below the cut-off threshold. These changes in regulation of cell cycle control genes were associated with restoration of cell proliferation control. Specifically, each transfectant was capable of only 15 population doublings compared with unlimited population doublings for parental A549. This change was associated with an approximate 2-fold increase in population doubling time to 38.4 hours (from 22.3 hrs), resumption of contact-inhibition, and reduced dividing cell fraction as measured by flow cytometric DNA analysis.ConclusionThese results, likely due to increased p21-mediated down-regulation of E2F1 TA at the G1/S phase transition, are consistent with our hypothesis. Specifically, they provide experimental confirmation that a line with slope of 7,000 on the p21 versus [MYC × E2F1] bivariate plot quantifies the difference between normal and malignant BEC at the level of transcript abundance.

[1]  J. Willey,et al.  Differential effects of 12-O-tetradecanoylphorbol-13-acetate on cultured normal and neoplastic human bronchial epithelial cells. , 1984, Cancer research.

[2]  A. Iavarone,et al.  Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta. , 1995, Genes & development.

[3]  A. Haugen,et al.  Induction of squamous differentiation of normal human bronchial epithelial cells by small amounts of serum. , 1984, Differentiation; research in biological diversity.

[4]  G. Hannon,et al.  The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA , 1994, Nature.

[5]  M. Zajac-Kaye,et al.  Myc oncogene: a key component in cell cycle regulation and its implication for lung cancer. , 2001, Lung cancer.

[6]  N. Hayashi,et al.  Ninjurin1 increases p21 expression and induces cellular senescence in human hepatoma cells. , 2004, Journal of hepatology.

[7]  G. Hannon,et al.  Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair , 1994, Nature.

[8]  N. Dyson,et al.  The E2F transcriptional network: old acquaintances with new faces , 2005, Oncogene.

[9]  D. Gutmann,et al.  T-Cadherin-Mediated Cell Growth Regulation Involves G2 Phase Arrest and Requires p21CIP1/WAF1 Expression , 2003, Molecular and Cellular Biology.

[10]  N. L. Thangue,et al.  p14ARF regulates E2F activity , 2002, Oncogene.

[11]  A. Prescott,et al.  Gadd45 is a nuclear cell cycle regulated protein which interacts with p21Cip1. , 1995, Oncogene.

[12]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[13]  R. Spang,et al.  Role for E2F in Control of Both DNA Replication and Mitotic Functions as Revealed from DNA Microarray Analysis , 2001, Molecular and Cellular Biology.

[14]  J. Trent,et al.  WAF1, a potential mediator of p53 tumor suppression , 1993, Cell.

[15]  Stephen J. Elledge,et al.  p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest , 1994, Cell.

[16]  J. Willey,et al.  Variation in transcriptional regulation of cyclin dependent kinase inhibitor p21waf1/cip1 among human bronchogenic carcinomas , 2005, Molecular Cancer.

[17]  D. Olson,et al.  Use of standardized reverse transcription-polymerase chain reaction and the standardized expression measurement center in multi-institutional trials to develop meaningful lung cancer classification based on molecular genetic criteria. , 2004, Chest.

[18]  Robert B Boxer,et al.  Lack of sustained regression of c-MYC-induced mammary adenocarcinomas following brief or prolonged MYC inactivation. , 2004, Cancer cell.

[19]  David Beach,et al.  p21 is a universal inhibitor of cyclin kinases , 1993, Nature.

[20]  S. Elledge,et al.  The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases , 1993, Cell.

[21]  J. Modiano,et al.  Sustained nuclear localization of p21/WAF-1 upon growth arrest induced by contact inhibition. , 2000, Cancer letters.

[22]  J. R. Smith,et al.  Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. , 1994, Experimental cell research.

[23]  T. Ma,et al.  The macromolecular state of the transcription factor E2F and glucocorticoid regulation of c-myc transcription. , 1994, The Journal of biological chemistry.

[24]  Jiandong Chen,et al.  Expression of p14ARF overcomes tumor resistance to p53. , 2002, Cancer research.

[25]  H. Yoshikawa,et al.  Reciprocal Regulation via Protein-Protein Interaction between c-Myc and p21 cip1/waf1/sdi1 in DNA Replication and Transcription* , 2000, The Journal of Biological Chemistry.

[26]  W. Thilly,et al.  Quantitative RT-PCR measurement of cytochromes p450 1A1, 1B1, and 2B7, microsomal epoxide hydrolase, and NADPH oxidoreductase expression in lung cells of smokers and nonsmokers. , 1997, American journal of respiratory cell and molecular biology.

[27]  M. Nakanishi,et al.  Direct interaction of p21 cyclin-dependent kinase inhibitor with the retinoblastoma tumor suppressor protein. , 1999, Biochemical and biophysical research communications.

[28]  T. Tsuchiya,et al.  Decreased Tumorigenicity In Vivo When Transforming Growth Factor β Treatment Causes Cancer Cell Senescence , 2003, Bioscience, biotechnology, and biochemistry.

[29]  K. Kinzler,et al.  Cooperative effects of genes controlling the G(2)/M checkpoint. , 2000, Genes & development.

[30]  B. Clurman,et al.  Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination. , 2000, Molecular cell.

[31]  J. Willey,et al.  ABCC5, ERCC2, XPA and XRCC1 transcript abundance levels correlate with cisplatin chemoresistance in non-small cell lung cancer cell lines , 2005, Molecular Cancer.

[32]  Charles J. Sherr,et al.  Nucleolar Arf sequesters Mdm2 and activates p53 , 1999, Nature Cell Biology.

[33]  Alicia Samuels,et al.  Cancer Statistics, 2003 , 2003, CA: a cancer journal for clinicians.

[34]  M. Itoh,et al.  Involvement of the Interaction between p21 and Proliferating Cell Nuclear Antigen for the Maintenance of G2/M Arrest after DNA Damage* , 2001, The Journal of Biological Chemistry.

[35]  C. Turck,et al.  Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit , 1993, Nature.

[36]  I. Christensen,et al.  A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. , 1983, Cytometry.

[37]  T. Tsuji,et al.  Cigarette smoke induces senescence in alveolar epithelial cells. , 2004, American journal of respiratory cell and molecular biology.

[38]  K. Kinzler,et al.  Requirement for p53 and p21 to sustain G2 arrest after DNA damage. , 1998, Science.

[39]  J. Willey,et al.  Standardized RT-PCR and the standardized expression measurement center. , 2004, Methods in molecular biology.

[40]  W. Thilly,et al.  Normal bronchial epithelial cell expression of glutathione transferase P1, glutathione transferase M3, and glutathione peroxidase is low in subjects with bronchogenic carcinoma. , 2000, Cancer research.

[41]  L. Delavaine,et al.  Control of E2F activity by p21Waf1/Cip1 , 1999, Oncogene.

[42]  A. Gartel,et al.  Transcriptional regulation of the p21((WAF1/CIP1)) gene. , 1999, Experimental cell research.

[43]  A. Fornace,et al.  The differentiation primary response gene MyD118, related to GADD45, encodes for a nuclear protein which interacts with PCNA and p21WAF1/CIP1. , 1996, Oncogene.

[44]  D. Olson,et al.  The c-myc x E2F-1/p21 interactive gene expression index augments cytomorphologic diagnosis of lung cancer in fine-needle aspirate specimens. , 2003, The Journal of molecular diagnostics : JMD.

[45]  M. Ikeda,et al.  Distinct recruitment of E2F family members to specific E2F-binding sites mediates activation and repression of the E2F1 promoter , 2003, Oncogene.

[46]  J. Willey,et al.  Expression measurement of many genes simultaneously by quantitative RT-PCR using standardized mixtures of competitive templates. , 1998, American journal of respiratory cell and molecular biology.

[47]  M. Kirschner,et al.  Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA , 1995, Nature.

[48]  J. Willey,et al.  The gene expression index c-myc x E2F-1/p21 is highly predictive of malignant phenotype in human bronchial epithelial cells. , 1998, American journal of respiratory cell and molecular biology.