Immortalization of Human Bronchial Epithelial Cells in the Absence of Viral Oncoproteins

Ramirez, Ruben D.; Sheridan, Shelley; Girard, Luc; Sato, Mitsuo; Kim, Young; Pollack, Jon; Peyton, Michael; Zou, Ying; Kurie, Jonathan M.; DiMaio, J. Michael; Milchgrub, Sara; Smith, Alice L.; Souza, Rhonda F.; Gilbey, Laura; Zhang, Xi; Gandia, Kenia; Vaughan, Melville B.; Wright, Woodring E.; Gazdar, Adi F.; Minna, John D.

doi:10.1158/0008-5472.can-04-3703

Cited by 590 publications

(633 citation statements)

References 25 publications

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“…MRC5 cells were grown in Minimal Essential Medium (Biological Industries) supplemented with 10% non-heatinactivated fetal bovine serum, MEM-Eagle's nonessential amino acids, sodium pyruvate, L-glutamine and antibiotics. HBECs immortalized with hTERT+Cdk4 51 were grown in keratinocyte serum-free medium (Invitrogen, Grand Island, NY, USA) supplemented with 50 μg/ml bovine Pituitary extract and 5 ng/ml hEGF (Invitrogen). 52 5′aza-deoxycytidine (5′aza; MP Bio-Med, Santa Ana, CA, USA) was dissolved in acetic acid:H2O.…”

Section: Methodsmentioning

confidence: 99%

Reactivation of epigenetically silenced miR-512 and miR-373 sensitizes lung cancer cells to cisplatin and restricts tumor growth

et al. 2015

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MicroRNAs (miRs) regulate a variety of cellular processes, and their impaired expression is involved in cancer. Silencing of tumorsuppressive miRs in cancer can occur through epigenetic modifications, including DNA methylation and histone deacetylation. We performed comparative miR profiling on cultured lung cancer cells before and after treatment with 5′aza-deoxycytidine plus Trichostatin A to reverse DNA methylation and histone deacetylation, respectively. Several tens of miRs were strongly induced by such 'epigenetic therapy'. Two representatives, miR-512-5p (miR-512) and miR-373, were selected for further analysis. Both miRs were secreted in exosomes. Re-expression of both miRs augmented cisplatin-induced apoptosis and inhibited cell migration; miR-512 also reduced cell proliferation. TEAD4 mRNA was confirmed as a direct target of miR-512; likewise, miR-373 was found to target RelA and PIK3CA mRNA directly. Our results imply that miR-512 and miR-373 exert cell-autonomous and non-autonomous tumor-suppressive effects in lung cancer cells, where their re-expression may benefit epigenetic cancer therapy.

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Section: Methodsmentioning

confidence: 99%

Reactivation of epigenetically silenced miR-512 and miR-373 sensitizes lung cancer cells to cisplatin and restricts tumor growth

et al. 2015

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“…Primary podocytes, after two passages, were infected with retrovirus carrying CDK4 (22) or SV40 large T antigen mutant tsA58U19 (11,27) with 8 g/ml polybrene. Beginning after 72 h, cells were treated with 0.2 mg/ml G418 at 37°C (CDK4) or 33°C (tsA58U19) for 14 days.…”

Section: Glomerular Isolationmentioning

confidence: 99%

“…The kinase activity of CDK4 is specifically involved in progression through G1, via phosphorylation of retinoblastoma (Rb) family members (4). Recently, Ramirez et al (22) immortalized human bronchial epithelial cells by ectopic expression of CDK4 and human telomerase (hTERT). Microarray analysis showed that CDK4/hTERTimmortalized cells showed significantly different gene expression profiles compared with cells immortalized with human papiloma viral oncoproteins E6/E7 or SV40T and that CDK4-transformed cells clustered with primary bronchial epithelial cells.…”

mentioning

confidence: 99%

Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes

Sakairi

Abe

Jat

et al. 2010

American Journal of Physiology-Renal Physiology

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We transformed mouse podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor 1, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo.cyclin-dependent kinase 4; SV40 large T antigen; nephrin; synaptopodin; Wilms tumor-1; P-cadherin PODOCYTE CULTURES, ESPECIALLY podocyte cell lines transformed by thermosensitive SV40 large T antigen (SV40 T), have been widely used to study podocyte biology (19,25). However, as we recently reported in studies on a generation of mouse and human podocyte cell lines, podocytes transformed by thermosensitive SV40 T (tsT) exhibit phenotypic differences compared with podocytes in vivo, with reduced expression of certain differentiation marker genes (13, 24).Cyclin-dependent kinase 4 (CDK4), a catalytic subunit of the cyclin D-CDK4 serine/threonine kinase complex, is a critical regulator of the cell cycle. CDK4 is the first kinase activated by mitogenic signals. The kinase activity of CDK4 is specifically involved in progression through G1, via phosphorylation of retinoblastoma (Rb) family members (4). Recently, Ramirez et al. (22) immortalized human bronchial epithelial cells by ectopic expression of CDK4 and human telomerase (hTERT). Microarray analysis showed that CDK4/hTERTimmortalized cells showed significantly different gene expression profiles compared with cells immortalized with human papiloma viral oncoproteins E6/E7 or SV40T and that CDK4-transformed cells clustered with primary bronchial epithelial cells. In addition, recent studies showed that cells immortalized with CDK4 are useful tools to study physiology and pathophysiology (8,23,30).We set out to develop an alternative approach to transforming podocytes by comparing the phenotype among primary cells, CDK4-transformed podocytes (CDK4-podocytes) and thermosensitive SV40 T mutant tsA58U19 (tsT-podocytes). We found that CDK4-podocytes and tsT-podocyt...

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“…Over-representation of the entire 5p arm was a recurrent event in both cell types with the telomeric end of 5p15.33 showing the greatest amount of change. This region contains the telomerase reverse transcriptase (hTERT) gene which has been implicated in cell immortalisation in numerous cancers (Tomoda et al, 2002;Ramirez et al, 2004). Gain of the 11q13.1 -11q14.1 region was present in 450% of the lung cancer cell lines with the highest degree of concordance at 11q13.3 (Figure 2).…”

Section: Regions Of Similaritymentioning

confidence: 99%

Differential disruption of cell cycle pathways in small cell and non-small cell lung cancer

Coe¹,

Lockwood²,

Girard

et al. 2006

Br J Cancer

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Lung cancer is the leading cause of cancer-related mortality in the world, with small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) comprising the two major cell types. Although these cell types can be distinguished readily at the histological level, knowledge of their underlying molecular differences is very limited. In this study, we compared 14 SCLC cell lines against 27 NSCLC cell lines using an integrated array comparative genomic hybridisation and gene expression profiling approach to identify subtypespecific disruptions. Using stringent criteria, we have identified 159 of the genes that are responsible for the different biology of these cell types. Sorting of these genes by their biological functions revealed the differential disruption of key components involved in cell cycle pathways. Our novel comparative combined genome and transcriptome analysis not only identified differentially altered genes, but also revealed that certain shared pathways are preferentially disrupted at different steps in these cell types. Small cell lung cancer exhibited increased expression of MRP5, activation of Wnt pathway inhibitors, and upregulation of p38 MAPK activating genes, while NSCLC showed downregulation of CDKN2A, and upregulation of MAPK9 and EGFR. This information suggests that cell cycle upregulation in SCLC and NSCLC occurs through drastically different mechanisms, highlighting the need for differential molecular target selection in the treatment of these cancers.

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Immortalization of Human Bronchial Epithelial Cells in the Absence of Viral Oncoproteins

Abstract: By expressing two genes (hTERT andCdk4

Cited by 590 publications

References 25 publications

Reactivation of epigenetically silenced miR-512 and miR-373 sensitizes lung cancer cells to cisplatin and restricts tumor growth

Reactivation of epigenetically silenced miR-512 and miR-373 sensitizes lung cancer cells to cisplatin and restricts tumor growth

Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes

Differential disruption of cell cycle pathways in small cell and non-small cell lung cancer

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