Microarray analysis of early and late passage chicken embryo fibroblast cells.

Primary cultured cells derived from normal tissue have a limited lifespan due to replicative senescence and show distinct phenotypes such as irreversible cell cycle arrest and enlarged morphology. Studying senescence-associated genetic alterations in chicken cells will provide valuable knowledge of cellular growth characteristics, when compared with normal and rapidly growing cell lines. Microarray analysis of early- and late-passage (passage 4 and 18, respectively) primary chicken embryo fibroblast (CEF) cells was performed with a 4X44K chicken oligo microarray. A total of 1,888 differentially expressed genes were identified with a 2-fold level cutoff that included 272 upregulated and 1,616 downregulated genes in late-passage senescent CEF cells. Bioinformatic analyses were performed using Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com). Of the 1,888 differentially expressed genes in senescent CEF cells, 458 were identified as functionally known genes and only 61 genes showed upregulation. Because senescent cells generally showed the deactivated states of most cellular mechanisms for proliferation and energy metabolism, intensified analysis on upregulated genes revealed that the molecular mechanisms in senescent CEF cells are characterized by the suppression of cell cycle and proliferation, progression of cell death including apoptosis, and increased expression of various secreting factors. These regulatory pathways may be opposite to those found in the immortal CEF cell line, such as the DF-1 immortal line. Further comparison of differentially expressed genes between senescent and immortal DF-1 CEF cells showed that 35 genes overlapped and were oppositely regulated. The global gene expression profiles may provide insight into the cellular mechanisms that regulate cellular senescence and immortalization of CEF cells.

Establishment of an immortal chicken embryo liver-derived cell line.

A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>10(5) pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CEL-im cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21(WAF)), and CDK inhibitor p16 (p16(INK4)) were downregulated in the CEL-im cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16(INK4) (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV.