Overexpressing eukaryotic translation initiation factor 4E stimulates bovine mammary epithelial cell proliferation Ezhou Long a , Anthoula Lazaris-Karatzas b , Costas Karatzas b , Xin Zhao a, * a Department of Animal Science, Macdonald Campus of McGill Uniersity, 21,111 Lakeshore Rd, Ste -Anne -de -Belleue, Quebec, H9X 3V9, Canada b Nexia Biotechnologies Inc., Ste -Anne -de -Belleue, Quebec, H9X 3R2, Canada Received 4 August 2000; received in revised form 15 November 2000; accepted 5 December 2000 Abstract Background and aims: The eukaryotic translation initiation factor 4E regulates the proliferation of many cell types. In the present study, the effect of its overexpression on the growth of an immortalized bovine mammary epithelial cell line, MAC-T, has been investigated. Since involvement of cyclin D1 in growth regulation of other cell types has been suggested previously, the differences in cyclin D1 expression among the 4E-overexpressing and parental cells were also investigated. Methods: The cDNA of mouse eukaryotic translation initiation factor 4E coding region (either wild-type or mutant, where Trp–56 was mutated to Ala) was transfected into MAC-T cells, and its protein expression was detected by Western blot analysis. Growth rates and saturation densities were calculated based on the cell counting data at desired time points. Key results: The cells overexpressing wild-type 4E displayed higher growth rates and saturation densities compared to the parental cells (P 0.05), whereas cells expressing mutant 4E showed lower growth rates and saturation densities than the parental controls (P 0.05). The amounts of cyclin D1 mRNA and protein were higher in the wild-type transfectants than in the parental controls, whereas the mutant transfectants contained lower amounts of cyclin D1 mRNA and protein compared to the parental cells. Conclusion: Our results suggest that overexpression of eukaryotic translation initiation factor 4E leads to increased cyclin D1 expression at the transcriptional level, which consequently stimulates the proliferation of MAC-T cells. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bovine mammary epithelial cell proliferation; Overexpressing eukaryotic translation initiation factor 4E 1. Introduction In eukaryotes, mRNA translation is initiated by the association of 40S ribosomal subunit with the mRNA through the eukaryotic translation initia- tion factor 4F (eIF-4F). eIF-4F is a ternary com- plex consisting of eIF-4A, 4E, and 4G, among Abbreiations: BSA, bovine serum albumin; EGF, epidermal growth factor; eIF-4E, eukaryotic translation initiation factor 4E; HA, human influenza hemagglutanin; pRb, retinoblas- toma protein; TGF-, transforming growth factor alpha. * Corresponding author. Tel.: +1-514-3987975; fax: +1- 514-3987964. E-mail address: zhao@macdonald.mcgill.ca (X. Zhao). 1357-2725/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII:S1357-2725(00)00089-3