Expression of human eukaryotic initiation factor 3f oscillates with cell cycle in A549 cells and is essential for cell viability

Abstract

Background: Transcriptional and postranslational regulation of the cell cycle has been widely studied. However, there is scarce knowledge concerning translational control of this process. Several mammalian eukaryotic initiation factors (eIFs) seem to be implicated in controlling cell proliferation. In this work, we investigated if the human eIF3f expression and function is cell cycle related.

Results: The human eIF3f expression has been found to be upregulated in growth-stimulated A549 cells and downregulated in G0. Western blot analysis and eIF3f promotor-luciferase fusions revealed that eIF3f expression peaks twice in the cell cycle: in the S and the M phases. Deregulation of eIF3f expression negatively affects cell viability and induces apoptosis.

Conclusions: The expression pattern of human eIF3f during the cell cycle confirms that this gene is cell division related. The fact that eIF3f expression peaks in two cell cycle phases raises the possibility that this gene may exert a differential function in the S and M phases. Our results strongly suggest that eIF3f is essential for cell proliferation.

Figure 1

Human eIF3f expression is cell growth related in A549 cells. A) Western blot (WB) analysis of eIF3f expression in quiescent A549 cells (G0) and proliferating asynchronous cultures (A). Tubulin WB is shown to document equal protein loading. B) Promoter activity of the upstream sequences of eIF3f by promoter driven luciferase activity in quiescent A549 cells (G0) and proliferating asynchronous cultures (A). Luciferase activity with the empty vector pGL3 was below 10 RLU/μg of protein. Data expressed as mean ± SE; * p < 0.01 with respect to G0, n ≥ 3.

Figure 2

Transcriptional activity of human eIF3f gene. Promoter activity of the upstream sequence of eIF3f copy in chromosomes 2 is transcriptionally inactive in both 293-H and A549 asynchronous cultures, as exhibited by luciferase activity; in contrast, the eIF3f gene in chromosome 11 is active. Luciferase activity with the empty vector pGL3 was below 10 RLU/μg of protein. Data expressed as mean ± SE; * p < 0.01 with respect to control (pGL3), n ≥ 3.

Figure 3

Human eIF3f expression profile in A549 cells throughout the cell cycle. A) Expression of eIF3f protein (relative intensity units normalized with tubulin, panel B of this Figure), and mitotic index (%, verified with Cyclin B1 expression shown in panel B of this Figure) in a 24 hour cell cycle in A549 cells after HU release (t = 0 h). B) WB analysis of eIF3f (top), tubulin (middle), and Cyclin B1 (bottom) along the 24 h cell cycle. All proteins visualized by their respective specific antibodies. Only some of the timepoints represented in panel A are shown in panel B. The same membrane was used for eIF3f and tubulin blots. A second membrane was used for Cyclin B1, where protein concentration load was verified by another tubulin blot (not shown). C) Expression of eIF3f mRNA (relative intensity units normalized with 28S ribosomal RNA), and mitotic index (%) in a 24 hour cell cycle in A549 cells after HU release (t = 0 h). D) Northern blot analysis of eIF3f mRNA (top) and 28S ribosomal RNA (bottom) along the 24 h cell cycle. Only some of the timepoints represented in panel C are shown in panel D.

Figure 4

Promoter activity of the upstream sequence of functional eIF3f in quiescent A549 cells (G0), M, G1, and S synchronous cultures. A) Cell cultures in G0, M or S were transiently transfected with the promoter plasmid constructs and harvested 48 (for M and S cells) or 54 (for G0 cells) hours after transfection for luciferase activity assays. For cells in G1, cell cultures were transiently transfected with the promoter plasmid constructs for 20 hours, then arrested in M phase with nocodazole for 24 hours, and finally released from nocodazole to harvest cells 4 hours later for luciferase activity assays in G1. Luciferase activity with the empty vector pGL3 was below 10 RLU/μg of protein. Data expressed as mean ± SE; * p < 0.01 with respect to control (pGL3), n ≥ 3. B) Cell cycle distribution for nocodazole, HU and serum deprivation after release from arrest in M, S and G0, respectively. Cells were stained with DAPI at different timepoints to assess mitotic index. Micrographs of mitotic and interphase A549 cells are shown (top) at a magnification of 264×.

Figure 5

Ectopic expression of human eIF3f in A549 cells. A) WB analysis of eIF3f expression in transfected pMSG (control) and pFH11S (ectopic expression) A549 cultures. Tubulin WB is shown to document equal protein loading. B) Difference in cell growth in control and overexpressed eIF3f cultures after 24 and 48 hours of transfection. Timepoint cero represents cells before transfection. Cells were stained with acridine orange and ethidium bromide to visualize cell morphology and death by necrosis or apoptosis. Final magnification: 40×. C) Percent of apoptosis in control (water and pMSG) and overexpressed eIF3f (pFH11S) transfected cells, estimated from cell counts in microscopic fields similar to those shown in panel B. Data expressed as mean ± SE; * p < 0.01 with respect to control (water or pMSG), n ≥ 3.

Figure 6

Expression silencing of human eIF3f in A549 cells. A) WB analysis of eIF3f expression in transfected pMSG (control) and pFH11A (antisense) A549 cultures. Tubulin WB is shown to document equal protein loading. B) WB analysis of eIF3f expression in transfected scrambbled (control) and eIF3f siRNAs A549 cultures. Tubulin WB is shown to document equal protein loading. C) Difference in cell growth in control (water and scrambled siRNAs) and eIF3f siRNA treated A549 cultures after 24 and 48 hours of transfection. Left: suspended and adherent cells stained with trypan blue to assess cell viability after 24 hours of transfection. Right: Cells were stained with acridine orange and ethidium bromide to visualize cell morphology and death by necrosis or apoptosis. Timepoint cero represents cells before transfection. Final magnification: 40×. D) Percent of apoptosis in control (water and pMSG), antisense eIF3f (pFH11A), scrambled siRNAs and eIF3f siRNAs transfected cells, estimated from cell counts in microscopic fields similar to those shown in panel C. Data expressed as mean ± SE; * p < 0.01 with respect to their respective control (water, scrambled siRNAs or pMSG), n ≥ 3.