Heat stress‐induced loss of eukaryotic initiation factor 5A (eIF‐5A) in a human pancreatic cancer cell line, MIA PaCa‐2, analyzed by two‐dimensional gel electrophoresis

Abstract

Alterations of intracellular proteins during the process of heat stress-induced cell death of a human pancreatic cancer cell line, MIA PaCa-2, were investigated using two-dimensional gel electrophoresis (2-DE), agarose gel electrophoresis, and cell biology techniques. Incubation of MIA PaCa-2 at 45 degrees C for 30 min decreased the cell growth rate and cell viability without causing chromosomal DNA fragmentation. Incubation at 51 degrees C for 30 min suppressed cell growth and again led to death without DNA fragmentation. The cell death was associated with the loss of an intracellular protein of M(r) 17,500 and pI 5.2 on 2-DE gel. This protein was determined to be eukaryotic initiation factor SA (eIF-5A) by microsequencing of the N-terminal region of peptide fragments obtained by cyanogen bromide treatment of the protein blotted onto a polyvinylidene difluoride (PVDF) membrane. The sequences detected were QXSALRKNGFVVLKGRP and STSKTGXHGHAKVHLVGID, which were homologous with the sequence of eIF-5A from Gln 20 to Pro 36 and from Ser 43 to Asp 61, respectively. Furthermore, the result of sequencing suggested that the protein was an active form of hypusinated eIF-5A, because Lys 46 could be detected but not Lys 49, which is the site for hypusination. These results suggest that loss of the active form of eIF-5A is an important factor in the irreversible process of heat stress-induced death of MIA PaCa-2 cells.

The expression of genes encoding ribosomal subunits and eukaryotic translation initiation factor 5A depends on biotin and bisnorbiotin in HepG2 cells

“…This is consistent with the following observations from previous studies. First, eIF5A is known to play a role in promoting cell proliferation and preventing apoptosis [24,25]. Second, we have provided evidence that human cells maintain normal rates of cell proliferation and apoptosis if cultured in biotin-deficient medium for several weeks [26,36].…”

“…In the studies described below, we will repeatedly refer to eIF5A. Evidence has been provided that eIF5A plays a role in the translation of proteins that promote cell proliferation and inhibit apoptosis [24,25].…”

The unique hypusine modification of eIF5A promotes islet β cell inflammation and dysfunction in mice

“…eIF5A and its hypusinated form exhibit prolonged half-lives (≥24 hours) in many mammalian cells (40,41); strikingly, however, pulsechase studies revealed that eIF5A Hyp exhibits only approximately 6 hours of half-life in primary islets and islet-derived cell lines (present study). Interestingly, in some cell types, the half-life of eIF5A acutely diminishes to as little as 30 minutes in response to stressors such as heat shock (60,61), suggesting that both the cell type and environmental conditions can significantly influence the stability of the protein. In our case, therefore, the islet β cell may represent a unique case study for eIF5A Hyp biology.…”

Thermal killing of human colon cancer cells is associated with the loss of eukaryotic initiation factor 5A

“…Since one of the earliest response of cells to heat stress is the translation repression and loss of active polysomal ribosomes (Panniers and Henshaw, 1984; Mariano and Siekierka, 1986; Duncan and Hershey, 1989), we wonder whether eIF5A, an essential protein that recently reported to bind translational active ribosomes (Jao and Chen, 2006), may be involved in heat induced cell death. In this regard, it can be noted that acute heat stress of pancreatic cancer cells at high temperature (51°C) has been reported to cause a decrease of eIF5A (Takeuchi et al, 2002). We therefore examined the effect of severe heat stress on the level of eIF5A in HeLa and Caco‐2 cells.…”