All-trans-Retinoic Acid-mediated Growth Inhibition Involves Inhibition of Human Kinesin-related Protein HsEg5

Abstract

In this study we used differential display reverse transcription-polymerase chain reaction to search for differentially expressed all-trans-retinoic acid (ATRA)-responsive genes in pancreatic carcinoma cells. We identified the kinesin-related protein HsEg5, which plays an essential role in spindle assembly and spindle function during mitosis, as a novel molecule involved in ATRA-mediated growth inhibition. Using Northern and Western blot analysis we demonstrated that ATRA significantly inhibits HsEg5 expression in various pancreatic carcinoma cell lines as well as in HaCat keratinocytes. Inhibition of HsEg5 expression by ATRA occurs at the posttranscriptional level. As a consequence, tumor cells synchronized in S-phase revealed a retarded progression through G 2 /M phase of the cell cycle indicating that HsEg5 inhibition results in a delayed progression through mitosis. Furthermore, a significant decrease of HsEg5 protein expression achieved by antisense transfection revealed a significant growth inhibition compared with control cells. Therefore, HsEg5 represents a novel molecule involved in ATRA-mediated growth inhibition, suggesting that vitamin A derivatives can interact with the bipolar spindle apparatus during mitosis.Retinoic acid (RA) 1 modulates cellular proliferation and differentiation in a variety of tissues in the adult organism as well as during embryogenesis and development (1-3). The biological effects of RA are mediated by nuclear retinoic acid receptors and retinoid X receptors. Each receptor family consists of three receptor subtypes (␣, ␤, ␥) encoded by independent genes (4 -6). Modulation of gene expression occurs through binding of ligand-activated retinoic acid receptor/retinoid X receptor heterodimers to retinoid responsive elements located in the regulatory regions of target genes (5,7,8). These pleiotropic effects of retinoids are brought about by changes in the expression of numerous proteins such as transcription factors, enzymes, cytokines, growth factors, extracellular matrix, cell cycle, and Hox proteins (9 -12). One of the most widespread effects of RA is the ability to arrest growth in a large number of different cell types such as melanoma, lymphoma, neuroblastoma, embryonic stem, and carcinoma cells. In some cell types, RA-mediated growth inhibition is associated with decreased expression of transcription factors c-myc and c-myb, tumor suppressors p53 and pRB as well as epidermal growth factor receptor (9, 13-15). However, the search for factors responsible for the antiproliferative effects of retinoids has so far been unrewarding in the majority of cell types and tissues.We have previously demonstrated that various retinoids inhibit growth and induce cellular differentiation in a broad panel of human pancreatic carcinoma cells in vitro and in vivo (16 -19). A subsequent clinical trial has shown significant efficacy for retinoic acid in pancreatic cancer patients (20). We were therefore interested in identifying RA-regulated genes to delineate the molecules involved in the antip...

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