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J Biol Chem, Vol. 274, Issue 27, 18925-18931, July 2, 1999


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

Astrid KaiserDagger , Felix H. BrembeckDagger , Barbara NickeDagger , Bertram Wiedenmann§, Ernst-Otto RieckenDagger , and Stefan Rosewicz§

From the Dagger  Department of Gastroenterology, Klinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany and § Medizinische Klinik m. S. Hepatologie und Gastroenterologie, Charité, Campus Virchow Klinikum, Augustenburgerplatz 1, 13353 Berlin, Germany

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 G2/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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (alpha , beta , gamma ) 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 antiproliferative effects of retinoic acid.

Differential display RT-PCR has been demonstrated as a suitable approach to isolate differentially regulated genes in a variety of cells (21-23). We therefore used this technique to detect changes in gene expression due to retinoic acid treatment. We now report the identification of the kinesin-related protein HsEg5 as a central molecule involved in the antiproliferative action of all-trans-retinoic acid.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Growth Assay-- All cell lines were grown as subconfluent monolayer cultures either in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (v/v; Panc-1, MCF-7), in Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) supplemented with 10% fetal calf serum (v/v; BON), or in RPMI 1640 supplemented with 10% fetal calf serum (v/v; DAN-G, HL-60), penicillin (100 units/ml), and streptomycin (100 µg/ml). HaCat cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) under serum-free conditions with penicillin (100 units/ml) and streptomycin (100 µg/ml). Cells were kept under 95% air and 5% CO2 at 37 °C. All experiments were carried out in the log phase of growth after the cells had been plated for 24 h. For growth assays, cells were plated in 96-well dishes at a density of 5,000 cells/well. At the indicated times, cells were washed in phosphate-buffered saline (PBS) and then obtained by trypsinization. Viable cells were counted in a hemocytometer by trypan blue exclusion. Triplicate wells were analyzed for each time point.

RNA Isolation-- Poly(A)+ enriched RNA was extracted from DAN-G, Panc-1, and BON cells using the Poly AT tract kit following the instructions of the supplier. From DAN-G cells, total RNA was isolated by the method described by Chomczynski (24) using the RNAzolTMB reagent (Wak-Chemie, Bad Homburg, Germany). For quantitative analysis, RNA was analyzed in a 1% agarose, 2% formaldehyde gel, blotted onto Hybond N+ membranes (Amersham Pharmacia Biotech, Braunschweig, Germany) and immobilized by UV cross-linking. The BamHI/HindIII cDNA insert of HsEg5 was eluted by glasmilk with the Geneclean II kit (Dianova, Hamburg, Germany) and labeled using the Megaprime labeling kit with [alpha -32P]dCTP following the instructions of the supplier. Hybridization was carried out using the QuickHyb reagent (Stratagene, Heidelberg, Germany).

Differential Display RT-PCR (DDRT-PCR)-- Differential display RT-PCR was performed according to the method described by Liang et al. (21, 23) using the RNAmapTM kit (WAK-Chemie). Total RNA was isolated from DAN-G cells treated for 4 h either with 10 µM all-trans-retinoic acid or vehicle. 0.7 µg of RNA were treated with DNase I, reverse transcribed, and the reverse transcription mixture was then used for polymerase chain reaction in a dilution of 1:10. Subsequent PCR reaction was performed in 1× PCR buffer (2 µM dNTP, 10 mM Tris-HCL, pH 8.4, 50 mM KCL, 1.5 mM MgCl2, 0.001% gelatin) containing 1 µM of T12M (G/A/T/C) primers, 0.2 µM AP-12 primer, 0.6 µl of [33P]dATP (1,000-3,000 Ci/mmol) and 1 unit of AmpliTaq Polymerase (Perkin-Elmer). The amplification products were separated on a 6% denaturating polyacrylamide gel. The gel was blotted onto Whatman 3 MM paper, vacuum-dried, and exposed to BioMaxMR film (Eastman Kodak) overnight. Differentially expressed bands were excised from the dried gel and incubated in 100 µl of H2O for 10 min. After rehydration of the polyacrylamide gel, DNA was eluted by boiling the gel slice for 15 min. Subsequently DNA was ethanol precipitated with 50 µg of glycogen as carrier and redissolved in 10 µl of H2O. Reamplification was carried out with the same primers, and PCR conditions as previously used except that 20 µM dNTP and no labeled dATP were used. Reamplified cDNA probes were cloned into the PCRTM2.1 vector using the TA cloning system from Invitrogen (San Diego, CA, USA) and subsequently sequenced by the dideoxy-sequencing technique.

Nuclear Run-on Transcription Assay-- DAN-G cells were incubated in the absence or presence of 10 µM ATRA for 4 and 24 h. Nuclei were prepared by resuspending cells in a lysis buffer containing 0.25 M sucrose, 10 mM Hepes, pH 8.0, 10 mM MgCl2, 2 mM dithiothreitol, and 0.1% Triton X-100 and homogenized two to three times with 30 strokes of a tight fitting pestle of a Dounce homogenizer on ice. In vitro transcription was carried out using a modified protocol by Nelson & Groudine (25) exactly as described previously (26).

Western Blot Analysis-- DAN-G cells were resuspended in a buffer containing 1% SDS (w/v), 0.1 M Tris, pH 7.5, 0.05 M EDTA, 0.02 M EGTA, 0.1 M saccharose, 0.1 M beta -mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride and homogenized using an ultrasound cell disruptor. 20 µg of protein/lane were run on a 12% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with a rabbit antiserum raised against human HsEg5 (27) which was used at a dilution of 1:3,000. Detection was performed using goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Dianova) and the ECL detection system (Amersham Pharmacia Biotech).

Stable Transfection-- A 664-base pair fragment encoding the 5' region of human HsEg5 was isolated from the vector pCMVDW2-1 and subcloned in antisense orientation into the mammalian expression vector pRc/CMV. The construct was named pHsEg5-AS and verified by restriction analysis. DAN-G cells (approximately 2 × 106 cells/100-mm dish) were transfected with 5 µg of pHsEg5-AS plasmid using the LipofectAMINETM Reagent (Life Technologies, Inc.). Control cells were transfected with pRc/CMV vector (mock-transfected) only. 24 h after transfection cells were diluted 1:10 and plated in medium containing 1.0 mg/ml G418 (Life Technologies, Inc.). Resistant cell clones appeared after approximately 21 days and were picked for expansion at 35 days. The transfected and mock-transfected cells were kept under selective pressure at all times.

Flow Cytometry-- 5 × 105 DAN-G cells were preincubated with 10 µM ATRA for 48 h and synchronized by incubation with 2 mM thymidine for 24 h in early S-phase. Cells were released from S-phase by washing three times with PBS followed by addition of fresh medium containing either 10 µM ATRA or Me2SO. 12, 15, 18, and 21 h after release, cells were trypsinated and resuspended in PBS. Cells were fixed in 70% ethanol and incubated in PBS containing 40 µg/ml RNaseA for 30 min at room temperature. DNA content of the cells was analyzed in PBS containing 0.1% Triton X-100, 1 µM EDTA, 1.5 µg of propidium iodide on a FACScan (Becton Dickinson).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ATRA Mediates Inhibition of HsEg5 Expression in DAN-G Cells-- To detect early differences in gene expression mediated by ATRA in pancreatic carcinoma cells, we compared the mRNA expression pattern before and after treatment (4 h) with ATRA using DDRT-PCR. Subsequent DDRT-PCR was performed using a total of 60 primer combinations yielding 11 differentially expressed cDNA bands. Fig. 1 shows the sequencing gel of amplification products obtained with a primer combination of T12MG and AP12. One cDNA band that was only present in the control lane was therefore excised from the gel, reamplified, and analyzed for size determination in a 1% agarose gel. The 230-base pair reamplification product was then subcloned into a TA-cloning vector and subsequently sequenced. Comparison with the nucleotide sequence data base revealed that the isolated cDNA corresponds to the human kinesin-related protein (GenBankTM accession number X85137) as well as to the human kinesin-like spindle protein (GenBankTM accession number U37426). Both nucleotide sequences encode for the human HsEg5 protein (27, 28).


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  		Fig. 1.   DDRT-PCR analysis. 0.7 µg of RNA were reverse transcribed and subsequently subjected to PCR using the primers T12MG and AP12. The amplification products were separated on a 6% denaturing polyacrylamide gel. The arrowhead indicates the differentially expressed band, which was reamplified with the same primers by PCR and analyzed in a 1% agarose gel.

To confirm ATRA-mediated inhibition of HsEg5 expression discovered by DDRT-PCR, we performed Northern and Western blot analysis in DAN-G cells. Hybridization with the radioactively labeled HsEg5 cDNA revealed two bands at 4.8 and 3.7 kilobase pairs corresponding to both existing HsEg5 mRNA species. ATRA resulted in a rapid and significant decrease of HsEg5 mRNA and protein concentrations, reaching a maximal reduction of 50% of control at 48 h (Fig. 2, A and B).


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  		Fig. 2.   Effects of ATRA on HsEg5 expression in DAN-G cells. DAN-G cells were incubated for the indicated time points with 10 µM ATRA. A, 8 µg of poly(A)+ enriched RNA were separated on a 1% agarose-formaldehyde gel, transferred to Hybond N+ membrane, and hybridized with the radioactively labeled cDNA for HsEg5 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A representation of four experiments is shown. B, protein extracts were prepared, and 20 µg of protein for each condition were separated in a 10% SDS-polyacrylamide gel electrophoresis. HsEg5 protein was detected using antiserum raised against human HsEg5 and developed by the ECL system. A representation of two independent experiments yielding identical results is shown.

To elucidate by which mechanism ATRA inhibits HsEg5 expression, we investigated the effects of ATRA on HsEg5 gene transcription as well as mRNA stability. Using nuclear run-on analysis we were unable to detect an inhibition of HsEg5 gene transcription by ATRA (Fig. 3A). In contrast, mRNA decay studies revealed that pretreatment with ATRA for 12 h significantly decreases HsEg5 mRNA stability (t1/2, 5.6 h versus 14.0 h in untreated controls) suggesting a primarily posttranscriptional inhibitory mechanism (Fig. 3B).


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  		Fig. 3.   Effects of ATRA on HsEg5 gene transcription. A, DAN-G cells were treated with 10 µM ATRA or vehicle for either 4 or 24 h, and nuclei were isolated. In a nuclear run-on assay, the radioactively labeled RNA was hybridized to 2.5 µg of immobilized cDNA for HsEg5 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In one set of nuclei (24 h control) alpha -amanitin was added at a concentration of 2 µg/ml. A representation of three independent experiments yielding identical results is shown. B, DAN-G cells were either pretreated with 10 µM ATRA or vehicle for 12 h before incubation with actinomycin D (10 µg/ml) for 2, 4, and 6 h. Poly(A)+ RNA was then isolated and analyzed by Northern blot analysis. HsEg5 hybridization signals were normalized to 28 S RNA and quantitated using a laser scanner. The lines were fitted to the data using linear regression analysis. The correlation coefficients were r = -0.99 for actinomycin D (act.D) and r = -0.92 for act.D + RA. The mean ± S.E. of three independent experiments is shown.

Effects of ATRA on HsEg5 Expression in Various Cell Lines-- To examine whether the ATRA-mediated inhibition of HsEg5 expression was cell type specific, we investigated a panel of different cell types that are growth inhibited by ATRA. Northern blot analysis revealed a decrease of HsEg5 mRNA expression in human pancreatic carcinoma cells, BON and Panc-1, in response to ATRA (Fig. 4A). In addition, Western blot analysis revealed a significant decrease of HsEg5 protein expression in human HaCat keratinocytes (Fig. 4B). In contrast, ATRA at a concentration of 10 µM had no effect on HsEg5 expression in breast carcinoma MCF-7 and lymphoma HL-60 cells (Fig. 4B), although both cell lines were profoundly growth inhibited at a concentration range of 10 nM to 10 µM ATRA (data not shown).


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  		Fig. 4.   Effects of ATRA on HsEg5 gene expression in various cell types. BON, Panc-1, HaCat, MCF-7, and HL-60 cells were incubated for the indicated time periods with 10 µM ATRA. A, Northern blot analysis. A representation of two independent experiments is shown. B, Western blot analysis. HsEg5 protein was detected using antiserum raised against human HsEg5 and developed by the ECL system. A representation of two independent experiments is shown.

HsEg5 mRNA Expression Is Specifically Inhibited by ATRA-- To evaluate whether HsEg5 expression is specifically mediated by ATRA and to exclude that HsEg5 inhibition fortuitously coincides with inhibition of growth, we investigated the effects of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate (TPA) on growth and HsEg5 expression. Treatment of DAN-G cells with 1 mM sodium butyrate as well as with 1 µM TPA resulted in a pronounced and time-dependent growth inhibition (Fig. 5A). Western blot analysis revealed that neither sodium butyrate nor TPA had inhibitory effects on HsEg5 protein expression (Fig. 5B), suggesting that HsEg5 expression is not a nonspecific epiphenomenon associated with growth inhibition.


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  		Fig. 5.   HsEg5 gene expression is specifically correlated to ATRA-mediated growth inhibition. DAN-G cells were incubated either with sodium butyrate (NaBt), TPA, or vehicle for the indicated time points. A, cells were plated at a density of 10,000 cells/well, and the viable cell number was determined by counting cells after trypan blue exclusion at the indicated time points. B, Western blot. Protein extracts were prepared, and 10 µg of protein for each condition were separated in a 12% SDS-polyacrylamide gel electrophoresis. HsEg5 protein was detected using antiserum raised against human HsEg5 and developed by the ECL system. A representation of two independent experiments is shown.

Biological Significance of ATRA-mediated Inhibition of HsEg5 Expression-- HsEg5 is essential for the proper coordination of spindle assembly and spindle stabilization during mitosis. If HsEg5 plays a role in ATRA-mediated growth inhibition, we would therefore expect a retarded progression through the G2/M phase of the cell cycle upon ATRA treatment. After pretreatment with 10 µM ATRA, DAN-G cells were blocked in early S-phase with 2 mM thymidine. Cell cycle kinetics were performed after release from S-phase and compared with untreated control cells. Compared with controls, ATRA treatment of DAN-G cells resulted in a significant increase in the G2/M phase population accompanied by a decrease in the S-phase population for up to 21 h (Fig. 6). These results indicate that ATRA treatment interferes with the cell cycle by a delayed progression through G2/M phase supporting the hypothesis that inhibition of HsEg5 expression results in a transient arrest of DAN-G cells in mitosis.


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  		Fig. 6.   Effects of ATRA on cell cycle distribution in DAN-G cells. DAN-G cells were pretreated for 48 h with either 10 µM ATRA or Me2SO before blocking cells in early S-phase by 2 mM thymidine for 24 h. Cells were then released from S-phase block, and cell cycle distribution was determined after the indicated time periods by fluorescence-activated cell sorter analysis. One of two experiments yielding identical results is shown.

Effects of HsEg5 Depletion-- To further corroborate the functional biological correlation between ATRA-mediated inhibition of HsEg5 expression and growth inhibition, we established HsEg5-depleted DAN-G cell clones by stable transfection of a HsEg5 antisense cDNA. Five independent HsEg5-antisense (AS-11, AS-22, AS-26, AS-30, AS-34) and two mock-transfected (CMV6, CMV18) DAN-G cell clones were generated, and HsEg5 expression was investigated using Western blot analysis (Fig. 7A). All HsEg5 antisense clones showed a significant reduction of HsEg5 expression (AS-11, 17%; AS-34, 23%; AS-26, 43%; AS-30, 52%; AS-22, 63% of control) as compared with mock-transfected and wild type cells. We then explored whether depletion of HsEg5 expression was capable and sufficient to mediate growth inhibition in DAN-G cells. Therefore, all five HsEg5 antisense clones were investigated for cellular proliferation by determining viable cell number at various time points. Reduction of HsEg5 expression results in a significant decrease of cellular proliferation in all antisense clones compared with mock-transfected control cells (Fig. 7B). Furthermore, when we compared the rate of cellular proliferation with the intracellular HsEg5 concentration, we observed a tight linear correlation (Fig. 7C). In addition, the values obtained for ATRA-mediated HsEg5 reduction and growth inhibition fit well to the regression curve generated with HsEg5 antisense clones.


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  		Fig. 7.   Effects of HsEg5 antisense transfection on tumor cell proliferation. A, protein extracts of DAN-G wild type (WT), mock-transfected (CMV6, CMV18), and HsEg5 antisense clones (AS-11, AS-22, AS-26, AS-30, AS-34) were prepared, and 10 µg of protein were separated in a 10% SDS-polyacrylamide gel electrophoresis. HsEg5 protein was detected using antiserum raised against human HsEg5. HsEg5 signal was quantitated using laser densitometry. B, DAN-G mock-transfected (CMV6, CMV18) and HsEg5 antisense clones (AS-11, AS-22, AS-26, AS-30, AS-34) were plated at a density of 5,000 cells/well in triplicates, and cell number was determined by counting cells after trypan blue exclusion at the indicated time points. A representation of three experiments yielding identical results (mean ± S.E.; * p < 0.001 compared with mock-transfected cells CMV6 and CMV18) is shown. C, the linear correlation between HsEg5 protein concentration of ATRA-incubated DAN-G cells (RA), mock-transfected (CMV), and HsEg5 antisense clones (11, 22, 26, 30, 34) and growth at 96 h after plating is shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Based on their ability to inhibit growth and induce differentiation, retinoids have recently received considerable attention in medical oncology. In this context, we have recently established retinoids in preclinical and clinical trials as an effective novel treatment strategy for human pancreatic cancer (16-20). The identification of retinoid-responsive genes that are involved in retinoid-mediated growth inhibition might therefore provide new insights into the molecular mechanism of action and potentially reveal novel target molecules for the design of antiproliferative therapeutic strategies in cancer therapy.

Based on our previous work (16), we chose the human pancreatic carcinoma cell line DAN-G as a representative in vitro model, in which treatment with ATRA results in a dose-dependent profound growth inhibition with maximal effects observed at a concentration of 10 µM ATRA. Using DDRT-PCR in this system, we have identified for the first time the human kinesin-related protein HsEg5 whose gene expression is inhibited by ATRA in human pancreatic cancer cells. HsEg5 belongs to the superfamily of kinesin-related microtubule-associated motor proteins, which hydrolyze ATP to move toward the plus ends of microtubules (29). The kinesin-related proteins have been divided into at least seven different families and have been proposed to function in mitotic spindle assembly and dynamics (29, 30). HsEg5 belongs to the bimC family of kinesin-related proteins that are conserved throughout evolution, because homologs have been isolated from widely divergent organisms (27, 31-35).

Segregation of chromosomes during mitosis requires formation of the mitotic spindle apparatus, which consists of microtubules and associated proteins that force the movement of spindle assembly and chromosome distribution (36).Two mechanisms of action responsible for the regulation of kinesin-related motor proteins have recently been elucidated, phosphorylation and the level of intracellular concentration. First, HsEg5 has been shown to be associated with centrosomes during early prophase, which depends on specific phosphorylation of Thr-927 by the p34cdc2-cyclin B complex which might trigger the onset of mitosis (27). Second, microinjection of specific anti-HsEg5 antibodies into human HeLa cells resulted in a transient arrest with monoastral microtubule arrays in about 80% of injected cells, indicating that HsEg5 is required for centrosome separation and the assembly of bipolar spindles (27). Taken together, these data suggest that aside from cdc2-mediated phosphorylation, the cellular concentration of HsEg5 critically determines the mitotic process.

Confirming the differential expression of HsEg5 identified by DDRT-PCR, we observed a profound inhibition of HsEg5 mRNA and protein levels by ATRA in DAN-G cells, indicating a pretranslational regulation of HsEg5 expression. Although dose-response experiments were not performed in this study, it appears highly unlikely that the observed inhibition of HsEg5 expression is because of nonspecific effects due to relatively high ATRA concentrations, because we have previously shown that under identical experimental conditions, matrix-metalloprotease-1 gene expression is in fact stimulated in the same cell line by identical RA concentrations (37). We observed that ATRA did not alter the transcription rate of the HsEg5 gene but decreased HsEg5 mRNA stability compared with untreated cells. These results indicate that HsEg5 gene expression is regulated by ATRA at a posttranscriptional level. Over the last years, experimental evidence has accumulated that in addition to transcriptional control, modulation of mRNA stability by retinoids can function as an alternative molecular mechanism to control gene expression of retinoid-regulated genes (38-41). The underlying molecular mechanisms are currently poorly understood. In the case of connexin 43 gene expression, retinoic acid is able to influence mRNA stability via elements located in the 3'-untranslated region (39). Whether this observation also applies for retinoic acid-mediated HsEg5 mRNA stability is currently under investigation.

In addition, we observed that HsEg5 gene expression is specifically inhibited by ATRA and that other growth inhibitory agents, such as the phorbol ester TPA and sodium butyrate, had no effect on HsEg5 expression, although they resulted in pronounced growth inhibition. It is therefore unlikely that inhibition of HsEg5 expression by ATRA is a nonspecific epiphenomenon associated with growth inhibition.

If inhibition of HsEg5 expression indeed plays a central role in ATRA-mediated growth inhibition, it should be expected, based on the analogy to HsEg5 immunodepletion experiments performed in HeLa cells (27), that ATRA-treated cells display retarded progression through mitosis. In fact, when we performed cell cycle analysis in pancreatic tumor cells, which were synchronized in S-phase, we observed that ATRA treatment results in a significant increase of cells retarded in G2/M phase compared with untreated controls. The decrease in HsEg5 concentrations in response to ATRA treatment therefore most likely results in retardation of centrosome separation and bipolar spindle formation leading to a prolonged mitotic phase. Because we did not observe a significant increase of polyploid cells upon ATRA treatment (data not shown), we conclude that completion of mitosis was not completely inhibited by ATRA but strongly retarded. In analogy, Blangy et al. (27) demonstrated that HeLa cells injected with neutralizing HsEg5 antibody accumulated as mitotic cells for up to 40 h before completing the mitotic process.

To evaluate whether HsEg5 depletion per se is sufficient to mediate growth inhibition, we determined the proliferation rate of HsEg5 antisense clones over a period of 5 days. We demonstrated that all five transfected cell clones expressing reduced levels of HsEg5 protein were significantly growth inhibited as compared with mock-transfected controls. Furthermore, in the HsEg5 antisense clones as well as in the ATRA-treated tumor cells the level of HsEg5 expression correlated in a linear manner with the extent of growth inhibition. We therefore conclude that inhibition of HsEg5 expression is a crucial step in growth inhibition mediated by ATRA in pancreatic carcinoma cells.

The antiproliferative mechanism of action appears to be cell type specific for ATRA. Although ATRA inhibits HsEg5 expression in several pancreatic carcinoma cells as well as in keratinocytes, ATRA treatment in breast carcinoma MCF-7 cells and lymphoma HL-60 cells had no effect on HsEg5 expression although these cell lines are also growth inhibited by ATRA. This is most likely explained by the fact that retinoids cause an accumulation of cells in G1 phase of the cell cycle in MCF-7 and HL-60 cells due to down-regulation of cyclin E (HL-60) or cyclin D1, cdk 2 and pRB protein levels (MCF-7) (10, 11). Thus, retinoids might control different subsets of retinoid-responsive genes to induce growth inhibition in different cell types. Moreover, it has been suggested that mediation of the pleiotropic effects of retinoids are based on the cell-specific expression pattern and dimeric combinations of retinoic acid receptors and retinoid X receptors subtypes (6, 42). In addition, the recently discovered co-activators and co-repressors of retinoid receptors might display further cell type specific regulatory factors of retinoid-mediated gene expression (43-46).

In summary, we have identified the kinesin-related protein HsEg5 as a novel molecule involved in the antiproliferative effects of ATRA. To our knowledge, this observation represents the first experimental evidence that ATRA acts as a cytostatic drug by interference with the bipolar spindle apparatus, which might classify ATRA as a "chemotherapeutic vitamin."

    ACKNOWLEDGEMENTS

We are grateful to Dr. M. Kress (Villjuif, France) for providing the HsEg5 plasmid and to Dr. E. A. Nigg (Geneve, Switzerland) for providing the HsEg5 antiserum. We would like to thank Dr. C. Zouboulis (Berlin, Germany) for providing the HaCat keratinocytes.

    FOOTNOTES

* This work was supported by a Grant from the Deutsche Krebshilfe (10-0954-Ro2) and Deutsche Forschungsgemeinschaft (Ro674/10-1/2).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-30-45053733; Fax: 49-30-45053902; E-mail: stefan.rosewicz{at}charite.de.

    ABBREVIATIONS

The abbreviations used are: RA, retinoic acid; ATRA, all-trans-RA; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; DDRT-PCR, differential display RT-PCR; PBS, phosphate-buffered saline; TPA, 12-O-tetradecanoylphorbol-13-acetate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. De Luca, L. M. (1991) FASEB J. 5, 2924-2933[Abstract]
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