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Bcl-2 accelerates retinoic acid-induced growth arrest and recovery in human gastric cancer cells.

ABSTRACT: The role of Bcl-2 as an anti-apoptotic protein has been well documented. In the present work, we present evidence that Bcl-2 may also be involved in cell growth regulation. SC-M1 is an unique cell line which responds to retinoic acid (RA) treatment with reversible growth arrest [Shyu, Jiang, Huang, Chang, Wu, Roffler and Yeh (1995) Eur. J. Cancer 31, 237-243]. In this study, when treated with RA, SC-M1/Bcl2 cells, which were generated by transfecting SC-M1 cells with bcl-2 DNA, were growth-arrested two days earlier than SC-M1/neo cells, which were generated by transfecting SC-M1 cells with vector DNA. This indicates that Bcl-2 accelerates RA-induced growth arrest. In addition to the accelerated growth arrest, RA-treated SC-M1/Bcl2 cells also recovered from growth arrest two days faster than SC-M1/neo cells after the removal of RA. Previously, we had identified the cyclin-dependent kinase inhibitor p21((WAF1/CIP1)) (p21) as a mediator of RA-induced growth arrest [Tsao, Li, Kuo, Liu and Chen (1996) Biochem. J. 317, 707-711]. In a search for the mechanism by which Bcl-2 affects growth regulation, we found that p21 gene expression was more prominent in SC-M1/Bcl2 cells than in SC-M1/neo cells in the presence of RA, but when RA was removed, p21 gene expression levels in SC-M1/Bcl2 cells were also reduced earlier than in SC-M1/neo cells. The present report is the first to show that Bcl-2 accelerates not only growth arrest but also recovery from growth arrest. Moreover, the close correlation between the effect of Bcl-2 on both RA-induced growth arrest and RA-induced p21 gene expression suggests the possibility that Bcl-2 affects cell growth through the mechanism of p21.

SUBMITTER: Chou HK

PROVIDER: S-EPMC1221088 | biostudies-other | 2000 Jun

REPOSITORIES: biostudies-other

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Project description:Neuroblastoma cells having stem cell-like qualities are widely employed models for the study of neural stem/progenitor cell proliferation and differentiation. We find that human BE(2)C neuroblastoma cells possess a signaling cascade initiated by Ca(2+) influx via voltage-dependent calcium channels and the N-methyl-D-aspartate (NMDA) receptor and culminating in nuclear calmodulin-dependent protein kinase IV (CaMKIV)-mediated phosphorylation and activation of the transcription factors Ca(2+)/cyclic AMP-response element-binding protein (CREB) and ATF1 (activating transcription factor-1). This pathway functions to maintain BE(2)C cells in an undifferentiated, proliferative state. Parallel to this Ca(2+)-dependent pathway is a hormone-responsive program by which retinoic acid (RA) initiates the differentiation of BE(2)C cells toward a neuronal lineage. This is evidenced by RA-dependent induction of the cell cycle inhibitor p21/Cip1 (Cdk-interacting protein 1) and cell cycle arrest, induction of the neuroblastic marker doublecortin and of the neuron-specific intermediate filament protein, peripherin, and by RA-stimulated extension of neuritic processes. During neuronal differentiation there is a complex antagonistic interplay between these two major signaling pathways. RA down-regulates expression of CaMKIV and one of its upstream activators, CaMKK1 (calmodulin-dependent protein kinase kinase 1). This is accompanied by RA-induced suppression of activating phosphorylation of CREB with a time course paralleling that of CaMKIV down-regulation. RA-induced repression of the Ca(2+)/calmodulin-dependent protein kinase kinase/CaMKIV/CREB pathway appears to be involved in regulating the timing of neuronal differentiation, as shown by the effect of RNA interference of CaMKIV to markedly accelerate RA-dependent up-regulation of p21/Cip1 and doublecortin expression and RA-promoted neurite outgrowth. RA-induced repression of the CaMKIV signaling pathway may represent an early event in retinoid-dependent neuronal differentiation.

Project description:The mechanism of action of retinoic acid (RA) is of broad relevance to cell and developmental biology, nutrition, and cancer chemotherapy. RA is known to induce expression of the Burkitt's lymphoma receptor 1 (BLR1) gene which propels RA-induced cell cycle arrest and differentiation of HL-60 human myeloblastic leukemia cells, motivating the present analysis of transcriptional regulation of blr1 expression by RA. The RA-treated HL-60 cells used here expressed all RA receptor (RAR) and retinoid X receptor (RXR) subtypes (as detected by Northern analysis) except RXRgamma. Treatment with RAR- and RXR-selective ligands showed that RARalpha synergized with RXRalpha to transcriptionally activate blr1 expression. A 5'-flanking region capable of supporting RA-induced blr1 activation in HL-60 cells was found to contain a 205-bp sequence in the distal portion that was necessary for transcriptional activation by RA. Within this sequence DNase I footprinting revealed that RA induced binding of a nuclear protein complex to an element containing two GT boxes. Electromobility shift assays (EMSAs) and supershift assays showed that this element bound recombinant RARalpha and RXRalpha. Without RA there was neither complex binding nor transcriptional activation. Both GT boxes were needed for binding the complex, and mutation of either GT box caused the loss of transcriptional activation by RA. The ability of this cis-acting RAR-RXR binding element to activate transcription in response to RA also depended on downstream sequences where an octamer transcription factor 1 (Oct1) site and a nuclear factor of activated T cells (NFATc) site between this element and the transcriptional start, as well as a cyclic AMP response element binding factor (CREB) site between the transcriptional start and first exon of the blr1 gene, were necessary. Each of these sites bound its corresponding transcription factor. A transcription factor-transcription factor binding array analysis of nuclear lysate from RA-treated cells indicated several prominent RARalpha binding partners; among these, Oct1, NFATc3, and CREB2 were identified by competition EMSA and supershift and chromatin immunoprecipitation assays as components of the complex. RA upregulated expression of these three factors. In sum the results of the present study indicate that RA-induced expression of blr1 expression depends on a novel RA response element. This cis-acting element approximately 1 kb upstream of the transcriptional start consists of two GT boxes that bind RAR and RXR in a nuclear protein complex that also contains Oct1, NFATc3, and CREB2 bound to their cognate downstream consensus binding sites.

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Bcl-2 accelerates retinoic acid-induced growth arrest and recovery in human gastric cancer cells.

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