Retinoid-induced Apoptosis Research Articles

Effect of retinoid X receptor-α nuclear export inhibition on apoptosis of neurons invivo and invitro.

Alzheimer's disease (AD), which is characte-rized by excessive apoptosis of neurons, is considered to be a global public health crisis. Retinoid-induced apoptosis is dependent on the orphan nuclear receptor Nur77, a transcription factor that is expressed predominantly in brain tissues. Nur77 nuclear export requires retinoid X receptor‑α (RXRα) as a carrier. However, the involvement of Nur77 in mediating β‑amyloid (Aβ)‑induced neuronal apoptosis has not yet been elucidated. The primary aim of the present study was to investigate the potential of Nur77 in Aβ‑induced neuron apoptosis, and to evaluate the effect of RXRα nuclear export inhibition on neuronal apoptosis. Mouse neuroblastoma Neuro‑2a (N2a) cells and mouse hippocampi were treated with Aβ25‑35 or Aβ25‑35 combined with a RXRα ligand, 9‑cis‑retinoid acid (9‑cis‑RA), while untreated cells and mice served as controls. The expression of RXRα and Nur77 was determined using western blotting and reverse transcription‑quantitative polymerase chain reaction analyses, and the translocation of RXRα and Nur77 was detected using confocal microscopy. In addition, the apoptosis and viability of N2a cells was detected using flow cytometry and MTT assays, respectively, and the expression of B cell lymphoma 2 (Bcl‑2) and Bcl‑2 associatedX (Bax) was quantified by western blotting. No significant alterations in the protein or mRNA expression levels of RXRα and Nur77 in N2a cells or mouse hippocampi among the three groups were observed. Aβ25‑35 treatment resulted in elevated cytoplasmic protein ratios of RXRα and Nur77 in N2a cells when compared with controls, while combined treatment with Aβ25‑35 and 9‑cis‑RA reduced cytoplasmic protein ratios of RXRα and Nur77 to 6.67 and 5.44% in N2a cells, respectively. The MTT assay results revealed a significant reduction in the viability of N2a cells following treatment with Aβ25‑35 for 24h when compared with the controls, while the viability of N2a cells treated with Aβ25‑35 plus 9‑cis‑RA significantly increased from 53.65 to 84.10%. Western blotting revealed elevated Bax expression and reduced Bcl‑2 expression in Aβ25‑35‑treated N2a cells when compared with controls, while combined treatment with Aβ25‑35 and 9‑cis‑RA recovered Bcl‑2 expression from 0.46‑fold in cells treated with Aβ25‑35 alone to 2.44‑fold (relative to the control) and decreased Bax expression from 2.52‑fold in cells treated with Aβ25‑35 alone to 0.99‑fold (relative to the control). Flow cytometry analysis revealed that the apoptotic rate of untreated N2a cells was 4.36%, while a 15.1% apoptotic rate was detected in cells exposed to Aβ25‑35 for 24h and a ~5.31% apoptotic rate was observed in N2a cells treated with Aβ25‑35 plus 9‑cis‑RA. In conclusion, treatment with Aβ25‑35 or Aβ25‑35 plus 9‑cis‑RA demonstrated no significant effect on the protein and mRNA expression levels of RXRα and Nur77. In addition, inhibition of RXRα nuclear export reduced neuronal apoptosis. The results of the present study may provide novel insight into the development of novel anti-AD agents.

Retinoids induce Nur77-dependent apoptosis in mouse thymocytes

Nur77 is a transcription factor, which plays a determinant role in mediating T cell receptor-induced cell death of thymocytes. In addition to regulation of transcription, Nur77 contributes to apoptosis induction by targeting mitochondria, where it can convert Bcl-2, an anti-apoptotic protein into a proapoptotic molecule. Previous studies have demonstrated that retinoids are actively produced in the mouse thymus and can induce a transcription-dependent apoptosis in mouse thymocytes. Here we show that retinoic acids induce the expression of Nur77, and retinoid-induced apoptosis is completely dependent on Nur77, as retinoids were unable to induce apoptosis in Nur77 null thymocytes. In wild-type thymocytes retinoids induced enhanced expression of the apoptosis-related genes FasL, TRAIL, NDG-1, Gpr65 and Bid, all of them in a Nur77-dependent manner. The combined action of these proteins led to Caspase 8-dependent Bid cleavage in the mitochondria. In addition, we could demonstrate the Nur77-dependent induction of STAT1 leading to enhanced Bim expression, and the mitochondrial translocation of Nur77 leading to the exposure of the Bcl-2/BH3 domain. The retinoid-induced apoptosis was dependent on both Caspase 8 and STAT1. Our data together indicate that retinoids induce a Nur77-dependent cell death program in thymocytes activating the mitochondrial pathway of apoptosis.

HOXA5 Acts Directly Downstream of Retinoic Acid Receptor β and Contributes to Retinoic Acid–Induced Apoptosis and Growth Inhibition

The promise of retinoids as chemopreventive agents in breast cancer is based on the differentiation and apoptosis induced upon their binding to the retinoic acid (RA) receptor beta (RARbeta). We have previously shown that HOXA5 induces apoptosis in breast cancer cells. In this study, we investigated whether RA/RARbeta and HOXA5 actions intersect to induce apoptosis and differentiation in breast cancer cells. We found that HOXA5 expression can be induced by RA only in RARbeta-positive breast cancer cells. We have, for the first time, identified the RA response element in HOXA5, which was found to be located in the 3' end of the gene. Chromatin immunoprecipitation assays showed that RARbeta binds directly to this region in vivo. Overexpression of RARbeta strongly enhances RA responsiveness, and knocking down RARbeta expression abolishes RA-mediated induction of HOXA5 expression in breast cancer cells. In addition, there is coordinated loss of both HOXA5 and RARbeta expression during neoplastic transformation and progression in the breast epithelial cell model, MCF10A. Knockdown of HOXA5 expression partially abrogates retinoid-induced apoptosis and promotes cell survival upon RA treatment. These results strongly suggest that HOXA5 acts directly downstream of RARbeta and may contribute to retinoid-induced anticancer and chemopreventive effects.

Retinal Pigment Epithelium Rescues Vascular Endothelium from Retinoic Acid-Induced Apoptosis

To determine whether retinoids are capable of inducing vascular endothelial cell apoptosis and whether the presence of an intact RPE monolayer can block retinoid-induced vascular endothelial cell death. Confluent fetal bovine aortic endothelial (FBAE) cells were incubated with various concentrations of all-trans or 9-cis retinoic acid (an analogue of 11-cis retinoic acid). Apoptosis rates were determined at 24 hours, and the effect of inhibition of protein synthesis and activation of protein kinase C on apoptosis was investigated by supplying culture medium with 0.1 mg/mL cycloheximide and 10 nM phorbol myristate acetate. To investigate the impact of RPE on retinoid-induced apoptosis, confluent FBAE cells were cultured with a confluent layer of RPE in inserts where retinoids were added to the upper compartment. A confluent bovine corneal endothelium monolayer was used as the control. The permeabilities of the RPE and bovine corneal endothelium monolayers to fluorescein (20 microg/mL) and 9-cis retinoic acid (3 x 10(-4) M) were also determined. 9-cis Retinoic acid induced higher rates of apoptosis in FBAE cells than did all-trans retinoic acid and the control (P = 0.004). This effect was dose-dependent, with an ED(50) of 1.4 microM (r = 0.99, P = 0.004). Cycloheximide did not inhibit 9-cis retinoic acid-induced apoptosis, but phorbol myristate acetate significantly decreased the apoptosis rate (P = 0.005). The presence of a confluent RPE monolayer reduced the 9-cis retinoic acid-induced apoptosis rate (P = 0.002), but the presence of a bovine corneal endothelial monolayer did not (P > 0.05). Both cell types established a similar diffusion barrier against fluorescein and 9-cis retinoic acid. 9-cis Retinoic acid is an important mediator of vascular endothelial apoptosis. A confluent monolayer of RPE can prevent endothelial cell apoptosis, and this effect is not due simply to establishment of a diffusion barrier by the RPE.

Retinoids Induce Growth Inhibition and Apoptosis in Mast Cell Tumor Cell Lines

Retinoids are well recognized as promising antitumor agents in humans. However, there have only been a few reports about the effect of retinoids in canine cancers. To investigate the antitumor effect of retinoids on mast cell tumors (MCT), inhibitory effect on cell growth and induction of apoptosis were examined in vitro. Although sensitivity of these cells differed among the cells, the growth of three MCT cell lines (CoMS, CM-MC and VI-MC) were inhibited dose dependently when they were treated with retinoids. FACS analysis of PI-stained nuclei revealed an apoptotic fraction in CM-MC cells about 30% when treated with retinoids, while those of control cells were less than 5%. Caspase-3 activation was observed after retinoid treatment in CM-MC cells. This was confirmed by inhibiting the retinoid-induced apoptosis using the pan-caspase inhibitor, ZVAD-FMK. Both retinoid receptors, RARs and RXRs, were detected by immunoprecipitation followed by western blot analysis in all the three MCT cells. These data suggests that retinoids inhibit the growth of MCTs partly through apoptosis, and this growth inhibition by retinoids may be mediated by RARs and RXRs. We conclude that retinoid may be a potential adjunctive chemotherapeutic agent for the treatment of canine MCT.

Retinoid-Induced Apoptosis in HL-60 Cells Is Associated with Nucleolin Down-Regulation and Destabilization of Bcl-2 mRNA

All-trans retinoic acid (ATRA) induces differentiation of promyelocytic leukemia cells, but the mechanisms by which cellular differentiation leads to apoptosis are not well understood. Studies were done to address the question whether ATRA-induced apoptosis is a consequence of destabilization of bcl-2 mRNA and decreased cellular levels of the anti-apoptotic protein, bcl-2. ATRA induced differentiation of HL-60 cells along the granulocytic pathway within 48 h. The half-lives of bcl-2 mRNA in HL-60 cells incubated with ATRA for 48 or 72 h were reduced to 39 and 7% of the corresponding untreated control values, respectively. Cellular differentiation was accompanied by down-regulation of the cytoplasmic levels of nucleolin, a bcl-2 mRNA-stabilizing protein. Binding of a bcl-2 mRNA instability element (AU-rich element-1) to nucleolin in S100 extracts from ATRA-treated cells was decreased to 15% of control within 72 h. The decay of 5' capped, polyadenylated bcl-2 mRNA transcripts containing ARE-1 was more rapid in S100 extracts from ATRA-treated cells compared with untreated cells. However, when recombinant nucleolin was added to extracts of ATRA-treated cells, the rate of bcl-2 mRNA decay was similar to the rate in extracts of untreated cells. These results provide evidence that ATRA-induced apoptosis is a consequence of cellular differentiation, which leads to nucleolin down-regulation and bcl-2 mRNA instability.

Enhanced retinoid-induced apoptosis of MDA-MB-231 breast cancer cells by PKC inhibitors involves activation of ERK.

Retinoids are vitamin A derivatives, which cause growth inhibition, differentiation and/or apoptosis in various cell types, including some breast cancer cells. In general, estrogen receptor (ER)-positive cells are retinoic acid (RA) sensitive, whereas ER-negative cells are resistant. In this report, we show that ER-negative MDA-MB-231 cells are strongly growth inhibited by retinoids in combination with a PKC inhibitor. While neither RA nor GF109203X (GF) has a significant growth inhibitory effect in these cells, RA+GF potently suppress proliferation. We found that RA+GF induce apoptosis, as shown by an increase in fragmented DNA, Annexin-V-positive cells and caspase-3 activation. Apoptosis was also induced by GF in combination with two synthetic retinoids. Expression of phosphorylated as well as total PKC was decreased by GF and this was potentiated by RA. In addition, treatment with GF caused a strong and sustained activation of ERK1/2 and p38-MAPK, as well as a weaker activation of JNK. Importantly, inhibition of ERK but not p38 or JNK suppressed apoptosis induced by RA+GF, indicating that activation of ERK is specifically required. In support of this novel finding, the ability of other PKC inhibitors to cause apoptosis in combination with RA correlates with ability to cause sustained activation of ERK.

BMP-2 mediates retinoid-induced apoptosis in medulloblastoma cells through a paracrine effect.

The mechanisms of retinoid activity in tumors remain largely unknown. Here we establish that retinoids cause extensive apoptosis of medulloblastoma cells. In a xenograft model, retinoids largely abrogated tumor growth. Using receptor-specific retinoid agonists, we defined a subset of mRNAs that were induced by all active retinoids in retinoid-sensitive cell lines. We also identified bone morphogenetic protein-2 (BMP-2) as a candidate mediator of retinoid activity. BMP-2 protein induced medulloblastoma cell apoptosis, whereas the BMP-2 antagonist noggin blocked both retinoid and BMP-2-induced apoptosis. BMP-2 also induced p38 mitogen-activated protein kinase (MAPK), which is necessary for BMP-2- and retinoid-induced apoptosis. Retinoid-resistant medulloblastoma cells underwent apoptosis when treated with BMP-2 or when cultured with retinoid-sensitive medulloblastoma cells. Retinoid-induced expression of BMP-2 is thus necessary and sufficient for apoptosis of retinoid-responsive cells, and expression of BMP-2 by retinoid-sensitive cells is sufficient to induce apoptosis in surrounding retinoid-resistant cells.

Real Time RT-PCR Shows Correlation between Retinoid-Induced Apoptosis and NGF-R mRNA Levels

Neurotrophins and retinoic acid have a critical role in the differentiation and the survival of neurons. All-trans-, 9-cis-retinoic acid (10−6 M) or NGF (50–100 ng/ml) induced morphologic differentiation and inhibited cell growth in SH-SY5Y neuroblastoma cells after 7 days of culture. Continuous treatment of undifferentiated cells with all-trans- or 9-cis-retinoic (10−6 M) did not induce apoptosis, whereas NGF-differentiated cells showed dramatic apoptosis after 2 to 4 days of retinoic acid treatment as evidenced by TUNEL reaction and flow cytometry analysis following propidium iodide staining. Addition of Ro41-5253 blocked all-trans-retinoic-induced apoptosis, suggesting that the apoptotic signaling pathway was mediated by RARs. The effects of all-trans- or 9-cis-retinoic acid on the expression of NGF receptors was evaluated using real-time fluorescence reverse transcription-PCR. A slight transient increase in the expression of p75NGFR mRNA was observed by 2 to 4 h after retinoid treatment of undifferentiated cells, whereas a larger increase in the expression of both TrkA and p75NGFR mRNA up to threefold the basal level, was observed by 2 to 6 h after retinoid treatment of NGF-differentiated cells. Our results suggest that NGF-differentiated cells may be more susceptible to retinoid-induced apoptosis that undifferentiated cells.

Retinoids, apoptosis and cancer.

Retinoids are a class of natural and synthetic vitamin A analogs structurally related to all-trans-retinoic acid (ATRA). Natural retinoids are involved in the physiology of vision and as morphogenic agents during embryonic development; they are also known to play a major role in regulating growth and differentiation of a wide variety of normal and malignant cell types, and, indeed, they can in various ways inhibit cell proliferation, induce differentiation and cell death by apoptosis. The development of new active retinoids and the identification of two distinct families of retinoid receptors has led to an increased understanding of the cellular effects of activation of these receptors and of mechanisms involved in the retinoid-induced apoptosis. In this review a brief summary of cellular pathways relevant to programmed cell death is given together with therapeutic potentialities of retinoids having apoptotic activity. Structure-activity relationship studies concerning the importance of different stereochemistry at the C9 double bond of retinoids in conferring apoptotic activity will be described. It will be also described the preparation and the potent cytotoxic and apoptotic activity of a novel class of heterocycle-bridged arotinoids.

Apoptosis induction by acyclic retinoid: a molecular basis of 'clonal deletion' therapy for hepatocellular carcinoma.

We have shown previously that administration of acyclic retinoid to cirrhotic patients who had undergone curative treatment of preceding hepatocellular carcinoma (HCC) induced the disappearance of serum lectin-reactive alpha-fetoprotein (AFP-L3) and subsequently reduced the incidence of second liver cancers. AFP-L3 is a tumor marker that indicates the presence of occult tumors below the detection limit by diagnostic images. Therefore, we have proposed a new concept of 'clonal deletion' therapy with acyclic retinoid for the cancer chemoprevention against HCC. Such eradication of AFP-L3-producing latent malignant (or premalignant) cells from the liver suggested a new strategy to prevent HCC, which may be involved in the same category as cancer chemotherapy. In the present series of studies, we explored the molecular mechanism of 'clonal deletion' and found a novel mechanism of apoptosis induction by the retinoid. We have demonstrated a modification of a retinoid receptor, RXRalpha, by mitogen-activated protein (MAP) kinase-dependent phosphorylation, resulting in the loss of transactivating activity. This may lead HCC cells to be resistant to natural retinoic acid. However, acyclic retinoid restored the function of phosphorylated RXRalpha and induced its downstream pro-apoptotic genes including tissue transglutaminase, an enzyme that is implicated in apoptosis. Tissue transglutaminase-dependent apoptosis in HCC cells was independent of the activation of caspases. This novel mechanism of retinoid-induced apoptosis may give a clue to understand the molecular mechanism of clonal deletion.

Retinoid-dependent growth inhibition, differentiation and apoptosis in acute promyelocytic leukemia cells. Expression and activation of caspases.

In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARalpha-selective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARbeta agonist and an anti-AP1 retinoid have very limited activity, ATRA- and AM580-dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARalpha. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD.

Down-regulation of thediphthamide biosynthesis protein 2-like gene during retinoid-induced differentiation and apoptosis: Implications against its tumor-suppressor activity

Down-regulation of thediphthamide biosynthesis protein 2-like gene during retinoid-induced differentiation and apoptosis: Implications against its tumor-suppressor activity

Molecular determinants of AHPN (CD437)-induced growth arrest and apoptosis in human lung cancer cell lines.

6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437), originally identified as a retinoic acid receptor gamma-selective retinoid, was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study, we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis, a process that is accompanied by rapid induction of c-Jun, nur77, and p21(WAF1/CIP1). In addition, we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein, a dominant-negative inhibitor of c-Jun, in A549 cells, nur77 expression and apoptosis induction by AHPN/CD437 were impaired, whereas p21(WAF1/CIP1) induction and G0/G1 arrest were not affected. Furthermore, overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus, expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together, our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer.

Retinoid-induced apoptosis in normal and neoplastic tissues.

Vitamin A and its derivatives (collectively referred to as retinoids) are required for many fundamental life processes, including vision, reproduction, metabolism, cellular differentiation, hematopoesis, bone development, and pattern formation during embryogenesis. There is also considerable evidence to suggest that natural and synthetic retinoids have therapeutical effects due to their antiproliferative and apoptosis-inducing effects in human diseases such as cancer. Therefore it is not surprising that a significant amount of research was dedicated to probe the molecular and cellular mechanisms of retinoid action during the past decade. One of the cellular mechanisms retinoids have been implicated in is the initiation and modulation of apoptosis in normal development and disease. This review provides a brief overview of the molecular basis of retinoid signaling, and focuses on the retinoid-regulation of apoptotic cell death and gene expression during normal development and in pathological conditions in vivo and in various tumor cell lines in vitro.

Retinoid-induced apoptosis and Sp1 cleavage occur independently of transcription and require caspase activation.

Vitamin A and its derivatives, the retinoids, are essential regulators of many important biological functions, including cell growth and differentiation, development, homeostasis, and carcinogenesis. Natural retinoids such as all-trans retinoic acid can induce cell differentiation and inhibit growth of certain cancer cells. We recently identified a novel class of synthetic retinoids with strong anti-cancer cell activities in vitro and in vivo which can induce apoptosis in several cancer cell lines. Using an electrophoretic mobility shift assay, we analyzed the DNA binding activity of several transcription factors in T cells treated with apoptotic retinoids. We found that the DNA binding activity of the general transcription factor Sp1 is lost in retinoid-treated T cells undergoing apoptosis. A truncated Sp1 protein is detected by immunoblot analysis, and cytosolic protein extracts prepared from apoptotic cells contain a protease activity which specifically cleaves purified Sp1 in vitro. This proteolysis of Sp1 can be inhibited by N-ethylmaleimide and iodoacetamide, indicating that a cysteine protease mediates cleavage of Sp1. Furthermore, inhibition of Sp1 cleavage by ZVAD-fmk and ZDEVD-fmk suggests that caspases are directly involved in this event. In fact, caspases 2 and 3 are activated in T cells after treatment with apoptotic retinoids. The peptide inhibitors also blocked retinoid-induced apoptosis, as well as processing of caspases and proteolysis of Sp1 and poly(ADP-ribose) polymerase in intact cells. Degradation of Sp1 occurs early during apoptosis and is therefore likely to have profound effects on the basal transcription status of the cell. Interestingly, retinoid-induced apoptosis does not require de novo mRNA and protein synthesis, suggesting that a novel mechanism of retinoid signaling is involved, triggering cell death in a transcriptional activation-independent, caspase-dependent manner.

Retinoid-induced apoptosis in human metastatic melanoma cells

Retinoid-induced apoptosis in human metastatic melanoma cells

Altered Sensitivity to Retinoid-Induced Apoptosis Associated with Changes in the Subcellular Distribution of Bcl-2

In the acute promyelocytic leukemia cell line NB4, Bcl-2 downregulation occurred as a late event of retinoid-induced differentiation. In the maturation-resistant NB4-R1 subclone, retinoids failed to downregulate Bcl-2 even in the situation of apoptosis massively induced by pan-agonists and RXR-selective agonists. We observed that NB4 and NB4-R1 cells differed with respect to the intracellular localization of Bcl-2 which showed a perinuclear localization in NB4-R1 cells, while Bax was broadly expressed in the cytoplasm and to only a minor extent in the perinuclear area. Therefore, the distinct intracellular localization of Bcl-2 and Bax was in general nonoverlaping. Bcl-2 remained massively expressed until cell disruption. Bax was not significantly upregulated in cells committed to death. However, Bax localization changed from a diffuse pattern to concentrate in few specific cytoplasmic area at a stage preceding the formation of apoptotic bodies. A human Bcl-2 transgene was transiently overexpressed in NB4-R1 cells which showed increased resistance to apoptosis induced by retinoids. Stably transfected clones of NB4-R1 cells showed an increased expression of Bcl-2 and a marked resistance to apoptosis. Interestingly, the overexpression of Bcl-2 restored a pattern of uniform Bcl-2 labeling in the cytoplasm and, remarkably, the colocalization of Bcl-2 with Bax. This work demonstrates that the ability of retinoid-induced cells to undergo apoptosis depends on the level of expression and the functional interaction between Bcl-2 and Bax.

Retinoid-regulated expression of BCL-2 and tissue transglutaminase during the differentiation and apoptosis of human myeloid leukemia (HL-60) cells

Retinoids induce terminal differentiation and subsequent apoptosis in the human myeloid leukemia (HL-60) cell line. We have previously shown that in HL-60 cells, ligand activation of retinoic acid receptors (RARs) is sufficient to induce differentiation but ligand activation of retinoid X receptors (RXRs) is necessary for the retinoid-induced apoptosis of these cells. In the present studies we have characterized the effect of retinoids on the expression of two apoptosis-linked gene products, BCL-2 and tissue transglutaminase. BCL-2 is a membrane-associated protein whose expression has been linked to the suppression of apoptosis in many cells. Tissue transglutaminase is a protein cross-linking enzyme that accumulates in many cells undergoing apoptotic cell death. Our data suggest that ligand activation of RARs in HL-60 cells results in a global suppression of BCL-2 expression whereas ligand activation of both RARs and RXRs triggers the selective accumulation of tissue transglutaminase in the apoptotic HL-60 cells.

Induction of Apoptosis by Acyclic Retinoid in the Human Hepatoma-Derived Cell Line, HuH-7

HuH-7 cells, a human hepatoma-derived cell line, underwent apoptosis in response to all- trans 3, 7, 11, 15-tetramethyl-2, 4, 6, 10, 14-hexadecapentaenoic acid, or acyclic retinoid. The retinoid-induced apoptosis was verified by a characteristic step-wise fragmentation of genomic DNA and chromatin condensation. The induction of apoptosis was detected as early as 8 hrs after the addition of the retinoid and was concentration dependent(0.1∼10 μM). Neither the natural retinoid all- trans retinoic acid nor 9- cis retinoic acid induced apoptosis. These data strongly indicate that the antitumor activity of the acyclic retinoid may be partly explained by the induction of apoptosis in tumor cells.