Retinoic Acid-induced Apoptosis Research Articles

TRAF4 Silencing Induces Cell Apoptosis and Improves Retinoic Acid Sensitivity in Human Neuroblastoma.

Neuroblastoma (NB) is a pediatric malignancy that arises in the peripheral nervous system, and the prognosis in the high-risk group remains dismal, despite the breakthroughs in multidisciplinary treatments. The oral treatment with 13-cis-retinoic acid (RA) after high-dose chemotherapy and stem cell transplant has been proven to reduce the incidence of tumor relapse in children with high-risk neuroblastoma. However, many patients still have tumors relapsed following retinoid therapy, highlighting the need for the identification of resistant factors and the development of more effective treatments. Herein, we sought to investigate the potential oncogenic roles of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family in neuroblastoma and explore the correlation between TRAFs and retinoic acid sensitivity. We discovered that all TRAFs were efficiently expressed in neuroblastoma, but TRAF4, in particular, was found to be strongly expressed. The high expression of TRAF4 was associated with a poor prognosis in human neuroblastoma. The inhibition of TRAF4, rather than other TRAFs, improved retinoic acid sensitivity in two human neuroblastoma cell lines, SH-SY5Y and SK-N-AS cells. Further in vitro studies indicated that TRAF4 suppression induced retinoic acid-induced cell apoptosis in neuroblastoma cells, probably by upregulating the expression of Caspase 9 and AP1 while downregulating Bcl-2, Survivin, and IRF-1. Notably, the improved anti-tumor effects from the combination of TRAF4 knockdown and retinoic acid were confirmed in vivo using the SK-N-AS human neuroblastoma xenograft model. In conclusion, the highly expressed TRAF4 might be implicated in developing resistance to retinoic acid treatment in neuroblastoma, and the combination therapy with retinoic acid and TRAF4 inhibition may offer significant therapeutic advantages in the treatment of relapsed neuroblastoma.

Hepatitis B virus X protein suppresses all-trans retinoic acid-induced apoptosis in human hepatocytes by repressing p14 expression via DNA methylation.

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to activate p14 expression via promoter hypermethylation to induce p53-dependent apoptosis in human hepatocytes. In this study, we found that the oncogenic hepatitis B virus (HBV) X protein (HBx) of HBV, derived from both overexpression and 1.2-mer replicon systems, suppresses ATRA-induced apoptosis in p53-positive human hepatocytes. For this effect, HBx upregulated both protein and enzyme activity levels of DNA methyltransferase 1, 3a and 3b, in the presence of ATRA and thereby inhibited p14 expression via promoter hypermethylation, resulting in inactivation of the p14-mouse double minute 2 pathway and subsequent downregulation of p53 levels. As a result, HBx was able to impair the potential of ATRA to activate apoptosis-related molecules, including Bax, p53-upregulated modulator of apoptosis, caspase-9, caspase-3 and poly (ADP-ribose) polymerase. In conclusion, the present study provides a new oncogenic action mechanism of HBx, namely by suppressing the anticancer potential of ATRA to induce p53-dependent apoptosis in HBV-infected hepatocytes.

Hepatitis C virus Core overcomes all-trans retinoic acid-induced apoptosis in human hepatoma cells by inhibiting p14 expression via DNA methylation.

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to induce p14 expression via promoter hypomethylation to activate the p14-MDM2-p53 pathway, which leads to activation of the p53-dependent apoptotic pathway and subsequent induction of apoptosis in human hepatoma cells. In the present study, we found that hepatitis C virus (HCV) Core derived from ectopic expression or HCV infection overcomes ATRA-induced apoptosis in p53-positive hepatoma cells. For this effect, HCV Core upregulated both protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b and thereby repressed p14 expression via promoter hypermethylation, resulting in inactivation of the pathway leading to p53 accumulation in the presence of ATRA. As a result, HCV Core prevented ATRA from activating several apoptosis-related molecules, including Bax, p53 upregulated modulator of apoptosis, caspase-9, caspase-3, and poly (ADP-ribose) polymerase. In addition, complementation of p14 in the Core-expressing cells by either ectopic expression or treatment with 5-Aza-2′dC almost completely abolished the potential of HCV Core to suppress ATRA-induced apoptosis. Based on these observations, we conclude that HCV Core executes its oncogenic potential by suppressing the p53-dependent apoptosis induced by ATRA in human hepatoma cells.

Cold-inducible protein RBM3 protects neuroblastoma cells from retinoic acid-induced apoptosis via AMPK, p38 and JNK signaling

As a result of vitamin A abuse, excessive retinoic acid can induce apoptosis in neural cells. Mild-hypothermia and cold-inducible protein RBM3 protect neural cells from apoptosis. However, whether hypothermia/RBM3 protect neural cells from retinoic acid-induced apoptosis remains unclear. The SH-SY5Y neuroblastoma cell line, a common neural cell model of apoptosis, was employed here. Interestingly, retinoic acid overdose induced apoptosis in SH-SY5Y cells, which was prevented by mild-hypothermia. When RBM3 was silenced, hypothermia-related protection was completely abolished. RBM3 overexpression significantly prevented retinoic acid-induced apoptosis. Screening of signaling pathways revealed that RBM3 overexpression markedly inhibited retinoic acid-induced upregulation of p38 and JNK signaling, and antagonized downregulation of AMPK signaling. Subsequent assays demonstrated that p38 and JNK signaling were pro-apoptotic, while AMPK was anti-apoptotic. Overall, these data showed that mild-hypothermia protects neural cells from retinoic acid-induced apoptosis via induction of RBM3, which subsequently mediates the neuroprotective effects via AMPK, p38 and JNK signaling.

α-Mangostin, a Natural Agent, Enhances the Response of NRAS Mutant Melanoma to Retinoic Acid.

BackgroundThe identification and use of novel compounds alone or in combination hold promise for the fight against NRAS mutant melanoma.Material/MethodsWe screened a kinase-specific inhibitor library through combining it with α-Mangostin in NRAS mutant melanoma cell line, and verified the enhancing effect of α-Mangostin through inhibition of the tumorigenesis pathway.ResultsWithin the kinase inhibitors, retinoic acid showed a significant synergistic effect with α-Mangostin. α-Mangostin also can reverse the drug resistance of retinoic acid in RARa siRNA-transduced sk-mel-2 cells. Colony assay, TUNEL staining, and the expressions of several apoptosis-related genes revealed that α-Mangostin enhanced the effect of retinoic acid-induced apoptosis. The combination treatment resulted in marked induction of ROS generation and inhibition of the AKT/S6 pathway.ConclusionsThese results indicate that the combination of these novel natural agents with retinoid acid may be clinically effective in NRAS mutant melanoma.

Apoptosis related protein 3 is a lysosomal membrane protein

Apoptosis Related Protein 3 (APR3) is an important protein which is involved in retinoic acid-induced apoptosis, osteoblast differentiation and cervical squamous cell carcinoma progression. Although it was predicted to be a trans-membrane protein, its cellular localization is not clear. In this study, we analyzed APR3 with bioinformatic tools and found that APR3 contains a potential signal peptide, a transmembrane region and 3 N-glycosylation sites, all of which are characteristics of lysosomal proteins. Western blot with isolated lysosomes demonstrated that APR3 was mainly present in lysosomes, specially in the lysosomal membrane fraction, but not in endoplasmic reticulum. Concomitantly, double immunofluorescence confirmed that APR3 co-localized with lysosomal membrane protein, LAMP1, as well as lysosomal specific marker, Lyso-Tracker Red. Moreover, we showed that APR3 was highly expressed in the lung, liver, spleen, kidney and adipose tissue, but expressed at the low level in the heart, pancreas, stomach and intestine. Interestingly, APR3 expression was elevated in multiple hepatocellular carcinoma cell lines comparing to normal liver cells. Collectively, our results proved that APR3 is a novel lysosomal membrane protein and shed light on its possible functions.

MicroRNA-302b-inhibited E2F3 transcription factor is related to all trans retinoic acid-induced glioma cell apoptosis.

All-trans retinoic acid (ATRA), a derivative of retinoid, is involved in the onset of differentiation and apoptosis in a wide variety of normal and cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression. Several miRNAs were identified to participate in ATRA-mediated cell differentiation. However, no studies have demonstrated whether miRNA can enhance ATRA cytotoxicity, thereby resulting in cell apoptosis. This study investigated the effects of ATRA-mediated miRNA expression in activating apoptotic pathways in glioblastoma. First, we found that high-dose ATRA treatment significantly reduced cell viability, caspase-dependent apoptosis, endoplasmic reticular (ER) stress activation, and intracellular reactive oxygen species accumulation. From microarray data, miR-302b was analyzed as a putative downstream regulator upon ATRA treatment. Furthermore, we found that ATRA up-regulated miR-302b expression in a dose- and time-dependent manner through retinoic acid receptor α-mediated pathway. Overexpression and knockdown of miR-302b significantly influenced ATRA-mediated cytotoxicity. E2F3, an important transcriptional regulator of glioma proliferation, was validated to be a direct target gene of miR-302b. The miR-302b-reduced E2F3 levels were also identified to be associated with ATRA-mediated glioma cell death. These results emphasize that an ATRA-mediated miR-302b network may provide novel therapeutic strategies for glioblastoma therapy. We propose that high-dose all-trans retinoic acid (ATRA) treatment, a derivative of retinoid, significantly induces glioblastoma cell apoptosis via caspase-dependent apoptosis, endoplasmic reticular (ER) stress, and intracellular reactive oxygen species (ROS) accumulation. The miR-302b overexpression enhanced by ATRA-mediated retinoic acid receptor (RAR)α pathway was also identified. The E2F3 repression, a novel target gene of miR-302b, was involved in ATRA-induced glioblastoma cell cytotoxicity.

NGAL Can Alternately Mediate Sunitinib Resistance in Renal Cell Carcinoma

Serum NGAL is highly expressed in patients with advanced renal cancer treated with sunitinib. We investigated the role of NGAL in sunitinib resistance in renal cell carcinoma to identify potential tactics to overcome it. NGAL expression was correlated with sunitinib sensitivity. Vascular endothelial growth factor related upstream Ras, Erk1/2 and STAT1 phosphorylation activity in Caki-1 and NGAL transfected Caki-1 cells after sunitinib treatment was analyzed using Western blot. NGAL and vascular endothelial growth factor-A interaction with sunitinib therapeutic efficacy was monitored in renal cell carcinoma tumor xenografted mice by tumor growth inhibition, serum NGAL and vascular endothelial growth factor-a levels, and microscopic examination of tumor microvascular density. Sunitinib cytotoxicity in various renal cell carcinoma cell lines was reversibly related to NGAL expression. Sunitinib showed the lowest 50% inhibitory concentration (5.53 μM) in Caki-1 cells, which had the lowest NGAL expression of these renal cell carcinoma cell lines. After sunitinib treatment adding NGAL inhibited Ras and Erk1/2 phosphorylation but activated STAT1α phosphorylation in Caki-1 cells and Caki-1 cells transfected with NGAL. In a xenograft mouse model sunitinib significantly inhibited tumor growth in Caki-1 mice. NGAL transfected Caki-1 mice had higher serum NGAL and lower vascular endothelial growth factor-A than Caki-1 mice. Microvascular density was decreased in Caki-1 mice with sunitinib treatment. NGAL in tumor cells may show crosstalk with vascular endothelial growth factor-a and alternative activation in stimulating tumor growth during sunitinib treatment. It may become a therapeutic target to reverse sunitinib resistance in renal cell carcinoma.

Reactive oxygen species-regulated glycogen synthase kinase-3β activation contributes to all-trans retinoic acid-induced apoptosis in granulocyte-differentiated HL60 cells

All-trans retionic acid (ATRA) treatment confers disease remission in acute promyelocytic leukemia (APL) patients by inducing granulocytic differentiation, which is followed by cell apoptosis. Although glycogen synthase kinase (GSK)-3β is known to be required for spontaneous cell death in neutrophils, the requirement of GSK-3β activation for the apoptotic effects remains unknown. This question is addressed in the present study using a model of ATRA-induced granulocytic differentiation and apoptosis in APL HL60 cells. ATRA at a therapeutic concentration (1μM) induced granulocytic differentiation, followed by apoptosis. ATRA treatment caused decreased Mcl-1, caspase-3 activation, and PARP cleavage following the inactivation of phosphatidylinositol 3-kinase/AKT and the activation of GSK-3β. Pharmacologically and genetically inhibiting GSK-3β effectively retarded ATRA-induced Mcl-1 degradation and apoptosis. Additional differentiation inducers, phorbol 12-myristate 13-acetate and dimethyl sulfoxide, also triggered GSK-3β-dependent apoptosis. Mechanistically, ATRA caused the generation of reactive oxygen species (ROS) through increased expression of NADPH oxidase subunits (p47phox and p67phox) to facilitate ATRA-induced GSK-3β activation and cell apoptosis. This study indicates that ROS initiate GSK-3β-dependent apoptosis in granulocyte-differentiated cells after long-term ATRA treatment.

Characterization of retinoic acid–induced neurobehavioral effects in developing zebrafish

Retinoic signaling plays an important role in cell proliferation and differentiation. Disruption of retinoic signaling via excessive or deficient retinoic acid can cause teratogenic effects on developing embryos. Similar to retinoic acid, many xenobiotic environmental pollutants have been found to disrupt retinoic signaling through binding and eliciting agonistic activity on retinoic acid receptors. Currently, studies of retinoic acid or retinoic acid-like compounds in aquatic organisms have mainly focused on teratogenicity and few studies have explored their neurobehavioral toxicity. In the present study, the authors used retinoic acid as an example to explore the neurobehavioral toxicity associated with developmental exposure of retinoic acid-like compounds in zebrafish. The findings confirmed retinoic acid's teratogenic effects such as bent spine, malformed tail, and pericardial edema in developing zebrafish with a median effective concentration of 2.47 nM. Retinoic acid-induced cell apoptosis at 24 h postfertilization was consistently found in the eye and tail regions of embryos. Spontaneous movement as characterized by tail bend frequency was significantly increased in zebrafish embryos following exposure to 2 nM and 8 nM retinoic acid. Relatively low-dose retinoic acid exposure of 2 nM led to fast locomotion behavior in the dark period and hyperactivity during light-dark photoperiod stimulation. The 2-nM retinoic acid exposure also led to alterations of neurobehavior- and optic nerve-related genes, with the transforming growth factor-β signal transduction inhibitor noggin (nog) and the spinal cord marker homeobox c3a (hox) being underexpressed and the retinal G protein-coupled receptor a (rgr), the photoreceptor cell marker rhodopsin (rho), and the short wave-sensitive cone pigment opsin 1 (opn1sw1) being overexpressed. Increased expression of opn1sw1 and rho was confirmed by whole-mount in situ hybridization. Whether the misexpression of these genes leads to the neurobehavioral changes merits further study. The findings demonstrated that low-dose retinoic acid exposure perturbed the visual system and optic nerve development and caused hyperactivity in developing zebrafish.

Growth and Differentiation Factor 3 Induces Expression of Genes Related to Differentiation in a Model of Cancer Stem Cells and Protects Them from Retinoic Acid-Induced Apoptosis

Misexpression of growth factors, particularly those related to stem cell-like phenotype, is often observed in several cancer types. It has been found to influence parameters of disease progression like cell proliferation, differentiation, maintenance of undifferentiated phenotype and modulation of the immune system. GDF3 is a TGFB family member associated with pluripotency and differentiation during embryonic development that has been previously reported to be re-expressed in a number of cancer types. However, its role in tumor development and progression has not been clarified yet. In this study we decipher the role of GDF3 in an in vitro model of cancer stem cells, NCCIT cells. By classical approach to study protein function combined with high-throughput technique for transcriptome analysis and differentiation assays we evaluated GDF3 as a potential therapeutic target. We observed that GDF3 robustly induces a panel of genes related to differentiation, including several potent tumor suppressors, without impacting the proliferative capacity. Moreover, we report for the first time the protective effect of GDF3 against retinoic acid-induced apoptosis in cells with stem cell-like properties. Our study implies that blocking of GDF3 combined with retinoic acid-treatment of solid cancers is a compelling direction for further investigations, which can lead to re-design of cancer differentiation therapies.

Epstein–Barr Virus latent membrane protein 1 overcomes all-trans retinoic acid-induced apoptosis by inhibiting retinoic acid receptor-β2 expression

Nasopharyngeal carcinoma is closely associated with infection with Epstein–Barr Virus (EBV); however, the mechanism is still unclear. Here, we report that the EBV oncoprotein, latent membrane protein 1 (LMP1), suppresses apoptotic cell death provoked by all-trans retinoic acid (ATRA) in NPC cells. For this purpose, LMP1 downregulated levels of Bak whilst it upregulated levels of Bcl2, lowering the ratio of Bak to Bcl2. In addition, LMP1 suppressed ATRA-mediated activation of Caspase 9, Caspase 3, and PARP but not Caspase 8 in Ad-AH cells, suggesting that LMP1 acts by blocking the activation of intrinsic apoptosis pathway by ATRA. These effects were almost completely abolished when levels of retinoic acid receptor-β2 (RAR-β2) in the LMP1-expressing cells were recovered by either exogenous gene expression or treatment with a universal DNMT inhibitor, 5-Aza-2′dC, indicating that LMP1 executes its antiapoptotic effects by downregulating levels of RAR-β2 via DNA methylation.

Inhibition of the redox function of APE1/Ref-1 in myeloid leukemia cell lines results in a hypersensitive response to retinoic acid-induced differentiation and apoptosis

The standard of care for promyelocytic leukemia includes use of the differentiating agent all-trans retinoic acid (RA) and chemotherapy. RA induces cell differentiation through retinoic acid receptor (RAR) transcription factors. Because redox mechanisms influence how readily transcription factors bind to DNA response elements (RARE), the impact of small molecule (E3330) inhibition of the redox regulatory protein, apurinic-apyrimidinic endonuclease/redox effector factor (APE1/Ref-1) on RAR DNA binding and function in RA-induced myeloid leukemia cell differentiation and apoptosis was investigated. The redox function of APE1 was studied using the small molecule inhibitor E3330 in HL-60 and PLB acute myeloid leukemia cells. Electrophoretic mobility shift assays were employed to determine effect of inhibitor on APE1/Ref-1 redox signaling function. Trypan blue assays, Annexin-V/propidium iodide and CD11b staining, and real-time polymerase chain reaction analyses were employed to determine survival, apoptosis, and differentiation status of cells in culture. RARα binds to its RARE in a redox-dependent manner mediated by APE1/Ref-1 redox regulation. Redox-dependent RAR-RARE binding is blocked by E3330, a small molecule redox inhibitor of APE1/Ref-1. Combination treatment of RA + E3330 results in a profound hypersensitivity of myeloid leukemia cells to RA-induced differentiation and apoptosis. Additionally, redox inhibition by E3330 results in enhanced RAR target gene, BLR-1, expression in myeloid leukemia cells. The redox function of APE1/Ref-1 regulates RAR binding to its DNA RAREs influencing the response of myeloid leukemia cells to RA-induced differentiation. Targeting of APE1/Ref-1 redox function may allow manipulation of the retinoid response with therapeutic implications.

The Role of Cdk5 in Retinoic Acid-Induced Apoptosis of Cervical Cancer Cell Line

Cdk5 is a small serine/threonine protein kinase which belongs to Cdk family. Unlike other Cdk members, so far Cdk5 is known to be irrelevant in cell cycle. Cdk5 kinase activity is regulated by binding with its activator, p35. Our previous results indicate that CdkS and p35 are involved in drugs-induced apoptosis of prostate cancer cells. Retinoic acid (RA) is one of the vitamin A-related compounds. Because of its potency on biological functions, it has been widely studied in its novel actions including the ability to inhibit cancer cell growth and to induce apoptosis. Here, we report that RA treatment decreased the growth of human cervical cancer cell line, HeLa, and Cdk5 contributed to this effect. The involvement of Cdk5 in RA-reduced cell survival was performed by treatments of Cdk5 inhibitor and siRNA. We further identified that RA-induced growth inhibition was partly correlated to Cdk5 activity-related apoptosis by detecting cell cycle distribution of sub G1 phase and the signals of Annexin V staining. In addition, our results also indicated that Cdk5 activity was involved in RA-induced HeLa apoptosis by detecting cleavages of caspase-3 and its substrate, PARP (poly (ADP-ribose) polymerases) Interestingly, the nuclear localizations of Cdk5 and p35 proteins were increased by RA treatment, which, again, suggests the involvement of Cdk5 and p35 in RA-induced apoptotic effects. In conclusion, we provide evidence to suggest that Cdk5 and p35 might play important roles in RA-induced HeLa apoptosis.

Reelin signals survival through Src‐family kinases that inactivate BAD activity

Reelin plays an important role in the migration of embryonic neurons, but its continuing presence suggests additional functions in the brain. We now report a novel function where reelin protects P19 embryonal cells from apoptosis during retinoic acid-induced neuronal differentiation. This increased survival is associated with reelin activation of the phosphatidyl-inositol-3-kinase (PI3 K)/Akt pathway. When PI3 K was inhibited with LY294002, reelin failed to protect against this retinoic acid-induced apoptosis. The protective effect of reelin includes activating the Src-family kinases/PI3 K/Akt pathway which then led to selective phosphorylation of Bcl-2/Bcl-XL associated death promoter (BAD) at serine-136, while the phosphorylation-incompetent mutation of BAD (S136A) suppressed this protection. These and additional studies define a novel pathway where reelin binds apoE receptors, significantly activates the PI3 K/Akt pathway causing phosphorylation of BAD which helps to protect cells from apoptosing, thus serving an important role in promoting the survival of maturing neurons in the brain.

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.

13-cis Retinoic Acid Induces Apoptosis and Cell Cycle Arrest in Human SEB-1 Sebocytes

Isotretinoin (13-cis retinoic acid (13-cis RA)) is the most potent inhibitor of sebum production, a key component in the pathophysiology of acne, yet its mechanism of action remains largely unknown. The effects of 13-cis RA, 9-cis retinoic acid (9-cis RA), and all-trans retinoic acid (ATRA) on cell proliferation, apoptosis, and cell cycle proteins were examined in SEB-1 sebocytes and keratinocytes. 13-cis RA causes significant dose-dependent and time-dependent decreases in viable SEB-1 sebocytes. A portion of this decrease can be attributed to cell cycle arrest as evidenced by decreased DNA synthesis, increased p21 protein expression, and decreased cyclin D1. Although not previously demonstrated in sebocytes, we report that 13-cis RA induces apoptosis in SEB-1 sebocytes as shown by increased Annexin V-FITC staining, increased TUNEL staining, and increased cleaved caspase 3 protein. Furthermore, the ability of 13-cis RA to induce apoptosis cannot be recapitulated by 9-cis RA or ATRA, and it is not inhibited by the presence of a retinoid acid receptor (RAR) pan-antagonist AGN 193109. Taken together these data indicate that 13-cis RA causes cell cycle arrest and induces apoptosis in SEB-1 sebocytes by a RAR-independent mechanism, which contributes to its sebosuppressive effect and the resolution of acne.

Increased expression of cyclin A1 protein is associated with all-trans retinoic acid-induced apoptosis

Deregulated cell growth and inhibition of apoptosis are hallmarks of cancer. All-trans retinoic acid induces clinical remission in patients with acute promyelocytic leukemia by inhibiting cell growth and inducing differentiation and apoptosis of the leukemic blasts. An important role of the cell cycle regulatory protein, cyclin A1, in the development of acute myeloid leukemia has previously been demonstrated in a transgenic mouse model. We have recently shown that there was a direct interaction between cyclin A1 and a major all-trans retinoic acid receptor, RARα, following all-trans retinoic acid treatment of leukemic cells. In the present study, we investigated whether cyclin A1 might be involved in all-trans retinoic acid-induced apoptosis in U-937 leukemic cells. We found that all-trans retinoic acid-induced apoptosis was associated with concomitant increase in cyclin A1 expression. However, there was no induction of cyclin A1 mRNA expression following the all-trans retinoic acid-induced apoptosis. Treatment of cells with a caspase inhibitor was not able to prevent all-trans retinoic acid-induced up-regulation of cyclin A1 expression. Interestingly, induced cyclin A1 expression in U-937 cells led to a significant increase in the proportion of apoptotic cells. Further, U-937 cells overexpressing cyclin A1 appeared to be more sensitive to all-trans retinoic acid-induced apoptosis indicating the ability of cyclin A1 to mediate all-trans retinoic acid-induced apoptosis. Induced cyclin E expression was not able to initiate cell death in U-937 cells. Our results indicate that cyclin A1 might have a role in apoptosis by mediating all-trans retinoic acid-induced apoptosis.

Transforming growth factor-β1 inhibits all -trans retinoic acid-induced apoptosis

The interaction between retinoids and transforming growth factor-β1 (TGF-β1) leading to regulation of proliferation, differentiation and apoptosis is not still fully understood. In this study, we demonstrated that a combination treatment with all- trans retinoic acid (ATRA) and TGF-β1 led to the enhancement of ATRA-induced suppression of cell proliferation, which is accompanied by inhibition of ATRA-induced apoptosis in human leukemia HL-60 cells. This effect was preceded by the arrest of cells in G0/G1 cell cycle phase linked with pRb protein dephosphorylation, continuous accumulation of p21 and transiently increased level of p27, inhibitors of cyclin-dependent kinases. Inhibition of ATRA-induced apoptosis by TGF-β1 was associated with an increased level of Mcl-1 protein, an anti-apoptotic member of Bcl-2 family, but not with inhibition of mitochondrial membrane depolarization. Levels of other Bcl-2 family proteins (Bcl-2, Bcl-X L, Bad, Bak, Bax) were unaffected by simultaneous ATRA and TGF-β1 treatment, when compared to ATRA alone. Upregulation of c-FLIP L protein, an inhibitor of apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), correspond with inhibition of ATRA-induced (autocrine TRAIL-mediated) caspase-8 activation and apoptosis. These results suggest that apoptosis inhibition associated with proliferation block could depend on modulation of the TRAIL apoptotic pathway and regulation of the Mcl-1 protein level. In summary, we demonstrate that the balance of processes leading to regulation of proliferation and differentiation of myeloid cells can modulate cell sensitivity to apoptosis-inducing stimuli.

Structural and functional preservation of specific sequences of DNA and mRNA in apoptotic bodies from ES cells

Retinoic acid-induced apoptosis of embryonic stem (ES) cells is an experimental system which resembles the physiological programmed cell death that occurs during differentiation in embryonic development. Our aim was to analyze the involvement of epigenetic modifications such as DNA methylation and chromatin structure in the apoptotic process and to investigate the metabolic activity of apoptotic bodies. We found a relationship between DNA methylation and apoptosis, shown by a dose-dependent induction of apoptosis after treatment with the inhibitor of DNA methylation 5-aza-2'-deoxycytidine. Interestingly, we found a slight demethylation of specific sequences of the U2afl-rs1 imprinted gene in those RA treated cells which were specifically undergoing apoptosis. In addition, apoptotic bodies exhibited an unexpected open chromatin conformation accessible to the endonuclease DNase-I. Furthermore, we observed a structural and functional preservation of specific DNA sequences and mRNA. These results suggest that biological activities, such as transcription or protein synthesis, could be maintained even towards the end of the apoptotic process.