Arg278, but not Lys229 or Lys236, plays an important role in the binding of retinoic acid by retinoic acid receptor gamma

The diverse biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, beta and gamma) and retinoid X receptors (RXR alpha, beta, and gamma). Although the ligand-binding domains of RARs share the same novel folding pattern, many RAR subtype-specific retinoids have been synthesized indicating that the ligand-binding pocket of each RAR subtype has unique features. Previously we have demonstrated the importance for RA binding and RA-dependent transactivation of Arg276 of RARalpha alone and in RARbeta Arg269 in conjunction with Lys220. In this study, we have examined the role of the homologous amino acid residues (Lys229 and Arg278) in RARgamma for these activities. Like RARalpha but dissimilar to RARbeta, Arg278 in RARgamma alone was found to play an important role in RA binding and RA-dependent transactivation. Since Lys236 in RARgamma was suggested from the crystal structure of holo-RARgamma to interact with RA, we also examined its role and that of its homologs in RARalpha and RARbeta. Despite the suggestion from the crystal structure, neither Lys236 nor its homologs in RARalpha and RARbeta play a role in the binding of RA or RA-dependent transactivation. It is likely that Lys236 in RARgamma and its homologs in RARalpha and RARbeta are solvent exposed rather than pointing into the RA-binding pocket.     

Altered retinoic acid receptors

Structurally and functionally altered retinoic acid receptors have been associated with rare human neoplasms: acute promyelocytic leukemia and hepatocellular carcinoma. Whereas the retinoic acid receptor beta (RARbeta) rearrangement in hepatocellular carcinoma is unique, in acute promyelocytic leukemia (APL), RARalpha fusion to the promyelocytic leukemia (PML) gene by the t(15;17) translocation is a general feature of the disease. APL is an important model in cancer biology because retinoic acid induces complete remissions in this malignancy, providing the first example of differentiation therapy and of an antineoplastic drug directly targeted at the underlying genetic lesion. The molecular basis of PML/RARalpha fusion leukemogenesis is discussed with respect to dominant negative inhibition of nuclear receptor and PML functions.     

Greater synergism of retinoic acid receptor (RAR) agonists with vitamin D3 than that of retinoid X receptor (RXR) agonists with regard to growth inhibition and differentiation induction in monoblastic leukemia cells

Retinoids and 1alpha,25-dihydroxyvitamin D3 (VD3) cooperatively induce the differentiation of myeloid leukemia cells. We investigated the role of retinoid receptors (RARs and RXRs) in the combined effects of retinoids and VD3 on growth inhibition and differentiation induction in human monoblastic leukemia U937 cells by using RAR- or RXR-selective retinoids. An isobologram analysis showed that both combinations were synergistic with regard to inhibiting the proliferation, and RAR agonists exhibited greater synergism with VD3 than did RXR agonists. RXR agonists alone induced nitroblue tetrazolium (NBT) reduction and expression of CD11b in U937 cells, whereas RAR agonists alone did not. On the other hand, RAR agonists and RXR agonists enhanced the differentiation induced by VD3, but RXR agonists required higher concentrations. An RAR antagonist inhibited the differentiation induced by RAR agonists plus VD3, but not that induced by RXR agonists plus VD3. Thus, RARs and RXRs act differently in their synergism with VD3. RAR agonists are more potent than RXR agonists with regard to synergism with VD3, and their combination may be useful in differentiation therapy against myeloid leukemia.     

Differential effects of synthetic nuclear retinoid receptor-selective retinoids on the growth of human non-small cell lung carcinoma cells

Retinoids are promising agents for cancer chemoprevention and therapy. Nuclear retinoic acid receptors (RARs; RARalpha, -beta, and -gamma) and retinoid X receptors (RXRs; RXRalpha, -beta, and -gamma) are thought to mediate most of retinoids' effects on cell growth and differentiation. Because the majority of human non-small cell lung carcinoma (NSCLC) cell lines are resistant to all-trans-retinoic acid, we searched for more potent retinoids. Therefore, we examined the effects of 37 natural and synthetic retinoids that exhibit specific binding to and transactivation of individual RARs or RXRs on the proliferation of eight human NSCLC cell lines. All of these cells expressed mRNAs of the three RXRs; however, they expressed varying levels of RARalpha and RARgamma, and only three of the eight cell lines expressed RARbeta mRNA. Cellular retinoic acid-binding proteins (CRABPs) I and II were detected in one and three of the eight cell lines, respectively. Only 8 of the 37 retinoids exhibited growth-inhibitory activity (IC50, < 10 microM) against at least two of the eight NSCLC cell lines. The active retinoids included one (TD550) of five RARalpha-selective, one (Ch55) of three RARbeta-selective, three (CD437, CD2325, and SR11364) of six RARgamma-selective, and one (CD271) of four RARbeta/gamma-selective retinoids. The potency of these retinoids was low (IC50, > 1 microM), except for CD437, which was very potent (IC50, 0.1-0.5 microM). The six RXR-selective retinoids were mostly inactive even at 10 microM. However, combinations of RAR-selective and RXR-selective retinoids exhibited additive effects. There appeared to be no simple correlation among the histological type of the NSCLC (adeno- or squamous), the levels of nuclear receptors or CRABPs, and the response of the cells to the growth-inhibitory effects of retinoids. Nevertheless, in contrast with former studies with natural retinoids, these results suggest that several synthetic retinoids do exhibit inhibitory activity against NSCLC cells, and some of them may be useful clinically.     

The PML/RARalpha fusion protein inhibits tumor necrosis factor-alpha-induced apoptosis in U937 cells and acute promyelocytic leukemia blasts

We investigated the effect of the acute promyelocytic leukemia (APL) specific PML/RARalpha fusion protein on the sensitivity to TNF-alpha-mediated apoptosis. The U937 leukemia cell line was transduced with PML/RARalpha cDNA. PML/RARalpha expression caused a markedly reduced sensitivity to TNF-alpha, even if apoptosis was triggered by agonistic antibodies to TNF-alpha receptors I and II (TNF-alphaRI, II). PML/RARalpha induced a 10-20-fold decrease of the TNF-alpha-binding capacity via downmodulation of both TNF-alphaRI and TNF-alphaRII: this may mediate at least in part the reduced sensitivity to TNF-alpha. Furthermore, the fusion protein did not modify Fas expression (CD95) or sensitivity to Fas-mediated apoptosis. The pathophysiological significance of these findings is supported by two series of observations. (a) Fresh APL blasts exhibit no TNF-alpha binding and are resistant to TNF-alpha-mediated apoptosis. Conversely, normal myeloblasts-promyelocytes show marked TNF-alphaR expression and are moderately sensitive to TNF-alpha-mediated cytotoxicity. Similarly, blasts from other types of acute myeloid leukemia (AML M1, M2, and M4 FAB types) show an elevated TNF-alpha binding. (b) The NB4 APL cell line, which is PML/RARalpha+, shows low TNF-alphaR expression capacity and is resistant to TNF-alpha-triggered apoptosis; conversely a PML/RARalpha- NB4 subclone (NB4.306) exhibits detectable TNF-alpha-binding capacity and is sensitive to TNF-alpha-mediated cytotoxicity. These studies indicate that the PML/RARalpha fusion protein protects against TNF-alpha-induced apoptosis, at least in part via downmodulation of TNF-alphaRI/II: this phenomenon may play a significant role in APL, which is characterized by prolonged survival of leukemic blasts.     

Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARalpha fusion protein

All-trans-retinoic acid (RA) treatment induces morphological remission in acute promyelocytic leukemia (APL) patients carrying the t(15;17) and expressing the PML/RARalpha product by inducing terminal differentiation of the leukemic clone. RA treatment induces downregulation of PML/RARalpha and reorganization of the PML-nuclear bodies. These events have been proposed to be essential for the induction of APL cell differentiation by RA. Here, we show that in the APL-derived NB4 cell line as well as in myeloid precursor U937 cells expressing the PML/RARalpha (U937/PR9) and in blasts from APL patients, the PML/RARalpha fusion protein is cleaved by a caspase 3-like activity induced by RA treatment. In fact, a caspase 3-like activity is detectable in PML/RARalpha expressing cells after RA treatment, and selective caspase inhibitor peptides are able to prevent the RA-induced degradation of the fusion protein in vivo and in vitro. Using recombinant caspases and PML/RARalpha deletion mutants we mapped a caspase 3 cleavage site (Asp 522) within the alpha-helix region of the PML component of the fusion protein. The extent of PML/RARalpha cleavage directly correlates with the ability of RA to restore the normal PML nuclear bodies (NBs) pattern. However, RA-induced differentiation is not prevented by the persistence of the fusion product and occurs in the absence of normally structured PML NBs. These results indicate that PML/RARalpha is directly involved in conferring RA sensitivity of APL cells and that the RA-induced reassembly of PML NBs is the consequence of the disappearance of PML/RARalpha.     

Combined arsenic and retinoic acid treatment enhances differentiation and apoptosis in arsenic-resistant NB4 cells

In the acute promyelocytic leukemia (APL) cell line NB4, as well as in APL patients' cells, arsenic trioxide (As2O3) leads to incomplete cell maturation, induction of apoptosis, as well as to the degradation of the oncogenic PML/RARalpha fusion protein. We have isolated an arsenic-resistant NB4 subline (NB4-AsR), which fails to undergo apoptosis, but maintains the partial differentiation response to this drug. When grown in the presence of As2O3, NB4-AsR cells degrade PML/RARalpha, slightly differentiate, and become more sensitive to serum deprivation-induced apoptosis. Similarly, in RA-resistant NB4-R1 cells, RA induced a significant PML/RARalpha degradation and yet failed to induce cell maturation. Thus, As2O3- or retinoic acid (RA)-induced PML/RARalpha degradation may be a prerequisite, but is not sufficient for the full differentiative/apoptotic response to these drugs. Strikingly, RA-triggered differentiation and apoptosis were greatly accelerated in As2O3-treated NB4-AsR cells. The synergism between these two agents in this setting could provide an experimental basis for combined or sequential RA/As2O3 therapies.     

Effects of prostaglandin E2 and all-trans retinoic acid combination on induced differentiation of human acute promyelocytic leukemia NB4 cells

OBJECTIVE: A human promyelocytic leukemia (APL) cell line NB4 was used to demonstrate the synergistic effects between all-trans retinoic acid (ATRA) and prostaglandin E2 (PGE2) on growth inhibition and cytodifferentiation induction. METHODS: NB4 cells were cultured in the presence of either ATRA or PGE2 as a single agent or in combinations at various ratio. Cell growth was measured and myeloid differentiation was tested on consecutive days over the whole course of culture. RESULTS: PGE2 and ATRA synergistically induced the myeloid differentiation of NB4 cells. In comparison with ATRA alone, the combination of PGE2 with ATRA caused an almost 20-fold decrease of effective concentration of ATRA. CONCLUSION: The combination of ATRA and PGE2 at an appropriate ratio may provide a convenient oral regimen of combined differentiation therapy for a better clinical outcome in APL patients.     

All-trans-retinoic acid counteracts endothelial cell procoagulant activity induced by a human promyelocytic leukemia-derived cell line (NB4)

Therapy with all-trans-retinoic acid (ATRA) can rapidly improve the coagulopathy of acute promyelocytic leukemia (APL). This study was designed to evaluate whether the APL cell line NB4 induces the procoagulant activity (PCA) of human endothelial cells (ECs) in vitro, and whether this property is modified after ATRA-induced NB4 maturation. EC monolayers were incubated for 4 hours at 37 degrees C with the conditioned media (CM) of NB4 treated with 1 mumol/L ATRA (ATRA-NB4-CM) or the vehicle (control-NB4-CM). EC lysates were tested for PCA. ATRA-NB4-CM induced significantly more PCA:tissue factor (TF) than control-NB4-CM (P < .01). To identify the cause of TF induction, interleukin (IL)-1 beta antigen levels were measured in CM samples. ATRA-NB4-CM contained significantly more IL-1 beta than control-NB4-CM. EC PCA was significantly inhibited by an anti-IL-1 beta antibody. The addition to the media of 10 mumol/L ATRA counteracted the EC TF expression induced by NB4-CM. These data indicate that ATRA increases the promyelocyte-induced EC TF, partly through increased IL-1 beta production. However, ATRA can protect the endothelium from the procoagulant stimulus of leukemic cells.     

Effect of acute acipimox administration on the rates of lipid and glycogen synthesis in cachectic tumor-bearing rats

2'-Deoxycoformycin (dCF), a specific and potent inhibitor of adenosine deaminase, has demonstrated significant antitumor effect on lymphoid malignancies. The drug induced functional and morphologic differentiation of myeloid leukemia cells in combination with 2'-deoxyadenosine (dAd), but not dCF alone. NB4, a cell line derived from a patient with t(15; 17) acute promyelocytic leukemia (APL) underwent granulocytic differentiation when treated with all-trans retinoic acid (ATRA) or dCF plus dAd, but not with cytosine arabinoside. Pre-exposure of NB4 cells to ATRA greatly potentiated differentiation induced by dCF plus dAd, but pretreatment with dCF plus dAd before exposure to ATRA was less effective. Differentiation of NB4 cells was effectively induced by clinically applicable concentrations of dCF in combination with dAd. These findings may provide useful information about induction of differentiation in vivo.     

Cross-talk between retinoic acid and interferons: molecular mechanisms of interaction in acute promyelocytic leukemia cells

All-trans retinoic acid (ATRA) and interferons (IFNs) are active anticancer agents. ATRA is capable of inducing complete remission in acute promyelocytic leukemia (APL) patients, whereas IFNalpha is successfully used in the treatment of the stable phase of chronic myeloid leukemia. ATRA and IFNs have shown synergistic interactions in various experimental conditions and represent a potentially useful therapeutic combination in the treatment of various types of leukemias and solid tumors. The molecular basis of these interactions are poorly understood and need to be elucidated. In this review, we summarize a series of recent observations concerning the molecular mechanisms underlying the cross-talk between the intracellular pathways activated by ATRA and IFNs in APL cells. In APL blasts, IFNs regulate the expression of retinoic acid receptors, and ATRA, in turn, modulates the levels and the state of activation of members of the Jak-STAT second messenger pathway. This demonstrates a two-way interaction between ATRA and IFNs, which leads to cross-modulation of genes normally under the control of the retinoid and the cytokine. These data may be relevant in the context of a rational use of the combination between ATRA and IFNs in the clinical management of myeloid leukemias.     

Differentiating therapy in acute myeloid leukemia

Differentiating therapy is a new antineoplastic strategy which has received increasing attention due to the remarkable activity of the vitamin A derivative, all-trans retinoic acid (ATRA) in patients with acute promyelocytic leukemia (APL). Although it has been known for years that a variety of agents, including retinoids, could induce leukemic cells to differentiate in vitro, it was not until the initial report from Shanghai in 1988 that laboratory studies translated into clinical activity and benefit in patients. Since this initial report, a number of studies have confirmed that the majority of patients with both newly diagnosed and previously chemotherapy-treated patients with APL achieve complete remission (CR) with ATRA. In addition, the characteristic life-threatening coagulopathy resolves quickly. Several limitations to this approach have emerged, including the development of retinoid resistance, hyperleukocytosis and the retinoic acid syndrome, a constellation of findings including unexplained fever, fluid retention, pleuropericardial effusions and pulmonary infiltrates. Although ATRA is very effective in inducing CR, its benefits compared to conventional chemotherapy are only now being addressed. The first prospective randomized trial comparing ATRA plus chemotherapy to chemotherapy alone was terminated early because of an improved event-free survival for patients receiving ATRA. The benefit was attributable to a difference in relapse rate. A large, intergroup, prospective, randomized trial comparing conventional chemotherapy to ATRA for induction and ATRA to observation for maintenance has recently completed accrual and will provide insight into the emerging role of ATRA in patients with APL. ATRA represents the first example of a specific form of antileukemic therapy targeting a specific genetic abnormality and may serve as a paradigm for the development of differentiating therapy for patients with other hematologic malignancies.