Inhibition of intracellular Ca2+ signalling, cytotoxicity and antitumor activity of the herbicide oryzalin and its analogues

Purpose: Studies were conducted on oryzalin (3,5-dinitro-N,N-di(n-propyl)sulfanilamide), a widely used dinitroaniline sulfonamide herbicide, which was identified from plant extracts as an inhibitor of mitogen- and growth factor-mediated intracellular free Ca²⁺ ([Ca²⁺]_i) signalling in mammalian cells. Methods and Results: Oryzalin inhibited vasopressin, bradykinin and platelet-derived growth factor [Ca²⁺]_i signalling in Swiss 3T3 fibroblasts with IC₅₀ values of 14, 16 and 18 μM, respectively. ⁴⁵Ca²⁺ uptake into nonmitochondrial stores of saponin-permeabilized Swiss 3T3 cells was inhibited by oryzalin with an IC₅₀ of 34 μM. Oryzalin inhibited colony formation of HT-29 colon carcinoma cells with an IC₅₀ of 8 μM and inhibited the growth of a number of other cancer cell lines and primary human tumors in vitro with IC₅₀ values in the range 3 to 22 μM. A number of oryzalin analogues were studied and an association was found between the ability to inhibit [Ca²⁺]_i signalling and inhibition of the growth of HT-29 human colon cancer cells (P=0.001) and of CCRF-CEM human leukemia cells (P=0.016). Oryzalin at doses up to 600 mg/kg administered orally or subcutaneously daily to mice for 3 to 10 days beginning a day after tumor inoculation inhibited the growth of murine B16 melanoma by 63% but showed no appreciable activity when administered subcutaneously or intraperitoneally to mice beginning a number of days after tumor inoculation against a variety of human tumor xenografts. The peak plasma concentration of oryzalin following repeated subcutaneous administration of oryzalin at 600 mg/kg per day to mice was 37 μM and of its major metabolite N-depropyl oryzalin was 53 μM. Conclusion: It is unlikely that the absence of significant antitumor activity of oryzalin is a result of the inability to achieve adequate plasma concentrations. Received: 24 December 1996 / Accepted: 20 March 1997     

Release of Ca2+ from the endoplasmic reticulum and its subsequent influx into mitochondria trigger celastrol-induced paraptosis in cancer cells

Celastrol, a triterpene extracted from the Chinese “Thunder of God Vine”, is known to have anticancer activity, but its underlying mechanism is not completely understood. In this study, we show that celastrol kills several breast and colon cancer cell lines by induction of paraptosis, a cell death mode characterized by extensive vacuolization that arises via dilation of the endoplasmic reticulum (ER) and mitochondria. Celastrol treatment markedly increased mitochondrial Ca²⁺ levels and induced ER stress via proteasome inhibition in these cells. Both MCU (mitochondrial Ca²⁺ uniporter) knockdown and pretreatment with ruthenium red, an inhibitor of MCU, inhibited celastrol-induced mitochondrial Ca²⁺ uptake, dilation of mitochondria/ER, accumulation of poly-ubiquitinated proteins, and cell death in MDA-MB 435S cells. Inhibition of the IP₃ receptor (IP₃R) with 2-aminoethoxydiphenyl borate (2-APB) also effectively blocked celastrol-induced mitochondrial Ca²⁺ accumulation and subsequent paraptotic events. Collectively, our results show that the IP₃R-mediated release of Ca²⁺ from the ER and its subsequent MCU-mediated influx into mitochondria critically contribute to celastrol-induced paraptosis in cancer cells.     

Increased intracellular Ca2+ signaling caused by the antitumor agent helenalin and its analogues

The antitumor sesquiterpene lactone helenalin, which is found in species of the plant genusHelenium, cause a marked potentiation of the increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) produced by mitogens such as vasopressin, bradykinin, and platelet-derived growth factor in Swiss mouse 3T3 fibroblasts. Removing external Ca²⁺ partly attenuated the increased [Ca²⁺]_i response. caused by helenalin. The increased [Ca²⁺]_i responses occurred at concentrations of helenalin that inhibited cell proliferation. At higher concentrations, helenalin inhibited the [Ca²⁺]_i responses. No change in resting [Ca²⁺]_i was caused by helenalin even at high concentrations. Other helenalin analogues also increased the [Ca²⁺]_i response. Helenalin did not inhibit protein kinase C (PKC) and PKC appeared to play a minor role in the effects of helenalin on [Ca²⁺]_i responses in intact cells. Studies with saponin-permeabilized HT-29 human colon carcinosarcoma cells indicated that helenalin caused an increased accumulation of Ca²⁺ into nonmitochondrial stores and that the potentiating effect of helenalin on mitogen-stimulated [Ca²⁺]_i responses was due in part to an increase in the inositol-(1,4,5)-trisphosphate-mediated release of Ca²⁺ from these stores.     

Retinoic acid signaling cascade in differentiating murine epidermal keratinocytes: Alterations in papilloma- and carcinoma-derived cell lines

The retinoic acid (RA) signaling pathway was investigated by transient transfection of a chloramphenicol acetyltransferase (CAT) reporter gene construct containing the RA response element (RARE) of the murine (m) RARβ2 gene into murine primary epidermal keratinocytes (PEK), papilloma-derived SP1 cells, and carcinoma-derived 3P2 cells. Murine PEK transfected in a low-Ca²⁺ medium (0.05 mM Ca²⁺) exhibited a strong transactivation of the CAT gene after exposure of the cells to 0.1 μM RA. Transactivation of the CAT gene could, however, also be achieved by shifting RAREβ2-transfected low-Ca²⁺ PEK to high-Ca²⁺ conditions (0.15–1.2 mM Ca²⁺). Concomitantly, the Ca²⁺ raise also led to the induction of both cellular retinol (ROL)-binding protein I (CRBPI) and cellular RA-binding protein II (CRABPII), whereas expression of cellular RA-binding protein I (CRABPI) was not observed. Moreover, induction of in vitro differentiation also activated the ROL→RA converting enzyme system in PEK. These findings suggest the following sequence of events involved in the high Ca²⁺–mediated activation of RAREβ2. First, high Ca²⁺ induces the synthesis of mCRBPI, which binds ROL released from retinyl ester stores and makes it accessible to the ROL→RA converting enzyme system. Enzymatically generated RA is taken over by mCRABPII and transported to the nucleus, where it acts as ligand for nuclear receptors, which complex with RAREβ2 to activate the reporter gene. This hypothetical cascade of RA signaling was supported by our findings that inhibition of the ROL→RA converting enzyme system by citral abolished the Ca²⁺-mediated transactivation of the CAT gene in a nontoxic manner. Studies in transformed murine cell lines revealed that Ca²⁺-induced activation of RAREβ2 was essentially maintained in papilloma-derived SP1 cells, although all parameters of the Ca²⁺-dependent RAREβ2 activation cascade were induced to a much lower extent. In contrast, strong RAREβ2 activity was already observed in low-Ca²⁺ carcinoma-derived 3P2 cells. Low-Ca²⁺ 3P2 cells also expressed high levels of both mCRBPI and mCRABPII and possessed a highly active ROL→RA converting enzyme system. Again, inhibition of the enzyme by citral abolished RAREβ2 activity in low-Ca²⁺ 3P2 cells. Our data show that Ca²⁺-induced differentiation in cultured murine PEK entails a series of events that ultimately lead to the activation of RARE-containing genes. These properties are maintained in transformed epidermal keratinocytes. However, with increasing malignant potential of the cells, the respective signaling pathway becomes independent from a differentiation stimulus and leads to constitutive activation of RARE-controlled genes. Mol. Carcinog. 20:58–67, 1997. © 1997 Wiley-Liss, Inc.     

Involvement of Ca2+ and ROS in α‐tocopheryl succinate‐induced mitochondrial permeabilization

Release of mitochondrial proteins such as cytochrome c, AIF, Smac/Diablo etc., plays a crucial role in apoptosis induction. A redox‐silent analog of vitamin E, α‐tocopheryl succinate (α‐TOS), was shown to stimulate cytochrome c release via production of reactive oxygen species (ROS) and Bax‐mediated permeabilization of the outer mitochondrial membrane. Here we show that α‐TOS facilitates mitochondrial permeability transition (MPT) in isolated rat liver mitochondria, Tet21N neuroblastoma cells and Jurkat T‐lymphocytes. In particular, in addition to ROS production, α‐TOS stimulates rapid Ca²⁺ entry into the cells with subsequent accumulation of Ca²⁺ in mitochondria—a prerequisite step for MPT induction. Alteration of mitochondrial Ca²⁺ buffering capacity was observed as early as 8 hr after incubation with α‐TOS, when no activation of Bax was yet detected. Ca²⁺ accumulation in mitochondria was important for apoptosis progression, since inhibition of mitochondrial Ca²⁺ uptake significantly mitigated the apoptotic response. Importantly, Ca²⁺‐induced mitochondrial destabilization might cooperate with Bax‐mediated mitochondrial outer membrane permeabilization to induce cytochrome c release from mitochondria.     

Inhibition of store-operated Ca entry suppresses EGF-induced migration and eliminates extravasation from vasculature in nasopharyngeal carcinoma cell

Store-operated Ca²⁺ entry (SOCE) mediates Ca²⁺ responses evoked by extracellular signaling molecules to promote increases in cytosolic Ca²⁺, thereby triggering downstream signal transduction. Here we demonstrated that either the pharmacological blockage of Ca²⁺ influx through SOCE or the knockdown of Orai1, a key molecule of SOCE, suppressed the epidermal growth factor-induced migration by disturbing Ca²⁺ signaling in nasopharyngeal carcinoma (NPC) cell. Furthermore, Orai1 depletion led to a delayed cell attachment to the extracellular matrix surface in vitro and eliminated the extravasation of microinjected cells from vasculature in a zebrafish hematogenous metastasis model. Our findings thus indicate that SOCE acts as a predominant Ca²⁺ signaling involved in NPC cell metastasis, and may serve as a candidate target for anti-metastasis therapy in NPC.     

Calcium insensitivity of FA-6, a cell line derived from a pancreatic cancer associated with humoral hypercalcemia, is mediated by the significantly reduced expression of the Calcium Sensitive Receptor transduction component p38 MAPK

The Calcium-Sensing Receptor is a key component of Calcium/Parathyroid hormone homeostatic system that helps maintain appropriate plasma Ca²⁺ concentrations. It also has a number of non-homeostatic functions, including cell cycle regulation through the p38 MAPK pathway, and recent studies have indicated that it is required for Ca²⁺ mediated growth arrest in pancreatic carcinoma cells. Some pancreatic cancers produce pathogenic amounts of parathyroid like hormones, however, which significantly increase Ca²⁺ plasma concentrations and might be expected to block further cell growth. In this study we have investigated the expression and function of the p38 MAPK signaling pathway in Ca²⁺ sensitive (T3M-4) and insensitive (FA6) pancreatic cancer cell lines. FA-6 cells, which are derived from a pancreatic adenocarcinoma that secretes a parathyroid hormone related peptide, exhibit only very low levels of p38 MAPK expression, relative to T3M-4 cells. Transfecting FA-6 cells with a p38 MAPK expression construct greatly increases their sensitivity to Ca²⁺. Furthermore, the reduction of p38 MAPK in T3M-4 cells significantly reduces the extent to which high levels of Ca²⁺ inhibit proliferation. These results suggest that the low levels of p38 MAPK expression in FA-6 cells may serve to reduce their sensitivity to high concentrations of external Ca²⁺ that would otherwise block proliferation.     

Intracellular Ca2+ release mediates ursolic acid–induced apoptosis in human leukemic HL-60 cells

The effect of ursolic acid (UA) on tumor cell apoptosis was investigated using HL-60 human promyelocytic leukemia cells as a model cellular system. Treatment with UA resulted in a concentration-dependent decreased cell viability assessed by MTT assay. UA also induced genomic DNA fragmentation, a hallmark of apoptosis, indicating that the mechanism by which UA induced cell death was through apoptosis. The intracellular Ca²⁺ level was increased by treatment with UA. Intracellular Ca²⁺ inhibitors, such as intracellular Ca²⁺-release blockers (dantrolene, TMB-8 and ruthenium red) and an intracellular Ca²⁺ chelator (BAPTA/AM), significantly blocked the UA-induced increased intracellular Ca²⁺ concentration. These inhibitors also blocked the effects of UA on cell viability and apoptosis. These results suggest that enhanced intracellular Ca²⁺ signals may be involved in UA-induced apoptosis in HL-60 cells. Int. J. Cancer 73:725–728, 1997. © 1997 Wiley-Liss, Inc.     

Verapamil upregulates sensitivity of human colon and breast cancer cells to LAK-cytotoxicity in vitro

Pretreatment of human colon cancer LoVo-H cells and human breast cancer ZR-75 1A cells with low doses of verapamil, a Ca²⁺ channel blocker, for 48 h has a slight growth stimulatory effect and substantially increases cell sensitivity to lymphokine-activated killer (LAK) mediated cytotoxicity in the standard ⁵¹Cr release assay. The role of intracellular Ca²⁺ levels in determining verapamil effect is demonstrated by cytochemical evidence of intracellular Ca²⁺ lowering in verapamil-treated cells and by the reversal by the Ca²⁺ ionophore A-23187 of verapamil-induced sensitivity to LAK-mediated cytotoxicity.     

Colon-cancer cell variants producing regressive tumors in syngeneic rats,unlike variants yielding progressive tumors,attach to interstitial collagens through integrin α2β1

Nine clones of tumor cells, derived from a single rat colon carcinoma, were analyzed for their adhesive properties and in vivo growth patterns. Four clones (denoted REG) gave rise to regressively growing tumors. Cells from the 4 REG clones attached significantly better to collagen types I and III than did cells from the 5 clones (denoted PRO) which grew progressively in vivo. In contrast, REG and PRO clones did not differ in their attachment to collagen type IV, laminin or fibronectin. The attachment of REG cells to collagen was dependent on Mg²⁺, but not Ca²⁺. Monospecific rabbit IgG to rat integrin β₁-chain inhibited REG cell attachment to collagen, demonstrating involvement of a β₁ integrin in this process. PRO and REG cells expressed an underglycosylated β₁ chain (M_r ∼ 105,000) that was somewhat smaller than β₁-chains described previously on rat fibroblasts and hepatocytes (M_r ∼ 115,000). Monoclonal IgG to rat integrin α₂β₁, but not to α₁β₁, readily inhibited REG cell attachment to collagen, demonstrating the involvement of integrin α₂β₁. However, β₁ and α₂ integrin subunits were found in purified glycoproteins from both PRO and REG cells. This suggests that α₂β₁ integrin is expressed by both cell variants, but is functional as a collagen receptor on REG cells only. In this system of tumor-cell variants, the clear-cut differences in attachment to interstitial collagens of the 9 clones suggest a possible relationship between this attachment and the capacity to induce progressive or regressive tumors. © 1996 Wiley-Liss, Inc.     

No evidence of correlation between mutation at codon 531 of src and the risk of colon cancer in Chinese

The protein tyrosine kinase activity of c-src proto-oncogene product, pp60^c-src, is elevated in a number of human cancers, including colon cancer. Phosphorylation of human pp60^c-src carboxy-terminal tyrosine 530 suppresses its kinase activity. A recent report suggested that the risk of colon cancer is higher for those who carry a C→T transition mutation on codon 531 (Gln-531→Amber-531) of src gene. This mutation caused a prematured translation termination and up-regulated the kinase activity. To examine whether this mutation could be a risk factor for colon carcinoma in the Chinese population, we used the same PCR-based assay to analyze src genotypes of 131 colon cancers and other various types of carcinoma. No mutation was detected in all specimens that were screened in this study. Thus, mutation at Gln-531 of src gene does not seem to be involved in the development of colon cancer in Chinese ethnicity.     

In vivo and in vitro invasiveness of a rat colon-cancer cell line maintaining E-cadherin expression: An enhancing role of tumor-associated myofibroblasts

In various cell systems, an inverse relationship was found between expression of E-cadherin, a molecule involved in the Ca²⁺-dependent homophylic cell-to-cell attachment of epithelial cells, and the capacity to invade extracellular matrix gels or normal tissues in vitro., DHD/K12/TRb (PROb) cells, maintained as a cell line derived from a rat colon carcinoma, homogeneously expressed. in vitro immunoreactive E-cadherin, which was functional as shown in cell dissociation-reassociation assays. PROb cells were found to be non-invasive in 3 different assays in vitro., However, tumors resulting from a s.c. injection of PROb cells into syngeneic BD-IX rats were invasive, although PROb cells maintained E-cadherin expression in the tumors. Cells from a freshly dissociated PROb tumor showed, not only PROb cells but also tumor-associated myofibrobfasts and were able to cross a Matrigel-coated filter. PROb tumors were indeed infiltrated by numerous myofibroblasts, mainly located at the invasive edge of the tumor. Cells from an established culture of tumor-infiltrating myofibroblasts were able to confer upon PROb cells invasiveness through Matrigel-coated filter or into chick-heart fragments. PROb cells maintained their capacity to express E-cadherin after myofibroblast-enhanced Matrigel invasion. Tumor-associated myofibroblasts, but not PROb cells, secreted a 72-kDa collagenase that could play a role in tumor-cell invasion. These results strongly suggest that cells from the tumor stroma, and more specifically myofibroblasts, may be involved in the invasiveness of epithelial tumor cells in vivo, even when E-cadherin expression prevents tumor-cell invasiveness in different in vitro assays.     

Caffeine enhances radiosensitization to orthotopic transplant LM3 hepatocellular carcinoma in vivo

The aim of this study was to determine whether caffeine enhanced radiosensitization in an orthotopic transplant of LM3 human hepatocellular cancer in nude mice. LM3 hepatocellular carcinoma cells were infected with red fluorescent protein and irradiated, and cell cycle distribution and survival fraction were detected. A nude mouse model of orthotopic transplant of red fluorescent protein‐expressing LM3 hepatocellular cancer was established. Nude mice were divided into four groups: control (NS); caffeine (Caff) alone; irradiation (IR) alone; and caffeine + IR (Caff + IR). Tumor growth curves were described. Expression of cyclin and apoptosis were evaluated by analysis of phosphorylated cyclin dependent kinase 1 (CDC2) Tyr15 (CDC2‐Tyr15‐P), cyclinB1, TUNEL staining, and caspase‐3. Caffeine abrogated IR‐induced G₂ phase arrest and decreased survival of irradiated LM3 cells. Caffeine enhanced radiosensitivity of LM3 hepatocellular cancer in vivo. Tumor growth delay time in the Caff + IR group was 14.3 days compared with the NS group, 14.1 days compared with the Caff alone group, and 7.2 days compared with the IR alone group. At 15 Gy, expression of CDC2‐Tyr15‐P in the Caff + IR group (26.0 ± 8.9%) was significantly lower than in the IR alone group (68.4 ± 10.6%), expression of cyclinB1 and proportion of TUNEL‐positive cells in the Caff + IR group (30.4 ± 8.7% and 59.2 ± 9.5%, respectively) was significantly higher than in the IR alone group (7.0 ± 3.7% and 24.2 ± 7.2%, respectively), expression of caspase‐3 was consistent with the TUNEL staining results. This study suggested that caffeine might enhance the radiosensitivity of LM3 hepatocellular cancer in vivo, and may be feasible for further clinical applications. (Cancer Sci 2010)     

Early signals for keratinocyte differentiation: role of Ca2+-mediated inositol lipid metabolism in normal and neoplastic epidermal cells

Differentiation of cultured keratinocytes is regulated by theCa²⁺ concentration of the culture medium. Below 0.1 mM Ca²⁺,a monolayer of basal cells is formed which fully differentiatesin response to a rise in medium Ca²⁺. A role for protein kinaseC in this differentiation program has been suggested becausephorbol esters induce epidermal differentiation in cells grownin reduced Ca²⁺ medium, and exogenously added phospholipaseC (which increases cellular diacylglycerol) mimics phorbol esteraction. These findings suggested that the external Ca²⁺ signalmay lead to protein kinase C activation via stimulation of cellularphospholipase C activity. The effect of the external Ca²⁺ signalon phospholipase C was studied in cultures prelabeled with [³H]inositol. Within 2 min after addition of Ca²⁺ to 1 mM, an increasein inositol phosphates was measured. This correlated with adecrease in radiolabeled phosphoinositides, suggesting thatthese were the source of the increased inositol phosphates.After 3 h in 1 mM Ca²⁺ medium, each of the inositol phosphatesremained increased to 130–140% of control levels. Inositolphosphate metabolism in neoplastic epidermal cells was quantitativelysimilar to normal cells in response to the Ca²⁺ signal. Stimulationof phosphatidylinositol (PIP) metabolism appears to be mediatedby a rise in intracellular free Ca²⁺ because Ca²⁺ ionophoresA23187 and ionomycin also cause a similar rise in inositol phosphatelevels. Phorbol esters did not increase PIP turnover but insteadstimulated phosphatidylcholine metabolism. The induction ofepidermal differentiation by phorbol esters was enhanced byionomycin, suggesting that both protein kinase C activation,elevation of intracellular calcium and PIP turnover were importantcomponents of the signal for epidermal differentiation. Theseresults demonstrate that the second messenger system for Ca²⁺-mediated keratinocyte differentiation may be through a directeffect on phospholipase C activity.     

Putrescine-stimulated Intracellular Ca2+ Release for Invasiveness of Rat Ascites Hepatoma Cells

Our previous study showed that treatment of highly invasive rat ascites hepatoma (LC-AH) cells with α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, decreased both their intracellular level of putrescine and their in vitro invasion of a monolayer of calf pulmonary arterial endothelial (CPAE) cells, and that both these decreases were completely reversed by exogenous putrescine, but not spermidine or spermine. Here we show that all adhering control (DFMO-untreated) cells migrated beneath CPAE monolayer with morphological change from round to cauliflower-shaped cells (migratory cells). DFMO treatment increased the number of cells that remained round without migration (nonmigratory cells). Exogenous putrescine, but not spermidine or spermine, induced transformation of all nonmigratory cells to migratory cells with a concomitant increase in their intracellular Ca²⁺ level, [Ca²⁺]_i. The putrescine-induced increase in their [Ca²⁺]_i preceded their transformation and these effects of putrescine were not affected by antagonists of the voltage-gated Ca²⁺ channel, but were completely suppressed by ryanodine, which also suppressed the invasiveness of the control cells. The DFMO-induced decreases in both [Ca²⁺]_i and the invasiveness of the cells were restored by thapsigargin, which elevated [Ca²⁺]_i by inhibiting endoplasmic Ca²⁺-ATPase, indicating that thapsigargin mimics the effects of putrescine. These results support the idea that putrescine is a cofactor for Ca²⁺ release through the Ca²⁺ channel in the endoplasmic reticulum that is inhibited by ryanodine, this release being initiated by cell adhesion and being a prerequisite for tumor cell invasion.     

SOCE and cancer: Recent progress and new perspectives

Ca²⁺ acts as a universal and versatile second messenger in the regulation of a myriad of biological processes, including cell proliferation, differentiation, migration and apoptosis. Store‐operated Ca²⁺ entry (SOCE) mediated by ORAI and the stromal interaction molecule (STIM) constitutes one of the major routes of calcium entry in nonexcitable cells, in which the depletion of intracellular Ca²⁺ stores triggers activation of the endoplasmic reticulum (ER)‐resident Ca²⁺ sensor protein STIM to gate and open the ORAI Ca²⁺ channels in the plasma membrane (PM). Accumulating evidence indicates that SOCE plays critical roles in cancer cell proliferation, metastasis and tumor neovascularization, as well as in antitumor immunity. We summarize herein the recent advances in our understanding of the function of SOCE in various types of tumor cells, vascular endothelial cells and cells of the immune system. Finally, the therapeutic potential of SOCE inhibitors in the treatment of cancer is also discussed.     

Effect of all-trans-retinoic acid alone or in combination with chemotherapy in newly diagnosed acute promyelocytic leukaemia

Between February 1992 and November 1996 we treated 30 newly diagnosed acute promyelocytic leukaemia (APL) patients either with oral all-trans-retinoic acid (ATRA) alone (45 mg m^-2) or with a simultaneous combination of ATRA (45 mg m^-2), daunorubicin (DNR, 50 mg/m^-2 for 3 days) and cytosine arabinoside (ARA-C, 200 mg m^-2 for 7 days). There were 15 patients in each group. Patients with a white blood cell count < 5 x 10⁹/l at diagnosis received only ATRA as an induction therapy. Patients with initial white blood cell count > 5 x 10⁹/l received a combination of ATRA, DNR and ARA-C as an induction therapy. Within the first 20 days of induction, there were two early deaths in the group of patients receiving only ATRA, and six early deaths in the group of patients treated with a combination of ATRA and chemotherapy. Ten out of 13 patients (76.9%) receiving ATRA only achieved complete remission (CR) whereas seven out of nine patients (77.8%) receiving ATRA with chemotherapy achieved CR. Initial median peripheral white blood cell counts were significantly lower in the group of patients treated with ATRA alone (2.3 x 10⁹/l) than in the group of patients receiving ATRA and chemotherapy (14.0 x 10⁹/l). Morphological evidence of differentiation was noted in all patients entering CR. Patients in both groups who achieved CR received one course of standard ′3+7′ chemotherapy (DNR 45 mg m^-2,1 -3 days, ARA-C 200 mg m^-2,1-7 days) followed by two courses of standard ′2+5′ chemotherapy (DNR 50 mg m^-2 1-2 days, ARA-C 200 mg m^-2 1-5 days) as a consolidation therapy. Patients not achieving remission (three out of 13 in the ATRA group and two out of nine in ATRA+chemotherapy group) did not respond to salvage chemotherapy and all died within 3 months of diagnosis. Only one out of 10 patients (10%) in CR, treated with ATRA is in relapse after 18 months. In patients treated with ATRA alone two out of 10 (20%) survived 58 months following diagnosis whereas in the ATRA+chemotherapy group one out of seven has already survived their 58th month since diagnosis. Four out of eight patients with an early death died of retinoic acid syndrome. Other toxicities due to ATRA were minimal (cheilitis, xerosis, dermatitis, diarrhoea, liver damage or pseudotumor cerebri).