Granulocytic differentiation of human NB4 promyelocytic leukemia cells induced by all-trans retinoic acid metabolites.

The metabolism of all-trans retinoic acid (ATRA) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia (APL). However, ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro. The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [4-hydroxy-retinoic acid (4-OH-RA), 18-hydroxy-retinoic acid (18-OH-RA), 4-oxo-retinoic acid (4-oxo-RA), and 5,6-epoxy-retinoic acid (5,6-epoxy-RA)] in NB4, a human promyelocytic leukemia cell line that exhibits the APL diagnostic t(15;17) chromosomal translocation and expresses the PML-RAR alpha fusion protein. We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro. NB4 cell growth was inhibited (69-78% at 120 h) and cell cycle progression in the G1 phase (82-85% at 120 h) was blocked by ATRA and all of the metabolites at 1 microM concentration. ATRA and its metabolites could induce NB4 cells differentiation with similar activity, as evaluated by cell morphology, by the nitroblue tetrazolium reduction test (82-88% at 120 h) or by the expression of the maturation specific cell surface marker CD11c. In addition, nuclear body reorganization to macropunctated structures, as well as the degradation of PML-RAR alpha, was found to be similar for ATRA and all of its metabolites. Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required to differentiate 50% of cells in 72 h as follows: ATRA, 15.8 +/- 1.7 nM; 4-oxo-RA, 38.3 +/- 1.3 nM; 18-OH-RA, 55.5 +/- 1.8 nM; 4-OH-RA, 79.8 +/- 1.8 nM; and 5,6-epoxy-RA, 99.5 +/- 1.5 nM. The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors, because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells. These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway, and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo.

A mutated PML/RARA found in the retinoid maturation resistant NB4 subclone, NB4-R2, blocks RARA and wild-type PML/RARA transcriptional activities.

The fusion protein PML/RARA, associated with acute promyelocytic leukemia behaves as an abnormal retinoic acid (RA) receptor with altered transactivation properties but is still inducible by RA. The chimeric protein is thought to promote leukemogenesis but also paradoxically to mediate the sensitivity to ATRA of APL cells. This has been supported by works reporting that in vitro ATRA resistance is characterized by defects in the RARA/E-domain of PML/RARA. In the present report, we identified a new mutation in the E domain of PML/RARA which is associated with a RA-resistant subline of NB4 cells; NB4-R2. This mutation, identical to the Gln411 mutation found in HL60-R, changes the amino acid Gln903 to an in-phase stop codon, generating a truncated form of PML/RARA which has lost 52 amino acids at its C-terminal end. We have studied the effect of the truncated PML/RARA protein on PML NB formation and RARA and PML/RARA transcriptional activity. We show here that the fusion mutant exerts a dominant negative effect on wild-type PML, PML/RARA and RARA transcription activity. These findings highlight the important role of the RARA E-domain of PML/RARA in mediating RA sensitivity in APL cells.

An interstitial 11q23 deletion proven to be a rearrangement interrupting the MLL gene in an infant with acute myeloblastic leukemia.

Chromosome studies of an infant with acute myeloblastic leukemia (AML), classified as M2 in the FAB nomenclature revealed an unusual karyotype with del(11)(q23) and a marker chromosome resembling a small chromosomal fragment present in all metaphase cells examined. Fluorescence in situ hybridization (FISH) showed the splitting of a YAC probe containing a part of MLL between the del(11) and mar chromosomes. Painting showed that the mar chromosome contained DNA sequences from chromosome 11, but that the centromeric region was not marked by a chromosome 11-specific alphoid probe. The chromosomal breakpoint was located within the MLL gene by Southern blot experiments. The deletion of 11q was thus interstitial. This case illustrates the importance of associating cytogenetics, several FISH techniques, and molecular studies to analyze unusual karyotypes in leukemia.

Distinct MLL gene rearrangements associated with successive acute monocytic and lymphoblastic leukemias in the same patient.

A patient with acute monocytic leukemia (AMoL) and t(6;11)(q27;q23) developed acute lymphoblastic leukemia (ALL) and t(4;11)(q21;23), 10 months after complete remission of the AMoL. The MLL gene, normally located at band 11q23, appeared differently rearranged in the cells of these two leukemias, showing a different origin for the two malignant clones. The responsibility of etoposide, used in treatment of the AML, in the occurrence of the ALL is probable in this patient.

Autonomous rexinoid death signaling is suppressed by converging signaling pathways in immature leukemia cells.

On their own, retinoid X receptor (RXR)-selective ligands (rexinoids) are silent in retinoic acid receptor (RAR)-RXR heterodimers, and no selective rexinoid program has been described as yet in cellular systems. We report here on the rexinoid signaling capacity that triggers apoptosis of immature promyelocytic NB4 cells as a default pathway in the absence of survival factors. Rexinoid-induced apoptosis displays all features of bona fide programmed cell death and is inhibited by RXR, but not RAR antagonists. Several types of survival signals block rexinoid-induced apoptosis. RARalpha agonists switch the cellular response toward differentiation and induce the expression of antiapoptosis factors. Activation of the protein kinase A pathway in the presence of rexinoid agonists induces maturation and blocks immature cell apoptosis. Addition of nonretinoid serum factors also blocks cell death but does not induce cell differentiation. Rexinoid-induced apoptosis is linked to neither the presence nor stability of the promyelocytic leukemia-RARalpha fusion protein and operates also in non-acute promyelocytic leukemia cells. Together our results support a model according to which rexinoids activate in certain leukemia cells a default death pathway onto which several other signaling paradigms converge. This pathway is entirely distinct from that triggered by RAR agonists, which control cell maturation and postmaturation apoptosis.

Long lasting effects of intrauterine growth retardation on basal and 5-HT-stimulated Na(+)/K(+)-ATPase in the brain of developing rats.

Intrauterine growth retardation induced by ligation of the uterine vessels in pregnant rats on the 5th day before delivery was associated with brain and body weights of hypotrophic offspring significantly lower than those of pair-aged control rats, even after 6 weeks of postnatal rearing under normal conditions. In vitro measurements in homogenates indicated that Na(+)/K(+)-ATPase in the forebrain, cerebellum and hippocampus was less active in hypotrophic rats than in pair-aged controls for at least the first month after birth. However, 5-HT and related agonists (RU-24969, bufotenine, and to a lower extent, tryptamine) stimulated Na(+)/K(+)-ATPase activity more efficiently in tissues from hypotrophic rats than in those from control animals. Opposite changes were noted in the brain stem: basal Na(+)/K(+)-ATPase activity was higher in hypotrophic rats during the second half of the first postnatal month but the stimulatory effect of 5-HT was lower than in pair-aged control animals. Since potent 5-HT antagonists such as cinanserin, methiothepin and methysergide, prevented the 5-HT induced-activation of Na(+)/K(+)-ATPase in brain homogenates, these results are discussed in relation with the possible existence of a specific 5-HT receptor controlling Na(+)/K(+)-ATPase activity in the rat brain.

Na(+) K(+) ATPase activity in synaptosomes and myelin of developing control and intra-uterine growth retarded rats: effects of lead and serotonin.

An intrauterine growth retarded (IUGR) model based on restriction of blood supply to fetuses at 17 days of pregnancy in rats was studied. We investigated in vitro the effects of lead on Na(+)K(+) ATPase activity in synaptosomes and myelin of IUGR and control rats from 6 to 60 days after birth. In both groups an age-dependent effects existed in synaptosomes for the lowest doses of lead. The experimental group tended to be more sensitive to the metal than the control group and the Na(+)K(+)ATPase activity was less inhibited in the younger rats as compared to mature rats. Serotonin (5-HT) added to the subcellular preparations produced different changes in Na(+)K(+)ATPase activity. In synaptosomes, 5-HT stimulated the enzyme activity in a dose-related manner and apparently reversed the inhibiton induced by lead up to 22 days after birth in the control group. This action was less marked in the IUGR group. In myelin fractions, the Na(+)K(+)ATPase activity was inhibited by lead in both groups but the "protective effect of monoamines" was never observed. The Na(+)K(+) ATPase activity was modulated by monoamines in synaptosomes and not in myelin, perhaps through a mechanism involving soluble factor(s).

Loss of chromosome 22 in patients with refractory anemia with excess of blasts (RAEB) in transformation and acute leukemia after RAEB.

We report studies of 12 patients with refractory anemia and excess of blasts in transformation (RAEB-t) and 17 with acute myeloblastic leukemia (AML) after RAEB. Besides chromosome 5 and 7 abnormalities, five patients with complex karyotypic changes had monosomy 22. This association is discussed in relation to the hypothesis of a suppressor gene located on chromosome 22.

Translocation (2;3)(p22;q28) is associated with myeloid disorders.

Chromosome studies carried out in two children with acute myeloblastic leukemia (AML, M2) showed a t(2;3)(p22;q28). This abnormality was associated with monosomy 7 and del(12)(p12) in the first patient and was found only in relapse in the second patient. Comparison with the other previously published t(2;3) suggests that this translocation is a nonrandom abnormality involving a pluripotent stem cell and occurring as a secondary chromosome abnormality in AML.

Studies of BCR rearrangements in Philadelphia-positive acute leukemia.

Five patients with Philadelphia-positive acute leukemia were cytogenetically and molecularly investigated in order to determine the localization of the breakpoints on chromosome 22. Rearrangements of the bcr segment were detected in one case with acute mixed leukemia in a child. Rearrangements in the BCR gene first intron, the so-called bcr2 and bcr3 regions, were detected in two other cases, one with an acute lymphoblastic leukemia (ALL) and one with mixed acute leukemia. No molecular rearrangement could be detected in the last two cases, an ALL and a T-cell acute lymphoblastic leukemia with a t(2;22) translocation.

Presence of three recurrent chromosomal reaarrangements, t(2;3)(p12;q37), del(8)(q24), and t(14;18), in an acute lymphoblastic leukemia.

A complex chromosomal abnormality associating three recurrent rearrangements, t(2;3)((p12;q37), del (8)(q24) and t(14;18)(q32;q21), was detected in a patient with acute lymphoblastic leukemia of the Burkitt type. Southern blot studies showed rearrangements of the MYC, BCL2, and JH genes, thus confirming the cytogenetic data. However, no rearrangement of the LAZ3/BCL6 gene, normally localized on band 3q27, could be detected. The simultaneous presence of three recurrent rearrangements specific for lymphoid malignancies addresses the question of their timing in the malignant process and the prognostic significance of the association of such anomalies.

Abnormalities of chromosome 18 in myelodysplastic syndromes and secondary leukemia.

Monosomy 18 and partial deletion of 18q are nonrandom events in myelodysplastic syndromes (MDS) and secondary acute myeloblastic leukemia (sAML). They are part of complex chromosome abnormalities, as shown in the present study of six patients with MDS and sAML. We compared occurrence of chromosome 18 abnormalities in these syndromes with that in de novo AML.

Effect of intrauterine growth retardation on developmental changes in DNA and [14C]thymidine metabolism in different regions of rat brain: histological and biochemical correlations.

Intrauterine growth retardation was induced in the rat by clamping the uterine artery on day 17 of gestation. The effect of hypotrophy on DNA synthesis was studied in two different cerebral structures: hippocampus and cerebellum. Accumulation of DNA in these structures was biochemically measured in parallel to the incorporation of methyl-[14C]thymidine into nucleic acid at different ages and correlated with autoradiography. The various metabolites of thymidine in acid-soluble fraction were determined by using chromatographic procedures. Phosphorylation defects or reduced utilization of thymidine were found in hypotrophic rats and may delay the DNA synthesis. An essay of catch-up occurred with a different timing according to the cerebral region studied. A morphological and DNA synthesis. An essay of catch-up occurred with a different timing according to the cerebral region studied. A morphological and autoradiographic study after incorporation of [3H]thymidine was carried out in parallel. The neuronal and glial components of cytogenesis were analyzed separately and a good correlation was observed between histological and biochemical data in both groups of animals.

Long lasting effects of intrauterine growth retardation of 5-HT metabolism in the brain of developing rats.

Intrauterine growth retardation (IUGR) was achieved by ligating the artery and vein supplying one uterine horn in pregnant rats on the 5th day before delivery. At birth, the weight of the whole body and of the forebrain (but not that of the brain stem of about half of the offspring) were significantly lower than those of normal controls. This deficit persisted for at least the first 3 postnatal weeks. During the immediate period following birth, the concentrations of norepinephrine, dopamine, serotonin and 5-hydroxyindoleacetic acid (5-HIAA) were significantly higher in IUGR rats than in controls. Increased levels of serotonin and 5-HIAA were still observed in the forebrain and brain stem of 15-day-old IUGR rats and were associated with parallel increases in brain and serum free tryptophan levels. These results indicate that a transient insult in the fetal life could result in long-lasting alterations in 5-HT metabolism in CNS of developing rats.

Development of the cholinergic system in control and intra-uterine growth retarded rat brain.

The activity of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and muscarinic receptors was studied in control rats and in rats growth-retarded in utero because of reduction of the blood supply 5 days before birth. The different markers of the cholinergic system were estimated at P (postnatal day) 6, 9, 12, 15, 22 and 60 in cerebellum, hypothalamus, septum, striatum and CA1, CA3 and fascia dentata of the hippocampus. In control rats, there was a transient increase in ChAT activity in the septum during the second week of postnatal development. In the intrauterine growth retarded rats there was a marked delay in this developmental rise in CA1, CA3 at P6 and P9 and in the fascia dentata at P14 respectively. This delayed rise enzyme activity was associated with a significant reduction of muscarinic binding sites [( 3H]QNB) in the hippocampus. AChE staining showed a similar development in both groups. Therefore, the undernutrition produced by a reduction of the blood supply 5 days before birth is associated with a delayed maturation of cholinergic functions.

Effect of organic and inorganic mercuric salts on Na+K+ATPase in different cerebral fractions in control and intrauterine growth-retarded rats: alterations induced by serotonin.

An intrauterine growth-retarded (IUGR) model based on restriction of blood supply to the rat fetus at the 17th day of pregnancy was studied. We investigated in vitro the effects of thimerosal and mercuric chloride on Na+K+ATPase activity in total brain homogenate, synaptosomes and myelin at weaning. In addition, we evaluated the reversal effect of serotonin on mercury-inhibited Na+K+ATPase activity. The toxicity, in terms of inhibition of Na+K+ATPase activity was greater with mercuric chloride than with thimerosal. Synaptosomes and principally myelin were more sensitive to the metal salts than total homogenate. Serotonin stimulated the Na+K+ATPase activity in total brain homogenate and synaptosomes but inhibited the enzyme in the myelin fraction. This effect was more marked in the IUGR group than in the control group. Serotonin (1 mM) added to total homogenate pretreated with the mercury salts produced variable reversal effects. In the synaptosomal fraction reverse effect was noted with serotonin. In myelin fraction, added serotonin increased inhibition caused by thimerosal.

A PARP-1/JNK1 cascade participates in the synergistic apoptotic effect of TNFalpha and all-trans retinoic acid in APL cells.

When administrated by isolated limb perfusion, tumor necrosis factor alpha (TNFalpha) is an efficient antitumor agent that improves drug penetration and destroys angiogenic vessels. Moreover, the pronounced potentiation of TNFalpha-induced apoptosis by NF-kappaB inhibitors suggest that these compounds could enhance TNFalpha antitumor efficacy through direct induction of tumor cell apoptosis. Therefore, attempts at amplifying signaling pathways that mediate TNFalpha antitumor effects could help to design combination therapies improving its efficiency. We report that nanomolar concentrations of all-trans retinoic acid (ATRA) amplify TNFalpha-induced apoptosis in APL cells expressing a specific repressor of NF-kappaB activation. This effect is abolished by the pan-caspase inhibitor, Z-VAD-fmk and by caspase-8 and -9 inhibitors. Cell death is accompanied by a drop of mitochondrial potential and by poly (ADP-ribose) polymerase (PARP) activation. Using specific PARP-1 inhibitors and siRNAs, we show that PARP-1 is essential for the synergistic apoptotic effect and c-Jun N-terminal kinase 1 (JNK1) activation triggered by the ATRA/TNFalpha combination. JNK1 siRNAs reduce ATRA/TNFalpha-induced apoptosis, mitochondrial release of cytochrome c and caspase-9 activation. Altogether, these results identify a novel mechanism of PARP-1-induced apoptosis, in which JNK1 provides a link between PARP-1 activation and mitochondrial pathway of caspase-9 activation. This study also suggests that inclusion of nanomolar doses of ATRA could be clinically beneficial in amplifying TNFalpha-induced antitumor signals.

Effect of lead on Na+,K+ATPase activity in the developing brain of intra-uterine growth-retarded rats.

Lead (Pb) intoxication in developing mammals, including humans, produces serious brain damage. In addition, it is known that nutritional status influences the susceptibility to Pb toxicity. We developed an in utero undernutrition model based on restriction of blood supply to fetuses on d 17 of pregnancy (IUGR rats). The aim of this study was to investigate in vitro the possible effect of Pb on Na+, K+ATPase activity in the brain of developing IUGR and control rats from 6 to 60 d after birth. In addition, we measured the stimulation of Na+, K+ATPase by the monoamines noradrenaline and serotonin. Our results show that: The neurotoxic effect of Pb is an age-related phenomenon. Both IUGR and control rats were more sensitive to Pb in the first week of life. In adults, Pb had a weak inhibitory potency; the delayed matured brain in IUGR animals seemed less sensitive to Pb when compared to age-paired control rats; in the IUGR group, at 15 and 22 d, low doses of Pb had a stimulatory effect on Na+, K+ATPase instead of an inhibitory effect; noradrenaline and serotonin stimulated Na+, K+ATPase activity to an equivalent extent, but this was greater in IUGR than control rats; and at low Pb concentrations, the studied monoamines reversed Pb-induced inhibition.

Retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation.

Human telomerase, a cellular reverse transcriptase (hTERT), is a nuclear ribonucleoprotein enzyme complex that catalyzes the synthesis and extension of telomeric DNA. This enzyme is specifically activated in most malignant tumors but is usually inactive in normal somatic cells, suggesting that telomerase plays an important role in cellular immortalization and tumorigenesis. Terminal maturation of tumor cells has been associated with the repression of telomerase activity. Using maturation-sensitive and -resistant NB4 cell lines, we analyzed the pattern of telomerase expression during the therapeutic treatment of acute promyelocytic leukemia (APL) by retinoids. Two pathways leading to the down-regulation of hTERT and telomerase activity were identified. The first pathway results in a rapid down-regulation of telomerase that is associated with retinoic acid receptor (RAR)-dependent maturation of NB4 cells. Furthermore, during NB4 cell maturation, obtained independently of RAR by retinoic X receptor (RXR)-specific agonists (rexinoids), no change in telomerase activity was observed, suggesting that hTERT regulation requires a specific signaling and occurs autonomously. A second pathway of hTERT regulation, identified in the RAR-responsive, maturation-resistant NB4-R1 cell line, results in a down-regulation of telomerase that develops slowly during two weeks of all-trans retinoic acid (ATRA) treatment. This pathway leads to telomere shortening, growth arrest, and cell death, all events that are overcome by ectopic expression of hTERT. These findings demonstrate a clear and full dissociation between the process of tumor cell maturation and the regulation of hTERT mRNA expression and telomerase activity by retinoids. We propose telomerase expression as an efficient and selective target of retinoids in the therapy of tumors.

Translocation t(10;11) involving the MLL gene in acute myeloid leukemia. Importance of fluorescence in situ hybridization (FISH) analysis.

Fluorescence in situ hybridization analysis in an infant with acute monocytic leukemia revealed a complex translocation, t(10;11;4) (p12; q23;q26). Southern blot analysis confirmed the existence of rearrangement of the MLL gene. The frequent occurrence of complex translocations involving 10p12 and 11q23 is discussed in function of the opposite orientation of the AF10 and MLL genes on 10p and 11q. The importance of FISH analysis in t(10; 11) is emphasized.