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.

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.

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.

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.

RAR-independent RXR signaling induces t(15;17) leukemia cell maturation.

Although retinoic acid receptor alpha (RARalpha) agonists induce the maturation of t(15;17) acute promyelocytic leukemia (APL) cells, drug treatment also selects leukemic blasts expressing PML-RARalpha fusion proteins with mutated ligand-binding domains that no longer respond to all-trans retinoic acid (ATRA). Here we report a novel RARalpha-independent signaling pathway that induces maturation of both ATRA-sensitive and ATRA-resistant APL NB4 cells, and does not invoke the ligand-induced alteration of PML-RARalpha signaling, stability or compartmentalization. This response involves a cross-talk between RXR agonists and protein kinase A signaling. Our results indicate the existence of a separate RXR-dependent maturation pathway that can be activated in the absence of known ligands for RXR heterodimerization partners.

FISH analysis of translocations involving the short arm of chromosome 9 in lymphoid malignancies.

Deletion of the short arm of chromosome 9 (9p), resulting in the loss of the p16INK4a/MTS1 gene, now called CDKN2, has been found to occur frequently in acute lymphoblastic leukemia, even in the absence of a microscopically visible deletion. In this study, we have used YAC probes encompassing the CDKN2 locus to analyze by fluorescence in situ hybridization patients with leukemia and lymphoma and translocations involving 9p in order to establish the CDKN2 status in relation to the karyotype. We found that, in leukemic cells exhibiting loss of heterozygosity at the CDKN2 locus, the deleted allele was from the cytogenetically normal chromosome 9, whereas the other allele was located on a rearranged chromosome. This finding suggests that CDKN2 gene loss is nonrandomly associated with 9p translocation in lymphoid proliferations. Genes Chromosom.

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.

Chromosome microdissection in leukemia: a powerful tool for the analysis of complex chromosomal rearrangements.

In many human cancers the presence of marker chromosomes or unbalanced translocations prevents complete karyotypic analysis. Chromosome microdissection has become an increasingly important method for assessing chromosome rearrangements. However, most studies using chromosome microdissection have been carried out on established cancer cell lines that provide an unlimited supply of abnormal metaphase cells. We have routinely performed microdissection of as few as three marker chromosome copies from short-term cultures of acute myeloid leukemias, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH) to normal metaphase spreads. Using this type of "reverse chromosome painting," we were able to characterize precisely the chromosomal constitution of each marker chromosome in the samples, confirming the diagnostic usefulness of microdissection in cancer cytogenetics. In addition, in one leukemia with atypical cytological features, microdissection enabled us to detect a novel retinoic acid receptor alpha gene rearrangement.

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.

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.

High frequency of t(12;21) in childhood B-lineage acute lymphoblastic leukemia.

The recurrent t(12;21)(p12;q22) translocation fuses two genes, TEL and AML1, that have previously been shown to be independently involved in myeloid malignant proliferations. A search for rearrangement of the TEL locus in the region known to be involved in t(12;21) was performed by Southern blotting in a panel of hematopoietic malignancies. The presence of a t(12;21) was confirmed by fluorescence in situ hybridization (FISH) and/or reverse transcriptase (RT)-polymerase chain reaction (PCR). We report that fusion of TEL to AML1 is specifically observed in at least 16% of the childhood B-lineage acute lymphoblastic leukemia (ALL) investigated, none of which had been previously identified as harboring t(12;21).

Leukaemic non-Hodgkin's lymphomas with hyperdiploid cells and t(11;14)(q13;q32): a subtype of mantle cell lymphoma?

The present study describes five patients with leukaemic non-Hodgkin's lymphoma (NHL) detected on the basis of particular morphology and cytogenetic findings. With respect to histological, immunological and cytogenetic features these NHL are closely related to mantle cell lymphoma/intermediate differentiated lymphocytic lymphoma. However, the presence of unusual large cells associated with the t(11;14)(q13;q32) translocation and numerical chromosome changes, in the near triploid or near tetraploid range, could delineate a particular subtype of mantle cell lymphoma.

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.

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.

Alterations of the putative tumor suppressor gene p16/MTS1 in human hematological malignancies.

The chromosome band 9p21-22 is frequently rearranged or deleted in a variety of tumors including hematological malignancies. This supports the notion of a tumor suppressor gene in this chromosome region. Indeed, the p16/MTS1 gene encoding a cyclin-dependent kinase (CDK) inhibitor has been shown to be frequently deleted and/or inactivated by nonsense mutations in a number of tumors. We have examined 98 DNA samples from blood, bone marrow cells and lymph node biopsies of patients with leukemia (ALL and AML) or lymphoma (follicular lymphoma and T-cell lymphoma), using Southern blot hybridization and a p16/MTS1-specific probe. Molecular abnormalities, mainly homozygous deletions, were found principally in ALL (8 out of 22 patients), much less frequently in AML (2/32) and lymphoma (2/32). While these data argue in favor of a large involvement of p16/MTS1 in ALL, AML and lymphomas appear to be less frequently implicated.

In situ hybridization to interphase nuclei in acute leukemia.

Numerical chromosome abnormalities were studied in 17 acute lymphoblastic leukemias and one hyperdiploid acute myeloblastic leukemia by fluorescence in situ hybridization (FISH) using YAC clones specific to chromosomes 21 and 6. The results agreed well with cytogenetic findings. Hyperdiploid leukemias with more than 50 chromosomes usually had 4 copies of chromosome 21 and three of chromosome 6, while diploid and pseudodiploid cases were confirmed to have two copies of the two chromosomes. Interesting discrepancies were also observed. In one patient, trisomy 6 was detected by FISH but not by cytogenetics because of the probable inclusion of a chromosome 6 segment within a marker chromosome. The percentages of nuclei with 3 or 4 spots (chromosome 21) and three spots (chromosome 6) in hyperdiploid cells were significantly different in some patients, whereas they might be identical from cytogenetic data.

A novel translocation, t(9;11)(q33;q23) involving the HRX gene in an acute monocytic leukemia.

A t(9;11)(q33;q23) has been detected by chromosome painting with chromosome 11- and chromosome 9-specific probes in blast cells of a child with acute monocytic leukemia. Using a YAC clone spanning the usual breakpoint region of translocations of acute leukemias, it was shown that the breakpoint was effectively within the same region of the band 11q23. This was confirmed by Southern blot studies that showed the localization of the translocation breakpoint between the 6th and 8th exons of the HRX gene. The implication of the HRX gene in t(9;11)(q33;q23) is a novel example of the diversity of translocations involving this gene in hemopoietic disorders. Sequencing DNA in the vicinity of the breakpoints should help to understand the reason of the localization of the recombination hot spot at band 11q23.

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.

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.