Jian-Hua Tong

Fusion between a novel Krüppel-like zinc finger gene and the retinoic acid receptor-alpha locus due to a variant t(11;17) translocation associated with acute promyelocytic leukaemia.

We have identified a unique case of acute promyelocytic leukaemia (APL) with a t(11;17) reciprocal chromosomal translocation involving the retinoic acid receptor alpha (RAR alpha) and a previously uncharacterized zinc finger gene. As a result of this translocation, mRNAs containing the coding sequences of the new gene, fused in‐frame either upstream of the RAR alpha B region or downstream from the unique A1 and A2 regions of the two major RAR alpha isoforms, are expressed from the rearranged alleles. The above gene, which we have termed PLZF (for promyelocytic leukaemia zinc finger), encodes a potential transcription factor containing nine zinc finger motifs related to the Drosophila gap gene Krüppel and is expressed as at least two isoforms which differ in the sequences encoding the N‐terminal region of the protein. Within the haematopoietic system the PLZF mRNAs were detected in the bone marrow, early myeloid cell lines and peripheral blood mononuclear cells, but not in lymphoid cell lines or tissues. In addition, the PLZF mRNA levels were down‐regulated in NB‐4 and HL‐60 promyelocytic cell lines in response to retinoic acid‐induced granulocytic differentiation and were very low in mature granulocytes. Our results demonstrate for the first time the association of a variant chromosomal translocation involving the RAR alpha gene with APL, further implicating the RAR alpha in leukaemogenesis and also suggesting an important role for PLZF as well as retinoic acid and its receptors in myeloid maturation.

Arsenic Trioxide Controls the Fate of the PML-RARα Oncoprotein by Directly Binding PML

Arsenic on the Fingers Arsenic, an ancient drug used in traditional Chinese medicine, has attracted wide interest because it has therapeutic activity in patients with acute promyelocytic leukemia (APL). The drug acts by promoting degradation of an oncogenic protein, PML-RARα, a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor α, which is found specifically in APL cells and helps drive their growth. Zhang et al. (p. 240; see the Perspective by Kogan) now explain how arsenic initiates the molecular events leading to PML-RARα degradation. Arsenic was found to bind directly to cysteine residues within zinc finger domains of PML. Arsenic binding then induced oligomerization of PML, which in turn enhanced its association with an enzyme that helps catalyze SUMOylation, a posttranslational modification that can target proteins for degradation. Arsenic, a drug used clinically for leukemia, binds directly to an oncogenic protein, thereby promoting its degradation. Arsenic, an ancient drug used in traditional Chinese medicine, has attracted worldwide interest because it shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL). Arsenic trioxide (As2O3) exerts its therapeutic effect by promoting degradation of an oncogenic protein that drives the growth of APL cells, PML-RARα (a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor alpha). PML and PML-RARα degradation is triggered by their SUMOylation, but the mechanism by which As2O3 induces this posttranslational modification is unclear. Here we show that arsenic binds directly to cysteine residues in zinc fingers located within the RBCC domain of PML-RARα and PML. Arsenic binding induces PML oligomerization, which increases its interaction with the small ubiquitin-like protein modifier (SUMO)–conjugating enzyme UBC9, resulting in enhanced SUMOylation and degradation. The identification of PML as a direct target of As2O3 provides new insights into the drug’s mechanism of action and its specificity for APL.

Immunodetection of human telomerase reverse-transcriptase (hTERT) re-appraised: nucleolin and telomerase cross paths

The involvement of telomerase in cellular immortalization and senescence has often been assessed by means of telomerase expression at the RNA level and quantification of telomerase activity by the telomeric repeat amplification protocol assay. However, these methods either neglected the existence of various telomerase splice variants, or ignored the nonconventional functions of telomerase independent of its ability to elongate and maintain telomere length. Immunodetection of telomerase is now being recognized as a necessary approach to precisely elucidate its roles in oncogenesis and senescence. A few antibodies directed against the catalytic subunit of the human telomerase (hTERT) are currently used but their specificity is not always demonstrated. A survey of the literature showed inconsistencies and led us to comparatively re-evaluate the most frequently used antibodies. Surprisingly, mass spectrometry, two-dimensional gel analysis and immunofluorescent experiments revealed that the most frequently used hTERT immunoprobe, a mouse monoclonal antibody that was claimed to be directed against an hTERT protein epitope, in fact recognizes nucleolin rather than telomerase. Our findings have interesting implications regarding the biology of nucleolin and telomerase in the context of pathophysiological investigations recently carried out.

Clinical and cytogenetic features of 508 Chinese patients with myelodysplastic syndrome and comparison with those in Western countries

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis and leukemia progression. Racial differences may exist on clinical pictures and the molecular events leading to MDS, which are heterogeneous. To better define the clinical and cytogenetic features in Chinese patients, a retrospective multicentric study was performed in 508 MDS cases. Compared with Western countries, Chinese patients showed younger age (median: 49 vs 65–73 years), lower percentages of RARS (2.8 vs 6.6–15.3%), and CMML (5.2 vs 11.7–30.6%). Cytogenetically, among 367 cases with evaluable data, abnormal karyotypes were found in 136 cases, including 56 numerical and 80 structural changes. Incidences of single chromosome 5 and 7 abnormalities were lower than those in Western countries (2.2 vs 17.8–42.5%). However, complex cytogenetic aberrations and chromosome translocations were frequently observed and related to poor prognosis. Both multiple chromosome deletions and translocations were detected in advanced subtypes (RAEB and RAEB-T). Analysis of 200 cases revealed a higher incidence of hepatitis-B-virus infection than that in non-MDS population (21.00 vs 9.75%). This study further confirmed: (1) different genetic/environmental backgrounds between Asian and Western MDS populations; (2) a strong predictive value of cytogenetic abnormalities on disease outcome and involvement of genomic instability in leukemia clone development.

Cobalt chloride and low oxygen tension trigger differentiation of acute myeloid leukemic cells: possible mediation of hypoxia-inducible factor-1α

Cellular and systemic O2 concentrations are tightly regulated to maintain delicate oxygen homeostasis. Although the roles of hypoxia in solid tumors have been widely studied, few studies were reported regarding the possible effects of hypoxia on leukemic cells. Here, we showed for the first time that low concentrations of cobalt chloride (CoCl2), a hypoxia-mimicking agent, and 2–3% O2 triggered differentiation of various subtypes of human acute myeloid leukemic (AML) cell lines, including NB4, U937 and Kasumi-1 cells, respectively, from M3, M5 and M2b-type AML, but CoCl2 did not modulate AML subtype-specific fusion proteins promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) and AML1-ETO. Treatment with CoCl2 also induced primary leukemic cells from some AML patients to undergo differentiation. Similar to what occurs in solid tumor cells, CoCl2-mimicked hypoxia also increased the level of hypoxia-inducible factor (HIF)-1α protein and its DNA-binding activity in leukemic cells. The CoCl2 induction of HIF-1α protein and its DNA-binding activity were inhibited by 3-morpholinosydnonimine, which also blocked CoCl2-induced cell differentiation in leukemic cells. These results provide an insight into a possible link of hypoxia or HIF-1α and leukemic cell differentiation, and are possibly of significance to explore clinical potentials of hypoxia or hypoxia-mimicking agents and novel target-based drugs for differentiation therapy of leukemia

RIG-G as a key mediator of the antiproliferative activity of interferon-related pathways through enhancing p21 and p27 proteins.

The RIG-G gene, originally isolated from an acute promyelocytic leukemia cell line NB4, codes for a 60-kDa cytoplasmic protein that is induced by all-trans retinoic acid (ATRA) treatment along with the induction of morphological differentiation of NB4 cells. Here, we provide evidence that ectopic expression of Rig-G in U937 cells can lead to a significant accumulation of cells at G1/S transition. Growth arrest seems to occur by modulating several major cell cycle regulatory players. Interestingly, Rig-G alters JAB1 cellular distribution through interacting with this protein and increases the intracellular level of p27 by preventing it from the JAB-1-dependent and ubiquitin/proteasome-mediated degradation. Furthermore, we demonstrate a role of Rig-G for c-myc down-regulation that results in an up-regulation of p21, tightly associated with cell cycle arrest. In addition, our studies reveal that Rig-G is a direct target of STAT1, a key transcription factor in regulating IFN responses, and may be one of the first experimentally proven molecular mediators for the antiproliferative effect of IFN-α. Considering that IFN-α and ATRA synergistically inhibit growth along the intracellular pathways triggered by the two compounds in many cell types, we suggest that Rig-G may also represent one of the key molecular nodes of signaling cross-talk between ATRA and IFN-α.

Desferrioxamine induces leukemic cell differentiation potentially by hypoxia-inducible factor-1α that augments transcriptional activity of CCAAT/enhancer-binding protein-α

We reported recently that cobalt chloride-simulated hypoxia and mild hypoxia modified the differentiation of human acute myeloid leukemic (AML) cells, probably acting via a hypoxia-inducible factor-1 alpha (HIF-1α)-dependent mechanism. In this study, we investigated the effect of desferrioxamine (DFO), an iron chelator with ‘hypoxia-mimetic’ activity, on the differentiation of AML cells. The results showed that DFO at nontoxic concentrations induced the differentiation of AML cell lines NB4 and U937, as assessed by morphological criteria and differentiation-associated antigens. DFO-induced differentiation parallel to the rapid accumulation of HIF-1α protein in these two cell lines. Of importance, the transient transfection of HIF-1α cDNA induced U937 cells to develop the differentiation-related alterations such as growth arrest and increased CD11b expression. Furthermore, the inducible expression of chromosome translocation t(8;21)-generated leukemogenic AML1-ETO fusion gene attenuated DFO-induced differentiation of U937 cells with the decrease of CCAAT/enhancer-binding protein alpha (C/EBPα), a critical factor for granulocytic differentiation. Using immunoprecipitation and luciferase reporter assay, HIF-1α was also shown to interact physically with and to increase the transcriptional activity of C/EBPα. Taken together, these results provided novel evidence for a role of HIF-1α in AML cell differentiation, and suggested that C/EBPα might be a downstream effector for HIF-1α-mediated differentiation.

Arsenic trioxide-induced mitotic arrest and apoptosis in acute promyelocytic leukemia cells.

Arsenic trioxide (As2O3), an effective drug for the treatment of acute promyelocytic leukemia (APL), can induce apoptosis and partial differentiation in APL cells in vitro and in vivo. However, As2O3 also induces apoptosis in cancer cells other than APL with complex mechanisms, which seem to be cell type dependent. In this study, we report that APL cells (NB4 cell line) are arrested at early mitotic phase before the collapse of mitochondrial transmembrane potential (Δϕm) and apoptosis after treatment with pharmacological concentrations (1.0–2.0 μM) of As2O3. We have also made the following new discoveries: (1) 0.5 μM As2O3 that fails to induce apoptosis has no effects on cell cycle distribution. (2) With inhibition of As2O3-induced Δϕm collapse and apoptosis, dithiothreitol also effectively inhibits As2O3-induced mitotic arrest, suggesting that both As2O3-induced apoptosis and mitotic arrest involve proteins with thiol groups. (3) 1.5 mM caffeine that relieves cells from G2/M arrest also inhibits As2O3-induced Δϕm collapse and apoptosis, (4) 1.0 μM As2O3 increases the expression of both cyclin B1 and hCDC20 whereas it inhibits Tyr15 phosphorylation of p34cdc2. In conclusion, our results strongly support that there is a tight link between As2O3-induced apoptosis and mitotic arrest, the latter being one of common mechanisms for As2O3-induced apoptosis in cancer cells.

Treatment of Acute Promyelocytic Leukemia with ATRA and As2O3: A Model of Molecular

Most acute promyelocytic leukemia (APL) cases have t(15;17)(q22;q21) chromosomal translocation and PML-RARα chimeric gene which blocks granulocytic differentiation. The introduction of all-trans-retinoic acid (ATRA) and arsenic compounds, especially arsenic trioxide ( As2O3), has provided good models to study not only differentiation and/or apoptosis therapy but also molecular target-based cancer treatment. In vivo and in vitro investigations have shown that both agents are able to induce differentiation of APL cells: ATRA tends to induce terminal differentiation, while low-dose As2O3 can induce partial differentiation. Significant progress has been made in understanding the molecular mechanisms of APL pathogenesis and differentiation therapy. Pharmacological concentrations (0.1~1μM) of ATRA derepresses transcription by releasing CoR from, and recruiting CoA to PML-RARα, whereas As2O3 triggers a rapid degradation of PML-RARα. In fact, the two drugs act on the same oncoprotein through targeting different moieties and in distinct ways and thereby abrogate its dominant-negative effects on regulatory pathways necessary for granulocytic differentiation. As to apoptosis, it is clear that high-dose As2O3 can induce mitochondria-mediated cell death pathway in a thiol-dependent manner, while the mechanism of ATRA-induced apoptosis needs further elucidation. Transcriptomic and proteomic analysis are also expected to find new molecular targets. It is the hope that what we have learnt from APL will benefit further developments of anti-leukemia therapy.

Retinoic acid regulatory pathways, chromosomal translocations, and acute promyelocytic leukemia

Retinoic acids (RAs) exert a broad range of physiologic actions during embryonic development and adult life. Two families of RA receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), have been identified. The therapeutic effect of all‐trans‐RA (ATRA) in induction of remission for acute promyelocytic leukemia (APL) has largely been proved, and this has, over the past 10 years, greatly stimulated research on oncogenesis and RA‐regulated differentiation pathways. In APL, one of the RAR genes, RARA, is fused to PML in the great majority of patients as a result of the chromosomal translocation t(15;17). However, a small subset of APL patients have a different fusion gene, PLZF‐RARA, resulting from the variant translocation t(11;17). A third translocation, t(5;17), in which the NPM gene is fused to RARA, has been described. Current data suggest that PML‐RARα and PLZF‐RARα fusion receptors may play an important role in the development of APL and that PML‐RARα could be the target of ATRA differentiation therapy. Characterization of the genes regulated by retinoic acid may open up new prospects for an understanding of the mechanisms of ATRA differentiation therapy for APL and may help to extend the concept of cancer‐targeting treatment to other types of leukemias or solid tumors. Genes Chromosom Cancer 15:147–156 (1996).