The oncogenic TEL/PDGFR beta fusion protein induces cell death through JNK/SAPK pathway.

The TEL/PDGFR beta (T/P) fusion protein isolated from patients bearing a t(5;12) translocation is transforming when expressed in haematopoietic cells. To examine the signal transduction events activated by this protein, we measured the effect of T/P on activation of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) in mouse bone marrow-derived Ba/F3 cells. Significant increase in the activity of JNK/SAPK1 was observed in transient transfection as well as in Ba/F3 cells stably expressing T/P. This activation was abrogated when the T/P-expressing cells were treated with a specific inhibitor of the PDGFR beta tyrosine kinase, indicating that the activity of the PDGFR beta part of the fusion protein was involved in JNK/SAPK activation. Expression of a dominant negative mutant of mitogen-activated protein kinase kinase 4 (MKK4), a direct activator of JNK/SAPK, prevented T/P-induced JNK/SAPK activation. In addition, inhibition of phosphoinositide-3 OH kinase (PI-3 kinase), a promoting survival factor, potentiated the effect of T/P on JNK/SAPK activation. Interestingly, expression of T/P was shown to initiate an apoptotic response that was enhanced by treatment of cells with the PI-3 kinase inhibitor LY294002, suggesting that T/P mediated cell death through activation of JNK/SAPK signalling pathway. Consistent with this hypothesis, expression of the dominant negative mutant of MKK4 decreased T/P-mediated apoptosis, while a dominant-negative mutant of PI-3 kinase enhances cell death. These findings indicate that activation of JNK/SAPK by T/P is related to apoptosis rather than cell proliferation and transformation.

Signaling revisited in acute promyelocytic leukemia.

Although transcription factors are still the main focus to understanding leukemogenesis, recent results strongly suggest that alteration of a receptor and/or subsequent signaling plays a critical and co-operative role in the pathogenesis of acute myeloid leukemia (AML). The t(15;17) translocation, found in 95% of APL, encodes a PML-RARalpha fusion protein. A main model proposed for acute promyelocytic leukemia (APL) is that PML-RARalpha exerts its oncogenic effects by repressing retinoic acid-inducible genes critical to myeloid differentiation. Dysregulation of these genes may result in abnormal signaling, thereby freeing pre-leukemic cells from controls which normally induce the onset of differentiation. It is also likely that treatment of APL cells by retinoic acid induces de novo up-regulation of the same genes which are dominantly repressed by PML-RARalpha and whose expression is required for reactivation of the differentiation program. Identification of such genes together with the signaling pathways interrupted at the early stages of leukemia transformation and reactivated during retinoic acid-induced differentiation in APL cells will contribute to the development of new molecular targets for treatment of leukemia.

Structural study of hemoglobin Hazebrouck, beta 38(C4)Thr----Pro. A new abnormal hemoglobin with instability and low oxygen affinity.

A new beta-variant has been detected and structurally defined in a French male, with a life-long history of hemolytic anemia. This variant is moderately unstable and has a low oxygen affinity. The abnormal hemoglobin was not detected by standard electrophoretic procedures. It moved slightly slower than Hb A during isoelectric focusing (IEF). Two minor fractions were also seen; the first migrated just cathodal to Hb F, as did partially oxidized Hb A or hemichrome derivatives of some unstable hemoglobins; the second in the position of free alpha-chains. The abnormal beta-chain was readily separated from both beta A- and alpha A-chains by acid-urea-Triton globin chain electrophoresis. Structural study was conducted simultaneously by fingerprinting and high-performance liquid chromatography (HPLC) of tryptic peptides. A new mutation beta 38(C4)Thr----Pro was found, which was named Hb Hazebrouck.

Proteinase 3 mRNA expression is induced in monocytes but not in neutrophils of patients with cystic fibrosis.

Proteinase 3 (PR3), a serine proteinase which can degrade lung tissue, is present in the cystic fibrosis (CF) sputum. In the present study, PR3 protein and mRNA expression was determined in circulating neutrophils and monocytes. CF neutrophils contained similar PR3 concentrations as healthy controls and poorly expressed PR3 mRNA. In contrast, CF monocytes showed significantly higher PR3 concentrations than controls, together with an upregulation of PR3 mRNA expression especially during pulmonary exacerbation. Interestingly, antibiotic treatment fully abrogated PR3 mRNA expression and decreased PR3 protein in monocytes. Our findings highlight a potential role of monocyte-derived PR3 in CF-associated airway inflammation.

HIV-1 p17 and IFN-gamma both induce fructose 1,6-bisphosphatase.

The p17 matrix protein of the human immunodeficiency virus type 1 (HIV-1) plays a crucial role in AIDS pathogenesis. It orchestrates viral assembly and directs the preintegration complex to the nucleus of infected cells. Recently, the three-dimensional structure of p17 was shown to resemble that of interferon-gamma (IFN-gamma), suggesting that both proteins might share analogous functions. We demonstrate that in monocytes, p17 shares with IFN-gamma the ability to induce 1alpha-hydroxylase activity and to activate fructose 1,6-bisphosphatase gene expression in the presence of 25-hydroxyvitamin D3. However, p17 does not bind to the IFN-gamma cell membrane receptor and fails to increase expression of IFN-gamma-induced proteins, such as tryptophanyl-tRNA synthetase, Fc gammaRI, and HLA DR or B7/BB1 antigens. Altogether, our results raise the possibility that the structural resemblance between p17 and IFN-gamma causes the selective activation of a common pathway resulting in the production of 1,25-dihydroxyvitamin D3. We also found that unlike IFN-gamma, p17 increases the intracellular ATP content. Since transport of the HIV-1 preintegration complex through the nuclear membrane is an ATP-dependent process, our observation suggests that p17 plays a double role in this active transport, not only by acting as a chaperone molecule but also by recruiting the necessary energy for this process.

1,25-Dihydroxyvitamin D3 and fetal lung maturation: immunogold detection of VDR expression in pneumocytes type II cells and effect on fructose 1,6 bisphosphatase.

Lung maturation before birth includes type II pneumocyte differentiation with progressive disappearance of glycogen content and onset of surfactant synthesis. We have shown previously that 1,25-(OH)2D3 increases surfactant synthesis and secretion by type II cells and decreases their glycogen content in fetal rat lung explants. Recently, the gene coding fructose 1,6 bisphosphatase (F1,6BP), a regulatory enzyme of gluconeogenesis, has been identified in type II cells and its promoter bears a Vitamin D response element. Present results show:The coexistence of type II cells at different stages of maturation. in rat fetal lung on day 21 of gestation (electron microscopy), and the association between maturation of type II cells and disappearance of their glycogen content. The immunogold labeling of all type II cells when using the 9A7g VDR-antibody, with significantly more abundant gold particles in cells exhibiting an intermediate glycogen content. The expression of F1,6BP mRNA in a human type II cell line (NCI-H441) and the increase of this expression after 18h incubation with 1,25-(OH)2D3 (10(-8)M). These results bring further evidence for a physiological role of 1,25-(OH)2D3 during type II pneumocyte maturation. Activation of F1,6BP may participate to the 1,25-(OH)2D3 action on surfactant synthesis via the gluconeogenesis pathway.

Determination of myeloid antigen expression on childhood acute lymphoblastic leukaemia cells: discrepancies using different monoclonal antibody clones.

Prospective clinical studies including large numbers of patients have led to the conclusion that co-expression of myeloid antigens in childhood acute lymphoblastic leukaemia (My+ ALL) does not have prognostic significance. However, reports of the frequency of My+ ALL in children vary widely across laboratories using different mAb clones and staining and analysing procedures. Taking two commonly accepted thresholds of positivity for myeloid antigens (20 and 30%), we analysed the immunoreactivity of the most widely employed mAb clones against CD13 (SJ1D1, L138 and My7) and CD33 (My9, P67.6 and D3HL60) and compared the proportions of My+ ALL detected by these clones in childhood ALL. The correlation between myeloid antigen expression and the presence of the t(12;21) translocation was analysed concomitantly in the same samples. The percentage of ALL cases positive for myeloid markers varied significantly depending on the mAb clone and the positive threshold. Among patients with B-ALL, the proportion of CD13+ ALL was significantly lower using SJ1D1 than using L138 or My7, while the proportion of CD33+ ALL was significantly higher for My9 than for P67.6 or D3HL60. Analysis of the co-expression of CD13 and CD33 on B-ALL cells using combinations of mAb clones showed that this frequency was either underestimated by the SJ1D1/D3HL60 or overestimated by the L138/P67.6 and My7/My9 combinations. A correlation between CD13/CD33 positivity and the t(12;21) translocation was uniformly observed in B-ALL patients for a positive threshold of 30%, whereas SJ1D1/D3HL60 detected no correlation between t(12;21) and CD13/CD33 positivity when the threshold was lowered to 20%. These data show that the mAb clones commonly used to detect the CD13 and CD33 surface antigens have variable immunoreactivity against childhood ALL cells, which may partly explain the conflicting reports concerning the prognostic significance of myeloid antigen expression in paediatric ALL and its association with different translocations. The present findings may also be of clinical importance for therapeutic choices.

Determination of myeloid antigen expression on childhood acute lymphoblastic leukaemia cells: discrepancies using different monoclonal antibody clones.

Prospective clinical studies including large numbers of patients have led to the conclusion that co-expression of myeloid antigens in childhood acute lymphoblastic leukaemia (My+ ALL) does not have prognostic significance. However, reports of the frequency of My+ ALL in children vary widely across laboratories using different mAb clones and staining and analysing procedures. Taking two commonly accepted thresholds of positivity for myeloid antigens (20 and 30%), we analysed the immunoreactivity of the most widely employed mAb clones against CD13 (SJ1D1, L138 and My7) and CD33 (My9, P67.6 and D3HL60) and compared the proportions of My+ ALL detected by these clones in childhood ALL. The correlation between myeloid antigen expression and the presence of the t(12;21) translocation was analysed concomitantly in the same samples. The percentage of ALL cases positive for myeloid markers varied significantly depending on the mAb clone and the positive threshold. Among patients with B-ALL, the proportion of CD13+ ALL was significantly lower using SJ1D1 than using L138 or My7, while the proportion of CD33+ ALL was significantly higher for My9 than for P67.6 or D3HL60. Analysis of the co-expression of CD13 and CD33 on B-ALL cells using combinations of mAb clones showed that this frequency was either underestimated by the SJ1D1/D3HL60 or overestimated by the L138/P67.6 and My7/My9 combinations. A correlation between CD13/CD33 positivity and the t(12;21) translocation was uniformly observed in B-ALL patients for a positive threshold of 30%, whereas SJ1D1/D3HL60 detected no correlation between t(12;21) and CD13/CD33 positivity when the threshold was lowered to 20%. These data show that the mAb clones commonly used to detect the CD13 and CD33 surface antigens have variable immunoreactivity against childhood ALL cells, which may partly explain the conflicting reports concerning the prognostic significance of myeloid antigen expression in paediatric ALL and its association with different translocations. The present findings may also be of clinical importance for therapeutic choices.

[Effect of translocation t(15;17) on the gene expression regulation of myeloblastin during all trans retinoic acid induced myeloid differentiation in human leukemic cells]. / Effets de la translocation t(15;17) sur la régulation de l'expression du gène myéloblastine--mbn--au cours de la différenciation myéloïde induite par l'acide rétinoïque tout-trans des cellules leucémiques humaines.

Myeloblastin (mbn) is a serine protease involved in the control of growth and differentiation of human leukemic cells. In the promyelocytic-like human leukemia cell line HL-60 this protease is inhibited during retinoic acid (RA) induced differentiation. The t(15;17) translocation, specifically associated with the human acute promyelocytic leukemia (APL), fuses the retinoic acid receptor alpha (RAR alpha) to a novel gene PML generating the hybrid protein PML-RAR. We have shown that while mbn was early down-regulated in HL60 cells treated with all trans RA, the inhibition of this gene was considerably delayed in NB4 cells, which carry the t(15;17) translocation, upon treatment with the same inducer. This observation suggested that the changes in the myeloblastin regulation by RA found in NB4 cells could be ascribed to the presence of the fusion protein PML-RAR. To verify this hypothesis we have cloned the putative promoter region of mbn gene. Transactivation properties of endogenous retinoic acid receptors on this region have been tested in transfection experiments of HL60 and NB4 cell lines before and after treatment with all trans RA. We found that RA induced a significant inhibition of the luciferase reporter gene in HL60 cells. In contrast, a strong stimulation of luciferase activity was observed in NB4 cells treated with RA. The analysis of the promoter region allowed us to identify a new response element for retinoic acid receptors, named mREpal, which is probably affected by the product of t(15;17) translocation.

Wegener autoantigen and myeloblastin are encoded by a single mRNA.

Myeloblastin is a serine protease that has been identified in the human leukemia cell line HL-60. Down-regulation of this protease can inhibit proliferation and induce differentiation of promyelocyte-like human leukemic cells. Proteinase 3, a serine protease of human neutrophils, has been identified as the Wegener autoantigen. A high level of homology between myeloblastin and proteinase 3 has suggested that they may be a single serine protease. We have recently completed the 5'-terminal nucleotide sequence of proteinase 3 and shown that its mRNA was also expressed in HL-60 cells and in cells from patients with acute myeloid leukemia. Here we demonstrate that myeloblastin and proteinase 3 are encoded by a single mRNA.

Immunochemical evidence that three protein kinase C isozymes increase in abundance during HL-60 differentiation induced by dimethyl sulfoxide and retinoic acid.

Activity of the Ca2+/phospholipid-dependent protein kinase C has been shown to increase during differentiation of the human promyelocytic leukemia cell line HL-60 by dimethyl sulfoxide and retinoic acid (Zylber-Katz, E., and Glazer, R. I. (1985) Cancer Res. 45, 5159-5164). Antipeptide antibodies were prepared that specifically recognize the alpha, beta, and gamma isozymes of protein kinase C in rat brain cytosol and HL-60 cell extracts. The three isozymes do not share a common tissue distribution pattern. The gamma enzyme is abundant in brain but a relatively minor component in HL-60 cells; the opposite is true for the alpha enzyme. All three isozymes increase at least 2-fold in abundance in HL-60 cells exposed to 1.2% dimethyl sulfoxide for 48 h. The increase in abundance of the alpha and beta isoforms reaches 7- and 5-fold, respectively, by 96 h without further increase in the abundance of the gamma isozyme. Similarly, all three isozymes increase at least 1.5-fold in abundance after 48 h and 3-fold after 96 h with 1 microM retinoic acid. No further increase in the abundance of any of the isozymes is seen between 96 and 144 h of incubation with retinoic acid. The increase in protein kinase C activity is not limited to the cytosolic forms of the enzyme; a parallel increase in membrane-associated protein kinase C is also observed during differentiation. Approximately 10% of total protein kinase C activity is membrane-associated in both control and differentiating cells. These studies provide the first immunochemical evidence that all three protein kinase C isozymes increase during HL-60 cell differentiation, and they suggest that the increase in the isozyme levels may be coordinately regulated.

Myc is essential for transformation by TEL/platelet-derived growth factor receptor beta (PDGFRbeta).

The t(5;12) translocation identified in patients with chronic myelomonocytic leukemia (CMML) encodes a TEL/platelet-derived growth factor receptor beta (PDGFRbeta) fusion protein. A key hypothesis for how the TEL/PDGFRbeta fusion protein would function as an oncogene is that it represents a constitutively active version of the normal PDGFRbeta. A link between the function of the t(5;12)-encoded TEL/PDGFRbeta fusion protein and Myc expression is suggested by the fact that Myc is induced by PDGF and is essential for entry of cells into the S phase of the cell cycle. We here show that the kinase activity of TEL/PDGFRbeta is necessary for Ba/F3 cells to acquire interleukin-3 (IL-3) independence and that, in contrast to their untransfected counterpart, Ba/F3 cells stably transfected with TEL/PDGFRbeta maintain a high level of Myc expression after removal of IL-3. Using dominant negative mutants of Myc, we show that a threshold of active Myc is essential for TEL/PDGFRbeta to transform Ba/F3 and Rat-1 cells. The findings that the kinase activity of TEL/PDGFRbeta and a threshold of active Myc are involved in TEL/PDGFRbeta transformation may allow for the development of therapeutic strategies in patients with t(5;12)+ CMML using specific inhibitors of the PDGFRbeta kinase as well as compounds designed to interfere specifically with Myc.

Isolation of a subset of thymocytes inducible for terminal transferase biosynthesis.

A new technique using direct binding of nucleated hematopoietic cells to PNA-coated rabbit red blood cell monolayers was used to separate PNA + and PNA - cells from murine thymus. The rigorously purified PNA - thymocyte population was found to lack TdT and to be low in TL. Incubation of the negative fraction with TP 5 resulted in the synthesis of TdT in a large number of cells in the fraction, and the appearance of TL on the surface of about 20% of the cells. Isolation of this inducible population has led us to propose a new class of cells in intrathymic T cell development.

Evidence for a novel RasGAP-associated protein of 105 kDa in both mature trophoblasts and differentiating choriocarcinoma cells.

A novel tyrosine-phosphorylated, RasGAP-associated protein of 105 kDa (p105) is found in normal human term placental trophoblasts, as well as in JEG-3 human choriocarcinoma cells induced to differentiate by okadaic acid (OA). This p105 RasGAP-associated protein is distinct from other RasGAP-associated proteins described so far, none of which has either a molecular size close to p105 or a trophoblastic cell origin. The p105 appears, accompanied by p120 and p100 RasGAP expression, after OA treatment of JEG-3 cells but is almost undetectable in the absence of stimulation. Moreover, the p105 is the first discovered RasGAP-associated protein bound to p100 RasGAP. The natural occurrence of the p105 in normal mature trophoblasts isolated from human term placenta suggests that it may be linked to the differentiation state of human trophoblasts. Hence, this p105 RasGAP-associated protein might be considered a marker of human trophoblast differentiation.

Structure and expression of the mouse beta 2-microglobulin gene isolated from somatic and non-expressing teratocarcinoma cells.

Mouse teratocarcinoma cells express neither H-2 heavy chains nor beta 2-microglobulin (beta 2-m). We have constructed two genomic libraries, one from PCC4-aza-RI embryonal carcinoma cells and the other from their adult syngenic counterpart 129/Sv liver cells (H-2bc). The libraries were screened with a full length mouse beta 2-m cDNA probe which we isolated and sequenced. Two cosmid clones carrying the entire beta 2-m gene were isolated, one from each library. There was no detectable difference in structure between the two genes. Furthermore, both were shown to be active and to restore beta 2-m synthesis upon transfer into mutant cells deficient in beta 2-m. Irreversible DNA alterations in or around the beta 2-m gene are thus unlikely to account for the lack of beta 2-m gene expression in embryonal teratocarcinoma cells.

Activation of the fructose 1,6-bisphosphatase gene by 1,25-dihydroxyvitamin D3 during monocytic differentiation.

Cells from the human leukemia cell line HL-60 undergo terminal monocyte-like differentiation after exposure to either the active circulating form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or phorbol 12-myristate 13-acetate. Little is known about the genes that regulate monocytic differentiation. Using clonal variant cells of HL-60 origin, we constructed a cDNA library enriched for genes that are induced by 1,25-(OH)2D3. We now report that in HL-60, the fructose 1,6-bisphosphatase (FBPase; D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) gene is activated during 1,25-(OH)2D3-induced monocytic differentiation. This gene encodes two closely related mRNAs; one, activated by 1,25-(OH)2D3 at an early stage of HL-60 differentiation, encodes a protein that has homology to mammalian FBPase, a key enzyme in gluconeogenesis, although it does not exhibit its classical enzymatic activity. A second mRNA is activated by 1,25-(OH)2D3 mainly in peripheral blood monocytes. This mRNA is present in kidney as a unique transcript and encodes a protein with FBPase activity. Our data also show that this FBPase-encoding mRNA can be activated during monocytic maturation since it was detected in human alveolar macrophages.

1 alpha,25-dihydroxyvitamin D3-induced regulation of protein kinase C gene expression during HL-60 cell differentiation.

The human promyelocytic leukemia cell line HL-60 differentiates in vitro when treated with various inducers. It has previously been shown that protein kinase C (PKC) isozymes are modulated during granulocytic differentiation of HL-60 cells induced by dimethyl sulfoxide or retinoic acid (M. Makowske, R. Ballester, Y. Cayre, and O.M. Rosen, J. Biol. Chem., 263: 3402-3410, 1988; K. Hashimoto, A. Kishimoto, H. Aihara, I. Yasuda, K. Mikawa, and Y. Nishizuka, FEBS Left., 263: 31-34, 1990). HL-60 responds to 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) or to 12-O-tetradecanoylphorbol-13-acetate by giving rise to monocytic cells. In the present study, we demonstrate that treatment of HL-60 cells with 1,25-(OH)2D3 causes dramatic increases in PKC-alpha and PKC-beta protein levels detected by immunoblotting with PKC isoform-specific antibodies and in Ca(2+)- and phospholipid-dependent protein kinase activity. We also observed a transient increase in the steady-state levels of PKC-alpha and PKC-beta mRNA species in Northern blotting experiments, with maximal induction occurring 48 h after addition of 1,25-(OH)2D3. Analyses of 1,25-(OH)2D3-induced PKC mRNA expression by nuclear run-on transcription experiments suggest that the observed increases in PKC mRNA levels may occur by a posttranscriptional mechanism(s). In contrast to the transient increases in PKC mRNA levels, the increases in PKC Mr 80,000 protein species and in PKC enzyme activity were progressive in HL-60 cells treated with 1,25-(OH)2D3 between 1 and 5 days, thus implying the existence of a further up-regulation of PKC proteins occurring at the translational and/or posttranslational levels.(ABSTRACT TRUNCATED AT 250 WORDS)

A protein kinase C-independent pathway leading to c-Jun-dependent expression of 100-kDa Ras GTPase-activating protein in JEG-3 human choriocarcinoma cells.

Although the 100-kDa Ras GTPase-activating protein (p100 RasGAP) has been reported to exist specifically in human placental trophoblasts, the molecular mechanisms responsible for regulating its expression remain unclear. In this study we used okadaic acid, an inhibitor of serine/threonine phosphatase 1 and 2 A, as a probe to explore the signaling pathway regulating the expression of p100 RasGAP in JEG-3 human placental choriocarcinoma cells. Treatment of JEG-3 cells with okadaic acid provoked dose- and time-dependent stimulation of p100 RasGAP expression without marked modification of expression of p120 RasGAP, another isoform of RasGAP. Co-treatment of cells with okadaic acid and the protein kinase C activator, phorbol 12-myristate 13-acetate, exerted an additive effect on p100 RasGAP induction. Moreover, the response of the p100 RasGAP de novo synthesis to okadaic acid was not affected by the selective inhibitor of protein kinase C, GF 109203X. Thus this study identified a novel signaling pathway regulating p100 RasGAP expression, which is independent of protein kinase C. In addition, okadaic acid treatment resulted in the activation of ERK2 (p42 MAP kinase) and the induction of both c-Jun and c-Fos proteins without activating JNK (c-Jun NH2-terminal kinase). Significantly, blockade of c-Jun expression with antisense c-jun oligonucleotides suppressed p100 RasGAP expression. Taken together, it is concluded that okadaic acid induces the expression of p100 RasGAP protein in JEG-3 cells preceded by activation of ERK and AP-1 cascade, and that this okadaic acid-induced p100 RasGAP expression is independent of protein kinase C-mediated pathway but requires c-Jun/AP-1 function.