Theodore G. Gabig

Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation

A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) π, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.

Expression and chromosomal localization of the Requiem gene

Abstract. Apoptosis in murine myeloid cell lines requires the expression of the Requiem gene, which encodes a putative zinc finger protein. We detected the protein in both cytoplasmic and nuclear subcellular fractions of murine myeloid cells and human K562 leukemia cells, which suggests that the protein might have a function distinct from a transcription factor. This distribution did not alter upon apoptosis induction by IL-3 deprivation. As an approach to investigate its role in development, we determined the spatio-temporal expression pattern in the mouse. Expression was detected in various tissues in earlier gestational age; however, confined to testes, spleen, thymus, and part of the hippocampus in the adult mouse. The expression profile is consistent with a functional role during rapid growth and cell turnover, and in agreement with a regulatory function for hematopoietic cells. The human cDNA clone sequenced showed high homology to its murine counterpart and extended the open reading frame by 20 codons upstream. The gene is located in the proximal region of mouse Chromosome (Chr) 19. In the homologous human region at 11q13, it is located at about 150 kb centromeric from MLK3.

Isolation of the respiratory burst oxidase: the role of a flavoprotein component.

The article reviews the enzymatic and electron transfer properties of a low-potential FAD-dependent flavoprotein that is a component of the NADPH-dependent O2− · -generating respiratory burst oxidase of phagocytes. Current methods available for isolation of the respiratory burst oxidase and the flavo-protein component of the complex are also reviewed. These studies and data obtained from affinity-labeling of respiratory burst oxidase components, suggest that the flavoprotein has a molecular weight of 65–67 kD. The prevailing evidence suggests that the flavoprotein functions as a dehydrogenase/electron transferase and can directly catalyse NADPH-dependent O2− · formation when isolated. However, in neutrophil plasma membranes, the prevailing evidence suggests that the flavoprotein functions primarily to transfer electrons from NADPH to cytochromeb−245 and that this latter redox component is the catalytic side of O2− · formation. A working model for the arrangement of the flavorprotein and cytochromeb−245 components of the respiratory burst oxidase in neutrophil membranes is proposed.

Combined heterozygosity of factor V leiden and the G20210A prothrombin gene mutation in a patient with cerebral cortical vein thrombosis.

Cerebral venous thrombosis (CVT) is a rare type of stroke with a variety of causes. Several reports have suggested that either factor V Leiden or G20210A prothrombin gene mutation is associated with an increased risk of CVT. The genetic thrombophilias are typically associated with other predisposing factors. We report a unique case of CVT in a patient with both the factor V Leiden and the G20210A prothrombin gene mutations without other identifiable precipitating factors in a 28‐year‐old white male in good health. MRI and cerebral arterial angiography showed cerebral cortical venous thrombosis. This case suggests that combined heterozygous individuals may be particularly prone to spontaneous thrombosis, like CVT. Am. J. Hematol. 64:226–228, 2000.

Cell-free activation of phagocyte NADPH-oxidase: Tissue and differentiation-specific expression of cytosolic cofactor activity

We examined a variety of tissues for the presence of cytosolic cofactor activity that would support arachidonate-dependent cell-free activation of NADPH-oxidase in isolated human neutrophil membranes. Cofactor activity was not found in cytosol isolated from erythrocytes, lymphocytes, placenta, brain, liver, or the human promyelocytic leukemic cell line HL-60. Induction of differentiation in HL-60 cells led to expression of cytosolic cofactor activity. In dimethylsulphoxide-induced HL-60 cells the level of cytosolic cofactor activity was closely correlated with phorbol myristate acetate-stimulated whole cell superoxide production. These results strongly suggest that the cytosolic cofactor is a phagocyte-specific regulatory protein of physiologic importance in NADPH-oxidase activation.

Expression of val-12 mutant RAS P21 in an IL-3-dependent murine myeloid cell line is associated with loss of serum-dependence and increases in membrane PIP2-specific phospholipase C activity

We previously showed that the proliferative response of a serum- and interleukin-3 (IL-3)-dependent murine myeloid cell line, NFS/N1-H7, was partially inhibited by pertussis toxin as a result of toxin-induced increased adenylate cyclase activity. In the present studies, we examined the role of the phosphoinositide cycle in the proliferative response of these cells and demonstrated that there was no change in PIP (phosphatidylinositol bisphosphate)-specific phospholipase C activity in response to IL-3 alone. However, serum caused a pertussis toxin-insensitive increase in PIP2-specific phospholipase C activity as reflected by decreased cellular levels of 32P-labelled PIP2. Proliferation of a subline selected from val-12-mutant H-ras-transfected NFS-H7 cells, clone E5, was insensitive to pertussis toxin, occurred in the absence of serum but remained serum-stimulatable and absolutely dependent on IL-3. This val-12 mutant ras-expressing cell line showed an increase in 32P-labelled PIP (phosphatidylinositol phosphate) in response to serum whereas the parent cell line did not. Membrane fractions from 32P-labelled ras-transfected cells displayed higher GTP gamma S-, GTP-, or F(-)-stimulated PIP2-specific phospholipase C activity compared to membranes from the parent cell line. Thus serum-dependence and adenylate cyclase-mediated pertussis toxin-sensitivity of the parent cell line was bypassed by val-12 mutant ras p21, possibly as a result of increased PIP2-specific phospholipase C activity.

[18] NADPH oxidase from polymorphonuclear cells

Publisher Summary This chapter presents nicotinamide adenine dinucleotide phosphate (NADPH) oxidase from polymorphonuclear cells. The NADPH oxidase enzyme system from polymorphonuclear leukocytes is responsible for the sudden burst of oxygen consumption that follows the exposure of these cells to certain activating agents. The enzyme system catalyzes the univalent reduction of molecular oxygen at the expense of pyridine nucleotide (NADPH) oxidation. The membrane-associated enzyme system has a vectorial orientation in the plasma membrane or phagosomal membrane of the cell. Oxidase activation is a highly specific process controlled by complex mechanisms at the cellular level. Polymorphonuclear leukocytes are prepared from fresh whole blood anticoagulated with acid-citrate-dextrose by dextran sedimentation followed by centrifugation over lymphoprep and hypotonic lysis of red blood cells. Cell suspension is disrupted by sonication for three 15 seconds intervals at 20 W power with the standard probe tip of a Branson 200 sonifier set on pulsed power, 50% duty cycle. For different sonifiers, the power and time of sonification should be adjusted so that only 70–80% of the cells are completely disrupted.

Molecular heterogeneity in chronic granulomatous disease: a human model of defective phagocyte superoxide production.

Chronic granulomatous disease (CGD) is a genetically transmitted disorder thought to result from defect(s) in the activation or turnover of the NADPH dependent O2- generating oxidase enzyme system of human neutrophils and monocytes. The normal oxidase may be a flavoprotein-cytochrome b559 complex; therefore, these components of the oxidase were quantitated in the neutrophils from patients and family members of two unrelated CGD kindreds. The male propositus from an X-linked recessive kindred had a neutrophil oxidase fraction with low FAD content (26 pmol/mg protein) and undetectable cytochrome b559 (less than 5 pmol/mg protein). The male propositus from an autosomal recessive kindred had a neutrophil oxidase fraction with low FAD content (34 pmol FAD/mg protein), but normal cytochrome b559 content (170 pmol cytochrome b559/mg protein). Both parents of this latter CGD patient had normal FAD and cytochrome b559 content in their neutrophil oxidase fraction. We conclude that the carrier state in certain X-linked recessive female carriers of CGD can be detected by partial deficiencies of both flavoprotein and cytochrome b559 components of the oxidase, whereas presumed heterozygous carriers of certain autosomal recessive CGD kindreds cannot be detected by this means.