Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1.

Nox1, a homologue of gp91phox, the catalytic moiety of the superoxide (O(2)(-))-generating NADPH oxidase of phagocytes, causes increased O(2)(-) generation, increased mitotic rate, cell transformation, and tumorigenicity when expressed in NIH 3T3 fibroblasts. This study explores the role of reactive oxygen species (ROS) in regulating cell growth and transformation by Nox1. H(2)O(2) concentration increased approximately 10-fold in Nox1-expressing cells, compared with <2-fold increase in O(2)(-). When human catalase was expressed in Nox1-expressing cells, H(2)O(2) concentration decreased, and the cells reverted to a normal appearance, the growth rate normalized, and cells no longer produced tumors in athymic mice. A large number of genes, including many related to cell cycle, growth, and cancer (but unrelated to oxidative stress), were expressed in Nox1-expressing cells, and more than 60% of these returned to normal levels on coexpression of catalase. Thus, H(2)O(2) in low concentrations functions as an intracellular signal that triggers a genetic program related to cell growth.

Extracellular signal-regulated kinase activates topoisomerase IIalpha through a mechanism independent of phosphorylation.

The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, regulate cellular responses by mediating extracellular growth signals toward cytoplasmic and nuclear targets. A potential target for ERK is topoisomerase IIalpha, which becomes highly phosphorylated during mitosis and is required for several aspects of nucleic acid metabolism, including chromosome condensation and daughter chromosome separation. In this study, we demonstrated interactions between ERK2 and topoisomerase IIalpha proteins by coimmunoprecipitation from mixtures of purified enzymes and from nuclear extracts. In vitro, diphosphorylated active ERK2 phosphorylated topoisomerase IIalpha and enhanced its specific activity by sevenfold, as measured by DNA relaxation assays, whereas unphosphorylated ERK2 had no effect. However, activation of topoisomerase II was also observed with diphosphorylated inactive mutant ERK2, suggesting a mechanism of activation that depends on the phosphorylation state of ERK2 but not on its kinase activity. Nevertheless, activation of ERK by transient transfection of constitutively active mutant MAP kinase kinase 1 (MKK1) enhanced endogenous topoisomerase II activity by fourfold. Our findings indicate that ERK regulates topoisomerase IIalpha in vitro and in vivo, suggesting a potential target for the MKK/ERK pathway in the modulation of chromatin reorganization events during mitosis and in other phases of the cell cycle.

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

Analysis of conserved binding proteins for nuclear localization sequences.

Correct targeting of nuclear proteins is mediated by nuclear localization sequences (NLS) which permit specific binding to the nucleus and subsequent translocation across the nuclear envelope via the nuclear pore complex. It is proposed that nuclear import is facilitated by NLS-receptors which reside in the cytoplasm and at the nuclear pore. These NLS-receptors could facilitate an early step of nuclear protein import, i.e. targeting and binding of nuclear proteins at the nuclear pore. We have generated anti-idiotype antibodies against the SV40 T-antigen nuclear localization sequence that allowed us to study NLS-binding proteins in a variety of different organisms. Proteins of similar size are recognized by these antibodies in yeast, Drosophila, rat and human cells. Cytological analysis indicates that the NLS-binding proteins reside in part at nuclear pores. One of the proteins recognized by anti-idiotype antibodies is identical to a previously identified NLS-binding protein. Using isolated yeast nuclei we demonstrate that the anti-idiotype antibodies compete for binding of nuclear proteins in vitro. We show that the yeast mutant npl3, which is defective in nuclear protein localization, has an altered distribution of antigens recognized by these anti-idiotype antibodies, at the semi-permissive temperature. Our results suggest that a set of proteins common to various eukaryotes recognizes nuclear localization sequences.

In vivo self-association of the Drosophila rel-protein dorsal.

The Drosophila morphogen dorsal, KBF1, NF-kappa B, and the proto-oncogene c-rel belong to the rel family of transcription factors whose function is regulated post-translationally by selective nuclear import. In the early Drosophila embryo, dorsal protein is proposed to be retained in the cytoplasm through its interaction with cactus protein. The maternal dorsal group genes constitute a signal transduction pathway, which results in targeting cytoplasmic dorsal protein into the nuclei of the syncytial blastoderm embryo, in a ventral-to-dorsal gradient. The asymmetric transcriptional regulation of zygotic genes along the dorsoventral axis by the dorsal morphogen gradient establishes embryonic dorsoventral polarity. In the lymphocytes, the functional equivalent of cactus is I kappa B, which appears to retain NF-kappa B in the cytoplasm. This retention is relieved by extracellular signals in tissue culture. NF-kappa B and rel proteins each are known to function as oligomeric complexes. Here we present genetic and biochemical evidence for the existence and functional importance of an oligomeric dorsal complex in vivo.

Developmental regulation of Drosophila DNA topoisomerase II.

Affinity-purified polyclonal antibodies were used to quantitate steady-state levels of DNA topoisomerase II (topo II) throughout Drosophila development. Although wide fluctuations were recorded at different stages, these fluctuations were paralleled by changes in levels of the nuclear lamin, a nuclear structural protein used as an internal standard. The exception to this was adult males where lamin levels were significantly elevated relative to topo II. Northern blot analyses of topo II and lamin mRNA, performed in conjunction with immunoblot analyses of protein revealed fluctuations in levels of the two different messages that paralleled changes in each other and in their respective translation products. Biochemical and immunochemical analyses were complemented by indirect immunofluorescence and immunoperoxidase experiments performed in situ. topo II was found distributed throughout nuclei in most but not all cell types examined. These results for Drosophila topo II are apparently at odds with those obtained by others working in vertebrate systems (see, for example, Heck, M. M. S. and W. C. Earnshaw. 1986. J. Cell Biol. 103:2569-2581; Heck, M. M. S., W. N. Hittelman, and W. C. Earnshaw. 1988. Proc. Natl. Acad. Sci. USA. 85:1086-1090) and suggest that in Drosophila, topo II may not be a useful marker for the proliferative state.

Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ.

Karyoskeletal protein fractions prepared from Drosophila melanogaster embryos contain morphologically identifiable remnants of nuclear pore complexes and peripheral lamina as well as what appears to be an internal nuclear "matrix" (Fisher, P. A., M. Berrios, and G. Blobel, 1982, J. Cell Biol., 92: 674-686). Structural stability of these proteinaceous assemblies is dependent on thermal incubation in vitro (37 degrees C, 15 min) before subfractionation of nuclei. In the absence of such incubation, greater than 90% of the total karyoskeletal protein including major polypeptide components of internal "matrix," pore complexes, and the peripheral lamina, is solubilized by 1 M NaCl. In vivo heat shock induces karyoskeletal stabilization resembling that resulting from thermal incubation in vitro. Immunocytochemical studies have been used to establish the effects of heat shock on the organization and distribution of major karyoskeletal marker proteins in situ. Taken together, these results are consistent with the notion that in vivo, regulation of karyoskeletal plasticity (and perhaps form) may be a functionally significant component of the Drosophila heat shock response. They also have broad practical implications for studies pertaining to the structure and function of karyoskeletal protein (nuclear "matrix") fractions isolated from higher eukaryotic cells.