The pathways that regulate the S-phase events associated with the control of DNA replication are poorly understood. The bone marrow megakaryocytes are unique in that they leave the diploid (2C) state to differentiate, synthesizing 4 to 64 times the normal DNA content within a single nucleus, a process known as endomitosis. Human erythroleukemia (HEL) cells model this process, becoming polyploid during phorbol diesterinduced megakaryocyte differentiation. The mitotic arrest occurring in these polyploid cells involves novel alterations in the cdkl/cyclin Bi complex: a marked reduction in cdkl protein levels, and an elevated and sustained expression of cyclin Bi. Endomitotic cells thus lack cdkl/cyclin Bl-associated Hl-histone kinase activity. Constitutive overexpression of cdkl in endomitotic cells failed to re-initiate normal mitotic events even though cdkl was present in a 10-fold excess. This was due to an inability of cyclin-Bl to physically associate with cdkl. Nonetheless, endomitotic cyclin Bi possesses immunoprecipitable Hl-histone kinase activity, and specifically translocates to the nucleus. We conclude that mitosis is abrogated during endomitosis due to the absence of cdkl and the failure to form M-phase promoting factor, resulting in a disassociation of mitosis from the completion of S-phase. Further studies on cyclin and its interacting proteins should be informative in understanding endomitosis and cell cycle control. INTRODUCTION Two classes (or families) of proteins make up the protein-kinase complexes involved in the biochemical control of the cell cycle. The cell division kinases (CDKs), also referred to as cyclin-dependent kinases, are the catalytic subunits of these complexes, whereas the cyclins function as the regulatory subunits. Cyclins are proteins that undergo dramatic fluctuations in abundance as a function of cell cycle progression, and thus regulate the activation of the holoenzyme (Draetta and Beach, 1988;Draetta et al., 1989;Murray et al., 1989). Over the past few years, understanding of the biochemical control of the cell cycle has markedly improved, but is more complex than originally thought. For example, recent work demonstrated the existence of a family of related CDKs, each homologous to cdc2, the parent member of the CDK-family (cdc2 is also known as cdkl, which is the term used in this paper) (Meyerson et al., 1992). Likewise, there are at least eight members of the cyclin gene family Matsushime et al., 1991). Cyclin Bi is the best understood of the cyclins. It complexes with cdkl to form M-phase promoting factor (MPF), the mitosis-initiating protein kinase complex (Pines and Hunter, 1989;Riabowol et al., 1989). A-and E-type cyclins physically associate with cdkl and/or other cell cycle kinases (e.g., cdk2), and function at both G1/S and G2/M transitions (Dulic et al., 1992;Koff et al., 1992;Pagano et al., 1992). Finally, a family of G1 cyclins (D cyclins in mammals and Cln in yeast) recently were identified that are important in timing G1 progression as well as G1 /S trans...
The metabolic function and GM-CSF production rates of adherent human bone marrow stromal cells were investigated as functions of medium and serum feeding rates. A range of medium exchange schedules was studied, ranging from a typical Dexter culture protocol of one weekly medium exchange to a full media exchange daily, which more closely approximates what bone marrow cells experience in situ. Glucose consumption was found to be significantly higher at full daily exchange rate than at any other exchange schedule examined. However, the lactate yield on glucose was a constant, at 1.8 mol/mol, under all conditions considered. Differential serum vs. medium exchange experiment showed that both serum supply and medium nutrients were responsible for the altered behavior at high exchange rates. Glutamine consumption was found to be insignificant under all culture conditions examined. A change in exchange schedule from 50% daily medium exchange to full daily medium exchange after 14 days of culture was found to result in a transient production of GM-CSF and a change in metabolic behavior to resemble that of cultures which had full daily exchange from day one. These results suggest that both stromal cell metabolism and GM-CSF production are sensitive to medium exchange schedules. Taken together, the data presented indicate that attempts to model the function of human bone marrow in vitro may be well served by beginning with medium exchange schedules that more closely mimic the in vivo physiologic state of bone marrow.
Human bone marrow stromal cells repond to stimulation by the monokines IL-1 and TNF by producing colony-stimulating factors such as GM-CSF and G-CSF. In this study we show that I L -l a and TNFa act synergistically to stimulate GM-CSF and G-CSF production by cultured marrow stromal cells. We further show that I L -l a and TNFa synergistically stimulate production of GM-CSF and G-CSF by a clonal stroma-derived cell strain. Although IL-1 and TNF share many of the same biological activities, we show that I L -l a and TNFa have an unequal ability to induce myeloid-CSF production by both cultures, with I L -l a being the more potent inducer. We found that induction by I L -l a and TNFa was independent of cell proliferation. The effect of I L -l a and TNFa on production of the two myeloidCSFs by the clonal cells was significantly greater than the unfractionated passaged strornal cultures, having the greater effect on G-CSF production. The clonally derived stromal cells constitutively produced colony-stimulating activity, in particular GM-CSF, at levels easily detected by ELISA. These findings show that, in addition to the overlapping and additive activities of IL-la and TNFa, they can interact synergistically. Our findings further suggest that a small subpopulation of stroma cells may be the major producer of G-CSF in the marrow microenvironment during immune response. o 1994 ~i l e y -~i s s , Inc.
The metabolic and secretory characteristics of NIH-3T3 fibroblasts transfected with a cDNA encoding human granulocyte-macrophage colony stimulating factor (GM-CSF) were examined as a function of the culture medium exchange schedule. The rates of glucose and glutamine consumption and of lactate and ammonia production were measured over exchange schedules ranging from complete medium replacement weekly (1/week) to complete medium replacement daily (7/week). All measured metabolic rates increased with increased medium exchange rates and accelerated sharply between exchange rates of 3.5/week and 7/week. The lactate/glucose and ammonia/glutamine yield coefficients, however, remained invariant at about 1.9 and 1.0 mol/mol, respectively, under all medium perfusion conditions. A shift-up in medium perfusion rates from 3.5/week to 7/week resulted in increased metabolic rates that resembled those observed in the cultures that were exchanged at the 7/week rate throughout, showing that the metabolic rates could be directly controlled by the perfusion rate. Differential regulation of medium versus serum perfusion demonstrated that increased NIH-3T3 cell metabolism was directly proportional to the serum flux to which the cells were exposed. Thus a limiting serum component is responsible for the altered metabolic and growth rates. The GM-CSF production by the transfected 3T3 cells was stable but exhibited substantial transient increases during periods of cell proliferation, demonstrating that the secretion of transfected gene products can be highly modulated even when the cDNA is driven from a constitutive promoter. These studies show that the metabolic and secretory behavior of genetically engineered cells is influenced by the medium exchange schedule.
To explore the biochemical and physiologic basis of the overlapping effects of interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha) on myeloid cytokine production, we have studied the dynamics of granulocyte colony-stimulating factor (G-CSF) and granulocyte-monocyte colony-stimulating factor (GM-CSF) production as well as IL-1 receptor and TNF receptor expression in a clonally derived bone marrow stromal cell strain (CDCL). IL-1 alpha and TNF alpha act in a synergistic manner to stimulate G-CSF and GM-CSF production by CDCL, resulting in an increase in CSF secretion that is 250-fold greater than that observed with either cytokine alone. This synergism in protein secretion is paralleled by synergistic increases the steady-state level of GM- and G-CSF mRNA, with supra-additive levels achieved by 24 hours. Coincident with this synergistic induction of myeloid CSFs, treatment of CDCL cells with IL-1 alpha induces a 300% increase in the expression of TNF receptors. IL-1 alpha induction of TNF receptors reaches a peak after 6 hours and gradually returns to baseline level by 24 hours. IL-1 alpha does not affect TNF receptor ligand binding affinity. A kinetic study comparing IL-1/TNF synergistic induction of growth factor secretion with IL-1 alpha induction of TNF receptors shows that these events occur in parallel. In contrast with the induction of TNF receptors by IL-1 alpha, treatment with TNF alpha has no effect on either the number of IL-1 receptors expressed by CDCL cells or IL-1 receptor ligand binding affinity. Brief treatment of IL-1 alpha/TNF alpha-stimulated CDCL cells with cycloheximide before receptor induction reduces the synergistic increase in growth factor mRNA by 40% to 60% compared with cells not treated with CHX. Taken together, these results raise the possibility that IL-1 alpha cross-induction of TNF receptors may contribute to the biochemical mechanisms underlying the synergistic stimulation of G-CSF and GM-CSF production by IL-1 alpha and TNF alpha.
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