A B S T R A C T Monoclonal antibodies were prepared against pyruvate kinase (PyKi; ATP: pyruvate phosphotransferase, EC 2.7.1.40) and used to quantitate PyKi content in L2 lung cells and WI-38 fibroblasts cultivated under hypoxic and normoxic conditions. After 96 h of hypoxic cultivation, PyKi activity was significantly increased in both cell types (L2: normoxia [Po2 = 142 torr], 0.11+0.01 [SD]; hypoxia [Po2 = 14 torr], 0.25 ±0.04 U/,ug DNA, P < 0.01). PyKi content increased proportionately in both cell lines (L2: normoxia, 0.44 +0. 13; hypoxia, 0.94±0.13 ,g enzyme protein/,Lg DNA). Specific activity was not significantly different after 96h (L2: normoxia, 261±11; hypoxia, 261+14 U/mg enzyme protein). These results indicate that regulation of glycolysis during chronic hypoxia occurs at the level of enzyme content. Chronic 02 depletion leads to either an increased rate of biosynthesis or a decreased rate of biodegradation of PyKi, causing augmented glycolytic capacity. Monoclonal antibodies provide a highly specific, convenient approach to characterizing enzymes, as well as quantitating cellular enzyme content. INTRODUCTIONChronic 02 depletion is an important clinical problem, but the cellular mechanisms responsible for adaptation to chronic hypoxia are obscure. The increased rate of glycolysis following acute hypoxic exposure was first described by Pasteur [3][4][5]. This stimulation ofglycolysis by chronic hypoxic exposure may also explain the bioenergetic differences observed between pulmonary artery endothelial cells (Po2 = 40 torr) and aortic endothelial cells (Po2 = 100 torr), as well as the differences observed in fetal and neonatal brain (6,7). Increased activity of rate-limiting glycolytic enzymes has also been found in fibroblast, endothelial, and kidney cell lines cultured under hypoxic conditions. Therefore, the phenomenon appears to be a common cellular response to 02 depletion. To investigate the mechanism by which the activity of PyKi is increased following chronic hypoxic exposure, we determined the isozyme type and content of PyKi present in a cloned cell line derived from rat lung (L2) and in WI-38 fibroblasts following hypoxic or nonnoxic cultivation. The results show an increase in the intracellular content of PyKi, with no significant change in specific activity (PyKi activity/PyKi content) or isozyme type present after hypoxic cultivation. This suggests that the availability of molecular 02 modulates the content of key glycolytic enzymes, which in turn regulate the generation of ATP through glycolysis. METHODSCell culture. L2 clone derived from rat lung was obtained from Dr. William Douglas in 36th population doubling (8) and maintained in cell culture by using Ham's F-12 medium containing 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 ,ug/ml streptomycin. Confluent plates were trypsinized and replated weekly with a 1:4 split, and experiments were performed on cells between the 40th and 55th population doubling.WI-38 fibroblasts were obtained from the American Type Culture Collect...
The human promyelocytic leukemia cell line HL-60 is reactive with an antiserum raised against normal human granulocytes (AGS). Immunoprecipitation with AGS on [ S]methioninelabeled HL-60 cell lysates with subsequent analysis by NaDodSO4/polyacrylamide slab gel electrophoresis shows a major antigenic doublet with molecular weights-of 88,000 and 86,000, together with some minor antigens of lower molecular weight. Upon stimulation with dimethyl sulfoxide or 12-O-tetradecanoylphorbol 13-acetate, which induces HL-60 to differentiate to mature granulocytes or monocytes/macrophages, respectively, this antigenic doublet disappears. 12-O-Tetradecanoylphorbol 13-acetate induces the synthesis of an antigen, molecular weight 83,000, reactive with an antimonocyte serum. Neutrophil-specific alloantigens were not detected on HL-60 or its differentiated derivatives.With the aid of the appropriate human antisera in leukoagglutination or indirect immunofluorescence assays, several neutrophil-specific alloantigen systems have been detected on human granulocytes (1-4). In addition, neutrophil-specific xenogeneic antisera have been produced (5-7).Recently, a human leukemic cell line (HL-60) has been established from the peripheral blood of a patient with acute promyelocytic leukemia, which shows myeloid characteristics (8). Morphologically, the cultured HL-60 cells are predominantly promyelocytes, although some mature myelocytic cells are also present. Furthermore, cells of this cell line can be induced to differentiate in culture. Incubation with dimethyl sulfoxide (Me2SO) induces HL-60 to maturate into myelocytes and granulocytes (9) which have acquired functional properties characteristic of these cells (10). In contrast, the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) induces differentiation ofHL-60 along the monocyte/macrophage pathway, with a concomitant increase in phagocytic capacity and adherence to plastic surfaces (11).Other cell lines of putative myeloid origin include K562 (12) which, by both immunological (13) and biochemical (14) criteria, is an erythroleukemic cell line, and the early myeloid cell line KG-1, which expresses the HLA-DR antigen (15, 16). The relatively mature character of HL-60 is further evidenced by its lack of HLA-DR antigen normally present on myeloblasts (17).In this study, antigens present on HL-60 at its various stages of differentiation were detected by immunofluorescence with a set of reagents that included xenogeneic-antisera with specificity for granulocytes and monocytes, respectively. We further analyzed these antigens by immunoprecipitation on radioactively labeled cell lysates, followed by NaDodSOjpolyacrylamide gel electrophoresis. This study has potentially important consequences for analysis of antigens on leukemic cells present in patients with chronic myeloid leukemia and acute myeloblastic leukemia. MATERIALS AND METHODS Cell Lines and Conditions for Growth and Differentiation.The continuous suspension cell lines used in this study were HL-60 (8), K562 (12), ...
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