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The MTT assay, which is widely used to measure cell proliferation and to screen for anticancer drugs, is based on reduction of the tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by actively growing cells to produce a blue formazan product. Despite broad acceptance of this assay, neither the subcellular localization, nor the biochemical events involved in MTT reduction are known. Mitochondrial involvement in MTT reduction has been inferred from studies with respiratory inhibitors using succinate as a substrate, but the contribution of this activity to overall cellular MTT reduction is unknown. Using the bone marrow-derived cell line, 32D, we investigated the subcellular localization of MTT reduction using succinate, NADH, and NADPH as substrates. At optimum substrate concentrations, MTT reduction by whole cell homogenates was greatest with NADH and least with succinate, which accounted for less than 10% of the combined activities. Using succinate, 96% of recoverable MTT reducing activity was in particulate fractions of the cell and 77% in the mitochondrial and light mitochondrial/lysosomal fractions. When NADH and NADPH were used as substrates, increased amounts of MTT reducing activity were associated with soluble fractions of the cell and association with mitochondrial fractions was less pronounced. To further characterize MTT reduction by the mitochondrial fraction, respiratory chain inhibitors were used to explore involvement of electron transport in MTT reduction. Succinate-dependent mitochondrial MTT reduction was inhibited by 80% with chlorpromazine, 70% by antimycin A, and 85-90% by thenoyltrifluoroacetone (TTFA), but inhibition was not observed with rotenone at ≤2 μM, Amytal, or azide. These results suggest that when succinate is used as an electron donor, 70-80% of mitochondrial MTT reduction occurs subsequent to transfer of electrons from cytochrome c to cytochrome oxidase, but prior to the point of azide inhibition. In contrast to succinate, NADPH-dependent mitochondrial MTT reduction was not affected by any of the respiratory inhibitors tested, and NADH-dependent reduction was only inhibited by chlorpromazine (40-50% at plateau concentrations). These results suggest that most cellular MTT reduction occurs outside the mitochondrial inner membrane and involves NADH and NADPH-dependent mechanisms that are insensitive to respiratory chain inhibitors. This interpretation is supported by whole cell studies in which rotenone failed to affect basal and interleukin-3-stimulated MTT reduction at times up to 4 h but strongly inhibited DNA synthesis. We conclude that most cellular reduction of MTT occurs extramitochondrially and probably involves the pyridine nucleotide cofactors NADH and NADPH.

The stem cell-platelet lineage is uniquely defined by platelet cell-lineage antigens. These antigens are present on all stem cells measured by the spleen colony assay and become restricted to the platelet cell lineage as differentiation proceeds. In this study, anti-platelet serum (APS) has been used to identify cells in the bone marrow that express platelet cell-lineage antigens and to identify platelet cell surface molecules expressing these antigens. Anti-platelet IgG extensively absorbed with brain, thymus, and peritoneal cells bound selectively to stem cells, megakaryocyte progenitor cells (Mk-CFC), and megakaryocytes in CBA mouse bone marrow and to blood platelets. No other hemopoietic cell type, tissue, cell line, or tumor cell bound significant amounts of antibody against platelet cell-lineage antigens as determined by ability to absorb the anti-stem cell activity in APS. Studies with lactoperoxidase-labeled platelets showed that two major iodinated proteins of Mr= 114,000 and 138,000 were immunoprecipitated with APS and with antiserum that had been extensively absorbed. These proteins correspond to the platelet llb-llla glycoprotein complex, which is known to express receptors for collagen and fibrinogen, molecules known to influence hemopoietic cell proliferation and tumor cell growth. A panel of six monoclonal antibodies against human llb-llla inhibited spleen colony formation by 17% to 100%, J15 and A5.15 also being cytotoxic for granulocyte-macrophage progenitor cells and Mk-CFC. Other platelet monoclonal antibodies did not inhibit spleen colony formation. Although APS inhibited fibrinogen binding to platelets and platelet aggregation, these activities were greatly reduced with absorbed antiserum. Furthermore, fibrinogen treatment of bone marrow did not block the anti-stem cell activity in APS. Thus the evidence is consistent with expression of platelet cell-lineage antigens on the platelet llb-llla glycoprotein complex at a site removed from the fibrinogen binding site. © 1985 by Grune & Stratton, Inc.