Rhodamine 123, a fluorescent dye which binds as a result of the transmembrane potential, was used to stain the mitochondria of HL-60 cells, a cell line established from human promelocytic leukemia cells. The DMSO-induced differentiation of promyelocytic cells into mature granulocytes caused a fourfold decrease in fluorescence intensity that paralleled the disappearance of S-phase and G2M cells. This suggests that upon myeloid differentiation whereby the cells enter an irreversible quiescent state, the mitochondrial mass of the cells has decreased. This suggestion is corroborated by electron microscopy, which shows a decrease in the number of mitochondria, and by decreases in total mitochondrial protein and cytochrome oxidase activity. The respiratory rate of isolated mitochondria did not change, suggesting that the transmembrane potential remained the same. Undifferentiated cells in exponential phase of growth exhibit an intracellular heterogeneity of fluorescence intensity. This heterogeneity appears to have a cell age basis, as late S/G2M cells, obtained by centrifugal elutriation, yielded twice the fluorescence intensity of early G1 cells.

A flow cytometric method for quantitation of glutathione (GSH) was applied to simultaneous analysis of the major leukocyte types in peripheral blood. Cellular thiols (predominantly GSH) were stained with monochlorobimane (MCIB), and thiol fluorescence was measured with a flow cytometer. The fluorescence of the thiols closely reflected the GSH content, as measured by a specific glutathione reductase assay. Fluorescence of individual cell types could be measured after delineating those cells by their light-scatter characteristics, utilizing dual-angle light scatter for discrimination. By this means, GSH contents of 12.5 +/- 2.0 nmol/10(7) neutrophils, 14.5 +/- 2.7 nmol/10(7) monocytes, and 5.0 +/- 1.0 nmol/10(7) lymphocytes were found. The results obtained for neutrophils with the flow cytometer were virtually identical with those obtained with chemical assay in purified samples of neutrophils, indicating the validity of the flow cytometric method.

A new marker for young oligodendrocytes has been identified by a monoclonal antibody (mOg-1, IgM isotype) prepared from cerebellar plasma membrane stimulated mouse lymphocytes. mOg-1 reactive cells in the mouse cerebellum first appear at day 19 of gestation. Future white matter layers of fixed sections of neonatal rat cerebellum were labeled with mOg-1. Although EM analysis has shown cell-surface binding by presumptive oligodendroglia in neonatal cerebellum, the antibody does not bind to compact myelin. In cell cultures prepared from L-d-old mice, 1.1% of the cells bound mOg-1 after 3 d in culture, but up to 5% of the cells bound mOg-1 after 2 weeks in culture. Of these same Og-l-positive cells, 69% bound anti-gabtctocerebroside and 65% bound anti-myelin basic protein. After a week in culture Og-l-positive cells often produced lamellar sheets extending a millimeter over the polylysine substratum in the absence of normal myelin formation. mOg-1 recognizes a cell-surface determinant distinct from well-characterized oligodendroglial molecules (galactocerebroside, sulfatide and myelin basic protein) that is expressed early in oligodendrocyte development. The antibody has been used to follow the maturation of oligodendrocytes in cultures of both normal and jimpy mouse cerebellum.The nervous system is a complex network derived from multiple associations between different cell types. The limited number of distinct cell types and the availability of neurological mutants with defective cerebellar histogenesis make the mouse cerebellum an attractive model for studying cell-cell interactions.The development of antibodies toward neural cell-surface molecules could be instrumental in establishing the role of specific determinants in histogenesis. We have used the hybridoma technology of Milstein (1975, 1976) In this report, we describe a monoclonal antibody (mOg-1) reactive with the surface of young oligodendrocytes, prior to the Received Dec. 19, 1985; revised Mar. 4, 1986; accepted Mar. 4, 1986. We woula like to thank Dr. Robert Lasher for his helpful discussions and S.Hat&e for her expert assistance. We thank Drs. Dahl, De.Vellis, Evans, Habig, Qua&s, Ranscht, and Rapport for their generous girt of antisera and tetanus toxin used in these studies. Some of these studies were submitted in partial fulfillment of the requirementi for the Doctor of Philosophy degree from the University of Colorado (1983). This work was su~ucwted in nart bv USPHS Grants NS-098 18and T32-bM-07342.

In the design of first-in-human (FIH) studies in oncology, some changes are incremental; others are major shifts in goals and approaches. The contemporary emphasis on molecularly targeted agents has accelerated the trend toward biomarker studies, but these investigations remain as secondary findings in phase I behind the classic end points of toxicity and occasional tumor shrinkage. In contrast, evaluation of target modulation is the primary goal of phase 0 and is intended to inform decision making regarding further development of the relevant compound. Moving PharMacodynaMics FroM a correlative to a Pivotal PositionThere is always some blurring of the stages of a compound's journey toward the clinic. For many investigators, the firstin-human (FIH) experience is viewed as the end of discovery and the beginning of development. Phase 0 studies are explicitly designed to extend the process of discovery into FIH studies to obtain information that will shape the beginning of the development process that will follow if the FIH results are satisfactory. 1,2 As indicated in Table 1, the term "phase 0" was chosen to signify a major change in the structure of FIH studies of anticancer drugs. Prior to the 1980s, FIH studies in oncology focused on determination of toxicity, with only occasional pharmacokinetic (PK) or pharmacodynamic (PD) investigations. PK studies became more common in the 1980s, eventually leading to exploration of PK-guided dose-escalation designs. 3 The 1990s brought more extensive PK and PD studies. Attempts to associate PK/PD with clinical outcomes were designated as "correlative" studies, although many were ancillary to the main development path.Phase 0 studies were introduced in the current decade and are more than mere extensions of the trend toward more biomarker studies in phase I. The fundamental difference as regards phase 0 is that PD or biomarker studies are the primary outcome, not simply correlative or ancillary. Indeed, correlations with clinical outcome are not feasible, because neither toxicity nor activity is expected in phase 0.The most important change in drug development in oncology over the past decade has been the type of compound entering the clinic. For a compound in the "molecularly targeted" category, there is often excellent preclinical data to formulate a hypothesis regarding its mechanism of action. The most relevant test of the hypothesis would be an examination of the compound's behavior in patients. It therefore seems reasonable to expect early human studies to be specifically designed to test mechanistic hypotheses. Further, the early testing of hypotheses should shape development decisions.The key to decision making is the identification of the pivotal information for a "stop-or-go" decision. The design of the first clinical study must be customized to obtain that information. Because each compound and each target is different, the most important information sought from each trial will vary. For most target-oriented programs, PD measurements are the most important. If th...

A commerical cell sorter was used to obtain preparations of cells in various stages of granulocyte development from rabbit marrows stimulated by inflammatory response. Marrow cells were fractionated on density gradients of Ficoll/Hypaque and each fraction sorted using light scatter. Trial and error selection of appropriate gradient fractions and light scatter windows allowed sorting of early (blast cells, promyelocytes), intermediate (myelocytes, metamyelocytes) and late stage (band cells, polys) granulocytes with enhanced purity.