Apoptosis is regulated by the rate of glucose transport in an IL-3-dependent haemopoietic cell line.

In the absence of a survival stimulus such as a growth factor, haemopoietic progenitor cells undergo the process of programmed cell death known as apoptosis [ I ,2]. Suppression of apoptosis by specific oncogenes also appears to represent a key facet of leukaemic transformation. For example, progenitor cells from patients with chronic myeloid leukaemia, which is associated with an activated form of the Abelson protein tyrosine kinase [3], exhibit an enhanced survival potential in the absence of haemopoeitic growth factors [4]. To study the molecular mechanisms governing suppression of apoptosis by growth factors and oncogenes, we have employed an interleukin-3 (IL3)-dependent haemopoietic cell line (IC.DP) which expresses a temperature-sensitive mutant of the v-abl tyrosine kinase (v-ABL) [5]. Activation of the kinase at the permissive temperature permits cell survival in the absence of IL-3 by suppression of apoptosis, although the growth factor is still required for proliferation [6]. Glucose transport is known to be stimulated by IL-3 and other growth factors that suppress apoptosis. In view of the partial abrogation of IL-3-dependence by activation of V-ABL, we have assessed the effects of this activation on glucose uptake by IC.DP cells, and have investigated the relationship between glucose transport and apoptotic suppression. Glucose uptake into IC.DP cells grown at either the restrictive temperature (39'C) orthe permissive temperature (32°C) for V-ABL was found to be almost completely inhibitable by cytochalasin B, indicating that transport in these cells is catalysed by a member of the GLUT family of passive transporters [7]. Western blotting of IC.DP membranes using isoform-specific anti-peptide antibodies showed that of the known transporter isoforms, only GLUT1 was present. IL-3-deprivation of IC.DP cells grown at the restrictive temperature (39'C) was found to lead to a substantial decrease (>50% after 8 h deprivation of the growth factor) in hexose transport, as measured by the uptake of 'H-labelled 2-deoxy-D-glucose (1 mM). Readdition of IL-3 caused a rapid return of uptake rates to the original levels, with a t% of about 1 %h. However, in contrast to this behaviour at 3 9 T , IL-3 deprivation of cells incubated at the permissive temperature (32°C) did not result in the loss of transport activity. Furthermore, switching IL-3deprived cells from 39'C to 32'C reversed the previous decline in transport activity. These findings suggest that activation of the kinase can, at least in part, mimic the effect of IL-3 on glucose transport. Measurements of initial rates of sugar uptake over a range of 2-deoxyglucose concentrations showed that stimulation of transport by activation of the kinase for 2 h in IL-3-deprived cells was associated with a significant increase in the V, for transport, from 1.52 f 0.09 to 2.48 f 0.15 nmollminll0' cells (mean f SEM, p<O.OI), and with a significant decrease in the K,,,, from 4.71 f 0.28 mM to 2.78 f 0.30 mM (p<O.OI). This increase transport activity was independent of protein synthesis and was not mirrored by any change in the total GLUT1 content of the cells as measured by quantitative Western blotting using an antipeptide antibody raised against the C-terminus of the transporter [8]. However, confocal immunofluorescence microscopy provided evidence of a significant translocation of GLUT1 from a cytoplasmic, vesicular pool to the cell surface both upon activation of the kinase and upon readdition of growth factor to IL-bdeprived cells incubated at the nonpermissive temperature for the kinase. These results suggested that suppression of apoptosis upon activation of v-ABL might be mediated, at least in part, via its effect upon sugar uptake by the IC.DP cells. To investigate this hypothesis further, we examined the effects of two inhibitors of glucose transport, cytochalasin B and maltose. Both of these agents inhibited the suppression of apoptosis brought about by activation of V-ABL, whereas the non-transport-inhibitory analogues dihydrocytochalasin B and sucrose had much lesser effects. Furthermore, the effects of both cytochalasin B and maltose on the rate of apoptosis could be reversed, at least in part, by the provision of glutamine or pyruvate as alternative energy sources. These findings provide strong support for the hypothesis that deprivation of glucose, a major energy source for haemopoietic cells, plays a part in the induction of apoptosis by IL-3 deprivation. In summary, our results indicate that the supply of energy in the form of glucose or other substances directly influences the rate of apoptosis in IC.DP cells. Both IL-3 and activation of the kinase were found to stimulate the rate of sugar uptake by the cells by a protein synthesisindependent process involving translocation of intracellular GLUT1 to the cell surface. Regulation of glucose transport by oncogenes and cytokines therefore probably represents a key control point in the suppression of apoptosis. We do not suggest that this is the sole regulatory point governing apoptosis in haemopoietic cells: cytokines and other proteins such as bcl-2 probably elicit a complex set of effects to suppress apoptosis. However, our findings do implicate one aspect of cellular metabolism that may be disregulated in multi-stage leukaemogenesis.