Mitochondrial recoupling: a novel therapeutic strategy for cancer?

Recent findings link metabolic transformation of cancer cells to aberrant functions of mitochondrial uncoupling proteins (UCPs). By inducing proton leak, UCPs interfere with mitochondrial synthesis of adenosine 5'-triphosphate, which is also a key determinant of glycolytic pathways. In addition, UCP suppress the generation of superoxide, a byproduct of mitochondrial electron transport and a major source of oxidative stress. The near ubiquitous UCP2 becomes highly abundant in some cancers and may advance metabolic reprogramming, further disrupt tumour suppression, and promote chemoresistance. Here we review current evidence to suggest that inhibition of mitochondrial uncoupling may eliminate these responses and reveal novel anti-cancer strategies.

G-protein specificity in signaling pathways that mobilize calcium in insulin-secreting beta-TC3 cells.

Fuel- and receptor-induced signal transduction pathways were investigated in beta-TC3 cells, an insulin-secreting cell line. An increase of glucose concentration from 0 to 15 mM and stimulation with 0.5 mM carbachol resulted in up to a twofold increase in insulin secretion by beta-TC3 cells, and their simultaneous addition increased insulin release eightfold. In single fura 2-loaded cells, a potentiating effect of carbachol was also observed on glucose-induced intracellular Ca2+ mobilization. Immunoblotting with antibodies raised to the COOH-terminal of G-protein alpha-subunits showed that G alpha i, G alpha o, and G alpha q are present in beta-TC3 cells in commensurable quantities. The novel technique of microinjection of anti-G alpha antibodies into single beta-cell was used to probe the functional role of these G-proteins. Microinjection of anti-G alpha i antibodies into beta-TC3 cells had no effect on glucose- and carbachol-induced Ca2+ mobilization. However, anti-G alpha q completely inhibited the Ca(2+)-mobilizing effect of carbachol, but not of glucose, within 5 min. Microinjection of anti-G alpha o antibodies had no effect on carbachol-induced Ca2+ mobilization. Microinjection of anti-G alpha i and anti-G alpha q antibodies had a minimal effect on glucose-induced Ca2+ mobilization (< 8% of cells nonresponding), but microinjection of anti-G alpha o increased the proportion of nonresponding cells to 37%. The results suggest that, in beta-TC3 cells, distinct signal transduction pathways with specific G-protein involvement may interact with secretagogue-induced Ca2+ mobilization and, ultimately, with insulin secretion.

Disturbed intracellular calcium-related processes of hepatocytes and neutrophils in human alcoholic liver disease.

Isolated human hepatocytes and separated neutrophils of 11 patients with alcoholic liver disease (ALD) were used to study some aspects of cellular calcium-related processes compared to nonalcoholic controls. 45Ca2+ efflux from the cells decreased in ALD and the calmodulin-inhibitor trifluoperazine did not influence it further. The intracellular free calcium concentration [( Ca2+]i) of nonstimulated hepatocytes and neutrophils proved to be higher in ALD with the Quin2/AM loading technique. However, the [Ca2+]i rise in hepatocytes and neutrophils, with stimulation by low density lipoprotein (LDL) and N-formyl-methionyl-leucyl phenylalanine (FMLP), respectively, was diminished in ALD compared to appropriate controls. The slower 45Ca2+ extrusion rate, higher basal [Ca2+]i levels, and the diminished [Ca2+]i elevation of activated hepatocytes and neutrophils, suggest disturbed calcium-related intracellular processes in ALD, in particular, impaired regulation of the plasma membrane Ca2(+)-ATPase.

Association of Zollinger-Ellison syndrome with pancreatitis: report of five cases.

In a retrospective analysis, five cases of Zollinger-Ellison syndrome were found in a typical urban inner-city teaching hospital. Chronic alcohol abuse and heavy smoking characterized these patients, and four of them also had pancreatitis, suggesting an association of gastrin-producing tumors and pancreatic inflammation. Ductal obstruction by neuroendocrine tumors has been reported to cause pancreatitis in a few cases. In this analysis, however, a nonobstructive gastrinoma was the surgical diagnosis in three patients, and it was suggested by imaging studies in the two other cases. The potential other pathomechanisms for a dual cause-effect relationship of gastrinoma and pancreatitis are discussed.

Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production.

The regulation of intracellular pools of Ca2+ was investigated in an interleukin-3 (IL-3)-dependent hematopoietic cell line 32D that undergoes programmed cell death ("apoptosis") when deprived of lymphokine. Comparisons were made with 32D cells that had been stably transfected with a bcl-2 expression plasmid that encodes a 26-kDa intracellular integral-membrane protein known to abrogate apoptosis resulting from IL-3 withdrawal. Removal of IL-3 from cultures of 32D cells or control-transfected 32D-NEO cells for 1-2 days led to cell cycle arrest and oligonucleosomal DNA fragmentation and was associated with lower cytosolic free Ca2+ concentrations ([Ca2+]i), as measured by Indo-1 fluorescence of viable cells in Ca(2+)-containing media. In bcl-2-expressing 32D-BCL2 cells, IL-3 withdrawal also resulted in cessation of proliferation, but [Ca2+]i levels were not decreased and DNA fragmentation was markedly suppressed. Nonmitochondrial stores of Ca2+ were also significantly diminished in IL-3-deprived 32D-NEO but not in 32D-BCL2 cells, based on measurements of Ca2+ release into the cytosol following exposure of cells to thapsigargin (an inhibitor of endoplasmic reticulum Ca(2+)-ATPases) under Ca(2+)-free conditions. In contrast, estimates of mitochondrial Ca2+ stores using an uncoupler of oxidative phosphorylation 1799 (2,6-dihydroxy-1,1,1,7,7,7-he xafluoro-2,6-bis(trifluoromethyl)heptan-4-one[bis(he xafluoroacetonyl)]acetone) suggested that IL-3 deprivation leads to an increase in this intracellular pool of Ca2+ in 32D-NEO but not in 32D-BCL2 cells. Re-addition of IL-3 to factor-deprived 32D-NEO cells reversed the changes in thapsigargin- and 1799-releasable Ca2+ pools and rescued many of the cells from death. Measurements of total cellular Ca2+ revealed no difference in 32D-NEO cells before and after IL-3 withdrawal, suggesting that the observed alterations in mitochondrial and nonmitochondrial Ca2+ pools result from intracellular repartitioning of Ca2+. Treatment of IL-3-deprived 32D-NEO cells with Ca2+ ionophores blocked DNA fragmentation and prolonged cell survival, whereas addition of Ca2+ chelators to IL-3-stimulated 32D cells resulted in oligonucleosomal DNA fragmentation and cell death, suggesting that diminutions in the concentrations of Ca2+ in cytosol, endoplasmic reticulum, or other intracellular compartments either directly or indirectly regulate apoptosis in these lymphokine-dependent hematopoietic cells.

Growth arrest of a murine mesangial cell line by transforming growth factor beta 1 is associated with inhibition of mitogen-induced Ca2+ mobilization.

Transforming growth factor-beta (TGF beta) is a multifunctional cytokine showing growth effects on many cell types. In the present study effects of TGF beta 1 on mitogen-induced Ca2+ responses were investigated in an immortalized murine mesangial cell line where TGF beta 1 effects on growth are inhibitory. TGF beta 1 was found to inhibit intracellular Ca2+ mobilization induced by platelet-derived growth factor (PDGF). This effect was completely reversed by previous addition of the non-specific serine/threonine kinase inhibitor H-7, but was unaffected by GF 109203X, a specific inhibitor of protein kinase C (PKC). These findings suggest that inhibition of mitogen-induced Ca2+ mobilization by TGF beta 1 appears not to involve prior activation of PKC, but may participate in the mechanisms whereby mesangial cell growth is inhibited by TGF beta 1.

G protein coupling to the thrombin receptor in Chinese hamster lung fibroblasts.

The specific involvement of G proteins in thrombin receptor-mediated Ca2+ mobilization and DNA synthesis has been studied in single Chinese hamster lung fibroblasts (CCL39 cells) activated by the hexapeptide SFLLRN. Immunoblots performed with antibodies directed against the COOH terminus of G protein alpha subunits revealed that alpha q, alpha i, and alpha o were each present in CCL39 cells. The Ca2+ response to SFLLRN was measured after microinjection of anti-alpha q or anti-alpha o antibodies, which produced a total blockade in 71 and 46% of cells, respectively. A partial inhibition of the SFLLRN-induced Ca2+ response was observed in the remaining cells. The lag time between exposure of the cells to SFLLRN and the onset of Ca2+ mobilization was significantly longer (20-24 s) in cells microinjected with anti-alpha q- or anti-alpha o-antibodies than in control cells microinjected with preimmune serum (9 +/- 1 s). Moreover, the peak height of the Ca2+ response to SFLLRN was decreased by 36 and 73%, respectively in cells microinjected with anti-alpha q or anti-alpha o antibodies. SFLLRN-induced DNA synthesis in growth-arrested CCL39 cells was also inhibited (44-78%) by prior microinjection of anti-alpha q or anti-alpha o antibodies. Anti-alpha 1 antibodies had no effect on the SFLLRN-induced Ca2+ response or on DNA synthesis. These results provide direct evidence that the thrombin receptor in CCL39 cells is coupled to two different types of G proteins, Gq and Go, both causing Ca2+ mobilization and mitogenesis.

Hepatocyte growth factor induces calcium mobilization and inositol phosphate production in rat hepatocytes.

The effects of hepatocyte growth factor (HGF) on intracellular Ca2+ mobilization were studied using fura-2-loaded single rat hepatocytes. Hepatocytes microperfused with different amounts of HGF responded with a rapid concentration-dependent rise in the cytosolic free Ca2+ concentration with a maximum increase of 142% at 80 ng/ml of HGF. The lag period of the Ca2+ response was decreased with increasing HGF concentrations, being 64 +/- 12 s, 42 +/- 6 s, and 14 +/- 2 s, respectively, with 8, 20, and 80 ng/ml of HGF. The detailed pattern of Ca2+ transients, however, was variable. Out of 16 cells tested using 20 ng/ml of HGF, 68% showed sustained oscillatory responses, whereas other cells showed a sustained increase in the cytosolic-free Ca2+ upon exposure to HGF, which was dependent on the presence of extracellular Ca2+. HGF also induced Ca2+ entry across the plasma membrane. Mobilization of Ca2+ by HGF was accompanied by a rapid accumulation of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3). The effects of HGF and epidermal growth factor (EGF) were comparable and partly additive for Ins 1,4,5-P3 production and for the sustained phase of Ca2+ mobilization. Preincubation of cells with 10 microM of genistein to inhibit protein tyrosine kinases abolished the HGF-induced Ca2+ response and also inhibited HGF-induced Ins 1,4,5-P3 production in rat liver cells. These data indicate that early events in the signal transduction pathways mediated by HGF and EGF have in common the requirements for tyrosine kinase activity, Ins 1,4,5-P3 production, and Ca2+ mobilization.

The nature of after-hours telephone medical practice by GI fellows.

OBJECTIVE: Gastroenterology fellows on-call often serve as the initial or only contact for patients calling "after hours" with questions and symptoms. These fellows are rarely trained specifically in how to handle these calls. The aim of this study was to determine whether there are particular topics in telephone medicine that ought to be covered in new fellow training. Therefore, we sought to evaluate the nature of after-hours pages initiated by patients and to document the advice given by fellows. METHODS: The content of 100 patient-initiated telephone calls with GI fellows was recorded prospectively over 7 months. We included pages received between 5 PM and 8 AM daily as well as daytime calls on weekends. Fellows documented the time and length of the call, the issue raised by the patient, the advice given, and the patient's gender and attending gastroenterologist. When a particular patient paged more than once in a 24-h period, the repeat calls were not counted toward the 100-call tally. RESULTS: Twenty-two percent of calls occurred between 11 PM and 7 AM. Eighty-three percent of calls lasted less than 10 min. Sixty-seven percent of patients called because of symptoms. Only 30% of patients calling with symptoms were referred to the emergency room. Although only 1 of 13 patients with procedure-related (i.e., postendoscopy) symptoms required admission to the hospital, 18 of 54 (33%) patients with nonprocedure-related symptoms required admission either immediately or within a month of calling after hours. CONCLUSIONS: Most after-hours calls from patients are related to symptoms. Patients calling with postprocedure symptoms rarely require admission to the hospital. Conversely, a significant number of patients calling with non-procedure-related symptoms require admission within 30 days. Fellowship directors should consider providing training to fellows in the evaluation of symptoms over the telephone.

Epidermal growth factor-mediated signaling of G(i)-protein to activation of phospholipases in rat-cultured hepatocytes.

Hepatocytes were established in tissue culture in order to study the effects of pertussis toxin (PT) on epidermal growth factor (EGF)-mediated cellular responses under in vitro conditions. EGF caused a 3-fold increase of myo-inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) mass and a 50% increase of diacylglycerol mass within the first minute, with the change of diacylglycerol content being 100-fold greater than that of Ins-1,4,5-P3. Diacylglycerol, but not Ins-1,4,5-P3, continued to accumulate over several hours, indicating that EGF increased the hydrolysis of lipids other than phosphatidylinositol 4,5-bisphosphate (PIP2). EGF increased phosphoinositide-specific phospholipase C-gamma (PLC-gamma) tyrosine phosphorylation within 1 min, but no effect was observed with vasopressin, insulin, or glucagon after 5 min. EGF also caused a rapid, tyrosine kinase-dependent association of G(i) alpha with PLC-gamma, which was maximal within 10 min. In contrast to our previous data on fresh hepatocytes, PT had no effect on the EGF-induced tyrosine phosphorylation of PLC-gamma, although Ins-1,4,5-P3 and diacylglycerol production were inhibited. The role of G-proteins in EGF signaling was investigated further by microinjection of G alpha antibodies into single fura-2-loaded hepatocytes. Anti-G(i) alpha (common) antibodies prevented EGF-induced but not vasopressin-induced Ca2+ transients. These results strengthen previous observations that a PT-sensitive G-protein is involved in EGF-mediated phospholipid metabolism in hepatocytes and show that tyrosine phosphorylation of PLC-gamma is an insufficient signal for activation of PIP2 hydrolysis.

Pertussis toxin-sensitive Gi protein involvement in epidermal growth factor-induced activation of phospholipase C-gamma in rat hepatocytes.

Treatment of rat hepatocytes with epidermal growth factor (EGF) produced an enhanced tyrosine phosphorylation of the EGF receptor and phospholipase C-gamma (PLC-gamma) in conjunction with the mobilization of Ca2+. Approximately 30% of the total PLC-gamma was tyrosine-phosphorylated with a maximum being reached after 30 s of incubation with EGF. Pretreatment of the rats with pertussis toxin prior to isolation of the hepatocytes blocked EGF-induced tyrosine phosphorylation of PLC-gamma and Ca2+ mobilization but had no effect on autophosphorylation of the EGF receptor or Ca2+ responses elicited by angiotensin II or phenylephrine. Under these conditions Gi protein alpha subunits were fully ADP-ribosylated. A 41-kDa Gi protein alpha subunit was found to be present in the anti-PLC-gamma immune complex after EGF stimulation as shown by in vitro ADP-ribosylation using [32P]NAD+ and activated pertussis toxin. The kinetics of association between PLC-gamma with Gi alpha protein reached a maximum after 1 min of incubation with EGF. Antibodies specific for the EGF receptor also coimmunoprecipitated a Gi protein alpha subunit. Treatment of hepatocytes with EGF caused first an increase and then a decrease in the amount of Gi protein alpha subunit associated with the EGF receptor. In contrast, studies with cultured rat liver (WB) cells, a cell line in which EGF stimulation of phosphoinositide hydrolysis is not inhibited by pertussis toxin, showed that a stable complex of Gi alpha was not formed with either PLC-gamma or EGF receptor immunoprecipitates. These results indicate that a pertussis toxin-sensitive Gi protein is uniquely involved in the signal transduction pathway mediating EGF-induced activation of PLC-gamma and Ca2+ mobilization in hepatocytes.

Mitochondrial protein p26 BCL2 reduces growth factor requirements of NIH3T3 fibroblasts.

The BCL2 (B cell lymphoma/leukemia-2) proto-oncogene encodes a 26-kDa protein that has been localized to the inner mitochondrial membrane and that has been shown to enhance the survival of some types of hematopoietic cells. Here we show that NIH3T3 fibroblasts stably transfected with a BCL2 expression plasmid exhibit reduced dependence on competence-inducing growth factors (platelet-derived growth factor, PDGF; epidermal growth factor, EGF) for initiation of DNA synthesis. The importance of BCL2 for growth factor-induced proliferation of these cells was further confirmed by the useage of BCL2 antisense oligodeoxynucleotides. The mechanisms by which overexpression of p26 BCL2 contributes to fibroblast proliferation are unknown, but do not involve alterations in: (a) the production of inositol triphosphates (IP3), (b) PDGF-induced transient elevations in cytosolic Ca2+ ions, or (c) the activity of protein kinase C enzymes in these transfected cells. The results imply that changes in mitochondrial functions play an important role in the early stages of the cell cycle that render 3T3 cells competent to respond to the serum progression factors that stimulate entry into S-phase.

Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes.

beta cells sense glucose through its metabolism and the resulting increase in ATP, which subsequently stimulates insulin secretion. Uncoupling protein-2 (UCP2) mediates mitochondrial proton leak, decreasing ATP production. In the present study, we assessed UCP2's role in regulating insulin secretion. UCP2-deficient mice had higher islet ATP levels and increased glucose-stimulated insulin secretion, establishing that UCP2 negatively regulates insulin secretion. Of pathophysiologic significance, UCP2 was markedly upregulated in islets of ob/ob mice, a model of obesity-induced diabetes. Importantly, ob/ob mice lacking UCP2 had restored first-phase insulin secretion, increased serum insulin levels, and greatly decreased levels of glycemia. These results establish UCP2 as a key component of beta cell glucose sensing, and as a critical link between obesity, beta cell dysfunction, and type 2 diabetes.