Mitochondrial activation by inhibition of PDKII suppresses HIF1a signaling and angiogenesis in cancer.

Most solid tumors are characterized by a metabolic shift from glucose oxidation to glycolysis, in part due to actively suppressed mitochondrial function, a state that favors resistance to apoptosis. Suppressed mitochondrial function may also contribute to the activation of hypoxia-inducible factor 1α (HIF1α) and angiogenesis. We have previously shown that the inhibitor of pyruvate dehydrogenase kinase (PDK) dichloroacetate (DCA) activates glucose oxidation and induces apoptosis in cancer cells in vitro and in vivo. We hypothesized that DCA will also reverse the 'pseudohypoxic' mitochondrial signals that lead to HIF1α activation in cancer, even in the absence of hypoxia and inhibit cancer angiogenesis. We show that inhibition of PDKII inhibits HIF1α in cancer cells using several techniques, including HIF1α luciferase reporter assays. Using pharmacologic and molecular approaches that suppress the prolyl-hydroxylase (PHD)-mediated inhibition of HIF1α, we show that DCA inhibits HIF1α by both a PHD-dependent mechanism (that involves a DCA-induced increase in the production of mitochondria-derived α-ketoglutarate) and a PHD-independent mechanism, involving activation of p53 via mitochondrial-derived H(2)O(2), as well as activation of GSK3ß. Effective inhibition of HIF1α is shown by a decrease in the expression of several HIF1α regulated gene products as well as inhibition of angiogenesis in vitro in matrigel assays. More importantly, in rat xenotransplant models of non-small cell lung cancer and breast cancer, we show effective inhibition of angiogenesis and tumor perfusion in vivo, assessed by contrast-enhanced ultrasonography, nuclear imaging techniques and histology. This work suggests that mitochondria-targeting metabolic modulators that increase pyruvate dehydrogenase activity, in addition to the recently described pro-apoptotic and anti-proliferative effects, suppress angiogenesis as well, normalizing the pseudo-hypoxic signals that lead to normoxic HIF1α activation in solid tumors.

Hemorrhagic cystitis and possible neurologic disease from BK virus infection in a patient with AIDS.

BK virus (BKV)-associated hemorrhagic cystitis occurs in bone marrow transplant recipients but is rare among other immunosuppressed patients. We present a rare case of BKV-associated hemorrhagic cystitis in a 48-year-old man with AIDS and previously diagnosed progressive multifocal leukoencephalopathy.

Similar coronary vascular effects in the rat perfused heart of platelet-activating factor structural analogues with agonist and antagonist properties.

1. Selective blockade of platelet-activating factor (PAF) receptor subtypes by PAF receptor antagonists has been demonstrated. However, selective activation of PAF receptor subtypes by PAF receptor agonists has not been reported. 2. When structural analogues of PAF that have been shown to possess either agonist or antagonist effects were administered by a bolus injection in the rat perfused heart, they all showed agonist effects. Lower amounts produced vasodilation while higher amounts produced vasodilation followed by vasoconstriction. These coronary vascular effects were typical of that observed with PAF. Lyso-PAF did not show the same typical pattern of coronary vascular effect, confirming that the detergent effect of PAF structural analogues did not play a role in the coronary vascular effects. Other PAF antagonists, CV-6209 and WEB 2170, also did not produce the PAF-like response in the rat perfused heart. 3. The coronary vascular effects of hexanolamine-PAF (H-PAF, putative antagonist) and ethanolamine-PAF (E-PAF, agonist) were further studied. Pretreatment with FR-900452 (a PAF receptor antagonist) or MK-886 (a leukotriene synthesis inhibitor) significantly reduced the vasodilator and vasoconstrictor effects of H-PAF and E-PAF. 4. Pretreatment of rat perfused hearts with low concentrations of H-PAF and E-PAF blocked the response to PAF administration in a dose- and time-dependent manner. However, the pretreatment with either H-PAF or E-PAF did not result in a coronary vascular effect expected of a PAF receptor agonist. These results were compatible with H-PAF and E-PAF behaving as PAF receptor antagonists. 5. In summary, our results demonstrate that several PAF structural analogues possess agonist action in the rat perfused heart. Like the coronary vascular effects of PAF, the effects of H-PAF and E-PAF were blocked by a PAF antagonist (FR-900452) and a leukotriene synthesis inhibitor (MK-886). This suggests that both H-PAF and E-PAF mediate their effect through activation of PAF receptors with a subsequent release of leukotrienes that produced vasodilatation and vasoconstriction. Furthermore, pretreatment of perfused hearts with these compounds blocked the response to PAF in these hearts. Thus these compounds can also behave like a PAF receptor antagonist. This latter action may be due to a gradual receptor inactivation or desensitization by the pretreatment of H-PAF and E-PAF through a PAF receptor agonist effect rather than being a PAF receptor antagonist.

Electrophysiological effects of cesium and tetraethylammonium in canine cardiac Purkinje fibers.

Cesium (Cs) and tetraethylammonium (TEA) have been shown to increase action potential duration. However, action potential duration is known to be influenced by the rate of stimulation. In this study, the effect of stimulation rate on action potential characteristics was studied in Cs-treated and TEA-loaded canine Purkinje fiber preparations. Action potentials of Purkinje fibers from Cs-treated and TEA-loaded preparations had longer durations than action potentials of Purkinje fibers from normal preparations. Greater prolongation of action potential duration was observed when the rate of stimulation was reduced in Purkinje fibers from Cs-treated and TEA-loaded preparations than those from normal preparations. Whereas the increase in action potential duration of Purkinje fibers from Cs-treated preparations was accompanied by a significant membrane depolarization, no change in membrane potential was observed in Purkinje fibers from TEA-loaded preparations. In some Cs-treated and TEA-loaded preparations, the prolonged duration observed at slow stimulation rates was associated with the appearance of early afterdepolarizations. Lidocaine and cromakalim, agents known to reduce action potential duration in normal Purkinje fibers, also shortened action potential duration in Purkinje fibers from both Cs-treated and TEA-loaded preparations. However, lidocaine and cromakalim caused a significant membrane depolarization in Cs-treated Purkinje fibers but not in TEA-loaded Purkinje fibers. Our results suggested that although Cs and TEA are capable of producing rate-dependent prolongation of action potential duration and the occurrence of bradycardia-dependent early afterdepolarization, differences exist in Cs-treated Purkinje fibers in terms of the appearance of membrane depolarization at reduced stimulation rate and in the presence of lidocaine and cromakalim.

Mechanisms of the coronary vascular effects of platelet-activating factor in the rat perfused heart.

1. In a previous study it was demonstrated that bolus injections of platelet-activating factor (PAF) in the rat perfused heart resulted in coronary vasodilatation, vasoconstriction or the combination of both, depending on the amount of PAF that was injected. In the present study, the mechanisms of these coronary vascular effects of PAF in the rat perfused heart were investigated. 2. Pretreatment of the rat perfused heart with the PAF antagonists FR-900452 or BN-52021 did not affect the vasodilator effect of PAF but eliminated the vasoconstrictor effect of PAF. FR-900452 had no effect on the vasoconstrictor response to leukotriene C4 (LTC4) or LTD4. 3. The cyclo-oxygenase inhibitor, indomethacin, did not modify the coronary vascular effects of PAF. However L-649,923 (a leukotriene antagonist) and MK-886 (a leukotriene synthesis inhibitor) eliminated both the vasodilator and vasoconstrictor effects of PAF. 4. When leukotrienes were administered by bolus injection in the rat perfused heart, LTB4 produced vasodilatation while LTC4 and LTD4 produced vasoconstriction. L-649,923 blocked both the vasodilator and vasoconstrictor effects of the leukotrienes tested. 5. The results suggest that lipoxygenase products are responsible for both the vasodilator and vasoconstrictor actions of PAF in the coronary vasculature of the rat perfused heart while the cyclo-oxygenase products do not play a significant role. The ineffectiveness of PAF antagonists in blocking the vasodilatation produced by PAF is compatible with the concept that there may be multiple PAF receptors.

Effects of palmitoyl carnitine and structural analogs on arterial blood pressure in the rat. A comparison with platelet-activating factor.

Palmitoyl carnitine and lysophosphatidylcholine have been implicated in the generation of cardiac arrhythmias in the ischemic myocardium. These amphiphilic compounds are structurally similar to platelet-activating factor (PAF). The present study compared the hypotensive effect of these compounds to PAF in the anesthetized rat. Palmitoyl carnitine was about 1000 times less potent than PAF in lowering the blood pressure. Lysophosphatidylcholine and other structurally related compounds, in dosages similar to that of palmitoyl carnitine, had no significant hypotensive action. CV 3988, a PAF antagonist, blocked the hypotensive action of PAF but had no effect on the hypotensive action of palmitoyl carnitine. This suggested the effect of palmitoyl carnitine was not associated with the same site or mechanism as PAF. The results also ruled out the involvement of prostaglandin formation and of the sympathetic nervous system since indomethacin, phenoxybenzamine and propranolol did not affect the hypotensive action of palmitoyl carnitine. In addition, it is unlikely that palmitoyl carnitine exerted its effect by a direct membrane-perturbing action because lysophosphatidylcholine, which possesses similar amphiphilic properties, does not share the same hypotensive effect.

Production of interleukin-1 by draining lymph node cells during the induction phase of contact sensitization in mice.

Biologically active interleukin-1 (IL-1) has been detected in supernatants of draining lymph node cells isolated from contact-sensitized mice. Induction of IL-1 was dependent upon the concentration of sensitizer applied and occurred within 2 hr of exposure. The IL-1 activity could not be attributed to other interleukins and was neutralized by a specific antiserum. Reduced concentrations of IL-1 were produced by cells isolated from the draining nodes of mice that had been pre-exposed to the sensitizer. Since IL-1 has the potential to influence several aspects of lymphocyte activation, the production of significant concentrations of biologically active IL-1 by draining lymph node cells indicates that it is likely to play an important role in the afferent phase of contact sensitization.

The association of lysophosphatidylcholine with isolated cardiac myocytes.

The ability of exogenous lysophosphatidylcholine to produce electrophysiological derangements and cardiac arrhythmias in the heart has been documented. The action of lysophosphatidylcholine is thought to be mediated via its association with the membrane. The present study examined the nature of the association of lysophosphatidylcholine with isolated rat myocyte membrane. The association was studied by incubating myocytes in a lysophosphatidylcholine-containing medium. The association of lysophosphatidylcholine with the myocyte sarcolemma was not affected by palmitic acid and glycerophosphocholine but was reduced by platelet-activating factor (PAF). The addition of albumin (5 mg/mL) at the end of the incubation period effectively removed the lysophosphatidylcholine from the myocytes. Our results suggest that most of the lysophosphatidylcholine in isolated myocytes was associated preferentially with the outer leaflet of the myocyte sarcolemma. This type of association might be responsible for the lysophosphatidylcholine-induced electrophysiological alterations in the heart.

Coronary vascular response to platelet-activating factor in the perfused rat heart.

The coronary vascular responses to platelet-activating factor (PAF) were studied in isolated perfused rat hearts. Constant flow rates were maintained and the changes in perfusion pressure were recorded following bolus injections of PAF. The effect of PAF on the perfusion pressure was variable and depended on certain conditions. Firstly, when the starting baseline pressure was 65-75 mm Hg, bolus injections of PAF resulted in an initial decrease followed by an increase in perfusion pressure. However, the decrease in perfusion pressure was less pronounced when the starting baseline pressure was 40 mm Hg. Secondly, the response varied with the amount of PAF that was injected. Injections of 10 fmol-1 pmol of PAF prepared in a solution containing bovine serum albumin resulted in decreases in perfusion pressure that were not followed by increases. A biphasic response, consisting of an initial decrease followed by an increase in perfusion pressure, was observed following injections of 10 and 100 pmol of PAF. Only increases in perfusion pressure were recorded following injections of 1000 pmol of PAF. Thirdly, the response to injection of PAF was dependent on whether it was prepared in a solution containing serum albumin. This study demonstrates that it is possible to observe vasodilation, vasoconstriction and biphasic responses to PAF in the isolated rat heart model.

Correlation between lymphocyte proliferative responses and dendritic cell migration in regional lymph nodes following skin painting with contact-sensitizing agents.

We have investigated whether there exists a correlation between the induction of draining lymph node cell (LNC) proliferation in contact allergy and the accumulation of dendritic cells (DC) within such lymph nodes. CBA/Ca mice, which compared with mice of BALB/c strain, mount a more vigorous lymphocyte proliferative response following sensitization, also exhibited a more marked accumulation of DC in draining lymph nodes 24 h following skin painting. Moreover, studies with the skin-sensitizing fluorochromes fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RITC) revealed that DC-enriched fractions of draining LNC prepared from CBA/Ca mice contained a higher percentage of antigen-bearing cells than did those from BALB/c mice. A relationship between DC migration into lymph nodes and the magnitude of the induced LNC proliferative response was also indicated by experiments performed in BALB/c mice with a variety of contact allergens. It was observed that there was a direct correlation between the vigour of the proliferative response measured 3 days following exposure and the frequency of DC in draining nodes at 24 h. Collectively these data suggest that following skin sensitization the migration of DC into the draining lymph nodes influences quantitatively the primary immune response and the development of contact allergy.

Comparison of the sodium currents in normal Purkinje fibres and Purkinje fibres surviving infarction--a pharmacological study.

1. Purkinje fibres surviving infarction showed a lower maximum upstroke velocity (Vmax) and a longer action potential duration when compared to normal Purkinje fibers. A reduction in the fast sodium current and an increase in the sodium 'window' current may be responsible for the observed alterations in Vmax and action potential duration respectively. 2. Since voltage clamp studies were not feasible, a pharmacological approach was used. The responses to tetrodotoxin (TTX) and lignocaine in normal Purkinje fibres and Purkinje fibres surviving infarction were used to examine the sodium currents in these fibres. 3. Vmax, an indirect measure of the fast sodium current, was more sensitive to lignocaine in Purkinje fibres surviving infarction than in normal Purkinje fibres. The reduction in Vmax by lignocaine was more prominent at the shorter stimulation cycle length. Significant reduction of Vmax was observed with the higher concentration of TTX and no differential effect on Vmax between normal Purkinje fibres and Purkinje fibres surviving infarction was detected. 4. Reduction of action potential duration in the presence of TTX or lignocaine was used as a measure of the sodium 'window' current. A greater reduction of action potential duration by TTX and lignocaine was observed in normal Purkinje fibres than in Purkinje fibres surviving infarction. 5. The results suggested that the fast sodium current in Purkinje fibres surviving infarction is more sensitive to pharmacological agents with local anaesthetic properties and the prolonged action potential duration in these Purkinje fibres cannot be due to an increase in the sodium 'window' current. The results are compatible with an enhanced effect of antiarrhythmic drugs on Vmax and conduction in the ischaemic heart.

Electrophysiological effects of encainide and its metabolites in normal canine Purkinje fibers and Purkinje fibers surviving infarction.

In this study, we assessed the effects of O-demethyl encainide (0.5 microM), the most active metabolite of encainide, and the combination with 3-methoxy-O-demethyl encainide (0.5 microM) and encainide (0.1 microM) on cardiac action potential characteristics in normal canine Purkinje fibers and Purkinje fibers surviving 24 h of myocardial ischemia. O-demethyl encainide decreased Vmax and conduction in normal Purkinje fibers and Purkinje fibers surviving infarction. Further decreases were observed with the combination. Action potential duration at both 50 and 95% repolarization was decreased by O-demethyl encainide. The combination of O-demethyl encainide, 3-methoxy-O-demethyl encainide, and encainide had no further effect. The combination of O-demethyl encainide, 3-methoxy-O-demethyl encainide, and encainide produced a smaller change in effective refractory period than O-demethyl encainide in normal Purkinje fibers and in Purkinje fibers surviving infarction. O-demethyl encainide and the combination shifted the membrane responsiveness curve to more negative potentials in both normal Purkinje fibers and Purkinje fibers surviving infarction. It is apparent from this study that there are differences in the effects of O-demethyl encainide and the combination of O-demethyl encainide, 3-methoxy-O-demethyl encainide, and encainide in normal Purkinje fibers compared with Purkinje fibers surviving infarction. Also, the combination used in this study had different electrophysiological effects than those of O-demethyl encainide alone.

Dendritic cell accumulation in draining lymph nodes during the induction phase of contact allergy in mice.

Draining lymph node cells isolated from mice 24 h following topical exposure to a variety of contact-sensitizing chemicals, including the dinitrobenzene derivatives, 2,4-dinitrochlorobenzene and 2,4-dinitrothiocyanobenzene, contained increased numbers of dendritic cells (DCs). The increase in frequency of DCs was time-dependent and preceded significant changes in either lymph node cellularity or lymph node cell proliferative activity. The degree of DC accumulation was also influenced by the chemical used and the concentration employed for sensitization. In the context of contact allergy, the biological relevance of this phenomenon to the induction of hapten-specific responses is indicated by the fact that relatively small numbers of DC--enriched fractions of lymph node cells (comprising approximately 70% DCs), but not unfractionated or DC--depleted populations, transferred sensitization to naive animals. Moreover, using the skin-sensitizing fluorochrome, fluorescein isothiocyanate, it was observed that 24 h following exposure the majority of lymph node cells bearing high concentrations of antigen were within the DC-rich fraction.

Lysophosphatidylcholine accumulation in the ischemic canine heart.

The production of cardiac arrhythmias and the elevation of lysophosphatidylcholine level in the ischemic myocardium have been well-documented in a number of studies. However, the relationship between the production arrhythmias and the elevation of tissue lysophosphatidylcholine level was not reported. In this study, the lysophosphatidylcholine level and the occurrence of cardiac arrhythmias in the ischemic canine heart were monitored. A temporal relationship between the accumulation of lysophosphatidylcholine and the occurrence of arrhythmias was established after five hr of ischemia. A significant elevation of lysophosphatidylcholine was detected at three hr of ischemia without the occurrence of arrhythmias. The results indicate that cardiac arrhythmias did not cause the elevation of lysophosphatidylcholine and if lysophospholipids are causally related to the arrhythmias that a critical level of the lysophospholipid must accumulate in order to elicit electrophysiological alterations.

Depression of lymph node cell proliferation induced by oxazolone.

The influence of topical exposure to two sensitizing chemical on draining lymph node cell proliferative responses in BALB/c mice has been examined. Conventional contact sensitization with 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone) has been shown to induce a rapid and systemic suppression of subsequent proliferative responses to topically applied chemical which can be adoptively transferred to recipient mice with immune lymph node cells. In contrast to some previous reports in which such suppression was found to be largely antigen-specific in nature, we report that, at least initially, the inhibition of lymphocyte proliferation induced by skin sensitization is hapten-non-specific. The relevance of this phenomenon to the regulation of contact sensitization is discussed.

Electrophysiological effects of tocainide on canine subendocardial Purkinje fibers surviving infarction.

The effects of 10 and 20 mg/l of tocainide on transmembrane action potential characteristics were examined in Purkinje fibers surviving infarction. Infarcted tissue was obtained from canine hearts 24 h after coronary artery ligation. The preparation was stimulated at basic cycle lengths (BCL) of 1000-300 ms. Tocainide reduced the overshoot and amplitude of Purkinje fibers surviving infarction. The maximum upstroke velocity (Vmax) was decreased by tocainide in a dose dependent manner. This effect was more prominent at the shorter BCL. Statistical analysis revealed a significant interaction of the BCL with the drug effect on overshoot, amplitude, Vmax and action potential durations (APD50% and APD90%). Tocainide reduced the effective refractory period (ERP) at the BCL of 1000 ms, but had no significant effect at the BCL of 300 ms. Membrane responsiveness and steady state characteristics of Vmax were shifted significantly to more negative membrane potentials by tocainide. Investigation of the recovery kinetics of Vmax in the presence of tocainide showed an exponential recovery of Vmax with a time constant of 514 ms. These results support the finding that the effect of tocainide on Vmax and conductions is enhanced at faster rates of stimulation. Thus tocainide may be able to depress conduction to produce bidirectional block in the termination of ventricular tachycardia caused by reentry, while having minimal effect on conduction at normal heart rates.

The effect of lidocaine compared with verapamil on the enhanced ventricular rhythm in the infarcted canine heart.

Myocardial ischemia was produced in dogs by the occlusion of the left anterior descending (LAD) coronary artery for 24 or 48 h. After complete atrioventricular block was produced, enhanced ventricular rhythm was observed in all animals. The enhanced ventricular rhythm showed multiple QRS configurations and had spontaneous cycle lengths (SCL) of 397 +/- 18 ms (n = 20) after 24 h of LAD occlusion and 446 +/- 23 ms (n = 20) after 48 h of LAD occlusion. Overdrive pacing did not result in the termination of the enhanced ventricular rhythm in any experiment. Propranolol, as a cumulative dose of 1.5-2.0 mg/kg i.v., also did not abolish the enhanced ventricular rhythm. In 24-h infarcted hearts, lidocaine abolished the enhanced ventricular rhythm in 1 of 11 experiments. In the remaining 10 experiments, the ventricular SCL was increased from 401 +/- 22 to 491 +/- 26 ms after a cumulative dose of 8.8 +/- 0.7 mg/kg of lidocaine. In the presence of verapamil, given as a cumulative dose of 0.60 +/- 0.11 mg/kg, the ventricular SCL was increased from 401 +/- 33 to 482 +/- 64 ms (n = 9). In 48-h infarcted hearts, lidocaine abolished the enhanced ventricular rhythm in 5 of 11 experiments. Both lidocaine and verapamil increased the SCL of hearts in which the enhanced ventricular rhythm persisted. Analysis of variance showed that only the increase in SCL by lidocaine in 48-h infarcted hearts was statistically significant. The atrial and idioventricular rhythms in noninfarcted hearts responded differently to lidocaine and verapamil. The results suggest that some electrophysiological effects of antiarrhythmic drugs in the normal heart may not be applicable to those in the diseased situation.

Kinetics of responses of IL3 dependent lines to growth factors: implications for the assay and mechanism of cell promotion.

We have investigated the promotion of two IL3 dependent cell lines by IL3 and a rat factor derived from stimulated spleen cells. Both lines respond to purified IL3, but do not respond to IL2, GM-CSF contained in lung conditioned medium nor to Human IL2. Both lines respond to a factor in rat lymphocyte conditioned media as determined by a standard assay used to detect IL3. However, 123 cell line cells continue to proliferate whereas AC2 cells decline, due to different sensitivities of the lines to rat factor. 123 cells can be re-cloned in the rat factor and exhibit enhanced responses to IL3. The changing kinetics of cell promotion during the first 48 hours question the validity of the standard assay which is widely used, and the specificity of the mechanism by which the cells are promoted.