Combination of dihydroxyanthraquinone and radiation on L1210 murine leukemia

A new synthetic anthraquinone, dihydroxyanthraquinone (DHAQ), was tested as a single agent and in combination with whole-body x-irradiation in mice bearing L1210 leukemia in the ascites form. DHAQ alone had significant antitumor activity as evidenced by prolonged mean survival times; radiation was ineffective. The combination of the two modalities produced a therapeutic benefits greater than that produced by either agent alone: mean survival times were increased and a number of cures (animals alive at 30 days) produced. These results suggest the potential of using DHAQ and radiation in a combined modality therapeutic approach.     

Mechanisms of inhibition of amido phosphoribosyltransferase from mouse L1210 leukemia cells

Amido phosphoribosyltransferase (amido PRTase) catalyses the first step of the pathway for de novo biosynthesis of purine nucleotides. The enzyme is subject to inhibition by purine nucleoside 5'-monophosphates (AMP, IMP, and GMP), by dihydrofolate polyglutamates, and by the antifolate piritrexim [Sant, M. E., Lyons, S. D., Phillips, L., & Christopherson, R. I. (1992) J. Biol. Chem. 267, 11038-11045). Using a coupled radioassay, we have determined the substrate dissociation constants as 80.4 +/- 13.2 microM for 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) and 421 +/- 193 microM for L-glutamine with P-Rib-PP bound first with positive cooperativity for interaction with a second site on the catalytically active dimer (interaction factor of 0.247 +/- 0.042). Analysis of inhibition patterns for amido PRTase shows that the antifolate piritrexim is a noncompetitive inhibitor bound with positive cooperativity at two allosteric sites of an inactive dimer with a dissociation constant of 66.0 +/- 17.8 microM for interaction with the free enzyme and an interaction factor of 0.187 +/- 0.113 with P-Rib-PP as the varied substrate. With L-glutamine as the varied substrate, a dissociation constant of 62.3 +/- 15.6 microM for interaction with the enzyme-P-Rib-PP complex and an interaction factor of 0.0958 +/- 0.0585 microM were obtained. AMP binds as a competitive inhibitor with respect to P-Rib-PP with a dissociation constant of 40.0 +/- 8.1 microM for interaction with the free enzyme and as a noncompetitive inhibitor with respect to L-glutamine with a dissociation constant of 16.4 +/- 5.2 mM for interaction with the enzyme-P-Rib-PP complex. Sucrose density gradient centrifugation of partially purified amido PRTase showed three molecular forms of the enzyme: an inactive tetramer (10.2 S) formed in the presence of AMP, an active dimer (6.7 S) formed with P-Rib-PP, and an inactive dimer (7.2 S) with piritrexim. The latter species may predominate in cells containing high levels of dihydrofolate polyglutamates.     

Phospholipase D activity in L1210 cells: a model for oleate-activated phospholipase D in intact mammalian cells

The effect of 2-aminobicyclo[2.2.1]heptan-2-carboxylic acid (BCH), an L-leucine nonmetabolizable analogue and an allosteric activator of glutamate dehydrogenase, on glucose and glutamine synthesis was studied in rabbit renal tubules incubated with alanine, aspartate or proline in the presence of glycerol and octanoate, i.e. under conditions of efficient glucose formation. With alanine+glycerol+octanoate the addition of BCH resulted in a stimulation of alanine and glycerol consumption, accompanied by an increased glucose, lactate and glutamine synthesis. In contrast, when alanine was substituted by either aspartate or proline, BCH altered neither glucose formation nor glutamine and glutamate synthesis, while an accelerated glycerol utilization was accompanied by a small increase in lactate production. In view of the BCH-induced changes in intracellular metabolite levels the acceleration of gluconeogenesis by BCH in the presence of alanine+glycerol+octanoate is probably due to (i) increased uptake of alanine via alanine aminotransferase, (ii) stimulation of phosphoenolpyruvate carboxykinase, a key-enzyme of gluconeogenesis, (iii) rise of glucose-6-phosphatase activity, as well as (iv) activation of the malate-aspartate shuttle resulting in an augmented glycerol utilization for lactate and glucose synthesis.     

Plasminogen activator system modulates invasive capacity and proliferation in prostatic tumor cells

The malignant phenotype of prostatic tumor cells correlates with the expression of both uPA and its cell-membrane receptor (uPAR); however, there is little information concerning the role of cell-bound uPA in matrix degradation and invasion. Our results suggest that cell-associated uPA plays a key role in regulating the amount of plasmin present at the surface of prostatic carcinoma (PRCA) cells and show that differential production of uPA corresponds with the capacity to bind and activate plasminogen. In addition, we provide direct evidence that both uPA secretion and the presence of uPA-uPAR complexes characterize the invasive phenotype of PRCA cells and suggest the existence of several pathways by which tumor cells acquire plasmin activity. LNCaP cells (which do not produce uPA but express uPAR) may activate plasmin through exogenous uPA. In vivo, the source of uPA may be infiltrating macrophages and/or fibroblasts as observed in several other systems. PAI-1 accumulation in the conditioned medium (CM) limits plasmin action to the pericellular microenvironment. Our results indicate that MMP-9 and MMP-2 are also activated by plasmin generated by cell-bound but not by soluble, extracellular uPA. Plasmin activation and triggering of the proteolytic cascade involved in Matrigel invasion is blocked by antibodies against uPA (especially by anti- A-chain of uPA which interacts with uPAR) and by PA inhibitors such as p-aminobenzamidine which may regulate levels of cell-bound uPA. uPA may also regulate growth in PRCA cells. Indeed, antibodies against uPA A-chain (and also p-aminobenzamidine treatment) interfere with the ATF domain and inhibit cell growth in uPA-producing PC3 and DU145 prostate cancer cell lines, whereas exogenous uPA (HMW-uPA with ATF) induces growth of LNCaP prostate tumor cell line. These data support the hypothesis that in prostatic cancer patients at risk of progression, uPA/plasmin blockade may be of therapeutic value by blocking both growth of the primary tumor and dissemination of metastatic cells.     

Prostaglandin A2 and delta 12-prostaglandin J2 induce apoptosis in L1210 cells

Combined in vitro radioreceptor binding and autoradiographic techniques with [3H]SCH 23390 as a ligand were used to analyze the pharmacological profile and anatomical localization of dopamine D1 receptor sites in sections of human umbilical artery. The ligand was bound to sections of the artery in a manner consistent with the labelling of D1 receptors. These receptors, which show a fetal-to-maternal gradient with the highest concentration near the placenta, are located within the smooth muscle layer of the umbilical artery. The above findings suggest that endogenous dopamine may be involved in the control of the tone of the umbilical artery through the interaction with D1 receptors.     

Effect of methotrexate on the cell cycle of L1210 leukemia

The influence of methotrexate (MTX) on the proliferative activity of cells in different phases of cell cycle has been studied. MTX (5 mg/kg) was injected i.p. 3 days after the inoculation of 5 X 10(6) leukemia cells, into F1 (DBA X C57 BL) mice. It was shown that MTX causes degeneration of cells, being in G1- as well as in S-phase at the time of drug injection. Incorporation of 3H-TdR was suppressed for a period ranging from 2 to 12 hr after MTX administration, which is demonstrated by the decrease in the number of grains per cell. The number of cells labeled after 3H-TdR injection was also sharply decreased during this period. For a period of 3 until 15 hr after MTX administration the mitotic index decreased significantly as a result of inhibition of DNA synthesis. The blocking of the G1-S transition was evident during 4 hr after MTX. Thereafter the G1-S transition proceeds at a rate which is practically equal to that for nontreated controls. MTX did not inhibit transition to mitosis of cells being in G2-phase and in a very late S-phase at the time of drug injection. The sensitivity of G1-cells to the cytocidal effect of MTX shows that for L1210 leukemia cells MTX can be classified as a cycle-specific drug killing both G1-and S-cells rather than S-phase specific agent with self-limitation.     

Increased generation of lipid-derived and ascorbate free radicals by L1210 cells exposed to the ether lipid edelfosine

Using the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, we have detected a lipid-derived carbon-centered free radical generated from intact L1210 lymphoblastic leukemia cells that were exposed to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine or ET-18-OCH3) and oxidative stress. The spectral characteristics, including hyperfine splitting constants of aN = 15.61G and aH = 2.65G, were consistent with the spin trapping of an alkyl radical. Radical detection required iron and prior enrichment of cellular components with the polyunsaturated fatty acid docosahexaenoic acid; unmodified cells failed to generate detectable free radical. Ascorbate further enhanced radical generation. The detection of lipid-derived free radicals when intact cells are exposed to edelfosine provides further evidence that oxidative stress may play an important role in the cytotoxic mechanism of this class of anticancer drug.     

SCH56592 treatment of murine invasive aspergillosis

We analyzed the clinical presentation of 800 severe malaria cases six months to 15 years of age (mean +/- SD = 4.3 +/- 3.0) recruited at the pediatric ward of the Ouagadougou University Hospital, and at the Sourou and Nayala District Hospitals in Burkina Faso. Inclusion criteria followed the World Health Organization (WHO) definition of severe and complicated malaria. The children were treated according to WHO guidelines with a complete regimen of drugs that were provided free of charge as part of the study. The case fatality rate of each sign and symptom of severe malaria was calculated on the 686 children whose outcomes were known. A total of 95 patients (13.8%) died while in the hospital; the mean +/- SD age of these children was 3.2 +/- 2.1 years. The age distribution and the clinical patterns of severe malaria was compared in patients from the urban areas of Ouagadougou characterized by relatively low transmission, and from rural areas where the mean inoculation rates are at least 20-fold higher. The mean +/- SD age of the urban and rural patients was 4.8 +/- 3.0 and 2.2 +/- 1.9 years, respectively (P < 0.001). The prevalence of coma was higher in the urban subsample (53.6% versus 28.9%; P < 0.001) while that of severe anemia (hemoglobin < 5 g/dL) was higher in rural patients (47.4% versus 14.8%; P < 0.001). Our data, in line with previous results obtained comparing rural areas characterized by different inoculation rates, show that the epidemiologic context influences the clinical presentation of severe malaria.     

Pepstatin, an ascites retardant of L1210 tumor-bearing mice

The effect of pepstatin on the kinetics of ascitic fluid accumulation in L1210 tumor-bearing mice (DBA/2) was observed. Following inoculation of 1.5x10(6) tumor cells, untreated mice reached a peak of fluid accumulation on day 6 and remained at this level until death on day 9. A "lag" phase of 4 days occurred before fluid accumulation was seen. Pepstatin administered SC in a single dose of 80 mg/kg during the lag phase, significantly retarded fluid accumulation as compared to untreated animals. Pepstatin administered following fluid accumulation was much less effective. We concluded that pepstatin prevents fluid accumulation rather than acts as a diuretic agent. The term "ascites retardant" is suggested for the pharmacologic actions of pepstatin, since it prevents fluid accumulation without diminishing the cell count.     

Acute hypercapnia increases the oxygen-carrying capacity of the blood in ventilated dogs

OBJECTIVE: To test the hypothesis that PaCO2 levels generated during permissive hypercapnia may enhance arterial oxygenation, when ventilation is maintained. DESIGN: Prospective study. SETTING: Research laboratory in a hospital. SUBJECTS: One group of eight mongrel dogs (four male; four female). INTERVENTIONS: The dogs were anesthetized (30 mg/kg iv pentobarbital), intubated, and cannulated in one femoral artery and vein. While paralyzed with 0.1 mg/kg/hr iv vecouronium bromide, all subjects were ventilated with room air. Anesthesia was maintained, using 2 to 3 mg/kg/hr iv pentobarbital. Arterial hypercapnia at the levels generated during permissive hypercapnia was produced by stepwise increases in the dry, inspired Pco2 (PiCO2) (0, 30, 45, 60 and 75 torr [0, 4, 6, 8, and 10 kPa]; 15 mins each). MEASUREMENTS AND MAIN RESULTS: Blood gas profiles were determined at each level of hypercapnia. The minute volume was maintained at the baseline level during all exposures. Arterial hypercapnia produced gradual and significant increases in the hemoglobin concentration. These increases were approximately 6%, 7%, 11%, and 14% at PiCO2 of 30, 45, 60, and 75 torr (4, 6, 8, and 10 kPa), respectively (p < .05; repeated analysis of variance followed by Dunnett multiple comparisons test). In parallel, the oxygen content increased by approximately 6%, 7%, 11%, and 13%, respectively. During hypercapnic trials, the PaO2 remained at the normal range, whereas the dry, inspired PO2 (PiO2) was reduced from 150 to 138 torr (20 to 18.4 kPa). The average PaO2 at the highest investigated level of arterial hypercapnia was at a normal range. The hemoglobin concentration and oxygen content returned to baseline values 30 mins after hypercapnic trials. The PaCO2 and pH became normalized 15 mins after hypercapnic trials. Indirect evidence for a similar response to hypercapnia in humans is presented. CONCLUSIONS: Permissive hypercapnia due to inhaled CO2 increases oxygen-carrying capacity in dogs. The PaO2 remains at normal range even at a PiCO2 of 75 torr (10 kPa). The benefits of these effects during permissive hypercapnia, due to controlled hypoventilation, warrants investigation.     

Evidence that brain capillary endothelial cells can be infected with murine leukaemia retrovirus, LP-BM5

The effect of intraportal administrations of interleukin 1 beta (IL-1 beta) on the afferent activity of the hepatic branch of the vagus nerve was observed in urethane anesthetized rats. An intraportal injection of IL-1 beta in doses of 10 pg and 100 pg per animal (300-400 g body wt.) resulted in dose-dependent increase in the afferent activity, which lasted about 70-100 min. Further, intraportal injection of IL-1 beta (100 pg) induced reflex activation of efferent activity of the splenic (sympathetic) nerve and vagal thymic nerve. This reflex activation was not observed in hepatic vagotomized rat and after an administration of the same dose of IL-1 beta into the systemic vein in normal rat. The results suggest the existence of sensors for IL-1 beta which send information on IL-1 beta in the portal venous blood to the central nervous system through the hepatic branch of the vagus nerve and play some role in reflex regulation of immune function.     

Antibody-induced killing in vivo of L1210/MTX-R cells quantitated in passively immunized mice with 131I-iododeoxyuridine-labeled cells and whole-body measurement of retained radioactivity

The killing of the LR subline of the DBA/2J leukemia L1210/MTX by passive antibody was followed in vivo with 131I-iododeoxyuridine-labeled cells and whole-body measurement of retained radioactivity. The in vivo killing of LR cells was proportional to the in vitro 2-mercaptoethanol resistant titer, independent of the complement system, and radioresistant. Although a large percentage of the leukemic cells was killed in passively immunized mice, the protective effect of the passive antiserum was dependent on the active immune response of the host.     

Effects of 4'-modified analogs of aristeromycin on the metabolism of S-adenosyl-L-homocysteine in murine L929 cells

(1'R,2'S,3')-9-(2',3'-Dihydroxycyclopentan-1'-yl)adenine (DHCaA), (1'R,2'S,3'R)-9-(2',3'-dihydroxycyclopentan-1'-yl)-3-deazaadenine (3-deaza-DHCaA), (4'R)-4'-methyl-DHCaA, and (4'R)-4'-vinyl-DHCaA, which are analogs of the carbocyclic nucleoside aristeromycin, were synthesized earlier by our laboratory and were shown to be potent inhibitors of purified bovine liver S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1). In the present study, these analogs were shown to produce rapid (within 15 min) and concentration-dependent (0.03-10 microM) inhibition of AdoHcy hydrolase in cultured murine L929 cells [relative order of inhibitory activity, DHCaA = 3-deaza-DHCaA > (4'R)-4'-vinyl-DHCaA = (4'R)-4'-methyl-DHCaA]. The relative potencies of these inhibitors on the L929 AdoHcy hydrolase were consistent with their inhibitory effects on the recombinant forms of rat liver and human placental enzymes. This inhibition of L929 cellular AdoHcy hydrolase persisted for up to 48 hr. The inhibition of the L929 AdoHcy hydrolase resulted in a significant increase in the cellular concentrations of AdoHcy, whereas the cellular S-adenosylmethionine (AdoMet) levels remained relatively constant, thereby elevating the AdoHcy/AdoMet ratios. Maximum increases in AdoHcy levels and AdoHcy/AdoMet ratios occurred within 6 hr of exposure to the inhibitors and persisted for at least 24 hr. At a concentration of 1 microM, DHCaA and 3-deaza-DHCaA increased AdoHcy/AdoMet ratios to approximately 0.8 (after 24 hr of exposure to the inhibitors), whereas (4'R)-4'-vinyl-DHCaA and (4'R)-4'-methyl-DHCaA elevated AdoHcy/AdoMet ratios to approximately 0.15, compared with control levels of 0.05. Treatment of L929 cells with concentrations of DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA up to 10 microM did not result in changes in cellular levels of endogenous nucleotides (e.g., CTP, UTP, ATP, and GTP). In contrast, cells treated with 10 microM aristeromycin for 6 hr contained reduced cellular levels of CTP, ATP, and GTP and significant levels of aristeromycin triphosphate and a GTP metabolite of this carbocyclic nucleoside. These data clearly show that the 4'-modified analogs [DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA] retain inhibitory activity toward cellular AdoHcy hydrolase, causing elevated levels of AdoHcy and elevated AdoHcy/AdoMet ratios. However, these analogs are devoid of substrate or inhibitory activity toward cellular adenosine kinase. In addition, aristeromycin is rapidly metabolized in murine L929 cell lysates, i.e., > 60% of the aristeromycin had been metabolized in 6 hr. In contrast, neither DHCaA nor 3-deaza-DHCaA showed any decrease in concentration after incubation with cell lysates for up to 6 hr.     

Transforming growth factor-beta1 increases mRNA levels of osteoclastogenesis inhibitory factor in osteoblastic/stromal cells and inhibits the survival of murine osteoclast-like cells

Osteoclastogenesis inhibitory factor (OCIF), also termed osteoprotegerin (OPG), is a secreted member of the tumor necrosis factor (TNF) receptor family. It inhibits bone resorption in vivo and osteoclast-like cell (OCL) formation in vitro. To better understand the biological role of OCIF, we first examined the effects of various osteotropic agents on OCIF mRNA levels in mouse calvarial osteoblasts. Northern blot analysis showed that stimulators of OCL formation such as 1,25-(OH)2D3, prostaglandin E2 (PGE2), parathyroid hormone (PTH), and interleukin 1 (IL-1) decreased OCIF mRNA levels. In contrast, transforming growth factor (TGF)-beta1 increased OCIF mRNA levels in primary osteoblasts as well as in osteoblastic/stromal cell lines. Since it was reported that both TGF-beta1 and OCIF not only inhibited OCL formation but also impaired the survival of OCL by inducing apoptosis in vitro, we next examined the possible involvement of OCIF in TGF-beta1-induced impairment of OCL survival. In a mouse bone marrow culture, we confirmed that addition of OCIF or TGF-beta1 decreased the number of surviving OCL. Anti-OCIF IgG, which completely neutralized the effect of OCIF, partially prevented the TGF-beta1-induced decrease in the number of OCL. Our results suggest that (i) downregulation of OCIF expression is one of the mechanisms for the stimulatory effects of 1,25(OH)2D3, PGE2, PTH, and IL-1 on osteoclastogenesis; and (ii) the TGF-beta1-induced apoptosis of OCL is mediated, at least in part, by upregulation of OCIF expression.     

Reduced visual capacity increases the risk of accidents in street traffic]

Six patients diagnosed with obstructive sleep apnea completed titration of an adjustable oral appliance, called the Silencer, during a single night of polysomnography. This protocol allowed for rapid titration of the oral appliance and effective treatment of sleep apnea. Variables which showed improvement included frequency of obstructive events, oxyhemoglobin saturation and snoring. Dental appliance adjustments with the silencer device can be made within three minutes. We have demonstrated that incremental mandibular advancement and repositioning allow us to determine the most effective jaw position to treat sleep apnea and snoring, which is also most likely to be tolerated by the patient.     

Dose-dependent selective cytotoxicity of extracts from marine green alga, Cladophoropsis vaucheriaeformis, against mouse leukemia L1210 cells

The cases of four children who presented with respiratory distress unresponsive to bronchodilator treatment and who were subsequently diagnosed with severe anemia were retrospectively reviewed from charts at a children's hospital with 110 beds and an emergency department census of 32,000 per year. The four children, age range 11 months to 2 years, each initially presented with tachypnea. Three had wheezing, three were pale, and each received outpatient bronchodilator treatment. Three to 4 days later, each patient was hospitalized for continued respiratory distress. Each child was pale, and hemoglobin levels ranged from 2.5 to 5.2 g/dL. In three children, respiratory distress resolved with blood transfusions. Final diagnoses were transient erythroblastopenia of childhood in 2, acute lymphoblastic leukemia in 1, and iron deficiency anemia in 1. Severe anemia is a known cause of respiratory distress. These cases remind the clinician to consider severe anemia in children who are tachypneic, pale, and unresponsive to treatment of respiratory disorders.