Ribose 1-phosphate metabolism in Ehrlich ascites tumor cells in vitro.

Ribose 1-phosphate concentrations have been measured in tumor cells incubated with purine and pyrimidine nucleosides and with glucose. Highest concentrations (0.15 to 0.2 mumol/ml of cells) were attained in cells incubated with inosine. Although uridine was cleaved at approximately the same rate as inosine, as judged by lactate accumulation, concentrations of ribose 1-phosphate that accumulated were only approximately 0.06 mumol/ml. Ribose 1-phosphate accumulation in tumor cells incubated with inosine was dependent on the phosphate concentration of the medium up to at least 25 mM. Ribose 1-phosphate formed from inosine was readily converted both to phosphoribosyl pyrophosphate and to lactate.

Plasma of preeclamptic women stimulates and then inhibits endothelial prostacyclin.

We propose that the dichotomy between the in vivo reduction in intravascular prostacyclin production that occurs in preeclampsia and the in vitro stimulatory effect of plasma from preeclamptic patients on endothelial cell prostacyclin production is due to differential effects of chronic versus acute exposure to the plasma. We studied the acute versus chronic effects of 2% plasma from healthy pregnant and preeclamptic subjects by measuring endothelial prostacyclin production at different time periods after exposure to plasma. To determine whether such effects were specific to prostacyclin, we also measured prostaglandin E2 production. To determine whether chronic changes in prostacyclin production resulted from altered cellular responsiveness, we stimulated cells that had been exposed to plasma for 72 hours with arachidonic acid and measured prostaglandin production. Preliminary characterization of the plasma factor or factors responsible for alterations in prostaglandin production was performed. After 24 hours cells exposed to plasma from preeclamptic women produced more prostacyclin and prostaglandin E2 than cells exposed to plasma from healthy pregnant women. In contrast, after 72 hours exposure to plasma from preeclamptic women resulted in less endothelial cell prostacyclin production than exposure to plasma from healthy pregnant women, but there were no such differences in prostaglandin E2 production. Cells that had been exposed to plasma from preeclamptic women for 72 hours produced less prostacyclin but the same quantity of prostaglandin E2 after stimulation with arachidonic acid than cells exposed to plasma from healthy pregnant women. The plasma factor or factors responsible for altered prostacyclin production were sensitive to heat, acid, and proteases. In contrast to acute exposure, chronic exposure to plasma from preeclamptic women alters endothelial cells to result in decreased prostacyclin production, an observation consistent with in vivo findings.

Guanosine triphosphate catabolism in purine nucleoside phosphorylase deficient human B lymphoblastoid cells.

GTP catabolism induced by sodium azide or deoxyglucose was studied in purine nucleoside phosphorylase (PNP) deficient human B lymphoblastoid cells. In PNP deficient cells, as in control cells, guanylate was both dephosphorylated and deaminated but dephosphorylation was the major pathway. Only nucleosides were excreted during GTP catabolism by PNP deficient cells and the main product was guanosine. The level of nucleoside excretion was largely affected by intracellular orthophosphate (Pi) level. In contrast, normal cells excreted nucleosides only at low Pi level while at high Pi levels, purine bases (guanine and hypoxanthine) were exclusively excreted. PNP deficiency had no effect on the extent of GMP deamination.

Impairment of nucleotide metabolism by iron-chelating deferoxamine.

The effect of deferoxamine on nucleotide metabolism in HL-60 leukemic cells was studied to explore the mechanism of its antiproliferation activity. It was found that in intact cells deferoxamine markedly inhibited the ribonucleotide reduction and incorporation of bases (adenine, hypoxanthine), ribonucleosides (inosine, guanosine) and deoxyribonucleosides (thymidine, deoxyadenosine, deoxyguanosine) into nucleic acids. Although deferoxamine did not inhibit thymidine and uridine incorporation into free nucleotides, inhibition of hypoxanthine and adenine incorporation into nucleotides as well as inhibition of nucleotide biosynthesis de novo was found. Nucleotide catabolism, protein synthesis, and intracellular levels of ribonucleotides were not affected significantly by deferoxamine. These results showed that deferoxamine selectively affects several specific reactions of nucleotide metabolism. Inhibition of ribonucleotide reduction, inhibition of ribonucleotide and deoxyribonucleotide incorporation into nucleic acids, as well as inhibition of purine biosynthesis, may alter significantly cellular physiology and, therefore, contribute significantly to the antiproliferative activity of deferoxamine.

Selective protection of tubercidin toxicity by nitrobenzyl thioinosine in normal tissues but not in human neuroblastoma cells.

Tubercidin, an adenosine analogue, is toxic to human neuroblastoma cell lines, to peripheral blood mononuclear cells (PBMCs), and to myeloid colony-forming cells (CFU-C) as tested by a short-term labeled precursor uptake and by a clonogenic assay. When it was co-administered with a potent purine transport inhibitor, nitrobenzyl thioinosine (NBTI), the cytotoxic effect of tubercidin was abolished in PBMCs but not in neuroblastoma cells. Studies of nucleoside transport in neuroblastoma cells demonstrate that although [3H]NBTI binds to the plasma membrane of these cells, the transport of thymidine into the cells is only partially inhibited in the presence of excess NBTI. These data imply that neuroblastoma cells contain a nucleoside transport mechanism which is insensitive to NBTI. "Host protection" with a nucleoside transport inhibitor such as NBTI, may allow effective therapy with otherwise toxic dosages of tubercidin and other cytotoxic nucleosides in patients with neuroblastoma.

Adenine cycle in hepatopancreocytes of Helix pomatia (Gastropoda).

Intact hepatopancreocytes were obtained from hibernating or active purinotelic snails, H. pomatia (Gastropoda). When incubated with [14C]glycine or [14C]formate, they synthesized de novo purine compounds, including also adenylates, adenosine and adenine. Hepatopancreocytes resynthesized also adenylates and other purine compounds from [3H]adenine or from [3H]adenosine split by the H. pomatia cell enzyme to adenine; the resynthesis of ADP+ATP was proportional to adenine concentration. Thus all reactions of the postulated adenine cycle: AMP leads to adenosine leads to adenine leads to AMP occur in the intact hepatopancreocytes; this cycle could probably be responsible for maintenance of the high level of adenylates during winter sleep.

GRP78-targeting subtilase cytotoxin sensitizes cancer cells to photodynamic therapy.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER)-resident chaperone and a major regulator of the unfolded protein response (UPR). Accumulating evidence indicate that GRP78 is overexpressed in many cancer cell lines, and contributes to the invasion and metastasis in many human tumors. Besides, GRP78 upregulation is detected in response to different ER stress-inducing anticancer therapies, including photodynamic therapy (PDT). This study demonstrates that GRP78 mRNA and protein levels are elevated in response to PDT in various cancer cell lines. Stable overexpression of GRP78 confers resistance to PDT substantiating its cytoprotective role. Moreover, GRP78-targeting subtilase cytotoxin catalytic subunit fused with epidermal growth factor (EGF-SubA) sensitizes various cancer cells to Photofrin-mediated PDT. The combination treatment is cytotoxic to apoptosis-competent SW-900 lung cancer cells, as well as to Bax-deficient and apoptosis-resistant DU-145 prostate cancer cells. In these cells, PDT and EGF-SubA cytotoxin induce protein kinase R-like ER kinase and inositol-requiring enzyme 1 branches of UPR and also increase the level of C/EBP (CCAAT/enhancer-binding protein) homologous protein, an ER stress-associated apoptosis-promoting transcription factor. Although some apoptotic events such as disruption of mitochondrial membrane and caspase activation are detected after PDT, there is no phosphatidylserine plasma membrane externalization or DNA fragmentation, suggesting that in DU-145 cells the late apoptotic events are missing. Moreover, in SW-900 cells, EGF-SubA cytotoxin potentiates PDT-mediated cell death but attenuates PDT-induced apoptosis. In addition, the cell death cannot be reversed by caspase inhibitor z-VAD, confirming that apoptosis is not a major cell death mode triggered by the combination therapy. Moreover, no typical features of necrotic or autophagic cell death are recognized. Instead, an extensive cellular vacuolation of ER origin is observed. Altogether, these findings indicate that PDT and GRP78-targeting cytotoxin treatment can efficiently kill cancer cells independent on their apoptotic competence and triggers an atypical, non-apoptotic cell death.