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Introduction: Prognosis of patients with newly diagnosed multiple myeloma (MM), a third most common hematological cancer, is dependent on baseline cytogenetics. However, little is known about the prognostic significance of cytogenetic evolution (CE) at the time between the diagnosis and relapse of MM., Objectives: Here, we retrospectively analyzed the prognostic impact of CE detected in a routine interphase fluorescence in situ hybridization (FISH) test in a cohort of patients with MM., Patients and Methods: Among 650 patients evaluated with the FISH MM panel at our center between 2014 and 2019, we identified 29 individuals with MM who had been tested twice, at the time of diagnosis and relapse. Cytogenetic evolution was defined as the acquisition or loss of at least 1 cytogenetic abnormality at relapse (FISH2) compared with the baseline test result (FISH1)., Results: Cytogenetic evolution was seen in 14 patients (48%), whereas 15 had stable cytogenetics. Acquired chromosome 17p deletion (del[17p]) was the most common type of CE, found in 7 patients (24%). In univariable analysis, stable cytogenetics predicted longer overall survival (median not reached vs 3.8 years; hazard ratio [HR], 0.15; P = 0.04; median follow‑up of 3.1 years) and longer overall survival after FISH2 (median not reached vs 0.8 years; HR, 0.13; P = 0.002; median follow‑up of 0.6 years). In multivariable analysis, acquired del(17p) predicted shorter progression‑free survival and the overall survival after FISH2 (HR, 9.3 and 18.8; P = 0.005 and P = 0.004, respectively)., Conclusions: Presence of CE and, particularly, the acquisition of new del(17p) at relapse, negatively affect the outcome of MM. Therefore, re‑evaluation of FISH at MM relapse should be included in routine clinical practice.

Acid sphingomyelinase (aSMase) is involved in the generation of metabolites that function as part of the sphingolipid signaling pathway. It catalyzes the breakdown of sphingomyelin into ceramide, a bioactive lipid that, among other roles, is involved in regulation of apoptosis. Dry drop blood test (DBS) and colorimetric 2-step enzymatic assay were used to assess the activity of human blood aSMase, beta-galactosidase, and beta-glucosidase, these enzymes are lysosomal hydrolases that catalyze the degradation of related sphingolipids, of sphingolipid signaling molecules. Blood was collected from a group of healthy volunteers and patients that were diagnosed with multiple myeloma (MM) in various stages of the disease. Additionally, activity of those enzymes in patients diagnosed with other hematological cancers was also assessed. We found that aSMase activity in the blood of patients with MM (at the time of diagnosis) was 305.43 pmol/spot*20 h, and this value was significantly lower ( p < 0.030) compared to the healthy group 441.88 pmol/spot*20 h. Our collected data suggest a possible role of aSMase in pathogenesis of MM development.

Introduction: The risk of HBV reactivation is important in lymphoma patients receiving immunosuppressive chemotherapy containing steroids or anti-CD20 antibodies. We aimed to establish the prevalence of HBV Ag and anti-HBc serologic positive results in patients with non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) in Poland before anti-CD20 therapy initiation; to assess the frequency of insufficient HBV screening; and to assess the association between inadequate HBV screening and diagnosis according to the WHO classification and age or gender., Material and Methods: We retrospectively collected data from 805 patients with non-Hodgkin lymphoma and chronic lymphocytic leukemia treated in 2016-2018., Results: We found a positive result of HBsAg in 13 (1.16%), and a negative result in 633 (78.64%) patients. The test was not done in 159 (19.75%) patients. In the HBsAg negative subgroup of 633 patients, we found that the anti-HBc was positive in 52 (8.22%), negative in 303 (47.87%) and not done in 278 patients. In 136 out of 805 (16.9%) patients diagnostics tests were not performed before therapy initiation. We found that age is significantly associated ( p = 0.0002) with the lack of HBV infection screening, and in CLL this risk is significantly ( p = 0.024) higher (by 49%) compared with other WHO diagnosis subgroups., Conclusions: In Polish lymphoma patients the incidence of positive HBsAg and/or anti-HBc results is consistent with the prevalence in the United States or Australia. The adherence to appropriate HBV screening guidelines in Polish centers is not sufficient. We should intensify educational strategies in the global oncohematologic medical community., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2019 Termedia & Banach.)

L-ascorbate (L-ASC) is a widely-known dietary nutrient which holds promising potential in cancer therapy when given parenterally at high doses. The anticancer effects of L-ASC involve its autoxidation and generation of H 2 O 2 , which is selectively toxic to malignant cells. Here we present that thioredoxin antioxidant system plays a key role in the scavenging of extracellularly-generated H 2 O 2 in malignant B-cells. We show that inhibition of peroxiredoxin 1, the enzyme that removes H 2 O 2 in a thioredoxin system-dependent manner, increases the sensitivity of malignant B-cells to L-ASC. Moreover, we demonstrate that auranofin (AUR), the inhibitor of the thioredoxin system that is used as an antirheumatic drug, diminishes the H 2 O 2 -scavenging capacity of malignant B-cells and potentiates pharmacological ascorbate anticancer activity in vitro and in vivo. The addition of AUR to L-ASC-treated cells triggers the accumulation of H 2 O 2 in the cells, which results in iron-dependent cytotoxicity. Importantly, the synergistic effects are observed at as low as 200 µM L-ASC concentrations. In conclusion, we observed strong, synergistic, cancer-selective interaction between L-ASC and auranofin. Since both of these agents are available in clinical practice, our findings support further investigations of the efficacy of pharmacological ascorbate in combination with auranofin in preclinical and clinical settings., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1 . Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene ( MS4A1 ) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors., (© 2017 by The American Society of Hematology.)

Gene transfer into normal quiescent human B cells is a challenging procedure. The present study aimed to investigate whether it is possible to increase the levels of transgene expression by using various types of promoters to drive the expression of selected genes‑of‑interest. To produce lentiviral particles, the present study used the 2nd generation psPAX2 packaging vector and the vesicular stomatitis virus ‑expressing envelope vector pMD2.G. Subsequently, lentiviral vectors were generated containing various promoters, including cytomegalovirus (CMV), elongation factor‑1 alpha (EF1α) and spleen focus‑forming virus (SFFV). The present study was unable to induce satisfactory transduction efficiency in quiescent normal B cells; however, infection of normal B cells with Epstein‑Barr virus resulted in increased susceptibility to lentiviral transduction. In addition, the SFFV promoter resulted in a higher level of transgene expression compared with CMV or EF1α promoters. As a proof‑of concept that this approach allows for stable gene expression in normal B cells, the present study used bicistronic lentiviral vectors with genes encoding fluorescent reporter proteins, as well as X‑box binding protein‑1 and binding immunoglobulin protein.

Photodynamic therapy (PDT) exerts direct cytotoxic effects on tumor cells, destroys tumor blood and lymphatic vessels and induces local inflammation. Although PDT triggers the release of immunogenic antigens from tumor cells, the degree of immune stimulation is regimen-dependent. The highest immunogenicity is achieved at sub-lethal doses, which at the same time trigger cytoprotective responses, that include increased expression of glucose-regulated protein 78 (GRP78). To mitigate the cytoprotective effects of GRP78 and preserve the immunoregulatory activity of PDT, we investigated the in vivo efficacy of PDT in combination with EGF-SubA cytotoxin that was shown to potentiate in vitro PDT cytotoxicity by inactivating GRP78. Treatment of immunocompetent BALB/c mice with EGF-SubA improved the efficacy of PDT but only when mice were treated with a dose of EGF-SubA that exerted less pronounced effects on the number of T and B lymphocytes as well as dendritic cells in mouse spleens. The observed antitumor effects were critically dependent on CD8+ T cells and were completely abrogated in immunodeficient SCID mice. All these results suggest that GRP78 targeting improves in vivo PDT efficacy provided intact T-cell immune system.

Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease.

Adenanthin has been recently shown to inhibit the enzymatic activities of peroxiredoxins (Prdx) I and II through its functional α,β-unsaturated ketone group serving as a Michael acceptor. A similar group is found in SK053, a compound recently developed by our group to target the thioredoxin-thioredoxin reductase (Trx-TrxR) system. This work provides evidence that next to Prdx I and II adenanthin targets additional proteins including thioredoxin-thioredoxin reductase system as well as protein disulfide isomerase (PDI) that contain a characteristic structural motif, referred to as a thioredoxin fold. Adenanthin inhibits the activity of Trx-TR system and PDI in vitro in the insulin reduction assay and decreases the activity of Trx in cultured cells. Moreover, we identified Trx-1 as an adenanthin binding protein in cells incubated with biotinylated adenanthin as an affinity probe. The results of our studies indicate that adenanthin is a mechanism-selective, rather than an enzyme-specific inhibitor of enzymes containing readily accessible, nucleophilic cysteines. This observation might be of importance in considering potential therapeutic applications of adenanthin to include a range of diseases, where aberrant activity of Prdx, Trx-TrxR and PDI is involved in their pathogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Photodynamic therapy (PDT) of tumours is based on administration of a photosensitiser followed by irradiation of the tumour with visible light leading to production of reactive oxygen species that cause direct tumour cell death and vascular damage. PDT also initiates acute local inflammation, which facilitates the development of adaptive antitumour immunity. It has recently been reported that PDT can induce strong antitumour immunity towards tumours cells expressing P1A, tumour-associated antigen. Using four different tumour models, we show that antitumour immune response can be further improved when PDT is combined with a clinically approved epigenetic agent that induces expression of a silenced P1A antigen. Induction of P1A with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, resulted in potentiated antitumour effects in mice with Lewis lung carcinoma and 4T1 mammary carcinoma when combined with PDT treatment. In CT26 colon carcinoma and EMT6 mammary carcinoma models the combination therapy resulted in complete responses and long-term survival. All long-term surviving mice were resistant to re-inoculation with the same tumour cells. Antitumour efficacy of the combination treatment was severely impaired by depletion of CD8(+) cytotoxic T cells, whereas adoptive transfer of CD8(+) T cells from long-term surviving mice allowed for significant tumour growth delay in tumour-bearing mice. Taken together, these findings show that PDT leads to strong specific antitumour immune responses, and that epigenetic modification of tumour antigens levels may be a novel approach to further enhance the effectiveness of PDT. The present results provide a strong rationale for clinical development of this therapeutic approach., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Adenosine levels increase at seizure foci as part of a postulated endogenous negative feedback mechanism that controls seizure activity through activation of A1 adenosine receptors. Agents that amplify this site- and event-specific surge of adenosine could provide antiseizure activity similar to that of adenosine receptor agonists but with fewer dose-limiting side effects. Inhibitors of adenosine kinase (AK) were examined because AK is normally the primary route of adenosine metabolism. The AK inhibitors 5'-amino-5'-deoxyadenosine, 5-iodotubercidin, and 5'-deoxy-5-iodotubercidin inhibited maximal electroshock (MES) seizures in rats. Several structural classes of novel AK inhibitors were identified and shown to exhibit similar activity, including a prototype inhibitor, 4-(N-phenylamino)-5-phenyl-7-(5'-deoxyribofuranosyl)pyrrolo[2, 3-d]pyrimidine (GP683; MES ED50 = 1.1 mg/kg). AK inhibitors also reduced epileptiform discharges induced by removal of Mg2+ in a rat neocortical preparation. Overall, inhibitors of adenosine deaminase or of adenosine transport were less effective. The antiseizure activities of GP683 in the in vivo and in vitro preparations were reversed by the adenosine receptor antagonists theophylline and 8-(p-sulfophenyl)theophylline. GP683 showed little or no hypotension or bradycardia and minimal hypothermic effect at anticonvulsant doses. This improved side effect profile contrasts markedly with the profound hypotension, bradycardia, and hypothermia and greater inhibition of motor function observed with the adenosine receptor agonist N6-cyclopentyladenosine and opens the way to clinical evaluation of AK inhibitors as a novel, adenosine-based approach to anticonvulsant therapy.

The physiological role of adenosine (Ado) is well known. Although a number of pharmacological attempts have been made to manipulate Ado concentrations in ischemic conditions in different tissues, none have been clinically accepted up to now, mostly due to insufficient elevation of Ado concentrations or unacceptable toxicity. In this study, we evaluated the biochemical and pharmacological actions of several novel erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) analogs to identify new reversible adenosine deaminase (ADA) inhibitors with potential clinical utility. In cell culture experiments, these compounds elevate cellular Ado concentrations under conditions of simulated ischemic stress but very little, if any, under normoxic conditions. Two compounds were selected for study: 9'-chloro-EHNA (CPC-405) and 9'-phthalimido-EHNA (CPC-406), which specifically inhibit ADA in cell-free preparations as well as in intact cells. CPC-405 and CPC-406 do not affect adenosine kinase activity, and they do not affect adenosine transport (influx). CPC-405 and CPC-406 are also more potent than EHNA in elevating adenosine release from human astrocytoma cells and bovine heart microvascular endothelial cells in 2-deoxyglucose-simulated ischemia or under anaerobic conditions. Inhibition of adenosine deaminase by CPC-405 or CPC-406, as well as the 2'-deoxyadenosine toxicity expressed in the presence of these ADA inhibitors, is reversed when the inhibitors are removed by washing the cells. In the isolated rat heart model of ischemia, these novel ADA inhibitors showed enhanced recovery of left ventricular end-diastolic pressure, left ventricular developed pressure, +dP/dtmax and -dP/dtmax. In the rat hippocampal slice model of hypoxia, these compounds also showed neuroprotective effects on CA1 hypoxic injury. In conclusion, these novel ADA inhibitors may represent clinically useful Ado elevating compounds that show cardioprotective, as well as neuroprotective, effects. Also, their potential for immunotoxicity, if any, appears to be transient in nature, representing an important clinical advantage compared with tight-binding ADA inhibitors such as deoxycoformycin.

Objective: Oxidative stress from increased production of reactive oxygen species or decreased efficiency of inhibitory and scavenger systems may contribute to vascular injury. In this study, we developed an in vitro model of vascular injury by menadione-induced oxidative stress in bovine heart microvascular endothelial cells., Methods: Oxidative stress was induced by exposure to menadione. Superoxide, hydrogen peroxide and hydroxyl radical formation was measured by superoxide dismutase-inhibitable cytochrome c reduction, the dichlorofluorescin technique and the salicylate method, respectively. Electron paramagnetic-spin resonance spectroscopy employing 5-5'-dimethyl-l-pyrroline-N-oxide for superoxide trapping was used. Endothelial cell cytotoxicity was assessed by lactate dehydrogenase release., Results: Superoxide and hydroxyl radical were produced in a time- and concentration-dependent fashion. Fluorescence in the presence of dichlorofluorescin confirmed hydrogen peroxide formation. Endothelial cell cytotoxicity became evident after 5 h of menadione treatment at concentrations of 100 microM. 3-Aminobenzamide, a poly(ADP-ribose)polymerase inhibitor, and dimethylthiourea, a hydrogen peroxide and hydroxyl radical scavenger, decreased menadione cytotoxicity, whereas deferoxamine, an inhibitor of hydroxyl radical formation, did not., Conclusions: The results suggest that menadione toxicity is mediated by poly(ADP-ribose)polymerase activation via hydrogen peroxide formation and that menadione-treated bovine heart microvessel endothelial cells provide a suitable in vitro model to study oxidative stress in endothelial cells.

The effect of 5-amino-4-imidazole-carboximide (AI-CA)-riboside on different pathways of purine metabolism (biosynthesis de novo, salvage pathways, adenosine metabolism, ATP catabolism) was studied in human B lymphoblasts (WI-L2). AICA-Riboside markedly decreased intracellular levels of 5-phosphoribosyl-1-pyrophosphate and in consequence affected purine biosynthesis de novo and purine salvage pathways. AICA-riboside inhibited incorporation of glycine into purine nucleotides, but when formate was used as the precursor of purine biosynthesis de novo, a biphasic effect was observed. The incorporation of formate into purine nucleotides was increased by AICA-riboside at concentrations up to 2 mM but decreased at higher concentrations. Salvage of the purine bases adenine, hypoxanthine, and guanine was markedly inhibited and utilization of extracellular adenosine in B lymphoblasts was reduced by AICA-riboside. AICA-riboside increased ribose 1-phosphate concentrations and increased degradation of prelabeled ATP. No effect on the intracellular levels of orthophosphate was found. Proliferation of WI-L2 lymphoblasts was only slightly affected at concentrations of AICA-riboside below 500 microM but markedly inhibited by higher concentrations.

The conceptual framework which underlies many studies of purine and pyrimidine metabolism in intact cells has been critically evaluated. The model that is implicit in many such studies is the single, partially purified enzyme. This paper gives examples both of instances in which the extrapolation of results of enzymes studies to intact cells has been successful and of instances in which enzymes behave differently in the intact cell than in cell extracts. Pitfalls in the extrapolation of results of enzyme studies to intact cells concern (a) metabolic pathways, (b) intracellular enzyme activities, (c) enzyme regulation, and (d) intracellular metabolite concentrations. Examples are also given of situations in which perturbations in one aspect of purine or pyrimidine metabolism lead to changes in other aspects, often distant in the network of reactions.

Deficiency of either one of the subsequent purine catabolic enzymes adenosine deaminase or purine nucleoside phosphorylase results in immunodeficiency disease in humans. However, the mechanism by which impairment of purine metabolism may cause immunodeficiency is unclear. In the present work we have studied the catabolism of purine ribonucleotides and deoxyribonucleotides in T lymphocytes to better understand the role of purine nucleoside phosphorylase and adenosine deaminase in the immune function. It was found that purine deoxyribonucleotides are degraded via catabolic pathways distinctly different from those used for purine ribonucleotide degradation. Thus both adenine and guanine ribonucleotides are deaminated to IMP whereas purine deoxyribonucleotides are exclusively dephosphorylated to the corresponding deoxyribonucleosides. These findings may explain the relatively higher degradation rates of purine deoxyribonucleotides in mammalian cells as compared to purine ribonucleotides. The catabolism of purine nucleotides is tightly linked to the active purine nucleoside cycles which consist of the phosphorolysis of purine nucleosides and deoxyribonucleosides to their corresponding bases, their salvage to monophosphates and back to the corresponding ribonucleosides. The above observations also imply that a possible role of the purine nucleoside cycles is to convert purine deoxyribonucleotides into their corresponding ribonucleotide derivatives. Deficiencies of purine nucleoside phosphorylase or of adenosine deaminase activities, enzymes which participate or lead to the purine nucleoside cycles, thus result in a selective impaired deoxyribonucleotide catabolism and immunodeficiency.

The overall purine metabolism was studied in detail in resident peritoneal macrophages (M phi) and in thioglycolate elicited peritoneal M phi in vitro. The salvage of purine bases (adenine, hypoxanthine and guanine) was active in both M phi populations, whereas purine biosynthesis de novo was low. Purine nucleosides (inosine, guanosine and adenosine) were efficiently degraded to uric acid and only adenosine was directly salvaged into nucleotides. Purine salvage was markedly increased in elicited M phi as compared to resident M phi whereas purine degradation pathways were enhanced only slightly. These results clearly indicate that salvage of purine bases is the main source for purine nucleotide biosynthesis in M phi, but nucleotide catabolism is the predominant pathway.

The effects of lowered intracellular ATP and GTP concentrations on enzymes of purine ribonucleotide synthesis and intercoversion were studied using intact Ehrlich ascites tumor cells. The apparent rates of phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1) and of inosinate dehydrogenase (EC 1.2.1.14) were increased in cells containing lowered purine nucleotide concentrations, but apparent activities of amidophosphoribosyltransferase (EC 2.4.2.14), the purine phosphoribosyltransferases, and other enzymes of purine ribonucleotide interconversion were not affected.

Human intrathymic T lymphocytes were separated by a bovine serum albumin density gradient into a population of G1-phase small thymocytes and a population of S-phase-enriched large thymocytes. Purine metabolism was studied in these thymocyte populations, representing immature T lymphocytes, and compared with the metabolism of mature T lymphocytes isolated from the peripheral blood. De novo purine biosynthesis was highly cell cycle dependent; i.e., de novo purine biosynthetic activity was found only in large S-phase thymocytes, whereas both G1 T-cell populations lacked any significant activity. Thus G1-phase small thymocytes and G1-phase peripheral blood T lymphocytes have only salvage pathways to maintain their purine nucleotide pools. Despite the similarity of purine salvage activities in G1 thymocytes and in peripheral blood T lymphocytes, small thymocytes have fourfold lower levels of purine nucleoside triphosphates. The decreased levels of purine nucleotides in G1 thymocytes may be the result of increased purine efflux. It was found that an unusually large proportion (24-48%) of hypoxanthine incorporated by G1 thymocytes is excreted into the medium in the form of inosine.

Concentrations of intracellular orthophosphate were determined in Ehrlich ascites tumor cells incubated with glucose, inosine, or uridine in media of different orthophosphate concentration. The effects of orthophosphate concentration on the accumulation of lactate and of phosphoribosyl pyrophosphate and on concentrations of ribose 1-phosphate and ribose 5-phosphate in tumor cells incubated with glucose were also determined. Both the phosphorolysis of inosine and the rate of catabolism of ATP in cells incubated with 2-deoxyglucose were also influenced by the orthophosphate concentration of the medium.

The effect of recombinant interferon-alpha 2 (IFN-alpha 2) (50 U/ml) on the cell cycle, nucleotide metabolism, and protein and nucleic acid synthesis was studied in human B-lymphoblastoid (Daudi) cells. Cell cycle analysis showed that IFN treatment resulted in G0/G1 arrest (69%) as compared to control cells (42% at G0/G1). IFN inhibited the incorporation of radioactive thymidine and uridine into DNA and RNA, respectively, but had only slight effect on incorporation of radioactive threonine, leucine, or valine into proteins. IFN inhibited ribonucleotide biosynthesis by de novo and salvage pathways and decreased level of the P-ribose-PP. Both pathways of deoxyribonucleotide biosynthesis, ribonucleotide reduction and deoxyribonucleoside salvage, were also markedly inhibited by IFN., In contrast, ribonucleotide catabolism was significantly increased in the presence of IFN. No changes in ribonucleotide interconversion were found. Intracellular concentrations of both ribonucleotides and deoxyribonucleotides were markedly diminished by IFN. These results suggest that inhibition of both ribonucleotide and deoxyribonucleotide biosynthesis, together with increased rate of nucleotide catabolism, may significantly decrease intracellular nucleotide availability. Decrease of the supply of nucleic acid precursors, as well as limitation of nucleotides for energy metabolism and other processes, may result in the inhibition of cell multiplications.

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.

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.

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.

The effect of DNA damage caused by N-methyl-N'-nitro-nitrosoguanidine (MNNG) on poly(ADP-ribose) synthesis, NAD levels, and purine nucleotide metabolism was studied in human T-lymphoblasts. Excessive DNA breaks caused by MNNG activated poly(ADP-ribose) polymerase and rapidly consumed intracellular NAD. NAD depletion was followed by rapid catabolism of ATP as well as induction of total purine nucleotide catabolism leading to excretion of purine catabolic products. MNNG-treated cells were not able to replenish the intracellular nucleotide pools due to the depletion of intracellular ATP and phosphoribosylpyrophosphate pools which are required for de novo purine biosynthesis. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide prevented both the depletion of NAD pools and the associated changes in purine nucleotide metabolism.