Enhanced production of intracellular ara-CTP after sequential use of methotrexate and 1-beta-D-arabinofuranosylcytosine

Synergistic antiproliferative effect has been proven in vitro when mouse leukemic cells were sequentially treated with MTX and ara-C. The mechanism of this combination effect not well elucidated but the intracellular uptake of ara-C was higher when cells were pre-exposed to MTX. In this experiment, the intracellular ara-CTP was measured by HPLC after MTX and ara-C were sequentially administered to BDF1 mice bearing L1210 leukemic cell, either being sensitive or resistant to MTX. When MTX at the dose of 12 mg/kg was preceded 6 h and 3 h to ara-C at the dose of 25 mg/kg, the intracellular levels of ara-CTP were found to be significantly higher as compared with those of ara-C alone as control group. At 1 h after ara-C, ara-CTP was measured about 165 and 130% of the control levels, respectively, and at 12 h, ara-CTP was over 4 times higher of control level with group of mice to which MTX was preceded 6 h prior to MTX. On the contrary, the enhancement of ara-CTP production was definitely diminished with MTX-resistant cells in the same administrative model. From our present experiment, the time sequential modulation of intracellular ara-CTP production by MTX was reconfirmed in vivo, and this modulation might depend upon the sensitivity of MTX of leukemic cells.     

Integration of biochemical, cellular, and animal pharmacology of antineoplastic agents to design new model drug combinations for treatment of acute leukemia

An attempt was made to integrate the biochemical, cellular, and animal pharmacology of certain antineoplastic agents in order to design a model chemotherapeutic regimen that was based on scientific rationale. The agents that were chosen for this investigation were 5-fluorodeoxyuridine (FUdR), 5-bromodeoxyuridine (BUdR), and β-deoxythioguanosine (TGdR) because the biochemical interactions between these antimetabolites suggested that this drug combination would be synergistic with respect to its antineoplastic activity. The combination FUdR plus BUdR was found to be synergistic with respect to its in vitro cytotoxicity against tumor cells and its in vivo activity in mice with L1210 leukemia. When FUdR, BUdR, and TGdR were used in combination its in vitro cytotoxicity was synergistic. This 3 drug combination was not synergistic in vivo, but did produce a very potent antileukemic effect in the mice. Although more data are needed before it can be determined if this model drug combination has any future clinical application, this overall approach shows that it is possible to integrate the pharmacological data of antineoplastic agents and if used with some of the conventional antileukemic agents may possibly lead to the rational design of some very effective drug combinations for the treatment of acute leukemia.     

Regulation of cyclic adenosine 3',5'-monophosphate in relation to growth of dimethylbenz[a]anthracene-induced mammary tumors in rats

In this study, adenylate cyclase, phosphodiesterase and cyclic adenosine 3',5'-monophosphate (cAMP) levels were measured in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors with different growth characteristics in both intact and ovariectomized rats. Tumors were classified as growing, stable, or regressing over a 10-14-day period before excision. Regressing or static tumors had a higher cAMP concentration relative to tumors that were growing actively. This was due to high adenylate cyclase activity and low phosphodiesterase (both high and low Km) activities. Although estrogen deprivation resulted in a much greater rate of tumor regression than would occur spontaneously under normal conditions, the levels of adenylate cyclase, phosphodiesterases and cAMP were the same in tumors obtained from either intact or ovariectomized rats, when comparisons were made within the same category of tumor. These observations suggest that cAMP metabolism is independent of estrogen, since it is related to enhancement or retardation of growth rather than to the presence of absence of estrogen.     

Effect of cytosine arabinoside on nuclear labelling of leukaemic myeloblasts with tritiated thymidine triphosphate

The incorporation of ³H-thymidine-5-triphosphate (³H-TTP) into nuclei of leukaemic myeloblasts (MB) was studied together with various other cytokinetic parameters. In 13 cases with acute myeloid leukaemia (AML) the ³H-TTP labelling index (³H-TTP LI) exceeded the fraction in DNA synthesis by a factor 3–85. After a single i.v. bolus injection of cytosine arabinoside (ARA-C) an initial decrease of ³H-TTP LI after $\text{1}\text{2}$ h and a nadir after 6 h was seen in short-term studies. Long-term studies showed a decrease in the ³H-TTP LI up to 120 h after ARA-C. The data suggest that ARA-C acts on MB also outside of S-phase.     

Enzymological studies in chronic lymphocytic leukemia

Adenosine deaminase (ADA), 5'nucleotidase (5'NT), ecto-5'NT, purine nucleoside phosphorylase (PNP), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), adenosine kinase (AK), AMP-deaminase (AMPD) and adenylate kinase (AdKin) activities were assayed in peripheral blood lymphoid cells from 20 patients with B-cell type chronic lymphocytic leukemia (CLL). Significantly decreased mean activities of ADA, 5'NT, ecto-5'NT, PNP and AMPD were observed when comparing B-CLL lymphoid cells with control peripheral blood lymphocytes (PBL). AK and AdKin activities however, were found to be higher in B-CLL. Relatively wide ranges of ADA and 5'NT activity were observed. In patients with paraproteinaemia, 5'NT activity was found to be relatively high and in the range of the activities in normal PBL. ADA activity seemed to be slightly higher in patients without paraproteinaemia. No correlation could be found between the enzyme activities and the number of cells rosetting with sheep erythrocytes or bearing surface immunoglobulin (sIg). A relationship was suggested between 5'NT activity and Ig production.     

Down regulation of c-Myc and induction of an angiogenesis inhibitor, thrombospondin-1, by 5-FU in human colon cancer KM12C cells

5-FU is commonly used for treatment of various solid tumors including colon carcinoma. We have previously demonstrated that Egr-1 induced by 5-FU enhanced TSP-1 expression in human colon cancer KM12C cells. In this study, a Genechip analysis of KM12C cells treated with 5-FU revealed down-regulation of 924 genes and up-regulation of 460 genes. The decreased expression of c-Myc mRNA and phosphorylated c-Myc were detected and confirmed by RT-PCR and immunoblotting. Since 5-FU induced the expression of TSP-1, we examined the effect of c-Myc on the TSP-1 promoter. Deletion of the TSP-1 promoter region in which binding sites for c-Myc reside had no effect on the TSP-1 promoter activity induced by 5-FU. Meanwhile, 5-FU dose-dependently decreased the expression of miR-17-92 cluster. These findings suggest that 5-FU decreased the expression of c-Myc and consequently miR-17-92 cluster and increased the expression of TSP-1 mRNA.     

Purine metabolism in childhood acute lymphoblastic leukemia: biochemical markers for diagnosis and chemotherapy

Adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), 5'nucleotidase (5'NT), ecto-5'NT, hypoxanthine-guanine phosphoribosyltransferase(HGPRT), adenine phosphoribosyltransferase(APRT), adenosine kinase(AK), AMP deaminase (AMPD) and adenylate kinase(AdKin) activities were assayed in leukemic cells from bone marrow and/or peripheral blood of 43 newly diagnosed children with acute lymphoblastic leukemia(ALL). These enzyme activities have been investigated in relation to some immunological markers. ADA activity was higher in E-rosette positive leukemia(E+ ALL), while HGPRT, APRT, PNP, 5'NT, ecto-5'NT and AdKin activities were found to be lower in E+ ALL as compared to E- ALL. In common ALL (cALL) antigen positive leukemia, mean ADA activity was significantly lower as compared to cALL- leukemia, whereas PNP, 5'NT, ecto-5'NT and AdKin activities were significantly higher. cALL cells with cytoplasmic immunoglobulin M(IgM) heavy chains were found to have mean 5'NT activities twice as high as cALL cells lacking cytoplasmic IgM heavy chains. In two patients who had surface immunoglobulins on their cell membranes, low 5'NT activities were found. When measuring enzyme activities after 2-4 days of prednisone monotherapy, only mean ADA and HGPRT activities decreased in non-B, non-T ALL. These decreases were not significant in T-ALL patients. Mean enzyme activities in the leukemic cells of five patients with relapse were comparable to those in newly diagnosed patients, except for 5'NT, which was found to be within the activity range of control peripheral blood lymphocytes. It is concluded that ADA and AdKin activities are suitable as markers for E+ ALL and cALL+ leukemias respectively. 5'NT might help to distinguish between cALL cells having and lacking pre-B characteristics. Since 5'NT activity may also be decreased in B-ALL, it is not suitable as a T-ALL marker. Enzymes of purine metabolism in leukemic relapse need further investigation.     

Antagonistic action between cyclic AMP and estrogen in phosphorylation of mammary tumor nuclear proteins.

A new protein kinase-dependent phosphorylation occurs in the nuclei of hormone-dependent, 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced mammary carcinoma following preincubation of tumor slices with cyclic adenosine 3',5'-monophosphate (cAMP). The presence of 17beta-estradiol in the medium inhibits this effect. Both events have been observed in vivo in the nuclei of DMBA-induced tumors. The phosphorylation pattern of nuclei in hormone-independent mammary tumor, DMBA No. 1, however, is not affected by preincubation with either cAMP or estrogen. These findings suggest that the antagonistic effect of cAMP and estrogen in the growth control of mammary tumors is exerted through a specific action on nuclear protein phosphorylation and that these events correlate with the hormone-dependency of the tumors.     

S1PR5 regulates NK cell responses in preventing graft-versus-host disease while preserving graft-versus-tumour activity in a murine allogeneic haematopoietic stem cell transplantation model

Graft-versus-host disease (GVHD) remains a major complication following allogeneic haematopoietic stem cell transplantation (allo-HSCT) leading to high transplant-related mortality. Natural killer (NK) cells have been found to mitigate GVHD without attenuating the graft-versus-tumour (GVT) activity in the murine model of haematopoietic stem cell transplantation. Sphingosine-1-phosphate receptor 5 (S1PR5) is a very important chemokine receptor on NK cells that governs NK cell distribution in vivo and trafficking at lesion sites. Our preliminary studies showed that the incidence of GVHD was negatively correlated with S1PR5 expression in the NK cells of patients after allo-HSCT. In the present study, we found that S1PR5 deficiency in murine NK cells blocked the migration of NK cells from the bone marrow to the GVHD target organs and attenuated the inhibitory effects on the alloreactive T cells, especially CD3⁺CD8⁺T cells, which may be the reason why the loss of S1PR5 in NK cells could aggravate GVHD in recipient mice. Furthermore, we also demonstrated that the absence of S1PR5 expression in NK cells did not interfere with the antitumour effects of NK cells and T cells in vivo. Taken together, our data indicate that S1PR5 plays an essential role in balancing GVHD and GVT activity.     

Phorbol ester-induced loss of colchicine ultrasensitivity in chronic lymphocytic leukaemia lymphocytes

On exposure to the phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) the pathological (non-dividing) lymphocytes of B-cell chronic lymphocytic leukaemia (CLL) lose their characteristic ultrasensitivity to the cytocidal action of colchicine in vitro. They are no longer killed in 1 day by the drug at 10(-6)M-concentration. The effect was the same whether the cells were incubated in the continuous presence of TPA, or subjected instead to pulse-treatment with it (for as little as 5 min.). Colchicine at one thousand times greater concentration was now needed to kill the cells. CLL lymphocytes already primed to undergo interphase death by pretreatment with colchicine could be prevented from doing so by early addition of TPA. A marked proportion of those CLL lymphocytes destined to undergo early spontaneous death in vitro in the absence of colchicine could be prevented from doing so by TPA. The loss of colchicine ultrasensitivity applied to cells which had not yet undergone TPA-induced morphological transformation to blast-like cells or differentiation to cells containing abundant cytoplasmic immunoglobulins (CIg). These transformed cells materialised in greatest incidence (70-80%) after 3 days of culture, an observation in agreement with others workers.     

三氧化二砷联合5-杂氮-2′-脱氧胞苷对U937细胞SHP-1、JAK3、TYK2基因表达的影响

目的 探讨甲基化抑制剂5-杂氮-2′-脱氧胞苷（5-Aza-2′-deoxycytidine,5-aza-CdR）联合三氧化 二砷（As2O3）对白血病细胞株U937中JAK3、TYK2和造血细胞磷酸酶SHP-1表达水平的影响,并探讨它 们在白血病发病中的作用。方法 5-aza-CdR、As2O3单用及联合处理U937细胞,5-aza-CdR浓度为0.5、1、2 μmol/L,As2O3的浓度为1、2.5、5 μmol/L,As2O3 1μmol/L + 5-aza-CdR 0.5μmol/L、As2O32.5 μmol/L + 5-aza- CdR 1 μmol/L、As2O3 35 μmol/L + 5-aza-CdR 2 μmol/L 及不加药物组,分别处理24、48、72 h后提取细胞总 RNA,荧光实时定量PCR（Real-time Quantitative Polymerase Chain Reaction,RQ-PCR）检测JAK3、TYK2 及SHP-1的表达。结果 As2O3和5-aza-CdR单独作用及两药合用时,SHP-1 mRNA在U937细胞中的表达呈 剂量及时间依赖性,其表达逐渐升高;JAK3 mRNA的表达呈剂量及时间依赖性,其表达逐渐降低;TYK2 mRNA的表达呈剂量及时间依赖性,其表达逐渐降低;SHP-1与JAK3、TYK2负相关,JAK3所受影响较 TYK2更为显著。结论 （1）甲基化抑制剂5-aza-CdR和As2O3可使SHP-1表达升高,JAK3、TYK2表达降 低,与浓度及作用时间有关。（2）SHP-1基因的重新表达与其发生去甲基化有关,对JAK/STAT通路有负调 控作用。     

Glutamine requirements for purine metabolism in leukemic lymphoblasts

The two pathways of purine metabolism that include glutamine-dependent reactions, purine synthesis de novo and guanine nucleotide synthesis, were studied in cultured lymphoblasts derived from patients with T cell (JM), B cell (BALL) or null cell (NALL) acute lymphoblastic leukemia (ALL). When glutamine was omitted from the incubation medium, purine, synthesis de novo, measured by the incorporation of ¹⁴C-formate into purine compounds, was depressed to barely measurable rates in BALL and NALL cells, but proceeded at moderate through reduced rates in JM cells, when compared to synthesis in the presence of 2 mM glutamine. Similarly, the incorporation of ¹⁴C-hypoxanthine into guanine nucleotides was arrested at the glutanine-requiring XMP-aminase reaction in the BALL and NALL lines but not in the JM line, when exogenous glutamine was absent. The data suggest that glutamine deprivation, whether by omission from the culture medium in vitro or by glutaminase treatment in vivo, will have more profound biochemical consequences in B and null cell-derived ALL than in T ALL.     

Glucose-6-phosphate dehydrogenase and NADPH oxidase 4 control STAT3 activity in melanoma cells through a pathway involving reactive oxygen species,c-SRC and SHP2

Background: Glucose-6-phosphate dehydrogenase (G6PD) participates in glucose utilization by catalysing the first step of the pentose-phosphate pathway in mammalian cells. Previous studies have shown that changes in G6PD levels can promote tumor cell proliferation or apoptosis via the STAT3/5 pathway in a human melanoma xenograft model. G6PD cooperates with NADPH oxidase 4 (NOX4) in the cellular metabolism of reactive oxygen species (ROS) and in maintaining the intracellular redox state. Methods: In this study, the effect of G6PD or NOX4 silencing in the melanoma line A375 was examined in terms of redox state, proto-oncogene tyrosine-protein kinase Src (c-Src) and the tyrosine-specific protein phosphatase SHP2 expression as well as cell cycle progression. Results: The results demonstrate that: (1) Downregulation of cyclin D1 and CDK4 and up-regulation of p53 and p21 occurred in response to silencing of G6PD and NOX4 thus resulting in G1/S cell cycle arrest and inhibition of A375 cell proliferation. (2) The blockade of cell proliferation is primarily due to a reduced DNA-binding activity of STAT3. (3) The DNA-binding activity of STAT3 was regulated by the upstream factors, c-SRC and SHP2. Silencing of NOX4 in A375 cells inhibited c-SRC and SHP2 regulated STAT3 activity. Conclusion: The data are consistent with a novel G6PD-NOX4-NADPH-ROS-c-SRC/SHP2 pathway controlling STAT3 activity in A375 melanoma cells.     

Targeting autophagy using natural compounds for cancer prevention and therapy

Autophagy, also known as macroautophagy, is a tightly regulated process involved in the stress responses, such as starvation. It is a vacuolar, lysosomal pathway for the degradation of damaged proteins and organelles in eukaryotic cells. Autophagy also plays a key role in various tissue processes and immune responses and in the regulation of inflammation. Over the past decade, three levels of autophagy regulation have been identified in mammalian cells: 1) signaling, 2) autophagosome formation, and 3) autophagosome maturation and lysosomal degradation. Any deregulation of the autophagy processes can lead to the development of diverse chronic diseases, such as diabetes, obesity, cardiovascular disease, neurodegenerative disease, and malignancies. However, the potential role of autophagy in cancer is rather complex and has been associated with both the induction and the inhibition of neoplasia. Several synthetic autophagy modulators have been identified as promising candidates for cancer therapy. In addition, diverse phytochemicals derived from natural sources, such as curcumin, ursolic acid, resveratrol, thymoquinone, and γ-tocotrienol, also have attracted attention as promising autophagy modulators with minimal side effects. In this review, the authors discuss the importance of autophagy regulators and various natural compounds that induce and/or inhibit autophagy in the prevention and therapy of cancer.     

Genetic variation in Transaldolase 1 and risk of squamous cell carcinoma of the head and neck

Background: The Pentose Phosphate Pathway (PPP) is involved in the body's protection against oxidative stress and resistance/susceptibility to apoptosis and thus has been implicated in tumor development and progression. Here we present data examining the association of genetic variation in one of the key enzymes of the PPP, Transaldolase 1 (TALDO1) with squamous cell carcinoma of the head and neck (SCCHN). Methods: We performed sequencing analysis to identify common genetic variations in TALDO1 and then investigated their association with SCCHN using samples from a population-based case/control study with both European American (EA) and African American (AA) former and current smokers. Results: We identified three polymorphisms in TALDO1 that were associated with SCCHN risk in our EA study population. Specifically the 5′ upstream variant −490C > G or T (rs10794338), which we identified as tri-allelic, showed a reduced risk compared with any presence of the common allele, odds ratio (OR) [95% confidence interval (95% CI)]: 0.57 (0.38-0.86). Additionally two intronic high frequency polymorphisms demonstrated a positive association with disease, with the presence of the variant IVS1 + 1874T > A (rs3901233), 1.76 (1.19-2.61) and IVS4 + 2187A > C (rs4963163), 1.71 (1.16-2.53). Conclusion: These results provide preliminary evidence that genetic polymorphisms in TALDO1 are associated with SCCHN.     

Modulation of the Wnt/beta-catenin pathway in human oligodendroglioma cells by Sox17 regulates proliferation and differentiation

Oligodendrogliomas originate from oligodendrocyte progenitor cells (OPCs), whose development is regulated by the Sonic hedgehog and Wnt/beta-catenin pathways. We investigated the contribution of these pathways in the proliferation and differentiation of human oligodendroglioma cells (HOG). Inhibition of Hedgehog signaling with cyclopamine decreased cell survival and increased phosphorylated beta-catenin without altering myelin protein levels. Conversely, treatment of HOG with the Wnt antagonist secreted frizzled related protein (SFRP1), led to increased myelin protein levels and reduced cell proliferation, suggesting cell cycle arrest and differentiation. Unlike normal primary human OPCs, beta-catenin in HOG cells is not associated with endogenous Sox17 protein despite high levels of both proteins. Retroviral overexpression of recombinant Sox17 increased HOG cell cycle exit and apoptosis, and raised myelin protein levels and the percentage of O4⁺ cells, indicating increased differentiation. Recombinant Sox17 also increased beta-catenin-TCF4-Sox17 complex formation and decreased total cellular levels of beta-catenin. These changes were associated with increased SFRP1, and reduced expression of Wnt-1 and Frizzled-1, -3 and -7 RNA, indicating that Sox17 induced a Hedgehog target, and regulated Wnt signaling at multiple levels. Our studies indicate that Wnt signaling regulates HOG cell cycle arrest and differentiation, and that recombinant Sox17 mediates modulation of the Wnt pathway through changes in beta-catenin, SFRP1 and Wnt/Frizzled expression. Our results thus identify Sox17 as a potential molecular target to include in HOG therapeutic strategies.