Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023473 (chronic myeloid leukemia)
 18,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thymidylate kinase derived from the blast cells of human chronic myelocytic leukemia was purified 2186-fold to near homogeneity by means of alcohol precipitation, alumina-Cgamma gel fractionation, calcium phosphate gel fraction, ultrafiltration, and affinity column chromatography. The molecular weight was estimated by glycerol gradient centrifugation to be 50,000. This enzyme had an optimal activity at pH 7.1 and required a divalent cation in order to catalyze the reaction. Mg2+ and Mn2+ were found to be the preferential divalent cations. The activation energy was estimated to be 19.1 kcal/mol at pH 7.2. Initial velocity study suggested that the reaction followed a sequential mechanism. Mg2+ ATP had a Km of 0.25 mM and dTMP had a Km of 40 micrometer. The enzyme was unstable even at 4 degrees. In the presence of ATP or dTMP the enzyme maintained its activity. Purine triphosphate nucleosides were found to be better phosphate donors than the pyrimidine triphosphate nucleosides. ATP and dATP had a lower Km and a higher Vmax than GTP and dGTP. dTMP was the only preferred phosphate receptor among all the monophosphate nucleotides tested dTTP and IdUTP competed with both substrates and inhibited the reaction with a Ki of 0.75 mM and 1.1 mM, respectively.
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PMID:Human thymidylate kinase. Purification, characterization, and kinetic behavior of the thymidylate kinase derived from chronic myelocytic leukemia. 1 69

To determine whether decreased alkaline phosphatase activity in the granules from neutrophils of patients with chronic myelogenous leukemia (CML) was due to an absence of enzyme or the production of defective enzyme, we compared the immunologic properties of granule alkaline phosphatase derived from patients with CML with that of normal subjects and patients with polycythemia vera (PRV). Antisera prepared in rabbits against granule alkaline phosphatase purified from the neutrophils of a patient with PRV produced a single precipitin line of antigenic identity when reacted with extracts of normal, PRV, and CML neutrophil granules. A histochemical stain for alkaline phosphatase activity (alpha-naphthyl acid phosphate coupled with Fast Blue RR) specifically stained the precipitin line. A variety of quantitative precipitin techniques failed to produce satisfactory precipitation of alkaline phosphatase activity. Comparative analyses were therefore performed by affinity chromatography using goat antirabbit-gammaglobulin linked to Sepharose 4B to adsorb alkaline phosphatase complexed with rabbit gamma globulin. With this method, 100% of CML, normal, and PRV alkaline phosphatase could be adsorbed. Using limiting concentrations of antibody, a proportionally smaller fraction of enzyme activity was absorbed as the concentration of PRV alkaline phosphatase or normal alkaline phosphatase was increased. Extracts of CML granules containing comparable amounts of protein but 200-fold less alkaline phosphatase activity per milligram did not specifically reduce adsorption. Thus, in CML, we found no evidence that the granulocytes contained a large amount of antigenically normal but enzymatically defective alkaline phosphatase. Examination of electron micrographs revealed no significant differences in the number or distribution of granules in the granulocytes of normal subjects or patients with PRV or CML. This suggests that the low level of neutrophil alkaline phosphatase in CML granulocytes is the result of decreased enzyme content and not a consequence of synthesis of catalytically defective enzyme.
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PMID:Neutrophil alkaline phosphatase: comparison of enzymes from normal subjects and patients with polycythemia vera and chronic myelogenous leukemia. 4 59

Deoxycytidine kinase, which phosphorylates deoxycytidine (CdR) and its analog, cytosine arabinoside (ara-C), has been purified 71-fold from human leukemic cells. Biochemical properties of the partially purified enzyme included a molecular weight of 68,000, Kms of 7.8 muM for CdR and 25.6 muM for ara-C, and optimal activity with ATP and GTP as phosphate donors. Ara-C phosphorylation was strongly inhibited by CdR (Ki = 0.17 muM) and dCTP (Ki = 7.3 muM) and was weakly inhibited by ara-CTP (Ki = 0.13 mM). Purification by calcium phosphate gel elution and DEAE chromatography effectively separated this enzyme from cytidine deaminase, which deaminates both CdR and ara-C, and from uridine-cytidine kinase, the enzyme which phosphorylates 5-azacytidine. CdR kinase activity was found to decrease and cytidine deaminase to increase with maturation of normal and leukemic granulocytes. Myeloblasts purified by Ficoll sedimentation revealed an average kinase activity of 15.4 U/mg protein in acute myelocytic leukemia and 12.3 U/mg protein in blastic crisis of chronic myelocytic leukemia (CML). The average ratio of CdR kinase to deaminase activity in crude cell extracts varied from 0.197 in AML and 0.089 in blastic crisis to 0.0004 in normal granulocytes, reflecting the changes which take place with cellular maturation. The absolute levels of kinase and deaminase and the ratio of these two enzymes varied considerably among patients with AML, indicating that quantitative differences may be found in the metabolism of CdR and its analogs in leukemic cells.
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PMID:Deoxycytidine kinase: properties of the enzyme from human leukemic granulocytes. 5 55

Folate-binding protein(s) from chronic myelogenous leukemia cells have been purified using acid dialysis, ammonium sulfate fractionation and affinity chromatography. The purified preparation which migrates as a single band on disc electrophoresis could be separated by DEAE agarose chromatography into two folate-binding proteins (binders I and II) which bind molar equivalents of folic acid. One binder (I) eluted from DEAE at 1 mM sodium phosphate, pH 6.0, and the other (II) at 100 mM sodium phosphate, pH 7.4. Analysis of the purified mixture, which contained more than 90% binder II, by sedimentation equilibrium centrifugation indicated a homogeneous protein with a calculated molecular weight of 44000. Antiserum raised against the purified mixture gave a single precipitin line by immunodiffusion against a preparation of partially purified cell lysate. Hydrolysis of the more acidic binder (II) with neuraminidase converted it to a weakly acidic protein similar to binder I, suggesting that these binders are glycoproteins which differ in sialic acid content. With isoelectric focusing, the binding of folic acid could be demonstrated at pH 6.7, 7.3, 7.8 and 8.2 for binder I, and at pH 5.1, 5.8, and 6.5 for binder II. Binders I and II had equally high affinity for folic acid and dihydrofolate, lower affinity for N5-methyl-tetrahydrofolate, and no apparent affinity for N5-formyltetrahydrofolate or methotrexate.
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PMID:Properties of purified folate-binding proteins from chronic myelogenous leukemia cells. 28 Mar 77

Chronic granulocytic leukaemia (CGL) cells which contained a high concentration of unsaturated folate binding protein were incubated in suspension culture for a period of 5 h. Cell samples were periodically assayed for binder and these demonstrated active synthesis which was inhibited by puromycin, cyclo heximide, N-ethylmaleimide, and by incubation at 4 degrees C, but not by actinomycin D. Folate binding activity could also be demonstrated in the culture medium and this increased with the duration of incubation. This release of binder was inhibited by culturing the cells at 4 degrees C and by the addition of N-ethylmaleimide, but not by actinomycin D, puromycin, or cycloheximide. When the pre- and post-culture cell lysates were saturated with tritiated folic acid ([3H]PteGlu) and subjected to chromatography on DEAE-agrarose, approximately half of the bound folate eluted with 0.001 M phosphate buffer at pH 6.0 and the other half eluted with 0.2 M buffer at pH 7.2. The culture medium and plasma from this patient with CGL was well as serum from two normal subjects saturated with [3H]PteGlu and similarly chromatographed contained primarily the acidic binder and much less of the binder eluting with the low molarity buffer. Since a folate binding protein immunochemically similar to the binder in CGL cells has been identified in the serum of non-leukaemic subjects, these experiments suggest that the source of circulating folate binding protein may be the immature granulocyte.
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PMID:Studies with the folate binding protein in chronic granulocytic leukaemia cells. I. Synthesis and release of binder by cells in short-term culture. 106 9

Cancer cells have an increased ability to synthesize GTP (guanosine triphosphate) because of increased activity of IMP DH (inosine 5'-phosphate dehydrogenase, EC 1.1.1.205). Because IMP DH activity is rate limiting for de novo biosynthesis of GTP, this enzyme was suggested as a sensitive target for chemotherapy. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is converted in the cells into the active metabolite, TAD, (thiazole-4-carboxamide adenine dinucleotide) which potently inhibits IMP DH activity. By adding TAD to tissue extracts one can determine the extent of inhibition of IMP DH. We applied the IMP DH assay method to extracts of normal ovaries (N = 11) and epithelial ovarian carcinomas (N = 10). The IMP DH activity (mean +/- SE) in ovarian carcinoma was 21.1 +/- 5.8 which was markedly higher than that observed in normal ovaries (2.9 +/- 0.7 nmol/hr/mg protein) (P < 0.05%). The inhibition by TAD of IMP DH activity in ovarian carcinomas (N = 4) was 81%. The results indicate that IMP DH activity is elevated sevenfold in ovarian carcinomas as compared to normal ovary and can be inhibited by exposure to tiazofurin (TAD). Similar high IMP DH activity and inhibition of the activity by TAD was observed in patients with chronic granulocytic leukemia in blast crisis among whom 70 to 80% remissions were reported. Since there is increased IMP DH activity in human ovarian carcinomas and in OVCAR-5 cells and tiazofurin and TAD inhibit IMP DH activity of these cells and the proliferation of human ovarian carcinoma xenografts in the mouse, tiazofurin may merit serious consideration for a Phase II trial for patients with recurrent/refractory epithelial ovarian carcinoma.
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PMID:Inhibition by tiazofurin of inosine 5'-phosphate dehydrogenase (IMP DH) activity in extracts of ovarian carcinomas. 135 69

Implantation of genetically manipulated fibroblasts is now coming considered to be one of the important methods for gene therapy. Before the clinical application of this method, we still need to resolve several problems encountered. We have recently developed a model system for the fibroblast-mediated cytokine supplementation gene therapy. BMGNeo (bovine papilloma virus-derived plasmid) (gifted from Dr. Karasuyama) was used for expression of hG-CSF cDNA or hIFN-alpha cDNA (gifted from Dr. Nagata). The two plasmid DNAs (BMGNeoG-CSF and BMGNeoIFN) were individually transfected into NIH/3T3 fibroblasts by the calcium phosphate coprecipitation method. Cell clones producing a large amount of G-CSF or IFN-alpha were selected by the enzyme immunoassay methods and were called G-CSF3T3 or IFN3T3 respectively. Nude mice implanted with G-CSF3T3 highly produced G-CSF in vivo. Remarkable increases in both blood neutrophils and spleen hematopoietic stem cells/progenitor cells (CFU-S, BFU-E, CFU-E, CFU-GM and CFU-MK) were observed. To regulate the production of G-CSF by G-CSF3T3 in vivo, we developed a diffusion chamber system as the cells can be treated easily. We could control the peripheral neutrophil count in nude mice. In the same manner, IFN3T3 was implanted in nude mice bearing a CML cell line, KU812. KU812 tumor growth was significantly suppressed by implantation of IFN3T3 into the chamber. The fibroblast-mediated cytokine supplementation gene therapy might be useful for the treatment of patients requiring for continuous dosing of cytokines.
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PMID:[Implantation of genetically manipulated fibroblasts into mice as a model of gene therapy--supplementations of human granulocyte colony-stimulating factor (hG-CSF) and interferon-alpha (IFN-alpha)]. 165 96

Tiazofurin is an oncolytic agent which has shown therapeutic activity in end-stage acute nonlymphocytic leukemia (ANLL) and blast crisis of chronic granulocytic leukemia (CGL-BC). Tiazofurin is anabolized to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), which inhibits IMP dehydrogenase activity, leading to reduction of guanylate pools and cessation of cancer cell proliferation. The concentration of TAD in neoplastic cells of patients treated with tiazofurin should be a good indicator of sensitivity to the drug and also might herald the emergence of drug-resistant cells. Therefore, the precise quantitation of TAD in cancer cells during tiazofurin treatment is essential. In this paper we report a highly sensitive method for the determination of TAD in biological samples. With this technique, in addition to TAD, thirteen other biologically relevant adenine, guanine, cytosine and uridine nucleotides can be separated and quantitated accurately. TAD standard was separated on a Waters Partisil 10-SAX column in a RCM-10 module using an ammonium phosphate buffer system. TAD eluted at 21 min with a limit of detection of 15 pmol and linearity up to 3 nmol. The coefficient of variation was 0.6 +/- 0.1% for retention time and 2 +/- 0.3% for TAD concentration. Recovery of TAD was 96% with reproducibility of 98%. To examine the applicability of this method to a clinical setting, blood samples were obtained from a patient with CGL-BC and leukocytes were separated on a Ficoll-Hypaq gradient, extracted with trichloroacetic acid, and an aliquot was analyzed on HPLC. The TAD peak was identified by comparing the retention time and spectral analysis of the standard. After the patient was treated with a 2200 mg/m2 (12.7 mM) dose of tiazofurin, the TAD concentrations in the mononuclear cells at 2, 6, and 24 hr were 23.1, 13.6, and 0.8 microM. TAD levels at 2, 6, and 24 hr after a tiazofurin dose of 3300 mg/m2 (21.1 mM) were 42.8, 26.1, and 1.4 microM respectively.
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PMID:Determination of thiazole-4-carboxamide adenine dinucleotide (TAD) levels in mononuclear cells of leukemic patients treated with tiazofurin. 198 37

Long-term parenteral administration of human alpha-interferon (HuIFN-alpha) is effective in the treatment of several malignancies, including chronic myelocytic leukemia. In the present study, a model for fibroblast-mediated HuIFN-alpha gene therapy for the treatment of chronic myelocytic leukemia is described. Human IFN-alpha 5 complementary DNA was inserted into a bovine papilloma virus plasmid vector (BMGNeo) containing a neomycin resistance gene. The recombinant plasmid (BMGNeo-IFN) was transfected into NIH/3T3 fibroblasts by the calcium phosphate coprecipitation method, and stably transformed cells were isolated by G418 selection. A fibroblast clone secreting a large amount of HuIFN into the culture supernatant was selected by radioimmunoassay using anti-HuIFN-alpha monoclonal antibodies. Southern blot analysis revealed that the transformed cells contained approximately ten copies of the BMGNeo-IFN plasmid per cell, and Northern blot analysis demonstrated high expression of HuIFN-alpha mRNA in the cells. This fibroblast clone strongly suppressed proliferation of a HuIFN-alpha-sensitive chronic myelocytic leukemia cell line (KU812) during cocultivation in vitro. When the HuIFN-alpha-producing fibroblasts were implanted into nude mice bearing KU812 tumors by the subcutaneous diffusion chamber method, tumor growth in vivo was also significantly suppressed. This study suggests the clinical potential of fibroblast-mediated gene therapy in the future.
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PMID:Implantation of genetically manipulated fibroblasts into mice as antitumor alpha-interferon therapy. 216 55

The chronic myelogenous leukemia-associated P210 BCR-ABL oncogene protein product has been produced using the baculovirus expression system. High-level expression of the P210 BCR-ABL protein required the removal of GC rich 5' non-coding sequences. P210 BCR-ABL synthesized in insect cells is an active tyrosine protein kinase indistinguishable from P210 BCR-ABL isolated from human cells. Both proteins utilize angiotensin II as a phosphate acceptor in vitro with a Km for ATP of approximately 1.5 microM. P210 BCR-ABL produced in insect cells undergoes autophosphorylation in vitro and in vivo. Gel filtration of P210 BCR-ABL reveals that the protein elutes as a high molecular weight complex of about 800 kD. Approximately 4 to 5 mg of P210 BCR-ABL is produced in one liter of infected insect cells. Following cell disruption and a three-step ion exchange and gel filtration purification procedure, 0.4 mg of soluble P210 BCR-ABL is obtained per liter of suspension culture. An alternative procedure employing detergent extraction and immunoaffinity chromatography gave higher yields and purity from smaller amounts of infected cell extracts. The availability of intact, soluble and enzymatically active P210 BCR-ABL represents a significant advance for studying the biochemical and biophysical properties of the ABL oncogene family of proteins.
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PMID:Baculovirus expression of functional P210 BCR-ABL oncogene product. 249 63


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