Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0006826 (cancer)
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The effects of fludarabine triphosphate (Fara-ATP), 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), and aphidicolin on primer RNA and DNA synthesis in human CCRF-CEM leukemia cells were investigated. RNA-primed Okazaki fragment synthesis was monitored by first incubating whole cell lysates for 10 min in the presence or absence of the compound and then following the incorporation of [alpha-32P]ATP and [3H]dTTP into the primer RNA and DNA portions, respectively, of the Okazaki fragments. In whole cell lysates the degree of DNA synthesis inhibition induced by Fara-ATP was directly related to the extent of primer RNA synthesis inhibition over the entire range of Fara-ATP concentrations tested (10-50 microM). In contrast, primer RNA formation was stimulated by concentrations of ara-CTP (25-200 microM) and aphidicolin (0.5-5 micrograms/ml) that inhibited DNA synthesis. The primer RNA recovered from cell lysates incubated with either Fara-ATP, ara-CTP, or aphidicolin was of normal length, predominately 11 nucleotides. Fara-ATP was a more potent inhibitor of the polydeoxythymidylate primase activity than of the DNA polymerase alpha/delta activities present in the 100,000 x g supernatants of CCRF-CEM cells. Fara-ATP was a noncompetitive inhibitor of DNA primase with respect to ATP [50% inhibitory concentration, 2.3 +/- 0.3 (SD) microM, Ki = 6.1 +/- 0.3 (SE) microM] and the Km(ATP)/Ki (Fara-ATP) was 25. The 50% inhibitory concentration values of Fara-ATP for DNA polymerases alpha/delta activities on calf thymus DNA were 43 +/- 1.6 (SD) microM and greater than 100 microM with respect to dATP and dTTP. The effects of ara-CTP and aphidicolin on these enzymes were opposite those seen with Fara-ATP, since 50% inhibitory concentrations of either ara-CTP or aphidicolin for DNA polymerases alpha/delta did not inhibit polydeoxythymidylate primase activity. The results provide evidence that fludarabine phosphate blocks DNA synthesis in CCRF-CEM cells through inhibition of primer RNA formation. In contrast, the accumulation of primer RNA and RNA-primed Okazaki fragments that is induced by ara-CTP and aphidicolin could lead to the rereplication and amplification of chromosomal DNA segments.
Cancer Res 1991 Apr 01
PMID:Inhibition of primer RNA formation in CCRF-CEM leukemia cells by fludarabine triphosphate. 170 19

(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid [(6R)DDATHF] is a folate antimetabolite with activity specifically directed against de novo purine synthesis, primarily through inhibition of glycinamide ribonucleotide transformylase. This inhibition resulted in major changes in the size of the nucleotide pools in CCRF-CEM cells. After a 4-h incubation with 1 microM (6R)DDATHF, dramatic reductions in the ATP and GTP pools were observed, with almost no effect on CTP, UTP, and deoxyribonucleotide pools. When the incubation was continued in drug-free medium, recovery of ATP and GTP pools was protracted. ATP did not return to normal until 24-36 h, and GTP pools were only partially repleted by 48 h. The ATP and GTP pools were not affected when the initial 4-h incubation with (6R)DDATHF was conducted in the presence of 100 microM hypoxanthine. Addition of hypoxanthine to the medium after a 4-h incubation with (6R)DDATHF caused rapid recovery of the ATP and GTP pools. Similar effects were seen when the purine precursor aminoimidazole carboxamide was used in place of hypoxanthine. The effect of (6R)DDATHF on nucleotide pools and the capability of hypoxanthine or aminoimidazole carboxamide to prevent or reverse this phenomenon correlated directly with the inhibition of cell growth. Presumably as a consequence of the decrease in purine nucleotide triphosphate levels, the conversion of exogenously added uridine, thymidine, and deoxyuridine to nucleotides was markedly decreased. These effects were protracted for almost 48 h and were also reversed by hypoxanthine. Differential repletion of ATP and GTP pools after (6R)DDATHF pre-treatment demonstrated that diminished precursor phosphorylation is primarily a consequence of GTP rather than ATP starvation.
Cancer Res 1991 May 01
PMID:(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid effects on nucleotide metabolism in CCRF-CEM human T-lymphoblast leukemia cells. 170 49

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)
Cancer Res 1991 May 01
PMID:Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate. 170 52

Experimental tumors growing in irradiated tissue have been used to study the biological differences characteristic of locally recurrent tumors. Since the hypoxic cell fraction of tumors growing in irradiated tissue is increased and growth rate is slowed, these tumors are assumed to be metabolically deprived with hypoperfusion. In this study, we directly measured the effect of tumor bed irradiation on blood flow, growth rate, rate of nucleoside triphosphate (NTP) turnover, and metabolic state using 31P and 2H nuclear magnetic resonance, and an intradermal assay for angiogenesis. (NTP turnover refers to ATP-synthetase mediated NTP turnover that is visible to 31P nuclear magnetic resonance using the technique of saturation transfer.) A decrease in the number of small blood vessels perfusing tumors in a preirradiated bed was found. Most of the decrease was due to a loss of vessels with diameters less than 0.04 mm. When tumors growing in preirradiated tissue reached approximately 100 mm3 in volume, a high frequency of gross and microscopic necrosis and hemorrhage was already observed in most tumors. Consistent with these observations, the phosphocreatine/inorganic phosphate and nucleoside triphosphate/inorganic phosphate ratios were significantly lower in the tumors growing in a preirradiated bed compared with tumors in a nonirradiated bed. The blood flow rate was similar to control for tumors less than 100 mm3 (45.8 versus 40.5 ml/100 g/min, P = not significant), but was significantly lower than control for tumors greater than 100 mm3 (40.4 versus 12.2 ml/100 g/min, P less than 0.01). The NTP turnover rates correlated (P less than 0.005, r = 0.66) with the volume doubling rate (1/tumor volume doubling time), but for tumors approximately 100 mm3 in size neither the volume doubling rate nor the NTP turnover rate of tumors growing in an irradiated bed was statistically lower than control [NTP turnover: 14 +/- 3%/s versus 9 +/- 2%/s; volume doubling rate: 0.47 +/- 0.07/day versus 0.33 +/- 0.04/day (mean +/- SE)]. A large intertumor variability of all metabolic parameters was observed.
Cancer Res 1991 Jun 15
PMID:Angiogenesis determines blood flow, metabolism, growth rate, and ATPase kinetics of tumors growing in an irradiated bed: 31P and 2H nuclear magnetic resonance studies. 171 Jan 69

Porin is the pore-forming protein involved in the movement of adenine nucleotides across the outer mitochondrial membrane (OMM). Hexokinase and glycerol kinase interact with porin on the outer surface of the OMM in a manner which provides these enzymes with preferred access to the ATP generated in the mitochondrion. We review recent evidence which permits refinement of our knowledge of these proteins and their interactions at the OMM. The involvement of this system in metabolic microcompartmentation is discussed, as well as possible pathological consequences of its disruption in malignancy and genetic deficiencies of hexokinase, glycerol kinase, and porin.
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PMID:Porin interaction with hexokinase and glycerol kinase: metabolic microcompartmentation at the outer mitochondrial membrane. 171 Sep 14

The expression of protein-tyrosine kinases (PTKs; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112) was studied in normal human lung and various tumors by PCR followed by molecular cloning and sequence analysis. Six known PTKs (YES, FGR, LYN, HCK, PDGFB-R, and CSF1-R), as well as two additional members of this enzyme family, were detected in lung. One of the newly discovered sequences appears to represent a group of cytosolic PTKs. The cDNA sequence of the second unknown PTK revealed that it is a fourth member of the fibroblast growth factor receptor family. It was therefore called TKF (tyrosine kinase related to fibroblast growth factor receptor). Among a wide variety of cells and tissues tested, including human lymphocytes and macrophages, TKF was only found expressed in lung. Apart from normal lung, TKF expression could be demonstrated in some tumors of lung origin, but also in malignancies not derived from lung tissues. As fibroblast growth factors are generally involved in a variety of functions such as mitogenesis, angiogenesis, and wound healing, the specific expression of a receptor-related gene in lung only may point to yet another special function of this group of proteins.
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PMID:Two additional protein-tyrosine kinases expressed in human lung: fourth member of the fibroblast growth factor receptor family and an intracellular protein-tyrosine kinase. 172 May 39

The purpose of this study was to investigate whether substrate deprivation acutely and selectively decreases ATP concentration in an experimental sarcoma. Two methods of substrate deprivation were examined: glycolysis was inhibited using 2-deoxyglucose (2DG), and plasma substrate levels were reduced using insulin. The effects of treatment on tumor ATP, inorganic phosphate, and pH were studied by 31P nuclear magnetic resonance spectroscopy. 2DG (2 g/kg) was administered i.p. to rats bearing s.c. methylcholanthrene-induced sarcomas. Inhibition of glycolysis by 2DG caused a 52 +/- 13% (SE) decrease in the tumor ATP to inorganic phosphate ratio, associated with a decrease in pH of 0.38 +/- 0.10 unit. The same dose of 2DG caused no significant change in the ratio of phosphocreatine to ATP in brain. Insulin (125 units/kg, i.p.) caused a 68% decline in plasma glucose and a 71% decline in betahydroxybutyrate compared to saline-treated animals. Concomitantly, 31P nuclear magnetic resonance spectroscopy detected a 48 +/- 13% decrease in sarcoma ATP, with a reciprocal elevation of inorganic phosphate in insulin-treated animals. In contrast, the brain phosphocratine/ATP ratio was unaffected by insulin. These results suggest that large tumors are acutely sensitive to inhibition of glycolysis and reductions in plasma levels of substrates for oxidative phosphorylation and glycolysis, while the brain is unaffected. In addition, this work provides support for the use of 31P nuclear magnetic resonance spectroscopy to monitor tumor response to therapy.
Cancer Res 1992 Jan 01
PMID:Selective depletion of tumor ATP by 2-deoxyglucose and insulin, detected by 31P magnetic resonance spectroscopy. 172 88

Although various anti-cancer drugs have widely differing primary modes of action, the mechanisms of cell death appear similar but are not well understood. To investigate this problem we exposed cultured human leukemic T-lymphoblasts to 1-hr pulse doses of an alkylating agent (mafosfamide) and a topoisomerase II inhibitor (etoposide) that cause delayed cell death. The effects of these drugs on nucleotide content, poly (ADP-ribosyl)ation and DNA strand breakage were assessed. Both drugs caused DNA strand breakage, and although the pattern differed, this seemed to be the major mechanism by which cells were killed. The degree and time course of the NAD and ATP depletion that mafosfamide and etoposide caused were similar. Both drugs caused a nadir in cellular nucleotide levels 2 hr after exposure but between 2 and 6 hr there was a partial recovery. This correlates with the time course of the DNA damage they caused and appeared to result from poly (ADP-ribosyl)ation. Both drugs were shown to cause apoptotic cell death associated with endonucleolytic DNA fragmentation. We suggest that DNA damage, as a primary or secondary effect, associated with poly (ADP-ribosyl)ation and apoptotic cell death may be a common pathway of cytotoxic drug action.
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PMID:DNA damage, poly (ADP-ribosyl)ation and apoptotic cell death as a potential common pathway of cytotoxic drug action. 174 64

The presence of covalent modifications in DNA obtained from exfoliated urothelial cells of smokers and nonsmokers was determined using 32P postlabeling methods. Urine and blood samples were procured from 73 persons. Cells were removed from the urine by filtration. DNA was isolated using an enzyme-solvent extraction method and then coprecipitated with glycogen. Sufficient DNA to detect 1 carcinogen-DNA adduct/10(9) normal nucleotides was obtained from 40 of the 73 samples. DNA was hydrolyzed to 3'-phosphodeoxynucleotides and then 32P postlabeled under conditions of excess [32P]ATP. Carcinogen-DNA adducts were resolved using anion-exchange thin-layer chromatography and visualized by autoradiography; film exposures lasted as long as 7 days. Twelve different carcinogen-DNA adducts and a diagonal zone of radioactivity could be found, but no sample contained all adducts. At least four adducts appeared to be cigarette smoking related. These adducts were from 2 to 20 times higher in the smokers than the nonsmokers. Two carcinogen-DNA adducts were qualitatively very similar to adducts described earlier in a study of human bladder biopsies. One of these corresponded to N-(deoxyguanosin-8-yl)-4-aminobiphenyl. Adducts were correlated significantly with the levels of 4-aminobiphenyl hemoglobin adducts and number of cigarettes smoked. In addition, levels of the putative N-(deoxyguanosin-8-yl)-4-aminobiphenyl adduct and a measure of total adducts were correlated with the mutagenic activity of the individual's urine. These data suggest that noninvasive, biological monitoring techniques can be applied to the study of carcinogen-DNA adducts in humans at high risk for bladder cancer.
Cancer Epidemiol Biomarkers Prev
PMID:Detection of carcinogen-DNA adducts in exfoliated urothelial cells of cigarette smokers: association with smoking, hemoglobin adducts, and urinary mutagenicity. 184 72

Inherited susceptibility to a wide variety of neoplasias (Li-Fraumeni syndrome), has been shown in studies of one cancer-prone family, to have an intriguing association with an aberrant c-raf-1 gene and inheritance of a radioresistant phenotype in their non-cancerous skin fibroblasts. This association together with observations that DNA topoisomerases, when defective, can introduce errors into DNA and that these enzymes are perturbed in vitro by serine/threonine kinases similar to raf encoded proteins, prompted investigation of DNA topoisomerase activity of the family's fibroblasts. Since radioresistance was transferred to murine cells (NIH-3T3) when the aberrant c-raf-1 gene from this family was transfected, we also examined transformants containing this and other oncogenes. V-raf/c-myc and EJ-ras transformants were examined, the former because the family's skin fibroblasts also have 3-8-fold elevated myc expression (not apparently relevant to radioresistance) and the latter because ras, like raf, conveys radioresistance. The family members' fibroblasts and the three transfected murine lines, showed a similar perturbation of a spermidine and ATP-dependent DNA catenation activity (typical of DNA topoisomerase II). There was a significant positive correlation (r = 0.93; P = 0.0026) between the degree of activation of topoisomerase II and one measure of radioresistance (the Dq value). Relaxation of DNA supercoiling (topoisomerase I activity and other DNA nicking enzymes) was not abnormal. Cytotoxicity assays and evaluation of the influence of topoisomerase II inhibitors on DNA/protein complex formation, corroborated the existence of a qualitative topoisomerase II defect in the family's cells and transfectants. Although the contention that the qualitative topoisomerase II abnormalities observed here may be associated with malfunction is highly speculative, these findings may be relevant to the mechanism of oncogenesis, not only in this family, but with raf and ras type oncogenes.
Br J Cancer 1991 Jan
PMID:Aberrant DNA topoisomerase II activity, radioresistance and inherited susceptibility to cancer. 184 52


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