Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In our laboratory, we have studied the mechanism of action of tumor-inhibitory antibiotics, including bleomycin, phleomycin, adriamycin, aclarubicin, neothramycin, macromomycin, auromomycin, chartreusin, pluramycin, neopluramycin, xanthomycin A, angustmycins A and C, blasticidin S and phenomycin. The recent advances are summarized. Screening of microorganism for new antitumor antibiotics based upon our studies on mechanism of action are currently ongoing. We are interested in drug-resistance of tumor cells, and have obtained drug-resistant sublines of murine lymphoblastoma L5178Y cells. We have found that glycoprotein synthesis and alkaline phosphodiesterase (APD) activity of the plasma membrane are higher in adriamycin (ADM)-, aclarubicin (ACR)- and bleomycin (BLM)-resistant cell sublines than in the parental cells. An inhibitor of APD has been isolated from a soil Streptomyces, and identified with 2-crotonyloxymethyl-4,5,6-trihydroxycyclohex-2-enone (COTC). COTC inhibits growth of the drug-resistant cells more significantly than the parental cells, and exhibits synergistic activity with ACR against ACR-resistant cells. COTC is a SH inhibitor. Although COTC is a multifunctional drug, the inhibition of DNA polymerase alpha and some mitotic process may be related to its lethal action. In the course of our screening, we have found that a strain of Sterptomyces hygroscopicus produces two substances: one inhibits thymidine and uridine uptake of human leukemic K562 cells, and the other stimulates it. The inhibiting substance has been identified with tubercidin, and the stimulating one has been found to be a novel pyrrolo [2,3-d] pyrimidine antibiotic, cadeguomycin. Cadeguomycin shows low acute toxicity in mice, enhances DTH reaction, and inhibits Ehrlich ascitic carcinoma in mice. The antibiotic exhibits synergistic effects with arabinosylcytosine against growth of K562 cells. Saframycin, discovered by Prof. Arai, Chiba University, is effective against Ehrlich ascitic carcinoma, P388 and L1210 leukemia, and B16 melanoma in mice. The target is DNA. Stubomycin, discovered by Dr. Umezawa, Kitasato Institute, is effective against Sarcoma 180, Ehrlich carcinoma, P388 leukemia, IMC carcinoma and Meth-A tumor in mice, and shows low acute toxicity. The target is plasma membrane.
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PMID:[Study of new antineoplastic antibiotics based on newly discovered action mechanisms]. 619 73

The mechanisms of action of, and resistance to, important anticancer agents are briefly described. Their selective toxicity is considerably high, and is chiefly due to the distribution and metabolism in the body. The selective toxicity of some DNA-binding drugs may be attributed to the structural difference of DNA, nucleosome and/or chromatin between neoplastic and normal cells. Some studies of reducing side effects are summarized. In our laboratory, we are studying drug-resistance and metastasis of tumor cells. Since the mechanism of natural resistance of gastric cancer, pulmonary cancer, and other refractory cancers may be related to acquired resistance of leukemia, studies on new agents against drug-resistant tumor cells are important. In our laboratory, we have selected cell sublines of murine T-lymphoblastoma L5178Y for resistance to adriamycin (ADM), aclarubicin (ACR) or bleomycin (BLM), and have observed that the resistance is attributed to decreased influx and increased efflux of the antibiotic, resulting in lowered retention of the drug in the cells. Each resistant subline shows a characteristic cross-resistant pattern, suggesting that membrane alteration involved differs each other. We have also found that glycoprotein-synthesizing activity and alkaline phosphodiesterase activity of plasma membrane are higher in the three resistant sublines than in the parental cell line. We obtained a number of hybridomas producing antibodies to plasma membrane of an ACR-resistant subline of L5178Y cells. Among the syngeneic monoclonal antibodies, one was found by agglutination tests to react with the ACR-resistant cell line, but not significantly with the parental and ADM-, BLM-and MCR-resistant cell lines. Fluorographs of [14C] leucine-labeled ACR-resistant cells demonstrates two protein bands of 230 K and 20 K daltons, which are precipitated by the monoclonal antibody. The former seems to be specific to the ACR-resistant cells. Based on the results so far obtained, the 230 K protein may be related to the drug resistance and may be TATA (tumor-associated transplantation antigen). The results suggest that isolation of drug-resistant neoplastic cells is a novel method of finding TATA.
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PMID:[Mechanism of action and resistance of antineoplastic agents]. 619 71

A one-step radioassay for cyclic AMP phosphodiesterase was optimised for human polymorphonuclear leukocytes. Kinetic studies indicated the presence of two forms of phosphodiesterase activity with apparent Km values of 0.015 mmol/l and 0.98 mmol/l for cyclic AMP. Control neutrophils were homogenised in isotonic sucrose and, after low speed centrifugation, the supernatant was subjected to analytical subcellular fractionation. Gradient fractions were assayed for principal marker enzymes and for cyclic AMP phosphodiesterase. Both forms of phosphodiesterase activity were located in the cytosol. Polymorphonuclear leukocyte homogenates were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. A portion of each homogenate was used for enzyme analysis and the remainder assayed for cyclic AMP content. The specific activity (mUnits/mg protein) of the low Km phosphodiesterase was reduced in both patient groups compared with control values, whilst that of the high Km phosphodiesterase was unchanged. Leukocytes from patients with chronic granulocytic leukaemia had only a fifth of the cyclic AMP content of control neutrophils, whilst leukocytes from patients in the third trimester of pregnancy had an elevated cyclic AMP content.
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PMID:Studies on the activities, kinetic properties and subcellular localisation of cyclic AMP phosphodiesterase in human neutrophil leukocytes. 624 17

The adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), at low concentrations (less than 10 microM), enhances the inhibitory activity of adenosine against lymphocyte-mediated cytolysis (LMC) without itself being inhibitory. At higher concentrations, EHNA alone is inhibitory to LMC with an IC50 of 160 microM. This inhibition is reversible upon washout, appears to affect an early stage of the lytic process, and does not appear to involve changes in basal levels of cyclic AMP (cAMP), ribonucleoside 5'-triphosphate pool sizes, S-adenosylhomocysteine levels, or protein carboxymethylation. EHNA does enhance the cAMP response of cytolytic lymphocytes (CL) to activators of adenylate cyclase such as prostaglandin E1. EHNA inhibits lymphocyte high-affinity cAMP phosphodiesterase at immunosuppressive levels, exhibiting hyperbolic mixed-type inhibition (Ki = 83 microM, alpha = 0.47, beta = 0.18). Whereas inhibition of intralymphocytic ADA is complete at low concentrations (less than 25 microM) of EHNA, inhibition of LMC and intralymphocytic cAMP phosphodiesterase increases linearly with EHNA concentration to at least 200 microM. The presence of 200 microM EHNA during the centrifugation of mixtures of CL and EL4 leukemia target cells leads to increased CL cAMP levels. 2'-Deoxycoformycin, a more potent ADA inhibitor than EHNA, is not inhibitory to LMC and shows none of these cAMP-related effects. These results suggest that CL-target cell contact stimulates adenylate cyclase in the CL and that EHNA inhibits LMC due to its enhancement of this target cell-stimulated elevation of cAMP.
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PMID:Inhibition of lymphocyte-mediated cytolysis and cyclic AMP phosphodiesterase by erythro-9-(2-hydroxy-3-nonyl)adenine. 629 34

Calmodulin (Cam) was isolated from normal and from transformed human lymphocytes by affinity chromatography on CAPP-Sepharose 4B, followed by chromatofocusing. In the presence of Ca2+, lymphocyte Cam migrated as a single protein on 2-DE, and was located on the same position as Cam extracted from dog brain and rat testis; its MW was 17,500, with a pI of 3.9. In the presence of Ca2+, lymphocyte Cam stimulated activator-depleted dog brain phosphodiesterase; this effect was inhibited by trifluoperazine (TFP) or by EGTA. By the RIA technique, the EGTA-soluble Cam content of resting lymphocytes constituted 0.58% of the total protein; the total Cam was comparable to the content of other major proteins in lymphocytes, such as actin, tubulin, and intermediate filament protein. The amount of Cam per cell and the rate of incorporation of L-[35S]methionine into Cam increased after mitogen-induced transformation. Immunofluorescence labeling of normal, mitogen-transformed, and EBV-genome-positive lymphocytes with affinity-purified anti-Cam antibodies showed bright fluorescence in the region of the Golgi apparatus, as well as diffuse cytoplasmic but scant nuclear staining. Similar patterns were observed in T suppressor, T helper, and B cells. Normal lymphocytes cultured in the presence of 2 X 10(-5) M TFP remained viable, but failed to undergo blastogenic transformation after stimulation with allogeneic cells, concanavalin A (Con A), or pokeweed mitogen (PWM). The same concentration of TFP inhibited the replication of EBV-genome-positive and of leukemia cells. Exposure of natural killer cells or allospecific killer cells to this concentration of TFP inhibited the effector phase of killing in a dose-dependent manner. In the presence of lower concentrations of TFP (0.25-1.0 X 10(-5) M) strong MLC responses were inhibited, while weak reactions were markedly amplified. A similar effect was not observed in lymphocytes stimulated into blastogenesis by lectins, suggesting that different Cam-dependent secondary messenger(s) may be involved in the blastogenic responses evoked by alloantigenic determinants. The amplification of weak MLC responses by 0.25-1.0 X 10(-5) M TFP constitutes the first biological illustration of the capacity of a Cam-binding agent to enhance as well as to inhibit cellular activation. The paradoxical effect may have been a consequence of a shift in the relative concentrations of the four known molecular Cam X Ca2+ conformers. The results are also consistent with the suggested capacity of Cam X Ca2+ conformers to activate different enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The role of calmodulin in the regulation of human lymphocyte activation. 632 21

We examined the in vitro effects of 8-chloro-adenosine 3':5'-monophosphate (8-Cl-cAMP), a reportedly stable, potent and site-selective analogue of cAMP, on the proliferation and sensitivity to doxorubicin (DXR) of two mouse cell lines, the B16 melanoma and Friend leukaemia, both as wild-type (B16, FLC) and DXR-resistant (B16/DXR, FLC/DXR) variants. The latter strains had characteristics of 'typical' multidrug resistance (MDR), including the over-expression of P-glycoprotein. Encouragingly, 8-Cl-cAMP affected almost equally the growth of the chemosensitive and chemoresistant variants of both cell lines. Its activity proved to be much more elevated on cells cultivated with fresh rather than heat-inactivated calf serum. In fact, the IC50 values for B16 and B16/DXR were about 4.7 microM in fresh serum and 215 microM in heat-inactivated serum; the IC50 values for FLC and FLC/DXR were about 12 microM in fresh serum and 70 microM in heat-inactivated serum. Furthermore, experiments with B16 showed that cotreatments with isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, or adenosine deaminase (ADA) greatly reduce the activity of 8-Cl-cAMP bringing it to comparable levels in fresh and heat-inactivated serum. These results indicate that the antiproliferative effects of 8-Cl-cAMP may be due principally to metabolites formed by the enzymic activities of the serum, most probably including 8-chloro-adenosine (8-Cl-adenosine), as suggested by other authors. Moreover, the dose-response curves and the IC50 values of the latter compound for the various cell lines were compatible with those observed for 8-Cl-cAMP in fresh serum. Finally, there was no evidence that 8-Cl-cAMP, either in the presence of fresh or heat-inactivated serum, or 8-Cl-adenosine may increase the sensitivity to DXR of the MDR variants of B16 melanoma and Friend leukaemia.
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PMID:Effects of 8-chloro-cyclic adenosine monophosphate on the growth and sensitivity to doxorubicin of multidrug-resistant tumour cell lines. 783 Nov 98

Tiazofurin exhibits antitumor activity in murine and human tumor cells. In a recent phase I/II trial in patients with end-stage leukemia, tiazofurin showed good response; however, repeated treatment resulted in clinical resistance to the drug. To elucidate the mechanisms of resistance in human leukemic cells, two variants of human myelogenous leukemia K652 cells resistant to tiazofurin were developed by drug-selection pressure. Compared to a concentration producing 50% cell proliferation reduction that was 9.1 microM in sensitive cells, the resistant variants displayed concentrations producing 50% cell proliferation reductions of 12 and 16 mM. The activity of the target enzyme, IMP dehydrogenase, was not altered in the resistant cells. Studies on tiazofurin metabolism revealed that resistant variants formed < 10% of the active metabolite, thiazole-4-carboxamide adenine dinucleotide. This correlated with the activity of NAD pyrophosphorylase, the enzyme that synthesizes thiazole-4-carboxamide adenine dinucleotide, which was reduced to 10% in the resistant lines. Concurrently, the activity of thiazole-4-carboxamide adenine dinucleotide phosphodiesterase was elevated in the refractory cells. Compared to the sensitive counterpart, the levels of GMP and NAD were lower in the resistant lines. Guanine salvage activity was decreased in the resistant cells. Basal dGTP and dATP concentrations were elevated in the resistant line; nevertheless, tiazofurin incubation decreased dGTP levels in only the sensitive cells. Although there was no difference in the Km of tiazofurin transport or efflux, the Vmax of uptake of the drug was reduced in the resistant lines. Sensitive and resistant cells exhibit similar cytotoxicity to agents which do not share the mechanism of action of tiazofurin, suggesting that refractory cells are still sensitive to other standard antileukemic drugs.
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PMID:Biochemical consequences of resistance to tiazofurin in human myelogenous leukemic K562 cells. 809 64

Incorporation of the anticancer drug fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate; F-ara-AMP) into the 3'-end of DNA during replication causes termination of DNA strand elongation and is strongly correlated with loss of clonogenicity. Because the proofreading mechanisms that remove 3'-F-ara-AMP from DNA represent a possible means of resistance to the drug, the present study investigated the excision of incorporated F-ara-AMP from DNA by the 3' --> 5'-exonuclease activity of DNA polymerase epsilon from human leukemia CEM cells. Using the drug-containing and normal deoxynucleotide oligomers (21-base) annealed to M13mp18(+) DNA as the excision substrates, we demonstrated that DNA polymerase epsilon was unable to effectively remove F-ara-AMP from the 3'-end of the oligomer. However, 3'-terminal dAMP and subsequently other deoxynucleotides were readily excised from DNA in a distributive fashion. Kinetic evaluation demonstrated that although DNA polymerase epsilon has a higher affinity for F-ara-AMP-terminated DNA (Km = 7.1 pM) than for dAMP-terminated DNA of otherwise identical sequence (Km = 265 pM), excision of F-ara-AMP proceeded at a substantially slower rate (Vmax = 0.053 pmol/min/mg) than for 3'-terminal dAMP (Vmax = 1.96 pmol/min/mg). When the 3'-5' phosphodiester bond between F-ara-AMP at the 3'-terminus and the adjacent normal deoxynucleotide was cleaved by DNA polymerase epsilon, the reaction products appeared to remain associated with the enzyme but without the formation of a covalent bond. No further excision of the remaining oligomers was observed after the addition of fresh DNA polymerase epsilon to the reaction. Furthermore, the addition of DNA polymerase alpha and deoxynucleoside triphosphates to the excision reaction failed to extend the oligomers. After DNA polymerase epsilon had been incubated with 3'-F-ara-AMP-21-mer for 10 min, the enzyme was no longer able to excise 3'-terminal dAMP from a freshly added normal 21-mer annealed to M13mp18(+) template. We conclude that the 3' --> 5' exonuclease of human DNA polymerase epsilon can remove 3'-terminal F-ara-AMP from DNA with difficulty and that this excision results in a mechanism-mediated formation of "dead end complex."
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PMID:Inhibition of the 3' --> 5' exonuclease of human DNA polymerase epsilon by fludarabine-terminated DNA. 870 31

We show that the adenylate cyclase activating diterpine, forskolin, the phosphodiesterase inhibitor, aminophylline, and the permeant cAMP analog dibutyryl cAMP inhibit the in vitro clonogenic growth of leukemic B-cell precursors. We also used a SCID mouse xenograft model of refractory human B-cell precursor leukemia to evaluate the anti-leukemic effect of aminophylline in vivo. Treatment with aminophylline (6 mg/kg bolus followed by 0.1-0.5 mg/kg/hour x 7 days) significantly prolonged the event-free survival of SCID mice (median survival of control mice, 39 days, N = 79; median survival of aminophylline-treated mice, 60 days, N = 10; P < 0.0001 by log-rank test) and it was more effective than treatment with vincristine (median survival = 51 days, N = 5) or L asparaginase (median survival = 44 days, N = 5). However, aminophylline was not as effective as methylprednisolone (median survival: 103 days, N = 5). These results indicate that cAMP modulating agents may be useful in treatment of refractory human B-cell precursor leukemia.
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PMID:In vitro and in vivo anti-leukemic efficacy of cyclic AMP modulating agents against human leukemic B-cell precursors. 881 74

B cell chronic lymphocytic leukemia (B-CLL) is an incurable clonal disease which shows initial responsiveness to a number of chemotherapeutic drugs. However, in most patients the disease becomes resistant to treatment. Rolipram, a specific inhibitor of phosphodiesterase (PDE) type 4, the PDE predominantly expressed in B-CLL cells, has been shown to induce cAMP-dependent apoptosis in these cells. In the present study, we demonstrate that the extent of rolipram-induced apoptosis is similar to fludarabine-induced apoptosis in vitro. The combination of rolipram and fludarabine results in an enhancement in the number of apoptotic cells compared to apoptosis induced by either agent alone. Second, rolipram suppresses the expression of anti-apoptotic members of the Bcl-2 family and induces the pro-apoptotic protein Bax, thereby shifting the balance between pro- and anti-apoptotic members of the Bcl-2 family towards a pro-apoptotic direction. Finally rolipram-induced apoptosis is caspase-dependent. PDE 4 inhibitors are currently under investigation for chronic obstructive pulmonary disease and asthma in phase III clinical trials showing promising results with tolerable side-effects. In conclusion, by inducing apoptosis, by enhancing apoptosis induced by fludarabine, by suppressing Bcl-2, Bcl-X and by inducing Bax expression, PDE 4 inhibitors may add a new therapeutic option for patients with B-CLL.
Leukemia 2001 Oct
PMID:Phosphodiesterase type 4 inhibitor suppresses expression of anti-apoptotic members of the Bcl-2 family in B-CLL cells and induces caspase-dependent apoptosis. 1158 14


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