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Query: UNIPROT:P06889 (Mol)
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Recent work suggests a participation of mitochondria in apoptotic cell death. This role includes the release of apoptogenic molecules into the cytosol preceding or after a loss of mitochondrial membrane potential DeltaPsim. The two uncouplers of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2, 4-dinitrophenol (DNP) reduce DeltaPsim by direct attack of the proton gradient across the inner mitochondrial membrane. Here we show that both compounds enhance the apoptosis-inducing capacity of Fas/APO-1/CD95 signaling in Jurkat and CEM cells without causing apoptotic changes on their own account. This amplification occurred upstream or at the level of caspases and was not inhibited by Bcl-2. The effect could be blocked by the cowpox protein CrmA and is thus likely to require caspase 8 activity. Apoptosis induction by staurosporine in Jurkat cells as well as by Fas in SKW6 cells was unaffected by CCCP and DNP. The role of cytochrome c during Fas-DNP signaling was investigated. No early cytochrome c release from mitochondria was detected by Western blotting. Functional assays with cytoplasmic preparations from Fas-DNP-treated cells also indicated that there was no major contribution by cytochrome c or caspase 9 to the activation of effector caspases. Furthermore, an increase of rhodamine-123 uptake into intact cells, which has been explained by mitochondrial swelling, occurred considerably later than the caspase activation and was blocked by Z-VAD-fmk. These data show that uncouplers of oxidative phosphorylation can presensitize some but not all cells for a Fas death signal and provide information about the existence of separate pathways in the induction of apoptosis.
Mol Cell Biol 1999 May
PMID:Uncouplers of oxidative phosphorylation can enhance a Fas death signal. 1020 55

In clones of the CEM human acute lymphoblastic leukemic cell line, glucocorticoids, oxysterols and activators of the cAMP pathway acting synergistically with glucocorticoids, each can cause apoptotic cell death. Morphologically and kinetically, these deaths resemble one another. The kinetics are striking: in each case, after addition of the lethal compound(s), an interval of approximately 24 h follows, during which cell growth continues unabated. During this "prodromal" period, removal of the apoptotic agent leaves the cells fully viable. We hypothesize that a sequence of biochemical events occurs during the prodrome which eventually results in the triggering of the full apoptotic response as evidenced by the activation of caspases and DNA fragmentation. At some point, the process is irreversible and proceeds relatively rapidly to cell death. Suppression of c-Myc seems a universal early event evoked by each of these lethal compounds or combinations, and we conclude that the negative regulation of this proto-oncogene is an important aspect of the critical pre-apoptotic events in these cells.
J Steroid Biochem Mol Biol
PMID:Glucocorticoids, oxysterols, and cAMP with glucocorticoids each cause apoptosis of CEM cells and suppress c-myc. 1041 25

The phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase.
Mol Pharmacol 1999 Oct
PMID:Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine. 1049 51

Mercaptopurine and thioguanine are anticancer and immunosuppressive agents that exert their primary cytotoxic effects via incorporation of deoxythioguanosine (dG(s)) into DNA, but the precise mechanism(s) by which this causes cytotoxicity remains unknown. We initially determined that the level of dG(s) incorporation into DNA of human T- and B-lineage leukemia cell lines did not correlate significantly with the extent of cytotoxicity (IC(50)), except that there was no cytotoxicity in the absence of dG(s) incorporation. To elucidate biological processes perturbed by dG(s) incorporation into DNA, we chemically synthesized oligodeoxyribonucleotides containing a single dG(s) (11 mer and 19 mer), which decreased the melting temperature (T(m)) of DNA-DNA duplexes without major structural changes, as evidenced by circular dichroism spectra. Using nuclear extracts from human lymphoblastic leukemia cells (CCRF-CEM, NALM6, and Molt4), we documented that dG(s) incorporation into the DNA strand of DNA-RNA heteroduplexes significantly inhibited human RNase H-catalyzed RNA cleavage (80-90% inhibition) and that a similar inhibition was evident with bacterial RNase H. These data provide the first evidence that thiopurines inhibit the function of RNase H, indicating that their mechanism of cytotoxicity may involve interference with this component of the replication machinery.
Mol Pharmacol 1999 Oct
PMID:Human RNase H-mediated RNA cleavage from DNA-RNA duplexes is inhibited by 6-deoxythioguanosine incorporation into DNA. 1049 69

Heptachlor is an organochlorine insecticide used worldwide for the control of pests both agriculturally and domestically. Its lipophilic structure allows it to bioaccumulate and pass through the food chain, exposing those who come in contact with it to its tumor promoting and possible carcinogenic effects. As a mechanism of tumor promotion, we explored the possibility of heptachlor suppressing the apoptotic process in human CEM x 174 lymphocytes. In this article, we describe the effect of heptachlor on the activity of the apoptosis protease CPP32. We show that heptachlor by itself was able to stimulate CPP32 activity at relatively high concentrations. When combined with the chemotherapeutic agent doxorubicin, a known CPP32 activator, a dual effect was observed. Low concentrations of heptachlor (5 microM-10 microM) suppressed doxorubicin-induced CPP32 activity, and high concentrations of heptachlor (80 microM-120 microM) augmented it. We also showed that heptachlor alone at relatively high concentrations induced apoptosis-associated changes in CEM x 174 cells including high molecular weight (HMW) DNA cleavage and chromatin condensation. From these results, it appears that heptachlor has tumor promoting-like effects at lower concentrations, and at higher concentrations induces apoptosis as a mechanism of cytotoxicity.
J Biochem Mol Toxicol 2000
PMID:Modulation of CPP32 activity and induction of apoptosis in Human CEM X 174 lymphocytes by heptachlor, a chlorinated hydrocarbon insecticide. 1056 Oct 81

Incubation of CEM cells for 24 h with the guanine, 2,6-diaminopurine, and adenine nucleotide analogs of the 9-(2-phosphonylmethoxyethyl) series, 9-(2-phosphonylmethoxyethyl)guanine (PMEG), 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), was found to inhibit DNA synthesis 50% at concentrations of 1, 6, and 25 microM, respectively. Possible reasons for the marked differences were investigated, including cellular transport of the analogs, different efficiencies of intracellular phosphorylation, differential effects on 2'-deoxynucleotide (dNTP) pools, and differences in the affinities of the cellular DNA polymerases for the diphosphate derivatives of the drugs. No significant differences in cellular uptake were found among the analogs; however, they did differ in the efficiency of phosphorylation, i.e., CEM cells were found to accumulate higher levels of PMEG-diphosphate (PMEGpp) than PMEDAP-diphosphate (PMEDAPpp) or PMEA-diphosphate (PMEApp). Treatment of cells with any of the nucleotide analogs resulted in increased dNTP pools, with PMEG producing the greatest increase. All three analogs had the greatest effect on the dATP pool size, whereas the dGTP pool size was not significantly affected. Comparison of the ratios of nucleotide analog diphosphates to their corresponding dNTPs under conditions where DNA synthesis is inhibited 50% suggested that cellular DNA polymerases were approximately twice as sensitive to PMEGpp than to PMEDAPpp and 5-fold more sensitive to PMEGpp than to PMEApp. Consistent with this hypothesis, examination of the efficiencies with which the replicative DNA polymerases alpha, delta, and epsilon incorporated the analogs showed that DNA polymerase delta, the most sensitive of the DNA polymerases, incorporated PMEGpp twice as efficiently as PMEDAPpp and 7-fold more efficiently than PMEApp.
Mol Pharmacol 1999 Dec
PMID:9-(2-phosphonylmethoxyethyl) derivatives of purine nucleotide analogs: A comparison of their metabolism and interaction with cellular DNA synthesis. 1057 54

The administration of CycloSaligenyl 3'-azido-2',3'-dideoxythymidine monophosphate (CycloSal-AZTMP) to CEM cells resulted in a concentration- and time-dependent conversion to the 5'-monophosphate (AZTMP), 5'-diphosphate (AZTDP), and 5'-triphosphate (AZTTP) derivatives. High ratios of AZTMP/AZTTP were found in the CEM cell cultures treated with CycloSal-AZTMP. The intracellular T(1/2) of AZTTP in CEM cell cultures treated with either AZT and CycloSal-AZTMP was approximately 3 h. A variety of human T- and B-lymphocyte cell lines efficiently converted the prodrug to the AZT metabolites, whereas peripheral blood lymphocytes and primary monocyte/macrophages showed at least 10-fold lower metabolic conversion of the prodrug. CycloSal-AZTMP failed to generate marked levels of AZT metabolites in thymidine kinase-deficient CEM/TK(-) cells, an observation that is in agreement with the substantial loss of antiviral activity of CycloSal-AZTMP in CEM/TK(-) cells. The inability of CycloSal-AZTMP to generate AZTMP in CEM/TK(-) cells is presumably due to a relatively high hydrolysis rate of AZTMP to the parent nucleoside AZT, combined with the inability of CEM/TK(-) cells to phosphorylate AZT to AZTMP through the cytosolic salvage enzyme thymidine kinase.
Mol Pharmacol 1999 Dec
PMID:Intracellular metabolism of CycloSaligenyl 3'-azido-2', 3'-dideoxythymidine monophosphate, a prodrug of 3'-azido-2', 3'-dideoxythymidine (zidovudine). 2008 Sep 48

7-Alkyl, 7-alkyl-10-hydroxy, 7-alkyl-10-methoxy, and 7-alkyl-10, 11-methylenedioxy analogs of camptothecin have been synthesized and evaluated for their ability to trap human DNA topoisomerase I in cleavable complexes. The 7-alkyl chain lengths varied linearly from methyl to butyl. The concentration required to produce cleavable complexes with purified topoisomerase I in 50% of the plasmid DNA (EC(50)) was reduced by 1 order of magnitude by the introduction of a 10-methoxy or 7-alkyl group compared with camptothecin. The EC(50) values were reduced by 2 orders of magnitude with a 10-hydroxy or 10, 11-methylenedioxy moiety compared with camptothecin. The steady-state EC(50) concentrations for all of the analogs tested were slightly dependent on substitution at the 7-position, but this dependence was least with the 10-methoxy series. The kinetics of the reversibility of the complexes formed with all analogs was only slightly influenced by the length of the 7-substitution, with the trend that ethyl or greater lengths led to slightly reduced rate constants for cleavable complex reversal. These results were also observed for DNA-protein cross-link formation by the analogs in isolated CEM cell nuclei. Our data indicate that in vitro cleavable complex stability, as determined by the apparent rate constants for complex dissociation, does not reflect the in vitro biological activity of these camptothecin analogs. However, complex stability in vivo may be important for the antitumor activity of the compounds.
Mol Pharmacol 2000 Feb
PMID:7- and 10-substituted camptothecins: dependence of topoisomerase I-DNA cleavable complex formation and stability on the 7- and 10-substituents. 1064 33

ICRF-187 is a bisdioxopiperazine anticancer drug that inhibits the catalytic activity of DNA topoisomerase (topo) II without stabilizing DNA-topoII cleavable complexes. To better understand the mechanisms of action of and resistance to topoII catalytic inhibitors, human leukemic CEM cells were selected for resistance to ICRF-187. The clones CEM/ICRF-8 and CEM/ICRF-18 are approximately 40- and 69-fold resistant to ICRF-187, and 12- and 67-fold cross-resistant to ICRF-193, respectively, but are sensitive to other topoII catalytic inhibitors (merbarone and aclarubicin), as well as collaterally sensitive to the DNA-topoII complex-stabilizing drug etoposide (VP-16). Both the number of VP-16- induced DNA-topoII complexes formed and the amount of in vitro topoII catalytic activity are enhanced in the drug-resistant cells. The ICRF-187-resistant clones contain approximately 5-fold increase in topoIIalpha protein levels and approximately 2.2-fold increase in topoIIalpha mRNA levels. Furthermore, CEM/ICRF-8 expresses approximately 3.5-fold increase in topoIIalpha promoter activity, suggesting that up-regulation of topoIIalpha in this clone occurs at the transcriptional level. Treatment of the drug-resistant or -sensitive cells with equitoxic doses of merbarone or teniposide results in a G(2)/M arrest. In marked contrast, when treated with equitoxic ICRF-187 doses, the drug-resistant clones exhibit either a transient arrest or completely lack the G(2)/M checkpoint compared with the drug-sensitive cells. This aberrant cell cycle profile is associated with a 48-h delay in drug-induced apoptotic cell death, as revealed by fluorescent-end labeling of DNA and poly (ADP-ribose) polymerase cleavage. In summary, resistance to ICRF-187 in CEM cells is associated with increased levels of catalytically active topoIIalpha and altered G(2)/M checkpoint and apoptotic responses.
Mol Pharmacol 2000 Feb
PMID:Selection of human leukemic CEM cells for resistance to the DNA topoisomerase II catalytic inhibitor ICRF-187 results in increased levels of topoisomerase IIalpha and altered G(2)/M checkpoint and apoptotic responses. 1064 39

We have shown that the TARDIS assay (trapped in agarose DNA immunostaining) can be used to detect DNA-topoisomerase I (topo I) cleavable complexes in situ in individual cells following treatment with topo I-targeting drugs. This assay is a modification of the assay for DNA-topoisomerase II (topo II) cleavable complexes (Willmore et al., Mol Pharmacol 53: 78-85, 1998). Drug-stabilised topo I-DNA complexes were detected in situ by topo I-specific primary antibodies and then visualised using fluorescein isothiocyanate conjugated second antibodies. Immunofluorescence was then quantified using a cooled slow-scan coupled device camera and image analysis procedures. Camptothecin (CPT) was shown to stabilise topo I-DNA cleavable complexes in whole cells in a dose-dependent manner in both CCRF-CEM and K562 cells and in lymphoblasts from an adult with newly diagnosed acute myeloid leukaemia treated ex vivo with CPT. In K562 cells, cleavable complexes were found to be maximal between 30 and 90 minutes continuous exposure of CPT, and approximately 78% of cleavable complexes formed in these cells were found to be reversed within 5 minutes of drug removal. It has also been shown that the immunofluorescence detected by the TARDIS assay was specific for topo I-targeting agents. Hence, the TARDIS assay provides a powerful tool to determine the levels of drug-stabilised cleavable complexes in whole cells and thereby aid in the understanding of the mechanism of interaction between topo I-targeting drugs and their target.
 ...
PMID:Camptothecin-stabilised topoisomerase I-DNA complexes in leukaemia cells visualised and quantified in situ by the TARDIS assay (trapped in agarose DNA immunostaining). 1067 79


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