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C V Catapano

Publications and source records attributed to C V Catapano.

At least 19 recordsLinked to original sources
PubMed2002

Design of a novel triple helix-forming oligodeoxyribonucleotide directed to the major promoter of the c-myc gene.

Altered expression of c-myc is implicated in pathogenesis and progression of many human cancers. Triple helix-forming oligonucleotides (TFOs) directed to a polypurine/polypyrimidine sequence in a critical regulatory region near the c-myc P2 promoter have been shown to inhibit c-myc transcription in vitro and in cells. However, these guanine-rich TFOs had moderate binding affinity and required high concentrations for activity. The 23 bp myc P2 sequence is split equally into AT- and GC-rich tracts. Gel mobility analysis of a series of short TFOs directed in parallel and anti-parallel orientation to the purine strand of each tract showed that only parallel CT and anti-parallel GT TFOs formed stable triplex on the AT- and GC-rich tracts, respectively. A novel full-length GTC TFO was designed to bind simultaneously in parallel and anti-parallel orientation to the polypurine strand. Gel-shift and footprinting assays showed that the new TFO formed a triple helix in physiological conditions with significantly higher affinity than an anti-parallel TFO. Protein-binding assays showed that 1 microM GTC TFO inhibited binding of nuclear transcription factors to the P2 promoter sequence. The novel TFO can be developed into a potent antigene agent, and its design strategy applied to similar genomic sequences, thus expanding the TFO repertoire.

AT Rich Sequence↗
PubMed2001

Imbalanced DNA synthesis induced by cytosine arabinoside and fludarabine in human leukemia cells.

Previous studies have demonstrated that cytosine arabinoside (araC) induces an accumulation of Okazaki fragments, while fludarabine (FaraA) inhibits Okazaki fragment synthesis. We extended these observations in the present study to provide insights into various mechanisms by which these anticancer drugs affect DNA replication and induce genomic instability in human CEM leukemia cells. Neither araC nor FaraA induced a detectable amount of re-replicated DNA in S-phase cells, which indicated that drug-induced alterations in Okazaki fragment synthesis were not accompanied by DNA re-replication. Synthesis on both leading and lagging DNA strands within the c-myc locus was measured in cells incubated with equitoxic concentrations of araC or FaraA. In araC-treated cells, nascent DNA from the lagging strand was enriched about 5-fold compared with the leading strand. In contrast, FaraA did not induce any replication imbalance. AraC- and FaraA induced changes in the frequency of N-(phosphonacetyl)-l-aspartate (PALA) resistance and the extent of CAD gene amplification were monitored as markers of drug-induced genomic instability. At concentrations that reduced cloning efficiency by 50% (IC(50)), araC increased the frequency of PALA resistance about 4-fold, while FaraA did not have a significant effect on the frequency of PALA resistance. Pretreatment with araC also increased the extent of CAD gene amplification. We propose that the imbalanced DNA synthesis induced by araC leads to the accumulation of Okazaki fragments on the lagging arms and single-stranded DNA regions on the leading arms of replication forks. The formation of these abnormal replication structures was associated with the generation of genomic instability.

Antineoplastic Agents↗
PubMed2001

Synergistic interaction between topotecan and microtubule-interfering agents.

PURPOSE: Topotecan is a topoisomerase I inhibitor with demonstrated anticancer activity in preclinical and clinical studies. The purpose of the present study was to evaluate drug-drug interactions in therapeutic regimens that would combine topotecan with microtubule-interfering agents, such as Taxol and vinblastine. METHODS: The cytotoxic activities of various drug combinations and schedules of administration were measured in a colon cancer cell line using the MTT assay. Western blot and flow cytometry were performed to determine the effects of Taxol and vinblastine on topoisomerase I and Bcl-xL protein levels and cell cycle distribution. RESULTS: Brief incubation of colon cancer cells with low concentrations of either Taxol or vinblastine increased the efficacy of a subsequent treatment with topotecan. Preincubation of cells with vinblastine or Taxol reduced by 10- to 40-fold the concentration of topotecan necessary to induce a 50% decrease in cell survival. The effects were maximal when the cells were treated for 5 h with microtubule-interfering agents and then incubated for 19 h in drug-free medium before the addition of topotecan. Under these conditions, both Taxol and vinblastine caused an increase in topoisomerase I protein levels, fraction of S phase cells, and extent of Bcl-xL phosphorylation immediately prior to the addition of topotecan. All these factors may contribute to the increased efficacy of topotecan observed with sequential therapy. CONCLUSION: Combinations of topotecan and microtubule-interfering agents result in synergistic anticancer activity when the drugs are administered sequentially. The promising preclinical data presented here encourage clinical testing of these drug combinations using a sequential schedule of administration.

Antineoplastic Agents↗
PubMed2000

Antigene and antiproliferative effects of a c-myc-targeting phosphorothioate triple helix-forming oligonucleotide in human leukemia cells.

The c-myc gene is frequently deregulated and overexpressed in human cancers, and strategies designed to inhibit c-myc expression in cancer cells may have considerable therapeutic value. The purpose of the present work was to characterize the antigene and antiproliferative activity of a triple helix-forming oligonucleotide (TFO) targeted to a homopurine-homopyrimidine sequence in the P2 promoter of the c-myc gene. The TFO was synthesized with phosphorothioate (PS) internucleotide linkages to confer resistance to intra- and extracellular nucleases. This property is required of oligonucleotides designed for in vivo testing and therapeutic applications. The PS-TFO was found to form triplex DNA with affinity and specificity comparable with that of the corresponding phosphodiester TFO, as shown by gel mobility shift and footprinting assays. Fluorescence microscopy and polyacrylamide gel analysis showed that the fluorescein-labeled PS-TFO accumulated in nuclei of CEM leukemia cells and remained intact for at least 72 h. Incubation of CEM cells with PS-TFO reduced c-myc RNA and protein levels. A single exposure of leukemia cells to the PS-TFO was sufficient to induce dose-dependent growth inhibitory effects. Growth inhibition correlated with accumulation of cells in S phase and with induction of cell death by apoptosis. The PS-TFO was also effective in other leukemia and lymphoma cell lines. Control oligonucleotides had minimal effects in all assays. These data indicate that the c-myc-targeted PS-TFO is an effective antigene and antiproliferative agent, with potential for testing in vivo as a novel approach to cancer therapy.

Antigens↗
PubMed2000

Inhibition of gene expression and cell proliferation by triple helix-forming oligonucleotides directed to the c-myc gene.

Triple helix-forming oligonucleotides (TFOs) bind with high affinity and specificity to homopurine-homopyrimidine sequences in DNA and have been shown to inhibit transcription of target genes in various experimental systems. In the present study, we evaluated the ability of 3'-amino-modified phosphodiester TFOs directed to four sites in the c-myc gene to inhibit gene expression and proliferation of human leukemia (CEM, KG-1, and HL-60) and lymphoma (Raji and ST486) cells. GT-rich TFOs were designed to target sequences located either upstream (myc1 and -2) or downstream (myc3 and -4) of the P2 promoter, which is the major c-myc promoter. Myc2, which was directed to a site immediately upstream of this promoter, inhibited c-myc expression and proliferation of CEM cells. The effects of this TFO were sequence- and target-specific, since control oligonucleotides and TFOs directed to other sites were less or not active. Myc2 was also effective in KG-1, HL-60, and Raji cells. In contrast, ST486 cells were more sensitive to myc3, which targets a sequence in intron 1 upstream of the P3 promoter, than myc2. As result of a chromosomal translocation, P3 is the active promoter in ST486 cells. This study demonstrates the activity and specificity of TFOs designed to act as repressors of c-myc gene expression in human leukemia and lymphoma cells. Our results suggest that this is a valid approach to selectively inhibit gene expression and cancer cell growth, and encourage further investigation of its potential applications in cancer therapy.

Cell Division↗
PubMed1999

Mode of action of thiocoraline, a natural marine compound with anti-tumour activity.

Thiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina, was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase alpha at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase alpha activity.

Anti-Bacterial Agents↗
PubMed1997

Arrest of replication fork progression at sites of topoisomerase II-mediated DNA cleavage in human leukemia CEM cells incubated with VM-26.

Recent studies have shown that the anticancer drugs VM-26 and mitoxantrone stabilize preferentially the binding of topoisomerase IIalpha to replicating compared to nonreplicating DNA. To further understand the mechanisms by which cleavable complex-forming topoisomerase II inhibitors interfere with DNA replication, we examined the effects of VM-26 on this process in human leukemia CEM cells. Both the inhibition of DNA synthesis and cell survival were directly related to the total amount of drug-stabilized cleavable complexes formed in VM-26-treated cells. DNA chain elongation was also inhibited in a concentration-dependent fashion in these cells, which suggested that VM-26-stabilized cleavable complexes interfered with the movement of DNA replication forks. To test this hypothesis directly, we monitored replication fork progression at a specific site of VM-26-induced DNA cleavage. A topoisomerase II-mediated cleavage site was detected in the first exon of the c-myc gene in VM-26-treated cells. This cleavage site was downstream of a putative replication origin located in the 5' flanking region of the gene. Replication forks, which moved through this region of the c-myc gene in the 5' to 3' direction, were specifically arrested at this site in VM-26-treated cells, but not in untreated or aphidicolin-treated cells. These studies provide the first direct evidence that a VM-26-stabilized topoisomerase II-DNA cleavable complex acts as a replication fork barrier at a specific genomic site in mammalian cells. Furthermore, the data support the hypothesis that the replication fork arrest induced by cleavable complex-forming topoisomerase II inhibitors leads to the generation of irreversible DNA damage and cytotoxicity in proliferating cells.

DNA Damage↗
PubMed1996

Formation of topoisomerase II alpha complexes with nascent DNA is related to VM-26-induced cytotoxicity.

Several clinically active anticancer drugs are known to interfere with DNA topoisomerase II activity. However, the importance of the individual alpha (170 kDa) and beta (180 kDa) isozymes as targets of topoisomerase II-active drugs is not clear. To address this question, human CCRF-CEM leukemia cells were incubated with bromodeoxyuridine, and either the nascent DNA or bulk DNA not undergoing replication was purified by immunoprecipitation with an anti-bromodeoxyuridine antibody. The topoisomerase II isozymes that coprecipitated with either the nascent DNA or bulk DNA were analyzed by Western blotting. The alpha isozyme formed complexes with nascent DNA in cells pretreated with either VM-26 or mitoxantrone, while the beta isozyme was only bound to bulk DNA. At moderately cytotoxic concentrations, VM-26 enhanced the binding of topoisomerase II alpha to nascent DNA at least 5.2-fold compared to bulk DNA. However, in VM-26 resistant CEM/VM-1 cells incubated with equitoxic concentrations of VM-26, topoisomerase II alpha complex formation with nascent DNA was decreased at least 5.5-fold compared to bulk DNA. Drug-induced binding of topoisomerase II beta with bulk DNA in CEM/VM-1 cells did not correlate with cytotoxicity. Collectively, these results indicate that the formation of VM-26 stabilized complexes of topoisomerase II alpha with nascent DNA are critical to the development of cytotoxicity, and that resistance of CEM/VM-1 cells to VM-26 is related to impaired formation of these complexes. The results also provide indirect evidence that topoisomerase II alpha is involved in DNA, replication.

Antineoplastic Agents, Phytogenic↗
PubMed1995

Increased rate of adenosine triphosphate-dependent etoposide (VP-16) efflux in a murine leukemia cell line overexpressing the multidrug resistance-associated protein (MRP) gene.

WEHI-3B/NOVO is a cloned murine leukemia cell line selected for resistance to novobiocin that is cross-resistant to the cytotoxic action of etoposide (VP-16) and to a lesser extent to a variety of other topoisomerase II (topo II)-reactive drugs. We have reported previously (Cancer Res. 52: 2782-2790, 1992) that WEHI-3B/NOVO cells exhibit a pronounced decrease in VP-16 induced DNA-topo II cross-link formation compared to the parental WEHI-3B/S cell line in intact cells, in the absence of a significant difference in the P4 unknotting activity of topo II assayed in nuclear extracts. Because the pattern of cross-resistance was suggestive of a topo II-mediated mechanism, we have ascertained whether a change in topo II can account for the multidrug-resistant phenotype of WEHI-3B/NOVO cells. No differences existed between WEHI-3B/S and WEHI-3B/NOVO cells in topo II mRNA and protein levels, as well as in the amount of topo II associated with the nuclear matrix. Neither sensitive nor resistant cells expressed detectable levels of the MDR1 gene; however, VP-16 accumulation in WEHI-3B/NOVO cells was 3-4-fold less than that present in WEHI-3B/S cells, whereas doxorubicin accumulation was the same in both cell lines. Over the first 60 s, no difference existed in the rate of uptake of VP-16 between parental and resistant cells; however, beyond the first 60 s of incubation, [3H]VP-16 accumulated to a greater extent in parental sensitive cells. Thus, an increased rate of efflux of VP-16 was responsible for the lower steady-state concentration of the drug in resistant cells. The efflux Km for VP-16 in WEHI-3B/NOVO cells was 254.7 microM and the Vmax was 10.4 pmol/s/10(7) cells. In the presence of the inhibitors of energy metabolism, sodium azide and deoxyglucose, the efflux of VP-16 was markedly inhibited; readdition of glucose restored the original efflux rate. Northern blot analyses using the human 10.1 probe for the 3'-terminal region of the multidrug-resistance protein (MRP) cDNA revealed a mRNA species of approximately 6 kb in WEHI-3B/NOVO cells but not in WEHI-3B/S cells. Overexpression was associated with amplification of the cognate gene. To ascertain whether the overexpressed gene in WEHI-3B/NOVO cells was the murine MRP or a different member of the same superfamily of ATP-binding ABC cassette transporters, a 341-bp MRP cDNA probe was generated from a murine genomic library.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1995

DNA topoisomerase II isozymes involved in anticancer drug action and resistance.

DNA topoisomerase II is a major protein of the nuclear matrix. The enzyme appears to have a central role in both DNA organization and replication. The importance of nuclear matrix topoisomerase II alpha as a target for certain anticancer agents was evaluated in CEM human leukemia cells. Studies were done to determine the extent to which the alpha (170 kDa) and beta (180 kDa) isozymes of topoisomerase II form covalent enzyme-DNA complexes in whole cells and in the nuclear matrix and nonmatrix fractions of CEM cells that are either sensitive or resistant to topoisomerase II-active anticancer agents. Topoisomerase II alpha was detected in both the high salt-soluble (nonmatrix) and matrix fractions of nuclei from parental CEM cells. Most of the matrix topoisomerase II alpha was tightly bound to DNA in cells incubated with VM-26. In contrast, topoisomerase II beta was detected only in the high salt-soluble (nonmatrix) fraction of the nucleus. The subnuclear distribution of the alpha and beta topoisomerase II isozymes in CEM/VM-1 cells resistant to topoisomerase-active drugs was similar to that in drug-sensitive CEM cells. However, the amount and activity of topoisomerase II alpha in nuclear matrices of CEM/VM-1 cells were decreased 3- to 6-fold relative to that of CEM cells. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function. Furthermore, these results provide evidence that topoisomerase II alpha is the nuclear matrix target for VM-26, and that depletion of the nuclear matrix isozyme contributes to cellular resistance to this anticancer agent.

Adenosine Triphosphate↗
PubMed1995

The nuclear matrix as a site of anticancer drug action.

Many nuclear functions, including the organization of the chromatin within the nucleus, depend upon the presence of a nuclear matrix. Nuclear matrix proteins are involved in the formation of chromatin loops, control of DNA supercoiling, and regulation and coordination of transcriptional and replicational activities within individual loops. Various structural and functional components of the nuclear matrix represent potential targets for anticancer agents. Alkylating agents and ionizing radiation interact preferentially with nuclear matrix proteins and matrix-associated DNA. Other chemotherapeutic agents, such as fludarabine phosphate and topoisomerase II-active drugs, interact specifically with matrix-associated enzymes, such as DNA primase and the DNA topoisomerase II alpha isozyme. The interactions of these agents at the level of the nuclear matrix may compromise multiple nuclear functions and be relevant to their antitumor activities.

Animals↗
PubMed1995

GTP depletion induced by IMP dehydrogenase inhibitors blocks RNA-primed DNA synthesis.

Inhibitors of IMP dehydrogenase (EC 1.2.1.14), including mizoribine (Bredinin) and mycophenolic acid, have significant antitumor and immunosuppressive activities. Studies were aimed at determining the mechanism by which intracellular GTP depletion induced by these agents results in inhibition of DNA synthesis. Incubation of human CEM leukemia cells for 2 hr with IC50 concentrations of either mizoribine (4 microM) or mycophenolic acid (0.5 microM) reduced cellular GTP levels an average of 68% or 58%, respectively, compared with the levels in control cells. Under similar conditions, mizoribine and mycophenolic acid decreased the amount of [3H]adenosine incorporated into primer RNA by 75% and 70%, respectively, relative to the untreated controls, but had no significant effect on total RNA synthesis. Repletion of the guanine nucleotide pools by coincubation of CEM cells with guanosine plus 8-aminoguanosine prevented both the inhibition of primer RNA synthesis and the inhibition of tumor cell growth induced by these agents. Additional studies demonstrated that GTP depletion alone was capable of directly inducing inhibition of primer RNA synthesis. Primer RNA synthesis was inhibited an average of 84% in whole-cell lysates that lacked GTP but contained all remaining ribo- and deoxyribonucleoside triphosphates. On an M13 DNA template, RNA-primed DNA synthesis catalyzed by the purified complex of DNA primase (EC 2.7.7.6) and DNA polymerase alpha (EC 2.7.7.7) was decreased an average of 70% in the absence of GTP, compared with synthesis in the presence of 0.5 mM GTP. These results provide evidence that mizoribine and mycophenolic acid inhibit DNA replication by inducing GTP depletion, which suppresses the synthesis of RNA-primed DNA intermediates.

Adenosine Triphosphate↗
PubMed1994

Subcellular distribution of the alpha and beta topoisomerase II-DNA complexes stabilized by VM-26.

Studies were done to determine (a) the subcellular distribution of the alpha (170 kDa) and beta (180 kDa) isozymes of topoisomerase II, and (b) the extent to which each isozyme forms complexes with DNA in tumor cells incubated with and without VM-26. Western blotting revealed that topoisomerase II beta was highly unstable during cell fractionation. However, preincubation of human CEM leukemia cells with 5-100 microM VM-26 for 30 min protected the beta isozyme from degradation by progressively increasing the amount of this isoform bound to DNA. The amount of topoisomerase II beta detected in nuclei of CEM cells incubated for 30 min with 25 microM VM-26 was 7-fold greater than in nuclei from untreated control cells. VM-26 also had a protective effect on topoisomerase II beta in HL-60 leukemia and WiDR colon carcinoma cells. In contrast, the intercalating agents mitoxantrone and m-AMSA did not protect topoisomerase II beta from degradation during cell fractionation. The stabilization of topoisomerase II beta by VM-26 allowed subsequent studies of the subcellular distribution of the topoisomerase II isozymes. Both isozymes were detected in the nonmatrix (high salt-soluble) fraction of nuclei from CEM cells, but only topoisomerase II alpha was present in the nuclear matrix. VM-26 stabilized binding of the alpha and beta topoisomerase II isoenzymes to nonmatrix DNA and topoisomerase II alpha to matrix DNA. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function, and that both the alpha and beta isozymes are potential targets for VM-26 in intact cells. In addition, the results demonstrated that pretreatment of various cell lines with VM-26 is a useful way to stabilize topoisomerase II beta during cell fractionation.

Amsacrine↗
PubMed1993

Primer RNA chain termination induced by 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate. A mechanism of DNA synthesis inhibition.

The studies described herein were aimed at defining the mechanism by which 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (FaraATP), the active intracellular metabolite of fludarabine phosphate, inhibits the synthesis of primer RNA and RNA-primed DNA by the polymerase alpha-primase complex. Incubation of the purified DNA polymerase alpha-primase complex with a poly(dT) template, 500 microM ATP, and increasing concentrations of FaraATP from 2.5 to 50 microM resulted in the progressive accumulation of smaller oligoribonucleotides (2-6 nucleotides) at the expense of the full-length products of DNA primase (7-10 nucleotides). Comparison of the kcat/KM values for incorporation of FaraATP and ATP into oligoribonucleotides revealed that DNA primase incorporated FaraATP 30-fold more efficiently than ATP. FaraAMP was present exclusively at the 3'-termini of the growing primer RNA chains, which prevented further elongation of the primers by DNA primase (primer RNA chain termination). At all FaraATP concentrations tested, inhibition of RNA-primed DNA synthesis was accompanied by primer chain termination. In contrast, DNA polymerase alpha added FaraATP onto full-length primer RNAs about 8-fold less efficiently than dATP, and the incorporation of FaraAMP at the 3'-termini of the primers did not prevent further elongation of these primers by DNA polymerase alpha. These results indicate that primer RNA chain termination is the major effect responsible for the inhibition of RNA-primed DNA synthesis by fludarabine phosphate.

Adenosine Triphosphate↗
PubMed1993

Cytotoxic activity and mechanism of action of 5-Aza-2'-deoxycytidine in human CML cells.

We investigated the cytotoxic activity and some aspects of the mode of action of 5-aza-2'-deoxycytidine (Aza-dC) in 21 primary cultures of leukemic cells freshly obtained from patients with chronic myeloid leukemia (CML) in blast crisis. The cytotoxic potency of Aza-dC was comparable or even greater than that of 1-beta-D-arabinofuranosylcytosine (Ara-C) in most cases, suggesting that this drug has potential in the therapy of blast crisis of CML. Drug incorporation into DNA was evaluated by exposing leukemic cells simultaneously to 3H-Aza-dC at the concentration of 0.1 micrograms/ml and 14C-thymidine (TdR) used as internal standard. Incorporation of Aza-dC into DNA was detectable in all cases. In 17 samples we evaluated the DNA integrity of leukemic cells exposed to Aza-dC using alkaline elution techniques. The drug caused a detectable amount of DNA alkali labile sites (ALS). DNA-ALS increased in cells exposed to Aza-dC concentrations from 0.1 to 1 microgram/ml but did not further increase at 10 micrograms/ml. A plateau in the levels of DNA-ALS was also seen in human K562 cells exposed to increasing concentrations of Aza-dC from 5 to 10 micrograms/ml, whereas in these cells Aza-dC incorporation into DNA increased with increasing Aza-dC concentrations. Therefore, DNA-ALS caused by Aza-dC are not simply the result of the chemical decomposition of azacytosine molecules incorporated into DNA, but are presumably the result of a saturable DNA repair mechanism (e.g., glycosylases) leading to formation of the apyrimidinic sites.

Antineoplastic Agents↗
PubMed1992

Influence of O6-methylguanine on DNA damage and cytotoxicity of temozolomide in L1210 mouse leukemia sensitive and resistant to chloroethylnitrosoureas.

Temozolomide is a new anticancer agent which in the early clinical investigation has shown promising antitumor activity. It decomposes spontaneously to the active metabolite of DTIC (MTIC). Temozolomide is more cytotoxic against L1210 than against a subline L1210/BCNU, resistant to chloroethylnitrosoureas. Using [methyl-3H] temozolomide we found that after 1 h exposure the amount of O6-methylguanine (O6mGua) was twice as high in L1210 than in L1210/BCNU whereas the amount of N7 mGua was approximately the same in the two cell lines. O6-alkylguanine DNA alkyltransferase (AT) levels were higher in L1210/BCNU than in L1210, supporting the view that the resistance to methyltriazenes is probably related to the efficient repair of O6mGua in L1210/BCNU. Exposure of L1210/BCNU cells to 0.4 mM O6mGua for 24 h resulted in a depletion of AT and in a higher temozolomide-induced cytotoxicity. In the sensitive cell line L1210, temozolomide activity was not potentiated by O6mGua pretreatment. Moreover, in L1210/BCNU, O6mGua increased DNA single-strand breaks caused by temozolomide, suggesting that O6-guanine alkylation induces an excision repair mechanism in cells depleted in AT.

Alkalies↗
PubMed1991

Inhibition of primer RNA formation in CCRF-CEM leukemia cells by fludarabine triphosphate.

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.

Aphidicolin↗