aphidicolin and fludarabine

Purine analogs are among the most effective chemotherapeutic drugs for the treatment of chronic lymphocytic leukemia (CLL). However, chemoresistance and toxicity limit their clinical use. Here, we report that the DNA polymerase inhibitor aphidicolin, which displayed negligible cytotoxicity as a single agent in primary CLL cells, markedly synergizes with fludarabine and cladribine via enhanced apoptosis. Importantly, synergy was recorded regardless of CLL prognostic markers. At the molecular level, aphidicolin enhanced purine analog-induced phosphorylation of p53 and accumulation of γH2AX, consistent with increase in DNA damage. In addition, aphidicolin delayed γH2AX disappearance that arises after removal of purine analogs, suggesting that aphidicolin causes an increase in DNA damage by impeding DNA damage repair. Similarly, aphidicolin inhibited UV-induced DNA repair known to occur primarily through the nucleotide excision repair (NER) pathway. Finally, we showed that fludarabine induced nuclear import of XPA, an indispensable factor for NER, and that XPA silencing sensitized cell lines to undergo apoptosis in response to fludarabine. Together, our data indicate that aphidicolin potentiates the cytotoxicity of purine analogs by inhibiting a DNA repair pathway that involves DNA polymerases, most likely NER, and provide a rationale for manipulating it to therapeutic advantage.

Topics: Antineoplastic Agents; Aphidicolin; Apoptosis; Cladribine; DNA Damage; DNA Repair; Drug Synergism; Enzyme Inhibitors; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Vidarabine

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A.

Topics: Antineoplastic Agents; Aphidicolin; Biotransformation; Cell Line, Tumor; Cell Survival; Cladribine; Deoxycytidine; Deoxycytidine Kinase; Enzyme Activation; Gemcitabine; HCT116 Cells; HT29 Cells; Humans; Kinetics; Mutation; Phosphorylation; Purine Nucleosides; Pyrimidine Nucleosides; Serine; Structure-Activity Relationship; Substrate Specificity; Vidarabine

The inhibition of UV-initiated DNA repair by 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A), the nucleoside of fludarabine, induces apoptosis in quiescent human lymphocytes. The sensing and signaling mechanisms after DNA repair inhibition by F-ara-A are unknown. The purpose of this study was 2-fold: (a) determine the importance of the inhibition of DNA repair processes for F-ara-A cytotoxicity and (b) identify the apoptotic signaling mechanism(s) that respond to DNA repair inhibition by F-ara-A.. Lymphocytes were treated with F-ara-A to accumulate the active triphosphate metabolite and subsequently DNA repair was activated by UV irradiation. Cell viability was quantitated with respect to the treatments alone and in combination to evaluate the actions of F-ara-A inhibition of DNA repair on p53 status and Fas death receptor ligand expression and function.. Preincubation of lymphocytes with 3 micro M F-ara-A inhibited DNA repair initiated by 2 J/m(2) UV and induced greater than additive apoptosis after 24 h. After equivalent repair inhibition with 0.1 micro M aphidicolin, there was apparently lesser p53 activation and significantly less apoptosis in irradiated lymphocytes than after 3 micro M F-ara-A. Blocking the incorporation of F-ara-A nucleotide into repairing DNA using 30 micro M aphidicolin lowered the apoptotic response to that observed with aphidicolin and UV. p53 serine 15 phosphorylation and protein accumulation were detected 2 h after treatment. Fas and Fas ligand mRNA expression and protein levels increased significantly after repair inhibition. Neutralizing antibodies against Fas or Fas ligand significantly reduced apoptosis.. These results suggest that inhibition of UV-induced DNA repair by F-ara-A is critical for cytotoxicity and that induction of apoptosis may be conducted by a p53-mediated signaling mechanism to the Fas death pathway.

Topics: Aphidicolin; Apoptosis; Caspase 3; Caspase 8; Caspases; Cell Survival; Cells, Cultured; DNA Repair; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fas Ligand Protein; fas Receptor; Humans; Immunoblotting; Lymphocytes; Membrane Glycoproteins; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine; Signal Transduction; Time Factors; Tumor Suppressor Protein p53; Ultraviolet Rays; Vidarabine

Alkylating agents or platinum analogues initiate several excision repair mechanisms, which involve incision of the DNA strand, excision of the damaged nucleotide, gap filling by DNA resynthesis, and rejoining by ligation. The previous study described that nucleotide excision repair permitted incorporation of fludarabine nucleoside (F-ara-A) into the repair patch, thereby inhibiting the DNA resynthesis. In the present study, to clarify the repair kinetics in view of the inhibition by F-ara-A, normal lymphocytes were stimulated to undergo nucleotide excision repair by ultraviolet C (UV) irradiation in the presence or absence of F-ara-A. The repair kinetics were determined as DNA single strand breaks resulting from the incision and the rejoining using the alkaline single cell gel electrophoresis (comet) assay. DNA resynthesis was evaluated in terms of the uptake of tritiated thymidine into DNA. The lymphocytes initiated the incision step maximally at 1 h, and completed the rejoining process within 4 h after UV exposure. UV also initiated thymidine uptake, which increased time-dependently and reached a plateau at 4 h. A 2-h pre-incubation with F-ara-A inhibited the repair in a concentration-dependent manner, with the maximal inhibition by 5 mM. This inhibitory effect was demonstrated by the reduction of the thymidine uptake and by the inhibition of the rejoining. A DNA polymerase inhibitor, aphidicolin, and a ribonucleotide reductase inhibitor, hydroxyurea, were not so inhibitory to the repair process as F-ara-A at equimolar concentrations. The present findings suggest that inhibition of nucleotide excision repair may represent a novel therapeutic strategy against cancer, especially in the context of resistant cells with an increased repair capacity.

Topics: Antineoplastic Agents; Aphidicolin; Apoptosis; Comet Assay; DNA Repair; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Humans; Hydroxyurea; Kinetics; Lymphocytes; Time Factors; Ultraviolet Rays; Vidarabine

Cleavage of cellular DNA into high molecular weight (predominantly 50 kb) fragments is an early event during apoptosis. We previously reported that this fragmentation was a Ca2+-independent process during apoptosis, which was induced by anticancer agents in human leukemia cells. The present study demonstrated that a high molecular weight DNA fragmentation activity (HDFA) was induced in the drug-treated cells and, upon fusion of the drug-treated cells with untreated target cells prelabeled with [14C]thymidine, caused fragmentation of the labeled DNA in the target cells. Furthermore, extracts of the drug-treated cells caused high molecular weight DNA fragmentation in nuclei isolated from untreated cells. Biochemical characterization of HDFA revealed the following properties: HDFA was proteinaceous in nature, as evidenced by its inactivation by heating or by digestion with proteinase K; HDFA required Mg2+ for optimal activity but was inhibited by Zn2+ and K+; HDFA was active in vitro at pH 6.0-8.0 and was inactive under more acidic conditions (pH < 6.0); addition of ATP (0.5-2 mM) substantially potentiated HDFA activity in isolated nuclei; and HDFA was not inhibited by actin (an inhibitor of DNase I) but was inhibited by the extracts from K562 cells, which were resistant to drug-induced apoptosis. The specific inhibitor of cysteine proteases (interleukin 1beta-converting enzyme protease family) blocked the generation of drug-induced high molecular weight DNA fragmentation in whole cells, whereas in isolated nuclei, the cysteine protease inhibitors did not prevent the cleavage of chromatin by exogenous HDFA. These results suggest that, once HDFA is activated during apoptosis, it does not require the presence of cysteine proteases for its endonucleolytic activity and that the cysteine proteases may be involved in the apoptotic process upstream of the activation of HDFA in whole cells.

Topics: Antineoplastic Agents; Aphidicolin; Apoptosis; Cell Fusion; Cell Line; Cysteine Endopeptidases; Deoxycytidine; DNA Fragmentation; DNA, Neoplasm; Electrophoresis, Gel, Pulsed-Field; Gemcitabine; Humans; In Vitro Techniques; Molecular Weight; Nucleosomes; Vidarabine

The nucleoside analogs fludarabine and gemcitabine inhibit cellular DNA synthesis by two different mechanisms: (1) direct termination of DNA strand elongation after the triphosphate of each drug is incorporation into DNA; and (2) indirect inhibition of DNA synthesis by decreasing cellular dNTPs through inhibition of ribonucleotide reductase. The present study demonstrated that incorporation of the analogs into DNA is critical for the cytotoxic action of these drugs in human T lymphoblastoid CEM cells. S phase cells, which actively incorporated the analogs into DNA, were most sensitive to the cytotoxic action of these compounds. A relatively short-term (5-24 h) cessation of cellular DNA synthesis without analog incorporation was not sufficient to cause cell death. The drug-treated cells died through apoptosis characterized by generation of internucleosomal DNA fragmentation and apoptotic morphology. Induction of high molecular mass (50-500 kb) DNA fragmentation was also observed in cells undergoing apoptosis; this type of DNA degradation was strongly correlated with the analog-induced cell death process. Inhibition of the analog incorporation into DNA by aphidicolin blocked both types of DNA fragmentation and apoptotic morphology, indicating the essential role of analog incorporation into DNA in drug-induced cell death.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents; Aphidicolin; Apoptosis; Cell Cycle; Cell Line; Cell Membrane; Deoxycytidine; DNA Replication; DNA, Neoplasm; Gemcitabine; Humans; Kinetics; T-Lymphocytes; Time Factors; Tumor Cells, Cultured; Vidarabine

Cleavage of DNA into internucleosomal fragments is one of the characteristics of apoptosis. However, searches for in vivo evidence of nucleosomal DNA fragmentation in leukemia cells freshly obtained from patients during chemotherapy frequently failed to reveal nucleosomal multimers (DNA ladders). It is not clear whether this type of DNA cleavage is an essential event in drug-induced apoptosis and thus a denominator of cell killing, or whether the internucleosomal DNA fragments are merely the by-products of the apoptotic process. Here, we report our investigation into the role of DNA fragmentation in apoptotic cell death induced by anticancer nucleoside analogues, both in cell culture and in leukemia patients undergoing chemotherapy. Using a 5'-end DNA-labeling technique and pulsed field gel electrophoresis, we detected fragmentation of DNA in two distinct size classes, internucleosomal and high molecular weight (predominantly 50 kb) DNA fragments, in a human leukemia cell line exposed to the nucleoside analogues fludarabine and gemcitabine. We further demonstrated that the two types of DNA fragmentation were separate events, distinguishable by their requirements for Ca2+ and responses to phorbol ester treatment. The drug-treated cells underwent morphological changes of apoptosis even after internucleosomal DNA fragmentation was selectively inhibited by intracellular Ca2+ chelation, or by treatment with phorbol ester. In contrast, neither apoptotic morphology nor internucleosomal DNA fragmentation was observed when the high molecular weight DNA fragmentation was blocked by inhibition of nucleoside analogue incorporation into DNA. These results suggest that cleavage of DNA into large fragments may be an initial event that is critical for drug-induced apoptosis, whereas activation of a Ca2+-dependent endonuclease to cleave DNA at internucleosomal sites is not an absolute requirement for the execution of the apoptotic cell death program. Further studies of leukemic lymphocytes obtained from 9 patients with chronic lymphocytic leukemia during therapy with fludarabine revealed high molecular weight DNA fragmentation, which was correlated with a decrease of peripheral lymphocytes in 6 patients, whereas only 1 of the 15 patients evaluated for nucleosomal DNA fragments showed the DNA ladders. These results indicate that high molecular weight DNA fragmentation occurs in vivo, and may be correlated with the cytotoxic action of the anticancer drugs. Further study of

Topics: Antineoplastic Combined Chemotherapy Protocols; Aphidicolin; Apoptosis; Chelating Agents; Deoxycytidine; DNA Fragmentation; DNA, Neoplasm; Egtazic Acid; Enzyme Inhibitors; Gemcitabine; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Molecular Weight; Tumor Cells, Cultured; Vidarabine

The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on DNA synthesis was evaluated both in whole cells and in vitro. 9-beta-D-Arabinofuranosyl-2-fluoroadenine was converted to its 5'-triphosphate 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) in cells and then incorporated into DNA in a self-limiting manner. More than 94% of the analogue was incorporated into DNA at the 3' termini, indicating a chain termination action. In vitro DNA primer extension experiments further revealed that F-ara-ATP compared with dATP for incorporation into the A site of the extending DNA strand. The incorporation of F-ara-AMP into DNA resulted in termination of DNA strand elongation. Human DNA polymerase alpha incorporated more F-ara-AMP into DNA than polymerase epsilon (proliferating cell nuclear antigen-independent DNA polymerase delta) and was more sensitive to inhibition by F-ara-ATP. On the other hand, DNA polymerase epsilon was able to excise the incorporated F-ara-AMP from DNA in vitro. The incorporation of F-ara-AMP into DNA was linearly correlated both with inhibition of DNA synthesis and with loss of clonogenicity; thus it may be the mechanism of cytotoxicity.

Topics: Aphidicolin; Arabinonucleotides; Cell Line; Cell Survival; Clone Cells; Diterpenes; DNA Replication; DNA-Directed DNA Polymerase; Guanosine Monophosphate; Humans; Kinetics; Nucleic Acid Synthesis Inhibitors; Vidarabine; Vidarabine Phosphate