Characterization of protein kinase chk1 essential for the cell cycle checkpoint after exposure of human head and neck carcinoma A253 cells to a novel topoisomerase I inhibitor BNP1350.

Cellular topoisomerase I is an important target in cancer chemotherapy. A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity against human head and neck carcinoma A253 cells in vitro. As a basis for future clinical trials of BNP1350 in human head and neck carcinoma, in vitro studies were carried out to investigate its effect on DNA damage and cell cycle checkpoint response. The treatment of A253 cells with BNP1350 caused biphasic profiles of DNA fragmentation displayed from 0 to 48 h after 2-h exposure. Pulsed-field gel electrophoresis demonstrated that the first wave of DNA damage was mainly megabase DNA fragmentation, but the second wave of DNA damage was 50- to 300-kb DNA fragmentation in addition to megabase DNA damage. The cell cycle checkpoint response was characterized after exposure to 0.07 and 0.7 microM concentrations of BNP1350, the IC(50) and IC(90) values, respectively. After exposure to a low concentration of BNP1350 (IC(50)), A253 cells accumulated primarily in G(2) phase. In contrast, treatment with a high concentration of BNP1350 (IC(90)) resulted in S phase accumulation. The concentration-associated cell cycle perturbation by BNP1350 was correlated with different profiles of cell cycle-regulatory protein expression. When treated with the low concentration of BNP1350, cyclin B/cdc2 protein expression was up-regulated, whereas with the high concentration, no significant change was observed at 24 and 48 h. In addition, increased phosphorylation of a G(2) checkpoint kinase chk1 was observed when cells were treated with a low concentration of BNP1350, whereas only slight inhibition of chk1 activity was found in the cells treated with the higher concentration. Altered chk1 phosphorylation after DNA damage appears to be associated with specific phases of cell cycle arrest induced by BNP1350. Because A253 cells do not express the p53 protein, the drug-induced alterations of the G(2) checkpoint kinase chk1 are not p53-dependent.

Mechanism of action of the dual topoisomerase-I and -II inhibitor TAS-103 and activity against (multi)drug resistant cells.

UNLABELLED: TAS-103 is a recently developed dual inhibitor of topoisomerase-I (topo-I) and topoisomerase-II (topoII). TAS-103 has documented cytotoxicity in vitro and antitumor activity against a variety of mouse, rat, and human xenografts in vivo. PURPOSE: To determine TAS-103 activity against (multi)drug resistant cells in vitro and to delineate its mechanism of action. METHODS: TAS-103 was evaluated for activity against three human multidrug-resistant cell lines representing resistance mediated by P-glycoprotein (Pgp)-, multidrug resistance protein (MRP), and lung resistance protein (LRP) as well as one camptothecin-resistant cell line associated with a mutated topo-I enzyme. Drug sensitivity following short (2 h), intermediate (6-8 h) and long term (24 h) exposures were compared. The mechanism of action was studied by evaluating inhibition of topoisomerase-I and -II specific DNA relaxation assays, drug-induced DNA/protein cross-link formation, and competitive DNA intercalation with ethidium bromide. RESULTS: Increasing the exposure time only modestly potentiated TAS-103 cytotoxicity (3-5 fold) demonstrating a lack of strong exposure duration dependency. TAS-103 cytotoxicity was not affected by the presence of any of the drug resistance mechanisms studied. TAS-103 inhibits topo-I and -II activity in DNA relaxation assays, but in our assay system TAS-103 was found to have only a weak ability to induce DNA-protein crosslinks. DNA migration patterns in agarose gel electrophoresis indicate that TAS-103 can interact directly with DNA. Also its ability to displace ethidium bromide which has intercalated into the DNA provides an indication on the nature of drug-DNA interaction. CONCLUSIONS: TAS-103 cytotoxicity is not affected by the presence of Pgp, MRP, LRP or mutations in the CAM binding region of the topo-I enzyme and its growth-inhibitory effect appears to be weakly dependent on exposure duration. The presented evidence suggest that the inhibitory effects of TAS-103 on topo-I and -II may in part be related to its DNA binding rather than primarily through stabilization of topo-I or -II intermediates with DNA through specific binding to the enzymes.

Characterisation of a synergistic interaction between a thymidylate synthase inhibitor, ZD1694, and a novel lipophilic topoisomerase I inhibitor karenitecin, BNP1100: mechanisms and clinical implications.

We developed a combination protocol for inhibitors of thymidylate synthase (TS) and DNA topoisomerase I (Topo I) that can exert highly lethal effects in vitro against HCT-8 human colorectal cancer cells. The specific schedule was constructed so that a TS inhibitor could induce not only primary DNA damage but also cellular conditions optimal for the efficient action of a Topo I inhibitor. The initial drug treatment consisted of a brief exposure to a quinazoline-based antifolate, ZD1694. After an interval of approximately one cell-doubling time, cells were exposed for 8-24 h to BNP1100, a Karenitecin-class 7-thiomethyl-camptothecin, in the presence of 1-10 microM thymidine; the latter acted as a crucial factor to promote the collision of moving replication forks with the drug-stabilised DNA-Topo I cleavable complexes even under continuous TS inhibition. Clonogenic analyses confirmed that these mechanistically distinct drugs at clinically achievable concentrations worked in a highly synergistic manner, with a maximum effect abolishing the viability of virtually all cancer cells (> 99.9%). The pretreatment with ZD1694 increased the amount of DNA-bound Topo I by up to 4-fold and the DNA-damaging capability of BNP1100 by up to 15-fold. The possibility of at least four DNA-damaging pathways is proposed which might have resulted from the individual actions of TS and Topo I inhibitors as well as their concerted actions. Taken together, the present findings provided a logically permissible explanation as to why TS and Topo I inhibitors in concerted interactions induced a highly lethal effect which was more than a simple additive effect. Since these drugs are effective specifically on actively proliferating cancer cells, but not on non-cycling G0/G1 cells, this mechanism-based protocol may warrant consideration for clinical verification.

Cyclin E-cdk2 activation is associated with cell cycle arrest and inhibition of DNA replication induced by the thymidylate synthase inhibitor Tomudex.

Tomudex (ZD1694) is a specific antifolate-based thymidylate synthase inhibitor active in a variety of solid tumor malignancies. Studies were carried out in vitro to evaluate downstream molecular alterations induced as a consequence of the potent and sustained inhibition of thymidylate synthase by Tomudex. Twenty-four hours following the initial 2-h treatment with Tomudex, human A253 head and neck squamous carcinoma cells, not expressing p53 and p21(WAF1), were accumulated with DNA content characteristic of early S phase of the cell cycle with a concomitant reduction of cells in G1 and G2/M phases. The changes in cyclin and cdk protein expression and their kinase activities were examined in control and drug-treated A253 cells. Tomudex treatment resulted in the decrease in p27(kip1) expression, with an increase in cyclin E and cdk2 protein expression and kinase activities 24 h after a 2-h exposure. Although cyclin A protein expression was markedly increased, cyclin A kinase activity was only slightly increased. Cyclin D1, cyclin B, cdk4, and cdc2 protein expression and kinase activities remain constant. Lack of activation of cyclin A- and B-cdc2 was associated with a reduced proportion of cells in G2/M phases. Increased cyclin E-cdk2 protein expression was accompanied by the inhibition of DNA synthesis, with a decrease in E2F-1 expression. These results propose that cyclin E-cdk2 kinase can negatively regulate DNA replication. The studies with dThyd rescue from cyclin E-cdk2 protein overexpression and growth inhibition by Tomudex indicate that increased cyclin E-cdk2 protein expression is associated with effective inhibition of thymidylate synthase and resultant dNTP pool imbalance. Provision of dThyd more than 24 h after exposure to Tomudex allowed cells to replicate DNA for a single cycle back to G1, but did not prevent the profound growth-inhibitory effect manifested in the following 5 days. Tomudex treatment resulted in a time-dependent induction of the megabase DNA fragments, followed by secondary 50- to 300-kb DNA fragmentation. The 50- to 300-kb DNA fragmentation may be derived from the inhibition of DNA synthesis associated with cyclin E-cdk2 activation. These results suggest that the megabase DNA fragmentation is induced as a consequence of inhibition of thymidylate synthase by Tomudex and kilobase DNA fragmentation may correlate with the reduction of p27(kip1) expression and the increase in cyclin E and cdk2 kinase activities. Activation of cyclin E and cdk2 kinases allows cells to transit from G1 to S phase accompanied by the inhibition of DNA synthesis. The changes in cell cycle regulatory proteins associated with growth inhibition and DNA damage by Tomudex are not p53 dependent.

Novel cellular determinants for reversal of multidrug resistance in cells expressing P170-glycoprotein.

The newly synthesized calcium channel blocker, Ro44-5912, significantly potentiates doxorubicin (Dox)-induced cytotoxicity at non-cytotoxic concentrations in Dox-resistant human ovarian cell line, A2780/DX5, overexpressing P170-glycoprotein (Pgp). Induction of DNA single- and double-strand breaks (ssbs and dsbs) was measured using alkaline elution and constant-field gel electrophoresis (CFGE) assays. The results indicate that potentiation of the cytotoxicity of Dox by Ro44-5912 was accompanied by significant increases in both, Dox-induced DNA ssbs and dsbs in the resistant cells. Pulsed-field gel electrophoresis (PFGE) analysis showed that Dox induced DNA fragments in the 50-800 kilobase (kb) and 0.8-5.7 megabase (Mb) ranges. The majority of the newly synthesized DNA fragments were in the 50-800 kb range. Ro44-5912 treatment resulted in significant potentiation of DNA fragmentation in the 50-800 kb range with a minor increase in 0.8-5.7 Mb DNA fragments, suggesting that the modulator functions by potentiating nascent DNA fragmentation in the resistant cells. Exposure to Dox with Ro44-5912 was associated with a prolonged blockage of cells in the S-phase. In contrast, exposure to Dox alone resulted in temporary blockage of cells in G2/M phase (approximately 24 h) followed by restoration of cell proliferation and normal DNA histograms at 48 h after 2 h drug exposure. Incorporation of BrdUrd by flow cytometric analysis was inhibited by Dox in the presence of Ro44-5912, showing that there is a block of DNA replication. An increased damage in newly synthesized DNA could concur with a blocked DNA replication. Moreover, slowing progression through the S-phase in cells exposed to Dox in combination with Ro44-5912 is accompanied by increased sensitivity of Dox poisons, indicating a correlation of specific S-phase perturbation with the reversal of Dox resistance by Ro44-5912 in cells expressing Pgp. The results suggest that drug-induced augmentation of nascent DNA fragmentation and specific cell-cycle perturbation are potentially important molecular determinants for reversal of multidrug resistance in addition to restoration of intracellular drug retention.

DNA damage and p53 induction do not cause ZD1694-induced cell cycle arrest in human colon carcinoma cells.

Using four complementary approaches, ie., cell synchronization, bromodeoxyuridine labeling, and DNA and Western blot analyses, we investigated the underlying mechanism of cell cycle perturbation in response to ZD1694, a quinazoline-based antifolate thymidylate synthase inhibitor. With a single exposure at a concentration of 1 microM for 2 h, ZD1694 completely inhibits thymidylate synthase over 72 h and causes a sustained growth for at least 120 h, DNA damage, and p53 induction in human carcinoma cells. Although these cells displayed an S-phase block with the precise terminal arrest point depending on the timing of drug treatment in the cell cycle, their DNA-replicating machinery associated with polymerase alpha was preserved intact. When supplemented with exogenous dThd, these cells resumed an apparently normal S-phase progression for at least 4 h. Kinetic analyses based on synchronized cells indicate that S-phase arrest occurs first, preceding the induction of DNA double strand breaks and p53/p21. SW480 cells, in which p53mu failed to transduce p21, also exhibited the mode of S-phase arrest, essentially indistinguishable from that displayed by HCT-8 cells expressing the functional p53 (p53wt). That the DNA replication process is prerequisite for DNA double strand breaks was indicated by the following: (a) DNA damage occurred only when cells treated with ZD1694 progressed through S phase; and (b) the inhibition of DNA polymerase alpha by aphidicolin-blocked DNA damage. Based on the above, we conclude that S-phase arrest by ZD1694, with a subsequent damage of DNA double strands, is caused by the block of DNA synthesis in the middle of replication due to dTTP depletion and not by p53-mediated G1-G2 checkpoint mechanisms or p21-induced inactivation of the DNA-replicating machinery.

DiOC2(3) is not a substrate for multidrug resistance protein (MRP)-mediated drug efflux.

Multidrug resistance (MDR) is often related to expression of P-glycoprotein (Pgp) or Multidrug Resistance Protein (MRP). Pgp-mediated MDR can be evaluated by determining cellular retention of fluorescent substrates by flow cytometry. This study determined if agents used to evaluate Pgp function also can be used to evaluate MRP function. Cellular retention of doxorubicin (Dox), Rhodamine-123 (Rh-123), and 3,3'-diethyloxacarbocyanine iodide (DiOC2(3)) were studied in MRP-expressing cell lines (HL60/Adr and HT1080/DR4), whereas a Pgp expressing cell line (A2780/Dx5) served as a positive control. Overexpression of Pgp correlated inversely with retention of Dox, Rh-123, and DiOC2(3); however, under identical experimental conditions (1 h reincubation in drug-free medium), no retention difference of the three agents was detected between parental and MRP-expressing resistant cells. Upon extending the reincubation time to 4 h, an efflux of Rh-123 and Dox in the resistant lines became apparent and even more pronounced after 24h; however, still no efflux was detectable for DiOC2(3). Incubation of the cells with a modulator of MDR, PAK-104P, negated the observed drug efflux in Pgp and MRP expressing cells, which correlated with increased sensitivity of the MDR lines to doxorubicin. Thus both Dox and Rh-123 can be used to evaluate MRP-function, but DiOC2(3) can not.

Collateral sensitivity of human melanoma multidrug-resistant variants to the polyamine analogue, N1,N11-diethylnorspermine.

Certain N-alkylated analogues of the natural polyamine spermine, such as N1,N11-diethylnorspermine (DENSPM), rapidly deplete intracellular polyamine pools by down-regulating the biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, and by potently up-regulating the polyamine catabolizing enzyme, spermidine/spermine N1-acetyltransferase. On the basis of previously reported antitumor activity in human tumor xenograft model systems, DENSPM is currently undergoing Phase I clinical trials against human melanoma and other solid tumors. The antiproliferative activity of this analogue against the multidrug resistance (MDR) phenotype was examined in three MDR sublines of human melanoma RPMI-7932 cells, which were shown to be 2-to 10-fold resistant to classical MDR agents. These MDR lines had been separately derived using different selecting agents (Lemontt et al., Cancer Res., 48: 6344-6353, 1988). Subline functional resistance due to P-glycoprotein was confirmed by decreased retention of rhodamine 123 relative to parent cells as detected by flow cytometry. Although the three sublines were 2- to 10-fold less sensitive than the parent line to classical MDR-type agents, they were found in dose-response studies to be significantly more sensitive to DENSPM than the parent line. In addition, they showed a distinct cytotoxic response after a 48-h treatment with 10 microM DENSPM, which was not apparent in the parent line. Growth sensitivity of the sublines to the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine, or the S-adenosylmethionine decarboxylase inhibitor, CGP-48664, was found to be similar to parent cells. The ratio of the key biosynthetic enzyme activities for ornithine decarboxylase and S-adenosylmethionine decarboxylase was found to be 3.5- to 5-fold higher in all three sublines, due mainly to increases in the former enzyme. This imbalance produced unusually high putrescine pools. Although DENSPM down-regulation of decarboxylase activities and potent up-regulation of spermidine/spermine N1-acetyltransferase activity occurred similarly in both parent and variant lines, polyamine depletion was greater in the variant lines. Collateral sensitivity of the MDR sublines to DENSPM is partially attributable to the finding that analogue (and spermidine) uptake in the sublines was about 2-fold higher (after 2 h) than in the parent cells. The presence of disturbances in polyamine homeostasis and increased sensitivity to DENSPM in three independently selected cell line variants suggests that they may be generally associated with the MDR phenotype in human melanoma and possibly other tumor cells. The collateral sensitivity of human melanoma MDR variants to DENSPM represents a possible therapeutic indication which should be considered during the ongoing clinical evaluation of this drug.

Relationship between plasma Ara-C and intracellular Ara-CTP pools under conditions of continuous infusion and high-dose Ara-C treatment.

Plasma level Ara-C and Ara-U in vivo and intracellular Ara-CTP pools in vivo and in vitro were measured using high-performance liquid chromatography and radioimmunoassay. Plasma Ara-C during High Dose therapy was found in two phases; one, a peak at t1/2 of approximately 5-8 minutes, the other approaching that of Continuous Infusion with a t1/2 of about 6 to 8 hours. There appears to be no relationship between peak levels of plasma Ara-C and intracellular Ara-CTP formed during High Dose therapy. Intracellular Ara-CTP pools were found to be higher in peripheral blood than in bone marrow during High Dose treatment and were also higher in bone marrow of patients treated with High Dose rather than conventional dose Ara-C. In vitro experiments with various concentrations of Ara-C on patient cells prior to treatment suggest that patients may benefit from High Dose therapy when an increase in intracellular Ara-CTP occurred with higher extracellular concentrations of Ara-C. In patients with metabolically sensitive cells containing the necessary enzymes to activate Ara-C to Ara-CTP, where resistance is due to limited drug transport, High Dose therapy may prove beneficial. Conversely, in patients with no increase in Ara-CTP pools in vitro due to a depletion of activating enzymes, High Dose treatment may not be warranted. Therefore, it may be possible to determine patients most likely to benefit from High Dose chemotherapy by measuring pre-treatment in vitro intracellular Ara-CTP.