Preferential potentiation of topoisomerase I poison cytotoxicity by PARP inhibition in S phase.

BACKGROUND: Topoisomerase I (Topo I) poisons (e.g., camptothecin (CPT)), used to treat cancer, cause DNA breaks that are most cytotoxic during S phase. PARP-1 promotes DNA repair and PARP inhibitors (PARPi) sensitise cells to Topo I poisons. We aimed to determine whether chemosensitisation is also S phase specific using rucaparib, a potent PARPi in advanced clinical evaluation. METHODS: The impact of rucaparib, on CPT-induced cytotoxicity was measured in human colon cancer (LoVo) and leukaemic (K562) cells in asynchronous and cell cycle phase-separated cultures. Topoisomerase I and PARP levels and activity and the effect of rucaparib on DNA single-strand breaks (SSBs), double-strand breaks (DSBs) and collapsed replication fork induction and repair were determined in cell cycle phase-separated cells. RESULTS: The cytotoxicity of CPT was greatest during S phase, partially attributable to high Topo I activity, and rucaparib preferentially sensitised S-phase cells. Rucaparib increased CPT-induced DNA SSBs in all phases of the cell cycle, and increased DSB and γH2AX foci in S and G2, with γH2AX foci being highest in S-phase cells. Repair of SSBs and DSBs was most rapid during S then G2 phases and was substantially hindered by rucaparib. CONCLUSIONS: Rucaparib preferentially sensitises S-phase cells by increasing the frequency of collapsed replication forks.

NF-κB mediates radio-sensitization by the PARP-1 inhibitor, AG-014699.

The stress-inducible transcription factor, nuclear factor (NF)-κB induces genes involved in proliferation and apoptosis. Aberrant NF-κB activity is common in cancer and contributes to therapeutic-resistance. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated during DNA strand break repair and is a known transcriptional co-regulator. Here, we investigated the role of PARP-1 function during NF-κB activation using p65 small interfering RNA (siRNA), PARP siRNA or the potent PARP-1 inhibitor, AG-014699. Survival and apoptosis assays showed that NF-κB p65(-/-) cells were more sensitive to ionizing radiation (IR) than p65(+/+) cells. Co-incubation with p65 siRNA, PARP siRNA or AG-014699 radio-sensitized p65(+/+), but not p65(-/-) cells, demonstrating that PARP-1 mediates its effects on survival via NF-κB. Single-strand break (SSB) repair kinetics, and the effect SSB repair inhibition by AG-014699 were similar in p65(+/+) and p65(-/-) cells. As preventing SSB repair did not radio-sensitize p65(-/-) cells, we conclude that radio-sensitization by AG-014699 is due to downstream inhibition of NF-κB activation, and independent of SSB repair inhibition. PARP-1 catalytic activity was essential for IR-induced p65 DNA binding and NF-κB-dependent gene transcription, whereas for tumor necrosis factor (TNF)-α-treated cells, PARP-1 protein alone was sufficient. We hypothesize that this stimulus-dependent differential is mediated via stimulation of the poly(ADP-ribose) polymer, which was induced following IR, not TNF-α. Targeting DNA damage-activated NF-κB using AG-014699 may therefore overcome toxicity observed with classical NF-κB inhibitors without compromising other vital inflammatory functions. These data highlight the potential of PARP-1 inhibitors to overcome NF-κB-mediated therapeutic resistance and widens the spectrum of cancers in which these agents may be utilized.

Murine transgenic cells lacking DNA topoisomerase IIbeta are resistant to acridines and mitoxantrone: analysis of cytotoxicity and cleavable complex formation.

Murine transgenic cell lines lacking DNA topoisomerase II (topo II)beta have been used to assess the importance of topo IIbeta as a drug target. Western blot analysis confirmed that the topo IIbeta -/- cell lines did not contain topo IIbeta protein. In addition, both the topo IIbeta +/+ and topo IIbeta -/- cell lines contained similar levels of topo IIalpha protein. The trapped in agarose DNA immunostaining assay (TARDIS) was used to detect topo IIalpha and beta cleavable complexes in topo IIbeta -/- and topo IIbeta +/+ cells. These results show that both topo IIalpha and beta are in vivo targets for etoposide, mitoxantrone, and amsacrine (mAMSA) in topo IIbeta +/+ cells. As expected, only the alpha-isoform was targeted in topo IIbeta -/- cells. Clonogenic assays comparing the survival of topo IIbeta -/- and topo IIbeta +/+ cells were carried out to establish whether the absence of topo IIbeta caused drug resistance. Increased survival of topo IIbeta -/- cells compared with topo IIbeta +/+ cells was observed after treatment with amsacrine (mAMSA), methyl N-(4'-[9-acridinylamino]-2-methoxyphenyl) carbamate hydrochloride (AMCA), methyl N-(4'-[9-acridinylamino]-2-methoxyphenyl)carbamate hydrochloride (mAMCA), mitoxantrone, and etoposide. These studies showed that topo IIbeta -/- cells were significantly more resistant to mAMSA, AMCA, mAMCA, and mitoxantrone, than topo IIbeta +/+ cells, indicating that topo IIbeta is an important target for the cytotoxic effects of these compounds.

Nuclear distribution of human DNA topoisomerase IIbeta: a nuclear targeting signal resides in the 116-residue C-terminal tail.

We have analyzed the subcellular distribution of the beta isoform of human topoisomerase II using both isoform-specific antisera and an epitope-tagging approach. Previous immunocytochemical studies have yielded differing results with one reporting this isoform to be predominantly nucleolar. Later studies seem to refute this finding, as do our results with isoform-specific antisera reported here. Epitope tagging minimizes potential complications arising from the use of anti-topoisomerase II antisera that may recognize epitopes that are modified or masked in vivo and could lead to misleading results in immunocytochemical studies. A second strength of this approach is that it allowed a comparison with similarly tagged control proteins (derived from the nucleolar transcription factor UBF) that were known to localize unambiguously to the cytoplasmic, nucleoplasmic, or nucleolar compartments. We report that the C-terminal domain of topoisomerase IIbeta fused to a beta-galactosidase tag localizes to the nucleus (but not the nucleolar compartment) and that this is indistinguishable from the localization of native topoisomerase IIbeta detected by isoform-specific antisera. Further analysis revealed that the nuclear localization determinant lies within the 116-residue C-terminal tail of human topoisomerase IIbeta.

Etoposide targets topoisomerase IIalpha and IIbeta in leukemic cells: isoform-specific cleavable complexes visualized and quantified in situ by a novel immunofluorescence technique.

We have shown that both DNA topoisomerase (topo) IIalpha and beta are in vivo targets for etoposide using a new assay which directly measures topo IIalpha and beta cleavable complexes in individual cells after treatment with topo II targeting drugs. CCRF-CEM human leukemic cells were exposed to etoposide for 2 hr, then embedded in agarose on microscope slides before cell lysis. DNA from each cell remained trapped in the agarose and covalently bound topo II molecules from drug-stabilized cleavable complexes remained associated with the DNA. The covalently bound topo II was detected in situ by immunofluorescence. Isoform-specific covalent complexes were detected with antisera specific for either the alpha or beta isoform of topo II followed by a fluorescein isothiocyanate-conjugated second antibody. DNA was detected using the fluorescent stain Hoechst 33258. A cooled slow scan charged coupled device camera was used to capture images. A dose-dependent increase in green immunofluorescence was observed when using antisera to either the alpha or beta isoforms of topo II, indicating that both isoforms are targets for etoposide. We have called this the TARDIS method, for trapped in agarose DNA immunostaining. Two key advantages of the TARDIS method are that it is isoform-specific and that it requires small numbers of cells, making it suitable for analysis of samples from patients being treated with topo II-targeting drugs. The isoform specificity will enable us to extend our understanding of the mechanism of interaction between topo II-targeting agents and their target, the two human isoforms.

Drug sensitivity and sequence specificity of human recombinant DNA topoisomerases IIalpha (p170) and IIbeta (p180).

Effective anticancer agents, such as epipodophyllotoxins and anthracyclines, exert their antitumor activity through stabilization of cleavable topoisomerase II/DNA complexes, which may result in DNA breakage on detergent addition. Two isozymes (alpha and beta) of DNA topoisomerase II are present in human cells; however, their roles as drug targets have not been completely defined. We determined the in vitro isoenzyme sensitivities to VM-26 (teniposide) and 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (an anthracycline analog) and established the sequence selectivity of isoenzyme-mediated DNA cleavage. Human topoisomerases IIalpha and IIbeta were purified from yeast cells overexpressing the corresponding plasmid-borne cDNA. Enzyme sensitivities to drugs were measured by a DNA cleavage assay using 32P-labeled simian virus 40 DNA fragments, and cleavage sites were mapped using agarose and sequencing gels. Both isozymes were sensitive to the studied poisons. They stimulated similar cleavage intensity patterns in agarose and sequencing gels; however, minor differences could be detected. The results showed that local base preferences for DNA cleavage without drugs were different at positions -2 and -1. On the other hand, sequence specificities of VM-26 and 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin were identical for both isozymes and corresponded to those of the native murine enzyme. The identical drug sequence specificities suggested that molecular interactions of the tested drugs in the ternary complex are likely similar between the two isozymes. The current findings indicate that both topoisomerase IIalpha and IIbeta may be in vivo targets of antitumor poisons.

Amsacrine-promoted DNA cleavage site determinants for the two human DNA topoisomerase II isoforms alpha and beta.

Site-specific DNA cleavage by topoisomerase II (EC 5.99.1.3) is induced by many antitumour drugs. Although human cells express two genetically distinct topoisomerase II isoforms, thus far the role and determinants of drug-induced DNA cleavage have been examined only for alpha. Here we report the first high-resolution study of amsacrine (mAMSA) induced DNA breakage by human topoisomerase II beta (overexpressed and purified from yeast) and a direct comparison with the recombinant alpha isoform. DNA cleavage in plasmid pBR322 and SV40 DNA was induced by alpha or beta in the absence or presence of the antitumour agent mAMSA, and sites were mapped using sequencing gel methodology. Low-resolution studies indicated that recombinant human alpha promoted DNA breakage at sites akin to those of beta, although some sites were only cleaved by one enzyme and different intensities were observed at some sites. However, statistical analysis of 70 drug-induced sites for beta and 70 sites for alpha revealed that both isoforms share the same base preferences at 13 positions relative to the enzyme cleavage site, including a very strong preference for A at +1. The result for recombinant alpha isoform is in agreement with previous studies using alpha purified from human cell lines. Thus, alpha and beta proteins apparently form similar ternary complexes with mAMSA and DNA. Previous studies have emphasized the importance of DNA topoisomerase II alpha; the results presented here demonstrate that beta is an in vitro target with similar site determinants, strongly suggesting that beta should also be considered a target of mAMSA in vivo.

Position-specific effects of base mismatch on mammalian topoisomerase II DNA cleaving activity.

To further define the nucleic acid determinants of DNA site recognition by mammalian topoisomerase II, base mismatch effects on the enzyme DNA cleavage activity were determined in a 36-bp synthetic oligonucleotide corresponding to SV40 DNA. DNA cleavage sites induced by topoisomerase II without or with the antitumor drugs teniposide, idarubicin, or amsacrine were mapped using sequencing gels. Selected mismatches were studied, and always one of the two strands had the wild-type sequence. The effects of base mismatches were independent from the studied drugs. Mismatches introduced at the -4, -3, -2, or -1 positions, relative to the enzyme cleavage site, often abolished, or much reduced, DNA cleavage, whereas those at +1 and +2 positions often increased DNA breakage or were without influence. Mismatches at more distant positions, e.g., -7, -8, etc., had no effect. Those at positions -5 and -6 sometimes increased cleavage levels. These effects were always observed at sites already cleaved in the wild-type oligomer; new sites of cleavage were not induced by the studied mismatches. These results were obtained both for the native murine topoisomerase II and for the two recombinant human isozymes. No difference between topoisomerases II alpha(p170) and beta(p180) was seen in their response to mismatches. The results demonstrate that topoisomerase II recognition of the DNA site of cleavage requires fully paired nucleotides at the 3' terminus. Nevertheless, similarly to other DNA strand transferase enzymes, both topoisomerase II isoforms may have a sequence-specific nicking activity at the 5' side of unpaired bases.

Expression, domain structure, and enzymatic properties of an active recombinant human DNA topoisomerase II beta.

Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target. Here we report for the first time the successful overexpression of human topoisomerase II beta in yeast by cloning a topoisomerase II beta cDNA in a yeast shuttle vector under the control of a galactose-inducible promoter. Recombinant human topoisomerase II beta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase II alpha. Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains. Recombinant human topoisomerase II alpha and beta induced DNA breakage which was promoted by a variety of agents. Isoform differences in drug-induced DNA breakage were observed. These studies of human topoisomerase II beta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.

The effects of 5-fluoropyrimidines on nascent DNA synthesis in Chinese hamster ovary cells monitored by pH-step alkaline and neutral elution.

Nascent DNA (nDNA) replication intermediates can be isolated and quantified by pH-step alkaline elution (L.C. Erikson et al., Chromosoma, 74, 125-139, 1979). The effects of 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine (FdU) on the formation and persistence of nascent DNA structures were studied in Chinese hamster ovary K1 cells. Exogenous thymidine (dT) rescued FdU-induced, but had little effect on FU-induced, cytotoxicity, indicating DNA- and RNA-directed cytotoxic mechanisms respectively. Drug-treated cells were pulse labelled with [3H]dT and the percentage of total counts eluting from and retained by filters estimated at successive pH increments (11.0, 11.3, 11.5 and 12.1). A 1 min pulse of control cells resulted in > 75% of the DNA eluting at pH 12.1 or being retained by the filter. By 1 h, > 90% of the DNA was retained by the filter. FdU treatment resulted in the persistence of DNA in all four pH bands. More than 95% of total DNA synthesized during a 5 min pulse eluted as discrete peaks at each pH. This DNA was processed to higher M(r) species during a 1 h chase, but following a 24 h chase, 68% of the DNA still eluted at pH 12.1. In contrast, FU treatment caused only a transient accumulation (< or = 10 min) of the DNA (e.g. approximately 26% at 5 min) in the pH 11.0 and 11.3 bands only, indicating a selective effect of FU on the maturation of very short size classes of DNA. Neutral elution was used to assess the effect of FdU on double-strand break (dsb) formation in nDNA. Whereas no dsbs were evident in untreated cells, dsbs appeared in FdU-treated cells even following the shortest [3H]dT pulse times (1-10 min). Although these were processed into higher M(r) species with longer pulse times, dsbs were still evident at 2 h.

Cytotoxic mechanisms of 5-fluoropyrimidines. Relationships with poly(ADP-ribose) polymerase activity, DNA strand breakage and incorporation into nucleic acids.

A comparative study of the cytotoxic mechanisms of 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine (FdUrd) was carried out in Chinese hamster ovary K1 (CHO-K1) cells. The poly(ADP-ribose) polymerase (PADPRP) inhibitor, 3-aminobenzamide (3AB, 3 mM) enhanced the cytotoxicity of FU with a dose enhancement factor at 10% survival of 2. This enhancement was also evident when cells were grown in dThd-free medium, but the IC50 for FU was reduced from 50 to 35 microM. In contrast, 3AB did not enhance the cytotoxicity of FdUrd but exerted a small protective effect. The IC50 for FdUrd was reduced from 35 to 1.25 microM in dThd-free medium. A 55% reduction in NAD levels was seen within 6 hr of 5.0 microM FdUrd treatment in dThd-free medium, and this reduction persisted over 24 hr. This drop was prevented by co-incubation with 3AB, indicating that PADPRP activation was the cause of the NAD depletion. In contrast, FU treatment had little or no effect on NAD levels. Alkaline elution analysis of cells treated with up to 150 microM FU revealed no DNA strand breaks in mature DNA, but an increase in breaks in nascent DNA. Co-incubation with 3AB had little or no effect on strand break levels. FdUrd (up to 40 microM) produced a dose-dependent increase in both mature and nascent DNA strand breaks. Analysis using a "relative elution" formula demonstrated that 3AB increased the amount of FdUrd-induced strand breaks (at doses < or = 5-100 microM) in mature DNA. Whereas FU elution profiles for nascent DNA were biphasic, those for FdUrd were linear. Co-incubation with 3AB increased [3H]FU incorporation into both RNA (by 50%) and DNA (45%). 3AB also enhanced [3H]FdUrd incorporation (by 40%) into RNA but had no effect on incorporation into DNA. These data indicate that in addition to acting as an inhibitor of PADPRP, 3AB exerts other metabolic effects.

Correlation of enhanced 6-mercaptopurine cytotoxicity with increased phosphoribosylpyrophosphate levels in Chinese hamster ovary cells treated with 3-aminobenzamide.

3-Aminobenzamide (3AB) has been used widely to inhibit the nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) and study the involvement of poly(ADP-ribose) synthesis in DNA repair and other cellular functions. 3AB (3 mM) potentiates the cytotoxicity of 6-mercaptopurine (MP) and azathioprine in CHO-K1 cells with dose enhancement factors at 10% survival of 30-fold. In synchronized cells, 3AB is required during G1 and early S phase to obtain potentiation of MP cytotoxicity. There is a small but significant depletion of cellular NAD in MP-treated cells. As demonstrated by flow cytometric analysis, 20-40 microM MP causes an accumulation of cells in early S phase of the cell cycle. 3AB (3 mM) has no effect on cell cycle distribution; however, in the presence of MP, a similar accumulation is seen by 2-5 microM MP. 3AB and MP per se have no effect on phosphoribosylpyrophosphate levels, but coincubation causes a 30-fold increase in phosphoribosylpyrophosphate levels, reaching a maximum by 1.5 microM MP and declining to basal levels by 10 microM MP. There was a good correlation between the 3AB dose-dependent increase in cell killing and rise in phosphoribosylpyrophosphate levels.