Pharmacogenomic analyses of targeting the AT-rich malaria parasite genome with AT-specific alkylating drugs.

UNLABELLED: Human malaria parasites, including the most lethal Plasmodium falciparum, are increasingly resistant to existing antimalarial drugs. One remarkable opportunity to selectively target P. falciparum stems from the unique AT-richness of its genome (80% A/T, relative to 60% in human DNA). To rationally explore this opportunity, we used drugs (adozelesin and bizelesin) which distinctly target AT-rich minisatellites and an in silico approach for genome-wide analysis previously experimentally validated in human cells [Woynarowski JM, Trevino AV, Rodriguez KA, Hardies SC, Benham CJ. AT-rich islands in genomic DNA as a novel target for AT-specific DNA-reactive antitumor drugs. J Biol Chem 2001;276:40555-66]. Both drugs demonstrate a potent, rapid and irreversible inhibition of the cultured P. falciparum (50% inhibition at 110 and 10+/-2.3 pM, respectively). This antiparasital activity reflects most likely drug binding to specific super-AT-rich regions. Relative to the human genome, the P. falciparum genome shows 3.9- and 7-fold higher frequency of binding sites for adozelesin and bizelesin, respectively. The distribution of these sites is non-random with the most prominent clusters found in large unique minisatellites [median size 3.5 kbp of nearly pure A/T, with multiple converging repeats but no shared consensus other than (A/T)(n)]. Each of the fourteen P. falciparum chromosomes contains only one such "super-AT island" located within approximately 3-7.5 kbp of gene-free and nucleosome-free loci. Important functions of super-AT islands are suggested by their exceptional predicted potential to serve as matrix attachment regions (MARs) and a precise co-localization with the putative centromeres. CONCLUSION: Super-AT islands, identified as unique domains in the P. falciparum genome with presumably crucial functions, offer therapeutically exploitable opportunity for new antimalarial strategies.

Caspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells.

Irofulven (hydroxymethylacylfulvene) is a novel antitumor drug, which acts by alkylating cellular macromolecular targets. The drug is a potent inducer of apoptosis in various types of tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to irofulven involving mitochondrial dysfunction and leading to death of prostate tumor LNCaP-Pro5 cells. Irofulven caused early (2-5 hours) translocation of the proapoptotic Bax from cytosol to mitochondria followed by the dissipation of mitochondrial membrane potential and cytochrome c release at 4 to 12 hours. These effects preceded caspase activation and during the first 6 hours were not affected by caspase inhibitors. Processing of caspase-9 initiated the caspase cascade at approximately 6 hours and progressed over time. The activation of the caspase cascade provided a positive feedback loop that enhanced Bcl-2-independent translocation and cytochrome c release. General and specific caspase inhibitors abrogated irofulven-induced apoptotic DNA fragmentation with the following order of potency: pan-caspase > or = caspase-9 > caspase-8/6 > caspase-2 > caspase-3/7 > caspase-1/4. Abrogation of caspase-mediated DNA fragmentation failed to salvage irofulven-treated cells from growth inhibition and loss of viability, demonstrating a substantial contribution of a caspase-independent cell death. Monobromobimane, an inhibitor of alternative caspase-independent apoptotic pathway that is mediated by mitochondrial permeability transition, antagonized both apoptosis, measured as phosphatidylserine externalization, and cytotoxicity of irofulven. Collectively, the results indicate that irofulven-induced signaling is integrated at the level of mitochondrial dysfunction. The induction of both caspase-dependent and caspase-independent death pathways is consistent with pleiotropic effects of irofulven, which include targeting of cellular DNA and proteins.

Enhanced topoisomerase II targeting by annamycin and related 4-demethoxy anthracycline analogues.

Targeting topoisomerase II (topo II) is regarded as an important component of the pleiotropic mechanism of action of anthracycline drugs. Here, we show that 4-demethoxy analogues of doxorubicin, including annamycin, exhibit a greater ability to trap topo II cleavage complexes than doxorubicin and some other 4-methoxy analogues. In leukemic CEM cells with wild-type topo II, annamycin induced substantial levels of topo II-mediated DNA-protein cross-links (15-37% of total DNA for 0.5-50 micromol/L drug), whereas doxorubicin-induced DNA-protein cross-links were marginal (0-4%). In CEM/VM-1 cells that harbor mutated, drug-resistant topo II, both 4-methoxy and 4-demethoxy drugs produced marginal DNA-protein cross-links. Annamycin, but not doxorubicin, formed topo II-mediated DNA-protein cross-links also in isolated CEM nuclei. In disparity with the unequal DNA-protein cross-link induction, both drugs induced comparable levels of DNA strand breaks in CEM cells. Compared with CEM, drug cytotoxicity against CEM/VM-1 cells was reduced 10.5- to 13.8-fold for 4-demethoxy analogues but only 3.8- to 5.5-fold for 4-methoxy drugs. Hence, growth inhibition by 4-demethoxy analogues seems more dependent on the presence of wild-type topo II. The enhanced topo II targeting by 4-demethoxy analogues was accompanied by a profound induction of apoptotic DNA fragmentation in leukemic CEM cells. Normal WI-38 fibroblasts, however, were markedly more resistant to annamycin-induced DNA-protein cross-links, apoptosis, and growth inhibition. The enhanced topo II targeting by 4-demethoxy doxorubicin analogues underscores the mechanistic diversity of anthracycline drugs. This diversity needs to be recognized as a factor in responses to drugs such as annamycin and doxorubicin.

Cell cycle effects and induction of premitotic apoptosis by irofulven in synchronized cancer cells.

Unlike postmitotic cell death, direct premitotic apoptosis diminishes the risk of clonal selection and allows for the elimination of slowly growing cancer cells. This study characterized the ability to induce premitotic apoptosis by irofulven (hydroxymethylacylfulvene), a novel alkylating drug which targets cellular DNA and proteins. Irofulven effects were examined in HeLa-derived BH2 cancer cells with conditional overexpression of antiapoptotic Bcl-2. Cells were synchronized in either early S or in G(1). Following 12 h exposure to irofulven, cells that were originally in early S accumulated in late S or remained in early S phase (at 0.5 and 2.5 muM drug, respectively). Drug treatment of cells in the G(1) cohort prevented their entry into the S phase. Significant apoptosis was detected based on the appearance of sub-G(1) particles and cells with DNA strand breaks in both G(1) and S cohorts. Apoptotic cells were mostly recruited from the G(1)/S border ("G(1)" cohort) and from the S phase ("early S" cohort). All the cell cycle and apoptotic effects were only marginally affected by Bcl-2 overexpression. Similar results were obtained with irofulven-treated synchronized cultures of leukemic CEM cells. Collectively, these observations indicate that irofulven-treated cells become committed to death early. Neither active DNA replication nor traverse through mitosis are necessary for irofulven-induced cell death. The ability to promote direct premitotic apoptosis is likely to play a role in the consistently potent apoptotic effects of irofulven and its ability to cause tumor regression in vivo.

AT islands - their nature and potential for anticancer strategies.

The human genome contains a unique class of domains, referred to as AT islands, which consist typically of 200-1000 bp long tracts of up to 100% A/T DNA. The significance of AT islands as potential targets for chemotherapeutic intervention stems from two main aspects. First, AT islands are inherently unstable (expandable) minisatellites that are found in various known loci of genomic instability, such as AT-rich fragile sites. Second, AT islands are involved in the organization of the genomic DNA on the nuclear matrix by acting as scaffold/matrix attachment regions, S/MARs. DNA duplexes of AT islands are unusually flexible and prone to base unpairing, which are crucial MAR attributes. Various AT islands show high binding affinity for isolated nuclear matrices and associate with the nuclear matrix in the cell. The cellular MAR function of AT islands may differ in cancer and normal cells. The abnormally expanded AT islands in the FRA16B fragile site in leukemic CEM cells act as strong, permanent MARs, while their unexpanded counterparts in normal cells are loop localized. Given their instability and involvement in the remodeling of the nuclear architecture, AT islands may be a factor in cancerous phenotypes. AT islands are preferentially targeted by the extremely potent DNA-alkylating antitumor drugs, bizelesin and U78779. High lethality of lesions in AT islands is consistent with the critical role of MAR domains in DNA replication. The abnormal structure/function of AT islands, such as their expansion and acquired strong MAR properties, may sensitize cancer cells to AT island targeting drugs.

Matrix attachment region (MAR) properties and abnormal expansion of AT island minisatellites in FRA16B fragile sites in leukemic CEM cells.

AT-rich minisatellites (AT islands) are sites of genomic instability in cancer cells and targets for extremely lethal AT-specific drugs, such as bizelesin. Here we investigated the AT islands in the FRA16B fragile site region for their possible roles in the organization of DNA on the nuclear matrix. The FRA16B AT island nominally spans approximately 3 kb of mostly >90% A/T DNA. In silico analysis indicates that this domain exhibits characteristics of nuclear matrix attachment regions (MARs): an exceptionally intense computed 'MAR potential' and profound duplex destabilization and flexibility. FRA16B repeats specifically bind to isolated nuclear matrices, which indicates their in vitro MAR function. This binding is several-fold greater than that of a known MAR in the c-myc gene. AT islands in fragile sites FRA16B and FRA16D are significantly more abundant in CEM cells that are hypersensitive to bizelesin compared to normal WI-38 cells. FRA16B overabundance in CEM is due to an approximately 10-fold expansion of FRA16B repeats. The expanded FRA16B minisatellites in CEM cells preferentially localize to the nuclear matrix-associated DNA indicating their in vivo MAR function. The unexpanded repeats in WI-38 cells localize to the loop DNA. The c-myc MAR is also matrix-associated in CEM cells while localizing to loop DNA in WI-38 cells. These results are the first to demonstrate that AT islands in fragile sites can function as MARs both in vitro and in vivo. The ability of FRA16B-mediated MAR sites to rearrange depending on the repeat expansion status could be relevant to both genomic instability of cancer cells and their sensitivity to AT-island targeting drugs.

DNA strand breaks and apoptosis induced by oxaliplatin in cancer cells.

Platinum anticancer drugs, such as cisplatin, are thought to exert their activity by DNA damage. Oxaliplatin, a clinically active diaminocyclohexane platinum compound, however, requires fewer DNA-Pt adducts than cisplatin to achieve cell growth inhibition. Here we investigated whether secondary DNA damage and apoptotic responses to oxaliplatin compensate for the reduced formation of DNA adducts. Oxaliplatin treatment of leukemic CEM and ovarian A2780 cancer cells resulted in early (4 hr) induction of DNA single-strand breaks measured by nucleoid sedimentation. These infrequent early lesions progress with time into massive double-stranded DNA fragmentation (fragments >50k bp) paralleled by characteristic apoptotic changes revealed by cell morphology and multivariate flow cytometry. Profound oxaliplatin-induced apoptotic DNA fragmentation was detectable following a 24 hr treatment of A2780 and CEM cells with 2 and 10 microM oxaliplatin, respectively. This DNA fragmentation was inhibited completely by the broad-spectrum caspase inhibitor Z-VAD-fmk. Cisplatin, which forms markedly more DNA-Pt adducts in CEM and A2780 cells than equimolar oxaliplatin, was similarly potent as oxaliplatin in terms of early strand breaks and later apoptotic responses. Oxaliplatin was also profoundly apoptotic in several other tumor cell lines of prostate origin but had only a marginal effect in normal prostate PrEC cells. Collectively, the results demonstrate that, relative to the magnitude of the primary DNA-Pt lesions, oxaliplatin is disproportionately more potent than cisplatin in the induction of apoptosis. Apoptosis induction, possibly enhanced by a contribution of targets other than DNA, seems to be an important factor in the mechanism of action of oxaliplatin.

Apoptosis induction by the dual-action DNA- and protein-reactive antitumor drug irofulven is largely Bcl-2-independent.

The overexpression of Bcl-2 is implicated in the resistance of cancer cells to apoptosis. This study explored the potential of irofulven (hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863), a novel DNA- and protein-reactive anticancer drug, to overcome the anti-apoptotic properties of Bcl-2 in HeLa cells with controlled Bcl-2 overexpression. Irofulven treatment resulted in rapid (12hr) dissipation of the mitochondrial membrane potential, phosphatidylserine externalization, and apoptotic DNA fragmentation, with progressive changes after 24hr. Bcl-2 overexpression caused marginal or partial inhibition of these effects after treatment times ranging from 12 to 48hr. Both Bcl-2-dependent and -independent responses to irofulven were abrogated by a broad-spectrum caspase inhibitor. Despite the somewhat decreased apoptotic indices, cell growth inhibition by irofulven was unaffected by Bcl-2 status. In comparison, Bcl-2 overexpression drastically reduced apoptotic DNA fragmentation by etoposide, acting via topoisomerase II-mediated DNA damage, but had no effect on apoptotic DNA fragmentation by helenalin A, which reacts with proteins but not DNA. Irofulven retains its pro-apoptotic and growth inhibitory potential in cell lines that have naturally high Bcl-2 expression. Collectively, the results implicate multiple mechanisms of apoptosis induction by irofulven, which may differ in time course and Bcl-2 dependence. It is possible that the sustained ability of irofulven to induce profound apoptosis and to block cell growth despite Bcl-2 overexpression may be related to its dual reactivity with both DNA and proteins.

Targeting critical regions in genomic DNA with AT-specific anticancer drugs.

Cellular DNA is not a uniform target for DNA-reactive drugs. At the nucleotide level, drugs recognize and bind short motifs of a few base pairs. The location of drug adducts at the genomic level depends on how these short motifs are distributed in larger domains. This aspect, referred to as region specificity, may be critical for the biological outcome of drug action. Recent studies demonstrated that certain minor groove binding (MGB) drugs, such as bizelesin, produce region-specific lesions in cellular DNA. Bizelesin binds mainly T(A/T)(4)A sites, which are on average scarce, but occasionally cluster in distinct minisatellite regions (200-1000 bp of approximately 85-100% AT), herein referred to as AT islands. Bizelesin-targeted AT islands are likely to function as strong matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. Distortion of MAR-like AT islands may be a basis for the observed inhibition of new replicon initiation and the extreme lethality of bizelesin adducts (<10 adducts/cell for cell growth inhibition). Hence, long AT-islands represent a novel class of critical targets for anticancer drugs. The AT island paradigm illustrates the potential of the concept of regional targeting as an essential component of the rational design of new sequence-specific DNA-reactive drugs.

Preferential targeting of apoptosis in tumor versus normal cells.

Elimination of cancer cells by early apoptosis is preferred over other forms of cell growth inhibition. Apoptosis directly leads to tumor regression and reduces risks of selecting more aggressive and/or drug-resistant phenotypes that are often responsible for tumor regrowth and treatment failure. Although DNA damage by anticancer drugs is commonly recognized as an apoptotic stimulus, there is enormous variability in the magnitude and timing of such effects. Especially potent and rapid apoptosis seems to be a hallmark of various alkylating anticancer drugs that are regarded as DNA-reactive agents but are observed to react mainly with cellular proteins. Our studies with such dual-action drugs (irofulven, oxaliplatin) suggest that not only DNA damage, but also protein damage, contributes to apoptosis induction. DNA damage is well known to initiate death-signaling pathways leading to mitochondrial dysfunction. Protein damage, in turn, can distort cell redox homeostasis, which facilitates apoptosis execution. Such dual effects can be particularly lethal to tumor cells, which tend to function under pro-oxidative conditions. In contrast to tumor cells that are highly susceptible, normal cells show marginal apoptotic responses to the dual action drugs. This protection of normal cells might reflect their greater ability to buffer pro-oxidative changes and quickly restore redox homeostasis, despite substantial drug uptake and macromolecular binding. Importantly, by targeting the death process at multiple points, DNA- and protein-damaging drugs can be less vulnerable to various bypass mechanisms possible with single targets. The reviewed studies provide a proof of concept that differential apoptosis targeting in cancer versus normal cells can be a basis for tumor selectivity of anticancer drugs.

Irofulven induces apoptosis in breast cancer cells regardless of caspase-3 status.

Caspase-3 deficiency can limit the efficiency of pro-apoptotic anticancer treatments. Irofulven (hydroxymethylacyl-fulvene, HMAF. MGI 114, NSC 683863) is an antitumor drug, currently in a Phase III and multiple Phase II trials, which can differentiate between tumor and normal cells in apoptosis induction. This study investigated whether apoptosis induced by irofulven requires caspase-3. Irofulven action was compared in breast cancer cells differing in caspase-3 status: deficient MCF-7 cells and proficient MDA-MB-231 cells and in normal human mammary epithelial cells, HMEC. Irofulven induces significant, concentration and time-dependent apoptotic DNA fragmentation in breast cancer cell lines, regardless of caspase-3 status. After 12, 24 and 48 h incubation at 1 microM irofulven (approximately 3 x GI50), fragmented DNA comprised 3.7, 14.1 and 34.6% and 8.4, 12.6 and 20.3% of total DNA in MCF-7 and MDA-MB-231 cells, respectively. Cell viability (trypan blue exclusion) remained largely unaffected during the first 24 h but decreased markedly after 48 h, indicating secondary necrosis. Net losses in cell numbers were apparent at 48 h. Normal HMEC cells were refractory to 1 microM drug with only approximately 3-9% fragmented DNA after 12-48 h, although apoptosis was observed at drug levels >3 microM. The broad-spectrum caspase inhibitor Z-VAD-fmk inhibited irofulven-induced apoptosis of all cell lines at 20 microM with nearly complete abrogation of apoptosis at 100 microM. Irofulven treatment resulted in marginal caspase-3 processing in MDA-MB-231 and HMEC cells. These results indicate that whereas the caspase cascade mediates irofulven- induced apoptosis, caspase-3 is dispensable (supported by NIH CA70091 and CA78706).

The genome factor in region-specific DNA damage: the DNA-reactive drug U-78779 prefers mixed A/T-G/C sequences at the nucleotide level but is region-specific for long pure AT islands at the genomic level.

Bizelesin is the first anticancer drug capable of damaging specific regions of the genome with clusters of its binding sites T(A/T)(4)A. This study characterized the sequence- and region-specificity of a bizelesin analogue, U-78779, designed to interact with mixed A/T-G/C motifs. At the nucleotide level, U-78779 was found to prefer runs of A/Ts interspersed with 1 or 2 G/C pairs, although 25% of the identified sites corresponded to pure AT motifs similar to bizelesin sites. The in silico computational analysis showed that the preferred mixed A/T-G/C motifs distribute uniformly at the genomic level. In contrast, the secondary, pure AT motifs (A/T)(6)A were found densely clustered in the same long islands of AT-rich DNA that bizelesin targets. Mapping the sites and quantitating the frequencies of U-78779 adducts in model AT island and non-AT island naked DNAs demonstrated that clusters of pure AT motifs outcompete isolated mixed A/T-G/C sites in attracting drug binding. Regional preference of U-78779 for AT island domains was verified also in DNA from drug-treated cells. Thus, while the primary sequence preference gives rise to non-region-specific scattered lesions, the clustering of the minor pure AT binding motifs seems to determine region-specificity of U-78779 in the human genome. The closely correlated cytotoxic activities of U-78779 and bizelesin in several cell lines further imply that both drugs may share common cellular targets. This study underscores the significance of the genome factor in a drug's potential for region-specific DNA damage, by showing that it can take precedence over drug binding preferences at the nucleotide level.