The radiomimetic enediyne, 20'-deschloro-C-1027 induces inter-strand DNA crosslinks in hypoxic cells and overcomes cytotoxic radioresistance.

The ability of the radiomimetic anti-tumor enediyne C-1027 to induce DNA inter-strand crosslinks (ICLs), in addition to the expected DNA strand breaks, is unique among traditional DNA targeted cancer therapies. Importantly, radiation therapy and most radiomimetic drugs have diminished effect in hypoxic environments due to decreased induction of DNA strand breaks, which is an oxygen requiring process. However, C-1027's induction of ICLs is enhanced under hypoxia and it is actually more potent against hypoxic cells, overcoming this common tumor resistance mechanism.

DNA damage by C1027 involves hydrogen atom abstraction and addition to nucleobases.

C1027 is a potent antitumor agent that damages DNA. It has the unusual ability to produce double strand breaks and interstrand cross-links (ICLs) intracellularly, which enable it to initiate concurrent ataxia-telangiestasia mutated (ATM) and Rad-3 related (ATR) independent damage responses. The latter form of damage is not well characterized.

An extraction-free method by which a single slot blot can be used to quantify intracellular DNA damage (crosslinks or strand breaks) and changes in DNA damage response proteins or replication.

We report an extraction-free assay in which the same slot blot membrane can be used to assess total genomic DNA damage (i.e., crosslinks or strand breaks) and DNA replication (i.

C-1027, a radiomimetic enediyne anticancer drug, preferentially targets hypoxic cells.

The hypoxic nature of cells within solid tumors limits the efficacy of anticancer therapies such as ionizing radiation and conventional radiomimetics because their mechanisms require oxygen to induce lethal DNA breaks. For example, the conventional radiomimetic enediyne neocarzinostatin is 4-fold less cytotoxic to cells maintained in low oxygen (hypoxic) compared with normoxic conditions. By contrast, the enediyne C-1027 was nearly 3-fold more cytotoxic to hypoxic than to normoxic cells.

Designer enediynes generate DNA breaks, interstrand cross-links, or both, with concomitant changes in the regulation of DNA damage responses.

The ability of the radiomimetic anticancer enediyne C-1027 to induce ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR)-independent damage responses was discovered to reside in its unique ability to concurrently generate robust amounts of double-strand breaks (DSBs) and interstrand cross-links (ICLs) in cellular DNA. Furthermore, a single substitution to the chromophore's benzoxazolinate moiety shifted DNA damage to primarily ICLs and an ATR- but not ATM-dependent damage response. In contrast, single substitutions of the chromophore's beta-amino acid component shifted DNA damage to primarily DSBs, consistent with its induction of conventional ATM-dependent damage responses of the type generated by ionizing radiation and other radiomimetics.

Single chemical modifications of the C-1027 enediyne core, a radiomimetic antitumor drug, affect both drug potency and the role of ataxia-telangiectasia mutated in cellular responses to DNA double-strand breaks.

The radiomimetic enediyne C-1027 induces almost exclusively DNA double-strand breaks (DSB) and is extremely cytotoxic. Unique among radiomimetics, ataxia-telangiectasia mutated (ATM) is dispensable for cellular responses to C-1027-induced DNA damage. This study explores the biological activity of three recently bioengineered C-1027 analogues: 7''-desmethyl-C-1027 (desmethyl), 20'-deschloro-C-1027 (deschloro), and 22'-deshydroxy-C-1027 (deshydroxy).

Inhibition of RecBCD enzyme by antineoplastic DNA alkylating agents.

To understand how bulky adducts might perturb DNA helicase function, three distinct DNA-binding agents were used to determine the effects of DNA alkylation on a DNA helicase. Adozelesin, ecteinascidin 743 (Et743) and hedamycin each possess unique structures and sequence selectivity. They bind to double-stranded DNA and alkylate one strand of the duplex in cis, adding adducts that alter the structure of DNA significantly.

Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate.

The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate.

The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks.

Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent.

Hedamycin, a DNA alkylator, induces (gamma)H2AX and chromosome aberrations: involvement of phosphatidylinositol 3-kinase-related kinases and DNA replication fork movement.

Genotoxic treatments, such as UV light, camptothecin, and adozelesin, stall DNA replication and subsequently generate DNA strand breaks. Typically, DNA breaks are reflected by an increase in ataxia and Rad-related kinase (ATR)-regulated phosphorylation of H2AX (gammaH2AX) and require replication fork movement. This study examined the potential of the monofunctional DNA alkylating agent hedamycin, a powerful inhibitor of DNA replication, to induce DNA strand breaks, phosphorylated H2AX (gammaH2AX) foci, and chromosome aberrations.

The antitumor enediyne C-1027 alters cell cycle progression and induces chromosomal aberrations and telomere dysfunction.

This study examined the extent of chromosome instability induced in cultured human colon carcinoma HCT116 cells by the antitumor radiomimetic enediyne antibiotic C-1027. Spectral karyotype analysis showed frequent intrachromosomal fusions and fragmentations 26 hours after addition of as little as 0.035 nmol/L C-1027.

Molecular mechanism of inhibition of survivin transcription by the GC-rich sequence-selective DNA binding antitumor agent, hedamycin: evidence of survivin down-regulation associated with drug sensitivity.

Expression of the antiapoptotic protein survivin is associated with cancer cell viability and drug resistance. Thus, control of its expression in cancer cells has significant consequences for cancer therapeutics. Here we have shown that hedamycin, a GC-rich DNA binding drug, down-regulated survivin expression.

DNA damage effects of a polyamide-CBI conjugate in SV40 virions.

Polyamides are a class of synthetic molecules that exhibit high-affinity, sequence-specific reversible binding in the DNA minor groove but are incapable of inducing DNA damage. In cell-free systems, polyamides have been shown to regulate gene expression by activation, repression, and antirepression. However, effectiveness in cell culture has met with limited success and seems to be cell-dependent.

SV40 DNA replication inhibition by the monofunctional DNA alkylator Et743.

Ecteinascidin 743 (Et743) is a highly cytotoxic anticancer agent isolated from the squirt Ecteinascidia turbinate, which alkylates DNA in the minor groove at GC-rich sequences resulting in an unusual bending toward the major groove. The ability of Et743 to block DNA replication was studied using the well-established simian virus (SV40) model for mammalian DNA replication in cells and cell-free extracts. Intracellular SV40 DNA isolated from Et743-treated BSC-1 cells was analyzed by native, two-dimensional agarose gel electrophoresis.

DNA damage responses triggered by a highly cytotoxic monofunctional DNA alkylator, hedamycin, a pluramycin antitumor antibiotic.

Long-term exposure (72 h) to hedamycin, a monofunctional DNA alkylator of the pluramycin class of antitumor antibiotics, decreased growth of mammalian cells by 50% at subnanomolar concentrations. Short-term treatment (4 h) rapidly reduced DNA synthesis by 50% also at subnanomolar concentrations, but substantially higher levels were needed to block RNA synthesis while protein synthesis even at very high hedamycin concentrations remained unaffected. Hedamycin treatment at concentrations below its growth IC(50) induced only a transient and temporary accumulation of cells in G(2).

The DNA minor groove-alkylating cyclopropylpyrroloindole drugs adozelesin and bizelesin induce different DNA damage response pathways in human colon carcinoma HCT116 cells.

As members of the cyclopropylpyrroloindole family, adozelesin and bizelesin cause genomic DNA lesions by alkylating DNA. Adozelesin induces single-strand DNA lesions, whereas bizelesin induces both single-strand lesions and double-strand DNA cross-links. At equivalent cytotoxic concentrations, these agents caused different biological responses.

DNA crosslinking and biological activity of a hairpin polyamide-chlorambucil conjugate.

A prototype of a novel class of DNA alkylating agents, which combines the DNA crosslinking moiety chlorambucil (Chl) with a sequence-selective hairpin pyrrole-imidazole polyamide ImPy-beta-ImPy-gamma-ImPy-beta-Dp (polyamide 1), was evaluated for its ability to damage DNA and induce biological responses. Polyamide 1-Chl conjugate (1-Chl) alkylates and interstrand crosslinks DNA in cell-free systems. The alkylation occurs predominantly at 5'-AGCTGCA-3' sequence, which represents the polyamide binding site.

Induction of DNA damage responses by adozelesin is S phase-specific and dependent on active replication forks.

Adozelesin is an alkylating minor groove DNA binder that is capable of rapidly inhibiting DNA replication in treated cells through a trans-acting mechanism and preferentially arrests cells in S phase. It has been shown previously that in cells treated with adozelesin, replication protein A (RPA) activity is deficient, and the middle subunit of RPA is hyperphosphorylated. The adozelesin-induced RPA hyperphosphorylation can be blocked by the replicative DNA polymerase inhibitor, aphidicolin, suggesting that adozelesin-triggered cellular DNA damage responses require active DNA replication forks.

Cell-free and cellular activities of a DNA sequence selective hairpin polyamide-CBI conjugate.

Alkylating agents are generally highly reactive with DNA but demonstrate limited DNA sequence selectivity. In contrast, synthetic pyrrole-imidazole polyamides recognize specific DNA sequences with high affinity but are unable to permanently damage DNA. An eight-ring hairpin polyamide conjugated to the alkylating moiety cyclopropylpyrroloindole, related to the natural product CC-1065, affords a conjugate 1-CBI (polyamide 1-CBI (1-(chloromethyl)-5-hydroxyl-1,2-dihydro-3H-benz[e]indole) conjugate), which binds to specific sequences in the minor groove of DNA and alkylates a single adenine flanking the polyamide binding site.

Cellular uptake of N-methylpyrrole/N-methylimidazole polyamide-dye conjugates.

The cellular uptake and localization properties of DNA binding N-methylpyrrole/N-methylimidazole polyamide-dye conjugates in a variety of living cells have been examined by confocal laser scanning microscopy. With the exception of certain T-cell lines, polyamide-dye conjugates localize mainly in the cytoplasm and not in the nucleus. Reagents such as methanol typically used to fix cells for microscopy significantly alter the cellular localization of these DNA-binding ligands.

Inhibiting transcription factor/DNA complexes using fluorescent microgonotropens (FMGTs).

Fluorescent microgonotropens (FMGTs) are A/T selective, minor groove-binding bisbenzimidazole ligands. Basic side chains extending from these agents electrostatically contact the major groove side of the phosphodiester backbone of DNA, endowing them with high binding affinity. Here, we evaluate the potential of these agents as inhibitors of transcription factor (TF) binding to DNA and explore whether their ability to contact both grooves enhances their inhibitory activity.

Cellular responses to the DNA strand-scission enediyne C-1027 can be independent of ATM, ATR, and DNA-PK kinases.

The current paradigm based upon ionizing radiation (IR) studies states that cells deficient in either ataxia-telangiectasia-mutated kinase (ATM) or related phosphatidylinositol 3 (PI 3) -kinases (ATR and DNA-PK) are hypersensitive to DNA strand breaks because they are unable to rapidly activate downstream effectors such as p53. Here we have contrasted cell responses to IR and C-1027, a radiomimetic antibiotic that induces DNA strand breaks. At equal levels of DNA double strand breaks, cell lines with inactive ATM or other phosphatidylinositol 3-kinases displayed classical hypersensitivity to IR but not to C-1027.