Induction of AT-specific DNA-interstrand crosslinks by bizelesin in genomic and simian virus 40 DNA

Interstrand cross-links at T(A/T) 4 A sites in cellular DNA are associated with hypercytotoxicity of an anticancer drug, bizelesin. Here we evaluated whether these lethal effects reflect targeting critical genomic regions. An in silico analysis of human sequences showed that T(A/T) 4 A motifs are on average scarce and scattered. However, significantly higher local motif densities were identified in distinct minisatellite regions (200 -1000 base pairs of ϳ85-100% AT), herein referred to as "AT islands." Experimentally detected bizelesin lesions agree with these in silico predictions. Actual bizelesin adducts clustered within the model AT island naked DNA, whereas motif-poor sequences were only sparsely adducted. In cancer cells, bizelesin produced high levels of lesions (ϳ4.7-7.1 lesions/kilobase pair/M drug) in several prominent AT islands, compared with markedly lower lesion levels in several motif-poor loci and in bulk cellular DNA (ϳ0.8 -1.3 and ϳ0.9 lesions/kilobase pair/M drug, respectively). The identified AT islands exhibit sequence attributes of matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. The computed "MAR potential" and propensity for supercoiling-induced duplex destabilization (both predictive of strong MARs) correlate with the total number of bizelesin binding sites. Hence, MAR-like ATrich non-coding domains can be regarded as a novel class of critical targets for anticancer drugs.

Section: Discussionmentioning

confidence: 99%

AT-rich Islands in Genomic DNA as a Novel Target for AT-specific DNA-reactive Antitumor Drugs

Woynarowski¹,

Treviño²,

Rodriguez³

et al. 2001

“…We have found that this is also true for bizelesin. 4 Since aphidicolin is a DNA polymerase (␣, ␦, and ⑀) inhibitor, it was suggested that replication fork passage through a camptothecin-DNA adduct may be required to generate recognizable DNA damage (31). Hyperphosphorylation of RPA32 induced by 20 nM adozelesin can also be blocked by treatment with aphidicolin, suggesting that replication fork passage through an adozelesin-DNA adduct may also be required to generate recognizable DNA damage (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…CPI drugs are currently in clinical trials for several types of solid tumors (1,2). The CPI drug, adozelesin, carries a single cyclopropyl group and alkylates a single adenine (3)(4)(5)(6). CPI adduct formation on naked DNA is able to block progression of DNA polymerases and helicases (7)(8)(9).…”

mentioning

confidence: 99%

Adozelesin Triggers DNA Damage Response Pathways and Arrests SV40 DNA Replication through Replication Protein A Inactivation

Liu

Kuo

McHugh

et al. 2000

The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA. Treatment of human cells with low levels of adozelesin results in potent inhibition of both cellular and simian virus 40 (SV40) DNA replication. Extracts were prepared from adozelesintreated cells and shown to be deficient in their ability to support SV40 DNA replication in vitro. This effect on in vitro DNA replication was dependent on both the concentration of adozelesin used and the time of treatment but was not due to the presence of adozelesin in the in vitro assay. Adozelesin treatment of cells was shown to result in the following: induction of p53 protein levels, hyperphosphorylation of replication protein A (RPA), and disruption of the p53-RPA complex (but not disruption of the RPA-cdc2 complex), indicating that adozelesin treatment triggers cellular DNA damage response pathways. Interestingly, in vitro DNA replication could be rescued in extracts from adozelesin-treated cells by the addition of exogenous RPA. Therefore, whereas adozelesin and other anti-cancer therapeutics trigger common DNA damage response markers, adozelesin causes DNA replication arrest through a unique mechanism. The S phase checkpoint response triggered by adozelesin acts by inactivating RPA in some function essential for SV40 DNA replication.The cyclopropylpyrroloindole (CPI) 1 drugs are a group of DNA sequence-specific minor groove binders that alkylate the N-3 of adenine at the 3Ј end of the binding sites. CPI drugs are currently in clinical trials for several types of solid tumors (1, 2). The CPI drug, adozelesin, carries a single cyclopropyl group and alkylates a single adenine (3-6). CPI adduct formation on naked DNA is able to block progression of DNA polymerases and helicases (7-9). Previous studies have shown that two different CPI drugs, adozelesin and bizelesin, inhibit both the initiation and elongation stages of cellular and viral DNA replication in cultured cells (10 -12). However, the concentrations of these drugs required to cause S phase arrest are 2-4 orders of magnitude lower than levels of drug required to cause detectable adducts and to block polymerase or helicase progression. These results suggest that inhibition of DNA replication and cell cycle progression in cultured cells upon treatment with CPI drugs occurs via a trans-acting mechanism rather than by directly blocking DNA replication fork progression. The most likely explanation for this trans-inhibition of DNA replication is through cellular DNA damage response pathways or checkpoints.Since both viral, simian virus 40 (SV40), and cellular DNA replication are inhibited at similar CPI levels, it is possible to use the more easily studied viral system to elucidate how CPI treatment results in DNA replication arrest. SV40 DNA replication is the most well studied model for eukaryotic DNA replication. An in vitro system was developed that requires only one viral protein, SV40 large T antigen, an exogenous plasmid DNA template containing the SV40 origin sequence, and primate...

“…Repetitive Primer Extension (RPE) Assay-The reaction was carried out according to the published procedure (22,23). The oligonucleotide primer (5Ј-GCAGCTCCAAAGGTTCCC-3Ј) starting from position 1991 (reverse) on the SV40 genome was synthesized by the Biopolymer Facility at Roswell Park Cancer Institute.…”

Section: Methodsmentioning

confidence: 99%

“…To further test this characteristic on a more complex DNA molecule (5243 bp), the RPE assay was performed using agent-treated SV40 DNA as a template. In this assay, DNA nascent chain elongation driven by polymerase stops at sites of the damage on template DNA, and the partial DNA synthesis products indicate the positions of DNA damage (22).…”

Section: -Cbimentioning

confidence: 99%

Cell-free and Cellular Activities of a DNA Sequence Selective Hairpin Polyamide-CBI Conjugate

Wang

Dzięgielewski

Chang

et al. 2002

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. In this study, we show that 1-CBI alkylates DNA in both plasmid and intracellular minichromosomal form and inhibits DNA replication under both cell-free and cellular conditions. In addition, it inhibits cell growth and arrests cells in the G 2 /M phase of the cell cycle.