Human SNM1A and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair.

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.

Modulation of topoisomerase IIalpha expression by a DNA sequence-specific polyamide.

Topoisomerase IIalpha (topo IIalpha) is an important target for several chemotherapeutic agents, including etoposide and doxorubicin. Confluent cells express low levels of topo IIalpha and are resistant to etoposide treatment. Repression of transcription in confluent cells is mediated by binding of the transcription factor NF-Y to inverted CCAAT motifs within the topo IIalpha promoter. To block the repressive binding of NF-Y, a polyamide (JH-37) was designed to bind to the flanking regions of selected CCAAT sites within the topo IIalpha promoter. Electrophoretic mobility shift assays and DNase I footprinting assays showed occupancy of the inverted CCAAT sites by JH-37. Chromatin immunoprecipitation assays confirmed in vivo inhibition of NF-Y binding to the topo IIalpha promoter. Following incubation of confluent NIH3T3 cells with JH-37, increased expression of topo IIalpha mRNA and protein was detectable. This correlated both with increased DNA double-strand breaks as shown by comet assay and decreased cell viability following exposure to etoposide. Polyamides can modulate gene expression and chemosensitivity of cancer cells.

Novel nitrogen mustard-armed combi-molecules for the selective targeting of epidermal growth factor receptor overexperessing solid tumors: discovery of an unusual structure-activity relationship.

To enhance the potency of "combi-molecules", we designed 6a-d and 18 to release an inhibitor of EGFR TK and a bifunctional alkylator. The combi-molecules blocked EGFR TK with potency increasing with the basicity of the mustard moiety. They selectively killed cells transfected with EGFR and were potent against the DU145 prostate cancer cells. Combi-molecule 6a blocked EGFR phosphorylation in an irreversible manner, induced DNA-cross-links, and arrested the cells in mid-S.

Evodiamine, a dual catalytic inhibitor of type I and II topoisomerases, exhibits enhanced inhibition against camptothecin resistant cells.

DNA topoisomerases are nuclear enzymes that are the targets for several anticancer drugs. In this study we investigated the antiproliferative activity against human leukaemia cell lines and the effects on topoisomerase I and II of evodiamine, which is a quinazolinocarboline alkaloid isolated from the fruit of a traditional Chinese medicinal plant, Evodia rutaecarpa. We report here the anti-proliferative activity against human leukaemia cells K562, THP-1, CCRF-CEM and CCRF-CEM/C1 and the inhibitory mechanism on human topoisomerases I and II, important anti-cancer drugs targets, of evodiamine. Evodiamine failed to trap [Topo-DNA] complexes and induce any detectable DNA damage in cells, was unable to bind or intercalate DNA, and arrested cells in the G(2)/M phase. The results suggest evodiamine is a dual catalytic inhibitor of topoisomerases I and II, with IC(50) of 60.74 and 78.81 µM, respectively. The improved toxicity towards camptothecin resistant cells further supports its inhibitory mechanism which is different from camptothecin, and its therapeutic potential.

Measurement of DNA damage in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.

Measurement of DNA interstrand crosslinking in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

The Single Cell Gel Electrophoresis (Comet) assay, originally developed to allow visualisation of DNA strand break damage in individual cells, has been adapted to measure DNA interstrand cross-links. DNA interstrand cross-links are formed in cells by a number of commonly used cancer chemotherapy agents and are considered to be the critical lesion formed by such agents. This technique allows the analysis of DNA interstrand cross-link formation and repair at a single cell level, requires few cells, allows the determination of heterogeneity of response within a cell population and is sensitive enough to measure DNA interstrand cross-links at pharmacologically relevant doses. The method can be applied to any in vitro or in vivo application where a single cell suspension can be obtained. The method has also become invaluable in studies using human tissue and can be used as a method for pharmacodynamic analysis in early clinical trials.

Measurement of DNA interstrand crosslinking in naked DNA using gel-based methods.

Bifunctional DNA damaging agents continue to be the mainstay in various chemotherapeutic regimens used in the clinic. DNA interstrand crosslinks are considered to be the critical cytotoxic lesions for the biological activity of such agents. Gel-based electrophoretic assays can efficiently separate denatured single-stranded DNA from double-stranded, covalently-linked DNA resulting from the presence of an interstrand crosslink. The methods described here offer a simple way for the assessment of crosslinking efficiencies of bifunctional agents in both long fragments of DNA (e.g. 1-5 kb) and short oligonucleotide DNA duplexes. As the repair of interstrand crosslinks is a key determinant of cellular and clinical chemosensitivity, these methods can be useful for the characterization and isolation of site-directed adducted substrates for use in subsequent biochemical analysis of cellular recognition and DNA repair processes.

SG2285, a novel C2-aryl-substituted pyrrolobenzodiazepine dimer prodrug that cross-links DNA and exerts highly potent antitumor activity.

The pyrrolobenzodiazepines (PBD) are naturally occurring antitumor antibiotics, and a PBD dimer (SJG-136, SG2000) is in phase II trials. Many potent PBDs contain a C2-endo-exo unsaturated motif associated with the pyrrolo C-ring. The novel compound SG2202 is a PBD dimer containing this motif. SG2285 is a water-soluble prodrug of SG2202 in which two bisulfite groups inactivate the PBD N10-C11 imines. Once the bisulfites are eliminated, the imine moieties can bind covalently in the DNA minor groove, forming an interstrand cross-link. The mean in vitro cytotoxic potency of SG2285 against human tumor cell lines is GI(50) 20 pmol/L. SG2285 is highly efficient at producing DNA interstrand cross-links in cells, but they form more slowly than those produced by SG2202. Cellular sensitivity to SG2285 was primarily dependent on ERCC1 and homologous recombination repair. In primary B-cell chronic lymphocytic leukemia samples, the mean LD(50) was significantly lower than in normal age-matched B and T lymphocytes. Antitumor activity was shown in several human tumor xenograft models, including ovarian, non-small cell lung, prostate, pancreatic, and melanoma, with cures obtained in the latter model with a single dose. Further, in an advanced-stage colon model, SG2285 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. Our findings define SG2285 as a highly active cytotoxic compound with antitumor properties desirable for further development.

Functionalised cyclopentadienyl titanium compounds as potential anticancer drugs.

A number of new ionic titanocene compounds have been isolated and characterised, which exhibit excellent cytotoxicity against different human tumour cell lines including a defined cisplatin resistant cell line. A range of biological assays have been carried out to determine levels of cytotoxicity and levels of DNA interstrand crosslinking.