Interstrand Cross-linking Agents Research Articles

KIAA1018/FAN1 nuclease protects cells against genomic instability induced by interstrand cross-linking agents

Fanconi anemia (FA) is a rare genetic disease characterized by congenital defects, bone marrow failure, chromosomal instability, and cancer susceptibility. One hallmark of cells from FA patients is hypersensitivity to interstrand cross-linking agents, such as the chemotherapeutics cisplatin and mitomycin C (MMC). We have recently characterized a FANCD2/FANCI-associated nuclease, KIAA1018/FAN1, the depletion of which sensitizes human cells to these agents. However, as the down-regulation of FAN1 in human cells was mediated by siRNA and thus only transient, we were unable to study the long-term effects of FAN1 loss on chromosomal stability. We now describe the generation of chicken DT40 B cells, in which the FAN1 locus was disrupted by gene targeting. FAN1-null cells are highly sensitive to cisplatin and MMC, but not to ionizing or UV radiation, methyl methanesulfonate, or camptothecin. The cells do not display elevated sister chromatid exchange frequencies, either sporadic or MMC-induced. Interestingly, MMC treatment causes chromosomal instability that is quantitatively, but not qualitatively, comparable to that seen in FA cells. This finding, coupled with evidence showing that DT40 cells deficient in both FAN1 and FANCC, or FAN1 and FANCJ, exhibited increased sensitivity to cisplatin compared with cells lacking only FAN1, suggests that, despite its association with FANCD2/FANCI, FAN1 in DT40 cells participates in the processing of damage induced by interstrand cross-linking-generating agents also independently of the classical FA pathway.

Correction of the Fanconi Anemia Phenotype at the Cellular Level by S Phase Delay: Implications for Etiopathogenesis and Treatment

AbstractAbstract 879Fanconi Anemia (FA) is a rare, complex, recessively inherited disease, with 13 known complementation groups, in which many patients develop bone marrow failure and/or leukemia, especially myelogenous leukemia, at an early age. Although bone marrow transplantation (BMT) has been beneficial, a high proportion of these patients go on to develop head and neck cancers and other solid malignancies, regardless of whether BMT has been carried out. Our laboratory, and others, have shown that FA is associated with a cell cycle defect in which FA cells fail to slow or arrest their rate of replicative, S-phase DNA synthesis as do normal cells following treatment with a DNA interstrand cross-linking (ICL) agent. FA cells are also markedly hypersensitive to the clastogenic and cytotoxic effects of such ICL agents, characteristics used to define the disease in the classic diepoxybutane, or DEB test. We wished to test whether, if the cell cycle defect were corrected by treating the cells with an inhibitor of DNA synthesis, the cytoclastic and cytotoxic effects of an ICL agent, psoralen plus ultraviolet A light (PUVA), would be affected in FA cells. Among DNA synthesis inhibitors, we were particularly interested in examining hydroxyurea and 5-fluorouracil (HU and 5-FU), because they have been used successfully to treat other blood diseases and cancers, respectively, such as sickle cell anemia and colon adenocarcinomas. Following treatment with PUVA, normal lymphoblastoid cells (two cell lines), FA lymphoblastoid cells (two FA-A, one FA-C and one FA-G lines), and two genetically corrected FA lymphoblastoid cell lines (FA-A and FA-C) were either mock treated or treated with an inhibitor of DNA synthesis (high dose thymidine, methotrexate, or HU) for 24 hours. Chromosome breaks and both short term (trypan blue exclusion) and long term cell viability (colony forming ability) were then measured. Except for one FA-A cell line that had low thymidine kinase activity, and therefore did not respond to thymidine, all uncorrected FA cell lines, but not corrected FA cell lines, showed dramatic reductions in clastogenicity and increases in viability following PUVA and treatment with any of the DNA synthesis inhibitors. Normal cells and corrected FA cells failed to show a comparable response.These results indicate that the S-phase cell cycle defect in FA is important in its etiopathogenesis. The stalled DNA replication forks documented in FA cells containing DNA ICLs may actually be due, in part or entirely, to a prior defect in cell cycle regulation when damaged FA cells enter S phase. These results also suggest at least two possible modes of therapeutic intervention, treatment of FA patients with HU or 5-FU to prevent or delay onset of complications including bone marrow failure, leukemia, or, particularly in patients who have undergone BMT, head, neck, and other tumors. Disclosures:No relevant conflicts of interest to declare.

Repair of DNA interstrand cross‐links during S phase of the mammalian cell cycle

DNA interstrand cross-linking (ICL) agents are widely used in anticancer chemotherapy regimens, yet our understanding of the DNA repair mechanisms by which these lesions are removed from the genome remains incomplete. This is at least in part due to the enormously complicated nature and variety of the biochemical pathways that operate on these complex lesions. In this review, we have focused specifically on the S-phase pathway of ICL repair in mammalian cells, which appears to be the major mechanism by which these lesions are removed in cycling cells. The various stages and components of this pathway are discussed, and a putative molecular model is presented. In addition, we propose an explanation as to how this pathway can lead to the observed high levels of sister chromatid exchanges known to be induced by ICLs.

The FANCM/FAAP24 Complex Is Required for the DNA Interstrand Crosslink-Induced Checkpoint Response

Cells from Fanconi anemia (FA) patients are extremely sensitive to DNA interstrand crosslinking (ICL) agents, but the molecular basis of the hypersensitivity remains to be explored. FANCM (FA complementation group M), and its binding partner, FAAP24, anchor the multisubunit FA core complex to chromatin after DNA damage and may contribute to ICL-specific cellular response. Here we show that the FANCM/FAAP24 complex is specifically required for the recruitment of replication protein A (RPA) to ICL-stalled replication forks. ICL-induced RPA foci formation requires the DNA-binding activity of FAAP24 but not the DNA translocase activity of FANCM. Furthermore, FANCM/FAAP24-dependent RPA foci formation is required for efficient ATR-mediated checkpoint activation in response to ICL. Therefore, we propose that FANCM/FAAP24 plays a role in ICL-induced checkpoint activation through regulating RPA recruiment at ICL-stalled replication forks.

Deficiency of FANCD2-Associated Nuclease KIAA1018/FAN1 Sensitizes Cells to Interstrand Crosslinking Agents

Cytotoxicity of cisplatin and mitomycin C (MMC) is ascribed largely to their ability to generate interstrand crosslinks (ICLs) in DNA, which block the progression of replication forks. The processing of ICLs requires the Fanconi anemia (FA) pathway, excision repair, and translesion DNA synthesis (TLS). It also requires homologous recombination (HR), which repairs double-strand breaks (DSBs) generated by cleavage of the blocked replication forks. Here we describe KIAA1018, an evolutionarily conserved protein that has an N-terminal ubiquitin-binding zinc finger (UBZ) and a C-terminal nuclease domain. KIAA1018 is a 5'-->3' exonuclease and a structure-specific endonuclease that preferentially incises 5' flaps. Like cells from FA patients, human cells depleted of KIAA1018 are sensitized to ICL-inducing agents and display chromosomal instability. The link of KIAA1018 to the FA pathway is further strengthened by its recruitment to DNA damage through interaction of its UBZ domain with monoubiquitylated FANCD2. We therefore propose to name KIAA1018 FANCD2-associated nuclease, FAN1.

Knockdown of μ-Calpain in Fanconi Anemia, FA-A, Cells by siRNA Restores αII Spectrin Levels and Corrects Chromosomal Instability and Defective DNA Interstrand Cross-Link Repair

We have previously shown that there is a deficiency in the structural protein, nonerythroid alpha spectrin (alphaIISp), in cells from patients with Fanconi anemia (FA). These studies indicate that this deficiency is due to the reduced stability of alphaIISp and correlates with a decreased level of repair of DNA interstrand cross-links and chromosomal instability in FA cells. An important factor in the stability of alphaIISp is its susceptibility to cleavage by the protease, mu-calpain. We hypothesized that an increased level of mu-calpain cleavage of alphaIISp in FA cells leads to an increased level of breakdown of alphaIISp and that knocking down expression of mu-calpain in FA cells should restore levels of alphaIISp and correct a number of the phenotypic defects observed. The results showed that there is increased mu-calpain activity in FA-A, FA-C, FA-D2, FA-F, and FA-G cells that could account for the deficiency in alphaIISp in these FA cells. Protein interaction studies indicated that FANCA and FANCG bind directly to mu-calpain. We hypothesize that this binding may lead to inhibition of mu-calpain activity in normal cells. Knocking down mu-calpain by siRNA in FA-A cells restored levels of alphaIISp to normal and reversed a number of the cellular deficiencies in these cells. It corrected the DNA repair defect and the chromosomal instability observed after exposure to a DNA interstrand cross-linking agent. These studies indicate that FA proteins may play an important role in maintaining the stability of alphaIISp in the cell by regulating its cleavage by mu-calpain. Thus, by reducing the level of breakdown of alphaIISp in FA cells, we may be able to reverse a number of the cellular deficiencies observed in this disorder.

Mutation of the RAD51C gene in a Fanconi anemia–like disorder

Fanconi anemia (FA) is a rare chromosomal-instability disorder associated with a variety of developmental abnormalities, bone marrow failure and predisposition to leukemia and other cancers. We have identified a homozygous missense mutation in the RAD51C gene in a consanguineous family with multiple severe congenital abnormalities characteristic of FA. RAD51C is a member of the RAD51-like gene family involved in homologous recombination-mediated DNA repair. The mutation results in loss of RAD51 focus formation in response to DNA damage and in increased cellular sensitivity to the DNA interstrand cross-linking agent mitomycin C and the topoisomerase-1 inhibitor camptothecin. Thus, biallelic germline mutations in a RAD51 paralog are associated with an FA-like syndrome.

Abstract 3506: Rules of DNA adduct formation for pyrrolobenzodiazepine (PBD) dimers

Abstract Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective DNA minor-groove cross-linking agents developed from the tricyclic anthramycin family of naturally-occurring antitumor agents. The “lead” agent, SJG-136, is about to enter Phase II clinical trials as a “first-in-class” sequence-selective DNA minor-groove binding agent. PBDs possess electrophilic imine or carbinolamine moieties that form covalent aminal linkages with guanine C2-NH2 functionalities within the DNA minor groove. Previous studies have shown that PBD dimers with odd numbers of methylenes (e.g., n = 3 or 5) in the -O-(CH2)n-O- linker connecting the two PBD units through their C8/C8′-positions are the most potent interstrand cross-linking agents. We have developed a reversed-phase ion pair HPLC/MS method as a tool to evaluate the interaction of DNA-binding molecules with oligonucleotides of varying length and sequence. Based on this methodology, we recently reported that SJG-136 (n = 3) can form three different types of adducts: inter- and intrastrand cross-linked duplex adducts, and mono-alkylated adducts) (J. Am. Chem. Soc., 2009, 131, 13756-13766). Extending these studies to PBD dimers with longer central linkers (e.g., ELB-21 [n = 5] and DRG-16 [n = 5]), we have found that the type and distribution of the various adducts appear to depend on (1) the positioning of the two reactive guanine bases on the same or opposite strands, (2) their separation (typically the number of base pairs, usually ATs, between them) and (3) the length of the C8/C8′-linker connecting the two PBD units in the dimers. Based on these data, a set of rules have emerged that can be used to predict the binding behaviour of a particular PBD dimer towards a given DNA sequence. For example, for PBD dimers containing an -O-CH2CH2CH2-O- linker (i.e., n = 3) between the C8/C8′-positions the following rules apply: Rule 1 - For binding sequences with two guanines separated by two A/T base pairs (ideal sequence length for “n = 3” dimers according to previous modeling studies), there is a preference for interstrand rather than intrastrand cross-link formation; Rule 2 - For shorter binding sequences (e.g., two guanines separated by a single A/T base pair), interstrand cross-linking is preferred over intrastrand, although the rate of reaction is significantly slower; Rule 3 - For longer sequences (e.g., two guanines separated by three A/T base pairs), intrastrand cross-link formation is preferred and the rate of formation is faster. Similar rules have been found to apply to “n = 5” PBD dimers (e.g., ELB-21 and DRG-16) in relation to sequences with two guanines separated by 2, 3 or 4 base pairs. These results, and the simple rules that appear to explain them, suggest that it may be possible to design PBD dimers to target specific DNA sequences. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3506.

FANCD2 Localizes to Cross-Linked Induced Nuclear Foci That Are Distinct From Those to Which αaII Spectrin and the Cross-Link Repair Protein, XPF, Co-Localize, Indicating An Involvement of These Proteins in Different Steps of the DNA Interstrand Cross-Link Repair Process.

Abstract 3196Poster Board III-133The hereditary bone marrow failure disorder, Fanconi anemia (FA), is characterized by a markedly increased incidence of acute myelogenous leukemia, diverse congenital abnormalities and a defect in ability to repair DNA interstrand cross-links. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid a spectrin (aSpII), which is involved in repair of DNA interstrand cross-links and binds to cross-linked DNA. aSpII co-localizes in nuclear foci with FANCA and the cross-link repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. One of the FA proteins which is thought to play an important role in the repair of DNA interstrand cross-links is FANCD2, which is known to form nuclear foci after cross-link damage. The present study was undertaken in order to get a better understanding of the relationship between aSpII and FANCD2, whether they interact with each other during the DNA repair process and co-localize in damage-induced nuclear foci. Immunofluorescence microscopy was carried out to determine whether these proteins co-localized in nuclear foci after cells were damaged with a DNA interstrand cross-linking agent, 8-methylpsoralen plus UVA light (8-MOP) or mitomycin C (MMC). Time course measurements showed that FANCD2 foci were first visible at 2 hours after damage and increased up to 16 hours and were still present at 72 hours after damage. This time course of foci formation correlated with levels of monoubiquitination of FANCD2. Measurement of gH2AX foci formation showed that the time course of foci formation was similar to that of FANCD2 measured up to 72 hours post damage. In contrast, aSpII foci were first visible between 8-10 hours after damage. The number of these foci peaked at 16 hours and by 24 hours foci were no longer observed. Co-localization studies showed that there was little co-localization of the FANCD2 and aSpII foci over this time course. This indicates that these two proteins may be involved in different steps in the DNA interstrand cross-link repair process. Based on models that have been proposed for the role of FANCD2 in the repair of DNA interstrand cross-links, we propose that, after DNA damage, FANCD2 localizes at DNA replication forks stalled at sites of interstrand cross-links and aids in the assembly of proteins at this site. This is followed by localization of aSpII and XPF and other proteins involved in the initial incision steps in DNA interstrand cross-link repair where they play a role in the unhooking of the cross-link. FANCD2 is then involved in subsequent steps in the repair process, which involve homologous recombination. Thus two proteins, FANCD2 and aSpII, both of which have been shown to be critical for the DNA interstrand cross-link repair process may be involved in different or distinct steps in this repair process. Deficiencies in these proteins would impact on DNA interstrand cross-link repair and, as we have shown for aIISp, would have an adverse effect on the genomic stability of FA cells. . DisclosuresNo relevant conflicts of interest to declare.

Restoring Levels of αII Spectrin in Fanconi Anemia Group A Cells Leads to Reversal of the Chromosomal Instability and to Enhanced Ability to Repair DNA Interstrand Cross-Links.

Abstract 493Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, a predisposition to cancer, congenital abnormalities, genomic instability, and a defect in ability to repair DNA interstrand cross-links. We have shown that the structural protein, nonerythroid a spectrin (aIISp), plays an important role in the repair of DNA interstrand cross-links and that there is a deficiency in aIISp in FA cells. We have recently demonstrated that aIISp is also important in chromosome stability and that its depletion in normal cells by siRNA, to levels found in FA cells, leads to a number of the defects observed in FA cells, such as increased chromosomal instability, increased cellular sensitivity to DNA interstrand cross-linking agents and decreased ability to repair DNA interstrand cross-links. We have shown that the reduced levels of αIISp in FA cells are due to its increased breakdown. The goal of the present study was to determine whether, by increasing the levels of aIISp in FA cells to normal, it would be possible to enhance chromosome stability and decrease the sensitivity to DNA interstrand cross-linking agents and the DNA repair defect. In the cell, aIISp is cleaved by the protease, m calpain, to give distinctive cleavage products. Cells from a number of FA complementation groups were examined for levels and activity of m calpain. The results showed that there was increased m calpain activity in FA cells, which included FA-A, FA-C, FA-D2, FA-F and FA-G, which correlated increased m calpain breakdown product of αIISp in these cells. However, levels of calpain in the FA cells were similar to normal. In FA cells corrected with the corresponding FANC cDNA, activity of m calpain was reduced to normal levels and αIISp levels returned to normal, indicating a role for the FA proteins in regulating calpain activity and αIISp stability. Studies were carried out to determine whether FA proteins bound directly to calpain. Co-immunoprecipitation and yeast two-hybrid analysis showed that FANCA and FANCG bound directly to calpain and thus could potentially play a role in its activity. Studies were also undertaken to determine whether knockdown of m calpain in FA cells by siRNA could lead to restoration of αIISp levels and reversal of some of the cellular phenotypic characteristics of FA. The results showed that when m calpain was knocked down in FA-A cells by siRNA, αIISp levels returned to normal. When these siRNA transfected FA-A cells were exposed to a DNA interstrand cross-linking agent, mitomycin C (MMC), cell survival increased to ∼85% of normal. After treatment of these cells with MMC, αIISp and a cross-link repair protein, XPF, nuclear foci were observed at levels similar to normal. This is in contrast to nontarget transfected FA-A cells in which few damage-induced αIISp or XPF nuclear foci were seen. In addition, in cells in which m calpain was knocked down, chromosomal stability in these cells was enhanced. Few of the chromosomal abnormalities usually observed in FA-A cells after MMC treatment, such as chromosome breaks and rearrangements, were observed. These studies thus indicate that increased calpain activity in FA cells could be responsible for decreased αIISp in these cells. Several of the FA proteins could play an important role in regulating activity of m calpain and thus in maintaining normal levels of αIISp, which in turn is critical for maintenance of chromosome stability and DNA repair. Knocking down m calpain in FA-A cells restores levels of αIISp to normal and enhances chromosome stability and DNA repair in these cells. This could potentially have important therapeutic relevance. Disclosures:No relevant conflicts of interest to declare.

Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells

Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells.

PALB2 Functionally Connects the Breast Cancer Susceptibility Proteins BRCA1 and BRCA2

BRCA1 and BRCA2 are prominently associated with inherited breast and ovarian cancer. The encoded proteins function in DNA damage responses, but no functional link between BRCA1 and BRCA2 has been established. We show here that PALB2 physically and functionally connects BRCA1 and BRCA2 into a DNA damage response network that also includes the RAD51 recombinase. PALB2 directly binds BRCA1, as determined with bacterially expressed fragments of each protein. Furthermore, PALB2 independently interacts with BRCA1 and BRCA2 through its NH2 and COOH termini, respectively. Critically, two point mutants (L21P and L24P) of the PALB2 coiled-coil domain or an NH2-terminal deletion (Delta1-70) disrupt its interaction with BRCA1. We have reconstituted PALB2-deficient cells with PALB2Delta1-70, PALB2-L21P, or PALB2-L24P, or with COOH-terminally truncated PALB2 that is deficient for interaction with BRCA2. Using extracts from these cells, we find that PALB2 mediates the physical interaction of BRCA2 with a COOH-terminal fragment of BRCA1. Analysis of the assembly of foci in these cells by BRCA1, PALB2, BRCA2, and RAD51 suggests that BRCA1 recruits PALB2, which in turn organizes BRCA2 and RAD51. Resistance to mitomycin C and the repair of DNA double-strand breaks by homologous recombination require the interaction of PALB2 with both BRCA1 and BRCA2. These results suggest that BRCA1 and BRCA2 cooperate in DNA damage responses in a PALB2-dependent manner, and have important implications for the genesis of breast/ovarian cancer and for chemotherapy with DNA interstrand cross-linking agents.

The Schizosaccharomyces pombe Checkpoint Kinases Chk1 and Cds1 Are Important for Cell Survival in Response to Cisplatin

BackgroundDNA damage checkpoints insure that the integrity of genomic DNA is faithfully maintained throughout the eukaryotic cell cycle. In the presence of damaged DNA, checkpoints are triggered to delay cell cycle progression to allow for DNA repair. In fission yeast, the kinases Chk1 and Cds1 are major components of these DNA damage checkpoint pathways. Both Chk1 and Cds1 are important for viability in the presence of several DNA damaging agents. In this study we hypothesized that Chk1 and Cds1 play a vital role in fission yeast cells ability to survive exposure to the DNA damaging agent cisplatin. Cisplatin is a potent chemotherapeutic drug that interacts with DNA and causes both inter- and intra-strand DNA cross-links.Methodology/Principal FindingsHere, we demonstrated that treatment with cisplatin in fission yeast causes a Chk1-dependent DNA damage signal. chk1 − cells were sensitive to cisplatin and Chk1 was phosphorylated in response to cisplatin treatment. We also showed that a Chk1-dependent DNA damage checkpoint pathway is activated in a dose-dependent fashion in cells challenged with cisplatin. Furthermore the Cds1 checkpoint kinase was also important for viability in cisplatin challenged cells. In cds1 − cells, cisplatin treatment reduced cell viability and this phenotype was exacerbated in a chk1 −/cds1 − background.Conclusions/SignificanceThus, we conclude that the concerted effort of both major checkpoint kinases in fission yeast, Chk1 and Cds1, protect cells from cisplatin induced DNA damage. These observations are significant because they suggest that various classes of inter-strand crosslinking agents may generate slightly different lesions as work by others did not observe loss of viability in cds1 − cells treated with other crosslinking agents like nitrogen mustard.

Potent antitumor bifunctional DNA alkylating agents, synthesis and biological activities of 3a-aza-cyclopenta[ a]indenes

A series of bifunctional DNA interstrand cross-linking agents, bis(hydroxymethyl)- and bis(carbamates)-8 H-3a-azacyclopenta[ a]indene-1-yl derivatives were synthesized for antitumor evaluation. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and antitumor therapeutic efficacy against human tumor xenografts in vivo. Furthermore, these derivatives have little or no cross-resistance to either Taxol or Vinblastine. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft by 13a, b and 14g, h and significant suppression against prostate adenocarcinoma PC3 xenograft by 13b were achieved at the maximum tolerable dose with relatively low toxicity. In addition, these agents induce DNA interstrand cross-linking and substantial G2/M phase arrest in human non-small lung carcinoma H1299 cells. The current studies suggested that these agents are promising candidates for preclinical studies.

FANCJ helicase operates in the Fanconi Anemia DNA repair pathway and the response to replicational stress.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk. Cells from FA patients exhibit spontaneous chromosomal instability and hypersensitivity to DNA interstrand cross-linking (ICL) agents. Although the precise mechanistic details of the FA/BRCA pathway of ICL-repair are not well understood, progress has been made in the identification of the FA proteins that are required for the pathway. Among the 13 FA complementation groups from which all the FA genes have been cloned, only a few of the FA proteins are predicted to have direct roles in DNA metabolism. One of the more recently identified FA proteins, shown to be responsible for complementation of the FA complementation group J, is the BRCA1 Associated C-terminal Helicase (BACH1, designated FANCJ), originally identified as a protein associated with breast cancer. FANCJ has been proposed to function downstream of FANCD2 monoubiquitination, a critical event in the FA pathway. Evidence supports a role for FANCJ in a homologous recombination (HR) pathway of double strand break (DSB) repair. In this review, we will summarize the current knowledge in terms of FANCJ functions through its enzymatic activities and protein interactions. The molecular roles of FANCJ in DNA repair and the response to replicational stress will be discussed.

Azinomycin bisepoxides containing rigid aromatic linkers: synthesis, cytotoxicity and DNA interstrand cross-linking activity

The synthesis of a series of azinomycin bisepoxides containing rigid linkers is achieved through two different strategies. Double copper-mediated C–N bond formation under Buchwald-type conditions can be realised but yields are poor with fully intact epoxide substrates. The bisepoxides are made in improved yields through the simultaneous formation of two amide bonds. Bioassays reveal that 5, containing a 1,3-diaminobenzene linker, is a potent in vitro DNA interstrand cross-linking agent with significant cytotoxicity in the NCI 60-human cancer cell panel (GI 50 = 0.15 μM).

KSR1 Is Required for Cell Cycle Reinitiation Following DNA Damage

KSR1 (kinase suppressor of Ras 1) is a molecular scaffold and positive regulator of the Raf/MEK/ERK phosphorylation cascade. KSR1 is required for maximal ERK activation induced by growth factors and by some cytotoxic agents. We show here that KSR1 is also required for maximal ERK activation induced by UV light, ionizing radiation, or the DNA interstrand cross-linking agent mitomycin C (MMC). We further demonstrate a role for KSR1 in the reinitiation of the cell cycle and proliferation following cell cycle arrest induced by MMC. Cells lacking KSR1 underwent but did not recover from MMC-induced G(2)/M arrest. Expression of KSR1 allowed KSR1(-/-) cells to re-enter the cell cycle following MMC treatment. However, cells expressing a mutated form of KSR1 unable to bind ERK did not recover from MMC-induced cell cycle arrest, demonstrating the requirement for the KSR1-ERK interaction. In addition, constitutive activation of ERK was not sufficient to promote cell cycle reinitiation in MMC-treated KSR1(-/-) cells. Only cells expressing KSR1 recovered from MMC-induced cell cycle arrest. Importantly, MMC-induced DNA damage was repaired in KSR1(-/-) cells, as determined by resolution of gamma-H2AX-containing foci. These data indicate that cell cycle reinitiation is not actively signaled in the absence of KSR1, even when DNA damage has been resolved. These data reveal a specific role for the molecular scaffold KSR1 and KSR1-mediated ERK signaling in the cellular response to DNA interstrand cross-links.

SNM1B/Apollo interacts with Astrin and is required for the prophase cell cycle checkpoint

Previously, we have shown that SNM1A is a multifunctional gene involved in both the DNA damage response and in an early mitotic checkpoint in response to spindle stress. Another member of the SNM1 gene family, SNM1B/Apollo, has been shown to have roles in both the response to DNA interstrand cross-linking agents and in telomere protection during S phase. Here, we demonstrate a novel role for SNM1B/Apollo in mitosis in response to spindle stress. SNM1B-deficient cells exhibit a defect in the prophase checkpoint. Loss of the prophase checkpoint induces an extended mitotic delay, which is due to prolonged activation of the spindle checkpoint. In addition, we show that SNM1B/Apollo interacts with the essential microtubule binding protein Astrin. SNM1B/Apollo interacts with Astrin through its conserved metallo-β-lactamase domain, and disruption of this interaction by point mutations results in a deficient prophase checkpoint. These findings suggest that SNM1B/Apollo and Astrin function together to enforce the prophase checkpoint in response to spindle stress.

Knockdown of αII spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair

Nonerythroid α-spectrin (αIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that αIISp plays in normal human cells and in the repair defect in FA, αIISp was knocked down in normal cells using siRNA. Depletion of αIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced αIISp and XPF nuclear foci. Thus depletion of αIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.

Parallel determination of phenotypic cytotoxicity with a micropattern of mutant cell lines

This work presents a novel tool, the Continuous Flow Microspotter (CFM) and its use in patterning cellular microarrays of multiple cell types into the bottom of a tissue culture well. The CFM uses a system of isolated microfluidic channels to make an array of localized microspots of adhesion dependent cells in the bottom of a conventional tissue culture well. With this device we have created micropatterns of multiple cell lines in a single tissue culture well and used this system to conduct simultaneous cytotoxicity tests and recover dose survival curves in a parallel study. This mechanism of parallel testing allows the researcher to employ the use of positive and negative controls, as well as compare the chemical response of phenotypes in a tightly controlled microenvironment. For the experiments presented in this paper we have fabricated a CFM with a set of ten microchannels (five inlet channels and five outlet channels) to pattern a row of five microspots consisting of four cellular microspots and one empty spot for background measurements. Micropatterns containing a set of four different Chinese hamster ovarian cell (CHO) mutant phenotypes were deposited into the bottom of commercially available tissue culture wells then interrogated with mitomycin C, a chemotherapeutic agent. This study shows statistically significant (P < 0.05) hypersensitivity of the UV20 CHO mutant to a DNA interstrand cross-linking agent (mitomycin C). Because the CFM is also capable of depositing proteins and other biomolecules to the individual microspots of the array we foresee capabilities of the 48 microspot CFM to multiplex 48 cell types with 48 chemical reagents all within the confines of a 60 mm(2) area.