Isolation and detection of DNA-protein crosslinks in mammalian cells.

DNA-protein crosslinks (DPCs) are toxic DNA lesions wherein a protein is covalently attached to DNA. If not rapidly repaired, DPCs create obstacles that disturb DNA replication, transcription and DNA damage repair, ultimately leading to genome instability. The persistence of DPCs is associated with premature ageing, cancer and neurodegeneration. In mammalian cells, the repair of DPCs mainly relies on the proteolytic activities of SPRTN and the 26S proteasome, complemented by other enzymes including TDP1/2 and the MRN complex, and many of the activities involved are essential, restricting genetic approaches. For many years, the study of DPC repair in mammalian cells was hindered by the lack of standardised assays, most notably assays that reliably quantified the proteins or proteolytic fragments covalently bound to DNA. Recent interest in the field has spurred the development of several biochemical methods for DPC analysis. Here, we critically analyse the latest techniques for DPC isolation and the benefits and drawbacks of each. We aim to assist researchers in selecting the most suitable isolation method for their experimental requirements and questions, and to facilitate the comparison of results across different laboratories using different approaches.

Topoisomerase Assays.

Topoisomerases are enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of DNA topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and decatenate double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. Provided in this article are protocols to assess activities of topoisomerases and their inhibitors. Included are an assay for topoisomerase I activity based on relaxation of supercoiled DNA; an assay for topoisomerase II based on the decatenation of double-stranded DNA; and approaches for enriching and quantifying DNA-protein covalent complexes formed as obligatory intermediates in the reactions of type I and II topoisomerases with DNA; and assays for measuring DNA cleavage in vitro. Topoisomerases are not the only proteins that form covalent adducts with DNA in living cells, and the approaches described here are likely to find use in characterizing other protein-DNA adducts and exploring their utility as targets for therapy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Assay of topoisomerase I activity Basic Protocol 2: Assay of topoisomerase II activity Basic Protocol 3: In vivo determination of topoisomerase covalent complexes using the in vivo complex of enzyme (ICE) assay Support Protocol 1: Preparation of mouse tissue for determination of topoisomerase covalent complexes using the ICE assay Support Protocol 2: Using recombinant topoisomerase standard for absolute quantification of cellular TOP2CC Basic Protocol 4: Quantification of topoisomerase-DNA covalent complexes by RADAR/ELISA: The rapid approach to DNA adduct recovery (RADAR) combined with the enzyme-linked immunosorbent assay (ELISA) Basic Protocol 5: Analysis of protein-DNA covalent complexes by RADAR/Western Support Protocol 3: Adduct-Seq to characterize adducted DNA Support Protocol 4: Nuclear fractionation and RNase treatment to reduce sample complexity Basic Protocol 6: Determination of DNA cleavage by purified topoisomerase I Basic Protocol 7: Determination of inhibitor effects on DNA cleavage by topoisomerase II using a plasmid linearization assay Alternate Protocol: Gel electrophoresis determination of topoisomerase II cleavage.

Pathways and signatures of mutagenesis at targeted DNA nicks.

Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at a targeted nick in human cells. Mutations were distributed asymmetrically around the nick site. BRCA2 inhibited all categories of mutational events, including indels, SNVs and HDR. DNA2 and RPA promoted resection. DNA2 inhibited 1 bp deletions but contributed to longer deletions, as did REV7. POLQ stimulated SNVs. Parallel analysis of DSBs targeted to the same site identified similar roles for DNA2 and POLQ (but not REV7) in promoting deletions and for POLQ in stimulating SNVs. Insertions were infrequent at nicks, and most were 1 bp in length, as at DSBs. The translesion polymerase REV1 stimulated +1 insertions at one nick site but not another, illustrating the potential importance of sequence context in determining the outcome of mutagenic repair. These results highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells, and identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ.

Treatment of human cells with 5-aza-dC induces formation of PARP1-DNA covalent adducts at genomic regions targeted by DNMT1.

The nucleoside analog 5-aza-2'-deoxycytidine (5-aza-dC) is used to treat some hematopoietic malignancies. The mechanism of cell killing depends upon DNMT1, but is otherwise not clearly defined. Here we show that PARP1 forms covalent DNA adducts in human lymphoblast or fibroblasts treated with 5-aza-dC. Some adducts recovered from 5-aza-dC-treated cells have undergone cleavage by apoptotic caspases 3/7. Mapping of PARP1-DNA adducts, by a new method, "Adduct-Seq", demonstrates adduct enrichment at CpG-dense genomic locations that are targets of maintenance methylation by DNMT1. Covalent protein-DNA adducts can arrest replication and induce apoptosis, and these results raise the possibility that induction of PARP1-DNA adducts may contribute to cell killing in response to treatment with 5-aza-dC.

POLQ suppresses interhomolog recombination and loss of heterozygosity at targeted DNA breaks.

Interhomolog recombination (IHR) occurs spontaneously in somatic human cells at frequencies that are low but sufficient to ameliorate some genetic diseases caused by heterozygous mutations or autosomal dominant mutations. Here we demonstrate that DNA nicks or double-strand breaks (DSBs) targeted by CRISPR-Cas9 to both homologs can stimulate IHR and associated copy-neutral loss of heterozygosity (cnLOH) in human cells. The frequency of IHR is 10-fold lower at nicks than at DSBs, but cnLOH is evident in a greater fraction of recombinants. IHR at DSBs occurs predominantly via reciprocal end joining. At DSBs, depletion of POLQ caused a dramatic increase in IHR and in the fraction of recombinants exhibiting cnLOH, suggesting that POLQ promotes end joining in cis, which limits breaks available for recombination in trans These results define conditions that may produce cnLOH as a mutagenic signature in cancer and may, conversely, promote therapeutic correction of both compound heterozygous and dominant negative mutations associated with genetic disease.

Rapid, direct detection of bacterial topoisomerase 1-DNA adducts by RADAR/ELISA.

Topoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by "RADAR fractionation", a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches.

The "adductome": A limited repertoire of adducted proteins in human cells.

Proteins form adducts with nucleic acids in a variety of contexts, and these adducts may be cytotoxic if not repaired. Here we apply a proteomic approach to identification of proteins adducted to DNA or RNA in normally proliferating cells. This approach combines RADAR fractionation of proteins covalently bound to nucleic acids with quantitative mass spectrometry (MS). We demonstrate that "RADAR-MS" can quantify induction of TOP1- or TOP2-DNA adducts in cells treated with topotecan or etoposide, respectively, and also identify intermediates in physiological adduct repair. We validate RADAR-MS for discovery of previously unknown adducts by determining the repertoires of adducted proteins in two different normally proliferating human cell lines, CCRF-CEM T cells and GM639 fibroblasts. These repertoires are significantly similar with one another and exhibit robust correlations in their quantitative profiles (Spearman r = 0.52). A very similar repertoire is identified by the classical approach of CsCl buoyant density gradient centrifugation. We find that in normally proliferating human cells, the repertoire of adducted proteins - the "adductome" - is comprised of a limited number of proteins belonging to specific functional groups, and that it is greatly enriched for histones, HMG proteins and proteins involved in RNA splicing. Treatment with low concentrations of formaldehyde caused little change in the composition of the repertoire of adducted proteins, suggesting that reactive aldehydes generated by ongoing metabolic processes may contribute to protein adduction in normally proliferating cells. The identification of an endogenous adductome highlights the importance of adduct repair in maintaining genomic structure and the potential for deficiencies in adduct repair to contribute to cancer.

G-quadruplexes Sequester Free Heme in Living Cells.

Heme is an essential cofactor for many enzymes, but free heme is toxic and its levels are tightly regulated. G-quadruplexes bind heme avidly in vitro, raising the possibility that they may sequester heme in vivo. If so, then treatment that displaces heme from quadruplexes is predicted to induce expression of genes involved in iron and heme homeostasis. Here we show that PhenDC3, a G-quadruplex ligand structurally unrelated to heme, displaces quadruplex-bound heme in vitro and alters transcription in cultured human cells, upregulating genes that support heme degradation and iron homeostasis, and most strikingly causing a 30-fold induction of heme oxidase 1, the key enzyme in heme degradation. We propose that G-quadruplexes sequester heme to protect cells from the pathophysiological consequences of free heme.

Activation-induced deaminase (AID) localizes to the nucleus in brief pulses.

Activation-induced deaminase (AID) converts C to U and 5-methyl-C to T. These mutagenic activities are critical to immunoglobulin (Ig) gene diversification and epigenetic reprogramming, but they must be tightly controlled to prevent compromising cell fitness. AID acts in the nucleus but localizes predominately to the cytoplasm. To address this apparent paradox, we have carried out time-lapse imaging of AID in single living B cells and fibroblasts. We demonstrate that AID enters the nucleus in brief (30 min) pulses, evident in about 10% of cells in the course of a single cell cycle (24 hr imaging). Pulses do not depend on AID catalytic activity, but they are coordinated with nuclear accumulation of P53. Pulsing may protect cells from pathologic consequences of excess exposure to AID, or enable AID to synchronize its activity with transcription of genes that are AID targets or with nuclear entry of factors that act at sites of AID-catalyzed DNA deamination to promote Ig gene diversification or epigenetic reprogramming.

Increased levels of RECQ5 shift DNA repair from canonical to alternative pathways.

RECQ5 (RECQL5) is one of several human helicases that dissociates RAD51-DNA filaments. The gene that encodes RECQ5 is frequently amplified in human tumors, but it is not known whether amplification correlates with increased gene expression, or how increased RECQ5 levels affect DNA repair at nicks and double-strand breaks. Here, we address these questions. We show that RECQ5 gene amplification correlates with increased gene expression in human tumors, by in silico analysis of over 9000 individual tumors representing 32 tumor types in the TCGA dataset. We demonstrate that, at double-strand breaks, increased RECQ5 levels inhibited canonical homology-directed repair (HDR) by double-stranded DNA donors, phenocopying the effect of BRCA deficiency. Conversely, at nicks, increased RECQ5 levels stimulated 'alternative' HDR by single-stranded DNA donors, which is normally suppressed by RAD51; this was accompanied by stimulation of mutagenic end-joining. Even modest changes (2-fold) in RECQ5 levels caused significant dysregulation of repair, especially HDR. These results suggest that in some tumors, RECQ5 gene amplification may have profound consequences for genomic instability.

Initiation of homologous recombination at DNA nicks.

Discontinuities in only a single strand of the DNA duplex occur frequently, as a result of DNA damage or as intermediates in essential nuclear processes and DNA repair. Nicks are the simplest of these lesions: they carry clean ends bearing 3'-hydroxyl groups that can undergo ligation or prime new DNA synthesis. In contrast, single-strand breaks also interrupt only one DNA strand, but they carry damaged ends that require clean-up before subsequent steps in repair. Despite their apparent simplicity, nicks can have significant consequences for genome stability. The availability of enzymes that can introduce a nick almost anywhere in a large genome now makes it possible to systematically analyze repair of nicks. Recent experiments demonstrate that nicks can initiate recombination via pathways distinct from those active at double-strand breaks (DSBs). Recombination at targeted DNA nicks can be very efficient, and because nicks are intrinsically less mutagenic than DSBs, nick-initiated gene correction is useful for genome engineering and gene therapy. This review revisits some physiological examples of recombination at nicks, and outlines experiments that have demonstrated that nicks initiate homology-directed repair by distinctive pathways, emphasizing research that has contributed to our current mechanistic understanding of recombination at nicks in mammalian cells.

Assaying Repair at DNA Nicks.

Nicks are the most common form of DNA damage, but they have only recently been shown to initiate damage that requires repair. Analysis of the pathways of nick repair in human cells has benefited from the development of enzymes that target nicks to specific sites in the genome and of reporters that enable rapid analysis of homology-directed repair and mutagenic end joining. Nicks undergo efficient repair by single-stranded oligonucleotide donors complementary to either the nicked or intact DNA strand, via pathways that are normally suppressed by RAD51. Here we discuss the details of reporter assays that take advantage of the convenience and sensitivity of flow cytometry to analyze pathways of repair at targeted DNA nicks. These assays are readily carried out in 96-well format cell culture plates, enabling mechanistic questions to be addressed by determining the contributions of specific factors by depletion and/or ectopic expression.

Two Distinct Pathways Support Gene Correction by Single-Stranded Donors at DNA Nicks.

Nicks are the most common form of DNA damage. The mechanisms of their repair are fundamental to genomic stability and of practical importance for genome engineering. We define two pathways that support homology-directed repair by single-stranded DNA donors. One depends upon annealing-driven strand synthesis and acts at both nicks and double-strand breaks. The other depends upon annealing-driven heteroduplex correction and acts at nicks. Homology-directed repair via these pathways, as well as mutagenic end joining, are inhibited by RAD51 at nicks but largely independent of RAD51 at double-strand breaks. Guidelines for coordinated design of targets and donors for gene correction emerge from definition of these pathways. This analysis further suggests that naturally occurring nicks may have significant recombinogenic and mutagenic potential that is normally inhibited by RAD51 loading onto DNA, thereby identifying a function for RAD51 in maintenance of genomic stability.

The Werner syndrome RECQ helicase targets G4 DNA in human cells to modulate transcription.

The Werner syndrome (WS) is a prototypic adult Mendelian progeroid syndrome in which signs of premature aging are associated with genomic instability and an elevated risk of cancer. The WRN RECQ helicase protein binds and unwinds G-quadruplex (G4) DNA substrates in vitro, and we identified significant enrichment in G4 sequence motifs at the transcription start site and 5' ends of first introns (false discovery rate < 0.001) of genes down-regulated in WS patient fibroblasts. This finding provides strong evidence that WRN binds G4 DNA structures at many chromosomal sites to modulate gene expression. WRN appears to bind a distinct subpopulation of G4 motifs in human cells, when compared with the related Bloom syndrome RECQ helicase protein. Functional annotation of the genes and miRNAs altered in WS provided new insight into WS disease pathogenesis. WS patient fibroblasts displayed altered expression of multiple, mechanistically distinct, senescence-associated gene expression programs, with altered expression of disease-associated miRNAs, and dysregulation of canonical pathways that regulate cell signaling, genome stability and tumorigenesis. WS fibroblasts also displayed a highly statistically significant and distinct gene expression signature, with coordinate overexpression of nearly all of the cytoplasmic tRNA synthetases and associated ARS-interacting multifunctional protein genes. The 'non-canonical' functions of many of these upregulated tRNA charging proteins may together promote WS disease pathogenesis. Our results identify the human WRN RECQ protein as a G4 helicase that modulates gene expression in G4-dependent fashion at many chromosomal sites and provide several new and unexpected mechanistic insights into WS disease pathogenesis.

Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID.

AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.

G4-associated human diseases.

Recent research has established clear connections between G-quadruplexes and human disease. Features of quadruplex structures that promote genomic instability have been determined. Quadruplexes have been identified as transcriptional, translational and epigenetic regulatory targets of factors associated with human genetic disease. An expandable GGGGCC motif that can adopt a G4 structure, located in the previously obscure C9ORF72 locus, has been shown to contribute to two well-recognized neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This review focuses on these advances, which further dispel the view that genomic biology is limited to the confines of the canonical B-form DNA duplex, and show how quadruplexes contribute spatial and temporal dimensionalities to linear sequence information. This recent progress also has clear practical ramifications, as prevention, diagnosis, and treatment of disease depend on understanding the underlying mechanisms.

MRE11-deficiency associated with improved long-term disease free survival and overall survival in a subset of stage III colon cancer patients in randomized CALGB 89803 trial.

PURPOSE: Colon cancers deficient in mismatch repair (MMR) may exhibit diminished expression of the DNA repair gene, MRE11, as a consequence of contraction of a T11 mononucleotide tract. This study investigated MRE11 status and its association with prognosis, survival and drug response in patients with stage III colon cancer. PATIENTS AND METHODS: Cancer and Leukemia Group B 89803 (Alliance) randomly assigned 1,264 patients with stage III colon cancer to postoperative weekly adjuvant bolus 5-fluorouracil/leucovorin (FU/LV) or irinotecan+FU/LV (IFL), with 8 year follow-up. Tumors from these patients were analyzed to determine stability of a T11 tract in the MRE11 gene. The primary endpoint was overall survival (OS), and a secondary endpoint was disease-free survival (DFS). Non-proportional hazards were addressed using time-dependent covariates in Cox analyses. RESULTS: Of 625 tumor cases examined, 70 (11.2%) exhibited contraction at the T11 tract in one or both MRE11 alleles and were thus predicted to be deficient in MRE11 (dMRE11). In pooled treatment analyses, dMRE11 patients showed initially reduced DFS and OS but improved long-term DFS and OS compared with patients with an intact MRE11 T11 tract. In the subgroup of dMRE11 patients treated with IFL, an unexplained early increase in mortality but better long-term DFS than IFL-treated pMRE11 patients was observed. CONCLUSIONS: Analysis of this relatively small number of patients and events showed that the dMRE11 marker predicts better prognosis independent of treatment in the long-term. In subgroup analyses, dMRE11 patients treated with irinotecan exhibited unexplained short-term mortality. MRE11 status is readily assayed and may therefore prove to be a useful prognostic marker, provided that the results reported here for a relatively small number of patients can be generalized in independent analyses of larger numbers of samples. TRIAL REGISTRATION: ClinicalTrials.gov NCT00003835.

Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs.

Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathological features of Bloom syndrome, we analyzed mRNA and microRNA (miRNA) expression in fibroblasts from individuals with Bloom syndrome and in BLM-depleted control fibroblasts. We identified mRNA and miRNA expression differences in Bloom syndrome patient and BLM-depleted cells. Differentially expressed mRNAs are connected with cell proliferation, survival, and molecular mechanisms of cancer, and differentially expressed miRNAs target genes involved in cancer and in immune function. These and additional altered functions or pathways may contribute to the proportional dwarfism, elevated cancer risk, immune dysfunction, and other features observed in Bloom syndrome individuals. BLM binds to G-quadruplex (G4) DNA, and G4 motifs were enriched at transcription start sites (TSS) and especially within first introns (false discovery rate ≤ 0.001) of differentially expressed mRNAs in Bloom syndrome compared with normal cells, suggesting that G-quadruplex structures formed at these motifs are physiologic targets for BLM. These results identify a network of mRNAs and miRNAs that may drive the pathogenesis of Bloom syndrome.

Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

Enzymes that form transient DNA-protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA-protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine.

CpG island methylator phenotype is associated with response to adjuvant irinotecan-based therapy for stage III colon cancer.

BACKGROUND & AIMS: The CpG island methylator phenotype (CIMP), defined by a high frequency of aberrantly methylated genes, is a characteristic of a subclass of colon tumors with distinct clinical and molecular features. Cohort studies have produced conflicting results on responses of CIMP-positive tumors to chemotherapy. We assessed the association between tumor CIMP status and survival of patients receiving adjuvant fluorouracil and leucovorin alone or with irinotecan (IFL). METHODS: We analyzed data from patients with stage III colon adenocarcinoma randomly assigned to groups given fluorouracil and leucovorin or IFL after surgery, from April 1999 through April 2001. The primary end point of the trial was overall survival and the secondary end point was disease-free survival. DNA isolated from available tumor samples (n = 615) was used to determine CIMP status based on methylation patterns at the CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1 loci. The effects of CIMP on survival were modeled using Kaplan-Meier and Cox proportional hazards; interactions with treatment and BRAF, KRAS, and mismatch repair (MMR) status were also investigated. RESULTS: Of the tumor samples characterized for CIMP status, 145 were CIMP positive (23%). Patients with CIMP-positive tumors had shorter overall survival times than patients with CIMP-negative tumors (hazard ratio = 1.36; 95% confidence interval: 1.01-1.84). Treatment with IFL showed a trend toward increased overall survival for patients with CIMP-positive tumors, compared with treatment with fluorouracil and leucovorin (hazard ratio = 0.62; 95% CI: 0.37-1.05; P = .07), but not for patients with CIMP-negative tumors (hazard ratio = 1.38; 95% CI: 1.00-1.89; P = .049). In a 3-way interaction analysis, patients with CIMP-positive, MMR-intact tumors benefited most from the addition of irinotecan to fluorouracil and leucovorin therapy (for the interaction, P = .01). CIMP was more strongly associated with response to IFL than MMR status. Results for disease-free survival times were comparable among all analyses. CONCLUSIONS: Patients with stage III, CIMP-positive, MMR-intact colon tumors have longer survival times when irinotecan is added to combination therapy with fluorouracil and leucovorin.