thioguanine-anhydrous and Colorectal-Neoplasms

Colitis-associated cancer represents a long-standing problem, with two new factors adding to its importance: the diffusion of inflammatory bowel disease in developing countries, and the increased availability of effective drugs that control ulcerative colitis delaying or abrogating the need for a curative colectomy. The consolidated evidence that inflammation is the unique variable that factors in colitic cancer development has conferred impetus to the search and release of anti-inflammatory/immune suppressive molecules to pursue the goal of cancer chemoprevention. Cutting-edge research has provided breakthrough insights into the mechanism of the chemopreventive actions of mesalamines, thiopurines, and probiotics, and we expand on these topics. Despite these advancements, bedside evidence is still mixed and calls for further scrutiny. Nowadays, the clinician must continue to rely on classic preventive measures such as surveillance colonoscopy, and the early and aggressive use of drugs that permit to keep the degree of mucosal inflammation to a minimum.

Topics: Animals; Anti-Inflammatory Agents; Cell Transformation, Neoplastic; Chemoprevention; Colonoscopy; Colorectal Neoplasms; Humans; Inflammatory Bowel Diseases; Mesalamine; Probiotics; Thioguanine

Fifteen patients with advanced measurable colorectal carcinoma were treated with intravenous 6-thioguanine (6-TG) at a dosage of 55 mg/m2 for 5 consecutive days every 5 weeks. Only one patient had received prior adjuvant chemotherapy. No responses were detected, and eight patients had stable disease for a medium duration of three treatment cycles. Toxicity was tolerable. We conclude that 6-TG given by this dosage schedule is ineffective in treating metastatic colorectal carcinoma.

Topics: Adult; Aged; Colorectal Neoplasms; Drug Evaluation; Female; Humans; Injections, Intravenous; Male; Middle Aged; Remission Induction; Thioguanine

In a recent issue of Cancer Cell, Amodio and colleagues report an interesting method of modulating immunosurveillance in colorectal tumors with DNA mismatch repair (MMR) heterogeneity.

Topics: Animals; Colorectal Neoplasms; DNA Mismatch Repair; Mice; Thioguanine

(64)Cu-diacetyl-bis (N(4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a potential theranostic agent targeting the over-reduced state under hypoxia within tumors. Recent clinical Cu-ATSM positron emission tomography studies have revealed a correlation between uptake and poor prognosis; however, the reason is unclear. Here, using a human colon carcinoma HT-29 model, we demonstrated that the intratumoral (64)Cu-ATSM high-uptake regions exhibited malignant characteristics, such as upregulated DNA repair and elevated %CD133(+) cancer stem-like cells. Based on this evidence, we developed a strategy to enhance the efficacy of (64)Cu-ATSM internal radiotherapy (IRT) by inhibiting DNA repair with a nucleic acid (NA) antimetabolite. The results of the analyses showed upregulation of pathways related to DNA repair along with NA incorporation (bromodeoxyuridine uptake) and elevation of %CD133(+) cells in (64)Cu-ATSM high-uptake regions. In an in vivo(64)Cu-ATSM treatment study, co-administration of an NA antimetabolite and (64)Cu-ATSM synergistically inhibited tumor growth, with little toxicity, and effectively reduced %CD133(+) cells. (64)Cu-ATSM therapy targeted malignant tumor regions with activated DNA repair and high concentrations of CD133(+) cells in the HT-29 model. NA antimetabolite co-administration can be an effective approach to enhance the therapeutic effect of (64)Cu-ATSM IRT.

Topics: AC133 Antigen; Animals; Antimetabolites, Antineoplastic; Cell Proliferation; Colorectal Neoplasms; Coordination Complexes; Copper Radioisotopes; DNA Damage; DNA Repair; Fluorouracil; HT29 Cells; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Oligonucleotide Array Sequence Analysis; Organometallic Compounds; Pemetrexed; Phenotype; Radiopharmaceuticals; Theranostic Nanomedicine; Thioguanine; Thiosemicarbazones; Time Factors; Tumor Hypoxia; Tumor Microenvironment; Xenograft Model Antitumor Assays

Epigenetic changes in multiple genes are emerging as an important mechanism for tumour cells to acquire resistance to chemotherapy. In the present work, we test the hypothesis that epigenetic organisation in cancer cells can be affected by cytostatic drugs. Colorectal cancer cells were cultured for several weeks in the presence of 6-thioguanine. Bisulphite sequencing of the CpG-rich promoter regions of two expressed genes showed a significantly increased frequency of methylated CpG sites in drug-treated cells, as compared with controls: 4.7% and 1.7%, respectively, for the HPRT gene; and 11.1% and 8.2% for CDX1. Essentially, all of the increase for the CDX1 gene was in a four CpG sub-region previously found to correlate with gene activity (P=0.006). This pattern of sparse promoter methylation fits with a recently proposed 'seeding' two-step mechanism leading up to gene inactivation in cancer cells. Taken together, our findings suggest activation in cancer cells of an epigenetic process enabling a tumour to generate drug-resistant variant cells.

Topics: Antimetabolites, Antineoplastic; Colorectal Neoplasms; DNA Methylation; Drug Resistance, Neoplasm; Epigenesis, Genetic; Homeodomain Proteins; Humans; Hypoxanthine Phosphoribosyltransferase; Lymphocytes; Promoter Regions, Genetic; Sulfites; Thioguanine; Tumor Cells, Cultured

We investigate the roles of DNA mismatch repair (MMR) and p53 in mediating the induction of autophagy in human tumor cells after exposure to 6-thioguanine (6-TG), a chemotherapy drug recognized by MMR. We also examine how activation of autophagy affects apoptosis (type I cell death) after MMR processing of 6-TG.. Using isogenic pairs of MLH1(-)/MLH1(+) human colorectal cancer cells (HCT116) and MSH2(-)/MSH2(+) human endometrial cancer cells (HEC59), we initially measure activation of autophagy for up to 3 days after 6-TG treatment using LC3, a specific marker of autophagy. We then assess the role of p53 in autophagic signaling of 6-TG MMR processing using both pifithrin-alpha cotreatment to chemically inhibit p53 transcription and small hairpin RNA inhibition of p53 expression. Finally, we use Atg5 small hairpin RNA inhibition of autophagy to assess the effect on apoptosis after MMR processing of 6-TG.. We find that MMR is required for mediating autophagy in response to 6-TG treatment in these human tumor cells. We also show that p53 plays an essential role in signaling from MMR to the autophagic pathway. Finally, our results indicate that 6-TG-induced autophagy inhibits apoptosis after MMR processing of 6-TG.. These data suggest a novel function of MMR in mediating autophagy after a chemical (6-TG) DNA mismatch damage through p53 activation. The resulting autophagy inhibits apoptosis after MMR processing of 6-TG.

Topics: Adaptor Proteins, Signal Transducing; Autophagy; Cell Line, Tumor; Colorectal Neoplasms; DNA Mismatch Repair; Endometrial Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genes, p53; HCT116 Cells; Humans; MutL Protein Homolog 1; Nuclear Proteins; Thioguanine; Tumor Suppressor Protein p53

The DNA mismatch repair (MMR) system plays an important role in mediating a G2-M checkpoint arrest and subsequent cell death following treatment with a variety of chemotherapeutic agents. In this study, using 6-thioguanine (6-TG) as a mismatch-inducing drug, we examine the role of ataxia telangiectasia mutated (ATM)/CHK2 and ATM and Rad-3 related (ATR)/CHK1 signaling pathways in MMR-mediated cell cycle responses in MMR-proficient human colorectal cancer RKO cells. We show that, in response to 6-TG (3 micromol/L x 24 hours), activating phosphorylation of CHK1 at Ser317 [CHK1(pS317)] and CHK2 at Thr68 [CHK2(pT68)] are induced differentially during a prolonged course (up to 6 days) of MMR-mediated cell cycle arrests following 6-TG treatment, with CHK1(pS317) being induced within 1 day and CHK2(pT68) being induced later. Using chemical inhibitors and small interfering RNA of the signaling kinases, we show that a MMR-mediated 6-TG-induced G2 arrest is ATR/CHK1 dependent but ATM/CHK2 independent and that ATR/CHK1 signaling is responsible for both initiation and maintenance of the G2 arrest. However, CHK2(pT68) seems to be involved in a subsequent tetraploid G1 arrest, which blocks cells that escape from the G2-M checkpoint following 6-TG treatment. Furthermore, we show that CHK2 is hyperphosphorylated at later times following 6-TG treatment and the phosphorylation of CHK2 seems to be ATM independent but up-regulated when ATR or CHK1 is reduced. Thus, our data suggest that CHK1(pS317) is involved in a MMR-mediated 6-TG-induced G2 arrest, whereas CHK2(pT68) seems to be involved in a subsequent tetraploid G1-S checkpoint. The two signaling kinases seem to work cooperatively to ensure that 6-TG damaged cells arrest at these cell cycle checkpoints.

Topics: Antimetabolites, Antineoplastic; Ataxia Telangiectasia Mutated Proteins; Base Pair Mismatch; Caffeine; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Checkpoint Kinase 1; Checkpoint Kinase 2; Colorectal Neoplasms; DNA Repair; DNA-Binding Proteins; Humans; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; RNA, Small Interfering; Staurosporine; Thioguanine; Tumor Suppressor Proteins

The DNA mismatch repair (MMR) system plays an important role in mediating cell death after treatment with various types of chemotherapeutic agents, although the molecular mechanisms are not well understood. In this study, we sought to determine what signal is introduced by MMR after 6-thioguanine (6-TG) treatment to signal a G(2)-M arrest leading to cell death.. A comparison study was carried out using an isogenic MMR(+) and MMR(-) human colorectal cancer RKO cell system, which we established for this study. Cells were exposed to 6-TG (3 micro M x 24 h) and then harvested daily for the next 3-6 days for growth inhibition assays. Cell cycle effects were determined by flow cytometry, and DNA strand breaks were measured using pulsed-field gel electrophoresis and alkaline Comet assays.. We first established MMR(+) RKO cell lines by transfection of human MutL homologue 1 (hMLH1) cDNA into the hMLH1-deficient (MMR(-)) RKO cell line. The ectopically expressed hMLH1 protein restored a MMR-proficient phenotype in the hMLH1(+) transfectants, showing a significantly increased and prolonged G(2)-M arrest followed by cell death after 6-TG exposure, compared with the vector controls. The MMR-mediated, 6-TG-induced G(2)-M arrest started on day 1, peaked on day 3, and persisted to day 6 after 6-TG removal. We found that DNA double-strand breaks were comparably produced in both our MMR(+) and MMR(-) cells, peaking within 1 day of 6-TG treatment. In contrast, single-strand breaks (SSBs) were more frequent and longer lived in MMR(+) cells, and the duration of SSB formation was temporally correlated with the time course of 6-TG-induced G(2)-M arrest.. Our data suggest that MMR mediates 6-TG-induced G(2)-M arrest by introducing SSBs to signal a persistent G(2)-M arrest leading to enhanced cell death.

Topics: Adaptor Proteins, Signal Transducing; Base Pair Mismatch; Carrier Proteins; Cell Line, Tumor; Colorectal Neoplasms; DNA Damage; DNA Repair; DNA, Single-Stranded; Dose-Response Relationship, Drug; G2 Phase; Humans; Mitosis; MutL Protein Homolog 1; Neoplasm Proteins; Nuclear Proteins; Thioguanine; Transfection

Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G(2) arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch re pair (MMR) system mediates this cell cycle checkpoint. We confirmed this observation using two additional isogenetically matched human MLH1 (hMLH1)-deficient and -proficient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cells, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-deficient and -proficient human endometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR in general, is involved in IR-related G(2) arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arrest in response to IR (6 Gy). These differences in IR-induced G(2) arrest between MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G(0)/G(1) or S phase, indicating that MMR indeed dramatically affects the G(2)-M checkpoint arrest. However, unlike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cells compared with MMR-proficient cells was noted after high-dose-rate IR. In an attempt to define the signal transduction mechanisms responsible for MMR-mediated G(2) arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-M transition. Increased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-cdc2 rapidly decreased in MMR (hMLH1 or hMSH2)-deficient cell lines at times coincident with progress from the IR-induced G(2) arrest through M phase. Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rate IR correspond temporally with the observed differences in the IR-induced G(2) arrest, suggesting that MMR proteins may exert their effect on IR-induced G(2) arrest by signaling the cdc2 pathway. Although MMR status does not significantly affect the survival of cells after high-dose-rate IR, it seems to regulate the G(2)-M checkpoint and might affect overall mutation rates.

Topics: Adaptor Proteins, Signal Transducing; Base Pair Mismatch; Carrier Proteins; CDC2 Protein Kinase; Cell Survival; Colorectal Neoplasms; DNA Repair; Endometrial Neoplasms; Female; G2 Phase; Humans; MutL Protein Homolog 1; Neoplasm Proteins; Nuclear Proteins; Ovarian Neoplasms; Phosphorylation; S Phase; Signal Transduction; Thioguanine; Tumor Cells, Cultured

Loss of DNA mismatch repair is a common finding in hereditary non-polyposis colon cancer as well as in many types of sporadic human tumours. We compared the effect of loss of DNA mismatch repair on drug sensitivity as measured by a clonogenic assay with its effect on the ability of the same drug to enrich for mismatch repair-deficient cells in a proliferating tumour cell population. Mixed populations containing 50% DNA mismatch repair-deficient cells constitutively expressing green fluorescent protein and 50% mismatch repair-proficient cells were exposed to different chemotherapeutic agents. 6-Thioguanine, to which DNA mismatch repair-deficient cells are known to be resistant, was included as a control. The results in the cytotoxicity assays and in the enrichment experiments were concordant. Treatment with either carboplatin, cisplatin, doxorubicin, etoposide or 6-thioguanine resulted in enrichment for mismatch repair-deficient cells, and clonogenic assays demonstrated resistance to these agents, which varied from 1.3- to 4.8-fold. Treatment with melphalan, paclitaxel, perfosfamide or tamoxifen failed to enrich for mismatch repair-deficient cells, and no change in sensitivity to these agents was detected in the clonogenic assays. These results identify the topoisomerase II inhibitors etoposide and doxorubicin as additional agents for which loss of DNA mismatch repair causes drug resistance. The concordance of the results from the two assay systems validates the enrichment assay as a rapid and reliable method for screening for the effect of loss of DNA mismatch repair on sensitivity to additional drugs.

Topics: Adenocarcinoma; Antineoplastic Agents; Carboplatin; Cell Separation; Cisplatin; Colorectal Neoplasms; Cyclophosphamide; DNA Repair; DNA, Neoplasm; Doxorubicin; Enzyme Inhibitors; Etoposide; Humans; Melphalan; Paclitaxel; Tamoxifen; Thioguanine; Tumor Cells, Cultured; Tumor Stem Cell Assay

Loss of DNA mismatch repair is a common finding in many types of sporadic human cancers as well as in tumors arising in patients with hereditary nonpolyposis colon cancer. The effect of the loss of DNA mismatch repair activity on sensitivity to a panel of commonly used chemotherapeutic agents was tested using one pair of cell lines proficient or deficient in mismatch repair due to loss of hMSH2 function and another due to loss of hMLH1 function. 6-Thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine, to which these cells are known to be resistant, were included in the panel as controls. The results were concordant in both pairs of cells. Loss of either hMSH2 or hMLH1 function was associated with low level resistance to cisplatin, carboplatin, and etoposide, but there was no resistance to melphalan, perfosfamide, 5-fluorouracil, doxorubicin, or paclitaxel. The results are consistent with the concept that the DNA mismatch repair proteins function as a detector for adducts produced by 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, cisplatin, and carboplatin but not for melphalan and perfosfamide. They also suggest that these proteins play a role in detecting the DNA damage produced by the binding of etoposide to topoisomerase II and propagating signals that contribute to activation of apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Antineoplastic Agents; Carboplatin; Carrier Proteins; Cisplatin; Colorectal Neoplasms; Cyclophosphamide; DNA Adducts; DNA Damage; DNA Repair; DNA-Binding Proteins; DNA, Neoplasm; Doxorubicin; Drug Resistance, Neoplasm; Endometrial Neoplasms; Etoposide; Female; Fluorouracil; Humans; Melphalan; Methylnitronitrosoguanidine; Mutagenesis; MutL Protein Homolog 1; MutS Homolog 2 Protein; Neoplasm Proteins; Nuclear Proteins; Paclitaxel; Proto-Oncogene Proteins; Thioguanine; Tumor Cells, Cultured

Recent studies have revealed that tumors in patients with hereditary nonpolyposis colon cancer are associated with high-frequency alterations of microsatellite sequences. To investigate the mechanisms and consequences of this form of genetic instability, we identified three colorectal carcinoma cell lines that express dinucleotide-repeat instability like that found in hereditary nonpolyposis colon cancer tumors and show increased rates of spontaneous mutation at selectable loci. However, the pattern of hypermutation in these cell lines differed significantly. In one line (HCT116), microsatellite mutations occurred at a remarkably high rate (approximately 10(-2) mutations per cell per generation), whereas this rate was considerably lower in the two other lines (DLD-1 and HCT15). The rate of mutation at the locus encoding hypoxanthine guanine phosphoribosyltransferase was substantially elevated (200- to 600-fold) in all three tumor cell lines, yet the types of mutations arising differed. A specific frame-shift hotspot accounted for 24% of hypoxanthine guanine phosphoribosyltransferase mutations in HCT116. The frequency of mutations at this site was reduced significantly in DLD-1 and HCT15 lines. These data suggest that the mutatw phenotypes in the colorectal carcinoma cell lines could be the consequence of mutator genes affecting different repair or error-avoidance pathways.

Topics: Cell Line; Colorectal Neoplasms; Colorectal Neoplasms, Hereditary Nonpolyposis; DNA, Neoplasm; DNA, Satellite; Genetic Markers; Humans; Hypoxanthine Phosphoribosyltransferase; Mutagenesis; Mutation; Ouabain; Repetitive Sequences, Nucleic Acid; Thioguanine; Tumor Cells, Cultured