Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G 2 arrest in HCT116 human colon cancer cells, as well as Mlh1 ؊/؊ murine embryonic fibroblasts, after treatment with 5-fluoro-2-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2 In addition to its roles in correcting DNA replication errors and editing recombination intermediates, DNA mismatch repair (MMR) 1 can process numerous DNA lesions (1-4). In fact, an intact MMR system is required for the lethality of specific DNA-damaging agents such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), 6-thioguanine (6-TG), and cisplatin (5-7). MMR also mediates the lethality of fluoropyrimidines (FPs) such as 5-fluorouracil (FU) and 5-fluoro-2Ј-deoxyuridine (FdUrd) (8, 9). Inactivation of MMR allows resistance to the cytotoxic effects of these agents, a phenomenon referred to as "damage tolerance" (10 -13). Importantly, this enables cancer cells to uncouple persistent DNA damage from cell death, resulting in increased drug resistance (14 -16).The two major gene products that comprise MMR are MSH2 (which heterodimerizes with MSH3 or MSH6 to recognize mispairs and loops in DNA) and MLH1 (which heterodimerizes with PMS2 or MLH3 to act as a molecular matchmaker between the MSH2 complex and other DNA repair/replication and perhaps cell cycle factors) (17, 18). Defects in these two genes account for most cases of hereditary non-polyposis colorectal cancer, a familial condition with a predisposition to cancers of the colon, endometrium, stomach, ovary, and biliary tracts (19), as well as sporadic tumors of the colon (20), endometrium (21), stomach (22), head and neck (23), and prostate (24).Others and we (8, 9) have demonstrated that cells deficient in MLH1 are resistant to the cytotoxic effects of FU and FdUrd. Because FPs are the agents of choice in the treatment of colorectal cancer, understanding potential resistance mechanisms is important. FPs exert cytotoxic effects through incorporation into RNA and/or DNA, as well as inhibition of thymidylate synthase (TS). The inhibition of TS, which is the central enzyme of de novo pyrimidine synthesis, leads to decreases in intracellular dTTP pools; this depletion results in immediate cytostatic effects (via inhibition of DNA synthesis) and alters dNTP pool sizes (thus increasing the error rate of DNA polymerase) (25). A hallmark of MMR deficiency is instability in the length of repetitive sequences in DNA, referred to as microsatellite instability (MSI). This reflects the inability of MMRdeficient cells to correct insertions and deletions in their DNA that result from polymerase slippage at these sequences (26). It is also an easily measured clinical marker. Due to the resistance of MMR-deficient (i.e. MSI ϩ ) cancer cells to FU and FdUrd, one woul...

Tumor necrosis factor-␣ (TNF) is initially expressed as a 26-kDa membrane-bound precusor protein (pro-TNF) that is shed proteolytically from the cell surface, releasing soluble 17-kDa TNF. We have identified human ADAM 10 (HuAD10) from THP-1 membrane extracts as a metalloprotease that specifically clips a peptide substrate spanning the authentic cleavage site between Ala 76 and Val 77 in pro-TNF. To confirm that HuAD10 has TNF processing activity, we cloned, expressed, and purified an active, truncated form of HuAD10. Characterization of recombinant HuAD10 (rHuAD10) suggests that this enzyme has many of the properties (i.e. substrate specificity, metalloprotease activity, cellular location) expected for a physiologically relevant TNF-processing enzyme. Tumor necrosis factor-␣ (TNF)1 is a cytokine that is produced primarily by activated monocytes and macrophages in response to a variety of physiological stresses such as infection or injury (1). Clinical and experimental evidence has also identified TNF as a mediator of chronic autoimmune diseases such as rheumatoid arthritis (2) and Crohn's disease (3), as well as being involved in the pathology associated with sepsis (1). Accordingly, TNF has become a primary target for therapeutic intervention of several inflammatory diseases.TNF is initially synthesized as a 26-kDa membrane-bound protein (pro-TNF) that is subsequently cleaved to release soluble 17-kDa TNF with an NH 2 terminus of Val 77 (4). The identity of the protease(s) responsible for TNF processing remains controversial. Robache-Gallea et al. (5) detected a serine protease activity (PR3) in monocyte membrane preparations which was able to generate a 17-kDa active TNF with an NH 2 terminus of Arg 78 . In 1994, the partial isolation and characterization of a membrane-bound activity capable of generating the 17-kDa form of TNF were reported (6). The TNF-processing enzyme was thought to be a non-matrix metalloprotease since it was not inhibited by TIMP-1,2 or phosphoramidon, and no calcium requirement was detected. More recently, two members of the ADAM family (TNF-␣ converting enzyme (TACE) and bovine ADAM 10 (BoAD10)) have been shown to possess pro-TNF processing activity (7-9).In this report we describe the isolation, cloning, and characterization of a TNF-processing enzyme from the human monocytic cell line THP-1. The purified recombinant enzyme, rHuAD10, specifically recognizes the authentic cleavage site in pro-TNF and is sensitive to metalloprotease inhibitors that block soluble TNF production (6). MATERIALS AND METHODSReagents-Dinitrophenol-labeled polypeptides were synthesized by the Fmoc (N-(9-fluorenyl)methoxycarbonyl)/t-butyl-based solid phase peptide chemistry method using an Applied Biosystems, Inc. 431A peptide synthesizer (10). All peptides were purified by reversed phase HPLC, and molecular weights were verified by mass spectrometry.HPLC Peptide Assay-TNF processing activity was measured as the ability to cleave a 12-residue peptide spanning the Ala 76 -Val 77 site in pro-TNF. The chromatopho...

CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The chemotaxis (Che) proteins couple flagellar rotation to the environment by transducing chemotactic signals from specific transmembrane chemoreceptors to the flagellar switch apparatus. One of these proteins, CheY, is a small (14-kDa), single-domain protein homologous to the regulator proteins of bacterial two-component sensory transduction systems (3) and is the only two-component signaling protein for which the high-resolution crystal structure has been determined (47, 51). Like other regulators, CheY activity appears to be controlled by phosphorylation (reviewed in references 8 and 48); cheY function is required for CW flagellar rotation (34, 35); CheY is phosphorylated in vitro by CheA (16,17,33,54); and cheY or cheA mutations that disrupt phosphotransfer reactions also result in smoothswimming phenotypes, suggesting that CheY-P is the active CW generator (9,33). While CheY is thought to act directly on the switch apparatus to regulate flagellar rotation (11,38,39,53,55), exactly how CheY functions is not known.

Twelve x-ray-induced transcripts (xips), differentially expressed 8-to 230-fold in x-irradiated versus unirradiated radioresistant human melanoma (Ul-Mel) cells, were isolated as cDNA clones (xipl through xipl2) after four rounds of differential hybridization. Northern analyses revealed rare, medium, and abundant xips, ranging in size from 1.2 to 10 kb. All transcripts were transiently expressed and induced by low, but not by high (>600 cGy), doses of radiation. Three banscripts (xip4, -7, and -12) were induced only by ionizing radiation, and many (i.e.,

No abstract

The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA-and DNA-levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA-level antimetabolite formation and incorporation believed primarily responsible for tumour-selective responses. Attempts to direct FUra into DNA-level antimetabolites, based on mechanism-of-action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase-5-fluoro-2′-deoxyuridine 5′ monophoshate (FdUMP)-5,10-methylene tetrahydrofolate (5,10-CH2FH4) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR-dependent direct cell death signalling or futile cycle responses will be discussed. As 10-30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue-1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5-fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re-expression of hMLH1 and re-sensitization of tumours to FP therapy. (2009) Keywords: 5-fluorouracil; DNA mismatch repair; thymidylate synthase; hypermethylation; hMLH1; MMR/c-Abl/p73a/ GADD45a signalling Abbreviations: 5,10-CH2FH4, 5,10-methylene tetrahydrofolate; 5′-dRP, 5′-deoxyribose phosphate; AM, adaptors/mediators; AP, apyrimidinic/apurinic site; APE, apurinic/apyrimidinic endonuclease; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia-and-rad3-related; BER, base excision repair; CD, cytosine deaminase; dCMP, deoxycytidylate; dCMPD, deoxycytidylate deaminase; dH4Urd, deoxytetrahydrouridine; DS, damage sensors; DSBs, DNA double-strand breaks; dThyd, thymidine; ES, embryonic stem; FdCyd, 5-fluoro-2′-deoxycyticine; FdUDP, 5-fluoro-2′-deoxyuridine 5′-diphosphate; FdUMP, 5-fluoro-2′-deoxyuridine 5′-monophosphate; FdUTP, 5-fluoro-2′-deoxyuridine-5′-triphosphate; FdUrd, 5-fluoro-2′-deoxyuridine; FEN1, flap-endonuclease 1; FPs, fluorinated pyrimidines; FUDP, 5-fluorouridine 5′-diphosphate; FUra (or 5-FU), 5-fluorouracil; FUrd, fluorouridine; FUTP, 5-fluorouridine-5′-triphosphate; GADD45, growth arrest and DNA damage-inducible-45 gene/ protein; H4Urd, 3,4,5,6-tetrahydrouridine; hMLH1, human MutL homologue-1; hMSH2, human MutS homologue-2; hMSH3, human MutS homologue-3; hMSH6, human MutS homologue-6; hPMS2, postmeiotic segregation increased 2; IDL, insertion/deletion loop-type; MBD4, methyl-CpG binding domain protein 4; British Journal of Phar...

Deficiencies in DNA mismatch repair (MMR) have been found in both hereditary cancer (i.e., hereditary nonpolyposis colorectal cancer) and sporadic cancers of various tissues. In addition to its primary roles in the correction of DNA replication errors and suppression of recombination, research in the last 10 years has shown that MMR is involved in many other processes, such as interaction with other DNA repair pathways, cell cycle checkpoint regulation, and apoptosis. Indeed, a cell's MMR status can influence its response to a wide variety of chemotherapeutic agents, such as temozolomide (and many other methylating agents), 6-thioguanine, cisplatin, ionizing radiation, etoposide, and 5-fluorouracil. For this reason, identification of a tumor's MMR deficiency (as indicated by the presence of microsatellite instability) is being utilized more and more as a prognostic indicator in the clinic. Here, we describe the basic mechanisms of MMR and apoptosis and investigate the literature examining the influence of MMR status on the apoptotic response following treatment with various therapeutic agents. Furthermore, using isogenic MMR-deficient (HCT116) and MMR-proficient (HCT116 3-6) cells, we demonstrate that there is no enhanced apoptosis in MMR-proficient cells following treatment with 5-fluoro-2'-deoxyuridine. In fact, apoptosis accounts for only a small portion of the induced cell death response.

No abstract