Enzymatic bypass of an N6-deoxyadenosine DNA-ethylene dibromide-peptide cross-link by translesion DNA polymerases.

Unrepaired DNA-protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA-protein cross-links can be cleaved into DNA-peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide-DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (kcat/Km) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended.

A Wheat ß-Patchoulene Synthase Confers Resistance Against Herbivory in Transgenic Arabidopsis.

Terpenoids play important roles in plant defense. Although some terpene synthases have been characterized, terpenoids and their biosynthesis in wheat (Triticumaestivum L.) still remain largely unknown. Here, we describe the identification of a terpene synthase gene in wheat. It encodes a sesquiterpene synthase that catalyzes ß-patchoulene formation with E,E-farnesyl diphosphate (FPP) as the substrate, thus named as TaPS. TaPS exhibits inducible expression in wheat in response to various elicitations. Particularly, alamethicin treatment strongly induces TaPS gene expression and ß-patchoulene accumulation in wheat. Overexpression of TaPS in Arabidopsis successfully produces ß-patchoulene, verifying the biochemical function of TaPS in planta. Furthermore, these transgenic Arabidopsis plants exhibit resistance against herbivory by repelling beet armyworm larvae feeding, thereby indicating anti-herbivory activity of ß-patchoulene. The catalytic mechanism of TaPS is also explored by homology modeling and site-directed mutagenesis. Two key amino acids are identified to act in protonation and stability of intermediates and product formation. Taken together, one wheat sesquiterpene synthase is identified as ß-patchoulene synthase. TaPS exhibits inducible gene expression and the sesquiterpene ß-patchoulene is involved in repelling insect infestation.

Pharmacologic properties of polyethylene glycol-modified Bacillus thiaminolyticus thiaminase I enzyme.

We have previously shown that the bacterial enzyme thiaminase 1 has antitumor activity. In an attempt to make thiaminase I a more effective pharmaceutical agent, we have modified it by adding polyethylene glycol (PEG) chains of various lengths. We were surprised to find that 5k-PEGylation eliminated thiaminase cytotoxic activity in all cell lines tested. Both native thiaminase and 5k-PEGylated thiaminase efficiently depleted thiamine from cell culture medium, and both could use intracellular phosphorylated thiamine as substrates. However, native enzyme more effectively depleted thiamine and thiamine diphosphate in RS4 leukemia cell cytosol, and native thiaminase depressed cellular respiration, whereas PEGylated thiaminase did not. Despite the lack of in vitro cytotoxicity, PEGylation markedly increased the in vivo toxicity of the enzyme. Pharmacokinetic studies revealed that the half-life of native thiaminase was 1.5 h compared with 34.4 h for the 5k-PEGylated enzyme. Serum thiamine levels were depleted by both native and 5k-PEGylated enzyme. Despite superior pharmacokinetics, 5k-PEGylated thiaminase showed no antitumor effect against an RS4 leukemia xenograft, in contrast to native thiaminase, which showed antitumor activity. PEGylation of thiaminase I has demonstrated that depression of mitochondrial function contributes, at least in part, to its anticancer activity. PEGylation also enhances plasma retention time, which increased its vivo toxicity and decreased its activity against a leukemia xenograft, the opposite of the desired effects. These studies suggest that the mechanism of anticancer cytotoxicity of thiaminase requires acute depression of cellular respiration, whereas systemic toxicity is related to the duration of extracellular thiamine depletion.

Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells.

The primary function of O(6)-alkylguanine-DNA alkyltransferase (AGT) is to maintain genomic integrity in the face of damage by both endogenous and exogenous alkylating agents. However, paradoxically, bacterial and mammalian AGTs have been shown to increase cytotoxicity and mutagenicity of dihaloalkanes and other bis-electrophiles when expressed in bacterial cells. We have extended these studies to mammalian cells using CHO cells that lack AGT expression and CHO cells stably transfected with a plasmid that expresses human AGT. The cytotoxicity of 1,2-dibromoethane, dibromomethane and epibromohydrin was significantly increased by the presence of AGT but cytotoxicity of butadiene diepoxide was not affected. Mutations caused by these agents were assessed using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a reporter gene. There was a small (c. 2-3-fold) but statistically significant AGT-mediated increase in mutations caused by 1,2-dibromoethane, dibromomethane and epibromohydrin. Analysis of the mutation spectrum induced by 1,2-dibromoethane showed that the presence of AGT also altered the types of mutations with an increase in total base substitution mutants due to a rise in transversions at both G:C and A:T sites. AGT expression also led to mutations arising from the transcribed strand, which were not seen in cells lacking AGT. Although the frequency of deletion mutations was decreased by AGT expression, the formation of large deletions (> or = 3 exons) was increased. This work demonstrates that interaction of AGT with some bis-electrophiles can cause mutagenicity and diminished cell survival in mammalian cells. It is consistent with the hypothesis that DNA-AGT cross-links, which have been characterized in experiments with purified AGT protein and such bis-electrophiles, can be formed in mammalian cells.

Antiulcerogenic and anti-inflammatory activities of the essential oil from Pterodon emarginatus seeds.

OBJECTIVES: The objective of this work was to investigate the antiulcerogenic and anti-inflammatory activities of the essential oil from Pterodon emarginatus seeds. METHODS: The following tests were used: ulcers induced by ethanol, indometacin and HCl/ethanol, and pleurisy induced by carrageenan in Swiss albino rats. The rats were treated by the oral route with essential oil of P. emarginatus seeds. KEY FINDINGS: The essential oil at 100, 300 and 500 mg/kg exhibited significant protection against ulcers induced by ethanol, indometacin and HCl/ethanol (P < 0.001). The essential oil caused a marked reduction in the exudate volume and inhibited leucocyte and neutrophil influx (P < 0.05) in carrageenan-induced pleurisy. Moreover, the essential oil significantly decreased nitric oxide (NO) and interleukin-1 (IL-1) levels, without affecting tumour necrosis factor-alpha production. CONCLUSIONS: The results demonstrated the marked antiulcerogenic and anti-inflammatory effects of the essential oil from P. emarginatus, which are, at least in part, a consequence of NO and IL-1 modulation. P. emarginatus or its constituents might represent new therapeutic options to treat gastric ulcers and inflammatory diseases.

Farnesyltransferase inhibitors and human malignant pleural mesothelioma: a first-step comparative translational study.

It is known that the potential clinical use of farnesyltransferase inhibitors (FTI) could be expanded to include cancers harboring activated receptor tyrosine kinases. Approximately 70% of malignant pleural mesotheliomas (MPM) overexpress epidermal growth factor receptors (EGFR) and a subset express both EGFR and transforming growth factor alpha (TGF-alpha), suggesting an autocrine role for EGFR in MPM. We checked on MPM cells (10 human cell lines, 11 primary cultures obtained by human biopsies, and 7 short-term normal mesothelial cell cultures) concerning the following: (a) the relative overexpression of EGFR (Western blotting, flow cytometry, immunohistochemistry), (b) the relative expression of EGFR ligands (EGF, amphiregulin, TGF-alpha, ELISA), (c) the relative increase of the activated form of Ras (Ras-bound GTP) after EGF stimulation (Ras activation assay), (d) the efficacy of five different FTIs (HDJ2 prenylation, cell cytotoxicity, and apoptosis using ApopTag and gel ladder). EGFR was overexpressed in MPM cells compared with normal pleural mesothelial cells in equivalent levels as in non-small cell lung cancer cells A459. MPM cells constitutively expressed EGFR ligands; however, Ras activation was attenuated at high EGF concentrations (100 ng/mL). Growth of MPM cells was substantially not affected by treatment with different FTIs (SCH66336, BMS-214662, R115777, RPR-115135, and Manumycin). Among these, BMS-214662 was the only one moderately active. BMS-214662 triggered apoptosis in a small fraction of cells (not higher than 30%) that was paralleled by a slight decrease in the levels of TGF-alpha secreted by treated MPM cells. Our data highlighted the concept that the same signaling pathway can be regulated in different ways and these regulations can differ between different cells of different origin.

Farnesyltransferase inhibitors as anticancer agents: critical crossroads.

Farnesyltransferase (FT) inhibitors were originally designed as anticancer agents, and were thought to act by inhibiting the farnesylation of mutant Ras proteins. However, these compounds were subsequently demonstrated to have antitumor effects even in the absence of Ras mutations and it has now become clear that other protein targets are involved. This article discusses the preclinical and clinical development of FT inhibitors. To date, tipifarnib (Zarnestra; Janssen Pharmaceutica NV) and lonafarnib (Sarasar; Schering-Plough Research Institute) are the only two FT inhibitors to have been evaluated in phase III clinical trials. The clinical results of these two compounds are presented below, with emphasis on ways of enhancing the possibility of a successful FT inhibitor anticancer drug. Details of new FT inhibitors disclosed since the beginning of 2003 are also included.

Investigation of the effect of the farnesyl protein transferase inhibitor R115777 on isoprenylation and intracellular signalling by the prostacyclin receptor.

The human (h) and mouse (m) prostacyclin receptors (IPs) undergo isoprenylation through attachment of a C-15 farnesyl moiety within their conserved carboxyl terminal -CSLC sequences. Herein, the effects of a novel farnesyl transferase inhibitor R115777 on signalling by the hIP and mIP, overexpressed in human embryonic kidney 293 cells, and by the hIP endogenously expressed in human erythroleukaemia cells were investigated. R115777 significantly impaired IP-mediated cyclic AMP generation (IC(50) 0.37-0.60 nm) and intracellular calcium ([Ca(2+)](i)) mobilization (IC(50) 37-65 nm), but had no effect on signalling by the control nonisoprenylated beta(2) adrenergic receptor or the alpha or beta isoforms of the human thromboxane A(2) receptor (TP). Additionally, R115777 significantly reduced IP-mediated cross-desensitization of signalling by the TP alpha, but not by the TP beta, isoform of the human TP and impaired the farnesylation-dependent processing of the chaperone HDJ-2 protein (IC(50) 4.5 nm). Furthermore, R115777 fully impaired isoprenylation of both the Ha-Ras(WT) and Ha-Ras(CSLC) in vitro and in whole cells confirming that, unlike N-Ras and Ki-Ras, the -CSLC motif associated with the IP cannot support alternative geranylgeranylation in the presence of R115777 and does not act as a substrate for geranylgeranyl transferase 1 in vitro or in whole cells. In conclusion, these data confirm that R115777 potently impairs IP isoprenylation and signalling, and suggest that clinically it may not only target Ras proteins but may also disrupt IP isoprenylation, events which could impact on physiologic processes in which prostacyclin and its receptor are implicated.

Phase II study of the farnesyl transferase inhibitor R115777 in patients with sensitive relapse small-cell lung cancer.

BACKGROUND: R115777 (tipifarnib, Zarnestra) is a farnesyl transferase inhibitor that blocks the farnesylation of proteins involved in signal transduction pathways critical for cell proliferation and survival. This multicenter phase II study was conducted to determine the efficacy, tolerability and pharmacokinetics of R115777 in patients with relapsed small-cell lung cancer (SCLC). PATIENTS AND METHODS: Patients who had a partial or complete response to their initial chemotherapy regimen, followed by at least 3 months off treatment before relapse (sensitive relapse) were eligible. R115777 was administered in 3-week cycles at a dose of 400 mg orally twice daily for 14 consecutive days followed by 7 days off treatment. RESULTS: Twenty-two patients were enrolled. The median progression-free survival was 1.4 months and median overall survival was 6.8 months. Non-hematological toxicities were predominantly grade 1-2 and included nausea (64%) and fatigue (60%). Grade 3-4 granulocytopenia and thrombocytopenia occurred in 27% and 23% of patients, respectively. Febrile neutropenia was not observed. Pharmacokinetic studies demonstrated peak plasma concentrations of R115777 2.6-4.5 h after oral dosing and no significant drug accumulation. The trial was terminated because no objective responses were observed in 20 patients evaluable for response. CONCLUSIONS: R115777 showed no significant antitumor activity as a single agent in sensitive-relapse SCLC.

[Farnesyl transferase inhibitors: one target may be found in another]. / Les inhibiteurs de farnésyl transférase: une cible peut en cacher une autre.

The fact that proteins such as Ras require farnesylation to induce malignant transformation prompted many investigators to design farnesyl transferase inhibitors (FTI) as novel anticancer drugs. FTIs inhibit the growth of ras transformed cells in vitro and induce tumor regression in ras dependent tumor in vivo. Moreover, FTIs inhibit tumor progression in human tumor xenograft models. Currently, FTIs are undergoing phase I and II trials in various cancer types. They show impressive antitumour efficacy and they lack toxicity. Despite these promising results, the development of such molecules in hindered by the absence of appropriate clinical endpoints and of surrogate biological markers. Indeed, it seems likely that Ras is not the critical target of FTIs and that inhibition of the farnesylation of proteins such as RhoB, might also contribute to the observed antitumour properties. Identification of targets that underlie their biological effect is essential in order to predict and evaluate their efficacy.

Targeted therapies for stage III non-small cell lung cancer: integration in the combined modality setting.

Combined modality therapy represents current standard therapy for locoregionally advanced non-small cell lung cancer. In particular, concomitant chemoradiotherapy has emerged as the preferred approach. At the same time, efforts to increase locoregional and systemic antitumor activity are necessary to further improve long-term survival rates for these patients. In recent years, multiple cellular targets have emerged in the development of novel antitumor therapies. Several of these are of high relevance in the carcinogenesis of lung cancer including the epidermal growth factor receptor (EGFR), the ras signaling pathway, tumor angiogenesis, and cyclooxygenase-2 (COX-2) expression. Novel agents directed against these targets are currently under development with promising early results in non-small cell lung cancer when administered as single agents or in combination with chemotherapy in stage IV or recurrent disease. Similarly their use with concurrent radiation therapy is supported by preclinical models. Selected early clinical trials utilizing these agents in combination with radiotherapy or chemoradiotherapy are discussed.

Phase II study of the farnesyl transferase inhibitor R115777 in patients with advanced non-small-cell lung cancer.

PURPOSE: This phase II study was undertaken to define the efficacy and pharmacodynamics of R115777, a farnesyl transferase inhibitor, in the first-line treatment of patients with advanced non-small-cell lung cancer. PATIENTS AND METHODS: Forty-four patients with measurable stage IIIB (pleural effusion) or stage IV disease received 193 courses of treatment (median, 2.0; range, 1 to 22) with R115777 300 mg administered orally twice daily for 21 of every 28 days. Buccal mucosa samples and peripheral blood mononuclear cells (PBMCs) were collected before and after 8 days of treatment to evaluate inhibition of farnesyl transferase in vivo. RESULTS: No objective complete or partial responses were documented. Seven patients (16%; 95% confidence interval [CI], 8% to 31%) had disease stabilization for greater than 6 months. Median survival was 7.7 months (95% CI, 6.5 to 10.5) and time to progression was 2.7 months (95% CI, 1.9 to 3.1). The most severe toxicity was neutropenia (9% grade 3, 7% grade 4) and the most common toxicities were anemia (50% grade 1 or 2, 5% grade 3) and anorexia (50% grade 1 or 2, 2% grade 3). Mild peripheral neuropathy occurred in 25% of patients. Evidence of farnesyl transferase inhibition was documented in 83% of patients. CONCLUSION: Single-agent R115777 was well tolerated in patients with advanced NSCLC, but demonstrated minimal clinical activity. Inhibition of farnesylation in vivo was consistently documented. On the basis of promising results of farnesyl transferase inhibitor combinations with standard chemotherapy agents, future studies of this agent in NSCLC should be in combination with systemic chemotherapy.

Dominant negative alpha-subunit of farnesyl- and geranylgeranyl-transferase I inhibits insulin-induced differentiation of 3T3-L1 pre-adipocytes.

OBJECTIVE: To investigate whether the expression of a dominant negative (DN) farnesyl- and geranygeranyl-transferase I (FTase/GGTase I) alpha-subunit in 3T3-L1 pre-adipocytes can inhibit insulin's ability to induce differentiation. DESIGN: 3T3-L1 pre-adipocytes were stably transfected with vector alone or vector expressing a mutated DN FTase/GGTase I alpha-subunit (S60A)(S62A) and incubated in serum-free medium in the absence and presence of insulin. MEASUREMENTS: Various assays were performed to determine the effect of DN FTase/GGTase I alpha-subunit expression in 3T3-L1 pre-adipocyte on insulin-induced DNA synthesis, cell count, phosphorylation of the FTase/GGTase I alpha-subunit, FTase and GGTase I activity, amounts of prenylated p21Ras and RhoA, phosphorylation of MAP kinase and Akt, and differentiation to mature fat cells. RESULTS: Expression of DN FTase/GGTase I alpha-subunit inhibited insulin's ability to increase DNA synthesis, cell count, FTase and GGTase I activity, amounts of prenylated p21Ras and RhoA, and magnitude of phosphorylation of MAP kinase. Expression of DN FTase/GGTase I alpha-subunit in 3T3-L1 pre-adipocytes was without effect on insulin-induced Akt phosphorylation. CONCLUSION: Expression of DN FTase/GGTase I alpha-subunit inhibits insulin-induced differentiation of 3T3-L1 pre-adipocytes to mature adipocytes, and thus could indicate potential therapeutic avenues to assuage the deleterious effects of obesity and type 2 diabetes.

Involvement of NMDA receptors and a p21Ras-like guanosine triphosphatase in the constitutive activation of nuclear factor-kappa-B in cortical neurons.

The transcription factor nuclear factor-kappa-B (NF-kappaB) is now recognised as a key mediator of physiological and pathological plasticity in the central nervous system (CNS), and ionotropic glutamate receptor stimulation potently triggers NF-kappaB activation. This study was designed to identify the mechanisms responsible for the high basal levels of activated NF-kappaB present in neurons in the cerebral cortex. In cultured cortical neurons, the basal levels of activated NF-kappaB were reduced by the glutamate receptor antagonists MK801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but were not affected by exposure to a mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor, a p38 MAP kinase inhibitor or a cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor. However, activated NF-kappaB levels were reduced by a guanylate cyclase inhibitor, the Src-family tyrosine kinase inhibitor PP1, or the farnesyl transferase inhibitors manumycin and farnesyl transferase (Ftase) inhibitor 1. There was no additive effect when MK801 was applied together with manumycin. These results suggest that the basal levels of activated NF-kappaB in cortical neurons are maintained partially by synaptic activity involving N-methyl- D-aspartate (NMDA) and AMPA/kainate glutamate receptors, coupled to activation of an Src-family tyrosine kinase and a p21(Ras)-like guanosine triphosphatase (GTPase) in a cGMP-dependent manner. The results are intriguing in the light of the recent identification of a synaptic p21(Ras) activator stimulated by cGMP.

[How I treat...advanced cancer of the pancreas with a novel approach directed against new targets]. / Comment je traite.... Le cancer avancé du pancréas par une approche innovante dirigée contre les nouvelles cibles.

A better knowledge of fundamental mechanisms of carcinogenesis allows the development of novel therapeutic tools specifically targeting the cancer cell. Our understanding of cellular and molecular mechanisms controlling cellular cycle and cell survival is an important step for new anti-cancer treatments. This review will focus on new therapeutic's strategies in advanced pancreatic cancer.

Biologic approaches to the treatment of gastrointestinal malignancy.

Though new agents have recently been approved for the treatment of gastrointestinal malignancies, cure rates remain low and survival times of patients short. Because of these discouraging numbers, there is a vigorous search for new agents and new strategies. This article reviews some of these strategies and the status of agents in clinical development. Though an exhaustive search is impossible and the field is changing so rapidly, the agents selected here for discussion may be considered representative of others. Discussed here are agents targeting the vascular endothelial growth factor as an example of an angiogenesis inhibitor, and agents targeting the epidermal growth factor receptor, the ras oncogene, and the immune system.

Effects of the farnesyltransferase inhibitor UCF-1C/manumycin on growth and p21-ras post-translational processing in NIH3T3 cells.

Examination of the effect of the farnesylprotein transferase (FPTase) inhibitor UCF1-C/manumycin on NIH3T3 cells transfected with a normal N-ras gene and expressing high levels of the corresponding p21-ras protein showed that 10 microm UCF1-C immediately and reversibly inhibited growth in these cells, without modifying cell-death rate, thus acting as a cytostatic. There was also a 98% reduction of p21-ras neofarnesylation and a 3-fold decrease in total content in p21-ras products, yet without gross modification of the relative content in the post-translational products and without accumulation of the native protein to detectable levels. UCF1-C likewise reversibly inhibited growth in parental NIH3T3 cells, as well as in sub-strains expressing a transfected normal or mutated H-ras gene. Together with the fact that the well-developed network of actin stress fibers present in the NIH3T3 (N-ras) cells was not affected by the FPTase inhibitor, these data indicate that its growth-inhibitory effect is not necessarily in direct relation with that exerted on p21-ras processing. Alternatively, it might be causally related to the decreased prenylation of other cellular proteins, perhaps included among the 13 proteins, unrelated to p21-ras, of which the farnesylation was also reduced under UCF1-C treatment. Some cells transformed by a ras or non-ras oncogene might exhibit higher susceptibility towards FPTase inhibitors than normal cells, but this might then be attributable to differences in the pattern of expression and/or in the functional importance of non-ras farnesylated proteins.

Retroviral transfer of a bacterial alkyltransferase gene (ada) into human bone marrow cells protects against O6-benzylguanine plus 1, 3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity.

The antitumor activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is limited by the O6-alkylguanine-DNA alkyltransferase (ATase) in tumor cells and by delayed myelosuppression. Inactivation of neoplastic ATase by O6-benzylguanine (BG) improves the therapeutic index for BCNU. We have demonstrated previously that BG + BCNU-induced myelosuppression in mice is reduced by expression of the BG-resistant ATase ada in murine bone marrow. We have now generated an amphotropic retrovirus containing the ada gene and tested the effectiveness of ada expression in preventing BG + BCNU cytotoxicity in human hematopoietic progenitor cells. A retroviral producer clone with a biological titer of 6.5 x 10(4) colony-forming units/ml and 4.4 pmol ATase/mg protein was used for transduction of bone marrow. Cocultivation of these ada producer cells with progenitor cells from six normal individuals resulted in 1.9-3. 9-fold protection against BG + BCNU-induced cytotoxicity in committed progenitor cell assays. Furthermore, this cytoprotective effect was associated with a high transduction efficiency (40%) and a 2-fold increase of ATase activity in the surviving committed progenitor cell colonies. These data provide a basis for testing the clinical effectiveness of retroviral ada gene transfer into hematopoietic cells to increase the therapeutic index of BG + BCNU.