Novel Mechanism Of Resistance Research Articles

Abstract 22: Cyclin E amplification, a novel mechanism of resistance to trastuzumab in HER2 amplified breast cancer

Abstract HER2 amplification/overexpression occurs in 20% of breast cancers and is associated with poor prognosis. Therapeutic agents against HER2, including monoclonal antibodies and tyrosine kinase inhibitors, have shown to improve the survival of these patients. However, primary and acquired resistance to these agents is a major barrier to the effective treatment of this disease. Aiming to identify the molecular pathways responsible for resistance to anti-HER2 therapy we have established HER2 positive breast cancer cell lines refractory to the anti-proliferative effects of the therapeutic antibody trastuzumab. We have studied their acquired genetic aberrations by performing gene expression microarray and genome single nucleotide polymorphisms analyses. In trastuzumab resistant cells we identified an increase in copy number variation in locus 19q12. This chromosome region comprises seven genes, among them cyclin E1 (CCNE1). We further confirmed Cyclin E over-expression in trastuzumab resistant cells by western blot. To test the hypothesis that increased cyclin E expression could play a role in the acquisition of trastuzumab resistance we have demonstrated that ablation of cyclin E expression by siRNA restores trastuzumab sensitivity in HER2 positive resistant cells. Furthermore, we have determined that trastuzumab resistant cells are highly sensitive to the specific CDK inhibitor seliciclib and its more potent derivative Cmpd. 5 (Cyclacel) both in vitro and in vivo. In addition, we have analyzed a cohort of HER2 positive breast cancer patients treated with trastuzumab-based therapy and identified a proportion of patients refractory to trastuzumab that harbor amplification of the cyclin E gene. Our results suggest that cyclin E amplification decreases trastuzumab sensitivity and may provide the rationale to test the efficacy of CDK2 inhibitors in breast cancer patients with cyclin E amplification that escaped trastuzumab therapy. 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 22.

Hepatocyte Growth Factor Reduces Susceptibility to an Irreversible Epidermal Growth Factor Receptor Inhibitor in EGFR-T790M Mutant Lung Cancer

The secondary T790M mutation in epidermal growth factor receptor (EGFR) is the most frequent cause of acquired resistance to the reversible EGFR tyrosine kinase inhibitors (EGFR-TKI), gefitinib and erlotinib, in lung cancer. Irreversible EGFR-TKIs are expected to overcome the reversible EGFR-TKI resistance of lung cancer harboring T790M mutation in EGFR. However, it is clear that resistance may also develop to this class of inhibitors. We showed previously that hepatocyte growth factor (HGF) induced gefitinib resistance of lung cancer harboring EGFR-activating mutations. Here, we investigated whether HGF induced resistance to the irreversible EGFR-TKI, CL-387,785, in lung cancer cells (H1975) harboring both L858R activating mutation and T790M secondary mutation in EGFR. CL-387,785 sensitivity and signal transduction in H1975 cells were examined in the presence or absence of HGF or HGF-producing fibroblasts with or without HGF-MET inhibitors. HGF reduced susceptibility to CL-387,785 in H1975 cells. Western blotting and small interfering RNA analyses indicated that HGF-induced hyposensitivity was mediated by the MET/phosphoinositide 3-kinase/Akt signaling pathway independent of EGFR, ErbB2, ErbB3, and ErbB4. Hyposensitivity of H1975 cells to CL-387,785 was also induced by coculture with high-level HGF-producing lung fibroblasts. The hyposensitivity was abrogated by treatment with anti-HGF neutralizing antibody, HGF antagonist NK4, or MET-TKI. We showed HGF-mediated hyposensitivity as a novel mechanism of resistance to irreversible EGFR-TKIs. It will be clinically valuable to investigate the involvement of HGF-MET-mediated signaling in de novo and acquired resistance to irreversible EGFR-TKIs in lung cancer harboring T790M mutation in EGFR.

Tamoxifen Resistance in Estrogen Receptor Positive (ER+) Breast Cancer Is Driven by Estrogen Receptor Negative (ER-) Cancer Stem-Like Cells.

Abstract Introduction: Intrinsic and acquired resistance to endocrine therapy significantly reduces survival rates of women with ER+ breast cancer. In the normal breast, multipotent stem cells are ER-. We hypothesised that tumourigenic breast cancer stem-like cells (CSCs) are ER- and therefore function independently of estrogen, representing a novel mechanism of resistance to endocrine therapy.Methods: Three ER+ cell lines, MCF-7, T47D and BT474 were assessed for ER expression by immunocytochemistry (ICC) in monolayer and after CSC enrichment. Putative CSC number was assessed by the non-adherent mammosphere (MS) assay, in the presence and absence of tamoxifen, and by Fluorescence Activated Cell Sorting (FACS) for the markers CD44+/CD24-/low/epithelial specific antigen(ESA)+. The effect of acquired Tamoxifen resistance on the CSC population of two independent Tamoxifen-resistant (TAM-R) MCF-7 variants, from Cardiff (TAM-RCARD) and Copenhagen (TAM-RCOP) was compared to parental, tamoxifen sensitive (TAM-S) controls. In the MS assay both the number and size of colonies were assessed.Results: A small sub-population of ER_ cells were demonstrated in MCF7 (6.1 ± 1.4%), T47D (6.8± 0.5) and BT474 (3.7±1.3) cells by ICC. However, the majority of putative breast CSCs (CD44+/CD24-/low/ESA-) in MCF7 cells were ER_ (73.2 ± 4.6 % p= 0.0007). There was a significant increase in the proportion of ER- cells in TAM-R cell lines (TAM-RCARD 11.6 ± 1.4% p=0.01 and TAM-RCOP 22 ± 4.8% p=0.02 respectively) compared to the parental TAM-S controls. Both TAM-R cell lines demonstrated increased mammosphere forming efficiency (MFE) compared to TAM-S cells (TAM-RCARD 4% vs. 2.5% p=0.03, and TAM-RCOP 8% vs. 3% p=0.007). The putative CSC population (CD44+/CD24-/low/ESA+) was enriched more than 2.5 fold in TAM-R compared to TAM-S parental controls. Compared with controls, the absolute MFE was not affected by Tamoxifen treatment, although mammospheres formed were significantly smaller.Conclusions: The majority of the putative CSC populations in ER+ breast cancer do not express ER. There is an increase in the proportion of putative ER- CSCs in TAM-R MCF7 cell lines. The reduction in MS size suggests that Tamoxifen targets transit amplifying cells but does not reduce the absolute number of CSC in ER+ breast cancers. These findings demonstrate a novel mechanism of endocrine resistance in ER+ breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5125.

Genetic basis of antifungal drug resistance

Antifungal resistance caused by mutations of the drug target, overexpression of the drug target, and drug efflux by the upregulation of transporters is increasingly common. Recently our understanding of fungal drug resistance has been advanced by the identification of three key transcriptional regulators of resistance: Tac1p, Upc2p, and Mrr1p. The discovery of hyperactive variants of these regulators in resistant clinical isolates confirms the importance of transcriptional regulation in the development of antifungal resistance. Alternative mechanisms of drug resistance including aneuploidy and biofilm formation have recently been documented in fungi; as well as the phenomenon of drug tolerance. Characterization of the transcriptional regulation of fungal drug resistance and the identification of novel mechanisms of resistance has implications for current therapy and for the development of future antifungal drugs.

Resistance to peroxynitrite in Neisseria gonorrhoeae

Neisseria gonorrhoeae encodes a number of important genes that aid in survival during times of oxidative stress. The same immune cells capable of oxygen-dependent killing mechanisms also have the capacity to generate reactive nitrogen species (RNS) that may function antimicrobially. F62 and eight additional gonococcal strains displayed a high level of resistance to peroxynitrite, while Neisseria meningitidis and Escherichia coli showed a four- to seven-log and a four-log decrease in viability, respectively. Mutation of gonococcal orthologues that are known or suspected to be involved in RNS defence in other bacteria (ahpC, dnrN and msrA) resulted in no loss of viability, suggesting that N. gonorrhoeae has a novel mechanism of resistance to peroxynitrite. Whole-cell extracts of F62 prevented the oxidation of dihydrorhodamine, and decomposition of peroxynitrite was not dependent on ahpC, dnrN or msrA. F62 grown in co-culture with E. coli strain DH10B was shown to protect E. coli viability 10-fold. Also, peroxynitrite treatment of F62 did not result in accumulation of nitrated proteins, suggesting that an active peroxynitrite reductase is responsible for peroxynitrite decomposition rather than a protein sink for amino acid modification.

Endotoxin, Capsule, and Bacterial Attachment Contribute to Neisseria meningitidis Resistance to the Human Antimicrobial Peptide LL-37

Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen Neisseria meningitidis and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 microM LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain. N. meningitidis bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes, siaC and siaD, which further results in upregulation of capsule biosynthesis.

Genome sequencing of linezolid-resistant Streptococcus pneumoniae mutants reveals novel mechanisms of resistance

Linezolid is a member of a novel class of antibiotics, with resistance already being reported. We used whole-genome sequencing on three independent Streptococcus pneumoniae strains made resistant to linezolid in vitro in a step-by-step fashion. Analysis of the genome assemblies revealed mutations in the 23S rRNA gene in all mutants including, notably, G2576T, a previously recognized resistance mutation. Mutations in an additional 31 genes were also found in at least one of the three sequenced genomes. We concentrated on three new mutations that were found in at least two independent mutants. All three mutations were experimentally confirmed to be involved in antibiotic resistance. Mutations upstream of the ABC transporter genes spr1021 and spr1887 were correlated with increased expression of these genes and neighboring genes of the same operon. Gene inactivation supported a role for these ABC transporters in resistance to linezolid and other antibiotics. The hypothetical protein spr0333 contains an RNA methyltransferase domain, and mutations within that domain were found in all S. pneumoniae linezolid-resistant strains. Primer extension experiments indicated that spr0333 methylates G2445 of the 23S rRNA and mutations in spr0333 abolished this methylation. Reintroduction of a nonmutated version of spr0333 in resistant bacteria reestablished G2445 methylation and led to cells being more sensitive to linezolid and other antibiotics. Interestingly, the spr0333 ortholog was also mutated in a linezolid-resistant clinical Staphylococcus aureus isolate. Whole-genome sequencing and comparative analyses of S. pneumoniae resistant isolates was useful for discovering novel resistance mutations.

Pharmacogenetics in lung cancer for the lay doctor

Lung cancer embodies the conundrum of cancer in general: why some patients with apparently similar tumors respond and others progress. In spite of a plethora of knowledge at the investigational level, in daily practice, the physician is still challenged by the lack both of prognostic markers to identify patients with a high risk of relapse and of predictive markers for customizing chemotherapy and targeted therapy. Multiple microarray studies have deciphered the important network of signaling pathways that can lead to the formation of metastasis, and we are not far from being able to predict the risk of metastasis at a particular site (bone, brain or others) according to specific gene profiles. Quantitative PCR has allowed the assessment of mRNA expression levels of key genes, identified mostly by transcriptome analysis. Growing evidence indicates that three-or five-gene signatures by quantitative PCR are highly predictive of metastasis and survival in early-stage non-small cell lung cancer, including stage IB, paving the way for the selection of high-risk patients for adjuvant chemotherapy and for the customization of treatment. A meaningful proportion of lung cancer patients are EGFR-driven, and abundant knowledge has enabled us to identify extensive networks of downstream signals that collapse when drug treatment is effective. Novel mechanisms of resistance to chemotherapy and targeted therapies can be useful for personalizing treatment and can lead to the implementation of novel targeted therapies.

Modulation of the Tumor Cell Phenotype by IFN-γ Results in Resistance of Uveal Melanoma Cells to Granule-Mediated Lysis by Cytotoxic Lymphocytes

IFN-gamma, a pleiotropic immune regulator, is implicated in both tumor immune surveillance and selection of tumor variants resistant to immune control, i.e., immunoediting. In uveal melanoma patients, elevated serum levels of IFN-gamma correlate with the spread of metastasis and represent a negative prognostic marker. Treatment with IFN-gamma boosted the MHC class I presentation machinery in uveal melanoma cells but suppressed their MHC class I-restricted CTL lysis. Tumor cells exposed to IFN-gamma efficiently activated specific CTL but were less susceptible to permeabilization by perforin and exhibited a decreased capacity to bind and incorporate granzyme B. These results define a novel mechanism of resistance to granule-mediated CTL lysis in human tumors. Furthermore, the data suggest that immunoediting is not limited to genetic or epigenetic changes resulting in stable cellular phenotypes but also involves an inducible modulation of tumor cells in response to a microenvironment associated with immune activation.

Topoisomerase IIβ Negatively Modulates Retinoic Acid Receptor α Function: a Novel Mechanism of Retinoic Acid Resistance

Interactions between retinoic acid (RA) receptor alpha (RARalpha) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In patients with acute promyelocytic leukemia (APL), the RARalpha gene is fused with the promyelocytic leukemia (PML) gene via the t(15;17) translocation, resulting in the expression of a PML/RARalpha fusion protein. Here, we report that topoisomerase II beta (TopoIIbeta) associates with and negatively modulates RARalpha transcriptional activity and that increased levels of and association with TopoIIbeta cause resistance to RA in APL cell lines. Knockdown of TopoIIbeta was able to overcome resistance by permitting RA-induced differentiation and increased RA gene expression. Overexpression of TopoIIbeta in clones from an RA-sensitive cell line conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicated that TopoIIbeta is bound to an RA response element and that inhibition of TopoIIbeta causes hyperacetylation of histone 3 at lysine 9 and activation of transcription. Our results identify a novel mechanism of resistance in APL and provide further insight to the role of TopoIIbeta in gene regulation and differentiation.

Résistance plasmidique aux quinolones

Objectives Analysis of resistance mechanisms and to determine the place of plasmidic mediated mechanism. Methods They are based on literature search and personal experience. During 30 years, the only known mechanisms of resistance to quinolones were chromosome borne. The first plasmid-mediated resistance determinant to quinolones, QnrA, was discovered in 1998, followed in 2004 by two other markers QnrB and QnrS in several enterobacterial species. These markers interact with quinolones, the topoisomerases, and DNA, thus limiting the binding of the quinolones to their target. In 2005, a second plasmid-borne mechanism, which independantly contributes to the quinolones resistance by modification of the antibiotic molecule was described. This protein, AAC (6’)-Ib-cr, was a variant of the 6’ acetyl transferase, which is known to modify the chemical structure of aminoglycosides, and presented an enlarged enzymatic spectrum toward ciprofloxacin and norfloxacin. Epidemiology of plasmidic resistance to quinolones The plasmid-borne mechanisms of resistance to quinolones are disseminated worldwide among enterobacterial isolates. They confer a high level of resistance to first generation quinolones and confer decreased susceptibility to fluoroquinolones. Conclusion There is no phenotypic criteria allowing difference between chromosome and plasmid-borne mechanisms of resistance to quinolones. A molecular-based approach is required to identify these novel mechanisms of resistance in clinical isolates.

713 POSTER Molecularly targeted immunochemotherapeutic formulation (SEVINA) composed of pegylated trispecific disulfide linked Fv (sdFv) targeting epitopes of EGFR, PTHrP and RANKL conjugated covalently with SATA to vinorelbine eradicated osteolytic metastases of multiple myeloma inhibiting proliferation of tumor associated and bone microvascular endothelial cells inducing ADCC, AMP and type I, II, III PCD

Highly promising preclinical data obtained in cultured cells and in nude mice bearing xenografts contrast with the rather modest clinical efficacy of Polo-like kinase 1 (Plk1) inhibitors. In the present study, we investigated if Plk1 might be a suitable target in hepatocellular carcinoma (HCC) and if a genetically engineered mouse tumor model that well reflects the tumor cell and micro-environmental features of naturally occurring cancers might be suitable to study anti-Plk1 therapy. Analysis of Plk1 expression in human HCC samples confirmed that HCC express much higher Plk1 levels than the adjacent normal liver tissue. Inhibition of Plk1 by an adenovirus encoding for a short hairpin RNA against Plk1 or by the small-molecule inhibitor BI 2536 reduced the viability of HCC cell lines and inhibited HCC xenograft progression in nude mice. Treatment of transforming growth factor (TGF) α/c-myc bitransgenic mice with BI 2536 during hepatocarcinogenesis reduced the number of dysplastic foci and of Ki-67-positive cells within the foci, indicating diminished tumorigenesis. In contrast, BI 2536 had no significant effect on HCC progression in the transgenic mouse HCC model as revealed by magnetic resonance imaging. Measurement of BI 2536 by mass spectrometry revealed considerably lower BI 2536 levels in HCC compared with the adjacent normal liver tissue. In conclusion, low intratumoral levels are a novel mechanism of resistance to the Plk1 inhibitor BI 2536. Plk1 inhibitors achieving sufficient intratumoral levels are highly promising in HCC treatment.

Transposon-mediated resistance to Bacillus sphaericus in a field-evolved population of Culex pipiens (Diptera: Culicidae)

The binary toxin is the major active component of Bacillus sphaericus, a microbial larvicide used for controlling some vector mosquito-borne diseases. B. sphaericus resistance has been reported in many part of the world, leading to a growing concern for the usefulness of this environmental friendly insecticide. Here we characterize a novel mechanism of resistance to the binary toxin in a natural population of the West Nile virus vector, Culex pipiens. We show that the insertion of a transposable element-like DNA into the coding sequence of the midgut toxin receptor induces a new mRNA splicing event, unmasking cryptic donor and acceptor sites located in the host gene. The creation of the new intron causes the expression of an altered membrane protein, which is incapable of interacting with the toxin, thus providing the host mosquito with an advantageous phenotype. As a large portion of insect genomes is composed of transposable elements or transposable elements-related sequences, this new mechanism may be of general importance to appreciate their significance as potent agents for insect resistance to the microbial insecticides.

Local inflammation as a possible mechanism of resistance to gastrointestinal nematodes in Angus heifers

Understanding mechanisms of resistance to gastrointestinal nematodes is important in developing effective and sustainable control programs. A resource population of Angus cattle consisting of approximately 600 animals with complete pedigree records has been developed. The majority of these animals were completely characterized for their resistance to natural challenge by gastrointestinal nematodes. As the first step towards understanding the molecular basis of disease resistance, we investigated expression profiles of 17 cytokines, cytokine receptors, and chemokines using real-time RT-PCR in animals demonstrating resistance or susceptibility to pasture challenge. The animals exposed to natural infection for approximately 6 months were treated to remove existing parasites and then experimentally challenged with both Ostertagia ostertagi and Cooperia oncophora. The mRNA expression profiles of these genes in abomasal and mesenteric lymph nodes (ALN, MLN), fundic and pyloric abomasa (FA, PA), and small intestine (SI) were compared between resistant and susceptible animals. Resistant heifers exhibited elevated expression of inflammatory cytokines such as TNFα, IL-1β, and MIP-1α in fundic and pyloric abomasa 7 days post infection. Expression levels of IL-10, polymeric immunoglobullin receptor gene (PIGR), and WSX-1 were also 2.7–19.9-folds higher in resistant than susceptible heifers in these tissues. No difference in expression of CXCL6, CXCL10, IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-12 p40, IL-13, IL-15 and IL-18 was observed between the two groups. The expression of MIP-1α, IL-6, and IL-10 was also elevated in small intestines in resistant animals. In contrast, little difference in expression of these genes was detected between resistant and susceptible groups in the draining lymph nodes. These data indicate that resistant animals can better maintain inflammatory responses at the site of infection, suggesting a possible novel mechanism of resistance.

High-Level Resistance to <I>Bacillus thuringiensis</I> Toxin Cry1Ac and Cadherin Genotype in Pink Bollworm

Resistance to transgenic cotton, Gossypium hirsutum L., producing Bacillus thuringiensis (Bt) toxin Cry1Ac is linked with three recessive alleles of a cadherin gene in laboratory-selected strains of pink bollworm, Pectinophora gossypiella (Saunders), a major cotton pest. Here, we analyzed a strain (MOV97-R) with a high frequency of cadherin resistance alleles, a high frequency of resistance to 10 microg of Cry1Ac per milliliter of diet, and an intermediate frequency of resistance to 1000 microg of Cry1Ac per ml of diet. We selected two strains for increased resistance by exposing larvae from MOV97-R to diet with 1000 microg of Cry1Ac per ml of diet. In both selected strains, two to three rounds of selection increased survival at 1000 microg of CrylAc per ml of diet to at least 76%, indicating genetic variation in survival at this high concentration and yielding >4300-fold resistance relative to a susceptible strain. Variation in cadherin genotype did not explain variation in survival at 1000 microg of Cry1Ac per ml of diet, implying that one or more other loci affected survival at this concentration. This conclusion was confirmed with results showing that when exposure to Cry1Ac stopped, survival at 1000 microg of Cry1Ac per ml of diet dropped substantially, but survival at 10 microg Cry1Ac per ml of diet remained close to 100% and all survivors had two cadherin resistance alleles. Although survival at 1000 microg of Cry1Ac per ml of diet is not required for resistance to Bt cotton, understanding how genes other than cadherin confer increased survival at this high concentration may reveal novel mechanisms of resistance.

Small-colony variants: a novel mechanism for triclosan resistance in methicillin-resistant Staphylococcus aureus

A little-understood mode of antimicrobial resistance in Staphylococcus aureus is the evolution of a sub-population of small-colony variants (SCVs). SCVs are a cause of persistent and recurring infections refractory to antimicrobial chemotherapy. Following the inadvertent isolation of suspected SCVs growing in the presence of triclosan we set out to evaluate the formation of these colonial mutants and assess their antimicrobial susceptibility. SCVs were isolated on Mueller-Hinton agar supplemented with 1 mg/L triclosan. SCV formation frequency was calculated using a selection of both clinical methicillin-resistant S. aureus (MRSA) isolates and methicillin-susceptible S. aureus strains. Antimicrobial susceptibility was assessed and the fabI gene of SCVs was sequenced to ensure resistance was not mediated by mutation of this gene. We have found in vitro that triclosan can select for S. aureus colonies showing the characteristic SCV phenotype with low-level triclosan resistance and which coincidently have reduced susceptibility to penicillin and gentamicin. Additionally, triclosan-isolated SCVs were shown to have an increased tolerance to the lethal effects of triclosan. We propose the formation of SCVs by S. aureus is a novel mechanism of resistance to low concentrations of triclosan, which for 25 years has been used widely in the domestic setting in various consumer healthcare products. More recently it has been recommended for the control of MRSA. Consequently, our results identify the potential for treatment failure in infections already notoriously difficult to eradicate. It remains unclear to what extent isolates with decreased susceptibility to triclosan would develop and have the fitness to survive under real world conditions.

Cases of typhoid fever imported into England, Scotland and Wales (2000–2003)

Although typhoid fever is no longer endemic in most of the developed world, it remains a major infectious disease in less developed regions and imported cases continue to occur in returning travellers, immigrants or migrant workers. We analysed all 692 isolates of Salmonella enterica subspecies enterica serovar Typhi from cases in England, Scotland and Wales that were sent to the Laboratory of Enteric Pathogens at the Health Protection Agency, Centre for Infections, London, UK between 2000 and 2003. The country of acquisition was known for 416 isolates (60%), and the majority of these (70%) came from India or Pakistan. Overall, 24 countries were listed, mainly in Asia and Africa. A total of 48 phage types were detected, 41% of which were Vi-phage type E1. Antimicrobial susceptibility testing revealed that 22% of isolates were multidrug resistant (MDR) (defined as resistance to chloramphenicol, ampicillin and co-trimoxazole) and 39% were quinolone resistant. A significant number of isolates (n=49) were sensitive to nalidixic acid by disk test but exhibited low-level ciprofloxacin resistance, suggesting a novel mechanism of resistance and reinforcing the need for minimum inhibitory concentration determination. Overall, 13% of isolates were both MDR and likely to show a poor response to a fluoroquinolone. A third-generation cephalosporin (e.g. ceftriaxone) should be considered as empirical therapy in regions of the Indian subcontinent where resistance is now at high levels as well as in patients returning from these areas. This study helps to describe the epidemiology of antimicrobial drug resistance in typhoid fever.

Novel Mechanism of Resistance to Glycopeptide Antibiotics in Enterococcus faecium

Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.

TXR1-mediated thrombospondin repression: a novel mechanism of resistance to taxanes?

The usefulness of chemotherapeutic agents for the clinical treatment of cancer relies on their toxic effects on dividing cells. While most anti-cancer drugs directly affect DNA synthesis or integrity, the vinca-alkaloids and taxanes act by disrupting the cellular microtubule network (depicted schematically in Fig. 1; for review, see Zhou and Giannakakou 2005). In addition to providing structural integrity as part of the cytoskeleton, microtubules function as the cell’s freeway system by providing the infrastructure along which cargo can be transported by motor proteins such as kinesin and dynein. During cell division, microtubules are critically important for the formation of the mitotic spindle, which dictates the proper segregation of chromosomes during mitosis. Microtubules are synthesized through the polymerization of and -tubulin subunits on their fast-growing, or plus, ends. At the same time, they are degraded on their slow-growing, or minus, ends by the release of / dimers into the cytosol. The combined processes of assembly and disassembly cause microtubules to be in a state of “dynamic instability,” which allows the growing or shortening of individual microtubules as required. Anti-microtubule agents exert their anti-cancer effect by disrupting normal microtubule dynamics in distinct fashions. Whereas vinca-alkaloids are able to bind to free tubulin, thereby preventing microtubule assembly and concomitant mitotic spindle formation, they cannot associate with polymerized tubulin present in microtubules. In contrast, taxanes interact with polymerized tubulin to cause microtubule stabilization. As a result, cells become overloaded with microtubules and are unable to resolve the mitotic spindle, causing a mitotic arrest or apoptosis. Taxol, the prototype chemotherapeutic of the taxane family, was isolated in the early 1970s from Taxus brevifolia. In recent years, taxol/paclitaxel and taxotere/docetaxel, a semisynthetic taxane identified in the 1980s, have become part of standard drug treatment regimens and are widely used in the clinic to treat a variety of human malignancies, including breast, ovarian, lung, gastric, and prostate cancers. The latter two are of special interest because taxanes may prove to offer some advances in the treatment of these otherwise refractory diseases.

Elevated Expression of Mitogen-Activated Protein Kinase Phosphatase 3 in Breast Tumors: A Mechanism of Tamoxifen Resistance

Antiestrogen resistance is a major clinical problem in the treatment of breast cancer. Altered growth factor signaling with estrogen receptor (ER)-alpha is associated with the development of resistance. Gene expression profiling was used to identify mitogen-activated protein kinase (MAPK) phosphatase 3 (MKP3) whose expression was correlated with response to the antiestrogen tamoxifen in both patients and in vitro-derived cell line models. Overexpression of MKP3 rendered ER-alpha-positive breast cancer cells resistant to the growth-inhibitory effects of tamoxifen and enhanced tamoxifen agonist activity in endometrial cells. MKP3 overexpression was associated with lower levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in the presence of estrogen but that estrogen deprivation and tamoxifen treatment decreased MKP3 phosphatase activity, leading to an up-regulation of pERK1/2 MAPK, phosphorylated Ser(118)-ER-alpha, and cyclin D1. The MAPK/ERK kinase inhibitor PD98059 blocked tamoxifen-resistant growth. Accumulation of reactive oxygen species was observed with tamoxifen treatment of MKP3-overexpressing cells, and antioxidant treatment increased MKP3 phosphatase activity, thereby blocking resistance. Furthermore, PD98059 increased the levels of phosphorylated c-Jun NH(2)-terminal kinase (JNK) in tamoxifen-treated MKP3-overexpressing cells, suggesting an interaction between MKP3 levels, activation of ERK1/2 MAPK, and JNK signaling in human breast cancer cells. MKP3 represents a novel mechanism of resistance, which may be a potential biomarker for the use of ERK1/2 and/or JNK inhibitors in combination with tamoxifen treatment.