Investigations on mechanisms of acquisition of drug resistance in trypanosomes

The widespread occurrence of drug resistant trypanosomes has constituted a major hindrance to the control of animal trypanosomosis. The study described here was an investigation into the mechanisms of acquisition of drug resistance by Trypanosoma evansi. The main aim was to determine the biochemical basis for the development of resistance to the melaminophenyl arsenical drugs, mel Cy and mel B. Experiments were designed to compare the mode of development of resistance in vitro with in v/'vo-derived resistance and to determine the effect of host immunity on resistance development. Resistance to mel Cy was induced in a sensitive stock of T. evansi by infection and treatment with sub-curative levels of drug, in immunosuppressed and immunocompetent mice. Induction was also carried out in vitro by cultivation of the culture-adapted T. evansi isolate in the continuous presence of increasing subinhibitory concentrations of mel Cy. Resistance was measured using in vivo drug sensitivity tests and, in vitro growth inhibition assays.High levels (up to 300-fold) of resistance were attained within 6 months of induction in immunosuppressed mice and in vitro. It was not possible to produce resistance in immunocompetent mice, even after 24 months. In vivo drug resistant trypanosomes maintained their infectivity but virulence was reduced. Resistance induced by mel Cy was characterised with respect to stability of resistance and cross resistance to other trypanocides. The resistance developed from either immunosuppressed mice or in vitro continued to be expressed in immunocompetent mice treated with the trypanocidal drug. After withdrawal of drug in vz'/ro-induced resistance remained stable whereas in vivoderived resistance was reduced by over 50%. Mel Cy-induced resistance conferred cross resistance to other trypanocidal drugs, namely, mel B, Berenil®and, to a limited extent, quinapyramine, but not suramin. Subpopulations of trypanosomes tolerant to the selection dose (20 ηg/ml) of mel Cy were not found in the unselected parent clone. Increased sensitivity to Summary quinapyramine and suramin was observed after adaptation of the drug sensitive isolate to in vitro culture.Uptake of melaminophenyl arsenicals into T. evansi was investigated using an in vitro lysis assay. Mel Cy and mel B caused lysis of drug sensitive and drug resistant trypanosomes in a dose-dependent and saturable manner. Mel B was less effective in lysis of drug sensitive trypanosomes but very potent in lysis of drug resistant parasites. The in vitro lysis assay differentiated between drug sensitive and drug resistant trypanosomes. Mel Cy lysis was blocked by adenosine, adenine and Berenil®, but not inosine. Mel B lysis was inhibited by (R) Berenil but not adenosine, adenine or inosine. These results indicate that mel Cy competes for an adenine/adenosine transporter whereas mel B does not.Uptake of adenosine into drug sensitive and drug resistant trypanosomes was investigated using competitive inhibition experiments. Adenine, inosine and Berenil® all inhibited adenosine uptake in a concentration-dependent and saturable manner. Percentage inhibitions in the drug sensitive parasites were 53.6, 23.2, and 33.2, respectively, for adenine, inosine and Berenil®. Combinations of adenine and inosine resulted in total inhibition of uptake of adenosine. These findings suggest that T. evansi possesses a bipartite P1/P2 adenosine transport system. Mel Cy was taken up by the P2 transporter. The P2 transporter was maintained in arsenical resistant T. evansi. However, uptake on the P2 was reduced 3-10 times in different resistant lines. Reduction in uptake at the P2 transporter resulted in reductions in total adenosine uptake by the resistant parasites. Moderate reductions in PI were also observed. Berenil also interacted with the P2 transporter in drug sensitive trypanosomes. Interaction of Berenil® with the P2 transporter was greatly reduced in the resistant population. Thus, the cross resistance that develops between melaminophenyl arsenicals and diamidines is a result of their common mechanisms of uptake.Kinetic studies on the PI and P2 adenosine transporters were carried out on both the drug sensitive and the drug resistant T. evansi. Uptake rates including Amax (maximum transport capacity), Rate constant (T), and initial rates of uptake, were determined. In the drug sensitive trypanosomes the P2 transporter was a larger transport process. Amax for P2 was 2 times greater than Amax for PI transporter. Rate constant and initial rates for PI and P2 were similar. The Amax at the P2 was decreased 3.4 times in drug resistant trypanosomes. The Rate constant at both the PI and P2 were increased. Initial rate at the P2 was decreased 2-fold. Reduced Amax accounted for the reduced uptake at the P2 transporter. Michaelis-Menten kinetic parameters were also determined for the PI and P2 transporters. In drug sensitive trypanosomes the Umax at the P2 (28.9 pmol/108cells/5min.) was 2-fold greater than the Umax (12.4 pmol/108cells/5min) at the PI. The Km for the PI (0.07 pM) was 10-fold lower than the Km for P2 (0.74 pM). The Umax (3.3 pmol/108cells/5min.) for the P2 in drug resistant trypanosomes was reduced 9-fold. The Km ( 0.1 μM) was decreased 7-fold. Kinetic changes for total adenosine uptake in trypanosomes were also determined. It was found that reduction in uptake capacity of P2 in arsenical resistant T. evansi was not due to decreased affinity. Rather a decrease in numbers of transporter molecules was responsible for the decrease in adenosine uptake at the P2. The changes in kinetic parameters of adenosine transport suggested that the P2 in drug resistant trypanosomes had greatly undergone mutation (P2R), as such it interacted with Berenil® in a different way.The kinetics of inhibition of adenosine uptake showed that inosine, adenine and Berenil inhibited uptake on either the PI or P2 transporters with very low K; values. In the drug sensitive trypanosomes K, for inosine or adenine alone were 0.002 μM or 0.28 μM, respectively. K, for Berenil® alone was 0.00 μM indicating that there was little or no interaction. On the PI the K, for inosine was (ft) 0.03 μM. There was no interaction with Berenil on the PI. The K, for adenine and Berenil® on the P2 were 0.3 and 0.01 pM, respectively. In the drug resistant line the Kj for inosine on PI (0.06 pM) was similar to that of the drug sensitive line. However there was a 7-fold increase (1.94 μM) in K, for adenine on the P2. Based on changes in all the kinetic parameters of what should be the P2 it was concluded that a new transporter (P3) was present in the arsenical resistant trypanosomes. This transporter may have been selected for during induction of resistance to mel Cy, or may be an adaptation by the resistant trypanosomes following gross reductions in activity of the P2 transporterMolecular studies involving isolation and characterisation of the P2 and P3 transporters may fully elucidate the specific mutations responsible for arsenical resistance in trypanosomes. It will also be interesting to study the interaction of the P3 transporter with other alternative substrates, including toxic adenosine analogues. This may provide useful information for improved chemotherapy of trypanosomes resistant to arsenical compounds

Novel DNA-binding properties of the RNA-binding protein TIAR

TIA-1 related protein binds avidly to uridine-rich elements in mRNA and pre-mRNAs of a wide range of genes, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). The protein has diverse regulatory roles, which in part depend on the locus of binding within the transcript, including translational control, splicing and apoptosis. Here, we observed selective and potent inhibition of TIAR–RNP complex formation with IL-8 and VEGF 3′-untranslated regions (3′-UTRs) using thymidine-rich deoxyoligonucleotide (ODN) sequences derived from the VEFG 3′-UTR. We show by ultraviolet crosslinking and electrophoretic mobility shift assays that TIAR can bind directly to single-stranded, thymidine-rich ODNs but not to double-stranded ODNs containing the same sequence. TIAR had a nearly 6-fold greater affinity for DNA than RNA ([Formula: see text] versus 9.4 × 10(−9) M). Truncation of TIAR indicated that the high affinity DNA-binding site overlaps with the RNA-binding site involving RNA recognition motif 2 (RRM2). However, RRM1 alone could also bind to DNA. Finally, we show that TIAR can be displaced from single-stranded DNA by active transcription through the binding site. These results provide a potential mechanism by which TIAR can shuttle between RNA and DNA ligands

Ornithine Decarboxylase mRNA is Stabilized in an mTORC1-dependent Manner in Ras-transformed Cells

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3′UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3′UTR that may act as regulatory sequences. Analysis of ODC 3′UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development

Drosophila SPF45: A Bifunctional Protein with Roles in Both Splicing and DNA Repair

The sequence of the SPF45 protein is significantly conserved, yet functional studies have identified it as a splicing factor in animal cells and as a DNA-repair protein in plants. Using a combined genetic and biochemical approach to investigate this apparent functional discrepancy, we unify and validate both of these studies by demonstrating that the Drosophila melanogaster protein is bifunctional, with independent functions in DNA repair and splicing. We find that SPF45 associates with the U2 snRNP and that mutations that remove the C-terminal end of the protein disrupt this interaction. Although animals carrying this mutation are viable, they are nevertheless compromised in their ability to regulate Sex-lethal splicing, demonstrating that Sex-lethal is an important physiological target of SPF45. Furthermore, these mutant animals exhibit phenotypes diagnostic of difficulties in recovering from exogenously induced DNA damage. The conclusion that SPF45 functions in the DNA-repair pathway is strengthened by finding both genetic and physical interactions between SPF45 and RAD201, a previously uncharacterized member of the RecA/Rad51 protein family. Together with our finding that the fly SPF45 protein increases the survival rate of mutagen-treated bacteria lacking the RecG helicase, these studies provide the tantalizing suggestion that SPF45 has an ancient and evolutionarily conserved role in DNA repair

Tristetraprolin regulates interleukin‐6, which is correlated with tumor progression in patients with head and neck squamous cell carcinoma

BACKGROUND: Tumor‐derived cytokines play a significant role in the progression of head and neck squamous cell carcinoma (HNSCC). Targeting proteins, such as tristetraprolin (TTP), that regulate multiple inflammatory cytokines may inhibit the progression of HNSCC. However, TTP's role in cancer is poorly understood. The goal of the current study was to determine whether TTP regulates inflammatory cytokines in patients with HNSCC. METHODS: TTP messenger RNA (mRNA) and protein expression were determined by quantitative real‐time–polymerase chain reaction (Q‐RT‐PCR) and Western blot analysis, respectively. mRNA stability and cytokine secretion were evaluated by quantitative RT‐PCR and enzyme‐linked immunoadsorbent assay, respectively, after overexpression or knockdown of TTP in HNSCC. HNSCC tissue microarrays were immunostained for interleukin‐6 (IL‐6) and TTP. RESULTS: TTP expression in HNSCC cell lines was found to be inversely correlated with the secretion of IL‐6, vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE 2 ) . Knockdown of TTP increased mRNA stability and the secretion of cytokines. Conversely, overexpression of TTP in HNSCC cells led to decreased secretion of IL‐6, VEGF, and PGE 2 . Immunohistochemical staining of tissue microarrays for IL‐6 demonstrated that staining intensity is prognostic for poor disease‐specific survival ( P = .023), tumor recurrence and development of second primary tumors ( P = .014), and poor overall survival ( P = .019). CONCLUSIONS: The results of the current study demonstrated that down‐regulation of TTP in HNSCC enhances mRNA stability and promotes secretion of IL‐6, VEGF, and PGE 2 . Furthermore, high IL‐6 secretion in HNSCC tissue is a biomarker for poor prognosis. In as much as enhanced cytokine secretion is associated with poor prognosis, TTP may be a therapeutic target to reduce multiple cytokines concurrently in patients with HNSCC. Cancer 2011. © 2011 American Cancer Society. Tristetraprolin (TTP), a protein that decreases the stability of messenger RNA (mRNA) of cytokines and proinflammatory factors, is reduced in patients with head and neck squamous cell carcinoma with a corresponding increase in interleukin‐6 (IL‐6), vascular endothelial growth factor, and cyclooxygenase‐2 secretion. One of these tumor‐derived cytokines, IL‐6, is prognostic for poor disease‐specific survival, tumor recurrence, second primary lesions, and poor overall survival.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86915/1/25859_ftp.pd

Wnt-5a mRNA translation is suppressed by the Elav-like protein HuR in human breast epithelial cells

Wnt-5a is a non-transforming Wnt protein. Since Wnt-5a mRNA and protein levels differ within and between tumours, the potential of Wnt-5a as a prognostic factor has been debated. We have previously shown that the lack of Wnt-5a protein is a predictor of shorter disease-free survival in human breast cancer. Recently, however, we also showed that the breast tumours lacking Wnt-5a protein had a high or normal level of Wnt-5a mRNA that might explain the discrepancies in previous studies. We here report that Wnt-5a is regulated at the post-transcriptional level. The regulation was mediated by the Embryonic Lethal Abnormal Vision (ELAV)-like protein HuR, which inhibited translation of Wnt-5a when bound to highly conserved AU-rich sequences in the 3′-untranslated region (3′-UTR) of the Wnt-5a mRNA molecule, as shown by both HA-tagged Wnt-5a- and Luciferase-Wnt-5a-3′-UTR reporter assays. The HuR-dependent inhibition of Wnt-5a was supported by the fact that active HuR is located in the cytoplasm in invasive human breast tumours and that hypoxia-induced activation of HuR inhibits translation of both Luciferase-Wnt-5a-3′-UTR and endogenous Wnt-5a protein. We propose that the lack of Wnt-5a protein expression in invasive human breast tumours is caused by a HuR-mediated suppression of Wnt-5a mRNA translation

Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance

© 2014 The Authors. The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A 165 b. Whereas flTIA-1 selectively bound VEGF-A 165 mRNA and increased translation of VEGF-A 165 b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy

Confirmation of emergence of mutations associated with atovaquone-proguanil resistance in unexposed Plasmodium falciparum isolates from Africa

BACKGROUND: In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb) gene (Tyr268Ser and Tyr268Asn). However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa. METHODS: The prevalence of codon-268 mutations in the cytb gene of African P. falciparum isolates from Nigeria, Malawi and Senegal, where atovaquone-proguanil has not been introduced for treatment of malaria was assessed. Genotyping of the cytb gene in isolates of P. falciparum was performed by PCR-restriction fragment length polymorphism and confirmed by sequencing. RESULTS: 295 samples from Nigeria (111), Malawi (91) and Senegal (93) were successfully analyzed for detection of either mutant Tyr268Ser or Tyr268Asn. No case of Ser268 or Asn268 was detected in cytb gene of parasites from Malawi or Senegal. However, Asn268 was detected in five out of 111 (4.5%) unexposed P. falciparum isolates from Nigeria. In addition, one out of these five mutant Asn268 isolates showed an additional cytb mutation leading to a Pro266Thr substitution inside the ubiquinone reduction site. CONCLUSION: No Tyr268Ser mutation is found in cytb of P. falciparum isolates from Nigeria, Malawi or Senegal. This study reports for the first time cytb Tyr268Asn mutation in unexposed P. falciparum isolates from Nigeria. The emergence in Africa of P. falciparum isolates with cytb Tyr268Asn mutation is a matter of serious concern. Continuous monitoring of atovaquone-proguanil resistant P. falciparum in Africa is warranted for the rational use of this new antimalarial drug, especially in non-immune travelers

Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1α), and other RDM1 isoforms. We show that RDM1α undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response

Different modes of interaction by TIAR and HuR with target RNA and DNA

TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U–rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2′-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways