Gaining insights into the Bcr-Abl activity-independent mechanisms of resistance to imatinib mesylate in KCL22 cells: A comparative proteomic approach

Imatinib mesylate is a potent inhibitor of Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. Here we used the imatinib-resistant KCL22R and imatinib-sensitive KCL22S cells in which none of the known resistance mechanisms has been detected and hence novel Bcr-Abl activity-independent mechanisms could be envisaged. We characterized proteins that were differentially expressed between the KCL22R and KCL22S cells. Using two-dimensional differential gel electrophoresis coupled with mass spectrometry and Western blot analysis we identified 51 differentially expressed proteins: 27 were over-expressed and 24 were under-expressed in KCL22R versus KCL22S cells. Several of these proteins are likely to be involved in such survival mechanisms as modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-κB and Ras-MAPK signaling. The data reported may be useful for further studies on mechanisms of imatinib resistance and for the screening of biomarkers to develop new combinatorial therapeutic approaches.     

Comparison of Three Different LCIA Methods: EDIP97, CML2001 and Eco-indicator 99

Goal, Scope and Background

A number of impact assessment methodologies are available to the LCA practitioner. They differ, and often there is not one obvious choice among them. The question therefore naturally arises: ‘Does it make any difference to my conclusions which method I choose?’ To investigate this issue, a comparison is performed of three frequently applied life cycle impact assessment methods.

Methods

The three life cycle impact assessment methods EDIP97 [1], CML2001 [2] and Eco-indicator 99 [3] are compared on their performance through application to the same life cycle inventory from a study of a water-based UV-lacquer. EDIP97 and CML2001 are both midpoint approaches and hence quite similar in their scope and structure, and this allows a comparison during both characterisation and normalisation. The third impact assessment method Eco-indicator 99 is an endpoint method and different in scope and structure from the other two. A detailed comparison can not be done but a comparative analysis of the main contributors to the Eco-indicator 99 results and the weighted and aggregated EDIP97 results is performed.

Results and Discussion

Following a translation into common units of the EDIP97 and CML2001 output, differences up to two orders of magnitude are found for some of the indicator results for the impact categories describing toxicity to humans and ecosystems, and there is little similarity in the patterns of major contributors among the two methods. For human toxicity the CML2001 score is dominated by contribution from metals while the EDIP97 score is caused by a solvent and nitrogen oxides. For aquatic ecotoxicity, metals are the main contributors for both methods but while it is vanadium for CML2001, it is strontium for EDIP97. After normalisation, the differences are reduced but still considerable. For the other impact categories, the two methods show only minor differences. The comparison of the main contributors to the Eco-indicator 99 results and the weighted and aggregated EDIP97 results identifies nitrogen oxides as the main contributor for both methods. It is, however, much more dominant for Eco-indicator 99 while the EDIP97 score represents important contributions from a number of different substances, and furthermore, the analysis reveals that the aggregated scores for the two methods come from different impacts. It is thus difficult to extend the findings for these two methods to other inventories.

Conclusion

For EDIP97 and CML2001, it mainly matters which method is used if the chemical impacts on human health and ecosystem health are important for the study. For the other impact categories, the differences are minor for these two methodologies. For EDIP97 and Eco-indicator 99, the patterns of most important contributors to the weighted and aggregated impact scores are rather different, and considering the known differences in the underlying framework and models, the results of the two methods may well go in opposite directions for some inventories even if the conclusion is the same for the inventory studied in this paper.

Recommendations and Oudook

Particularly for the impact categories representing toxic impacts from chemicals, the study demonstrates the need for more a detailed analysis of the causes underlying the big differences revealed between the methods.

     

RAGE-mediated MAPK activation by food-derived AGE and non-AGE products

Investigating the cellular effects of food compounds formed by heat treatment during processing, we recently demonstrated the expression of the receptor for advanced glycation endproducts (RAGE) and the p44/42 MAP kinase activation by casein-N(epsilon )-(carboxymethyl)lysine (casein-CML), a food-derived AGE, in the intestinal cell line Caco-2. In this work, we report a Caco-2 p44/42 MAP kinase activation by bread crust and coffee extract. After identification, quantification, and synthesis of two key compounds formed in association with the process-induced heat impact applied to bread dough and coffee beans, those compounds, namely the AGE pronyl-glycine and the non-AGE N-methylpyridinium, were also demonstrated for the first time to activate the p44/42 MAP kinase through binding to RAGE in Caco-2 cells. Blocking of RAGE by an antagonistic antibody and expression of C-terminally truncated RAGE resulted in a reduced Caco-2- and HEK-293-MAP kinase activation. These findings unequivocally point to a RAGE-mediated activating effect of chemically defined food-derived, thermally generated products, both, AGEs and non-AGEs, on cellular signal transduction pathways involved in inflammatory response and cellular proliferation.     

Nightmare frequency as a function of age, gender, and September 11, 2001: Findings from an Internet questionnaire.

Retrospective estimates of nightmare frequency for a sample of 23,990 respondents to an Internet questionnaire (female: N = 19,367, mean age = 24.9 ± 10.14 years; male: N = 4,623; mean age = 25.5 ± 10.81) were evaluated as a function of age, gender, and pre- versus post-September 11, 2001. Female respondents reported more frequent monthly nightmares (4.44 ± 6.71) than did male respondents (3.39 ± 6.07), and this result was seen for all age strata younger than 60. Also, for female respondents, nightmare frequency increased from ages 10-19 to 20-39 then decreased monotonically to ages 50-59. For male respondents, nightmare frequency was stable from ages 10-19 to 30-39 then decreased to ages 50-59. An increase in nightmare frequency was observed post-September 11 only for male respondents-particularly for 10- to 29-year-olds. This increase was sustained 2 years later. These effects were maintained when dream recall was held constant. Results replicate, in a single sample, previously published gender and age effects and provide new evidence that the nightmares of males may be differentially sensitive to traumatic events for which victims and/or perpetrators are primarily male. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Oxidized SOD1 alters proteasome activities in vitro and in the cortex of SOD1 overexpressing mice

Premature ageing, one of the characteristics of Down syndrome (DS), may involve oxidative stress and impairment of proteasome activity. Transgenic mice overexpressing the human copper/zinc superoxide dismutase (SOD1) gene are one of the first murine models for DS and it has been shown that SOD1 overexpression might be either deleterious or beneficial. Here, we show a reduction in proteasome activities in the cortex of SOD1 transgenic mice and an associated increase in the content of oxidized SOD1 protein. As we demonstrate that in vitro oxidized SOD can inhibit purified proteasome peptidase activities, modified SOD1 might be partially responsible for proteasome inhibition shown in SOD1 transgenic mice.     

The novel Aurora A kinase inhibitor MLN8237 is active in resistant chronic myeloid leukaemia and significantly increases the efficacy of nilotinib

Novel therapies are urgently needed to prevent and treat tyrosine kinase inhibitor resistance in chronic myeloid leukaemia (CML). MLN8237 is a novel Aurora A kinase inhibitor under investigation in multiple phase I and II studies. Here we report that MLN8237 possessed equipotent activity against Ba/F3 cells and primary CML cells expressing unmutated and mutated forms of breakpoint cluster region-Abelson kinase (BCR-ABL). Notably, this agent retained high activity against the T315I and E255K BCR-ABL mutations, which confer the greatest degree of resistance to standard therapy. MLN8237 treatment disrupted cell cycle kinetics, induced apoptosis, caused a dose-dependent reduction in the expression of the large inhibitor of apoptosis protein Apollon, and produced a morphological phenotype consistent with Aurora A kinase inhibition. In contrast to other Aurora kinase inhibitors, MLN8237 did not significantly affect BCR-ABL activity. Moreover, inhibition of Aurora A with MLN8237 significantly increased the in vitro and in vivo efficacy of nilotinib. Targeted knockdown of Apollon sensitized CML cells to nilotinib-induced apoptosis, indicating that this is an important factor underlying MLN8237's ability to increase the efficacy of nilotinib. Our collective data demonstrate that this combination strategy represents a novel therapeutic approach for refractory CML that has the potential to suppress the emergence of T315I mutated CML clones.     

Recombination drives genetic diversification of Streptococcus dysgalactiae subspecies equisimilis in a region of streptococcal endemicity

Infection of the skin or throat by Streptococcus dysgalactiae subspecies equisimilis (SDSE) may result in a number of human diseases. To understand mechanisms that give rise to new genetic variants in this species, we used multi-locus sequence typing (MLST) to characterise relationships in the SDSE population from India, a country where streptococcal disease is endemic. The study revealed Indian SDSE isolates have sequence types (STs) predominantly different to those reported from other regions of the world. Emm-ST combinations in India are also largely unique. Split decomposition analysis, the presence of emm-types in unrelated clonal complexes, and analysis of phylogenetic trees based on concatenated sequences all reveal an extensive history of recombination within the population. The ratio of recombination to mutation (r/m) events (11:1) and per site r/m ratio (41:1) in this population is twice as high as reported for SDSE from non-endemic regions. Recombination involving the emm-gene is also more frequent than recombination involving housekeeping genes, consistent with diversification of M proteins offering selective advantages to the pathogen. Our data demonstrate that genetic recombination in endemic regions is more frequent than non-endemic regions, and gives rise to novel local SDSE variants, some of which may have increased fitness or pathogenic potential.     

Optimal control of treatment in a mathematical model of chronic myelogenous leukemia

We consider a mathematical model of drug therapy for chronic myelogenous leukemia for an individual patient over a fixed time horizon. The disease dynamics are given by a system of ordinary differential equations that describe the interaction between naive T cells, effector T cells and leukemic cancer cells in a hypothetical patient. We introduce two drug therapies into this model, one a targeted therapy, and the other a broad cytotoxic therapy. Our goal is to find treatment regimens that minimize the cancer cell count and the deleterious effects of the drugs for a given patient. We examine the control setting analytically, and include numerical solutions to illustrate the optimal regimens under various assumptions.     

Targeting of gene expression by siRNA in CML primary cells

Development of array methods contributes to elucidation of many genes expressed during oncogenesis. Our array-based analyses of gene expression in patients with chronic myeloid leukemia (CML) revealed several genes (MMP8, MMP9, PCNA, JNK2, MAPK p38) with significant increased expression. We suppose that the genes may be implicated in the disease development and a siRNA-suppression can elucidate their functions in leukemogenesis. One of the crucial requirements for this purpose is a high efficiency of siRNA delivery into CML primary cells. Using fluorescein-labeled siRNAs we systematically tested a variety of physical and chemical non-vector based transfection methods in order to evaluate which of them gave the most suitable transfer. Chemically synthesized siRNAs against mentioned genes were transfected into the cells and level of knockdown was determined by real time RT-PCR. Chemical transfection reagents (Oligofectamine, Metafectene, siPORT Amine) commonly used to transfect siRNAs in CML cell lines showed very low siRNA delivery in CML primary cells—mRNA levels decreased at the most to 76%. Electroporation achieved better results (suppression to 63%) but it was associated with high degree of cell death (more than 60%). In the study we obtained the best transfection efficiency using nucleofector technology. Gene expressions ranged 22–37% that remained from original levels. According to our results, nucleofection appears to be the only suitable non-viral method for siRNA delivery into the hard-to-transfect CML primary cells.     

Hypochlorous acid generates N epsilon-(carboxymethyl)lysine from Amadori products

Since the accumulation of N^ε-(carboxymethyl)lysine (CML), a major antigenic advanced glycation end product, is implicated in tissue disorders in hyperglycemia and inflammation, the identification of the pathway of CML formation will provide important information regarding the development of potential therapeutic strategies for these complications. The present study was designed to measure the effect of hypochlorous acid (HOCl) on CML formation from Amadori products. The incubation of glycated human serum albumin (glycated-HSA), a model of Amadori products, with HOCl led to CML formation, and an increasing HOCl concentration and decreasing pH, which mimics the formation of these products in inflammatory lesions. CML formation was also observed when glycated-HSA was incubated with activated neutrophils, and was completely inhibited in the presence of an HOCl scavenger. These data demonstrated that HOCl-mediated CML formation from Amadori products plays a role in CML formation and tissue damage at sites of inflammation.