BMS-354825: a novel drug with potential for the treatment of imatinib-resistant chronic myeloid leukaemia

The BCR-ABL tyrosine kinase inhibitor imatinib has greatly improved the outcome for patients with chronic myeloid leukaemia (CML). Unfortunately, mutations causing resistance to imatinib are leading to relapses in some patients. In addition to inhibiting the wild-type BCR-ABL, BMS-354825 inhibited 14 of 15 BCR-ABL mutants. BMS-354825 treatment of immunodeficient mice prevented the progression of the disease in mice treated with the most clinical common imatinib-resistant mutant Met351Thr. The safety and efficacy of BMS-354825 is presently being evaluated in a phase I/II clinical trial in CML patients with imatinib resistance. The frequency of clinical use of SMS-3548125 in CML patients will depend on its efficacy/safety profile in clinical trial.

Applying Mathematical Models to Study the Role of the Immune System in Chronic Myelogenous Leukemia

Although tyrosine kinase inhibitors (TKIs) such as imatinib have transformed chronic myelogenous leukemia (CML) into a chronic condition, these therapies are not curative in the majority of cases. Most patients must continue TKI therapy indefinitely, a requirement that is both expensive and that compromises a patient's quality of life. While TKIs are known to reduce leukemic cells' proliferative capacity and to induce apoptosis, their effects on leukemic stem cells, the immune system, and the microenvironment are not fully understood. A more complete understanding of their global therapeutic effects would help us to identify any limitations of TKI monotherapy and to address these issues through novel combination therapies. Mathematical models are a complementary tool to experimental and clinical data that can provide valuable insights into the underlying mechanisms of TKI therapy. Previous modeling efforts have focused on CML patients who show biphasic and triphasic exponential declines in BCR-ABL ratio during therapy. However, our patient data indicates that many patients treated with TKIs show fluctuations in BCR-ABL ratio yet are able to achieve durable remissions. To investigate these fluctuations, we construct a mathematical model that integrates CML with a patient's autologous immune response to the disease. In our model, we define an immune window, which is an intermediate range of leukemic concentrations that lead to an effective immune response against CML. While small leukemic concentrations provide insufficient stimulus, large leukemic concentrations actively suppress a patient's immune system, thus limiting it's ability to respond. Our patient data and modeling results suggest that at diagnosis, a patient's high leukemic concentration is able to suppress their immune system. TKI therapy drives the leukemic population into the immune window, allowing the patient's immune cells to expand and eventually mount an efficient response against the residual CML. This response drives the leukemic population below the immune window, causing the immune population to contract and allowing the leukemia to partially recover. The leukemia eventually reenters the immune window, thus stimulating a sequence of weaker immune responses as the two populations approach equilibrium. We hypothesize that a patient's autologous immune response to CML may explain the fluctuations in BCR-ABL ratio that are regularly seen during TKI therapy. These fluctuations may serve as a signature of a patient's individual immune response to CML. By applying our modeling framework to patient data, we are able to construct an immune profile that can then be used to propose patient-specific combination therapies aimed at further reducing a patient's leukemic burden. Our characterization of a patient's anti-leukemia immune response may be especially valuable in the study of drug resistance, treatment cessation, and combination therapy.

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family.

Philadelphia chromosome-positive leukemias result from the fusion of the BCR and ABL genes, which generates a functional chimeric molecule. The Abr protein is very similar to Bcr but lacks a structural domain which may influence its biological regulatory capabilities. Both Abr and Bcr have a GTPase-activating protein (GAP) domain similar to those found in other proteins that stimulate GTP hydrolysis by members of the Rho family of GTP-binding proteins, as well as a region of homology with the guanine nucleotide dissociation-stimulating domain of the DBL oncogene product. We purified as recombinant fusion proteins the GAP- and Dbl-homology domains of both Abr and Bcr. The Dbl-homology domains of Bcr and Abr were active in stimulating GTP binding to CDC42Hs, RhoA, Rac1, and Rac2 (rank order, CDC42Hs > RhoA > Rac1 = Rac2) but were inactive toward Rap1A and Ha-Ras. Both Bcr and Abr acted as GAPs for Rac1, Rac2, and CDC42Hs but were inactive toward RhoA, Rap1A, and Ha-Ras. Each individual domain bound in a noncompetitive manner to GTP-binding protein substrates. These data suggest the multifunctional Bcr and Abr proteins might interact simultaneously and/or sequentially with members of the Rho family to regulate and coordinate cellular signaling.

Cell cycle-related shifts in subcellular localization of BCR: association with mitotic chromosomes and with heterochromatin.

The disruption of the BCR gene and its juxtaposition to and consequent activation of the ABL gene has been implicated as the critical molecular defect in Philadelphia chromosome-positive leukemias. The normal BCR protein is a multifunctional molecule with domains that suggest its participation in phosphokinase and GTP-binding pathways. Taken together with its localization to the cytoplasm of uncycled cells, it is therefore presumed to be involved in cytoplasmic signaling. By performing a double aphidicolin block for cell cycle synchronization, we currently demonstrate that the subcellular localization of BCR shifts from being largely cytoplasmic in interphase cells to being predominantly perichromosomal in mitosis. Furthermore, with the use of immunogold labeling and electron microscopy, association of BCR with DNA, in particular heterochromatin, can be demonstrated even in quiescent cells. Results were similar in cell lines of lymphoid or myeloid origin. These observations suggest a role for BCR in the phosphokinase interactions linked to condensed chromatin, a network previously implicated in cell cycle regulation.

BCR and TLR signalling pathways are recurrently targeted by genetic changes in splenic marginal zone lymphomas.

The genetics and pathogenesis of splenic marginal zone lymphoma are poorly understood. The lymphoma lacks chromosome translocation, and ~30% of cases are featured by 7q deletion, but the gene targeted by the deletion is unknown. A recent study showed inactivation of A20, a 'global' NF-kB negative regulator, in 1 of 12 splenic marginal zone lymphoma. To investigate further whether deregulation of the NF-kB pathway plays a role in the pathogenesis of splenic marginal zone lymphoma, we screened several NF-kB regulators for genetic changes by PCR and sequencing. Somatic mutations were found in A20 (6/46=13%), MYD88 (6/46=13%), CARD11 (3/34=8.8%), but not in CD79A, CD79B and ABIN1. Interestingly, these genetic changes are largely mutually exclusive from each other and MYD88 mutation was also mutually exclusive from 7q deletion. These results strongly suggest that deregulation of the TLR (toll like receptor) and BCR (B-cell receptor) signalling pathway may play an important role in the pathogenesis of splenic marginal zone lymphoma.

Otimização das condições de pré-redução do As(V) em extratos do método BCR para quantificação de arsênio por HG-AAS

A determinação de As por espectrometria de absorção atômica com geração de hidretos (HG-AAS) constitui um método simples, sensível, preciso e de baixo custo. Entretanto, essa técnica requer a pré-redução das espécies de As(V), o que se obtém através do uso de agentes redutores como o KI. Em extratos contendo agentes oxidantes, a pré-redução do As é comprometida, como acontece em extratos obtidos pela aplicação do método BCR (acrônimo francês para Community Bureau of Reference) para a extração sequencial de As em sedimentos. O objetivo deste trabalho foi avaliar as condições de redução do As(V) a As(III), de modo a permitir o uso da HG-AAS para a quantificação do arsênio em extratos obtidos a partir do método BCR. Foram avaliadas condições reacionais utilizando KI, L-cisteína e ácido ascórbico. Para cada uma das etapas de extração do método BCR, diferentes condições de pré-redução possibilitaram a detecção quantitativa do As presente. O uso do método BCR para a extração de arsênio em amostras de sedimentos contaminadas e a aplicação das condições de pré-redução do As(V) selecionadas, seguida pela detecção por HG-AAS, forneceram percentagens de recuperação entre 91 e 99 %.

Gleevec, an Abl family inhibitor, produces a profound change in cell shape and migration (in press)

The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.

A combination of STI571 and BCR-ABL1 siRNA with overexpressed p15INK4B induced enhanced proliferation inhibition and apoptosis in chronic myeloid leukemia

p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.

La muerte de Abél : poéma moral en prosa, en cinco cantos

UANL

Characteristics of ABL during active and weak phases of monsoon

Understanding of the Atmospheric Boundary Layer (ABL) is imperative in the arena of the monsoon field. Here, the features of the ABL are studied employing Conserved Variable Analysis (CVA) using equivalent potential temperature and humidity. In addition, virtual potential temperature and wind are used during active and weak phases of monsoon. The analysis is carried out utilising the radiosonde observations during the monsoon months for two stations situated in the west coast of India. All these parameters show considerable variations during active and weak monsoon phases in both the stations. The core speed and core height vary with these epochs. The core speed is found to be more than 38 knots in the active monsoon phase around 1.2 km over Trivandrum and around 2 km over Mangalore. But during weak monsoon phase the core wind speed is decreased and core height is elevated over both stations. The wind direction shows an additional along shore component during weak monsoon period. The Convective Boundary Layer (CBL) height shows increase during weak monsoon phase over both stations due to less cloudiness and subsequent insolation. The CBL height during the southwest monsoon is more over Mangalore and is attributed by the orographic lifting in the windward side of the Western Ghats while the influence of the Ghats is less over Trivandrum.

A standardised BCR resistance test for all anticoagulant rodenticides

This paper presents a reappraisal of the blood clotting response (BCR) tests for anticoagulant rodenticides, and proposes a standardised methodology for identifying and quantifying physiological resistance in populations of rodent species. The standardisation is based on the International Normalised Ratio, which is standardised against a WHO international reference preparation of thromboplastin, and allows comparison of data obtained using different thromboplastin reagents. ne methodology is statistically sound, being based on the 50% response, and has been validated against the Norway rat (Rattus norvegicus) and the house mouse (Mus domesticus). Susceptibility baseline data are presented for warfarin, diphacinone, chlorophacinone and coumatetralyl against the Norway rat, and for bromadiolone, difenacoum, difethialone, flocoumafen and brodifacoum against the Norway rat and the house mouse. A 'test dose' of twice the ED50 can be used for initial identification of resistance, and will provide a similar level of information to previously published methods. Higher multiples of the ED50 can be used to assess the resistance factor, and to predict the likely impact on field control.