Focal adhesion kinase inhibition decreases cell viability and induces apoptosis of JAK2 V617F positive cells

Abstract Focal Adhesion Kinase (FAK) protein participates in proliferation, migration, cell survival, and apoptosis process. It has been described as overexpressed in several neoplasms being a promising target for therapy. BCR-ABL negative chronic Myeloproliferative Neoplasms (MPN) are clonal disorders characterized by the excess of proliferation and apoptosis resistance. The identification of the acquired JAK2 V617F mutation in MPN patients allowed a better understanding of pathogenesis. However, there is still no pharmacological treatment that leads all patients to molecular remission, justifying new studies. The present study aimed to evaluate FAK involvement in the viability and apoptosis of HEL and SET-2 cells, both JAK2 V617F positive cell lines. The FAK inhibitor PF 562,271 was used. Cell viability was determined using MTT assay and apoptosis verified by cleaved PARP, cleaved Caspase 3 and Annexin-V/PI staining detection. FAK inhibition significantly reduced HEL and SET-2 cells viability and induced apoptosis. Considering the role of JAK/STAT pathway in MPN, further investigation of FAK participation in the MPN cells proliferation and apoptosis resistance, as well as possible crosstalk between JAK and FAK and downstream pathways may contribute to the knowledge of MPN pathophysiology, the discovery of new molecular targets, and JAK inhibitors resistance mechanisms.

Anomalously phosphorylated tau and Abeta fragments in the CSF correlates with cognitive impairment in MCI subjects.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence of extracellular amyloid deposits, consisting largely of Abeta peptide and the presence of intraneuronal aggregates of neurofibillary tangles formed by tau. Development of cerebrospinal fluid (CSF) biomarkers has become a rapidly growing research field, considering the need for diagnostic tools for AD, thus allowing therapeutic compounds to have the greatest potential for being effective. We have focused on the relationships between critical biomarkers such as tau and Abeta in the CSF and the cognitive impairment of patients, as assessed by a battery of neuropsychological tests derived from CDR and CERAD, of value in the evaluation of AD patients. As part of a longitudinal study, we analyzed by ELISA and Western blots the levels and molecular patterns of hyperphosphorylated tau in the CSF of three different groups of patients: AD patients between 69- and 73-years-old, a group characterized with mild cognitive impairment (MCI) between 65- and 70-years-old, and a non-demented neurological control group of comparable ages. The levels of AT8-reactive phosphorylated tau were significantly higher (P<0.05) in AD patients (0.604+/-0.078, n=23) as compared with the control group (0.457+/-0.086, n=25). No differences between the levels of AT8-reactive tau of MCI patients (0.510+/-0.090, n=45) and controls were observed. However, when the MCI group was divided on the basis of the total box score (TBS) from CDR, those subjects with a TBS<1.5 presented tau levels (0.456+/-0.032, n=31) similar to controls, whereas those patients with TBS>or=1.5 displayed tau levels (0.590+/-0.086, n=14) comparable with those of AD. Western blot analyses revealed a higher AT8 reactivity in CSF samples of AD patients as compared with MCI and control samples, indicating higher levels of AD tau phosphoepitopes in the CSF. Tau heterogeneity was observed in samples of AD and MCI with higher impairment as compared with controls. As expected from previous reports, levels of Abeta (1-42) were lower (0.052+/-0.005) than controls (0.070+/-0.010), whereas the levels of MCI group were 0.060+/-0.007. The MCI group with a TBS>or=1.5 presented Abeta levels of 0.053+/-0.005 similar to those of AD patients, whereas the MCI group with TBS<1.5 exhibited Abeta levels (0.066+/-0.007) similar to controls. Studies highlight the relationships between anomalously phosphorylated tau markers in CSF with the information from TBS analysis of the different groups of patients.

ApoE alleles and tau markers in patients with different levels of cognitive impairment.

BACKGROUND: The presence of brain hyperphosphorylated tau constitutes a hallmark of neurodegenerative disorders of the Alzheimer's type. This report describes the relationships between tau markers in the cerebrospinal fluid (CSF), the degree of cognitive impairment and the predictive value of genetic markers such the alleles of apolipoprotein E, namely, the presence of Apo-epsilon4, as part of a longitudinal study. METHODS: Three major groups of patients with ages ranging from 65-73 years were evaluated in this study (n=72): Alzheimer's disease patients (AD), a group with mild cognitive impairment (MCI) and normal senile patients (NS). Hyperphosphorylated tau and tau dephosphorylated species at the Alzheimer-type epitopes in CSF samples were analyzed by ELISA assays using a battery of different monoclonal antibodies. ApoE was analyzed by PCR in blood samples. RESULTS: The levels of hyperphosphorylated tau were significantly higher in AD patients, but no statistical differences were found between the MCI and NS groups. However, the analysis of tau markers and cognitive impairment indicated the existence of two main subgroups within this population: MCI patients with a higher cognitive impairment as revealed by the total box score (TBS) >1.5 who exhibited phosphorylated tau patterns similar to the AD group, and patients with a mild impairment (TBS <1.5) with tau patterns similar to normal patients. In regard to ApoE, epsilon4/epsilon4 genotype was absent in the Chilean population analyzed, and only the epsilon2/epsilon4 genotype was significantly increased in both MCI and AD patients. A detailed analysis of the ApoE alleles, particularly epsilon3 and epsilon4, indicated a tendency to increase the epsilon4 allele in the MCI group with higher cognitive impairment and in AD patients. CONCLUSIONS: Studies indicate that hyperphosphorylated tau is a good indicator of the degree of cognitive disorders in early stages of AD and that no clear correlation exists with the epsilon4/epsilon4 and epsilon3/epsilon4 genotypes, even though a higher proportion of epsilon4 allele in the MCI group with a more significant level of impairment and in AD patients was evidenced.

Determination of cobalt in urine by FI-ICP-AES with online preconcentration.

A method for the preconcentration and determination of cobalt in human urine samples was developed. The online preconcentration and determination were attained using inductively coupled plasma atomic emission spectromety (ICP-AES) coupled to a flow injection (FI) method. Cobalt was retained on an Amberlite XAD-7 resin as cobalt-2-(5-bromo-2-pyridylazo)-5-diethylaminophenol complex at pH 9.5. Cobalt was removed from the microcolumn with perchloric acid. A sensitivity enhancement factor of 90 was obtained with respect to cobalt determination by ICP-AES without preconcentration. The value of detection limit for the preconcentration method proposed was 25 ng/L. The precision for 10 replicate determinations at the 5 mg/L (mean +/- SD, 5.1 +/- 0.14) Co level was 2.7% relative standard deviation, calculated from the peak heights obtained. The calibration graph preconcentration method for cobalt was linear with a correlation coefficient of 0.9994 from approximately 0.25 mg/L up to at least 100 mg/L. The method was successfully applied to the determination of cobalt in human urine samples.

Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies.

The intracellular polymerization of cytoskeletal proteins into their supramolecular assemblies raises many questions regarding the regulatory patterns that control this process. Binding experiments using the ELISA solid phase system, together with protein assembly assays and electron microscopical studies provided clues on the protein-protein associations in the polymerization of tubulin and actin networks. In vitro reconstitution experiments of these cytoskeletal filaments using purified tau, tubulin, and actin proteins were carried out. Tau protein association with tubulin immobilized in a solid phase support system was inhibited by actin monomer, and a higher inhibition was attained in the presence of preassembled actin filaments. Conversely, tubulin and assembled microtubules strongly inhibited tau interaction with actin in the solid phase system. Actin filaments decreased the extent of in vitro tau-induced tubulin assembly. Studies on the morphological aspects of microtubules and actin filaments coexisting in vitro, revealed the association between both cytoskeletal filaments, and in some cases, the presence of fine filamentous structures bridging these polymers. Immunogold studies showed the association of tau along polymerized microtubules and actin filaments, even though a preferential localization of labeled tau with microtubules was revealed. The studies provide further evidence for the involvement of tau protein in modulating the interactions of microtubules and actin polymers in the organization of the cytsokeletal network.