l-Amino acid oxidase isolated from calloselasma rhodostoma snake venom induces cytotoxicity and apoptosis in JAK2V617F-positive cell lines

BACKGROUND: Myeloproliferative neoplasms are Philadelphia chromosome-negative diseases characterized by hyperproliferation of mature myeloid cells, associated or not with the Janus kinase 2 tyrosine kinase mutation, JAK2V617F. As there is no curative therapy, researchers have been investigating new drugs to treat myeloproliferative neoplasms, including l-amino acid oxidase from Calloselasma rhodostoma snake venom (CR-LAAO), which is a toxin capable of eliciting apoptosis in several tumor cell lines. OBJECTIVE: To evaluate the effects of l-amino acid oxidase from C. rhodostoma snake venom in the apoptotic machinery of JAK2-mutated cell lines. METHODS: The HEL 92.1.7 and SET-2 cell lines were cultured with l-amino acid oxidase and catalase for 12 h at 37 °C in 5% carbon dioxide. The cell viability was assessed by the multi-table tournament method, the level of apoptosis was measured by flow cytometry, and the expression of cysteine-dependent aspartate-specific proteases and cleaved Poly(ADP-ribose) polymerase were analyzed by Western blotting. RESULTS: l-Amino acid oxidase from C. rhodostoma snake venom was cytotoxic to HEL 92.1.7 and SET-2 cells (50% inhibitory concentration = 0.15 µg/mL and 1.5 µg/mL, respectively) and induced apoptosis in a concentration-dependent manner. Cell treatment with catalase mitigated the l-amino acid oxidase toxicity, indicating that hydrogen peroxide is a key component of its cytotoxic effect.The activated caspases 3 and 8 expression and cleaved PARP in HEL 92.1.7 and SET-2 cells confirmed the apoptosis activation by CR-LAAO. CONCLUSIONS: l-Amino acid oxidase from C. rhodostoma snake venom is a potential antineoplastic agent against HEL 92.1.7 and SET-2 JAK2V617F-positive cells as it activates the extrinsic apoptosis pathway.

Correlation of JAK2V617F mutational status in primary myelofibrosis with clinico-hematologic characteristics and international prognostic scoring system scoring: A single center experience.

Introduction: Somatic mutation in the exon 14 of Janus Kinase 2 gene is an established diagnostic marker in bcr-abl negative myeloproliferative neoplasms, especially primary idiopathic myelofi brosis (PIMF). Aim: Our primary aim was to fi nd out the correlation between the JAK2V617F mutational status and the clinico-hematologic characteristics, as well as the international prognostic scoring system (IPSS) scoring of patients with PIMF. Materials and Methods: Clinical and hematologic features were reviewed for 68 patients with primary idiopathic myelofi brosis (PIMF). JAK2V617F mutation status was analyzed by amplifi cation refractory mutation screening-polymerase chain reaction. The patients were further stratifi ed into low, intermediate-1, intermediate-2 and high-risk groups on the basis of IPSS scoring. Results: The JAK2V617F mutation was detected in 58.8% patients. Univariate analysis of variables at presentation identifi ed that JAK2V617F negative patients were signifi cantly associated with more severe anemia (P = 0.045), younger age (P = 0.008), higher transfusion requirement (P = 0.017), and thrombocytopenia (P = 0.015). Patients who were homozygous for JAK2V617F mutation were associated with thrombocytosis (P = 0.014) and also had higher median total leucocyte count (P = 0.20) than the other groups. No signifi cant correlation was detected between JAK2V617F mutational status and the presence of constitutional symptoms, spleen size, grade of bone marrow fi brosis or prognostic risk stratifi cation of the PIMF patients. Conclusion: The variations in the prognostic implication of PIMF patients with mutation status as stated by various publications worldwide, reinstates the need for larger prospective studies using standardized JAK2V617F quantifi cation methods as well as estimation of other newer molecular markers to develop deeper insight into various molecular alterations involving PIMF patients in India as well as worldwide.

Prevalence of JAK2 V617F mutation in Indian patients with chronic myeloproliferative disorders.

Background & objectives: The Janus-associated Kinase-2 mutation JAK2 V617F in chronic myeloproliferative disorders (CMPDs) has been described as a frequent genetic event in majority of patients with polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Its frequency varies in different populations but there are no data from India. We therefore, looked for JAK2 V617F mutation in Indian patients with chronic myeloproliferative disorders. Methods: Mutation screening for JAK2 V617F in patients with polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis was performed in 75 patients attending Haematology clinic in a tertiary care hospital in north India, by polymerase chain reaction and restriction enzyme-based assay. Results: JAK2 V617F mutation was found in 51 of 75 cases (68%) of CMPD, 82 per cent in PV, 70 per cent in ET and 52 per cent of IMF. The presence of JAK2 V617F mutation was associated with a higher haemoglobin level (P<0.05), a higher white blood cell count (P<0.01) and higher age (P<0.05). Interpretation & conclusion: The JAK2 V617F mutation was detected in 86 per cent of patients with CMPD disorders. Peripheral blood mutation screening for JAK2 V617F can be incorporated into the initial evaluation of patients suspected to have CMPD.

Diagnostic Usefulness of the Janus Kinase 2 Mutation in non BCR/ABL Myeloproliferative Disorders

BACKGROUND: We investigated the Janus kinase 2 (JAK2) mutation and its diagnostic value in patients suffering with non BCR/ABL myeloproliferative diseases (nMPD) or other reactive conditions. METHODS: We reviewed the clinical records of 83 patients who underwent bone marrow (BM) examinations with suspect of nMPD. The diagnoses of nMPD were made based on the WHO criteria since 2001 and the PVSG criteria before 2001. The JAK2 mutation was examined by PCR in 54 patients whose BM samples were available. RESULTS: The JAK2 mutation was detected in 25 patients (46%); 12 of 26 patients with essential thrombocythemia (ET), 9 of 12 patients with polycyhtemia vera (PV), one of 7 patients with chronic idiopathic myelofibrosis (CIM) and one patient with unclassifiable MPD. Additionally, JAK2 mutation was detected in each one patient with secondary polycythemia and reactive thrombocytosis. These two patients and two other patients among the JAK2 mutated ET did not meet the WHO PV criteria due to their initial low hemoglobin levels. These patients had liver cirrhosis and hypersplenism due to Budd-Chiari syndrome (1), gastrointestinal bleeding (1) or the initial hemoglobin level was slightly below the level as provided by the criteria, but the level showed a rising pattern despite cytoreductive therapy (2). With the results of the JAK2 mutation available, 4 patients' disease could be re-diagnosed as PV. Finally, the positive rate of the JAK2 mutation was 81% in PV, 48% in ET and 14% in CIM. The presence of JAK2 mutation closely correlated with PV (p=0.001), leukocytosis (p=0.001) and an increased cellularity of BM (p=0.024). CONCLUSIONS: The JAK2 mutation may help differentiate nMPD from secondary cytosis. Therefore, it should be incorporated into the guidelines for the nMPD work-up for making a more accurate diagnosis and administering proper treatment.