Evaluation of protein tyrosine phosphatase activity in patients with acute leukemia.

Protein tyrosine phosphatases regulate physiological processes including growth, differentiation, metabolism and the cell cycle. Together with tyrosine kinases, they control the phosphorylation state of tyrosine residues of signaling proteins. An increased level of protein phosphorylation results in abnormal proliferation and many cancer types show a mutation or deletion of a protein tyrosine phosphatase gene. In this study we evaluated the protein tyrosine phosphatase activity in acute leukemia patients. Tyrosine phosphatase activity in bone marrow mononuclear cells of acute leukemia patients was measured using a tyrosine phosphatase assay system kit and compared with a control group. We found that tyrosine phosphatase activity in acute leukemia patients was high compared to the controls. According to subgroups of acute leukemia, tyrosine phosphatase activity in the AML-M2 subgroup was high compared to the controls. The effect of increased level of protein tyrosine phosphatase activity on leukemogenesis needs further evaluation. Studies in a large group of patients are needed to emphasize the importance of tyrosine phosphatase activity in acute leukemia patients.

Effect of early or delayed administration of warfarin with heparin on thrombosis in pulmonary thromboembolism.

OBJECTIVE: The aim of this study was to investigate the effect of early or delayed warfarin administration with unfractionated heparin (UFH) on coagulation parameters in pulmonary thromboembolism (PTE). PATIENTS AND METHODS: This study was performed between November 2006 and July 2007. Thirty-three patients with PTE were sequentially slotted to early (n = 16) and delayed (n = 17) warfarin treatment groups. In the early group, both UFH infusion and warfarin were started simultaneously and in the delayed group, warfarin was added (1-3 days later) based on when partial thromboplastin time reached the therapeutic level with UFH. The proteins C and S, D-dimer, hematocrit levels, and platelet counts for all patients were studied prior to treatment and 6, 24, and 48 h after warfarin treatment. In order to determine the overall effect of early and delayed warfarin treatment on clot formation, a thromboelastogram was performed simultaneously. RESULTS: In both groups, a similar chronological decrease in protein C levels reaching maximum at 24 h with warfarin treatment was observed. However, intragroup or intergroup decreases in protein S levels were not different. On thromboelastogram, INTEM and EXTEM clotting times were significantly prolonged chronologically, but this prolongation was not different between groups. CONCLUSION: The suppressor effect of warfarin on proteins C and S in the early period of double anticoagulant treatment did not appear to aggravate the risk of thrombosis in patients with PTE in whom warfarin was started simultaneously with UFH.

Association of gastrointestinal stromal tumor and acute myeloid leukemia preceded by myelodysplastic syndrome with refractory anemia.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. GISTs are believed to be related to mutational activation of receptor tyrosine kinases, KIT, or platelet-derived growth factor receptor-alpha. The coexistence of GISTs with other neoplasms has been extensively addressed in the literature. The most common second neoplasms are colorectal cancer, prostate cancer, and neoplasms derived from lymphoid tissue. In this case report, we describe a patient affected by GIST and acute myeloid leukemia preceded by myelodysplastic syndrome with refractory anemia. The clinicopathological characteristics of the patient are discussed and the literature is reviewed.

Peripheral blood natural killer cells in Crimean-Congo hemorrhagic fever.

BACKGROUND: The relationship of changes in the peripheral blood lymphocyte subgroups during Crimean-Congo hemorrhagic fever (CCHF) to prognosis has not been reported. OBJECTIVES: To determine peripheral blood lymphocyte subgroups in CCHF patients at the time of diagnosis and relate these to clinical outcome. STUDY DESIGN: Peripheral blood samples were obtained from the patients treated at the Karadeniz Technical University Hospital for CCHF in 2004. Lymphocyte subgroups were analyzed by flow cytometry on these samples and their association with patients' risk group (severe vs. non-severe) and mortality was recorded. RESULTS: There were significantly more peripheral blood natural killer (NK) cells in severe risk CCHF patients than in non-severe risk group CCHF patients. A positive correlation was found between NK cell count and aspartate transferase (AST), alanine transferase (ALT) and activated partial thromboplastin times (aPTT). In addition, NK cell counts were observed to be higher in two patients who died. CONCLUSION: Elevated NK cell counts may be a prognostic marker in CCHF patients.

Voriconazole-induced neuropathy.

BACKGROUND: Fungal infections are common and life threatening among immunosupressive patients. Rare side effects may occur related to the use of voriconazole, which is the drug of choice in invasive aspergillosis. PATIENTS AND METHODS: Neuropathy was determined through clinical and electromyographic findings during the course of voriconazole therapy in 2 patients developing invasive aspergillosis. RESULTS: Since examinations revealed no neuropathy capable of ascription to any other cause and improvement followed the cessation of the drug, this suggested that neuropathy may be linked to voriconazole use. CONCLUSION: Neuropathy may be seen as a side effect during voriconazole treatment. Voriconazole-induced side effects should be borne in mind and patients carefully monitored during its use.

Change of coagulation parameters after double plateletpheresis.

In the previous studies, some authors reported that automated apheresis leads to a hypercoagulable state. We tried to find out changes in coagulation parameters after double plateletpheresis in this study. Forty-five donors were recruited to the study, and coagulation parameters were assessed before and after double plateletpheresis. After double plateletpheresis, fibrinogen, factor V, factor VIII and factor IX were decreased compared with the values before apheresis. Although serum levels of this coagulation parameters are decreasing, they are still in the normal limits. Therefore, we suggest that double plateletpheresis is a safe procedure for healthy volunteers taking into account these coagulation parameters.

The relation of lymphoma and hepatitis B virus/hepatitis C virus infections in the region of East Black Sea, Turkey.

AIM AND BACKGROUND: Hepatitis B virus (HBV) and hepatitis C virus (HCV) are not only hepatotropic, but also lymphotropic viruses. Infections with these viruses induce chronic antigenicity and may stimulate clonal expansion of malignant B-cell neoplasms. Moreover, these viruses can proliferate in lymphatic structures and bone marrow. However, the relationship between lymphomas and HBV/HCV infections is not clear. In our region of the East Black Sea, Turkey (the city of Trabzon), we intended to demonstrate a relation of lymphoma and HBV/HCV infections with a case-controlled study. METHODS: A total of 109 patients diagnosed with lymphoma between 2002-2005 in the Black Sea Technical University Hospital was investigated. Seventy-one patients had a high-grade and 38 patients a low-grade lymphoma. Hepatitis B surface antigen (HBsAg) and anti-HCV antibodies (anti-HCV Ab) were screened. The control group consisted of patients (n = 551) from other departments with diagnoses other than lymphoma. RESULTS: HBsAg was 3.7% and anti-HCV Ab positivity was 2.8% in lymphoma patients, compared with control of 5.3%, 5.1%, respectively. There was no statistically significant difference between two groups (P = 0.7, OR = 0.69, 95% CI, 0.20-2.10; P = 0.4, OR = 0.53, 95% CI, 0.13-1.86, respectively). CONCLUSION: Our findings suggest that the incidence of HBV and HCV infections in lymphoma patients is no different than that of nonlymphoma patients. Therefore, no direct correlation can be deduced between lymphoma and HBV-HCV infections in our East Black Sea region of Turkey.

Red grape seed extract and its compound resveratrol exert cytotoxic effect to various human cancer lines.

Modern medicinal agents currently available for treatment of cancers are very expensive, toxic, and less effective in treating some of the disease. Thus, one must investigate further in detail the agents derived from natural sources, such as grape seed, for the prevention and treatment of cancer and disease. In recent years interest of researchers has focused on grape seed and nowadays scientists have used extracts of grape seed to treat different health problems including cancer. We examined the cytotoxic effect of red grape seed extract (GSE) and its main compound resveratrol (RES) on different human cancer cell lines representing various solid tumors and hematological malignancies at the same time. Red GSE was prepared by using 1, 1, 1, 2- Tetrafluoroethane extraction method. Cytotoxicity of the extract and RES was evaluated by using trypan blue dye exclusion method and MTT assay. The results of our study show that GSE and RES have cytotoxic activities in varying degree in several cancer cell lines. There has not been any study evaluating the GES and RES in the same cell lines and in the same conditions. But, it is still needed to have more pre-clinical and laboratory studies to validate the usefulness of these agents either alone or in combination with existing therapy.

Evaluation of Manisa propolis effect on leukemia cell line by telomerase activity.

Propolis is a resinous substance which is used by bees to repair and maintain their hives. It has more than 180 compounds including flavonoids, phenolic acids and its esters which have anti-inflammatory, antibacterial, antiviral, immunomodulatory, antioxidant and antiproliferative effects. Propolis is shown to inhibit cell division and protein synthesis. However the exact mechanism underlying antitumor effect is not clearly described. On the other hand progressive telomere shortening to a critical level results with senescence of normal cells by inducing apoptosis and telomerase prevents erosion of telomeres. In this study we aimed to evaluate hTERT ratios in propolis-treated T-cell acute lymphoblastic leukemia (CCFR-CEM) cell line. Cell counts and cell viability of propolis-treated and propolis-free T-cell acute lymphoblastic leukemia (CCFR-CEM) cell line were assessed by trypan blue dye exclusion test and MTT assay. The LightCycler instrument was used (online real-time PCR) for the quantification of hTERT in CCFR-CEM cell line. The hTERT ratio significantly decreased 60 and 93% after 24 and 72 h respectively compared to the initial value of the cells incubated with propolis. It had almost no cytotoxic effect and caused 30, 30, 22 and 12% decrease in cell counts after 24, 48, 72 and 96 h respectively which is statistically significant. In conclusion propolis may show antitumor and apoptotic effect via inhibiting telomerase expression besides the mechanisms which have been described previously.

Thrombin activatable fibrinolysis inhibitor in Behçet's disease.

INTRODUCTION: Thrombin activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase downregulating plasmin formation, thereby causing a tendency for thrombosis development. Since, Behçet's disease (BD) is a systemic vasculitis, which is commonly complicated by arterial and venous thrombosis, we aimed to find out plasma TAFI levels in BD, compared with healthy controls. We also searched whether plasma TAFI levels were significantly different between Behçet's subgroups with and without thrombosis. MATERIALS AND METHODS: In this study, 105 BD patients (M/F: 64/41; mean age 36+/-1 years), followed up by Ege University Rheumatology Department were enrolled. The exclusion criteria were hemophilia, hyperlipidemia, diabetes mellitus, hepatic diseases renal failure, antiphospholipid positivity, oral contraceptive use and pregnancy. Age-and sex-matched healthy controls (n=53) were also included. Plasma TAFI levels were measured by ELISA. Since TAFI is also an acute-phase reactant, we also measured other inflammatory markers such as C-reactive protein (CRP). RESULTS: Plasma TAFI levels were significantly higher in Behçet's patients (91.1+/-7.4 ng/ml) compared with healthy controls (14.3+/-4.5 ng/ml) (P<0.001), but there were no significant difference between the subgroups with and without thrombosis. In BD, there was no correlation between plasma TAFI levels and CRP. CONCLUSIONS: Regardless of manifest thrombosis, plasma TAFI levels in BD were significantly higher than in healthy controls. High TAFI levels might possibly contribute to the thrombotic tendency in BD. Future studies investigating TAFI gene polymorphism and functional activity are clearly needed, to clarify the exact role of TAFI in Behçet's thrombosis.

Arsenic trioxide and methylprednisolone use different signal transduction pathways in leukemic differentiation.

Certain cell lines like HL 60 and K 562 are utilised as leukemic cell models for leukemogenesis research, which differentiate along the granulocytic and/or monocytic pathway when treated with certain inducer molecules. High dose methylprednisolone treatment has been shown to induce in vivo and in vitro differentiation of myeloid leukemia cells to mature granulocytes in patients with acute promyelocytic leukemia (APL) and other subtypes of acute myeloid leukemia (AML). Arsenic trioxide (As(2)O(3)) has been confirmed to have remission induction effects on APL. However, there are conflicting results on the effects with other AML subtypes. Also, it has been well established that the reversible phosphorylation of proteins is a major regulatory mechanism in the signal transduction pathways that control cell growth and differentiation. Serine/threonine protein phosphatases (PP) are major components of phosphorylation. In this study, we investigated the effect of As(2)O(3) on HL 60 and K 562 myeloid leukemic differentiation and compared the signalling cascades of the two inducers with respect to serine/threonine PP 1 and 2A. We utilised PP1 and PP2A inhibitors okadaic acid and calyculin A. In contrast to methylprednisolone, there was no effect of phosphatase inhibitors on As(2)O(3)-induced leukemic differentiation. Incomplete leukemic differentiation occurred with lower As(2)O(3) concentration as 10(-6)M. Unlike As(2)O(3), methylprednisolone induced complete granulocytic and/or monocytic differentiation of HL 60 and K 562 cells via upregulation of PP2A regulatory subunits. Therefore, As(2)O(3) and methylprednisolone are promising agents that have the potential to be used together in myeloid leukemic differentiation therapy.

Involvement of protein phosphatase 2A in interferon-alpha-2b-induced apoptosis in K562 human chronic myelogenous leukaemia cells.

Interferon-alpha (IFN-alpha)-2b is known to have antiproliferative effects on hematological malignant cells, especially chronic myelogenous leukaemia (CML). However, it can induce cytogenetical remissions in a very small percentage of the patients. Also during interferon therapy, resistance can emerge in the CML clones. K562 is an in vitro model cell line transformed from a Ph positive CML patient. It can be induced to differentiate to granulocytic and/or monocytic lineages with certain molecules. IFN-alpha-2b generally exerts its effects on CML cells by Janus family kinases (Jak/Stat) pathway, mostly through tyrosine kinase system. However, there is almost no data on the relevance of serine/threonine (Ser/Thr) protein phosphatase (PP) system in the interferon induced signal transduction pathways. In this study, we investigated serine/threonine protein phosphatases in the IFN-alpha-2b induced K562 cytotoxicity. Trypan blue dye exclusion test and MTT assay were utilised for determining cytotoxicity. IC(50) of IFN-alpha-2b on K562 cells was found to be 600IU/ml. However, no differentiation was determined by analysis of cell surface antigen expressions. Serine/threonine protein phosphatase inhibitors calyculin A (Cal A) and okadaic acid (OKA) augmented the IFN-alpha-2b induced cytotoxicity. Apoptosis assay by the mono-oligonucleosome detection and acridine orange/propidium iodide dye revealed marked apoptosis underlying cytotoxicity. Phosphatase enzyme assay revealed a gradual increase in protein phosphatase 2A (PP2A) activity during interferon induced cytotoxicity. Conversely, immunoblots showed no change in the expression of PP2A catalytic and regulatory subunits. In conclusion, PP2A plays a role in IFN-alpha-2b induced apoptosis of K562 cells and should be investigated as a new window furthermore.

Cytotoxic and inhibitory effects of 4,4'-dihydroxy chalcone (RVC-588) on proliferation of human leukemic HL-60 cells.

Chalcones have been identified as interesting compounds with cytotoxic and tumor reducing properties. In the present study, the biological activity of synthetic chalcones on myeloid leukemic cells was investigated. Human myeloid HL-60 leukemia cells were exposed to 1-20 micro M chemicals for 0-96h. The viability of the cells was measured using trypan blue dye exclusion method. 4,4'-Dihydroxy chalcone (RVC-588) was selected for further experiments to determine characteristics of cytotoxicity among other compounds. The data show that cell viability decreased after treatment and IC(50) value was approximately 2 micro M for RVC-588. Cell differentiation was analyzed with cytofluorometry by changes in expression of glicoprotein surface markers CD11b/Mac-1, CD11c and CD14 together with morphological analysis. A maximum level of expression changes was determined at 72h but these changes were not statistically significant to show the differentiation of HL-60 cells to mature myeloid and/or monocytoid cells. Apoptotic DNA degradation was evaluated and quantitated using sensitive enzyme-linked immunoabsorbant (ELISA) method. Using this technique, a maximum level of apoptosis 1.2-fold higher than control was observed in cultures exposed for 48h to 2 micro M RVC-588. The DNA ladder assay was subsequently used to determine DNA breaks qualitatively. After 24h, the cells exposed to 2 micro M RVC-588 was shown to have cytotoxic-late apoptotic ladder pattern compared to control cells. These data demonstrate that RVC-588 has a high cytotoxic and antitumor activity in HL-60 cells among other chemicals we synthesized. Although the mechanism by which RVC-588 initiated cell death in these cells is presently not known and apoptotic mechanisms are likely to play less role compared to other chalcone analogues reported previously.

Differential effects of doxorubicin and docetaxel on nitric oxide production and inducible nitric oxide synthase expression in MCF-7 human breast cancer cells.

Anthracyclines and docetaxel are frequently used agents in the chemotherapy of breast cancer. In this study we examined the role of inducible nitric oxide synthase (iNOS) expression during the cytotoxicity of these drugs in the MCF-7 breast cancer cell line. Cell viability and cytotoxicity were evaluated by the trypan blue dye exclusion method. Apoptosis and necrosis were determined by the acridine orange/ethidium bromide dye method. The percentage of apoptotic cells was significantly higher with doxorubicin. However, total cytotoxic cell numbers were higher in the docetaxel group compared with doxorubicin, with respect to the control. Most of the cells were seen to be necrotic with the dye method. Cell extracts during the apoptotic process were applied to immunoblot by anti-iNOS monoclonal antibodies. While there was an increase in iNOS expression during docetaxel induced-cytotoxicity, a significant decrease in iNOS expression was detected during doxorubicin-induced cytotoxicity. The Griess method was used for detection of nitrate levels. It was compatible with immunoblot results. These data open a window for further studies to understand the mechanism underlining the cytotoxicity of docetaxel and doxorubicin.

Alterations in the activity and expression of serine/threonine protein phosphatases during all trans retinoic acid-induced apoptosis in breast cancer cells.

Retinoids exert different effects on malignant cells with various phenomena. They can induce differentiation and apoptosis in various cancer cells. However, the underlying mechanism of these effects is not clear. There are data related to the role of protein phosphatases during retinoid-induced leukemic cell differentiation. The aim of this study was to evaluate effects of the All trans retinoic acid (ATRA) on protein/serine phosphatases during ATRA induced apoptosis in the breast cancer cells. The MTT assay was used to determine drug-mediated cytotoxicity. A cell death detection ELISA kit was used for detection of the DNA fragments. The activity of serine/threonine protein phosphatases was assayed by the serine/threonine phosphatase system. The expression of serine/threonine protein phosphatases was evaluated by Western blot. During ATRA treatment, a significant decrease in the activity of serine/threonine phosphatases 2A, B and C occurred. The decreased activity of PP2A correlated with the up-regulation of PP2A catalytic and PP2A/B gamma, PP2A/B alpha regulatory subunits. The decrease in activity of the PP2B correlated with down-regulation of PP2B catalytic and up-regulation of PP2B regulatory subunit expression. In addition, there was an up-regulation in PP4C and down regulation in PP2C alpha/beta subunits protein expression. We demonstrated clear alteration in the activity and expression of serine/threonine protein phosphatases in breast cancer cells during ATRA treatment, and we suggest that the ATRA-induced apoptosis of the MCF-7 cells is significantly related to the phosphorylation dynamics.

The effect of type II diabetes mellitus on platelet aggregation in patients who underwent percutaneous transluminal coronary angioplasty.

BACKGROUND: In noninsulin-dependent type II diabetic (tIIDM) patients it was reported that ADP-induced platelet aggregation response was increased with decreased level of platelet guanylate cyclase. This study was therefore designed to examine the effects of tIIDM on collagen-induced, in-vitro platelet aggregation in percutaneous transluminal coronary angioplasty (PTCA) patients. METHODS: Twenty patients with tIIDM and 30 nondiabetic patients who had successful PTCA were included in the study. Platelet-rich plasma samples from the patients before and after PTCA were treated with in-vitro collagen and platelet aggregation waves were calculated via the turbidometric method of Born. The maximum amplitude (%) and the ratio of changes after PTCA in the study participants were measured by these waves and data were compared by student's t tests and nonparametric methods. The maximum amplitude of collagen-induced platelet aggregation before and after the procedure was also compared using variant analysis. RESULTS: The change in collagen-induced maximum amplitude of platelet aggregation in both wave 1 and wave 2 was significantly more (P < 0.001- P < 0.001) in the tIIDM group. The ratio of restenosis seen in the control coronary angiography made 6 months after intervention was found to be significantly more in the tIIDM group (P < 0.05). CONCLUSIONS: Collagen-induced platelet aggregation response was greater in patients with tIIDM than in nondiabetic patients. This makes us think that tIIDM patients could need more potent antiplatelet therapy before PTCA after the blood glucose levels have been regulated.

Potential Involvement of Calcineurin in Regulating the State of Differentiation and Apoptosis of HL-60 Cells During Methylprednisolone-Treatment.

To evaluate the role of calcineurin (protein phosphatase type 2B, PP2B) in methylprednisolone-induced differentiation and apoptosis of leukaemic cells, we have investigated the induction of apoptosis, calcineurin specific protein phosphatase activity and expression of regulatory and catalytic subunits of calcineurin and calmodulin after induction of HL-60 leukaemic cells with methylprednisolone. The cells underwent differentiation and apoptosis within 72 hours time period after methylprednisolone added to cell culture media. Before apoptosis occurred, the specific calcineurin enzyme activity revealed gradual increase during the differentiation process. However, immunoblots of catalytic and regulatory subunits of calcineurin showed no amplification in the amount of these cellular signaling mediators during methylprednisolone-induced differentiation and apoptosis but calmodulin expression gradually increased during the process. Significant increase in the specific calcineurin enzyme activity during differentiation and apoptosis might be crucial to the posttranslational modifications of calcineurin during methylprednisolone-induced differentiation.

Effect of LMO2 protein expression on survival in chronic myeloid leukemia patients treated with imatinib mesylate.

LIM domain only-2 (LMO2) is an important regulator of hematopoietic stem cell development. LMO2 protein is expressed in all three hematopoietic lineages precursors of the hematopoietic system, and its expression has been shown to decrease gradually during differentiation. Chronic myeloid leukemia (CML) is a malignant clonal myeloproliferative disorder in which the terminal differentiation is not altered until the appearance of an accelerated or blast phase. We examined whether LMO2 protein expression can predict outcome CML patients undergoing tyrosine kinase inhibitor therapy, imatinib mesylate (IM). Immunohistochemistry on bone marrow biopsy material for LMO2 protein was performed in 47 CML patients. We report that the LMO2 protein expression is correlated with improved hematologic remission and overall survival in the CML patients treated with IM. The immunohistologic analysis of LMO2 protein expression may become a predictive factor for anticipating the treatment responses of CML patients.

The interaction between taxoids and serine/threonine protein phosphatase activities during taxan-induced apoptosis of HL 60 leukemic cells.

Paclitaxel and docetaxel (taxoids) are chemotherapy agents whose mode of action is through an effect on cellular microtubules. Several studies have investigated their potential in the treatment of myeloid malignancies. The aim of our study was to investigate the potential role of the serine/threonine protein phosphatase system in docetaxel/paclitaxel induced cytotoxicity on HL 60 cells. The IC50 dose of paclitaxel and docetaxel were found as 20 and 5 nM respectively using trypan blue dye exclusion and XTT assays. Treating HL 60 cells with docetaxel and paclitaxel resulted in dose and time dependent cytotoxicity. Docetaxel induced the decrease in the activity of protein phosphatase 1 (PP1) and increase in the activity of PP2 subgroups, while paclitaxel induced the increase in the activity of PP1 and decrease in the activity of PP2 subgroups. Potential use of specific protein phosphatase inhibitors or activators in combination with taxoids will open new windows in the treatment of myeloid leukemias.

Effect of pathologic fractures on survival in multiple myeloma patients: a case control study.

BACKGROUND: Multiple Myeloma (MM) is a B cell neoplasm characterized by the clonal proliferation of plasma cells. Skeletal complications are found in up to 80% of myeloma patients at presentation and are major cause of morbidity. METHODS: 49 patients were enrolled with MM admitted to Black Sea Technical University Hospital between 2002-2005. Pathologic fractures (PFs) were determined and the patients with or without PF were followed up minimum 3 years for survival analysis. RESULTS: PF was observed in 24 patients (49%) and not observed in 25 patients (51%). The risk of death was increased in the patients with PF compared with patients who had no fractures. While overall survival was 17.6 months in the patients with PFs, it was 57.3 months in the patients with no PFs. CONCLUSION: These findings suggest that PFs may induce reduced survival and increased mortality in the MM patients, however, larger sample size is essential to draw clearer conclusions added to these data.