Dual specificity phosphatase 7 drives the formation of cardiac mesoderm in mouse embryonic stem cells.

Dual specificity phosphatase 7 (DUSP7) is a protein belonging to a broad group of phosphatases that can dephosphorylate phosphoserine/phosphothreonine as well as phosphotyrosine residues within the same substrate. DUSP7 has been linked to the negative regulation of mitogen activated protein kinases (MAPK), and in particular to the regulation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). MAPKs play an important role in embryonic development, where their duration, magnitude, and spatiotemporal activity must be strictly controlled by other proteins, among others by DUSPs. In this study, we focused on the effect of DUSP7 depletion on the in vitro differentiation of mouse embryonic stem (ES) cells. We showed that even though DUSP7 knock-out ES cells do retain some of their basic characteristics, when it comes to differentiation, they preferentially differentiate towards neural cells, while the formation of early cardiac mesoderm is repressed. Therefore, our data indicate that DUSP7 is necessary for the correct formation of neuroectoderm and cardiac mesoderm during the in vitro differentiation of ES cells.

Roscovitine sensitizes leukemia and lymphoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand with selective antitumor activity. However, many primary tumors are TRAIL resistant. Previous studies reported that roscovitine, a cyclin-dependent kinase inhibitor, sensitized various solid cancer cells to TRAIL. We show that roscovitine and TRAIL demonstrate synergistic cytotoxicity in hematologic malignant cell lines and primary cells. Pretreatment of TRAIL-resistant leukemia cells with roscovitine induced enhanced cleavage of death-inducing signaling complex-bound proximal caspases after exposure to TRAIL. We observed increased levels of both pro- and antiapoptotic BCL-2 proteins at the mitochondria following exposure to roscovitine. These results suggest that roscovitine induces priming of cancer cells for death by binding antiapoptotic BCL-2 proteins to proapoptotic BH3-only proteins at the mitochondria, thereby decreasing the threshold for diverse proapoptotic stimuli. We propose that the mitochondrial priming and enhanced processing of apical caspases represent major molecular mechanisms of roscovitine-induced sensitization to TRAIL in leukemia/lymphoma cells.

PBPC collections: Management, techniques and risks.

We evaluated the efficiency, safety and risks of three techniques which were used for autologous PBPC collections: (a) large-volume leukapheresis (LVL), (b) standard collections, and (c) a new modified technique which was named as "Mixed" collections. In spite of the fact that the standard and LVL collection techniques are used routinely, there may occur special conditions in which the procedures cannot be recommended. Some patients may suffer from serious clinical complications and they cannot tolerate either standard procedures with administration of higher doses of ACD-A, or the high extent of procedure in the course of LVL. We tried to find the safe and efficient collection technique which could help this group of patients to overcome their problems. The "Mixed" collection technique could be such a choice. The numbers of 136 autologous PBPC collections were performed in 98 patients who suffered from hemato-oncological diseases. We evaluated the results of (a) 93 LVL (more than 3 TBV, total blood volumes of the patients were processed; anticoagulation: ACD-A and Heparin), (b) 16 Standard procedures (less than 3 TBV were processed; anticoagulation: ACD-A), and (c) 27 "Mixed" collections (less than 3 TBV of patients were processed; anticoagulation: ACD-A+ Heparin). Collections were performed by the use of separator Cobe Spectra, Caridian. In patients (a) with a good effect of mobilization (precollection CD 34+ cells in blood higher than 20×10(3)/mL) we prepared almost the same median dose of CD 34+ cells from the standard and "Mixed" collections, 3.8 and 4×10(6)/kg, respectively. In LVL the median yield of CD 34+ cells was 8.2×10(6)/kg. In patients (b) who were mobilized weakly (precollection CD 34+ cells in blood lower than 20×10(3)/mL), LVL enabled to prepare 1.5×10(6) of CD 34+/kg from one collection, while the median yield of CD 34+ cells from the standard and "Mixed" collections was 0.9 and 1.2×10(6)/kg. All the standard, LVL and "Mixed" procedures were tolerated well without any serious adverse reactions. We detected 22 adverse reactions, but only three reactions were associated directly with the procedure. Mild hypocalcemia (2) and hypotensive reaction (1) were transient and treated efficiently. Procedures could continue and were finished according to the planned programme. Other reactions were related either to the insufficient function of central venous catheter or to the poor clinical condition of the patients. LVL enabled to get a higher yield of CD 34+ cells than the Standard and "Mixed" collections in well mobilized patients as well as in weakly mobilized patients. We observed the similar efficiency in standard and "Mixed" collections in well mobilized and weakly mobilized patients. We can recommend LVL in all patients who can tolerate it due to a greater chance of collecting higher yields of progenitor cells. In the weakly mobilized patients LVL offers a greater chance of collecting at least a minimum amount of CD 34+ cells needed for transplantation. "Mixed" collections may be used as an alternative technique under the circumstances in which standard or LVL cannot be recommended - like in patients who do not tolerate a high amount of citrate or a high extent of the procedure, e.g. patients with cardiac arrhytmia, impaired liver or renal function or unstable vital signs.

PBPC collection techniques: standard versus large volume leukapheresis (LVL) in donors and in patients.

Transplantations of autologous and allogeneic peripheral blood progenitor cells (PBPC) are able to assure a complete hematopoietic and immunologic reconstitution in patients. PBPC are collected by leukapheresis technique after prior mobilization therapy, but procedures and results remain still highly variable and are poorly characterized. An optimum regimen for PBPC collections has not yet been recommended, but 2-3 total blood volumes (TBV) of the donor or patient are regarded as a standard. Another promising technique is large volume leukapheresis (LVL) with processing of 3-6 TBV of donor or patient. The aim of this paper is to find the most efficient and safe collection technique for an individual donor or patient and, consequently minimize the number of procedures required. Finding the optimal collection procedure would be helpful while considering which method would be preferred in an individual donor or patient with respect to the result of mobilization, health state and required yield of CD 34+ cells for transplantation. We evaluated the results in a total of 134 standard and LVL procedures, which were performed in 21 well mobilized donors (Group I), in 65 well mobilized patients (Group II), and in 14 weakly mobilized patients (Group III) with hemato-oncological diseases. A precollection concentration of CD 34+ cells in peripheral blood higher than 20 x 10(3)/mL was considered to be the criterion for efficient mobilization. Such levels of concentration indicating the start of PBPC collections could be easily reached in Group I of donors and Group II of well mobilized patients. Heavily pretreated patients at advanced stages of disease (Group III) did not respond to mobilization sufficiently and had a concentration of CD 34+ cells lower than 20x10(3)/mL. LVL technique made it possible to obtain higher numbers of CD 34+ cells than in the standard collection in well mobilized donors (Group I), well mobilized patients (Group II), and even in weakly mobilized patients in Group III. In donors and well mobilized patients (Group I and Group II) it was possible to collect sufficient amounts of CD 34+ cells for allogeneic or for autologous transplantation from one LVL collection. The median yield of CD 34+ cells from one LVL collection was 5.5 x 10(6)/kg b.w. in donors, and 6.0 x 10(6)/kg b.w. in well mobilized patients. Due to the linear dependence of the yield of collected CD 34+ cells on the concentration of CD 34+ cells in blood, it can be used as a simple prediction of the success of collection in Group II (correlation coefficient 0.93 for standard procedures, and correlation coefficient 0.88 for LVL). In Group III of weakly mobilized patients the standard collections were usually ineffective and the relationship between the yield of CD 34+ cells/kg in the product and the precollection concentration of CD 34+ cells was much less significant (correlation coefficient 0.56 for standard procedures and correlation coefficient 0.66 for LVL). The median of CD 34+ cells collected from one standard procedure was only 0.7 x 10(6)/kg but using LVL the median increased to 1.4 x 10(6)/kg. Our results prove that the yield of CD 34+ cells in the product can be enhanced by large volume leukapheresis (LVL). Based on the results obtained, we recommend LVL in all donors and patients who can tolerate it due to a greater chance of collecting higher yields of progenitor cells while minimizing adverse reactions. LVL procedures should also be preferred in weakly mobilized patients where it is not possible to collect sufficient amounts of CD 34+ cells for transplantation using the standard regime. In weakly mobilized patients LVL provides a greater chance to at least collect a minimum amount of CD 34+ cells necessary. LVL should be used in circumstances where extremely high doses of CD 34+ cells has to be prepared, e.g. planned "tandem" transplantations or manipulations with a graft in which a significant loss of cells is expected.