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For the purpose of ascertaining parameters to embryo transfer on some domestic animals, mouse morulae were used as a model to investigate the effect of in-straw thawing on in vitro and in vivo-development of vitrified embryos. Embryos were vitrified in 0.25 ml straws preloaded with dilution solution (0.5 M Sucrose) and thawed in the straw by mixing the vitrification solution (Ethylene glycol + Ficoll 70 + Sucrose) and the dilution solution at 25 degrees C. The embryos were randomly divided into six groups and expelled from the straws after they had been suspended in the in-straw mixture for 3 min, 5 min, 8 min, 12 min, 16 min, and 20 min, respectively, and then they were collected under a microscope for in vitro culture or direct transfer. The in vitro developmental rates of the embryos were 92.3% to 98.4% and hatching rates were 64.1% to 75.6% for the groups of 3 min to 16 min, showing no significant differences with those of nonfrozen controls (100%, 76.2%; P > 0.05). While embryos were suspended in the straw for 20 min, the developmental rate (86.6%) and hatching rate (52.4%) were significant lower than those of the control (100%, 76.2%; P < 0.01). When the 168 frozen-thawed embryos (in-straw thawing for 5 min) and 168 fresh embryos were transferred, respectively, the proportion of live fetuses in the pregnant recipients between them (58.7% vs. 54.5%) showed no significant difference (P > 0.05). The data indicate that vitrification with EFS30 and suspension in the in-straw mixture for 3 min to 16 min, when thawing, did not affect the in vitro developmental rate and hatching rate. Moreover, the in vivo developmental rate between vitrified embryos and fresh embryos did not differ significantly. It can be concluded that this method is fit for nonsurgical embryo transfer in some domestic animals with a suggestion that the operation of embryo transfer should be accomplished within 16 min.

Mutations in transcription factors (TFs) and protein tyrosine kinases (PTKs), which result in inhibition of differentiation/apoptosis or enhanced proliferative/survival advantage of hematopoietic stem/progenitor cells, are two classes of the most frequently detected genetic abnormalities in leukemias. The critical roles for mutant TFs and/or PTKs to play in leukemogenesis, and the absence of mutant TFs/PTKs in normal hematopoietic cells, suggest that the two types of aberrant molecules may serve as ideal therapeutic targets. The great success of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) in treating acute promyelocytic leukemia through modulation of the causative PML-RARalpha oncoprotein represents the first two paradigms of mutant TFs-targeting therapeutic strategies for leukemia. More recently, tyrosine kinase inhibitor STI-571/Imatinib mesylate/Gleevec in the treatment of Breakpoint Cluster Region-Abelson (BCR-ABL) positive leukemia elicits paradigm of mutant PTKs as ideal antileukemia targets. Thus to further improve clinical outcome of leukemia patients, elucidation of pathogenesis of leukemia, screening for oncoprotein-targeting small molecules, as well as rationally designed combination of drugs with potential synergy are of importance.

This study was first employed to investigate the developmental potential of mouse hatched blastocyts (HBs) vitrified by a two-step open-pulled straw (OPS) method. HBs were obtained by culture of morulae in vitro. First, the embryos were placed in four cryprotectant solutions - that is, 10% ethylene glycol (EG), 10%E + 10%D (10% EG and 10% dimethyl sulphoxide (DMSO) in mPBS), EFS30 (30% EG, Ficoll, and sucrose) and EDFS30 (15% EG, 15% DMSO, Ficoll, and sucrose)--at 25 degrees C for 0.5 to 10 min, respectively, to determine their optimal survival after rapid dilution in 0.5 M sucrose. Secondly, based on the above best survival, the embryos were plunged into liquid nitrogen after first pretreatment in 10%E for 0.5 min and then 0.5 min equilibration in EFS30 (Group 1), or 10%E + 10%D and EDFS30 for 0.5 min, respectively (Group 2). When warming, three methods were used to dilute the cryoprotectants from the vitrified embryos. The embryos were assessed by the re-expansion of the blastocoel or development to term. The result showed that all the vitrified-warmed HBs got high in vitro survival rates (83.7% to 98.9%). The highest in vitro survival rates (87.8% in Group 1, 98.9% in Group 2) were obtained when the vitrified embryos were diluted first in 0.3 M sucrose for 5 min, then in 0.15 M sucrose for 2 min (method C). When the vitrified embryos diluted with method C were transferred, their survival rate in vivo (35.5% to 42.2% of the total) were similar to (P > 0.05) that of control (45.7%). These results demonstrate OPS method was highly efficient for the cryopreservation of mouse HBs.

Objective: To explore the mechanism of apoptosis of myeloid leukemia cells induced by Bortezomib, a proteasome inhibitor., Methods: Human acute myeloid leukemia cells of the line HL60 were cultured and treated with Bortezomib of the concentrations of 0, 10, 20, 30, and 40 micromol/L for 24 hours. MTT assay and flow cytometry were used to detect the proliferation inhibition and apoptosis. Hoechst 33342 staining was used to observe the morphology of the cells. Western blotting was used to detect the protein expression of Bcl-2, Caspase-9, Caspase-3, and poly ADP-ribose polymerase (PARP)., Results: Bortezomib could induce HL60 cell apoptosis dose- and time-dependently. After treated for 24 hours by 30 nmol/L Bortezomib the HL60 cells' proliferation was significantly inhibited, the inhibition rate was 76%, and the cell nuclei became progressively pyknotic and were extensively fragmented. FCM showed apoptosis peaks 24 hours after treatment of Bortezomib of the concentrations of were 10 and 20 nmol/L, the apoptosis rate was 62.6%. Bcl-2 protein expression was down-regulated and the protein expressions of Caspase-9, Caspase-3, and PARP were all up-regulated., Conclusion: The mechanism of Bortezomib to induce apoptosis of myeloid leukemia cells is associated with down-regulation of Bcl-2 protein expression and cleaved activation of Caspase-9, Caspase-3 and PARP proteins.

To examine the efficiency of EDFS and EFS for blastocyst cryopreservation and to provide information for preparation of vitrification medium at different temperature, the present investigation was conducted to establish a model for vitrification of domestic animal's blastocyst and human blastocyst using mouse blastocyst as a model and OPS (open pulled straw) as the container. When ambient temperature was 25 degrees C and heating plate temperature was 37 degrees C, mouse blastocysts were vitrified using 1-step OPS method, after thawing, the expanded blastocyst (EB) rates of EDFS40 and EFS40 group (92.31%, 92.30%) were not different from that of the control (97.26%) (P > 0.05), however, the hatched blastocyst (HB) rate of EDFS40 group (59.62%) was significantly lower than that of the control (83.56%); When 2-step method was adopted at the same temperature, mouse blastocyst was successfully cryopreserved with EDFS30 and EFS40, the post-thawing EB rate (95.69%, 95.05%) and HB rate(80.48%, 78.95%) of both EDFS30 and EFS40 groups did not differ from those of the control (P > 0.05). When ambient temperature was 25 degrees C and heating plate was free, the EB rate (85.96%) and HB (75.44%) of embryos vitrified with EDFS40 using 1-step OPS method were not statistically different from those of the control (96.05% and 82.89%, respectively) (P > 0.05); the EB and HB rates (92.03%-95.31%, 67.19%-76.76%) of embryos vitrified with EDFS30, EDFS40 and EFS40 using 2-step OPS method were similar to those of the control (P > 0.05). According to in vitro development of vitrified embryos, the promising group was chosen for embryo transfer to examine in vivo development of vitrified embryos. When vitrified blastocysts were transferred into uterus of pseudopregnant mouse, the pregnancy rate of receipts (90.90%) and birth rate of foster mother (37.33%) were similar to those of fresh embryos (91.67% and 42.33%) (P > 0.05). All data demonstrated that mouse blastocyst was successfully cryopreserved with EDFS30, EDFS40 and EFS40 at different temperature and using different procedures.

To investigate the potential role and the mechanism of PLZF-RARalpha/RARalpha-PLZF double fusion gene in the pathogenesis of acute promyelocytic leukemia (APL) in vivo at systematic biological level, PLZF-RARalpha/RARalpha-PLZF double transgenic mouse model was established by intercross; the integration and expression of fusion genes were analyzed by PCR and RT-PCR; the disease phenotype was detected by morphological and pathological examination of peripheral blood and bone marrow cells, as well as flow cytometry assays; the effects of ATRA with or without tricostatin A on bone marrow blast cells from PLZF-RARalpha/RARalpha-PLZF double TM were observed. The results showed that leukemia occurred in 5 PLZF-RARalpha/RARalpha-PLZF double TM 7, 7, 9, 11 and 11 months respectively, out of them two (40%) with classic APL features, the others (60%) with chronic myeloid leukemia through an observation period of 18 months. The leukemia occurrence of PLZF-RARalpha/RARalpha-PLZF TM was about 10%, which was similar to PLZF-RARalpha TM as that reported before. The latency was over 6 months, not earlier than PLZF-RARalpha TM only. No morphologic changes of PLZF-RARalpha/RARalpha-PLZF double TM blast cells to ATRA were observed, but increased cytoplasmic-nuclear ratio and nuclear condensation in bone marrow blast cells were found in combination of ATRA with tricostatin A. It is concluded that PLZF-RARalpha/RARalpha-PLZF double fusion gene transgenic mice have heterogeneity of pathogenesis. HDAC inhibitors such as trichostatin A, in combination with ATRA, induce differentiation of the blast/promyelocytic cells from PLZF-RARa/RARa-PLZF double TM, but not ATRA alone.

To explore the genetic abnormalities that cooperate with AML1-ETO (AE) fusion gene to cause acute myeloid leukemia (AML) with t(8;21), we screened a number of candidate genes and identified 11 types of mutations in C-KIT gene (mC-KIT), including 6 previously undescribed ones among 26 of 54 (48.1%) cases with t(8;21). To address a possible chronological order between AE and mC-KIT, we showed that, among patients with AE and mC-KIT, most leukemic cells at disease presentation harbored both genetic alteration, whereas in three such cases investigated during complete remission, only AE, but not mC-KIT, could be detected by allele-specific PCR. Therefore, mC-KIT should be a subsequent event on the basis of t(8;21). Furthermore, induced expression of AE in U937-A/E cells significantly up-regulated mRNA and protein levels of C-KIT. This may lead to an alternative way of C-KIT activation and may explain the significantly higher C-KIT expression in 81.3% of patients with t(8;21) than in patients with other leukemias. These data strongly suggest that t(8;21) AML follows a stepwise model in leukemogenesis, i.e., AE represents the first, fundamental genetic hit to initiate the disease, whereas activation of the C-KIT pathway may be a second but also crucial hit for the development of a full-blown leukemia. Additionally, Gleevec suppressed the C-KIT activity and induced proliferation inhibition and apoptosis in cells bearing C-KIT N822K mutation or overexpression, but not in cells with D816 mC-KIT. Gleevec also exerted a synergic effect in apoptosis induction with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.

Leukemia, a group of hematological malignancies characterized by clonal expansion of hematopoietic cells with uncontrolled proliferation, decreased apoptosis and blocked differentiation, is one of the most notorious enemies of mankind which accounts for some 300,000 new cases and 222,000 deaths each year worldwide. Leukemia can be divided into acute or chronic, lymphoid or myeloid types, based on the disease progression and hematopoietic lineages involved 5. The responses of leukemia to therapies differ from one type or subtype to another. Hence, to improve the clinical outcome, the therapeutic strategies should be disease pathogenesis-based and individualized. The close collaboration between bench and bedside may not only shed new lights on leukemogenesis, gain insights into therapeutic mechanisms, but also provide opportunities for designing more rational therapies. The development of curative approaches for acute promyelocytic leukemia (APL) may serve as a paradigm.

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure-that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method-that is, embryos were first pretreated in 10%E + 10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E + 10%D for 0.5 min, exposed to EDFS30for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.

Background: A nonsteroidal anti-inflammatory drug, indomethacin, has been shown to have anti-leukemic activity and induce leukemic cell apoptosis. This study was to elucidate the mechanism of indomethacin-induced K562 cell apoptosis., Methods: K562 cells were grown in RPMI 1640 medium and treated with different doses of indomethacin (0 micromol/L, 100 micromol/L, 200 micromol/L, 400 micromol/L, 800 micromol/L) for 72 hours. The cells were harvested, and cell viability or apoptosis was analyzed using MTT assay and AO/EB stain, combining laser scanning confocal microscopy (LSCM) technique separately. For the localization and distribution of intracellular caspase-3 or caspase-8 protein, immunofluorescence assay was carried out. To reveal the activation of caspase-3 or caspase-8 in indomethacin-treated cells, Western blot detection was used. The change in intracellular free calcium was determined by Fluo-3/Am probe labeling combined with LSCM., Results: Indomethacin could lead to K562 cell apoptosis and inhibit cell viability in a concentration-dependent manner. An increased expression of intracellular caspase-3 or caspase-8 was observed at higher doses of indomethacin (400 - 800 micromol/L). Western blot results showed upregulation and activation in both caspase-3 and caspase-8 protein. Under indomethacin intervention, the levels of intracellular free calcium showed a significant increase. Blocking the activity of cyclooxygenase did not abolish the effects of indomethacin on K562 cell apoptosis., Conclusions: Activation and upregulation of caspase-3 or caspase-8 protein were responsible for Indomethacin-induced K562 cell apoptosis. Variation of intracellular free calcium might switch on the apoptotic pathway and the proapoptotic effect of indomethacin might be cyclooxygenase-independent.