ALBERT

Description: p16 is a tumor suppressor gene frequently affected by hypermethylation of promoter CpG island in malignant disorders. Phenylhexyl isothiocyanate (PHI) has been shown to induce p16 hypomethylation in leukemia cells. However, the methylation status is usually studied qualitatively or semi-quantitatively at the best. In this study, we developed a novel method to quantify p16 methylation in clinical specimen from patients with hematological malignancies as well as in U266, a multiple myeloma cell line. To establish a standard curve of p16 promoter methylation, methylated and demethylated PCR products were subcloned into plasmids respectively. The clones were selected out as completely methylated and demethylated clones, respectively. The PCR products of the two clones were mixed at different ratios and analyzed on a mciroarray chip to produce a standard curve of methylation. p16 promoter methylation was determined in U266 cells. The baseline p16 methylation was 78.2% in U266 cells. The methylation was decreased to 61.7% and 54.8%, respectively in the presence of PHI of 5 and 10 uM, respectively. We further analyzed p16 methylation status in 7 patients’ specimen, two from blood, 5 from bone marrow. Two patients had ALL, two had AML, one CLL, one MM, and one CMML. 3 patients have p16 gene hypermethylation at 17.7%, 47.5% and 84.3%, respectively. Hypomethylation occurred in two of the three patients’ cells after PHI treatment in vitro (84.3% to 64.8%, and 47.5% to 25.4%). In conclusion, a quantitative microarray method was established for quantitation of p16 methylation status. This will be used for laboratory screening of hypomethylation drugs, such as PHI, and for clinical application to monitor therapeutic efficacy of hypomethylating drugs, such as azacitidine, and to prognostify patients based on p16 methylation level.

Description: OBJECTIVE: Myelodysplastic syndromes (MDS) are a heterogeneous group with the expansion of a malignant clone. No satisfactory treatment for MDS is available. Sodium valproate (VPA), can induce G0–G1 phase arrest and cell apoptosis, inhibit proliferation of tumor cells in vitro significantly. The effects and possible mechanism of VPA on MUTZ-1 cell line of MDS were studied in this experiment. METHODS: Cell proliferation was determined by MTT assay. Apoptotic morphological features were observed under microscope and transmission electronmicroscope. Cell apoptosis and cell cycle were analyzed by flow cytometry (FCM). The expression of p21WAF1(cyclin-dependent kinase inbihitor) was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. RESULTS: VPA could inhibit the proliferation of MUTZ-1 cells in dose-and time-dependent manners. The typical apoptotic morphological features appeared in cells treated with 4 mmol/L VPA for 72 hours. Condensation of cells and nuclear chromatin, disintegration of nuclear chromatin, and apoptotic body could be observed under microscope. Aggregation and margination of apoptotic nuclear chromatin, cytoplasm condensation, and irregular chromatin masses could be observed under transmission electronmicroscope. The percentage of apoptotic cells which were treated with 1,2 and 4 mmol/L VPA for 72 hours increased from 1.39% to 2.18%, 16.03%, 22.02%, and cell cycle arrest at G0–G1 phase could be caused by VPA as shown by FCM. The expression level of p21WAF1 mRNA and p21WAF1 protein were up-regulated in a dose dependent manner in MUTZ-1 treated with VPA for 72 hours (P

Description: Objective: To prepare functionalized Fe3O4-magnetic nanoparticles(Fe3O4-MNPs) loaded with adriamycin(ADM) or Fe3O4-MNPs co-polymerized with ADM and tetrandrine(Tet) to investigate whether the temperature, time or ratio of drug to nanoparticles influences the polymerization. To study the reversal role that the drug-loaded nano-composites play in K562 and one of its resistant cell line K562/A02 and to learn the reversal mechanism of this kind of treatments so as to provide theoretic evidence for the clinical application of them as resistance modifying agents. Method: The drug-loaded nanoparticles were prepared using mechanical absorption polymerization process in different condition of 4° or 37° for 24h or 48h. To investigate whether Fe3O4-MNPs loaded with ADM and Tet would play a synergetic reverse role in multidrug resistant cell, the drug-loaded nanoparticles by mechanical absorption polymerization were prepared to act with K562 and one of its resistant cell line K562/A02. The survival of cells which were cultured with drug-loaded nano-composites for 48h was observed through MTT assay, the growth inhibition efficacy of cells was calculated then. Using cells under the same condition described before, we took use of fluorescence microscope to measure fluorescence intensity of intracellular ADM at a wavelength of 488nm. P-glycoprotein (P-gp) was analyzed with flow cytometer. The expression of mdr1 mRNA was measured by RT-PCR. Results: The results showed that the growth inhibition efficacy of both the two cells increased with augmenting concentration of Fe3O4-MNPs which were loaded with drugs. The condition of 4° and 48h was significantly better than that of 37° and 24h respectively. Both Fe3O4-MNPs loaded with ADM or Fe3O4-MNPs co-polymerized with ADM and Tet elevated the intracellular ADM accumulation in K562/A02. However, no linear correlation was found between fluorescence intensity of intracellular adriamycin and augmenting concentration of Fe3O4-MNPs in K562/A02. Tet could downregulate the level of mdr-1 gene and decrease the expression of P-gp. Furthermore, Tet polymerized with Fe3O4-MNPs reinforced this downregulation, causing a 100-fold more decrease in mdr1 mRNA level, but did not reduce total P-gp content. Conclusions: Fe3O4-MNPs can load ADM or both ADM and Tet by mechanical absorption polymerization, which depends on proper temperature and time. Furthermore, the drug-loaded nano-composites have the ability in multidrug resistance reversal. Fe3O4-MNPs loaded with ADM or Tet can enhance the effective accumulation of the drugs in K562/A02 and Fe3O4-MNPs loaded with Tet obviously reverse multidrug resistance by reinforcing mdr1 gene downregulation. Functionalized Fe3O4-MNPs loaded with Tet probably have synergetic effect on reversal in multidrug resistance.

Description: Objective The aim of this study is to investigate and analysis the effect of ultrasound potentiate the cytotoxicity of adriamycin and reverse drug resistance on leukemia drug resistance K562/Adm cell line in vitro, to find out the mechanism of the reverse effect of ultrasound exposure. Methods Human leukemia adriamycin resistant strain K562/Adm as target cells, were treated by adriamycin singly in group Adm, by ultrasound exposure singly in group US and by adriamycin prior to ultrasound exposure in group Adm+US. The dosages that didn’t attribute to cell killing immediately were detected. Trypan blue dye exclusion test and MTT assay were used to determine the sensitivity of K562/Adm. Wright’s staining and transmission electron microscope were used to detect the apoptosis and structure change of K562/Adm cells. Flow cytometry was used to analysis intracellular drug concentration and electron microscopic scanning was used to observe the membrane change. Immunocytochemistrial method was used to evaluate the expressions of P-gp. Results At 0.17W•cm−2 and lower acoustic intensity, ultrasound didn’t result in K562/Adm acute cells destruction; and 0.5 W•cm−2 ultrasonic intention could make cell killed rapidly after K562/Adm cells were irritated by ultrasound exposure singly. Significant differences were obtained between ultrasound treated and untreated cells in the presence of various concentrations of adriamycin. If the same concentration of cytotoxic agents were used, more cells were killed if sonication was applied. ultrasound for 30s at 20kHz, 0.17W•cm−2 intensity almost could not damage K562/Adm cells but dramatically decrease adriamycin concentration which induce cell achieve IC50;There were some morphological alterations in cells irradiated by ultrasound, Nearly all the treated cells by ultrasound exhibited small holes with diameter about 1~2 μm in the K562/Adm cell surface; the intracellular adriamycin accumulation in group Adm+US were prompted compared with Adm group and controlled group. Many apoptotic phenomena were observed in Adm+US group, show many vesicle and the form of apoptotic body, but there were no change in US group compared with controlled group. And the expression of P-gp protein had no significant difference between before and after ultrasound eradiated. Conclusions Higher ultrasound intensity could make K562/Adm cells killed rapidly, and lower ultrasonic level could potentiate the cytotoxicity of adriamycin to K562/Adm cells and reverse drug resistance on K562/Adm cells. Ultrasonic cavitation and sonoporation are the main mechanisms of the synergism between adriamycin and low-level sonication; the ultrasonically induced increase in intracellular drug accumulation. The expression of P-gp had no change in US group compare with controlled group.

Description: Objective: To establish a method for quantitative analysis of hematopoietic chimerism by polymerase chain reaction (PCR) based on short tandem repeat (STR) locies. To investigate the correlation between the kinetics of chimerism and hematologic engraftment, graft rejection, disease relapse and graft versus host disease (GVHD) after allogeneic nonmyeloablative peripheral blood stem cell transplantation. To guide implementation of therapy at an early stage and to improve patients life quality. Method: Cell dilution experiments were performed by mixing mononuclear cells (MNCs) obtained from peripheral blood samples of unrelated individuals to test the sensitivity, accuracy and linearity of the assay. Quantitative assessment of hematopoietic chimerism was performed by short tandem repeat-polymerase chain reaction(STR-PCR), polyacrylamide gels, silver staining and analyzed by Image Analysis System. 28 patients received nonmyeloablative stem cell transplantation were evaluated. The conditioning regimen included fludarabine 30mg/(m2·d)×6d, busulphan 4mg/(kg·d)×2d, CTX 600mg·d−1×2d, ±Ara-C. Peripheral blood were collected before and after transplantation in different period and the chimerism were analysed by this method. Results: Sensitivity varied from 1.25% to 5% depending on the STR locies being tested. vWA and D16S539 appeared better sensitivity from 1.25% to 2.5% than other locies. The quantitative results showed a linear correlation between the percent chimerism calculated and the DC proportion mixed(R2 =0.97~0.98, P

Description: This study was aimed to investigate functions of glycosylation cooled rabbit platelets in vivo and in vitro and the method to store cold platelets with UDP-gal. We collected rabbit heart blood, prepared concentrated platelet suspensions in a normal way to which we added UDP-Gal, and then stored them for ten days in 4° refrigerator. Thereafter platelet counts, mean platelet volume, platelet distributing width, platelet aggregation function, the activity to urge coagulation including PF3aT and APCT and apoptosis were determine- d. Meanwhile, survival time in vivo was tested after cold-stored rabbit platelets labeled with Cr51 were transfused into rabbits. Rabbit ear bleeding time and percentage plate recovery(PPR) were determined 1 hour and 24 hour after they were transfused into rabbit thrombocytopenia model. Results show that there was not significant difference in PLT counts, MPV, PDW, PF3aT and APCT between UDP-Gal cold-stored platelet group and fresh platelet group(p〉0.05). On the contrary, platelet counts decreased significantly, MPV, PDW jumped and PF3aT and APCT went down in cold control group compared to fresh platelet group(p

Description: E2A, a basic helix-loop-helix transcription factor, plays a crucial role in determining tissue-specific cell fate, including differentiation of B-cell lineages. In 5% of childhood acute lymphoblastic leukemia (ALL), the t(1,19) chromosomal translocation specifically targets the E2A gene and produces an oncogenic E2A-PBX1 fusion protein. Although previous studies have shown the oncogenic functions of E2A-PBX1 in cell and animal models, the E2A-PBX1–enforced cistrome, the E2A-PBX1 interactome, and related mechanisms underlying leukemogenesis remain unclear. Here, by unbiased genomic profiling approaches, we identify the direct target sites of E2A-PBX1 in t(1,19)–positive pre-B ALL cells and show that, compared with normal E2A, E2A-PBX1 preferentially binds to a subset of gene loci cobound by RUNX1 and gene-activating machineries (p300, MED1, and H3K27 acetylation). Using biochemical analyses, we further document a direct interaction of E2A-PBX1, through a region spanning the PBX1 homeodomain, with RUNX1. Our results also show that E2A-PBX1 binding to gene enhancers is dependent on the RUNX1 interaction but not the DNA-binding activity harbored within the PBX1 homeodomain of E2A-PBX1. Transcriptome analyses and cell transformation assays further establish a significant RUNX1 requirement for E2A-PBX1–mediated target gene activation and leukemogenesis. Notably, the RUNX1 locus itself is also directly activated by E2A-PBX1, indicating a multilayered interplay between E2A-PBX1 and RUNX1. Collectively, our study provides the first unbiased profiling of the E2A-PBX1 cistrome in pre-B ALL cells and reveals a previously unappreciated pathway in which E2A-PBX1 acts in concert with RUNX1 to enforce transcriptome alterations for the development of pre-B ALL.

Description: Objective This paper aims to investigate the reversal multiple of daunomycin(DNR) with different concentration of tetrandrine(tet) at different time, to study the variation of Soluble resistance related calcium binding protein (sorcin)’s expression in the reversion of multidrug resistance of K562/A02 leukemic line with different concentration of tet at different time, and to provide new theoretic evidence for the clinical application of tet as resistence modifying agents. Method The reversal multiple of DNR with different concentration of tet at different time was assayed by MTT (concentration of tet is 0.5mg/l or1mg/l or 2mg/l, incubation time is 48h or 72h). The variation of the gene’s expression of sorcin with different concentration of tet at different time wasassayed by RT-PCR(grouping like MTT). Result MTT analysis demonstrated that the reversal concentration was 1.81(0.5mg/l,48h),3.62(1.0mg/l,48h),6.14(2.0mg/l,48h),2.8(0.5mg/l,72h),(1.0mg/l,48h),7.12(2.0mg/l,72h) respectively. RT-PCR analysis demonstrated that sorcin gene was lowly displayed in K562 cells and highly displayed in K562/A02 cells. The variation was first accentuation and then attenuation with the concentration accrescence of tet, this tendency had no obviously difference between 48h and 72h. Conclusion 1 The result of the experiment shows that tet may reverse multidrug resistance of K562/A02 cells by the down regulation of the expression of sorcin gene and proteinum; 2. It may have the concentration dependence and the time dependence in the reversion of multidrug resistance of K562/A02 cells with tet.

Description: Objective: This paper was to study the reversal effect of magnetic Nano-Fe3O4 or Nano-Au with DNR, on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this combination, and to provide theoretic evidence for the clinical application of them as resistance modifying agents. Method: The IC50 (the concentration causing 50% inhibition of cell growth) of DNR, Nano-Fe3O4 and Nano-Au respectively were assayed by MTT method.The drug-loaded nanoparticles were prepared by solvent diffusion method.Some nanoparticales, volume ratio from 1.5% to 50%, were combined with some DNR to find the best combination to prepare best drug-loaded nanopartilces.At last, the K562/A02 cells was treated with the composite of 25% nanoparticales and 10mg/L DNR, which MDR1 mRNA was assayed by RT-PCR;intracellular drug concentration and the apoptosis was determined by fluorometry and confocal fluorescence microscope. Results: The IC50 of DNR for K562/A02 and K562 cells were 23.23mg/L and0.307mg/L respectively.Two nanoparticles themselves have not evident cytotoxic effect to K562/A02 and K562 cells.Pretreating K562/A02 cells with the composite of 25% nanoparticales and 10mg/L DNR for 48 hours partially restored the sensitivity of K562/A02 cells to DNR;K562/A02 showed apoptotic characteristics after treated with this composite;drug-loaded nanopartilces elevated the intracellular DNR accumulation in K562/A02 and its MDR1 mRNA were down regulated.Data was analyzed by SPSS 11.5 software and expressed as mean ± SD. Conclusions: Nano- Fe3O4 or Nano-Au can increases the intracellular free DNR concentration of the K562/A02 cells, which lead to more K562/A02 cells apoptosis. Two nanoparticles themselves could not lower the MDR1 gene expression of the K562/A02 cells, but they degraded the MDR1 gene level with combine DNR. These results suggested that Nano- Fe3O4 or Nano-Au with DNR can reverse the resistance of K562/A02 cells significantly.

Description: Objective: To investigate a new concept aiming for induction of graft-vs-leukemia (GVL) effect prior to stem cell transplantation (SCT). Mismatched lymphocytes given pre-SCT will be followed by selective elimination of alloreactive donor lymphocytes, thus avoiding lethal graft-vs-host disease (GVHD). Methods: Female (BALB/c×C57BL/6)F1 mice (H-2d/b) as recipients received sublethal total body irradiation (TBI) of 4 Gy (60Coγ-ray) on day 0 followed by being inoculated with 0.5×107 P388D1 leukemia cell line on day 1, injection of 1.5×107 allogeneic splenocytes supplied by C57BL/6 male mice(H-2b)for induction of GVHD, intraperitoneally injection of cyclophosphamide (Cy) (200 mg/kg) or TBI (9 Gy) were given on day 7, one day later, treated mice were rescued with 3×107 syngeneic bone marrow cells supplied by (BALB/c×C57BL/6)F1 male mice(H-2d/b). Recipients were observed clinical manifestation, phenotype, re-establishment of haematogenesis, histopathologic changes of internal organs suffered from GVHD and investigated donor chimerism by the semi-quantitate analyses of polymerase chain reaction (PCR). Data was analyzed by SPSS 10.0 software and expressed as mean ± SD. Results: Recipients had no occurrence of leukemia and GVHD by selective elimination of alloreactive donor lymphocytes by Cy and TBI, survived more than 210 days, to become complete-donor chimerism on day +21. The ratio of chimerism descended subsequently, but still displayed mixed-chimerism on day +90. Control mice died of evident GVHD, leukemia or other death-related-transplantation within 20 to 36 days(p