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Leukemia stem cells (LSCs) reside in bone marrow niche and receive important signals from the microenvironment that support self-renewal, maintain quiescence and endow LSC with the ability of chemotherapy resistance. Rac1 belongs to the small GTP-binding protein superfamily and is implicated in the interactions of hematopoietic progenitors and bone marrow niche. Our previous studies have shown that Rac1 is over-expressed in leukemia patients and activation of Rac1 GTPase is closely associated with the efficient migration of leukemia cells. However, the potential functions for Rac1 GTPase in LSCs behaviors and in the residence of leukemia cells in niche remain unknown. In this study, by forced expression of a dominant-negative form of Rac1 GTPase in a CD34(+) myeloid leukemia cell line, as well as bone marrow cells from leukemia patients, we show that inactivation of Rac1 GTPase causes impaired migration and enhances chemotherapeutic sensitivity. Inactivation of Rac1 in leukemia cells also lead to a reduction in the frequency of cells in quiescent state and inhibition of homing to bone marrow niche. Gene expression analysis shows that inactivation of Rac1 down-regulates the expression of several cell intrinsic cell cycle inhibitors such as p21, p27, and p57, as well as the extrinsic molecules that mediated the interaction of LSC with osteoblastic niche. Furthermore, we show that Rac1 mediated the localization in niche is further attributed to the maintenance of quiescence. Our results provide evidence for the critical role of Rac1 GTPase in leukemia cell chemotherapy resistance, quiescence maintenance and the interaction with bone marrow microenvironment.

Periplocymarin, a cardiac glycoside isolated from Periploca sepium (P. sepium) and Periploca graeca (P. graeca), is a potential anti-cancer compound. The aim of the study was to investigate the potential for periplocymarin to interact with P-glycoprotein (P-gp) and to inhibit cytochrome P450s known to be expressed in the human small intestine. The in vitro and in situ permeability of periplocymarin were studied using Madin-Darby canine kidney (MDCK-II-WT) cells transfected with or without the human multidrug resistance (MDR1) gene and the single-pass perfused rat intestinal model. The cell system exhibited high functional activity and a net efflux ratio (NER) of 4.32 after transport of Rhodamine 123 (R123) (the P-gp substrate). Periplocymarin is highly permeable (Papp 10 × 10(-6) cm/s; Peff(rat) 5.09 × 10(-5) cm/s) and independent of P-gp influences. The NER at 100 μm periplocymarin (0.8) was unchanged in the presence of cyclosporine A (a non-specific P-gp inhibitor) (0.82). In the single-pass intestinal model, the Peff (rat) of 5 µg/ml periplocymarin (5.490 × 10(-5) cm/s) did not change in the presence of cyclosporine A (5.394 × 10(-5) cm/s). In the R123-inhibition assay, periplocymarin did not competitively inhibit P-gp. The inhibitory potential of periplocymarin on cytochrome P450 (CYP450s) was also studied. Periplocymarin (5, 50 μm) did not inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6 or CYP3A4. Thus, periplocymarin is unlikely to encounter drug-drug interactions with P-gp and CYP450s. Periplocymarin could be taken forward for further studies in drug development to test bioavailability, Phase II enzyme interactions and additional transporter interactions.

In order to explore various factors to the aluminum surface self-assembly process on film forming properties, this study tested a large number of single-factor method to determine the optimal experimental conditions: film-forming time was 3min; pH was 6; PESA was 2.5mL/L; organic phosphorus compounds was 5mL/L; nitrilotriacetic acid was 5g/L; fluoride was 10g/L; processing time was 3min. Results showed that under these conditions the SAMs had good overall performance with strong adhesion.

G-rich oligonucleotides (GROs) can inhibit cell proliferation by inducing cell cycle arrest at S phase in tumor cell lines. GROs bind specific cellular proteins, such as nucleolin, a crucial protein interacting with P53; however, little is known about the relationship between GROs and P53. In this study, we have shown that GROs inhibited the proliferation of U937 cells (a human monocytic leukemia cell line without P53 expression) by inducing S-phase arrest. We also showed that GRO colocalized with nucleolin in U937 cells. GRO treatment induced alteration of a series of cell cycle regulatory proteins in U937 cells. Increased Cdk2 expression might promote the cells to enter S phase and subsequent decrease of Cdk2 might induce cell cycle arrest in S phase. Transfection of U937 cells with a wild-type p53 gene caused the formation of nucleolin-P53 complex, which alleviated the effect of GRO on leukemia cells. This alleviated effect is probably due to the decreased uptake of GRO.

To investigate the relationship between coxsackievirus infection and type 1 diabetes mellitus (T1DM), and observe the changes of T lymphocyte subsets in the development of T1DM.We detected Coxsackievirus RNA by reverse transcription PCR, and measured the change in T-lymphocyte subsets by flow cytometry in 22 cases of newly diagnosed T1DM (group I), 30 patients with diabetes for some time (group II), and 30 healthy subjects (group III).The positivity rate of coxsackie virus RNA in groups I, II, and III was 55.55%, 23.33%, and 6.67%, respectively, showing a significant difference among the 3 groups (P0.01). Patients with upper respiratory tract infection had a higher positivity rate for coxsackie virus RNA than those without upper respiratory tract infection in group I (P0.05). Compared with the control group, the percentage of CD3, CD4 and CD4/CD8 ratio decreased significantly in groups I and II (P0.01 or P0.05). CD3, CD4 and CD4/CD8 tended to increase in group II in comparison with group I, and there was an significant difference in CD3 and CD4 between the two groups (P0.01 or P0.05). Compared with the control group and CVBRNA-negative group, CVBRNA-positive group showed significantly lowered CD3, CD4, CD8 and CD4/CD8 (P0.01 or P0.05).The occurrence and development of type 1 diabetes is closely related to coxsackie virus infection, and the changes in T lymphocyte subsets serves as a probable mechanism of its pathogenicity.

E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.

Midkine (MK), a heparin-binding growth factor, has been reported to be overexpressed in a variety of human solid tumors. In the previous study, we found that MK was overexpressed in bone marrow samples derived from acute leukemia (AL) patients. To elucidate the role of MK, we stably transfected MK in IL-3-dependent BA/F3 cells. The results indicated that the capacity of proliferation and colony formation was significantly increased in the MK-transfected subclones than in the empty vector-transfected subclones. MK potentiated proliferation of BA/F3 cells by promoting cell cycle progression. Apoptosis assays showed a remarkable reduction of apoptosis in MK expressing subclones. Exogenous MK could induce the phosphorylation of Raf-1, and inhibit the expression of Bax in BA/F3 cells. These results indicate that MK might be involved in the pathogenesis of leukemia and could be taken as an ideal diagnostic marker and molecular target for the treatment of acute leukemia.

AML1/RUNX1 is a critical transcription factor in hematopoietic cell differentiation and proliferation. From the AML1 gene, at least three isoforms, AML1a, AML1b and AML1c, are produced through alternative splicing. AML1a interferes with the function of AML1b/1c, which are often called AML1. In the current study, we found a higher expression level of AML1a in ALL patients in comparison to the controls. Additionally, AML1a represses transcription from promotor of macrophage-colony simulating factor receptor (M-CSFR) mediated by AML1b, indicating that AML1a antagonized the effect of AML1b. In order to investigate the role of AML1a in hematopoiesis and leukemogenesis in vivo, bone marrow mononuclear cells (BMMNCs) from mice were transduced with AML1a and transplanted into lethally irradiated mice, which develop lymphoblastic leukemia after transplantation. Taken together, these results indicate that overexpression of AML1a may be an important contributing factor to leukemogenesis.

iASPP is an inhibitory member of ASPP (apoptosis stimulating protein of p53, or Ankyrin repeats, SH3 domain and proline-rich region contain Protein) family. As reported previously, it at least includes two isoforms, one is iASPP/RAI (351 amino acids, aa) and the other is iASPP (828 aa).Here, we identified a novel open reading frame of human iASPP, which encodes a 407 aa protein and highly matches with the C terminus of iASPP (828 aa, CAI60219). Hereafter, iASPP (407 aa) will be referred to as iASPP-SV (iASPP splice variant). In further study, we found that iASPP-SV is a nuclear protein, and is capable of binding to p53 in vivo. Moreover, overexpression of iASPP-SV can inhibit the transcriptional activity of p53 on the promoters of both Bax and p21.

Acute myeloid leukemia (AML) is a hematological malignancy resulting from the transformation of normal hematopoietic stem cell (HSC). Except for the intrinsic factors, it is acceptable that some extrinsic events from microenvironment could be the important co-factors in the development of leukemia. In addition to the specific component, as an extrinsic factor, interaction between HSC and bone marrow niche regulates HSCs fate. Disruption on the interactions also influences hematopoiesis. It has become evident that Rac members of Rho GTPases family are important molecules regulating HSCs interactions with hematopoietic microenvironment and activation of Rac1 are observed in a serials of leukemia cells. We previously reported that Rac1 is highly expressed in leukemia cells and found that activation of Rac1 GTPase lead to an increase in leukemia cells migration, chemotherapy resistance, quiescence and trafficking to bone marrow niche. Furthermore, we showed that Rac1 mediated the localization in niche is further attributable to the maintenance of LSC quiescence. In this study, we investigated the effects of active Rac1 GTPase in the transformation of HSC and determined if the activation of Rac1GTPase could promote the interaction of HSC with osteoblastic niche and further contribute to the leukomogenesis. By forced expression of a constitutively active form of Rac1 GTPase (Rac1 V12）in c-Kit+ hematopoietic stem/progenitor cell, we show that activation of Rac1 GTPase promotes cell migration, adhesion and colony formation, and also lead to an increase in the frequency of cells in quiescent state. Gene expression analysis shows that activation of Rac1 up-regulates the expression of several molecules that mediated the interaction of LSC with osteoblastic niche, as well as the cell cycle inhibitors such as p21, p27, and p57. Furthermore, we established a mouse model of acute myeloid leukemia by transduction murine c-kit+HSPC with Rac1 V12 combined with AML1-ETO9a, followed by transplantation into lethally irradiated mice. To investigate the role of Rac1 activation in leukemogenesis in vivo, we treated the AML1-ETO-Rac1 leukemia cells with Rac1 GTPase inhibitor EHT1846 and then transplanted into recipient mice. After 40 μM EHT1846 treatment, no engraftment of AML cells in recipient mice was observed. Kaplan-Meier analyses indicate that treatment with EHT1846 significantly prolongs survival of the transplanted mice. 20μM dose of EHT1846 was less effective. These data indicated that active Rac1 might be an important contributing factor to leukemogenesis. In addition, short-term homing assays showed that EHT 1846 treatment causes a marked inhibition of AML cell homing into both bone marrow and spleen as compared with controls, indicating that Rac1 mediated homing could be an important step and participated in the leukemogensis. Altogether, our data suggest that activation of Rac1 GTPase is critical for the interaction between HSCs with BM niche and even be contributed to leukemia development. Disclosures Wang: Novartis: Consultancy; Bristol Myers Squibb: Consultancy.

Introduction: As the important suprressor of P53, iASPP was found to be overexpressed in leukemia, and functioned as oncogene that inhibited apoptosis of leukemia cells. Sertad1 is identified as one of the proteins that can bind with iASPP in our previous study by two-hybrid screen. Sertad1 is highly expressed in carcinomas from pancreatic, lung and ovarian tissues, which considered Sertad1 as an oncoprotein. In this study, our findings revealed that Sertad1 could interact with iASPP in the cytoplasm near nuclear membrane, which could block iASPP to enter into nucleus to interact with P53, and inhibited the function of iASPP eventually. Methods: Co-immunoprecipitation and fluorescence confocal microscopic imaging were used to confirm the interaction between iASPP and Sertad1, the exact binding domains and the subcellular colocalization.The plasmids of iASPP and Sertad1 were transfected alone or co-transfected into K562 cells, the stable subclones that highly expressed iASPP, Sertad1 or both of them were then established by limiting dilution and named as K562-iASPPhi, K562-Sertad1hi, and K562-Douhi, respectively. The cell proliferation, cell cycle and apoptosis of above subclones were investigated by flow cytometry. Further, silence of the above two proteins was performed to confirm their functions. Immunoblotting analysis and immunofluorescence were performed to explore the possible mechanisms of difference between the biological functions of the above subclones. Results: Sertad1 expression level varied in leukemic cell lines and AML patients irrespectively of iASPP and P53. Interaction between iASPP and Sertad1 did exist in 293 cell and leukemic cells, both iASPP and Sertad1 scattered in the cytoplasm and nucleus, and their colocalizations were mainly in the cytoplasm, which encircled the nucleus. iASPP binds directly to Sertad1 through its PHD-bromo domain, C-terminal domain and Cyclin-A domain in a reduced order, and Serta domain failed to bind to iASPP. Overexpression of iASPP in K562 cells (iASPPhi) could result in the increased cell proliferation, cell cycle arrest in G2/M phase and resistance to apoptosis induced by chemotherapy drugs. While overexpression of iASPP and Sertad1 at the same time (Douhi) could slow down the cell proliferation, lead the cells more vulnerable to the chemotherapy drugs. As figure showed, in K562-Douhi cells, both iASPP and Sertad1 were obviously located in the cytoplasm, which encircled the nuclei, the subcellular colocalization was nearly outside the nuclei. The immunoblotting analysis further supported the conclusions. The resistance of iASPP to chemotherapeutic drug was accompanied by Puma protein expression in a p53-independent manner. By knocking down the expersssion of iASPP and Sertad separately, we found that iASPP is dispensable for maintenance of anti-apoptotic function and Sertad1 is indispensable for cell cycle in leukemic cells. Conclusions: In normal situation, the protein iASPP and Sertad1 scatter in the nucleus and cytoplasm, mainly in the cytoplasm. As convinced by our study, iASPP was overexpressed in the leukemia cell lines and primary AML patients, it could function as oncogene through its binding with P53 protein in the nucleus, inhibit the function of P53. When iASPPhi cells were exposed to apoptosis stimuli, Puma protein could play an important role in this process, irrespective of the expression level of P53. But when iASPP and Sertad1 were both overexpressed in the leukemic cells, Sertad1 could tether iASPP outside the nucleus mainly through its PHD-bromo domain, prevent it from inhibiting P53 function, suppress the leukemic cell growth and stimulate cell apoptosis by rescuing the P53 eventually. Our data provided a new insight to overcome iASPP protein, namely through its binding partners, when the similar proteins or drugs that can tether iASPP outside the nucleus such as Sertad1 are transfected into the leukemic cells, it may restore p53 function to eliminate the leukemic cells. Figure 1 Figure 1. Disclosures Wang: Novartis: Consultancy; Bristol Myers Squibb: Consultancy.

The metal magnetic memory (MMM) technology, a new nondestructive evaluation (NDE) method, is an effective means for early damage prediction. However, there is so much work to do, such as the relationship between the magnetic field density and stress state. This paper aims at finding out MMM signal characteristics of critical damage stress state by tension experiments. Comparisons between MMM testing and traditional NDE method are presented. The principle of MMM testing is investigated. Different materials, low carbon steal X70 and medium carbon Q235B, are detected on-line and off-line respectively. It is found that MMM signal rules are gradually increasing up to fluctuating on the verge of yield and sharp changing of magnetic polarity on the verge of fracture. With the increase in material strength, magnetic field density of low carbon steel X70 is lower than that of medium carbon steel Q235B. This offers fundamental study for the quantitative MMM testing of critical damage stress state.Copyright © 2005 by ASME

To determine the ETO-interaction domain of nuclear receptor co-repressor (N-CoR) for abolishing the biological function of AML1-ETO.Ten different regions of N-CoR (N-CoRYs) were generated by means of polymerase chain reaction (PCR), and cloned into yeast expression plasmid pGADGL to construct pGADGL/N-CoRYs. The yeast two-hybrid technique and X-gal staining were used to determine the binding between the 10 different regions of N-CoR and ETO.It was shown that the co-existence of 988-1,126 and 1,551-1,803 amino acid residues of N-CoRY was the ETO-interaction domains required for the binding with ETO.Two domains of N-CoR that interact with two zinc fingers of ETO, and keep stable binding between the two proteins were identified.

Sheng Liang,* Fengchun Deng,* Haiyan Xing, He Wen, Xiaoyan Shi, Orleans Nii Martey, Emmanuel Koomson, Xin HeSchool of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin, People's Republic of China*These authors contributed equally to this workAbstract: Periplocin, an active and toxic component of the traditional Chinese herbal medicine Periploca sepium Bge, is a cardiac glycoside compound that has been implicated in various clinical accidents. This study investigated the role of transporters in the intestinal absorption and biliary excretion of periplocin, as well as the possible metabolic mechanism of periplocin in liver S9. In a bidirectional transport assay using Madin–Darby canine kidney (MDCK) and MDCK multidrug-resistance protein (MRP)-1 cell monolayers, both in situ intestinal and liver-perfusion models were used to evaluate the role of efflux and uptake transporters on the absorption and biliary excretion of periplocin. In addition, in vitro metabolism of periplocin was investigated by incubating with human/rat liver S9 homogenate fractions to evaluate its metabolic mechanisms in liver metabolic enzymes. The results showed that P-glycoprotein (P-gp) was involved in the intestinal absorption of periplocin, whereas MRP2 and breast cancer-resistance protein were not. The efflux function of P-gp may be partly responsible for the low permeability and bioavailability of periplocin. Moreover, both inhibitors of P-gp and organic anion-transporting polypeptides (OATPs) increased periplocin biliary excretion. No obvious indications of metabolism were observed in the in vitro incubation system, which suggests that periplocin did not interact with the hepatic drug metabolic enzymes. The results of this study showed that the efflux and uptake transporters P-gp and OATPs were involved in the absorption and biliary excretion of periplocin, which may partially account for its low permeability and bioavailability. As a toxic compound, potential drug–herb/herb–herb interactions based on OATPs and P-gp should be taken into account when using P. sepium Bge in the clinic.Keywords: periplocin, P-gp, OATPs, toxicity, interactions

Feng-jun Sun , Xiao-bing Zhang , Yadong Fang , Jianhong Chen , Haiyan Xing , Huiqing Shi , Wei Feng , Peiyuan Xia * Department of Pharmacy, Southwest Hospital, The Third Military Medical University, Chongqing 400038, PR China Department of Laboratory Medicine, Southwest Hospital, The Third Military Medical University, Chongqing 400038, PR China c Institute of Burn Research Southwest Hospital, The Third Military Medical University, Chongqing 400038, PR China