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A multisignal method for the sensitive detection of norovirus based on Mn paramagnetic relaxation and nanocatalysis was developed. This dual-modality sensing platform was based on the strong relaxation generated by cracked Au@MnO2nanoparticles (NPs) and their intrinsic enzyme-like activity. Ascorbic acid rapidly cracked the MnO2layer of Au@MnO2NPs to release Mn(II), resulting in the relaxation modality being in a “switch-on” state. Under the optimal conditions, the relaxation modality exhibited a wide working range (6.02 × 103–3.01 × 107copies/μL) and a limit of detection (LOD) of 2.29 × 103copies/μL. Using 4,4′,4″,4″′-(porphine-5,10,15,20-tetrayl) tetrakis (benzenesulfonic acid) (tpps)-β-cyclodextrin (tpps-β-CD) as a T1relaxation signal amplification reagent, a lower LOD was obtained. The colorimetric modality exploited the “peroxidase/oxidase-like” activity of Au@MnO2NPs, which catalyzed the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB, which exhibited a working range (6.02 × 104–6.02 × 106copies/μL) and an LOD of 2.6 × 104copies/μL. In addition, the rapid amplification reaction of recombinase polymerase enabled the detection of low norovirus levels in food samples and obtained a working range of 101–106copies/mL and LOD of 101copies/mL (relaxation modality). The accuracy of the sensor in the analysis of spiked samples was consistent with that of the real-time quantitative reverse transcription polymerase chain reaction, demonstrating the high accuracy and practical utility of the sensor.

In this study, we introduced a Raman detection technique based on a combination of functionalized magnetic beads and surface-enhanced Raman scattering (SERS) tags to develop a rapid and sensitive strategy for the detection of Staphylococcus aureus(S. aureus), a typical foodborne pathogen. Polyethylene glycol (PEG) and bovine serum albumin (BSA) dual-mediated teicoplanin functionalized magnetic beads (TEI-BPBs) were prepared for separation of target bacteria. SERS tags were used to immobilize antibodies on gold surfaces with bifunctional linker proteins to ensure specific recognition of S. aureus. Under optimal conditions, the combination of TEI-BPBs and SERS tags showed reliable performance, exhibiting good capture efficiency even in the presence of 106CFU mL−1of non-target bacteria. The SERS tag provided an effective hot spot for subsequent Raman detection, presenting good linearity in the range of 102–107CFU mL−1. Good performance has also been shown in detecting target bacteria in milk samples, where it has a recovery of 95.5–101.3%. Thus, the highly sensitive Raman detection technique combined with TEI-BPBs capture probes and SERS tags is a promising method for the detection of foodborne pathogens in food or clinical samples.

Food safety has become a topic of global concern in the recent decades. The significant food safety incidents occur from time to time around the world, seriously threatening the public health and causing extensive economic losses. In particular, the occurrence of COVID-19 highlights the importance of the food safety for the public health. Therefore, there is an urgent need to establish a fast, simple, sensitive, and efficient method for the detection of food safety. In recent years, the upconversion (UC) nanotechnology has been widely used in the field of food detection. The UC fluorescence analysis technology possesses the advantages of ultra-sensitivity detection, non-invasiveness, light stability, etc., and has broad application prospects in the field of food safety. After cladding and surface modification, it can be combined with other substances through a variety of mechanisms, such as electrostatic interaction, thereby expanding its application in the food safety detection. Thus, overall, there is a vital need to evaluate and utilize the potential of UC nanoparticles in the field of rapid detection of food safety.

Surface siloxane (3-aminopropyl triethoxysilane hydrolyzates)-modified silica materials were used as “initiators”, which resulted in the release and desorption of intact molecules adsorbed on the surface of a matrix. A covalently cross-linked MIL-53(Al) material was used to enhance the ionization of analytes. Herein, we have provided an efficient matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) matrix strategy, which responded to both ion and laser irradiation with low background interference in the low-molecular-weight (MW) region. The matrixes MIL-53(Al), SBA-15@APTES, SiO2@APTES, SBA-15@APTES@MOF, and SiO2@APTES@MOF were synthetized and used for the analysis of a series of low-MW compounds to verify the effectiveness of the strategies. Compared to conventional matrixes, the surface-modified SBA-15@APTES@MOF and SiO2@APTES@MOF had low background, high sensitivity, extensive applicability, good stability, and ultrahigh tolerance of salt concentrations. The detection limits of standard analytes were determined to range from 0.1 to 1 × 10–5mg/mL for 16 amino acids as well as citric acid, reserpine, tetraethylammonium chloride, melamine, bisphenol A, and malachite green. These results could help in designing more efficient nanostructure-initiator materials and further promote the application of MALDI-TOF MS.

Self-assembled polydiacetylene (PDA) vesicles, with the distinct advantages of low-cost materials, simple preparation, and excellent chromatic properties, can be perfectly combined with a colorimetric strip for on-site inspection. Herein, without involving expensive reagents and instruments, a visual colorimetric strip based on well-prepared PDA vesicles was developed to analyze and monitor histamine in deep-sea fish and its canned food. The standard calorimetric card for semiquantitative detection of histamine was successfully prepared and the quantitative detection can be further realized by analyzing the gray value using ImageJ and “Color Grab” in a smart phone. After optimizing the assembly conditions, this assay exhibited a linear response to histamine within the range from 70 to 2240 ppm. With excellent stability and sensitivity, this strip can be used to monitor the quality change of canned fish at different temperatures, so that people can avoid suffering from histamine poisoning, suggesting that it holds great potential in the intelligent system for on-site detection and real-time monitoring.

Mycotoxin pollutants removal using adsorbents has been studied extensively in recent years, although the development of low-cost, recyclable, and highly efficient adsorbents remains a challenge to date. Covalent Organic Frameworks (COFs) represent a novel kind of porous crystalline material, which have been applied widely. In this work, a flower-like COF fiber membrane (PAN@COF FM) for adsorbing Aflatoxin B1was fabricated using a simple approach. The COF was grown on the surface of polyacrylonitrile (PAN) skeleton using the in situ growth method. Then, the AFB1adsorption performance of the prepared membrane and the underlying adsorption mechanism were studied. The results revealed that the activated PAN@COF FM reached adsorption equilibrium within 30 min. The used membranes were regenerated by washing with an alkaline solution and then tested for adsorption efficiency again. The PAN@COF FM could adsorb above 98% Aflatoxin B1after 10 cycles, indicating its good reusability. The well adsorption performance of the membrane was attributed to the π-π stacking, hydrogen bonding, and hydrophobic interactions between PAN@COF FM and Aflatoxin B1. In summary, the as-prepared PAN@COF FM possesses the advantage of high reusability and good adsorption performance, and has the promising application in pollutant removal.

The BZ19-6 condensate gas field is the largest metamorphic gas field of the Bohai Bay Basin in China and contains typical fractured reservoirs within the inner parts of an Archean metamorphic buried hill. However, the mechanisms driving its creation and evolution are not well explored. Based on core and sidewall samples, thin sections, X-ray diffraction (XRD) analysis, U–Pb age dating, fluid inclusions, and imaging logging and seismic data, the lithologies, mineral contents and reservoir characteristics of the inner parts of a metamorphic buried hill were evaluated and then the main factors controlling the fractured reservoir were further explored in the Bozhong area. The results show that the lithology mainly includes granitic gneiss, cataclastic rocks and Mesozoic intrusions, among which the granitic gneiss is widely distributed and contains a large number of light-colored minerals. The reservoir spaces are mainly composed of tectonic fractures (TFs), cleavage fractures (CFs), introduced fractures (IFs) and dissolved pores (DPs). Numerous tectonic fractures provide the main reservoir spaces and can be divided into two groups, namely those with nearly WE- and NS-oriented strikes, which are closely related to the stress fields produced by Indosinian and Yanshan movements, respectively. IFs and DPs were developed by magmatic activities, which occurred during the Mesozoic, while magmatic hydrothermal fluid had an obvious negative impact on reservoir quality, resulting in fracture fillings with minerals such as ankerite and calcite. In this study area, reservoir spaces underwent various changes, from creation to development and from filling to dissolution. Finally, we proposed an integrated reservoir model to describe the distribution and evolution of high-quality reservoirs. This study explores the formation mechanisms of fractured reservoirs within metamorphic rocks and makes practical contributions to oil and gas exploration of buried hills.

Although there is considerable interest in self-assembly of ordered, porous “inverse opal” structures for optical, electronic, and chemical applications, uncontrolled defect formation limits the usefulness of such materials. Herein, we develop a highly ordered and plasmonic enhanced sensing inverse opal photonic crystal (IOPC) material. The co-assembly of the colloidal template with the matrix material avoids the need for liquid penetration into the preassembled colloidal crystals and minimizes the associated rupture and inhomogeneity of the resulting IOPC. Au nanoparticles (Au NPs) not only act as a “bridge” between recognition elements (aptamers) and IOPCs, but also can amplify optical signals. Furthermore, the enhancement mechanism of Au NPs is simulated by COMSOL. During the detection process, the optical signal of the sensing Au–Apt IOPC responds to the Staphylococcal enterotoxin B with a concentration ranging from 10–2to 103pg mL–1, and the limit of detection is 2.820 fg mL–1. Spiked real sample detection indicates that the as-proposed method possessed good accuracy. The sensing Au–Apt IOPC provides an extensive biosensor platform to detect a variety of toxic and harmful substances through replacing the aptamer by other recognition elements, such as antibodies or receptors.

In recent years, the combination of DNA nanotechnology and biosensing has been extensively reported. Herein, we attempted to develop a dual sensitization smartphone colorimetric strategy based on rolling circle amplification (RCA) coils gathering Au tetrahedra and explore its application. The dual sensitization effect of this strategy was achieved by rolling circle amplification (RCA) and Au tetrahedra. Under the initiation of the complementary DNA, a large number of ssDNA were generated, achieving amplification of the reaction signal. At the same time, due to the formation of Au tetrahedra, more gold nanoparticles could be gathered under the same conditions, and the signal would be amplified again. Using software ImageJ, the gray value of the reaction solution can be analyzed, detecting the target timely under the practical conditions of lack of equipment. By selecting aptamers with strong binding affinity, we applied this strategy to detect creatine kinase isoenzymes (CK-MB), showing a limit of detection of 0.8 pM, which performed well in actual detection and can meet the needs for real-time detection of CK-MB. Therefore, a universal detection platform was developed, which has broad application prospects in biosensing, clinical diagnosis, food detection, and other fields.

No relevant conflicts of interest to declare.

Abstract 3272

We showed before that cells transformed by BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2 and TEL/PDGFR, inducing chronic myeloproliferative disorders (MPDs), and CD34+ chronic myeloid leukemia (CML) stem/ progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain an excess of DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress [Blood, 2005; Cell Cycle, 2006; DNA Repair, 2006; Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cellenriched populations seem to display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Elevated levels of DSBs were also observed in leukemia cells expressing imatinib-resistant BCR/ABL kinase mutants. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. Numerous ROS- and radiation- induced DSBs are not lethal for BCR/ABL-positive leukemia cells; instead, they induce chromosomal instability implicating enhanced, but unfaithful repair [Leukemia, 2008]. The previous report [Mol. Cell, 2001] and ongoing studies demonstrated that BCR/ABL kinase (non-mutated and imatinib-resistant mutants) modulates expression of the mammalian RecA homologs RAD51, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which are responsible for homologous recombination repair (HRR) of DSBs. RAD51 plays a key role in HRR in cells transformed by BCR/ ABL and other FTKs [Mol. Cell, 2001; Mol. Cell. Biol., 2002]. BCR/ABL stimulates the expression of, interacts with and phosphorylates RAD51 on Y315, which is located in a critical fragment of RAD51 essential for its filament formation on DNA. Accordingly, our recent results indicated that BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for nuclear RAD51 foci formation in response to DNA damage. In addition to RAD51, BCR/ABL interacts directly with and phosphorylates RAD51B and XRCC2, but not other RecA homologs. Altogether, it appears that BCR/ABL can deregulate the expression and phosphorylation of some RecA homologs, which may have a significant impact on the efficiency and fidelity of DSB repair resulting in protection from apoptosis and chromosomal instability. Therefore, disassembly of BCR/ABL from RecA homologs should reduce the capability of CML cells to repair numerous ROS-induced DSBs and eventually trigger apoptosis. Based on this hypothesis we investigated the mechanisms of association between BCR/ABL and RecA homologs. Interactions between BCR/ ABL and RAD51 or RAD51B depend on the proline- rich (PP) regions of RAD51 and RAD51B, and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL, which form a pocket binding the PP regions. Disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) abrogated direct interaction with the BCR/ABL SH3-SH2-CD pocket. On the other hand, single amino acid substitutions in the BCR/ABL SH3-SH2-CD pocket, which eliminated its capability of binding the PP regions, prevented complex formation with RAD51 and RAD51B. In addition, RAD51 and RAD51B may interact with members of the BCR/ABL proteome such as Grb2 and Shc (RAD51 and RAD51B), and c-CrkL (only RAD51B), but not Gab2 and c-Cbl. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3-SH2-CD pocket mutant, where single amino acid substitutions disrupted its direct interaction with RAD51, displayed a slower proliferation rate and responded poorly to genotoxic stress despite intact kinase activity in comparison to cells transformed with non-mutated BCR/ABL. Interestingly, expression of RAD51 PP-LL mutant eliminated BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. These results suggest that the interaction between BCR/ABL and RAD51 may be targeted for selective elimination of leukemia cells and/or suppression of genomic instability. To test this hypothesis we are employing the peptide aptamer strategy targeting RAD51 PP regions in CD34+ cells obtained from imatinib-sensitive and imatinib-resistant CML patients and healthy volunteers in vivo and in vitro. In summary, we hypothesize that mechanisms regulating the association of BCR/ABL with RAD51 and other mammalian RecA homologs may be explored for the planning of more effective anti-tumor modalities.

We showed before that cells transformed by BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2 and TEL/PDGFR, inducing chronic myeloproliferative disorders (MPDs), and CD34+ chronic myeloid leukemia (CML) stem/ progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain an excess of DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress [Blood, 2005; Cell Cycle, 2006; DNA Repair, 2006; Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cellenriched populations seem to display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Elevated levels of DSBs were also observed in leukemia cells expressing imatinib-resistant BCR/ABL kinase mutants. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. Numerous ROS- and radiation- induced DSBs are not lethal for BCR/ABL-positive leukemia cells; instead, they induce chromosomal instability implicating enhanced, but unfaithful repair [Leukemia, 2008]. The previous report [Mol. Cell, 2001] and ongoing studies demonstrated that BCR/ABL kinase (non-mutated and imatinib-resistant mutants) modulates expression of the mammalian RecA homologs RAD51, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which are responsible for homologous recombination repair (HRR) of DSBs. RAD51 plays a key role in HRR in cells transformed by BCR/ ABL and other FTKs [Mol. Cell, 2001; Mol. Cell. Biol., 2002]. BCR/ABL stimulates the expression of, interacts with and phosphorylates RAD51 on Y315, which is located in a critical fragment of RAD51 essential for its filament formation on DNA. Accordingly, our recent results indicated that BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for nuclear RAD51 foci formation in response to DNA damage. In addition to RAD51, BCR/ABL interacts directly with and phosphorylates RAD51B and XRCC2, but not other RecA homologs. Altogether, it appears that BCR/ABL can deregulate the expression and phosphorylation of some RecA homologs, which may have a significant impact on the efficiency and fidelity of DSB repair resulting in protection from apoptosis and chromosomal instability. Therefore, disassembly of BCR/ABL from RecA homologs should reduce the capability of CML cells to repair numerous ROS-induced DSBs and eventually trigger apoptosis. Based on this hypothesis we investigated the mechanisms of association between BCR/ABL and RecA homologs. Interactions between BCR/ ABL and RAD51 or RAD51B depend on the proline- rich (PP) regions of RAD51 and RAD51B, and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL, which form a pocket binding the PP regions. Disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) abrogated direct interaction with the BCR/ABL SH3-SH2-CD pocket. On the other hand, single amino acid substitutions in the BCR/ABL SH3-SH2-CD pocket, which eliminated its capability of binding the PP regions, prevented complex formation with RAD51 and RAD51B. In addition, RAD51 and RAD51B may interact with members of the BCR/ABL proteome such as Grb2 and Shc (RAD51 and RAD51B), and c-CrkL (only RAD51B), but not Gab2 and c-Cbl. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3-SH2-CD pocket mutant, where single amino acid substitutions disrupted its direct interaction with RAD51, displayed a slower proliferation rate and responded poorly to genotoxic stress despite intact kinase activity in comparison to cells transformed with non-mutated BCR/ABL. Interestingly, expression of RAD51 PP-LL mutant eliminated BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. These results suggest that the interaction between BCR/ABL and RAD51 may be targeted for selective elimination of leukemia cells and/or suppression of genomic instability. To test this hypothesis we are employing the peptide aptamer strategy targeting RAD51 PP regions in CD34+ cells obtained from imatinib-sensitive and imatinib-resistant CML patients and healthy volunteers in vivoand in vitro. In summary, we hypothesize that mechanisms regulating the association of BCR/ABL with RAD51 and other mammalian RecA homologs may be explored for the planning of more effective anti-tumor modalities.

Bcl-2 expression is high in CLL and this appears to participate in disease progression. Zap70 expression is a poor prognostic marker for the disease whose function may be to contribute in a pathway promoting heightened bcl-2 levels. We recently identified a pathway to heightened bcl-2 transcriptional expression involving the bcl-2 promoter CRE (TGACGTCA) element and c-jun/ATF-2 downstream of JNK1. Indeed, JNK activation is a known survival pathway in B cell lymphoma (Gururajan, et al. Blood106: 1382, 2005). We confirmed a strict correspondence of Zap70 expression with JNK activity in CLL cell lysates by immunoblot identification of Zap70 and of phospho-S73-c-jun; and we found bcl-2 levels to be high in cells with Zap70 and JNK activity. We uncovered an adaptor/scaffolding pathway physically linking these mediators. By immunoprecipitation from lysates in a series of Zap70+ CLL’s we found JNK1 to coimmunoprecipitate with CrkL, a JNK1 scaffolding that binds by its SH3 domain to polyproline sequences of JNK1, and with p85 and Cbl and Zap70 as well as Syk. By contrast, using GST-p85 as bait in pull-down experiments, a selective interaction was found for Zap70 with p85, particularly of the p85 C-terminal SH2 domain, in lysates with heavy Y319 phosphorylation of Zap70. In parallel pull-downs with such lysates, exclusion of Cbl N-terminal TKB domain binding from Zap70 but not Syk was observed. Zap70+ CLL cell growth stimulated by the cytokine April was inhibited by the novel JNK inhibitor, CC-401, 5 uM, without significant effect on inhibition of NFkB or AKT activations. Such inhibition of JNK1 also led to upregulation of bim mRNA and downregulation of bcl-2 mRNA, and modest-to-moderate bcl-2 protein downregulation, in cells whose nuclear proteins contained abundant phospho-ATF2/c-jun binding activity for the bcl-2 promoter CRE site indicative of a transcriptional connection. Thus, Zap70 represents a point of origin in CLL cells for parallel pathways of Zap70 and Syk signaling to JNK1, involving specific complexes of Zap70 and Syk with p85/CrkL/JNK1(Cbl), respectively. These dual routes to JNK activation promote cell survival, in part, through the ability of JNK to contribute in a bcl-2 regulatory mechanism.

Bcl-2 expression is high in CLL and this appears to participate in disease progression. Zap70 expression is a poor prognostic marker for the disease whose function may be to contribute in a pathway promoting heightened bcl-2 levels. We recently identified a pathway to heightened bcl-2 transcriptional expression involving the bcl-2 promoter CRE (TGACGTCA) element and c-jun/ATF-2 downstream of JNK1. Indeed, JNK activation is a known survival pathway in B cell lymphoma (Gururajan, et al. Blood 106: 1382, 2005). We confirmed a strict correspondence of Zap70 expression with JNK activity in CLL cell lysates by immunoblot identification of Zap70 and of phospho-S73-c-jun; and we found bcl-2 levels to be high in cells with Zap70 and JNK activity. We uncovered an adaptor/scaffolding pathway physically linking these mediators. By immunoprecipitation from lysates in a series of Zap70+ CLL's we found JNK1 to coimmunoprecipitate with CrkL, a JNK1 scaffolding that binds by its SH3 domain to polyproline sequences of JNK1, and with p85 and Cbl and Zap70 as well as Syk. By contrast, using GST-p85 as bait in pull-down experiments, a selective interaction was found for Zap70 with p85, particularly of the p85 C-terminal SH2 domain, in lysates with heavy Y319 phosphorylation of Zap70. In parallel pull-downs with such lysates, exclusion of Cbl N-terminal TKB domain binding from Zap70 but not Syk was observed. Zap70+ CLL cell growth stimulated by the cytokine April was inhibited by the novel JNK inhibitor, CC-401, 5 uM, without significant effect on inhibition of NFkB or AKT activations. Such inhibition of JNK1 also led to upregulation of bim mRNA and downregulation of bcl-2 mRNA, and modest-to-moderate bcl-2 protein downregulation, in cells whose nuclear proteins contained abundant phospho-ATF2/c-jun binding activity for the bcl-2 promoter CRE site indicative of a transcriptional connection. Thus, Zap70 represents a point of origin in CLL cells for parallel pathways of Zap70 and Syk signaling to JNK1, involving specific complexes of Zap70 and Syk with p85/CrkL/JNK1(Cbl), respectively. These dual routes to JNK activation promote cell survival, in part, through the ability of JNK to contribute in a bcl-2 regulatory mechanism.

BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells, and that p85 subunit of PI-3k co-immunoprecipitates with BCR/ABL (Skorski et al., (1995) Blood 86, 726–36; Skorski et al., (1997) Embo J 16, 6151–61; Klejman et al., (2002) Oncogene 21, 5868–76). Therefore, we made an attempt to better characterize the p85 - BCR/ABL interactions. Here we show that SH3 domain of p85 (p85-SH3) pulls-down the p210BCR/ABL kinase from hematopoietic cell lysates. In addition, we characterize the p85-SH3 mutants, which abrogate or enhance this interaction. The results of pull-down assays of the p85-SH3 mutants seem to support the assumption that p85-SH3 interacts with the BCR/ABL protein network via the proline-rich (PxxP) region. One of the surprising findings was the enhanced binding affinity of the tyrosine to phenylalanine p85-SH3 domain mutants in comparison to the wild-type p85-SH3. Based on these results we speculate on the capability of p85-SH3 to interact with BCR/ABL and on the p85-SH3 conformational requirements necessary for this reaction. PxxP - binding appears to be required for the interaction of p85-SH3 with BCR/ABL protein complex and activation of the catalytic activity of PI-3k, whereas subsequent BCR/ABL-dependent phosphorylation of the tyrosines may facilitate the release of activated PI-3k from the complex.

BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells, and that p85 subunit of PI-3k co-immunoprecipitates with BCR/ABL (Skorski et al., (1995) Blood 86, 726–36; Skorski et al., (1997) Embo J 16, 6151–61; Klejman et al., (2002) Oncogene 21, 5868–76). Therefore, we made an attempt to better characterize the p85 - BCR/ABL interactions. Mutagenesis approach combined with pull-down assays indicated that both N-terminal and C-terminal SH2 domains (nSH2 and cSH2, respectively) and SH3 domain of p85 play an important role in the interaction with BCR/ABL. SH2 domains exerted their function through binding to the tyrosine-phosphorylated motifs, and SH3 domain recognized proline-rich regions. Disruption of these functions by introduction of point-mutations in nSH2, cSH2 and SH3 domains abrogated their interaction with BCR/ABL. p85 mutant protein (p85-mut) bearing these mutations was not able to interact with BCR/ABL, while its binding to the p110 catalytic subunit of PI-3k was intact. In addition, binding of Shc and Gab2, but not Crk-L, to p85-mut was abrogated. When expressed in BCR/ABL-transformed hematopoietic cells p85-mut diminished activation of Akt kinase, the downstream effector of PI-3k. This effect was associated with the inhibition of BCR/ABL-dependent growth of hematopoeitic cells line and murine bone marrow cells. Interestingly, addition of IL-3 rescued BCR/ABL-transformed cells from the inhibitory effect of p85-mut. SCID mice injected with BCR/ABL-positive hematopoietic cells expressing p85-mut survived longer in comparison to the animals inoculated with BCR/ABL-transformed counterparts transfected with empty plasmid. In conclusion, we have identified the domains of p85 responsible for the interaction with BCR/ABL. Moreover, we demonstrated that expression of p85-mut, which binds to the p110 catalytic subunit of PI-3k but is not able to interact with BCR/ABL, affected the growth ability of BCR/ABL-positive leukemia cells.

BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells, and that p85 subunit of PI-3k co-immunoprecipitates with BCR/ABL (Skorski et al., (1995) Blood 86, 726–36; Skorski et al., (1997) Embo J 16, 6151–61; Klejman et al., (2002) Oncogene 21, 5868–76). Therefore, we made an attempt to better characterize the p85 - BCR/ABL interactions. Here we show that SH3 domain of p85 (p85-SH3) pulls-down the p210BCR/ABL kinase from hematopoietic cell lysates. In addition, we characterize the p85-SH3 mutants, which abrogate or enhance this interaction. The results of pull-down assays of the p85-SH3 mutants seem to support the assumption that p85-SH3 interacts with the BCR/ABL protein network via the proline-rich (PxxP) region. One of the surprising findings was the enhanced binding affinity of the tyrosine to phenylalanine p85-SH3 domain mutants in comparison to the wild-type p85-SH3. Based on these results we speculate on the capability of p85-SH3 to interact with BCR/ABL and on the p85-SH3 conformational requirements necessary for this reaction. PxxP - binding appears to be required for the interaction of p85-SH3 with BCR/ABL protein complex and activation of the catalytic activity of PI-3k, whereas subsequent BCR/ABL-dependent phosphorylation of the tyrosines may facilitate the release of activated PI-3k from the complex.

BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells, and that p85 subunit of PI-3k co-immunoprecipitates with BCR/ABL (Skorski et al., (1995) Blood 86, 726–36; Skorski et al., (1997) Embo J 16, 6151–61; Klejman et al., (2002) Oncogene 21, 5868–76). Therefore, we made an attempt to better characterize the p85 - BCR/ABL interactions. Mutagenesis approach combined with pull-down assays indicated that both N-terminal and C-terminal SH2 domains (nSH2 and cSH2, respectively) and SH3 domain of p85 play an important role in the interaction with BCR/ABL. SH2 domains exerted their function through binding to the tyrosine-phosphorylated motifs, and SH3 domain recognized proline-rich regions. Disruption of these functions by introduction of point-mutations in nSH2, cSH2 and SH3 domains abrogated their interaction with BCR/ABL. p85 mutant protein (p85-mut) bearing these mutations was not able to interact with BCR/ABL, while its binding to the p110 catalytic subunit of PI-3k was intact. In addition, binding of Shc and Gab2, but not Crk-L, to p85-mut was abrogated. When expressed in BCR/ABL-transformed hematopoietic cells p85-mut diminished activation of Akt kinase, the downstream effector of PI-3k. This effect was associated with the inhibition of BCR/ABL-dependent growth of hematopoeitic cells line and murine bone marrow cells. Interestingly, addition of IL-3 rescued BCR/ABL-transformed cells from the inhibitory effect of p85-mut. SCID mice injected with BCR/ABL-positive hematopoietic cells expressing p85-mut survived longer in comparison to the animals inoculated with BCR/ABL-transformed counterparts transfected with empty plasmid. In conclusion, we have identified the domains of p85 responsible for the interaction with BCR/ABL. Moreover, we demonstrated that expression of p85-mut, which binds to the p110 catalytic subunit of PI-3k but is not able to interact with BCR/ABL, affected the growth ability of BCR/ABL-positive leukemia cells.