Naoki Uemura

The Adapter Protein Crkl Links Cbl to C3G after Integrin Ligation and Enhances Cell Migration

Crkl, an SH2-SH3-SH3 adapter protein, is one of the major tyrosine phosphoproteins detected in cells from patients with chronic myelogenous leukemia. Crkl binds to BCR/ABL through its N-terminal SH3 domain and is known to interact with several signaling proteins that have been implicated in integrin signaling, including Cbl, Cas, Hef-1, and paxillin. We have previously shown that overexpression of Crkl enhances adhesion to extracellular matrix proteins through β1 integrins. In this study, the effects of Crkl on spontaneous and chemokine-directed migration of the hematopoietic cell line Ba/F3 were examined. Full-length, SH2-, and SH3(N)-domain deletion mutants of Crkl were expressed transiently as fusion proteins with green fluorescent protein. Successfully transfected cells were isolated by fluorescence-activated cell sorting. The ability of these cells to migrate across a fibronectin-coated membrane, either spontaneously or in response to the chemokine stromal-derived factor-1α, was determined. Cells expressing green fluorescent protein alone were not distinguishable from untransfected or mock transfected Ba/F3 cells. However, Ba/F3 cells overexpressing full-length Crkl were found to have an increase in spontaneous migration of 2.8 ± 0.6-fold in seven independent assays. The enhancement of migration required both the SH2 domain and the N-terminal SH3 domain. Migration in response to stromal-derived factor-1α was not significantly enhanced by overexpression of Crkl. Overexpression of Crkii also augmented spontaneous migration but to a lesser degree than did Crkl. Because the SH2 domain was required for enhanced migration, we looked for changes in phosphotyrosine containing proteins coprecipitating with Crkl, but not Crkl ΔSH2, after integrin cross-linking. Full-length Crkl, but not CrklΔSH2, coprecipitated with a single major tyrosine phosphoprotein with anM r of approximately 120 kDa, identified as Cbl. The major Crkl SH3-binding protein in these cells was found to be the guanine nucleotide exchange factor, C3G. Interestingly, overexpression of C3G also enhanced migration, suggesting that a Cbl-Crkl-C3G complex may be involved in migration signaling in Ba/F3 cells. These data suggest that Crkl is involved in signaling pathways that regulate migration, possibly through a complex with Cbl and C3G.

p130CAS Forms a Signaling Complex with the Adapter Protein CRKL in Hematopoietic Cells Transformed by the BCR/ABL Oncogene

The Philadelphia chromosome (Ph) translocation generates a chimeric tyrosine kinase oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML) and a type of acute lymphoblastic leukemia (ALL). In primary samples from virtually all patients with CML or Ph+ALL, the CRKL adapter protein is tyrosine phosphorylated and physically associated with p210BCR/ABL. CRKL has one SH2 domain and two SH3 domains and is structurally related to c-CRK-II (CRK) and the v-Crk oncoprotein. We have previously shown that CRKL, but not the related adapter protein c-CRK, is tyrosine phosphorylated in cell lines transformed by BCR/ABL, and that CRKL binds to BCR/ABL through the CRKL-SH3 domains. Furthermore, the CRKL-SH2 domain has been shown to bind one or more cellular proteins, one of which is p120CBL. Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130CAS. p130CAS was found to be tyrosine phosphorylated and associated with CRKL in BCR/ABL expressing cell lines and in samples obtained from CML and ALL patients, but not in samples from controls. In both normal and BCR/ABL transformed cells, p130CAS was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125FAK, and paxillin constitutively associated with p130CAS. However, in BCR/ABL transformed cells, the interaction between p130CAS and tensin was disrupted, while the associations between p130CAS, p125FAK, and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130CAS in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130CAS containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130CAS with the focal adhesion protein tensin. These alterations in the structure of signaling proteins in focal adhesion like structures could contribute to the known adhesion abnormalities in CML cells.

The BCR/ABL oncogene alters interaction of the adapter proteins CRKL and CRK with cellular proteins

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary leukemic neutrophils from patients with CML, the major tyrosine phosphorylated protein is CRKL, an SH2-SH3-SH3 adapter protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene. In cell lines transformed by BCR/ABL, CRKL was tyrosine phosphorylated, while CRK was not. We looked for changes in CRK- and CRKL-binding proteins in Ba/F3 hematopoietic cell lines which were transformed by BCR/ABL. Anti-CRK II or anti-CRKL immunoprecipitates were probed by far Western blotting with CRK II- or CRKL-GST fusion proteins to display CRK- and CRKL-coprecipitating proteins. There was a striking qualitative difference in the proteins coprecipitating with CRKL and CRK II. In untransformed cells, three major proteins coprecipitated with CRKL, identified as C3G, SOS and c-ABL. Each of these proteins was found to interact with the CRKL-SH3 domains, but not the SH2 domain. After BCR/ABL transformation, the CRKL SH3-domain binding proteins did not change, with the exception that BCR/ABL now coprecipitated with CRKL. Compared to CRKL, very few proteins coprecipitated with CRK II in untransformed, quiescent cells. After BCR/ABL transformation, both the CRKL- and CRK-SH2 domains bound to a new complex of proteins of approximate molecular weight 105–120 kDa. The major protein in this complex was identified as p120CBL. Thus, in these hematopoietic cell lines, CRKL is involved to a greater extent than CRK II in normal signaling pathways that involve c-ABL, C3G and SOS. In BCR/ABL-transformed cells, CRKL but not CRK II, appears to form complexes which potentially link BCR/ABL, c-ABL, C3G, and SOS to the proto- oncoprotein, p120CBL.

Pure red cell aplasia caused by parvovirus B19 infection in a renal transplant recipient

To the Editor: Human parvovirus B 19, which is the etiologic agent of transient aplastic crisis in hemolytic anemia, is known to cause chronic bone marrow failure in immunocompromised hosts (1-4). To our knowledge, there have been 2 previous case reports describing parvovirus B 19 infection in renal transplant recipients (5 ,6 ) . We report here another case of severe anemia caused by parvovirus B 19 infection in a renal transplant recipient. Although humoral immune response to the virus was absent for a while due to immunosuppressive therapy, treatment with a regimen of intravenous commercial immunoglobulin resulted in rapid elevation of the reticulocyte count and resolution of the anemia. A 48-year-old man with chronic renal failure secondary to diabetic nephropathy, who had been undergoing chronic hemodialysis and administration of recombinant human erythropoietin (rhEPO) for the treatment of renal anemia, was hospitalized to receive a cadaveric kidney transplant. On the 1st hospital day (d 0), he underwent cadaver donor kidney transplantation, and immunosuppressive therapy with cyclosporin (3 mg/kg/d), prednisolone (70 mg/d), mizoribine (200 mg/d), and antilymphocyte globulin (1000 mg/d) was started. The donor was a 52-year-old man who had died of subarachnoid hemorrhage. Laboratory examination of the recipient on admission showed a white blood cell count of 5300/mm3, a hemoglobin level of 10.6 g/dl, and a platelet count of 204 000/mm3. After transplantation, his anemia began to progress without improvement after rhEPO treatment. On d 18, his hemoglobin level dropped to 5.0 g/dl, reticulocyte count was 0.08 %, white blood cell count was 2500/mm3, and platelet count was 172000/mm3. Bone marrow aspiration revealed severe erythroid hypoplasia with the appearance of many giant proerythroblasts, suggesting the presence of parvovirus B 19 infection. Thereafter, rhEPO was discontinued and the patient was treated with a 7-d course of intravenous commercial immunoglobulin preparations ( 5 g/d) and red cell transfusion. Thereafter, the reticulocyte count increased and his anemia improved. Normal hematopoiesis was observed on bone marrow examination on d 38. The serial serum specimens during the course of illness were subjected to enzyme-linked immunosorbent assay (ELISA) for detection of virus antigen, and polymerase chain reaction (PCR) for detection of virus DNA. B19 antigen became positive on d 2, reached a peak on d 5 , and lasted 18 d. However, viral DNA was detected by PCR in the serum specimen taken just before transplantation, and was negative or faintly positive on d 48. Interestingly, the amount of viral DNA was transiently elevated on d 153 (Fig. 1). The presence of a replicative form of B 19 virus was demonstrated by Southern blot analysis in the bone marrow specimen (Fig. 2) (7). In the serum specimen from the donor, viral DNA was not detected. The assay of virusspecific antibody revealed that IgM and IgG response was not observed until d 153 while the appearance of IgG response was detected in the sample

RNAi-mediated gene silencing of ST6GalNAc I suppresses the metastatic potential in gastric cancer cells

BackgroundST6GalNAc I is a sialyltransferase controlling the expression of sialyl-Tn antigen (STn), which is overexpressed in several epithelial cancers, including gastric cancer, and is highly correlated with cancer metastasis. However, the functional contribution of ST6GalNAc I to development or progression of gastric cancer remains unclear. In this study, we investigated the effects of suppression of ST6GalNAc I on gastric cancer in vitro and in vivo.MethodsGastric cancer cell lines were transfected with ST6GalNAc I siRNA and were examined by cell proliferation, migration, and invasion assays. We also evaluated the effect of ST6GalNAc I siRNA treatment in a peritoneal dissemination mouse model. The differences in mRNA levels of selected signaling molecules were analyzed by polymerase chain reaction (PCR) arrays associated with tumor metastasis in MKN45 cells. The signal transducer and activator of transcription 5b (STAT5b) signaling pathways that reportedly regulate the insulin-like growth factor-1 (IGF-1) were analyzed by Western blot.ResultsST6GalNAc I siRNA inhibited gastric cancer cell growth, migration, and invasion in vitro. Furthermore, intraperitoneal administration of ST6GalNAc I siRNA- liposome significantly inhibited peritoneal dissemination and prolonged the survival of xenograft model mice with peritoneal dissemination of gastric cancer. PCR array confirmed that suppression of ST6GalNAc I caused a significant reduction in expression of IGF-1 mRNA. Decreased IGF-1 expression in MKN45 cells treated with ST6GalNAc I siRNA was accompanied by reduced phosphorylation of STAT5b.ConclusionST6GalNAc I may regulate the gene expression of IGF-1 through STAT5b activation in gastric cancer cells and may be a potential target for treatment of metastasizing gastric cancer.

Relationship Between Increased Fucosylation and Metastatic Potential in Colorectal Cancer

BACKGROUND Fucose is utilized for the modification of different molecules involved in blood group determination, immunological reactions, and signal transduction pathways. We have recently reported that enhanced activity of the fucosyltransferase 3 and/or 6 promoted TGF-ß-mediated epithelial mesenchymal transition and was associated with increased metastatic potential of colorectal cancer (CRC), suggesting that fucose is required by CRC cells. With this in mind, we examined requirement of L-fucose in CRC cells and developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specific to CRC. METHODS In this study, we first examined the expression of fucosylated proteins in 50 cases of CRC by immunochistochemical staining with biotinylated Aleuria aurantia lectin (AAL). Then we carried out an L-fucose uptake assay using three CRC cell lines. Finally, we developed fucose-bound nanoparticles as vehicles for the delivery of an anticancer drug, SN38, and examined tumor growth inhibition in mouse xenograft model (n = 6 mice per group). All statistical tests were two-sided. RESULTS We found a statistically significant relationship between vascular invasion, clinical stage, and intensity score of AAL staining (P≤ .02). L-fucose uptake assay revealed that L-fucose incorporation, as well as fucosylated protein release, was high in cells rich in fucosylated proteins. L-fucose-bound liposomes effectively delivered Cy5.5 into CRC cells. The excess of L-fucose decreased the efficiency of Cy5.5 uptake through L-fucose-bound liposomes, suggesting an L-fucose receptor dependency. Intravenously injected, L-fucose-bound liposomes carrying SN38 were successfully delivered to CRC cells, mediating efficient tumor growth inhibition (relative tumor growth ratio: no treatment group [NT], 8.29 ± 3.09; SN38-treated group [SN38], 3.53 ± 1.47; liposome-carrying, SN38-treated group [F0], 3.1 ± 1.39; L-fucose-bound, liposome-carrying, SN38-treated group [F50], 0.94 ± 0.89; F50 vs NT,P= .003; F50 vs SN38,P= .02, F50 vs F0,P= .04), as well as prolonging survival of mouse xenograft models (log-rank test,P< .001). CONCLUSIONS Thus, fucose-bound liposomes carrying anticancer drugs provide a new strategy for the treatment of CRC patients.

Anti-erythropoietin receptor antibody-associated pure red cell aplasia accompanied by Coombs-negative autoimmune hemolytic anemia in a patient with T cell/histiocyte-rich large B cell lymphoma

A 79-year-old female diagnosed with T cell/histiocyte-rich large B cell lymphoma in complete remission after six cycles of rituximab-combined chemotherapy developed severe anemia, reticulocytopenia, and bone marrow erythroid hypoplasia. She was diagnosed with pure red cell aplasia (PRCA) accompanied by Coombs-negative autoimmune hemolytic anemia evidenced by a lack of glycophorin-A-positive cells in the bone marrow, haptoglobin under the detection level, and a high titer of RBC-bound IgG. Anti-erythropoietin receptor (EPOR) antibody was detected in the serum, and oligoclonal α/β and γ/δ T cells were also detected in her peripheral blood by Southern blotting analysis. Parvovirus B19 DNA was not detected by PCR. Although the treatment with rituximab had limited efficacy (specifically, only for hemolysis), subsequent cyclosporine therapy led to prompt recovery of erythropoiesis with the disappearance of anti-EPOR antibody and oligoclonal T cells. This is the first case report of anti-EPOR antibody-associated PRCA in a patient with malignant lymphoma treated successfully with cyclosporine.

Spontaneous cholesterol crystal embolism to lymph node

A 65-year-old male diagnosed with hypertension and hypertrophic cardiomyopathy in April 2010 at a different hospital was administered angiotensin II receptor blocker and low-dose aspirin. Although laboratory data at that time showed eosinophilia (2,860/lL), further examination was not performed. He had a history of smoking 1.5 packs of cigarettes a day for 45 years, but no history of diabetes mellitus. He developed cerebral infarction in January 2012, but recovered uneventfully with conservative treatment, including statins for dyslipidemia. He was subsequently referred to our hospital to investigate the eosinophilia. On physical examination, he had several swollen lymph nodes in bilateral inguinal regions, but no cutaneous lesion was observed. Laboratory data were as follows: WBC 10,600/ lL, eosinophil 840/lL, Hb 11.2 g/dL, Plt 8.8 9 10/lL, FDP 12.0 lg/mL, LDH 352 U/L, BUN 17.5 mg/dL, Cr 1.00 mg/dL, IgE 8,600 IU/mL, ACTH 15.3 pg/mL and cortisol 9.4 lg/dL, as well as negative test results for ANA and MPO-ANCA. The urinalysis showed proteinuria and microhematuria. Parasite eggs were not detected in the feces. Bone marrow examination showed 9.1 % eosinophils among all nucleated cells without dysplasia, and FIP1L1-PDGFRa and BCR-ABL chromosomal aberrations were not detected by FISH analysis. Chest and abdominal CT showed several enlarged inguinal lymph nodes up to 18 mm in the minor axis. Although he stated that he had recognized these inguinal masses about 10 years previously and that they had not changed markedly in size, we performed biopsy from the right inguinal lymph node. Histopathological findings revealed needle-shaped clefts in the lumen of arterioles with multinucleated giant cell infiltration surrounded by normal lymphoid follicles (Fig. 1a–c). Perivascular inflammatory cell infiltration, mainly of eosinophils, was also observed. Flow cytometric analysis of lymph node showed no abnormality. The diagnosis of cholesterol crystal embolism (CCE) to lymph node was made. As he presented no other clinical manifestations of CCE, no further therapeutic intervention was performed. CCE is a rare systemic disease caused by occlusion of small arteries by cholesterol crystals released from atheromatous plaques of the aorta or major branches. Chest CT in this patient also showed calcification and wall thickness of the thoracic aorta, which can be a source of cholesterol crystals (Fig. 2). The common manifestations of CCE are characteristic skin lesions, such as livedo reticularis, cyanosis or ulceration, renal impairment, and gastrointestinal disorder. CCE involvement of lymph node is extremely rare. Only a few preand postmortem cases of CCE to lymph node have been reported to date [1, 2]. CCE usually occurs following an invasive vascular procedure, or anticoagulant or thrombolytic therapy, but it can also occur spontaneously. We surmised that the CCE in this patient was spontaneous, as he had not undergone any such intervention during this clinical course. The exact time at which the CCE developed was unclear, but pathological findings of lymph nodes showing CCE with giant cell infiltration and no signs of fibrosis suggested that it had been a relatively recent event. Hence, we suspect that the A. Fujimi (&) A. Hashimoto Y. Kanisawa Department of Hematology and Oncology, Oji General Hospital, 3-4-8 Wakakusa-cho, Tomakomai 053-8506, Japan e-mail: Akihito.fujimi@ojihosp.or.jp

Targeting Notch-1 positive acute leukemia cells by novel fucose-bound liposomes carrying daunorubicin.

Complete remission by induction therapy in acute myelogenous leukemia (AML) can be achieved due to improvements in supportive and optimized therapy. However, more than 20% of patients will still need to undergo salvage therapy, and most will have a poor prognosis. Determining the specificity of drugs to leukemia cells is important since this will maximize the dose of chemotherapeutic agents that can be administered to AML patients. In turn, this would be expected to lead to reduced drug toxicity and its increased efficacy. We targeted Notch-1 positive AML cells utilizing fucose-bound liposomes, since activation of Notch-1 is required for O-fucosylation. Herein, we report that intravenously injected, L-fucose-bound liposomes containing daunorubicin can be successfully delivered to AML cells that express fucosylated antigens. This resulted in efficient tumor growth inhibition in tumor-bearing mice and decreased proliferation of AML patient-derived leukemia cells. Thus, biological targeting by fucose-bound liposomes that takes advantage of the intrinsic characteristics of AML cells could be a promising new strategy for Notch-1 positive-AML treatment.

Sustained c-kit expression in a human erythroleukemia cell line (HEL) after megakaryocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA)

Changes in c-kit proto-oncogene expression were examined in a human erythroleukemia cell line, HEL, during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced megakaryocytic differentiation. When HEL cells were treated with 10(-7) M TPA, glycophorin A expression and hemoglobin synthesis were reduced, while the expression of GP IIb/IIIa was induced in association with the morphological changes. Northern blot analysis showed that, during this megakaryocytic differentiation of HEL cells, c-kit mRNA expression persisted even after there was an apparent reduction in c-myc mRNA. This finding supports the idea that the expression of c-kit, a marker of primitive hematopoietic progenitors, may persist along with differentiation toward a megakaryocytic lineage.