Changes in cell morphology and cytoskeletal organization are induced by human mitotic checkpoint gene, Bub1.

The mitotic spindle checkpoint prevents the onset of anaphase and subsequent cell division until chromosomes are properly aligned on a bipolar spindle. Thus, it regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. The budding uninhibited by benzimidazole (Bub1) is a key component of mitotic checkpoint. Bub1 encodes a serine/threonine kinase required for mitotic spindle checkpoint function. The regulation of cell morphology in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). Here we show that Bub1 directly affects the structural integrity of IFs. Constitutive expression of Bub1 caused disappearance of filamentous vimentin, a type III IF, and consequently changed cell morphology. Expression of kinase domain-deleted Bub1 induced neither morphological change nor disappearance of vimentin. These observations suggest that Bub1 not only regulates the cell cycle, but also may be involved in the cytoskeletal control in interphase cells.

Epstein-Barr virus infection of human natural killer cell lines and peripheral blood natural killer cells.

Although considerable part of natural killer (NK) cell neoplasms possess EBV genome, there has been no direct evidence that EBV infects human NK cells in vitro. In this study, we demonstrated EBV entry into NK cells using a recombinant EBV, which contains enhanced green fluorescent protein (EGFP) gene in its genome (EGFP-EBV). After 48 h of exposure to EGFP-EBV, we detected EGFP signals in approximately 30% of NK-92 and NKL cells and >40% of peripheral blood NK cells from three healthy volunteers. Reverse transcription-PCR analysis of various EBV-associated genes confirmed EBV infection. In situ hybridization for EBERs and BHLFs showed that latent and lytic infections coexisted at the early phase of EBV infection in two NK cell lines. Although BHLF-positive cells in the early lytic phase were round-shaped, EBER-positive cells in latent EBV infection tended to show a bizarre shape. Flow cytometric analysis of EGFP-EBV-exposed NK cell lines showed that most of EBV-infected cells entered early apoptosis after 72 h of EBV exposure, which explains the difficulties to establish EBV-carrying NK clones. Flow cytometry and reverse transcription-PCR analysis indicated that two NK cell lines may fuse with EBV using HLA class II after binding to the virus through a distinct molecule from CD21. We established two EBV-carrying NKL clones showing latency types I and II, both of which are recognized in EBV-associated NK cell neoplasms. Because EBV-infected NKL cells showed only type I latency during the early phase of infection, the temporal profile of latent gene expression is similar to that of T cells. We first report in vitro EBV infection of human NK cells and establishment of EBV-carrying NK clones, which should contribute to elucidate the role of EBV in the development of NK cell neoplasms.

Adeno-associated virus 2-mediated intratumoral prostate cancer gene therapy: long-term maspin expression efficiently suppresses tumor growth.

Maspin is a member of the serine protease inhibitors and the maspin gene, a tumor suppressor gene, is down-regulated in a large fraction of prostate cancers. We evaluated the use of adeno-associated virus (AAV, serotype 2) vector encoding maspin as a means for in vivo gene therapy for human prostate cancer. TUNEL assay of subcutaneously formed LNCaP or DU145 tumors in nude mice showed that intratumoral AAV-mediated maspin expression significantly upregulated the number of apoptotic cells compared with AAV-LacZ treatment. Immunofluorescence double staining for maspin protein and apoptosis in LNCaP tumors showed that the percentage of apoptotic cells in AAV-maspin-mediated maspin-expressing cells was significantly high compared with that in AAV-GFP-mediated GFP-expressing cells. Moreover, significantly fewer CD31-positive microvessels were observed in AAV-maspin-treated tumors compared with the control tumors. These therapeutic responses were highly correlated to persistent maspin expression in tumors, confirmed by Western blot analysis until at least day 56 after treatment. Finally, intratumoral delivery of AAV-maspin significantly suppressed growth of LNCaP and DU145 tumors and improved survival of mice. We conclude that AAV-mediated prolonged maspin expression efficiently suppresses human prostate tumor growth in vivo by apoptosis induction and inhibition of angiogenesis.

Isolation of bone marrow stromal cell-derived smooth muscle cells by a human SM22alpha promoter: in vitro differentiation of putative smooth muscle progenitor cells of bone marrow.

BACKGROUND: Bone marrow stromal cells (BMSCs) have many characteristics of mesenchymal stem cells that can differentiate into smooth muscle cells (SMCs). However, there have been few studies closely following the cell development of smooth muscle lineage among BMSCs. METHODS AND RESULTS: To investigate the possible existence of a cell population committed to the SMC lineage among bone marrow adhesion cells, we tried to detect and follow the in vitro differentiation of such a cell type by using a promoter-sorting method with a human SM22alpha promoter (-480 bp)/green fluorescent protein (GFP) construct. The construct was transfected to adhesion cells that appeared 5 days after the seeding of mononuclear cells from bone marrow. GFP was first detectable 5 days after the transfection in a cell population [Ad(G) cells], which expressed PDGF-beta but neither mature (calponin) nor immature (SMemb) SMC-specific proteins at that time. However, the cells were eventually grown into individual clones that expressed SMC-specific proteins (alpha-smooth muscle actin, calponin, and SM-1), suggesting that Ad(G) cells have partly at least progenitor properties. Because early studies have reported that PDGF-beta signaling plays pivotal roles in the differentiation of mesenchymal smooth muscle progenitor cells, Ad(G) cells might be putative mesenchymal smooth muscle progenitors expressing PDGF-beta. CONCLUSIONS: We demonstrated the presence of a cell population fated to become SMCs and followed their differentiation into SMCs among BMSCs.

More than 13 years of hypereosinophila associated with clonal CD3-CD4+ lymphocytosis of TH2/TH0 type.

A 65-year-old Japanese woman was referred to our hospital because of hypereosinophilia lasting for more than 10 years, and skin ulceration, especially on the hands. Closer examination revealed the clonal proliferation of CD3-CD4+T-lymphocytes. The patient had generalized pruritus without severe end-organ involvement and high serum levels of IgE. A diagnosis of monoclonal CD3-CD4+ T-lymphocyte-associated idiopathic hypereosinophilic syndrome (HES) was made based on these findings. This case showed that this newly recognized entity of HES is not restricted to Western countries. The abnormal T-cell clone was not merely TH2 type but was clearly TH2/TH0 type. Although this disease is considered prelymphoma, this patient did not develop lymphoma during more than 13 years of follow-up. Therefore, in some patients, clonal CD3-CD4+ lymphocyte-associated HES may take a more indolent course. In this subgroup, the control of clinical manifestations seems very important. In the present case, treatment with hydroxyurea quite dramatically improved the intractable skin manifestations, although the treatment lessened only the number of peripheral eosinophils and not the number of clonal CD3-CD4+ T-lymphocytes.

Late appearance of t(1;19)(q11;q11) in myelodysplastic syndrome associated with dysplastic eosinophilia and pulmonary alveolar proteinosis.

We report a case of myelodysplastic syndrome (MDS), which developed marked eosinophilia and pulmonary alveolar proteinosis after the appearance of t(1;19)(q11;q11). Chromosomal analysis of the peripheral eosinophils identified the same chromosome abnormality in all metaphases to that of bone marrow blast cells. Review of the literature revealed three reported cases of concurrent MDS and pulmonary alveolar proteinosis. We reviewed four cases of concurrent MDS and pulmonary alveolar proteinosis, including the present case. Interestingly, all but one of these patients also had evidence of eosinophilia and abnormality of chromosome 1p. These findings, together with morphologic abnormalities of eosinophils observed in this case, indicate clonal involvement of eosinophils in the MDS clone, and that the eosinophilia was derived from the neoplastic clone with the translocation. We postulate that this chromosomal rearrangement is involved in the development of eosinophilia and pulmonary alveolar proteinosis in MDS.

Improvement of idiopathic thrombocytopenic purpura by antithyroid therapy.

Here we report a case of idiopathic thrombocytopenic purpura accompanied by Graves' disease. Improvement in thyroid function with methimazole led to the spontaneous recovery of the platelet count from 8 x 10(9)/L to 84 x 10(9)/L. Furthermore, the second fall and recovery of the platelet count well coincided with the recurrence of hyperthyroidism after the discontinuation of methimazole and its normalization by resumption of the drug, respectively. These parallel fluctuations of platelet and thyrotropin because of the cessation and resumption of antithyroid therapy suggests that correction of hyperthyroidism may be beneficial to the control of an imbalance in the immune system which impairs not only thyroid but also the platelet.

Hepatocyte growth factor receptor is a coreceptor for adeno-associated virus type 2 infection.

After the first attachment of virus to the cell surface through a primary receptor, efficient entry of virus requires the presence of a coreceptor. For adeno-associated virus type 2 (AAV2) infection, heparan sulfate proteoglycan is supposed as the primary receptor, and alphavbeta5 integrin and FGFR1 are reported to act as coreceptors. In this study, we were able to demonstrate that hepatocyte growth factor receptor, c-Met, is also a coreceptor for AAV2 infection. AAV2-mediated transgene analyses revealed that c-Met expression significantly up-regulated transgene expression without increasing AAV2 cell binding. Moreover, a viral overlay assay elucidated the physical interaction between AAV2 and the beta subunit of c-Met. These data suggest that c-Met plays the role of coreceptor for AAV2 infection by facilitating AAV2 internalization into the cytoplasm.

Mononucleosis syndrome and acute monocytic leukemia.

The association of infectious mononucleosis and an immunocompromised host such as occurs in acute leukemia is reported. The most common cause of infectious mononucleosis is Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Patients with mononucleosis syndrome caused by other agents are rare. We report a case of acute monocytic leukemia (AMoL) who developed varicella zoster virus (VZV) mononucleosis syndrome in the bone marrow recovery phase after myelosuppression due to high-dose cytarabine. Mononuclear leukocytes appearing during the mononucleosis syndrome were very similar to the initial leukemic cells. Varicella zoster virus mononucleosis syndrome was confirmed by delayed herpes zoster rash with dermatomal distribution.

CD3-negative, CD20-positive T-cell prolymphocytic leukemia: case report and review of the literature.

We report a case of CD3-negative, CD20-positive T-cell prolymphocytic leukemia (T-PLL). The leukemic cells were of medium-to-large size, mature-looking, and did not have cytoplasmic granules. The leukemic cells were negative for surface CD3, CD2, and CD7 and strongly positive for CD20. T-cell lineage markers such as CD4, CD5, and cytoplasmic CD3 were also positive. A monoclonal rearrangement of the T-cell receptor (TCR) beta chain gene was detected. CD3-negative T-PLL has been reported often, but CD20-positive T-PLL has not. We reviewed seven cases of CD20-positive immature and mature T-cell leukemias, including the present case. Three were immature T-cell leukemias (acute lymphoblastic leukemia), and four were mature T-cell leukemias (granular lymphocytic leukemia, small lymphocytic lymphoma/chronic lymphocytic leukemia, adult T-cell leukemia, and the present case). Splenomegaly was a common feature. However, our case alone had "bright" CD20 expression on the leukemic cells. This is the first report of CD20(+) T-PLL.

Low-dose doxorubicin-induced necrosis in Jurkat cells and its acceleration and conversion to apoptosis by antioxidants.

We treated rapidly growing Jurkat cells with 40 nmol/l of doxorubicin for 72 h. After 36 h, the G2-arrested cells became larger and some of them started endoreplication. Nuclear staining with Hoechst 33342 combined with propidium iodide (PI) exclusion revealed that about 90% of the cells were necrotic at 72 h, although apoptotic cells accounted for only 8%. Incubation with 40 nmol/l of aclarubicin or cytosine beta-d-arabinofuranoside for 60 h induced necrosis both in Jurkat and ml-1 cells. Pre-necrotic Jurkat cells incubated with 40 nmol/l of doxorubicin had much higher intracellular reactive oxygen species (ROS) levels than pre-apoptotic ones. Addition of Tempol or Desferal accelerated doxorubicin-induced necrosis and partially converted it into apoptosis. Both antioxidants reduced surviving colony numbers of prenecrotic Jurkat cells. n-acetyl-l-cysteine had little effect on the apoptotic conversion but profoundly accelerated necrosis. Because an apoptosis-resistant Jurkat subclone was also refractory to doxorubicin-induced necrosis, apoptosis and necrosis might share some common pathways. Low-dose doxorubicin increased micronuclei-positive cell percentages and also suppressed high-dose doxorubicin-induced apoptosis in Jurkat and ml-1 cells. Some of the prenecrotic cells, therefore, might survive and obtain genomic instability. Antioxidants may be useful to suppress, at least to some extent, this vicious consequence.

Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3.

Modification of the cytoplasmic tails of the integrin alpha(IIb)beta(3) plays an important role in the signal transduction in platelets. We searched for proteins that bind to the alpha(IIb) cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous protein 1 (Aup1), that is ubiquitously expressed in human cells. Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm. Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that approximately 40% of alpha(IIb) is complexed with Aup1. Binding study with an alpha(IIb) cytoplasmic tail peptide and glutathione S-transferase-Aup1 fusion protein revealed a low affinity (K(d) = 90 microm). Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin alpha subunits. Binding study with the purified Aup1 and various glutathione S-transferase-alpha(IIb) cytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin alpha subunits and plays a crucial role in the alpha(IIb)beta(3) inside-out signaling. As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.