[A case of sarcoidosis following chemotherapy for follicular lymphoma].

A 53-year-old man, who had received salvage chemotherapy for follicular lymphoma, complained of fever and dry cough. High-resolution computed tomography of the chest showed bilateral diffuse ground-glass opacities with weak F18-fluorodeoxyglucose uptake on positron emission tomography. Transbronchial lung biopsy specimens revealed noncaseating epithelioid cell granulomas. His serum angiotensin-converting enzyme (ACE) level was elevated and ophthalmologic examination showed uveitis. Sarcoidosis was diagnosed, he was treated with corticosteroid eye drops, and his chest ground-glass opacities spontaneously resolved after 2 months. Here, we report a rare case of sarcoidosis with a review of the literature.

[A case of tumor microembolism diagnosed by perfusion scan and transbronchial lung biopsy].

A 70-year-old woman with breast cancer treated with hormonal therapy had progressive shortness of breath for one month. Chest radiograph and computed tomography showed mild interstitial changes, but could not account for her respiratory failure. Lymphangitic carcinomatosis, drug-induced pneumonitis, idiopathic interstitial pneumonitis, opportunistic infection, and pulmonary edema were considered in the differential diagnosis of the CT findings. A perfusion scan revealed numerous small subsegmental perfusion defects in both lung fields. Bronchoalveolar lavage fluid (BALF) contained some cancer cells, suggesting lymphangitic carcinomatosis. Transbronchial biopsy (TBLB) specimen showed tumor emboli in small pulmonary arteries. Immunohistochemical findings of TBLB specimen were consistent with breast cancer cells. A diagnosis of tumor microembolism caused by breast cancer metastasis was made. Antemortem diagnosis of tumor microembolism is very difficult. Here, we report a case of tumor microembolism diagnosed by perfusion scan and TBLB.

Ultrastructural study of the morphogenesis of human herpesvirus 6 type B in human T-lymphotropic virus type I-producing lymphoid cells.

A few studies of the morphogenesis of human herpesvirus (HHV) 6 type A and B (HHV-6A, -6B) have been performed using neurogenic, lymphoid, or epithelial cells. When human MT-4 T-lymphotropic virus type I (HTLV-I)-producing lymphoid cells were coinfected with HHV-6B in vitro, viral-specific proteins were clearly detected. We therefore attempted to detect virus particles at the ultrastructural level, focusing on the morphogenesis of such particles. Ultrastructurally, HHV-6B virus particles could be observed in the nuclei, cytoplasm, and extracellular spaces of MT-4 cells, in addition to extracellular HTLV-I particles of C type. In the nuclei, dense-cored or doughnut-shaped viral capsids were found, as well as peculiar tubular rods. When budding to perinuclear spaces, these intranuclear capsids exhibited a thin tegument on their surfaces. Distinct teguments were found in the intracytoplasmic particles, which budded into cytoplasmic vacuoles during the process of maturation. The mature particles were detected in the extracellular spaces and the intracytoplasmic vacuoles, with a distinct tegument and surface spikes. An electron-dense layer in the outer part of the tegument was found in some mature particles located in the extracellular space, but no such layer was detected in mature particles in intracytoplasmic vacuoles. No annulate lamellae, but intranuclear tubular rods, were found in the cytoplasm of MT-4 cells. These observations indicate that HHV-6B in MT-4 cells is similar to HHV-6A in fine structure, but differs from HHV-7 and HHV-8 in ultrastructural characteristics. Further comparisons of HHV-6B with HHV-6A, HHV-7, and HHV-8 are needed with regard to functional activity.

Histone deacetylase inhibitors induce growth arrest and apoptosis of HTLV-1-infected T-cells via blockade of signaling by nuclear factor kappaB.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive disease with a poor prognosis in which nuclear factor kappa B (NF-kappaB) is thought to play a role. This study explored the effects of histone deacetylase inhibitors (HDACIs) MS-275, suberoylanilide hydroxamic acid (SAHA), and LBH589 on both human T-cell lymphotropic virus type I (HTLV-1)-infected T cells (MT-1, -2, -4, and HUT102) and freshly isolated ATL cells harvested from patients. HDACIs effectively inhibited the proliferation of these cells. For example, MS-275, SAHA, and LBH589 effectively inhibited the proliferation of MT-1 cells with ED(50s) of 6microM, 2.5microM, and 100nM, respectively, as measured by 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay on day 2 of culture. In addition, HDACIs induced cell cycle arrest at the G2/M phase and apoptosis of HTLV-1-infected T-cells in conjunction with regulation of apoptosis-related proteins. Electrophoretic mobility shift assay showed that exposure of HTLV-1-infected T-cells to HDACIs for 48h inhibited formation of the NF-kappaB/DNA binding complex. Moreover, we found that HDACIs accumulated NF-kappaB and inhibitory subunit of NF-kappaB in the cytoplasm in conjunction with the down-regulation of NF-kappaB in the nucleus, suggesting that HDACIs blocked nuclear translocation of NF-kappaB. Based on these findings, we believe HDACIs can be useful for treating patients with ATL or other types of cancer in which NF-kappaB plays a role.

Promoter hypermethylation of the bone morphogenetic protein-6 gene in malignant lymphoma.

PURPOSE: Bone morphogenetic proteins (BMP), belonging to the transforming growth factor-beta superfamily, are important regulators of cell growth, differentiation, and apoptosis. The biological effects of BMPs on malignant lymphoma, however, remain unknown. Promoter methylation of the BMP-6 gene in lymphomas was investigated. EXPERIMENTAL DESIGN: We investigated BMP-6 promoter methylation and its gene expression in various histologic types of 90 primary lymphomas and 30 lymphoma cell lines. The effect of BMP-6 promoter hypermethylation on clinical outcome was also evaluated. RESULTS: BMP-6 was epigenetically inactivated in subsets of lymphomas. The silencing occurred with high frequency in diffuse large B-cell lymphoma (DLBCL) and Burkitt's lymphoma in association with aberrant BMP-6 promoter methylation. The methylation was observed in 60% (21 of 35) of DLBCL cases and 100% (7 of 7) of DLBCL cell lines, and in 83% (5 of 6) of Burkitt's lymphoma cases and 86% (12 of 14) of Burkitt's lymphoma cell lines. In contrast, other histologic types of primary lymphomas studied had little or no detectable methylation (1 of 49; 2%). The presence of BMP-6 promoter hypermethylation in DLBCL statistically correlated with a decrease in disease-free survival (P = 0.014) and overall survival (P = 0.038). Multivariate analysis showed that the methylation profile was an independent prognostic factor in predicting disease-free survival (P = 0.022) and overall survival (P = 0. 046). CONCLUSION: BMP-6 promoter was hypermethylated more often in aggressive types of lymphomas, and the hypermethylation is likely to be related to the histologic type of lymphomas. BMP-6 promoter methylation may be a potential new biomarker of risk prediction in DLBCL.

A novel treatment strategy targeting Aurora kinases in acute myelogenous leukemia.

The Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. Aberrant expression of these kinases occurs in solid tumors and is associated with aneuploidy and carcinogenesis. We found in this study that Aurora kinase A and B were aberrantly expressed in a variety of types of human leukemia cell lines (n = 15, e.g., PALL-1, PALL-2, HL-60, NB4, MV4-11, etc.), as well as freshly isolated leukemia cells from individuals with acute myelogenous leukemia (n = 44) compared with bone marrow mononuclear cells from healthy volunteers (n = 11), as measured by real-time PCR. ZM447439 is a novel selective Aurora kinase inhibitor. The compound induced growth inhibition, caused accumulation of cells with 4N/8N DNA content, and mediated apoptosis of human leukemia cells as measured by thymidine uptake, cell cycle analysis, and annexin V staining, respectively. Especially profound growth inhibition occurred with the PALL-1 and PALL-2 cells, which possess wild-type p53 gene. In contrast, ZM447439 did not inhibit clonogenic growth of myeloid committed stem cells harvested from healthy normal volunteers. Taken together, inhibition of Aurora kinases may be a promising treatment strategy for individuals with leukemia.

Idiopathic hypereosinophilic syndrome presenting acute abdomen.

We describe a 27-year-old man with hypereosinophilc syndrome (HES) presenting acute abdomen due to acute thrombosis of the mesenteric artery, who had a past history of eosinophilic pneumonia followed by multiple arterial thromboses of the extremities. At the recurrence of eosinophilia, he was treated with high-dose corticosteroids. Immediately after the reduction of peripheral blood eosinophils, he suddenly developed perforation of the intestine due to acute thromboses of mesenteric arteries despite sustained anticoagulation therapy. Molecular analysis demonstrated that the FIP1L1-PDGFRA fusion gene was negative. Histopathology showed thrombi and eosinophilic inflammation of arteries. It is important to recognize that HES could be a cause of acute abdomen.

AZD1152, a novel and selective aurora B kinase inhibitor, induces growth arrest, apoptosis, and sensitization for tubulin depolymerizing agent or topoisomerase II inhibitor in human acute leukemia cells in vitro and in vivo.

Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases, and ZM447439, a potent inhibitor of Aurora kinases, effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152, a highly selective inhibitor of Aurora B kinase, on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60, NB4, MOLM13), ALL line (PALL-2), biphenotypic leukemia (MV4-11), acute eosinophilic leukemia (EOL-1), and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM, as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis, as measured by cell-cycle analysis and annexin V staining, respectively. Of note, AZD1152 synergistically enhanced the antiproliferative activity of vincristine, a tubulin depolymerizing agent, and daunorubicin, a topoisomerase II inhibitor, against the MOLM13 and PALL-2 cells in vitro. Furthermore, AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together, AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation.

Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on lung cancer: roles of cyclooxygenase-2.

We examined the effects of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the lung cancer cell lines PC-9, LA-1 and A549. In addition, we examined if the effects of the cytokines on the cell lines are mediated by activation of cyclooxygenase (COX)-2. The three cell lines did not constitutively produce either G-CSF or GM-CSF. G-CSF did not influence cell growth in the three cell lines, while GM-CSF increased cell growth in the A549 and LA-1 lines. G-CSF and GM-CSF dose-dependently decreased cell death in the three cell lines. RT-PCR demonstrated GM-CSF receptor expression in the three lung cancer cell lines, whereas the G-CSF receptor exists only in the PC-9 line. We suggest that G-CSF might rescue the tumor cells from cytotoxicity due to serum deprivation through cellular pathways independent of the G-CSF receptor. G-CSF and GM-CSF increased cyclooxygenase-2 (COX-2) expression in PC-9 and LA-1 cells whereas they decreased COX-2 expression in A549 cells. The COX-2 inhibitor NS-398 increased cell death in PC-9 and LA-1 cells, whereas it decreased cell death in A549 cells. PC-9 and LA-1 clones transfected with sense G-CSF- or GM-CSF showed an increase in COX-2 expression, while COX-2 expression was decreased in transfected A549 clones. COX-2 expression was increased in anti-sense G-CSF- and GM-CSF-transfected A549 clones. Thus, although COX-2 activation seems to induce different biological behavior depending on the cell type, we propose that G-CSF and GM-CSF might accelerate tumor progression by directly regulating COX-2 expression, independently of an autocrine mechanism.

Longitudinal inhibition of PI3K/Akt/mTOR signaling by LY294002 and rapamycin induces growth arrest of adult T-cell leukemia cells.

This study found that phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling was activated in human T-cell lymphotropic virus type I (HTLV-1)-infected leukemia cells. Rapamycin (1-100 nM, 48h), the inhibitor of mTOR and its analog RAD001 (1-100 nM, 48 h)-induced growth inhibition and G0/G1 cell cycle arrest of these cells in association with de-phosphorylation of p70S6K and 4E-BP-1, although IC50 was not achieved. Paradoxically, rapamycin-stimulated phosphorylation of Akt at Ser473. Blockade of Akt signaling by the PI3K inhibitor LY294002 (1-20 microM, 48 h) also resulted in the growth inhibition and G0/G1 cell cycle arrest of HTLV-1-infected cells, with IC50 ranging from 5 to 20muM, and it caused de-phosphorylation of p70S6K and 4E-BP-1. Of note, when rapamycin was combined with LY294002, rapamycin-induced phosphorylation of Akt was blocked, and the ability of rapamycin to induce growth arrest of HTLV-1-infected T-cells and suppress the p-p70S6K and p-4E-BP-1 proteins was potentiated. Moreover, both LY294002 and rapamycin down-regulated the levels of c-Myc and cyclin D1 proteins in these cells, and their combination further decreased levels of these cell cycle-regulating proteins. Taken together, longitudinal inhibition of PI3K/Akt/mTOR signaling represents a promising treatment strategy for individuals with adult T-cell leukemia.

Effect of SU11248 on gastrointestinal stromal tumor-T1 cells: enhancement of growth inhibition via inhibition of 3-kinase/Akt/mammalian target of rapamycin signaling.

SU11248 is an orally available type III and V receptor tyrosine kinase inhibitor. Clinical studies have shown the efficacy of SU11248 in individuals with gastrointestinal stromal tumors (GIST); however, the molecular mechanisms by which SU11248 inhibits the proliferation of these tumor cells remains to be fully elucidated. Taking advantage of GIST-T1 cells, which possess an activating mutation in exon 11 of the c-KIT gene, we examined the medicinal action of SU11248 in GIST cells. Clonogenic and MTT assays showed that SU11248 potently inhibited the proliferation of GIST-T1 cells with IC50 of approximately 1 nM and 40 nM, respectively. SU11248 (10 or 20 nM, 48 h) activated caspase-3 and induced apoptosis of GIST-T1 cells as measured by caspase assay, annexin V staining and cleavage of poly (ADP-ribose) polymerase. Western blot analyses found that SU11248 blocked autophosphorylation of c-KIT in association with inhibition of its downstream effectors, including Akt and extracellular signal-regulated kinase, but not signal transducers and activators of transcription. Interestingly, when phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling was blocked simultaneously by either LY294002 or rapamycin, growth inhibition mediated by SU11248 was potentiated. Taken together, this study supports clinical studies of SU11248 for individuals with GIST, and the combination of SU11248 and inhibitors of 3-kinase/Akt/mammalian target of rapamycin signaling represents a promising novel treatment strategy.

Effects of GM-CSF and M-CSF on tumor progression of lung cancer: roles of MEK1/ERK and AKT/PKB pathways.

Several studies have demonstrated that colony-stimulating factors (CSFs) are closely associated with tumor progression, metastasis and invasion through autocrine or paracrine mechanism in lung cancer. However, biologic roles of CSFs are still unknown. Elucidating the biologic roles of CSFs and the regulatory mechanisms of tumor-specific behavior by CSFs raises the possibility of having a new therapeutic approach for lung cancer. We previously established two adenocarcinoma cell lines, A924 and A964 and a large cell carcinoma cell line MI-4. MI-4 and A924 constitutively produced an abundant dose of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). We examined the effects of GM-CSF and M-CSF on tumor growth, death, and invasion in CSF-producing (A924 and MI-4) and non-producing lung cancer cells (A549 and A964). These cell lines demonstrated both GM-CSF and M-CSF receptor mRNA expression. In our study, GM-CSF seemed to have advantage for tumor proliferation and invasion in lung cancer cells. M-CSF seemed to have advantage for tumor invasion, but not proliferation. The tumor-specific phenotypes (proliferation, invasion and survival) up-regulated by GM-CSF and M-CSF were mediated through MEK/ERK and PI3k/Akt pathways. However, when MEK/ERK was activated by transfection of active form of MEK1 cDNA, the tumor-specific behavior was promoted in CSF-non-producing cells, whereas inhibited in CSF-producing cells though MEK/ERK activation increased constitutive GM-CSF production. MEK/ERK signaling regulated differently tumor-specific behavior between CSF-producing cells and CSF-non-producing cells.

Diffuse micronodular pulmonary metastasis of lung adenocarcinoma predicts gefitinib response in association with epidermal growth factor receptor mutations.

Female, non-smoker, Asian ethnicity and adenocarcinoma histology are the major clinical predictors of gefitinib response in non-small cell lung cancer, as shown in previous studies. Recently, response to gefitinib has been associated with epidermal growth factor receptor (EGFR) mutations. Higher rates of mutation were seen in females, patients with adenocarcinomas, the Asian population and never-smokers, which may explain the clinical response predictors. The presence of diffuse micronodular pulmonary metastasis on chest imaging as a novel clinical predictor of its response is proposed here. Two cases of lung adenocarcinomas in men presenting with diffuse micronodular pulmonary metastasis were encountered. Both patients showed a major response to gefitinib. The dramatic reduction of micronodular pulmonary nodules throughout both lungs on computed tomography scans was achieved after treatment for a couple of months with 250 mg of oral gefitinib. In the molecular analysis, one patient had a heterozygous delL746-A750 mutation and the other had a heterozygous L858R EGFR mutation. In conclusion, patients with lung adenocarcinoma, even men, who presented with bilateral diffuse micronodular metastatic spread to the lungs tended to have an activated EGFR mutation. Therefore, they are most likely to receive benefits from molecular target drugs such as gefitinib and possibly erlotinib.

HIV-1 protease inhibitor ritonavir potentiates the effect of 1,25-dihydroxyvitamin D3 to induce growth arrest and differentiation of human myeloid leukemia cells via down-regulation of CYP24.

HIV-1 protease inhibitor, ritonavir (RTV) is a potent inhibitor of cytochrome p450 (CYPs) enzymes. This study explored the effects of RTV on CYP24 which converts 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to its inactive form 1,24,25,(OH)(3). Real-time RT-PCR showed that exposure of HL-60 cells to 1,25(OH)(2)D(3) induced expression of CYP24, and pre-incubation of these cells with RTV decreased this transcripts, resulting in increased intracellular levels of 1,25(OH)(2)D(3) and potentiation of the ability of 1,25(OH)(2)D(3) to induce growth arrest and differentiation of these cells. Taken together, inhibition of CYP24 might open a new paradigm for therapy using Vitamin D compounds.

Role of protein kinase C in expression of granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor in lung cancer cells.

We examine the role of protein kinase C (PKC) pathways in the constitutive expression of granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF) in lung cancer cells. Two cell lines, OKa-C-1 and MI-4, constitutively produce an abundant dose of G-CSF and GM-CSF. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated the production of GM-CSF in a dose-dependent manner and reduced G-CSF in the cell lines. The PKC inhibitor staurosporine had effects opposite to those of PMA in the cell lines. Another PKC activator (4beta-phorbol 12, 13-dibutyrate) and six specific PKC inhibitors (bisindolylmaleimide I, calphostin C, chelerythrine chloride, Gö 6976, PKC inhibitor 19-27, and Ro-32-0432) also worked as well as PMA and staurosporine, respectively. The induction of GM-CSF expression via PKC activation was mediated by the activation of nuclear factor-kappaB. The induction of G-CSF expression via PKC inhibition was mediated by p44/42 mitogen-activated protein kinase and c-Jun N-terminal kinase pathway signaling. GM-CSF may accelerate cell growth and inhibit cell death via PKC activation in the cell lines. G-CSF also seems to reverse growth suppression and cell death induced by PKC inhibition.