IMiD/CELMoD-induced growth suppression of adult T-cell leukemia/lymphoma cells via cereblon through downregulation of target proteins and their downstream effectors.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell neoplasia associated with human T-cell leukemia virus type 1 (HTLV-1) infection and has an extremely poor prognosis. Lenalidomide (LEN; a second-generation immunomodulatory drug [IMiD]) has been employed as an additional therapeutic option for ATL since 2017, but its mechanism of action has not been fully proven, and recent studies reported emerging concerns about the development of second primary malignancies in patients treated with long-term IMiD therapy. Our purpose in this study was to elucidate the IMiD-mediated anti-ATL mechanisms. Thirteen ATL-related cell lines were divided into LEN-sensitive or LEN-resistant groups. CRBN knockdown (KD) led to a loss of LEN efficacy and IKZF2-KD-induced LEN efficacy in resistant cells. DNA microarray analysis demonstrated distinct transcriptional alteration after LEN treatment between LEN-sensitive and LEN-resistant ATL cell lines. Oral treatment of LEN for ATL cell-transplanted severe combined immunodeficiency (SCID) mice also indicated clear suppressive effects on tumor growth. Finally, a novel cereblon modulator (CELMoD), iberdomide (IBE), exhibited a broader and deeper spectrum of growth suppression to ATL cells with efficient IKZF2 degradation, which was not observed in other IMiD treatments. Based on these findings, our study strongly supports the novel therapeutic advantages of IBE against aggressive and relapsed ATL.

The Interferon-Inducible Human PLSCR1 Protein Is a Restriction Factor of Human Cytomegalovirus.

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and it has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the levels of human cytomegalovirus (HCMV) plaque formation in OUMS-36T-3 (36T-3) cells with high basal expression of PLSCR1 were significantly lower than those in human embryonic lung (HEL) cells with low basal expression of PLSCR1. In addition, the levels of HCMV plaque formation and replication in PLSCR1-knockout (KO) 36T-3 cells were significantly higher than those in parental 36T-3 cells and were comparable to those in HEL cells. Furthermore, compared to that in PLSCR1-KO cells, the expression of HCMV major immediate early (MIE) proteins was repressed and/or delayed in parental 36T-3 cells after HCMV infection. We also showed that PLSCR1 expression decreased the levels of the cAMP-responsive element (CRE)-binding protein (CREB)•HCMV immediate early protein 2 (IE2) and CREB-binding protein (CBP)•IE2 complexes, which have been suggested to play important roles in the IE2-mediated transactivation of the viral early promoter through interactions with CREB, CBP, and IE2. Interestingly, PLSCR1 expression repressed CRE- and HCMV MIE promoter-regulated reporter gene activities. These observations reveal, for the first time, that PLSCR1 negatively regulates HCMV replication by repressing the transcription from viral MIE and early promoters, and that PLSCR1 expression may contribute to the IFN-mediated suppression of HCMV infection. IMPORTANCE Because several IFN-stimulated genes (ISGs) have been reported to suppress HCMV replication, HCMV replication is thought to be regulated by an IFN-mediated host defense mechanism, but the mechanism remains unclear. PLSCR1 expression is induced in response to viral infection and IFN treatment, and PLSCR1 has been reported to play an important role in IFN-dependent antiviral responses. Here, we demonstrate that HCMV plaque formation and major immediate early (MIE) gene expression are significantly increased in PLSCR1-KO human fibroblast cells. PLSCR1 reduces levels of the CREB•IE2 and CBP•IE2 complexes, which have been suggested to play important roles in HCMV replication through its interactions with CREB, CBP, and IE2. In addition, PLSCR1 expression represses transcription from the HCMV MIE promoter. Our results indicate that PLSCR1 plays important roles in the suppression of HCMV replication in the IFN-mediated host defense system.

Regulation of Endothelium-Reticulum-Stress-Mediated Apoptotic Cell Death by a Polymethoxylated Flavone, Nobiletin, Through the Inhibition of Nuclear Translocation of Glyceraldehyde 3-Phosphate Dehydrogenase in Retinal Müller Cells.

In the early stages of diabetic retinopathy (DR), subtle biochemical and functional alterations occur in Müller cells, which are one of the components of the blood-retinal barrier (BRB). Müller cells are the principal glia of the retina and have shown a strong involvement in the maintenance of homeostasis and the development of retinal tissue. Their functional abnormalities and eventual loss have been correlated with a decrease in the tight junctions between endothelial cells and a consequent breakdown of the BRB, leading to the development of DR. We demonstrated that the endothelium reticulum (ER) triggers Müller cell death and that nuclear accumulation of glyceraldehyde 3-phosphate dehydrogenase is closely associated with ER-induced Müller cell death. In addition, induction of ER stress in Müller cells increased vascular endothelial growth factor expression but decreased pigment-epithelium-derived factor (PEDF) expression in Müller cells. We found that nobiletin, a polymethoxylated flavone from citrus explants, exerts protective action against ER-stress-induced Müller cell death. In addition, nobiletin was found to augment PEDF expression in Müller cells, which may lead to the protection of BRB integrity. These results suggest that nobiletin can be an attractive candidate for the protection of the BRB from breakdown in DR.

RLTPR Q575E: A novel recurrent gain-of-function mutation in patients with adult T-cell leukemia/lymphoma.

OBJECTIVES: Adult T-cell leukemia/lymphoma (ATL) is an intractable T-cell malignancy caused by long-term infection with human T-cell leukemia virus type-1 (HTLV-1). While ATL pathogenesis has been associated with HTLV-1-derived oncogenic proteins, including Tax and HBZ, the contribution of genomic aberrations remains poorly defined. METHODS: To elucidate the genomic basis of ATL, whole exome sequencing was performed on cells from 47 patients with aggressive ATL. RESULTS: We discovered the novel mutation RLTPR Q575E in four patients (8.5%) with a median variant allele frequency of 0.52 (range 0.11-0.68). Despite being reported in cutaneous T-cell lymphoma, three ATL patients carrying RLTPR Q575E lacked skin involvement. Patients carrying RLTPR Q575E also harbored CARD11 (75%), PLCG1 (25%), PRKCB (25%), or IKBKB (25%) mutations related to TCR/NF-κB signaling. Jurkat cells transfected with RLTPR Q575E cDNA displayed increased NF-κB activity and significantly increased IL-2 mRNA levels under stimulation. RLTPR Q575E increased the interaction between RLTPR and CARD11, while RLTPR directly interacted with Tax. CONCLUSIONS: We identified, and functionally validated, a novel gain-of-function mutation in patients with aggressive ATL. During TCR activation by Tax or gain-of-function mutations, RLTPR Q575E selectively upregulates NF-κB signaling and may exert oncogenic effects on ATL pathogenesis.

Interaction of phospholipid scramblase 1 with the Epstein-Barr virus protein BZLF1 represses BZLF1-mediated lytic gene transcription.

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and PLSCR1 has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the basal expression of PLSCR1 was significantly elevated in Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC). PLSCR1 was observed to directly interact with the EBV immediate-early transactivator BZLF1 in vitro and in vivo, and this interaction repressed the BZLF1-mediated transactivation of an EBV lytic BMRF1 promoter construct. In addition, PLSCR1 expression decreased the BZLF1-mediated up-regulation of lytic BMRF1 mRNA and protein expression in WT and PLSCR1-knockout EBV-infected NPC cells. Furthermore, we showed that PLSCR1 represses the interaction between BZLF1 and CREB-binding protein (CBP), which enhances the BZLF1-mediated transactivation of EBV lytic promoters. These results reveal for the first time that PLSCR1 specifically interacts with BZLF1 and negatively regulates its transcriptional regulatory activity by preventing the formation of the BZLF1-CBP complex. This interaction may contribute to the establishment of latent EBV infection in EBV-infected NPC cells.

Complete Sequences of the Human T-Cell Leukemia Virus Type 1 Proviral Genomes from Newly Established Adult T-Cell Leukemia Cell Lines in Oita Prefecture, Japan.

We report two complete proviral genome sequences of human T-cell leukemia virus type 1 (HTLV-1) isolated from the peripheral blood specimens of acute type adult T-cell leukemia (ATL) patients in Oita Prefecture, Japan.

Fermented Citrus reticulata (ponkan) fruit squeezed draff that contains a large amount of 4'-demethylnobiletin prevents MK801-induced memory impairment.

A previous study reported biotransformation of a citrus peel polymethoxyflavone, nobiletin, by Aspergillus enabling production of 4'-demethylnobiletin, and the product's antimutagenic activity. However, the effects of fermented citrus peel on the basal forebrain-hippocampal system remain unidentified. Citrus reticulata (ponkan) fruit squeezed draffs are generated as mass waste in beverage factories. In this study using PC12D cells and cultured central nervous system neurons, we therefore examined whether Aspergillus kawachii-fermented citrus fruit squeezed draff could affect cAMP response element (CRE)- and choline acetyltransferase gene (ChAT) promoter region-mediated transcriptional activities relevant to memory formation and cholinergic function. Our current fermentation yielded approximately 80% nobiletin bioconversion, and a sample of hot-water extract of the fermented fruit squeezed draff was stronger than that of the unfermented one in facilitating CRE-mediated transcription in cultured hippocampal neurons as well as in PC12D cells. A sample of 0-80% ethanol-eluted fraction of Diaion HP-20 column-adsorbed components of the preparation obtained by the fermentation concentration-dependently and more strongly facilitated CRE-mediated transcription than did the fraction of the unfermented one in both cell culture systems. In a separate study, this polymethoxyflavone-rich fraction of the fermented fruit squeezed draff showed a potent ability to facilitate CRE-mediated and ChAT transcription in a co-culture of hippocampal neurons and basal forebrain neurons. Repeated oral gavage of mice with the fermented fraction sample prevented MK801-impaired memory formation in mice. These findings suggest that the 4'-demethylnobiletin-rich fraction prepared from the Aspergillus-fermented ponkan squeezed draff has a potential anti-dementia effect.

I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions.

The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner. Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability.

Interaction of the phospholipid scramblase 1 with HIV-1 Tat results in the repression of Tat-dependent transcription.

Human phospholipid scramblase 1 (PLSCR1) is an interferon (IFN)-stimulated gene and possesses an IFN-mediated antiviral function. We show here that PLSCR1 directly interacts with human immunodeficiency virus type-1 (HIV-1) Tat. This interaction occurs both in vitro and in vivo through amino acids 160-250 of PLSCR1. Overexpression of PLSCR1 efficiently represses the Tat-dependent transactivation of the HIV-1 long terminal repeat (LTR) and reduces the nuclear translocation of Tat. In addition, shRNA-mediated suppression of endogenous PLSCR1 expression enhances the levels of gag mRNA in an HIV-1-infected T-cell line. These findings indicate that PLSCR1 negatively regulates the Tat-dependent transactivation of the HIV-1 LTR during HIV-1 infection.

Human phospholipid scramblase 1 interacts with and regulates transactivation of HTLV-1 Tax.

Human phospholipid scramblase (PLSCR) 1 expression is strongly activated in response to interferon (IFN) treatment and viral infection, and PLSCR1 is necessary for the IFN-dependent induction of gene expression and antiviral activity. We show here that PLSCR1 directly interacts with human T-cell leukemia virus type-1 (HTLV-1) Tax in vitro and in vivo. This interaction reduced the cytoplasmic distribution of Tax. PLSCR1 efficiently repressed the Tax-mediated transactivation of the HTLV-1 long terminal repeat and the NF-κB binding site reporter constructs in an interaction-dependent manner in COS-1 and Tax-producing HTLV-1-infected T cell lines. Furthermore, we show that PLSCR1 repressed the homodimerization of Tax in vitro. These data reveal for the first time that PLSCR1 specifically interacts with HTLV-1 Tax and negatively regulates its transactivation activity by altering the subcellular distribution and the homodimerization of Tax. PLSCR1 may play an important role in the IFN-mediated repression of Tax-dependent transactivation during HTLV-1 infection.

I-mfa domain proteins specifically interact with SERTA domain proteins and repress their transactivating functions.

The I-mfa domain proteins I-mfa and HIC are considered to be candidate tumor suppressor genes and have been shown to be involved in transcriptional regulation. We show here that I-mfa and HIC specifically interact with SEI-1 through their C-terminal I-mfa domains in vivo. This interaction affects the intracellular localization of I-mfa and requires the region of SEI-1 between 30 and 90 amino acids, which includes its SERTA domain, and results in repression of its intrinsic transcriptional activity. I-mfa also decreases the levels of the SEI-1·DP-1 complex and endogenous Fbxw7 mRNA, the expression of which is coregulated by E2F·DP-1 and SEI-1 in an interaction-dependent manner in vitro. In addition, I-mfa also specifically interacts with other SERTA domain-containing proteins, including SEI-2, SEI-3, SERTAD3 and SERTAD4, through its I-mfa domain in vivo. This interaction also affects the intracellular localization of I-mfa and represses the intrinsic transcriptional activities of SEI-2 and SERTAD3, which are also involved in the E2F-dependent transcription. These data reveal for the first time that I-mfa domain proteins interact with SERTA domain proteins and negatively regulate their transcriptional activity. Because SEI-1, SEI-2 and SERTAD3, whose intrinsic transcriptional activities are repressed by I-mfa, are suggested to be oncogenes, I-mfa domain proteins may be involved in their oncogenic functions by negatively regulating their transcriptional activities.

[Clinical efficacy of octreotide acetate in cancer patients with malignant bowel symptoms depend on terminal stage].

There are many reports that octreotide acetate(SMS)is effective for terminally ill cancer patients with malignant bowel obstructions such as nausea, vomiting and abdominal distension. We retrospectively found that the clinical efficacy of SMS in 23 patients with these symptoms depended on the early terminal stage(about six months until death)or middle terminal stage(within one month until death). SMS was more effective to relieve abdominal distension(p=0. 01)and these bowel symptoms occurred among cancer patients in the early terminal stage rather than in the middle terminal stage(p<0. 001).

[A case of recurrent breast cancer with lung metastasis resection showing four disease-free years under trastuzumab treatment].

We report a case of recurrent breast cancer with solitary lung metastasis that has shown no recurrence with treatment by trastuzumab alone after partial resection of the right lung upper lobe. A 56-year-old woman, who presented with left breast cancer, underwent quadrantectomy and axillar lymph node dissection in March 2004. Pathological findings were as follows: invasive ductal carcinoma, 3. 7 cm in size, histological grade 3, positive invasion of lymphatic and blood vessels, negative nodal status, negative ER/PgR status, and overexpression of HER2/ neu. She had received adjuvant radiotherapy followed by cyclophosphamide, methotrexate and fluorouracil combination chemotherapy; however, a lung nodule developed 14 months after first operation, which had grown gradually. Partial resection of the lung with thoracoscope assistance revealed metastatic lung cancer from breast cancer. Trastuzumab treatment for 6 months after second operation has maintained no recurrence for 4 years.

Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription.

Kaposi's sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3beta (GSK-3beta) and to negatively regulate its activity, leading to stimulation of GSK-3beta-dependent beta-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a beta-catenin-regulated reporter construct, and decreased the level of the LANA.GSK-3beta complex. These data reveal for the first time that I-mfa domain proteins interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA.GSK-3beta complex.

[A case of gastric gastrointestinal stromal tumor operated after low-dose chemotherapy with imatinib mesylate].

A 76-year-old man was admitted to our hospital because of tarry motions. Endoscopic findings showed an ulcer on a large submucosal tumor in the stomach. Abodminal CT scan showed a protruding lesion of approximately 13 cm at the lumen of the gastric body. FDG-PET imaging revealed FDG uptake in the gastric body and abdominal cavity. We diagnosed it as GIST with peritoneal dissemination clinically, and treatment with 300 mg of imatinib mesylate was started in December 2006. The main tumor was reduced(reduction rate of 27%)and FDG-PET imaging revealed a decrease in FDG uptake in the main tumor and all disseminated tumors after 5 months of treatment. However, the drug was discontinued for arthritis(grade 3). Partial gastrectomy with sampling peritoneal nodules was performed in June 2007. The present case suggests that low-dose chemotherapy with imatinib mesylate may be useful as a preoperative therapy for a minimal surgery.

HPLC determination of chondrosine in mouse blood plasma after intravenous or oral dose.

The bioavailability of chondrosine was evaluated by its direct measurement as found in the blood plasma following removal of plasma proteins by perchloric acid. The postcolumn HPLC determination of chondrosine was performed on an SCX column (6 mm i.d.x 150 mm), 0.35 mol/l boric acid (pH 5.2 adjusted by 0.1 mol/l NaOH) as an eluent (0.9 ml/min), 0.5% 2-cyanoacetamide and 1.0 M NaOH as fluorogenic reagents (0.25 ml/min each) with a fluorescence detector (ex. 331nm, em. 383nm). Two separate animal studies were conducted. In study 1, adult male ddY mice (n=6) received i.v. chondrosine (1.0 mg/kg body weight) and the plasma samples were collected. In the second study, 6 adult male ddY mice received p.o. chondrosine (400 mg/kg body weight) and the plasma samples were collected. Blood plasma samples were deproteinized by perchloric acid, analyzed and the bioavailability of chondrosine was determined. Twenty five to fifty microliters of blood plasma were required for the assay. Chondrosine was absorbed after oral administration with two phases having two maximum values, 7.8+/-5.4 and 4.0+/-1.9 at 15 microg/ml and 120 min, respectively; it disappeared from the blood flow very quickly after intravenous administration. This study provides the first report of the bioavailability of orally administered chondrosine in mice.

[Good response in ARDS treated with sivelestat sodium hydrate during chemotherapy for cholangiocarcinoma].

We experienced a case of good response in acute respiratory distress syndrome (ARDS) treated with sivelestat sodium hydrate during chemotherapy for cholangiocarcinoma. A 66-year-old male treated with combined paclitaxel (PTX) and S-1 suffered from ARDS following neutropenia. Sputum and blood culture examinations demonstrated an unknown origin, so sivelestat sodium hydrate was considered more effective than antibiotics. Sivelestat sodium hydrate ought to be used for ARDS treatment even during administration of anti-cancer agent.

[The up-regulation of vimentin and ezrin in the Epstein-Barr virus LMP1 gene transfected cells].

Epstein-Barr virus (EBV) is associated with the development of a variety of highly metastatic carcinomas, including nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1) is essential for B-cell transformation. In this study, we used two-dimensional differential gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) to study the mechanism behind tumor invasion and metastasis. Eight proteins, including Vimentin and Ezrin, were identified from the alteration of expressed proteins in HEK-293 cells responding to LMP1 gene transfection. Vimentin is a major protein of the mesenchymal intermediate filament, which maintains the cytoskeleton conformation. Ezrin is also an essential protein that links the cell membrane to the actin cytoskeleton. The up-regulation of Vimentin and Ezrin in the LMP1 gene-transfected cells suggests that EBV LMP1 is involved in the progression and metastasis of NPC.

Structure-activity relationships of cyclic peptide-based chemokine receptor CXCR4 antagonists: disclosing the importance of side-chain and backbone functionalities.

Previously, we have identified a highly potent CXCR4 antagonist 2 [cyclo(-D-Tyr1-Arg2-Arg3-Nal4-Gly5-)] and its Arg2 epimer 3 [cyclo(-D-Tyr1-D-Arg2-Arg3-Nal4-Gly5-)] by the screening of cyclic pentapeptide libraries that were designed based on the structure-activity relationship studies on 14-residue peptidic CXCR4 antagonist 1. In the present study, a new series of analogues of 2 and 3 were synthesized to evaluate the influences of peptide side-chain and backbone modification on bioactivities. Based on the Ala-scanning study, in which each residue in 2 and 3 was replaced with Ala having the identical chirality, substitution of Arg3 and Nal4 [Nal = L-3-(2-naphthyl)alanine] with Ala (compounds 6, 7, 10, 11) led to significant loss of the potency, indicating these amino acids are more important contributors to the bioactivity. For the cyclic peptide backbone, several modifications including d/l-Ala or cyclic amino acids substitution at the Gly5 position and sequential N-methylation on amide nitrogens were conducted. Among the analogues, compounds 13 [cyclo(-D-Tyr1-Arg2-Arg3-Nal4-D-Ala5-)] and 32 [cyclo(-D-Tyr1-D-MeArg2-Arg3-Nal4-Gly5-)] were close in potency to the most potent lead 2. NMR and conformational analysis indicated that both of these analogues favor the same backbone conformation as 2, whereas similar analysis of less potent analogues indicates that an altered backbone conformation is favored. The conformational analysis showed that steric repulsion by a 1,3-allylic strain-like effect across the planar peptide bond might contribute to the conformational preferences of cyclic pentapeptides.

[Effect of orally administered chondrosine on uptake of 35S sulfate into rat cartilage].

Chondroitin sulfate is widely distributed in animal tissues and possibly plays an important role in different types of metabolic reactions as well as protecting joints, the internal wall of blood vessels, skin, bone, etc. In cartilage, glycosaminoglycans have a protective function; in particular, chondroitin sulfate stabilizes fibrous and cellular elements of the connective tissue and, at the same time, lubricates and protects the membranes in joints. Recently, chondroitin sulfate has been used as a nutraceutical for the treatment of joint diseases such as osteoarthritis, although acidic and large molecules such as chondroitin sulfate might not be able to be absorbed through digestive apparatus such as the intestine. In this study, we investigated the effects of orally administered chondrosine derived from shark chondroitin sulfate on the uptake of inorganic (35)S sulfate into rat cartilage and found that chondrosine stimulates the incorporation of (35)S sulfate into cartilage compared with intact chondroitin sulfate.