Lower expression of activating transcription factors 3 and 4 correlates with shorter progression-free survival in multiple myeloma patients receiving bortezomib plus dexamethasone therapy.

Bortezomib (BTZ), a proteasome inhibitor, is widely used in the treatment of multiple myeloma (MM), but a fraction of patients respond poorly to this agent. To identify factors predicting the duration of progression-free survival (PFS) of MM patients on BTZ treatment, the expression of proteasome and endoplasmic reticulum (ER) stress-related genes was quantified in primary samples from patients receiving a combination of BTZ and dexamethasone (BD). Fifty-six MM patients were stratified into a group with PFS<6 months (n=33) and a second group with PFS⩾6 months (n=23). Of the 15 genes analyzed, the expression of activating transcription factor 3 (ATF3) and ATF4 was significantly lower in patients with shorter PFS (P=0.0157 and P=0.0085, respectively). Chromatin immunoprecipitation analysis showed that these ATFs bind each other and transactivate genes encoding the pro-apoptotic transcription factors, CHOP and Noxa, which promote ER stress-associated apoptosis. When either ATF3 or ATF4 expression was silenced, MM cells partially lost sensitivity to BTZ treatment. This was accompanied by lower levels of Noxa, CHOP and DR5. Thus low basal expression of ATF3 and ATF4 may attenuate BTZ-induced apoptosis. Hence, ATF3 and ATF4 could potentially be used as biomarkers to predict efficacy of BD therapy in patients with MM.

Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing.

The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy.

Potent antitumor effects of bevacizumab in a microenvironment-dependent human lymphoma mouse model.

We established a mouse model of microenvironment-dependent human lymphoma, and assessed the therapeutic potential of bevacizumab, an antitumor agent acting on the microenvironment. NOD/Shi-scid, IL-2Rγ(null) (NOG) mice were used as recipients of primary tumor cells from a patient with diffuse large B-cell lymphoma (DLBCL), which engraft and proliferate in a microenvironment-dependent manner. The lymphoma cells could be serially transplanted in NOG mice, but could not be maintained in in vitro cultures. Injection of bevacizumab together with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone) significantly increased necrosis and decreased vascularization in the tumor, compared with CHOP alone. Levels of human soluble interleukin-2 receptor (sIL2R) in the serum of bevacizumab+CHOP-treated mice (reflecting the DLBCL tumor burden) were significantly lower than in CHOP recipients. Mice receiving bevacizumab monotherapy also showed significant benefit in terms of tumor necrosis and vascularization, as well as decreased serum sIL2R concentrations. The present DLBCL model reflects the human DLBCL in vivo environment more appropriately than current mouse models using established tumor cell lines. This is the first report to evaluate the efficacy of bevacizumab in such a tumor microenvironment-dependent model. Bevacizumab may be a potential treatment strategy for DLBCL patients.

Evaluation of the efficacy and safety of the combination of gemcitabine and nedaplatin for elderly patients with advanced non-small-cell lung cancer.

OBJECTIVE: The aim of the present study was to retrospectively assess the safety and efficacy of the combination of gemcitabine and nedaplatin in elderly patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Patients ≥75 years with previously untreated NSCLC who underwent chemotherapy consisting of gemcitabine (800 mg/m(2) on days 1 and 8) and nedaplatin (80 mg/m(2) on day 1) every 3 weeks were retrospectively analyzed. RESULTS: Of the 35 patients, 28 were men and 7 were women, with a mean age of 78 years (range 75-87); 10 patients had stage IIIB disease and 25 patients had stage IV disease. The overall response rate was 45.7% (95% confidence interval 28.8-63.4). The median survival time was 14 months (range 3-44). Grade 3-4 toxicities included neutropenia in 74.3%, thrombocytopenia in 48.6%, anemia in 34.3%, hepatic dysfunction in 11.4%, and infection in 2.9%. There were no treatment-related deaths. There were no differences in response rate and survival between patients aged 75-79 years and patients ≥80 years, although grade 3-4 thrombocytopenia and anemia were significantly more frequent in patients ≥80 years. CONCLUSION: Our results suggest that the combination of gemcitabine and nedaplatin is effective and well tolerated for selected elderly patients with advanced NSCLC.

Bortezomib-resistant myeloma cell lines: a role for mutated PSMB5 in preventing the accumulation of unfolded proteins and fatal ER stress.

Bortezomib is an effective agent for treating multiple myeloma (MM). To investigate the underlying mechanisms associated with acquired resistance to this agent, we established two bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, the 50% inhibitory concentration values of which were respectively 24.7- and 16.6-fold higher than their parental cell lines. No activation of caspase and BH3-only proteins such as Noxa was noted in bortezomib-resistant cells after exposure to the drug. The accumulation of polyubiquitinated proteins was reduced in bortezomib-resistant cells compared with the parental cells, associated with avoidance of catastrophic ER stress as assessed by downregulation of CHOP expression. These resistant MM cells have a unique point mutation, G322A, in the gene encoding the proteasome beta5 subunit (PSMB5), likely resulting in conformational changes to the bortezomib-binding pocket of this subunit. KMS-11 parental cells transfected to express mutated PSMB5 also showed reduced bortezomib-induced apoptosis compared with those expressing wild-type PSMB5 or the parental cells. Expression of mutated PSMB5 was associated with the prevention of the accumulation of unfolded proteins. Thus, a fraction of MM cells may acquire bortezomib resistance by suppressing apoptotic signals through the inhibition of unfolded protein accumulation and subsequent excessive ER stress by a mutation of the PSMB5 gene.

The CCR4 as a novel-specific molecular target for immunotherapy in Hodgkin lymphoma.

Here, we report that tumor cells from some patients (23.8%) with Hodgkin lymphoma (HL) are positive for CC chemokine receptor 4 (CCR4). We therefore tested the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to enhance antibody-dependent cellular cytotoxicity (ADCC), as a novel immunotherapy for refractory HL. KM2760 demonstrated a promising antitumor activity in the CCR4-positive HL-bearing mouse model in the therapeutic setting. Although KM2760 did not induce any ADCC mediated by mouse natural killer (NK) cells, it significantly enhanced phagocytosis mediated by mouse monocytes/macrophages against the CCR4-positive HL cell line in vitro. Together with the findings that KM2760 did not exhibit any complement-dependent cytotoxicity or direct antiproliferation activity in vitro, these data indicated that KM2760 exerted its robust in vivo antitumor activity via monocytes/macrophages in mice. In the human system, KM2760 enhanced phagocytic activity mediated by monocytes/macrophages. Furthermore, it induced robust ADCC mediated by NK cells against the CCR4-positive HL cell line in vitro. Thus, it is conceivable that KM2760 would have much more potent antitumor activity in humans than in mice. Collectively, this study strongly indicates that anti-CCR4 mAb could be a novel treatment modality for patients with CCR4-positive HL.

Toll-like receptors, NOD1, and NOD2 in oral epithelial cells.

Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR)4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of beta-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.

Chemically synthesized pathogen-associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll-like receptors, NOD1 and NOD2 in human oral epithelial cells.

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti-bacterial factors. In humans, four kinds of PGRPs (PGRP-L, -Ialpha, -Ibeta and -S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized pathogen-associated molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli-type tryacyl lipopeptide (Pam3CSSNA), E. coli-type lipid A (LA-15-PP), diaminopimelic acid containing desmuramyl peptide (gamma-D-glutamyl-meso-DAP; iE-DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP-Ialpha and -Ibeta, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll-like receptor (TLR)2, TLR4, nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA-15-PP, iE-DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)-kappaB in the epithelial cells, and suppression of NF-kappaB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.

Muramyldipeptide and diaminopimelic acid-containing desmuramylpeptides in combination with chemically synthesized Toll-like receptor agonists synergistically induced production of interleukin-8 in a NOD2- and NOD1-dependent manner, respectively, in human monocytic cells in culture.

Two types of synthetic peptidoglycan fragments, diaminopimelic acid (DAP)-containing desmuramylpeptides (DMP) and muramyldipeptide (MDP), induced secretion of interleukin (IL)-8 in a dose-dependent manner in human monocytic THP-1 cells, although high concentrations of compounds are required as compared with chemically synthesized Toll-like receptor (TLR) agonists mimicking bacterial components: TLR2 agonistic lipopeptide (Pam3CSSNA), TLR4 agonistic lipid A (LA-15-PP) and TLR9 agonistic bacterial CpG DNA. We found marked synergistic IL-8 secretion induced by MDP or DAP-containing DMP in combination with synthetic TLR agonists in THP-1 cells. Suppression of the mRNA expression of nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the synergistic IL-8 secretion induced by DMP and MDP with these TLR agonists respectively. In accordance with the above results, enhanced IL-8 mRNA expression and the activation of nuclear factor (NF)-kappaB induced by MDP or DMP in combination with synthetic TLR agonists were markedly suppressed in NOD2- and NOD1-silenced cells respectively. These findings indicated that NOD2 and NOD1 are specifically responsible for the synergistic effects of MDP and DMP with TLR agonists, and suggested that in host innate immune responses to invading bacteria, combinatory dual signalling through extracellular TLRs and intracellular NODs might lead to the synergistic activation of host cells.

Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype.

Point mutations of the transcription factor AML1 are associated with leukemogenesis in acute myeloblastic leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and mutations in the second tyrosine kinase domain of the Fms-like tyrosine kinase 3 (FLT3) gene represent the most frequent genetic alterations in AML. However, such mutations per se appear to be insufficient for leukemic transformation. To evaluate whether both AML1 and FLT3 mutations contribute to leukemogenesis, we analyzed mutations of these genes in AML M0 subtype in whom AML1 mutations were predominantly observed. Of 51 patients, eight showed a mutation in the Runt domain of the AML1 gene: one heterozygous missense mutation with normal function, five heterozygous frameshift mutations and two biallelic nonsense or frameshift mutations, resulting in haploinsufficiency or complete loss of the AML1 activities. On the other hand, a total of 10 of 49 patients examined had the FLT3 mutation. We detected the FLT3 mutation in five of eight (63%) patients with AML1 mutation, whereas five of 41 (12%) without AML1 mutation showed the FLT3 mutation (P=0.0055). These observations suggest that reduced AML1 activities predispose cells to the acquisition of the activating FLT3 mutation as a secondary event leading to full transformation in AML M0.

Prognostic significance of the null genotype of glutathione S-transferase-T1 in patients with acute myeloid leukemia: increased early death after chemotherapy.

We investigated the prognostic significance of genetic polymorphism in glutathione-S transferase mu 1 (GSTM1), glutathione-S transferase theta 1 (GSTT1), NAD(P)H:quinone oxidoreductase (NQO1) and myeloperoxidase (MPO), the products of which are associated with drug metabolism as well as with detoxication, in 193 patients with de novo acute myeloid leukemia (AML) other than M3. Of the patients, 64.2% were either homozygous or heterozygous for GSTT1 (GSTT1(+)), while 35.8% showed homozygous deletions of GSTT1 (GSTT1(-)). The GSTT1(-) group had a worse prognosis than the GSTT1(+) group (P = 0.04), whereas other genotypes did not affect the outcome. Multivariate analysis revealed that GSTT1(-) was an independent prognostic factor for overall survival (relative risk: 1.53; P = 0.026) but not for disease-free survival of 140 patients who achieved complete remission (CR). The rate of early death after the initiation of chemotherapy was higher in the GSTT1(-) group than the GSTT1(+) group (within 45 days after initial chemotherapy, P = 0.073; within 120 days, P = 0.028), whereas CR rates and relapse frequencies were similar. The null genotype of GSTT1 might be associated with increased toxicity after chemotherapy.

Human MD-2 confers on mouse Toll-like receptor 4 species-specific lipopolysaccharide recognition.

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS). MD-2 is associated with TLR4 and imparts LPS responsiveness to it. Little is known, however, as to whether MD-2 directly regulates LPS recognition by TLR4. To address the issue, we took advantage of a species-specific pharmacology of lipid IVa, an analogue of lipid A. Lipid IVa acted agonistically on mouse (m) TLR4/MD-2 but not on human (h) TLR4/MD-2. Lipid IVa antagonized the agonistic effect of lipid A on hTLR4/MD-2. We examined the chimeric complex consisting of mTLR4 and hMD-2 to ask whether species specificity is conferred by TLR4 or MD-2. hMD-2 was clearly distinct from mMD-2 in the way of influencing LPS recognition by mTLR4. hMD-2 conferred on mTLR4 responsiveness to lipid A but not to lipid IVa. Moreover, lipid IVa acted as a lipid A antagonist on mTLR4 that is associated with hMD-2. Collectively, MD-2 directly influences the fine specificity of TLR4.

[CD7(+) acute myeloid leukemia (M0) associated with a mediastinal bulky mass lesion].

A 41-year-old man visited his doctor in May 2000 because of a sore throat and high fever. His symptoms did not improve, despite administration of antibiotics and nonsteroidal anti-inflammatory drugs. Since a chest X-ray examination revealed an anterior mediastinal bulky tumor, he was referred and admitted to our hospital on June 21, 2000. The peripheral white blood cell count was 44,540/microliter with 74% myeloblasts. Bone marrow aspiration revealed a hypercellular marrow with 82% myeloblasts, which were negative for peroxidase and alpha-naphthyl butylate esterase staining. Blast cells were positive for CD7, CD13, CD33, CD34, and HLA-DR, and negative for CD56. A needle biopsy specimen of the mediastinal tumor consisted of myeloblasts. We diagnosed the patient as having CD7 (+) acute myeloid leukemia (AML) (M0) with a bulky mediastinal mass based on the surface marker analysis, although the clinical features resembled myeloid/NK precursor acute leukemia. The patient achieved a complete remission after two courses of induction therapy. We are planning an allogeneic stem cell transplantation during his first remission because of the high risk of relapse.

Multicenter prospective study of interferon-alpha versus bone marrow transplantation for newly diagnosed patients with chronic myelogenous leukemia: a preliminary analysis.

Interferon-alpha (IFN-alpha) therapy was compared with bone marrow transplantation (BMT) in patients with chronic myelogenous leukemia (CML) in a multicenter, prospective study. Of 254 evaluable patients, 175 received IFN-alpha and 79 received allogeneic BMT, 50 of whom received transplants from human leukocyte antigen (HLA)-identical related donors and 29 from HLA-matched unrelated donors. Complete hematologic response was achieved by 148 patients (89%) in the IFN-alpha group and 53 (78%) in the BMT group. In the IFN-alpha group, a complete cytogenetic response was induced in 25 patients (15%), a partial cytogenetic response in 37 (23%), and a minor cytogenetic response in 41 (25%). At a median follow-up of 38 months, in the IFN-alpha group the predicted 5-year survival rate was 79%, and the predicted 5-year rate of remaining in chronic phase was 66%. In the BMT group the predicted 5-year survival rate was 72% for related-donor BMT and 67% for unrelated-donor BMT. Among low Sokal-risk patients, 5-year survival did not differ between IFN-alpha therapy and BMT, irrespective of age. In higher Sokal-risk patients, survival for related-donor BMT and unrelated-donor BMT tended to be better than that with IFN-alpha therapy in younger patients. On the other hand, in older patients, survival in the BMT group, especially for those receiving unrelated-donor BMT, appeared to be inferior to that in the IFN-alpha group. Unrelated-donor BMT can be recommended for high-risk younger patients. However, for older patients, it should be performed after careful consideration of prognostic factors such as age, Sokal score, and response to IFN-alpha.

Chemical structure and biological activity of a lipid A component from Helicobacter pylori strain 206.

The chemical structure of a lipid A, which was obtained as a minor component from lipopolysaccharide of Helicobacter pylori strain 206-1, was determined to be a glucosamine beta-(1 -6) disaccharide 1-(2-aminoethyl)phosphate acylated by (R)-3-hydroxyoctadecanoic acid, (R)-3- hydroxyhexadecanoic acid, and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2-, 3- and 2'-positions, respectively. Compared with the other major lipid A from the same strain, which was previously reported [Suda Y, Ogawa T, Kashihara W et al. Chemical structure of lipid A from Helicobacter pylori strain 206-1 lipopolysaccharide. J Biochem 1997; 121: 1129--1133], the structure was very similar with one exception. An (R)-3-hydroxyhexadecanoic acid was present at the 3-position of the novel lipid A component. The structure is apparently identical to one of the proposals by Moran et al. [Moran AP, Lindner B, Walsh EJ. Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides. J Bacteriol 1997; 179: 6453--6463], who concluded the same structure as the so-called major lipid A from the H. pylori strain NCTC 11637 but without isolating a homogeneous component. The endotoxic properties and pro-inflammatory cytokine-inducing activities of this novel tetra-acyl type lipid A were lower than those of previously reported major tri-acyl type lipid A.

Structural elucidation of a capsular polysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn's disease.

The structure of a capsular polysaccharide (CPS) from a clinical isolate of Bacteroides vulgatus was elucidated. B. vulgatus IMCJ 1204 was isolated from feces of a patient with Crohn's disease. CPS was prepared by phenol/water extraction of the bacterial cells followed by hydrophobic interaction chromatography and then gel filtration chromatography of the extract. The structure of CPS was determined by chemical analysis and NMR spectroscopy including DQF-COSY, TOCSY, ROESY, HSQC-TOCSY, HMQC and HMBC to be a polysaccharide composed of the following repeating unit: -->3)beta-D-Glcp(1-->6)[alpha-D-GalpNAc(1-->2)beta-D-Galp(1-->4)]beta-D-GlcpNAc(1-->3)alpha-D-Galp(1-->4)beta-D-Manp(1-->.

Heparin modulates integrin function in human platelets.

PURPOSE: Heparin binds to human platelets and can cause activation and aggregation, although the mechanisms are unknown. To determine how heparin alters platelet function, we identified platelet-binding sites for heparin and measured heparin's influence on the function of platelet integrin alpha(IIb)beta(3) (glycoprotein IIb/IIIa). METHODS: Photoaffinity cross-linking and affinity chromatography experiments were performed to identify platelet membrane proteins that bind heparin. Heparin's effect on fibrinogen binding to platelets was measured with a radioligand-binding assay. The translocation to the cytoskeleton of Rap2, a guanosine triphosphate-binding protein, was measured from platelets aggregating in response to heparin and other agonists. RESULTS: Cross-linking and affinity chromatographic experiments positively identified the integrin alpha(IIb)beta(3) as a heparin-binding site. Heparin aggregation was calcium dependent. Low concentrations of unfractionated porcine mucosal heparin (2-5 U/mL) significantly increased fibrinogen I 125 binding to activated platelets, whereas higher doses did not. Heparin-mediated platelet aggregation was completely blocked by GRGDS peptide (5 mmol/L), a competitive inhibitor of fibrinogen binding, and was blocked by EDTA (2 mmol/L), which dissociates the functional integrin complex. Aggregation was associated with Rap2 translocation to the cytoskeleton, a sign of outside-in signaling. CONCLUSIONS: Heparin binds to the alpha(IIb)beta(3) integrin in vitro and ex vivo, and heparin increases fibrinogen binding to the integrin. Heparin-mediated aggregation requires an intact integrin and ligand and leads to Rap2 translocation to the cytoskeleton-an outside-in signal of ligand engagement. Heparin may directly modulate platelet integrin function, most likely through direct binding and modulation of integrin function.