|
|
|
|
4. Research projects
1)Role of the Ikaros Gene in Human Hematologic Malignancies
Previously, we demonstrated overexpression of the dominant-negative isoform of the transcription factor Ikaros, IK-6, in patients with B cell malignancies including blast crisis of chronic myelogenous leukemia and acute lymphoblastic leukemia. To investigate the consequence of overexpression of IK-6 in B cells, we made IK-6 transfectants of FDH-1 and Ramos, and analyzed the effect on apoptosis. FDH-1, which was established from a patient with early pre-B acute lymphoblastic leukemia, undergoes apoptosis with treatment of dexamethasone, whereas Ramos undergoes apoptosis following anti-IgM antibody treatment. Compared with the wild type, overexpression of IK-6 rendered the transfectants more resistant to dexamethasone-induced and anti-IgM-induced apoptosis in FDH-1 and Ramos, respectively. These results suggest a possible mechanism of leukemogenesis by overexpression of IK-6.
2)Biology of Epstein-Barr Virus-positive Natural Killer Cell Lymphoma
Nasal angiocentric natural killer (NK) cell lymphoma is a novel clinicopathological entity which is common in Asian countries and associated with Epstein-Barr virus(EBV) infection. Based upon experiments in EBV- transformed B cells, latent membrane protein (LMP-1) has been believed to play critical roles in bcl-2 expression, growth promotion and apoptosis resistance. We have recently established a novel cell line, NK-YS, from a patient with nasal angiocentric EBV-positive NK cell lymphoma, which maintains characteristics of NK cell in morphology, surface marker analysis and cytotoxic activity.
We examined the function of LMP-1 through suppressing LMP-1 expression by antisense deoxyoligonucleotide to LMP-1(AS-oligo) in two EBV-positive NK cell lines, NK-YS and YT, and an EBV-transformed B-cell line, CMG-1. The AS-oligo suppressed LMP-1 mRNA and protein expression in all these lines. Bcl-2 protein expression was suppressed, proliferation was inhibited, and apoptosis was induced in CMG-1 cells, but none of these changes was observed in NK-YS or YT cells. These results suggest that proliferation, inhibition of apoptosis and bcl-2 expression in EBV-positive NK cell lymphoma cells are not intimately regulated by LMP-1 as in EBV-transformed B-cell lines, and are mediated through other signal transducing systems.
3)Analysis and Modulation of Differentiation in Leukemic Cell Line
We have established granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3(IL-3)-dependent monoblastic cell line UG3, which is inducible to osteoclasts by macrophage colony-stimulating factor(M-CSF) and osteoclast differentiation factor(ODF) and to dendritic cells by GM-CSF and inteeerleukin-4(IL-4). We are trying to induce cytotoxic T cells against various tumor antigens using GM-CSF/IL-4-treated UG3 as antigen presenting cells, and to clone the genes playing critical roles in regulating differentiation by cDNA arrays.
UT-7 is a well characterized GM-CSF-dependent megakaryoblastic cell line. Since STAT5A is a signal-transducing transcription factor activated by GM-CSF, IL-3 thrombopoietin and erythropoietin, and is considered to have regulatory roles in growth and differentiation, a vector expressing constitutively active STAT5A mutant was transduced into UT-7. We found that transduced UT-7 acquired cytokine- independent growth and spontaneous differentiation into megakaryocytic lineage.
4)Basic and Clinical Studies on Lung Cancer
We have been contributing to show clinical evidence in the treatment of small-cell lung cancer (SCLC). The chest radiotherapy and prophylactic cranial irradiation improve survival among patients with SCLC (N Engl J Med 316(15): 912,1987; 341(7): 476, 1999). Both of these prospective randomized studies from Japan cited in NEJM were carried out by our group. Now we are trying to demonstrate the superiority of high-dose ICE chemotherapy supported by peripheral blood stem cell transplantation (PBSCT) in a multicenter phase II study. Recently we have reported on concurrent chemoradiotherapy(cisplatin+5-fluorouracil+radiation) which improves disease-free survival(40% at 3 year) in inoperable patients with non-small cell lung cancer (NSCLC) in a phase II study (Br J Cancer, 2000). We have established a variety of lung cancer cell lines, to elucidate mechanisms of drug-resistance in vitro. Using these cell lines, in vivo drug-resistant mechanisms are being investigated. Recently we have established a drug-sensitivity panel to detect new drugs and the best combination. The combination effect of two drugs is confirmed by median effect plot analysis. According to this strategy, we are treating patients with metastatic NSCLC with a triplet chemotherapy (cisplatin/docetaxel, day1 & irinotecan day 2) in a phase I/II study.
5)Immunotherapyfor lung cancer
We demonstrated loss of an HLA haplotype in three out of seven NSCLC cell linesand showed the possibility that tumors escape T-cell surveillance. We generatedautologous tumor-specific CTL lines from PBMCs using these cell lines asstimulator cells. Now we are trying to sort new tumor antigens in this systemby using expression screening methods.
6)Detectionof Early Lung Cancer
We have performed a cohort study of population-based lung cancer screening,which consisted of annual chest radiograph for all participants and sputumcytology examination for high-risk participants having either smoking index> 600 or history of bloody sputum, in collaboration with Okayama Institutefor Health and Prevention, Okayama. Cases analyzed in this study included 412individuals aged between 40 and 79 years old who had died of lung cancer. Withthe conditional logistic regression analysis, smoking adjusted odds ratio ofdeath from lung cancer was estimated between screened vs unscreened individualswithin 12 months before the diagnosis; the odds ratio was calculated as 0.59(95%CI: 0.46-0.74; p=0.0001). When analysis was limited to individuals thathave never or once been screened for evaluation of the optimal screeninginterval, odds ratios for individuals having the most recent examination during0-12 months and 13-24 months before the diagnosis were 0.54 (95%CI: 0.31-0.92;p=0.023) and 0.74 (95%CI: 0.33-1.67; p=0.47), respectively. These observationssuggest that lung cancer screening may be contributory to reduce lung cancermortality by 41%, but the effect is limited within 12 months. Necessity of the annualscreening is indicated.
7)Chemopreventionfor Lung Cancer
Green tea contains a variety of polyphenols known as cathechins.(-)-Epigallocatechin gallate (EGCG) is a major component of polyphenols ingreen tea. Polyphenols in green tea have anti-oxidative, anti-mutagenic,anti-carcinogenic and antitumor activities in experimental animals.Epidemiological studies have shown a lower risk of cancer among people whoconsume a large amount of green tea. In our institute, an in vivo model isestablished to test chemopreventive agents. Using this model, we confirmed thatEGCG completely inhibited tobacco-specific nitrosamine-induced lungtumorigenesis in A/J mice and demonstrated that EGCG could inhibitcisplatin-induced lung tumorigenesis in A/J mice (Carcinogenesis 21: 915, 2000).We are investigating the carcinogenesis of human adenocarcinoma of the lungcomparing with tumorigenesis in A/J mice and exploring effectivechemopreventive agents for lung cancer.
8)Autologous and AllogeneicHematopoietic Stem Cell Transplantation
From 1994 to 2000 we have performed more than 300transplants of allogeneic bone marrow and autologous and allogeneic peripheralblood stem cells(auto-PBSCT and allo-PBSCT) in the treatment of hematologicmalignancies and solid tumors at Okayama University Hospital and relatedinstitutions.High-dose chemotherapy(HDCT) facilitated by auto-PBSCT has produced encouragingresults; 5-year disease-free survival(DFS) is estimated to be 59% in patientswith aggressive non-Hodgkinﾕs lymphoma(NHL) who received auto-PBSCT duringfirst complete remission(n=41). In 25 NHL patients who developed sensitiverelapse, 4-year DFS is 41% after auto-PBSCT. Similarly, 23 patients with acute myelogenous leukemia were treated with HDCTand auto-PBSCT during first complete remission. Their DFS is estimated to be66% at a median follow-up of 376 days. HDCT with auto-PBSCT has beenincreasingly used for treatment of solid tumors including small cell lungcancer(SCLC), breast cancer, ovarian cancer. 17 SCLC patients achieving good orcomplete responses were treated with HDCT plus auto-PBSCT, and 6 of them(335%)are now surviving more than 3 years posttransplant. These observations clearlyindicate that HDCT with auto-PBSCT can be used as a postremission therapy ordose-intensified curative treatment in selected patients with malignancy.In 1996, we conducted a multicenter phase II study of allo-PBSCT. 26 patientswith standard-risk leukemia received allo-PBSCT from their HLA-identicalsibling donors, and 4-year DFS is estimated to be 72%. In our institution, we have experienced 31 allo-PBSCT for treatment ofleukemia. Although the incidences of acute and chronic GVHD were increased, thetreatment outcome is encouraging with 69% DFS at 4 year for standard-riskleukemia. Similar results are confirmed in 1997 nationwide survey(n=103) andl999 survey(n=275) of allo-PBSCT. Thus, allo-PBSCT can be used as analternative to allogeneic bone marrow transplantation.
9)Role of Human Basophils inAllergic Inflammation
Human basophils, as well as mast cells, are known to play a crucial role inallergic inflammation. However, the exact mechanism of basophil activation atthe site of allergic inflammation has not been well understood mainly due totheir low number in blood and the difficulty in obtaining a sufficient numberof highly purified basophils for investigation.We could expand FcεRI-positivehuman basophils in vitro with high yield and purity by culturing G-CSFmobilized peripheral blood stem cell (PBSC) from patients with malignanciessuch as malignant lymphoma and lung cancer. For PBSC mobilization, G-CSF wasadministered during hematopoietic recovery after chemotherapy, and collectionof PBSC enriched with CD34+ cells was performed by leukapheresis with acontinuous blood cell separator. Mononuclear cells from separated PBSC harvestswere cultured in the presence of rhIL-3. After 3 weeks of culture, 35-80% ofthe cells were identified as FcεRI-positive basophils by flow cytometry(FCM).These cells resembled mature peripheral blood basophils morphologically whenexamined by light and electron microscopy. Two-color analysis of FCM showedthat they expressed both FcεRI and FcεRII. FcεRIcrosslinking resulted in intracellular calcium mobilization, histamine releaseand synthesis of sulfido-leukotrienes. The intracellular histamine content andthe release of these chemical mediators triggered by anti-IgE antibodies werecomparable to those of peripheral blood basophils. These findings suggest thatPBSC-derived basophils expanded in vitro are morphologically and functionallymature and will be a useful tool for studying basophil functions.We are now investigating the function of FcγRII on these basophils,especially the ability of superoxide anion and/or cytokine production, and theeffect of FcγRIIon FcεRI-mediatedreactions(signaling and mediator release).
10)Regulation of urokinas-type plasminogen activator and its inhibitor gene expressions
Urokinase-type plasminogen activator(uPA) catalyses theconversion of zymogen plasminogen to plasmin and plays a central role in manyaspects of cellular regulation such as fibronolysis, tissue remodeling,inflammation, cell migration, and tumor metastasis. Therefore, it is ofessential importance to identify molecules regulating uPA and its inhibitorexpression. We have been investigating these gene regulations in humanlymphoma, leukemia and lung cancer cell lines that show the constitutive expressionof uPA or its inhibitors. Using nuclear run-on and northem blot analyses, wenow know that the stimulation with inflammatory cytokines and oxidative stressup-regulates transcriptionally the uPA expression. Furthermore, the microarrayanalysis using the Human Cancer CHIP on which 532 human ﾒcancer-relatedﾓ geneswere spotted revealed that uPA is one of three genes clearly up-regulated bythe stimulation with oxidative stress in a small cell lung carcinoma cell line.These data suggest that the oxidative stress induces uPA in human malignantcells through activating the gene transcription. Therefore, by up-regulatinguPA expression, the oxidative stress may influence many biologicalcell-functions mediated by the uPA/plasmin system.
11)Role of eosinophils and T cells in the development of allergic disease in theairways
Bronchial asthma is a syndrome associated withallergen-induced airway hyperresponsiveness(AHR) and chronic airwayinflammation. However, the mechanisms leading to AHR are still poorlyunderstood and can be attributed to immune-dependent and immune-independentmechanisms. We established several kinds of mouse models ofovalbumin(OVA)-induced AHR (J Exp Med 186:449-454,1997; J Clin Invest104:301-308,1999; Am J Respir Crit Care Med, In press, 2000). These studiesconfirm that IL-5 dependent eosinophilic inflammation plays an essential rolein the development of changes in airway function following re-challenge ofallergen sensitized mice. We demonstrated the critical role of STAT6 in the developmentof allergic airway eosinophilia and AHR after allergen sensitization andchallenge. STAT6 deficient mice did not develop a Th2 cytokines response or anallergen-specific IgE response and had a reduced constitutive andallergen-induced expression of CD23 as well as lower mucus production in theairway epithelium(Am J Respir Crit Car Med 160:1283-1291,1999). Further, wealso identify that IL-10 is necessary for the expression of AHR but notpulmonary inflammation after allergic sensitization using IL-10 deficientmice(Proc Natl Acad Sci USA 97:6007-6012,2000). In a recent study, we found forthe first time γδT cells downregulate AHRindependently of αβT cells(Nature Med5:1150-1156,1999). Exposure to nebulized OVA alone caused increased AHR after removingγδT cells either by using TCR-δdeficient mice or antibody depletion. However, this dramatic change in AHR by asmall T cell population was not dependent on the generation of OVA specificimmunoglobulins in the serum or associated with the production of Th2 cytokinesin the bronchoalveolar lavage fluid. This suggests that the increase of AHR isdue to a local response in the lung which is controlled by γδT cells. These observations underscore theimportance of targeting these pathways in new antiallergic asthma drugdevelopment.
12)Mechanisms of alloimmune responses. Pathophysiology of graft-versus-host disease and graft-versus-thumor effects
Graft-versus-host disease (GVHD) is one of the major toxicity of allogeneic bone marrow transplantation (BMT). The pathophysiology of GVHD involves donor T cell responses to the host alloantigens and donor T cell attack to target organs such as liver, skin, and gut.
Alloantigens appear to be presented by host antigen-presenting cells (APCs) within the secondary lymphoid organs (J.EXP.Med. 1114,1980;Science 285:412,1999;Nat Med 8:575,2002). We recently found that alloantigen expression is not necessary on GVHD target cells (Nat Med 8:575, 2002). On the other hand, clinical evidences suggested close association of benefical graft-versus-tumor (GVT) effects with GVHD. We will further investigate immunopathophysiologic mecanisms of GVHD and GVT, and explore novel strategy for GVHD prevention and augmentation of GVT. |
|