野村 純

Mechanisms of molecular responses of human cells to gravity change and/or space radiation are one of the most important physiological problems in space science. We have previously reported that expression levels of several genes are changed in cultured human cells after UVC irradiation, and a few of those genes are responsible for UVC sensitivity. In this study, to find candidates for genes that play roles in susceptibility of human cells to gravity stressors, including those responsible for genetic stability in humans, we analyzed genes expressed differentially after gravity stress in human cells, using a PCR-based mRNA differential display (D.D.) method. Cells used were RSa and its variant cell lines, with discrepant sensitivity to radiation cell-killing and mutagenicity [correction of mutagenecity].

In some v-Ha-ras-transfected cell lines, serum deprivation results in apoptosis, Clarification of the molecular mechanisms by which oncogenic Ras controls susceptibility to apoptosis may assist in the development of effective therapies against human cancer with oncogenic ras gene. In this report, we established a v-Ha-ras-transfected human fibroblast clone, R1. In R1 cells, induction of v-Ha-Ras enhanced susceptibility to cell death under serum-deprived conditions. Ladders of cellular DNA were identified only when oncogenic ras was induced under serum-deprived conditions. Platelet-derived growth factor (PDGF) precluded DNA fragmentation of serum-deprived v-Ha-ras-transformed cells. Under serum-depleted conditions, the amounts of activated ERK and Akt decreased as compared with those under serum-containing conditions, The decreased levels of activated ERK and Akt were restored by the addition of PDGF., Inhibition of phosphorylated-ERK and Akt resulted in renewed susceptibility to cell death. These results indicate that failure of signal transduction of oncogenic Ras by the deficiency of growth factors such as PDGF causes v-Ha-Ras-dependent apoptosis, (C) 2000 Academic Press.

Proteolytic systems have various involvements in apoptotic pathways. To understand the role of calpain in apoptosis, calpastatin, a specific inhibitor of calpain, was overexpressed in human UVr-1 fibroblasts by transfection of its cDNA, The elevated expression of calpastatin resulted in decreased survival in the presence of okadaic acid (OA) but in no apparent alteration in the sensitivity toward other drugs such as 5-fluorouracil, mitomycin C and methotrexate, After treatment with OA, a typical apoptotic DNA ladder was observed in control vector-transfected cells but not in calpastatin-transfected cells. This indicates that OA-indnced apoptosis was suppressed by overexpression of calpastatin, Further immunoblot analysis showed that the OA-induced hyperphosphorylation of c-Jun was inhibited in calpastatin-transfected cells. This might be involved in the resistance to OA-induced cell death in calpastatin-overproducing cells. (C) 1999 Federation of European Biochemical Societies.

Gorlin syndrome (GS) is an autosomal dominant disorder in which patients are abnormally susceptible to ionizing radiation with radiotherapeutic doses. Radiogenic basal cell carcinomas may develop with a short latent period in patients. The mechanisms underlying the abnormal radiosusceptibility of cells in patients with GS has not been well characterized. In this study we report an increase in the number of nucleoli in fibroblast cells from 3 patients with GS after x-radiation. In GS fibroblasts, the increase in nucleolus number concomitant with the increase of ribonucleoprotein immunoreactive aggregates within the nucleus was observed after x-radiation, whereas significant change was not found in normal fibroblasts derived from healthy donors. This increase disappeared when cells were cultured with the RNA synthesis inhibitor actinomycin D after x-radiation but not when they were cultured with cycloheximide or aphydicolin, which are protein and DNA synthesis inhibitors, respectively. Ultraviolet exposure did not induce remarkable changes in the GS nucleoli. Thus the increase in nucleoli was induced after x-radiation of GS fibroblasts, and this increase seemed to be related to RNA synthesis metabolism.

To study cellular signaling factors responsible for the susceptibility of human cells to cell proliferation inhibition by anticancer drugs, human RSa cell line and its ultraviolet-resistant derivative UV(r)-1 were compared with respect to their sensitivity to the anti-proliferative effects of mitomycin C (MMC), 5-fluorouracil, nimustine (ACNU), cisplatin, pirarubicin (THP), bleomycin, methotrexate and ifosfamide. RSa cells were found to be highly sensitive to MMC by MTT assay compared to UV(r)-1 cells. The half maximum inhibition concentration of MMC against proliferation of RSa cells was approximately 100 ng/ml while that of UV(r)-1 cells was greater than 1 μg/ml. There was no significant difference observed between RSa and UV(r)-1 cells in the sensitivity to other seven drugs examined. Analysis by flow cytometry revealed that the cell cycle of RSa was completely blocked at the G2/M phase 40 h after treatment with MMC at a concentration of 100 ng/ml whereas a substantial proportion of UV(r)-1 cells was not arrested at that phase even in the presence of MMC. Further immunoblot analysis on MMC-induced signal transduction showed that the amounts of phosphorylated ERK MAP kinases were increased in UV(r)-1 cells to a greater extent than those in RSa cells after treatment with MMC for longer than 2 h. However, the increase in p21(Cip1) was observed in RSa cells 1 h after addition of MMC but was not observed in UV(r)-1 cells. These distinct signalling pathways might account for the differences in sensitivity to MMC between RSa and UV(r)-1 cells.

UCr-1 UV-resistant cells were established from UV-sensitive human RSa cells. We looked for genes expressed differentially between UVr-1 and RSa cells using PCR-based mRNA differential display to elucidate the molecular mechanisms underlying UV resistance. The transcription levels of syndecan-1 mRNA were increased in UVr-1 cells compared with those of RSa cells. Syndecan-1 is a transmembrane heparan sulfate proteoglycan and associates with cell adhesion and the cytoskeleton. Flow cytometric analysis using anti-syndecan-1 monoclonal antibody revealed that syndecan-1 was more abundant in UVr-1 cells than in RSa cells. The MTT method revealed that UVr-1 cells treated with the antibody showed higher sensitivity to UV cell killing than mock-treated cells. Studies using antisense oligonucleotides for syndecan-1 showed that antisense-treated UVr-1 cells became sensitive to UV cell killing. Thus, syndecan-1 might be involved in UV resistance in UVr-1 cells. (C) 1998 Academic Press.

A search for serum factors that modulate the mutability of human cells has been attempted in the peripheral blood of lung cancer patients. Factors were separated by dye-ligand chromatography and first identified as those exhibiting the ability to enhance the frequency of drug-resistance mutations in human RSa cells. The frequency was assessed by estimation of the cloning efficiency of mutant cells resistant to ouabain-mediated cell killing (Oua(R)) after irradiation with far-ultraviolet light (UV, mainly 254-nm wavelength), Pre-culture of cells with medium containing the factors prior to UV irradiation led to about a 19- to 37-fold increase in the OuaR mutation frequency compared with that of cells irradiated but not treated with the factors, The enhancing activity was detected in the serum of all 7 lung cancer patients, although the serum itself, which had not been treated with chromatography, had little or no enhancing activity in all patients. No enhancing activity was detected in serum preparations from healthy donors. The enhancing activity of lung cancer serum factors on UV-induced mutagenicity was next confirmed by detecting an enhancement of K-ras codon 12 base substitution mutations in human RSb cells, as analyzed by polymerase chain reaction (PCR) and differential dot-blot hybridization. Our results, together with previous findings; on suppression of mutagen-induced mutagenicity by human interferons, suggest the existence of extracellular factors that modulate the mutability of human cells. (C) 1998 Wiley-Liss, Inc.

Expression of the surrogate light (psi L) chain genes encoding the VpreB and lambda 5/14.1 proteins is restricted to B lineage cells, Pro-B and pre-B cells produce psi L chains, but whether both employ these as cell surface receptor components remains enigmatic. Recombinant human VpreB protein was used to generate a large panel of monoclonal anti-VpreB Abs to examine this issue. Native psi L chain proteins within pro-B cells as web as those serving as receptor components on pre-B cells were precipitated by 16 of the 26 anti-VpreB Abs. Surrogate light chains were easily detected on pre-B cell lines! whereas these anti-VpreB Abs reacted with pro-B cell lines only after plasma membrane permeabilization. The subpopulation of normal bone marrow cells bearing pre-B receptors included large and small pre-B cells exclusively, although pro-B cells also contained intracellular VpreB, VpreB proteins were not detected on or within B cells in bone marrow or the circulation, but a subpopulation of B cells in germinal centers was found to express the VpreB proteins intracellularly. Surrogate I, chains are thus intermittently produced during human B-lineage differentiation, while their role as receptor components appears limited to the pre-B cell stage.

Signals through the B cell antigen receptor lead to a variety of cellular events such as activation, anergy, and apoptosis. B cells select these outcomes to establish and maintain self-tolerance, and to mount adequate antibody responses. However, it is not fully understood how one and the same signal causes such different consequences. in the present study, we have studied the effect of activation signals on the outcome of responses to antigen receptor Ligation. Two distinct growth-promoting signals were used to activate B cells. Ligation of either RP105, a newly discovered B cell surface molecule, or the CD40 molecule, drove B cells to proliferate. Resultant blastic cells were then exposed to anti-immunoglobulin M (IgM). Blast cells that had been stimulated with anti-RP105 ceased growing and underwent apoptosis after cross-linking of surface IgM. Coligation of the Fc gamma receptor IIB with surface IgM augmented, rather than aborted, this response. In contrast to RP105-activated B cells, blast cells that had been activated by CD40 Ligation were unaltered by anti-IgM. On the other hand, CD40-activated B cells became extremely susceptible to Fas-mediated apoptosis, whereas RP105-activated B cells were much less sensitive. Anti-IgM-induced apoptosis in RP105 blasts was independent of Fas, because it was demonstrable with Fas-deficient MRL-lpr/lpr mice. These results demonstrate that the nature of an initial activation signal has a great influence on the fate of activated B cells after (re)engagement of the antigen receptor. RP105, as well as CD40, may be important in this life/death decision.

Most T(h)2 clones, when activated, produce IL-4 and express CD40 ligand (CD40L) on their cell surface. Therefore, they can induce growth and differentiation of B cells by cognate help, In contrast, activated T(h)1 clones, which produce IFN-gamma and express both CD40L and Fas ligand (Fast) on their cell surface, often induce B cell apoptotic cell death, To understand the mechanism by which T(h)2 cells can induce a cell growth and differentiation in the presence of Fast-positive cells, we stimulated a cells with IL-4, anti-IgM and/or anti-CD40 in the presence of anti-Fas, We report here that addition of anti-Fas strongly inhibited anti-CD40-induced B cell proliferation without affecting anti-IgM-induced a cell proliferation, Furthermore we showed that stimulation of B cells with anti-CD40 induced the expression of Fas molecules on the B cells (similar to 30%) and rendered them highly sensitive to anti-fas-mediated apoptotic cell death, Indeed, over 23% of anti-CD40-stimulated B cells showed hypodiploid DNA after being incubated with anti-Fas, while <2% of anti-CD40-stimulated B cells showed hypodiploid DNA after being incubated with medium alone, We also showed that IL-4 enhanced expression of Fas on anti-CD40-induced a cells (similar to 50%), although co-stimulation with anti-CD40 and IL-4 protected B cells from anti-fas-mediated apoptotic cell death and induced their growth and differentiation, Our present result might suggest that T(h)2 cells could dominate over Fast-positive T(h)1 cells by production of CD40L and IL-4, which in combination induce antibody production and inhibit the T(h)1 cell-mediated immune response.

To study the activation and differentiation of murine B cells, we prepared a hybridoma secreting monoclonal antibody, LB429, which can directly induce the proliferation of murine B cells in vitro. LB429 recognizes a B cell-specific surface molecule of 45 kDa. It recognizes an epitope of murine CD40 produced as a soluble fusion protein with glutathione S-transferase. LB429 stains COS-7 transfectant with murine CD40 cDNA and mature B-cell lines but does not stain pre-B cell lines. Two-color staining demonstrated that the epitope recognized with LB429 appears on the surface of B220(+) cells of spleen and bone marrow. LB429 can induce a strong proliferation of murine B cells from spleen in the absence of initial triggering with anti-IgM antibody or with anti-IgM antibody + IL-4. LB429 induced the cell size enlargement and the cell cycle transition of resting B cells as well as lipopolysaccharide (LPS). LB429 and LPS stimulate B cells synergistically in vitro by accumulating 44.7% of cells in S/G2/M phases of cell cycle. However, stimulation of spleen B cells with LB429 resulted in the increase of sIgM(high+) sIgD(high+) B cells, in contrast LPS showed the proliferation of both sIgM(high+) sIgD(high+) B cells and sIgM(low+) sIgD(high+) B cells. These results suggested that LB429 and LPS cause the proliferation of B cells through different stimulatory pathways. This anti-mouse CD40 antibody (LB429) is a very useful reagent to study the activation and differentiation of B cells in vitro.

Ig receptor (IgR) on the surface of B cells mediates the Ag-specific stimulatory signal for B cell proliferation and differentiation. In immature B cells, the stimulatory signal causes an inhibitory effect which is believed to be a key phenomenon in B cell tolerance or B cell anergy. Here, we studied the molecular mechanism of the inhibitory response of the IgR-mediated signal transduction that results in the programmed cell death of immature B cells. To analyze the downstream molecules oi the IgR-mediated signal transduction, we prepared a mAb against a 160-kDa membrane protein (p160) that can coprecipitate the kinase molecule(s) acting on serine, threonine, and tyrosine residues. Anti-IgR stimulation induces the increase of the kinase activity coprecipitated with the p160 protein in mature B cell BAL17 and normal adult spleen B cells. This result suggests that the p160-associated kinase activity is one of the downstream events of the IgR-mediated signal transduction cascade. Interestingly, immature B cell lymphoma WEHI-231 and the neonatal spleen B cells showed the adverse reaction of the p160-associated kinase which results in the transient loss of the kinase activity. Moreover, the transient decrease of the p160-associated kinase was caused by the tyrosine phosphatase activity induced by the stimulation of IgR in WEHI-231. The results suggest that this molecular difference in the downstream events of the IgR-mediated signal transduction between immature B cells and mature B cells already begins at the transmembrane level in the IgR-mediated signal transduction pathway.

Triggering of the Ig receptor (IgR) induces the activation in multiple intracellular signal transduction reactions including protein tyrosine phosphorylation, activation of phospholipase C, increased inositoltriphosphate, increased diacylglycerol, intracellular Ca2+ mobilization, and activation of protein kinase C. The IgR-complex, composed of mu-chain, L chain, Ig-alpha (MB-1), and Ig-beta (B29) proteins, is a functional unit both for expression of IgR and for signal transduction into cells, possibly by physical association with the down-stream functional molecules. An important functional motif ((D or E)-X(7)-(D or E)-Y-X(3)-L-X(7)-Y-X(2)-(L or I)) in the cytoplasmic domain of MB-1 molecule was shown to bind with several phosphoprotein components including src-type tyrosine kinases and phosphatidylinositol-3 kinase. To further study the functional components, we analyzed the phosphoprotein molecules coprecipitated with MB-1 protein. We found that a 52-kDa protein is coprecipitated with MB-1 protein and is inducibly phosphorylated by the stimulation with PMA. A rat mAb, prepared by immunizing the 52-kDa protein purified from SDS-PAGE, could detect the similar 52-kDa phosphoprotein (p52) expressed on the cell surface. In comparison with the 52-kDa protein in the immunoprecipitate of MB-1, the p52 migrated to the same position on 2-D gel electrophoresis (nonequilibrium pH gradient gel electrophoresis/SDS-PAGE). An in vitro kinase reaction analysis demonstrated that the p52 is tightly associated with the tyrosine kinase molecule(s), one of which is an 80-kDa protein containing an apparent autophosphorylation activity. These molecules would provide the informations of the down-stream molecules in the cascade reactions of the IgR-mediated signal transduction.

Cross-linking of surface B cell Ag receptor (BCR) induces tyrosine phosphorylation of BCR-associated components through a receptor-mediated signal transmission pathway. B cell-specific mb-1 and B29 genes encode the alpha/beta components of the BCR-associated complex in mature sIgM+ B cells. Here, we studied the involvement of the mb-1 gene product, MB-1, in the BCR-related structure of immature B cells. Affinity-purified anti-MB-1 antibody coprecipitated mu chain/20-kDa/15-kDa proteins together with monomer MB-1 and Ig-alpha/Ig-beta heterodimer components from digitonin lysates of the pre-B cell line 18.81. The monomer MB-1 and Ig-alpha in the pre-B cell line were shown to migrate with identical patterns in nonequilibrium pH gradient gel electrophoresis/SDS-PAGE. Western blot analysis showed that MB-1 protein is coprecipitated with mu chain from the pre-B cell line. We studied the tyrosine phosphorylation response of bone marrow B lineage cells as well as spleen B cells after cross-linking of BCR-related components with anti-mu, anti-kappa, and anti-MB-1 antibodies. We identified the activation of tyrosine kinase by direct cross-linking of MB-1 expressed on the surface of early B lineage cells. Anti-mu antibody stimulation induced the activation of tyrosine kinase in early (5- to 10-min) and late (30- to 120-min) responses in bone marrow early B lineage cells. Anti-MB-1 mAb (11-18-5) induced the late response exclusively but anti-kappa antibody induced only the early response. These results clearly indicate that MB-1 acts in the BCR-mediated signal transmission in early B lineage cells. To explore the molecular mechanism of protein tyrosine phosphorylation in bone marrow B lineage cells, we studied associated components of the BCR complex by using an in vitro kinase reaction and observed the phosphorylation of a 60-kDa protein in pre-B cell lines. The 60-kDa phosphoprotein coprecipitated with MB-1 and the BCR-related complex is very similar to the Src-type Fyn tyrosine kinase or a Fyn-related protein.

Specific binding of antigens to the surface immunoglobulin M (slgM) triggers B cells with several biochemical events involved in receptor-mediated signal transmission for proliferation and differentiation into antibody-producing cells. Recent studies with the Digitonin lysis method identified the slgM-associated component, IgM-alpha (B34)/Ig-beta, as the possible candidate for the transducer molecule(s) in the immunoglobulin receptor-mediated signal transmission. The 34 kd protein (B34 or IgM-alpha) of this component is suggested to be encoded by the B cell-specific mb-1 gene. We prepared monoclonal antibodies which recognize the mb-1 gene product (MB-1) and studied the functional role of MB-1 in the signal transmission in B lineage cells. Using murine pre-B lymphoma cells (18-81 and 70Z/3), we demonstrated the early phase increase of the intracellular [Ca2+]i concentration and the subsequent inhibition of the proliferation by the monoclonal anti-MB-1 antibody (11-18-5). These results clearly demonstrate signal transmission through the surface MB-1 molecule on B lineage lymphomas. This MB-1-mediated signal transmission in pre-B cell lines would suggest an alternative function of MB-1 acting at the pre-B cell stage.

A 57-year-old woman who suffered from acute myeloblastic leukemia during the course of chronic thyroiditis, is described. The patient was diagnosed as having chronic thyroiditis in 1984 when she was 53 year-old, and was treated with L-T<sub>4</sub>·Na. She admitted in July 1988 because of general fatigue, fever, cough and sore throat. On admission, hematological examination in the peripheral blood showed marked anemia and increased leukocytes with 20.5% leukemic cells positive for peroxidase staining. Bone marrow aspiration showed 38.8% leukemic cells. She was diagnosed acute myeloblastic leukemia. She reached complete remission after combination chemotherapy.<br>The case of acute myeloblastic leukemia associated with chronic thyroiditis is rarely reported. We reviewed the literature and discussed acute myeloblastic leukemia associated with chronic thyroiditis including this case.