Pupil noise is a discriminator between narcoleptics and controls

In previous studies, our laboratory demonstrated that Rat 6 (R6) fibroblasts which stably overproduce high levels of PKCepsilon display abnormalities in growth control that are characteristic of malignant transformation (Cacace et al., 1993, Oncogene, 8:2095-2104). The R6-PKCepsilon overproducing cell lines also exhibited a decreased growth factor requirement. The present study demonstrates that conditioned medium (CM) from two individual clones, R6-PKCepsilon 10 and 30, stimulates DNA synthesis in control R6-C1 cells. Maximal DNA synthesis and morphologic transformation was achieved in control cells when they were treated with medium from R6-PKCepsilon cells grown in the presence of TPA (TPA-CM). Size fractionation of the TPA-CM from PKCepsilon 30 cells revealed that this activity is due to a factor(s) that has an apparent molecular weight in the range of 10-30 kD and is heat and acid stable. This factor, like TGFbeta1, stimulated anchorage-independent growth of NRK cells. Western blot analysis (under nonreducing conditions) of the TPA-CM from R6-PKCepsilon 30 and R6-PKCepsilon 10 cells revealed the presence of the 25 kD active forms of TGFbeta2 and 3. These active forms of TGFbeta were not found in the CM of control R6 cells, or R6 cells that overexpress PKCalpha or PKCbeta1. The addition of a pan-specific TGFbeta antibody to NRK cells treated with the 10-30 kD fraction of TPA-CM from PKCepsilon 30 cells blocked the ability of this material to stimulate thymidine incorporation. Taken together, these studies suggest that the oncogenic activity of PKCepsilon in R6 cells is due, at least in part, to its ability to induce production of the active forms of TGFbeta2 and 3.     

Periodontal ligament cell culture on the hydrophobic substrate coated with proteins of periodontal ligament fibroblast-conditioned medium

In regenerating periodontal ligament (PDL) around the root of an artificial tooth, an important role is played by some physiologically active substance that promotes adhesion of the cells to the surface of the tooth root and induces cell proliferation and differentiation. In this study, the supernatant of the conditioned medium (CM) of dog periodontal ligament fibroblast (DPLF) was fractionated using an ion exchange chromatography-diethylaminoethyl (IEC-DEAE) column. DPLFs were cultured on hydrophobic dishes coated with each fraction. Cell proliferative activity and alkaline phosphatase (ALPase) activity, including electron microscopic features of the contact surface between the cells and the dish, were investigated. The DPLF-CM was separated by IEC-DEAE column into six fractions. Each fraction promoted an increase in DNA content and ALPase activity of the cultured DPLF, and especially remarkable were fractions 2 and 3. Fraction 2 at a molecular weight (Mw) of 210, 160, 85, 50 and 22 kD, and fraction 3 at Mw = 21 and 23 kD contained the type of proteins not found in other fractions. Electron microscopic analysis revealed that the cells in the coating group were in close contact with the surface of the dishes and that fine fibers protruding from the cell membrane clinged to the dishes. In the control group, a wide gap between the cells and the dishes was observed. These findings suggest that the DPLF-CM fractions contain specific physiological activating factors that induce proliferation and differentiation as well as cell adhesion of the DPLF cells.     

Polymorphism of the HLA-C promotor region

We have studied how keratinocytes cultured under hyperthermal conditions modulate skin fibroblast growth potential and their biosynthetic phenotypes in vitro. When keratinocytes were cultured at 30, 34, 37 or 39 degrees C, the conditioned medium of the keratinocytes cultured at 39 degrees C showed a greater inhibitory activity for fibroblast proliferation and greater synthetic activities of collagen and glycosaminoglycans than those incubated at 30, 34, or 37 degrees C. Transforming growth factor (TGF) beta 1 production in skin fibroblasts was also stimulated by the keratinocyte conditioned medium cultured at 39 degrees C. The stimulating activity of collagen and glycosaminoglycan syntheses of keratinocyte conditioned medium may be explained at least partly by enhanced TGF beta 1 production. The results indicate that keratinocytes cultured at a higher temperature (39 degrees C) may secrete factor(s) which modulate both fibroblast growth and matrix synthesis. This may provide evidence that under hyperthermal conditions epidermis can influence the functions of skin fibroblasts.     

A glutamate-sensitizing activity in conditioned media derived from rat cerebellar granule cells

When cerebellar granule cells that had been cultured in vitro for 8 days were subjected to a cytotoxic glutamate pulse (100 microM, 30 min incubation), the response varied according to cell density and the volume of medium in which cells were grown. Thus, lowering the cell density by a factor of 4 compared with usual conditions (2.6 x 10(5) cells/cm2) or increasing the volume by an identical 4-fold factor reduced cell death from 90-95% to 20-30%. Addition of a conditioned medium derived from high-density to low-density cultures or to high-volume cultures markedly increased the sensitivity of the cells to glutamate. This glutamate-sensitizing activity, which accelerated by several days the onset of the response of cerebellar cultures to glutamate, was inhibited by actinomycin D and was not detectable in conditioned medium derived from confluent cultures of cerebellar astroglia, or from cell lines such as PC12, GT1-7, 3T3 and CHP 100. Glutamate-sensitizing activity was not mimicked by trilodo-L-thyronine, insulin-like growth factor-I (IGF-I), truncated IGF-I, GPE [a tripeptide (gly-pro-glu) derived from IGF-I], brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor or tumour necrosis factor-alpha. However, IGF-I added to cultures of granule cells plated at high density and grown in basal medium Eagle's without serum or any other constituent of chemically defined media was capable of supporting production of glutamate-sensitizing activity to an extent similar to that shown by whole fetal calf serum. Under the same conditions triiodo-L-thyronine and BDNF did not support the production of glutamate-sensitizing activity. Glutamate-sensitizing activity was not mimicked by glutamate, NMDA, glycine or lactate, and was not inhibited by glucose, haemoglobin or N-omega-nitro-L-arginine methyl ester. At variance with the response of granule cells, the response to glutamate of GABAergic cells present in the same culture was not affected by cell density or by glutamate-sensitizing activity.     

Tumor-associated cysteine proteinase activities in human melanoma cells and fibroblasts of different origin

Specific catalytic activities of cysteine proteinases including cathepsins B (EC 3.4.22.1) and L (EC 3.4.22.15) in human melanoma cell lines SK-MEL-28, SK-MEL-30, MEL-HO and in fibroblasts of different origin are reported. Cell line-specific pH profiles of these cysteine proteinases were determined fluorometrically with benzyloxycarbonyl-phenylalanyl-arginine-amidomethylcoumarine (Z-Phe-Arg-AMC) under saturated conditions. Single activities of cathepsins B and L were inactivated by urea and by benzyloxycarbonyl-phenylalanyl-phenylalanine-diazomethylketone (Z-Phe-Phe-CHN2) in order to describe the activities of these enzymes separately. The melanoma cell line MEL-HO, which originated from a primary lesion, showed highest activity of an unknown cysteine proteinase. This enzyme is not inactivated by urea and Z-Phe-Phe-CHN2 and has a Michaelis constant (K(M) value) of approximately 1 mM. The specific characteristics suggest that it is a tumor-associated cathepsin B. In addition, high invasive subpopulations of SK-MEL-28 and SK-MEL-30 cell lines isolated by an invasion assay showed higher proteinase activities than the low invasive subpopulations. Furthermore, in fibroblasts originating from melanoma tissue cysteine proteinase activities were increased compared to normal skin fibroblasts. In conclusion, these results indicate that these cysteine proteinases shown here are tumor-associated proteinases, possibly facilitating invasion and dissemination of melanoma cells.     

Regulation of matrix-degrading enzymes in gynecologic cancer tissues and cells

1. INTRODUCTION: Studies of tumor invasion and metastases have focused on the degradation of the basement membrane, which is predominantly made up of type IV collagen, laminin, and heparan sulfate proteoglycans. Matrix metalloproteinase-2 (MMP-2) and MMP-9, which can degrade type IV collagen, are implicated in cancer invasion and metastasis. Released and activated MMPs are controlled by specific tissue inhibitors of metalloproteinase (TIMP). In the present study, we have examined gelatinolytic and TIMP activity in the conditioned medium of human normal and cancer tissues by zymography and reverse zymography. 2. MATERIALS AND METHODS: 1) Tissues. Tissues were obtained at operation after informed consent was got from each patient. Sliced tissues were incubated in serum-free medium for 4 or 24 h at 37 degrees C. Human ovarian cancer cells (SAOV) were cultured for 24 h in serum-free medium containing conditioned medium of stromal tissues. After washing by PBS 3 times, SAOV cells were cultured for a further 24 h. 2) Zymography. Conditioned medium was subjected to SDS polyacrylamide gel containing 0.3 mg/ml of gelatin in zymography, and purified MMPs were added further in reverse zymography. After electrophoresis the gel was washed with Triton X-100, and incubated for 20 h at 37 degrees C in the reaction buffer. The gel was then stained with Coomassie brilliant blue. The gelatinase and TIMP activities were detected as unstained and stained bands, respectively. The photographs of the gels were scanned with a densitometer. 3) Other method. TIMP-1 levels of conditioned medium were assayed by ELISA kit. 4) Statistics. Statistical comparisons were made by Mann-Whiteny U test. 3. RESULTS AND DISCUSSION: We have examined the gelatinolytic activity in gynecologic normal and cancer tissues by zymography and reverse zymography. Ovarian, cervical, and endometrial cancer tissues demonstrated higher gelatinolytic activity than normal tissues. The major gelatinases were those with molecular weight of 92 and 72kD, which corresponded to MMP-9 and MMP-2, respectively. The ratio of MMP 9 to MMP-2 was significantly higher in 3 types of cancer tissues than in normal tissues. Reverse zymography demonstrated that TIMP-1 and TIMP-2 were present in all tissues, and the ratio of TIMP-1 to TIMP-2 was significantly higher in 3 types of cancer tissues than in normal tissues. These findings suggested that MMP-9 and TIMP-1 were more associated with cancer phenotype than other types of MMP and TIMP. The influence of human stromal tissues (peritoneum, myometrium, ovary) on the secretion of MMPs and TIMPs was examined by addition of these stromal tissues culture medium to human ovarian cancer cells (SAOV). All conditioned medium of stromal tissues could increase in both MMP-2, MMP-9, TIMP-1, and TIMP-2 activity in SAOV cells. Fraction (> 100kD) of conditioned medium of peritoneum could increase remarkably in MMP-9, and this increase could be inhibited by anti alpha 5 antibody, which is the most popular receptor of fibronectin. Furthermore, the addition of fibronectin to SAOV cells induced increase in the secretion of MMP-9. These results demonstrated that one of the factors included in conditioned medium of peritoneum was fibronectin. We found that interferon beta could suppressed the secretion of MMP-2 and invasion in choriocarcinoma cells. However, no effect of interferon beta was observed in SAOV cells. Several flavonoids were screened to have ability to suppress the secretion of MMPs. All trans retinoic acid (RA) could suppress the secretion of MMPs in SAOV cells in time and concentration dependent manners. Further, RA could inhibited the invasion of SAOV cells by invasion assay using boyden chamber coated with matrigel.     

Identification of an inhibitor targeting macrophage production of monocyte chemoattractant protein-1 as TGF-beta 1

Monocyte chemoattractant protein-1 (MCP-1) is expressed in a diverse range of cells in response to various pathologic stimuli, whereas little is known about endogenous inhibitors of MCP-1 expression. I sought negative regulators of MCP-1 in culture medium conditioned by several cell lines and found that glomerular mesangial cells exclusively secrete a factor that inhibits expression of MCP-1 by activated macrophages. Treatment of J774.2 macrophages with conditioned medium from mesangial cells blunted the induction of MCP-1 by LPS. Even after the induction, subsequent treatment of macrophages with the conditioned medium down-regulated the MCP-1 expression. Medium conditioned by normal rat glomeruli contained a similar inhibitory activity that was enhanced in regenerating glomeruli, where mesangial cells are activated. The activity of the conditioned medium was not diminished, but enhanced by heat treatment, which was consistent with the unique property of TGF-beta family of molecules. Indeed, the mesangial cell-derived medium contained active TGF-beta 1. An anti-TGF-beta 1 neutralizing Ab abolished the inhibitory effect exerted by the mesangial cell medium, and externally added TGF-beta 1 suppressed the MCP-1 expression by macrophages at both mRNA and protein levels. The inhibitory effect of TGF-beta 1 on MCP-1 was observed in other macrophage cell lines, RAW264.7 and NR8383, and peritoneal macrophages, but not in fibroblastic cell line NRK49F. Treatment of J774.2 macrophages with TGF-beta 1 inhibited LPS induction of c-jun that was found to be crucial for the MCP-1 expression. These data demonstrate that TGF-beta 1 functions as an inhibitor of MCP-1 expression in macrophages possibly via down-regulation of c-Jun/activator protein-1.     

Stimulation of the protein tyrosine kinase c-Yes but not c-Src by neurotrophins in human brain-metastatic melanoma cells

The c-Yes proto-oncogene (pp62c-Yes) encodes a non-receptor-type protein tyrosine kinase (NRPTK) of the Src family. c-Yes activities and protein levels are elevated in human melanoma and melanocyte cell lines. Because the neurotrophins (NT) are important in the progression of melanoma to the brain-metastatic phenotype, we determined whether NT stimulate c-Yes activity in human MeWo melanoma cells and two variant sublines with opposite metastatic capabilities, 3 S 5 and 70W. The highly brain-metastatic 70W subline had an intrinsically higher c-Yes activity than parental MeWo or poorly metastatic 3 S 5 cells. c-Yes kinase was further induced by the prototypic human NT, nerve growth factor (NGF) in a dose and time-dependent manner. In contrast, c-Src activity (pp60-Src) was similar in all these cells and unaffected by NGF exposure. Additionally, human NGF and neurotrophin-3 stimulated c-Yes in brain-metastatic 70W cells. The magnitude of c-Yes activation correlated with the degree of invasion of 70 W cells following incubation of these neurotrophins. To further examine NT stimulation of c-Yes in melanoma cells, three additional cell lines were examined. Metastatic TXM-13 and TXM-18 increased c-Yes activity in response to NGF. In contrast, no increase was observed in low-metastatic TXM-40 cells. Together, these data suggest that altered c-Yes expression may play a role in the malignant progression of the human melanocyte towards the brain-metastatic phenotype and that NT enhance the activity of c-Yes in signaling penetration into the matrix of NT-rich stromal microenvironments such as the brain.     

Stimulatory effect of lymphocyte-derived factor on catecholamine efflux from cultured bovine adrenal medullary cells

The effects of lymphocytes and their conditioned medium on catecholamine efflux and uptake were examined in cultured bovine adrenal medullary cells. Co-culture of adrenal medullary cells with lymphocytes for 3 days caused an increase in appearance of catecholamines in the culture medium. Treatment of adrenal medullary cells with a conditioned medium prepared from lymphocytes also enhanced the appearance of catecholamines in culture medium in time- (8-48 h) and concentration-dependent manners. Heat treatment of the conditioned medium at 60 and 100 degrees C for 10 min reduced its stimulatory effect to 59 and 20% of control, respectively. After gel filtration on a Sephadex G-25 column or dialysis (<8 kDa molecular mass cutoff), the stimulatory activity of the conditioned medium was found in a high molecular fraction. The conditioned medium had little effect on the activity of lactate dehydrogenase in the medium of cultured adrenal medullary cells and on desipramine-sensitive [3H]norepinephrine uptake by the cells. These findings suggest that lymphocytes release a heat-sensitive factor(s) (molecular mass of more than 8 kDa) which increases efflux of catecholamines from cultured adrenal medullary cells.     

In vitro wound repair by human gastric fibroblasts: implications for ulcer healing

Fibroblasts modulate epithelial biological activities and play a key role in the ulcer healing process. There is no information regarding the biological response of human gastric fibroblasts to regulatory compounds. The aim of this study was to assess the effects of growth factors and prostaglandins on an in vitro model of human gastric fibroblast wound repair. Subconfluent fibroblast cultures were used to study proliferative responses, determined by [3H]thymidine incorporation into DNA. In vitro wound repair was determined in confluent fibroblast monolayers after mechanical denudation. The presence of putative growth factors secreted by fibroblasts was studied in conditioned medium by heparin-affinity chromatography and immunodetection with specific antibodies. Serum and platelet-derived growth factor (PDGF) -BB induced a dramatic increase in both gastric fibroblast proliferation and closure of wounded cell monolayers, whereas these activities were inhibited by both transforming growth factor (TGF) -beta1 and prostaglandin E1. Basal activities in unstimulated gastric fibroblasts were lower than those obtained in skin fibroblasts. Conditioned medium stimulated fibroblast proliferation and wound repair activity, which was inhibited by the addition of suramin, and was partially dependent on the presence of PDGF-like factor. PDGF is a major, autocrine promotor of human gastric fibroblast-dependent wound repair activities, which are inhibited by prostaglandins and TGF-beta. These findings might be important for future therapeutic ulcer healing approaches.     

Human bone marrow-derived mitogenic stimulation selective for breast carcinoma and neuroblastoma cells

In patients with neuroblastoma (NB) or breast carcinoma (BC), metastatic disease in the bone marrow (BM) is observed more frequently than at any other site, and a high incidence of BM metastases in these patients is associated with advanced disease and poor prognosis. These observations suggest the presence of BM micro-environmental elements that are favorable for NB and BC tumor cell growth. The influence of normal human BM cell-derived conditioned medium (CM) on clonogenic growth of BC and NB cell lines was investigated in vitro. The effects obtained were compared with those on tumor cells with a lower potential for BM metastasis. CM from unstimulated cultures of normal, healthy, low-density BM cells reproducibly and markedly augmented clonogenic growth of 3 BC and 3 NB cell lines. In contrast, growth of cell lines established from human tumors with differing metastatic propensity was unaffected by BM CM. Initial characterization, using crude BM CM, indicated that mitogenic activity (i) is mediated by peptides released by the non-adherent fraction of low-density BM cells and (ii) is not abolished by neutralizing antibodies against various cytokines known to be produced by BM cells and to regulate hematopoietic cell growth. Our observations suggest that certain specific peptides in the BM micro-environment may be responsible for the preferential growth of NB and BC metastases in BM.     

Mechanisms of cytotoxic effects of heavy water (deuterium oxide: D2O) on cancer cells

Heavy water (deuterium oxide: D2O) contains a neutron and a proton in its hydrogen atoms and shows a variety of biologic activities different from normal light water. In the present study the cytotoxic and cytostatic activity of D2O was assessed using a BALB/c-3T3 fibroblast cell line and four human digestive organ cancer cell lines, i.e. HepG2 hepatic, Panc-1 pancreatic, KATO-3 gastric and Colo205 colonic cancer cell lines. Against four cancer cell lines, D2O showed significant cytotoxic and cytostatic effects in a MTT assay and a Trypan blue dye exclusion assay, at concentrations higher than 30% D2O. These effects were time and dose dependent, and the IC50 after 72 h of culture ranged from 20 to 30% D2O in the Trypan blue dye exclusion assay and from 30 to 50% D2O in the MTT assay. By contrast, IC50 for the 3T3 fibroblast cell line after 72 h of culture was about 15% in the Trypan blue dye exclusion assay and 50% inhibition was not achieved in the MTT assay. Furthermore, D2O was found to significantly inhibit the invasion of tumor cells in a Matrigel invasion chamber assay at concentrations higher than 10% D2O. Incubation with D2O resulted in enlargement of cells, nuclear pyknosis and vacuolization, and immunostaining studies demonstrated that D2O treatment resulted in an increase in nuclear nick-end-labeling, which indicates DNA fragmentation, in KATO-3 and HepG2 cell lines. Furthermore, the nucleic acids and protein synthesis inhibition assay suggested that the inhibition of DNA synthesis may be one of the mechanisms responsible for the antitumor effects of D2O. Furthermore, oral administration of D2O resulted in a significant inhibition of the growth of Panc-1 tumor xenografted s.c. in nude mice, but survival was not prolonged. In conclusion, D2O has cytotoxic and cytostatic activities against human digestive organ cancer cell lines, and D2O may be a potential anticancer agent.     

Heparin stimulates the proliferation of bovine aortic endothelial cells probably through activation of endogenous basic fibroblast growth factor

We investigated the effect of heparin on the proliferation of cultured bovine aortic endothelial (BAE) cells. Heparin increased DNA synthesis in BAE cells in a concentration-dependent manner. The DNA synthesis increased by 2 to 2.5-fold with 1 mg/ml of heparin after 48 h incubation without serum and exogenous fibroblast (heparin-binding) growth factors. The stimulating effect of heparin decreased with the diminishing number of monosaccharide units which constitute heparin. By the addition of a neutralizing antibody to basic fibroblast growth factor (bFGF), the stimulating effect of heparin decreased, whereas an antibody to acidic fibroblast growth factor (aFGF) had no effect. The culture medium conditioned by heparin-treated BAE cells stimulated DNA synthesis in Balb/3T3 fibroblasts that proliferate in response to bFGF. The mitogenic activity of the conditioned medium was suppressed by the antibody to bFGF. However, heparin did not increase bFGF mRNA level in BAE cells. These results suggest that heparin stimulates the proliferation of BAE cells by the activation of endogenous bFGF, but not by the induction of its synthesis.     

Acidic and basic fibroblast growth factors in human breast tissue

Previously we have reported changes in fibroblast growth factors (FGF) in conditioned medium (CM) derived from rat mammary tumours undergoing remission. We have used a similar approach to assay for the presence of FGFs in human breast tissue and cell lines. The majority of cancer tissues (35/50), benign tissues (8/9) and all cancer adjacent normal tissues (20/20) released heat labile, NR6 transforming activity which coeluted from heparin with acidic FGF (aFGF) at 0.9-1.1 M NaCl and was neutralised by antibodies to aFGF. The conclusion that the majority of breast cancers contain active aFGF was supported by immunoblotting. The CM of a minority (15/50) of cancers and one benign tissue had highly transforming activity for NR6 cells, and was mitogenic for a breast cancer cell line, was heat labile, and strongly heparin binding, eluting at 1.5-2.0 M salt. It was not immunoreactive with antibodies to aFGF, basic FGF (bFGF) or Kaposi's FGF (kFGF) and its activity was reduced by the presence of aFGF, suggesting competition for the same receptor. Very little aFGF was observed in the CM of these tumours, and neither aFGF nor other FGF activity was detected in CM of breast cell lines.     

Characterization of human malignant melanoma cell lines. VII. Glycoprotein synthesis and shedding as revealed by [3H]glucosamine labeling

We have established conditions for the study of membrane glycoprotein synthesis and turnover in cultured human malignant melanoma cell lines using the labeled precursor [3H]glucosamine. Uptake of label increased parallel with cell growth, reaching a steady state in resting cultures. Fifteen to 30% of incorporated label can be released from the cells by trypsin treatment depending on the conditions of exposure to the enzyme, and about 50% of the incorporated label is spontaneously shed from the cells within 96 hr of incubation. Labeling in exhausted medium gave a 5- to 8-fold increase in uptake which was inhibited by addition of glucose (2 mg per ml) into the culture medium. The percentage of trypsin-releasable material was identical in fresh and exhausted medium; however, the percentage shed was less in cells initially labeled in exhausted medium. These data provide background information for further studies on the antigenic composition of the glycoproteins of cultured melanoma cells.     

Biologically active Fas antigen and its cognate ligand are expressed on plasma membrane-derived extracellular vesicles

Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas-mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.     

Proprotein-processing endoprotease furin controls growth of pancreatic beta-cells

We have previously reported that in the well-differentiated beta-cell line MIN6 cells, the beta-cell-specific differentiated characteristics, such as insulin content, expression of prohormone convertases PC2 and PC3, and glucose-regulated insulin secretion, diminished when the proprotein-processing endoprotease furin was highly expressed. Since furin converts many growth-related protein precursors to their bioactive forms, we compared the four pancreatic islet cell lines RINm5F, betaTC3, betaHC9, and MIN6 with respect to cell growth rate, furin expression, endoprotease activity, and insulin content. RINm5F cells exhibited the strongest furin expression, higher furin-type endoprotease activity, and the fastest cell growth, but had the least insulin content. In contrast, MIN6 cells exhibited only a weak furin expression, little furin-type endoprotease activity, and the slowest cell growth, but had the highest insulin content. To test whether furin-expressing cells secrete growth-promoting factors cleaved by furin, we prepared conditioned media from RINm5F and furin cDNA-introduced MIN6 (MIN6-F) cells. The conditioned media from RINm5F and MIN6-F induced increased DNA synthesis and promoted the growth of normal MIN6 cells, compared with the medium from the empty vector-introduced MIN6-0 cells. We then examined the effect of the protease inhibitors alpha1-antitrypsin and its variants by infecting their vaccinia recombinants to the four cell lines. All conditioned media from each cell line expressing the furin-specific alpha1-antitrypsin variant exhibited the least DNA synthetic capacity on normal MIN6 cells. Furthermore, all three sublines of MIN6-F grew faster than MIN6-0 and MIN6. Thus, we suggest that the islet cells with higher furin expression may induce increased production of growth factors, which result in an increase in cell growth, through an autocrine/paracrine mechanism.     

Alpha-melanocyte-stimulating hormone blocks invasion of reconstituted basement membrane (Matrigel) by murine B16 melanoma cells

We have examined the effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on invasive ability of murine melanoma cell lines with different metastatic potential in a Matrigel invasion assay. alpha-MSH potently blocked the invasion of B16-BL6 cells with highly metastatic potential in a concentration-dependent manner, whereas it was less effective in inhibiting the invasion of weakly metastatic B16-F1 cells. Pretreatment of B16-BL6 cells with alpha-MSH resulted in a decrease of the adhesiveness to fibronectin and laminin substrates in a time-dependent fashion. As assessed by zymographic analysis, alpha-MSH partially inhibited the production of matrix metalloproteinase (MMP)-2 and -9 from both cell lines to a similar degree without affecting the degradative activity of these MMPs. alpha-MSH was more potent in inhibiting the migration of B16-BL6 cells towards both fibronectin- and laminin-coated substrates than that of B16-F1 cells. The growth and morphology of B16-BL6 cells were not changed after a 7-day incubation with alpha-MSH. The number of lung tumor colonies markedly decreased when B16-BL6 cells were coinjected intravenously with 10(-6) M alpha-MSH. However, alpha-MSH had no effect on the experimental lung metastases by B16-F1 cells. These results suggest that alpha-MSH suppressed the invasive and metastatic properties of B16 melanoma cells, and the degree of inhibition was associated with metastatic potential of B16 melanoma cells.     

Treatment of normal skeletal muscle with FK506 or rapamycin results in halothane-induced muscle contracture

Cytotoxic effects of normal mouse serum on mouse tumor cells were investigated in vitro. When FE melanoma cells of C57BL/6 mouse origin, were cultured in medium containing 1% fetal calf serum (FCS) and 10-30% C57BL/6 mouse serum, number of viable FE cells markedly decreased after a little increase in their number, indicating cell death of FE cells in culture with mouse serum. Phase-contrast microscopic examination showed appearance of fatty degeneration in FE cells after 24 h, and an increase in cell death after 48 h. Electron microscopic examination, and agarose gel electrophoresis of DNA at 72 h of culture showed that their cell death occurred as necrosis. This cytotoxic effect of mouse serum was also found in culture of combinations of C57BL/6 mouse serum and C57BL/6 mouse melanoma cells (G6 cells), and BALB/c mouse serum and various BALB/c mouse tumor cells (G-5 and G-1 liver tumor cells, and Colon 26 cells). Furthermore, sera of BALB/c and B10D2 mice also showed the cytotoxic effect on FE cells. The cytotoxic effect of mouse serum was not ascribed to complement activity because all mouse sera were treated at 56 degrees C for 30 min before use, and this heat treatment completely abolished complement activity, and because serum of C5-deficient mice also showed the cytotoxic effect. This cytotoxic activity was stable at heat treatment at 100 degrees C for 10 min, and was in a serum fraction of molecular weights more than 30,000 dalton. The present results show that normal mouse serum has a factor(s) inducing fatty degeneration and necrosis of mouse tumor cells.