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M H Wang

Publications and source records attributed to M H Wang.

At least 73 records · Page 4Linked to original sources
PubMed1996

Expression and coupling of human cytochrome P450 2E1 and NADPH-cytochrome P450 oxidoreductase in dual expression and co-infection systems with baculovirus in insect cells.

In order to study the interaction between human cytochrome P450 2E1 (h2E1) and NADPH-P450 oxidoreductase (hOR) in a native membrane environment, we used two approaches to express both h2E1 and hOR in a baculovirus expression system. For a dual-expression system, h2E1 and hOR were coexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus carrying both h2E1 and hOR cDNAs (v-h2E1-hOR). The h2E1 cDNA was expressed under the control of the polyhedrin promoter P(Polh), whereas hOR cDNA was expressed under the control of the P10 promoter. The expressed enzymes were catalytically active in the cell membrane preparations. The estimated molar expression ratio of h2E1 to hOR in the membranes was 1:5. The apparent Km and kcat for N-nitrosodimethylamine (NDMA) demethylase activity were 145 microM and 2.4 min-1, respectively. When Sf9 cells were co-infected with the dual-expression virus (v-h2E1-hOR) and human cytochrome b5 recombinant virus (v-hb5), a 9-fold decrease in the Km of NDMA demethylase activity (16 microM) was observed in the membrane preparations, whereas the kcat was increased to 4 min-1. In the second approach, recombinant viruses of h2E1 and hOR (v-h2E1 and v-hOR) were used to co-infect the Sf9 cells. In this double-expression system, with a fixed amount of v-h2E1, the expression of h2E1 in the Sf9 cells decreased as the amount of v-hOR increased. Western blot analysis of the membrane preparations showed that the level of hOR increased, but the level of h2E1 decreased with increasing amounts of v-hOR. A corresponding decrease in h2E1 mRNA, however, was not observed. In the presence of human cytochrome b5 (hb5), the optimal h2E1:hOR molar ratio for h2E1 catalytic activity was 1:1. In order to further investigate the hb5 effect on h2E1-catalyzed reactions in the native membranes, we co-infected Sf9 cells with v-h2E1, v-hOR, and v-hb5 and obtained a membrane preparation containing h2E1, hOR, and hb5. Stoichiometric analysis with membrane preparations from double-infection and triple-infection systems revealed that the presence of hb5 decreased NADPH oxidation and H202 formation by 72 and 80%, respectively, but increased product formation from NDMA 13-fold. These results suggest that hb5 enhances the coupling between h2E1 and hOR for product formation. These studies also demonstrate that the baculovirus-insect cell system can produce high levels of expression of functional h2E1, hOR, and hb5 for mechanistic studies.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1996

Kinetic analysis of the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by heterologously expressed human P450 enzymes and the effect of P450-specific chemical inhibitors on this activation in human liver microsomes.

The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is enzymatically activated by the hydroxylation of the alpha-methyl and alpha-methylene groups, leading to the formation of reactive species which can pyridyloxobutylate and methylate DNA, respectively. The present study examined the kinetic parameters of NNK-derived keto alcohol (alpha-methyl hydroxylation), and keto aldehyde (alpha-methylene hydroxylation) formation catalyzed by human P450s heterologously expressed by either the baculovirus-insect cell expression system (P450s 2A6, 2D6, 2E1, and 3A4) or by stable expression in CHO cells (P450s 3A4 and 2D6) and human B-lymphoblastoid cells (P450 2D6). Membrane preparations of the expressed P450s catalyzed the alpha-hydroxylation of NNK, leading to the formation of keto aldehyde and keto alcohol. Human P450 2A6 showed the lowest KM (118 microM) for the formation of keto aldehyde. A similar KM was observed for keto alcohol formation by expressed P450 2A6, but the kcat was lower than the value obtained for keto aldehyde formation. The addition of exogenous b5 increased the expressed 2A6-dependent NNK hydroxylation activity 2.5-fold for both alpha-hydroxylation products. Human P450s 2E1 and 2D6 exhibited a high capacity for keto alcohol formation; however, their KM values for this reaction were in the millimolar range. Expressed human P450 3A4 oxidized NNK to keto aldehyde also with a high KM. Ten human liver microsomal samples were each shown to activate NNK to keto aldehyde and keto alcohol. A positive correlation coefficient of 0.74 was found between keto aldehyde formation and both coumarin 7-hydroxylation (P450 2A6) and 6 beta-testosterone hydroxylation (3A4) activity in characterized human liver microsomes. Keto alcohol formation showed a significant correlation with ethoxyresorufin O-dealkylation (P450 1A2) in human liver microsomes. Both coumarin and troleandomycin, specific inhibitors of P450 2A6 and 3A4, respectively, inhibited the formation of keto aldehyde, but inhibited the formation of keto alcohol only slightly in human liver microsomes. Both furafylline, a P450 1A2 inhibitor, and N-nitrosodimethylamine, a P450 2E1 substrate, inhibited the formation of keto alcohol but not keto aldehyde in human liver microsomes. Quinidine, a specific inhibitor of P450 2D6, was not an effective inhibitor of NNK metabolism. These results demonstrate that P450s 2A6 and 3A4 may be important P450s for the activation of NNK to a DNA-methylating agent and keto aldehyde via the alpha-methylene hydroxylation pathway. P450s 1A2, 2E1, and 2D6 are shown to be selective for alpha-methyl hydroxylation of NNK leading to keto alcohol and a DNA-pyridyloxobutylating agent.

Animals↗
PubMed1996

Medulloblastoma: long-term results for patients treated with definitive radiation therapy during the computed tomography era.

PURPOSE: We performed a retrospective evaluation of the patterns of failure and outcome for medulloblastoma patients treated with craniospinal irradiation therapy during the computed tomography (CT) era. MATERIALS AND METHODS: The records of 100 patients treated at Memorial Sloan-Kettering Cancer Center between 1979 and 1994 were reviewed. CT scans or magnetic resonance imaging were used to guide surgical intervention and evaluate the extent of resection postoperatively. All patients were treated with conventional fractionation (1.8 Gy/day) and the majority received full-dose neuraxis radiation therapy and > 50 Gy to the primary site. RESULTS: With a median follow-up of 100 months, the median, 5-year, and 10-year actuarial overall survival for the entire group were 58 months, 50%, and 25%, respectively. The median, 5- and 10-year actuarial disease-free survivals were 37 months, 41%, and 27%, respectively. Patients with localized disease (no evidence of disease beyond the primary site) had significantly improved overall (p < 0.02) and disease-free (p < 0.02) survivals compared to those with nonlocalized disease. For patients with localized disease, the 5- and 10-year overall survival rates were 59% and 31%, whereas the disease-free survivals were 49% and 31%, respectively. Disease-free and overall survivals at similar intervals for patients with nonlocalized disease were 29% and 30% (5 years), and 29% and 20% (10 years), respectively. Sixty-four of 100 patients failed treatment. Local failure as any component of first failure occurred in 35% of patients or 55% (35 of 64) of all failures and as the only site of first failure in 14% or 22% (14 of 64) of all failures. For patients presenting with localized disease (n = 68), local failure as any component of first failure occurred in 32% (22 of 68) and in 18% (12 of 68) as the only site. A multivariate analysis showed that M stage was the only prognostic factor to influence overall survival. For disease-free survival, M stage and the extent of resection were prognostic factors. Ventriculoperitoneal shunting and the use of chemotherapy were associated with a poor outcome; however, these results were confounded by the positive impact of chemotherapy in decreasing the risk of extraneural metastases and the use of these therapies in the more advanced patients. CONCLUSION: These long-term follow-up data represent one of the largest series of patients with complete follow-up who were treated with a consistent radiation therapy treatment policy during the CT era. Local failure in patients with localized disease, the persistent risk of late failures, treatment-related toxicity, and the ever-present risk of secondary malignancies demonstrate the limitations of standard therapies. Strategies used to increase the total dose to the primary site should be pursued along with other adjuvant therapies such as intensive chemotherapy.

Adolescent↗
PubMed1996

Macrophage-stimulating protein induces proliferation and migration of murine keratinocytes.

Macrophage stimulating protein (MSP) is a chemotactic factor for murine peritoneal macrophages. The receptor for human MSP was recently identified as the ron gene product, a transmembrane protein tyrosine kinase cloned from a human keratinocyte cDNA library. Here we report that MSP induced proliferation of murine primary keratinocytes and established keratinocyte cell lines in a concentration-dependent manner. The growth efficacy of MSP was comparable to that of epidermal growth factor and keratinocyte growth factor. In three of four cell lines tested in a chemotaxis chamber, MSP also stimulated migration of keratinocytes on a collagen type IV substratum. The action of MSP was mediated by specific binding of MSP to the STK gene product, a murine homologue of the RON MSP receptor. Binding of MSP to keratinocyte STK induced phosphorylation of the 150 kDa STK beta chain. Herbimycin A, a protein tyrosine kinase inhibitor, blocked MSP-mediated phosphorylation of the STK receptor as well as proliferation of keratinocytes, suggesting the importance of tyrosine kinase activity for transduction of the message delivered by MSP. Previously, the only known target cell for MSP was the resident peritoneal macrophage. These studies establish the keratinocyte as a new target cell for MSP. The action of MSP on keratinocytes may have implications for tissue repair, wound healing, and tumor growth.

Animals↗
PubMed1996

Proteolytic cleavage and activation of pro-macrophage-stimulating protein by resident peritoneal macrophage membrane proteases.

Macrophage stimulating protein (MSP), which is secreted as biologically inactive pro-MSP, is activated to MSP by cleavage at a single peptide bond. Our objectives were to determine the form of MSP in circulating blood and to study proteolytic activation of pro-MSP by its target cell. Western blot of immunoaffinity-purified serum MSP showed that all the protein was pro-MSP, without detectable MSP. The circulating form of the protein is therefore pro-MSP, and conversion to MSP does not occur when blood is shed. Incubation of radiolabeled pro-MSP with murine peritoneal macrophages caused proteolytic cleavage to predominantly inactive fragments. Among several protease inhibitors, soybean trypsin inhibitor was one of two that inhibited nonspecific cleavage and revealed a macrophage proteolysis of pro-MSP, and certain concentrations enhanced cleavage to mature MSP. Macrophage membranes had nonspecific and specific pro-MSP proteolytic activity, which was not present in macrophage culture fluids. The results suggest that control of MSP activity can occur at the level of the target cell by proteolytic cleavage of pro-MSP to mature MSP or to inactive fragments.

Animals↗
PubMed1996

Epidermal growth factor modulates cell attachment to hyaluronic acid by the cell surface glycoprotein CD44.

Cell adhesion to and migration through extracellular matrices (ECM) are critical events in tumor invasion and metastasis. Previous work by us had demonstrated that signaling of epidermal growth factor receptor (EGFR) confers an oncogenic phenotype on NR6 cells and that these cells when transfected with holo EGFR demonstrate greater motility and invasiveness than cells carrying a carboxy-terminal truncated EGFR. Recently, a cell surface glycoprotein, CD44, has been implicated in cell-ECM adhesion involved in tumor cell migration, signal transduction, and metastasis. We investigated whether EGF regulates cellular interactions with ECM components, and in particular, hyaluronate, by modulating CD44 expression. In vitro cell attachment assays on hyaluronate-coated plates demonstrated similar basal level of binding (approximately 33%) for murine NR6 parental cells devoid of endogenous EGFR (P) or expressing wild-type EGFR (WT), while a time-dependent increase in binding was observed in WT cells stimulated with EGF. Additionally, utilizing monoclonal antibody blocking assays, CD44, but not EGFR, was shown to be directly involved in this attachment. Both WT and P cells possessed equivalent 95 kDa bands on immunoblots, corresponding to CD44. The existence of CD44 mRNA was verified by RT-PCR using synthetic oligonucleotides in which a 1.1 kb cDNA was detected in both cell lines and confirmed by DNA sequencing. After 24-h exposure to exogenous EGF, an increase in CD44 protein and mRNA expression was found in WT cells, but not in P cells, supporting the contention that a functional EGFR signaling pathway is required for CD44 regulation. Thus, EGF stimulates cell binding to hyaluronate in vitro by regulating CD44 expression.

Animals↗
PubMed1996

Macrophage-stimulating protein, a ligand for the RON receptor protein tyrosine kinase, suppresses myeloid progenitor cell proliferation and synergizes with vascular endothelial cell growth factor and members of the chemokine family.

Macrophage-stimulating protein (MSP), originally identified as an inducer of murine resident macrophage responsiveness to chemoattractants, is a ligand for human RON/murine STK receptor protein tyrosine kinases. Since STK was cloned from populations enriched for hematopoietic stem cells, we initiated studies on the effects of MSP on colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) myeloid progenitor cells. MSP alone had no colony stimulating activity. However, MSP caused about a 50% suppression of CFU-GM colony formation induced by synergistic combinations of SLF or Flt-L plus GM-CSF, G-CSF, or IL-3 and of BFU-E and CFU-GEMM colonies induced by SLF or Flt3-L plus Epo or Epo and IL-3. In contrast, MSP had no effect on progenitors stimulated by one growth factor. MSP also suppressed colony formation by stimulated cord blood progenitors, but only after preinduction to a rapidly cycling state. It was previously reported that several members of the chemokine family synergistically suppress myeloid progenitor proliferation. Likewise, synergistic suppression was observed when MSP was paired with VEGF, MIP-1 alpha, IL-8, PF4, MCP-1, IP-10, or ENA-78, or when VEGF was paired with the chemokines; and the required MSP concentration was more than 100-fold less than for MSP alone. Additionally, MSP or VEGF inhibited proliferation of the human myeloid growth factor-dependent cell line, M07e, but a sustained effect required multiple additions over time. At the least, some of the MSP suppressive effects on myeloid progenitors, as assessed on single isolated CD34 marrow cells, appeared to be directly on the progenitors; sustained additions of MSP were required to see this effect. The suppressive action of MSP and its synergism with proteins of the chemokine family may be of relevance to regulation of blood cell production.

Bone Marrow Cells↗
PubMed1996

Antiplatelet and calcium inhibitory properties of eugenol and sodium eugenol acetate.

1. Eugenol (3-methoxy-4-hydroxy-propenylbenzene) or sodium eugenol acetate (4-O-acetic acid sodium-3-methoxy-1-propenylbenzene) (0.25, 0.5, 1 mM) concentration-dependently inhibited arachidonic acid (AA)-, collagen-, epinephrine- and ADP-induced platelet aggregation. 2. Eugenol or sodium eugenol acetate inhibited collagen-induced aggregation of washed rabbit platelets synergistically with creatine phosphate/creatine phosphokinase (CP/CPK, 5 mM/10 U/ml) or p-bromophenacyl bromide (p-BPB, 10 microM), and they also potentiated the inhibitory action of imidazole (0.5 mM) on AA-induced aggregation. 3. Eugenol or sodium eugenol acetate (0.25, 0.5, 1 mM) concentration-dependently inhibited AA-induced thromboxane B2 and prostaglandin E2 formation. 4. The rise of intracellular Ca2+ caused by collagen, epinephrine, ADP, and AA were inhibited by eugenol or sodium eugenol acetate (1 mM).

Animals↗
PubMed1996

Inhibition of N-nitrosodimethylamine demethylase in rat and human liver microsomes by isothiocyanates and their glutathione, L-cysteine, and N-acetyl-L-cysteine conjugates.

Natural and synthetic isothiocyanates and their conjugates were examined for their inhibitory effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd) activity using a radiometric NDMAd assay. Substrate concentrations of 30 and 60 microM were used to probe the activity of cytochrome P4502E1 isozyme through the alpha-hydroxylation of NDMA. It was found that alkyl isothiocyanates such as sulforaphane and allyl isothiocyanate displayed very weak inhibition, whereas the arylalkyl isothiocyanates such as benzyl and phenethyl isothiocyanate showed significant inhibition toward rat liver NDMAd activity with IC50 values of 9.0 and 8.3 microM, respectively. More interestingly, glutathione conjugates of benzyl, phenethyl, and 6-phenylhexyl isothiocyanates all inhibited NDMAd at the comparable concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked differences in their inhibitory activity; i.e., its conjugates with L-cysteine (IC50 = 4.3 microM) and with glutathione (IC50 = 4.0 microM) are more potent than its conjugate of N-acetylcysteine (IC50 = 24.0 microM). The same trend was also observed for the human liver microsomal NDMAd activity. The half-lives of these conjugates were determined in the presence of other free thiols from L-cysteine or glutathione using an HPLC system. It was shown that isothiocyanates are released from their conjugates and react with the free thiols present in the solution. The longer half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other conjugates is consistent with its lower inhibitory activity. The inhibition of NDMAd, and therefore cytochrome P4502E1, by isothiocyanate conjugates is most likely due to the action of the free isothiocyanates released from the conjugates. Since cytochrome P4502E1 and other isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these results provide a basis for investigating the potential of isothiocyanate conjugates as chemopreventive agents.

Acetylcysteine↗
PubMed1995

Macrophage-stimulating protein activates Ras by both activation and translocation of SOS nucleotide exchange factor.

Macrophage-stimulating protein (MSP) is a chemotactic factor that activates the receptor tyrosine kinase RON. The involvement of Ras in MSP-induced signal transduction was investigated. Here we demonstrate that, in RON-transfected MDCK cells, an active GTP-bound form of Ras was rapidly accumulated by MSP treatment and the Ras-guanine nucleotide exchange activity in SOS immunoprecipitates was concomitantly increased. GAP activity was not changed under the same conditions used. Furthermore, the SH2 domain of adaptor protein GRB2, but not Shc, associated with the activated RON-beta chain, and GRB2-SOS complexes translocated from the cytosol to the membrane upon MSP treatment. These results strongly suggest that MSP activates Ras through RON, and that MSP-induced activation of Ras might be controlled by both the enhancement of catalytic exchange activity of SOS and its translocation to the membrane where its target Ras is localized.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Terminal differentiation of murine resident peritoneal macrophages is characterized by expression of the STK protein tyrosine kinase, a receptor for macrophage-stimulating protein.

STK, a new member of the hepatocyte growth factor receptor family, is the receptor for macrophage-stimulating protein (MSP), which acts on murine resident peritoneal macrophages. We established polyclonal and monoclonal antibodies against STK and characterized the structure of STK protein and STK expression on cells of the mononuclear phagocyte system. Western blotting showed that the STK transcript is translated into a single-chain precursor and then cleaved into a 165-kD disulfide-linked heterodimer composed of a 35-kD alpha-chain and a 144-kD beta-chain. Western blotting detected STK protein on resident peritoneal macrophages, a target of MSP, and showed that it was autophosphorylated in cells stimulated by MSP. By flow cytometric analysis using a monoclonal anti-STK antibody, we showed that STK protein is expressed on restricted macrophage populations such as resident peritoneal macrophages, but not on exudate peritoneal macrophages or mononuclear phagocytes of the bone marrow, peripheral blood, spleen, or alveoli. Resident peritoneal macrophages were classified into two fractions according to their reactivity with an anti-STK antibody and a marker antibody for macrophages: STKhigh-F4/80high cells and STKnegative-F4/80low cells. Acute exudative macrophages were all STKnegative-F4/80low, but they gradually became predominantly STKhigh-F4/80high several days after entrance into the peritoneal cavity. These results showed that after monocytes migrate into the peritoneal cavity, they undergo terminal differentiation in the peritoneal microenvironment. This is the first evidence of tissue-specific terminal differentiation of peritoneal macrophages, and this terminal differentiation can be characterized by the expression of STK receptor tyrosine kinase.

Animals↗
PubMed1995

The murine stk gene product, a transmembrane protein tyrosine kinase, is a receptor for macrophage-stimulating protein.

Macrophage-stimulating protein (MSP) was originally identified as an inducer of murine resident peritoneal macrophage responsiveness to chemoattractants. We recently showed that the product of RON, a protein tyrosine kinase cloned from a human keratinocyte library, is the receptor for MSP. Similarity of murine stk to RON led us to determine if the stk gene product is the murine receptor for MSP. Radiolabeled MSP could bind to NIH 3T3 cells transfected with murine stk cDNA (3T3/stk). Binding was saturable and was inhibited by unlabeled MSP but not by structurally related proteins, including hepatocyte growth factor and plasminogen. Specific binding to STK was demonstrated by cross-linking of 125I-labeled MSP to membrane proteins of 3T3/stk cells, which resulted in a protein complex with a molecular mass of 220 kDa. This radiolabeled complex comprised 125I-MSP and STK, since it could be immunoprecipitated by antibodies to the STK beta chain. Binding of MSP to stk cDNA-transfected cells induced tyrosine phosphorylation of the 150-kDa STK beta chain within 1 min and caused increased motile activity. These results establish the murine stk gene product as a specific transmembrane protein tyrosine kinase receptor for MSP. Inasmuch as the stk cDNA was cloned from a hematopoietic stem cell, our data suggest that in addition to macrophages and keratinocytes, a cell in the hematopoietic lineage may also be a target for MSP.

3T3 Cells↗
PubMed1995

Probing the active sites of rat and human cytochrome P450 2E1 with alcohols and carboxylic acids.

Cytochrome P450 2E1 (P450 2E1) catalyzes the biotransformation of many low-molecular-weight compounds including industrial solvents, indoor pollutants, alcohol, and drugs. In order to understand the nature of the P450 2E1 active site, we studied the competitive inhibition of the P450 2E1-catalyzed N-nitrosodimethylamine demethylation by alcohols and carboxylic acids with different alkyl chain lengths. Using microsomes from acetone-treated rats as the enzyme source of P450 2E1, the Ki value for each compound was measured. With primary alcohols and secondary alcohols, the Ki decreased with the increase in the carbon chain length until the carbon number reached 6 or 7; the free energy increment of binding was 0.28 kcal/mol CH2 group. Similar inhibitory effects were also observed with human P450 2E1 heterologously expressed in Hep G2 cells. These results suggest that both rat and human P450 2E1 contain a pocket with hydrophobicity that serves as a binding site for low-molecular-weight substrates. Among a series of carboxylic acids and omega-hydroxycarboxylic acids investigated, dodecanoic acid was the strongest inhibitor (Ki = 22 microM), and 12-hydroxydodecanoic acid had the lowest Ki (320 microM) within the series of omega-hydroxycarboxylic acids. The free energy increment of binding for carboxylic acids was 0.35 kcal/mol CH2 group. 1,10-Decanedicarboxylic acid, which contains a carboxylic group at each end, showed a Ki > 20 mM. We suggest that for optimal interaction of a carboxylic acid moiety with the active site of P450 2E1, the hydrocarbon end of the molecule binds to the substrate binding site, leaving the carboxylic acid group outside of a proposed substrate access channel. The length of optimal substrates such as dodecanoic acid may reflect the length of the substrate access channel and the size of the active site. We estimate that the distance from the opening of the access channel to the oxygenation site is about 15 A. Based on the structural features of P450 2E1 substrates and competitive inhibitors, we propose a conceptual model to illustrate the binding of these molecules in the active site of P450 2E1.

Alcohols↗
PubMed1995

In vitro invasiveness of DU-145 human prostate carcinoma cells is modulated by EGF receptor-mediated signals.

Prostate carcinomas often present an autocrine stimulatory loop in which the transformed cells both express the EGF receptor (EGFR) and produce activating ligands (TGF alpha and EGF forms). Up-regulated EGFR signalling has been correlated with tumor progression in other human neoplasia; however, the cell behaviour which is promoted remains undefined. To determine whether an EGFR-induced response contributes to cell invasiveness, we transduced DU-145 human prostate carcinoma cells with either a full-length (WT) or a mitogenically-active but motility-deficient truncated (c'973) EGFR. The DU-145 Parental and two transgene sublines all produced EGFR and TGF alpha, but the transduced WT and c'973 EGFR underwent autocrine downregulation to a lesser degree, with more receptor remaining intact. DU-145 cells transduced with WT EGFR transmigrated a human amniotic basement membrane matrix (Amgel) to a greater extent than did Parental DU-145 cells (175 +/- 22%). Cells expressing the c'973 EGFR invaded through the Amgel only to about two thirds the extent of the Parental cells (62 +/- 23%). A monoclonal antibody which prevents ligand-induced activation of EGFR decreased the invasiveness of WT-expressing cells by half and Parental cells by a fifth, but had little effect on the invasiveness of c'973-expressing cells; with the result that in the presence of antibody, all three cell lines transmigrated the Amgel to the same extent. The different levels of invasiveness between the three sublines were independent of cell proliferation. These findings demonstrated that EGFR-mediated signals increase tumor cell invasiveness and suggested that domains in the carboxy-terminus are required to signal invasiveness. As an initial investigation into the mechanisms underlying the EGFR-mediated enhanced invasiveness, we determined whether these cells presented different collagenolytic activity, as the major constituents of Amgel are collagen types I and IV. All three sublines secreted easily detectable levels of gelatin-directed proteases and TIMP-1, with WT cells secreting equivalent or lower levels of proteases. The proteolytic balance in these cells did not correlate with invasiveness. These data suggest that the TGF alpha-EGFR autocrine loop promotes invasiveness and that this is accomplished by signalling cell properties other than differential secretion of collagenolytic activity.

Carcinoma↗
PubMed1995

Targeted tumor killing via an intracellular antibody against erbB-2.

Specific killing of erbB-2-overexpressing tumor cells can be achieved using expression of an intracellular antibody directed against the erbB-2 oncoprotein. We have developed a strategy using a recombinant adenovirus encoding an anti-erbB-2 single chain antibody to achieve targeted tumor cell killing in vivo and can show significantly prolonged survival of animals carrying a human ovarian carcinoma tumor burden within their peritoneal cavities. This strategy of gene therapy for ovarian carcinoma offers the potential to achieve highly specific, targeted killing of human tumor cells and thus establishes the rationale to undertake human clinical trials on this basis.

Adenoviridae↗
PubMed1995

Stearate inhibits human tumor cell invasion.

Tumor growth and metastasis are affected by changes in membrane lipid composition, however, little is known regarding the role of specific fatty acids in these pathological events. We investigated the effects of the long-chain saturated fatty acids (LCSFA), myristate (C14:0), palmitate (C16:0) and stearate (C18:0) on two key steps of metastasis: cell adhesion and invasion into extracellular matrix (ECM). Using a new 72-hour ECM (Amgel) invasion assay, we demonstrated that the exposure of highly invasive human fibrosarcoma HT-1080 cells to 0.3 mM stearate inhibited their ability to traverse Amgel by 59.4 +/- 8%. In contrast, treatment of tumor cells with 0.3 mM myristate or palmitate had no effect. Microscopic examination revealed a time-dependent inhibition of tumor cell adhesion to the Amgel in the stearate-treated group. Cell adhesion assays further showed a series of rapid morphological cellular changes, i.e. retraction of processes, cell rounding, and subsequent detachment in the presence of stearate. These morphological events were both dose- and time-dependent. Viability of LCSFA-treated cells exceeded 80%. This stearate inhibition of HT-1080 cell adhesion was also observed with two other invasive human tumor cell lines. Similar treatment of HT-1080 cell with the unsaturated long-chain fatty acid oleate (C18:1) did not alter tumor cell adhesiveness. In contrast, nontransformed human fibroblasts (Hs-68) were unaffected by stearate treatment. This inhibition of cell adhesion by stearate was determined to be dependent upon laminin-containing ECM. Pretreatment of HT-1080 cells with stearate dramatically abolished their capacity to attach to laminin but not to collagen type IV or fibronectin matrices. Immunofluorescent studies with anti-beta 1 integrin receptor and antivinculin antibodies demonstrated beta 1 subunit and vinculin colocalization to focal adhesions in untreated HT-1080 cells adherent to laminin, in contrast to stearate-treated tumor cells. Further, stearate-induced changes were shown to be functionally coupled to integrins as an anti-beta 1 antibody markedly diminishes the adhesive ability of tumor cells to laminin. These data demonstrate stearate inhibits tumor cell adhesion, and therefore invasion, via a mechanism involving a laminin integrin receptor.

Cell Adhesion↗
PubMed1994

Identification of the ron gene product as the receptor for the human macrophage stimulating protein.

Macrophage-stimulating protein (MSP) is a member of the hepatocyte growth factor-scatter factor (HGF-SF) family. Labeled MSP bound to Madin-Darby canine kidney (MDCK) cells transfected with complementary DNA encoding Ron, a cell membrane protein tyrosine kinase. Cross-linking of 125I-labeled MSP to transfected cells (MDCK-RE7 cells) and immunoprecipitation by antibodies to Ron revealed a 220-kilodalton complex, a size consistent with that of MSP (80 kilodaltons) cross-linked to the beta chain of Ron (150 kilodaltons). The binding of 125I-labeled MSP to MDCK-RE7 cells was inhibited by unlabeled MSP, but not by HGF-SF. MSP caused phosphorylation of the beta chain of Ron and induced migration of MDCK-RE7 cells. These results establish the ron gene product as a specific cell-surface receptor for MSP.

Animals↗