Biomedical subjects

M Park

Publications and source records attributed to M Park.

At least 163 records · Page 9Linked to original sources

PubMed1995

Bombesin stimulates c-fos and c-jun mRNAs in small cell lung cancer cells.

The effects of bombesin/gastrin-releasing peptide (BN/GRP) on c-fos and c-jun gene expression were investigated using small cell lung cancer (SCLC) cells. BN (10 nM) increased c-fos mRNA fivefold using NCI-H345 or NCI-H510 cells. The increase was concentration dependent with 1 nM BN half-maximally increasing c-fos mRNA. Also, the increase in c-fos mRNA caused by BN was time dependent, being maximal after 1 h and returning to basal values after 4 h. GRP and GRP(14-27) but not GRP(1-16) increased c-fos mRNA. BW2258U89 (1 microM), a GRP receptor antagonist, had no effect on basal c-fos but inhibited the increase in c-fos mRNA caused by 10 nM BN. Also, BN transiently increased c-jun mRNA twofold and the increase caused by BN was blocked by BW2258U89. These data suggest that GRP receptors may regulate nuclear oncogene gene expression in SCLC cells.

Bombesin↗

PubMed1995

Spontaneous release of interleukin-6 by primary cultures of lymphoid and tumor cell populations purified from human ovarian carcinoma.

Interleukin-6 (IL-6) is a cytokine that has been implicated as a growth factor in human ovarian carcinoma, yet the in vivo source of IL-6 in patients remains undefined. We measured IL-6 by ELISA in cell-free ascites (CFA) of 19 patients with ovarian carcinoma. IL-6 was detectable in all samples (mean level 3.3 ng/ml). To identify the cellular source of IL-6, we measured this cytokine by ELISA in 24-48 h supernatants of cultured lymphocyte-, macrophage-, and tumor cell-enriched populations purified from three solid ovarian carcinomas by centrifugal elutriation. All cell populations spontaneously released IL-6; however, tumor cells and tumor-associated macrophage released levels of IL-6 that greatly exceeded those released by tumor-associated lymphocytes. Kinetic studies revealed that IL-6 was detectable at 6 h and that levels increased in all cultures examined over a 48 h time course. These data suggest that both tumor and infiltrating host cells may be the source of the high levels of IL-6 found in carcinomatous ascites. Furthermore, although all three cell types examined may contribute to IL-6 production in patients with ovarian carcinoma, tumor cells are perhaps the most clinically significant source.

Adenocarcinoma↗

PubMed1995

Expression of the hepatocyte growth factor/scatter factor receptor tyrosine kinase is localized to epithelia in the adult mouse.

The met proto-oncogene receptor tyrosine kinase has been identified as a receptor for hepatocyte growth factor (HGF)/scatter factor (SF). HGF/SF is a multifunctional cytokine that stimulates mitogenesis, dissociation, and motility of a broad spectrum of epithelial and endothelial cells in culture, promotes the progression of carcinoma cells to a more invasive phenotype, and acts as a morphogenic factor for tubular epithelia. HGF/SF is predominantly expressed by mesenchymal cells, whereas the met/HGF/SFR is predominantly expressed by epithelial and carcinoma cells in culture. We have shown by Northern analyses that the met/HGF/SFR is expressed in many adult mouse tissues. To elucidate the normal physiologic role for the met/HGF/SFR and the possible pathologic consequences of deregulation of this pathway, we have examined the expression of the met/HGF/SFR in adult mouse tissue by in situ hybridization. We show that the met/HGF/SFR is generally expressed in epithelia, including hepatocytes, epithelial cells that line the proximal and distal convoluted tubules of the kidney, epithelia of stomach, esophagus, uterus, lung and skin, as well as in granulosa cells of developing and mature oocytes. By reverse transcriptase PCR amplification, we show that the HGF/SF gene is expressed at low levels in many of these tissues. Our data support a possible role for the met/HGF/SFR in epithelial cell growth and tissue organization.

Animals↗

PubMed1994

Three forms of gonadotropin-releasing hormone characterized from brains of one species.

Most vertebrate species have more than one form of gonadotropin-releasing hormone (GnRH) in their brains, but it is not clear whether each form has a distinct function. We report that sea bream (Sparus aurata) brains have three forms of GnRH, one of which is described herein and is called sea bream GnRH (sbGnRH). The primary structures of two forms were determined by Edman degradation and mass spectral analysis. The amino acid sequence of sbGnRH is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Ser-Pro-Gly-NH2. The second peptide is identical to a form originally isolated from chicken brains (cGnRH-II): pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2. cGnRH-II is the most ancient form of GnRH identified to date in jawed fish and the most prevalent form throughout the vertebrates. The third form of GnRH has previously been identified as salmon GnRH by cDNA studies and is confirmed here by chromatographic and immunological studies. Phylogenetic distribution of GnRH peptides suggests sbGnRH arose in the perch-like fish as a gene duplication of the existing cGnRH-II or salmon GnRH genes. All three identified GnRH peptides were synthesized and shown to release gonadotropin in vivo in the sea bream. The dominant form of GnRH stored in the pituitary was sbGnRH. Not only was the content of sbGnRH 500-fold greater than that of salmon GnRH but also cGnRH-II was not detected in the pituitary. The latter evidence suggests that sbGnRH is the endogenous releaser of gonadotropin II.

Amino Acid Sequence↗

PubMed1994

Tyrosine 1356 in the carboxyl-terminal tail of the HGF/SF receptor is essential for the transduction of signals for cell motility and morphogenesis.

The met proto-oncogene is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF is a multifunctional cytokine that stimulates mitogenesis, motility, invasion, and tubulogenesis of a spectrum of epithelial and endothelial cells in culture. Using a chimeric receptor (CSF-MET), containing the extracellular domain of the colony stimulating factor-1 (CSF-1) receptor fused to the transmembrane and intracellular domain of the Met receptor, we have previously demonstrated that activation of the Met kinase domain is sufficient to mediate the motility, invasion and morphogenic signals of HGF/SF in Madin-Darby canine kidney epithelial cells (MDCK). In this study we have analyzed the role of tyrosine phosphorylation of the Met receptor in the transmission of these signals by site-directed mutagenesis of specific tyrosine residues. Mutation of two tyrosine residues (tyrosine 1234 and tyrosine 1235), involved in activation of the catalytic activity of the kinase, abrogates the biological activity of the chimera. In addition, we have identified a single noncatalytic tyrosine residue (tyrosine 1356) in the carboxyl terminus of the Met receptor, that is essential for the biological activity of the chimeric receptor. Mutation of tyrosine 1356 to a nonphosphorylatable phenylalanine residue does not affect the exogenous kinase activity of the receptor toward enolase, but it impairs the ability of the mutant protein to associate with the adaptor protein Grb2, and MDCK cells expressing this mutant fail to scatter, invade, and form branching tubules in response to CSF-1. These results support a crucial role for tyrosine 1356 in activation of signaling pathways involved in the biological activity of the Met receptor in response to HGF/SF.

Animals↗

PubMed1994

Purification of a novel 43-kDa protein (p43) intermediary in the activation of steroidogenesis from rat adrenal gland.

In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five-step procedure that includes DEAE-cellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains the capacity to stimulate steroid synthesis in an in vitro recombination assay. This activity is inhibited by the use of phospholipase A2 inhibitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDS/PAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearance of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N-terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein. We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin-induced stimulation of arachidonic acid release and steroid synthesis.

Adrenal Cortex↗

PubMed1994

Catalysis and energy coupling of H(+)-ATPase (ATP synthase): molecular biological approaches.

The molecular biological approach has provided important information for understanding the F0F1 H(+)-ATPase. This article focuses on our recent results on the catalytic site in the beta subunit, and the roles of alpha/beta subunit interaction and amino/carboxyl terminal interaction of the gamma subunit in energy coupling. Extensive mutagenesis of the beta subunit revealed that beta Lys-155, beta Thr-156, beta Glu-181 and beta Arg-182 are essential catalytic residues. beta Glu-185 is not absolutely essential, but a carboxyl residue may be necessary at this position. A pseudo-revertant analysis positioned beta Gly-172, beta Ser-174, beta Glu-192 and beta Val-198 in the proximity of beta Gly-149. The finding of the roles of beta Gly-149, beta Lys-155, and beta Thr-156 emphasized the importance of the glycine-rich sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser, E. coli beta residues between beta Gly-149 and beta Thr-156) conserved in many nucleotide binding proteins. The A subunits of vacuolar type ATPases may have a similar catalytic mechanism because they have conserved glycine-rich and Gly-Glu-Arg (corresponding to beta Gly-180-beta Arg-182) sequences. The results of these mutational studies are consistent with the labeling of beta Lys-155 and beta Lys-201 with AP3-PL, and of beta Glu-192 with DCCD [15]. The DCCD-binding residue of a thermophilic Bacillus corresponds to beta Glu-181, an essential catalytic residue discussed above. The defective coupling of the beta Ser-174-->Phe mutant was suppressed by the second mutation alpha Arg-296-->Cys, indicating the importance of alpha/beta interaction in energy coupling. The gamma subunit, especially its amino/carboxyl interaction, seems to be essential for energy coupling between catalysis and transport judging from studies on gamma Met-23-->Lys or Arg mutation and second-site mutations which suppressed the gamma Lys-23 mutation. Thus the conserved gamma Met-23 is not absolutely essential but is located in the important region for amino/carboxyl interaction for energy coupling.

Amino Acid Sequence↗

PubMed1994

Monomeric activin A retains high receptor binding affinity but exhibits low biological activity.

Activins are multipotent hormones/growth factors that belong to the transforming growth factor-beta (TGF-beta) superfamily. Like TGF-beta s, activins have 9 conserved cysteine residues and are disulfide-bonded dimers. Based on the three-dimensional structure of TGF-beta 2, we deduced Cys80 in activin A to form the intermolecular disulfide bond. To obtain a monomeric form of activin, Cys80 was exchanged for a serine residue by polymerase chain reaction mutagenesis. The mutant protein was expressed in a baculovirus/insect cell expression system. The molecular mass of this mutant activin was determined to be 13 kDa (consistent with a single chain form of the protein) by SDS-polyacrylamide gel electrophoresis and by laser desorption mass spectroscopy. When this mutant monomeric activin was incubated with cells that expressed either the activin type IIB receptor or both the type I and type IIB receptors, its affinity was found to be 20% of that of native activin on a mass basis. Binding affinity determined using the mouse pituitary cell line AtT 20 was 10% of that of native activin A. Biological potency, however, as determined by the mutant protein's ability to release FSH from anterior pituitary cells in primary culture and by its ability to suppress basal ACTH secretion form AtT 20 cells, was only 1% of that of the native protein. This discrepancy of an order of magnitude between binding and biological activity is consistent with a model in which dimerization of the hormone is not necessary for high affinity binding to its receptor(s) while being essential for efficient signal transduction.

Activin Receptors↗

PubMed1994

Role of scatter factor in the pathogenesis of AIDS-related Kaposi sarcoma.

Kaposi sarcoma (KS) is a complex multicellular neoplasm that is commonly associated with AIDS. The pathogenesis of KS is not well understood. KS tumor cells grow poorly in vitro and require medium conditioned by retrovirus-infected T lymphocytes. We observed that conditioned medium (CM) from type II human T-cell leukemia virus (HTLV-II)-infected T cells (HTLV-II CM) induces conversion of endothelial cells (ECs) to a KS tumor cell-like phenotype. ECs grown in HTLV-II CM acquired a spindle-shaped morphology, the ability to express factor XIIIa and other KS cell markers, and a cytokine production profile similar to that of KS cells. We found that HTLV-II CM contains large quantities of scatter factor (SF), an angiogenic cytokine that stimulates cell motility. SF induced ECs to become spindle-shaped and express factor XIIIa. Moreover, SF was found to be a mitogen for KS cells in vitro and was identified within KS lesions in vivo. SF mRNA was present in KS cells in vitro, and antibodies against SF inhibited the growth of KS cells. The receptor for SF, the c-met protein, was expressed by ECs, dermal dendrocytes, and KS tumor cells in vitro and in vivo. HTLV-II CM was highly angiogenic in vivo, which was blocked by antibodies against SF. Based on these findings, we suggest that SF plays a role in the initiation and maintenance of KS lesions.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Assignment of disulfide bonds in corticotropin-releasing factor-binding protein.

We have previously isolated, cloned, and characterized a protein that specifically binds and inactivates the peptide corticotropin-releasing factor. The integrity of the disulfide bonds in the binding protein is essential for this activity as reduction abolishes the protein's ability to bind corticotropin-releasing factor. The disulfide arrangement of the 10 cysteines present in the mature protein was established by analysis of proteolytically cleaved protein and sequence analysis of cystine containing fragments. A pattern is observed where each cysteine is connected to the next one in a sequential manner. Inspection of the genomic DNA encoding for this protein reveals that four of the domains defined by disulfide linkage coincide with four different exons.

Amino Acid Sequence↗

PubMed1994

Involvement of hepatocyte growth factor in kidney development.

Using PCR, hepatocyte growth factor (HGF) and c-met (HGF receptor) expression were analyzed in the developing mouse kidney. Both HGF and c-met were expressed from Gestational Day 11.5 onward, the time at which branching morphogenesis of ureteric bud occurs. Coculturing the embryonic kidney with MDCK cells seeded in Type I collagen induced branching morphogenesis of the MDCK cells. When a monospecific antiserum against HGF was included in the culture medium, the branching morphogenesis induced by the embryonic kidney was markedly inhibited (73%). This anti-HGF serum also inhibited metanephric growth when incubated with 11.5- to 12.5-day embryonic kidneys in an organ culture assay. No inhibition was observed by nonimmune serum. Perturbation of ureteric duct development as well as extraductal metanephric development was observed in embryonic kidneys incubated with the anti-HGF serum. Together, our data indicates an important role for HGF in kidney development.

Animals↗

PubMed1994

NADH and NAD modulates Ca(2+)-activated K+ channels in small pulmonary arterial smooth muscle cells of the rabbit.

We have investigated the effect of NADH and NAD on the gating of large conductance Ca(2+)-activated K(KCa) channels in arterial smooth muscle cells isolated from small pulmonary artery (outer diameter < 300 microns) and ear artery, using the patch clamp technique. In the inside-out configuration, intracellularly applied 2 mM NADH inhibited the activity of KCa channels in pulmonary arterial smooth muscle cells, while it had no significant effect on ear arterial smooth muscle cells. On the other hand, 2 mM NAD increased the opening of KCa channels in pulmonary arterial smooth muscle cells. The effects of another intracellular redox couple, glutathione(GSH) and glutathione disulfide(GSSG) were also dependent on their redox potentials. GSH(5 mM) inhibited KCa channels activity, while GSSG(5 mM) increased the activity of pulmonary arterial smooth muscle cells. It could be concluded that the modulation of KCa channels by intracellular redox state contributes, at least in part, to the hypoxic suppression of outward current in pulmonary arterial smooth muscle cells.

Animals↗

PubMed1994

Breast carcinoma: a collective disorder.

The development and differentiation of the epithelial component of glandular tissues such as the breast is regulated by two apparently unrelated processes. One of these is presumed to be epithelial cell collective autonomous, that is, it is mediated by gene products which act directly on the epithelial cells. An important component of autonomous regulation is the functional expression of homotypic cell-cell adhesion molecules such as cadherins. The second process is non-autonomous and involves an inductive effect of the neighboring mesenchymal cell collective. An important component of non-autonomous regulation is the aggregation/condensation of mesenchyme closely associated with the epithelium. We propose that molecular alterations in autonomous and non-autonomous pathways are important causes and indicators respectively of breast cancer progression and that these two fundamental regulators of epithelial collective organization are in fact inter-dependent. For example, we show that the expression of hepatocyte growth factor (HGF), an epithelially targeted mesenchymally derived morphogenic factor is regulated by mesenchymal cell density (condensation) and by factors released from epithelial cells. Breast epithelial cells produce factors which inhibit and stimulate HGF expression. The inhibitory factor is transforming growth factor beta (TGF-beta) and the activation state of TGF-beta is a crucial element in HGF homeostasis. The balance of negative and positive HGF regulators is markedly affected by the growth conditions and differentiation state of the epithelial cells. The expression of the HGF receptor, met, is high in normal breast epithelial cells and in dedifferentiated (ER negative) tumor cells but is reduced or lost in ER positive well differentiated epithelial cells. Our results indicate that the expression of at least one epithelial morphogen, HGF, is inter-dependently regulated by mesenchymal condensation and by factors released by neighboring epithelial cells.

Breast Neoplasms↗

PubMed1994

Two tightly-linked Drosophila male accessory gland transcripts with the same developmental expression derive from independent transcription units.

Acp26Aa and Acp26Ab are Drosophila male accessory gland transcripts that are tightly linked and transcribed from the same DNA strand. Despite their being separated by 20 base pairs, the transcripts show identical responses to several developmental signals. These observations make it important to determine whether the 26A region contains two separable genes with the same developmental expression or a single developmentally regulated transcription unit whose product is processed to yield Acp26Aa and Acp26Ab. We show that Acp26Aa and Acp26Ab are separate mRNAs using a reverse transcription-polymerase chain reaction assay and reporter gene fusions. We also show that the regulatory elements for Acp26Ab lie within a fragment containing the intergenic region and transcribed sequences of Acp26Aa and Acp26Ab.

Animals↗

PubMed1994

Oncogenic activation of tyrosine kinases.

Tyrosine kinases comprise the largest group of oncoproteins, a fact that underscores the importance of reversible tyrosine phosphorylation in the regulation of essential cellular functions. Oncogenic activation of tyrosine kinases results in the constitutive activation of what is normally a conditionally regulated enzyme activity. Studies of tyrosine kinase oncoproteins, and a comparison with their corresponding proto-oncogene products, have identified important functional and regulatory domains within these proteins, positive and negative regulators of their enzyme activities and signalling cascades that control cell growth and differentiation.

Animals↗

PubMed1994

Role of hepatocyte growth factor in breast cancer: a novel mitogenic factor secreted by adipocytes.

Stromal cells can dramatically affect the growth and metastatic capability of breast carcinoma cells. Growth factors, considered to be important mediators of this process, act as either mitogenic or mito-inhibitory regulators. We have developed an in vitro coculture system to examine the influence of adipocytes, a dominant mammary stromal cell type, on the growth of a murine mammary carcinoma, SP1. Previously, we have reported that conditioned medium (CM) from 3T3-L1 adipocytes can promote in vitro growth of SP1 cells. We now show that the major mitogenic signal derived from 3T3-L1 adipocyte CM is mediated by hepatocyte growth factor (HGF). Neutralizing antibody against HGF at 15 micrograms/ml completely abrogated mitogenic activity of 3T3-L1 CM. Furthermore, heparin, an inhibitor of biological activity of HGF, inhibited the mitogenic activity of 3T3-L1 CM. Western blot analysis also confirmed the presence of HGF in 3T3-L1 CM. Although basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I) were mitogenic for SP1 cells, neutralizing antibodies against IGF-I, bFGF, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) did not inhibit the mitogenic activity of 3T3-L1 CM. Immunoprecipitation and immunoblotting of HGF receptor/c-met showed that c-met is expressed at high level in SP1 cells, and is phosphorylated following HGF ligation. Together, our present data demonstrate that 3T3-L1 adipocytes secrete HGF, which stimulates SP1 cell growth by a paracrine mechanism. Furthermore, the mitogenic effect of 3T3-L1 CM requires HGF receptor ligation and activation of tyrosine kinase signaling cascades in SP1 cells. These results highlight the importance of stromal-tumor cell interactions and suggest that HGF secreted by adipocytes may be a key regulator of mammary tumor growth.

3T3 Cells↗

PubMed1994

Receptor chimeras indicate that the met tyrosine kinase mediates the motility and morphogenic responses of hepatocyte growth/scatter factor.

The met protooncogene is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF is a multifunctional cytokine secreted mainly by mesenchymal cells that stimulates movement, invasion, and morphogenesis of some epithelial and endothelial cells and mitogenicity of others. Although the met receptor tyrosine kinase is a high affinity receptor for HGF/SF, it is not known whether this receptor can mediate the pleiotropic functions of HGF/SF. To investigate this in epithelial cells that normally respond to HGF/SF, we generated a chimeric receptor containing the extracellular domain from the colony stimulating factor 1 (CSF-1) receptor fused to the transmembrane and cytoplasmic domain of the met receptor. We show that the CSF-MET chimera, when expressed in Madin-Darby canine kidney (MDCK) epithelial cells, is fully functional. Treatment of MDCK cells expressing the chimera with CSF-1 leads to cell dissociation and scattering, as well as invasion and tubule formation of cells grown in collagen matrices. This effect is dependent on a functional met kinase. Stimulation of the receptor chimera with CSF-1 leads to activation of the met kinase and tyrosine phosphorylation of the chimeras in vivo, whereas a kinase inactive mutant chimera shows no biological response to CSF-1. These findings demonstrate that stimulation of the met kinase is sufficient and essential to mediate the motogenic, invasive, and morphogenic responses of MDCK cells to HGF/SF and that this is a suitable system for a detailed analysis of the molecular signaling events involved in these responses.

Amino Acid Sequence↗

PubMed1994

Autophosphorylation modulates the kinase activity and oncogenic potential of the Met receptor tyrosine kinase.

The met oncogene is activated by a genomic rearrangement that generates a hybrid protein containing tpr sequences at its amino terminus fused directly to the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase domain. The resultant p65Tpr-Met hybrid protein possesses tyrosine kinase activity and is constitutively phosphorylated on tyrosine in vivo. A substitution of the conserved lysine in the catalytic domain abolished kinase activity and the transforming potential of Tpr-Met demonstrating the requirement for kinase activity for transformation. To study the role of autophosphorylation at particular tyrosine residues on the transforming activity of Tpr-Met, the major autophosphorylation sites were identified by two dimensional phosphopeptide mapping. Two tyrosine residues in the catalytic domain, tyrosine 365 and tyrosine 366, were found to be the major autophosphorylation sites both in vitro and in vivo. Mutation of these sites singly or together modulates the biochemical and biological properties of Tpr-Met. Compared with the wild-type Tpr-Met, mutants have a reduced in vitro kinase activity when measured by levels of autophosphorylation and by their ability to phosphorylate an exogenous substrate and have little or no transforming activity. These results indicate that autophosphorylation at critical tyrosine residues is required for activation of the kinase activity of Tpr-Met and for cell transformation by this oncogene.

3T3 Cells↗