guanosine-5--o-(3-thiotriphosphate) and Melanoma

Uveal melanoma is the most common intraocular tumor in adults and often metastasizes to the liver, leaving patients with few options. Recurrent activating mutations in the G proteins, Gα

Topics: Animals; Cell Growth Processes; Cell Line, Tumor; Depsipeptides; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Insecta; MAP Kinase Signaling System; Melanoma; Rats; Recombinant Proteins; Uveal Neoplasms

We investigated the effects of morphine and other agonists on the human mu opioid receptor (MOP) expressed in M2 melanoma cells, lacking the actin cytoskeleton protein filamin A and in A7, a subclone of the M2 melanoma cells, stably transfected with filamin A cDNA. The results of binding experiments showed that after chronic morphine treatment (24 h) of A7 cells, MOP-binding sites were down-regulated to 63% of control, whereas, unexpectedly, in M2 cells, MOP binding was up-regulated to 188% of control naive cells. Similar up-regulation was observed with the agonists methadone and levorphanol. The presence of antagonists (naloxone or CTAP) during chronic morphine treatment inhibited MOP down-regulation in A7 cells. In contrast, morphine-induced up-regulation of MOP in M2 cells was further increased by these antagonists. Chronic morphine desensitized MOP in A7 cells, i.e., it decreased DAMGO-induced stimulation of GTPgammaS binding. In M2 cells DAMGO stimulation of GTPgammaS binding was significantly greater than in A7 cells and was not desensitized by chronic morphine. Pertussis toxin treatment abolished morphine-induced receptor up-regulation in M2 cells, whereas it had no effect on morphine-induced down-regulation in A7 cells. These results indicate that, in the absence of filamin A, chronic treatment with morphine, methadone or levorphanol leads to up-regulation of MOP, to our knowledge, the first instance of opioid receptor up-regulation by agonists in cell culture.

Topics: Blotting, Western; Cell Line; Cell Line, Tumor; Contractile Proteins; Data Interpretation, Statistical; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Filamins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Melanoma; Microfilament Proteins; Morphine; Narcotic Antagonists; Narcotics; Pertussis Toxin; Radioligand Assay; Receptors, Opioid, mu; Tubulin; Up-Regulation

Phospholipase D (PLD) is a highly regulated enzyme involved in lipid-mediated signal transduction processes affecting vesicular trafficking and cytoskeletal reorganization. It is regulated by protein kinase C, adenosine diphosphate (ADP)-ribosylation factors and Rho family proteins, and both protein kinase C and Rho family proteins have been implicated in the metastatic potential of melanoma. We analysed PLD in four human melanoma cell lines and in primary human melanocytes. Melanoma cell lines showed phosphatidylcholine-hydrolysing, phosphatidylinositol 4,5-bisphosphate-dependent PLD activity, which was activated by phorbol ester and a non-hydrolysable guanosine triphosphate (GTP) analogue in a dose-dependent and synergistic manner, whereas primary melanocytes exhibited only low PLD activity compared with the melanoma cell lines. As determined by reverse transcription polymerase chain reaction, both splicing variants of PLD1, PLD1a and PLD1b, and the isoenzyme PLD2, are expressed in melanoma cells and melanocytes. Western blot analysis showed that PLD1 expression was low in primary melanocytes in contrast to melanoma cells, which is in agreement with our finding of low activity. Interestingly, Rho protein mRNA was elevated in all melanoma cell lines. We conclude that in human melanoma cells, the PLD activity that is stimulated by phorbol ester requires ADP-ribosylation factor, protein kinase C and Rho proteins for full activity, and most probably represents the isoenzyme PLD1.

Topics: Adenosine Diphosphate; ADP-Ribosylation Factors; Alternative Splicing; Blotting, Western; Cell Division; Cell Line, Tumor; Cells, Cultured; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Activation; Gene Expression Regulation, Enzymologic; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Immunoblotting; Melanocytes; Melanoma; Neoplasm Metastasis; Oleic Acid; Phorbol Esters; Phospholipase D; Protein Isoforms; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA; RNA, Messenger

Using cDNA expression cloning, a cDNA encoding a novel human melanoma Ag, MART-2 (melanoma Ag recognized by T cells-2), recognized by HLA-A1-restricted CD8(+) T cells from tumor-infiltrating lymphocytes (TIL1362) was isolated from an autologous melanoma cell line, 1362 mel. Homologous sequences to the cDNA had been registered in the EST database. This gene encoded an uncharacterized protein expressed ubiquitously in most normal and cancer cells. A mutation (A to G transition) was found in the cDNA obtained from the1362 mel melanoma cell line in the sequences encoding the phosphate binding loop (P-loop) that resulted in loss of the ability to bind GTP. Transfection of NIH-3T3 with the mutated MART-2 did not result in the development of significant foci. By screening 36 various cancer cell lines using single-strand conformation polymorphism, a possible mutation in the P-loop of MART-2 was found in one squamous cell lung cancer cell line, EBC1. The T cell epitope for TIL1362, FLEGNEVGKTY, was identified to be encoded by the mutated sequence of the MART-2 Ag. The mutation substituted glycine in the normal peptide with glutamic acid at the third amino acid of the epitope, which is an important primary anchor amino acid for HLA-A1 peptide binding. The normal peptide, FLGGNEVGKTY, was not recognized by TIL1362, suggesting that this T cell response was specific for the autologous tumor. Although transforming activity was not detected in the NIH-3T3 assay, MART-2 with the mutation in the P-loop may be involved in the generation of melanoma through a loss of GTP binding activity.

Topics: 3T3 Cells; Adult; Amino Acid Sequence; Animals; Antigens, Neoplasm; Base Sequence; COS Cells; DNA, Complementary; Epitopes, T-Lymphocyte; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); HLA-A1 Antigen; Humans; Lymphocytes, Tumor-Infiltrating; Melanoma; Mice; Molecular Sequence Data; Mutation; Neoplasm Proteins; Protein Structure, Secondary; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured

The class II IL-8 receptor (IL-8R) binds both melanoma growth stimulatory activity (MGSA) and IL-8 with high affinity. Reverse transcriptase polymerase chain reaction demonstrates that the class II IL-8R mRNA, which has previously been detected only in cells of hematopoietic lineage, is also expressed in non-hematopoietic cell types shown to respond to MGSA or IL-8. To study the signaling mechanism by MGSA through the class II IL-8R in non-hematopoietic cells, this receptor was overexpressed in the 3ASubE human placental and the 293 human kidney cell lines. Membrane preparations of the class II IL-8R expressing 3ASubE transfectants exhibited a 2.3 +/- 0.2-fold increase in GTP gamma 35S binding, which was sensitive to pertussis toxin, in response to MGSA treatment (0.2 microM). This MGSA response was not observed in cells transfected with the parental expression vector. In vivo phosphorylation studies demonstrated that the class II IL-8R was basally phosphorylated in the untreated transfectants, and MGSA (5 nM) treatment markedly enhanced the phosphorylation of this receptor. The MGSA-induced receptor phosphorylation was both time and concentration dependent and could be mimicked by treatment with the calcium ionophore A23187. Phosphoamino acid analysis indicated that the MGSA-induced receptor phosphorylation was on serine residue(s), suggesting that a serine kinase is activated in response to MGSA binding to the class II IL-8R in non-hematopoietic cells.

Topics: Base Sequence; Cell Line; Cell Membrane; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Cloning, Molecular; DNA Primers; Female; Growth Substances; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Keratinocytes; Kidney; Kinetics; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Open Reading Frames; Phosphorylation; Placenta; Polymerase Chain Reaction; Pregnancy; Receptors, Interleukin; Receptors, Interleukin-8A; RNA, Messenger; Transfection; Tumor Cells, Cultured