L-dopa binding sites in rodent melanoma cells.

Rapid, saturable, specific and stereoselective binding of L-dopa to crude membranes and purified nuclei from rodent amelanotic melanoma cells is reported. Cross-linking of [3H]dopa to melanoma cell surface emphasized proteins of approx. 55, 30, 25 and less than 20 kDa. It is suggested that these binding sites may regulate melanocyte activity.     

Calmodulin inhibits the phosphorylation of spectrin in vitro

In vitro phosphorylation of purified spectrin dimer was studied in the presence of Ca2+-calmodulin (CaM). CaM inhibited autophosphorylation of the beta subunit of spectrin. The inhibitory effect (65% at a 32-fold molar excess) appeared to be due to a weak interaction of CaM with spectrin. CaM was similarly effective in a phosphatase-stimulated autothiophosphorylation of the beta subunit with [gamma-35S]ATP. Hence, its inhibitory effect was not due to stimulation of a spectrin-associated phosphatase activity. Phosphorylation of spectrin by the catalytic subunit of a cAMP-dependent protein kinase occurred in both subunits (1984, FEBS Lett. 169, 323). CaM selectively inhibited a cAMP-dependent phosphorylation of the alpha subunit of spectrin to 30% at two CaM per spectrin. It was ineffective on the cAMP-dependent phosphorylation of the beta subunit up to a 32-fold molar excess. These results yield functional evidence for a CaM-spectrin interaction. They further suggest that CaM can regulate the extent of a cAMP-dependent phosphorylation of the alpha subunit of spectrin.     

Neuronal tyrosine phosphorylation in growth cone glycoproteins

Neuronal glycoproteins derived from growth cones are enriched in protein tyrosine kinase activity. In contrast, glycoproteins obtained from mature synaptosomes are relatively lacking in this activity. At least three types of protein tyrosine kinases were detected in association with growth cone glycoproteins: 1) a Mn2(+)-dependent activity which phosphorylates several endogenous substrates; 2) a Mn2+/Mg2(+)-dependent activity which phosphorylates synthetic substrate and is stimulated by acidic brain extracts; and 3) protein tyrosine kinases corresponding to the insulin and insulin-like growth factor I receptors. These enzymes may be particularly important during earlier stages of neuronal maturation.     

Echistatin inhibits pp125FAK autophosphorylation, paxillin phosphorylation and pp125FAK-paxillin interaction in fibronectin-adherent melanoma cells.

Echistatin, a snake-venom RGD-containing protein, was previously shown to disrupt cell-matrix adhesion by a mechanism that involves the reduction of pp125FAK tyrosine phosphorylation levels. The aim of this study was to establish the sequence of events downstream pp125FAK dephosphorylation that could be responsible for echistatin-induced disassembly of actin cytoskeleton and focal adhesions in fibronectin-adherent B16-BL6 melanoma cells. The results obtained show that echistatin induces a decrease of both autophosphorylation and kinase activity of pp125FAK. One hour of cell exposure to echistatin caused a 39% decrease of pp125FAK Tyr397 phosphorylation and a 31% reduction of pp125FAK autophosphorylation activity as measured by immune-complex kinase assay. Furthermore, 1 h of cell treatment by echistatin produced a 63% decrease of paxillin phosphorylation, as well as a reduction in the amount of paxillin bound to pp125FAK. Immunofluorescence analysis of echistatin treated cells showed the concomitant disappearance of both paxillin and pp125FAK from focal adhesions. The reduction of paxillin phosphorylation may represent a critical step in the pathway by which disintegrins exert their biological activity, including the inhibition of experimental metastasis in vivo.     

Molecular mechanism of tyrosinase regulation by L-dopa in hamster melanoma cells

Exposure of hamster amelanotic melanoma cells to L-dihydroxyphenylalanine (L-DOPA) resulted in a time dependent increase of cell pigmentation, tyrosinase concentration and activity with peak after 24 hours. Northern blot analysis showed a small but reproducible increase of tyrosinase mRNA after 3 hours and a decrease below the control level after 9 hours. After 24 and 48 hours tyrosinase mRNA was undetectable. It is suggested that L-DOPA or its oxidation products can stimulate intracellular tyrosinase concentration and regulate tyrosinase mRNA level both in positive and negative fashion.     

Protein phosphorylation in irradiated human melanoma cells

In the present study, we examined the response of confluent, primary human fibroblasts and cells of a melanoma (YUSAC2) cell line to ionizing radiation mediated through post-translational protein phosphorylation. Since the purpose of our study was to identify novel radiation-induced phosphoproteins in the DNA damage stress response of melanoma cells, we were primarily interested in changes in protein phosphoserine expression at early times after irradiation. Our rationale was that by examining the overall protein phosphorylation profile (the phosphoproteome) in irradiated cells, we might discover novel radiation-induced phosphoproteins that distinguish fibroblasts from melanoma cells. Cell proteins were separated by gel electrophoresis and phosphoproteins were identified by Western blot analysis using nonspecific anti-phosphoamino acid antibodies. This approach was not pursued previously since adequate antibodies for examining global protein phosphoserine expression were unavailable. While some radiation-induced phosphoprotein changes in high-abundance proteins were identified, in general the sensitivity of this approach was not sufficient to detect changes in low-abundance, regulatory proteins. Characterization of these phosphoproteins will require greater enrichment of low-abundance proteins.     

Heparin inhibits metastatization of experimental melanoma

Heparin treatment, at human equivalent doses, modulates coagulation parameters in mice similarly to the human situation. Heparins were tested in various melanoma metastasis models for their antimetastatic activity. Heparins were active against melanoma metastasis without influencing the primary tumor. Tumor cell-induced platelet aggregation was not the primary target of heparins, since melanoma cells were not active in this respect. Our results support the notion that heparins have antimetastatic activity.     

Role of IIb-IIIa-like glycoproteins in cell-substratum adhesion of human melanoma cells

The platelet fibrinogen receptor, glycoprotein complex IIb-IIIa, was isolated from human platelets by lectin and monoclonal antibody affinity chromatography and a polyclonal antiserum (anti-IIb-IIIa) was generated and used to probe for the presence and function of IIb-IIIa-like molecules in two adherent human cell lines. Both C32 melanoma cells and WI38 fibroblasts expressed a IIb-IIIa-like complex on their surface as indicated by immunoprecipitation of detergent extracts of surface radiolabeled cells. When added to cells plated in medium containing 10% serum, the anti-IIb-IIIa antiserum perturbed the adhesion of C32 melanoma cells, but not of WI38 fibroblasts. In a serum-free system, anti-IIb-IIIa antibodies inhibited attachment and spreading of C32 cells to fibrinogen, vitronectin, and fibronectin adsorbed to glass. Anti-IIb-IIIa had no effect on the attachment and spreading of WI38 cells to the extracellular matrix proteins, however. Thus, the IIb-IIIa-like complex appears to play a predominant role in cell-substratum adhesion of C32 cells, but not WI38 cells, and may result from the fact that, on a protein basis, the C32 melanoma cells express approximately 3 times more complex on their surface than do WI38 fibroblasts. The results suggest that the relative abundance of a particular adhesion receptor on the cell surface may govern its importance to cell-substratum adhesion.     

Novobiocin inhibits passive chromatin assembly in vitro.

Novobiocin, an inhibitor of prokaryotic DNA gyrase and eukaryotic type II topoisomerase enzymes, interferes with in vitro chromatin assembly using purified histones, DNA and nucleoplasmin. The target of inhibition is not topoisomerase II; this energy-independent assembly system lacks any ATP and Mg2+-dependent type II topoisomerase or gyrase activities. Rather, novobiocin interacts with histones, disrupting histone-histone associations required for octamer formation, and causing histones to precipitate from both nucleoplasmin-histone and histone-DNA complexes. Thus, novobiocin is able to generate 'dynamic' chromatin in vitro in the absence of ATP and Mg2+ by removing histones from previously assembled static chromatin, so that the DNA supercoils, previously constrained by conventional nucleosomes, become susceptible to removal by topoisomerase I.     

cAMP inhibits the in vitro assembly of microtubules.

A new method for assaying microtubule assembly is described. The method utilizes the colchicine binding property of tubulin. This technique was used to study the effect of cyclic AMP on tubulin assembly using 100,000g supernatant and cycle-purified tubulin prepared from porcine brain. Cyclic AMP, in the presence of NaF, inhibited tubulin assembly from 100,000g supernatant but had no effect on cycle-purified tubulin.     

Antisense RNA inhibits splicing of pre-mRNA in vitro.

Antisense RNAs complementary to human beta-globin pre-mRNA or to a chimeric globin/adenovirus E2a pre-mRNA specifically and efficiently inhibit pre-mRNA splicing in vitro. The level of inhibition depends on the length, position and concentration of the antisense RNA relative to the pre-mRNA substrate. Antisense RNAs complementary to sequences greater than 80 nucleotides downstream of the globin 3' splice site inhibit at least as efficiently as those extending across the splice sites. Thus splicing is sensitive to perturbations involving exon sequences some distance from the splice sites. Inhibition is mediated by factors which affect the annealing of antisense and substrate RNAs. Direct analysis of RNA duplex formation demonstrates the presence of an activity in HeLa cell nuclear extract which promotes the rapid annealing of complementary RNAs in an ATP-independent manner. Both annealing and inhibition are greatly reduced when antisense RNA is added to the splicing reaction greater than or equal to 5 min after substrate. This result may reflect a transition between an open structure, in which annealing of antisense RNA with pre-mRNA is facilitated, and a closed complex in which pre-mRNA is sequestered at an early stage of spliceosome assembly.     

Frap-dependent serine phosphorylation of IRS-1 inhibits IRS-1 tyrosine phosphorylation

We have previously shown that interferon-alpha (IFN alpha)-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) is impaired by serine phosphorylation of IRS-1 due to the reduced ability of serine phosphorylated IRS-1 to serve as a substrate for Janus kinase 1 (JAK1). Here we report that FKBP12-rapamycin-associated protein (FRAP) is a physiologic IRS-1 kinase that blocks IFN alpha signaling by serine phosphorylating IRS-1. We found that both FRAP and insulin-activated p70 S6 kinase (p70(s6k)) serine phosphorylated IRS-1 between residues 511 and 772 (IRS-1(511-772)). Importantly, only FRAP-dependent IRS-1(511-772) serine phosphorylation inhibited by 50% subsequent JAK1-dependent tyrosine phosphorylation of IRS-1. Furthermore, treatment of U266 cells with the FRAP inhibitor rapamycin increased IFN alpha-dependent tyrosine phosphorylation by twofold while reducing constitutive IRS-1 serine phosphorylation within S/T-P motifs by 80%. Taken together, these data indicate that FRAP, but not p70(s6k), is a likely physiologic IRS-1 serine kinase that negatively regulates JAK1-dependent IRS-1 tyrosine phosphorylation and suggests that FRAP may modulate IRS-dependent cytokine signaling.     

Long-term in vitro cell culture of Sinclair swine melanoma.

The melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in cluture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell cultures of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18.     

Anandamide inhibits oxidative phosphorylation in isolated liver mitochondria

A study on the effect of anandamide (AEA) in energy coupling of rat liver mitochondria is presented. Micromolar concentrations of AEA, while almost ineffective on substrate supported oxygen consumption rate and on uncoupler stimulated respiration, strongly inhibited the respiratory state III. AEA did not change the rate and the extent of substrate generated membrane potential, but markedly delayed rebuilding by respiration of the potential collapsed by ADP addition. Overall, these data suggest that anandamide inhibits the oxidative phosphorylation process. Direct measurement of the F_oF₁ ATP synthase activity showed that the oligomycin sensitive ATP synthesis was inhibited by AEA, (IC₅₀, 2.5 μM), while the ATP hydrolase activity was unaffected. Consistently, AEA did not change the membrane potential generated by ATP hydrolysis.