Up-regulation of MSH receptors by MSH in Cloudman melanoma cells.

MSH can up-regulate MSH binding capacity of cultured Cloudman melanoma cells in a dose- and time-dependent fashion. Binding is mediated through proteins exhibiting an apparent molecular weight of 50-53kDa, consistent with previous studies implicating them as the principal MSH receptors on Cloudman cells. Pre-incubation of cells with MSH stimulates expression of the receptor proteins both on the plasma membrane surface as well as in internal sites associated with coated vesicles. The effects of MSH are additive with those of UV light, suggesting that UV and MSH might stimulate receptor expression through separate mechanisms.     

Melanotropic activity of gamma MSH peptides in melanoma cells.

We studied the pigmentary activity of the peptides gamma 1, gamma 2 and gamma 3 melanocyte stimulating hormone (MSH), which differ in the structure of their C-termini, using hamster and mouse melanoma cell lines responsive to beta-MSH by increasing tyrosinase activity. Gamma 1-MSH alone or in combination with beta-MSH had no effect on either cell line. Gamma 2-MSH alone was biologically inactive but potentiated beta-MSH stimulation of tyrosinase activity. Gamma 3-MSH at high concentration (10 microM) induced tyrosinase activity and dendrite formation in the hamster melanoma line. When added together with beta-MSH, gamma 3-MSH partially inhibited the tyrosinase activity response to beta-MSH. Thus, gamma-MSH peptides have low intrinsic melanotropic activity in mammalian melanoma cells; the specific pigmentary responses appear to be affected by the structure of the C-terminal portion.     

Effect of X rays on protein synthesis and RNA synthesis in adenovirus-infected cells.

Ionizing radiation exerts a deteriorating effect on the rate of protein and RNA synthesis in HeLa cells. This effect is higher in cells infected with adenovirus, thus pointing to a higher radiosensitivity of viral syntheses compared with the cellular ones.     

The role of protein synthesis in the decay of phosphoenolpyruvate carboxykinase messenger RNA.

The role of protein synthesis in the control of phosphoenolpyruvate carboxykinase (PEPCK; 4.1.1.32) mRNA turnover was studied in FTO-2B rat hepatoma cells. A previous study demonstrated that incubation of these cells with cAMP prolongs the half-life of the otherwise short-lived PEPCK mRNA. The decay rate of PEPCK mRNA was also slowed in cells incubated with cycloheximide, but not in cells incubated with other translation inhibitors, such as puromycin or pactamycin, even though protein synthesis was inhibited 85-95% by these agents. No correlation was noted between the rate of L-[3H]valine incorporation into cellular proteins and PEPCK mRNA half-life, suggesting that protein synthesis per se is not required for breakdown of the mRNA. Exposure of cells to the translation initiation inhibitor pactamycin together with cycloheximide abolished the "slowing" effect of cycloheximide, and PEPCK mRNA decayed at the same rate as in cells incubated in the presence of pactamycin alone. In contrast, pactamycin did not reverse the effect of cAMP, and the mRNA decayed at the same slow rate in cells incubated in the presence of either (Bu)2cAMP alone or (Bu)2cAMP together with pactamycin. Since pactamycin promotes polysomes dissociation, these results suggest that cAMP enhances the stability of a polysome-free PEPCK mRNA. Furthermore, these results strongly indicate that neither the rapid decay of PEPCK mRNA nor the cAMP-mediated stabilization of the mRNA requires on-going protein synthesis.     

The effect of retinoic acid on protein phosphorylation in mouse melanoma cells

Vitamin A inhibits growth and increases the activity of cAMP-dependent protein kinase in B16 mouse melanoma cells. In this report we show that retinoic acid (RA) treatment of intact cells alters their subsequent in vitro protein phosphorylation, but we could not demonstrate any changes in in vivo protein phosphorylation. A 48-h treatment with RA results in a concentration-dependent decrease of protein phosphorylation of a 95K molecular weight (MW) protein in both supernatant and particulate fractions. The phosphorylation of this protein does not appear to be regulated by cAMP. Proteins at 92K and 82K MW in the supernatant fraction are increased in phosphorylation. The former (but not the latter) is regulated by cAMP. In the particulate fraction a variety of proteins 12K-68K MW are increased in phosphorylation, as the cells are treated with increasing amounts of RA. The phosphorylation of most of these proteins is regulated by cAMP. Another inhibitor of B16 cell growth, melanocyte-stimulating hormone (MSH) also alters protein phosphorylation. At short incubation periods (1 h), this hormone stimulates phosphorylation of a number of proteins (17-40K MW), while in longer incubation periods (48 h) phosphorylation is inhibited. All of these phosphorylations appear to be regulated by cAMP. We attempted to repeat these observations using intact-cell phosphorylation with 32PO4. In two experiments we saw small changes in the phosphorylation of proteins. In most experiments, however, we could find no change in the phosphoproteins. Further experiments have led us to question the in vivo phosphorylation, since treatment of the cells with MSH, cholera toxin, or db-cAMP also did not affect intact-cell protein phosphorylation. We have previously documented that under these latter conditions cAMP levels are greatly elevated and cAMP-dependent protein kinase is activated. The in vitro phosphorylation results suggests that in RA-treated cells, kinase activities and/or protein substrate levels are changing. However, the physiological significance of the particular MW phosphoproteins changes we have described must await resolution of the in vivo phosphorylation data.     

In situ melanin assay for MSH using mouse B16 melanoma cells in culture

A sensitive in situ melanin assay using cultured mouse B16 melanoma cells is described for structure-activity studies with melanocyte-stimulating hormone (MSH) peptides. B16 Cells were seeded at a density of 2500 cells per well in 96-well microtest tissue culture plates; after 24 h the cells were incubated in the presence of serial dilutions of MSH peptides for 3 to 5 days. The melanin released into the medium of each well was then determined spectrophotometrically at a wavelength of 405 nm using an automatic microplate reader calibrated against synthetic melanin. Studies with alpha-MSH, [Nle4, D-Phe7]-alpha-MSH, [3'-iodo-Tyr2]-alpha-MSH, adrenocorticotropin (ACTH)(1-24), and ACTH(1-39) showed that the peptides had identical intrinsic activities and that the relative potencies were similar to those obtained with a tyrosinase assay. The EC50 of alpha-MSH was 27 pM, i.e., about five- to sevenfold lower than that in the assays for tyrosinase or intracellular melanin. Thus, the new assay represents the most sensitive melanoma cell assay for MSH available to date.     

RNA turnover and protein synthesis in fish cells

Protein synthesis in fish has been previously correlated with RNA content. The present study investigates whether protein and RNA synthesis rates are similarly related. Protein and RNA synthesis rates were determined from ³H-phenylalanine and ³H-uridine incorporation, respectively, and expressed as % · day⁻¹ and half-lives, respectively. Three fibroblast cell lines were used: BF-2, RTP, CHSE 214, which are derived from the bluegill, rainbow trout and Chinook salmon, respectively. These cells contained similar RNA concentrations (∼175 μg RNA · mg⁻¹ cell protein). Therefore differences in protein synthesis rates, BF-2 (31.3 ± 1.8)>RTP (25.1 ± 1.7)>CHSE 214 (17.6 ± 1.1), were attributable to RNA translational efficiency. The most translationally efficient RNA (BF-2 cells), 1.8 mg protein synthesised · μg⁻¹ RNA · day⁻¹, corresponded to the lowest RNA half-life, 75.4 ± 6.4 h. Translationally efficient RNA was also energetically efficient with BF-2 cells exploiting the least costly route of nucleotide supply (i.e. exogenous salvage) 3.5–6.0 times more than the least translationally efficient RNA (CHSE 214 cells). These data suggest that differential nucleotide supply, between intracellular synthesis and exogenous salvage, constitutes the area of pre-translational flexibility exploited to maintain RNA synthesis as a fixed energetic cost component of protein synthesis. Accepted: 12 November 1999     

On the mechanism of erythropoietin-induced differentiation. XII. A cytoplasmic protein mediating induced nuclear RNA synthesis

Erythropoietin, the primary inducer of red blood cell differentiation, has no effect on RNA synthesis by isolated bone marrow nuclei. A cytoplasmic fraction from marrow cells exposed to erythropoietin does, however, stimulate RNA synthesis by such nuclei. This marrow cell cytoplasmic factor (MCF) also stimulates RNA synthesis by liver and kidney nuclei, whereas erythropoietin has no effect on intact kidney or lung cells. MCF appears rapidly in cells after addition of erythropoietin, and its formation does not require protein synthesis. MCF is inactivated by trypsin, but not by ribonuclease. The data suggest that erythropoietin acts on the responsive cells to generate a cytoplasmic protein that mediates the effect of the hormone on nuclear RNA synthesis.     

Effect of reduced temperatures on protein synthesis in mouse L cells.

The rate of incorporation of leucine into protein, the rate of polypeptide elongation and termination, and the relative quantity and size of polysomes were analyzed in mouse L cells grown in suspension culture at various temperatures between 0 degrees C and 36 degrees C. Between 10 degrees C and 36 degrees C protein synthesis exhibited two different apparent activation energies (39 kcal/mole, 10-25 degrees C; 14 kcal/mole, 25-36 degrees C), whereas elongation and termination had only one (16 kcal/mole). Below 36 degrees C, the polysome level and size decreased, reaching a minimum of 30% of the control 36 degrees C values at 10 degrees C; below 10 degrees C the level increased again back to control values at 0 degrees C. The polysome decline was time dependent, requiring about 5 hr to reach the equilibrium value. This decline is completely reversible within 60 min, even in the presence of 4 mug/ml of actinomycin D, and even after 15 hr of incubation at the lower temperature. The results suggest that polypeptide initiation is rate limiting, particularly below 25 degrees C; whereas above this temperature, elongation or perhaps some other process may be limiting. These results are quite different from those obtained for E. coli and rabbit reticulocyte protein synthesis.     

RNA localization and its role in the spatially restricted protein synthesis

RNA localization is an evolutionarily conserved phenomenon that occurs in uni- and multi-cellular animal and plant species. Localized RNA plays a role in the establishment of cell polarity and/or the determination of cell fate. In recent years, it became evident that the major function of RNA localization is the creation of a high concentration of proteins in specific cellular compartments. The movement of RNA involves interactions between targeting signals within the RNA molecule, cytoskeleton, and molecular motors. Translocating RNA must be translationally silent, and on-site translation at the destination site requires a de-repression mechanism. This is probably achieved by sequestering RNA and the regulators of translation within the multiprotein RNP complexes that co-translocate all the components to the ultimate destination within the cell.     

Atypical protein kinase Czeta suppresses migration of mouse melanoma cells.

Mouse melanoma B16 F1 cells cultured in RPMI 1640 supplemented with the melanin precursors tyrosine and phenylalanine display increased melanin levels and elevated migration while down-regulating protein kinase C (PKC)zeta to low levels. Although control experiments rule out a direct role by melanin, PKCzeta down-regulation is shown to be a critical determinant of cell migration. Transfection of high-motility cells with either wild-type PKCzeta or its regulatory domain suppresses migration. Known to bind to the regulatory domain of PKCzeta, the proapoptotic protein prostate apoptosis response-4 (Par-4) coimmunoprecipitates with PKCzeta as a 47-kDa protein. Transfection of Par-4 (or its leucine zipper element) further suppresses migration of low-motility cells (which express high levels of PKCzeta), whereas high-motility cells (which express low levels of PKCzeta) are unaffected by Par-4 overexpression. It is proposed that in nonmetastatic cells, the PKCzeta Par-4 complex provides a brake on migration that is released by melanin precursors that initiate PKCzeta down-regulation. Elevation of PKCzeta in melanoma cells, or preventing its down-regulation through the dietary restriction of tyrosine and phenylalanine, may therefore control metastatic behavior.     

Interactions between ultraviolet light and interleukin-1 on MSH binding in both mouse melanoma and human squamous carcinoma cells

Interactions between beta-melanotropin (MSH), interleukin 1-a (IL-1), and ultraviolet light (UV) were examined in Cloudman S91 mouse melanoma and RHEK human squamous carcinoma cell lines. The following points were established: 1) both cell lines produced IL-1 and their production was stimulated by exposure of the cells to UV; 2) both cell lines possessed high affinity binding sites for MSH, and their ability to bind MSH was modulated by IL-1; 3) IL-1 exhibited both stimulatory and inhibitory effects on MSH binding to Cloudman cells; and 4) the stimulatory effect of IL-1 on MSH binding to melanoma cells was reflected in enhanced cellular responsiveness to MSH regarding tyrosinase activity (E.C. 1.14.18.1) and melanin content. The findings raise the possibility that interactions between keratinocytes and melanocytes may be regulated by IL-1 and MSH, and suggest a possible mechanism for stimulation of cutaneous melanogenesis by solar radiation: enhancement of MSH receptor activity by induction of IL-1.     

Ribonucleotide depletion in glucose-deprived tumor cells - the role of RNA synthesis.

Ribonucleoside triphosphate pools decreased rapidly and profoundly in Ehrlich ascites tumor cells incubated in glucose-free medium. Ribonucleoside triphosphate pools decreased to a lesser degree and total ribonucleotide pools remained normal in cells that were incubated with actinomycin D or with cycloheximide for 30 min before deprivation of glucose. RNA synthesis rates in glucose-deprived cells were 53–70% of the rates in glucose-supplemented cells. These findings suggest that ribonucleoside triphosphate consumption in RNA synthesis is an important factor in the depletion of ribonucleotide pools in tumor cells subjected to glucose starvation. Restriction of ATP regeneration is the other major factor.