The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 × 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5–13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [³⁵S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.     

The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 x 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5-13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.     

TGF-beta regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell proliferation; interestingly its action is clearly cell type-dependent. In particular, it inhibits epithelial and endothelial cells' proliferation, while its action on many mesenchymal cells has been reported to be stimulatory. In this direction, we have recently shown that TGF-beta regulates the proliferation of normal human skin fibroblasts according to their developmental origin: i.e. it inhibits fetal fibroblasts, while it stimulates the proliferation of adult ones. Here, we present evidence on the mechanisms underlying this differential action. Concerning fetal fibroblasts, we have found that TGF-beta activates Protein Kinase A (PKA) and induces the expression of the cyclin-dependent kinase inhibitors (CKIs) p21(CIP1/WAF1) and p15(INK4B). Moreover, the specific PKA inhibitor H-89 blocks the induction of both CKIs and annuls the TGF-beta-mediated inhibitory effect, indicating the central role of PKA in this process. In contrast, in adult cells no PKA activation is observed. Moreover, TGF-beta stimulates cell proliferation by activating the MEK-ERK pathway, as the MEK inhibitor PD98059 blocks this effect. A specific neutralizing antibody against Fibroblast Growth Factor-2 (FGF-2) inhibits both ERK activation and the mitogenic activity of TGF-beta, indicating that the latter establishes an autocrine loop, via FGF-2, leading to cell proliferation. This loop requires FGF receptor-1 (FGFR-1), as its down-regulation by siRNA approach prevents TGF-beta from stimulating ERK-1/2 activation and DNA synthesis. In conclusion, the differential proliferative response of fetal and adult normal human skin fibroblasts to TGF-beta is regulated by distinct signaling pathways and furthermore it may provide information on the bimodal effect of this factor on cell proliferation, in general.     

Deficit of complex I activity in human skin fibroblasts with chromosome 21 trisomy and overproduction of reactive oxygen species by mitochondria: involvement of the cAMP/PKA signalling pathway

DS (Down's syndrome) is the most common human aneuploidy associated with mental retardation and early neurodegeneration. Mitochondrial dysfunction has emerged as a crucial factor in the pathogenesis of numerous neurological disorders including DS, but the cause of mitochondrial damage remains elusive. In the present study, we identified new molecular events involved in mitochondrial dysfunction which could play a role in DS pathogenesis. We analysed mitochondrial respiratory chain function in DS-HSFs (Down's syndrome human foetal skin fibroblasts; human foetal skin fibroblasts with chromosome 21 trisomy) and found a selective deficit in the catalytic efficiency of mitochondrial complex I. The complex I deficit was associated with a decrease in cAMP-dependent phosphorylation of the 18 kDa subunit of the complex, due to a decrease in PKA (protein kinase A) activity related to reduced basal levels of cAMP. Consistently, exposure of DS-HSFs to db-cAMP (dibutyryl-cAMP), a membrane-permeable cAMP analogue, stimulated PKA activity and consequently rescued the deficit of both the cAMP-dependent phosphorylation and the catalytic activity of complex I; conversely H89, a specific PKA inhibitor, suppressed these cAMP-dependent activations. Furthermore, in the present paper we report a 3-fold increase in cellular levels of ROS (reactive oxygen species), in particular superoxide anion, mainly produced by DS-HSF mitochondria. ROS accumulation was prevented by db-cAMP-dependent activation of complex I, suggesting its involvement in ROS production. Taken together, the results of the present study suggest that the drastic decrease in basal cAMP levels observed in DS-HSFs participates in the complex I deficit and overproduction of ROS by DS-HSF mitochondria.     

Effects of all-trans retinoic acid on UVB-irradiated human skin substitute

Retinoids are frequently used for treatment of photodamaged skin. We wished to find out whether photodamage could be attenuated by applying all-trans retinoic acid (RA) during repetitive irradiation. For this purpose, we used human cutaneous cells and tissue: pure monolayer cultures containing either keratinocytes or fibroblasts, and human skin substitute (SS) containing both cell types. All cultures were exposed to 8 mJ/cm² of UVB and were immediately treated with RA (0, 1.5, or 3 μM). The irradiation and RA treatment protocol was repeated until the cells of the nonirradiated culture had reached confluence. In the irradiated SS, RA preserved the structure (epidermal stratification and differentiation) and ultrastructure (well-organized intermediate filaments and desmosomes) in a state comparable to that observed in nonirradiated SS. As well RA maintained secretion of basement membrane components (laminin and type-IV collagen). Following irradiation, cutaneous cells also displayed more proliferative capacity when SS was treated. In the irradiated monolayer cultures, RA maintained the proliferative capacity of fibroblasts and decreased their differentiation whereas the opposite effect was seen on keratinocytes. In conclusion, RA clearly helps protect human skin against photodamage induced by repeated exposure to UVB. J. Cell. Physiol. 181:14–23, 1999. © 1999 Wiley-Liss, Inc.     

Effects of 50 Hz electromagnetic field exposure on apoptosis and differentiation in a neuroblastoma cell line

Experiments were carried out to assess whether a magnetic field of 50 Hz and 1 mT can influence apoptosis and proliferation in the human neuroblastoma cell line LAN-5. TUNEL assays and poly-ADP ribose polymerase (PARP) expression analysis were performed to test apoptosis induction, and the WST-1 assay was used to calculate the proliferation index in a long term exposure. No alterations were found in cellular ability to undergo programmed cell death, but a small increase in the proliferation index was evidenced after 7 days of continuous exposure. Also, a slight and transient increase of B-myb oncogene expression was detected after 5 days of exposure. Combined exposures of cells to EMF and to chemical agents which interfere with proliferation, such as the differentiative agent retinoic acid and the apoptotic inducer camptothecin, showed an antagonistic effect of magnetic fields against the differentiation of the LAN-5 cells and a protective effect towards apoptosis.     

Nitric oxide regulates synthesis of gene products involved in keratinocyte differentiation and ceramide metabolism

During terminal differentiation of keratinocytes the expression of various proteins, which are required for the formation of the epidermal water barrier in the skin of land dwelling animals, is upregulated. Using a cell culture model for the differentiation of human keratinocytes and real-time PCR, we quantified the mRNA levels of several proteins involved in differentiation and ceramide metabolism. A calcium shift (1.1 mM CaCl2, 10 microM linoleic acid) for 8 days triggered an increase in mRNA levels of keratin 10 (75-fold), profilaggrin (55-fold), glucosylceramide synthase (40-fold), beta-glucocerebrosidase (30-fold), prosaposin (15-fold), acid sphingomyelinase (5-fold), and serine palmitoyltransferase (SPTLC2, 4-fold). However, mRNA levels of keratin 14 and acid ceramidase did not change significantly. On the other hand nitric oxide added at concentrations lower than 0.25mM stimulates proliferation of keratinocytes (Krischel et al., J. Invest. Dermatol. 111, 286-291, 1998). Accordingly, the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.2 mM) had no effect on the morphology of cultured keratinocytes, whereas in cultured human fibroblasts apoptosis was induced. The expression patterns obtained suggest that keratinocytes remain in a basal proliferative state, with a 3-fold increase in keratin 14 expression, a marked decrease in mRNA levels of differentiation markers and of most ceramide-metabolizing enzymes to negligible levels. The inhibitor of the NO synthase, N(G)-nitro-L-arginine-methyl ester (L-NAME, 10 mM), induced a transient increase in ceramide formation, followed by apoptosis in keratinocytes but not in fibroblasts. Both, SNAP and L-NAME, decreased the mRNA levels of all proteins involved in ceramide metabolism.     

Effects of a low-intensity electromagnetic field on fibroblast migration and proliferation

The aim of this study was to test if an extremely weak 1 GHz electromagnetic field (EMF), known to be in resonance with clusters of water molecules, has biological effects on human fibroblasts. We demonstrated that in an in vitro model of wound healing, this EMF can activate fibroblast migration. [³H]thymidine incorporation experiments demonstrated that the EMF could also activate fibroblast proliferation. Activation of the expression of human fibroblast growth factor 1 (HFGF1) after EMF exposure showed that molecular wound healing pathways are activated in response to this water-resonant EMF.     

Effects of a low-intensity electromagnetic field on fibroblast migration and proliferation

The aim of this study was to test if an extremely weak 1?GHz electromagnetic field (EMF), known to be in resonance with clusters of water molecules, has biological effects on human fibroblasts. We demonstrated that in an in vitro model of wound healing, this EMF can activate fibroblast migration. [(3)H]thymidine incorporation experiments demonstrated that the EMF could also activate fibroblast proliferation. Activation of the expression of human fibroblast growth factor 1 (HFGF1) after EMF exposure showed that molecular wound healing pathways are activated in response to this water-resonant EMF.     

Dual role of PKA in phenotypic modulation of vascular smooth muscle cells by extracellular ATP

Extracellular ATP is released from activated platelets and endothelial cells and stimulates proliferation of vascular smooth muscle cells (VSMC). We found that ATP stimulates a profound but transient activation of protein kinase A (PKA) via purinergic P2Y receptors. The specific inhibition of PKA by adenovirus-mediated transduction of the PKA inhibitor (PKI) attenuates VSMC proliferation in response to ATP, suggesting a positive role for transient PKA activation in VSMC proliferation. By contrast, isoproterenol and forskolin, which stimulate a more sustained PKA activation, inhibit VSMC growth as expected. On the other hand, the activity of serum response factor (SRF) and the SRF-dependent expression of smooth muscle (SM) genes, such as SM--actin and SM22, are extremely sensitive to regulation by PKA, and even transient PKA activation by ATP is sufficient for their downregulation. Analysis of the dose responses of PKA activation, VSMC proliferation, SRF activity, and SM gene expression to ATP, with or without PKI overexpression, suggests the following model for the phenotypic modulation of VSMC by ATP, in which the transient PKA activation plays a critical role. At low micromolar doses, ATP elicits a negligible effect on DNA synthesis but induces profound SRF activity and SM gene expression, thus promoting the contractile VSMC phenotype. At high micromolar doses, ATP inhibits SRF activity and SM gene expression and promotes VSMC growth in a manner dependent on transient PKA activation. Transformation of VSMC by high doses of ATP can be prevented and even reversed by inhibition of PKA activity. adenosine triphosphate; purinergic receptors; protein kinase A; serum response factor; proliferation; -actin; SM22     

Effects of Cigarette Smoke Extracts on the Growth and Senescence of Skin Fibroblasts In Vitro

Epidemiological studies have shown that cigarette smoke (CS), a very common environmental factor, plays an important role in skin aging. Although some in vivo studies have suggested that CS affects skin aging, the detailed effects of CS on skin cells in vitro remain largely unknown. In this study, we investigated the effects of cigarette smoke extract (CSE) on the growth, proliferation, and senescene of skin fibroblasts and the possible mechanism underlying these effects. Primary cultured human fibroblasts were exposed to a range of concentrations of CSE. Cell viability and cell proliferation after CSE exposure were analyzed with the methyl thiazolyl tetrazolium (MTT) assay and bromodeoxyuridine incorporation assay, respectively. Growth curves of fibroblasts exposed to different concentrations of CSE were developed and prolonged CSE-exposed cells were observed. Morphological and ultrastructural changes in fibroblasts were assessed by inverted light microscopy and transmission electron microscopy (TEM). Dying cells were stained with senescence-associated β-galactosidase (SA β-gal). Intracellular reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) activity were determined by a colorimetric method. We found that proliferative capacity and growth were inhibited by CSE exposure in a dose- and time-dependent manner. Fibroblasts exposed to even low concentrations of CSE for a long period of time (5 passages) showed significantly increased SA β-gal activity and typical features of aging cells. Meanwhile, CSE inhibited superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and augmented ROS levels. Our observations suggest that CSE exposure impairs fibroblast growth and proliferation and leads to features similar to those seen in senescent cells. Oxidative stress injury and inhibition of antioxidant defense activity may be involved in CSE-induced fibroblast senescence.     

Anti-apoptotic action of Wnt5a in dermal fibroblasts is mediated by the PKA signaling pathways

Wnts are secreted glycoproteins that control diverse biological processes, such as proliferation, differentiation, and apoptosis. We here found that Wnt5a inhibited apoptosis induced by serum deprivation in primary-cultured human dermal fibroblasts. Anti-apoptotic activity of Wnt5a was not inhibited by a dickkopf-1 (DKK), which blocks the canonical Wnt pathway. On the other hand, loss of function of protein kinase A (PKA), induced by treatment with PKA inhibitors, siRNA-mediated knocking down of endogenous PKA catalytic subunits, or enforced expression of dominant-negative PKA inhibited the Wnt5a anti-apoptotic activity, indicating the involvement of PKA in the Wnt5a anti-apoptotic activity. In agreement, phosphorylation levels of a cAMP response element binding protein (CREB), a representative downstream effector of PKA, the activation of which is known to lead to the pro-survival effects, was elevated by Wnt5a. In addition, Wnt5a increased the nuclear beta-catenin level and treatment with imatinib or ionomycin, either of which blocks the beta-catenin pathway, reduced the anti-apoptotic activity of Wnt5a, together suggesting the simultaneous involvement of the beta-catenin-mediated pathway in the Wnt5a anti-apoptotic activity. Based on another finding indicating that Wnt5a upregulated PKA-mediated phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at serine 9 that caused inactivation of GSK-3beta and subsequently resulted in activation of the beta-catenin pathway, we have speculated that the Wnt5a anti-apoptotic activity may be partially mediated by PKA-mediated phosphorylation of GSK-3beta and subsequent activation of the beta-catenin pathway.     

Low-energy electromagnetic fields promote proliferation of vascular smooth muscle cells

The rationale was to investigate the effects of low-energy electromagnetic fields (EMF) on the proliferation of bovine coronary and murine aortic smooth muscle cells (SMC). EMF were applied to SMC at field frequencies of 25, 50, or 100 Hz, and exposure time was set to 5, 15, or 30 minutes. Significant increases in SMC-counts compared with sham exposed controls were found for all EMF-frequencies tested. The effect was most pronounced for 50 Hz fields with maximum increases of 1.2-fold over controls. Sequential double exposure of mouse aortic SMC to 50 Hz fields revealed significantly enhanced cell proliferation by 1.2 fold compared with single exposure (p < 0.05). Experiments performed on bovine SMC also revealed significant increases in cell proliferation. The results demonstrate that EMF are capable of significantly enhancing the proliferation of vascular SMC. These results rise the question whether EMF would qualify as supportive means to angio-/arteriogenic approaches.     

EFFECTS OF BETA MERCAPTOETHANOL ON THE PROLIFERATION AND DIFFERENTIATION OF HUMAN OSTEOPROGENITOR CELLS

Antioxidants are known to influence metabolism and promote cell survival in a number of cell culture systems. However, their effects on the modulation of bone cell differentiationin vitroare not clearly defined. In the present studies we have investigated the effects of β-mercaptoethanol (βME) and ascorbate alone and in combination on human osteoprogenitors derived from bone marrow fibroblasts. In primary marrow cultures, βME stimulated colony formation (2-fold), alkaline phosphatase activity (3.5-fold) and, increased DNA synthesis (8-fold) after 21 days. Cell proliferation was increased significantly by βME during the first 4 days of a 10-day culture period, indicating stimulation of marrow osteoprogenitor proliferation. Ascorbate did not significantly augment the effects of βME in primary cultures or long-term cultures of passaged bone marrow fibroblasts. These findings indicate a potential beneficial role for βME addition for the optimal maintenance of colony formation, cell proliferation and differentiation of marrow osteoprogenitor cells in primary human bone marrow fibroblast cultures.     

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-β (TGF-β). Conversely, migration toward ABA, but not toward TGF-β, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.     

Melatonin protects rat cerebellar granule cells against electromagnetic field‐induced increases in Na+ currents through intracellular Ca2+ release

Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low‐frequency electromagnetic field (ELF‐EMF)‐induced Na_v activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF‐EMF for 60 min. significantly increased the Na_v current (I_Na) densities by 62.5%. MT (5 μM) inhibited the ELF‐EMF‐induced I_Na increase. This inhibitory effect of MT is mimicked by an MT₂ receptor agonist and was eliminated by an MT₂ receptor antagonist. The Na_v channel steady‐state activation curve was significantly shifted towards hyperpolarization by ELF‐EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF‐EMF. ELF‐EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK‐7 did not reduce the ELF‐EMF‐induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF‐EMF‐induced Na_v activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca²⁺ level, but it significantly elevated the intracellular Ca²⁺ level evoked by the high K⁺ stimulation in cerebellar GC that were either exposed or not exposed to ELF‐EMF. In the presence of ruthenium red, a ryanodine‐sensitive receptor blocker, the MT‐induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal I_Na that result from ELF‐EMF exposure through Ca²⁺ influx‐induced Ca²⁺ release.     

Interleukin-1 increases collagen production and mRNA levels in cultured skin fibroblasts

In the present study we show that highly purified human interleukin-1 increases collagen production nearly 2-fold and mRNA levels of type I and III collagen over 2.5-fold in cultured normal human dermal fibroblasts. To minimize the effects of transient prostaglanding E2 production in fibroblasts treated with interleukin-1, the cell cultures were preincubated for 24 h before these measurements were made. The effects of interleukin-1 were also tested on scleroderma fibroblasts exhibiting increased collagen production. Although collagen synthesis was stimulated by interleukin-1 to some degree, the cells grown from both affected and unaffected skin areas were found to be relatively unresponsive to the effects of interleukin-1, suggesting a role for this monokine in the earlier stages of the disease process. The results also suggest that interleukin-1 has a role in stimulation of collagen synthesis under certain normal and pathological conditions in addition to stimulating fibroblast proliferation.     

Overexpression of Glia Maturation Factor in C6 Cells Promotes Differentiation and Activates Superoxide Dismutase

In order to evaluate the intracellular function of glia maturation factor (GMF), we overexpressed GMF in C6 rat glioma cells using two methods: stable transfection using the pcDNA3 plasmid, and transient transfection using replication-defective human adenovirus. With both methods, C6 cells transfected with GMF and overexpressing the protein exhibit a lower saturation density in culture compared to non-transfected or vector alone controls. Transfected cells also exhibit morphological differentiation as shown by the outgrowth of cell processes. When inoculated into nude mice, transfected cells are less tumorigenic than controls, and express the mature astrocytic marker glial fibrillary acidic protein. In tissue culture, transfected cells show a 3.5-fold increase in CuZn-dependent superoxide dismutase (CuZnSOD) activity. Western blot analysis reveals a 3.5-fold increase in CuZnSOD protein, suggesting an induction of the enzyme. In view of recent findings that reactive oxygen species (ROS) and the antioxidant enzymes are intricately involved in key physiologic processes such as proliferation, differentiation and apoptosis, the study raises the possibility that CuZnSOD may be a mediator of GMF function.     

Murine melanoma cell differentiation and melanogenesis induced by poly(ADP-ribose) polymerase inhibitors.

Murine melanoma cells treated with the melanocyte-stimulating hormone (MSH) family of peptides undergo differentiation characterized by enhanced melanogenesis and altered morphology. These effects are mediated via the adenylate cyclase-cAMP pathway leading to activation of protein kinase A (PKA). We have discovered that inhibition of a post-translational modification of chromatin proteins, viz. poly(ADP-ribosylation), also induces melanogenesis and differentiation in these cells. A range of competitive inhibitors (benzamide and its derivatives) of the nuclear enzyme poly(ADP-ribose) polymerase (PADPRP; EC 2.4.2.30) was utilized, and their ability to induce melanogenesis reflected their potency as PADPRP inhibitors. These compounds induced melanogenesis at low doses (20 microM-2 mM) which did not affect cell growth or viability. Induction of melanogenesis was not attributable to inhibition of cyclic nucleotide phosphodiesterase by these compounds. MSH treatment caused a transient rise in cAMP levels (up to 200-fold by 5 min and returning to near basal levels by 5 h). It also stimulated PKA activity up to 5-fold, and the temporal kinetics of this activation mirrored the changes in cAMP levels. In comparison, the PADPRP inhibitors had no effect on either of these processes. These data constitute a novel demonstration of a cAMP-independent mechanism for the induction of melanoma cell differentiation, including melanogenesis.