Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. METHODS: Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. RESULTS: Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. CONCLUSIONS: Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

Vitamin D protects human endothelial cells from H2O2 oxidant injury through the Mek/Erk-Sirt1 axis activation.

Endothelium homeostasis alterations govern the pathogenesis of cardiovascular diseases. Several studies show that vitamins anti-oxidant proprieties rescue the endothelial functions adversely affected by oxidative stress in several diseases. We investigated the vitamin D anti-oxidant potential in human endothelial cells exposed to H2O2 oxidative stress. Vitamin D protected endothelial cells against H2O2 oxidative stress counteracting the superoxide anion generation, the apoptosis and blocking the extrinsic caspase cascade by positively controlling phospho-active ERKs level. MEKs/ERKs inhibitor U0126 reverted the vitamin D anti-oxidant effects. Characterizing the vitamin D downstream effector, we found that vitamin D up-regulated SirT-1 and reverted the SirT-1 down-regulation induced by H2O2. ERKs activation by vitamin D strictly correlated with SirT-1 protein accumulation since both MEKs/ERKs inhibition and ERK1/2 silencing decreased SIRT-1. SirT-1 inhibition by Sirtinol reverted the vitamin D anti-oxidant effects. Thus, vitamin D significantly reduced the endothelial malfunction and damage caused by oxidative stress, through the activation of MEKs/ERKs/SirT-1 axis.

Enhanced expression of myogenic regulatory genes in aging skeletal muscle.

MyoD, myogenin, myf-5, and MRF4, belonging to the family of basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs), control muscle cell differentiation, in concert with other transcription factors such as MEF-2, yet their role in age-related skeletal muscle alteration has not been addressed. We here report that MyoD and myogenin transcripts are expressed at high levels in the hind limb muscles of newborn mice and their level of expression continuously declines throughout postnatal life to become virtually undetectable in the adult mouse. However, these transcripts are again expressed at high levels in the muscles of older mice. MRF4 transcript, on the other hand, is present at a constant level throughout the life span of the animal. Conversely, the expressions of myf-5 and MEF-2C, components of the autoregulatory loop for the activation of bHLH gene expression, conspicuously increase in adult and senile muscle. In order to establish whether these transcripts are functioning in the aged muscle we investigated the expression of bHLH inhibitory factor Id mRNA showing that it does not present significant changes during aging. Immunofluorescence analysis with an anti-myogenin antibody revealed nuclear accumulation of the protein in the muscle fibers of old, but not of adult, mice. Muscle-specific genes transactivated by MyoD and myogenin such as AChR, MLC, and MCK are also up-regulated during aging, albeit at a lower level. Significant changes in the size and ratio of type I/type II fibers are detectable in senile muscle. These findings show that all members of the MRF family are expressed to a high extent and are likely active in senile muscle. It is conceivable that these changes might operate as a compensatory mechanism in maintaining the expression of differentiated muscle products in senile muscle at a steady-state level.

Calcitonin down-regulates immediate cell signals induced in human osteoclast-like cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism.

Calcitonin (CT) is a peptide hormone that interacts with the cAMP-and phospholipase C-associated CT receptor subtypes. We investigated whether CT modulates the interaction of human tumoral osteoclast-like (GCT23) cells with a protein of the bone matrix, bone sialoprotein-II (BSP-II). Single GCT23 cells loaded with the intracellular Ca2+ indicator fura-2 were treated with the maximal active dose (300 micrograms/ml) of the 18-mer Arg-Gly-Asp (RGD)-containing BSP-IIA fragment, and the cytosolic free Ca2+ concentration ([Ca2+]i) was measured by dual wavelength microfluorometry. BSP-IIA stimulated an elevation in [Ca2+]i, consisting mainly of a peak, followed by a rapid return toward baseline. Pretreatment with CT induced a modest elevation of [Ca2+]i. However, CT significantly inhibited the response to BSP-IIA in a dose-dependent manner. Maximal inhibition (90% vs. untreated) was observed in the micromolar range. The intracellular mechanisms leading to this effect were investigated by pretreatment of GCT23 cells with the cAMP permeant analog, (Bu2)cAMP, and the protein kinase-C-activating agent, 12-O-tetradecanoylphorbol 13-acetate. Similar to CT, both agents inhibited the response to 300 micrograms/ml BSP-IIA. The effect induced by CT was specific, because an increase in the extracellular Ca2+ concentration, which is also known to inhibit bone resorption, failed to modify the ability of BSP-IIA to alter [Ca2+]i in GCT23 cells. To investigate whether the CT-induced alteration of BSP-IIA-dependent cell signals was due to a modification in the synthesis of cell surface receptors (integrins) for the extracellular matrix macromolecules, 1-h CT-treated [35S]methionine metabolically labeled GCT23 cell lysates were immunoprecipitated with anti-alpha 3-, -alpha v-, -beta 1-, and -beta 3-integrin subunit antibodies. Autoradiography demonstrated that 10(-7)-10(-6) M CT did not alter new synthesis of the alpha v beta 3 and the alpha 3 beta 1 receptors. Similarly, CT did not affect surface expression of these receptors, assessed by enzyme-linked immunosorbent assay. Finally, no alteration of the adhesion rate and spreading of GCT23 cells onto BSP-IIA-coated substrates was observed. This indicates that CT-induced down-regulation of immediate cell signals prompted by BSP-IIA in GCT23 cells is a postintegrin receptor event.

TPA-induced differentiation of human rhabdomyosarcoma cells involves dephosphorylation and nuclear accumulation of mutant P53.

Previous studies have shown that human rhabdomyosarcoma cells are induced to differentiate by TPA, in the absence of appreciable alterations of the muscle regulatory genes and their products (1). The question was addressed whether the tumor suppressor p53 could be a target of TPA action in these cells. Genomic analysis by a Polymerase Chain Reaction/Single-Strand Conformation Polymorphism (PCR/SSCP) and direct sequencing indicate the presence of a mutation in exon VII at codon 248 (C to T transition) and a loss of heterozygosity of p53 gene in human rhabdomyosarcoma cell line (RD). It is here shown that transcription of p53 mRNA strongly decreases in RD cells induced to growth arrest and differentiate by TPA treatment. In these cells immunoprecipitation and immunoblot analysis show that both synthesis and total cellular concentration of the protein are also reduced by TPA. Nevertheless nuclear p53 accumulation is at much higher extent, whereas 32P-orthophosphate labelling, followed by immunoprecipitation, demonstrates a decrease of phosphorylation of both cytoplasmic and nuclear p53. These results indicate that TPA causes a number of alterations of mutant p53, likely mediated through a protein kinase C dependent mechanism, which might impair the transforming ability of mutant p53 in growth-arrested and differentiating RD cells.

Paracrine stimulation of senescent satellite cell proliferation by factors released by muscle or myotubes from young mice.

The proliferative potential of satellite cells undergoes a dramatic decrease in the early postnatal period and a more modest but continuous decrease throughout the life span of the animal. To address the problem of the mechanism regulating this phenomenon and to understand whether it is causally linked to senile muscle atrophy, we studied the response of aged satellite cells to serum and to different growth factors. The data reported indicate a generalised reduction in the response to all mitogens tested, which could not be compensated for by increased concentrations of serum or growth factors. On the other hand, conditioned medium of differentiated myotubes from young mice exhibited a strong mitogenic action on aged satellite cells, while conditioned media of myotubes from old mice or from a variety of non-muscle cells were ineffective. Furthermore, saline extracts from muscle of young mice are also able to exert this mitogenic action. Saline extracts of muscle from old mice were poorly mitogenic for satellite cells from young mice, and not at all for satellite cells from old mice. These data indicate that paracrine interactions operate inside the muscle tissue and are probably required for the normal replicative behaviour of satellite cells. The failure of such interactions may be among the causes leading to age-related muscle hypotrophy.

Spontaneous channel activity induced by tumor promoter TPA in chick myotubes.

Patch-clamp recordings were used to study the activation of ion channels in the cell membrane of cultured embryonic chick myotubes treated with the specific activator of protein kinase C, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 1 x 10(-7) M). Myotubes exhibited a spontaneous channel activity when the TPA-induced dedifferentiative processes developed. This consisted in the activation of inward current channels (approximately 35 pS conductance; approximately 6 ms open time). These spontaneously active channels were insensitive to alpha-bungarotoxin, curare, atropine and tetrodotoxin and were not inhibited by the withdrawal of TPA. It is suggested that a prolonged stimulation of the protein kinase C causes a irreversible deregulation of the membrane channel function during cell dedifferentiation.

Microwave effects on acetylcholine-induced channels in cultured chick myotubes.

The behavior of cultured myotubes from chick embryos exposed to microwaves has been experimentally analyzed. Recordings of acetylcholine-induced currents have been obtained via patch-clamp techniques using both cell-attached (single-channel current recording) and whole-cell (total current recording) configurations. During the exposure to low-power microwaves the frequency of the ACh-activated single channel openings decreased, while the ACh-induced total current showed a faster falling phase. Channel open time and conductance were not affected by microwave irradiation. It is concluded that the exposure to microwaves increases the rate of desensitization and decreases the channel opening probability. The nonthermal origin and the molecular interaction mechanisms governing these electromagnetic-induced effects are discussed.

Acetylcholine regulation of nicotinic receptor channels through a putative G protein in chick myotubes.

1. Single-channel currents induced by acetylcholine (ACh) were recorded from unstriated and non-innervated embryonic chick myotubes using the cell-attached patch-clamp technique. 2. ACh applied to the non-patched membrane decreased both channel opening probability and conductance. These ACh-induced effects occurred also when the non-patched membrane was exposed to nominally Ca2+-free extracellular medium, but were absent when it was treated with curare. 3. ACh-induced membrane current recorded under whole-cell patch-clamp conditions decreased in amplitude and time course when myotubes were intracellularly loaded with guanosine-5'-O-(3-thiotriphosphate) GTP gamma S), but not with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) or cyclic adenosine-5'-monophosphate (cyclic AMP). Internal perfusion of GTP gamma S affected the ACh-induced openings in a similar manner to the non-patch ACh application. 4. These results suggest that ACh, in addition to its direct effect, acts indirectly on the nicotinic receptor channels by delivering an intracellular messenger and through the activation of a putative G protein.

Membrane molecules involved in adhesion properties of cultured Sertoli cells.

Membrane components involved in adhesion properties of cultured Sertoli cells have been studied by a combination of immunological and biochemical methods. An antiserum prepared against Sertoli cells induced reversible rounding and detachment of the cells from the culture dishes. The cell surface morphology during detachment was studied by scanning electron microscopy and indirect immunofluorescence. A Triton soluble fraction of crude membrane preparations inhibited the antibody-induced detachment. The antibodies recognized a restricted number of membrane glycoproteins [detectable as prominent bands on Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), Mr 170, 140, 80, and 48K] both in the Triton soluble fraction of crude membrane preparation and on intact Sertoli cells. The data suggest that the molecules involved in adhesion properties of cultured Sertoli cells are integral membrane glycoproteins exposing antigenic determinants at the cell surface.

Acetylcholine may regulate its own nicotinic receptor-channel through the C-kinase system.

Acetylcholine (ACh)-activated channel properties were examined on an aneural culture of chick embryo myotubes by using patch-clamp techniques. Changes in conductance, open time and closed time were induced by the selective activator of the calcium- and phospholipid-dependent C-kinase (PKc), 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of TPA was mimicked by exogenous phospholipase C and was blocked by the PKc inhibitor, 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine. In addition to its gating action, ACh was shown to stimulate phosphoinositide turnover and to translocate PKc from the cytosol to the cell membrane. Both these ACh-induced effects were inhibited by curare and not substantially affected by atropine. Bath-applied ACh outside the patch-pipette in the cell-attached patch-clamp mode, had a strong effect on the ACh-activated channels in the patch membrane, in a way that resembled the action of TPA. These findings raise the possibility that ACh regulates its own nicotinic receptors through the C-kinase system.

Proteins of the membrane skeleton in rat Sertoli cells.

Analogues of the alpha, beta and gamma subunits of human spectrin and erythroid protein 4.1 have been detected in rat Sertoli cell primary cultures. Immunofluorescence of permeabilized cells showed that erythroid type spectrin, protein 4.1 and actin co-distribute within the cells as filamentous structures. Fodrin-like molecules were distributed in a diffuse manner, mostly associated with the plasma membrane. Immunoprecipitation and immunoblotting experiments indicated that the polypeptides present in rat Sertoli cells are immunologically related and display molecular weights similar to their analogues in the human erythroid and non-erythroid membrane skeleton.

Cyclic AMP regulates the life time of acetylcholine-activated channels in cultured myotubes.

'Giga-seal' patch-clamp recording was performed in embryonic chick myotubes at day 3 to 4 of culture. Myotubes were exposed to agents that enhance the concentration of cytosolic cyclic AMP (cAMPi) and their action on acetylcholine- (ACh) activated channels was investigated. While the conductance and the closed time was unaffected by forsokolin, cholera toxin, dibutyryl cyclic AMP and 8-bromo-cyclic AMP, these agents lengthened the ACh-activated channel life time with efficacy that paralleled with their capability to increase the cAMPi.

Acetylcholine stimulates phosphatidylinositol turnover at nicotinic receptors of cultured myotubes.

Acetylcholine treatment of [3H]inositol pre-labelled cultured chick embryo myotubes results in the stimulation of phosphatidylinositol breakdown, as shown by the measurement of inositol-1-phosphate accumulating in the presence of lithium. The described effect is dependent on agonist concentration and incubation time, and is inhibited by tubocurarine and alpha-bungarotoxin. The activation of phosphatidylinositol breakdown by acetylcholine at extrajunctional nicotinic receptors is likely to be involved in the modulation of the functional activity of the receptor.

Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes.

We have examined acetylcholine (ACh)-elicited potentials or currents in current- or voltage-clamped cultured myotubes exposed to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter that activates protein kinase C. Although this agent had little action on either membrane resting potential or electrical resistance, a reversible decrease in ACh sensitivity was induced on 3-4-d-old chick myotubes. Depression of transmitter action by TPA was extended to 7-8-d mouse myotubes only when they were treated with phosphatidylserine. Glyceryl dioleate had effects on myotubes similar to those of TPA but with a reduced efficacy. We conclude that the activation of protein kinase C might be involved with the capacity of ACh receptors to respond to transmitter stimulation.

Enhanced synthesis of a specific protein by 12-O-tetradecanoylphorbol-13-acetate in cultured chick embryo muscle cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces in cultured postmitotic myotubes specific alterations of synthesized proteins as revealed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis after pulse labeling with [35S]methionine. Synthesis of myosin heavy chain is remarkably inhibited after exposure to 1.6 X 10(-7) M TPA for periods of 9 hr or longer. During shorter periods of TPA treatment (2 hr), an enhanced synthesis of a Mr 31,000 polypeptide is observed, which is associated with the particulate fraction of cultured myotubes. "Pulse chase" experiments show that this polypeptide is not a degradation product induced by TPA. The stimulation of Mr 31,000 polypeptide requires simultaneous RNA synthesis, since actinomycin D completely and selectively abolishes [35S]methionine incorporation into this polypeptide. The stimulation of Mr 31,000 polypeptide is a transient biosynthetic event not detectable after prolonged incubation (24 hr) of myotubes with the tumor promoter. However, TPA-containing medium preincubated with cultures for up to 24 hr induces stimulation of Mr 31,000 polypeptide when administered to untreated cultures. The early stimulatory effect on Mr 31,000 polypeptide synthesis and the late inhibitory effects on contractile protein synthesis are also observed when postmitotic, unfused myoblasts, rather than myotubes, are treated with TPA.

Changes of Sertoli cell glycoproteins induced by removal of the associated germ cells.

Sertoli cell glycoproteins were studied in culture where these cells were in contact with germ cells (Sertoli cell enriched cultures, SCEC) and in pure Sertoli cell cultures. Sertoli cell only cultures (SCOC) were prepared by a short treatment of SCEC with hypotonic solution or by culturing seminiferous epithelium fragments from prenatally irradiated rats. After metabolic labeling with [3H]fucose. [14C]N-acetylglucosamine or [3H]leucine, SCEC and SCOC particulate fractions (105 000 g pellet) were analyzed by one-dimensional slab gel electrophoresis and fluorography. The comparison of the electrophoretic patterns obtained, demonstrated that a glycoprotein of MW 48 000, undetectable in SCEC, was present in SCOC after labelling with both sugar precursors. The MW 48 000 glycoprotein was also present in the electrophoretic profile of particulate fraction from [3H]fucos-labelled Sertoli cell cultures from prenatally irradiated rat. Such difference was not observed after labelling with [3H]leucine; in this experimental condition a MW 48 000 band was present in the electrophoretic profile of polypeptides from SCEC as well from SCOC. The synthesis of this glycoprotein represented a specific and stable cell response, since it occurred only a few hours after germ cell removal, and it was still detectable 3 days later. FSH stimulation did not influence the synthesis of the MW 48 000 glycoprotein, whereas it increased the synthesis of high MW glycoproteins. The hypothesis is discussed that the appearance of a new glycoprotein when Sertoli cells have lost their contact with germ cells could represent a product of glycosylation of preexisting molecules and their possible location in the Sertoli cell membrane. The results presented here provide additional evidence that Sertoli cell functions may be dependent on the association with the germ cell.

Early alteration induced by tumor promoters on chick embryo muscle cells in culture.

When differentiated, multinucleated cultured myotubes are treated with PMA (Phorbol-12-myristate-13-acetate), they display drastic morphological alterations and undergo inhibition of the expression of differentiative traits, without being induced to reenter the cell cycle. Differentiated myotubes, obtained after cytochalasin B treatment of primary chick embryo myoblast cultures, were treated with 1.6 x 10(7) M PMA for different times, labelled with 35S-methionine and different fractions of cell extracts were analysed by SDS-PAGE followed by fluorography. The data presented here indicate that PMA treatment induces in myotubes increased synthesis of a 31.000 Mr polypeptide (31 K) within 4 hr of treatment, while the inhibition of the synthesis of contractile proteins, such as myosin and actin, occurs only after 8 hr of treatment. Morphological alterations of myotubes require longer incubation with PMA (15-20 hr). The reported effects of PMA are not induced by non tumor promotor analogs of the drug, and pulse chase experiments indicate that 31 K stimulation is not the result of increased protein degradation induced by PMA. In addition the stimulation of 31K does not occur in cultured fibroblast indicating that this is a specific early response of differentiated myogenic cells preceeding the dedifferentiative effect of this tumor promotor.