Myc down-regulation induces apoptosis in M14 melanoma cells by increasing p27(kip1) levels.

In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the melanoma pathogenesis. We have previously demonstrated that treatment of melanoma cells with c-myc antisense oligodeoxynucleotides can inhibit cell proliferation and activate apoptosis. To gain insight into the mechanisms activated by Myc down-regulation, we have now developed an experimental model that allows modulating Myc protein expression in melanoma cells. This was achieved by originating stable melanoma cell clones expressing ecdysone-inducible c-myc antisense RNA. We show that the induction of c-myc antisense RNA in M14 melanoma cells leads to an inhibition of cell proliferation characterized by accumulation of cells in the G(1) phase of the cell cycle (up to 80%) and activation of apoptosis (50%). These data are associated with an increase of p27(kip1) levels and a significant reduction of the cdk2-associated kinase activity. In addition, we show that an ectopic overexpression of p27(kip1) in this experimental model can enhance the apoptotic rate. Our results indicate that down-regulation of Myc protein induces a G(1) arrest and activates apoptosis by increasing p27(kip1) content in melanoma cells, that are known to be defective for the p16-cyclinD/cdk4-pRb G(1) checkpoint. This is particularly relevant for identifying new therapeutic strategies based on the re-establishment of the apoptotic pathways in cancer cells.

The length of telomeric G-rich strand 3'-overhang measured by oligonucleotide ligation assay.

A typical G-rich telomeric DNA strand, which runs 5'-->3' toward the chromosome ends, protrudes by several nucleotides in lower eukaryotes. In human chromosomes long G-rich 3'-overhangs have been found. Apart from the standard G-rich tail, several non-canonical terminal structures have been proposed. However, the mechanism of long-tail formation, the presence and the role of these structures in telomere maintenance or shortening are not completely understood. In a search for a simple method to accurately measure the 3'-overhang we have established a protocol based on the ligation of telomeric oligonucleotide hybridized to non-denatured DNA under stringent conditions (oligonucleotide ligation assay with telomeric repeat oligonucleotide). This method enabled us to detect a large proportion of G-rich single-stranded telomeric DNA that was as short as 24 nt. Nevertheless, we showed G-tails longer than 400 nt. In all tested cells the lengths ranging from 108 to 270 nt represented only 37% of the whole molecule population, while 56-62% were <90 nt. Our protocol provides a simple and sensitive method for measuring the length of naturally occurring unpaired repeated DNA.

TNF-alpha and IFN-gamma regulate expression and function of the Fas system in the seminiferous epithelium.

Sertoli cells have long been considered to be involved in the regulation of the immune response in the testis. More recently, the Fas system has been implicated in the maintenance of the immune privilege in the testis as well as in the regulation of germ cell apoptosis. However, the control of Fas and Fas ligand (FasL) expression in the testis remains unknown. In the present study, we demonstrate that cultured mouse Sertoli cells constitutively express a low level of membrane-bound Fas protein, but not a soluble form of Fas. Sertoli cells stimulated with TNF-alpha and IFN-gamma markedly increase the expression of both soluble and membrane-bound Fas in a dose-dependent manner. The up-regulated membrane-bound Fas protein is functionally active because it induces a significant level of Sertoli cell death in the presence of Neuro-2a FasL+ effector cells. Interestingly, the soluble form of Fas, which is induced by the same cytokines but has an antiapoptotic effect, is also functional. In fact, conditioned media from TNF-alpha-stimulated Sertoli cell cultures inhibit Neuro-2a FasL+-induced cell death. Taken together, our data suggest a possible regulatory role of TNF-alpha and IFN-gamma on Fas-mediated apoptosis in the testis through disruption of the balance between different forms of Fas.

Arrest of G(1)-S progression by the p53-inducible gene PC3 is Rb dependent and relies on the inhibition of cyclin D1 transcription.

The p53-inducible gene PC3 (TIS21, BTG2) is endowed with antiproliferative activity. Here we report that expression of PC3 in cycling cells induced accumulation of hypophosphorylated, growth-inhibitory forms of pRb and led to G(1) arrest. This latter was not observed in cells with genetic disruption of the Rb gene, indicating that the PC3-mediated G(1) arrest was Rb dependent. Furthermore, (i) the arrest of G(1)-S transition exerted by PC3 was completely rescued by coexpression of cyclin D1 but not by that of cyclin A or E; (ii) expression of PC3 caused a significant down-regulation of cyclin D1 protein levels, also in Rb-defective cells, accompanied by inhibition of CDK4 activity in vivo; and (iii) the removal from the PC3 molecule of residues 50 to 68, a conserved domain of the PC3/BTG/Tob gene family, which we term GR, led to a loss of the inhibition of proliferation as well as of the down-regulation of cyclin D1 levels. These data point to cyclin D1 down-regulation as the main factor responsible for the growth inhibition by PC3. Such an effect was associated with a decrease of cyclin D1 transcript and of cyclin D1 promoter activity, whereas no effect of PC3 was observed on cyclin D1 protein stability. Taken together, these findings indicate that PC3 impairs G(1)-S transition by inhibiting pRb function in consequence of a reduction of cyclin D1 levels and that PC3 acts, either directly or indirectly, as a transcriptional regulator of cyclin D1.

Activation of Jun N-terminal kinase/stress-activated protein kinase pathway by tumor necrosis factor alpha leads to intercellular adhesion molecule-1 expression.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. We have previously shown that mouse Sertoli cells respond to TNF-alpha by increasing interleukin-6 production and intercellular adhesion molecule-1 (ICAM-1) expression (1). In this cell type TNF-alpha activates the mitogen-activated protein kinase (MAPK) pathways p42/p44 MAPK, JNK/SAPK, and p38, the last of which is responsible for interleukin-6 production (1). To determine which MAPK signaling pathway is required for TNF-alpha induction of ICAM-1 expression, we have utilized the protein kinase inhibitor dimethylaminopurine, demonstrating that treatment of Sertoli cells with such compound significantly reduced ICAM-1 expression and JNK/SAPK activation. Moreover, dimethylaminopurine treatment increased the expression of MAPK phosphatase-2, providing a possible mechanism of action of this compound. By using agonist antibodies to p55 and to p75 TNF-alpha receptors and both human and mouse TNF-alpha, we demonstrate that both TNF receptors are expressed and that only the p55 receptor is involved in ICAM-1 expression. The p55 receptor activates all of the three pathways, whereas p75 failed to activate any of the MAPKs. Altogether our results demonstrate that TNF-alpha up-regulates ICAM-1 expression through the activation of the JNK/SAPK transduction pathway mediated by the p55 receptor.

Activated alpha2macroglobulin increases beta-amyloid (25-35)-induced toxicity in LAN5 human neuroblastoma cells.

The presence of the alpha2macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2Mr/LRP) and its ligands alpha2macroglobulin (alpha2M), apoliprotein E, and plasminogen activators was detected in senile plaques of Alzheimer's disease (AD). To explore a possible role of alpha2M in neurodegenerative processes occurring in AD, we analyzed the effect of alpha2M on Abeta 25-35-induced neurotoxicity. Treatment of LAN5 human neuroblastoma cells with 10 microM beta-amyloid peptide fragment 25-35 (Abeta 25-35) for 72 h resulted in a 50% decrease in cell viability as determined by MTT incorporation and cell counts. The addition of alpha2M to the culture medium of these cells did not determine any effect, but when the activated form alpha2M* was used a dose-dependent decrease in cell viability was observed, the maximum effect being reached at 140 and 280 nM. Moreover, treatment of LAN5 cells with alpha2M* in combination with Abeta 25-35 increased the neurotoxicity of the amyloid peptide by 25%. This neurotoxic effect of alpha2M* seems to be related to its capability to bind and inactivate TGFbeta in the culture medium, since it was mimicked by a TGFbeta neutralizing antibody. A possible involvement of receptor-mediated endocytosis was ruled out, since alpha2M receptor is not present on LAN5, as revealed by RT-PCR and Western blotting experiments. The presence of alpha2M* in amyloid deposits of Alzheimer's disease has been recently reported and a possible impairment of LRP internalization processes has been hypothesized. Our data suggest that the local accumulation of alpha2M* in AD plaques may increase Abeta 25-35-induced neurotoxicity by neutralizing TGFbeta-mediated neuroprotective mechanisms.

Interferons antagonize gamma-ray-induced depression of natural immunity.

PURPOSE: The aim of this study was to determine the inhibitory effects of in vitro radiation on the number and function of natural killer (NK) cells and to investigate the capability of interferons (IFNs) to restore the activity of NK, depressed by gamma-rays. METHODS AND MATERIALS: Mononuclear cells (MNC) were obtained from intact or in vitro irradiated (20 Gy) peripheral blood collected from healthy donors. Alternatively, MNC were irradiated (20 Gy) after separation from intact whole blood. The in vitro treatment of MNC with IFNs (alpha, beta, or gamma, 200 UI/ml) was performed at different times after or before radiation. The NK activity (4 h-51Cr release test), the percentage of CD16+/CD56+ cells and apoptosis (cytometric analysis), and binding (microscopic observation) were evaluated on Days 0, 1, 2, and 5 from gamma-ray exposure and IFNs treatment. RESULTS: The in vitro treatment of irradiated MNC with betaIFN after radiation completely reverses the inhibitory effects of gamma-rays on human NK activity. BetaIFN do not reduce the apoptosis induction by radiation and don't modify the number of CD16- or CD56-positive cells. The binding between irradiated effectors and tumor cells (K562) appears partially increased in betaIFN-treated MNC. CONCLUSIONS: The results of the present investigation suggest a possible role of betaIFN in reversing the detrimental effect of radiation on human natural immunity and provide a rational basis for in vivo use of betaIFN in cancer radiotherapy.

Human CD4+ T cell clones produce and release nerve growth factor and express high-affinity nerve growth factor receptors.

BACKGROUND: Increasing evidence shows that nerve growth factor (NGF) plays a role in the complex and fascinating linkage between the nervous and the immune systems due to its ability to modulate functions of several inflammatory cells. OBJECTIVE: To investigate NGF receptor expression and NGF production and release by human CD4+ cells clones, which have primary relevance in modulating inflammatory events through their different subsets of functional phenotypes. METHODS: The expression of NGF and a transmembrane tyrosine kinase (TrkA) was evaluated by immunohistochemistry and flow cytometry analysis in five T(H0), six T(H1), and five T(H2) cell clones derived from human circulating mononuclear blood cells. Moreover, the amount of NGF protein was assessed by measuring the NGF levels in culture supernatants of the T cell clones before stimulation and 48 hours after phytohemagglutinin (PHA) activation by use of an immunoenzymatic assay. RESULTS: Our data have shown that in unstimulated conditions, human CD4+ T cell clones express both immunoreactivity for NGF and the TrkA NGF receptor irrespective of their cytokine profile. Moreover, T(H1) and T(H2) clones, but not T(H0) clones, secrete NGF in basal conditions. PHA activation induces NGF secretion in T(H0) clones and a significant increase of NGF levels in T(H2) (p < 0.05), but not in T(H1) culture supernatants. CONCLUSIONS: Results obtained represent the first evidence of TrkA expression and NGF production and release in human CD4+ cell clones and suggest a possible functional role of NGF in modulating the immune and inflammatory network.

Cell loss and the concept of potential doubling time.

The in vivo infusion of Bromodeoxyuridine (BrdUrd), followed by delayed biopsy and bivariate DNA-BrdUrd flow cytometry, allows the potential doubling time (Tpot) of human tumors to be estimated. According to Steel, the mathematical definition of Tpot is Tpot = ln 2/Kp, where Kp is the rate constant of cell production. All the operative formulas which allow the estimation of Tpot from flow cytometric data derive from this definition. Most authors, however, identify the potential doubling time as the doubling time that the same cell population would exhibit if cell loss were removed. We denote here as T(d)noloss this quantity. Although these two definitions are equivalent in the case of uniform random cell loss, we show, in the framework of Steel's theory of growing cell populations, that Tpot and T(d)noloss become distinct kinetic quantities when cell loss is not uniform, i.e., when loss differently affects the quiescent and the proliferative compartment. We discuss the validity of the two formulas currently used for the calculation of Tpot, one based on LI and the other on the v-function, in conditions of non-uniform cell loss. Moreover, we propose two formulas for the estimation of the cycle time T(C), which require, in addition to T(S) and LI, that a measure of the growth fraction be available.

Modulation of the alpha 2 macroglobulin receptor/low density lipoprotein receptor related protein by interferon-gamma in human astroglial cells.

Alpha 2 macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 Mr/LRP) is a multi-functional cell surface receptor that has been implicated in important processes, such as atherogenesis, cellular migration, immune response and degenerative diseases. Its expression increases in human brain during Alzheimer's disease, tissue injury and neoplastic transformation. In the present paper we studied the regulation of alpha 2 Mr expression by interferon-gamma (IFN gamma) in human astrocytoma cell lines and in fetal astrocytes. Western blots demonstrated an increase of the alpha 2 Mr expression after 24 h of IFN gamma treatment. This effect paralleled the up-regulation of alpha 2 Mr mRNA, as detected by PCR. By prolonging incubation with IFN gamma, we observed a decrement of alpha 2 Mr in IFN gamma treated cells, both by western blot and cytometric analysis. Since in the same cells IFN gamma also up-regulates alpha 2 macroglobulin, this effect may be due to an augmented degradation of the receptor during its recycling.

Overexpression of the nerve growth factor-inducible PC3 immediate early gene is associated with growth inhibition.

PC3 (pheochromocytoma cell-3) is an immediate early gene isolated as sequence induced in the rat PC12 cell line during neuronal differentiation by nerve growth factor (NGF). PC3, which is expressed in vivo in the neuroblast when it ceases proliferating and differentiates into a neuron, has partial homology with two antiproliferative genes, BTG1 and Tob. Here we report that overexpression of PC3 in NIH3T3 and PC12 cells leads to marked inhibition of cell proliferation. In stable NIH3T3 clones expressing PC3, the transition from G1 to S phase was impaired, whereas the retinoblastoma (RB) protein was detected as multiple isoforms of M(r) 105,000-115,000 (indicative of a hyperphosphorylated state) only in low-density cultures. Such findings are consistent with a condition of growth inhibition. Thus, PC3 might be a negative regulator of cell proliferation, possibly acting as a transducer of factors influencing cell growth and/or differentiation, such as NGF, by a RB-dependent pathway. This is the first evidence of a NGF-inducible immediate early gene displaying antiproliferative activity.

An N-terminal domain shared by Fas/Apo-1 (CD95) soluble variants prevents cell death in vitro.

Fas/Apo-1 molecule, also designated as CD95, is a member of the TNF receptor family. Fas cross-linking by its natural ligand or by agonistic mAbs results in rapid induction of apoptosis in susceptible cells. in addition to the Fas full-length mRNA, human activated PBMC and tumor cell lines express several mRNA Fas variants that derive from alternative splicing of the primary transcript. All five variants identified, two of which are newly described here, code for soluble proteins that, with the exception of FasTMDel, are truncated in the extracytoplasmic region and possess short C-terminal amino acid sequences corresponding to a different reading frame. We have identified Abs that recognize all splicing variants and established a sandwich ELISA by which the soluble Fas molecules could be detected in culture supernatants of transfected cell lines and in PBMC following T cell activation. Next, we have studied in detail the functional role of these variants by apoptosis inhibition studies. We found that all soluble proteins block the apoptosis induced by either an agonistic Ab or, more importantly, by the natural Fas ligand in Fas-positive sensitive cell lines. interestingly, this functional property can be assigned to the first 49 amino acids of the mature protein that is the only region shared by the five soluble Fas molecules.

Human microglia cultures: a powerful model to study their origin and immunoreactive capacity.

In this paper, we report that pure cultures of human microglia were obtained from long-term astrocytic cultures of human fetal brain. After five to six months and repeated cell passages, macrophage-like cells started to spontaneously form in vitro, so that in two to three weeks the whole culture was populated by them. These cells were grown up to over 50 passages in culture and analyzed for morphology, specific marker positivity, growth rate and major histocompatibility complex (MHC) antigen expression with or without gamma-interferon (IFN) stimulation. We found that, regardless of embryonic age of original cultures (10-15 weeks of gestation), cultures showed a remarkable homogeneity and purity and over 90 stained for typical microglial markers. Under basal conditions, two cell subpopulations similar to those described in vivo, we observed: the reactive 'ameboid' type and the resting 'ramified' one, the latter increasing with time in vitro and cell passages. Both cell subpopulations were capable of active phagocytosis and of high-rate proliferation. They spontaneously expressed low levels of MHC class II antigens, but were negative for MHC class I. Stimulation with gamma-interferon lymphokine upregulated the MHC class II expression as well as the MHC class I heavy chain form in ameboid, 'reactive' cells but not in the ramified ones. We also found that beta 2 microglobulin, already expressed in basal conditions, was dissociated from HLA A-B-C molecules in lymphokine-stimulated cells at early passages. The physiological significance of these data, as well as the possible correlation with in vivo ontogenetic modifications, are also discussed.

Inflammatory mediators increase surface expression of integrin ligands, adhesion to lymphocytes, and secretion of interleukin 6 in mouse Sertoli cells.

The expression of the cell adhesion molecules ICAM-1, ICAM-2, and VCAM-1 and the secretion of the cytokine interleukin 6 have been measured in mouse Sertoli cells cultured in vitro. Cytometric analysis revealed that, in basal conditions, low levels of ICAM-1 and VCAM-1 were present on the surface of the cells, whereas treatment with interleukin 1, tumor necrosis factor alpha, lipopolysaccharide, or interferon gamma induced, with different kinetics, increases in their expression. ICAM-2 was not detectable in basal conditions, nor was it inducible. Electron microscopic analysis and binding experiments using 51Cr-labeled lymphocytes demonstrated that increased expression of ICAM-1 and VCAM-1 on the surface of Sertoli cells, induced by inflammatory mediators, determines an augmented adhesion between the two cell types. The same stimuli, with the exception of interferon gamma, produced a rapid and remarkable increment of interleukin 6 production by Sertoli cells. These results suggest the presence of both direct and paracrine mechanisms of interaction between Sertoli and immune-competent cells, possibly involved in the control of immune reactions in the testis. Such mechanisms are of interest for the understanding of autoimmune pathologies of the testis and, if confirmed in humans, they could be involved in the sexual transmission of human immunodeficiency virus infection.

Cell cycle analysis by the relative movement approach: effect of variability across S-phase of DNA synthesis rate.

Cell populations pulse-labelled with BrdUrd, and sampled at increasing times after the pulse, yield DNA-BrdUrd distributions from which the relative movement (RM) and the depletion function (DF) of labelled, undivided cells can be calculated. In this paper we present an extension of the equation for the time course of RM, given by White and Meistrich (Cytometry 1986, 7, 486-490), to the case in which the rate of DNA synthesis changes across S-phase. Some modalities of cell loss were also considered. Computer simulations showed that different patterns of DNA synthesis rate across S-phase can result in appreciably different RM curves. An analytical expression of the RM curve, in which the variability across S-phase of the rate of DNA synthesis is accounted for by only one parameter, was proposed. This expression was used for the simultaneous fitting of time sequences of RM and DF data of U937 cells, in order to estimate the phase transit times TS and TG2+M, and the potential doubling time Tpot. The use of the extended model gave better results than those obtained under the assumption of constant rate of DNA synthesis across S-phase.

Cell-cycle progression and apoptosis in K562 and Molt-4 cells after cell-to-cell transmission of HTLV-I: modulation by interferons.

Human T-cell leukemia virus type I (HTLV-I) is mainly propagated by cell division and therefore the virus-driven proliferation of infected cells can represent a predisposing condition to final development of adult T-cell leukemia (ATL) in vivo. To correlate virus expression and cell cycle progression of recipient cells after acute infection with HTLV-I, K562 multipotent erytholeukemia and Molt-4 T-lymphoma cells were used as recipient cells in a cell-to-cell virus transmission model. Cell cycle progression was studied by flow cytometry during one duplication cycle of recipient cells and transcription of HTLV-I was evaluated during the same time course. The antiproliferative and antiviral effects of recombinant interferons alpha, beta and gamma were also evaluated on cell cycle progression and HTLV-I expression. Transcription of HTLV-I in immortalised virus-donor MT-2 T-cells was found to be related to cell cycle. After coculturing recipient K562 or Molt-4 cells with lethally irradiated, non-dividing virus-donor MT-2 cells, progression into cell cycle of recipient cells was delayed. A pre-G(1) peak, corresponding to 6-11 % apoptotic cells, was identified in cocultured Molt-4/MT-2 cells and not in Molt-4 controls, and was not affected by treatment with IFNs. Notably, no such peak was identified either in control or in cocultured K562 cells. During this time course, transcription of the viral subgenomic mRNA encoding for the env-pX region was prevalently observed. Treatment with IFNalpha and especially with IFNbeta at the onset of the cultures inhibited the growth of both control and virus-exposed recipient cells. IFNgamma was less effective. A clearcut reduction of the percentage of cells entering the S phase was observed only after treatment with IFNbeta. At the same time, in IFNbeta-treated cocultures a marked inhibition of transcription of viral mRNA was observed, suggesting that, during acute infection, treatment with IFNbeta contributes to reduce the infection of recipient cells by down-regulating both the cellular proliferation rate and virus transcription in infected cells.

Interferon gamma up-regulates alpha 2 macroglobulin expression in human astrocytoma cells.

An established human astrocytoma cell line (T67) was shown to constitutively produce the proteinase inhibitor alpha 2 macroglobulin (alpha 2M). Interferon gamma (IFN gamma), a potent immunoregulatory lymphokine, was able to increase the synthesis of alpha 2M by these cells, as measured by ELISA on cell supernatants. The alpha 2M induction was also observed in other human glioma cell lines (T70 and ADF) and in human fetal astrocyte cultures following IFN gamma treatment. In T67 cells this effect was dose-dependent and the maximum (2.7-fold increase) was obtained with 2000 U/ml of IFN gamma. A corresponding enhanced alpha 2M mRNA accumulation was demonstrated by PCR and Northern blot techniques. Our results suggest an important role of alpha 2M during inflammatory and immune processes in the CNS. An increased release of alpha 2M following IFN gamma stimulation may allow the removal of the bulk of proteases released at the site of inflammation, strengthening at the same time the antigen presentation processes.

The peptide binding specificity of HLA-B27 subtypes.

Five HLA-B27 subtypes, B*2701, B*2703, B*2704, B*2705, and B*2706, were tested for direct binding with twenty-six synthetic nonapeptides carrying the primary anchor residue motifs (combination of amino residues at positions 2 and 9) relevant to B*2705. The peptide sequences were derived from human HSP89 alpha, P53 and MBP. The alpha chains were immunospecifically isolated from LH (B*2701), CH (B*2703), WE1 (B*2704), BTB (B*2705), and LIE (B*2706) cells and their peptide binding was measured by the HLA class I alpha chain refolding assay. The data obtained indicated that the B27 subtypes tested can bind a common set of peptides carrying several different anchor residue motifs. The motifs, R-K and R-R, reported for B*2705 and a new motif H-R were accepted by B*2703, B*2704, and B*2706, but not by B*2701. However, other motifs, including known B*2702 and/or B*2705 motifs, R-H, R-L, R-A, and R-F, and a new motif found here, R-G, were apparently accepted by all B27 subtypes tested. The observed cross-peptide binding in the B27 subgroup is compatible with the so-called arthritogenic peptide hypothesis in the pathogenesis of ankylosing spondylitis.

Receptor-mediated endocytosis of alpha 2 macroglobulin by a glioma cell line.

Previous experiments have shown that human neoplastic and embryonic glial cell lines synthesize and secrete in culture, alpha 2 macroglobulin (alpha 2M), a broad spectrum proteinase inhibitor present in serum and extracellular fluids. The present study was aimed to investigate the presence of alpha 2M receptors on glial cell membrane, since several non-neural cell types producing alpha 2M also express alpha 2M receptors. By flow cytometric analysis, immunofluorescence and immunoelectronmicroscopy techniques we demonstrate an alpha 2M receptor-related immunoreactivity on the plasma membrane of a human glioma cell line. Ultrastructural experiments reveal a close colocalization of immunoreactivities for alpha 2M and its receptor in clathrin-coated pits and vesicles, structures typically involved in receptor-mediated endocytic pathways.