Antisense oligonucleotides as inhibitors of signal transduction: development from research tools to therapeutic agents.
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Animals↗
Biomedical subjects
D Fabbro
Publications and source records attributed to D Fabbro.
Second-generation antisense oligonucleotides: structure-activity relationships and the design of improved signal-transduction inhibitors.
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Animals↗
Inhibition of the growth of glioblastomas by CGP 41251, an inhibitor of protein kinase C, and by a phorbol ester tumor promoter.
Protein kinase C (PKC) plays a central role in signal transduction pathways that mediate the action of certain growth factors, tumor promoters, and cellular oncogenes. To explore whether PKC might be an appropriate target for the chemotherapy of human brain tumors, cell lines were established from five glioblastomas, one mixed gliosarcoma and glioblastoma, two astrocytomas, and one choroid plexus carcinoma. The staurosporine derivative CGP 41251, an inhibitor of PKC, inhibited cell proliferation in all nine cell lines with an IC50 in the range of 0.4 micrometer. Drug withdrawal and clonogenicity assays showed that CGP 41251 induced an irreversible growth arrest. Three cell lines were examined in detail: two human glioblastoma cell lines, GB-1 and GB-2, and one gliosarcoma cell line, GS-1. All of these three cell lines were highly aneuploid and displayed morphologies and immunohistochemical markers characteristic of the glial lineage. The compound 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter and activator of PKC, also inhibited the growth of these cell lines. CGP 41251 in combination with TPA caused further growth inhibition. Cultures treated with CGP 41251 displayed an increase in the fraction of cells in G2-M, a decrease of cells in S phase, and no consistent effect on G0-G1. Immunohistochemical analyses demonstrated that growth inhibition by CGP 41251 was associated with the formation of giant nuclei with extensive fragmentation and apoptotic bodies. These effects of CGP 41251 were abrogated by withdrawal of serum from the medium or by exposure of these cells to aphidicolin, actinomycin D, cycloheximide, or TPA. In contrast to the effects seen with the glioblastoma cell lines, nontransformed astrocyte lines remained viable in the presence of 0.4 and 0.8 micrometer CGP 41251 and displayed only a slight increase in the fraction of giant nuclei with fragmentation. The antitumor activity of CGP 41251 was demonstrated in vivo against xenografts of the glioblastoma cell lines U87 MG and U373 MG. These findings suggest that CGP 41251 might be a useful agent for the treatment of glioblastomas.
Aged↗
Different susceptibility of protein kinases to staurosporine inhibition. Kinetic studies and molecular bases for the resistance of protein kinase CK2.
A systematic analysis reveals that out of 20 protein kinases examined, specific for either Ser/Thr or Tyr, the majority are extremely sensitive to staurosporine, with IC50 values in the low nanomolar range. A few of them however, notably protein kinases CK1 and CK2, mitogen-activated protein (MAP) kinase and protein-tyrosine kinase CSK, are relatively refractory to staurosporine inhibition, exhibiting IC50 values in the micromolar range. With all protein kinases tested, namely PKA, CK1, CK2, MAP kinase (ERK-1), c-Fgr, Lyn, CSK and TPK-IIB/p38Syk, staurosporine inhibition was competitive with respect to ATP, regardless of its inhibitory power. In contrast, either uncompetitive or noncompetitive kinetics of inhibition with respect to the phosphoacceptor substrate were exhibited by Ser/Thr and Tyr-specific protein kinases, respectively, consistent with a different mechanism of catalysis by these two sub-families of kinases. Computer modeling based on PKA crystal structure in conjunction with sequence analysis suggest that the low sensitivity to staurosporine of CK2 may be accounted for by the bulky nature of three residues, Val66, Phe113 and Ile174 which are homologous to PKA Ala70, Met120 and Thr183, respectively. In contrast these PKA residues are either conserved or replaced by smaller ones in protein kinases highly sensitive to staurosporine inhibition. On the other hand, His160 which is homologous to PKA Glu170, appears to be responsible for the unique behaviour of CK2 with respect to a staurosporine derivative (CGP44171A) bearing a negatively charged benzoyl substituent: while CGP44171A is 10- 100-fold less effective than staurosporine against PKA and most of the other protein kinases tested, it is actually more effective than staurosporine for CK2 inhibition, but it looses part of its efficacy if it is tested on a CK2 mutant (H160D) in which His160 has been replaced by Asp. It can be concluded from these data that the catalytic sites of protein kinases are divergent enough as to allow a competitive inhibitor like staurosporine to be fairly selective, a feature that can be enhanced by suitable modifications designed based on the structure of the catalytic site of the kinase.
Alkaloids↗
The phosphodiesterase inhibitor SQ 20006 selectively blocks mitogen activation of p70S6k and transition to S phase of the cell division cycle without affecting the steady state phosphorylation of eIF-4E.
In quiescent cells high levels of protein synthesis are required in order to re-enter the cell cycle upon stimulation. Initiation of polypeptide synthesis is the step most often subject to regulation, controlled in part by phosphorylation of 40 S ribosomal protein S6 and a number of initiation factors. The kinase responsible for S6 phosphorylation is p70S6k. We now show that the p70S6k pathway can be selectively blocked by the aminopurine analogue, SQ 20006. This agent is known to raise cAMP levels, resulting in activation of protein kinase A. We present evidence that the increase in cAMP is not responsible for the inhibitory effect observed. We also show that SQ 20006 can prevent the activation of p70S6k in a rapid and reversible manner. The compound does not exert its inhibitory activity on p70S6k but can inhibit in vitro two protein kinase C isozymes (alpha and gamma). In a B lymphoblastoid cell line, treatment with SQ 20006 results in inhibition of protein synthesis at the initiation stage. In contrast, when tested directly upon the translational machinery in the reticulocyte lysate, inhibition is manifest at both the level of initiation and elongation. The role of protein kinase A in the modulation of p70S6k and the rate of translation is discussed.
1-Methyl-3-isobutylxanthine↗
Definition of the DNA-binding specificity of TTF-1 homeodomain by chromatographic selection of binding sequences.
The homeodomain of the thyroid transcription factor-1 (TTF-1HD) shows a peculiar DNA-binding specificity, preferentially recognizing sequences having the 5'-CAAG-3' core motif. In order to detail the DNA-binding specificity of this protein, a TTF-1HD-Sepharose column chromatography was used. A sequential selection and amplification of sequences was performed. TTF-1HD binding activity for selected and unselected sequences was measured. The presence of the 5'-CAAG-3' core motif was necessary, but not sufficient, to obtain the maximal binding activity for TTF-1HD. However, several of the selected sequences do not contain the 5'-CAAG-3' core motif and are bound by TTF-1HD only 2-fold less with respect to sequences bound with the highest affinity. Therefore, these data indicate that TTF-1HD specifically recognizes a spectrum of sequences wider than previously determined.
Base Sequence↗
PKC zeta is a molecular switch in signal transduction of TNF-alpha, bifunctionally regulated by ceramide and arachidonic acid.
Tumor necrosis factor (TNF-alpha) stimulates a number of signal transduction pathways in which phospholipases produce lipid second messengers. However, the immediate molecular targets of these messengers, in particular those of ceramide and arachidonic acid (AA) and their role in TNF signaling are not well defined. In this study we investigated the relationship of ceramide and AA in regulating an atypical PKC isozyme, PKC zeta. U937 cells responding to TNF-alpha treatment with NF kappa B activation displayed enhanced phosphorylation of PKC zeta, which is already detectable 30 s after stimulation. [14C]ceramide specifically binds to and regulates kinase activity of PKC zeta in a biphasic manner. Binding studies indicate high and low affinity binding with bmax values of 60 and 600 nM and Kd values of 7.5 and 320 nM respectively. At ceramide concentrations as low as 0.5 nM an up to 4-fold increase in autophosphorylation is obtained, which, at concentrations > 60 nM, again declines to basal levels. Interestingly, AA competes for ceramide binding and inhibits basal and ceramide-stimulated PKC zeta kinase activity at < 100 nM. Metabolism of [14C]ceramide in cells is slow and is inhibited in the presence of equimolar concentrations of lyso-phosphatidylcholine. Based on the bifunctional modulation of PKC zeta by the lipid messengers ceramide and AA, a model of TNF signal pathways is suggested in which PKC zeta takes a central position, acting as a molecular switch between mitogenic and growth inhibitory signals of TNF-alpha.
Arachidonic Acid↗
Platelet-derived growth factor and angiotensin II stimulate the mitogen-activated protein kinase cascade in renal mesangial cells: comparison of hypertrophic and hyperplastic agonists.
Exposure of mesangial cells to platelet-derived growth factor (PDGF) BB caused a significant stimulation of cell proliferation and protein synthesis, as measured by [3H]thymidine incorporation and [3H]leucine incorporation respectively. In contrast, cells treated with angiotensin II had no significant increase in [3H]thymidine incorporation, but demonstrated a marked increase in [3H]leucine incorporation. Furthermore, angiotensin II significantly increased total protein content per cell. These data show that, whereas PDGF-BB is a mitogen and stimulates mesangial-cell hyperplasia, angiotensin II causes hypertrophy of the cells without hyperplasia. Treatment of mesangial cells with PDGF and angiotensin II rapidly and dose-dependently stimulated mitogen-activated protein (MAP) kinase activity, as shown by an assay for activity in vitro using myelin basic protein as a substrate, and by immunoprecipitation of 32P-labelled cells with specific antibodies against the 42 kDa and 44 kDa mitogen-activated protein kinases p42mapk and p44mapk, respectively. Whereas stimulation with PDGF-BB caused a potent and sustained (for more than 30 min) phosphorylation and activation of p42mapk and p44mapk, as well as of the upstream activators MAP kinase kinase and c-Raf, the effect of angiotensin II was less potent, reaching a peak at 5-10 min and thereafter declining rapidly. In summary, these results suggest that PDGF-BB and angiotensin II differ in their potency and duration of activation of the MAP kinase cascade, which may explain why PDGF-BB is a potent mitogen for mesangial cells, whereas angiotensin II only triggers mesangial-cell hypertrophy.
Amino Acid Sequence↗
Platelet-derived growth factor stimulates de-novo synthesis of mitogen-activated protein kinase in renal mesangial cells.
Platelet-derived growth factor (PDGF) BB is a potent mitogen for renal mesangial cells and stimulates a biphasic mitogen-activated protein kinase (MAP kinase) activation. A rapid increase in activity (maximal at 10 min) is followed by a lower persistent level of activity which is maximal at 4-6 h. The second peak of MAP kinase activity is markedly attenuated by the protein synthesis inhibitor cycloheximide and, consequently, is paralleled by a marked de-novo synthesis of p42 and p44 MAP kinases, as measured by immunoprecipitation of [35S]methionine-labeled mesangial cells and by a 700% increase in total MAP kinase protein, as detected by Western-blot analysis. A 30-min treatment with PDGF-BB is sufficient to induce pronounced de-novo synthesis of MAP kinase. However, for maximal induction of MAP kinase synthesis, PDGF is required to be present for at least 4 h. In addition, an increased de-novo synthesis of MAP kinase kinase, the upstream activator of MAP kinase, is observed in response to PDGF stimulation. We propose that PDGF-induced de-novo synthesis of MAP kinase and MAP kinase kinase is important for the potent mitogenic activity of this growth factor.
Amino Acid Sequence↗
NDF/heregulin activates MAP kinase and p70/p85 S6 kinase during proliferation or differentiation of mammary epithelial cells.
Neu differentiation factors (NDF) are a novel family of polypeptide factors which activate sub-class I tyrosine kinase receptors. In all mammary epithelial cells analysed in this study, NDF activates the same signalling pathways while it induces different, cell-specific biological effects. In AU565 cells which are growth inhibited, as well as in T47D or HC11 cells which proliferate in response to NDF, the MAP kinase isoforms p44ERK1 and p42ERK2 and the p70/p85 S6 kinase are activated. NDF stimulates tyrosine phosphorylation and the in vitro kinase activity of ErbB-2. When PKC is activated by TPA, NDF is no longer able to activate ErbB-2 in T47D cells, leading to a blockage of cell proliferation. Activation of ErbB-2 by point mutation, or by monoclonal antibodies, also stimulates both the MAPK and the p70/p85 S6 kinase pathways. The same monoclonal antibodies can induce AU565 cell differentiation. In summary, during growth or differentiation of mammary epithelial cells, NDF stimulates several independent signalling pathways which can also be triggered by ErbB-2 stimulation alone. PKC activation blocks the biological effect induced by NDF through negative modulation of ErbB-2.
Amino Acid Sequence↗
Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.
The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells.
Amino Acid Sequence↗
[Congenital Chagas disease in the city of Santa Fé. Diagnosis and treatment].
We studied 6123 pregnant women and their 341 newborn (NB), from Santa Fe city, by the following serological tests for chagasic infection: Direct Agglutination with and without 2-mercaptoethanol, Indirect Hemagglutination and Indirect Immunofluorescence test, and by identification of parasites by Fresh drops, Strout and/or by Xenodiagnosis. The prevalence of seropositivity found in pregnant women was of 14.62% with a 73% of migratory history. The parasitological studies yielded 9/341 incidence of transplacentary infection. Clinical examinations were made in the infected newborn (NB). They were treated with Benznidazol or Nifurtimox, and post-treatment evolution was evaluated. We registered connatal infection in twin-brothers. Brothers/sisters (siblings) of infected NB were also studied. Some of them were seropositive and the others seronegative. Results here obtained show that this way of transmission is important, and should be considered even in low endemicity areas. The parasitological assays proved to be decisive for the NB infection diagnosis (Table 1). The serological assays enabled us to follow the non-infected NB up to their negativization. A 6 month follow-up is recommended. It is impossible to define only one clinical outline because both symptomatic and asymptomatic infected NB may be found with gestational age at term and pre-term and when born with a weight above or below 2000 g. We obtained parasitological and serological negativization in all cases. The chagasic pregnant woman does not necessarily transmit the infection to all her descendents. Only 2.64% are infected. It is possible to systematize the diagnosis without extra resources beyond the usual ones.
Animals↗
Expression of thyroid-specific transcription factors TTF-1 and PAX-8 in human thyroid neoplasms.
TTF-1 and PAX-8 are tissue-specific transcription factors expressed in the thyroid follicular cells, contributing to the maintenance of the differentiated phenotype. In fact, it has been demonstrated that TTF-1 and PAX-8 are able to activate transcription from thyroglobulin and thyroperoxidase (TPO) promoters, the transcriptional activity of which is in vivo restricted only to the thyroid follicular cell. In order to gain insight into how these transcription factors control in vivo the differentiation of the thyroid cell and to have a better molecular characterization of human thyroid tumors, TTF-1, PAX-8, thyroglobulin, and TPO mRNA levels were measured in nonmalignant and malignant human thyroid tissues. Results indicate that the expression of TTF-1 and PAX-8 is not sufficient per se for the expression of the thyroid-differentiated phenotype. Furthermore, in follicular adenomas, PAX-8 mRNA levels are strictly related to TPO mRNA levels, suggesting that the amount of PAX-8 could play a role in the modulation of TPO gene expression. TTF-1 mRNA is always well detectable in papillary carcinomas and, in contrast, always absent in anaplastic carcinomas. Identical results were obtained when the expression of TTF-1 protein was investigated using immunohistochemistry. Thus, TTF-1 gene expression could be a molecular marker in order to distinguish these two types of thyroid neoplasms.
Adenocarcinoma, Follicular↗
Comparison of different tumour promoters and bryostatin 1 on protein kinase C activation and down-regulation in rat renal mesangial cells.
The effects of a series of protein kinase C (PKC) activators with different spectra of biological activities and reportedly different patterns of PKC isoenzyme activation were examined in renal mesangial cells. Treatment of mesangial cells with the tumor promoters phorbol 12-myristate 13-acetate (PMA), debromoaplysiatoxin, dihydroteleocidin and thymeleatoxin, as well as with the marine natural product bryostatin 1, caused translocation and at least partial down-regulation of the PKC-alpha, -delta and -epsilon isoenzymes as assessed by immunoblot analysis. Bryostatin 1 mediates a faster depletion of PKC-alpha isoform than any of the other PKC activators. Thymeleatoxin, which has been reported to selectively activate PKC-alpha, -beta and -gamma, but not PKC-delta or -epsilon isoenzymes in vitro, turned out to exert the most potent effect on PKC-delta and -epsilon in mesangial cells and down-regulated these isotypes within 8-24 hr. None of the compounds tested affected cellular distribution or amount of PKC-zeta in mesangial cells. Thus, all of the PKC activators tested are able to translocate and down-regulate three of the four PKC isoenzymes present in mesangial cells although with different kinetics. All PKC activators stimulated a phospholipase A2-mediated arachidonic acid release, a phospholipase D-mediated phosphatidylcholine hydrolysis, a comparable small proliferative response and an inhibition of phospholipase C-mediated inositol trisphosphate generation. These results suggest: (i) that the PKC activators investigated in this study do not display any type of isotype-specificity that could be used to selectively activate or down-regulate PKC isoenzymes in intact cell-systems; (ii) that thymeleatoxin has a different isoenzyme selectivity in intact cells as compared to in vitro enzyme inhibition data; and (iii) PKC-zeta is resistant to all PKC activators investigated in this study.
Angiotensin II↗
Overexpression of protein kinase C beta I in a murine keratinocyte cell line produces effects on cellular growth, morphology and differentiation.
The present study demonstrates that the murine keratinocyte cell line 3PC expresses the Ca(2+)-insensitive isoforms of protein kinase C (PKC) delta, epsilon, zeta and (at both the mRNA and protein levels), but does not express the Ca(2+)-sensitive PKC isoforms alpha, beta or gamma. Recombinant retroviral gene transduction was used to develop derivatives of this cell line that stably express high levels of 1 PKC beta I-related transcripts and proteins, and have increased levels of Ca(2+)-stimulated PKC enzyme activity. Functional overexpression of the PKC beta I isoform in 3PC cells enhances both 12-O-tetradecanoyl phorbol-13-acetate-induced growth inhibition, and Ca(2+)-induced morphologic differentiation.
Animals↗
Sequence-specific DNA recognition by the thyroid transcription factor-1 homeodomain.
The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodomain (TTF-1HD) has been investigated. Methylation and ethylation interference experiments show that the TTF-1HD alone recapitulates the DNA binding properties of the entire protein. Studies carried out with mutant derivatives of TTF-1HD indicate a precise correspondence of some of its amino acid residues with specific bases in its binding site, allowing a crude orientation of the TTF-1HD within the protein-DNA complex. TTF-1HD shows an overall geometry of interaction with DNA similar to that previously observed for Antennapedia class HDs, even though the binding specificities of these two types of HDs are distinct. We demonstrate that the crucial difference between the binding sites of Antennapedia class and TTF-1 HDs is in the motifs 5'-TAAT-3', recognized by Antennapedia, and 5'-CAAG-3', preferentially bound by TTF-1. Furthermore, the binding of wild type and mutants TTF-1 HD to oligonucleotides containing either 5'-TAAT-3' or 5'-CAAG-3' indicate that only in the presence of the latter motif the Gln50 in TTF-1 HD is utilized for DNA recognition. Since the Gln at position 50 is an essential determinant for DNA binding specificity for several other HDs that bind to 5'-TAAT-3' containing sequences, we suggest that utilization by different HDs of key residues may depend on the sequence context and probably follows a precise hierarchy of contacts.
Antennapedia Homeodomain Protein↗
Hypoxia-induced accumulation of erythropoietin mRNA in isolated hepatocytes is inhibited by protein kinase C.
To define the role of protein kinase C (PKC) in oxygen-dependent production of erythropoietin (EPO) in the liver, we have determined EPO messenger ribonucleic acid (mRNA) expression in primary cultures of juvenile rat hepatocytes incubated at different oxygen tensions in the absence and presence of phorbol esters, vasopressin, and structurally different kinase inhibitors. Upon reduction of oxygen concentrations from 40% to 3% EPO mRNA in cultured hepatocytes increased markedly within 1.25 h, reached maximal values after 2.5 h and remained elevated for up to 72 h. Treatment of hepatocytes during 1.25-5 h of hypoxic exposure with phorbol 12-myristate-13 acetate (PMA) attenuated hypoxia-induced EPO mRNA levels dose-dependently by a maximum of approximately 50%. This inhibitory effect of PMA disappeared upon treatment for more than 5 h and was completely lost after incubation for 9 and 18 h in the presence of 10(-6) M and 10(-7) M PMA, respectively. Phorbol 12,13-dibutyrate and vasopressin also inhibited EPO mRNA accumulation, whereas 4 alpha-phorbol 12,13-didecanoate was ineffective. Western blot analysis of PKC isozymes revealed the presence of PKC alpha, beta II, delta, epsilon and zeta and provided no evidence that the PMA-induced inhibition of EPO expression was associated with depletion of any of these isozymes. Conversely, PMA-induced inhibition of EPO mRNA accumulation was paralleled by translocation of PKC alpha from cytosol to membranes and the time- and dose-dependent attenuation of the inhibitory effect of PMA on EPO mRNA levels was paralleled by down-regulation of PKC alpha. A dose-dependent inhibition of EPO mRNA formation, independent of effects on total RNA synthesis, as determined by [3H]uridine incorporation, was also found in the presence of the kinase inhibitor staurosporine (ED50 approximately 2 x 10(-8) M) and three structurally related derivatives with increased selectivity for PKC (RO 317549, ED50 approximately 1 x 10(-6) M; RO 318220, ED50 approximately 1 x 10(-6) M and CGP 41251, ED50 approximately 4 x 10(-6) M). The markedly lower potency of the latter three compounds as compared to staurosporine suggests that this suppression of EPO gene induction was not mediated by inhibition of PKC. In summary the data indicate that PKC alpha is a negative modulator of EPO gene expression in hepatocytes. A kinase other than PKC, however, appears to be an essential element of hypoxic signalling.
Alkaloids↗
Protein kinase C isozyme expression and down-modulation in growing, quiescent, and transformed renal proximal tubule epithelial cells.
Renal alpha-protein kinase C (PKC) is rapidly down-modulated modulated in animals treated with the renal toxin and tumor promoter, folic acid (Dong et al., Cancer Res., 53: 4542-4549, 1993). To further explore the role of PKC isozymes in renal growth and carcinogenesis, we compared phorbol ester receptor and PKC isozyme content, distribution, and regulation in primary and oncogene-altered rat renal proximal tubule epithelial cells (RPTE) in culture. Immunoblot analysis and RNase protection assays indicated that RPTE expressed at least four PKC isozymes, alpha, delta, epsilon, and zeta. Total phorbol ester receptors were decreased in primary proliferating, E1A-immortalized, and SV40-transformed RPTE compared to primary quiescent RPTE. The decrease in PDBu binding was largely due to a specific decrease in alpha-PKC protein content to approximately 50% of the level in quiescent RPTE. Degradation rates and message levels were compared to determine the mechanism for the decrease in alpha-PKC. Whereas alpha-PKC message levels in quiescent and proliferating primary RPTE were comparable, alpha-PKC degradation was increased in proliferating cells. These results indicate that the decreased alpha-PKC content was due largely to increased turnover. Phorbol ester stimulated the rate of degradation, thus demonstrating a link between degradation rate and PKC activation. These results suggest that the increased basal degradation rate in proliferating and oncogene-altered cells reflects an increase in activity of PKC in these cells.
Animals↗