Biomedical subjects

N Agell

Publications and source records attributed to N Agell.

At least 19 recordsLinked to original sources

PubMed2006

Glyceraldehyde 3-phosphate dehydrogenase is a SET-binding protein and regulates cyclin B-cdk1 activity.

We report here that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts in vitro and in vivo with the protein SET. This interaction is performed through the acidic domain of SET located at the carboxy terminal region. On analysing the functional relevance of SET-GAPDH interaction, we observed that GAPDH reverses in a dose-dependent manner, the inhibition of cyclin B-cdk1 activity produced by SET. Similarly to SET, GAPDH associates with cyclin B, suggesting that the regulation of cyclin B-cdk1 activity might be mediated not only by the interaction of GAPDH with SET but also with cyclin B. To analyse the putative role of GAPDH on cell cycle progression, HCT116 cells were transfected with a GAPDH expression vector. Results indicate that overexpression of GAPDH does not affect the timing of DNA replication but induces an increase in the number of mitosis, an advancement of the peak of cyclin B-cdk1 activity and an acceleration of cell cycle progression. All these results suggest that GAPDH might be involved in cell cycle regulation by modulating cyclin B-cdk1 activity.

CDC2 Protein Kinase↗

PubMed2006

Heterogeneous nuclear ribonucleoprotein A2 is a SET-binding protein and a PP2A inhibitor.

The oncoprotein SET participates in a diversity of cellular functions including cell proliferation. Its role on cell cycle progression is likely mediated by inhibiting cyclin B-cdk1 and the protein phosphatase 2A (PP2A). On identifying new SET cellular partners, we found that SET interacts in vitro and in vivo with the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2); a protein involved in various aspects of mRNA biogenesis. The SET-binding region of hnRNPA2 is the RNP1 sequence that belongs to the RNA-binding domain (RBD) of this protein. We also found that hnRNPA2 has much higher affinity for single-standed DNA than for SET. On analysing the effect of hnRNPA2 on PP2A inhibition by SET, we observed that hnRNPA2 cooperates with SET on PP2A inhibition. This is because we found that hnRNPA2 is also a PP2A inhibitor. HnRNPA2 interacts with PP2A by the RNP1 sequence; however, to inhibit PP2A activity, the complete RBD is needed. We also observed that overexpression of hnRNPA2 inhibits PP2A activity and stimulates cell proliferation. Interestingly, the overexpression of the complete RBD is sufficient to stimulate proliferation. As hnRNPA2 is overexpressed in a variety of human tumors, our results suggest that hnRNPA2 might participate in oncogenesis by stimulating cell proliferation.

Animals↗

PubMed2001

A new bis-indole, KARs, induces selective M arrest with specific spindle aberration in neuroblastoma cell line SH-SY5Y.

KARs, new semisynthetic antitumor bis-indole derivatives, were found to be inhibitors of tubulin polymerization with lower toxicity than vinblastine or vincristine, used in chemotherapy. Here, we compare the effect of KARs with those of vinblastine and vincristine on cell viability, cell proliferation, and cell cycle in neuroblastoma cell line (SH-SY5Y). At concentrations of the different compounds equivalent in causing 50% of inhibition of cell growth, KARs induced a complete arrest in the G2/M phase, whereas vinblastine and vincristine induced a partial arrest in both G0/G1 and G2/M. Moreover, a combination of KAR-2 and W13 (an anticalmodulin drug) qualitatively caused a similar arrest in both G0/G1 and G2/M than vinblastine. Levels of cyclin A and B1 were higher in KARs-treated cells than in vinblastine- or vincristine-treated cells. Cdc2 activity was much higher in KAR-2 than in vinblastine-treated cells, indicating a stronger mitotic arrest. The effect of KAR2 and vinblastine on microtubules network was analyzed by immunostaining with anti-tubulin antibody. Results indicated that KAR-2-induces the formation of aberrant mitotic spindles, with not apparent effect on interphase microtubules, whereas vinblastine partially destroyed interphase microtubules coexisting with normal and aberrant mitotic spindles.

Antineoplastic Agents, Phytogenic↗

PubMed2001

Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling.

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

3T3 Cells↗

PubMed2000

Osmotic stress regulates the stability of cyclin D1 in a p38SAPK2-dependent manner.

We report here that different cell stresses regulate the stability of cyclin D1 protein. Exposition of Granta 519 cells to osmotic shock, oxidative stress, and arsenite induced the post-transcriptional down-regulation of cyclin D1. In the case of osmotic shock, this effect was completely reversed by the addition of p38(SAPK2)-specific inhibitors (SB203580 or SB220025), indicating that this effect is dependent on p38(SAPK2) activity. Moreover, the use of proteasome inhibitors prevented this down-regulation. Thus, osmotic shock induces proteasomal degradation of cyclin D1 protein by a p38(SAPK2)-dependent pathway. The effect of p38(SAPK2) on cyclin D1 stability might be mediated by direct phosphorylation at specific sites. We found that p38(SAPK2) phosphorylates cyclin D1 in vitro at Thr(286) and that this phosphorylation triggers the ubiquitination of cyclin D1. These results link for the first time a stress-induced MAP kinase pathway to cyclin D1 protein stability, and they will help to understand the molecular mechanisms by which stress transduction pathways regulate the cell cycle machinery and take control over cell proliferation.

Arsenites↗

PubMed2000

[Lys61]N-Ras is able to induce full activation and nuclear accumulation of Cdk4 in NIH3T3 cells.

The elements of the cell cycle regulatory machinery activated by the oncogenic form of Ras, [Lys61]N-Ras, have been analysed in NIH3T3 cells. We demonstrate that [Lys61]N-Ras expression is able to induce full cdk4 activation. As already reported, oncogenic Ras expression was sufficient to induce cyclin D1 and p21cip1 expression and their association with cdk4. Furthermore, serum-starved [Lys61]N-Ras NIH3T3 cells showed nuclear accumulation of cyclin D1 and cdk4 not observed in serum-starved NIH3T3 cells. This accumulation of cdk4 into the cell nucleus observed in serum-starved [Lys61]N-Ras NIH3T3 cells was inhibited by a microinjection of neutralizing anti-Ras antibodies. Thus, active [Lys61]N-Ras was a sufficient signal to induce nuclear accumulation of cyclin D1/cdk4, leading to its full activation. Transfection of [Lys61]N-Ras NIH3T3 cells with an inactive form of MEK or their treatment with PD 98059, showed that nuclear translocation of cdk4 was MEK dependent. Interestingly, cells constitutively expressing [Lys61]N-Ras did not inactivate pRb and did not proliferate in the absence of serum. This may be due to the fact that although association of cdk2 with cyclin E and the translocation of those complexes to the nucleus were achieved, [Lys61]N-Ras expression was not sufficient to induce cdk2 activation. The high levels of p27(kip1) that were found in cyclin E/cdk2 complexes may be responsible for the inability of oncogenic Ras to activate this kinase. In consequence, oncogenic alterations that lead to a decrease in p27kip1 bound to cyclin E may cooperate with Ras to induce full cdk2 activation, pRb inactivation and thus cell proliferation.

3T3 Cells↗

PubMed2000

Branched-chain amino acids inhibit proteolysis in rat skeletal muscle: mechanisms involved.

Incubation of isolated rat soleus and EDL muscles in the presence of 10 mM leucine resulted in a decreased proteolytic rate as measured by the release of tyrosine into the incubation medium. The effect of this branched-chain amino acid (BCAA) is associated with a decreased activity of the lysosomal proteases and a decreased expression of the genes of the ATP-ubiquitin-dependent proteolysis (ubiquitin and C8). Incubation of muscles in the presence of actinomycin D revealed that the effects of the amino acid can be accounted for by an inhibition of the transcription rate. The presence of leucine did not influence the gene expression of other nonlysosomal (m-calpain) and lysosomal (cathepsin B) proteolytic systems. It is concluded that the well-known effect of BCAA on muscle proteolysis is mediated, in the short term, by the inhibition of lysosomal proteolysis. In a longer period, based on the inhibition of gene transcription observed, an involvement of the ATP-dependent proteolytic system is also likely to occur.

Adenosine Triphosphate↗

PubMed2000

Differential association of p21Cip1 and p27Kip1 with cyclin E-CDK2 during rat liver regeneration.

BACKGROUND/AIMS: The cell cycle inhibitors p21Cip1 and p27Kip1 regulate liver regeneration by modulating the activity of cyclin-dependent kinases (CDKs). However, the specific role of these inhibitors in the regulation of CDK2 activity during liver regeneration remains unknown. The aim of this study was to examine the association of p21Cip1 and p27Kip1 with cyclin E-CDK2 and cyclin A-CDK2 complexes during rat liver regeneration and to correlate the association of both inhibitors with CDK2 activity. METHODS: The association of p21Cip1 or p27Kip1 with cyclin E-CDK2 or cyclin A-CDK2 and the activities of these complexes were analyzed by immunoprecipitation of rat liver homogenates obtained at different times after a partial hepatectomy (PH), followed by Western blotting or kinase assays. RESULTS: High amounts of p27Kip1 bound to cyclin E-CDK2 were observed during the first 13 h after PH, when CDK2 activity was very low. At 24 h, when CDK2 activity was maximal, the amount of bound-p27Kip1 decreased strongly. The amount of p21Cip1 bound to these complexes was low during the first 13 h but subsequently increased. No cyclin A-CDK2 complexes were found during the first 13 h after PH. At 24 h, complexes containing low levels of both inhibitors were detected and at 28 h, a significant increase in p21Cip1 and p27Kip1 associated with cyclin A-CDK2 was observed. CONCLUSIONS: p27Kip1 acts as a brake on cyclin E-CDK2 activity during the first 13 h after a PH. Both p21Cip1 and p27Kip1 down-regulate cyclin A-CDK2 activity at 28 h after PH, after its maximal activation.

Animals↗

PubMed2000

Novel, potent calmodulin antagonists derived from an all-D hexapeptide combinatorial library that inhibit in vivo cell proliferation: activity and structural characterization.

Calmodulin is known to bind to various amphipathic helical peptide sequences, and the calmodulin-peptide binding surface has been shown to be remarkably tolerant sterically. D-Amino acid peptides, therefore, represent potential nonhydrolysable intracellular antagonists of calmodulin. In the present study, synthetic combinatorial libraries have been used to develop novel D-amino acid hexapeptide antagonists to calmodulin-regulated phosphodiesterase activity. Five hexapeptides were identified from a library containing over 52 million sequences. These peptides inhibited cell proliferation both in cell culture using normal rat kidney cells and by injection via the femoral vein following partial hepatectomy of rat liver cells. These hexapeptides showed no toxic effect on the cells. Despite their short length, the identified hexapeptides appear to adopt a partial helical conformation similar to other known calmodulin-binding peptides, as shown by CD spectroscopy in the presence of calmodulin and NMR spectroscopy in DMSO. The present peptides are the shortest peptide calmodulin antagonists reported to date showing potential in vivo activity.

Animals↗

PubMed1999

The protein SET regulates the inhibitory effect of p21(Cip1) on cyclin E-cyclin-dependent kinase 2 activity.

The cyclin-dependent kinase (CDK) inhibitor p21(Cip1) has a dual role in the regulation of the cell cycle; it is an activator of cyclin D1-CDK4 complexes and an inhibitor of cyclins E/A-CDK2 activity. By affinity chromatography with p21(Cip1)-Sepharose 4B columns, we purified a 39-kDa protein, which was identified by microsequence analysis as the oncoprotein SET. Complexes containing SET and p21(Cip1) were detected in vivo by immunoprecipitation of Namalwa cell extracts using specific anti-p21(Cip1) antibodies. We found that SET bound directly to p21(Cip1) in vitro by the carboxyl-terminal region of p21(Cip1). SET had no direct effect on cyclin E/A-CDK2 activity, although it reversed the inhibition of cyclin E-CDK2, but not of cyclin A-CDK2, induced by p21(Cip1). This result is specific for p21(Cip1), since SET neither bound to p27(Kip1) nor reversed its inhibitory effect on cyclin E-CDK2 or cyclin A-CDK2. Thus, SET appears to be a modulator of p21(Cip1) inhibitory function. These results suggest that SET can regulate G(1)/S transition by modulating the activity of cyclin E-CDK2.

Amino Acid Sequence↗

PubMed1999

Calmodulin binds to p21(Cip1) and is involved in the regulation of its nuclear localization.

p21(Cip1), first described as an inhibitor of cyclin-dependent kinases, has recently been shown to have a function in the formation of cyclin D-Cdk4 complexes and in their nuclear translocation. The dual behavior of p21(Cip1) may be due to its association with other proteins. Different evidence presented here indicate an in vitro and in vivo interaction of p21(Cip1) with calmodulin: 1) purified p21(Cip1) is able to bind to calmodulin-Sepharose in a Ca(2+)-dependent manner, and this binding is inhibited by the calmodulin-binding domain of calmodulin-dependent kinase II; 2) both molecules coimmunoprecipitate when extracted from cellular lysates; and 3) colocalization of calmodulin and p21(Cip1) can be detected in vivo by electron microscopy immunogold analysis. The carboxyl-terminal domain of p21(Cip1) is responsible for the calmodulin interaction, since p21(145-164) peptide is also able to bind calmodulin and to compete with full-length p21(Cip1) for the calmodulin binding. Because treatment of cells with anti-calmodulin drugs decreases the nuclear accumulation of p21(Cip1), we hypothesize that calmodulin interaction with p21(Cip1) is important for p21(Cip1), and in consequence for cyclin D-Cdk4, translocation into the cell nucleus.

Amino Acid Sequence↗

PubMed1999

Activation of cdk4 and cdk2 during rat liver regeneration is associated with intranuclear rearrangements of cyclin-cdk complexes.

Partial hepatectomy (PH) triggers the entry of rat liver cells into the cell cycle. The signals leading to cell-cycle activation converge into a family of kinases named cyclin-dependent kinases (cdks). Specific cyclin-cdk complexes are sequentially activated during the cell cycle. Cyclin D-cdk4 and cyclin E-cdk2 are activated during the G1 phase, cyclin A-cdk2 is activated during the S phase, and cyclin B-cdk1 during mitosis. In the present study, we have examined the timing of the activation of cdk4 and cdk2, the intracellular location of G1/S cyclins and cdks, and the relationship between location and cdk4 and cdk2 activities during rat liver regeneration after a PH. Results showed that the activity of both kinases started at 13 hours and showed maximal levels at 24 hours after hepatectomy. In quiescent cells, cyclin D3 and cdk4 were cytoplasmatic, whereas cyclin D1 was nuclear. At 5 hours after hepatectomy, cyclin D3 and cdk4 began to move into the nucleus, and at 13 hours, they were mostly nuclear. During the first 13 hours after hepatectomy, significant amounts of cyclin D1-cdk4 and cyclin D3-cdk4 complexes were formed, but they were mostly inactive. At 24 hours, these complexes were maximally activated. This activation was associated with the accumulation of cyclin D1, cyclin D3, and cdk4 in a nuclear subfraction extractable with nucleases. At 28 hours, the activity of cdk4 in this nuclear subfraction decreased when cyclin D1 moved from this fraction to the nuclear matrix (NM) and the levels of cyclin D3 diminished. The maximal activation of cdk2 at 24 hours was also associated with the accumulation of cyclin E, cyclin A, and cdk2 in this nuclease-sensitive fraction. The inactivation of cdk2 at 28 hours was associated with a strong decrease in cdk2 in this nuclear subfraction. Thus, results reported here indicate that the activation of cdk4 and cdk2 observed in rat liver cells after a PH is associated with a specific intranuclear location of these cdks and their associated cyclins.

Animals↗

PubMed1999

Ubiquitin gene expression is increased in human muscle undergoing neurogenic involvement.

Histological features of neurogenic muscle involvement included type grouping, muscle fiber atrophy, and target fibers. In muscles with myofiber atrophy and target fibers, we found an increased expression of the genes encoding for the ubiquitin-ATP-dependent proteolytic system. Thus, in patients with target fibers, a 5.2- and a 3.9-fold increase were observed for the 2.4 and 1.2 kb transcripts, respectively, while in those with atrophic angulated hyperoxidative fibers, a 3.9- and a 4.4-fold increase were observed for the 2.4 and 1.2 kb transcripts, respectively. It is suggested that the activation of this proteolytic system may be responsible for the skeletal muscle alterations that often accompany human muscle neurogenic involvement.

Blotting, Northern↗

PubMed1998

Calmodulin is essential for cyclin-dependent kinase 4 (Cdk4) activity and nuclear accumulation of cyclin D1-Cdk4 during G1.

Although it is known that calmodulin is involved in G1 progression, the calmodulin-dependent G1 events are not well understood. We have analyzed here the role of calmodulin in the activity, the expression, and the intracellular location of proteins involved in G1 progression. The addition of anti-calmodulin drugs to normal rat kidney cells in early G1 inhibited cyclin-dependent kinase 4 (Cdk4) and Cdk2 activities, as well as retinoblastoma protein phosphorylation. Protein levels of cdk4, cyclin D1, cyclin D2, cyclin E, p21, and p27 were not affected after CaM inhibition, whereas decreases in the amount of cyclin A and Cdc2 were observed. The decrease of Cdk4 activity was due neither to changes in its association to cyclin D1 nor to changes in the amount of p21 or p27 bound to cyclin D1-Cdk4 complexes. Calmodulin inhibition also produced a translocation of nuclear cyclin D1 and Cdk4 to the cytoplasm. This translocation could be responsible for the decreased Cdk4 activity upon calmodulin inhibition. Immunoprecipitation, calmodulin affinity chromatography, and direct binding experiments indicated that calmodulin associates with Cdk4 and cyclin D1 through a calmodulin-binding protein. The facts that Hsp90 interacts with Cdk4 and that its inhibition induced Cdk4 and cyclin D1 translocation to the cytoplasm point to Hsp90 as a good candidate for being the calmodulin-binding protein involved in the nuclear accumulation of Cdk4 and cyclin D1.

Animals↗

PubMed1998

Different cytokines modulate ubiquitin gene expression in rat skeletal muscle.

Intravenous administration of different cytokines caused important changes in the expression of ubiquitin genes in skeletal muscle. Tumour necrosis factor-alpha caused a 2.2- and 1.9-fold increase in the expression of the 2.4 and 1.2 kb transcripts, respectively. Administration of interferon-gamma also caused a 2.2- and 1.8-fold increase in the 2.4 and 1.2 kb transcripts, respectively. While administration of leukaemia inhibitory factor and interleukin-6 resulted in no changes in ubiquitin gene expression, interleukin-1 administration also caused an increase in both ubiquitin gene transcripts (2.8- and 1.9-fold for the 2.4 and 1.2 kb transcripts, respectively). The results suggest that some of the cytokine effects on the ubiquitin system gene expression could be related to the enhanced skeletal muscle proteolysis found during cancer cachexia and other pathological states.

Animals↗

PubMed1998

Disruption of the antiproliferative TGF-beta signaling pathways in human pancreatic cancer cells.

Resistance to TGF-beta1 occurred in pancreatic cancer cells suggesting that inactivation of TGF-beta inhibitory signaling pathways may play an important role in human pancreatic cancer. The aim of our study was to determine the presence of alterations in the main putative components of the TGF-beta inhibitory signaling pathways (p15, Smad4, Smad2, TGFbeta-RII, CDC25A). A panel of human carcinomas of the exocrine pancreas orthotopically implanted and perpetuated in nude mice and pancreatic cancer cell lines were studied. p15 gene alterations, mainly homozygous deletions that involved exons 1 and/or 2, were found in the 62.5% (5 of 8) of pancreatic xenografts whereas Smad4 gene aberrations were found in one of eight xenografts and in two of seven cell lines. Additional aberrations in these genes were acquired during in vivo perpetuation and distal dissemination. Paradoxically, TGFbeta-RII overexpression and a decrease in CDC25A protein levels were found in all tumors and cell lines. In one cell line, resistance to TGF-beta1 occurred in the absence of alterations in the genes analysed so far. We conclude that all human pancreatic tumor cells analysed herein have non-functional TGF-beta pathways. The majority of cells harbor alterations in at least one of the putative components of TGF-beta pathways, mainly in p15 and Smad4 genes. These results suggest that inactivation of TGF-beta signaling pathways plays an important role in human pancreatic tumorigenesis.

Animals↗

PubMed1998

Calmodulin inhibitor W13 induces sustained activation of ERK2 and expression of p21(cip1).

One of the major signaling pathways by which extracellular signals induce cell proliferation and differentiation involves the activation of extracellular signal-regulated kinases (ERKs). Because calmodulin is essential for quiescent cells to enter cell cycle, the role of calmodulin on ERK2 activation was studied in cultured fibroblasts. Serum, phorbol esters, or active Ras induced ERK2 activation in NIH 3T3 fibroblasts. This activation was not inhibited by calmodulin blockade. Surprisingly, inhibition of calmodulin prior to fetal bovine serum addition prolonged activation of ERK2. Furthermore, inactivation of calmodulin in serum-starved cells induced ERK2 phosphorylation that was dependent on MAP kinase kinase (MEK). Inactivation of calmodulin in serum-starved cells also induced activation of Ras, Raf, and MEK. On the contrary, tyrosine phosphorylation of tyrosine kinase receptors was not observed. These results indicate that calmodulin inhibits ERK2 activation pathway at the level of Ras. Calmodulin inhibition induced overexpression of p21(cip1) which was dependent on MEK activity. We propose that inhibition of Ras by calmodulin prevents the activation of ERK2 at low serum concentration. Thus, entering into the cell cycle after serum addition would imply the overcoming of the inhibitory effect of calmodulin and consequently ERK2 activation. Furthermore, down-regulation of Ras by calmodulin may be also important to determine the duration of ERK2 activation and to prevent a high p21(cip1) expression that would lead to an inhibition of cell proliferation.

3T3 Cells↗

PubMed1998

Protein turnover in skeletal muscle of tumour-bearing transgenic mice overexpressing the soluble TNF receptor-1.

The implantation of the Lewis lung carcinoma (a fast-growing mouse tumour that induces cachexia) to both wild-type and transgenic mice for the soluble TNF receptor type I protein (sTNF-R1) resulted in a considerable loss of carcass weight in both groups. However, while in the wild-type mice there was a loss of both fat and muscle, in the transgenic mice muscle waste was not affected to the same extent as in the wild-type group. Muscle waste in wild-type mice was accompanied by an increase in the fractional rate of protein degradation, while no changes were observed in protein synthesis. The result was a decreased rate of protein accumulation which accounted for the muscle weight loss observed as a result of the tumour burden. In contrast, transgenic mice did not have such low rates of protein accumulation after tumour implantation. The increase in protein degradation in the tumour-bearing transgenic mice was accompanied by a similar increase in protein synthesis which compensated for the loss of muscle protein by degradation. Both tumour-bearing groups showed an enhanced expression of ubiquitin and proteasome C8 subunit genes, all of them related to the activation of the ATP-dependent proteolytic system in skeletal muscle. It is suggested that TNF may, in part, be responsible for the loss of protein in skeletal muscle of tumour-bearing mice.

Animals↗