It has recently been reported (T. Shimizu et al., J. Biol. Chem., 273: 8669-8674, 1998) that the pro-apoptotic drug, camptothecin, an inhibitor of topoisomerase I, induces a protein kinase ...C-alpha-mediated phosphorylation of lamin B in HL-60 cells, which precedes both degradation of lamin B and fragmentation of DNA. In this paper, we report that, in HL-60 cells exposed to camptothecin, there is a rapid and sustained increase of nuclear protein kinase C-alpha activity that is due to an increase in the amount of protein kinase C-alpha present in the nucleus. The enhancement of nuclear kinase C activity is preceded by an increase in the mass of nuclear diacylglycerol. As demonstrated by its sensitivity to propranolol, the nuclear diacylglycerol mass increase is due to the activation of a phospholipase D. Indeed, inhibitors of neither phosphatidylcholine-specific phospholipase C nor phosphoinositide-specific phospholipase C blocked the rise in nuclear diacylglycerol. In vitro assays also demonstrated the activation of a nuclear phospholipase D, but not of a phosphoinositide-specific phospholipase C, after treatment with camptothecin. Propranolol was also able to block the rise in nuclear protein kinase C-alpha activity, thus suggesting that the increase in diacylglycerol mass is important for the activation of the kinase at the nuclear level. Moreover, propranolol was capable of drastically reducing the number of HL-60 cells that underwent apoptosis after treatment with camptothecin. Our results show the activation during apoptosis of a phospholipase D-mediated signaling pathway operating at the nuclear level. This pathway may represent an attractive therapeutic target for the modulation of apoptotic events in human disease.
Previous results from our laboratory have demonstrated that lamin B1 is a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-α that are important for this binding. By ...means of overlay assays and fusion proteins made of glutathione-
S-transferase (GST) fused to elements of the regulatory domain of rat PKC-α, we have established that binding occurs through both the V1 region and a portion of the C2 region (i.e., the calcium-dependent lipid binding CaLB domain) of the kinase. In particular, we have found that amino acids 200–217 of the CaLB domain are essential for binding lamin B1, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain of PKC-α and lamin B1. In agreement with the results of other investigators, we have determined that binding of regulatory elements of PKC-α to lamin B1 does not require the presence of cofactors such as PS and Ca
2+. We have also found that the binding site of lamin B1 for PKC-α is localized in the carboxyl-terminus of the lamin. Our findings may prove to be important in shedding more light on the mechanisms that regulate PKC functions within the nuclear compartment and may also lead to the synthesis of isozyme-specific pharmacological tools to attenuate or reverse PKC-dependent nuclear signalling pathways important for the pathogenesis of cancer.
The effect of pulsed electromagnetic fields (PEMFs) on the proliferation and survival of matrix-induced autologous chondrocyte implantation (MACI®)-derived cells was studied to ascertain the healing ...potential of PEMFs. MACI-derived cells were taken from cartilage biopsies 6 months after surgery and cultured. No dedifferentiation towards the fibroblastic phenotype occurred, indicating the success of the surgical implantation. The MACI-derived cultured chondrocytes were exposed to 12 h/day (short term) or 4 h/day (long term) PEMFs exposure (magnetic field intensity, 2 mT; frequency, 75 Hz) and proliferation rate determined by flow cytometric analysis. The PEMFs exposure elicited a significant increase of cell number in the SG2M cell cycle phase. Moreover, cells isolated from MACI® scaffolds showed the presence of collagen type II, a typical marker of chondrocyte functionality. The results show that MACI® membranes represent an optimal bioengineering device to support chondrocyte growth and proliferation in surgical implants. The surgical implant of MACI® combined with physiotherapy is suggested as a promising approach for a faster and safer treatment of cartilage traumatic lesions.
The existence of intranuclear lipid-dependent signal transduction systems has been demonstrated by several independent groups. Remarkably, intranuclear lipid-dependent signal transduction pathways ...are regulated independently from their membrane/cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation, differentiation, and apoptosis. Diacylglycerol (DG) is a fundamental lipid second messenger which is produced in the nucleus. Since the levels of nuclear DG fluctuate during the cell cycle progression, it has been suggested that this lipid second messenger has important regulatory roles. Most likely, nuclear DG serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases (DGKs), i.e. the enzymes that, by converting DG into phosphatidic acid (PA), terminate DG-dependent events. This review aims at highlighting the different isozymes of DGKs present within the nucleus as well as at discussing their potential functions with particular emphasis placed on DNA replication.
The existence of intranuclear lipid-dependent signal transduction systems has been demonstrated by several independent groups. Remarkably, intranuclear lipid-dependent signal transduction pathways ...are regulated independently from their membrane/cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation, differentiation, and apoptosis. Diacylglycerol (DG) is a fundamental lipid second messenger which is produced in the nucleus. Since the levels of nuclear DG fluctuate during the cell cycle progression, it has been suggested that this lipid second messenger has important regulatory roles. Most likely, nuclear DG serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases (DGKs), i.e. the enzymes that, by converting DG into phosphatidic acid (PA), terminate DGdependent events. This review aims at highlighting the different isozymes of DGKs present within the nucleus as well as at discussing their potential functions with particular emphasis placed on DNA replication.
D-3 phosphorylated inositides are a peculiar class of lipids, synthesized by phosphatidylinositol 3-kinase (PtdIns 3-K), which are also present in the nucleus. In order to clarify a possible role for ...nuclear D-3 phosphorylated inositides during human erythroid differentiation, we have examined the issue of whether or not, in K562 human erythroleukemia cells, erythropoietin (EPO) may generate nuclear translocation of an active PtdIns 3-K. Immunoprecipitation with an anti-p85 regulatory subunit of PtdIns 3-K, revealed that both the intranuclear amount and the activity of the kinase increased rapidly and transiently in response to EPO. Enzyme translocation was blocked by the specific PtdIns 3-K pharmacological inhibitor, LY294002, which also inhibited erythroid differentiation. In vivo, intranuclear synthesis of phosphatidylinositol (3,4,5) trisphosphate (PtdIns (3,4,5)P
3) was stimulated by EPO. Almost all PtdIns 3-K that translocated to the nucleus was highly phosphorylated on tyrosine residues of the p85 regulatory subunit. These findings strongly suggest that an important step in the signaling pathways that mediate EPO-induced erythroid differentiation may be represented by the intranuclear translocation of an active PtdIns 3-K.
Insulin-like growth factor-l (IGF-I) and its receptor (IGF-IR) have been implicated in the pathophysiology of many human cancers, including those of hematopoietic lineage. We investigated the ...therapeutic potential of the novel IGF-IR tyrosine kinase activity inhibitor, NVP-AEW541, on human acute myeloid leukemia (AML) cells. NVP-AEW541 was tested on a HL60 cell subclone, which is dependent on autocrine secretion of IGF-I for survival and drug resistance, as well as primary drug resistant leukemia cells. NVP-AEW541 treatment (24 h) induced dephosphorylation of IGF-IR. NVP-AEW541 also caused Akt dephosphorylation and changes in the expression of key regulatory proteins of the cell cycle. At longer incubation times (48 h), NVP-AEW541-induced apoptotic cell death, as demonstrated by caspase-3 cleavage. Apoptosis was accompanied by decreased expression of anti-apoptotic proteins. NVP-AEW541 enhanced sensitivity of HL60 cells to either cytarabine or etoposide. Moreover, NVP-AEW541 reduced the clonogenic capacity of AML CD34 super(+) cells cultured in the presence of IGF-I. Chemoresfstant AML blasts displayed enhanced IGF-I secretion, and were sensitized to etoposide-induced apoptosis by NVP-AEW541. Our findings indicate that NVP-AEW541 might be a promising therapeutic agent for the treatment of those AML cases characterized by IGF-I autocrine secretion.
BACKGROUND: A previous study showed that white cells in blood units undergo apoptosis during storage.
STUDY DESIGN AND METHODS: The present study attempts to show the release of nuclear matrix ...protein (NMP) in the supernatants of red cell units and to determine whether antibodies against nuclear components may be present in multiply transfused patients; the methods employed were enzyme‐linked immunosorbent assay, flow cytometry, microscopy, immunoblotting, immunofluorescence, and confocal laser‐scanning microscopy.
RESULTS: NMP is released from white cells in the supernatant of packed red cell units upon cold storage (1‐6°C). The concentration of NMP correlates well with the degree of apoptosis, as analyzed by flow cytometry, nuclear dye staining, and DNA gel electrophoresis. Immunofluorescence also shows that white cells undergoing apoptosis (pre‐G1 peak, as seen by propidium iodide staining and flow cytometry) have an NMP content lower than control cells, which confirms an actual release of NMP. Moreover, immunoblotting analysis and immunofluorescent staining showed that, in 4 of 38 multiply transfused patients, autoantibodies against NMPs were present without any clinical or laboratory sign of autoimmune disease. One of the sera, recognizing a 64‐kDa NMP, immunostained nuclear dots that were identified as coiled bodies because of their colocalization with p 80 coilin.
CONCLUSION: NMP is released in the supernatant of red cell units. The results obtained from patients suggest that nuclear proteins released during apoptosis, once transfused, may induce an immune response in multiply transfused patients.
The serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K), plays a pivotal role in tumorigenesis because it affects the growth and survival of cancer cells. ...Several laboratories have demonstrated that Akt inhibits transcriptional activation of a number of related forkhead transcription factors now referred to as FoxO1, FoxO3, and FoxO4. Akt-regulated forkhead transcription factors are involved in the control of the expression of both the cyclin-dependent kinase (cdk) inhibitor p27(Kip1) and proapoptotic Bim protein. Very little information is available concerning the importance of the PI3K/Akt pathway in HL60 human leukemia cells. Here, we present our findings showing that the PI3K/Akt axis regulates cell cycle progression of HL60 cells through multiple mechanisms also involving the control of FoxO1 and FoxO3. To this end, we took advantage of a HL60 cell clone (HL60AR cells) with a constitutively activated PI3K/Akt axis. When compared with parental (PT) HL60 cells, HL60AR cells displayed higher levels of phosphorylated FoxO1 and FoxO3. In AR cells forkhead factors localized predominantly in the cytoplasm, whereas in PT cells they were mostly nuclear. AR cells proliferated faster than PT cells and showed a lower amount of the cdk inhibitor p27(Kip1), which was mainly found in the cytoplasm and was hyperphosphorylated on threonine residues. AR cells also displayed higher levels of cyclin D1 and phosphorylated p110 Retinoblastoma protein. The protein levels of cdk2, cdk4, and cdk6 were not altered in HL60AR cells, whereas the activities of both ckd2 and cdk6 were higher in AR than in PT cells. These results show that in HL60 cells the PI3K/Akt signaling pathway may be involved in the control of the cell cycle progression most likely through mechanisms involving the activation of forkhead transcription factors.
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