Signal transduction by protein kinase C in mammalian cells

1. The past two decades have witnessed great advances in our understanding of the role of protein kinase C (PKC) in signal transduction. The Ca(2+)-activated, phospholipid-dependent protein kinase discovered by Nishizuka's group in 1977 is now a family of at least 11 isoforms. Protein kinase C isoforms exist in different proportions in a host of mammalian cells and each isoform has a characteristic subcellular distribution in each cell type. 2. Stimulation of a specific PKC isoform often causes redistribution of the isoform from one subcellular compartment to another compartments where it complexes with and phosphorylates a specific protein substrate. 3. The interaction of a specific PKC isoform with its protein substrate may directly activate a specific function of the cell or may trigger a cascade of protein kinases that ultimately stimulates a specific response in differentiated cells or regulates growth and proliferation in undifferentiated cells.     

Ubiquitination and proteasome mediated degradation of polo-like kinase

Endovascular infections after percutaneous transluminal renal angioplasty with stenting (PTRAS) are rarely reported. Because strict longitudinal follow-up of patients undergoing PTRAS is lacking, the true incidence of such complications remains obscure. We report the first case of a patient with an infected renal artery pseudoaneurysm and de novo mycotic aortic aneurysm after PTRAS. This case serves to illustrate several important points, including (1) the retrieval of renal function in patients with renal artery occlusion, (2) the pathogenesis of infection after PTRAS, (3) the diagnosis and management of endovascular infection after percutaneous vascular intervention, and (4) recommendations for periprocedural antibiotic prophylaxis during PTRAS.     

Evidence of morphological and physiological transformation of mammalian cells by strong magnetic fields

Cultures of L-929 and WI-38 cells, frozen to 4.2 degrees K and exposed for 4 to 8 hours to 5000-oersted magnetic fields, were markedly inhibited in their growth as compared to controls. In cultures grown on cover slips, approximately 7 days after exposure, morphologically distinct cells emerged and were propagated from generation to generation; 3 weeks later, in flask cultures, contact inhibition was abolished. It is concluded that under certain experimental conditions, strong magnetic fields induce morphological and physiological transformations of target cells.     

Malignant transformation of NIH3T3 cells by overexpression of mot-2 protein

The murine mortalin genes, mot-1 and mot-2, are members of the hsp70 family of proteins and differ from each other by only two amino acid residues. Mot-1 is expressed in normal cells and has pancytosolic cellular distribution whereas mot-2 is found in the perinuclear region of immortal cells. We report here that a high level of expression of mot-2 protein resulted in malignant transformation of cells as analysed by anchorage independent growth and nude mice assays. A high level of protein expression is attributed to the 900 bp 3' untranslated region of the cDNA which does not have any transforming activity per se. Mortalin cDNA clones isolated from human transformed cells were also found to have transforming activity in similar assays and a high level of expression was apparent in some of the human immortalized cells that showed non-pancytosolic mortalin immunofluorescence. Taken together, the data suggest that nonpancytosolic mortalin may have a role in tumorigenesis.     

Cell cycle regulation of the Saccharomyces cerevisiae polo-like kinase cdc5p

Progression through and completion of mitosis require the actions of the evolutionarily conserved Polo kinase. We have determined that the levels of Cdc5p, a Saccharomyces cerevisiae member of the Polo family of mitotic kinases, are cell cycle regulated. Cdc5p accumulates in the nuclei of G2/M-phase cells, and its levels decline dramatically as cells progress through anaphase and begin telophase. We report that Cdc5p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). We have determined that Cdc5p-associated kinase activity is restricted to G2/M and that this activity is posttranslationally regulated. These results further link the actions of the APC to the completion of mitosis and suggest possible roles for Cdc5p during progression through and completion of mitosis.     

Purification and cloning of a protein kinase that phosphorylates and activates the polo-like kinase Plx1

The Xenopus polo-like kinase 1 (Plx1) is essential during mitosis for the activation of Cdc25C, for spindle assembly, and for cyclin B degradation. Polo-like kinases from various organisms are activated by phosphorylation by an unidentified protein kinase. A protein kinase, polo-like kinase kinase 1 or xPlkk1, that phosphorylates and activates Plx1 in vitro was purified to near homogeneity and cloned. Phosphopeptide mapping of Plx1 phosphorylated in vitro by recombinant xPlkk1 or in progesterone-treated oocytes indicates that xPlkk1 may activate Plx1 in vivo. The xPlkk1 protein itself was also activated by phosphorylation on serine and threonine residues, and the kinetics of activation of xPlkk1 in vivo closely paralleled the activation of Plx1. Moreover, microinjection of xPlkk1 into Xenopus oocytes accelerated the timing of activation of Plx1 and the transition from G2 to M phase of the cell cycle. These results define a protein kinase cascade that regulates several events of mitosis.     

Selective suppression of stress-activated protein kinase pathway by protein phosphatase 2C in mammalian cells

Between 1985 and 1987, 1837 primary total knee arthroplasty (TKA) prostheses were implanted in 1503 patients. Group I included 843 knee with metal-backed patellar components (MBPC), and group II included 994 knees with all polyethylene patellar components (APPC). Follow-up averaged 5.7 years (range, 2 to 11 years). Twenty-four MBPC (2.9%) and 16 APPC TKA cases (1.6%) developed deep infection. In the time interval between arthroplasty and 2-year follow-up, eight MBPC and 11 APPC knees developed deep periprosthetic infection. The difference in the cumulative probability of infection between the two groups during this time interval was not significant (relative risk, 0.9; 95% confidence interval [CI], 0.3-2.1; P = 0.73). However, after the 2-year follow-up, 16 MBPC and 5 APPC knees developed late infection, and the difference in the cumulative probability of infection between the MBPC and APPC knees during this time interval was significant (relative risk, 3.1; 95% CI, 1.1-8.5; P = 0.02). Although the mechanism for this increased risk of these late infections is not well understood, the attendant synovitis, effusion, and relative hyperemia of these knees in the presence of the particulate metal and polyethylene debris may increase the potential of bacterial seeding to these prostheses. Particulate metal debris has been previously shown to suppress bacterial phagocytosis and may play a role in the pathogenesis of these infections. We propose that the presence of metal-backed patellar failure represents a "prosthesis at risk" for the development of late prosthetic infection.     

Cell-specific ecdysone-inducible expression of FLP recombinase in mammalian cells

The ability of site-specific recombinases, like FLP and Cre, to catalyze alterations in genomic DNA is well established, whereas their application to genetic engineering strategies has been restricted because of the inability to temporally regulate their expression and subsequent recombination events in specific populations of cells. We describe a regulatory system for ecdysone-controlled expression of FLP recombinase. Furthermore, we demonstrate that ecdysone-induced, FLP-mediated site-specific recombination events can be targeted to specific cells. This system can be applied to cell-lineage studies as well as to the design of gene-therapy strategies, particularly in stem cells.     

Neoplastic transformation of rat colon epithelial cells by expression of activated human K-ras

Somatic mutations of the K-ras oncogene play an important role in colorectal carcinogenesis. We determined whether rat colon epithelial cells could be transformed by introducing retroviruses carrying the activated human K-ras oncogene alone. Primary epithelial cells from the rat distal colon were infected with retroviruses carrying wild-type and two types of activated K-ras (asp and val at codon 12) cDNAs. Cells infected with the wild-type K-ras virus showed no change in morphology and died within 3 weeks, whereas the activated K-ras virus-infected cells underwent morphological changes within 3 days and continued to proliferate. From these cells, several cell lines were subsequently established. Epithelial cells transformed by activated K-ras formed colonies in soft agar culture and tumors in athymic nude mice. Multiple copies of human K-ras genes and large amounts of K-ras mRNAs and proteins were found in the transformed cells. These data suggest that overexpression of activated K-ras transforms rat colon epithelial cells.     

Downregulation of polo-like kinase correlates with loss of proliferative ability of cardiac myocytes

Cardiac myocytes rapidly increase the cell number during the fetal and early neonatal period, but they lose their proliferative ability soon after birth. To understand the mechanism of how cardiac myocytes exit from the cell cycle, we examined the role of a newly identified serine/threonine kinase, polo-like kinase (Plk), in the process of proliferation of cardiac myocytes. Northern blot analysis revealed that Plk gene was abundantly expressed in cardiac myocytes and non-myocytes of fetal and neonatal rats but not in cardiocytes of adult rats. Western blot analysis showed that Plk protein was also detected only in fetal and neonatal hearts. During the early stage of cardiac differentiation. Plk expression was well correlated with the proliferative ability of cardiocytes. Plk mRNA was most abundant in undifferentiated embryonic stem (ES) cells and the mRNA levels decreased along with cardiac differentiation in the developing ES cell system. Once serum was deprived from the culture media, expression levels of Plk were markedly decreased and DNA was not synthesized in both cardiac myocytes and non-myocytes of neonatal rats. Re-addition of serum stimulated Plk gene expression and DNA synthesis in non-myocytes but not in cardiomyocytes. All these results taken together with the critical role of Plk in DNA synthesis in many cell types suggest that downregulation of Plk is important for the permanent withdrawal of cardiomyocytes from the cell cycle.     

Inducible gene expression in mammalian cells and transgenic mice

The discovery of the superantigens (SAgs) offered new insights on the interaction between microorganisms and the host immune system. Associated to Major Histocompatibility Complex (MHC) class II molecules, SAgs bind to the variable domain of the beta chain (V beta) of the TCR alpha beta engaged in the family specificity of lymphocytes. Therefore, these molecules are able to activate a high number of T lymphocytes as well as surface MHC class II bearing cells, leading to an overriding release of cytokines and inflammatory mediators, which have been related to their toxic effects. Endogenous SAgs are encoded by murine tumor proviruses (Mtv) which are integrated in the genome of mice. Bacteria and viruses produce exogenous SAgs and those related to food poisoning have been widely studied. The presence of parasite SAgs is still unclear and further studies are required to establish their existence and effects on the corresponding infections.     

Malignant transformation by overproduction of translation initiation factor eIF4G

N4G3, a cell line that overexpresses translation initiation factor eIF4G, one of the components of eIF4F, was made by stable transfection of the human eIF4G cDNA into NIH3T3 cells. The cells expressed 80-100 times greater levels of eIF4G mRNA than did NIH3T3 cells. N4G3 cells formed transformed foci on a monolayer of cells, showed anchorage-independent growth, and formed tumors in nude mice. These results indicate that overexpression of eIF4G caused malignant transformation of NIH3T3 cells. It is also known that overexpression of eIF4E, another component of eIF4F, causes transformation of NIH3T3 cells. However, there was no difference in the amount of eIF4E protein between N4G3 and NIH3T3 cells, indicating that cell transformation does not involve a change in eIF4E levels. The results may be due to an effect of eIF4G on translational control of protein synthesis directed by mRNAs having long 5'-untranslated region.     

Malignant transformation of Recklinghausen disease in the small intestine

Neurofibromatosis is a relatively common genetic disorder of the peripheral and central nervous systems. Benign or malignant tumours may occur in different organs. Visceral tumours arising in the neural plexus of the intestinal wall may cause ulceration, bleeding, obstruction, perforation and palpable abdominal masses. In this paper a case of chronic anaemia due to a chronically bleeding intestinal neurofibroma is presented in a woman who had undergone intestinal resection. Histopathological analysis showed a neurofibroma with malignant transformation and a liver metastasis.     

Malignant transformation of duct-like cells originating from acini in transforming growth factor transgenic mice

BACKGROUND & AIMS: In transgenic mice overexpressing transforming growth factor (TGF)-alpha in the exocrine pancreas, progressive pancreatic fibrosis and a transdifferentiation of acinar cells to duct-like cells occurs. The present study was undertaken to analyze this transdifferentiation process. METHODS: Pancreatic specimens were characterized using light microscopy and immunohistochemistry. Expression of the epidermal growth factor receptor (EGFR) and TGF-alpha was evaluated with slot blot and Western analysis. To identify other generic events, K-ras mutations were screened with an enriched polymerase chain reaction approach and p53 expression was detected with immunohistochemistry. RESULTS: Morphological examination revealed an aggregation of interlobular fibroblasts and a decrease in acinar cell height starting at day 14 after birth. In older animals, these acinar cells change to duct-like cells, which form tubular structures and express ductal markers. Evidence for dysplastic changes was found in 12 of 21 TGF-alpha transgenic mice older than 1 year. We also observed four malignant pancreatic tumors, which were multicentric and originated from dysplastic tubular complexes. They displayed a mixed cystic-papillary phenotype strongly positive for carbonic anhydrase activity. EGFR expression progressively increased in the transition from acinar to duct-like and transformed cells. Activating K-ras mutations could not be detected; however, tubular complexes and tumors displayed increased immunoreactivity for nuclear p53. CONCLUSIONS: These data suggest an involvement of the TGF-alpha/EGFR pathway in conjunction with other yet unknown events in pancreatic tumor development. Furthermore, these observations are in favor of an acinar-ductal carcinoma sequence. Thus, these transgenic animals will be useful to define genetic alterations associated with a transition from acinar cells to a neoplastic ductal phenotype.     

Estrogen upregulates endothelial constitutive nitric oxide synthase expression in human osteoblast-like cells

The protective effects of estrogen on the cardiovascular system are thought to be mediated, in part, by nitric oxide (NO). Estrogen also has protective effects on bone although the mechanisms of action have not been fully established. Since nitric oxide synthase (NOS) inhibitors have been found to abrogate the protective effect of estrogen on bone in ovariectomised rats, we studied the effects of 17beta-estradiol on NOS activity and NOS mRNA levels in cultured human osteoblast-like cells. 17beta-Estradiol stimulated NOS activity by approximately 2.0 fold and this effect was reversed by the calcium chelator, EGTA, and the NOS inhibitor, L-NMMA, implicating activation of a constitutive, calcium-dependent isoform. Further studies using RT/PCR indicated that only the endothelial nitric oxide synthase (ecNOS) isoform was expressed and RNase protection assays showed that 17beta-Estradiol treatment resulted in a 2.2 fold increase in ecNOS mRNA levels. These findings suggest that estrogen stimulates NOS activity in osteoblastic cells by activation of the ecNOS pathway, and taken together with previous data, is consistent with the possibility that NO may act as a mediator of estrogen actions on bone.