Confluence of vascular endothelial cells induces cell cycle exit by inhibiting p42/p44 mitogen-activated protein kinase activity.
(1/378)
Like other cellular models, endothelial cells in cultures stop growing when they reach confluence, even in the presence of growth factors. In this work, we have studied the effect of cellular contact on the activation of p42/p44 mitogen-activated protein kinase (MAPK) by growth factors in mouse vascular endothelial cells. p42/p44 MAPK activation by fetal calf serum or fibroblast growth factor was restrained in confluent cells in comparison with the activity found in sparse cells. Consequently, the induction of c-fos, MAPK phosphatases 1 and 2 (MKP1/2), and cyclin D1 was also restrained in confluent cells. In contrast, the activation of Ras and MEK-1, two upstream activators of the p42/p44 MAPK cascade, was not impaired when cells attained confluence. Sodium orthovanadate, but not okadaic acid, restored p42/p44 MAPK activity in confluent cells. Moreover, lysates from confluent 1G11 cells more effectively inactivated a dually phosphorylated active p42 MAPK than lysates from sparse cells. These results, together with the fact that vanadate-sensitive phosphatase activity was higher in confluent cells, suggest that phosphatases play a role in the down-regulation of p42/p44 MAPK activity. Enforced long-term activation of p42/p44 MAPK by expression of the chimera DeltaRaf-1:ER, which activates the p42/p44 MAPK cascade at the level of Raf, enhanced the expression of MKP1/2 and cyclin D1 and, more importantly, restored the reentry of confluent cells into the cell cycle. Therefore, inhibition of p42/p44 MAPK activation by cell-cell contact is a critical step initiating cell cycle exit in vascular endothelial cells. (+info)
Proliferation and differentiation of unilocular fat cells in the bone marrow.
(2/378)
Fat cells contribute not only to systemic lipid metabolism, but also to osteogenesis and hemopoiesis in the bone marrow. The present study represents the first attempt to culture mature unilocular fat cells of the bone marrow. Two methods devised in our laboratory were employed: one is the "ceiling culture method" that utilizes the floating property of the cells; the other, a three-dimensional collagen gel matrix culture that captures unilocular fat cells in the gel matrix. Using these methods, the proliferation of unilocular fat cells from the bovine metacarpal bone marrow was demonstrated. First, we confirmed the proliferative ability of unilocular fat cells derived from the bone marrow, using autoradiography to study 3H-thymidine incorporation into the nuclei. The unilocular fat cells de-differentiated into fibroblast-like fat cells and then proceeded to proliferate. When they underwent a contact inhibition of growth, re-differentiation from fibroblast-like fat cells into unilocular fat cells occurred at a high rate. A specific enzymatic marker of the fat cell, alpha-glycerophosphate dehydrogenase activity related to lipogenesis, was then demonstrated in the cultured fat cells. We examined the functional reactivity of the fat cells by treatment with insulin and cyclic-AMP, and both lipogenesis and lipolysis were also confirmed in them. We concluded that unilocular fat cells from the bone marrow de-differentiated, proliferated and re-differentiated in culture. The present results may help to clarify the various functions of fat cells in the bone marrow. (+info)
Oncogenic potential of Hsp72.
(3/378)
Hsp72 is the major heat shock-inducible protein capable of protecting cells from a variety of stresses. In non-transformed cells at normal conditions Hsp72 is expressed at very low levels. It is, however, present at elevated levels in the major fraction of tumors and in many transformed cell lines. It is commonly assumed that in tumor cells the expression of Hsp72 at elevated levels is the consequence of oncogenic transformation. In the present study we addressed an alternative possibility that Hsp72 plays an active role in the process of oncogenic transformation. We report here that when Hsp72 was expressed in the Rat-1 fibroblasts either constitutively or from an adenovirus-based construct, cells become oncogenically transformed by the following criteria: loss of contact inhibition and formation of foci characteristic for oncogenically transformed cells; acquisition of the ability to grow in an anchorage-independent manner and to form colonies in soft agar; generation of tumors upon injection into mice. Furthermore, we also report that turning off the Hsp72 expression led to the reversal of the transformed phenotype. We also show that oncogenic potential of Hsp72 is confined in its peptide binding domain since the expression of this domain alone was sufficient for oncogenic transformation of Rat-1 cells. (+info)
A single amino acid substitution in the cyclin D binding domain of the infected cell protein no. 0 abrogates the neuroinvasiveness of herpes simplex virus without affecting its ability to replicate.
(4/378)
The infected cell protein no. 0 (ICP0) of herpes simplex virus 1 is a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection. The protein interacts with several viral and cellular proteins. Earlier studies have shown that ICP0 binds and stabilizes cyclin D3 but does interfere with the phosphorylation of retinoblastoma protein, its major function. Cyclin D3 plays a key role in the transition from G1 to S phase. To define the role of cyclin D3 in productive infection, the ICP0 binding site for cyclin D3 was mapped and mutagenized by substitution of aspartic acid codon 199 with the alanine codon. We report that the substitution precluded the interaction of this protein with cyclin D3 in the yeast two-hybrid system and the stabilization of cyclin D3 in infected cells. A recombinant virus carrying this mutation could not be differentiated from wild-type parent with respect to replication in dividing cells but yielded 10-fold less progeny from infected resting cells and serum-deprived or contact-inhibited human fibroblasts. In mice, the mutant was only slightly less pathogenic than the wild-type parent by intracerebral route but was significantly less neuroinvasive after peripheral inoculation. Replacement of the mutated amino acid with aspartic acid restored wild-type phenotype. Stabilization of cyclin D3 therefore is linked to higher virus yields in nondividing cells and potentially higher virulence in experimental and natural hosts. One function of ICP0 is to scavenge the cell for proteins that could bolster viral replication. (+info)
Point mutation causing constitutive signaling of CXCR2 leads to transforming activity similar to Kaposi's sarcoma herpesvirus-G protein-coupled receptor.
(5/378)
The chemokine receptor CXCR2 is the closest homologue to Kaposi's sarcoma herpesvirus-G protein-coupled receptor (KSHV-GPCR), which is known to be constitutively activated and able to cause oncogenic transformation. Among G protein-coupled receptors, a DRY sequence in the second intracellular loop is highly conserved. However, the KSHV-GPCR shows a VRY sequence instead. In this study, we exchanged Asp138 of the DRY sequence in the CXCR2 with a Val (D138V), the corresponding amino acid in KSHV-GPCR, or with a Gln (D138Q), and investigated the functional consequences of these mutations. In focus formation and soft agar growth assays in NIH 3T3 cells, the D138V mutant exhibited transforming potential similar to the KSHV-GPCR. Surprisingly, the CXCR2 wild type itself showed transforming activity, although not as potently, due to continuous autocrine stimulation, whereas the D138Q mutant formed no foci. In agreement with these results were high levels of inositol phosphate accumulation in the D138V mutant and the KSHV-GPCR, indicating constitutive activity. These data emphasize the importance of the DRY sequence for G protein-coupled signaling of the CXCR2. Either constitutive activation or persistent autocrine stimulation of the CXCR2 causes transformation similar to KSHV-GPCR-transfected cells, probably activating the same signal transduction cascade that can abrogate normal growth control mechanisms. (+info)
Exogenous expression of beta-catenin regulates contact inhibition, anchorage-independent growth, anoikis, and radiation-induced cell cycle arrest.
(6/378)
beta-Catenin is an important regulator of cell-cell adhesion and embryonic development that associates with and regulates the function of the LEF/TCF family of transcription factors. Mutations of beta-catenin and the tumor suppressor gene, adenomatous polyposis coli, occur in human cancers, but it is not known if, and by what mechanism, increased beta-catenin causes cellular transformation. This study demonstrates that modest overexpression of beta-catenin in a normal epithelial cell results in cellular transformation. These cells form colonies in soft agar, survive in suspension, and continue to proliferate at high cell density and following gamma-irradiation. Endogenous cytoplasmic beta-catenin levels and signaling activity were also found to oscillate during the cell cycle. Taken together, these data demonstrate that beta-catenin functions as an oncogene by promoting the G(1) to S phase transition and protecting cells from suspension-induced apoptosis (anoikis). (+info)
A monoclonal antibody directed against the murine macrophage surface molecule F4/80 modulates natural immune response to Listeria monocytogenes.
(7/378)
Whole spleen cell cultures from SCID mice release high levels of IFN-gamma when exposed to heat-killed Listeria monocytogenes (HKL). This microbe-induced and T cell-independent response depends on both macrophages (MPhi) and NK cells: HKL-stimulated MPhi release TNF-alpha and IL-12, which together activate NK cells for IFN-gamma release. We show here that this cytokine-mediated activation cascade can be modulated by a mAb against the MPhi surface glycoprotein F4/80. HKL-induced IL-12, TNF-alpha, and IFN-gamma in SCID whole spleen cell cultures was inhibited by coincubation with anti-F4/80 mAb whereas IL-1 and IL-10 were enhanced. Both effects were apparent at mRNA and protein release levels. Whereas inhibitory activities were F4/80 Ag specific, stimulatory effects were Fc dependent and nonspecific. Furthermore, cytokine inhibition by anti-F4/80 was only apparent when MPhi and NK cells were present simultaneously and in close vicinity, indicating that direct cell-to-cell contact is a prerequisite. These data suggest a novel pathway for microbe-induced MPhi/NK cell interaction involving direct cell-to-cell signaling and give the first evidence for a functional role of the MPhi surface glycoprotein F4/80. (+info)
Contact-dependent inhibition of cortical neurite growth mediated by notch signaling.
(8/378)
The exuberant growth of neurites during development becomes markedly reduced as cortical neurons mature. In vitro studies of neurons from mouse cerebral cortex revealed that contact-mediated Notch signaling regulates the capacity of neurons to extend and elaborate neurites. Up-regulation of Notch activity was concomitant with an increase in the number of interneuronal contacts and cessation of neurite growth. In neurons with low Notch activity, which readily extend neurites, up-regulation of Notch activity either inhibited extension or caused retraction of neurites. Conversely, in more mature neurons that had ceased their growth after establishing numerous connections and displayed high Notch activity, inhibition of Notch signaling promoted neurite extension. Thus, the formation of neuronal contacts results in activation of Notch receptors, leading to restriction of neuronal growth and a subsequent arrest in maturity. (+info)