Growth Of Mouse Embryo Fibroblasts Research Articles

Pt(II) complex of Schiff base derived from L-phenylalanine and furfuraldehyde in the presence of 8-hydroxyquinoline: Structural analysis, composition of complex and biological activity

In the present study, we synthesized a Schiff base (L 1 ) derived from L-phenylalanine with furfuraldehyde and its Pt(II) complex ([Pt(L 1 )(L 2 )]: [Pt(N-(furfuralidene)phenylalanine)(8-hydroxyquinoline)]) in the presence of 8-hydroxyquinoline (L 2 ). The chemical structures of the synthesized Schiff base and Pt(II) complex were characterized using ESI-MS, UV–Visible, FT-IR, 1H NMR, 13C NMR, and powder-XRD spectroscopy. The surface morphologies and elemental composition were determined by SEM and EDX. Thermal analysis was performed with TG-DTA. Mole ratio of Meyer and Job methods were used for the composition of complexes. According to these methods, the ratio [Pt 2+ ] : [L 1 +L 2 ] was found to be 1 : 1. In addition, the antimicrobial activities of L 1 and the complex showed that the compounds have significant antibacterial and antifungal properties. They also show important cytotoxic effects against the growth of mouse embryo fibroblasts (MEF) and human prostate adenocarcinoma (Du145) cancer cells. L 1 , L 2 , and the Pt(II) complex also displayed effective antioxidant activity.

HAUSP Stabilizes p53

The tumor suppressor p53 is regulated by phosphorylation and controlled proteolysis by the ubiquitin-proteasome system. Li et al. show that herpesvirus-associated ubiquitin-specific protease (HAUSP) interacted with p53 and promoted its accumulation, even in conditions where the p53-ubiquitin ligase Mdm2 is overexpressed. Overexpression of HAUSP inhibited colony formation by lung carcinoma cells and inhibited growth of mouse embryo fibroblasts. This growth inhibition was dependent on the cells expressing p53. In addition to growth inhibition, HAUSP promoted p53-dependent apoptosis. HAUSP is a member of the ubiquitin-specific processing protease (UBP) family of deubiquitination enzymes (DUBs). The ability of HAUSP to influence p53 stability required the deubiquitinating activity and HAUSP decreased the detectable ubiquitinated p53 in Western blots. In an in vitro assay, HAUSP was able to directly deubiquitinate p53. In normal cells, the expression of a dominant-negative form of HAUSP that can bind p53, but not deubiquitinate it, resulted in accumulation of ubiquitinated p53 species (when the proteasome was inhibited). In addition, p53 was destabilized compared with that in cells that did not express the mutated HAUSP. The concentration of p53 appears to be regulated both by processes that promote ubiquitination and subsequent degradation and by processes that promote deubiquitination and subsequent stabilization. M. Li, D. Chen, A. Shiloh, J. Luo, A. Y. Nikolaev, J. Qin, W. Gu, Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 416 , 648-653 (2002). [Online Journal]

HAUSP Stabilizes p53

The tumor suppressor p53 is regulated by phosphorylation and controlled proteolysis by the ubiquitin-proteasome system. Li et al. show that herpesvirus-associated ubiquitin-specific protease (HAUSP) interacted with p53 and promoted its accumulation, even in conditions where the p53-ubiquitin ligase Mdm2 is overexpressed. Overexpression of HAUSP inhibited colony formation by lung carcinoma cells and inhibited growth of mouse embryo fibroblasts. This growth inhibition was dependent on the cells expressing p53. In addition to growth inhibition, HAUSP promoted p53-dependent apoptosis. HAUSP is a member of the ubiquitin-specific processing protease (UBP) family of deubiquitination enzymes (DUBs). The ability of HAUSP to influence p53 stability required the deubiquitinating activity and HAUSP decreased the detectable ubiquitinated p53 in Western blots. In an in vitro assay, HAUSP was able to directly deubiquitinate p53. In normal cells, the expression of a dominant-negative form of HAUSP that can bind p53, but not deubiquitinate it, resulted in accumulation of ubiquitinated p53 species (when the proteasome was inhibited). In addition, p53 was destabilized compared with that in cells that did not express the mutated HAUSP. The concentration of p53 appears to be regulated both by processes that promote ubiquitination and subsequent degradation and by processes that promote deubiquitination and subsequent stabilization.M. Li, D. Chen, A. Shiloh, J. Luo, A. Y. Nikolaev, J. Qin, W. Gu, Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 416, 648-653 (2002). [Online Journal]

BASAL LEVELS OF METALLOTHIONEIN I AND II EXPRESSION IN MOUSE EMBRYO FIBROBLASTS ENHANCE GROWTH IN LOW FOLATE THROUGH A CELL CYCLE MEDIATED PATHWAY

The impact of basal (non-induced) expression levels of metallothionein I and II on the growth of mouse embryo fibroblasts in standard DMEM/F-12 containing 8.8μm folic acid, and in DMEM/F12 without hypoxanthine, thymidine or folic acid, containing 15n m or 15p m[6S]-folinic acid, was assessed by comparing wild-type MT (+/+) and homozygous null MT (−/−) cell lines. No difference in growth rate was observed between the two in DMEM/F12, although MT (−/−) cells displayed a 6-fold decrease in p27Kip1, a two fold increase in p53 and a slight increase in p21Waf1. After 6 days in culture, the growth rate for MT (−/−) cells in 15n m or 15p m[6S]-folinic acid was half that of MT (+/+). After an additional 6 days in 15n m folate, both MT (+/+) and (−/−) cells maintained their respective growth rates, while those in 15p m had ceased to grow. During the initial 6 days in 15n m folate, neither cell population displayed an increase in apoptosis or a change in cell cycle distribution, even though MT (−/−) cells sustained an additional 4-fold increase in p21Waf1and a 6-fold decrease in cyclin E expression. At day 12, however, the MT (−/−) population, but not MT (+/+), underwent a 7-fold increase in apoptosis coupled with a 3 fold increase in S phase cells. Hence, the basal level of MT I and II constitutively expressed in MT (+/+) cells enhances growth in 15nM [6S]-folinic acid by preventing S phase arrest and apoptosis.

Shared role of the pRB-related p130 and p107 proteins in limb development.

The p130 protein shares extensive sequence similarity with pRB, the product of the retinoblastoma gene, and is a major E2F-associated protein in quiescent cells. To investigate its biological function, we have mutated p130 via gene targeting in the mouse. Homozygous mutation of p130 had little discernible effect on development or on the growth of mouse embryo fibroblasts in culture. Much of the E2F activity that normally associates with p130 in serum-starved mouse embryo fibroblasts associated instead with the highly related p107 protein. To determine whether p130 and p107 have overlapping biological roles, we produced mice having simultaneous inactivation of the p130 and p107 genes. Such mice exhibited deregulated chondrocyte growth, defective endochondral bone development, shortened limbs, and neonatal lethality. These findings indicate that p130 and p107 play an important role in limb development through their abilities to control chondrocyte proliferation. Thus, in certain settings p107 and p130 perform growth-regulatory functions that are not fulfilled by pRB.

Dimethyl sulfoxide inhibits the expression of early growth-response genes and arrests fibroblasts at quiescence

We have previously shown that dimethyl sulfoxide (DMSO) treatment of mouse embryo fibroblasts (MEF) at the early hours of mitogenic stimuli resulted in the inhibition of DNA and protein synthesis; delayed treatment of serum-stimulated cells with DMSO had little effect on the synthesis of these macromolecules. Here, we demonstrate the specific inhibition of expression of early growth response genes by DMSO in serum-stimulated MEF. The expression of interleukin 6 and of oncogenes c- myc and c- fos were inhibited when the cells were treated with 2% DMSO from the beginning of serum-stimulated growth but not after 3 h of mitogenic stimuli. Although the actin gene is an early serum-response gene, its expression was not affected by DMSO. The synthesis of another serum-induced protein, the plasminogen activator inhibitor-1 was blocked during concurrent and delayed (after 3 h of stimulation) treatment of serum-stimulated fibroblasts with DMSO. The expression of glyceraldehyde-3-phosphate dehydrogenase gene was not affected by DMSO. These results indicate that the expression of non-growth-related genes are either not affected or affected nonspecifically both at early and late stages of serum-induced growth of mouse embryo fibroblasts. The serum-induced expression of c- fos gene was abolished by DMSO treatment of MEF while the phorbol 12-myristate 13-acetate-induced expression of fos gene was not, indicating that the PMA signaling pathway was refractory to DMSO. Treatment of cells with medium containing 2% DMSO for 24–48 h prevents them from progression into cell cycle by preventing the expression of genes involved in G 0–G 1 transition of quiescent cells.

Translocation of plasminogen activator inhibitor-1 during serum stimulated growth of mouse embryo fibroblasts

Serum-stimulated mouse embryo fibroblasts specifically secrete two proteins of molecular weights 48,000 and 26,000. The 48 kDa protein showed affinity to concanavalin A and was precipitated by antibody to plasminogen activator inhibitor. Immunoflowcytometry using anti plasminogen activator inhibitor-1 serum indicate the presence of the 48 kDa protein in quiescent cells; this protein was virtually absent in serum-stimulated cells. The presence of the plasminogen activator inhibitor-1 related protein in quiescent cells and its absence in serum-stimulated cells in combination with the observation on the absence of this protein, in the medium of quiescent cells and its presence in the medium of stimulated cells indicate that the 48 kDa protein was transferred from the cells into the medium upon serum-stimulation. The serum-mediated transfer of plasminogen activator inhibitor-1 from the cells into the medium was inhibited by actinomycin-D suggesting that the transfer process required actinomycin-D sensitive events. Treatment of pre-labelled quiescent cells with medium containing 20% fetal calf serum resulted in the gradual transfer of the labelled 48 kDa protein to the extra cellular matrix. These studies indicate that exposure of quiescent cells to fetal calf serum results in the transfer of plasminogen activator inhibitor-1 from the cells to the growth mediumvia extracellular matrix. The translocation of the protease inhibitor from the cells to the matrix and medium may enable the cellular and possibly the membrane proteases to act on growth factors or their receptors thereby initiating the mitogenic response.

Serum stimulated DNA synthesis in mouse embryo fibroblasts: Synergistic enhancement by staphylococcal lipoteichoic acid

Lipoteichoic acid (LTA) isolated from Staphylococcus aureus was found to exert a synergistic effect with calf serum to enhance DNA synthesis and growth of mouse embryo fibroblasts. LTA alone had no effect on DNA synthesis or cell multiplication. Mild base hydrolysis of LTA, which separates the molecule into its hydrophilic and hydrophobic moieties, completely destroyed its biological activity in this regard. Results are presented which suggest that LTA may enhance serum-induced DNA synthesis through interaction with cell surface components. The data indicate that LTA may prove to be a useful tool with which to probe cell membrane parameters involved in the regulation of cell proliferation.