PubMed Health⌕ Search

Biomedical subjects

J Boonstra

Publications and source records attributed to J Boonstra.

At least 19 recordsLinked to original sources
PubMed2006

Inhibitors of phosphatidylinositol 3-kinase activity prevent cell cycle progression and induce apoptosis at the M/G1 transition in CHO cells.

Phosphatidylinositol 3-kinase (PI3-kinase) activity has been implicated in regulating cell cycle progression at distinct points in the cell cycle by preventing cell cycle arrest or apoptosis. In this study, the role of PI3-kinase activity during the entire G1 phase of the ongoing cell cycle was studied in Chinese hamster ovary (CHO) cells synchronized by mitotic shake-off. We show that inhibition of PI3-kinase activity during and 2 h after mitosis inhibited cell cycle progression into S phase. In the presence of the PI3-kinase inhibitor wortmannin or LY294002, cells were arrested during early G1 phase, leading to the expression of the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PI3-kinase activity is required for progression through the M/G1 phase. In the absence of PI3-kinase activity, cells are induced for apoptosis in this particular phase of the cell cycle.

Actin Cytoskeleton↗
PubMed2004

Identification of a restriction point at the M/G1 transition in CHO cells.

The regulation of cell cycle progression in normal mammalian cells is dependent on the presence of growth factors. In their absence, non-transformed cells will stop dividing and enter the quiescent state (G0). We show here that in Chinese hamster ovary cells, at least two serum-dependent points exist during G1 that lead to different cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis. The second point is located late in G1, and probably corresponds with the 'classic' restriction point R. Cells depleted of serum after the first restriction point will not stop randomly in G1 but continue G1 progression until they reach the late restriction point, as marked by translocation of p42(MAPkinase) (ERK2) to the nucleus.

Animals↗
PubMed2002

Hydrogen peroxide inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells.

Hydrogen peroxide (H(2)O(2)) induces a number of events, which are also induced by mitogens. Since the progression through the G1 phase of the cell cycle is dependent on mitogen stimulation, we were interested to study the effect of H(2)O(2) on the cell cycle progression. This study demonstrates that H(2)O(2) inhibits DNA synthesis in a dose-dependent manner when given to cells in mitosis or at different points in the G1 phase. Interestingly, mitotic cells treated immediately after synchronization are significantly more sensitive to H(2)O(2) than cells treated in the G1, and this is due to the inhibition of the cell spreading after mitosis by H(2)O(2). H(2)O(2) reversibly inhibits focal adhesion activation and stress fiber formation of mitotic cells, but not those of G1 cells. The phosphorylation of MAPK is also reversibly inhibited in both mitotic and G1 cells. Taken together, H(2)O(2) is probably responsible for the inhibition of the expression of cyclin D1 and cyclin A observed in cells in both phases. In conclusion, H(2)O(2) inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. This may play a role in pathological processes in which H(2)O(2) is generated.

Animals↗
PubMed2002

Cytosolic phospholipase A2 and lipoxygenase are involved in cell cycle progression in neuroblastoma cells.

Arachidonic acid has been implicated in regulating cellular proliferation, and is preferentially released by the 85-kDa cytosolic phospholipase A2 (cPLA2). Recently, we demonstrated that cPLA2 is activated at distinct periods during the ongoing cell cycle of neuroblastoma cells. The purpose of the present study was to establish the role of these cPLA2 activity peaks in cell cycle progression. Inhibition of cPLA2 activity with arachidonyl trifluoromethylketone (ATK) in early G1 phase reduced DNA synthesis markedly. A 24-h incubation with ATK revealed no significant difference in cell number compared to untreated cells, although cPLA2 activity was still inhibited. This suggests redundancy of different PLA2 enzymes. Lipoxygenase inhibition in early G1 resulted in G1 phase arrest, whereas inhibitors for cyclooxygenase had no effect. Furthermore, cells stopped progressing through S phase when lipoxygenase was inhibited in early S phase, demonstrating the requirement of lipoxygenase products for S phase progression.

Animals↗
PubMed2002

Impaired cutinase secretion in Saccharomyces cerevisiae induces irregular endoplasmic reticulum (ER) membrane proliferation, oxidative stress, and ER-associated degradation.

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.

Carboxylic Ester Hydrolases↗
PubMed2001

Phosphorylation of p42/44(MAPK) by various signal transduction pathways activates cytosolic phospholipase A(2) to variable degrees.

Arachidonic acid has been implicated to play a role in physiological and pathophysiological processes and is selectively released by the 85-kDa cytosolic phospholipase A(2) (cPLA(2)). The activity of cPLA(2) is regulated by calcium, translocating the enzyme to its substrate, and by phosphorylation by a mitogen-activated protein kinase (MAPK) family member and a MAPK-activated protein kinase. In this study, the signal transduction pathways in growth factor-induced phosphorylation of p42/44(MAPK) and cPLA(2) activation were investigated in Her14 fibroblasts. p42/44(MAPK) in response to epidermal growth factor was not only phosphorylated via the Raf-MEK pathway but mainly through protein kinase C (PKC) or a related or unrelated kinase in which the phosphorylated p42/44(MAPK) corresponded with cPLA(2) activity. Serum-induced phosphorylation of p42/44(MAPK) also corresponded with cPLA(2) activity but is predominantly mediated via Raf-MEK and partly through PKC or a related or unrelated kinase. In contrast, activation of PKC by phorbol ester did not result in increased cPLA(2) activity, while p42/44(MAPK) is phosphorylated, mainly via Raf-MEK and through MEK. Moreover, p42/44(MAPK) phosphorylation is present in quiescent and proliferating cells, and p42/44(MAPK) is entirely phosphorylated via Raf-MEK, but it only corresponds to cPLA(2) activity in the former cells. Collectively, these data show that p42/44(MAPK) in proliferating, quiescent, and stimulated cells is phosphorylated by various signal transduction pathways, suggesting the activation of different populations of p42/44(MAPK) and cPLA(2).

3T3 Cells↗
PubMed2001

Cytosolic phospholipase A(2) activity during the ongoing cell cycle.

Cytosolic phospholipase A(2) (cPLA(2)) is of special interest because it selectively releases arachidonic acid from membrane phospholipids. Arachidonic acid has been implicated to play an important role in various cellular responses. Recently arachidonic acid release and prostaglandin synthesis have been shown to be cell cycle dependent and therefore the activity of cPLA(2) during the ongoing cell cycle was investigated, using the mitotic shake off method for cell synchronisation. cPLA(2) activity was high in mitotic cells and decreased rapidly in the early G1 phase. A strong increase in activity was measured following the G1/S transition in both neuroblastoma and Chinese hamster ovary cells. The changes in activity were not due to a difference in cPLA(2) expression but due to phosphorylation of cPLA(2). Phosphorylation of cPLA(2) occurs through MAPK since the use of a specific MAPK kinase inhibitor and serum depletion of synchronised cells inhibited cPLA(2) activity.

Animals↗
PubMed2001

Regulation of G1 phase progression by growth factors and the extracellular matrix.

Cell cycle progression is regulated by both intracellular and extracellular control mechanisms. Intracellular controls ensure that cell cycle progression is stopped in response to irregularities such as DNA damage or faulty spindle assembly, whereas extracellular factors may determine cell fate such as differentiation, proliferation or programmed cell death (apoptosis). When extracellular factors bind to receptors at the outside of the cell, signal transduction cascades are activated inside the cell that eventually lead to cellular responses. We have shown previously that MAP kinase (MAPK), one of the proteins involved in several signal transduction processes, is phosphorylated early after mitosis and translocates to the nucleus around the restriction point. The activation of MAPK is independent of cell attachment, but does require the presence of growth factors. Moreover, it appears that in Chinese hamster ovary cells, a transformed cell line, growth factors must be present early in the G1 phase for a nuclear translocation of MAPK and subsequent DNA replication to occur. When growth factors are withdrawn from the medium immediately after mitosis, MAPK is not phosphorylated, cell cycle progression is stopped and cells appear to enter a quiescent state, which may lead to apoptosis. Furthermore, in addition to this growth-factor-regulated decision point in early G1 phase, another growth-factor-sensitive period can be distinguished at the end of the G1 phase. This period is suggested to correlate with the classical restriction point (R) and may be related to cell differentiation.

Animals↗
PubMed2001

The effect of hydrogen peroxide on the cyclin D expression in fibroblasts.

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H2O2) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incubated with 3 mM or higher H2O2 concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expression of cyclin D1 and D2 upon H2O2 treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H2O2 was not due to induction of protein synthesis. These results indicate that H2O2 reversibly inhibits the ubiquitin-proteasome dependent degradation of cyclin D1 and D2, probably by transiently inhibiting ubiquitination and/or the proteasome.

3T3 Cells↗
PubMed2000

Hydrogen peroxide reversibly inhibits epidermal growth factor (EGF) receptor internalization and coincident ubiquitination of the EGF receptor and Eps15.

Recently, we demonstrated that hydrogen peroxide (H2O2) inhibits the internalization of the epidermal growth factor (EGF) receptor and the EGF-induced mono-ubiquitination of EGF receptor pathway substrate clone #15 (Eps15) in fibroblasts. In addition, it was suggested that EGF receptor internalization might be inhibited by H2O2 by inhibition of ubiquitination of proteins involved in endocytosis. Here, we show that H2O2 also inhibits the poly-ubiquitination of the EGF receptor in fibroblasts. Furthermore, recovery of the cells resulted in re-establishment of ubiquitination of both the EGF receptor and Eps15 and coincided with restoration of internalization of those receptors that had bound EGF in the presence of H2O2. In addition, EGF receptor internalization was inhibited by the sulphydryl reagent N-ethylmaleimide (NEM), indicating that intact SH groups might be required for receptor-mediated endocytosis. Furthermore, H2O2 rapidly induced an increase in the cellular ratio of GSSG:GSH (oxidized glutathione:reduced glutathione) and removal of H2O2 resulted in a fast restoration of the ratio of GSSG:GSH. Therefore, these results suggest a relation between the inhibition of internalization ubiquitination and an increase in GSSG:GSH ratio, which strengthens the hypothesis that H2O2 inhibits EGF receptor internalization by an inhibition of ubiquitination of proteins involved in EGF receptor-mediated endocytosis.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Regulation of cytosolic phospholipase A(2) in a new perspective: recruitment of active monomers from an inactive clustered pool.

cPLA(2) plays a key role in many signal transduction cascades by hydrolyzing arachidonic acid from membrane phospholipids. Tight control of cPLA(2) activity by a number of regulatory mechanisms is essential to its cellular function. We recently described the localization of cPLA(2) in clusters in fibroblasts and now propose that these clusters reflect a localized inactive pool from which active monomers can be recruited to keep cPLA(2) activity under control on the subcellular level. Using an electron microscopic in vitro approach, we show that cPLA(2) monomers, but not the clusters, bind to membranes in a Ca(2+)-dependent manner. This binding is accompanied by hydrolytic activity. The present data combined with our previous observation of a relative abundance of clusters over monomers in fixed fibroblasts [Bunt, G., de Wit, J., van den Bosch, H., Verkleij, A., and Boonstra, J. (1997) J. Cell Sci. 110, 2449-2459] gives rise to a concept of cPLA(2) regulation in which small amounts of active monomers are recruited to fulfill their function upon stimulation. This is in contrast to processes described for inflammatory cells, where a substantial part of the cytoplasmically localized cPLA(2) translocates to the perinuclear region upon stimulation to become active. Small-scale regulation of cPLA(2) by the proposed cluster-monomer cycle allows local and strictly confined control of cPLA(2) activity, apparently necessary for its cellular role in fibroblasts.

3T3 Cells↗
PubMed2000

Hydrogen peroxide inhibits epidermal growth factor receptor internalization in human fibroblasts.

Several cellular signal transduction cascades are affected by oxidative stress. In this study, the effect of hydrogen peroxide (H2O2) on the endocytosis of the epidermal growth factor (EGF) receptor was investigated. Exposure of HER14 cells to H2O2 resulted in a concentration-dependent inhibition of EGF receptor internalization. Binding studies demonstrated that this H2O2-induced inhibition in internalization was not due to altered binding of EGF to its receptor. Addition of H2O2 at different time points during internalization showed that EGF receptor internalization was rapidly reduced, suggesting that one of the first steps in the internalization process is inhibited. In addition, H2O2 inhibited the internalization of a different receptor, the chicken hepatic lectin receptor, in a concentration-dependent manner as well. Treatment of cells with another inducer of oxidative stress, cumene hydroperoxide, also resulted in a decreased internalization. Finally, we showed that H2O2 inhibited EGF-induced mono-ubiquitination of the EGF receptor pathway substrate clone 15, a process that normally occurs during EGF receptor endocytosis. These results clearly show that oxidative stress interferes with EGF signaling.

3T3 Cells↗
PubMed2000

Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts.

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama V(HH) antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.

Animals↗
PubMed2000

Large-scale screening assay to measure epidermal growth factor internalization.

Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H(2)O(2)) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the H(2)O(2)-induced inhibition of internalization, a screening assay was developed to measure the internalization in 96-well plates. In this assay, cells are exposed to biotin-conjugated EGF and the amount of internalized EGF is detected with horseradish peroxidase-conjugated streptavidin. We show that the results obtained by this new assay are comparable with those from internalization studies performed with radioactive labeled EGF. Therefore, the cellular internalization assay as presented here is a reliable method to measure EGF receptor internalization. Moreover, because elaborate processing of the cells is not required, the assay is a relatively fast and inexpensive method to study ligand-induced internalization in 96-well plates and thereby is suitable for large-scale screening of compounds or conditions interfering with this internalization.

3T3 Cells↗
PubMed1999

Integrin signaling at the M/G1 transition induces expression of cyclin E.

The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21(cip1/Waf1) and p27(Kip1) is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment.

Animals↗
PubMed1999

Nuclear translocation of mitogen-activated protein kinase p42MAPK during the ongoing cell cycle.

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated rapidly in cells stimulated by various extracellular signals. With stimulation of quiescent cells by growth factors, activated p42/p44 MAP kinases rapidly translocate to the nucleus, where they induce immediate early gene transcription. The MAP kinase signal transduction pathway represents an important mechanism by which growth factors regulate cellular events such as cell cycle progression or cell growth. In the present study, p42MAPK (ERK2) was studied during the ongoing cell cycle of Chinese hamster ovary cells synchronized by mitotic shake-off. We show that protein expression of p42MAPK increased in mid-G1 and that MAP kinase is phosphorylated during G1, as visualized by a gel-mobility shift and by the use of phosphospecific antibodies. This phosphorylation appeared to occur in the cytoplasm rather than at the plasma-membrane. In addition, phosphorylated p42MAPK was found to translocate to the nucleus during late/mid-G1. Treatment of cells with MEK inhibitor PD098059 prevented the phosphorylation and nuclear translocation of MAP kinase and DNA synthesis. Thus, nuclear translocation of p42MAPK is not restricted to the G0/G1 transition but occurs in every cell cycle and seems to be required for cell cycle progression.

Animals↗