Gert Rijksen

Polo-like kinase-1 is a target of the DNA damage checkpoint

Polo-like kinases (PLKs) have an important role in several stages of mitosis. They contribute to the activation of cyclin B/Cdc2 and are involved in centrosome maturation and bipolar spindle formation at the onset of mitosis. PLKs also control mitotic exit by regulating the anaphase-promoting complex (APC) and have been implicated in the temporal and spatial coordination of cytokinesis. Experiments in budding yeast have shown that the PLK Cdc5 may be controlled by the DNA damage checkpoint. Here we report the effects of DNA damage on Polo-like kinase-1 (Plk1) in a variety of human cell lines. We show that Plk1 is inhibited by DNA damage in G2 and in mitosis. In line with this, we show that DNA damage blocks mitotic exit. DNA damage does not inhibit the kinase activity of Plk1 mutants in which the conserved threonine residue in the T-loop has been changed to aspartic acid, suggesting that DNA damage interferes with the activation of Plk1. Significantly, expression of these mutants can override the G2 arrest induced by DNA damage. On the basis of these data we propose that Plk1 is an important target of the DNA damage checkpoint, enabling cell-cycle arrests at multiple points in G2 and mitosis.

c‐Src PROTEIN EXPRESSION IS INCREASED IN HUMAN BREAST CANCER. AN IMMUNOHISTOCHEMICAL AND BIOCHEMICAL ANALYSIS

In human breast cancer, c‐Src activity is elevated compared to normal breast tissue. It is not yet known whether this increase in c‐Src activity is accompanied by an increase in c‐Src protein expression. In this study, c‐Src activity and protein expression were determined in a series of human breast cancers and in normal breast tissue, using immune complex kinase assays and immunoblotting. As the heterogeneity of breast cancer is not taken into account in these biochemical experiments, immunohistochemistry was also used to distinguish between normal and malignant cells. In human breast cancers, the c‐Src activity is increased 4‐ to 30‐fold, compared with normal breast tissue. This enhanced activity is accompanied by an increase in c‐Src protein expression, as shown by both immunoblotting and immunohistochemistry. Immunohistochemistry indicates that the majority of c‐Src appears to be concentrated around the nucleus in malignant cells, whereas in normal cells, it is distributed more evenly in the cytoplasm. These data confirm that c‐Src activity is increased in human breast cancer. In addition, this study provides strong immunohistochemical evidence that the c‐Src protein is also overexpressed, enabling a distinction to be made between normal and malignant cells.

p21 Inhibits Thr161 Phosphorylation of Cdc2 to Enforce the G2 DNA Damage Checkpoint

The cyclin-dependent kinase inhibitor p21 is required for a sustained G2 arrest after activation of the DNA damage checkpoint. Here we have addressed the mechanism by which p21 can contribute to this arrest in G2. We show that p21 blocks the activating phosphorylation of Cdc2 on Thr161. p21 does not interfere with the dephosphorylation of two inhibitory phosphorylation sites on Cdc2, Thr14 and Tyr15, indicating that p21 targets a different event in Cdc2 activation as the well described DNA damage checkpoint pathway involving Chk1 and Cdc25C. Taken together our data show that a cell is equipped with at least two independent pathways to ensure efficient inhibition of Cdc2 activity in response to DNA damage, influencing both positive and negative regulatory phosphorylation events on Cdc2.

p21waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNA-replication or stress-activated kinases

p21waf1 has been shown to mediate the p53-dependent growth arrest induced by DNA-damaging agents. Several functions have been ascribed to p21waf1 that could be involved in this growth arrest. For one, p21waf1 is an efficient inhibitor of cyclin-dependent kinases (CDKs). Also, p21waf1 can interact with proliferating cell nuclear antigen (PCNA), and as such inhibit in vitro DNA-replication. Finally, p21waf1 has been reported to inhibit stress-activated protein kinases (SAPKs). In order to study these multiple functions of p21waf1 we have established U2OS-derived cell lines, in which the expression of p21waf1 can be regulated by the concentration of tetracycline in the culture medium. We observed a virtually complete, but reversible inhibition of cell growth upon induction of p21waf1-expression. Both [3H]thymidine-incorporation and CDK2-activity were strongly inhibited by p21waf1. Upon induction of p21waf1 cells accumulated with a 2N or 4N DNA content suggesting events in G1 and G2 can be inhibited by p21waf1. Indeed, kinase activity associated with cyclin B was reduced dramatically upon induction of p21waf1, although cyclin B continues to be expressed. In contrast, p21waf1 does not seem to inhibit the function of PCNA in ongoing DNA replication, since cells expressing high levels of p21waf1 apparently progressed normally through S-phase. Also, the activity of SAPKs was not substantially affected by the high levels of p21waf1. We conclude that, at least in these U2OS-derived cells, p21waf1 functions as an inhibitor of CDK-activity in G1 and G2, but not as an inhibitor of PCNA or SAPKs.

Overexpression of EGFR and c-erbB2 causes enhanced cell migration in human breast cancer cells and NIH3T3 fibroblasts

Overexpression of EGFR and c‐erbB2 frequently occurs in human breast cancers, correlating with poor prognosis. Here we show that overexpression of EGFR and c‐erbB2 in cell lines increases cell migration, an important step in metastasis formation. The effect of EGFR on migration is dependent on the addition of EGF to the cells. In contrast, c‐erbB2 seems to act independently of its ligand in these assays. Overexpression of this receptor is sufficient to induce cell migration. In addition, we investigated the involvement of a number of signal transduction pathways known to be activated by the EGFR. We found that inactivation of MAPKK results in a decreased migration, while inactivation of PI3K increases migration.

CD28 induces cell cycle progression by IL-2-independent down-regulation of p27kip1 expression in human peripheral T lymphocytes

CD28 is the primary T cell costimulatory receptor, and upon ligation with its ligands, it enhances T cell proliferation and IL‐2 synthesis. In this study we examined the role of CD28 in the initial proliferative response and cell cycle entry of T lymphocytes. Stimulation through CD3 alone resulted in a poor proliferative response, while in the presence of CD28 costimulation a strong increase in the number of cells in S‐phase could be detected after 48 h of stimulation. CD28 costimulation enhanced expression of cyclin D3 and induced down‐regulation of p27kip1 expression. Cross‐linking CD28 was much more effective in inducing cyclin D3 expression and in down‐regulating p27kip1 expression than addition of IL‐2. Blocking experiments, using antibodies that neutralize IL‐2 or the IL‐2 receptor, showed that the effects induced by CD28 are independent of endogenous IL‐2. Moreover, using a variety of immunosuppressants that interfere with IL‐2 signaling pathways, we were able to show that IL‐2 is not required for cell cycle entry induced by CD28 costimulation. From these experiments it can be concluded that CD28 and IL‐2 use different signaling pathways for down‐regulation of p27kip1 expression. We hypothesize that costimulation through CD28 is responsible for initial cell cycle entry of T lymphocytes, while IL‐2, which is produced after costimulation, might be involved in sustaining proliferation.

Quantitative erythrocyte membrane proteome analysis with Blue-Native/SDS PAGE

The erythrocyte membrane plays a pivotal role in erythrocyte functioning. Many membrane protein aberrations are known that result in hemolytic anemia, however, the origin of numerous disorders is not known to date. To extend the current set of diagnostic tools, we used a novel proteome-wide approach to quantitatively analyze membrane proteins of healthy donor and patient erythrocytes. Blue-native PAGE has proven to be a powerful tool for separation of membrane proteins and their complexes, but has hitherto not been applied to erythrocyte membranes to find biomarkers. Using this technique, we detected almost 150 protein spots, from which more than 500 proteins could be identified by LC-MS/MS. Further, we successfully assessed the potential of using CyDye labeling to quantify the membrane proteins. Our final goal was to determine if this approach is suited to detect protein level changes in disordered erythrocyte membranes, and we could successfully confirm that erythrocyte spectrin levels were dramatically decreased for a hemolytic anemia patient. This approach provides a new tool to detect potential biomarkers and can contribute to an improved understanding of the causes of erythrocyte membrane defects in patients suffering from hemolytic anemia.

Grb2 overexpression in nuclei and cytoplasm of human breast cells: a histochemical and biochemical study of normal and neoplastic mammary tissue specimens

In 20–30 per cent of human breast cancers, the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and c‐erbB2 are overexpressed. This overexpression leads to increased mitogenic signalling and is correlated with poor prognosis. Overexpression of associated adaptor proteins, like Grb2, can also induce upregulation of signalling pathways. In this study, the expression of the Grb2 adaptor protein was determined in both normal human breast tissue and mammary cancers, using immunoblotting experiments and immunostaining on paraffin‐embedded tissue sections. Both biochemical and immunohistochemical techniques revealed overexpression of Grb2 in all breast cancer specimens. In addition, although Grb2 protein is described as localized in the cytoplasm, it can also be detected in the nucleus, both in normal and in tumour breast tissue. In tumour breast tissue, 58 per cent of Grb2 protein is found in the nucleus, while 37 per cent is detected in the cytoplasm. In normal breast tissue, 22 per cent of Grb2 is found in the nucleus and 70 per cent in the cytoplasm. These findings indicate that in human breast cancer, Grb2 is overexpressed and appears to be predominantly localized in the nucleus.

Glycolytic Enzyme Activities in Breast Cancer Metastases

The activities of hexokinase, phosphofructokinase, aldolase, enolase and pyruvate kinase were studied in breast cancer metastases occurring at various sites and compared with the enzyme activities in a series of primary breast cancers. The activities of all enzymes studied were significantly higher in the metastases compared to the primary tumors (p less than or equal to 0.05). However, no changes in the isoenzyme patterns of enolase and pyruvate kinase were observed when the metastases were compared with primary breast cancers. Differences in location of the metastases did not lead to differences in enzyme activities. Our data suggest an association of an increasing rate of glycolysis with tumor progression.

Glycolytic Enzymes in Breast Cancer, Benign Breast Disease and Normal Breast Tissue

The activities of hexokinase, phosphofructokinase, aldolase, enolase and pyru-vate kinase were studied in breast cancer tissues, in comparison to benign breast disease and normal breast tissues. The e