Katrien Vermeulen

The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer

Abstract.  The cell cycle is controlled by numerous mechanisms ensuring correct cell division. This review will focus on these mechanisms, i.e. regulation of cyclin‐dependent kinases (CDK) by cyclins, CDK inhibitors and phosphorylating events. The quality checkpoints activated after DNA damage are also discussed. The complexity of the regulation of the cell cycle is also reflected in the different alterations leading to aberrant cell proliferation and development of cancer. Consequently, targeting the cell cycle in general and CDK in particular presents unique opportunities for drug discovery. This review provides an overview of deregulation of the cell cycle in cancer. Different families of known CDK inhibitors acting by ATP competition are also discussed. Currently, at least three compounds with CDK inhibitory activity (flavopiridol, UCN‐01, roscovitine) have entered clinical trials.

Cell cycle and apoptosis.

Abstract.  Apoptosis and proliferation are intimately coupled. Some cell cycle regulators can influence both cell division and programmed cell death. The linkage of cell cycle and apoptosis has been recognized for c‐Myc, p53, pRb, Ras, PKA, PKC, Bcl‐2, NF‐κB, CDK, cyclins and CKI. This review summarizes the different functions of the proteins presently known to control both apoptosis and cell cycle progression. These proteins can influence apoptosis or proliferation but different variables, including cell type, cellular environment and genetic background, make it difficult to predict the outcome of cell proliferation, cell cycle arrest or cell death. These important decisions of cell proliferation or cell death are likely to be controlled by more than one signal and are necessary to ensure a proper cellular response.

Apoptosis: mechanisms and relevance in cancer

Apoptosis or programmed cell death is a process with typical morphological characteristics including plasma membrane blebbing, cell shrinkage, chromatin condensation and fragmentation. A family of cystein-dependent aspartate-directed proteases, called caspases, is responsible for the proteolytic cleavage of cellular proteins leading to the characteristic apoptotic features, e.g. cleavage of caspase-activated DNase resulting in internucleosomal DNA fragmentation. Currently, two pathways for activating caspases have been studied in detail. One starts with ligation of a death ligand to its transmembrane death receptor, followed by recruitment and activation of caspases in the death-inducing signalling complex. The second pathway involves the participation of mitochondria, which release caspase-activating proteins into the cytosol, thereby forming the apoptosome where caspases will bind and become activated. In addition, two other apoptotic pathways are emerging: endoplasmic reticulum stress-induced apoptosis and caspase-independent apoptosis. Naturally occurring cell death plays a critical role in many normal processes like foetal development and tissue homeostasis. Dysregulation of apoptosis contributes to many diseases, including cancer. On the other hand, apoptosis-regulating proteins also provide targets for drug discovery and new approaches to the treatment of cancer.

Induction of complete and molecular remissions in acute myeloid leukemia by Wilms’ tumor 1 antigen-targeted dendritic cell vaccination

Active immunization using tumor antigen-loaded dendritic cells holds promise for the adjuvant treatment of cancer to eradicate or control residual disease, but so far, most dendritic cell trials have been performed in end-stage cancer patients with high tumor loads. Here, in a phase I/II trial, we investigated the effect of autologous dendritic cell vaccination in 10 patients with acute myeloid leukemia (AML). The Wilms’ tumor 1 protein (WT1), a nearly universal tumor antigen, was chosen as an immunotherapeutic target because of its established role in leukemogenesis and superior immunogenic characteristics. Two patients in partial remission after chemotherapy were brought into complete remission after intradermal administration of full-length WT1 mRNA-electroporated dendritic cells. In these two patients and three other patients who were in complete remission, the AML-associated tumor marker returned to normal after dendritic cell vaccination, compatible with the induction of molecular remission. Clinical responses were correlated with vaccine-associated increases in WT1-specific CD8+ T cell frequencies, as detected by peptide/HLA-A*0201 tetramer staining, and elevated levels of activated natural killer cells postvaccination. Furthermore, vaccinated patients showed increased levels of WT1-specific IFN-γ–producing CD8+ T cells and features of general immune activation. These data support the further development of vaccination with WT1 mRNA-loaded dendritic cells as a postremission treatment to prevent full relapse in AML patients.

A maximal exercise bout increases the number of circulating CD34+/KDR+ endothelial progenitor cells in healthy subjects. Relation with lipid profile

Mobilization of bone marrow-derived endothelial progenitor cells (EPC) might explain exercise-induced improvement of endothelial function. We assessed whether a maximal exercise bout could alter the number of circulating EPC in healthy subjects and whether this effect is related to their cardiovascular risk profile. Additionally, we investigated possible mediators of this effect, namely nitric oxide (NO) bioavailability and vascular endothelial growth factor (VEGF) release. Healthy subjects (group 1, n = 11; group 2, n = 14) performed a symptom-limited cardiopulmonary exercise test on a bicycle ergometer. Numbers of CD34+/kinase insert domain receptor (KDR)+ cells were determined by flow-cytometric analysis, either after magnetic separation of CD34+ cells (group 1) or starting from whole blood (group 2). Serum concentrations of VEGF and NO metabolites were measured by using ELISA. Following exercise, EPC increased by 76% (15.4 +/- 10.7 cells/ml vs. 27.2 +/- 13.7 cells/ml; P = 0.01) in group 1 and by 69% in group 2 (30.9 +/- 14.6 cells/ml vs. 52.5 +/- 42.6 cells/ml; P = 0.03). The increase in EPC correlated positively with LDL and total cholesterol/HDL ratio and negatively with peak oxygen consumption and oxygen consumption at anaerobic threshold. VEGF levels increased with exercise, with a strong trend toward significance (P = 0.055). NO levels remained unchanged. The present study demonstrates that a maximal bout of exercise induces a significant shift in CD34+ cells toward CD34+/KDR+ cells. This response was larger in subjects with a less favorable lipid profile.

Quantification of circulating endothelial progenitor cells: A methodological comparison of six flow cytometric approaches

OBJECTIVES The validity of endothelial progenitor cells as biomarkers and their therapeutic potential depend on the accuracy of techniques used for enumeration. This study assessed the agreement between 6 flow cytometric methods and a CFU assay used for EPC quantification. METHODS Two blood samples were obtained from 30 healthy volunteers (60 samples). CD34+/VEGFR2+ cells were analyzed with flow cytometry, starting from whole blood (A-C) or PBMC (D-F), using different gating strategies: A: lymphocyte gating; B and D: exclusion of autofluorescent cells (CD3 negative selection); C and E: exclusion of autofluorescence and cell aggregates (pulse shape analysis by FSCarea/FSCpeak); F: exclusion of autofluorescence, cell aggregates and non-nucleated cells (Draq 5). PBMC were cultured under endothelial cell conditions to assess CFU numbers. RESULTS Moderate agreement was found between methods B-C and D-E (ICC 0.647 and 0.530). Comparison of methods B-D and C-E showed poor agreement (ICC 0.178 and 0.249). This was also the case for techniques that considerably differed with regard to gating strategies (A-B, A-F, B-F). CFU numbers did not correlate with flow cytometric quantification (all p>0.05). CONCLUSIONS Agreement between methods for EPC quantification is moderate to poor, which may explain apparent controversies in literature. Although each protocol is highly reproducible, this study cautions against comparing study results gathered with different enumeration techniques.

Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro

PURPOSE The mechanism of radiosensitization by gemcitabine is still unclear. It has been hypothesized that the accumulation of cells in early S phase may play a role in enhancing radiosensitivity. METHODS AND MATERIALS The schedule dependency of the radiosensitizing effect was studied in ECV304, human bladder cancer cells, and H292, human lung cancer cells, by varying the incubation time and time interval between gemcitabine and radiation treatment. To determine the role of cell cycle perturbations in the radiosensitization, the influence of gemcitabine on the cell cycle at the moment of radiation was investigated by flow cytometry. RESULTS The radiosensitizing effect increased with a longer incubation period: Dose enhancement factors varied from 1.30 to 2.82 in ECV304 and from 1.04 to 1.78 in H292 after treatment during 8-32 h, respectively. Radiosensitization decreased with an increasing interval: Dose enhancement factors varied from 2.26 to 1.49 in ECV304 and from 1.45 to 1.11 in H292 after an interval 0-24 h, respectively. Cells were blocked in the early S phase of the cell cycle by gemcitabine. The highest percentage S-phase cells was observed after treatment with the schedules that resulted in the highest radiosensitizing effect. CONCLUSIONS We observed a clear schedule-dependent radiosensitization by gemcitabine. Our findings demonstrated a correlation between gemcitabine-induced early S-phase block and the radiosensitizing effect.

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord

BackgroundBone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord.ResultsFirst, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants.ConclusionIn this study, we demonstrate that genetically modified hMSC lines can survive in healthy rat spinal cord over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression. However, before genetically modified hMSC can potentially be used in a clinical setting to treat spinal cord injuries, more research on standardisation of hMSC culture and genetic modification needs to be done in order to prevent tumour formation and transgene silencing in vivo.

Antiproliferative effect of plant cytokinin analogues with an inhibitory activity on cyclin-dependent kinases

In this study, analogues of olomoucine, a previously described plant cytokinin analogue with cyclin-dependent kinase (CDK) inhibitory activity, were investigated for effect on CDK1 and CDK2 and for effect on cell proliferation. Eight new compounds exhibit stronger inhibitory activity on CDK1 and CDK2 and on cell proliferation than olomoucine. Some active compounds showed low inhibition of proliferation of normal myeloid growth. Improvement of inhibitory activity of known compounds with a C6-benzylamino group was brought about by substitution with one hydroxyl. Also, new C2 substituents associated with inhibitory activity on CDK and on cell proliferation are described. There was a significant correlation between effect on CDK and antiproliferative effect on the KG1 and Molt3 cell lines and on primary human lymphocytes, strongly suggesting that at least part of the antiproliferative effect of cytokinin analogues was due to inhibition of CDK activity. Cytokinin analogues induced apoptosis in a time- and concentration-dependent manner and changes in cell cycle distribution. The antiproliferative and pro-apoptotic effects of plant cytokinin analogues suggest that they are a new class of cytostatic agents and that they may find an application in the chemotherapy of cancer.

Plant cytokinin analogues with inhibitory activity on cyclin-dependent kinases exert their antiproliferative effect through induction of apoptosis initiated by the mitochondrial pathway: Determination by a multiparametric flow cytometric analysis

OBJECTIVE Regulation of the cell cycle by cyclin-dependent kinase (CDK) activity occurs at multiple levels and is often altered in human cancers. Therefore, CDK activity has been targeted for drug discovery, and a number of small molecules have now been identified as CDK inhibitors. Plant cytokinin analogues with CDK inhibitory activity and antiproliferative effects were studied to characterize the cellular basis of the cytotoxic effect. METHODS The IC(50) value (concentration at which 50% of the cell proliferation is inhibited) and AC(50) value (concentration at which 50% of the cell population is apoptotic) were determined by flow cytometry and microscopy, respectively. A new multiparametric flow cytometric analysis was used to study the sequence of different apoptotic events. In this assay, analysis of phosphatidylserine exposure, mitochondrial membrane depolarization, activation of caspases and DNA condensation were combined. RESULTS Treatment of Jurkat and KG1 cells with the CDK inhibitors results in a decrease of viable cells and a parallel increase in percentage of apoptotic cells. Apoptosis was accompanied by a rapid decrease of mitochondrial membrane potential, which precedes DNA condensation, exposure of phosphatidylserine and activation of caspases. CONCLUSIONS The main cellular mechanism of the antiproliferative effect of plant cytokinin analogues with CDK inhibitory activity is the induction of apoptosis. The multiparametric flow cytometric technique allowed to follow the kinetics of various aspects of apoptotic cell changes and demonstrated that cytokinin analogue-induced apoptosis starts through the mitochondrial pathway. This technique could also become of value for the rapid screening of pro-apoptotic properties of chemotherapeutic compounds.