Carmen Ranftler

Roscovitine-activated HIP2 kinase induces phosphorylation of wt p53 at Ser-46 in human MCF-7 breast cancer cells.

Human MCF‐7 breast cancer cells are relatively resistant to conventional chemotherapy due to the lack of caspase‐3 activity. We reported recently that roscovitine (ROSC), a potent cyclin‐dependent kinase 2 inhibitor, arrests human MCF‐7 breast cancer cells in the G2 phase of the cell cycle and concomitantly induces apoptosis. Exposure of MCF‐7 cells to ROSC also strongly activates the wt p53 tumor suppressor protein in a time‐ and dose‐dependent manner. The p53 level increased despite upregulation of Hdm‐2 protein and was attributable to the site‐specific phosphorylation at Ser‐46. The p53 protein phosphorylated at serine 46 causes the up‐regulation of the p53AIP1 protein, a component of mitochondria. In the present study we identified the pathway mediating ROSC‐induced p53 activation. Exposure of MCF‐7 cells to ROSC activated homeodomain‐intereacting protein kinase‐2 (HIPK2). The overexpression of wild‐type but not kinase inactive HIPK2 increased the basal and ROSC‐induced level of p53 phosphorylation at Ser‐46 and strongly enhanced the rate of apoptosis in cells exposed to ROSC. We show that HIPK2 is activated by ROSC and mediates ROSC‐induced P‐Ser‐46‐p53, thereby stabilizing wt p53 and increasing the efficacy of drug‐induced apoptosis in MCF‐7 cells. These results identify HIPK2 as a component of the ROSC‐induced signaling pathway leading to the stabilization and activation of wt p53 protein. J. Cell. Biochem. 100: 865–874, 2007.

A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death

The efficacy of distinct anti‐cancer drugs used in the chemotherapy of human malignancies varies between tumor tissues and depends largely on the ability of the therapeutic agents to simultaneously inhibit cell proliferation and to eliminate malignant cells by apoptosis. Especially, detection of early apoptotic changes seems to be important because early stages of apoptosis differ from those of necrosis. Therefore, the development of a novel test allowing fast and concomitant screening of the anti‐proliferative and pro‐apoptotic action of a number of anti‐cancer drugs is of great interest. For this purpose, we choose as an experimental model a well characterized anti‐proliferative and pro‐apoptotic effect of cisplatin (CP) on human cervical carcinoma HeLaS3 cells. As previously reported, exposure of HeLaS3 to CP resulted in a concomitant inhibition of cell proliferation and induction of apoptosis in a dose‐ and time‐dependent manner. In the present study we performed two independent approaches. In the first approach, we examined the cell proliferation and activity of caspases‐3/7 in two separate microtiter plates using the CellTiter‐Glo™ Luminescent Cell Viability Assay and the Caspase‐Glo™ 3/7 Assay, respectively. In the second approach, we determined the same parameters sequentially in one microtiter plate by a mutiplexing assay using CellTiter‐Blue™ Cell Viability Assay and Caspase‐Glo™ 3/7 Assay. The both approaches gave very similar results indicating that this new multiplexing assay offers an important advantage for simultaneous detection of cell number and activation of caspases‐3/7. The new multiplexing assay offers a range of benefits over standard assays.

Phenol red reduces ROSC mediated cell cycle arrest and apoptosis in human MCF-7 cells.

We reported recently that roscovitine (ROSC), a selective cyclin‐dependent kinase (CDK) inhibitor, arrested human MCF‐7 breast cancer cells in G2 phase of the cell cycle and concomitantly induced apoptosis. On the other hand, ROSC‐induced G1 arrest observed by another group has not been accompanied by apoptosis. Therefore, we decided to prove to which extent components of tissue culture media could affect the primary action of ROSC. For this purpose we compared the efficacy of the ROSC treatment on MCF‐7 cells cultivated in medium with and without phenol red. The kinetics of MCF‐7 cell proliferation strongly depended on the presence of phenol red that has been recognized previously as a weak estrogen. Exposure of MCF‐7 cells cultivated in phenol red‐deprived medium to ROSC resulted in a strong G2 arrest and apoptosis. However, the anti‐proliferative and pro‐apoptotic action of ROSC was strongly diminished in cells maintained in medium containing phenol red. The ratio of the G2 cell population after 12 h ROSC was reduced by approximately 20% in the latter and correlated with the lack of CDK2 inactivation. Moreover, the kinetics of ROSC‐induced apoptosis was delayed in the presence of phenol red. These results clearly evidence that the efficacy of the therapy of ER‐positive breast cancers by CDK inhibitors is diminished in the presence of estrogen‐mimicking compounds and indicate that phytoestrogens and xenoestrogens could interfere with the therapy. Therefore, the exposure of cancer patients to the estrogen mimics should be avoided at least during chemotherapy by CDK inhibitors. J. Cell. Biochem.

The ceramide-enriched trans-Golgi compartments reorganize together with other parts of the Golgi apparatus in response to ATP-depletion

In this study, the ceramide-enriched trans-Golgi compartments representing sites of synthesis of sphingomyelin and higher organized lipids were visualized in control and ATP-depleted hepatoma and endothelial cells using internalization of BODIPY-ceramide and the diaminobenzidine photooxidation method for combined light-electron microscopical exploration. Metabolic stress induced by lowering the cellular ATP-levels leads to reorganizations of the Golgi apparatus and the appearance of tubulo-glomerular bodies and networks. The results obtained with three different protocols, in which BODIPY-ceramide either was applied prior to, concomitantly with, or after ATP-depletion, revealed that the ceramide-enriched compartments reorganize together with other parts of the Golgi apparatus under these conditions. They were found closely associated with and integrated in the tubulo-glomerular bodies formed in response to ATP-depletion. This is in line with the changes of the staining patterns obtained with the Helix pomatia lectin and the GM130 and TGN46 immuno-reactions occurring in response to ATP-depletion and is confirmed by 3D electron tomography. The 3D reconstructions underlined the glomerular character of the reorganized Golgi apparatus and demonstrated continuities of ceramide positive and negative parts. Most interestingly, BODIPY-ceramide becomes concentrated in compartments of the tubulo-glomerular Golgi bodies, even though the reorganization took place before BODIPY-ceramide administration. This indicates maintained functionalities although the regular Golgi stack organization is abolished; the results provide novel insights into Golgi structure–function relationships, which might be relevant for cells affected by metabolic stress.

Phenol red in the culture medium strongly affects the susceptibility of human MCF-7 cells to roscovitine

Estrogens play an important role in the growth and terminal differentiation of the mammary gland. Prolonged exposure to estrogens seems to predispose women to breast cancer. It recently became evident that not only the intrinsic hormonal status but also external factors such as the occurrence of pharmaceuticals and chemicals with hormone activity in the environment may put women at greater risk of developing breast cancer. We focused on the interference of endocrine disruptors in breast cancer therapy. We observed that phenol red added to the culture medium strongly promoted the cell proliferation and cell cycle progression of human cells expressing the estrogen receptor, and affected their susceptibility to chemotherapy.

A new, unexpected action of olomoucine, a CDK inhibitor, on normal human cells: Up-regulation of CLIMP-63, a cytoskeleton-linking membrane protein

Inhibition of cyclin‐dependent kinases (CDKs) is a novel strategy in the therapy of human malignancies. The pharmacological CDK inhibitors representing a few distinct classes of compounds exert different target specificity. Considering the fact that dividing and quiescent cells differ in their CDK activity and in the pattern of their expression, one might expect that anti‐proliferative efficiency of the pharmacological CDK inhibitors would depend on the mitotic index of treated cells. The present article shows that olomoucine (OLO), a weak CDK2 inhibitor has new, unexpected activity. At concentrations up to 100 µM OLO did not inhibit proliferation of normal human cells, but arrested growth of human HL‐60 leukemia cells. The anti‐proliferative effect of OLO was clearly weaker than that of roscovitine (ROSC). Surprisingly, OLO at low doses strongly up‐regulated a cellular protein with approximately 65 kDa in normal, but not in immortalized and cancer cells. By mass spectrometric analysis CLIMP‐63, a cytoskeleton‐linking membrane protein was identified as the major component of the up‐regulated protein band. These results were subsequently confirmed by immunoblotting. Further experiments revealed that OLO, but not ROSC, strongly up‐regulates CLIMP‐63 in a dose‐ and time‐dependent manner solely in senescent cells. J. Cell. Biochem. 102: 1405–1419, 2007.

Characterization of the Antibodies to p62 Nucleoporin in Primary Biliary Cirrhosis Using Human Recombinant Antigen

Reactivity of sera from patients with primary biliary cirrhosis (PBC) with a 60 kDa component of nuclear pore complexes (NPCs), purified by affinity chromatography on wheat‐germ agglutinin (WGA)‐Sepharose, was previously detected. Recently, clinical significance of the anti‐NPC antibodies in PBC became evident. In the light of recent reports, indicating the correlation of the anti‐NPC antibodies with severity and progression of the disease, the characterization of the reactive antigens is becoming essential in the clinical management of patients with PBC. Since accurate autoantibody detection represents one of the fundamental requirements for a reliable testing, we have generated a human recombinant p62 protein and validated an immunoprecipitation assay for the detection of anti‐p62. We also demonstrated that the generated human recombinant p62 nucleoporin was modified by N‐acetylglucosamine residues. More than 50% of tested PBC sera precipitated 35S‐radioactively labeled p62 recombinant nucleoporin and 40% recognized this recombinant antigen by immunoblotting. We compared the reactivity of PBC sera with rat and human nucleoporin. The incidence of anti‐p62 nucleoporin positive PBC sera increased by 15% when human recombinant antigen was used. The titer of autoantibodies in p62‐positive PBC samples strongly varied. Preadsorption of the PBC sera with p62 recombinant protein completely abolished their reactivity with the antigen. In conclusion, this study unequivocally proves that autoantibodies reacting with the 60 kDa component of NPCs target p62 nucleoporin and, more importantly, provide a better antigen source for future evaluations of the clinical role of anti‐p62 in PBC. J. Cell. Biochem. 104: 27–37, 2008.

Electron microscopy of endocytic pathways.

Detailed insight into the fine structure and 3D-architecture of the complex and dynamic compartments of the endocytic system is essential for a morpho-functional analysis of retrograde traffic from the cell surface to different intracellular destinations. Here, we describe a cytochemical approach for electron microscopic exploration of endocytic pathways with the use of wheat germ agglutinin (WGA) in combination with either conventional chemical fixation or ultrafast physical fixation of the cells by high pressure-freezing. Horseradish peroxidase-labeled WGA endocytozed by human hepatoma cells for various periods of time served as a marker. Its intracellular routes were visualized by means of diaminobenzidine oxidation either done conventionally after chemical fixation or in living cells prior to physical fixation. The latter protocol permits the combination of peroxidase-catalyzed cytochemistry with high pressure-freezing (HPF), which is state of the art for ultrastructural studies of complex and dynamic organelles at high spatial and temporal resolutions. The technique yields distinct cytochemical reactions and excellently preserved fine structures well qualified for detailed electron microscopic and 3D-studies of the complex endocytic architectures.

Effect of Distinct Anticancer Drugs on the Phosphorylation of p53 Protein at Serine 46 in Human MCF-7 Breast Cancer Cells

Abstract:  Roscovitine (ROSC), a potent cyclin‐dependent kinase inhibitor (CDI), inactivates cyclin‐dependent kinase (CDK)2 resulting in the arrest of human MCF‐7 breast cancer cells in G2 phase of the cell cycle. We have recently observed a strong activation of wild‐type (wt) p53 protein in human MCF‐7 breast cancer cells upon treatment with ROSC implicating that upregulated p53 might additionally modulate the primary action of ROSC. ROSC stabilized wt p53 protein resulting in a marked extension of its half‐life. Since ROSC exhibits low cytotoxicity, it seems to upregulate p53 protein in a way different from DNA damage. ROSC induced phosphorylation of p53 protein at serine 46. Therefore, we decided to examine whether other anticancer drugs are also able to induce phosphorylation of wt p53 protein at serine 46. Exposure of MCF‐7 cells to doxorubicin (DOX) at doses inducing a strong G2 arrest resulted in a weak upregulation of p53. No site‐specific phosphorylation of p53 at serine 46 was detected. These results indicate that p53 activation is dispensable for DOX‐induced G2 arrest. Moreover, the pattern of p53 phosphorylation strongly depends on the type of the stimulating factor.

Golgi apparatus dis- and reorganizations studied with the aid of 2-deoxy-d-glucose and visualized by 3D-electron tomography

We studied Golgi apparatus disorganizations and reorganizations in human HepG2 hepatoblastoma cells by using the nonmetabolizable glucose analogue 2-deoxy-d-glucose (2DG) and analyzing the changes in Golgi stack architectures by 3D-electron tomography. Golgi stacks remodel in response to 2DG-treatment and are replaced by tubulo-glomerular Golgi bodies, from which mini-Golgi stacks emerge again after removal of 2DG. The Golgi stack changes correlate with the measured ATP-values. Our findings indicate that the classic Golgi stack architecture is impeded, while cells are under the influence of 2DG at constantly low ATP-levels, but the Golgi apparatus is maintained in forms of the Golgi bodies and Golgi stacks can be rebuilt as soon as 2DG is removed. The 3D-electron microscopic results highlight connecting regions that interlink membrane compartments in all phases of Golgi stack reorganizations and show that the compact Golgi bodies mainly consist of continuous intertwined tubules. Connections and continuities point to possible new transport pathways that could substitute for other modes of traffic. The changing architectures visualized in this work reflect Golgi stack dynamics that may be essential for basic cell physiologic and pathologic processes and help to learn, how cells respond to conditions of stress.