Analyses of calcium regulated cell proliferation and survival in mielo-erythroid cells

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1 Analyses of calcium regulated cell proliferation and survival in mielo-erythroid cells Ph. D. thesis Jánossy Judit PhD School in Biology, PhD program of Molecular Cell and Neurobiology University of Eötvös Loránd, Faculty of Science Leader of the Doctorate School: Erdei Anna Ph.D., D.Sc. Program vezető: Dr. Sass Miklós D.Sc. Supervisors: Dr. Péter Friedrich and Dr Péter Tompa Hungarian Academy of Sciences, Biological Research Center, Institute of Enzymology, Budapest, Hungary 2008.

2 INTRODUCTION The thesis summarizes the work carried out using TF-1 cells. This human hormone-dependent myelo-erithroid cell line is a suitable model to explore the signal transduction pathways of progenitor erythroid bone marrow cells. The effect of [Ca 2+ ] i -raising agents and calpain on TF-1 cells was investigated. Under physiological conditions, depending on the maturation state, progenitor cells may exhibit transient rises of [Ca 2+ ] i stimulated by Epo or GM-CSF hormones. In our model controlled elevations of the [Ca 2+ ] i led to hormone-independent survival and proliferation but not to erythroid differentiation. High levels of [Ca 2+ ] I induced apoptosis. Inhibition of calpain had no effect on the maturation or survival of cells, but reversibly arrested cell growth. Earlier, elevations of [Ca 2+ ] I inducing survival pathways was only observed in excitable cells. Thus, in-depth analyses were preformed on the processes underlying the phenomena. The first part of the thesis focuses on the activation of ERK1/2 mitogen activated proitein kinase (MAPK) pathway induced by rises of [Ca 2+ ] i as this route is commonly implicated in growth signaling. The second part of the thesis deals with the role of calpains in the cell cycle and to identify in which phase(s) are they involved. All of my work was carried out in close collaboration with the Signal transduction group, Institute of Hematology and Immunology Department of Molecular Cell Biology.

3 OBJECTIVES Changes in the intracellular Ca 2+ concentrations may initiate a number of cellular processes leading to cell survival, proliferation, differentiation or even cell death. Within my thesis I have investigated the different effects of Ca 2+ raising agents exhibited on hormone-dependent myelo-erithroid progenitor cells. I have focused on the calcium induced pathways leading to hormone-independent survival and proliferation, including the role of calpains, the calcium activated neutral proteases. My aim was to set up such a model system that allows the exploration of the pathways assisting the cell in decoding the different Ca 2+ -signals. I have challenged to answer the following questions in my thesis: What types of biological outcomes can we generate by different calcium-increasing agents in mielo-erithroid cells? What types of signals are required to bring about the biological effects? Which signal transduction pathway is activated by the increase of [Ca 2+ ] i? In what do the activation of ERK/MAPK pathways induced by different [Ca 2+ ] i increases differ? What role do calpains play in the proliferation and survival of TF-1 cells? Which phase(s) of the cell cycle is m- or µ-calpain activation implicated? Does the inhibition of calpain influence the ERK/MAPK pathway activated by [Ca 2+ ] I changes?

4 RESULTS We have established a complex model that allows the characterization of the biological outcomes induced by various Ca 2+ signals. The survival and proliferation of the model cell line was fully dependent on GM-CSF or IL-3. Hormone deprivation induced cell cycle arrest followed by apoptosis. Different [Ca 2+ ] i increases were stimulated, whereby controlled moderate elevation caused hormone-independent cell survival, controlled high increase even induced proliferation, whereas sustained high elevation induced apoptosis. The biological outcome depended on the amplitude and duration of the [Ca 2+ ] i elevation. Both hormone dependent and independent survival were accompanied by the rise of Bcl-2 levels, an antiapoptotic protein and the decrease of the Caspase-3 enzyme activity. Observed MEK/ERK1/2/Elk-1/c-Fos pathway activations correlated with the changes in the intracellular calcium concentrations. No activation was detected with control hormone-deprived cells, whilst stimulations causing survival and proliferation displayed a biphasic activation of the pathway. Furthermore, in accordance with the [Ca 2+ ] i changes, sustained activation of the pathway stimulated apoptosis. Calpain, a calcium-activated protease had no effect on the Ca 2+ stimulated pathway. However, we have shown that calcium is involved in the proliferation of our model hemopoetic cell line. Cell growth could be arrested reversibly by a specific calpain inhibitor without declining the viability of the cells. The detailed analyses of the effect of calpain inhibition on the cell cycle revealed for the first time that calpain is involved in all phases of the cell cycle, including S phase. Based on our observations and the literature it can be postulated that calpain may be activated by the MEK/ERK1/2 cascade either at a distal point or by another branch of the pathway. To sum it up, [Ca 2+ ] i changes may display completely different outcomes. In a human erythroid cell line controlled [Ca 2+ ] i elevations and MAPK activations resulted in hormoneindependent survival and proliferation, while sustained elevations induced apoptosis. We have concluded that calpain, another important member of the Ca 2+ signaling pathway plays a key role in cell proliferation. We suggest that our model can be useful in drug development for screening candidate compounds that target the Ca 2+ homeostasis of the cells.

5 METHODS Cell culture: We have characterized the hormone-dependent, myelo-erythroid cell line (TF-1), by monoclonal antibodies against CD34, CD38, CD33, CD14, CD15, and GPA cell surface markers. Cell number changes: Cell viability was determined by Trypan blue exclusion and MTT incorporation. Detection of the signs of apoptosis: Caspase-3 activity was measured by a colorimetric reaction. The DNA ladder formation was determined by agarose gel electrophoresis using ethidium bromide. The levels of PARP cleavage products and apoptotic proteins (Bcl-2, Bcl-xl és Bax) were followed by Westernblot analyses. Alteration of the intracellular free Ca 2+ : Changes of [Ca 2+ ] i were followed by fluorimetric method, using Fura-2AM. Activation of kinases and transcription factors: The activation of ERK 1/2 and Elk-1 was detected by a phospho-specific antibody. Study of transcription factors: The c-fos protein level was detected by Western-blot. Cell cycle analysis Cell cycle analyses were performed by both one-parametric staining using propidium iodide and pulse-chase analyses. Main Results and Conclusions: 1. I have shown for the first time that in human hematopoietic cells controlled transient intracellular calcium increases can lead to hormone-independent cell proliferation and survival, whereas sustained, high [Ca 2+ ]i elevation induces apoptosis. 2. I have shown that elevations of the expression of the anti-apoptotic protein, Bcl-2 plays an important role in Ca 2+ induced hormone independent cell survival. 3. I have demonstrated that the MEK/ERK/Elk-1 pathway plays a pivotal role in the hormone independent survival of TF-1 cells induced by CPA or ionomycin treatment. A correlation was observed between the kinetics of ERK1/2-MAPK activation and the biological outcome. 4. I have demonstrated that the sustained high activation of the ERK1/2-MAPK pathway leads to apoptosis independently from the mode of activation.

6 5. Inhibition of calpain by a specific inhibitor led to the reversible arrest of the proliferation of TF-1 cells without having an impact on the viability of cells. 6. The inhibition of calpain by the specific inhibitor affected all cell phases of the cell cycle. Besides controlling the G1-S transition and mitosis this was the first time that restriction of progression through the S phase was observed. 7. Calpain has not affected the activation of the ERK1/2-MAPK pathway in the model investigated. Publications included in the thesis: Janossy J, Ubezio P, Apati A, Magocsi M, Tompa P, Friedrich P. Calpain as a multi-site regulator of cell cycle Biochemical Pharmacology;67(8): Apati A, Janossy J, Brozik A, Magocsi M Effects of intracellular calcium on cell survival and the MAPK pathway in a human hormone-dependent leukemia cell line (TF- 1). Annals of New York Academia of Sciences. 1010:70-3. Apati, A.; Janossy, J.; Brozik, A.; Bauer, P. I.; Magocsi, M Calcium induces cell survival and proliferation through the activation of the MAPK pathway in a human hormonedependent leukemia cell line, TF-1. Journal of Biological Chemistry, 278(11): p Other related publications: Kolonics, A.; Apati, A.; Janossy, J.; Brozik, A.; Gati, R.; Schaefer, A.; Magocsi, M Activation of Raf/ERK1/2 MAP kinase pathway is involved in GM-CSF-induced proliferation and survival but not in erythropoietin-induced differentiation of TF-1 cells. Cellular Signaling. 13(10): p

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