Celestine Yeung and Dr. John Bell, Department of Physiology and Developmental Biology
This project studied and documented the membrane changes of cancer cells undergoing the complex cellular process apoptosis, or programmed cell suicide. More specifically, the purpose of this project was to achieve a visual comparison between S49 mouse lymphoma cells experiencing apoptosis induced by a variety of cytotoxic and chemotherapeutic drugs. This comparison was to be accomplished through cell imaging obtained by merocyanine (MC) 540 fluorescence confocal microscopy. Imaging accomplished in this project is an extension of ongoing research conducted by Dr. Bell and fellow colleagues concerning the action of the secretory phospholipase A2 (sPLA2) enzyme with respect to the effects of various chemotherapeutic drugs on lymphoma cell membranes. This project, along with the associated research concerning sPLA2, is intended to inform the scientific community of the potential implications of chemotherapeutic agents that promote apoptosis in cancer cells and why certain cell populations might exhibit resistance to sPLA2 despite treatment with such drugs. Answering these two questions relies on a sound, precise understanding of the process of programmed cell death that this project aimed to increase.
Though all apoptotic cells ultimately reach the same endpoint, the apoptotic process occurs through a variety of signaling cascades depending on the inducer. My project intended to determine physical distinctions, if any, between the cell membrane morphology of several of these apoptotic pathways. Originally, the project intended to include imaging and analysis of the physical effects of five apoptosis-inducing drugs: ionomycin, thapsisgargin, dexamethasone, methatrexate, and daunorubicin, on the cell surface membrane.
The details of the pathways induced by these five drugs are not entirely known, but current understanding indicates that each drug is unique in its respective stimulation of cell death. Ionomycin operates as a calcium ionophore, producing a dramatic and rapid influx of calcium ions (Ca2+) from the external environment into the cell cytosol (1). Elevated Ca2+ levels lead to activation of endoplasmic reticulum (ER) stress signaling pathways and consequently triggers cellular apoptosis. Thapsigargin is an inhibitor of the sacroplasmic/endoplasmic reticulum ATPase pump (2). Consequently, the presence of the drug results in a depletion of ER Ca2+ levels and simultaneous elevation of cytoplasmic Ca2+ levels, changes which also activate ER stress signaling pathways and induce apoptosis. Dexamethasone is a synthetic glucocorticoid that stimulates apoptosis through disruption of optimal intracellular Ca2+ levels. Specifically, the drug depletes ER Ca2+ and increases cytoplasmic Ca2+ levels (3). The mechanism of methatrexateinduced cellular apoptosis is not entirely known but the drug is thought to operate through a caspase-dependant apoptotic pathway (4). Daunorubicin is known to bind top isomerase II following cellular DNA replication. Disruption of the cell cycle by the drug therefore results in what is believed to be caspase-independent apoptosis (5). Ionomycin-, dexamethasone-, and daunorubicin-induced apoptosis are considered independent of caspase activity while thapsigargin- and methatrexate-induced apoptosis are considered dependent on caspase activity.
Image acquisition first commenced with microscopy of the effects of cytotoxic and caspaseindependent inducer ionomycin. Images were acquired on an Olympus FluoView FV 300 confocal laser scanning microscope equipped with five unique laser sources. The green Helium- Neon laser sources (TRITC) with an excitation wavelength of 543 nm were used to excite the merocyanine (MC) 540 probe. MC540 intercalates between the phospholipid heads of cell membranes (1). For imaging purposes, MC540 fluorescence indicated both phospholipid spacing and positioning as well as relative morphology of either surface membranes or membrane sections. Comparison of drug effects was both qualitative and quantitative; the comparison criteria included observations of apoptotic time frame, percent cells undergoing apoptosis at relative time frames, MC540 probe uptake and penetration of the cell surface membrane (fluorescence), and prevalence of membrane blebbing.
The imaging process continued using next the gluccocorticoid dexamethasone. Imaging of ionomycin treated- and dexamethasone treated- lymphoma cells demonstrated that apart from differences in time frame of the onset of membrane morphology (ionomycin approx. 20 min, dexamethasone approx. 24 hr), physical changes observed in the membrane were indistinguishable between the ionomycin-treated cell populations and the dexamethasone-treated ones. MC540 probe fluorescence, prevalence and size of blebbing were all highly comparable among the two treatments. After consideration, we concluded that this could be attributed to the principle that both ionomycin and dexamethasone were both caspase-dependant drugs.
The effects of caspase-independent drug thapsigargin were then imaged and documented. Results obtained were essentially analogous to those observed among respective ionomycin-treated and dexamethasone treated- populations, aside from the intermediate apoptotic onset time frame of 2- 4 hrs. At this stage in the process, it was determined that enough concerning the visual effects of drugs, whether caspase-dependant or caspase-independent, had been determined. Imaging of methatrexate-treated and daunorubicin-treated populations was therefore not conducted.
Though the results acquired and conclusions drawn were less informative than originally anticipated, the project was successful in eliminating membrane morphology observed by confocal microscopy as a means of differentiating between apoptotic pathways occurring within the cell. Also concluded was that the physical changes observed in lymphoma cell membranes via microscopy during apoptosis are not manifestations specific to the apoptotic inducer. This project allowed us to identify the limitations of confocal microscopy in studying apoptotic membranes, and future research is developing involving other experimental techniques.
References
- Bailey RW, et al. Relationship between membrane physical properties and secretory phospholipase A2 hydrolysis kinetics in S49 cells during ionophore-induced apoptosis. 2007. Biophys J. 93(7): 2350-62.
- Denmeade S, Isaacs J. The SERCA pump as a therapeutic target: making a “smart bomb” for prostate cancer. 2005. Cancer Biology & Therapy J. 4 (1): 14-22.
- Bian X, Hughes F, Huang Y, Cidlowski J, Putney J. Roles of cytoplasmic Ca2+ and intracellular Ca2+ stores in induction and suppression of apoptosis in S49 cells. 1997. American Journal of Physiology. 272 (4 Pt 1):C1241-9.
- Hattangadi D, DeMasters G, Walker T, et al. Influence of p53 and caspase 3 activity on cell death and senescence in response to methotrexate in the breast tumor cell. 2004. Biochemical Pharmacology J. 68 (9): 99-103
- Terrisse AD, Bezombes C, Lerouge S, et al. Daunorubicin and Ara-C induced interphasic apoptotic of human Type II leukemia cells is caspase-8-independent. 2002. Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids J. 1584 (2-3): 99-103.