Dr. John Bell, Department of Physiology & Developmental Biology

Abstract of original proposal

Many chemotherapeutic agents treat cancer by inducing programmed cell death (apoptosis) in the tumor. One of the consequences of apoptosis is changes in the physical properties of the cell membrane. Some of these changes are important for other phagocytic cells to recognize and remove the dying cells thereby preventing an inflammatory response. However, other physical changes convert the membrane from its normal state of resistance to hydrolytic attack by enzymes present in the blood to a state of susceptibility. If this susceptibility precedes removal of the apoptotic cells by phagocytes, a harmful inflammatory response could potentially occur. Whether cells become susceptible to hydrolysis early during chemotherapy-induced death is currently unknown. This project will test the hypothesis that apoptosis stimulated by cancer chemotherapeutic agents causes the membrane to become susceptible to hydrolytic attack before it is prepared for removal by phagocytes. It will also explore the nature of the membrane changes and whether they involve the action of intracellular enzymes (“caspases”) associated with the apoptosis process.

Hypothesis tested

Our original hypothesis contained three elements: 1) changes occur in the cell membrane that render it susceptible to hydrolytic attack during apoptosis initiated by cancer chemotherapy; 2) this is a general phenomenon regardless of the mechanism of action of the chemotherapeutic drug; 3) this event precedes other membrane changes associated with preparation of the cells for phagocytosis. A secondary hypothesis will also be tested by aim 3: these membrane changes are initiated by the action of intracellular proteases (“caspases”) that are known to be involved in apoptosis.

Specific aims

1. Assess the relative timing of the following three events during apoptosis of S49 lymphoma cells initiated by various cancer chemotherapeutic drugs: susceptibility of the membrane to hydrolysis by the lipolytic enzyme sPLA_{2, exposure of phosphatidylserine on the outer face of the cell membrane (loss of membrane asymmetry), and loss of membrane integrity.}
2. Usefluorescentprobesofmembranestructure(merocyanine540,laurdan,patman, and diphenylhexatriene) to identify changes in the cell membrane physical properties that coincide temporally with the three events investigated in aim 1. These probes have been selected based on their sensitivity to the following membrane properties: interlipid spacing, merocyanine 540; membrane hydration, laurdan and patman; membrane lipid fluidity and order, diphenylhexatriene.
3. Determinewhetherintracellularcaspasesareactivatedduringapoptosisinducedby cancer chemotherapeutic agents. Use a general caspase inhibitor to determine which of the events studied in aims 1 and 2 depend on caspase activation.

General evaluation

The project went very well. The necessary experiments have been completed and two papers detailing the results are in process. One has been reviewed and is now in process of revision for resubmission to a different journal that is a better fit with the work. The other article is in preparation for submission at the end of 2010 or early in 2011. Moreover, the results provided important data for submission of a grant proposal to NIH in June, 2010. The proposal has been declined and will be revised for resubmission early in 2011.

In addition to the investigations directly for this award, additional research applying these results to studies of normal human lymphocytes and neutrophils were initiated. Moreover, mentored students also participated in experiments, data analysis, and manuscript preparation for a project studying endoplasmic reticulum stress and apoptosis. This additional research helped meet the aims of a previous NIH grant, provided the basis for the next MEG proposal.

Results

Aim #1

Four chemotherapeutic drugs that operate through different mechanisms were tested: actinomycin D, paclitaxel, daunorubicin, and methotrexate. Each of these drugs caused S49 cells to enter a process of programmed cell death. Each was tested for the relative timing of lose of membrane integrity (permeability to the fluorescent dye propidium iodide) and susceptibility to hydrolysis by sPLA₂. In every case, a sizable proportion of the cells (up to 35% of the sample) became susceptible to enzymatic degradation prior to the time at which membrane integrity was compromised (Figure 1). It was also verified that each drug causes transmembrane movement of the phospholipid phosphatidylserine from its normal intracellular location to being exposed on the external face of the cell membrane, an event typical of apoptosis and part of the signal to empower macrophages to engulf the apoptotic cells.

Aim #2

The same four drugs were tested with S49 cells to complete this aim. Recent experiments from another project studying endoplasmic reticulum stress revealed that a different fluorescent probe, trimethylammonium diphenylhexatriene (TMA-DPH) would reveal more specific information about changes in the cell membrane than any of the other probes originally proposed for this MEG. We therefore included it with the experiments proposed in the MEG application. Figure 2 displays the results gathered thus far for TMA-DPH. In each case, TMA-DPH anisotropy decreased over time. This is the same result obtained during apoptosis initiated by endoplasmic reticulum stress. The interpretation is that lipid motion in the cell membrane increases during the process of programmed death regardless of the biochemical basis for starting the apoptotic events. Experiments with other fluorescent probes such as merocyanine 540 supported this conclusion (Figure 3).

Aim #3

Only a few (about 25%) of the proposed experiments were completed. These experiments demonstrated that the drug methotrexate leads to activation of caspases. Reports from other labs addressing this same question and recent technical difficulties with the measurements lessened the significance and our enthusiasm for completing this aim. The results of from the other two aims and reference to work from other laboratories will be sufficient to publish the findings and complete the overall objectives of the work in this proposal.

Students mentored

The following students were mentored at some level through this award.

Evaluation of mentoring environment

The original proposal was to fund seven undergraduate students. In fact, we were able to provide some level of funding to 11. Based on student comments, the mentoring environment was excellent. In addition to regular direct participation in planning experiments and in gathering and analyzing data, many of the students have had significant opportunity to assist with the process of producing abstracts for presentation and manuscripts for publication. Eight had the opportunity of attending the LFD at least once. Seven have or will present data in person at a national meeting of the Biophysical Society. Weekly lab meetings were held throughout the year in which students presented their research and received advice from each other and from the mentor. A social gathering was held in the mentor’s home three times per year. Two students received summer fellowships from the BYU Cancer Research Center to continue work associated with this MEG.

Publications resulting from students mentored during the period of this MEG (indicated in Bold):

Articles

• Bailey, R.W., Nguyen, T., Robertson, L., Gibbons, E., Nelson, J., Christensen, R.E., Bell, J.P., Judd, A.M., and Bell, J.D. (2009) Sequence of physical changes to the cell membrane during glucocorticoid-induced apoptosis in S49 lymphoma cells. Biophys. J. 96, 2709-2718.
• Gonzalez L.J., Gibbons E., Bailey R.W., Fairbourn J., Nguyen T., Smith S.K., Best K.B., Nelson J., Judd A.M., and Bell J.D. (2009) The influence of membrane physical properties on microvesicle release in human erythrocytes. PMC Biophys. 2:7.
• Olson, E.D., Nelson, J., Griffith, K., Nguyen, T., Streeter, M., Wilson-Ashworth, H.A., Gelb, M.A., Judd, A.M., and Bell, J.D. (2010) Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2. J. Biol. Chem. 285, 10993-11002.
• Franchino, H.A., Johnson, B.C., Neeley, S.K., Tajhya, R.B., Vu, M.P., Wilson- Ashworth, H.A., and Bell, J.D. Combined Use of Steady-State Fluorescence Emission and Anisotropy of Merocyanine 540 to Distinguish Crystalline, Gel, Ripple, And Liquid Crystalline Phases in Dipalmitoylphosphatidylcholine Bilayers. PMC Biophys. (revised version in review).
• Nelson, J., Berbert, A.M., Gibbons, E., Pickett, K.R., Streeter, M., Warcup, A.O., Yeung, C.H.-Y., Judd, A.M., and Bell, J.D. Relationship between Membrane Permeability and Specificity of Human Secretory Phospholipase A2 Isoforms during Cell Death. Biophys. Biochim. Acta (in revision for resubmission).

Three more papers including student co-authors mentored by this MEG are currently in preparation for submission in late 2010 or early 2011.

Abstracts at the Biophysical Society Meeting

• Franchino, H.A., Johnson, B.C., Neeley, S.K., Tajhya, R.B., and Bell, J.D. (2009) Assessment of merocyanine subpopulations in DPPC vesicles using anisotropy and lifetime measurements. Biophys. J. 96 (suppl), (#788).
• Gibbons, E., Askew, C.E., Griffith, K.R., Streeter, M.C., Warcup, A.O., Yeung, C.H.-Y., Judd, A.M., and Bell, J.D. (2009) Membrane changes during apoptosis: part of the process or characteristics of the corpse? Biophys. J. 96 (suppl), (#2191).
• Nelson, J., Barlow, K., Beck, D.O., Berbert, A., Damm, K., Eschenroder, N., Neeley, K., Pruitt, M., Thompson, K., Thurber, B., Yeung, C.H.-Y., Judd, A.M., and Bell, J.D. (2009) Chemotherapeutic apoptosis: who assailed the membrane, the inducer or the induced? Biophys. J. 96 (suppl), (#2192).
• Franchino, H., and Bell, J.D. (2010) Combined use of steady-state fluorescence emission and anisotropy of merocyanine 540 to distinguish crystalline, gel, ripple, and liquid crystalline phases in dipalmitoylphosphatidylcholine bilayers. Biophys. J. 98 (suppl), (#427).
• Anderson, L., Damm, K., Baker, R., Chen, J., Hamaker, A., Izidoro, I., Moss, E., Orton, M., Papworth, K., Sherman, L., Stevens, E., Yeung, C., Nelson, J., Judd, A.M., and Bell, J.D. (2010) Differential susceptibility of normal and transformed human leukocytes to hydrolytic attack by secretory phospholipase A2. Biophys. J. 98 (suppl), (#2410).
• Nelson, J., Olson, E., Griffith, K., Streeter, M., Judd, A.M., and Bell, J.D. (2010) Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2. Biophys. J. 98 (suppl), (#2411).

Accounting of Funds