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BCL-x_L-Dependent Light Scattering by Apoptotic Cells

Nada N. Boustany ^*, Yien-Che Tsai , Bryan Pfister , Wilsaan M. Joiner , George A. Oyler ^¶ || and Nitish V. Thakor 

^* Department of Biomedical Engineering, Rutgers University, Piscataway, New Jersey 08854; Laboratory of Protein Dynamics and Signaling, National Cancer Institute, Frederick, Maryland; Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland; ^¶ Department of Neurology, University of Maryland School of Medicine, Baltimore, Maryland; and ^|| Research Services, Baltimore VA Medical Center, Baltimore, Maryland

Correspondence: Address reprint requests to N. N. Boustany, PhD, Dept. of Biomedical Engineering, Rutgers University, 617 Bowser Rd., Piscataway, NJ 08854. Tel.: 732-445-5337; Fax: 732-445-3753; E-mail: nboustan{at}rci.rutgers.edu.

We measured the intensity ratio of wide-to-narrow angle scatter, optical scatter image ratio (OSIR), in single cells during apoptosis and after overexpression of the mitochondria-bound antiapoptotic protein BCL-x_L. OSIR is sensitive to particle size/shape for objects with wavelength-scale dimensions, and was used as a morphometric measure of cellular response. Three cell variants were treated with staurosporine (STS): nontransfected parental CSM14.1, CSM14.1 stably expressing yellow fluorescent protein (YFP) with diffuse YFP fluorescence, and apoptosis-resistant CSM14.1 stably expressing the fusion protein construct YFP-BCL-x_L with YFP fluorescence localized on the mitochondria. After treatment with 1 or 2 µM STS, the measured OSIR decreased monotonically by 25% in the nontransfected and YFP variants, and reached a steady-state value 40–60 min after STS treatment. The decrease in OSIR at the onset of apoptosis preceded phosphatidyl serine exposure by 5 h. In the YFP-BCL-x_L cell variant, the initial OSIR was already 24% lower than the initial OSIR in YFP and nontransfected cells, and only decreased by <10% after STS treatment. Alterations in light scattering by cells overexpressing BCL-x_L even before apoptosis induction raise interesting questions as to the role of BCL-x_L in conferring apoptosis resistance by preconditioning the cells and possibly altering mitochondrial morphology.

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