Deborah Finucane

Phosphatidylserine Externalization during CD95-induced Apoptosis of Cells and Cytoplasts Requires ICE/CED-3 Protease Activity

Phosphatidylserine (PS), a lipid normally confined to the inner leaflet of the plasma membrane, is exported to the outer plasma membrane leaflet during apoptosis to serve as a trigger for recognition of apoptotic cells by phagocytes. The mechanism of PS export during apoptosis is not known nor is it clear whether the nuclear changes that typify apoptosis contribute in any way to this event. Here, we demonstrate that ligation of the CD95 (Fas/APO-1) molecule on Jurkat cytoplasts induces dramatic PS externalization similar to that observed during apoptosis of intact cells. Apoptosis of both cells and cytoplasts was associated with proteolytic processing of CPP32, a member of the interleukin-1β converting enzyme (ICE)/CED-3 protease family, to its active form. Fodrin, a component of the cortical cytoskeleton, also underwent proteolytic cleavage during apoptosis of both cytoplasts and intact cells. Strikingly, CPP32 activation, fodrin proteolysis, and PS externalization were all inhibited in the presence of peptide inhibitors of ICE/CED-3 family proteases. These data provide strong support for the notion that the cell death machinery is extranuclear and is likely to be comprised of one or more members of the ICE/CED-3 family and that activation of this machinery does not require nuclear participation.

ANTI-APOPTOTIC ONCOGENES PREVENT CASPASE-DEPENDENT AND INDEPENDENT COMMITMENT FOR CELL DEATH

Apoptosis is a morphologically defined type of cell death associated with the activation of certain proteases belonging to the ICE/CED-3 family, known as caspases. Resistance to apoptosis has been implicated as one of the mechanisms that participates in oncogenesis. We found that the broad-spectrum peptide inhibitor of the caspases, zVAD-fmk, interferes in a dose-dependent way with all the morphological and biochemical changes associated with apoptosis induced by anti-CD95 mAb, staurosporine, VP-16 and Act-D. However, with the exception of anti-CD95-triggered apoptosis, the insulted cells lost their clonogenic potential, even when pre-treated with a high dose of zVAD-fmk. Under these circumstances, the dying cells displayed no signs of apoptosis, including activation of caspases, externalization of phosphatidylserine, nuclear condensation, or DNA fragmentation. Instead, this cell death was characterized by cytoplasmic and nuclear vacuolization followed by the loss of plasma membrane integrity. Thus, preventing the onset of apoptosis by blocking caspase activity did not rescue cells from dying in response to drugs such as staurosporine, VP-16 and Act-D. In comparison, ectopic expression of anti-apoptotic oncogenes such as bcl-2 and bcr-abl not only inhibited apoptosis but also preserved the clonogenic potential of the cells. Therefore, oncogenesis is promoted not by simply interfering with caspase-mediated apoptosis, but by preventing an upstream event which we define as the commitment point for cell death.

Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect Apoptosis

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane,Thomas Brunner, Ella Bossy-Wetzel and Douglas R. GreenThis protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector etal.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. Thisthree-volume set is now out of print; however, some of the microscopy methods wererepublished in Basic Methods in Microscopy, by David L. Spector and Robert D.Goldman.

Analysis of DNA Fragmentation Using Agarose Gel Electrophoresis

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane, Thomas Brunner, Ella Bossy-Wetzel and Douglas R. Green This protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This three-volume set is now out of print; however, some of the microscopy methods were republished in Basic Methods in Microscopy, by David L. Spector and Robert D. Goldman. INTRODUCTION

Staining of Suspension Cells with Hoechst 33258 to Detect Apoptosis

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane,Thomas Brunner, Ella Bossy-Wetzel and Douglas R. GreenThis protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector etal.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. Thisthree-volume set is now out of print; however, some of the microscopy methods wererepublished in Basic Methods in Microscopy, by David L. Spector and Robert D.Goldman.

Analysis of TUNEL Staining by Flow Cytometry to Detect Apoptosis

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane,Thomas Brunner, Ella Bossy-Wetzel and Douglas R. GreenThis protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector etal.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. Thisthree-volume set is now out of print; however, some of the microscopy methods wererepublished in Basic Methods in Microscopy, by David L. Spector and Robert D.Goldman.

Detection of phosphatidylserine externalization during apoptosis.

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane, Thomas Brunner, Ella Bossy-Wetzel and Douglas R. Green This protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This three-volume set is now out of print; however, some of the microscopy methods were republished in Basic Methods in Microscopy, by David L. Spector and Robert D. Goldman.

Biochemical analysis of cell death using colorimetric quantification of caspase activation.

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane,Thomas Brunner, Ella Bossy-Wetzel and Douglas R. GreenThis protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector etal.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. Thisthree-volume set is now out of print; however, some of the microscopy methods wererepublished in Basic Methods in Microscopy, by David L. Spector and Robert D.Goldman.

TUNEL Staining of Adherent Cells to Detect Apoptosis

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane,Thomas Brunner, Ella Bossy-Wetzel and Douglas R. GreenThis protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector etal.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. Thisthree-volume set is now out of print; however, some of the microscopy methods wererepublished in Basic Methods in Microscopy, by David L. Spector and Robert D.Goldman.

TUNEL Staining of Tissue Sections to Detect Apoptosis

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane,Thomas Brunner, Ella Bossy-Wetzel and Douglas R. GreenThis protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector etal.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. Thisthree-volume set is now out of print; however, some of the microscopy methods wererepublished in Basic Methods in Microscopy, by David L. Spector and Robert D.Goldman.