The role of mitochondria in apoptotic signaling
Recently a large amount of evidence has accumulated suggesting
that mitochondria are a central signalling step in some forms
of apoptosis. In this talk I will critically review the data
supporting this hypothesis.
Motoneurone survival factors
McLennan, I.S., Koishi, K. and Zhang, M.
Approximately half of immature motoneurones die during
development, through a process which involves a competition
for an anti-apoptotic factor. This search for this factor
has been long but recently over a dozen candidate factors
have been discovered. Surprisingly, none of these factors
seems physiologically important. They are, however, providing
important clues about the regulation of adult motoneurone
survival. We will briefly review this work, with reference
to our own search for anti-apoptotic factors.
Programmed cell death following mitochondrial
Department of Biochemistry, University of Otago.
Mitochondria are a major source of cellular oxidative
stress. Here I am investigating the induction of programmed
cell death mechanisms by oxidative stress. This follows from
work in my PhD project where I investigated the role of mitchondrial
oxidative stress in the opening of a mitochondrial inner membrane
pore. In particular I was interested in peroxynitrite, a potent
oxidant which is formed from superoxide and nitric oxide and
whose biological significance has recently been realised.
My PhD project showed that mitochondria can produce enough
peroxynitrite to open a pore in the mitochondral inner membrane,
an event called the mitochoridrial permeability transition.
The present project has looked at whether these processes
are involved in the programmed cell death of a PC12 cell culture
model. The role of the mitochondrial permeability transition
is unclear but recently there is considerable interest in
the possibility of the process being part of an active process
to release apoptogenic factors from mitochondria. Here PCI2
cell apoptotic death has been induced in a number of ways
and the role of the permeability transition in the death process
Release of apoptogenic proteins from the mitochondrial
intermembrane space during the mitochondrial permeability
Department of Biochemistry, University of Otago.
The Bc1-2-sensitive release of proteins such as cytochrome
c from the mitochondrial intermembrane space into the cytosol
is a critical early event in apoptosis. The mitochondrial
permeability transition is also an important event in many
forms of apoptotic cell death. To determine whether the permeability
transition led to the release of apoptogenic proteins from
mitochondria we induced the permeability transition in isolated
rat liver mitochondria and characterised the proteins which
were released. The permeability transition led to a generalised,
non-specific release of proteins, including cytochrome c,
from the mitochondrial intermembrane space which was prevented
by an inhibitor of the permeability transition. To determine
the mechanism of this protein release we measured both mitochondrial
matrix swelling, and protein release during the permeability
transition in media of different osmolarities. Protein release
correlated with mitochondrial matrix swelling, therefore the
permeability transition causes release of proteins from the
intermembrane space by rupturing the mitochondrial outer membrane.
Supporting an apoptotic role for the proteins released by
this mechanism, supernatants from mitochondria undergoing
the permeability transition caused apoptotic changes in isolated
nuclei. These data support the proposal that the mitochondrial
permeability transition can induce apoptosis by releasing
apoptogenic proteins into the cytoplasm
Tunel vision - detection of cell death in
Department of Biochemistry, Box 56, University of Otago, Dunedin,
The ability to detect apoptotic cells is of importance
in assessing the efficacy of therapy in many pathological
conditions such as osteosarcoma. In such cases information
on the extent of tissue damage due to chemotherapeutic agents
may be obtained from observations of tissue sections using
histochemical techniques. In this talk I will give an overview
of some of the methods used for detection of apoptosis in
histopathological sections. Particular attention will be given
to a method for detecting cleavage of DNA into oligonucleosome-sized
fragments, which is considered a major biochemical event in
apoptosis. This cleavage can be localised morphologically
by enzymatic in situ labelling of apoptosis-induced DNA strand
breaks using the TUNEL (terminal deoxynucleotidyl transferase
(TdT)-mediated d-UTP nick end labelling) technique.
Induction of apoptosis in endothelial cells
treated with hypochlorous acid
Free Radical Research Group, Department of Pathology, Christchurch
School of Medicine.
We are studying the, effects of the neutrophil oxidant
hypochlorous acid (HOCl) on cell metabolism. We have shown
that HOCl can cross cell membranes, and GSH and protein sulphydryls
are particularly sensitive targets. This raises the possibility
that HOCl may cause non-lethal oxidative stress to cells.
We plan to determine whether the induction of apoptosis is
one potential response of a cell to low levels of HOCl. I
will discuss our preliminary results with human umbilical
vein endothelial cells, and also our future plans for studying
apoptosis in these target cells.
Do viruses need p53 for efficient growth?
Pathology Dept, DSM.
There is now a long list of viruses that encode proteins
that can induce apoptosis as well as proteins that inhibit
apoptosis. Much literature, particularly dealing with the
DNA tumour viruses, suggests that apoptosis is not conducive
to efficient virus growth and that it therefore needs to be
inhibited. For the adenoviruses, the Ela gene products cause
cell cycle alterations, presumably to maximise the cellular
machinery for virus replication, and apparently as a consequence
of this, apoptosis is induced, often mediated through the
p53/pRb pathway. The virus has therefore evolved other proteins,
such as those encoded in the Elb gene, to prevent apoptosis.
In this way, the virus has time to replicate itself before
the cells die. One of the Elb proteins, called Elb58kDa, interacts
directly with p53 and is thought to block p53 mediated apoptosis.
Such data have led to the suggestion that adenoviruses defective
in Elb58kDa expression might selectively grow and kill p53
deficient cells (ie tumour cells) because the absence of p53
circumvents the need for interaction with p53 (Science 274,
3731 1996). Such viruses might therefore be useful in tumour
It seems curious however, that viruses have evolved a
set of proteins to induce apoptosis on the one hand, but on
the other hand have evolved a second set of proteins to deal
with the problem. In my laboratory, we have tested the model
by examining whether adenoviruses grow in p53 deficient cells
better that in normal p53 expressing cells. These experiments
were also done with adenoviruses defective in expression of
Elb58kDa. Our data suggest, in contrast to the above model,
that p53 mediated apoptosis is actually required by adenoviruses
for an efficient productive infection to occur. Consequently,
our data do not support an hypothesis that Elb mutants are
useful in tumour therapy.
Inhibitors of apoptosis from invertebrate
T. Maguire, 0. Hyink, J. Kalmakoff and V.K. Ward
Department of Microbiology, University of Otago, P.O. Box
The aims of our studies are to develop assays to measure
inhibition of apoptosis and to identify and isolate genes
in invertebrate viruses which code for inhibitors of apoptosis
(iap genes). Direct gene probing with known iap gene sequences
from four invertebrate viruses, in an attempt to demonstrate
sequence homology in a range of iridescent viruses, did not
prove successful. The first functional assay to be assessed
was based on the inhibition of apoptosis induced by chemicals
(actinomycin D, anisomycin, DRB and cyclohexamide). The purpose
of this assay was to standardise the time and the dose of
inducing agent which produces the maximum DNA fragmentation
as detected either by ELISA, or DNA laddering in agarose gels.
Having established these parameters, we are now transfecting
Spodoptera frugiperda cells and Trichoplusia ni
cells with plasmid DNA containing putative iap gene sequences,
and assessing the ability of these genes to inhibit chemically-induced
apoptosis. Secondly, we are examining the ability of transfected
putative iap gene-containing DNA to complement the loss of
apoptosis induction by an Autographa califomica nuclear
polyhedrosis virus, which has a functional iap gene (P35)
deleted. Both these assays will be used to screen restriction
enzyme libraries from some 20 invertebrate viruses in order
to determine how universal apoptosis inhibitors are in these
viruses and whether or not they function in similar ways.
"Sniff" sequencing of DNA from a Hind III library from the
leaf-roller caterpillar Epiphyas postvittana nuclear
polyhedrosis virus, has identified a homologue of the iap-2
gene from Autographa califomica nuclear polyhedrosis
virus. This homologue has now been fully sequenced. Plasmid
DNA containing the sequence, when transfected into Spodoptera
cells appears to be able to inhibit the ability of actinomycin
D to induce apoptosis in these cells, and the Epiphyas
gene thus seems to code for a functional apoptosis inhibitor.
Involvement of cytochrome c in caspase activation
by hydrogen peroxide
Mark Hampton* and Sten Orrenius
Institute of Environmental Medicine, Karolinska Institutet,
We have shown caspase activation to occur in Jurkat T-lymphocytes
3-4 hours after exposure to low concentrations of hydrogen
peroxide. This was followed by phosphatidylserine exposure
and other morphological changes associated with apoptosis.
At higher concentrations of hydrogen peroxide there was no
detectable caspase activity, and the cells died by necrosis.
The mitochondrial-to-cytoplasmic export of cytochrome c was
investigated as a potential mechanism for caspase activation
by hydrogen peroxide, Elevated levels of cytochrome c were
detected in the cytoplasm 2-3 hours after hydrogen peroxide
treatment, however identification and specific inhibition
of the mechanism of cytochrome c release will be necessary
to determine the involvement of this phenomenon in apoptosis.
We also undertook an in vitro investigation into the mechanism
by which cytochrome c activates the cytoplasmic pro-caspases.
We concluded that while specific allosteric interactions between
cytochrorne c and cytoplasmic factors were necessary, the
redox state of cytochrome: c was not important.
*now at Free Radical Research Group, Department of Pathology,
Christchurch School of Medicine.