University of Otago

Centre for Gene Research

Centre for Gene Research
University of Otago

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Centre for Gene Research

One Day Seminar on Apoptosis
October 13, 1997
Department of Biochemistry Seminar Room
Sponsored by Boehringer Mannheim and the Centre for Gene Research



The role of mitochondria in apoptotic signaling

Mike Murphy
Biochemistry Department

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 oxidative damage

Michael Packer,
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 explored.


Release of apoptogenic proteins from the mitochondrial intermembrane space during the mitochondrial permeability transition

Jared Scariett
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 tissue sections

Steve Shnyder
Department of Biochemistry, Box 56, University of Otago, Dunedin, New Zealand

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

Margaret Vissers
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?

Antony Braithwaite,
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 therapy.

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 viruses

T. Maguire, 0. Hyink, J. Kalmakoff and V.K. Ward
Department of Microbiology, University of Otago, P.O. Box 56, Dunedin.

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, Stockholm Sweden

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.


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