01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

chromatin condensation, drop in pH, and intranucleosomal
DNA degradation by which a cell actively commits
suicide. Virtually all tissues have apoptotic cells; salient
examples in the adult are: the eye lenses which consist of
apoptotic cells that replaced their cytoplasm with
crystallin; intestinal wall cells which migrate to the tip of
the finger-like projections over several days where they
die; ineffectual T cells which mature in thymus and which
would attack the body’s own tissues are eliminated by
apoptosis before entering the bloodstream; skin cells
migrate from the deepest layers to the surface where they
commit suicide forming the outer layer of the skin.
Apoptosis is an essential process during embryogenesis:
mammals eliminate neuron cells as the nervous system is
formed; tadpoles delete their tails by apoptosis (reviewed
by Duke et al, 1996).
Virus-transformed as well as severely X-ray-damaged
or UV-damaged cells are similarly eliminated from the
tissue via apoptosis; if they are left they can form
malignant cells. Initiated cancer cells may lead to tumor
development only when a dysfunction in their apoptotic
pathway takes place. Although the biochemical aspects of
cell death are fraught with the problem of cause versus
effect, the role of apoptosis in neoplasia and its regulation
by a number of oncogenes and p53 has emerged.
Apoptosis is essential for normal development and
homeostasis; deregulation in the positive control of
apoptosis is associated with cancer and autoimmune
disease whereas deregulation in the negative control of
apoptosis is associated with degenerative diseases
(reviewed by White, 1993; Duke et al, 1996).
B. Molecular mechanisms for apoptosis:
p53, Bax, Bcl-2, c-Myc and other proteins
Apoptosis is of special interest in gene therapy not
only of cancer but of other diseases such as arterial
disease. Apoptosis is a complex process involving a
significant number of apoptotic and antiapoptotic
mechanisms. The cytotoxic (killer) T lymphocytes of the
immune system of the infected organism bind to virusinfected
cells inflicting their eradication with two different
type of proteins: Perforin is a transmembrane molecule
transferred from the killer T cell to the membrane of the
infected cells forming holes on the membrane of the target
cell allowing uptake of proteases called granzymes that
activate ICE-like proteases to induce apoptosis. A number
of antiviral drug development strategies are based on
blockage of the activity of antiapoptotic viral proteins.
Expression of a number of genes induce apoptosis;
their protein products include adenovirus E1A (Debbas
and White, 1993; Lowe and Rudley, 1993) and c-Myc
(Hermeking and Eick, 1994; Wagner et al, 1994). A
number of proteins when expressed at sufficient amounts
block apoptosis; these include Bcl-2 and E1B 19 kDa
protein of adenovirus (Debbas and White, 1993; Chiou et
Gene Therapy and Molecular Biology Vol 1, page 65
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al, 1994). Exposure of cells to a variety of growth factors
including IL-3, IL-6, and erythropoietin, acting as survival
factors, inhibit induction of apoptosis (Johnson et al, 1993;
Yonish-Rouach et al, 1993; Canman et al, 1995).
The role of p53 in these molecular processes has been
discussed in previous pages in this review. The
involvement of p53 in apoptosis is thought to occur via
upregulation of bax and downregulation of bcl-2 genes by
wt p53 but not by mutated p53 proteins; Bax protein
induces apoptosis and its upregulation triggers the
apoptotic mechanism in cells which display elevated
levels of p53 as a result, for example, of DNA damage.
Down-regulation of Bcl-2 has a similar effect on the
induction of apoptosis. p53 may induce apoptosis
independently of transcription, although the G1 arrest by
p53 requires transcription of p53 targets (reviewed by Ko
and Prives, 1996). Induction of the apoptotic pathway by
p53 was proposed to involve: (i) transcriptional induction
of redox-related genes; (ii) formation of reactive oxygen
species; and (iii) the oxidative degradation of
mitochondrial components (Polyak et al, 1997). The
potential of p53 in cancer gene therapy is discussed above.
While p53 and E1A activate apoptosis, Bcl-2 and E1B
19k proteins inhibit apoptosis. All four protein molecules
act upstream of Bax which is a potent inducer of
apoptosis: both the cellular Bcl-2 and the 19 kDa protein
E1B of adenovirus are able to interact with Bax inhibiting
its involvement in induction of apoptosis (Han et al, 1996;
Figure 1 on page 9). E1A acts upstream of p53 by
increasing the half-life of p53 resulting in an accumulation
of p53 molecules in the nucleus (Lowe and Ruley, 1993);
increased levels of p53 are then believed to upregulate the
bax gene (Figure 1). The survival factors IL-3 and IL-6
appear to prevent p53-dependent apoptosis (see White,
1993).
p53 induces apoptosis after exposure to UV irradiation
(Ziegler et al, 1994) and hypoxia (Graeber et al, 1996);
this acts as a protective mechanism for the removal of
severely damaged cells from the body which could
become initiated cancer cells and progress to tumors.
Spontaneous or radiation-induced apoptosis mediated by
p53 has been shown to act for the removal of cells from
the gastrointestinal tract in mice (Merritt et al, 1994) and
the skin after sunburn (Ziegler et al, 1994). Epidermal
growth factor (EGF) has induced apoptosis in various
cancer cell lines via a novel signal transduction pathway
of EGF mediated through p53 (Murayama and Horiuchi,
1997).
c-myc expression, normally induced in proliferating
hematopoietic cells by mitogens, drops dramatically by
mitogen withdrawal leading to cell arrest in G1. During
deregulated c-myc expression, c-myc levels were not
down-regulated upon mitogen withdrawal; instead, DNA
synthesis continued resulting in apoptosis but not in