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

growth arrest. The transforming segment of c-Myc was
responsible for induction of apoptosis (see White, 1993).
Pax5 is a repressor of expression of the p53 gene
interacting directly with a regulatory region within exon 1
of the p53 gene. At early stages during pre-B cell
development the levels of Pax5 are high and p53 is downregulated;
however, later in development Pax5 levels drop
and the p53 gene is activated; this process was proposed to
lead to the decision of B cells to enter apoptosis or
differentiate into plasma cells (Stuart et al, 1995).
Down regulation of the Cu 2+
/Zn 2+
superoxide
dismutase (SOD1) induced oxidative stress and apoptosis
(Troy et al, 1996). A great deal of oxidative damage
during the procedures for ex vivo-modification of cells
induces their apoptosis; transfer of the Cu 2+
/Zn 2+
superoxide dismutase to ex vivo modified cells increased
their survival after implantation (see Nakao et al, 1995).
This demonstrates the importance of blocking apoptotic
pathways during cell manipulation for successful ex vivo
gene therapy.
Gene therapy for cancer could involve restoration of
the apoptotic pathway in cancer cells leading to their
suicidal death; this could be effected by overexpression of
the bax gene, by suppression of the endogenous bcl-2 gene
(see below), or by transfer of the wt p53 gene.
C. Role of tumor necrosis factor (TNF)
The tumor necrosis factor-α (TNF-α) is a cytokine
produced by macrophages, monocytes, lymphoid cells,
fibroblasts and other cell types in response to
inflammation and infection. TNF-α is produced by
lipopolysaccharide (LPS)-stimulated macrophages; the
molecular pathways leading to TNF-α production in these
specialized cells involves activation by LPS of several
kinases including the extracellular-signal-regulated
kinases 1 and 2 (ERK1 and ERK2), p38, Jun N-terminal
kinase/stress-activated protein kinase (JNK/SAPK), as
well as activation of the immediate upstream MAPK
activators MAPK/ERK kinases 1 and 4 (MEK1 and
MEK4) and of MEK2, MEK3, and MEK6 (Swantek et al,
1997).
TNF-α binds to two type of specific receptors, TNFR1
and TNFR2, causing their trimerization and leading to
activation of a number of kinases (ceramide-activated
kinase, IκB kinase, Raf-1, Jun N-terminal kinases or
JNKs, p38/Mpk2). Activation of Raf-1, JNK, and
p38/Mpk2 contribute to the induction of AP-1 whereas
activation of IκB kinase is leading to the activation of the
transcription factors NF-κB. This activation leads further
to upregulation of genes and induction of other cytokines,
metalloproteinases, and immunoregulatory proteins (see
Liu et al, 1996 and the references cited therein).
Boulikas: An overview on gene therapy
66
TNF can induce apoptotic death or necrosis in some
tumor cells; this effect of TNF could be mediated by
activation of sphingomyelinases and phospholipases,
synthesis of metabolites of arachidonic acid, generation of
free radicals, changes in intracellular calcium, generation
of DNA strand breaks and activation of poly(ADPribosyl)ation,
or activation of ICE-like proteases.
TNF-α, IL-1β, IFN-γ, and vitamin D3 after binding to
their transmembrane receptors stimulate the production of
the second messager ceramide from sphingomyelin in the
plasma membrane by activating sphingomyelinase; this
results in a cascade of signal transduction events that
result in down regulation of c-myc and induction of
apoptosis, to terminal differentiation, or to RB-mediated
cell cycle arrest (Figure 23).
IL-1 signaling leads to NF-κB activation and to
protection against TNF-induced apoptosis. The IL-1Rassociated
kinase (IRAK) is homologous to Pelle of
Drosophila. Two additional proximal mediators, both
associating with the IL-1R signaling complex, were
required for IL-1R-induced NF-κB activation: IRAK-2, a
Pelle family member, and MyD88, an adaptor molecule
containing a death domain (Muzio et al, 1997).
Treatment of different cell types with TNF-α results in
the activation of the MEKK1 pathway of protein kinases
ultimately resulting in AP-1 transcription factor activation
and in the upregulation of several cytokine genes. TNF-αstimulation
also results in the activation of NF-κB and
inhibition of apoptosis (Figure 24). A TNF-responsive