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

esistant to 1M guanidine extraction (Bodnar et al, 1989;
Schaack et al, 1990; Fredman and Engler, 1993).
Three types of internal matrix structures were
recognized in HeLa cells infected with adenovirus 2; an
amorphously dense region; granular regions representing
virus capsid assembly structures; and filaments connecting
these regions to one another and to the peripheral lamina
(Zhonghe et al, 1987); the perinuclear matrix was also
rearranged after adenovirus infection.
Electron micrographs of thin sections through nuclei of
adenovirus-infected HeLa cells showed that the 3
Hdeoxyuridine
grains were located at the periphery as well
as in the interior of nuclei. Simultaneous visualization of
adenovirus transcription and replication showed that the
two processes occurred in adjacent, yet distinct, foci
throughout the interior and periphery of nuclei presumably
in association with the nuclear matrix; DNA molecules
were found to be displaced from the replication foci and to
become spread in the surrounding nucleoplasm serving as
templates for transcription (Pombo et al, 1994).
Adenovirus infection provokes dramatic
rearrangements to the nuclear matrix. A reorganization in
both internal and peripheral NM was also observed in
HeLa cells after infection with adenovirus 2 giving
structures able to support the increased replication
demands and capsid assembly of the virus (Zhonghe et al,
1987). Splicing of adenoviral HnRNA takes place on the
nuclear matrix. All adenovirus 2 polyadenylated RNAs
could be UV crosslinked to two host HnRNP proteins that
are involved in the association of HnRNA to the matrix
(Mariman et al, 1982).
Adenovirus establishes foci called replication centers
within the nucleus, where adenoviral replication and
transcription occur; although the rAAV genome was
faintly detectable in a perinuclear distribution after
successfully entering the cell, AAV was mobilized to the
adenovirus replication centers when the cell was infected
with adenovirus; thus AAV colocalizes with the
adenovirus replication centers (Weitzman et al, 1996).
B. Adenovirus E1A and E1B proteins in
apoptosis and control of the host cell cycle
Viruses have developed strategies to shut down protein
synthesis in the host and subdue its protein synthesizing
machinery to produce progeny virus when infecting cells.
In response, many cell types commit suicide after viral
infection to protect the organism from further infection.
Striking back, viruses have evolved mechanisms to
prevent infected cells from perishing using mechanisms
that inhibit apoptosis of the host cell; adenoviruses
synthesize the 19 kDa E1B protein which has a domain
similar to that of the cellular protein Bcl-2, the apoptosis
inhibitor (Sarnow et al, 1982; van den Heuvel et al., 1990).
Boulikas: An overview on gene therapy
8
p53 can be complexed with adenovirus E1B (Sarnow et al,
1982; van den Heuvel et al., 1990).
Expression of the adenovirus E1A protein stimulates
host DNA synthesis and induces apoptosis; on the contrary
E1B 19 kDa associates with Bax protein and inhibits
apoptosis (Figure 1). The E1A oncogene of adenovirus
exerts its effect via p53 protein (Debbas and White, 1993;
White, 1993). Indeed, expression of E1A increases the
half-life of p53 resulting in accumulation of p53 molecules
in adenovirus-infected cells leading to apoptosis. Although
induction of host DNA synthesis by E1A provides a
suitable environment for virus replication, the induction of
apoptosis by the same protein impairs virus production
since virus-infected cells are eliminated (see Han et al,
1996 for references). p53-deficient cells are transformed
by E1A because of absence of the pathway for induction
of apoptosis by p53 (Lowe et al, 1994).
E1A represses HER-2/neu transcription and functions
as a tumor suppressor gene in HER-2/neu-overexpressing
cancer cells. Transfer the E1A gene into cancer cells that
overexpress HER-2/neu is an interesting aspect of gene
therapy (see E1A in gene therapy; Yu et al, 1995; Chang
et al, 1996; Ueno NT et al, 1997; Rodriguez et al, 1997;
Xing et al, 1997).
The E1B oncogene products inhibit apoptosis induced
by E1A expression thus preventing premature death of
host cells during adenovirus infection. This gives an
advantage to virus for its proliferation and E1B proteins
(19 kDa and 55 kDa) are necessary for transformation of
primary rodent cells by E1A. E1A alone is unable to
transform primary rodent cells (White, 1993).
The E1B 19K protein of adenovirus is the putative
viral homolog of the cellular Bcl-2 protein; using the yeast
two-hybrid system for the identification of proteins
interacting with E1B, Han and coworkers (1996) have
identified Bax as one of the seven 19k-interacting clones.
The 50-78 amino acid domain of Bax contains a conserved
region homologous to Bcl-2 which is able to interact
specifically with either Bcl-2 or E1B. In p53 mutant cells
expression of Bax induced apoptosis; inhibition of
apoptosis by Bcl-2 may proceed via its ability to bind the
death-promoting Bax protein (Han et al, 1996). The bax
gene is upregulated by p53.
Expression of p53 and of adenovirus E1A induce
apoptosis (Debbas and White, 1993; Lowe and Rudley,
1993). 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 al, 1994). 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). E1A acts upstream of p53 by increasing the
half-life of p53 resulting in an accumulation of p53