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

tested for its ability to eliminate bcl-2 expression from
hormone-refractory prostate cancer cells. When this
hammerhead ribozyme was directly transfected into
cultured prostate cancer cells (LNCaP derivatives), it
significantly reduced bcl-2 mRNA and protein levels
within 18 hr of treatment and induced apoptosis in a lowbcl-2-expressing
variant of LNCaP, but not in a high-bcl-
2-expressing LNCaP line (Dorai et al, 1997).
B. Bcl-xs
Many cancers overexpress a member of the Bcl-2
family of inhibitors of apoptosis, such as Bcl-2 and BclxL.
Members of the Bcl-2 family were found to be
essential for survival of cancer cells derived from solid
tissues including breast, colon, stomach, and neuroblasts
(Clarke et al, 1995). On the contrary, Bcl-xs is a dominant
negative repressor of Bcl-2 and Bcl-xL; thus, Bcl-xs
induces apoptosis. Transient overexpression of Bcl-xs in
MCF-7 human breast cancer cells, which overexpress BclxL,
with a replication-deficient adenoviral vector induced
apoptosis in vitro; intratumoral injection of the bcl-xs
adenovirus on solid MCF-7 tumors in nude mice showed a
50% reduction in size with evident apoptotic cells at sites
of injection (Ealovega et al, 1996).
An adenovirus vector expressing bcl-xs specifically
and efficiently killed carcinoma cells arising from multiple
organs including breast, colon, stomach, and neuroblasts
even in the absence of an exogenous apoptotic signal such
as x-irradiation. In contrast, normal hematopoietic
progenitor cells and primitive cells capable of
repopulating SCID mice were not killed by the bcl-xs
adenovirus. Thus, transfer of the bcl-xs gene could be used
in killing cancer cells contaminating the bone marrow of
patients undergoing autologous bone marrow
transplantation (Clarke et al, 1995).
C. E2F-1 and TNF-α gene transfer
E2F cooperates with p53 to induce apoptosis; high
levels of wild-type p53 potentiate E2F-induced apoptosis
in fibroblasts. The physiological relevance of E2F in the
apoptotic mechanism is thought to arise from the ability of
E2F to act as a functional link between p53 and RB; p53
levels increased in response to high levels of E2F.
Targeted disruption of the E2F-1 gene yields transgenic
animals with an excess of mature T cells due to a defect in
lymphocyte apoptosis (Field et al, 1996).
Overexpression of the transcription factor E2F-1 could
induce apoptosis in quiescent rat embryo fibroblasts in a
p53-dependent manner; however, Hunt et al (1997) have
shown that overexpression of the E2F-1 gene after
adenoviral transfer can mediate apoptosis in the absence of
wild-type p53: adenovirus-mediated transfer of the E2F-1
gene under control of the CMV promoter to human breast
and ovarian carcinoma cell lines resulted in the induction
Boulikas: An overview on gene therapy
70
of significant morphological changes in four of the five
cell lines that had mutations in the p53 gene within 48 h of
transduction characteristic of apoptosis.
Retroviral vector-mediated transfer of the TNF-α gene
into the DNA of human tumor cells induced apoptosis in
high- TNF-α-producing clones generated from a human
lymphoma T-cell line (ST4); the apoptotic death of the
cells was associated with a downregulation of the
apoptosis-preventing gene, bcl-2, while the expression of
bax and p53 genes persisted (Gillio et al, 1996).
D. E6, E7 of human papillomavirus (HPV)
E6 and E7 of HPV possess transforming ability, have
been shown to interact with the cellular tumor suppressors
p53 and RB (Werness et al, 1990; Dyson et al., 1989) and
are believed to play a central role in HPV-induction of
cervical carcinogenesis as well as in the maintenance of
the malignant phenotype. 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. Because virus-infected
cells commit suicide to protect the organism from further
infection viruses have evolved mechanisms to prevent
apoptosis of the host cell ensuring their propagation; E6
protein interacts with p53 to exclude p53 molecules from
their apoptotic functions and to inhibit apoptosis in HPVinfected
cells thus giving to HPV a proliferation
advantage.
27-mer phosphorothioate oligodeoxynucleotides
(oligos) targeting the ATG translational start region of
HPV-16 E6 and E7 sequences showed antiproliferative
effects in all HPV-16-positive cell lines tested and in
primary cervical tumor explants while the endometrial and
two ovarian primary tumors as well as the HPV-negative
C33-A cell line and HPV-18-positive cell line HeLa were
relatively insensitive to the HPV-16 oligos (Madrigal et al,
1997).
E. Prevention of apoptosis for gene therapy
of heart disease and for ex vivo manipulations
of therapeutic cells
As induction of apoptosis is the desired effect for the
gene therapy of cancer, prevention of apoptosis by gene
therapy can fight heart disease. Cardiomyocyte death
results from heart ischemia proceeding via necrosis and
from reperfusion which induces additional cardiomyocyte
death by apoptosis; prevention of apoptosis would
constitute an important target for fighting heart disease.
Prevention of apoptosis should also solve a major problem
in cell culture cells which are subject to oxidation damage
during their manipulation for ex vivo gene transfer and
most important during the step of reimplantation,
encapsulation in biopolymer membranes for surgical