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

mutation was designed in the ATF site (Okuyama et al,
1996).
pRB activates expression of the human transforming
growth factor-β2 gene through ATF-2; the human RB
gene promoter is autoregulated by RB protein via an ATF-
2-like binding site at the carboxyl-terminal domain of
pRB; overexpression of RB stimulates RB promoter
activity through the ATF binding site in a variety of
different cell types (Park et al, 1994).
The candidate oncoprotein Bcl-3, previously
characterized as a member of the IκB family, activated
transcription of the RB gene, whose promoter has no
typical NF-κB sites, via binding to a DNA element
identical to E4TF1/GABP site; Bcl-3 promoted
tetramerization of E4TF1. Expression of the antisense bcl-
3 RNA in myoblasts suppressed induction of RB and
myogenic differentiation whereas transient expression of
bcl-3 in myoblasts was shown to induce expression of the
endogenous RB (Shiio et al, 1996).
Two oncogenic point mutations at the Sp1 and ATF
sites of the RB gene promoter were identified in two
separate hereditary RB families. The Sp1 consensus site
mutation was blocking the action of RBF-1, recently
identified as the human GABP/E4TF1, a transactivator
from the adenovirus early-region 4 promoter. The human
GABP/E4TF1 protein enhanced the core RB promoter
activity, whereas it did not stimulate a mutant RBF-1 site
and was proposed to be the most essential transcription
factor for human RB gene activation (Clark et al, 1997).
Whereas binding of the Sp1 transcription factor is not
significantly affected by methylation of the CpG
dinucleotide within its binding site, 5'-GGGCGG (lower
strand, 5'-CCGCCC) methylation of the outer C is
inhibitory (mammalian cells also have the capacity to
methylate cytosines at CpNpG sites) and in particular
methylation of both cytosines m Cp m CpG inhibited binding
by 95%; endogenous m Cp m CpG methylation of an Sp1 site
in the CpG island promoter of the RB gene was identified
by genomic sequencing in a proportion of retinoblastoma
tumors which were extensively CpG methylated in the RB
promoter (Clark et al, 1997).
E. RB gene transfer
Functional loss of the RB gene has been implicated in
the initiation or progression of several human tumor types
including cancer of the eye, bone, bladder, and prostate.
The cancer suppressor activity of RB was directly
demonstrated by the introduction of a normal RB gene
into retinoblastoma cells that have lost the RB function
(inability to be phosphorylated because of mutations at the
appropriate sites) by mutation at both alleles; this led to
the suppression of the neoplastic phenotype and loss of the
tumorigenicity of RB cells in nude mice (Huang et al,
1988). Expression of the normal RB gene into the human
Boulikas: An overview on gene therapy
64
prostate carcinoma cell line DU145, mediated by
recombinant retrovirus integration, also resulted in loss of
its tumorigenic ability in nude mice (Bookstein et al,
1990). Studies with tumor cells reconstituted with RB ex
vivo and implanted into immunodeficient mice, as well as
with germline transmission of a human RB transgene into
tumor-prone Rb +/- mice have demonstrated cancer
suppression (see Riley et al, 1996).
DU145 cells express a shorter protein lacking 35
amino acids from exon 21 due to a 105 nucleotide inframe
deletion (Bookstein et al, 1990). The human bladder
carcinoma cell line J82 contains a mutated RB protein
with exactly these features (Horowitz et al, 1989); this 35
amino acid stretch is required for complexation with T
antigen and E1A. However, the two cell lines have lost
exon 21 of RB because of a different type of mutation: J82
cells have a point AG to GG mutation in the intron 20splice
acceptor site but the type of mutation in DU145
leading to exon 21 loss is different (Bookstein et al, 1990).
Intratumoral infection of spontaneous pituitary
melanotroph tumors arising in immunocompetent Rb +/-
mice with a recombinant adenovirus carrying the RB
cDNA inhibited the growth of tumors, re-established
innervation by growth-regulatory dopaminergic neurons,
and prolonged the life spans of treated animals (Riley et
al, 1996).
Retrovirus-mediated gene transfer of RB to the breast
carcinoma cell lines MDA-MB468 and BT549, both of
which harbor partial RB gene deletions as well as point
mutations of their p53 genes, restored its expression in
cells, reduced their ability to grow in soft agar, and their
tumorigenicity in nude mice, although it did not
significantly altered growth rate in culture (Wang et al,
1993).
Future therapeutic approaches using the RB gene are
directed toward inhibition in cell proliferation (such as to
inhibit neointima formation and smooth muscle cell
proliferation in arterial diseases, see Arterial injury below
and Chang et al, 1995) rather that aggressive suppression
and apoptosis of solid tumors; p53 is a better gene than
RB for tumor eradication.
XX. Induction of apoptosis for cancer
gene therapy
A. Apoptosis as an essential process
Apoptosis has become a basic tool in developing
cancer research in establishing new anticancer strategies.
The health of a multicellular organism depends both on
the ability of the body to produce new cells but also on the
ability of certain type of cells to perish, self-destruct, when
they become superfluous or severely damaged. Apoptosis,
or programmed cell death, is a biological process
associated with pronounced morphological changes,