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

glioblastoma, astrocytoma, and others), AIDS, cystic
fibrosis, adenosine deaminase deficiency, cardiovascular
diseases (restenosis, familial hypercholesterolemia,
peripheral artery disease), Gaucher disease, Hunter
syndrome, chronic granulomatous disease, PNP
deficiency, α1-antitrypsin deficiency, leukocyte adherence
deficiency, partial ornithine transcarbamylase deficiency,
Cubital Tunnel syndrome, Canavan disease and
rheumatoid arthritis. Several RAC-approved protocols use
gene marking rather than gene therapy . An important
number of protocols in cancer use ex vivo immunotherapy
(Appendix 1, pages 159-172 & 203-206).
XVII. Gene therapy strategies based on
p53
A. p53 as a tumor suppressor protein
The p53 has been a fascinating subject in cancer
biology since its discovery (Lane and Crawford, 1979;
Linzer and Levine, 1979). Originally assigned in the
constellation of oncogenes was later shown to exert
suppressive effects on cell growth (Finlay et al, 1989);
indeed, the mutated p53 has many characteristics of an
oncogene (Will and Deppert, 1998, this volume).
Mutations in the p53 gene contribute to the emergence of
the malignant phenotype (Diller et al., 1990; Baker et al.,
1990). Alterations in the p53 tumor suppressor gene
appear to be involved, directly or indirectly, in the
majority of human malignancies (Vogelstein, 1990). For
example, human lung cancer cell lines and specimens
showed allelic loss for chromosome regions 3p and 17p
(p53 is assigned to 17p13); these specimens displayed
homozygous deletions of p53, DNA rearrangements
involving the p53 gene, or expression of truncated p53
transcripts suggesting abnormal splicing, initiation, and
termination arising from point or other mutations
(Takahashi et al, 1989; Nigro et al, 1989).
An interesting approach to unravel the molecular
mechanism of action of p53 in restricting cell growth and
in inducing apoptosis was the cloning of genes induced by
p53 before the onset of apoptosis; this led to the
identification of a group of 14 genes (out of 7,202
transcripts examined) which were markedly increased in
p53-expressing cells compared with control cells many of
which were predicted to encode proteins that could
generate oxidative stress or respond to oxidative stress
(Polyak et al, 1997). Additional studies in this line have
suggested that the induction of the apoptotic pathway by
p53 involves (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).
p53 can inhibit transformation of rat embryo
fibroblasts mediated by adenovirus E1A plus activated ras
and can also suppress focus formation mediated by myc
Boulikas: An overview on gene therapy
52
plus activated ras (Finlay et al, 1989; Eliyahu et al, 1989).
Both alleles of p53 need to be mutated or altered for
transformation. Introduction of a null mutation by
homologous recombination in murine embryonic stem
cells gave mice which appeared normal but were
susceptible to a variety of neoplasms by 6 months of age
(Donehower et al, 1992).
The tumor suppressive activity of p53 seems to involve
at least six independent pathways: (i) induction by p53 of
the p21/Waf-1/Cip-1 gene which causes growth arrest both
via inhibition of cyclin-dependent kinases and via
inactivation of PCNA; PCNA is the accessory molecule to
DNA polymerases α and δ and its absence causes arrest of
DNA synthesis at the replication fork; (ii) induction of the
death-promoting bax gene by p53 as a mechanism which
eliminates oncogenic virus-infected and transformed cells;
(iii) by a direct interaction of p53 with origins or
replication preventing firing and initiation of DNA
replication; (iv) via binding of p53 to a number of
important molecules involved in transcription (TATA boxbinding
protein or TBP, TFIIH); (v) by the role of p53 in
DNA repair via its patrolling the genome for small
insertion deletion mismatches or free ends of DNA; (vi)
p53 is able to attract RPA, an accessory to DNA
polymerases α and δ as well as TFIIH and RAD51 at the
damaged DNA sites; TFIIH, RAD51, and RPA have a
demonstrated role in DNA repair (Figure 19). Additional
properties of p53 include the induction of Gadd45
involved in the arrest of the cell cycle and induction of
Mdm2 which, after exceeding a threshold value in the cell
associates with p53 to restrict its regulatory functions;
thus, Mdm2 acts as a feedback loop for p53 to moderate
its apoptotic and cell cycle restrictive functions (Figure
20).
B. Genes regulated by wild-type p53
Protein p53 appears to be a transcription factor able to
recognize specific regulatory regions in a number of genes
via its central DNA-binding domain; the DNA sequencespecific
binding of wt p53 is regulated by the C-terminal
domain of p53 and is activated by a variety of
posttranslational modifications (Hupp et al, 1992). p53 is
phosphorylated and is constitutively expressed at low
levels in most normal tissues (Lane and Crawford, 1979;
Linzer and Levine, 1979).
The sequence specificity of p53 has been determined
using random synthetic oligonucleotides followed by
selection by wtp53 and cloning; these studies revealed the
10 bp motif RRRC AA
GYYY (where R is purine and Y
TT
pyrimidine) as the binding and recognition site of wtp53
recognition (El-Deiry et al., 1992); two such 10 bp motifs
are required for p53 binding separated by up to 13 bp of
random sequence. Since the 10 bp motif is a palindrome,
the binding site of p53 comprises 4 copies of the half