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

C. Binding of p53 to viral oncoproteins
p53 was first detected in rodent cells transformed with
SV40 in a complex with T antigen (Lane and Crawford,
1979; Linzer and Levine, 1987). Subsequent studies have
shown that p53 can be complexed with adenovirus E1B
(Sarnow et al, 1982; van den Heuvel et al., 1990) and the
E6 oncoprotein of human papilloma virus (Werness et al.,
1990). SV40 T antigen was unable to act as an initiator of
SV40 DNA replication in vitro when complexed with wt
murine p53 (Wang et al, 1989) thought to act by blocking
the interaction of T antigen with DNA polymerase α
(Gannon and Lane, 1987; Braithwaite et al, 1987).
What appears to be important in understanding the
involvement of p53 in tumorigenesis is that p53 is unable
to transactivate the p53-inducible reporter genes in cells
that express one of these viral oncoproteins (Yew and
Berk, 1992). In addition, the growth suppressive effect of
p53 protein may be mediated by its association with
cellular proteins (Fields and Jang, 1990; Raycroft et al.,
1990). Negative elements that could be required for an
efficient growth shutdown leading to the reversible G 0
state or to irreversible out-of-cycle conditions such as
terminal differentiation, apoptosis, and senescence, may
be affected by p53 (Bargonetti et al., 1991).
D. Transcription repression by interaction
of p53 with TBP
Although p53 activates a number of promoters that
contain p53-responsive elements, it represses transcription
from many promoters that lack p53 binding sites; central
to the promoter repression by p53 was thought to be its
interaction with the TATA-box binding protein or TBP
(Seto et al, 1992; Mack et al, 1993; Truant et al, 1993).
This interaction may activate transcription when TBP
interacts with a preformed p53-DNA complex or may
repress transcription when p53 interacts with DNA-bound
TBP (Deb et al, 1994). However, p53 acts as a repressor
only in cells undergoing apoptosis and p53-mediated
transcriptional repression is released by adenovirus E1B or
cellular Bcl-2 (Shen and Shenk, 1994; Sabbatini et al,
1995).
Both wild-type and mutant p53 interact with C/EBP on
the human hsp70 promoter (Agoff, 1993), with TFIIH
(Xiao et al, 1994), holo-TFIID (Chen et al, 1993; Liu et al,
1993) and the TAFII40 and TAFII60 subunits of TFIID
(Thut et al, 1995).
E. Inhibition of DNA replication by wildtype
p53
Several lines of evidence suggested inhibition in DNA
replication by wild-type p53 but not by tumor-derived
Boulikas: An overview on gene therapy
56
mutant forms of p53. Indeed, SV40 T antigen was unable
to act as an initiator of SV40 DNA replication in vitro
when complexed with p53 (Wang et al, 1989); mutant p53
was unable to cause inhibition in the initiating functions of
T antigen in vitro (Friedman et al, 1990). Inhibition in
DNA replication in vivo by p53 (Braithwaite et al, 1987)
suggested that p53 might interact with cellular DNA
replication initiator proteins or other components of the
replication fork. p53 also interacts with replication protein
A (RPA) implicated in DNA replication and in repair;
interaction of p53 inhibits the replication functions of RPA
(Dutta et al, 1993) although interaction of p53 with RPA
via its acidic domains stimulates BPV-1 DNA replication
in vitro (Li and Botchan, 1993). Immunolocalization of
p53 (also of RB and host replication proteins) at foci of
viral replication in HSV-infected cells (Wilcock and Lane,
1991) provided further evidence for a direct interaction of
p53 with proteins (or DNA sequences) at the replication
fork.
According to a second model, p53 can cause inhibition
in DNA replication by a direct interaction with origins of
replication at the DNA sequence level rather than via its
interaction with replication initiator proteins. The potential
role of p53 as a down-regulator of DNA replication in a
DNA-binding-dependent manner has been suggested from
replication assays of polyoma virus in vitro (Miller et al,
1995) and from the inhibition in nuclear DNA replication
by a form of p53, truncated at its C-terminus, which is
constitutively active for DNA binding in transcription
incompetent extracts from Xenopus eggs (Cox et al, 1995).
In the experiments of Miller and coworkers (1995) wildtype
p53 suppressed DNA replication in vitro when the
p53 binding site (RGC) 16 from the ribosomal gene cluster
was cloned on the late side of the polyomavirus (Py) core
origin; when mutated p53-binding sites were used, the
inhibition in Py replication was not observed. In addition,
RPA (able to interact directly with p53) was unable to
relieve the p53-mediated repression in Py replication.
Furthermore, tumor-derived mutants of p53 that had lost
their sequence-specific DNA-binding capacity were
unable to inhibit Py replication of the construct with the
wild-type oligomerized RGC sites in vitro.
F. Differences in biological functions
between wild-type p53 and tumor-derived p53
mutants
Tumor-derived mutant forms of p53 have lost their
DNA sequence-specific binding capacities. For example
the Trp-248 and His-273 mutants of p53 have poor DNAbinding
abilities and are unable to activate transcription
from constructs containing p53 binding sites (Farmer et al,
1992).
Wild-type (wt) p53 tumor suppressor protein
negatively regulates cell growth (Hollstein et al, 1991;