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

(Bagchi et al., 1991; Chellapan et al., 1991) become
dissociated from the RB protein in the presence of these
viral proteins in the cell (E1A, T antigen, E7), leading to
cell cycle progression. This constitutes a mechanism (also
the interaction of viral proteins with p53, see above)
viruses use to render infected cells continuously cycling.
(iii) RB is able to repress directly c-fos gene
expression (Robbins et al., 1990) and has been proposed to
have a similar effect on c-myc expression (Pietenpol et al.,
1990). (iv) RB also suppresses cell growth by directly
repressing transcription of the rRNA and tRNA genes by
blocking the activity of RNA polymerase I transcription
factor UBF (Cavanaugh et al, 1995; reviewed by White,
1998).
Hypophosphorylated RB, but not mutant RB, was
associated with the nuclear matrix, particularly
concentrated at the nuclear periphery and in nucleolar
remnants, only during early G1; the peripheral matrix
proteins lamin A and C bound RB in vitro. This
association was thought to be important for the ability of
RB to regulate cell cycle progression (Mancini et al,
1994). It is interesting that mutated p53 but not wtp53
interacts with specific types of MARs (Will et al, 1998);
nuclear matrix is an essential structure for replication
transcription recombination and repair processes
intimately connected to mechanisms of carcinogenesis.
The tumor suppressor function of RB is believed to
occur by complex formation between E2F and RB or the
RB-related proteins p107 and p130, a complex that downregulates
the DNA-binding activities of E2F; the
transcription activating capacity of E2F on the genes it
regulates can be repressed by interaction with RB (Nevins,
1992). Cyclin A, believed to facilitate DNA replication,
also associates with E2F; both types of complexes, E2F-
RB and E2F-cyclin A, can be dissociated by the
adenovirus E1A protein. The release of E2F by E1A
results in cell cycling and this constitutes an additional
mechanism of interference of adenoviruses with the
proliferation of the infected cells; release of E2F from RB
induced by E1A is critical for transformation of cells by
E1A (for references see Hiebert et al, 1995).
The p107 protein with similarities in structure and
DNA-binding properties to RB also binds cyclin A;
whereas RB is complexed to E2F during G1 the p107cyclin
A complex interacted with E2F as cells entered S
phase (Shirodkar et al., 1992).
E2F is a transcription factor that activates the
adenovirus E2 gene and a number of cellular genes that
respond to proliferation signals and that control the
passage of the cell cycle through S phase such as myc and
DHFR genes and contributes to the uncontrolled
proliferation of adenovirus-transformed cells (Mudryj et
al., 1991; see White, 1998, this volume). It has been
speculated that the physiological function of RB (and also
of its similar protein p107) in negatively-regulating cell
Boulikas: An overview on gene therapy
62
growth and in acting as a tumor suppressor protein are
exerted via its ability to down-regulate the activity of E2F
(Shirodkar et al., 1992); this has been subsequently
confirmed by numerous studies. RNA ligands that bind to
E2F1 were selected from RNA libraries and were used to
inhibit the induction of S phase in cultured cells (Ishizaki
et al, 1996). Such molecules might find applications in
cancer therapy because of the important role of E2F
proteins in the regulation of cell cycling.
Retinoblastoma protein has a functional domain (the
pocket) for binding to transcription factor E2F implicated
in cell growth control. The same domain is responsible for
the association of RB with the adenovirus E1A, the SV40
large T, and the human papilloma virus E7 proteins
(Kaelin et al., 1992). Using an approach for screening
λgt11 expression libraries, clones encoding for RBbinding
proteins were identified; among those are RBAP-1
and 2, or retinoblastoma-associated proteins 1 and 2
(Kaelin et al., 1992) and RBP3 (Helin et al., 1992).
RBAP-1 binds to the RB pocket, copurifies with E2F,
contains a functional transactivation domain, and binds to
E2F cognate sequences (Kaelin et al., 1992).
E2F contains a RB-binding domain in its C-terminus
(Helin et al., 1992; Shan et al., 1992). RB binds directly to
the activation domain of E2F1 and silences it, thereby
preventing cells from entering S phase. To induce
complete G1 arrest, RB requires the presence of the
hbrm/BRG-1 proteins, which are components of the
coactivator SWI/SNF complex. This cooperation was
mediated through a physical interaction between RB and
hbrm/BRG-1. RB can contact both E2F1 and hbrm at the
same time, thereby targeting hbrm to E2F1 (Trouche et al,
1997).
E2F cooperates with p53 to induce apoptosis (Wu and
Levine, 1994) and high levels of wild-type p53 potentiate
E2F-induced apoptosis in fibroblasts (Qin et al, 1994). The
physiological relevance of E2F in the apoptotic
mechanism was thought to arise from the ability of E2F to
act as a functional link between p53 and RB; p53 levels
increase in response to high levels of E2F (DP is required
for the association of E2F with RB); overexpression of
both E2F-1 and DP-1 led to a rapid death of (IL-3)dependent
32D.3 myeloid cells even in the presence of
survival factors (Hiebert et al, 1995). Overexpression of
exogenous E2F-1 using a tetracycline-controlled
expression system in Rat-2 fibroblasts promoted S-phase
entry and subsequently led to apoptosis (Shan and Lee,
1994).
B. Phosphorylation of RB: the TGF-β1,
IL-1, and IL-6 connection