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

Work from several groups has shown that RB is un- or
under-phosphorylated in G0/G1 and becomes phosphorylated
in its N-terminal domain during S and G2/M
(Buchkovich et al., 1989; Chen et al., 1989; DeCaprio et
al., 1989; Mihara et al., 1989). Only under-phosphorylated
RB interacts with E2F (Chellappan et al., 1991).
Treatment with TGF-β1 maintained RB protein in its
active dephosphorylated form, thus providing a link
between RB growth suppression and growth inhibition by
TGF-β1.
Interleukin-6 (IL-6), known to mediate autocrine and
paracrine growth of multiple myeloma (MM) cells and to
inhibit tumor cell apoptosis was determined to exert this
function via phosphorylation of RB protein; this finding
could explain the abnormalities of RB protein and
mutations of RB gene associated with up to 70% of MM
patients and 80% of MM-derived cell lines. Culture of
MM cells with RB antisense, but not RB sense,
oligonucleotide triggered IL-6 secretion and proliferation
in MM cells; phosphorylated pRB was constitutively
expressed in MM cells and IL-6 shifted pRB from its
dephosphorylated to its phosphorylated form (Urashima et
al, 1996).
Interleukin-1 (IL-1) causes G0/G1 phase growth arrest
in human melanoma cells, A375-C6 via hypophosphorylation
of RB protein. Exposure to IL-1 caused a timedependent
increase in hypo-phosphorylated RB that
correlated with an accumulation of cells arrested in the
G0/G1 phase; this was abrogated by the SV40 large T
antigen which binds preferentially to hypo-phosphorylated
RB, but not by the K1 mutant of the T antigen, which is
defective in binding to RB (Muthukkumar et al, 1996).
C. Genes regulated by RB protein
RB represses a number of genes by sequestering or
inactivating the positive transcription factor E2F and
seems to activate some other genes by interacting with
factors like Sp1 or ATF-2 (Rohde et al, 1996). RB protein
is a master regulator of a complex network of gene
activities defining the difference between dividing and
resting or differentiated cells. Using the method of
differential display Rohde et al (1996) detected a number
of genes which were upregulated by ectopic expression of
the RB gene in RB-deficient mammary carcinoma cells
including the endothelial growth regulator endothelin-1
and the proteoglycans versican and PG40.
Introduction of the wild-type RB gene via retrovirusmediated
gene transfer has provided several RBreconstituted
retinoblastoma cell lines (Huang et al., 1988;
Chen et al., 1992). These RB +
cell lines showed little
difference in their growth rates in culture when compared
to the parental or revertant RB -
Gene Therapy and Molecular Biology Vol 1, page 63
cells; however, RB +
cells
invariably lost their tumorigenicity in nude mice assays
(Chen et al., 1992). RB protein down-regulates its own
63
gene and this negative autoregulation is mediated by the
transcription factor E2F; this was shown by inserting the
promoter of the RB gene 5' of the bacterial CAT reporter
gene followed by its transfection into RB +
and RB- retinoblastoma cells: RB promoter activity was significantly
decreased in RB +
cells (Shan et al., 1994).
D. Transcription factors (TFs) that
regulate the RB gene
Several mutations have been found in the promoter
region of the RB gene, suggesting that inappropriate
transcriptional regulation of this gene contributes to
tumorigenesis. The presence of E2F recognition sites in
promoters of a number of growth-related genes suggested
that expression of these genes might be affected by RB.
Understanding the nature and availability of TFs which
regulate the RB gene in particular cell types is instructive
for a successful gene therapy application involving
transfer of RB.
An E2F recognition site lies within a region critical for
RB gene transcription; binding of E2F-1 at this site
transactivates the RB promoter; striking back, the resulting
overexpression of RB suppresses E2F-1-mediated
stimulation of RB promoter activity and, thus, the
expression of RB is negatively autoregulated through E2F-
1 (Shan et al, 1994). Up-regulation of the RB gene by E2F
was shown by co-transfection of RB - osteosarcoma Saos2
cells in culture with a plasmid expressing E2F-1 under the
control of the CMV immediate-early gene promoter-CAT
construct: expression of E2F-1 stimulated RB promoter
activity 10-fold under conditions where E2F-1 had little
effect on c-jun, c-myc, and EGR-1 gene expression (Shan
et al., 1994). The autoregulation of RB gene by RB may
be accomplished via a direct protein-DNA complex
formation, via protein-protein interaction regulating the
activity of other transcription factors on the promoter of
the RB gene, or both.
Two distinct DNA-binding factors, RBF-1 and ATF,
play an important part in the transcription of the human
RB gene. The promoter of the human RB gene and of the
mouse RB1 gene (Zacksenhaus et al., 1993) contain
binding sites for ATF, and a Sp1-like transcription factor
(Mitchell and Tijan, 1989) where the RBF-1
(retinoblastoma binding factor 1) may bind (Sakai et al.,
1991). Human RB gene is also regulated by AP-1
(Linardopoulos et al, 1993), as well by the early response
transcription factor, nerve growth factor inducible A gene
(NGFI-A) which is expressed in prostate cells and binds to
the site GCGGGGGAG at -152 to -144 within the RB
gene promoter (Day et al, 1993). The ATF site of the RB
promoter is a responsive element during myogenic
differentiation; RB promoter activity increased about 4fold
during differentiation and was reduced when a point