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

master regulator of the cell cycle at the G1/S transition
point. Whereas cdk2 is expressed at constant levels
throughout the cell cycle, its activation by phosphorylation
is first detected a few hours before the onset of DNA
synthesis; furthermore, antibodies directed against Cdk2
blocked mammalian cells from entering S phase. D1
cyclin associates with Cdk2, Cdk4, and Cdk5 to control
the G1→S transition point; the genes of cyclins D1 and E
are overexpressed or rearranged in malignancies and
conditional overexpression of human cyclins D1 and E in
Rat-1 fibroblasts causes a decrease in the length of G1 and
an acceleration of the G1/S phase transition. D1 appears to
be specialized in the emergence of cells from quiescence
(Go→G1 transition) whereas cyclin E is more oriented
toward control of the G1/S transition. Cdc2, a close
relative of Cdk2 and whose pattern of phosphorylation is
cell cycle-regulated, becomes associated with cyclin B to
regulate the G2→M transition (see Boulikas, 1995a).
CDK activity is essential for the phosphorylation of
RB at the G1/S checkpoint of the cell cycle resulting in the
release of E2F transcription factor from RB-E2F
complexes and in the up-regulation of genes required for
DNA synthesis by the released E2F. p21 levels are
reduced considerably in tumor cells that have lost the p53
protein or contain a nonfunctional mutated form of p53
(El-Deiry et al, 1993).
Induction of the p21/Waf-1/Cip-1 gene also causes
growth arrest via inactivation of PCNA; indeed, the p21
inhibitor of cyclin-dependent kinases associates with
PCNA, the accessory of DNA polymerases α and δ , thus
blocking its ability to activate these DNA polymerases;
this could give rise to the abnormal control in DNA
replication or to the loss of coordination between DNA
replication and cell cycle progression seen in tumor cells
(Li et al, 1994).
B. p21 and p16 gene transfer
Introduction of the wt p53 or of the p21 downstream
mediator of p53-induced growth suppression into a mouse
prostate cancer cell line, deficient in p53, led to an
association of p21 with Cdk2; this interaction was
sufficient to downregulate Cdk2 by 65% (Eastham et al,
1995). The p21 gene, driven by CMV promoter into an
Adenovirus 5 vector, was more effective than the
AD5CMV-p53 vector, (harboring the p53 gene under
control of the same elements as p21), in reducing tumor
volume in syngeneic male mice with established s.c.
prostate tumors; tumors were induced by injection of 2
million cells in each animal. These studies suggested that
p21 expression might have more potent growth
suppressive effect than p53 in this tumor model and that
p21 may be seriously be included in the constellation of
anticancer arsenals.
Gene Therapy and Molecular Biology Vol 1, page 61
61
Transfer of p21 is an effective tool to lead carcinoma
cells with inactivated p53 into less malignant phenotypes.
p53 is frequently inactivated by papilloma viruses in
carcinomas of the uterine cervix. Transfer of the p21 gene
to HeLa cells, a widely used uterine cervix cell line,
resulted in a significant growth retardation by blockage of
G1 to S transition, reduced anchorage-independent growth
and attenuated telomerase activity (Yokoyama et al,
1997). Introduction of p21 with adenoviral vectors into
malignant cells completely suppressed their growth in
vivo and also reduced the growth of established preexisting
tumours (Yang et al, 1997).
Transfer of p21 was used to suppress neointimal
formation in the balloon-injured porcine or rat carotid
arteries in vivo (Yang et al, 1996; Ueno H et al, 1997a). A
combination therapy in mice with simultaneous transfer of
the p21 gene and of the murine MHC class I H-2Kb gene,
which induces an immune response that stimulates tumor
regression, was more effective than treatment with either
gene alone (Ohno et al, 1997).
Malignant gliomas extensively infiltrate the
surrounding normal brain and their diffuse invasion is one
of the most important barriers to successful therapy; one
of the most frequent abnormalities in the progression of
gliomas is the inactivation of the tumor-suppressor gene
p16, suggesting that loss of p16 is associated with
acquisition of malignant characteristics. Restoring wildtype
p16 activity into p16-null malignant glioma cells
modified their phenotype. Adenoviral transfer of the
p16/CDKN2 cDNA in p16-null SNB19 glioma cells
significantly reduced invasion into fetal rat-brain
aggregates and reduced expression of matrix
metalloproteinase-2 (MMP-2), an enzyme involved in
tumor-cell invasion (Chintala et al, 1997).
XIX. Gene therapies based on transfer
of the retinoblastoma (RB) gene
A. RB and E2F proteins in the control of
the cell cycle and apoptosis
Retinoblastoma protein is a transcription factor (Lee et
al, 1987) involved in the regulation of cell cycle
progression genes (reviewed by White, 1998, this
volume). The role of RB on cell proliferation and tumor
suppression arises (i) from its association with E2F, an
association disrupted by RB phosphorylation at the G1/S
checkpoint resulting in release of E2F and in the
upregulation of a number of genes required for DNA
replication; (ii) from the direct association of RB protein
with a number of viral oncoproteins or key regulatory
proteins including E1A of adenovirus (Whyte et al., 1988),
SV40 large T (Ludlow et al., 1990) and the human
papilloma virus E7 protein (Dyson et al., 1989). Normal
cellular targets of RB, such as the transcription factor E2F