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

injected with these cells; control animals in this highly
aggressive ovarian xenograft model died between 25-45
days from injection time (Mujoo et al, 1996). Adenoviral
transfer of a functional p53 gene into a radiation-resistant
SCCHN cell line that harbors mutant p53 restored the G1
block and apoptosis in these cells in vitro and sensitized
SCCHN-induced mouse xenografts to radiotherapy in vivo
(Chang et al, 1997).
The efficacy of a replication-deficient p53 adenovirus
construct was tested against three human breast cancer cell
lines expressing mutant p53, MDA-MB-231, -468, and -
435 and was found to be highly effective against 231 and
468 cells as well as their tumor xenografts in nude mice
but not against 435 cells probably due to their low
adenovirus transduction. 37% of growth inhibition of 231
cells was due to p53, while 49% was adenovirus-specific
(Nielsen et al, 1997).
Cytotoxic T lymphocytes (CTLs) recognizing a murine
wild-type p53 were able to discriminate between p53overexpressing
tumor cells and normal tissue and caused
complete and permanent tumor eradication without
damage to normal tissue after adoptive transfer into tumorbearing
p53 +/+ nude mice. CTLs, presented by the MHC
class I molecule H-2Kb, were generated by immunizing
p53 gene deficient (p53 -/- ) C57BL/6 mice with syngeneic
p53-overexpressing tumor cells (Vierboom et al, 1997).
L. Transfer of the p53 tumor suppressor
gene to prostate cancer cells
Although primary prostate tumors have few mutations
in the p53 gene (Voeller et al, 1994; Isaacs et al, 1994),
specimens from advanced stages of the disease and
metastases as well as their cell lines frequently display
mutations or deletions at both alleles of the p53 gene (Chi
et al, 1994; Dinjens et al, 1994). Three of five prostate
cancer cell lines examined (TSUPr-1, PC3, DU145) and
one out of two primary prostate cancer specimens were
found to harbor mutations altering the amino acid
sequence of the conserved exons 5-8 of the p53 gene;
transduction of the p53-defective cell lines with the wt p53
gene using lipofectin showed reduction in tumorigenicity
assayed from reduced colony formation and the cells
became growth arrested (Isaacs et al, 1991).
Endocrine therapy is ineffective once the prostate
cancer becomes androgen-independent; these cancers
remain unresponsive to conventional chemotherapy.
Androgen-independent and metastatic prostate cancers
were established in athymic male mice by co-inoculation
with the LNCaP human prostate cancer cell line and the
MS human bone stromal cell line; these tumors became
necrotic and were successfully eradicated by intratumoral
injection of a recombinant p53/adenovirus; the p53 gene
was driven by the CMV promoter and the SV40 poly(A)
signal placed in the E1 region of Ad5 (Ko et al, 1996). It
Boulikas: An overview on gene therapy
60
was suggested that in addition to the tumor suppressor,
apoptotic, and antiangiogenesis function of p53, tumor
necrosis was induced by a bystander effect or a general
immune response which attracted immune cells to cause
tumor cell killing (Ko et al, 1996).
M. Clinical trials using p53 gene transfer
A human clinical trial at M.D. Anderson Cancer
Center uses transfer of the wild-type p53 gene, in patients
suffering with non-small cell lung cancer and shown to
have p53 mutations in their tumors, using local injection
of an Ad5/CMV/p53 recombinant adenovirus at the site of
tumor in combination with cisplatin (Roth, 1996; Roth et
al, 1996; protocols #29 and 124, Appendix 1). A retroviral
vector containing the wild-type p53 gene under control of
a β-actin promoter was used for multiple percutaneous
injections or direct thoracoscopic injections at the site of
the tumor into nine patients, all in advanced stages, with
non-small cell lung cancers. Patients whose conventional
treatments failed were selected for a p53 mutation in the
lung tumor. Reduction in tumor volume was achieved via
apoptosis (assayed in posttreatment biopsies) in three
patients, and arrest in tumor growth in three other patients
(Roth et al, 1996).
RAC-approved clinical trials (Appendix 1) using p53
cDNA transfer are #29 (treatment of non-small cell lung
cancer with p53 and antisense K-ras), #124 (intratumoral
delivery of adenoviral p53 cDNA plus cisplatin), #130
(intratumoral injection of adenoviral p53 to treat head and
neck squamous cell carcinoma), #131 (primary and
metastatic malignant tumors of the liver), #147
(percutaneous injections of adenovirus p53 for
hepatocellular carcinoma), #148 (advanced or recurrent
adenocarcinoma of the prostate), #152 (intra-tumoral
injections of ad5cmv-p53 to patients with recurrent
squamous cell carcinoma of the head and neck), #153
(intralesional delivery of adenovirus p53 in combination
with chemotherapy in breast cancer), #154 (intratumoral
injection of adeno p53 to patients with advanced prostate
cancer), #155 (intratumoral injection of adeno p53 to
patients with advanced and metastatic bladder cancer), and
#156 (adeno p53 for non-small cell lung cancer).
XVIII. p21 and p16 in cancer gene
therapy
A. Molecular action of p21
p53 upregulates the p21/CIP1/WAF1 gene (simply
called p21) (ElDeiry et al, 1993). Induction of the
p21/Waf-1/Cip-1 gene causes growth arrest via inhibition
of cyclin-dependent kinases (CDKs). CDKs are
upregulated by cyclins which act as positive regulators of
cell cycle progression. Cdk2, also called p33 cdk2
, is the