In vitro and Clinical Studies of Gene Therapy with
Recombinant Human Adenovirus-p53 Injection for Oral
Clin Cancer Res 2009;15(21) November 1, 2009
Yi Li,1 Long-Jiang Li,1,2 Song-Tao Zhang,1 Li-Juan Wang,3 Zhuang Zhang,1
Ning Gao,1 Yuan-Yuan Zhang,1 and Qian-Ming Chen1
Authors' Affiliations: 1State Key Laboratory of Oral Diseases, 2Department of Head
and Neck Oncology, West China College of Stomatology, and 3West China Health
Hospital, Sichuan University, Chengdu, P.R. China
Purpose: Oral leukoplakia is a well-recognized precancerous lesion of squamous cell
carcinoma. When accompanied with abnormal p53 expression, it suffered a higher
risk of canceration. The present study was carried out to test whether the recombinant
human adenovirus-p53 could introduce wild-type p53 gene to oral leukoplakia
cellsand induce cell cycle arrest and apoptosis.
Experimental Design: We select p53(-) oral dysplastic keratinocyte POE-9n, to
observe the growth inhibition, cell cycle change, apoptosis-induced effects, and
elaborate the corresponding molecular mechanism of recombinant adenovirus-p53 on
POE-9n cells. Meanwhile, we evaluate the feasibility, safety, and biological activity of
multipoints intraepithelial injectionsof recombinant adenovirus-p53 in 22 patientswith
dysplastic oral leukoplakia.
Results: Exogenous p53 could be successfully transduced into POE-9n cells by
recombinant adenovirus-p53. The optimal infecting titer in thiss tudy wasmultiplicity
of infection (MOI) = 100. Recombinant adenovirus-p53 could strongly inhibit cell
proliferation, induce apoptosis, and arrest cell cycle in stage G1 in POE-9n cellsby
inducing p21CIP/WAF and downregulating bcl-2 expression. In the posttreatment
patients, p53 protein and p21CIP/WAF protein expression were significantly
enhanced, yet bcl-2 protein presented low expression. Sixteen patients showed clinical
response to the treatment, and 14 patients showed obvious histopathologic
improvement. Conclusion: Intraepithelial injectionsof recombinant human
adenovirus-p53 were safe, feasible, and biologically active for patients with dysplastic
Oral leukoplakia (OLK) is defined as a white patch or plaque that cannot be
clinically diagnosed as any other disease (1). Histologically, OLK is the epithelial
hyperkeratosis accompanied with pure proliferation or epithelial dysplasia. It is the
most common premalignant lesion of mucosa and 43% OLK develop into oral
squamous cell carcinoma (2, 3), which is the sixth common malignant carcinoma in
the world (4). It has been shown by previous studies (5, 6) that abnormal p53
expression was detected in 33% to 76% of oral squamous cell carcinoma patients and
20% of OLK ones, and OLK accompanied with abnormal p53 expression suffered a
higher risk of canceration.Therefore, the effective treatment toward OLK becomes
one of the preventive methods against oral squamous cell carcinoma.
The wild-type human tumor suppressor p53 (wt-p53) gene is a primary mediator
of cell cycle arrest, DNA repair, and apoptosis and is intimately involved in tumor
development. The mutation and inactivation of p53 is a critical event in the formation
and progression of head and neck carcinoma (7, 8). Transduction of the wt-p53 gene
into OLK cells is expected to restore the tumor suppressor functions and prevent the
Reintroduction of wt-p53 has been accomplished with recombinant human
adenovirus-p53 (rAd-p53), a replicationincompetent human type 5 adenovirus in
which the E1 region has been replaced with an expression cassette containing the
human wt-p53 cDNA (10). Adenovirus delivery of the wt-p53 gene results in strong
p53 protein expression in tumor cells with minimal toxicity to the hematopoietic
system (11–15). Clinical trials results showed that rAd-p53 is effective against a
variety of malignancies, including colon, glioma, lung, ovarian, and head and neck
tumors (11–17). In the clinical trials, the routes of administration typically used for
rAd-p53 were intratumoral injection (17), perfusion (18), and i.v. infusion (19).
These in vitro and clinical studies evaluate for the first time a novel strategy to
treat oral leukoplakia: gene therapy with recombinant human adenovirusp53 via the
multipoint intraepithelial injection. The results show that exogenous p53 can be
successfully transduced and inhibit cell proliferation, induce apoptosis, and arrest cell
cycle in stage G1, and the clinical method was both safe and effective. We conclude
that intraepithelial injection of recombinant adenovirus-p53 is an effective new
strategy for treating oral leukoplakia. Meanwhile, the present study provided a
feasible method to block the progression of the pre-malignant lesions.
In this article, we selected rAd-p53 as the intervention drug and POE-9n, an OLK
cell line with negative p53 expression as the intervention object. Aiming to explore
the molecular mechanism of rAd-p53, we observed changes of biological behaviors of
preinfection and postinfection cells and analyzed gene expressions relating with the
functional pathway of p53. Moreover, to evaluate the feasibility of rAd-p53 as a
clinical therapy, 22 patients with dysplastic OLK received multipoint intraepithelial
injections of rAd-p53. The results of in vitro and clinical studies are reported in this
Materials and Methods
POE-9n cell line was purchased from the Institute of Medical Research, Harvard
University. This p53 expression deletion cell line was derived from OLK lesion with
severe epithelial dysplasia.POE-9n cells were cultured in D-keratinocyte serum–free
medium supplemented with 5 ng/mL epidermal growth factor, 50 μg/mL bovine
pituitary extractive (Invitrogen-Life Technologies), 100 IU/mL penicillin, and 100
μg/mL streptomycin. Cells were amplificated by using routine cell culture techniques
at 37°C in 5% CO2.
A total of 22 patients with dysplastic OLK were enrolled in the clinical trial in
West China Hospital of Stomatology, Sichuan University. All the cases were
confirmed by clinical and pathologic diagnosis. The average area of the lesions was
4.1 cm2 with the largest of 7 cm2 and the smallest of 2 cm2. All the cases were
classified as mild (nine cases), moderate (eight cases), or severe dysplasia (five cases).
The patients were excluded of systemic diseases and did not receive any treatments
within 3 mo. The informed consents were signed by all patients before the therapy.
This study has been approved by our Institutional Review Board. The Declaration of
Helsinki protocols were followed during the whole study.
rAd-p53 (Shenzhen Sibiono Genetech Co. Ltd) was diluted by 0.9% saline solution
to the concentration of 4 × 109 vp/mL before using. The whole course of treatment
included 15 d, and multipoint intraepithelial injections were done on the 1st, 4th, 7th,
10th and 13th day under the local block anesthesia. With the identified best infection
titer of rAd-p53 (MOI = 100) and the reported maximum single tolerant dose (20, 21),
the injection specification was determined as 2 × 109 vp/cm2, i.e., one injection point
per square centimeter and 0.5 mL rAd-p53 solution for one point. The injection
needles should be pricked into mucosa at a 30- to 45-degree angle and the depths
were 2 to 3 mm according to the thickness of the lesion, to inject the solution
intraepithelially. On the 15th day of the course, samples were collected for biopsy
under local anesthesia. Therapeutic response of all the patients was monitored during
therapeutic course and 30 d after the therapy. All patients were followed up for 24 mo
and the therapeutic effects were recorded. All of the patients did not receive any
therapy during the follow-up except for recurrence and cancerization.
Infection efficiency of rAd-GFP. POE-9n cells in logarithmic growth phase were
seeded onto six-well plate with the concentration of 1 × 105 per well. After 24h,
rAd-GFP solution were added into the medium with the amount of MOI = 0, 25, 50,
100, 200, and 500. Cultured for another 72 h, fluorescence-activated cell sorting was
done for quantitation of infection efficiency.
POE-9n cells in logarithmic growth phase were seeded onto 25-mL culture flask.
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