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Original Studies |
Departments of Pharmacology 1 (Y.N., M.N.) and Nature Medicine (E.N., H.N., S.Y.), Nagasaki University School of Medicine, Nagasaki 852-8523; and DNAVEC Research Institute (H.Y., K.T., M.H.), Tsukuba 305-0856, Japan
Address all correspondence and requests for reprints to: Dr. Yuji Nagayama, M.D., Associate Professor, Department of Pharmacology 1, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail: nagayama{at}net.nagasaki-u.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the functional loss of p53 gene is now well recognized as the most common event in carcinogenesis (6), it is not surprising that wild-type (wt) p53 gene introduction has recently been used as a cancer gene therapy in numerous experimental cancer models and also in human clinical trials (7). Although carcinogenesis is a multistep genetic process, the efficacy of wt-p53 gene therapy has been described (8, 9). The effect of wt-p53 gene introduction includes not only induction of apoptotic cell death and/or cell growth arrest but also chemo- and radiosensitization (8, 10, 11), induction of senescence (12), inhibition of angiogenesis (13, 14), and secretion of growth inhibitors (15).
Our in vitro studies with permanent transfection of temperature-sensitive mutant p53 (ts-p53) gene have previously demonstrated wt-p53 induction of cell growth inhibition with G1 arrest and radiosensitization, but not apoptotic cell death or chemosensitization, in a p53-null thyroid carcinoma cell line FRO (16, 17). Moreover, we have recently found, using FRO cells stably expressing wt-p53 (18), that wt-p53 gene expression inhibits tumorigenesis and induces angiogenesis-restricted dormancy in an in vivo tumor model (19). The latter study suggests that wt-p53 gene therapy for anaplastic thyroid carcinoma may be much more efficacious than we have previously thought from the in vitro data in the former studies (16, 17).
Considering the clinical trial of wt-p53 gene therapy for anaplastic thyroid carcinoma, an adequate gene delivery system has to be used. The adenovirus vector may now be the best one, because of its broad host range, high viral titer, high gene transfer efficiency, and its ability to infect nonproliferating cells (9). In this study, we tested the potential of adenovirus-mediated wt-p53 gene therapy for anaplastic thyroid carcinoma in vitro and in vivo.
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Materials and Methods |
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Four human thyroid carcinoma cell lines with defective or mutant p53 (ARO, FRO, NPA, and WRO cells) were obtained from Prof. J. A. Fagin (University of Cincinnati College of Medicine) and were grown in RPMI 1640 medium supplemented with 10% FBS and ampicillin/streptomycin. ARO and FRO cells are both derived from anaplastic carcinomas and harbor defective and mutant (R273H) p53, respectively (3). WRO (20) and NPA cells, which are however from papillary and follicular carcinomas, respectively, were also used in this study to provide the proof-of-principle for feasibility of our approach because these cells also contain mutant p53 (G266E in NPA and P223L in WRO) (3).
Thyroid tissue samples were obtained, by subtotal thyroidectomy, from patients with Graves’ disease, and normal human thyroid follicular cells in primary culture were prepared as previously described (21) and maintained in Ham F12 medium with 5% FBS and ampicillin/streptomycin. The use of human tissues in this study was approved by the Clinical Research Committee in Nagasaki University School of Medicine. Written informed consent was also obtained from each patient.
In vitro LacZ gene expression with adenovirus infection
E1/E3-deleted, replication-deficient recombinant adenovirus vector expressing Escherichia coli ß-galactosidase (AxCALacZ) was the generous gift from Dr. I. Saito (Tokyo University) (22), in which ß-galactosidase expression is under the control of constitutive CAG (CMV-IE enhancer + chicken ß-actin promoter + rabbit ß-globin polyA signal) promoter (23). Adenovirus was propagated in 293 human embryonal kidney (HEK) cells and purified through 2 rounds of CsCl density gradient centrifugation (24). The multiplicity of infection (MOI) was defined as the ratio of total number of plaque-forming units used in a particular infection/number of cells. Adenovirus titers were determined by plaque formation after infection into 293 HEK cells. The cells, seeded at 1–3 x 105 cells/well in 24-well culture plates, were infected with AxCALacZ at the MOI indicated. Two days later, the cells were stained with 5-bromo-4-chloro-indolyl-ß-D-galactopyranoside, as previously described (25).
In vitro cytotoxic effect with adenovirus infection and anticancer agents
E1/E3-deleted, replication-deficient recombinant adenovirus expressing human wt-p53 (AxCAyp53), in which CAG promoter controls p53 expression, has been constructed by one of the authors (H. Yokoi, unpublished) on the basis of the COS-TPC method established previously (24).
In the cytotoxic assay for adenovirus infection alone (Fig. 1
), the cells in 6-well culture plates
were infected with either AxCALacZ or AxCAyp53 at the MOI indicated. On
the next day (day 1), the cells were seeded at a density of 5 x
104/well in 24-well culture plates. Viable cell
number was counted with a trypan blue exclusion test on day 3. Cell
number was expressed as percentage relative to that on day 1.
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), the cells were seeded at a density of 1–2 x
103/well in 96-well culture plates (day 0).
Anticancer drugs [cisplatin (Nihon Kayaku Co., Tokyo, Japan),
doxorubicin (Kyowa Hakko Co., Tokyo, Japan), 5-fluorouracil (5-FU)
(Kyowa Hakko), and paclitaxel (Bristol-Myers Squibb Co.,
Kanagawa, Japan)] were added on day 2. The cells were infected with
either AxCAyp53 at sublethal levels (MOI = 1 for ARO and NPA, and
MOI = 30 for FRO and WRO) or AxCALacZ at an MOI of 300 on day 1 or
day 3. Cell viability was quantitated on day 5 (3-day incubation with
anticancer drugs) with a WST-1 assay (Cell Counting Kit; Wako Pure Chemical Industries Ltd., Osaka, Japan). Survival
ratio was expressed as percentage relative to untreated controls.
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The subconfluent cells in a 10-cm dish were infected with either AxCAyp53 or AxCALacZ at the MOI indicated. Two days later, total cell lysates were prepared, and 40 µg protein was subjected to Western blot analysis in 7.5% SDS-PAGE electrophoresis under denaturing and reducing conditions with antihuman p53 monoclonal antibody (Clone no. 80, 1:500, Transduction Laboratories, Inc., Lexington, KY), as previously described (26).
FACScan analysis
The subconfluent cells in a 10-cm dish were infected with either AxCAyp53 or AxCALacZ at the MOI indicated. Two days later, the cells were trypsinized and fixed with 70% ethanol/PBS, followed by ribonuclease treatment and staining with 50 mg/L propidium iodide in PBS. Flow cytometry was performed on a FACScan flow cytometry (Becton Dickinson and Co., San Jose, CA), and the data were analyzed with Lysis II software (Becton Dickinson and Co.).
In vivo tumor growth
Six- to 7-week-old male nude mice (Charles River Laboratories, Inc.-Japan, Tokyo, Japan) were injected sc, on both sides of the flanks, with 5 x 106 FRO or NPA cells in 100 µL PBS (the same cells for both sides of each mouse). Two to 4 weeks later (when tumors were 7–10 mm in diameter), mice were left untreated or dosed with adenovirus, doxorubicin, or both. Adenovirus was directly injected into both sides of the tumors on days 1 and 2. The total amounts of injected virus were 1 x 109 plaque-forming units (pfu)/tumor. Doxorubicin (4 mg/kg) was given ip, thrice a week. Tumor sizes were monitored for 2 weeks. The perpendicular tumor diameters were measured using calipers, and tumor volumes (V) were calculated by the formula of rotational ellipsoid: V = A x B2/2 (A = longer diameter; B = smaller diameter) (25).
All mice were maintained in Nagasaki University Animal Facility. All animal studies were approved by the Animal Care Committee and were conducted in accordance with the principles and the procedures outlined in the Guide for the Care and Use of Laboratory Animals in Nagasaki University School of Medicine.
Statistical analysis was performed using an unpaired Student’s t test with StatView 4.02 software (Abacus Concepts Inc., Berkeley, CA).
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Results |
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The efficiency of adenovirus gene transfer into thyroid carcinoma cells and normal thyroid cells in primary culture was determined by 5-bromo-4-chloro-indolyl-ß-D-galactopyranoside staining, 48 h after infection with AxCALacZ. AxCALacZ efficiently transferred the LacZ gene into these cells; more than 95% staining was obtained at an MOI of 1 in normal thyroid cells, 3 in ARO and NPA cells, and 30 in FRO and WRO cells (data not shown).
In vitro effect of adenovirus infection on viability of thyroid carcinoma cell lines and normal thyroid cells
To evaluate the effect of p53 overexpression on in
vitro growth and viability of thyroid cells, four thyroid
carcinoma cell lines and normal thyroid cells in primary culture were
infected with either AxCAyp53 at MOIs ranging from 3–300 or AxCALacZ
at an MOI of 300. As shown in Fig. 1
, infection of AxCALacZ at an MOI
of 300 had little effect on cell growth, whereas infection of AxCAyp53
led to a dose-dependent decrease in cell number in all the cells. In
ARO and NPA cells infected with AxCAyp53 at higher MOIs, the viable
cell number was clearly decreased (Fig. 1, A and B), and the number of
trypan blue-staining cells (e.g. dead cells) was increased
(data not shown) on day 3, compared with day 1. In FRO and WRO cells
infected with AxCAyp53 at the highest MOI, although the viable cell
number was increased on day 3, compared with day 1 (Fig. 1, C and D),
trypan blue-staining cells could be readily detected on day 3 (data not
shown). These data indicate that adenovirus-mediated wt-p53
overexpression can induce cell death in thyroid carcinoma cells with
defective or mutant p53. This effect was, at least in part,
correlated with adenoviral infectivity in these carcinoma cells.
Normal thyroid cells in primary culture show little proliferation in
standard culture conditions (Fig. 1E). These cells die only with the
highest MOI of AxCAyp53 infection despite its highest susceptibility to
adenovirus infection (see above), suggesting that normal thyroid cells
are relatively resistant to wt-p53 mediated cell killing.
p53 expression in AxCAyp53-infected cells
The expression of wt-p53 protein by AxCAyp53 infection was
confirmed by Western blot analysis (Fig. 2). Uninfected ARO, NPA, and WRO (but not
FRO) cells expressed low levels of mutant p53 protein, data consistent
with their p53 gene status. Although infection of AxCALacZ
did not induce p53 expression, high levels of p53 protein expression
were achieved in all four carcinoma cell lines infected with
AxCAyp53 in a dose-dependent manner, and the expression levels were
also correlated with their adenoviral infectivity.
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Flow cytometric analysis was performed with the cells infected
with AxCAyp53 to analyze the cell death mechanism. Representative data
with ARO and FRO cells are shown in Fig. 3. AxCAyp53 infection induced a sub-G1
peak (hypodiploid fraction) in the flow cytometric histogram, which
corresponds to DNA fragmentation, a hallmark of apoptotic cell death,
in a dose-dependent manner. The percentage of cells in the sub-G1 peak
was also correlated with adenoviral infectivity. Thus, approximately
30% of the cells entered into the sub-G1 fraction with AxCAyp53
infection at an MOI of 30 in ARO (and NPA) cells that exhibited
high adenoviral infectivity, whereas FRO (and WRO) cells, which are
resistant to adenoviral infection, required an MOI of 300 to exhibit
the same degree of apoptotic change.
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The effect of combining wt-p53 and different chemotherapeutic
agents was investigated in four thyroid carcinoma cell lines. p53 was
expressed at sublethal levels (an MOI of 30 for FRO and WRO, and 1 for
ARO and NPA) in two different treatment regimens: 1) anticancer drugs
24 h before AxCAyp53 infection; and 2) anticancer drugs 24 h
after AxCAyp53 infection. Under both treatment regimens, wt-p53
enhanced chemosensitivity approximately 5- to 10-fold for doxorubicin
(a DNA damaging agent) in FRO and NPA cells (Fig. 4, A and C) and approximately 5-fold for
5-FU (an antimetabolic drug) in FRO cells (Fig. 4B), compared with
AxCALacZ-infected cells. Doxorubicin and 5-FU showed no statistically
significant schedule dependence.
No chemosensitizing effect was observed with doxorubicin or 5-FU in WRO and ARO cells, and also with cisplatin (a DNA damaging drug) or paclitaxel (a tubulin-polymerizing drug) in any cells (data not shown).
In vivo effect of adenovirus infection and doxorubicin on growth of thyroid tumors established in nude mice
In vivo efficacy of adenovirus infection was studied in
two thyroid carcinoma cell lines; one (FRO) is relatively resistant to
and the other (NPA) is highly susceptible to adenovirus infection and
wt-p53-mediated apoptosis. AxCALacZ or AxCAyp53 was injected directly
into the sc FRO and NPA tumors established in nude mice. As shown in
Fig. 5, A and B, growth of FRO and NPA
tumors was almost completely inhibited by 1 x
109 pfu/tumor AxCAyp53, compared with that with
AxCALacZ or control. Of interest, the antitumor effect of wt-p53
expression was virtually identical in both FRO and NPA tumors despite
their different responsiveness in vitro. The tumor sizes on
day 15 were 581.6 ± 165.4% (mean ±
SD) in the control group, 510.0 ± 157.4%
in the LacZ-treated group, and 130.0 ± 98.2% in the p53-treated
group (P < 0.01 vs. control group) in FRO
tumors; and 239.5 ± 30.4% in the control group, 200.2 ±
28.6% in the LacZ-treated group (P < 0.05
vs. control group), and 120.3 ± 19.6% in the
p53-treated group (P < 0.01 vs. control
group) in NPA tumors, compared with those on day 1.
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). Doxorubicin alone (4 mg/kg, thrice a week)
inhibited tumor growth slightly but significantly (406.2 ±
125.0%, P < 0.01 vs. control group). The
combination of two regimens led to tumor regression (79.2 ±
55.0%, P < 0.05 vs. p53-treated group).
Thus, the combination of doxorubicin and AxCAyp53 augmented the effect
of each single treatment. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to previous studies performed by us (16, 17) and also others (27), which have shown wt-p53-induced cell growth arrest, not apoptosis, in FRO and ARO cells by using ts-p53, the present study demonstrates that adenovirus-mediated wt-p53 expression can induce apoptotic cell death in thyroid carcinoma cells with defective or mutant p53 in vitro. The similar findings have recently been reported in other anaplastic thyroid carcinoma cell lines (28). The decision of a cell to undergo growth arrest or apoptosis, in response to p53 overexpression, is reported to depend on cell type, microenvironment, and/or expression level of p53 (29). At least in FRO and ARO cells, expression the level of wt-p53 is critical for this decision. We found that expression levels of p53 in FRO cells stably transfected with ts-p53 or wt-p53, which we have used in our previous studies (16, 17), are comparable with that obtained with AxCAyp53 infection at an MOI of 3 (data not shown).
Normal thyroid cells have similar or lower susceptibility to p53-mediated apoptosis but show the highest adenovirus infectivity, compared with thyroid carcinoma cells, suggesting that, although normal thyroid cells are relatively resistant to p53-mediated apoptosis, there is a risk for normal cells adjacent to carcinoma cells to be killed in wt-p53 gene therapy when adenovirus is directly injected into the primary tumor. However, this adverse effect may be minimal.
We also found wt-p53 mediated-chemosensitization to two out of four chemotherapeutic agents in two out of four carcinoma cell lines. The reason(s) for these variable effects are, at present, unclear. Whatever the reason, this chemosensitizing effect of wt-p53 in FRO cells treated with doxorubicin or 5-FU could not be observed in our previous study with FRO cells stably expressing ts-p53 (17). However, the experimental conditions are quite different between our present and previous studies; we believe that acute overexpression of wt-p53 in this study mimics the clinical setting of wt-p53 gene therapy to humans more than continuous expression of the relatively low dose of wt-p53 in the previous study.
There are two mutually exclusive points of view regarding the effect of wt-p53 on chemosensitivity (30): 1) wt-p53 could increase chemosensitivity because of apoptosis; or 2) wt-p53 could decrease chemosensitivity because of growth arrest and DNA repair. Although some inconsistent data have been reported in studies on chemosensitization in the cells with different p53 status (30, 31), most studies with adenovirus expressing wt-p53 have described the favorable combination of acute wt-p53 overexpression by adenovirus and chemotherapeutic drugs in many different cancer cells (10, 30, 31, 32, 33).
Consistent with the in vivo antitumor effects of wt-p53 reported in our recent study (inhibition of tumorigenesis and induction of angiogenesis-restricted dormancy) (19) and the in vitro ability of wt-p53 to induce apoptosis and chemosensitization observed in the present study, in vivo experiments demonstrate encouraging results. Thus, local treatment of the established sc tumors with intratumoral injection of adenovirus expressing wt-p53 led to almost complete inhibition of tumor growth. Furthermore, tumor regression was observed when combined with doxorubicin administration. Although complete tumor eradication may not be possible because intratumoral injection of adenovirus is reported to lead to uneven transgene expression in sc tumors (34), our results indicate that wt-p53 gene therapy, particularly when combined with chemotherapeutic drug(s) (e.g. doxorubicin), is efficacious for treatment of thyroid carcinomas harboring defective or mutant p53. Clinical application of p53 gene therapy for patients with anaplastic thyroid carcinoma will therefore be worth trying in the future.
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Acknowledgments |
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Received December 17, 1999.
Revised April 19, 2000.
Revised July 20, 2000.
Accepted August 2, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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