The poor prognosis for patients with metastatic osteosarcoma (OS) indicates that new therapeutic options should be explored. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. However, limited work has been carried out with pediatric cancers, including OS. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (Cys135Ser) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four OS cell lines: Saos-2 (p53−/−), HOS (R156P), KHOS/NP (R156P) and MNNG (R156P, F270L). We demonstrated that the virus efficiently enters the cells using the β-galactosidase assay. Using the MTT assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. We have also shown that treatment with Ad-wtp53 significantly increases sensitivity of the cell lines to cisplatin and doxorubicin, chemotherapeutic agents commonly used in the treatment of OS. Our results indicate that restoration of wt p53 function in OS cells provides a basis for novel approaches to treatment of this disease.
Osteosarcoma (OS) is the most frequent primary bone malignancy with an incidence of 5.6 per million children under 15 years of age.1, 2 Considerable progress has been made in the treatment of OS. Highly effective limb-sparing surgical techniques have been developed to avoid amputation without compromising therapeutic benefit. Multidrug chemotherapeutic regimens that typically include cisplatin and doxorubicin are used to combat micrometastatic disease. These advances have improved the 5-year survival rate for patients without visible metastases from 20% to approximately 70%.3
Treatment of metastatic OS offers a unique set of challenges as complete resection of all metastatic disease is a prerequisite for prolonged survival. Unfortunately, metastatic OS has a high rate of treatment failure and even with surgical intervention it is essentially incurable.1 Clearly, novel therapeutic approaches must be investigated.
Adenovirus-mediated delivery of p53 as a therapeutic strategy has been investigated in a variety of adult neoplasms but the extent of research in pediatric neoplasms is far more limited. Studies by Kawashima et al.4 examining the transduction efficiency of adenovirus in musculoskeletal tumors and OS suggests that this mode of therapy is likely to be suitable for the treatment of such tumors.
In this study, we examined the effect of adenovirus-mediated p53 gene transfer in four OS cell lines with various p53 status: Saos-2 (p53 deficient), HOS (Arg156Pro) and two of its derivatives, KHOS/NP (Arg156Pro) and MNNG (Arg156Pro, Phe270Leu).
Materials and methods
The cell line Saos-2 was established from the OS of an 11-year old female. The cell line HOS was established from the OS of a 13-year old female and the cell lines MNNG and KHOS/NP are derivatives of HOS. The MNNG line was derived from HOS by transformation with 0.01 μg/ml MNNG (a carcinogenic nitrosamine) and the KHOS/NP line was derived by transformation using Kirsten murine sarcoma virus (Ki-MSV). All cell lines are commercially available ATCC and maintained in α-MEM containing 10% foetal bovine serum (FBS) (Gibco).
All three viral vectors, Ad-wtp53, Ad-mutp53 and Ad-lacZ, were kind gifts from Dr Phillip Branton, McGill University, Montreal, Canada. Briefly, cells were grown in α-MEM culture medium with 10% FBS to approximately 75% confluence in a six-well tissue culture plate and treated with different doses of Ad-lacZ. After 1 h of incubation to allow viral entry, α-MEM containing 2% FBS was added to each well. After 24 h, cells were fixed and stained with X-gal. Cells infected with virus that express the lacZ gene could be visualized by their blue color and infection efficiency was determined by counting blue cells.
Cell growth assay
Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, the MTT assay involves preparing MTT solution (5 μg/ml in PBS without phenol red) and adding a volume equal to 1/10 the volume of the culture medium. After incubation for 3–4 h at 37°C, the medium is removed, the precipitate is dissolved with 10% HCl in isopropanol and the color intensity is measured at 570 nm. Cell numbers were quantified using a Coulter counter.
Cell death assay
Cell death was detected using the ApoPercentage Apoptosis Assay (Biocolor) and the TUNEL Assay (Roche; in situ cell death detection kit). Following an incubation period of 72 h, the ApoPercentage assay was performed according to the manufacturer's instructions. Briefly, the dye reagent was added to each well and the cultures were incubated at 37°C for 60 min. The medium was then removed and the cells were washed twice with PBS. Next, 40 μl of PBS were added to each well and the cells were photographed using a camera attached to an inverted microscope (Figure 1a). For TUNEL staining, cells were grown to subconfluence in a 10 cm dish and treated with the Ad-wtp53 and Ad-mutp53 vectors at a dose of 8 pfu/cell (16 pfu/cell for KHOS/NP). Following 72 h of incubation, the cells were fixed and stained according to the manufacturer's instructions. Briefly, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate and labelled with nucleotide polymers containing fluorescein. The nucleotide polymers will only react with free 3′-OH termini, a distinctive feature of cleaved DNA from apoptotic cells. The labelled cells were detected and quantified by flow cytometry.
All data is expressed as mean±s.d. The Webb's equation was used to determine synergism between Ad-wtp53 and the chemotherapeutic agents, cisplatin and doxorubicin. Percent cell viability is determined for the individual treatments (A% and B%) and if the percentage cell viability of the combination therapy (C%) is less than 70% of the product of the individual therapies, the combination therapy is deemed to synergistic (if C% <0.70[A% × B%] then combination therapy is synergistic).5
To determine the viral doses that should be employed to efficiently express p53 in the OS cell lines, a β-galactosidase assay was used to examine infection efficiency of adenovirus. The four OS cell lines were treated with Ad-lacZ at doses of 1–8 pfu/cell. Cells that took up the virus and expressed the lacZ gene would appear blue upon treatment with X-gal and were visually quantitated. Infection efficiency increased with viral dose with no cytopathic effect in any of the four cell lines. However, at any given dose, infection efficiency varied greatly among the cell lines. While an efficiency of close to 100% was achieved for MNNG at a dose of 4 pfu/cell, the infection efficiency for KHOS/NP was only just over 40% at a dose of 8 pfu/cell.
To determine if replacement of wild-type p53 affects the growth of OS tumor cells in vitro, Saos-2, HOS, MNNG and KHOS/NP cells were grown to a density of 4 × 104 cells/well in a 24-well tissue culture plate and treated with Ad-wtp53 in doses varying from 1 to 8 pfu/cell. The cells were also treated, at the same doses, with two negative control vectors. Ad-lacZ, adenovirus containing the β-galactosidase gene, and Ad-mutp53, adenovirus containing a mutant form of the p53 gene (Cys135Ser) producing nonfunctional protein. At 72 h postinfection, cells were visualized for morphological changes with confocal microscopy and an MTT assay was performed to quantify cell viability. Significant reduction of cell growth occurs even at the lowest dose of 1 pfu/cell with Ad-wtp53. This does not occur with Ad-lacZ and Ad-mutp53 even at the highest dose of 8 pfu/cell. This suggests that exogenous expression of p53 protein is causing growth suppression in the cell lines.
The most significant growth inhibition occurred in Saos-2, HOS and MNNG where treatment with Ad-wtp53 at a dose of 1 pfu/cell resulted in a 50% decrease in viable cells. In contrast, only about a 20% decrease in viable cells was observed at this dose with KHOS/NP. However, at a dose of 8 pfu/cell, a decrease of over 50% is seen with KHOS/NP. This can be attributed to the lower efficiency of infection with the KHOS/NP cell line.
We examined treated cells for evidence of apoptosis using the colorimetric ApoPercentage assay (Biocolor) and the TUNEL assay. The TUNEL assay was performed with the In Situ Cell Death Detection Kit (Roche). Cells were grown to a density of 104 cells/well in a 96-well tissue culture plate and treated with Ad-wtp53 and Ad-mutp53 at a dose of 8 pfu/cell (16 pfu/cell for KHOS/NP). Following an incubation period of 72 h, the ApoPercentage assay was performed according to the manufacturer's instructions and the cells were photographed using a camera attached to an inverted microscope (Figure 1a). For TUNEL staining, cells were grown to subconfluence in a 10 cm dish and treated with the Ad-wtp53 and Ad-mutp53 vectors at a dose of 8 pfu/cell (16 pfu/cell for KHOS/NP). Following 72 h of incubation, the cells were fixed and stained according to the manufacturer's instructions. The labelled cells were detected and quantified by flow cytometry. Approximately half the cell population in each cell line appears to be undergoing apoptosis as detected by TUNEL staining (Figure 1b). Both methods demonstrate extensive evidence of apoptosis in all OS cell lines 3 days after treatment with Ad-wtp53 but not with Ad-mutp53.
There is mounting evidence that p53 plays an important role in modulating drug sensitivity.6 We examined the effect of Ad-wtp53 on the sensitivity of the cells to the chemotherapeutic agents cisplatin and doxorubicin, which are known to be active in OS.
The OS cell lines were treated with various doses of Ad-wtp53. From previous experiments, doses of the chemotherapeutic agents cisplatin and doxorubicin were also chosen that, by themselves, would have a limited effect (20–30% cell death). The goal would be to combine low-dose treatments that would result in a synergistic cytotoxic effect on the cell lines.
Cells were grown to a density of 4 × 104 cells/well in a 24-well tissue culture plate and treated with Ad-wtp53 in doses that would cause a 20–50% decrease in cell viability after 72 h. Doses of cisplatin and doxorubicin were also used that would produce a similar outcome. Treatment with the chemotherapeutic agents involved growing the cells under normal growth conditions to a density of 4 × 104 cells/well. The medium was then replaced with α-MEM containing 2% FBS and the chemotherapeutic agent. The cells were exposed to the chemotherapy for the duration of the 72 h experiment. Cells were treated with combinations of Ad-mutp53 and chemotherapy as well.
The results of the treatment with Ad-wtp53, chemotherapy and combination therapy are shown in Figure 2. The doses that showed the greatest synergistic effect are shown. Synergy was evaluated using a simplified version of Webb's fractional product method.5 Our results indicate that a synergistic effect of Ad-wtp53 and each chemotherapy is seen in Saos-2, MNNG and KHOS/NP while the combination therapy seems to have a more additive effect in HOS cells. In Saos-2 cells, the most dramatic synergistic effect is seen when a dose of 0.3 pfu/cell Ad-wtp53 is combined with 1 μg/ml cisplatin or 0.12 μg/ml doxorubicin. While the combination effect in HOS cells did not meet the definition of synergy, a significant additive effect was observed by combining 0.25 pfu/cell of Ad-wtp53 with 1.2 μg/ml cisplatin or 0.4 μg/ml doxorubicin. While the treatments of virus or chemotherapy alone resulted in 70–80% cell viability, the combination therapies reduced this to approximately 40% viability with either cisplatin or doxorubicin. Both MNNG and KHOS/NP demonstrated synergy when doses of 0.2 and 1 pfu/cell, respectively, were used. When combined with cisplatin and doxorubicin at doses of 0.8 μg/ml for MNNG and 0.3 μg/ml for KHOS/NP, a synergistic effect was observed. With three out of the four cell lines demonstrating a synergistic killing effect and the other demonstrating a significant additive effect, it is clear that Ad-wtp53 sensitizes OS cell lines to cisplatin and doxorubicin.
p53 mutations are the most commonly observed gene alterations in human cancers. As a central regulator of cell growth, p53 is essential for the maintenance of the normal balance between cell proliferation and cell death. Given the importance of p53 in regulating cell growth and its frequent loss in human cancers, scientists began to explore the idea of reintroducing wild-type p53 into cancer cells to induce growth arrest, apoptosis and to increase sensitivity to chemotherapeutic agents. Adenovirus-mediated delivery of p53 has been investigated in a variety of cancer cell lines including cervical, ovarian, prostatic, lung, esophageal, and nasopharyngeal cancers, and squamous cell carcinoma of the head and neck, among others. In vivo animal studies with Ad-wtp53 have shown promise and phase I clinical trials have shown the treatment to be safe and well tolerated.7
Our results indicate that adenovirus efficiently enters OS cell lines using doses of 1–8 pfu/cell. Efficiency was lowest in KHOS/NP cells where viral entry occurred in just over 40% of cells as determined by X-gal staining. However, this is sufficient to demonstrate a biological effect. All OS cell lines exhibited a dose-dependent decrease in cell viability when treated with Ad-wtp53 but not with Ad-mutp53 or Ad-lacZ. These results indicate that it is the gene product of Ad-wtp53 rather than the viral vector itself that has a detrimental effect on the cells. Consistent with the viral entry data, higher doses of Ad-wtp53 are required in KHOS/NP cells to achieve a response similar to what is seen in the other cell lines. While a dose of 1–2 pfu/cell is sufficient to reduce cell viability to 40% in Saos-2, HOS and MNNG, a dose of 8 pfu/cell is required to achieve the same effect in KHOS/NP cells. This variability points to difficulty in determining appropriate doses when translating in vitro data into in vivo use. It is thought that coxsackie-adenovirus receptor and integrin expression levels are primarily responsible for these response variations. Adenovirus infection depends on coxsackievirus and adenovirus receptor (CAR)-mediated virus attachment to the cell surface. Kawashima et al.4 demonstrated that SaoS-2 and HOS express relatively high levels of CAR-receptor mRNA. In a study using primary tumors, Gu et al.8 showed that 75% (15/20) tumor samples expressed CAR. More recently, however, Graat et al.9 performed immunohistochemical studies on seven OS tumor specimens and found that 4/7 samples did not express CAR and the remaining three samples expressed the receptor in less than 10% of cells. It remains to be shown whether adenovirus is efficently taken up by OS cells in vivo.
MTT experiments and apoptosis assays demonstrate that Ad-wtp53 suppresses cell growth and induces apoptosis in four OS cell lines. The most promising aspect of p53 gene therapy is the potential to sensitize cells to chemotherapy that are otherwise drug resistant. We combined Ad-wtp53 treatment of our cell lines with cisplatin and doxorubicin, two agents commonly used in the treatment of OS. Metastatic OS often harbors alteration of p53 and has a high rate of treatment failure. Demonstrating that introduction of p53 into OS cell lines can sensitize them to chemotherapy would be an important step towards development of treatment strategies that would be effective for this subgroup of OS patients who do not respond to current therapies. Our results indicate that treatment of OS cells with Ad-wtp53 dramatically enhances their sensitivity to cisplatin and doxorubicin. Three of the four cell lines demonstrated a synergistic cytotoxic effect while the combination treatment showed a more additive effect in HOS cells. These results suggest that this combination therapy may hold promise for the treatment of pulmonary metastatic OS. Combining this therapy with conventional chemotherapeutic agents may yield a more beneficial response than conventional treatments alone and this regimen can be examined in an in vivo system.
With improving gene delivery systems and novel drugs that restore p53 function, it is likely that combining wild-type p53 with conventional therapies will be the most effective cancer therapy regimen in the future. Our results indicate that adenovirus-mediated p53 gene transfer could offer a powerful therapeutic approach in drug-resistant pulmonary metastatic OS where conventional treatments have failed.
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HG is supported by funds from the Canadian Institutes of Health Research. MG, MK and NP were funded through studentships of the Ontario Student Opportunity Trust Fund – Hospital for Sick Children Foundation Student Scholarship Program. AN is supported by a Research Scientist award from the Kids Cancer Care Foundation of Alberta. This work was supported in part by a Seed Grant of the Research Institute, The Hospital for Sick Children, the Andrew Mizzoni Cancer Research Fund, and the Harry and Hanna Fisher Research Fund.
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Ganjavi, H., Gee, M., Narendran, A. et al. Adenovirus-mediated p53 gene therapy in osteosarcoma cell lines: sensitization to cisplatin and doxorubicin. Cancer Gene Ther 13, 415–419 (2006). https://doi.org/10.1038/sj.cgt.7700909
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