Sarcomas, or tumors of connective tissue, represent roughly 20% of childhood cancers. Although the cure rate for sarcomas in general has significantly improved in the last 10 years, there continue to be subgroups that are difficult to treat. High-grade or metastatic soft-tissue sarcomas and rhabdomyosarcomas (RMS) of the extremities remain therapeutic challenges and their prognosis is often poor. The future of sarcoma therapy will likely include molecular approaches including gene/protein expression profiling and gene-based therapy. Most sarcomas harbor defects in the p53 or pRb pathways. The tumor suppressor p53 is central to regulation of cell growth and tumor suppression and restoring wild-type p53 function in pediatric sarcomas may be of therapeutic benefit. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. Studies of this approach, however, remain limited in pediatric cancers, including sarcomas. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (C135S) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four pediatric sarcoma cell lines, RD and Rh4 (RMS), Rh1 (Ewing's sarcoma) and A204 (undifferentiated sarcoma). Using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) 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. Cells treated with Ad-wtp53 show upregulation of the p53 downstream targets, p21CIP1/WAF1 and bax. Growth curves demonstrate suppression of cell growth over a period of 4 days and cells treated with Ad-wtp53 demonstrate a significant increase in sensitivity to the chemotherapeutic agents, cisplatin and doxorubicin. Our results indicate that restoration of wild-type p53 function in pediatric sarcoma cells could provide a basis for novel approaches to treatment of this disease.
Sarcomas represent 20% of pediatric malignancies and 5% of neoplasms in adults.1 Among the most common pediatric sarcomas are osteosarcoma (OS), rhabdomyosarcoma (RMS) and Ewing's sarcoma (ES). A spectrum of undifferentiated soft-tissue sarcomas (UND-STS) also exist whose tissue or cell of origin is unknown.2 However, recent advances in molecular profiling are refining the subclassification of these tumors. While overall survival in the pediatric sarcoma population has improved considerably in the last few decades, patients who present with advanced disease and have unfavorable prognostic indicators remain a challenge to clinical management.3,4,5
STS in children and adolescents are mesenchyme-derived connective tissue neoplasms that may arise in any region of the body.6 They are generally divided into two classes: RMS and nonRMS soft-tissue sarcomas (NRSTS) that consist of a diverse group of tumors including fibrosarcoma, leiomyosarcoma, liposarcoma, schwannoma, soft-tissue ES and synovial sarcoma.
RMS is the most common STS in children, accounting for approximately two-thirds of all sarcomas in children aged 0–14 years.7 It arises as a consequence of disruption of skeletal muscle progenitor cell growth and differentiation. Histologically, RMS can be divided into two major types: embryonal, accounting for approximately 75% of all cases, and alveolar, which accounts for most of the remaining cases. Embryonal RMS appears as spindle-shaped cells with a significant stromal component and are characterized by LOH at the 11p15 locus, a region harboring the insulin-like growth factor (IGF2) gene. Alveolar RMS appears as small round densely packed cells arranged around sinusoidal structures resembling pulmonary alveoli. These tumors harbor a characteristic translocation involving the FKHR gene, a member of the forkhead/HNF-3 transcription factor family, at chromosome 13p14.8,9,10 Most translocations involve fusion of the FKHR gene with the gene for the transcription factor PAX3 which is on chromosome 2 (t(2;13)(q35;q14)). FKHR can also be fused to PAX7 on chromosome 1 producing the rearrangement, t(1;13)(p36;q14).11 In either case, the gene fusion product is a potent oncogenic transcription factor.12 In addition to these characteristic genetic alterations, a subset of early-onset RMS is associated with germline mutations of the p53 tumor suppressor,13 and mutations of the Ras and Met oncogenes are commonly observed in sporadic RMS.14,15,16 Head and neck RMS is more often seen in younger children and is more likely to be embryonal, while tumors of the extremities are more common in adolescents and tend to be alveolar.17 Both RMS types are highly malignant, but alveolar RMS is associated with a poorer outcome.5 Approximately 25% of patients have metastatic disease at presentation with the lungs, bone, bone marrow and lymph nodes being the most common sites.18,19
ES is the second most common bone malignancy in children after OS.7 It can occur at any age but the vast majority (approximately 80%) occur before age 20 years. ES arises from neural crest cells20,21,22 and can occur in any region of any bone often exhibiting an extensive soft-tissue component.23 The hallmark genetic feature of ES is the t(11;22)(q24;q12) seen in 90–95% of cases or t(21;22)(q22;q12) translocation seen in 5–10% of cases.24,25 Both translocations involve the EWS transcription factor gene.26,27 The gene fusion product of t(11;22) is EWS-FLI1, a gene with potent transforming capability in NIH3T3 cells.28 p53 alterations in ES are not common (approximately 10%) but are associated with poor outcomes.29,30,31 As in RMS, 20–25% of ES patients present with metastatic disease, which reduces 2-year survival from approximately 70 to 40%.32,33 Cure rates of up to 70–75% have been reported in cases with localized disease,34 but patients presenting with metastatic disease almost always have a fatal outcome.35
STS whose tissue of origin cannot be identified are termed ‘undifferentiated’. UND-STS account for approximately 5–10% of STS36 and have similar clinical features and outcomes as other STS.4 A common feature of all pediatric sarcomas is the dismal prognosis for patients who present with advanced disease. Clearly, novel therapeutic approaches should be investigated.
Adenovirus-mediated delivery of p53 as a therapeutic strategy has been investigated in a variety of neoplasms including cervical cancer,37 ovarian cancer,38 prostatic cancer,39 lung cancer,40 esophageal cancer,41 nasopharyngeal cancer42 and squamous cell carcinoma of the head and neck43 among others. Currently (spring 2004), there are approximately 30 clinical trials being performed to evaluate adenovirus-mediated p53 gene therapy in a variety of adult cancer (Journal of Gene Medicine: http://www.wiley.co.uk/genetherapy/clinical/). The extent of research on adenovirus-mediated p53 gene therapy in pediatric neoplasms is far more limited.44,45 Recent work by Kawashima et al 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.46
In this study, we examined the effect of adenovirus-mediated p53 gene transfer in four sarcoma cell lines exhibiting a spectrum of p53 mutation status: two RMS (RD, Rh4), one Ewing's (Rh1) and one UND-STS (A204). We used three viral vectors (courtesy of Dr Phillip Branton, McGill University) containing wild-type p53, mutant (Cys135Ser) p53 and the lacZ reporter gene. Using Ad-lacZ with the β-galactosidase assay, we demonstrated that the virus efficiently enters the cells. RT-PCR analysis confirmed that Ad-wtp53 induced p21WAF1 and bax in all four cell lines, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay demonstrated a dose-dependent decrease in cell viability 72 hours post-treatment. TUNEL assay confirmed that cell death is occurring through apoptosis. Growth curves demonstrated suppression of cell growth over a period of 5 days. We have also shown that treatment with Ad-wtp53 significantly increases sensitivity of the cell lines to cisplatin and doxorubicin.
Materials and methods
The cell line RD was established from the embryonal RMS of a female fetus (kind gift from Dr Maria Zielenska, Hospital for Sick Children, Toronto, Canada). Rh4 is an alveolar RMS cell line. Rh1, previously identified as embryonal RMS, is an ES cell line and A204 is an undifferentiated STS cell line (Rh4, Rh1 and A204 were kind gifts from Dr Tom Look, St Jude Children's Research Hospital, Memphis, TN). The cell lines exhibited a spectrum of aberrant p53 mutation status. Rh1 harbors a homozygous Thr229Cys mutation at exon 6, Rh4 harbors a homozygous 13 base-pair deletion of amino acids 150–154 at exon 3, RD harbors a homozygous Arg248Trp mutation at exon 7 and A204 has wild-type p53. All cell lines are maintained in α-MEM containing 10% fetal 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.47 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. Treatment with virus involved removing the medium and adding a mixture of the viral preparation and enough phosphate-buffered saline (PBS) to cover the cell monolayer. After 1 hour of incubation to allow viral entry, α-MEM containing 2% FBS was added to each well. To determine infection efficiency, cells were fixed and stained with X-gal 24 hours later. 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. Subsequent experiments were performed without fixing and staining with X-gal.
Cell viability and cell growth assay
Cell viability was quantified using the MTT assay. Briefly, the MTT assay involves preparing MTT solution (5 μg/ml in PBS without phenol red) and adding a volume equal to the volume of the culture medium. After incubation for 3–4 hours 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. For cell growth assays, cell numbers were quantified using a Coulter counter.
Cell death assay
Cell death was detected using the TUNEL Assay (In situ cell death detection kit; Roche Applied Science, Laval, Quebec, Canada). 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 10 plaque-forming units (PFU)/cell (20 PFU/cell for RD). Following 72 hours 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 labeled 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 labeled cells were detected and quantified by flow cytometry.
Cells were grown to approximately 80% confluence in 10 cm plates and treated with Ad-wtp53 and Ad-mutp53 using a dose of 10 PFU/cell as described above. After 16 hours, RNA was isolated from the culture samples using the TRIzol method according to the manufacturer's protocol. cDNA was synthesized by reverse transcription RT-PCR carried out on 250 ng of RNA using the GeneAmp RNA PCR Kit by Applied Biosystems according to the manufacturer's instructions. PCR for bax, p21WAF1/CIP1 and GAPDH was performed using previously published primer sequences and cycle conditions.48,49 PCR products were visualized on 2% agarose gels.
All data are expressed as mean±SD. The Webb equation was used to determine synergism between Ad-wtp53 and the chemotherapeutic agents, cisplatin and doxorubicin.50 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).51,52
Effect of Ad-wtp53 on in vitro cell growth
To determine if replacement of wild-type p53 affects the growth of the sarcoma cells in vitro, RD, Rh1, Rh4 and A204 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 10 PFU/cell. 4 days post-infection, cells were visualised for morphological changes with confocal microscopy and an MTT assay was performed to quantify cell viability. As shown in Figure 1, significant reduction of cell growth occurs even at the lowest dose of 1 PFU/cell with Ad-wtp53. This does not occur with Ad-mutp53 even at the highest dose of 10 PFU/cell. This suggests that p53 protein is causing growth suppression in the cell lines.
The most significant growth inhibition occurred in Rh1 where treatment with Ad-wtp53 at a dose of 1 PFU/cell was sufficient to decrease cell viability by over 50% (Fig 1a). In contrast, only about a 12% decrease in viable cells was observed at this dose with RD. However, at a dose of 10 PFU/cell, a decrease of approximately 50% is seen with RD. This could be attributed to lower efficiency of infection from low expression of the CAR receptor.46
Figure 1b shows the morphologic changes that occur when the sarcoma cell lines are treated with Ad-wtp53. All four cells lines showed evidence of lower cell density, lifting of cells off plate and membrane blebbing, an indication of apoptosis. Cells treated with Ad-mutp53 did not show these characteristics even at the highest viral doses.
To determine the effect of gene transfer on the cell lines over time, a 5-day growth curve was performed on each. To allow growth over this period, cells were grown to a lower density of 104 cells/well in a 24-well tissue culture plate. RD, Rh1, Rh4 and A204 were treated with Ad-wtp53 at doses of 0.1, 0.2 and 0.4 PFU/cell. As RD requires higher doses of virus to achieve growth suppression, it was treated with doses of 2, 5 and 10 PFU/cell. As a control, the cells were also treated, at the same doses, with Ad-mutp53. Cells were trypsinized and a sample representing the total cell number in each well was counted daily by Coulter Counter for 5 days. Growth patterns are displayed in Figure 2. There was no significant growth suppression observed in any of the cell lines with the Ad-mutp53 virus regardless of the dose. In contrast, the Ad-wtp53 virus caused significant growth suppression over 5 days in a dose-dependent manner.
Detection of apoptosis in Ad-wtp53-treated sarcoma cells
We examined treated cells for evidence of apoptosis using the TUNEL assay. The assay was performed with the In Situ Cell Death Detection Kit (Roche Applied Science, Laval, Quebec, Canada). Approximately half the cell population in each cell line treated with Ad-wtp53 appears to be undergoing apoptosis as detected by TUNEL staining (Fig 3).
Expression of p53 downstream targets in treated cells
To demonstrate that functional p53 protein is being expressed in the cell lines and to further investigate the mechanism by which cell growth is inhibited by Ad-wtp53 treatment, induction of p21WAF1/CIP1 and bax expression was examined in all four cell lines. p21WAF1/CIP1 and bax are downstream targets of p53 and are important factors in cell cycle arrest and apoptosis, respectively. p53 acts as a transcription factor to induce p21 and bax, so we examined changes in expression levels by RT-PCR (Fig 4). Our data demonstrates that both p21WAF1/CIP1 and bax are induced by Ad-wtp53 in all sarcoma cell lines, indicating that p53 is being expressed and is functional.
Sensitization of sarcoma cell lines to chemotherapeutic agents by Ad-wtp53
There is mounting evidence that p53 plays an important role in modulating drug sensitivity.53 We examined the effect of Ad-wtp53 on the sensitivity of the cells to the chemotherapeutic agents cisplatin and doxorubicin, which are commonly used in the treatment of pediatric STS. The sarcoma cell lines were treated with various doses of Ad-wtp53. From our previous studies,54 doses of the chemotherapeutic agents cisplatin and doxorubicin were also chosen that, by themselves, would have a limited effect (20–30% cell death).
The results of the treatment with Ad-wtp53, chemotherapy and combination therapy are shown in Figure 5. Synergy was evaluated using a simplified version of Webb's fractional product method.50,51,52 Our results indicate that a synergistic effect between Ad-wtp53 and each chemotherapy is seen in Rh1, Rh4 and A204, while the effect is not seen in RD. The most dramatic effect is seen in Rh4 and A204, where combination therapy resulted in less than 30% cell viability with cisplatin and less than 15% cell viability with doxorubicin. Optimal synergy was achieved using 0.1 PFU/cell for Rh4 and 0.5 PFU/cell for A204. The concentrations of chemotherapy in the media were 2.5 μg/ml cisplatin and 1.0 μg/ml doxorubicin for Rh4 and 2.0 μg/ml cisplatin and 0.5 μg/ml doxorubicin for A204, respectively. Combination therapy also fit the definition of synergy with Rh1 cells as 30–35% cell viability was achieved using 0.3 PFU/cell Ad-wtp53 and 0.4 μg/ml cisplatin and 0.2 μg/ml doxorubicin. RD was treated with Ad-wtp53 using doses of 0.1–10 PFU/cell and with 0.1–2.0 μg/ml of cisplatin and doxorubicin, respectively. No combination of these treatments resulted in a synergistic killing effect for this cell line. These observations demonstrate that Ad-wtp53 can sensitize sarcoma cell lines to cisplatin and doxorubicin.
Over half of all human malignancies harbor mutations in the p53 gene55 and nearly every type of cancer demonstrates some alteration in the p53 pathway.56,57,58 The primary functions of p53 are cell cycle arrest, DNA repair and apoptosis,59 and proper function of the p53 pathway is crucial for the prevention of abnormal cellular proliferation and tumor prevention. The central role of p53 function in the prevention of tumorigenesis has been demonstrated in p53-deficient mice, almost all of which develop tumors in the first few months of life60,61,62 and in individuals with Li Fraumeni syndrome who have inherited germline p53 mutations and are susceptible to development of a variety of tumors.63,64
p53 mutations are common in RMS with nearly 50% of cases exhibiting a lesion in the p53 gene.63,65 p53 mutations are not as common in ES with frequencies of 3–16%;31,66 however, the subset of ES patients with mutant p53 have a markedly poorer outcome.30 The frequency of p53 mutations in undifferentiated STS in children is unknown. Given p53's central role in tumor prevention and emerging role in drug sensitivity, research on p53-based gene therapy as a novel therapeutic strategy has been gaining momentum in recent years. Owing to its diverse tropism, adenovirus is commonly used as the gene delivery vector. Expression of the gene product is transient and phase I clinical trials have shown the virus to be safe and well tolerated at the highest doses of 1011 PFU.67,68,69,70,71,72,73 Studies have demonstrated the feasibility of both intravascular74 and intratumoral75 administration.
The STS-derived cell lines that we examined exhibited a spectrum of aberrant p53 mutation status. Our results indicate that adenoviral-mediated p53 gene therapy could be a useful therapeutic strategy in aggressive sarcomas for which conventional treatments fail. p53 mRNA and transcripts of the downstream targets p21WAF1/CIP1 and bax were detected in all four sarcoma cell lines after treatment with Ad-wtp53, suggesting that the virus efficiently enters the cells and expresses its gene products. This is consistent with the findings of Kawashima et al, who demonstrated expression of coxsackie-adenovirus receptor (CAR), which is crucial for efficient viral entry into cells, was high in musculoskeletal tumor cell lines and tissue samples.46 All sarcoma cell lines exhibited a dose-dependent decrease in cell viability when treated with Ad-wtp53 but not with Ad-mutp53. These results indicate that it is the gene product of Ad-wtp53 rather than the viral vector itself that is cytotoxic. As would be expected, different doses of Ad-wtp53 are required in each cell line to achieve the same response. While a dose of 1–2 PFU/cell is sufficient to reduce cell viability to 50% in Rh1, Rh4 and A204, a dose of 10 PFU/cell is required to achieve the same effect in RD cells. This variability could be at least partially attributed to the variability in CAR and integrin expression levels that are required for viral uptake into the cells.76,77,78,79,80,81,82,83,84
To demonstrate suppression of cell growth in the sarcoma cell lines, lower doses of Ad-wtp53 were used so as not to produce an overt cytotoxic effect with massive cell death as seen in our previous experiments. Doses ranging from 0.1 to 0.4 PFU/cell (2–10 PFU/cell for RD) were used and a cell count was performed every 24 hours for 5 days. A dose-dependent suppression of cell growth was observed in all cell lines. Visual examination during the first 3 days ruled out cell death as the cause of decreased cell numbers in the Ad-wtp53-treated wells. Cell morphology was normal and all cells remained adhered to the plate. At higher doses, both cell cycle arrest and apoptosis are likely occurring in cells treated with Ad-wtp53 as both p21WAF1 and bax transcripts as detected by RT-PCR. The presence of apoptosis was confirmed by TUNEL assay and the colorimetric ApoPercentage assay.
The aspect of this study with the most clinical relevance is the sensitisation of the sarcoma cell lines to chemotherapy after treatment with Ad-wtp53. Doses of Ad-wtp53 were chosen that would produce a 20–30% decrease in cell viability at 3 or 4 days and combined with various doses of cisplatin and doxorubicin, two commonly used chemotherapeutic agents. The highest doses of cisplatin and doxorubicin used (2.5 and 2.0 μg/ml, respectively) were within pharmacological concentrations. Using the simplified Webb equation for synergy,50 three of the four sarcoma cell lines were found to experience a synergistic killing effect when Ad-wtp53 is combined with cisplatin or doxorubicin exhibiting >75% cell kill. These results suggest that this combination therapy may hold promise for the treatment of aggressive refractory pediatric sarcomas that are drug resistant. We suggest that these combination therapies be examined in in vivo models to evaluate their feasibility in biological systems.
Taken together, our results indicate that adenovirus-mediated p53 gene transfer could offer a powerful novel therapeutic approach in aggressive drug-resistant pediatric sarcomas.
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HG is supported by funds from the Canadian Institutes of Health Research. MG was funded by a Restracomp Studentship (Research Institute, The Hospital for Sick Children). AN is supported by a Research Scientist award from the Kids Cancer Care Foundation of Alberta. DM is a Research Scientist of the National Cancer Instititute of Canada/Canadian Cancer Society. 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 Hannah Fisher Research Fund.
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Ganjavi, H., Gee, M., Narendran, A. et al. Adenovirus-mediated p53 gene therapy in pediatric soft-tissue sarcoma cell lines: sensitization to cisplatin and doxorubicin. Cancer Gene Ther 12, 397–406 (2005). https://doi.org/10.1038/sj.cgt.7700798
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