Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Adenovirus-mediated p53 gene therapy in pediatric soft-tissue sarcoma cell lines: sensitization to cisplatin and doxorubicin

Abstract

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.

Main

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

Cell lines

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).

Adenovirus vector

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.

RT-PCR

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.

Statistical analysis

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

Results

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.

Figure 1
figure1

Effect of Ad-wtp53, Ad-mutp53 and Ad-lacZ on cell viability in sarcoma cell lines. RD, Rh4, Rh1 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. After 4 days, an MTT assay was performed to quantify cell viability (a) and a visual observation was made with regard to morphology and the cells were photographed using a camera attached to an inverted microscope (b).

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.

Figure 2
figure2

Growth inhibition of sarcoma cell lines by Ad-wtp53. Cells were grown to a density of 104 cells/well in a 24-well tissue culture plate. Rh4, Rh1 and A204 were treated with Ad-wtp53 at doses of 0.1, 0.2 and 0.4 PFU/cell. KHOS/NP 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 every day by Coulter Counter over a period of 5 days. Each data point represents the mean±SD from three independent experiments.

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).

Figure 3
figure3

Detection of apoptosis in sarcoma cells treated with Ad-wtp53. 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 PFU/cell (20 PFU/cell for RD). Following 72 hours of incubation, the cells were fixed and stained according to the manufacturer's instructions. The labeled 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.

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.

Figure 4
figure4

Detection of p21WAF1/CIP1 and bax mRNA in sarcoma cells treated with Ad-wtp53. 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 (20 PFU/cell for RD). 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 and PCR was performed to amplify p21WAF1/CIP1 and bax. Amplification of GAPDH was used as an internal control. PCR products were visualised on 2% agarose gels.

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.

Figure 5
figure5

Sensitization of sarcoma cell lines to cisplatin and doxorubicin by Ad-wtp53. 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 hours. Doses of cisplatin and doxorubicin were also used that would produce a similar outcome. Cells were also treated with combinations of Ad-wtp53 and chemotherapy and cell viability was determined by MTT assay.

Discussion

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.

References

  1. 1

    Mackall CL, Meltzer PS, Helman LJ . Focus on sarcomas. Cancer Cell. 2002;2:175–178.

    CAS  PubMed  Google Scholar 

  2. 2

    Gonzalez-Crussi F, Black-Schaffer S . Rhabdomyosarcoma of infancy and childhood. Problems of morphologic classification. Am J Surg Pathol. 1979;3:157–171.

    Google Scholar 

  3. 3

    Grier H, Krailo M, Link M, et al. Improved outcome in nonmetastatic Ewing's sarcoma and PNET of bone with the addition of ifosfamide and etoposide to vincristine, Doxorubicin, cyclophosphamide, and actinomycin: a Children's Cancer Group and Pediatric Oncology Group report. Proc Am Soc Clin Oncol. 1994;13:421.

    Google Scholar 

  4. 4

    Pawel BR, Hamoudi AB, Asmar L, et al. Undifferentiated sarcomas of children: pathology and clinical behavior — an Intergroup Rhabdomyosarcoma study. Med Pediatr Oncol. 1997;29:170–180.

    CAS  PubMed  Google Scholar 

  5. 5

    McDowell HP . Update on childhood rhabdomyosarcoma. Arch Dis Child. 2003;88:354–357.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Wexler LH, Helman LJ . Soft tissue sarcomas of childhood. In: Holland JF, Kufe DW, Pollock RE, et al, eds. Holland-Frei Cancer Medicine. 5th edn. Hamilton, ON: BC Decker Inc. Publisher; 2000.

    Google Scholar 

  7. 7

    NCI. Cancer Incidence and Survival among Children and Adolescents: United States SEER Program 1975–1995. Bethesda, MD: National Cancer Institute.

  8. 8

    Barr FG, Galili N, Holick J, Biegel JA, Rovera G, Emanuel BS . Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcoma. Nat Genet. 1993;3:113–117.

    CAS  PubMed  Google Scholar 

  9. 9

    Galili N, Davis RJ, Fredericks WJ, et al. Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat Genet. 1993;5:230–235.

    CAS  PubMed  Google Scholar 

  10. 10

    Davis RJ, D'Cruz CM, Lovell MA, Biegel JA, Barr FG . Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. Cancer Res. 1994;54:2869–2872.

    CAS  PubMed  Google Scholar 

  11. 11

    Sorensen PH, Lynch JC, Qualman SJ, et al. PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children's oncology group. J Clin Oncol. 2002;20:2672–2679.

    CAS  PubMed  Google Scholar 

  12. 12

    Barr FG . Gene fusions involving PAX and FOX family members in alveolar rhabdomyosarcoma. Oncogene. 2001;20:5736–5746.

    CAS  PubMed  Google Scholar 

  13. 13

    Diller L, Sexsmith E, Gottlieb A, Li FP, Malkin D . Germline p53 mutations are frequently detected in young children with rhabdomyosarcoma. J Clin Invest. 1995;95:1606–1611.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Pulciani S, Santos E, Lauver AV, Long LK, Aaronson SA, Barbacid M . Oncogenes in solid human tumours. Nature. 1982;300:539–542.

    CAS  PubMed  Google Scholar 

  15. 15

    Chardin P, Yeramian P, Madaule P, Tavitian A . N-ras gene activation in the RD human rhabdomyosarcoma cell line. Int J Cancer. 1985;35:647–652.

    CAS  PubMed  Google Scholar 

  16. 16

    Ferracini R, Olivero M, Di Renzo MF, et al. Retrogenic expression of the MET proto-oncogene correlates with the invasive phenotype of human rhabdomyosarcomas. Oncogene. 1996;12:1697–1705.

    CAS  PubMed  Google Scholar 

  17. 17

    Dagher R, Helman L . Rhabdomyosarcoma: an overview. Oncologist. 1999;4:34–44.

    CAS  PubMed  Google Scholar 

  18. 18

    Koscielniak E, Rodary C, Flamant F, et al. Metastatic rhabdomyosarcoma and histologically similar tumors in childhood: a retrospective European multi-center analysis. Med Pediatr Oncol. 1992;20:209–214.

    CAS  PubMed  Google Scholar 

  19. 19

    Raney Jr RB, Tefft M, Maurer HM, et al. Disease patterns and survival rate in children with metastatic soft-tissue sarcoma. A report from the Intergroup Rhabdomyosarcoma Study (IRS)-I. Cancer. 1988;62:1257–1266.

    PubMed  Google Scholar 

  20. 20

    Delattre O, Zucman J, Melot T, et al. The Ewing family of tumors — a subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med. 1994;331:294–299.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Scotlandi K, Serra M, Manara MC, et al. Immunostaining of the p30/32MIC2 antigen and molecular detection of EWS rearrangements for the diagnosis of Ewing's sarcoma and peripheral neuroectodermal tumor. Hum Pathol. 1996;27:408–416.

    CAS  PubMed  Google Scholar 

  22. 22

    West DC . Ewing sarcoma family of tumors. Curr Opin Oncol. 2000;12:323–329.

    CAS  PubMed  Google Scholar 

  23. 23

    Weber KL, Sim FH . Ewing's sarcoma: presentation and management. J Orthop Sci. 2001;6:366–371.

    CAS  PubMed  Google Scholar 

  24. 24

    Denny CT . Gene rearrangements in Ewing's sarcoma. Cancer Invest. 1996;14:83–88.

    CAS  Google Scholar 

  25. 25

    Massey GV, Dunn NL, Heckel JL, Davis EC, Jackson-Cook C, Russell EC . Unusual presentation of Ewing sarcoma with t(21;22) in a 3-year-old boy. J Pediatr Hematol Oncol. 1996;18:198–201.

    CAS  PubMed  Google Scholar 

  26. 26

    Bertolotti A, Lutz Y, Heard DJ, Chambon P, Tora L . hTAF(II)68, a novel RNA/ssDNA-binding protein with homology to the pro-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II. EMBO J. 1996;15:5022–5031.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Bertolotti A, Melot T, Acker J, Vigneron M, Delattre O, Tora L . EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: interactions between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes. Mol Cell Biol. 1998;18:1489–1497.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    May WA, Lessnick SL, Braun BS, et al. The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1. Mol Cell Biol. 1993;13:7393–7398.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Abudu A, Mangham DC, Reynolds GM, et al. Overexpression of p53 protein in primary Ewing's sarcoma of bone: relationship to tumour stage, response and prognosis. Br J Cancer. 1999;79:1185–1189.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    de Alava E, Antonescu CR, Panizo A, et al. Prognostic impact of P53 status in Ewing sarcoma. Cancer. 2000;89:783–792.

    CAS  PubMed  Google Scholar 

  31. 31

    Lopez-Guerrero JA, Pellin A, Noguera R, Carda C, Llombart-Bosch A . Molecular analysis of the 9p21 locus and p53 genes in Ewing family tumors. JA Lab Invest. 2001;81:803–814.

    CAS  Google Scholar 

  32. 32

    Wilkins RM, Pritchard DJ, Burgert Jr EO, Unni KK . Ewing's sarcoma of bone. Experience with 140 patients. Cancer. 1986;58:2551–2555.

    CAS  PubMed  Google Scholar 

  33. 33

    Cotterill SJ, Ahrens S, Paulussen M, et al. Prognostic factors in Ewing's tumor of bone: analysis of 975 patients from the European Intergroup Cooperative Ewing's Sarcoma Study Group. J Clin Oncol. 2000;18:3108–3114.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Burgert Jr EO, Nesbit ME, Garnsey LA, et al. Multimodal therapy for the management of nonpelvic, localized Ewing's sarcoma of bone: intergroup study IESS-II. J Clin Oncol. 1990;8:1514–1524.

    PubMed  PubMed Central  Google Scholar 

  35. 35

    Arndt CA, Crist WM . Common musculoskeletal tumors of childhood and adolescence. N Engl J Med. 1999;341:342–352.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Chung EB . Current classification of soft tissue tumors. In: Fletcher CDM, McKee PH eds. Pathobiology of Soft Tissue Tumors. New York, NY: Churchill; 1991.

    Google Scholar 

  37. 37

    Hamada K, Alemany R, Zhang WW, et al. Adenovirus-mediated transfer of a wild-type p53 gene and induction of apoptosis in cervical cancer. Cancer Res. 1996;56:3047–3054.

    CAS  Google Scholar 

  38. 38

    Kigawa J, Sato S, Shimada M, et al. p53 gene status and chemosensitivity in ovarian cancer. Hum Cell. 2001;14:165–171.

    CAS  PubMed  Google Scholar 

  39. 39

    Sweeney P, Pisters LL . Ad5CMVp53 gene therapy for locally advanced prostate cancer — where do we stand? World J Urol. 2000;18:121–124.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Swisher SG, Roth JA . Clinical update of Ad-p53 gene therapy for lung cancer. Surg Oncol Clin N Am. 2002;11:521–535.

    Google Scholar 

  41. 41

    Shimada H, Matsubara H, Ochiai T . p53 gene therapy for esophageal cancer. J Gastroenterol. 2002;37:87–91.

    CAS  PubMed  Google Scholar 

  42. 42

    Liu FF . Novel gene therapy approach for nasopharyngeal carcinoma. Semin Cancer Biol. 2002;12:505–515.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Frank DK . Gene therapy for head and neck cancer. Surg Oncol Clin N Am. 2002;11:589–606.

    PubMed  Google Scholar 

  44. 44

    Delatte SJ, Hazen-Martin DJ, Re GG, Kelly JR, Sutphin A, Tagge EP . Restoration of p53 function in anaplastic Wilms' tumor. J Pediatr Surg. 2001;36:43–50.

    CAS  PubMed  Google Scholar 

  45. 45

    Shetty S, Taylor AC, Harris LC . Selective chemosensitization of rhabdomyosarcoma cell lines following wild-type p53 adenoviral transduction. Anticancer Drugs. 2002;13:881–889.

    CAS  PubMed  Google Scholar 

  46. 46

    Kawashima H, Ogose A, Yoshizawa T, et al. Expression of the coxsackievirus and adenovirus receptor in musculoskeletal tumors and mesenchymal tissues: efficacy of adenoviral gene therapy for osteosarcoma. Cancer Sci. 2003;94:70–75.

    CAS  Google Scholar 

  47. 47

    Marcellus RC, Teodoro JG, Charbonneau R, Shore GC, Branton PE . Expression of p53 in Saos-2 osteosarcoma cells induces apoptosis which can be inhibited by Bcl-2 or the adenovirus E1B-55 kDa protein. Cell Growth Differ. 1996;7:1643–1650.

    CAS  PubMed  Google Scholar 

  48. 48

    Laffon B, Pasaro E, Mendez J . Effects of styrene-7,8-oxide over p53, p21, bcl-2 and bax expression in human lymphocyte cultures. Mutagenesis. 2001;16:127–132.

    CAS  PubMed  Google Scholar 

  49. 49

    Hodges NJ, Chipman JK . Down-regulation of the DNA-repair endonuclease 8-oxo-guanine DNA glycosylase 1 (hOGG1) by sodium dichromate in cultured human A549 lung carcinoma cells. Carcinogenesis. 2002;23:55–60.

    CAS  Google Scholar 

  50. 50

    Webb JL . Effect of more than one inhibitor. In: Webb JL, ed. Enzymes and Metabolic Inhibitors. Vol. 1. New York, NY: Academic Press; 1963: 66–79, 487–512.

    Google Scholar 

  51. 51

    Yeh YA, Herenyiova M, Weber G . Quercetin: synergistic action with carboxyamidotriazole in human breast carcinoma cells. Life Sci. 1995;57:1285–1292.

    CAS  Google Scholar 

  52. 52

    Zhang L, Hung MC . Sensitization of HER-2/neu-overexpressing non-small cell lung cancer cells to chemotherapeutic drugs by tyrosine kinase inhibitor emodin. Oncogene. 1996;12:571–576.

    CAS  Google Scholar 

  53. 53

    Chang EH, Pirollo KF, Bouker KB . Tp53 gene therapy: a key to modulating resistance to anticancer therapies? Mol Med Today. 2000;6:358–364.

    CAS  Google Scholar 

  54. 54

    Ganjavi H, Gee M, Narendran A, et al. Adenovirus-mediated p53 gene therapy in osteosarcoma cell lines: Sensitisation to cisplatin and doxorubicin. Hum Gene Ther. (under review). 2004.

  55. 55

    Levine AJ . p53, the cellular gatekeeper for growth and division. Cell. 1997;88:323–331.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Greenblatt MS, Bennett WP, Hollstein M, Harris CC . Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 1994;54:4855–4878.

    CAS  Google Scholar 

  57. 57

    Hollstein M, Sidransky D, Vogelstein B, Harris CC . p53 mutations in human cancers. Science. 1991;253:49–53.

    CAS  Google Scholar 

  58. 58

    Nigro JM, Baker SJ, Preisinger AC, et al. Mutations in the p53 gene occur in diverse human tumour types. Nature. 1989;342:705–708.

    CAS  PubMed  Google Scholar 

  59. 59

    Smith ND, Rubenstein JN, Eggener SE, Kozlowski JM . The p53 tumor suppressor gene and nuclear protein: basic science review and relevance in the management of bladder cancer. J Urol. 2003;169:1219–1228.

    CAS  Google Scholar 

  60. 60

    Donehower LA, Harvey M, Slagle BL, et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature. 1992;356:215–221.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Jacks T, Remington L, Williams BO, et al. Tumor spectrum analysis in p53-mutant mice. Curr Biol. 1994;4:1–7.

    CAS  PubMed  Google Scholar 

  62. 62

    Purdie CA, Harrison DJ, Peter A, et al. Tumour incidence, spectrum and ploidy in mice with a large deletion in the p53 gene. Oncogene. 1994;9:603–609.

    CAS  Google Scholar 

  63. 63

    Malkin D, Li FP, Strong LC, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science. 1990;250:1233–1238.

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    Varley JM . Germline TP53 mutations and Li-Fraumeni syndrome. Hum Mutat. 2003;21:313–320.

    CAS  PubMed  Google Scholar 

  65. 65

    Srivastava S, Zou ZQ, Pirollo K, et al. Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome. Nature. 1990;348:747–749.

    CAS  PubMed  Google Scholar 

  66. 66

    Tsuchiya T, Sekine K, Hinohara S, et al. Analysis of the p16INK4, p14ARF, p15, TP53, and MDM2 genes and their prognostic implications in osteosarcoma and Ewing sarcoma. Cancer Genet Cytogenet. 2000;120:91–98.

    CAS  PubMed  Google Scholar 

  67. 67

    Nielsen LL, Dell J, Maxwell E, et al. Efficacy of p53 adenovirus-mediated gene therapy against human breast cancer xenografts. Cancer Gene Ther. 1997;4:129–138.

    CAS  Google Scholar 

  68. 68

    Bookstein R, Demer W, Gregory R, et al. p53 gene therapy in vivo of hepatocellular and liver metastatic colorectal cancer. Semin Oncol. 1996;23:66–77.

    CAS  Google Scholar 

  69. 69

    Kock H, Harris MP, Anderson SC, et al. Adenovirus-mediated p53 gene transfer suppresses growth of human glioblastoma cells in vitro and in vivo. Int J Cancer. 1996;67:808–815.

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70

    Mujoo K, Maneval DC, Anderson SC, et al. Adenoviral-mediated p53 tumor suppressor gene therapy of human ovarian carcinoma. Oncogene. 1996;12:1617–1623.

    CAS  PubMed  PubMed Central  Google Scholar 

  71. 71

    Tursz T, Le Cesne A, Baldeyrou P, et al. Phase I study of a recombinant adenovirus-mediated gene transfer in lung cancer patients. J Natl Cancer Inst. 1996;88:1857–1863.

    CAS  Google Scholar 

  72. 72

    Clayman GL, el-Naggar AK, Lippman SM, et al. Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma. J Clin Oncol. 1998;16:2221–2232.

    CAS  PubMed  PubMed Central  Google Scholar 

  73. 73

    Clayman GL, Frank DK, Bruso PA, et al. Adenovirus-mediated wild-type p53 gene transfer as a surgical adjuvant in advanced head and neck cancers. Clin Cancer Res. 1999;5:1715–1722.

    CAS  PubMed  PubMed Central  Google Scholar 

  74. 74

    Reid T, Warren R, Kirn D . Intravascular adenoviral agents in cancer patients: lessons from clinical trials. Cancer Gene Ther. 2002;9:979–986.

    CAS  Google Scholar 

  75. 75

    Neyns B, Noppen M . Intratumoral gene therapy for non-small cell lung cancer: current status and future directions. Monaldi Arch Chest Dis. 2003;59:287–295.

    CAS  PubMed  Google Scholar 

  76. 76

    Asaoka K, Tada M, Sawamura Y, Ikeda J, Abe H . Dependence of efficient adenoviral gene delivery in malignant glioma cells on the expression levels of the Coxsackievirus and adenovirus receptor. J Neurosurg. 2000;92:1002–1008.

    CAS  PubMed  PubMed Central  Google Scholar 

  77. 77

    Hutchin ME, Pickles RJ, Yarbrough WG . Efficiency of adenovirus-mediated gene transfer to oropharyngeal epithelial cells correlates with cellular differentiation and human coxsackie and adenovirus receptor expression. Hum Gene Ther. 2000;11:2365–2375.

    CAS  Google Scholar 

  78. 78

    Turturro F, Seth P, Link Jr CJ . In vitro adenoviral vector p53-mediated transduction and killing correlates with expression of coxsackie-adenovirus receptor and alpha(nu)beta5 integrin in SUDHL-1 cells derived from anaplastic large-cell lymphoma. Clin Cancer Res. 2000;6:185–192.

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79

    Li D, Duan L, Freimuth P, O'Malley Jr BW . Variability of adenovirus receptor density influences gene transfer efficiency and therapeutic response in head and neck cancer. Clin Cancer Res. 1999;5:4175–4181.

    CAS  PubMed  PubMed Central  Google Scholar 

  80. 80

    Pearson AS, Koch PE, Atkinson N, et al. Factors limiting adenovirus-mediated gene transfer into human lung and pancreatic cancer cell lines. Clin Cancer Res. 1999;5:4208–4213.

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81

    Croyle MA, Walter E, Janich S, Roessler BJ, Amidon GL . Role of integrin expression in adenovirus-mediated gene delivery to the intestinal epithelium. Hum Gene Ther. 1998;9:561–573.

    CAS  PubMed  PubMed Central  Google Scholar 

  82. 82

    Hemmi S, Geertsen R, Mezzacasa A, Peter I, Dummer R . The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures. Hum Gene Ther. 1998;9:2363–2373.

    CAS  PubMed  PubMed Central  Google Scholar 

  83. 83

    Takayama K, Ueno H, Pei XH, Nakanishi Y, Yatsunami J, Hara N . The levels of integrin alpha v beta 5 may predict the susceptibility to adenovirus-mediated gene transfer in human lung cancer cells. Gene Ther. 1998;5:361–368.

    CAS  PubMed  PubMed Central  Google Scholar 

  84. 84

    Hamilton TE, McClane SJ, Baldwin S, et al. Efficient adenoviral-mediated murine neonatal small intestinal gene transfer is dependent on alpha(v) integrin expression. J Pediatr Surg. 1997;32:1695–1703.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Corresponding author

Correspondence to David Malkin.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Keywords

  • soft tissue sarcoma
  • p53
  • chemosensitization
  • gene replacement

Further reading

Search

Quick links