Adenovirus-mediated p53 gene therapy inhibits human sarcoma tumorigenicity

Abstract

Mutations of the p53 tumor-suppressor gene are the most frequent genetic abnormality in soft tissue sarcomas. Because these rare tumors also respond poorly to standard chemotherapy and bear a 50% 5-year mortality rate, we investigated the possible therapeutic benefits of p53 gene restoration in sarcomas. We constructed Ad5p53, which is an E1A-deleted, replication-deficient adenovirus expressing a cytomegalovirus promoter-driven wild-type p53 cDNA with a Flag sequence tag. SKLMS-1 human leiomyosarcoma cells containing a mis-sense p53 point mutation were effectively transduced with Ad5p53. Increasing levels of Flag-p53 protein, as well as dose-dependent p21Cip1 induction, were observed through a dose range of 10 -500 plaque-forming units (PFU)/cell. In vitro administration of Ad5p53 as a single 100 PFU/cell dose caused 40 -60% growth inhibition of SKLMS-1 cells at posttreatment days 4, 6, and 8 compared with untreated or viral control treated-cells (P Ͻ .05, Student's t test). Relative to these same controls, in vivo treatment of SKLMS-1-bearing severe combined immunodeficient mice with 6 ϫ 10 9 PFU of Ad5p53 by intratumoral injection resulted in a 35-day tumor growth delay and complete tumor regression in 40% of mice (P Ͻ .05, Student's t test). The expression of virally derived p53 mRNA in Ad5p53-treated tumor tissues was detected in treated tumor specimens by reverse transcriptase polymerase chain reaction. Reduced intratumoral cellularity and the presence of p53 staining in adjacent normal tissue, consistent with delivery of exogenous p53 to the tumor target, were evident only in Ad5p53-treated tumors after immunohistochemical staining for p53. These results indicate that wild-type p53 gene restoration in sarcomas retards tumor growth and may come to be usefully applied to the clinical treatment of this disease as a single regimen or in combination with conventional therapies. Cancer Gene Therapy (2000) 7, 422-429

Isolated limb perfusion: a novel delivery system for wild-type p53 and fiber-modified oncolytic adenoviruses to extremity sarcoma

“…[27][28][29] Our own investigations utilizing autologous human primary and metastatic sarcoma have demonstrated that clonal expansion of p53-mutated cells in STS confers a distinct metastatic advantage, 30 and we have previously shown that re-expression of wild-type p53 into STS cells harboring a p53 mutation can induce apoptosis, enhance chemosensitivity, inhibit angiogenesis and prolong survival in vitro and in vivo. [15][16][17] Taking these findings into account, it is logical to investigate the role of wild-type p53 as a therapeutic agent in STS, and results from our preclinical sarcoma models are encouraging. For instance, tumor cell transfection with wild-type p53 caused tumor regression in 40% of mice so treated 15 and also restored STS sensitivity to doxorubicin by downregulating the multidrug-resistance protein-1 (MDR1).…”

“…[15][16][17] Taking these findings into account, it is logical to investigate the role of wild-type p53 as a therapeutic agent in STS, and results from our preclinical sarcoma models are encouraging. For instance, tumor cell transfection with wild-type p53 caused tumor regression in 40% of mice so treated 15 and also restored STS sensitivity to doxorubicin by downregulating the multidrug-resistance protein-1 (MDR1). 16 Furthermore, ITI of FLAGp53 resulted in significant tumor growth inhibition.…”

“…Using a replicationdeficient, wild-type fiber adenovirus with cytomegalovirus (CMV) promoter-driven expression of FLAG tagged wild-type p53 (AdFLAGp53) we generated previously, 15 we infected SKLMS-1 cells at 1:5000 virus particle (VP) for 48 h in vitro to confirm viral function and transgene efficacy by Western blot analysis (Figure 1a). Wild-type p53 transgene expression was detected and associated with upregulation of 14-3-3s and p21 CIP1/WAF1 proteins in our mutant p53 STS cell line.…”

“…We sought to address the first issue for several reasons. First, p53 is the most frequently detected mutation in STS, and reconstitution of its wildtype function confers a number of well-documented anticancer outcomes such as growth arrest, 14,15 resensitization to chemotherapy, 16 suppression of invasion and attenuation of angiogenesis. 17 Second, we have previously shown that intra-tumoral injection (ITI) of adenoviral delivered wild-type p53 significantly inhibited STS growth in SCID mice.…”

“…17 Second, we have previously shown that intra-tumoral injection (ITI) of adenoviral delivered wild-type p53 significantly inhibited STS growth in SCID mice. 15 Third, adenovirus delivery of p53 is an accepted novel therapy currently in clinical trials for other cancers such as non-small-cell lung cancer, 18,19 liver tumors, 20 and glioma; 21 however, to the best of our knowledge this has not been attempted in STS patients. The second aim of this study undertaken was to improve the efficiency of gene delivery, STS targeting and tumor viral transduction.…”