Modulazione termica dell'espressione della subunità catalitica della tossina difterica e delle sue varianti CRM176 e CRM197 nella terapia genica del carcinoma del pancreas

Despite surgery and/or chemotherapy more than 80% of patients affected by pancreatic cancer do not survive more than 5 years after diagnosis. Gene therapy with bacterial toxins might be an effective approach for pancreatic cancer treatment. The aims of the present study were: 1. to create expression vectors, encoding highly toxic compounds, which can be activated and modulated by heat; 2. to verify, in vitro, their possible application in pancreatic cancer gene therapy. First objective The catalytic domain of diphtheria toxin (DTA) and of its variants CRM176 and CRM197 have been employed in this study as potent inhibitors of protein synthesis. To confine the expression of these cytotoxic agents to neoplastic tissue we focused our attention on heat inducible promoters, which allow a selective spatial and time control of gene induction by external heat. We chose the promoter of HSPA6 (encoding Hsp70B' protein) because it is strictly inducible, its basal expression levels being barely detectable in most tissues. We engineered three expression vectors with different heat-inducible HSPA6-derived promoter sequences driving the reporter gene eGFP: V1 containing a 473bp commercial sequence with a 91% homology to HSPA6; V2 with a 104bp sequence designed by us containing five Heat Shock Elements in tandem and the minimal promoter of HSPA6; V3 containing the 104bp sequence located downstream the 473bp promoter. In order to define the optimal heat shock temperature and exposure time we used four pancreatic cancer cell lines stably transfected with V1: the highest levels of eGFP expression (measured by Q-RT-PCR and FACS analysis) were obtained with a heat shock of 42,5°C for 1,5 hours. After setting the experimental conditions we compared the eGFP protein and mRNA levels in V1, V2 and V3 transfected pancreatic cancer cell lines. At 37°C there were minimal eGFP expression levels in cells transfected with all three vectors. After heat shock, however, V1, V2 and V3 transfected cells behaved differently: V1 and V2 transfected lines showed low and similar levels of expression (fold increase in mRNA about 8) while V3 transfected cells were highly induced by heat reaching up to 30 times the basal levels. V3, therefore, was shown to offer the best combination of high transciption efficiency and low background levels and was selected to drive the expression of DTA and its less toxic variants. Second objective The growth of all cell lines, transfected with DTA or its twenty fold less active variant CRM176, was significantly delayed even at 37°C. In other words the basal transcription levels of these toxins are sufficient to cause cell death; therefore DTA and CRM176 cannot be considered suitable candidates for gene therapy protocols using V3 promoter. At 37°C the supposedly inactive toxin CRM197 caused mild distress in transfected cells. After heat shock this phenomenon was amplified: cell growth was reduced in all CRM197 transfected cell lines. These findings indicate that the lethal effects of CRM197 are probably dose correlated. A spatial and time controlled expression of this toxin variant might offer the opportunity of combining its cytotoxic effects with its immunogenic properties, which may help antitumor immune system reaction. Conclusions The expression vector with the V3 heat-inducible promoter driving the CRM197 variant can be considered a promising starting point for future in vivo applications of pancreatic cancer gene therapy.