Heat-induced transcription of diphtheria toxin A or its variants, CRM176 and CRM197: implications for pancreatic cancer gene therapy.
Vectors combining the heat shock proteins (HSPs) promoter with the catalytic subunit A of the diphtheria toxin (DTA) or its variants, cross-reacting material (CRM) 176 and 197, were engineered to investigate the effect of bacterial toxins on pancreatic cancer (PC) cells. Three heat-inducible enhanced green fluorescent protein (eGFP)-expression vectors were obtained: V1 (91% homology to HSPA6), V2 (five heat shock elements upstream the minimal HSPA6 promoter) and V3 (V1 and V2 combined). The highest eGFP transcription and translation levels were found in V3 transfected PC cells. The V3 promoter was used to control DTA, CRM176 and CRM197 expression, treatment response being investigated in four PC cell lines. DTAwt or CRM176 transfected cell growth was completely arrested after heat shock. CRM197 toxin presumed to be inactive, caused mild distress at 37 degrees C and induced a 25-50% reduction in cell growth after heat shock. Preliminary in vivo findings showed that heat treatment arrests tumor growth in DTA197 stably transfected PSN1 cells. In conclusion, the efficient HSP promoter identified in this study may be extremely useful in controlling the transcription of toxins such as CRM197, which have lethal dose-related effects, and may thus be a promising tool in PC gene therapy in vivo.
Fogar
P
P
Department of Medical and Surgical Sciences, University of Padova, Padova, Italy.
Navaglia
F
F
Basso
D
D
Zambon
C-F
CF
Moserle
L
L
Indraccolo
S
S
Stranges
A
A
Greco
E
E
Fadi
E
E
Padoan
A
A
Pantano
G
G
Sanzari
M C
MC
Pedrazzoli
S
S
Montecucco
C
C
Plebani
M
M
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D045744:Cell Line, Tumor; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000235:genetics*; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D018869:Heat-Shock Response / Q000235:genetics; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014162:Transfection
D001426:Bacterial Proteins; D004167:Diphtheria Toxin; D018840:HSP70 Heat-Shock Proteins; C544598:HSPA6 protein, human; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A; C028581:CRM197 (non-toxic variant of diphtheria toxin)