Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer

Abstract

Nitric oxide synthase gene therapy has been shown to be effective at inducing apoptosis in experimental tumours and sensitizing them to radiotherapy. We have also shown that expression of inducible nitric oxide synthase (iNOS) can be effectively restricted to the tumour volume by the use of the radiation inducible promoter (WAF1) to drive the transgene in clinically relevant protocols. A synthetic construct (pE9), incorporating nine radiosensitive CArG elements from the Egr1 promoter, has recently been developed for cancer gene therapy. We have now investigated basal gene expression of transgenes driven by this promoter to assess its suitablility for use in iNOS gene therapy protocols in vivo. Transfection of human microvascular endothelial cells (HMEC-1) with pE9iNOS, using a cationic lipid vector, resulted in progressively increasing (o5-fold) levels of iNOS protein expression up to 8 h after transfection. Transfection of an ex vivo rat artery preparation with pE9iNOS caused 83% inhibition of response to the vasoconstrictor phenylephrine (PE). CMVi-NOS transfection also reduced response to PE, but by only 52%. A single injection of 25 mg of pE9iNOS DNA in a lipid vector into the centre of a murine sarcoma (RIF1) induced iNOS protein expression by four-fold and increased nitrite concentration eight-fold. This caused a 7-day delay in tumour growth and was more effective than the constitutive CMVdriven construct. Our data suggest that generation of NO K , as a result of iNOS overexpression, is capable of further activating the E9 promoter, through a positive feedback loop, yielding stronger and sustained levels of NO K . This pE9iNOS combination may, therefore, be particularly useful in an anticancer gene therapy strategy as its antitumour effect in vivo was clearly superior to that of the strong constitutive promoter, CMV.

p21(WAF1)-mediated transcriptional targeting of inducible nitric oxide synthase gene therapy sensitizes tumours to fractionated radiotherapy

“…We have also shown previously that WAF1 could be used to drive iNOS expression in vivo and could sensitize both HT29 and RIF1 tumours to a single therapeutic radiation dose. 7,18,19 We also demonstrated that the iNOS levels were increased following direct injection of this construct into RIF1 tumours within 24 h with levels significantly elevated up to 5 days; similar results were seen with the HT29 xenograft, both before and after an inducing dose of radiation, to activate the WAF1 promoter. 18,19 We now wanted to test this approach using fractionated radiation schedules in vivo at clinically relevant doses per fraction.…”

“…5,6 We have also shown that a synthetic construct (pE9), incorporating nine radiosensitive CArG elements to drive inducible nitric oxide synthase (iNOS), was more effective than a constitutive CMV-driven construct both in vitro and in vivo. 7 The tumour microenvironment itself also presents opportunities to specifically target tumour tissue. Due to the ineffectual blood vessel network that exists in tumours, there are areas of low oxygen tension that are problematic for conventional cancer treatments in that they are inherently resistant to radio-or chemotherapy.…”

Nitric Oxide Produced in Response to Engagement of β2 Integrins on Human Neutrophils Activates the Monomeric GTPases Rap1 and Rap2 and Promotes Adhesion

“…Nitrite Production-Nitrite production is measured using a nitric oxide quantitation kit (Active Motif, Ede, The Netherlands) (45). Nitrite is quantified by the addition of Griess reagent, which converts nitrite into a purple-colored azo compound.…”

The radiation-inducible pE9 promoter driving inducible nitric oxide synthase radiosensitizes hypoxic tumour cells to radiation

“…Our previous physiological study of an ex vivo rat tail artery treated with pE9/iNOS showed rapid and more pronounced vasodilatation compared to CMV/iNOS-treated preparations, supporting our hypothesis of a positive feedback loop increasing the activation of the pE9 promoter. 10 Furthermore, significant antitumour effects were reported after a single 25 mg injection of the pE9/iNOS construct into intradermal radiation-induced fibrosarcoma (RIF)-1 tumours. A fourfold increase in protein expression and an eightfold increase in nitrite concentration within the tumour resulted in a 7-day delay in tumour growth as compared with untreated tumours.…”

“…8,9 In a previous study we have shown that the CArG/iNOS construct may form a positive feedback loop, whereby the production of NO K , coupled with the elevated endogenous levels of NO K in tumours, resulted in the formation of ROS that in turn further activate the promoter. 10 Many tumours exhibit hypoxic regions reducing the effectiveness of radiotherapy. 6 Previously, we have shown that iNOS gene therapy diminished radioresistance observed under hypoxic conditions, thereby making it an ideal strategy for the treatment of solid tumours.…”