¹⁹ F‐NMR detection of lacZ gene expression via the enzymic hydrolysis of 2‐fluoro‐4‐nitrophenyl β‐D‐galactopyranoside in vivo in PC3 prostate tumor xenografts in the mouse ¹

Abstract

Gene therapy shows promise for treating prostate cancer and has been evaluated in several clinical trials. A major challenge that remains is to establish a method for verifying transgene activity in situ. The lacZ gene encoding beta-galactosidase historically has been the most popular reporter gene for molecular biology. We have designed a 19F NMR approach to reveal lacZ gene expression by assessing beta-galactosidase (beta-gal) activity in vivo. The substrate 2-fluoro-4-nitrophenyl beta-D-galactopyranoside (OFPNPG) is readily hydrolyzed by beta-gal with a corresponding decrease in the 19F-NMR signal from OFPNPG and the appearance of a new signal shifted 4-6 ppm upfield from the aglycone 2-fluoro-4-nitrophenol (OFPNP). We report proof of principle in cultures of PC3 prostate cancer cells using 19F NMR spectroscopy and 19F chemical shift imaging. More importantly, we demonstrate for the first time the ability to differentiate wild-type and lacZ-expressing prostate tumor xenografts in mice using this approach.

Harnessing the complexity of DNA-damage response pathways to improve cancer treatment outcomes

“…Similarly, 18-fluoro-L-thymidine uptake may be falsely low if cell cycle arrest is reversible. However, combining these imaging agents with other methods such as measurement SA-b-gal activity (Tung et al, 2004;Liu et al, 2007) and cell death (Al-Ejeh et al, 2007, 2009Thierry et al, 2009) can be used to monitor accurately the status of tumors, for example, in response to DNAdamaging chemotherapy. 'Certainly, mouse models of cancer have been used to address genetics of tumorigenesis for years, but they have been understudied as experimental platforms to investigate short-and long-term consequences of druginducible effector programs' (Kilic and Schmitt, 2008).…”

19F NMR applications in chemical biology

“…Cleavage of the glycosidic bond results in the formation of p-fluoroo-nitrophenol 17, which has a chemical shift difference of >3.6 ppm compared with the substrate (Scheme 5). This assay system has been applied to the detection of LacZ in transfected human PC3 prostate tumour cells [38]. Signal-to-noise was improved with the incorporation of a trifluoromethyl group instead of a single fluorine atom [39], but the relative chemical shift response to glycosidic bond cleavage was smaller (<1.2 ppm).…”

Detection of fluorine labeled Herceptin using cellular 19F MRI ex vivo

“…19 F SI was measured directly from axial slice of HFB device [11]. The changes in 19 F SI at different H8-H21 concentrations were observed and the calibration curves were prepared, as 19 F SI is linearly related with the amount of 19 F molecules [12]. The 19 F SI of pure PFOB was considered as 100% of SI.…”