Limits...
Photo-activation of the hydrophobic probe iodonaphthylazide in cells alters membrane protein function leading to cell death.

Viard M, Garg H, Blumenthal R, Raviv Y - BMC Cell Biol. (2009)

Bottom Line: Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced.Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor.It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nanobiology Program, Center of Cancer Research, National Cancer Institute, Frederick, Maryland, USA. viardm@mail.ncifcrf.gov

ABSTRACT

Background: Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310-380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated.

Results: INA treatment of various cell lines followed by irradiation with UV light (310-380 nm) resulted in loss of cell viability in a dose dependent manner. The mechanism of cell death appeared to be apoptosis as indicated by phosphatidylserine exposure, mitochondrial depolarization and DNA fragmentation. Inhibition by pan-caspase inhibitors and cleavage of caspase specific substrates indicated that at low concentrations of INA apoptosis was caspase dependent. The INA-UV treatment showed similar cell killing efficacy in cells over-expressing MRP1 function as control cells. Efflux of an MRP1 substrate was blocked by INA-UV treatment of the MRP1-overexpressing cells. Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced. Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor.

Conclusion: INA is a photo-activable agent that induces apoptosis in various cancer cell lines. It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis. This activity of INA maybe exploited for use as an anti-cancer agent.

Show MeSH

Related in: MedlinePlus

INA-UV treatment inhibits the calcein efflux of MRP1 expressing cells. The loading of calcein by 293/MRP1 and 293 cells was assessed by FACS analysis. A. Histogram of the calcein fluorescence of 293/MRP1 cells treated or not with 50 μM INA-UV or 40 μM verapamil. B. Histogram of the calcein fluorescence of 293 cells treated or not with 50 μM INA-UV or 40 μM verapamil.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2666636&req=5

Figure 6: INA-UV treatment inhibits the calcein efflux of MRP1 expressing cells. The loading of calcein by 293/MRP1 and 293 cells was assessed by FACS analysis. A. Histogram of the calcein fluorescence of 293/MRP1 cells treated or not with 50 μM INA-UV or 40 μM verapamil. B. Histogram of the calcein fluorescence of 293 cells treated or not with 50 μM INA-UV or 40 μM verapamil.

Mentions: MRP1 is a complex transmembrane protein with 17 membrane spanning domains that mediates efflux of various substrates from the cytoplasm of the cell to the extracellular milieu. Calcein is an anionic fluorescent probe that acts as a substrate for MRP1 and determination of its cellular accumulation and efflux allows the investigation of MRP1 activity [27]. As observed in figure 6A, after one hour incubation at 37°C, the 293 cells expressing MRP1 have released a significant portion of the entrapped calcein as evident from the lower fluorescence seen by flow cytometry. This efflux was prevented by incubation of the cells with verapamil (40 μM), an inhibitor of MRP1 function, yielding a labeling efficiency with calcein similar to the one obtained with 293 that do not express MRP1 (figure 6A). Interestingly pre-treatment of the 293/MRP1 cells with INA-UV induced a similar block in MRP1 function as observed with verapamil (figure 6A). On the other hand, in control cells lacking MRP1 neither verapamil nor INA-UV treatment had any significant effect on calcein efflux (figure 6B). This suggests that the increase in calcein labeling in INA-UV treated MRP1 positive cells was in fact due to a specific inhibition of MRP1 function. This is consistent with the inactivation of transmembrane proteins by INA labeling.


Photo-activation of the hydrophobic probe iodonaphthylazide in cells alters membrane protein function leading to cell death.

Viard M, Garg H, Blumenthal R, Raviv Y - BMC Cell Biol. (2009)

INA-UV treatment inhibits the calcein efflux of MRP1 expressing cells. The loading of calcein by 293/MRP1 and 293 cells was assessed by FACS analysis. A. Histogram of the calcein fluorescence of 293/MRP1 cells treated or not with 50 μM INA-UV or 40 μM verapamil. B. Histogram of the calcein fluorescence of 293 cells treated or not with 50 μM INA-UV or 40 μM verapamil.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666636&req=5

Figure 6: INA-UV treatment inhibits the calcein efflux of MRP1 expressing cells. The loading of calcein by 293/MRP1 and 293 cells was assessed by FACS analysis. A. Histogram of the calcein fluorescence of 293/MRP1 cells treated or not with 50 μM INA-UV or 40 μM verapamil. B. Histogram of the calcein fluorescence of 293 cells treated or not with 50 μM INA-UV or 40 μM verapamil.
Mentions: MRP1 is a complex transmembrane protein with 17 membrane spanning domains that mediates efflux of various substrates from the cytoplasm of the cell to the extracellular milieu. Calcein is an anionic fluorescent probe that acts as a substrate for MRP1 and determination of its cellular accumulation and efflux allows the investigation of MRP1 activity [27]. As observed in figure 6A, after one hour incubation at 37°C, the 293 cells expressing MRP1 have released a significant portion of the entrapped calcein as evident from the lower fluorescence seen by flow cytometry. This efflux was prevented by incubation of the cells with verapamil (40 μM), an inhibitor of MRP1 function, yielding a labeling efficiency with calcein similar to the one obtained with 293 that do not express MRP1 (figure 6A). Interestingly pre-treatment of the 293/MRP1 cells with INA-UV induced a similar block in MRP1 function as observed with verapamil (figure 6A). On the other hand, in control cells lacking MRP1 neither verapamil nor INA-UV treatment had any significant effect on calcein efflux (figure 6B). This suggests that the increase in calcein labeling in INA-UV treated MRP1 positive cells was in fact due to a specific inhibition of MRP1 function. This is consistent with the inactivation of transmembrane proteins by INA labeling.

Bottom Line: Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced.Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor.It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nanobiology Program, Center of Cancer Research, National Cancer Institute, Frederick, Maryland, USA. viardm@mail.ncifcrf.gov

ABSTRACT

Background: Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310-380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated.

Results: INA treatment of various cell lines followed by irradiation with UV light (310-380 nm) resulted in loss of cell viability in a dose dependent manner. The mechanism of cell death appeared to be apoptosis as indicated by phosphatidylserine exposure, mitochondrial depolarization and DNA fragmentation. Inhibition by pan-caspase inhibitors and cleavage of caspase specific substrates indicated that at low concentrations of INA apoptosis was caspase dependent. The INA-UV treatment showed similar cell killing efficacy in cells over-expressing MRP1 function as control cells. Efflux of an MRP1 substrate was blocked by INA-UV treatment of the MRP1-overexpressing cells. Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced. Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor.

Conclusion: INA is a photo-activable agent that induces apoptosis in various cancer cell lines. It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis. This activity of INA maybe exploited for use as an anti-cancer agent.

Show MeSH
Related in: MedlinePlus