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Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity.

Di Domenico F, Foppoli C, Blarzino C, Perluigi M, Paolini F, Morici S, Coccia R, Cini C, De Marco F - J. Exp. Clin. Cancer Res. (2009)

Bottom Line: E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions.However, the cellular effects of such an interaction are still under debate.These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemical Sciences, University of Rome La Sapienza, Rome, Italy. fabio.didomenico@uniroma1.it

ABSTRACT

Background: Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype.

Methods: The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored.

Results: We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs.

Conclusion: Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the cell phenotype and can induce a higher sensitivity to tyrosine related anti-blastic drugs.

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Effect of HPV-16 E5 expression on the sensitivity of melanoma cells to the tyrosine related antiblastic agents. M14 control cells (grey bars) or HPV-16 E5 expressing cells (black bars) were incubated with DHBA (up) or BSO (down) at a 30 μM concentration. After 48 h incubation, the cell number was determined using the CV assay as described in the methods section. The E5 expression is associated with a marked sensitivity of melanoma cells to the named anti-tumour agents. Similar results were obtained with FRM cells (data not shown). Reported values are expressed as A540 and are the mean ± SD. of eight independent replicas of a representative experiment in a set of four. Statistical comparison was made using the non parametric Mann – Whitney test * p < 0.05; ** p < 0.005.
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Figure 7: Effect of HPV-16 E5 expression on the sensitivity of melanoma cells to the tyrosine related antiblastic agents. M14 control cells (grey bars) or HPV-16 E5 expressing cells (black bars) were incubated with DHBA (up) or BSO (down) at a 30 μM concentration. After 48 h incubation, the cell number was determined using the CV assay as described in the methods section. The E5 expression is associated with a marked sensitivity of melanoma cells to the named anti-tumour agents. Similar results were obtained with FRM cells (data not shown). Reported values are expressed as A540 and are the mean ± SD. of eight independent replicas of a representative experiment in a set of four. Statistical comparison was made using the non parametric Mann – Whitney test * p < 0.05; ** p < 0.005.

Mentions: Subsequently we wondered whether the above reported endosomal alkalinisation and the reactivation of tyrosinase was associated with modifications in cell phenotype eventually resulting in an altered susceptibility to chemotherapeutic agents. Based on the notion that 3,4-DHBA, a dopamine mimetic pro-drug, is a substrate for tyrosinase with consequent production of toxic intermediates [40] we evaluated its cytotoxic effect in E5 expressing cells. Fig. 7 shows that a 30 μM concentration induced a much stronger impairment of cell viability on E5 expressing melanomas than on the control cells. The same figure shows also that BSO, a well-known inhibitor of glutathione synthesis whose cytotoxic effects are correlated with the level of tyrosinase activity [40], determined a drastic reduction of cell viability in E5 expressing cells, while control cells were scarcely affected.


Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity.

Di Domenico F, Foppoli C, Blarzino C, Perluigi M, Paolini F, Morici S, Coccia R, Cini C, De Marco F - J. Exp. Clin. Cancer Res. (2009)

Effect of HPV-16 E5 expression on the sensitivity of melanoma cells to the tyrosine related antiblastic agents. M14 control cells (grey bars) or HPV-16 E5 expressing cells (black bars) were incubated with DHBA (up) or BSO (down) at a 30 μM concentration. After 48 h incubation, the cell number was determined using the CV assay as described in the methods section. The E5 expression is associated with a marked sensitivity of melanoma cells to the named anti-tumour agents. Similar results were obtained with FRM cells (data not shown). Reported values are expressed as A540 and are the mean ± SD. of eight independent replicas of a representative experiment in a set of four. Statistical comparison was made using the non parametric Mann – Whitney test * p < 0.05; ** p < 0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654431&req=5

Figure 7: Effect of HPV-16 E5 expression on the sensitivity of melanoma cells to the tyrosine related antiblastic agents. M14 control cells (grey bars) or HPV-16 E5 expressing cells (black bars) were incubated with DHBA (up) or BSO (down) at a 30 μM concentration. After 48 h incubation, the cell number was determined using the CV assay as described in the methods section. The E5 expression is associated with a marked sensitivity of melanoma cells to the named anti-tumour agents. Similar results were obtained with FRM cells (data not shown). Reported values are expressed as A540 and are the mean ± SD. of eight independent replicas of a representative experiment in a set of four. Statistical comparison was made using the non parametric Mann – Whitney test * p < 0.05; ** p < 0.005.
Mentions: Subsequently we wondered whether the above reported endosomal alkalinisation and the reactivation of tyrosinase was associated with modifications in cell phenotype eventually resulting in an altered susceptibility to chemotherapeutic agents. Based on the notion that 3,4-DHBA, a dopamine mimetic pro-drug, is a substrate for tyrosinase with consequent production of toxic intermediates [40] we evaluated its cytotoxic effect in E5 expressing cells. Fig. 7 shows that a 30 μM concentration induced a much stronger impairment of cell viability on E5 expressing melanomas than on the control cells. The same figure shows also that BSO, a well-known inhibitor of glutathione synthesis whose cytotoxic effects are correlated with the level of tyrosinase activity [40], determined a drastic reduction of cell viability in E5 expressing cells, while control cells were scarcely affected.

Bottom Line: E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions.However, the cellular effects of such an interaction are still under debate.These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemical Sciences, University of Rome La Sapienza, Rome, Italy. fabio.didomenico@uniroma1.it

ABSTRACT

Background: Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype.

Methods: The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored.

Results: We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs.

Conclusion: Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the cell phenotype and can induce a higher sensitivity to tyrosine related anti-blastic drugs.

Show MeSH
Related in: MedlinePlus