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


CONCLUSIONS
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.
Di Domenico
Fabio
F
Department of Biochemical Sciences, University of Rome La Sapienza, Rome, Italy. fabio.didomenico@uniroma1.it
Foppoli
Cesira
C
Blarzino
Carla
C
Perluigi
Marzia
M
Paolini
Francesca
F
Morici
Salvatrice
S
Coccia
Raffaella
R
Cini
Chiara
C
De Marco
Federico
F
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D045744:Cell Line, Tumor; D004298:Dopamine / Q000031:analogs & derivatives / Q000494:pharmacology; D011992:Endosomes / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008543:Melanins / Q000378:metabolism; D018328:Melanoma, Amelanotic / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D054328:Proton Pump Inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D014162:Transfection; D000086082:Viroporin Proteins
D000964:Antimetabolites, Antineoplastic; D019328:Buthionine Sulfoximine; D004298:Dopamine; D008543:Melanins; D014442:Monophenol Monooxygenase; D009856:Oncogene Proteins, Viral; D054328:Proton Pump Inhibitors; D012333:RNA, Messenger; C054859:oncogene protein E5, Human papillomavirus type 16; D000086082:Viroporin Proteins; C022133:3,4-dihydroxybenzylamine