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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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Expression of HPV-16 E5 oncogene does not affect tyrosinase mRNA transcription and protein expression levels. Tyrosinase mRNA levels were evaluated by RT-PCR in FRM and M14 melanoma control cells (CTR), in cells treated with 20 nM Con-A (+ ConA) and in cell expressing the HPV-16 E5 (+ E5). Panel a) – Total mRNA (1 μg) was reverse transcribed and amplified with HuTyr-1/HuTyr-2. Four independent experiments gave similar results. All the samples showed similar levels of tyrosinase mRNA. Western blot analysis (panel b) and densitometric quantisation (panel c) of the chemo-luminescent signals of tyrosinase protein levels. No protein modulation was observed under any experimental condition. Results represent the mean ± standard deviation (SD) of four independent experiments. (A.U. = Arbitrary Unit).
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Figure 6: Expression of HPV-16 E5 oncogene does not affect tyrosinase mRNA transcription and protein expression levels. Tyrosinase mRNA levels were evaluated by RT-PCR in FRM and M14 melanoma control cells (CTR), in cells treated with 20 nM Con-A (+ ConA) and in cell expressing the HPV-16 E5 (+ E5). Panel a) – Total mRNA (1 μg) was reverse transcribed and amplified with HuTyr-1/HuTyr-2. Four independent experiments gave similar results. All the samples showed similar levels of tyrosinase mRNA. Western blot analysis (panel b) and densitometric quantisation (panel c) of the chemo-luminescent signals of tyrosinase protein levels. No protein modulation was observed under any experimental condition. Results represent the mean ± standard deviation (SD) of four independent experiments. (A.U. = Arbitrary Unit).

Mentions: In order to understand if the onset of melanotic phenotype and tyrosinase activation following the E5 expression depends on a modulation of tyrosinase transcription and/or protein expression, we determined the tyrosinase mRNA and protein levels by RT-PCR and by Western Blot (WB) analyses, respectively. Figure 6 shows that the expression of E5 oncogene had no effect on tyrosinase mRNA levels both in M14 and FRM cells and confirmed that in these cell lines the amelanotic phenotype is associated with a fair transcription of tyrosinase mRNA [27]. Moreover, WB analysis showed that tyrosinase protein levels were not modulated in E5 expressing cells in comparison with controls. These results, while confirming the poor connection between pigmentation genes expression and the pigmentary status of melanomas, indicate that the amelanotic phenotype of FRM and M14 cells is indeed related to post-translational regulatory process in melanocytes that express normal amounts of tyrosinase protein.


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)

Expression of HPV-16 E5 oncogene does not affect tyrosinase mRNA transcription and protein expression levels. Tyrosinase mRNA levels were evaluated by RT-PCR in FRM and M14 melanoma control cells (CTR), in cells treated with 20 nM Con-A (+ ConA) and in cell expressing the HPV-16 E5 (+ E5). Panel a) – Total mRNA (1 μg) was reverse transcribed and amplified with HuTyr-1/HuTyr-2. Four independent experiments gave similar results. All the samples showed similar levels of tyrosinase mRNA. Western blot analysis (panel b) and densitometric quantisation (panel c) of the chemo-luminescent signals of tyrosinase protein levels. No protein modulation was observed under any experimental condition. Results represent the mean ± standard deviation (SD) of four independent experiments. (A.U. = Arbitrary Unit).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Expression of HPV-16 E5 oncogene does not affect tyrosinase mRNA transcription and protein expression levels. Tyrosinase mRNA levels were evaluated by RT-PCR in FRM and M14 melanoma control cells (CTR), in cells treated with 20 nM Con-A (+ ConA) and in cell expressing the HPV-16 E5 (+ E5). Panel a) – Total mRNA (1 μg) was reverse transcribed and amplified with HuTyr-1/HuTyr-2. Four independent experiments gave similar results. All the samples showed similar levels of tyrosinase mRNA. Western blot analysis (panel b) and densitometric quantisation (panel c) of the chemo-luminescent signals of tyrosinase protein levels. No protein modulation was observed under any experimental condition. Results represent the mean ± standard deviation (SD) of four independent experiments. (A.U. = Arbitrary Unit).
Mentions: In order to understand if the onset of melanotic phenotype and tyrosinase activation following the E5 expression depends on a modulation of tyrosinase transcription and/or protein expression, we determined the tyrosinase mRNA and protein levels by RT-PCR and by Western Blot (WB) analyses, respectively. Figure 6 shows that the expression of E5 oncogene had no effect on tyrosinase mRNA levels both in M14 and FRM cells and confirmed that in these cell lines the amelanotic phenotype is associated with a fair transcription of tyrosinase mRNA [27]. Moreover, WB analysis showed that tyrosinase protein levels were not modulated in E5 expressing cells in comparison with controls. These results, while confirming the poor connection between pigmentation genes expression and the pigmentary status of melanomas, indicate that the amelanotic phenotype of FRM and M14 cells is indeed related to post-translational regulatory process in melanocytes that express normal amounts of tyrosinase protein.

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