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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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Presence of HPV-16 E5 DNA and expression of the specific mRNA in M14 and FRM cells after infection with HPV-16 E5 retroviral vector. The retroviral vector containing HPV-16 E5 gene was obtained by the transfection of Phoenix A retroviral producer cells with the LZRSpBMNZ-E5 plasmid. The control retroviral vector was obtained by the transfection of Phoenix cells with the empty LZRSpBMNZ plasmid. Cells were infected with either recombinant retrovirus or with the control retrovirus. Total DNA or RNA (1 μg) extracted from cells 96 h post infection were reverse transcribed and amplified with E5P65 sense (TGC ATC CAC AAC ATT ACT GGC G) and E5M3AS antisense (AAC ACC TAA ACG CAG AGG CTG C) primers. Upper panel: FRM cells; Lower panel: M14 cells. Lane 1: DNA from cells infected with the control retrovirus; Lane 2: DNA from cells infected with the HPV-16 E5 retrovirus; Lane 3: DNA digested total RNA from cells infected with the HPV-16 E5 retrovirus; Lane 4: Non retrotrascribed DNA digested total RNA from cells infected with the HPV-16 E5 retrovirus; Lane 5: No template negative control; Lane 6 positive control (0.5 μg Siha cell DNA). MW: DNA molecular weight marker VIII (Roche Biochemicals SpA): arrows on the left-hand side indicate the bp length of some reference bands. The band with size of 160 bp (left sided empty arrow) demonstrate the presence of viral E5 sequence and its transcription. Four independent experiments gave similar results.
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Figure 1: Presence of HPV-16 E5 DNA and expression of the specific mRNA in M14 and FRM cells after infection with HPV-16 E5 retroviral vector. The retroviral vector containing HPV-16 E5 gene was obtained by the transfection of Phoenix A retroviral producer cells with the LZRSpBMNZ-E5 plasmid. The control retroviral vector was obtained by the transfection of Phoenix cells with the empty LZRSpBMNZ plasmid. Cells were infected with either recombinant retrovirus or with the control retrovirus. Total DNA or RNA (1 μg) extracted from cells 96 h post infection were reverse transcribed and amplified with E5P65 sense (TGC ATC CAC AAC ATT ACT GGC G) and E5M3AS antisense (AAC ACC TAA ACG CAG AGG CTG C) primers. Upper panel: FRM cells; Lower panel: M14 cells. Lane 1: DNA from cells infected with the control retrovirus; Lane 2: DNA from cells infected with the HPV-16 E5 retrovirus; Lane 3: DNA digested total RNA from cells infected with the HPV-16 E5 retrovirus; Lane 4: Non retrotrascribed DNA digested total RNA from cells infected with the HPV-16 E5 retrovirus; Lane 5: No template negative control; Lane 6 positive control (0.5 μg Siha cell DNA). MW: DNA molecular weight marker VIII (Roche Biochemicals SpA): arrows on the left-hand side indicate the bp length of some reference bands. The band with size of 160 bp (left sided empty arrow) demonstrate the presence of viral E5 sequence and its transcription. Four independent experiments gave similar results.

Mentions: HPV 16 E5 is a small hydrophobic molecule expressed at very low levels in keratinocytes at early stages during viral infection and appearing to be critically linked to viral pathogenic potentials. Two amelanotic melanoma cell lines, FRM and M14, were infected with a HPV 16 E5 expressing retroviral vector and compared with the same lines infected with an "empty" retrovirus. After the infection with the E5 retroviral construct, the presence of cDNA for the E5 oncogene, as well as the corresponding mRNA, was shown by PCR and RT-PCR both in M14 and FRM cells (Fig. 1). Subsequently we investigated whether the E5 oncogene can be tolerated in these cells. Despite the high hydrophobic structure of the E5 protein would suggest a rather toxic effect, the expression of this viral oncogene had almost no effect on cell morphology (data not shown), cell proliferation and cell viability, while a clear increase of the cell specific metabolic activity (more evident in FRM than in M14) was seen in E5 expressing cells (Fig. 2). These characteristics were rather stable being observed in both cell lines as far as the HPV 16 E5 oncogene was retained (at least 4–6 passages in vitro). Taken together these data indicate that the isolated HPV 16 E5 oncogene can be expressed in amelanotic melanomas and that its expression, devoid of any immediate gross cell toxicity, induces the fine modulation of selective cell activities.


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)

Presence of HPV-16 E5 DNA and expression of the specific mRNA in M14 and FRM cells after infection with HPV-16 E5 retroviral vector. The retroviral vector containing HPV-16 E5 gene was obtained by the transfection of Phoenix A retroviral producer cells with the LZRSpBMNZ-E5 plasmid. The control retroviral vector was obtained by the transfection of Phoenix cells with the empty LZRSpBMNZ plasmid. Cells were infected with either recombinant retrovirus or with the control retrovirus. Total DNA or RNA (1 μg) extracted from cells 96 h post infection were reverse transcribed and amplified with E5P65 sense (TGC ATC CAC AAC ATT ACT GGC G) and E5M3AS antisense (AAC ACC TAA ACG CAG AGG CTG C) primers. Upper panel: FRM cells; Lower panel: M14 cells. Lane 1: DNA from cells infected with the control retrovirus; Lane 2: DNA from cells infected with the HPV-16 E5 retrovirus; Lane 3: DNA digested total RNA from cells infected with the HPV-16 E5 retrovirus; Lane 4: Non retrotrascribed DNA digested total RNA from cells infected with the HPV-16 E5 retrovirus; Lane 5: No template negative control; Lane 6 positive control (0.5 μg Siha cell DNA). MW: DNA molecular weight marker VIII (Roche Biochemicals SpA): arrows on the left-hand side indicate the bp length of some reference bands. The band with size of 160 bp (left sided empty arrow) demonstrate the presence of viral E5 sequence and its transcription. Four independent experiments gave similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Presence of HPV-16 E5 DNA and expression of the specific mRNA in M14 and FRM cells after infection with HPV-16 E5 retroviral vector. The retroviral vector containing HPV-16 E5 gene was obtained by the transfection of Phoenix A retroviral producer cells with the LZRSpBMNZ-E5 plasmid. The control retroviral vector was obtained by the transfection of Phoenix cells with the empty LZRSpBMNZ plasmid. Cells were infected with either recombinant retrovirus or with the control retrovirus. Total DNA or RNA (1 μg) extracted from cells 96 h post infection were reverse transcribed and amplified with E5P65 sense (TGC ATC CAC AAC ATT ACT GGC G) and E5M3AS antisense (AAC ACC TAA ACG CAG AGG CTG C) primers. Upper panel: FRM cells; Lower panel: M14 cells. Lane 1: DNA from cells infected with the control retrovirus; Lane 2: DNA from cells infected with the HPV-16 E5 retrovirus; Lane 3: DNA digested total RNA from cells infected with the HPV-16 E5 retrovirus; Lane 4: Non retrotrascribed DNA digested total RNA from cells infected with the HPV-16 E5 retrovirus; Lane 5: No template negative control; Lane 6 positive control (0.5 μg Siha cell DNA). MW: DNA molecular weight marker VIII (Roche Biochemicals SpA): arrows on the left-hand side indicate the bp length of some reference bands. The band with size of 160 bp (left sided empty arrow) demonstrate the presence of viral E5 sequence and its transcription. Four independent experiments gave similar results.
Mentions: HPV 16 E5 is a small hydrophobic molecule expressed at very low levels in keratinocytes at early stages during viral infection and appearing to be critically linked to viral pathogenic potentials. Two amelanotic melanoma cell lines, FRM and M14, were infected with a HPV 16 E5 expressing retroviral vector and compared with the same lines infected with an "empty" retrovirus. After the infection with the E5 retroviral construct, the presence of cDNA for the E5 oncogene, as well as the corresponding mRNA, was shown by PCR and RT-PCR both in M14 and FRM cells (Fig. 1). Subsequently we investigated whether the E5 oncogene can be tolerated in these cells. Despite the high hydrophobic structure of the E5 protein would suggest a rather toxic effect, the expression of this viral oncogene had almost no effect on cell morphology (data not shown), cell proliferation and cell viability, while a clear increase of the cell specific metabolic activity (more evident in FRM than in M14) was seen in E5 expressing cells (Fig. 2). These characteristics were rather stable being observed in both cell lines as far as the HPV 16 E5 oncogene was retained (at least 4–6 passages in vitro). Taken together these data indicate that the isolated HPV 16 E5 oncogene can be expressed in amelanotic melanomas and that its expression, devoid of any immediate gross cell toxicity, induces the fine modulation of selective cell activities.

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