Altered expression of the poly(ADP-ribosyl)ation enzymes in uveal melanoma and regulation of PARG gene expression by the transcription factor ERM.
OBJECTIVE
Poly(ADP-ribosyl)ation is a reversible post-translational modification that requires the contribution of the enzymes poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Our study explores expression and activity of PARP-1 and PARG in uveal melanoma cell lines with varying tumorigenic properties.


METHODS
Gene profiling on microarrays was conducted using RNA prepared from the uveal melanoma cell lines T97, T98, T108, and T115. The activity of PARP-1 and PARG was monitored by enzymatic assays, whereas their expression was measured by Western blot and PCR. The PARG promoter was analyzed using promoter deletions and site-specific mutagenesis in transfection analyses. The transcription factors binding the PARG promoter were studied by electrophoretic mobility shift assay (EMSA) analyses. Suppression of PARP-1 and PARG expression was performed in T97 and T115 cells by RNAi, and their tumorigenic properties monitored by injections into athymic mice.


RESULTS
Expression of PARP-1 was found to vary considerably between uveal melanoma cell lines with distinctive tumorigenic properties in vivo. Sp1 and the ETS protein ERM were shown to bind to the PARG gene promoter to ensure basal transcription in uveal melanoma. Importantly, suppression of PARG gene expression in T97 and T115 cells increased their capacity to form tumors in athymic mice, whereas suppression of PARP-1 significantly reduced or almost entirely abolished tumor formation.


CONCLUSIONS
Our results suggest that while overexpression of PARP-1 may confer a proliferative advantage to aggressive uveal melanoma tumors, PARG may, on the other hand, support a tumor suppressor function in vivo.
LOEX/CUO-Recherche, Hôpital du Saint-Sacrement, Centre de recherche du CHA, Québec, Canada.
Vanessa
V
Molloy-Simard
Jean-François
JF
St-Laurent
François
F
Vigneault
Manon
M
Gaudreault
Natasha
N
Dargis
Marie-Christine
MC
Guérin
Steeve
S
Leclerc
Mohib
M
Morcos
Daniel
D
Black
Yvonne
Y
Molgat
Dan
D
Bergeron
Yvan
Y
de Launoit
François
F
Boudreau
Serge
S
Desnoyers
Sylvain
S
Guérin
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000502:physiology*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D008545:Melanoma / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D046228:Microarray Analysis; D020411:Oligonucleotide Array Sequence Analysis; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014157:Transcription Factors / Q000502:physiology*; D014604:Uveal Neoplasms / Q000235:genetics / Q000378:metabolism*; D000098943:Uveal Melanoma
D004268:DNA-Binding Proteins; C513242:Etv5 protein, mouse; D020690:GTPase-Activating Proteins; D014157:Transcription Factors; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases