Intracellular distribution of the ΔNp73 protein isoform in medulloblastoma cells: a study with newly generated rabbit polyclonal antibodies
Renata Veselska1,2, Jakub Neradil1,3, Marta Nekulova3, Lucia Dobrucka1,2, Borivoj Vojtesek3, Jaroslav Sterba2,3 and Karel Zitterbart1,2
1Department of Experimental Biology, School of Science, Masaryk University, Brno, Czech Republic, 2Department of Pediatric Oncology, University Hospital Brno and School of Medicine, Masaryk University, Brno, Czech Republic and 3Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic.
Offprint requests to: Renata Veselska, Laboratory of Tumor Biology, Department of Experimental Biology, School of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic. e-mail: firstname.lastname@example.org
Summary. The p73 protein is a member of the p53 family of transcription factors that has two N-terminal isoforms: the TAp73 isoform is reported to have a tumor suppressor function, whereas the ΔNp73 isoform likely has oncogenic potential. The expression of these isoforms and the differences in their intracellular distribution have been described in many cancer types; however, little is known about the p73 isoforms in brain tumors. Our study is focused on the intracellular localization of ΔNp73 in medulloblastoma cell lines. Due to a lack of suitable anti-ΔNp73 antibodies, we developed two new rabbit polyclonal antibodies, ΔNp73-26 and ΔNp73-27, with sufficient specificity, as demonstrated by immunodetection methods using transiently transfected cell lines. Both of these new antibodies were subsequently used for analysis of the ΔNp73 distribution in medulloblastoma cells using immunofluorescence, immunoblotting and immunogold labeling for transmission electron microscopy. We found a nuclear localization of the ΔNp73 isoform in all of the medulloblastoma cell lines included in this study. Furthermore, a non-random accumulation of the ΔNp73 isoform near the cell nuclei was observable in all of these cell lines. By double-labeling with ΔNp73 and golgin-97, we showed the co-localization of the ΔNp73 isoform with the Golgi apparatus. Nevertheless, further detailed analyses of possible interactions of ΔNp73 with the proteins accumulated in the Golgi apparatus should be performed to explain the dynamics of ΔNp73 outside the cell nucleus. Histol Histopathol 28, 913-924 (2013)
Key words: Medulloblastoma, DeltaNp73, Immuno-cytochemistry, Immunoblotting, Transmission electron microscopy