Nuclear mRNA retention and aberrant doppel protein expression in human astrocytic tumor cells.
Doppel (Dpl) is a paralogue of the mammalian Prion (PrP) protein. It is abundant in testis and, unlike PrP, it is expressed at low levels in the adult central nervous system (CNS). Besides, Dpl overexpression correlates with some prion-disease pathological features, such as ataxia and death of cerebellar neurons. Recently, ectopic expression of doppel was found in two different tumor types, specifically in glial and haematological cancers. In this study the doppel gene (PRND) mRNA and protein expression in PRT-HU2 and IPDDC-A2 astrocytoma-derived cell lines was investigated. Northern blot analysis revealed two equally abundant PRND mRNA isoforms, while real-time PCR, on nuclear and cytoplasmic RNA fractions, and cRNA in situ hybridization, on astrocytoma cells and bioptical specimens, showed a nuclear retention of PRND transcripts. Western blot analysis showed that the amount of protein expressed is low compared to the level of mRNA. Moreover deglycosylation studies indicated that Dpl undergoes unusual glycosylation processes. Immunohistochemistry experiments demonstrated that Dpl was mainly localised in the cytoplasm of the astrocytic tumor cells, and that it failed to be GPI-anchored to the cell membrane. This unusual cellular localization was also confirmed through EGFP-Dpl expression in astrocytomas; on the contrary, HeLa cells exhibited the expected Dpl membrane localization. Our findings suggest an aberrant doppel gene expression pattern, characterized by a substantial nuclear retention of the transcript, an altered post-translational modification of the protein and an unusual cytoplasmic localization.
Comincini
Sergio
S
Dipartimento di Genetica e Microbiologia, Università di Pavia, 27100 Pavia, Italy. sergio.c@ipvgen.unipv.it
Chiarelli
Laurent R
LR
Zelini
Paola
P
Del Vecchio
Igor
I
Azzalin
Alberto
A
Arias
Agustina
A
Ferrara
Valentina
V
Rognoni
Paola
P
Dipoto
Antonella
A
Nano
Rosanna
R
Valentini
Giovanna
G
Ferretti
Luca
L
D001254:Astrocytoma / Q000235:genetics / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D058851:GPI-Linked Proteins; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D011328:Prions / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
D058851:GPI-Linked Proteins; C501125:PRND protein, human; D011328:Prions; D012333:RNA, Messenger