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.