Nuclear mRNA retention and aberrant doppel protein expression in human astrocytic tumor cells

Abstract

Abstract. 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. IntroductionIn 1999, the first prion-gene paralogue, doppel (PRND), was described in rodents (1,2). Subsequently, the doppel gene was confirmed in humans (3), cattle, sheep (4), and goats (5). Comparative gene expression analysis was reported on different patterns of temporal and spatial expression among prion and doppel. Thus, whereas prion protein (PrP) is widely expressed, showing the highest expression profiles within the CNS (6), doppel shows barely detectable levels in most tissue (4,7). Doppel protein (Dpl) was found highly expressed only in adult and fetal testis (8), and different groups recently proposed an involvement in male gametogenesis (9-11). Based on the structural similarities between PrP (12) and Dpl (13), a role of doppel in the development of prion neurodegenerative diseases was hypothesised (14). Specifically, when ectopically expressed in some Prnp 0/0 transgenic lines, Dpl causes Purkinje cell death and ataxia (reviewed in ref. 15). However, Dpl-induced neurodegeneration can be rescued by the introduction of a Prnp transgene (16), suggesting the possibility that the protein interacts or competes with PrP in a sort of molecular antagonism-model (17). Further studies were performed to demonstrate a direct involvement of doppel in prion-diseases in humans (7) as well as in animals (18,19). From these analyses, doppel seems not to be associated with the diseases, neither if one ...

Biochemical Signatures of Doppel Protein in Human Astrocytomas to Support Prediction in Tumor Malignancy

“…As expected [9, 10, 12], in the cortex section of peritumoral area, Dpl expression was faint compared to the neoplastic tissue (Figure 4(c)). …”

PRND (Prion Protein 2 (Dublet))

“…altered glycosilation pattern) different from Doppel as normally expressed in testis (Azzalin et al, 2006;Azzalin et al, 2008;Comincini et al, 2006;Comincini et al, 2004;Comincini et al, 2007;Rognoni et al, 2010;Sbalchiero et al, 2008).…”

Emerging Role of Cellular Prion Protein in the Maintenance and Expansion of Glioma Stem Cells

“…Intriguing differences between PrP C and Dpl concern also their intracellular localization in GBM cells. While PrP C displayed a typical expression around Golgi and at plasma membrane, Dpl showed mostly cytoplasmic distribution indicating retention into the endosomal-lysosomal system [201,202]. Such unconventional topology of Dpl is more evident in GBM than in low grade astrocytomas, in which most Dpl is tethered at plasma membrane as in normal adult testis [203].…”