Tumor Marker
Doppel possesses similar exon–intron architecture to that of the prion gene, as shown in Fig. 2.1a;
additionally, the two genes share a chromosomal synteny in different mammalian species (Comincini et al., 2006a). As previously stated, these evidences reinforced the concept that an ancestral gene duplica- tion event originated the prion-family gene chromoso- mal locus, designated Prn. Similarly to the prion gene,
the doppel coding region is contained within a sin- gle exon. However, the two genes exhibited marked different expression patterns, with prion gene being mostly expressed within the CNS tissues, while dop- pel is uniquely expressed in the adult testis tissue but, notably, at very low levels in nervous system.
Therefore, while prion gene expression is relevantly related to the prion disease onset and epidemiology, doppel related studies were targeted to male gameto- genesis and fertilization processes.
In addition to neurodegenerative and reproduc- tive investigations, a novel doppel-related branch of research was proposed (Comincini et al., 2004). The rationale was to investigate doppel expression per- turbation within CNS-related diseases such as brain cancers, where it is expected to be physiologically not expressed. These studies led to the identification and characterization of a novel expression marker within the human glial tumors and to the association with the malignant grading progression. Glial tumors are histo-pathologically divided into four grades of malig- nancy, according to the WHO classification (Louis et al.,2007): pilocytic (WHO I), low-grade (WHO II), anaplastic astrocytomas (WHO III) and glioblastoma multiforme (WHO IV). In contrast to the long- standing and well defined histo-pathological criteria, the underlying molecular and genetic basis emerged only recently. In particular, several genes and pathways have been identified as being associated with tumorige- nesis and with the anaplastic progression (Comincini, 2001). Adopting a high sensitive Real-time PCR based approach, doppel gene expression was investigated in large cohorts of patients with glial tumors. As a result, doppel expression was directly related to the malignancy of the tumor: highest in glioblastoma mul- tiforme, lower in anaplastic astrocytomas, and even lower in low grade astrocytoma specimens, as graph- ically reported in Fig.2.2(Comincini et al.,2004).
Extensive differences in doppel gene expression were also found within each grade of malignancy, sug- gesting that the quantification of doppel expression might be useful to distinguish astrocytoma subtypes.
In addition, it was demonstrated that its expression is helpful in disease stratification and in the iden- tification of patient subsets with specific molecular signatures (Comincini et al., 2007). Further molecu- lar doppel gene expression investigations were then performed within human glial tumor specimens and in derived cell lines. These studies revealed that the
upregulated doppel transcripts underwent a significant nuclear retention process within the glial tumor cells, as shown in the image of Fig. 2.2(Comincini et al., 2006b): this alternative post-transcriptional pathway might directly regulate the excess of potentially dele- terious transcripts. In addition, this aberrant nuclear retention may have a functional meaning, as other genes with this trademark were described. In fact, nuclear mRNA retention is increasingly recognized as an important mechanism to regulate the activity of transcription related proteins and to modulate cell growth and death. For this reason, the export of nuclear mRNA is constantly challenged by the opposing force of mRNA retention from one side, and its decay from the other. This balance ensures that only perfect tran- scripts persist and that nonfunctional and potentially deleterious transcripts are differently regulated in their biogenesis. In detail, doppel human mRNA underwent alternative maturation processes within glial tumor cells, thus originating an alternative shorter transcript of 1.9 kb, significantly different from that originated in testis tissue (Comincini et al., 2006b). Altogether, these data might suggest that glial tumor cells overpro- duce doppel transcripts in primis, and that these tran- scripts are then subjected to an initial quality/quantity control through a nuclear retention process.
In a similar manner, the expression pattern and the distribution of doppel were investigated in tumors with a non-glial origin and, in a former study, high levels of doppel transcripts were detected in gastric adeno- carcinoma and in anaplastic meningioma specimens (Comincini et al.,2004). Additionally, Travaglino et al.
(2005) investigated doppel expression in bone marrow- derived cells of patients with acute myeloid leukaemia (AML) and with myelo-dysplastic syndrome (MDS).
As a result, while doppel transcripts were barely detectable in normal bone marrow samples, AML and MDS cases exhibited a marked increase in dop- pel expression, particularly localized in blast-like cells with a significant phenomenon of nuclear retention of its transcripts. As a consequence of the ectopi- cally expression of doppel gene in different tumor biopsies, one may hypothesize that the corresponding gene product belongs to the group of the cancer-testis antigens (CTA), that recently captured considerable interest (Zendman et al., 2003). In fact, along with its physiological expression restricted to germ cells of the testis that exclusively reappears in neoplastic- transformed cells, the doppel gene reflects other typical
Fig. 2.2 Doppel molecular and cellular signatures in human astrocytoma. Doppel molecular and cellular signatures in human astrocytoma. As described, doppel gene and protein expres- sion levels increase following the glioma malignancy grading;
a significant nuclear retention of the doppel transcripts has been previously reported (inset a), as well as an increase of
the complexity in the glycan moiety composition (Comincini et al.,2006b). Additionally, as illustrated (inset b), the cellular localization of the doppel protein shifts from plasma mem- brane to the cytosol, particularly within lysosome organelles (Sbalchiero et al.,2008)
features that characterise CTA, such as its belonging to a gene family and the single-exon ORF gene structure.
To date, the molecular mechanisms responsi- ble for doppel over-expression in transformed cells remain summarily defined. Doppel altered expres- sion could merely be an epiphenomenon because of the widespread change in gene methylation patterns observed within different tumor types (Travaglino et al., 2005). To better delve into doppel gene regu- latory mechanisms, investigations in different species were performed; in general, these experimental and bioinformatics computational analysis supported the concept that doppel gene expression is tightly regu- lated through an interplay of positive and negative cis- acting factors that specifically recognize activating and
inhibiting elements in the promoter and in its surround- ing sequence (Del Vecchio et al.,2005). Furthermore, doppel expression is affected by the methylation sta- tus of its promoter sequence, differently from the prion paralogue gene (Comincini, unpublished data).
It is therefore conceivable that doppel is positively regulated in testis and negatively regulated in CNS.
Interestingly, as a conclusive remark, the high expres- sion profiles of doppel gene, physiologically in the first stage of the brain development and ectopically in glial tumor specimens, may indicate the neoplastic reacquisition of tumor cells of a primitive expression behaviour (Comincini et al.,2004).