Abstract
We have demonstrated previously that the Myc oncoprotein blocks cancer cell differentiation by forming a novel transcriptional repressor complex with histone deacetylase and inhibiting gene transcription of tissue transglutaminase (TG2). Moreover, induction of TG2 gene transcription and transamidase activity is essential for the differentiating effects of retinoids in cancer cells. Here, we show that two structurally distinct TG2 protein isoforms, the full-length (TG2-L) and the short form (TG2-S), exert opposing effects on cell differentiation. Repression of TG2-L with small interfering RNA, which did not affect TG2-S expression, induced dramatic neuritic differentiation in neuroblastoma cells. In contrast, overexpression of TG2-S or a GTP-binding-deficient mutant of TG2-L (R580A), both of which lack the GTP-binding Arg-580 residue, induced neuroblastoma cell differentiation, which was blocked by an inhibitor of transamidase activity. Whereas N-Myc repressed and retinoid activated both TG2 isoforms, repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhanced neuroblastoma cell differentiation due to N-Myc small interfering RNA or retinoid. Moreover, suppression of vasoactive intestinal peptide (VIP) expression alone induced neuroblastoma cell differentiation, and VIP was up-regulated by TG2-L, but not TG2-S. Taken together, our data indicate that TG2-L and TG2-S exert opposite effects on cell differentiation due to differences in GTP binding and modulation of VIP gene transcription. Our findings highlight the potential importance of repressing the GTP binding activity of TG2-L or activating the transamidase activity of TG2-L or TG2-S for the treatment of neuroblastoma, and possibly also other Myc-induced malignancies, and for enhancing retinoid anticancer effects.
Highlights
Council of Australia Grants 568753 and 459406
TG2-L and TG2-S Gene Expression Is Repressed by N-Myc Oncogene and Reactivated by Retinoid Differentiation Therapy— Through RT-PCR analysis using primers that target TG2 exons 4 and 6, which are shared by TG2-L and TG2-S, we have demonstrated previously that N-Myc oncoprotein represses expression of this mRNA sequence, which we called TG2-Total (TG2-T, TG2-L plus TG2-S) [9]
The induction of TG2 gene transcription and transamidase activity is essential for tumor cell differentiation due to N-Myc oncogene repression [9] or retinoid differentiation therapy (6 – 8) in neuroblastoma and other malignant cells
Summary
Immunoblot analysis roblastoma cell differentiation block, we reviewed the showed that transfection of the siRNA-resistant TG2-L expres- published cDNA microarray data that compared gene expression construct resulted in overexpression of TG2-L protein, sion in neuroblastoma SY-5Y cells stably transfected with a even when co-transfected with TG2-L siRNA (Fig. 2D). RT-PCR and immunoblot analyses apy induced gene expression of both TG2-L and TG2-S in neushowed that in BE[2]-C cells, the expression of VIP was reduced roblastoma cells (Fig. 1, B and C), we tested whether by Ͼ80% after repression of TG2-L expression by TG2-L transcriptional activation of TG2-L rendered neuroblastoma siRNA, but not after simultaneous repression of TG2-L and cells resistant to the anticancer efficacy of retinoid.
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