Cloning and characterization of a novel alpha-actinin gene from human neuroblastoma
Neuroblastoma is the most common extra-cranial malignant solid tumor in children. Established cell lines comprise three predominant cell types: a neuroblastic cell (N) with properties of an embryonic sympathoblast, a substrate-adherent cell (S) with properties of a Schwann/glial/melanocytic precursor cell, and a stem cell (I) with properties of both N and S cells. During studies of MYCN protooncogene expression in neuroblastoma cell variants, we identified a 98 kDa protein which cross-reacted with one anti-MYCN monoclonal antibody. This p98 protein was abundant in the S-type cells and weakly or not expressed in N- and I-type counterparts. Isolation, cloning and sequencing of the cDNA for this protein revealed that p98 is a novel $\alpha$-actinin protein. Molecular characterization studies have shown that the cDNA for the newly identified $\alpha$-actinin, which we named ACTN4, is structurally similar to all known $\alpha$-actinin genes. ACTN4 has a 71-83% amino acid identity to the previously characterized human nonmuscle gene (ACTN1) and the two skeletal muscle $\alpha$-actinin (ACTN2 and ACTN3) isoforms. Analysis of the amino acid composition of the EF-hand motifs of our $\alpha$-actinin isoform suggest that ACTN4 might have a lower calcium sensitivity to calcium than the other nonmuscle form. Northern blot analysis using a panel of human tumor cell lines showed that this new gene (ACTN4) is expressed in malignant cells of diverse differentiation lineages and/or tissue origins and that its expression generally is correlated with substrate adhesiveness. The ACTN4 has been localized to chromosome #4 with somatic cell hybrid panels. Transfection experiments explored the effect of expression of ACTN4 on differentiation and transformation state. In general, stable transfectants with 10- to 40-fold higher levels of $\alpha$-actinin protein exhibited a more S-like morphology and loss of neuronal-specific proteins. They also showed a significantly lower (than the controls) colony-forming efficiency in soft agar, the extent of which was inversely correlated with $\alpha$-actinin protein levels. These results suggest that this new member of the $\alpha$-actinin gene family may be influential in the regulation of differentiation and transformation in human neuroblastoma variants.
Nikolopoulos, Sotiris Nick, "Cloning and characterization of a novel alpha-actinin gene from human neuroblastoma" (1997). ETD Collection for Fordham University. AAI9719654.