HAMLET: Functional Properties and Therapeutic Potential

James Ho CS; Anna Rydström; Maria Trulsson; Johannes Bålfors; Petter Storm; Manoj Puthia; Aftab Nadeem; Catharina Svanborg

Disclosures

Future Oncol. 2012;8(10):1301-1313. 

In This Article

Cell Detachment Induced by HAMLET

Adherent tumor cells detach in response to HAMLET in vitro (Figure 3) and, in patients with bladder cancer, local HAMLET instillations have been shown to trigger rapid tumor cell detachment in vivo.[15,60] To identify molecules involved in cell detachment, cellular extracts were screened for HAMLET targets. One such screen identified α-actinin-1 and -4 as binding partners for HAMLET.[60] α-actinins are the major F-actin-binding and crosslinking proteins in human cells. Two α-actinin molecules form a functional antiparallel homodimer[61] and the cellular localization of α-actinin differs depending on the cell type.[62] α-actinin-1 has been shown to localize at focal adhesion plaques or adherence junctions and α-actinin-4 at points of cell–cell contact. However, α-actinin-4 has also been shown to interact with focal adhesion constituents, including vinculin and the cytoplasmic domain of β-integrins.[63–65]

Figure 3.

Cellular response to HAMLET visualized by holographic live-cell imaging.
(A) Kinetics of the morphological change in lung carcinoma cells exposed to HAMLET (35 µM). Cells start to round up after approximately 30 min, and, at 60 min, a reduction in cell number is observed. (B) Enlargement of a single cell.

The α-actinin-4 domains interacting with HAMLET were identified using a synthetic peptide library mapped onto a cryoelectron microscopy reconstruction representation of smooth muscle α-actinin.[60] Four peptides facing the cavity between the actin-binding domain and the central rod domain were identified, potentially forming a combined binding site for HAMLET. In addition, a potential β-integrin-binding site was identified and HAMLET bound to two peptides in the α-actinin-C-terminal. HAMLET triggered a rapid disruption of focal adhesion complexes and cytoskeletal structure and focal adhesion kinase phosphorylation and signaling. The effect was only observed in carcinoma cells, as normal differentiated cells retained their morphology in the presence of HAMLET.

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