Tumor suppressor tropomyosin Tpm2.1 regulates sensitivity to apoptosis beyond anoikis characterized by changes in the levels of intrinsic apoptosis proteins

Abstract

The actin cytoskeleton is a polymer system that acts both as a sensor and mediator of apoptosis. Tropomyosins (Tpm) are a family of actin binding proteins that form co-polymers with actin and diversify actin filament function. Previous studies have shown that elevated expression of the tropomyosin isoform Tpm2.1 sensitized cells to apoptosis induced by cell detachment (anoikis) via an unknown mechanism. It is not yet known whether Tpm2.1 or other tropomyosin isoforms regulate sensitivity to apoptosis beyond anoikis. In this study, rat neuroepithelial cells overexpressing specific tropomyosin isoforms (Tpm1.7, Tpm2.1, Tpm3.1, and Tpm4.2) were screened for sensitivity to different classes of apoptotic stimuli, including both cytoskeletal and non-cytoskeletal targeting compounds. Results showed that elevated expression of tropomyosins in general inhibited apoptosis sensitivity to different stimuli. However, Tpm2.1 overexpression consistently enhanced sensitivity to anoikis as well as apoptosis induced by the actin targeting drug jasplakinolide (JASP). In contrast the cancer-associated isoform Tpm3.1 inhibited the induction of apoptosis by a range of agents. Treatment of Tpm2.1 overexpressing cells with JASP was accompanied by enhanced sensitivity to mitochondrial depolarization, a hallmark of intrinsic apoptosis. Moreover, Tpm2.1 overexpressing cells showed elevated levels of the apoptosis proteins Bak (proapoptotic), Mcl-1 (prosurvival), Bcl-2 (prosurvival) and phosphorylated p53 (Ser392). Finally, JASP treatment of Tpm2.1 cells caused significantly reduced Mcl-1, Bcl-2 and p53 (Ser392) levels relative to control cells. We therefore propose that Tpm2.1 regulates sensitivity to apoptosis beyond the scope of anoikis by modulating the expression of key intrinsic apoptosis proteins which primes the cell for death.

Co-polymers of Actin and Tropomyosin Account for a Major Fraction of the Human Actin Cytoskeleton

“…Similar to the BJeLR cells, U2OS, MCF-10A, and MCF-7 cells also expressed relatively low levels of HMW tropomyosin isoforms, and Tpm3.1/3.2 also made up the majority of the tropomyosin in these cells (Table 1). This result may reflect the requirement in transformed cells for Tpm3.1/3.2 filaments in MAPK/ERK signaling [27], glucose transport [13], and the inhibition of apoptosis [28]. This result was expected for the U2OS and MCF-7 cells as these cell lines were known to be transformed, but not for the MCF-10A cell line that is typically used as a model for normal human mammary epithelial cells.…”

Tropomyosin isoforms have specific effects on the transcriptome of undifferentiated and differentiated B35 neuroblastoma cells

“…Within the protein C‐terminal‐binding protein pathway are genes, associated with apoptosis. Tpm2.1 is known to have tumor‐suppressing properties in breast and urinary bladder cancers and has recently been shown to increase cell sensitivity to apoptosis by detachment from the extracellular matrix, referred to as anoikis, and through the modulation of various apoptosis‐inducing proteins .…”

“…B35 rat neuroblastoma cells, stably overexpression of different Tpm isoforms, were previously described for the overexpression of Tpm1.12 and Tpm3.1 , Tpm2.1 , and Tpm4.2 . Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Life Technologies, Melbourne, Vic., Australia), 0.6% geneticin (Invitrogen, Life Technologies), and 10% heat‐inactivated fetal bovine serum (FBS; Invitrogen, Life Technologies) at 37 °C in a 5% CO 2 incubator.…”

“…In this study, we aimed to test whether the level of expression of Tpms determines the cell transcriptome in an isoform‐specific manner and whether these changes are dependent on the differentiation stage of these cells. Rat B35 neuroblastoma cells have extensively been used to study cellular processes of eukaryotic cells, including neuronal morphogenesis , cell motility , vesicular trafficking , and apoptosis . Our group has used the B35 cell system previously to study the role of different Tpm isoforms in neuronal cell morphogenesis .…”

“…Our group has used the B35 cell system previously to study the role of different Tpm isoforms in neuronal cell morphogenesis . Stable rat B35 neuroblastoma cell lines overexpressing the Tpm isoforms Tpm1.12, Tpm2.1, Tpm3.1, and Tpm4.2, from each of the Tpm genes 1‐4, were previously generated . Neuronal differentiation requires the coordinated reorganization of the actin cytoskeleton to facilitate the sprouting and elongation of neurites, which ultimately form axons and dendrites.…”