Panoply™ Human DMPK Over-expressing Stable Cell Line

The tumor suppressor p53 plays a crucial role in DNA damage-induced apoptosis, a biological process that inhibits tumor progression. During apoptosis, significant changes in cell morphology occur, closely related to actin-myosin contraction; however, how p53 regulates DNA damage-induced actin-myosin contraction remains completely unknown. To identify a novel p53-regulated gene encoding a myosin regulator, researchers performed DNA microarray analysis. They found that under the influence of the DNA damage agent doxorubicin, the expression of myotonic dystrophy protein kinase (DMPK), known to upregulate actin-myosin contraction, increased in a p53-dependent manner. The promoter region of the DMPK gene contains a potential p53-binding sequence, and overexpression of the p53 family protein p73 enhances its promoter activity; however, unexpectedly, overexpression of p53 did not have this effect. Furthermore, researchers found that doxorubicin treatment induced p73 expression, while p53 downregulation significantly attenuated p73 expression. These data indicate that p53 induces DMPK expression by upregulating p73 expression. DMPK overexpression promotes actin cortex contraction, leading to vesicle formation, loss of cell adhesion, and associated caspase activation. In summary, these results suggest the existence of a p53-p73-DMPK axis that mediates DNA damage-induced cortical actin contraction and associated cell death.

Here, researchers investigated the consequences of increased DMPK expression in MCF-7 cells. They examined the effects of overexpression of DMPK E containing the VSGGG motif on actin organization and myosin activity in MCF-7 cells. F-actin staining showed that overexpression of wild-type (WT) DMPK led to cortical actin disruption and the formation of vesicular protrusions on the plasma membrane (Figure 1a-c). Furthermore, it induced cell rounding and cell detachment from the extracellular matrix. Compared to non-overexpressing cells, DMPK-overexpressing cells showed higher levels of MLC2 phosphorylation in the cortex; MLC2 is a key step in the activation of non-muscle myosin (Figure 1a). The researchers then examined whether DMPK kinase activity was crucial to these effects. Unlike DMPK WT, overexpression of its kinase-inactivating mutant (MT) did not induce either vesicular protrusion formation or cell rounding. Myosin activity, assessed by MLC2 phosphorylation levels, was comparable between DMPK MT-overexpressing and non-overexpressing cells (Figure 1a). Furthermore, consistent with the effect of DMPK WT overexpression (rather than DMPK MT overexpression) on cell morphology, the accumulation of caspase biosensors in the nucleus was significantly higher in DMPK WT-overexpressing cells than in DMPK MT-overexpressing cells (Figure 1d, e). The researchers then examined whether DMPK induced cell rounding under caspase inhibition conditions. Thirty hours after transfection, DMPK WT-overexpressing cells were treated with the pan-caspase inhibitor Q-VD-OPh, and the results showed that cell rounding was unaffected (Figure 1f), indicating that caspase activity is not essential for DMPK-dependent cell rounding.

Figure 1. Overexpression of DMPK induces caspase activation in a kinase activity dependent manner. (Itoh K, et al., 2019)