ROCK inhibitor reduces Myc-induced apoptosis and mediates immortalization of human keratinocytes

Abstract

The Myc/Max/Mad network plays a critical role in cell proliferation, differentiation and apoptosis and c-Myc is overexpressed in many cancers, including HPV-positive cervical cancer cell lines. Despite the tolerance of cervical cancer keratinocytes to high Myc expression, we found that the solitary transduction of the Myc gene into primary cervical and foreskin keratinocytes induced rapid cell death. These findings suggested that the anti-apoptotic activity of E7 in cervical cancer cells might be responsible for negating the apoptotic activity of over-expressed Myc. Indeed, our earlier in vitro studies demonstrated that Myc and E7 synergize in the immortalization of keratinocytes. Since we previously postulated that E7 and the ROCK inhibitor, Y-27632, were members of the same functional pathway in cell immortalization, we tested whether Y-27632 would inhibit apoptosis induced by the over-expression of Myc. Our findings indicate that Y-27632 rapidly inhibited Myc-induced membrane blebbing and cellular apoptosis and, more generally, functioned as an inhibitor of extrinsic and intrinsic pathways of cell death. Most important, Y-27632 cooperated with Myc to immortalize keratinocytes efficiently, indicating that apoptosis is a major barrier to Myc-induced immortalization of keratinocytes. The anti-apoptotic activity of Y-27632 correlated with a reduction in p53 serine 15 phosphorylation and the consequent reduction in the expression of downstream target genes p21 and DAPK1, two genes involved in the induction of cell death.

Establishment of a nasopharyngeal carcinoma cell line capable of undergoing lytic Epstein–Barr virus reactivation

“…Viral replication may cause DNA damage in host cells and trigger apoptosis [24,27,34], which may be an explanation for the rapid loss of episomal viral genomes from cultured epithelial cells. ROCK inhibitor reverses blebbing of keratinocytes and inhibits apoptotic pathways [35]. This might be another reason why inclusion of ROCK inhibitor leads to establishment of EBVcontaining NPC cell lines.…”

Rho‐kinase inhibitor Y‐27632 facilitates the proliferation, migration and pluripotency of human periodontal ligament stem cells

“…In our study, 10 and 20 μM Y‐27632 promoted the proliferation and migration of PDLSCs while did not increase apoptosis at the same time. Previous studies demonstrated that Y‐27632 had anti‐apoptotic effect and inhibited the apoptosis of human keratinocytes , human corneal endothelial cells and human embryonic stem cells . The inconsistency with our study may be attributed to the effect of Y‐27632 on apoptosis varying amongst different cell types.…”

DlgR2 knockdown boosts dendritic cell activity and inhibits hepatocellular carcinoma tumor in-situ growth

“…The reverse transcription was performed using the SYBR Green kit (Applied Biosystems) [ 37 – 39 ]. The ABI-7600 Fast Real-Time PCR system was utilized to perform the quantitative real time-PCR (qRT-PCR) assay [ 40 , 41 ]. For each analysis, melt curve analysis was performed to calculate the melting temperature of the product.…”

“…GAPDH ( glyceraldehyde-3-phosphatedehydrogenase) mRNA was always tested as the reference gene. The 2 −ΔΔCt method was applied to quantify targeted mRNA change [ 40 , 41 ]. DIgR2 mRNA and GAPDH mRNA primers were described previously [ 12 , 40 , 41 ].…”