Silencing of protein kinase D2 induces glioma cell senescence via p53-dependent and -independent pathways

Abstract

BackgroundGlioblastoma multiforme (GBM) is a highly aggressive tumor of the central nervous system with a dismal prognosis for affected patients. Aberrant protein kinase C (PKC) signaling has been implicated in gliomagenesis, and a member of the PKC-activated protein kinase D (PRKD) family, PRKD2, was identified as mediator of GBM growth in vitro and in vivo.MethodsThe outcome of PRKD2 silencing and pharmacological inhibition on glioma cell proliferation was established with different glioma cell lines. Western blotting, senescence assays, co-immunoprecipitation, fluorescence activated cell sorting, quantitative PCR, and immunofluorescence microscopy were utilized to analyze downstream signaling.ResultsRNA-interference (21-mer siRNA) and pharmacological inhibition (CRT0066101) of PRKD2 profoundly inhibited proliferation of p53wt (U87MG, A172, and primary GBM2), and p53mut (GM133, T98G, U251, and primary Gli25) glioma cells. In a xenograft experiment, PRKD2 silencing significantly delayed tumor growth of U87MG cells. PRKD2 silencing in p53wt and p53mut cells was associated with typical hallmarks of senescence and cell cycle arrest in G1. Attenuated AKT/PKB phosphorylation in response to PRKD2 silencing was a common observation made in p53wt and p53mut GBM cells. PRKD2 knockdown in p53wt cells induced upregulation of p53, p21, and p27 expression, decreased phosphorylation of CDK2 and/or CDK4, hypophosphorylation of retinoblastoma protein (pRb), and reduced transcription of E2F1. In p53mut GM133 and primary Gli25 cells, PRKD2 silencing increased p27 and p15 and reduced E2F1 transcription but did not affect pRb phosphorylation.ConclusionsPRKD2 silencing induces glioma cell senescence via p53-dependent and -independent pathways.

Interference with distinct steps of sphingolipid synthesis and signaling attenuates proliferation of U87MG glioma cells

“…To elucidate the contribution of de novo SL synthesis to cell proliferation, SPTLC1, a catalytic long chain base subunit of SPT catalyzing the first and committed step of SL synthesis, was silenced in a panel of glioma cells. To get an indication whether the p53 status determines the outcome of SPTLC1 silencing on cell proliferation we used three established glioma cell lines (U87MG, U251, and GM133) and one low passage culture (GBM2) that was established by our group; of these cells, GM133 and U251 are p53 mut , while GBM2 and U87MG are p53 wt [34] . Despite efficient knockdown of SPTLC1 protein levels in response to RNAi ( Fig.…”

“…Primary GBM2 were established from glioblastoma multiforme tissue obtained during surgery and diagnosed according to the WHO classification. The p53 status of GBM2 cells was sequenced and described recently [34] . The protocol was approved by the local ethical review boards.…”

Estrogen Signaling Induces Mitochondrial Dysfunction-Associated Autophagy and Senescence in Breast Cancer Cells

“…The general consensus is that the expression and activation of P53 and Rb is critical for the induction of senescence [57,58]. Our data show that the expression of P53 and Rb expression increased, mainly in MCF-7 cells early on but then decreased afterwards.…”

“…Moreover, the data show that pRb was expressed at the background level when cells were treated with 20 nM E2 at 6 and 12 h, but then significantly decreased (Figure 3B). At the risk of over interpreting this set of data, it is possible that such time-dependent changes may reflective a dynamic and complex system that involves opposing players which employ gene suppression, protein degradation and/or activation/inactivation events [18,57]. Additionally, several studies have shown that while P53 is essential for the initiation of autophagy [57,58], its expression and/or structural integrity are compromised by the action of ER-α [54].…”

“…At the risk of over interpreting this set of data, it is possible that such time-dependent changes may reflective a dynamic and complex system that involves opposing players which employ gene suppression, protein degradation and/or activation/inactivation events [18,57]. Additionally, several studies have shown that while P53 is essential for the initiation of autophagy [57,58], its expression and/or structural integrity are compromised by the action of ER-α [54]. Additionally, several studies have suggested that loss or inactivation of Rb [58][59][60][61] and P53 [59], as tumor suppressors, induces degenerative autophagic responses.…”