Protein kinase C-eta regulates resistance to UV- and gamma-irradiation-induced apoptosis in glioblastoma cells by preventing caspase-9 activation.

Both increased cell proliferation and apoptosis play important roles in the malignant growth of glioblastomas. We have demonstrated recently that the differential expression of protein kinase C (PKC)-eta increases the proliferative capacity of glioblastoma cells in culture; however, specific functions for this novel PKC isozyme in the regulation of apoptosis in these tumors has not been defined. In the present study of several glioblastoma cell lines, we investigated the role of PKC-eta in preventing UV- and gamma-irradiation-induced apoptosis and in caspase-dependent signaling pathways that mediate cell death. Exposure to UV or gamma irradiation killed 80% to 100% of PKC-eta-deficient nonneoplastic human astrocytes and U-1242 MG cells, but had little effect on the PKC-eta-expressing U-251 MG and U-373 MG cells. PKC-eta appears to mediate resistance to irradiation specifically such that when PKC-eta was stably expressed in U-1242 MG cells, more than 80% of these cells developed resistance to irradiation-induced apoptosis. Reducing PKC-eta expression by transient and stable expression of antisense PKC-eta in wild-type U-251 MG cells results in increased sensitivity to UV irradiation in a fashion similar to U-1242 MG cells and nonneoplastic astrocytes. Irradiation of PKC-eta-deficient glioblastoma cells resulted in the activation of caspase-9 and caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and a substantial increase in subdiploid DNA content that did not occur in PKC-eta-expressing tumor cells. A specific inhibitor (Ac-DEVD-CHO) of caspase-3 blocked apoptosis in PKC-eta-deficient U-1242 MG cells. The data demonstrate that resistance to UV and gamma irradiation in glioblastoma cell lines is modified significantly by PKC-eta expression and that PKC-eta appears to block the apoptotic cascade at caspase-9 activation.

[1]  J. Koudstaal,et al.  Analysis of Proliferation and Apoptosis in Brain Gliomas: Prognostic and Clinical Value , 2004, Journal of Neuro-Oncology.

[2]  A. Basu,et al.  Overexpression of protein kinase C-η attenuates caspase activation and tumor necrosis factor-α-induced cell death , 2000 .

[3]  Tak W. Mak,et al.  Two Distinct Pathways Leading to Nuclear Apoptosis , 2000, The Journal of experimental medicine.

[4]  L D Lunsford,et al.  Effective treatment of experimental glioblastoma by HSV vector-mediated TNF alpha and HSV-tk gene transfer in combination with radiosurgery and ganciclovir administration. , 2000, Molecular therapy : the journal of the American Society of Gene Therapy.

[5]  S. Vandenberg,et al.  Phorbol 12-Myristate 13-Acetate Induces Protein Kinase Cη-specific Proliferative Response in Astrocytic Tumor Cells* , 2000, The Journal of Biological Chemistry.

[6]  Guido Kroemer,et al.  Apoptosis‐inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis , 2000, FEBS letters.

[7]  P. Majumder,et al.  Mitochondrial Translocation of Protein Kinase C δ in Phorbol Ester-induced Cytochrome c Release and Apoptosis* , 2000, The Journal of Biological Chemistry.

[8]  W. May,et al.  Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Y. Lazebnik,et al.  Caspase-9 and APAF-1 form an active holoenzyme. , 1999, Genes & development.

[10]  S. Sim,et al.  Brain Tumors with Cysts Treated with Gamma Knife Radiosurgery: Is Microsurgery Indicated? , 1999, Stereotactic and Functional Neurosurgery.

[11]  M. Kazanietz,et al.  New insights into the regulation of protein kinase C and novel phorbol ester receptors , 1999, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[12]  T. Ito,et al.  Ceramide Induces Bcl2 Dephosphorylation via a Mechanism Involving Mitochondrial PP2A* , 1999, The Journal of Biological Chemistry.

[13]  H. Um,et al.  Hydrogen peroxide suppresses U937 cell death by two different mechanisms depending on its concentration. , 1999, Experimental cell research.

[14]  X. Liu,et al.  An APAF-1·Cytochrome c Multimeric Complex Is a Functional Apoptosome That Activates Procaspase-9* , 1999, The Journal of Biological Chemistry.

[15]  A. Fields,et al.  Protein Kinase Cι Activity Is Necessary for Bcr-Abl-mediated Resistance to Drug-induced Apoptosis* , 1999, Journal of Biological Chemistry.

[16]  S H Kaufmann,et al.  Mammalian caspases: structure, activation, substrates, and functions during apoptosis. , 1999, Annual review of biochemistry.

[17]  B. Nickoloff,et al.  Protein Kinase Cδ Is Activated by Caspase-dependent Proteolysis during Ultraviolet Radiation-induced Apoptosis of Human Keratinocytes* , 1998, The Journal of Biological Chemistry.

[18]  J C Reed,et al.  Mitochondria and apoptosis. , 1998, Science.

[19]  Alan G. Porter,et al.  Caspase-3 Is Required for DNA Fragmentation and Morphological Changes Associated with Apoptosis* , 1998, The Journal of Biological Chemistry.

[20]  S. Nagata,et al.  Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis , 1998, Nature.

[21]  D. Yew,et al.  Apoptosis in Astrocytomas with Different Grades of Malignancy , 1998, Acta Neurochirurgica.

[22]  Y. Pommier,et al.  Activation of PKCα Downstream from Caspases during Apoptosis Induced by 7-Hydroxystaurosporine or the Topoisomerase Inhibitors, Camptothecin and Etoposide, in Human Myeloid Leukemia HL60 Cells* , 1997, The Journal of Biological Chemistry.

[23]  S. Srinivasula,et al.  Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade , 1997, Cell.

[24]  S. Ohno,et al.  The proteolytic cleavage of protein kinase C isotypes, which generates kinase and regulatory fragments, correlates with Fas-mediated and 12-O-tetradecanoyl-phorbol-13-acetate-induced apoptosis. , 1997, European journal of biochemistry.

[25]  D. Kufe,et al.  Caspase-3-mediated Cleavage of Protein Kinase C θ in Induction of Apoptosis* , 1997, The Journal of Biological Chemistry.

[26]  C. Thompson,et al.  Bcl-x(L) can inhibit apoptosis in cells that have undergone Fas-induced protease activation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[27]  F. Caponigro,et al.  Protein kinase C: a worthwhile target for anticancer drugs? , 1997, Anti-cancer drugs.

[28]  L. Zon,et al.  Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis , 1996, Nature.

[29]  W. Dewey,et al.  Cell cycle synchrony unmasks the influence of p53 function on radiosensitivity of human glioblastoma cells. , 1996, Cancer research.

[30]  W. Dewey,et al.  p53-dependent G1 arrest and p53-independent apoptosis influence the radiobiologic response of glioblastoma. , 1996, International journal of radiation oncology, biology, physics.

[31]  D. Kufe,et al.  Activation of protein kinase Cdelta in human myeloid leukemia cells treated with 1-beta-D-arabinofuranosylcytosine. , 1996, Blood.

[32]  J. Furuyama,et al.  Apoptosis of human glioma cells in response to calphostin C, a specific protein kinase C inhibitor. , 1995, Journal of neurosurgery.

[33]  J. Renart,et al.  Apoptosis induced by protein kinase C inhibition in a neuroblastoma cell line. , 1995, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[34]  M. Israel,et al.  Inactivation of p53 is associated with decreased levels of radiation-induced apoptosis in medulloblastoma cell lines. , 1995, Cell death and differentiation.

[35]  M. Prados,et al.  Constitutional p53 mutations associated with brain tumors in young adults. , 1995, Cancer genetics and cytogenetics.

[36]  R. Brown,et al.  Cell cycle arrests and radiosensitivity of human tumor cell lines: dependence on wild-type p53 for radiosensitivity. , 1994, Cancer research.

[37]  P. May,et al.  Concomitant p53 gene mutation and increased radiosensitivity in rat lung embryo epithelial cells during neoplastic development. , 1994, Cancer research.

[38]  John Calvin Reed,et al.  Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. , 1994, Oncogene.

[39]  Thomas C. Chen,et al.  Protein kinase C inhibitors induce apoptosis in human malignant glioma cell lines , 1994, FEBS letters.

[40]  B. Darras,et al.  Frequency of p53 tumor suppressor gene mutations in human primary brain tumors. , 1993, Neurosurgery.

[41]  R. Slebos,et al.  Loss of a p53-associated G1 checkpoint does not decrease cell survival following DNA damage. , 1993, Cancer research.

[42]  D. Brachman,et al.  p53 mutation does not correlate with radiosensitivity in 24 head and neck cancer cell lines. , 1993, Cancer research.

[43]  D. Ellison,et al.  Expression of the p53 protein in a spectrum of astrocytic tumours , 1992, The Journal of pathology.

[44]  M. Jung,et al.  Mutations in the p53 gene in radiation-sensitive and -resistant human squamous carcinoma cells. , 1992, Cancer research.

[45]  S. Korsmeyer,et al.  bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes , 1991, Cell.

[46]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.