Enhancement of radiosensitivity of wild-type p53 human glioma cells by adenovirus-mediated delivery of the p53 gene

View More View Less
  • 1 Departments of Neurosurgery, Neuro-Oncology, and Experimental Radiation Oncology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas
Restricted access

Purchase Now

USD  $45.00

JNS + Pediatrics - 1 year subscription bundle (Individuals Only)

USD  $515.00

JNS + Pediatrics + Spine - 1 year subscription bundle (Individuals Only)

USD  $612.00
Print or Print + Online

Object. The authors sought to determine whether combining p53 gene transfer with radiation therapy would enhance the therapeutic killing of p53 wild-type glioma cells. It has been shown in several reports that adenovirus-mediated delivery of the p53 gene into p53 mutant gliomas results in dramatic apoptosis, but has little effect on gliomas containing wild-type p53 alleles. Therefore, p53 gene therapy alone may not be a clinically effective treatment for gliomas because most gliomas are composed of both p53 mutant and wild-type cell populations. One potential approach to overcome this problem is to exploit the role p53 plays as an important determinant in the cellular response to ionizing radiation.

Methods. In vitro experiments were performed using the glioma cell line U87MG, which contains wild-type p53. Comparisons were made to the glioma cell line U251MG, which contains a mutant p53 allele. Monolayer cultures were infected with an adenovirus containing wild-type p53 (Ad5CMV-p53), a control vector (dl312), or Dulbecco's modified Eagle's medium (DMEM). Two days later, cultures were irradiated and colony-forming efficiency was determined. Transfection with p53 had only a minor effect on the plating efficiency of nonirradiated U87MG cells, reducing the plating efficiency from 0.23 ± 0.01 in DMEM to 0.22 ± 0.04 after addition of Ad5CMV-p53. However, p53 transfection significantly enhanced the radiosensitivity of these cells. The dose enhancement factor at a surviving fraction of 0.10 was 1.5, and the surviving fraction at 2 Gy was reduced from 0.61 in untransfected controls to 0.38 in p53-transfected cells. Transfection of the viral vector control (dl312) had no effect on U87MG radiosensitivity. In comparison, transfection of Ad5CMV-p53 into the p53 mutant cell line U251MG resulted in a significant decrease in the surviving fraction of these cells compared with controls, and no radiosensitization was detected.

To determine whether Ad5CMV-p53—mediated radiosensitization of U87MG cells involved an increase in the propensity of these cells to undergo apoptosis, flow cytometric analysis of terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridinetriphosphate nick-end labeling—stained cells was performed. Whereas the amount of radiation-induced apoptosis in uninfected and dl312-infected control cells was relatively small (2.1 ± 0.05% and 3.7 ± 0.5%, respectively), the combination of Ad5CMV-p53 infection and radiation treatment significantly increased the apoptotic frequency (18.6 ± 1.4%).

To determine whether infection with Ad5CMV-p53 resulted in increased expression of functional exogenous p53 protein, Western blot analysis of p53 was performed on U87MG cells that were exposed to 9 Gy of radiation 2 days after exposure to Ad5CMV-p53, dl312, or DMEM. Infection with Ad5CMV-p53 alone increased p53 levels compared with DMEM- or dl312-treated cells. Irradiation of Ad5CMV-p53—infected cells resulted in a further increase in p53 that reached a maximum at 2 hours postirradiation. To determine whether exogenous p53 provided by Ad5CMV-p53 had transactivating activity, U87MG cells were treated as described earlier and p21 messenger RNA levels were determined. Infection of U87MG cells with Ad5CMV-p53 only resulted in an increase in p21 compared with DMEM- and dl312-treated cells. Irradiation of Ad5CMV-p53—infected cells resulted in an additional time-dependent increase in p21 expression.

Conclusions. These data indicate that adenovirus-mediated delivery of p53 may enhance the radioresponse of brain tumor cells containing wild-type p53 and that this radiosensitization may involve converting from a clonogenic to the more sensitive apoptotic form of cell death. Although the mechanism underlying this enhanced apoptotic susceptibility is unknown, the Ad5CMV-p53—infected cells have a higher level of p53 protein, which increases further after irradiation, and this exogenous p53 is transcriptionally active. Thus, it is possible that the combination of Ad5CMV-p53 infection and radiation treatment increases p53 protein to a level that is sufficient to overcome at least partially the block in apoptosis existing in U87MG cells.

JNS + Pediatrics - 1 year subscription bundle (Individuals Only)

USD  $515.00

JNS + Pediatrics + Spine - 1 year subscription bundle (Individuals Only)

USD  $612.00
  • 1.

    Buchsbaum DJ, , Raben D, & Stackhouse MA, et al: Approaches to enhance cancer radiotherapy employing gene transfer methods. Gene Ther 3:10421068, 1996 Buchsbaum DJ, Raben D, Stackhouse MA, et al: Approaches to enhance cancer radiotherapy employing gene transfer methods. Gene Ther 3:1042–1068, 1996

    • Search Google Scholar
    • Export Citation
  • 2.

    Clarke AR, , Purdie CA, & Harrision DJ, et al: Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 362:849852, 1993 Clarke AR, Purdie CA, Harrision DJ, et al: Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 362:849–852, 1993

    • Search Google Scholar
    • Export Citation
  • 3.

    Clayman G, , El-Naggar AK, & Roth JA, et al: In vivo molecular therapy with p53 adenovirus for microscopic residual head and neck squamous carcinoma. Cancer Res 55:16, 1995 Clayman G, El-Naggar AK, Roth JA, et al: In vivo molecular therapy with p53 adenovirus for microscopic residual head and neck squamous carcinoma. Cancer Res 55:1–6, 1995

    • Search Google Scholar
    • Export Citation
  • 4.

    Eastham JA, , Hall SJ, & Sehgal I: In vivo gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res 55:51515155, 1995 Eastham JA, Hall SJ, Sehgal I: In vivo gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res 55:5151–5155, 1995

    • Search Google Scholar
    • Export Citation
  • 5.

    Fertil B, & Malaise EP: Inherent cellular radiosensitivity as a basic concept for human tumor radiotherapy. Int J Radiat Oncol Biol Phys 7:621629, 1981 Fertil B, Malaise EP: Inherent cellular radiosensitivity as a basic concept for human tumor radiotherapy. Int J Radiat Oncol Biol Phys 7:621–629, 1981

    • Search Google Scholar
    • Export Citation
  • 6.

    Frankel RH, , Bayona W, & Koslow M, et al: P53 mutations in human malignant gliomas: comparison of loss of heterozygosity with mutation frequency. Cancer Res 52:14271433, 1992 Frankel RH, Bayona W, Koslow M, et al: P53 mutations in human malignant gliomas: comparison of loss of heterozygosity with mutation frequency. Cancer Res 52:1427–1433, 1992

    • Search Google Scholar
    • Export Citation
  • 7.

    Gallardo D, , Drazan E, & McBride WH: Adenovirus-based transfer of wild-type p53 gene increases ovarian tumor radiosensitivity. Cancer Res 56:48914893, 1996 Gallardo D, Drazan E, McBride WH: Adenovirus-based transfer of wild-type p53 gene increases ovarian tumor radiosensitivity. Cancer Res 56:4891–4893, 1996

    • Search Google Scholar
    • Export Citation
  • 8.

    Gomez-Manzano C, , Fueyo J, & Kyritsis AP, et al: Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis. Cancer Res 56:694699, 1996 Gomez-Manzano C, Fueyo J, Kyritsis AP, et al: Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis. Cancer Res 56:694–699, 1996

    • Search Google Scholar
    • Export Citation
  • 9.

    Gorczyca W, , Bruno S, & Darzynkiewicz RJ, et al: DNA strand breaks occurring in apoptosis: their early detection in situ by deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int J Oncol 1:639648, 1992 Gorczyca W, Bruno S, Darzynkiewicz RJ, et al: DNA strand breaks occurring in apoptosis: their early detection in situ by deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int J Oncol 1:639–648, 1992

    • Search Google Scholar
    • Export Citation
  • 10.

    Hamada K, , Alemany R, & Zhang WW, et al: Adenovirus-mediated transfer of a wild-type p53 gene and induction of apoptosis in cervical cancer. Cancer Res 56:30473054, 1996 Hamada K, Alemany R, Zhang WW, et al: Adenovirus-mediated transfer of a wild-type p53 gene and induction of apoptosis in cervical cancer. Cancer Res 56:3047–3054, 1996

    • Search Google Scholar
    • Export Citation
  • 11.

    Harper JW, , Adami GR, & Wei N, et al: The p21 Cdk-interacting protein cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805816, 1993 Harper JW, Adami GR, Wei N, et al: The p21 Cdk-interacting protein cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805–816, 1993

    • Search Google Scholar
    • Export Citation
  • 12.

    Harris C: Structure and function of the p53 tumor suppressor gene: clues for rational cancer therapeutic strategies. J Natl Cancer Inst 88:14421455, 1996 Harris C: Structure and function of the p53 tumor suppressor gene: clues for rational cancer therapeutic strategies. J Natl Cancer Inst 88:1442–1455, 1996

    • Search Google Scholar
    • Export Citation
  • 13.

    James CD, , Carlbom E, & Dumanski JP, et al: Clonal genomic alterations in glioma malignancy stages. Cancer Res 48:55465551, 1988 James CD, Carlbom E, Dumanski JP, et al: Clonal genomic alterations in glioma malignancy stages. Cancer Res 48:5546–5551, 1988

    • Search Google Scholar
    • Export Citation
  • 14.

    Jayaraman L, & Prives C: Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus. Cell 81:10211029, 1995 Jayaraman L, Prives C: Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus. Cell 81:1021–1029, 1995

    • Search Google Scholar
    • Export Citation
  • 15.

    Köck H, , Harris MP, & Anderson SC, et al: Adenovirus-mediated p53 gene transfer suppresses growth of human glioblastoma cells in vitro and in vivo. Int J Cancer 67:808815, 1996 Köck H, Harris MP, Anderson SC, et al: Adenovirus-mediated p53 gene transfer suppresses growth of human glioblastoma cells in vitro and in vivo. Int J Cancer 67:808–815, 1996

    • Search Google Scholar
    • Export Citation
  • 16.

    Kuerbitz SJ, , Plunkett BS, & Walsh WV, et al: Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci USA 89:74917495, 1992 Kuerbitz SJ, Plunkett BS, Walsh WV, et al: Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci USA 89:7491–7495, 1992

    • Search Google Scholar
    • Export Citation
  • 17.

    Lang FF, , Miller DC, & Pisharody S, et al: High frequency of p53 protein accumulation without p53 gene mutation in human juvenile pilocytic, low grade and anaplastic astrocytomas. Oncogene 9:949954, 1994 Lang FF, Miller DC, Pisharody S, et al: High frequency of p53 protein accumulation without p53 gene mutation in human juvenile pilocytic, low grade and anaplastic astrocytomas. Oncogene 9:949–954, 1994

    • Search Google Scholar
    • Export Citation
  • 18.

    Lee Y, , Chen Y, & Chang LS, et al: Inhibition of mouse thymidylate synthase promoter activity by the wild-type p53 tumor suppressor protein. Exp Cell Res 234:270276, 1997 Lee Y, Chen Y, Chang LS, et al: Inhibition of mouse thymidylate synthase promoter activity by the wild-type p53 tumor suppressor protein. Exp Cell Res 234:270–276, 1997

    • Search Google Scholar
    • Export Citation
  • 19.

    Li R, , Waga S, & Hannon GJ, et al: Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature 371:534537, 1994 Li R, Waga S, Hannon GJ, et al: Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature 371:534–537, 1994

    • Search Google Scholar
    • Export Citation
  • 20.

    Liu TJ, , Zhang WW, & Taylor DL, et al: Growth suppression of human head and neck cancer cells by the introduction of wild-type p53 gene via a recombinant adenovirus. Cancer Res 54:36623667, 1994 Liu TJ, Zhang WW, Taylor DL, et al: Growth suppression of human head and neck cancer cells by the introduction of wild-type p53 gene via a recombinant adenovirus. Cancer Res 54:3662–3667, 1994

    • Search Google Scholar
    • Export Citation
  • 21.

    Lowe SW, , Schmitt EM, & Smith SW, et al: p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362:847849, 1993 Lowe SW, Schmitt EM, Smith SW, et al: p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362:847–849, 1993

    • Search Google Scholar
    • Export Citation
  • 22.

    Meyn RE, , Stephens LC, & Ang KK, et al: Heterogeneity in the development of apoptosis in irradiated murine tumours of different histologies. Int J Radiat Biol 64:583591, 1993 Meyn RE, Stephens LC, Ang KK, et al: Heterogeneity in the development of apoptosis in irradiated murine tumours of different histologies. Int J Radiat Biol 64:583–591, 1993

    • Search Google Scholar
    • Export Citation
  • 23.

    Miyashita T, , Krajewski S, & Krajewska M, et al: Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9:17991805, 1994 Miyashita T, Krajewski S, Krajewska M, et al: Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9:1799–1805, 1994

    • Search Google Scholar
    • Export Citation
  • 24.

    Miyashita T, & Reed JC: Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80:293299, 1995 Miyashita T, Reed JC: Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80:293–299, 1995

    • Search Google Scholar
    • Export Citation
  • 25.

    Nelson WG, & Kastan MB: DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol Cell Biol 14:18151823, 1994 Nelson WG, Kastan MB: DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol Cell Biol 14:1815–1823, 1994

    • Search Google Scholar
    • Export Citation
  • 26.

    Newcomb EW, , Madonia WJ, & Pisharody S, et al: A correlative study of p53 protein alteration and p53 gene mutation in glioblastoma multiforme. Brain Pathol 3:229235, 1993 Newcomb EW, Madonia WJ, Pisharody S, et al: A correlative study of p53 protein alteration and p53 gene mutation in glioblastoma multiforme. Brain Pathol 3:229–235, 1993

    • Search Google Scholar
    • Export Citation
  • 27.

    Nigro JM, , Baker SJ, & Preisinger AC, et al: Mutations in the p53 gene occur in diverse human tumour types. Nature 342:705708, 1989 Nigro JM, Baker SJ, Preisinger AC, et al: Mutations in the p53 gene occur in diverse human tumour types. Nature 342:705–708, 1989

    • Search Google Scholar
    • Export Citation
  • 28.

    Ramqvist T, , Magnusson KP, & Wang Y, et al: Wild-type p53 induces apoptosis in a Burkitt lymphoma (BL) line that carries mutant p53. Oncogene 8:14951500, 1993 Ramqvist T, Magnusson KP, Wang Y, et al: Wild-type p53 induces apoptosis in a Burkitt lymphoma (BL) line that carries mutant p53. Oncogene 8:1495–1500, 1993

    • Search Google Scholar
    • Export Citation
  • 29.

    Roth JA, , Nguyen D, & Lawrence DD, et al: Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nat Med 2:985991, 1996 Roth JA, Nguyen D, Lawrence DD, et al: Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nat Med 2:985–991, 1996

    • Search Google Scholar
    • Export Citation
  • 30.

    Seto E, , Usheva A, & Zambetti GP, et al: Wild-type p53 binds to the TATA-binding protein and represses transcription. Proc Natl Acad Sci USA 89:1202812032, 1992 Seto E, Usheva A, Zambetti GP, et al: Wild-type p53 binds to the TATA-binding protein and represses transcription. Proc Natl Acad Sci USA 89:12028–12032, 1992

    • Search Google Scholar
    • Export Citation
  • 31.

    Shaw P, , Bovey R, & Tardy S, et al: Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc Natl Acad Sci USA 89:44954499, 1992 Shaw P, Bovey R, Tardy S, et al: Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc Natl Acad Sci USA 89:4495–4499, 1992

    • Search Google Scholar
    • Export Citation
  • 32.

    Sidransky D, , Mikkelsen T, & Schwechheimer K, et al: Clonal expansion of p53 mutant cells is associated with brain tumour progression. Nature 355:846847, 1992 Sidransky D, Mikkelsen T, Schwechheimer K, et al: Clonal expansion of p53 mutant cells is associated with brain tumour progression. Nature 355:846–847, 1992

    • Search Google Scholar
    • Export Citation
  • 33.

    Smith ML, , Chen IT, & Zhan Q et al: Interaction of the p53-regulated protein GADD45 with proliferating cell nuclear antigen. Science 266:13761380, 1994 Smith ML, Chen IT, Zhan Q et al: Interaction of the p53-regulated protein GADD45 with proliferating cell nuclear antigen. Science 266:1376–1380, 1994

    • Search Google Scholar
    • Export Citation
  • 34.

    Spitz FR, , Nguyen D, & Skibber JM, et al: Adenoviral-mediated wild-type p53 gene expression sensitizes colorectal cancer cells to ionizing radiation. Clin Cancer Res 2:16651671, 1996 Spitz FR, Nguyen D, Skibber JM, et al: Adenoviral-mediated wild-type p53 gene expression sensitizes colorectal cancer cells to ionizing radiation. Clin Cancer Res 2:1665–1671, 1996

    • Search Google Scholar
    • Export Citation
  • 35.

    Velculescu VE, & El-Diery WS: Biological and clinical importance of the p53 tumor suppressor gene. Clin Chem 42:858868, 1996 Velculescu VE, El-Diery WS: Biological and clinical importance of the p53 tumor suppressor gene. Clin Chem 42:858–868, 1996

    • Search Google Scholar
    • Export Citation
  • 36.

    von Deimling A, , Eibl RH, & Ohgaki H, et al: p53 mutations are associated with 17p allelic loss in Grade II and Grade III astrocytomas. Cancer Res 52:29872990, 1992 von Deimling A, Eibl RH, Ohgaki H, et al: p53 mutations are associated with 17p allelic loss in Grade II and Grade III astrocytomas. Cancer Res 52:2987–2990, 1992

    • Search Google Scholar
    • Export Citation
  • 37.

    Walker MD, , Green SB, & Byar DP, et al: Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery. N Engl J Med 303:13231329, 1980 Walker MD, Green SB, Byar DP, et al: Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery. N Engl J Med 303:1323–1329, 1980

    • Search Google Scholar
    • Export Citation
  • 38.

    Wheeler JA, , Stephens LC, & Tornos C, et al: Apoptosis as a predictor of tumor response to radiation in stage 1B cervical adenocarcinoma. Int J Radiat Oncol Biol Phys 32:14871493, 1995 Wheeler JA, Stephens LC, Tornos C, et al: Apoptosis as a predictor of tumor response to radiation in stage 1B cervical adenocarcinoma. Int J Radiat Oncol Biol Phys 32:1487–1493, 1995

    • Search Google Scholar
    • Export Citation
  • 39.

    Wills KN, , Maneval DC, & Menzel P, et al: Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer. Hum Gene Ther 5:10791088, 1994 Wills KN, Maneval DC, Menzel P, et al: Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer. Hum Gene Ther 5:1079–1088, 1994

    • Search Google Scholar
    • Export Citation
  • 40.

    Yonish-Rouach E, , Resnitzky D, & Rotem J, et al: Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352:345347, 1991 Yonish-Rouach E, Resnitzky D, Rotem J, et al: Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352:345–347, 1991

    • Search Google Scholar
    • Export Citation
  • 41.

    Zhang W, , Fang X, & Mazur W, et al: High efficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus. Cancer Gene Ther 1:513, 1994 Zhang W, Fang X, Mazur W, et al: High efficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus. Cancer Gene Ther 1:5–13, 1994

    • Search Google Scholar
    • Export Citation
  • 42.

    Zhang WW, , Fang X, & Branch CD, et al: Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis. Biotechniques 15:868872, 1993 Zhang WW, Fang X, Branch CD, et al: Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis. Biotechniques 15:868–872, 1993

    • Search Google Scholar
    • Export Citation

Metrics

All Time Past Year Past 30 Days
Abstract Views 541 132 7
Full Text Views 144 9 0
PDF Downloads 89 4 0
EPUB Downloads 0 0 0