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Experimental Therapeutics |
Department of Molecular Biotherapy Research, Cancer Chemotherapy Center, Cancer Institute, 1–37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455 [N. S., Y. M., M. N., Y. Y., H. H.]; Department of Neurosurgery, Tokyo University, Tokyo 113-8655 [N. S., A. A., T. K.]; Department of Molecular Pathology, Tohoku University School of Medicine, Sendai 980-8575 [A. S., T. Y., T. F., A. H.]; Developmental Neurobiology Laboratory, RIKEN Brain Science Institute and Molecular Neurobiology Laboratory, RIKEN, Ibaraki 305-0074 [M. H.]; and Core Research for Evolutional Science and Technology [T. K.] and Department of Molecular Medicine [H. H.], Sapporo Medical University, Sapporo, 060-8556, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60% of the patients (6)
. However, some cancers were resistant to p53 gene therapy in vitro (5)
and in vivo (5
, 6)
. Therefore, radiotherapy (7, 8, 9)
and chemotherapy (10, 11, 12)
have been used to potentiate the cytotoxicity of p53 gene therapy. In addition to conventional therapies, treatment with 2-methoxyestradiol (13)
, which induces and stabilizes wild-type p53 protein, and adenovirus-mediated transfer of the p16INK4/CDKN2 (14)
or interleukin 2 gene (15)
, have each been combined with p53 gene therapy to augment its cytopathic effect in cancers. Proteins encoded by several tumor suppressor genes including IRF1 (16) , WT1 (17) , and p33ING1 (18) , have been demonstrated to physically associate with the p53 protein to exert their antiproliferative effects on cells. Garkavtsev et al. (18) reported that both the p33ING1 and p53 genes should be expressed to block cell proliferation. Overexpression of p33ING1 in cells that overexpressed a native human c-myc protein, conferred sensitivity to serum starvation-induced apoptosis (19) . Overexpression of p53 arrests the cell cycle via the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (20) ; it also induces apoptosis through either transcriptional activation (21) of the Bax (22) or Fas gene (23) or transcription-independent pathways (24) . Therefore, we hypothesized that the coinduction of p33ING1 and p53 would cooperate to promote apoptosis. In this study, we investigated whether Adv3 -mediated gene transfer of the p33ING1 gene, together with p53, enhances apoptosis. To evade damage to normal tissues, we used the MBP promoter, a glioma-specific promoter (25, 26, 27, 28) , for induction of p53.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of Recombinant Adenoviral Vectors.
We first isolated the cDNA of p33ING1 by reverse transcription-PCR as described previously (29)
using fetal brain cDNA library (Clontech, Palo Alto, CA) as the template. Nucleotide sequences of the primers were p33F (sense), 5'-TTTGGATCCATGTTGAGTCCTGCCAAC-3', and p33R (antisense), 5'-TTTCTCGAGTTGCACCTCAACAAAGGCAGC-3'. These primers harbor a BamHI and an XhoI site for p33F and p33R, respectively, to facilitate cloning. Conditions for the PCR amplification was as follows: 94°C for 2 min for the initial denaturation, followed by 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Final elongation at 72°C for 5 min was also performed. The amplified product was electrophoresed in a 2% agarose gel, purified, digested by BamHI and XhoI, and cloned in BamHI-XhoI-digested pBluescript II SK(+) (pSKII+hp33). The blunt-ended XbaI/KpnI fragment of human p33ING1 cDNA from pSKII+hp33 was inserted into the blunt-ended EcoRI site of pCAcc (30)
, which generated pCA-hp33. The cosmid pAxCA-hp33 was generated by inserting the ClaI expression cassette from pCA-hp33 to the ClaI site of the cosmid pAxcw (31)
.
The BamHI fragment of human p53 cDNA from pRx-hp53 (32) was inserted into the BglII site of pCALNLw (33) , which generated pCALNL-hp53. The cosmid pAxCALNL-hp53 was generated by inserting the blunt-ended SalI/HindIII expression cassette from pCALNL-hp53 to the SwaI site of the cosmid pAxcw (31) .
Each cosmid was cotransfected with the genomic DNA-terminal protein complex of adenovirus type 5 (Ad5dlX), and the recombinant adenoviruses were generated according to the method described by Miyake et al. (31) . The construction of Adv for p53 with fiber mutation of K20 (Adv-p53-F/K20) was described previously (32) .
The recombinant adenovirus AdexMBP-NL-Cre carries the Cre recombinase gene (34)
with nuclear localization signal under the control of the mouse MBP promoter (1.3 kbp from the BglII site at nucleotide -1297 to the AccIII site at nucleotide 34, relative to the transcription start position at nucleotide 1; Refs. 27
and 28
). Because the transduction efficiency of Adv vector for lacZ along with AdexMBP-NL-Cre was much higher compared with that of adenovirus vector for lacZ directly controlled by MBP promoter,4
we used AdexMBP-NL-Cre for the specific expression of p53 in glioma cells. Details of the generation of AdexMBP-NL-Cre will be described elsewhere.4
Adv-mediated gene transduction was performed as described previously (30) . AxCALNL-hp53 was always coinfected with AdexMBP-NL-Cre at a ratio of MOI of 1:5. The total MOI of adenovirus used to infect each cell was kept the same in all experiments by supplementing with the Adv for lacZ (Adv-lacZ; Refs. 31 and 33 ).
DNA Sequence and Analysis.
The mutation search of the p53 and p33ING1 genes was carried out essentially as described previously (35)
. Briefly, DNA sequences were determined using a Thermo Sequenase dye terminator cycle sequencing pre-mix kit (Amersham, Little Chalfont, United Kingdom) and ABI PRISMTM 310 Genetic Analyzer (Perkin-Elmer, Foster City, CA), according to the suppliers’ recommendations. Nucleotide sequence for both sense and antisense strands were determined to confirm the results. Nucleotide sequences of the primer sets for the mutation search of the p53 gene were described previously (35)
. Those for p33ING1 will be described elsewhere.5
Assessment of Cell Death.
The degree of cell death was assessed by determining the percentage of cells that had died, the percentage of hypodiploid cells, and the degree of DNA fragmentation.
To determine the percentage of cells that had died, cells that were adhered to the plate and those that had detached from the plate, were stained with 0.2% trypan blue. The cells were then counted using a hemocytometer.
The percentage of hypodiploid cells was determined by the method described previously (36) . Briefly, ethanol-permeabilized cells were stained with propidium iodide and then analyzed with CELLQuest software on a FACScan (Becton Dickinson, San Jose, CA). The DNA fluorescence gate was set up to exclude cell aggregates and debris. The percentage of cells that had undergone apoptosis was assessed to be the ratio of the fluorescent area smaller than the G0-G1 peak of the total area of fluorescence. Three samples of cells for each experimental condition were analyzed.
DNA fragments in apoptotic cells were detected using the APO-BRDU kit (PharMingen, San Diego, CA), according to the manufacturer’s instructions. Briefly, the 3'-hydroxyl ends of the DNA in apoptotic cells were labeled with Br-dUTP by terminal deoxynucleotidyl transferase, and Br-dUTP was stained by a FITC-labeled anti-BrdUrd monoclonal antibody. The samples were stained with propidium iodide and analyzed by FACScan.
Electron Microscopy.
For transmission electron microscopy, the cells were first fixed in 0.1 M sodium phosphate buffer containing 2.5% glutaraldehyde at a pH of 7.5. They were then fixed in 0.1 M sodium phosphate buffer containing 1% OSO4 at a pH of 7.2. The cells were embedded into Epon 812 (TAAB, Berkshire, United Kingdom) and sliced into 60-nm sections. The ultrathin sections were contrasted with uranyl acetate and lead citrate and then examined with a Hitachi H7000 transmission electron microscope (Tokyo, Japan).
Immunoblot Analysis.
Immunoblot analysis was performed using the ECL kit (Amersham, Buckinghamshire, United Kingdom), as described previously (36)
. Briefly, 106 cells were lysed by incubating them in lysis buffer [10 mM Tris-HCl (pH 8.0), 0.2% NP40, 1 mM EDTA] for 15 min on ice. The lysate was centrifuged at 18,500 x g for 2 min at 4°C, and the protein content of the supernatant was quantified using the DC protein assay kit (Bio-Rad, Hercules, CA), according to the manufacturer’s instructions. An equal volume of 2x Laemmli buffer was added to the supernatant, and this was boiled for 5 min. Equal amounts of protein from each extract (10 µg/lane) were separated by electrophoresis on 10–12% polyacrylamide gels and then transferred onto nitrocellulose membranes. After blocking with 5% dry milk in TBS [10 mM Tris-HCl (pH 7.5), 150 mM sodium chloride], the membranes were incubated with the primary antibody for 1 h at room temperature. The primary antibodies used were rabbit anti-human p33ING1 polyclonal antibody (PharMingen), mouse anti-human p53 monoclonal antibody (Oncogene Science, Cambridge, MA), mouse anti-human Bax monoclonal antibody (Medical and Biological Laboratories, Nagoya, Japan), and mouse anti-
-actin monoclonal antibody (Sigma Chemical Co., St. Louis, MO) as the control. After washing, the membranes were incubated for 1 h at room temperature with 30 µl (per 15 ml) of horseradish peroxidase-conjugated donkey anti-rabbit IgG [F(ab')2; Amersham) for p33ING1 or rabbit anti-mouse IgG+A+M (H+L) (ZYMED Laboratories, San Francisco, CA) for p53, Bax, and -actin. Staining was carried out using the ECL kit, according to the manufacturer’s instructions.
Detection of Fas.
For flow cytometric (FACS) analysis of Fas, 1 x 106 cells were incubated at 4°C for 20 min with 0.25 µg of FITC-conjugated mouse anti-human CD95 antibody (PharMingen). The cells were analyzed by FACScan. An isotype-matched control antibody was used for negative control staining.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of p33ING1 and p53 in Glioma Cells Infected with Adv for p33ING1 or p53.
For glioma-specific expression of p53, we used an Adv carrying the Cre recombinase gene controlled by the MBP promoter, together with the Adv for p53 in which a spacer DNA with poly(A) addition signal flanked by a pair of loxP sequence was excised by the Cre recombinase. Transfection of Adv for p53 controlled by MBP (Adv-MBP-p53) into U251 cells, as well as into U-373 MG cells, induced the expression of p53 (Fig. 1
, upper panel, Lanes 4 and 8). Transfection of Adv for p33ING1 (Adv-p33) into these two cell lines induced the expression of p33ING1 (Fig. 1
, middle panel, Lanes 3 and 7); endogenous p33ING1 expression was not seen in either cell line. As shown in Fig. 1
, the relative molecular weight (Mr) of the endogenous mutant p53 (Fig. 1
, upper panel, Lanes 1 and 5) is smaller than that of the wild-type p53 (Fig. 1
, upper panel, Lanes 4 and 8). Induction of the wild-type p53 gene in these two cell lines suppressed the endogenous mutant p53 gene from being expressed (Fig. 1
, upper panel, Lanes 4 and 8). Biosynthesis of wild-type p53 may effect a negative feedback regulation on the expression of the endogenous mutant p53 gene in these two cell lines (37)
.
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and U-373 MG cell lines. The percentage of cells that had died among U251 cells (74.7 ± 1.5%) and among U-373 MG cells (30.7 ± 1.1%) coinfected with Adv-p33 and Adv-MBP-p53 was much higher than the percentage of cells that had died among U251 cells and U-373 MG cells infected with Adv-p33 alone (13.8 ± 0.4% and 6.7 ± 1.5%, respectively) or Adv-MBP-p53 alone (22.0 ± 1.3% and 9.8 ± 1.0%, respectively; Fig. 3
). The percentage of hypodiploid cells reflects the degree of apoptosis. Coinfection of Adv-p33 and Adv-MBP-p53 induced drastically enhanced apoptosis in both cell lines, compared with that induced by the single infection of Adv-p33 or Adv-MBP-p53 alone (Fig. 4)
. The percentage of hypodiploid cells by coinfection with Adv-p33 and Adv-MBP-p53 in U251 cells and in U-373 MG cells was 43.6 ± 1.2% and 9.3 ± 0.1%, respectively, whereas that by single infection with Adv-p33 or Adv-MBP-p53 was 6.2 ± 0.4% or 7.6 ± 1.4%, respectively, in the U251 cells, and 1.6 ± 0.1% or 1.9 ± 0.2%, respectively, in the U-373 MG cells (Fig. 4)
. Similarly, the Br-dUTP uptake assay for the detection of DNA fragments revealed that 33% of U251 cells coinfected with Adv-p33 and Adv-MBP-p53 contained fragmented DNA, whereas only 0.2% of U251 cells infected with Adv-p33 and 0.5% of U251 cells infected with Adv-MBP-p53 contained fragmented DNA (Fig. 5A)
. Although the degree of apoptosis in U-251 cells was more remarkable than that in U-373 MG cells, similar results were obtained in the U-373 MG cells (Fig. 5)
. The percentage of cells with fragmented DNA among U-373 MG cells coinfected with Adv-p33 and Adv-MBP-p53 was 5.8%, whereas that among cells infected with Adv-p33 or Adv-MBP-p53 alone was 1.3 or 1.0%, respectively (Fig. 5B)
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Next, the MOI of the single infection of Adv-p33 or Adv-MBP-p53 was increased to 300 in U251 cells to evaluate whether a high level of expression of p33 or p53 protein induces apoptosis. Although infection of Adv-MBP-p53 induced apoptosis at a MOI of 300 (26%), infection of Adv-p33 did not show any cytopathic effect in U251 cells (1.5%; Fig. 5C
). Infection of Adv-p33 at a MOI of 300 did not induce apoptosis in NGF-treated PC-12 cells (1.7%), which are representative of normal neuronal cells (Ref. 38
; Fig. 5D
). Moreover, coinfection of Adv-p33 and Adv-MBP-p53 did not induce apoptosis in NGF-treated PC-12 cells (0.2%; Fig. 5D
). These results suggest that a high level of expression of p33 alone does not induce apoptosis in cells including neurons, and that coinfection of Adv-p33 and Adv-MBP-p53 does not induce apoptosis in neurons.
Electron microscopic analysis of U251 cells coinfected with Adv-p33 and Adv-MBP-p53 revealed condensed chromatin in the nuclei (Fig. 6
, lower middle panel) and apoptotic bodies (Fig. 6
, lower right panel) 3 days after infection. These are features of apoptotic cell death.
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, lower left panel), although the nucleus remained intact (Fig. 6
, upper right panel). On the other hand, infection of Adv-lacZ did not induce damage to the mitochondria (Fig. 6
, upper middle panel). Coinfection of Adv-p33 and Adv-MBP-p53 did not up-regulate the expression of Bcl-2 or Bcl-XL (data not shown). It did not up-regulate the expression of Bax (Fig. 7A)
nor Fas(Fig. 7B)
1 day after infection, which indicates that up-regulation of these two proteins did not precede the mitochondrial damage.
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, the increase in the expression level of p53 was not obvious after the Adv-MBP-p53 infection at MOI 50, whereas the expression level of p53 clearly increased after infection of Adv-MBP-p53 at MOI 300 compared with that of endogenous p53. This indicates that the infection of Adv-MBP-p53 at MOI 50 induced exogenous expression of p53, although its expression level was rather low compared with that after infection with Adv-MBP-p53 at MOI 300 or Adv-p53-F/K20 at MOI 50 (Fig. 7C)
. Because the exogenous expression of p53 after infection with Adv-MBP-p53 at MOI 50 was relatively low, it did not significantly up-regulate Bax or Fas protein. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Overexpression of p33ING1 inhibits cell growth (40)
and plays an important role in the process of cellular senescence (43)
. Moreover, overexpression of p33ING1 in cells that conditionally overexpress a native human c-myc confers sensitivity to serum starvation-induced apoptosis (19)
. The tumor suppression activity of p33ING1 requires an intact p53 tumor suppressor gene (18)
. Neither of these two genes can cause growth inhibition when the other one is suppressed (18)
. It has been demonstrated that coinduction of p33ING1 and p53 inhibits cell growth. However, no previous study on the cooperative effect of p33ING1 and p53 in inducing apoptosis has been reported.
In this study, we demonstrated that coinduction of the p33ING1 and p53 genes induced drastic apoptosis using Adv vectors, which can easily transduce various genes into cancer cells at high copy numbers. Which genes are involved in the apoptosis induced by overexpression of the p33ING1 and p53 genes? Expression of p33ING1 promotes p53-dependent transactivation of p21/WAF1 (18)
. Adenoviral gene transfer of p21/WAF1 inhibited tumor growth attributable to cell cycle arrest, but it was reported that it does not induce apoptosis in some cancers (44
, 45)
. Thus, we investigated whether coinfection of Adv-p33 and Adv-MBP-p53 increased the levels of expression of Bax (22)
and Fas (23)
, which have been reported to be up-regulated by induction of p53 expression. Bax and Fas expression were not up-regulated by coinduction of p33ING1 and p53, although most of the mitochondrias had been morphologically damaged. Polyak et al. (39)
investigated the change in level of transcripts induced by p53 expression before the onset of apoptosis and found that p53-expressing cells have markedly high expression of genes encoding proteins closely related to the formation of reactive oxygen species, which led to mitochondrial damage. Therefore, the levels of these proteins, instead of Bax and Fas proteins, might initially increase in response to coinduction of p33ING1 and p53. The combined transduction of p33ING1 and p53 did not up-regulate the expression of the oxidative stress-related genes such as manganese superoxide dismutase, heat shock protein 32, c-Jun, or glutathione S-transferase-
(data not shown). Further investigation is required to clarify the genes involved in the apoptosis induced by coinfection of Adv-p33 and Adv-MBP-p53.
Concerning the gene therapy of gliomas, there are several advantages to coinfecting gliomas with Adv-p33 and Adv-MBP-p53. The combined transduction of p33ING1 and p53 into glioma cells induced a highly intensified apoptotic effect. As shown in our study (Figs. 3
4
5)
, infection of Adv-MBP-p53 alone induced a very low level of apoptosis, if any, in U251 and U-373 MG cells. On the other hand, the coinfection of Adv-p33 and Adv-MBP-p53 dramatically enhanced the degree of apoptosis in both cell lines. Thus, this coinfection approach may induce apoptosis in gliomas that do not show apoptosis by overexpression of p53 alone.
Some glioma cells are refractory to p53-mediated gene therapy (5) . The combined transfer of the p33ING1 and p53 genes not only enhances the apoptosis of glioma cells but also might possibly induce apoptosis, even in gliomas that do not undergo apoptosis by p53-mediated gene therapy alone, because expression of both p33ING1 and p53 may overcome the apoptotic-resistant mechanisms in gliomas. Neuroblastoma cells often contain high levels of wild-type p53 (46) , and some harbor a rearranged p33ING1 gene (40) , suggesting that loss of p33ING1 function is a potential mechanism for inactivation of p53 in cancers. Indeed, U251 and U-373 MG cells showed low expression levels of p33ING1, which might render these cell lines antiapoptotic in p53-mediated gene therapy and highly susceptible to apoptosis induced by combined transfer of the p33ING1 and p53 genes. Although other glioma cell lines must be analyzed, if glioma cells containing wild-type p53 do not undergo apoptosis due to impairment of p33ING1 function, coexpression of the p33ING1 gene would bypass the resistance and consequently kill the glioma.
Because the combined transduction of the p33ING1 and p53 genes induces a synergistic apoptotic effect on glioma cells, the dosage of adenoviral vectors (Adv) of each of the two genes administered to patients can be decreased so as to reduce the opportunity of both the appearance of replication-competent Adv and the undesirable inflammatory and immune response related to Adv overdosage. Moreover, administering lower doses of Adv to patients could reduce the potential damage incurred to normal tissues. For example, in p53 gene therapy of gliomas, transfer of p53 to neighboring neurons should be reduced to the greatest extent possible, because it has been shown that p53 gene transduction into cultured neurons induces apoptosis (47) . Therefore, we used the MBP promoter, which has been demonstrated to transduce genes in a glioma-specific manner (25, 26, 27, 28) . Indeed, coinfection of Adv-p33 and Adv-MBP-p53 did not induce apoptosis in NGF-treated PC-12 cells, which are representative of neurons (38) . In addition, overexpression of p33ING1 did not induce apoptosis in glioma cells nor in NGF-treated PC-12 cells, suggesting that induction of p33ING1 protein alone would not induce apoptosis in normal tissues. Therefore, the cotransduction of p33ING1 and p53 genes could possibly lead to remarkable apoptosis of glioma cells, without damaging neurons.
In conclusion, the coinfection of Adv-p33 and Adv-MBP-p53 dramatically enhanced the degree of apoptosis in glioma cells. This gene therapy will be a practical and promising approach for the treatment of gliomas.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by a special grant for Advanced Research on Cancer from the Ministry of Education, Science, Sports and Culture of Japan and grants from the Ministry of Health and Welfare of Japan. 
2 To whom requests for reprints should be addressed, at Hirofumi Hamada, Cancer Chemotherapy Center, Cancer Institute, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan. Phone: 81-3-3918-0111, extension 4356; Fax: 81-3-3918-3716; E-mail: hhamada{at}jfcr.or.jp
3 The abbreviations used are: Adv, adenovirus; Br-dUTP, bromolated deoxyuridine triphosphate nucleotide; MBP, myelin basic protein; MOI, multiplicity of infection; NGF, nerve growth factor; FACS, fluorescence-activated cell sorter.
4 M. Hashimoto, unpublished data.
6 N. Shinoura and H. Hamada, unpublished data.
Received 1/12/99. Accepted 9/ 3/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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