Journal of Neuro-Oncology 63: 25–31, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Laboratory Investigation
Apoptotic DNA endonuclease (DNase-γ ) gene transfer induces cell death accompanying DNA fragmentation in human glioma cells Ryuta Saito1,3 , Masaaki Mizuno2 , Toshihiro Kumabe3 , Takashi Yoshimoto3 , Sei-ichi Tanuma4 and Jun Yoshida1 1 Department of Neurosurgery, 2 Department of Molecular Neurosurgery, Nagoya University Graduate School of Medicine, Nagoya, Japan; 3 Department of Neurosurgery, Tohoku University Graduate School of Medicine, Sendai, Japan; 4 Department of Biochemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Japan
Key words: apoptosis, DNase-γ , gene transfer, glioma Summary Aims: Both the genetic restoration of the apoptotic pathway and the introduction of proapoptotic molecules are now drawing attention. Concerning apoptosis of human glioma cells induced by human interferon-β protein, we found that DNA endonuclease (DNase-γ ) acts as an executive molecule. The authors investigated whether gene transfer of this DNase-γ exerts some therapeutic effects on human glioma cells. Methods: We transduced U251SP, U251MG, and T98G human glioma cells with DNase-γ gene via multilamellar cationic liposomes, monitored the growth of those cells, and carefully observed the cell-death pattern. Results: DNase-γ gene transfer resulted in an overexpression of DNase-γ protein and induced DNA fragmentation in gene-transferred cells. The cytotoxic effect rose with multiple inoculations of the liposome, suggesting a relationship between its expression and the therapeutic effect. Conclusions: These results demonstrate that DNase-γ gene transfer can induce apoptosis in human glioma cells, indicating its potential to become a future gene therapy strategy. Introduction Although malignant gliomas are the most common tumors in the central nervous system, multimodality treatments that include extensive tumor resection, megavoltage radiation, and chemotherapy have resulted in little improvement in the prognosis of patients with these disorders [1–3]. Thus, novel therapeutic strategies are necessary. Generally, malignancy results not only from unregulated cell proliferation but also from declining celldeath (apoptosis) signals [4]. Although, apoptosis is a process essential for normal development and homeostasis in multicellular organisms, providing a defense against oncogenesis or viral invasion [5], its pathway is often disrupted in tumor cells [6]. Therefore, a genetic restoration of the apoptotic pathway or introduction of proapoptotic molecules is a very attractive option for the treatment of tumors. Recently, gene therapeutic
strategies from this point of view such as overexpression of Bax [7,8], CPP32β gene [9], caspase-7 gene [10], or active caspase-6 gene [11] transfer have been reported. This study also focused on the apoptotic pathway. Apoptotic cell-death is characterized by the accompanying morphological changes such as cell shrinkage, condensation of nuclei, and loss of microvilli. On the other hand, the biochemical hallmark of apoptosis is the cleavage of chromosomal DNA into nucleosomal units, which appears to be the final stage in the cell-death process. DNA endonucleases that participate in this process are now attracting attention. Several Ca2+ /Mg2+ -dependent endonucleases and divalent cation-independent acidic endonucleases have been suggested as candidates for the apoptotic endonuclease. Among those DNA endonucleases, (DNase-γ ) is a novel endonuclease that was found in the nuclei of
26 apoptotic rat thymocytes. It is a Ca2+ /Mg2+ -dependent neutral endonuclease whose molecular mass is 33 kDa, and produces 3 -OH/5 -phosphate ends of nucleosomal DNA fragments. The expression of a 1.6 kb DNase-γ mRNA was detected at high levels in spleen, thymus, lymph nodes, and liver but little was seen in brain, heart, kidney, or testis. This might imply the stringent repression of DNase-γ gene expression in those latter organs. This enzyme is synthesized as an inactive precursor protein and converted into an active enzyme by the removal of an N-terminal precursor peptide. However, the inhibiting or activating factors that regulate the activity of DNase-γ have not yet been discovered, and it is suggested that the existence of these factors differs among cells and the apoptotic mechanism also differs among cells [12–17]. We have previously shown that this endonuclease is activated in the apoptotic process of Ltk-murine fibroblast mediated by tumor necrosis factor-α [18]. Similarly, our recent study on the apoptotic pathway mediated by Interferon (IFN)-β in human glioma cells revealed the activation of apoptotic endonuclease DNase-γ preceding DNA fragmentation in IFN-β susceptible cells, SK-MG-1, but not in resistant cells, U251SP. From these observations, we first hypothesized that DNase-γ participates in the apoptotic cell-death of glioma cells, and carried out this study to investigate whether overexpression of this DNA endonuclease bestows some therapeutic benefits on human glioma cells.
Materials and methods Cell culture Human glioma cell lines SK-MG-1, U251SP, U251MG, and T98G were maintained in Eagle’s minimum essential medium supplemented with 10% fetal calf serum, 5 mM l-glutamine, 2 mM non-essential amino acids, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) at 37◦ C in an atmosphere of 5% CO2 in air. Cells were plated into 250 ml tissue culture flasks and used at 70% confluence. Antibodies Antibodies used in this study are the anti-human DNase-γ monoclonal antibody hg302 (provided by us) and hg303 (Wako, Osaka, Japan).
Plasmids Plasmids designated pcDNA3.1–Myc–His C/DNase-γ (pcDNA3.1–hDNase-γ ) and pcDNA3.1–Myc–His C (pcDNA3.1) were used. The former plasmid contains the CMV early promoter and the C-terminal Myc and His tagged forms of human DNase-γ . pcDNA3.1–Myc–His C (Invitrogen Corp., Carlsbad, CA) was employed as a backborn plasmid. Preparation of multilamellar cationic liposome containing plasmids Liposome-entrapped plasmids were prepared by a procedure described in our previous papers [19–21]. The liposomes were prepared using the positively charged lipid N-(α-trimethylammonio-acetyl)didodecyl-d-glutamate chloride (TMAG), dilauroyl phosphatidylchorine (DLPC), and dioleoyl phosphatidylethanolamine (DOPE), and were composed in a molecular ratio of 1 : 2 : 2 as TMAG : DLPC : DOPE. We prepared two kinds of liposomes containing plasmids: one containing pcDNA–hDNase-γ (Lip(pcDNA3.1–hDNase-γ )) and another containing pcDNA3.1 (Lip(pcDNA3.1)). Gene transfer into cultured cells Aliquots of 4 × 104 human glioma cells were inoculated in each well of a Falcon plate (No. 3046) with 1 ml of medium and incubated for 12 h. Lip(pcDNA3.1– hDNase-γ ) or Lip(pcDNA3.1) (15 nmol of lipid/0.3 µg of DNA/ml) was added to the medium, and incubation was continued for 36 h. At the end of the incubation, cell numbers were counted using the trypan-blue exclusion method. For the detection of DNA fragmentation or Western-blotting, cells were cultured in a Falcon flask (No. 3110), and cells at 70% confluence were used. Observation of cell death induced by DNase-γ gene transfer under video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy To evaluate morphological changes in human glioma cells at high magnification, we employed a VEC-DIC microscopy system. The 15 × 104 cells were inoculated in a glass-bottomed culture dish (P35G-0-14-C, MatTec, USA) with 2 ml of culture medium, then incubated at 37◦ C in a humidified atmosphere of
27 5% CO2 and 95% air. After a 12-h incubation, Lip(pcDNA3.1–hDNase-γ ) was added to the dish. After another 24–48 h of incubation, cells were observed under the VEC-DIC microscope.
extracted using the same methods. One microgram of each purified DNA was loaded onto agarose gel in Tris-borate EDTA buffer and subjected to electrophoresis. After electrophoresis, DNA was visualized using ethidium bromide and a UV light source.
Detection of DNase-γ by Western-blotting Cells treated with Lip(pcDNA3.1–hDNase-γ ) were harvested 24 h after treatment, and total cell aliquots were assayed for protein content using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of protein were loaded onto gels in a sample buffer and subjected to electrophoresis in gels containing SDS and 15% (w/v) polyacrylamide. The protein was then transferred to a Hybond-P PDGF membrane (Amersham Bioscience, Piscataway, NJ), probed with anti-human DNase-γ monoclonal antibody hg303 (2 µg/ml), and visualized using an enhanced chemiluminescence (ECL) reagent (Amersham Bioscience). Detection of DNase-γ by anti-DNase-γ antibody Using an antibody recognizing activated-DNase-γ (hg302), an immunohistochemical study was performed. The staining procedure was as follows: cells in which apoptosis was induced with Lip(pcDNA3.1–hDNase-γ ) were washed with 0.1% Tween PBS and fixed with ice-cold methanol for 30 min at −20◦ C. Then washed with 0.1% Tween PBS three times and incubated with first antibodies: (×50 anti-DNase-γ antibody hg302 in 1% FBS, 0.1% Tween PBS) in a humidified chamber for 60 min at 37◦ C. The cells were again washed with 0.1% Tween PBS twice, and incubated with second antibodies: (×200 anti-mouse IgG antibody conjugated with FITC in 1% FBS, 0.1% Tween PBS) in a humidified chamber for 60 min at 37◦ C. They were washed with 0.1% Tween PBS again twice and observed under the fluorescence microscope.
Results Production of hDNase-γ in human glioma cells treated with liposome containing its gene After the liposomes containing pcDNA3.1–hDNase-γ were added to the cells and maintained at 37◦ C for 24 h, cells were harvested and subjected to Westernblotting for DNase-γ . Overexpression was confirmed in the gene-transferred cells (Figure 1). Cytotoxic effects on human glioma cells treated with Lip(pcDNA3.1–hDNase-γ ) Next, we investigated whether overexpression of DNase-γ conferred any therapeutic benefits on human glioma cells. As shown in Figure 2, lip(pcDNA3.1– hDNase-γ ) inhibited the growth of human glioma cells significantly. In contrast, empty liposomes (data not shown) as well as liposomes containing a backborn plasmid (Lip(pcDNA3.1)) did not significantly reduce the cell growth. Investigating the mechanism of growth suppression, we observed the morphological changes in DNase-γ -transferred U251SP cells. Those observations were made 24–48 h after gene transduction. We observed a cell death characterized by cytoplasmic shrinkage together with membrane blebbing and ballooning, which indicated apoptotic cell-death (Figure 3).
Detection of DNA fragmentation during apoptotic process To detect the DNA fragmentation during apoptosis, we used the DNA laddering kit (Roche Molecular Biochemicals, Mannheim, Germany). Forty-eight hours following the transfer of DNase-γ gene, cells were harvested and DNA was extracted. Simultaneously, DNA from non-treated cells was
Figure 1. Western-blotting confirming the overexpression of DNase-γ . Cells treated with Lip(pcDNA3.1–hDNase-γ ) were harvested after 6, 12, and 24 h of gene transfer. Cells without treatment or those treated with empty liposomes for 24 h were also harvested. Same amount of protein extracted from these cells was subjected to immuno-blotting of human DNase-γ .
28 To confirm DNA fragmentation in human glioma cells treated with Lip(pcDNA3.1–hDNase-γ ), we performed a DNA laddering assay. Figure 4 (right) shows the DNA fragmentation in U251SP cells observed
48 h after DNase-γ gene transfer. Subsequently, we performed immunohistochemistry to confirm the activation of DNase-γ (Figure 4, left). Positive staining was observed, suggesting the activation of DNase-γ protein. Efficacy of repeated DNase-γ gene transfer in glioma cells
Figure 2. Cytotoxic effects induced by Lip(pcDNA3.1– hDNase-γ ). Cell count was performed by trypan-blue exclusion methods. Aliquots of 4×104 cells were inoculated on Falcon plate (No. 3047) and, after 12 h of incubation, treated with liposomes containing plasmid encoding human DNase-γ or its backborn plasmid. Cell count was performed after 36 h of incubation of liposome administration. Growth reductions in DNase-γ -transferred cells were found, whereas no significant cytotoxity was observed in backborn transferred cells.
As shown in the previous literature, gene expression achieved by multilamellar cationic liposomes is only found in dividing cells, so that its efficacy is limited to some extent [19,22]. To overcome that limitation, we demonstrated that multiple injections of liposomes can enhance the level of gene expression in our previous studies [23]. Thus, we next tried 3 injections of Lip(pcDNA3.1–hDNase-γ ). As shown in Figure 5, a multiplicity-related enhancement of cytotoxicity was observed. Discussion Defects in apoptosis regulation are implicated in a variety of human diseases, including cancer [4]. It is likely that alterations in apoptotic pathways play a role
Figure 3. Morphological changes observed with VEC-DIC microscope. Upper figures were obtained after 36 h of liposome administration. Upper left: cells treated with empty liposome without plasmids (×200), upper middle: cells treated with liposomes containing human DNase-γ expression plasmid (×200), and upper right: ×400 magnification of cells observed in upper middle figures. Lower figures show the process of dying cells observed in DNase-γ -transferred cells. Morphological changes of apoptosis such as cell shrinkage, membrane blebbing, and ballooning can be seen.
29 control
DNase -γ
Control DNase -γ
Figure 4. Activation of DNase-γ in its gene-transferred U251SP cells. U251SP cells treated with liposomes containing DNase-γ expression plasmids were incubated for 36 h and its activation was investigated. The positive staining confirms the activation of DNase-γ in this DNase-γ -induced apoptotic process of U251SP cells. Upper left and upper right: immunohistochemistry of DNase-γ , lower left and lower right: same view as upper figures. Right: DNA laddering observed in DNase-γ -transferred U251SP cells. After gene transfer of DNase-γ , cells were harvested and DNA fragmentation was checked.
Figure 5. Enhanced cytotoxicity observed from multiple transfection methods. As efficacy of gene transfer is known from our previous studies to correlate with the number of injections, liposomes were injected to the culture medium of U251SP three times every 15 h (the doubling time of these cells). A cell count was performed after 72 h of first gene transfer using the same method as in Figure 2. The percentage growth inhibition against control cell numbers is shown with the data for cells injected with empty liposomes to rule out changes due to the toxicity of liposomes.
in the tumorigenesis of glioma and resistance to current therapies such as radio- and chemotherapy that induce apoptosis. Among new therapeutic strategies, gene transfer of proapoptotic molecules or molecules
that induce apoptosis are now being actively studied. Li et al. [7] demonstrated that the adenovirus-mediated Bax overexpression induces apoptosis in prostate cancer, Marcelli et al. [10] introduced the overexpression of caspase-7 as a new gene therapy strategy for prostate cancer, and Komata et al. [11] reported the effectiveness of a gene transfer of the gene coding activated caspase-6 in glioma cells. On the other hand, apoptotic signalings in tumor cells are now also arousing interest. Many reports regarding apoptosis pathways in leukemia cells or other cancer cells are continually being published (review in [24–26]). As for glioma cells, Knight et al. [27] reported that FasL and TRAIL induced an apoptotic pathway in glioma cells. These reports also describe the mechanisms of cellular resistance to apoptotic stimuli such as the lack of some components that participate in apoptotic signaling or the existence of some inhibitory proteins. One example of the former is the lack of caspase-8 in neuroblastoma cells [28], and an instance of the latter is the existence of a decoy receptor for FasL [29,30]. Taking these mechanisms of resistance to apoptotic stimuli into consideration, more downward molecules such as DNA endonucleases might have some stronger therapeutic benefits.
30 In our recent investigation of apoptotic pathways induced by IFN-β in human glioma cells, we discovered the activation of DNase-γ , a recently introduced apoptotic DNA endonuclease (unpublished data). DNase-γ protein is known to be trapped in the nuclear membrane of non-apoptotic cells, and though the precise mechanisms are not clear, it internalize to the nucleus by some apoptotic stimulation and fragments genome DNA [31]. Thus, we hypothesized that overexpression of this protein using CMV promoter can induce an overflow of DNase-γ so that such overflowing, un-trapped DNase-γ enters the nucleus. As research in our laboratory is concentrating on a gene transfer method using multilamellar cationic liposomes, and since IFN-β gene therapy using such a liposome has already begun for patients with malignant glioma, we utilized this liposomal gene transfer method [19–22,32]. By monitoring the cellular proliferation, we demonstrated a growth reduction in DNase-γ genetransferred cells (Figure 2), the mechanism of which was apoptotic cell-death as defined by morphological change and DNA fragmentation. An activated form of DNase-γ was also detectable in this cell death. From these observations, we conclude that overexpression of DNase-γ causes DNA fragmentation and induces apoptosis in human glioma cells. U251SP cells are cells whose DNA fragmentation can hardly be detected via chemotherapeutic agents CDDP, VP-16, and MCNU or by the apoptosis-inducing agent FasL (data not shown). Considering U251SP cells to be apoptosisresistant cells in the sense that DNA fragmentation can scarcely be detected, this finding suggests that the role of DNase-γ is very important in apoptotic DNA fragmentation. Moreover, although the precise mechanisms underlying apoptotic morphological changes such as cell shrinkage, membrane blebbing, and ballooning are yet to be defined, these findings also suggest the possibility of apoptosis occurring through DNA fragmentation even in apoptosis-resistant tumor cells. One limitation inherent in these kinds of therapeutic strategies is their relatively low efficiency of gene transfer. To overcome this problem, we showed the effectiveness of multiple injections (Figure 5). Moreover, apoptotic cells are reported to induce immune activation [33–35]. This mechanism may enhance the therapeutic effects in clinical use. Our findings support the idea that DNase-γ gene transfer can condemn the cells to death by apoptosis, and suggest that this gene is a promising candidate for future gene therapeutic strategy.
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Address for offprints: Jun Yoshida, Department of Neurosurgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; Tel.: +81-52-744-2355; Fax: +81-52-744-7261; E-mail:
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