Etoposide

Aims: In a variety of tumors, the susceptibility of the tumor cells to appptotic cell death following chemotherapy is a major determinant of therapeutic outcome. Gliomas are resistant to most chemotherapeutic agents, and its mechanism is not known in detail. In an attempt to understand the mechanism of chemo-resistance, we investigated the roles of insulin-like growth factor-I (IGF-I), IGF-I receptors (IGF-IR), and their relationship with the apoptotic response of two glioma cell lines to etoposide, a chemotherapeutic agent for malignant gliomas.

Methods: Two human glioma cell lines, U-87MG and KNS-42, were used. Etoposide-induced cell growth inhibition was quantified using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide), colorimetric assay. Hoechst 33258 staining, DNA fragmentation assay, and western blot were used for the evaluation of apoptosis. ApoAlert(TM) caspase assay was used for measuring the activity of caspase-3 (CPP32) and interleukin-1β-converting enzyme (ICE) protease. In addition, the effect of IGF-IR antisense was tested in U-87MG and KNS-42 glioma cell lines.

Results: Etoposide inhibited the growth of U-87MG and KNS-42 cells in a concentration-dependent manner. Etoposide increased the expression of wild-type p53, activated CPP32 (but not ICE) activity, and induced apoptosis in these cells. IGF-I prevented etoposide-induced apoptosis by increasing the expression of bcl-2 and decreasing the activity of CPP32. IGF-IR antisense enhanced the apoptotic effect of etoposide. Conclusions IGF-I decreased etoposide-induced apoptosis in glioma cells by increasing the expression of bcl-2 and decreasing the activity of CPP32. The antisense of IGF-IR increased etoposide-induced apoptosis. The anti-apoptotic effect of IGF-I and IGF-IR might be related to the chemo-resistance of glioma to chemotherapeutic agents. [Neural Res 2005; 27: 27-35]

Keywords: Antisense; apoptosis; etoposide; glimoa; IGF-I; IGF-IR

INTRODUCTION

Human malignant gliomas are among the most difficult tumors to treat successfully. Despite advances in the understanding of cancer molecular biology, gliomas are resistant to most chemotherapeutic agents, including etoposide, and the prognosis for patients with glioblastoma multiforme remains dismal1'2. It has been established that one of the most important actions of anticancer agents is their ability to induce apoptosis in the target cells. In a variety of tumors, the susceptibility of the tumor cells to apoptosis following chemotherapy is a major determinant of therapeutic outcome3"". However, the chemo-resistance mechanism in glioma cells is not known in detail. The relationship between insulin-like growth factor-l (IGF-I) and IGF-I receptor (IGF-IR) and the expression of wild-type p53 and bcl-2 and the activity of interleukin-1β-converting enzyme (ICE) or caspase-3 (CPP32) protease in etoposide-induced apoptosis in glioma cells has received little attention.

IGF-I is a major survival factor in serum and induces pleiotropic responses, including the regulation of growth and differentiation in many cell types. IGF-I elicits its responses by the activation of IGF-IR6'7, which in turn activates their kinase activity and results in the phosphorylation of the insulin receptor substrate I. IGF-I can prevent apoptosis under a wide variety of circumstances, including growth factor withdrawal, overexpression of myc, and especially in chemotherapy8. IGF-I is an important mitogen. It is highly expressed in many malignant gliomas9'10 and modulates the growth of glioblastomas multiforme10. IGF-I has been suggested as a putative autocrine stimulator of tumor cell proliferation and tumor growth in the central nervous system9"13. Expression of IGF-IR has been shown in human glioma9'14"16. Multiple transcripts from the IGF-I gene are seen in malignant glioma13. It is also reported that the pattern of IGF-I and IGF-IR expression has been associated with the development of malignant phenotypes in glioblastomas12. Thus, IGF-I and IGF-IR might interfere with the action of etoposide to induce apoptosis in tumor cells.

We used human LJ-87MG and KNS-42 glioma cell lines in the present study. The expression of apoptosis-related proteins, such as p53 and bcl-2 and the activity of ICE or CPP32 protease, were evaluated in the presence of etoposide with or without IGF-I. In addition, the effect of IGF-IR antisense was tested in U-87MG and KNS-42 glioma cell lines.

MATERIALS AND METHODS

Cells and drugs

Two human glioma cell lines (U-87MG and KNS-42), provided by the Cancer Research Resources Bank (Tokyo, Japan), were maintained in Dulbecco's modified Eagle's medium, containing glutamine, 10% fetal calf serum, and penicillin/streptomycin (100 U ml^sup -1^ : 10 µg ml^sup -1^). Cells were grown at 37°C in a 5% CO2 incubator.

Human recombinant IGF-I was purchased from Biomedical Technologies, Inc. (Stoughton, MA). Etoposide was purchased from Sandoz Pharmaceuticals (Tokyo, Japan) and was dissolved in dimethylsulfoxide (DMSO) as a 75 mg ml^sup -1^ stock solution. The final concentration of DMSO in the culture medium was

Cell growth

The inhibitory effect of etoposide in U-87MG and KNS-42 cells was quantified using a modified MTT colorimetric assay (Chemicon, Temecula, CA)17. Cells were seeded at 104 cells/well in 0.1 ml of medium in 96-well flat-bottomed plates (Corning, Corning, NY) and incubated overnight at 37°C. Then, various concentrations of etoposide, from 10 to 80 µM, were added to the wells. After incubation for 24-72 hours, 0.01 ml of the MTT reagent was added to each well for an additional 4-hour incubation at 37°C. lsopropanol (0.1 ml with 0.04 N HCI) was added to dissolve the precipitates, and the solvents were measured in an automated reader (ER8000, Sanko Junyaku Co., Tokyo, Japan) at 570 nm.

Hoechst 33258 staining

Etoposide-treated cells were stained with Hoechst 33258 as described previously18. The treated cells were fixed in 1.0% formaldehyde and 0.2% glutaraldehyde for 5 minutes, washed in phosphate-buffered solution (PBS) twice, and incubated with Hoechst 33258 (8 µg ml^sup -1^) for 15 minutes at room temperature. 500 cells were counted and scored for the incidence of apoptotic chromatin changes under fluorescence microscopy.

Analysis of DNA fragmentation

Analysis of DNA fragmentation was performed as described previously19. The harvested cells (1×10^sup 7^) were centrifuged and washed twice with cold PBS. The cell pellet was lysed in 1.OmI buffer consisting of 10 mM Tris-HCI, 10 mM ethylenediamine tetraacetic acid (EDTA), and 0.2% Triton X-100 (pH 7.5). After 10 minutes on ice, the lysate was centrifuged (13 000 g) for 10 minutes at 4°C. The RNA and fragmented DNA in the supernatant were extracted, first with phenol and then with phenol-chloroform-isoamyl alcohol (25:24:1, vol : vol). The aqueous phase was brought to 300 mM NaCI, and the nucleic acids were precipitated with 2 volumes of ethanol. The pellet was rinsed with 70% ethanol, air-dried, and then dissolved in 20 µl of 1OmM Tris-HCl-1 mM EDTA (pH 7.5). After digestion of the RNA with ribonuclease A (0.6 mg ml^sup -1^ at 37°C for 30 minutes), the samples were electrophoresed in a 2% agarose gel with Boyer's buffer (50 mM Tris-HCI, 20 mM sodium acetate, 2 mM EDTA, and 18 mM NaCI at pH 8.05). The DNA was visualized with ethidium bromide staining.

Western blotting assay

The expressions of IGF-IR, wild-type p53, and bcl-2 proteins in the U-87MG and KNS-42 cells were assessed by immunoblotting, using a monoclonal antibody to IGF-IR (Santa Cruz Biotechnology, Santa Cruz, CA), to the wild-type p53 protein (Oncogene Science, Uniondale, NY), and to the bcl-2 protein (DAKO, Glostrop, Denmark). Sub-confluent cells treated with or without etoposide were rinsed twice with ice-cold PBS, pelleted at 15 000 g for 5 minutes, and lysed in 500 µl of extraction buffer [1% Triton X-100, 1% sodium deoxycholate, 0.01% SDS, 0.15 M NaCl, 50 mM Tris-HCI (pH 7.4), and 2 mM phenylmethyl sulfonylfluoride]. Equal amounts (1 mg ml^sup -1^) of proteins were boiled for 3 minutes at 10O°C in a buffer containing 2.5% SDS, 5% 2-mercaptoethanol, and 1OmM Tris-HCI (pH 8.0), and subjected to SDS-PAGE. Electrophoresis was performed for 3 hours at 35 mA in a 0.4 mm thick, 10% polyacrylamide gel at room temperature. The proteins were transferred electrophoretically to nitrocellulose filters at a constant voltage of 8 V for 1 hour. The filters were blocked with 5% non-fat milk, after which specific monoclonal antibodies against IGF-IR, wild-type p53, and bcl-2 were used for immunoblotting. The signals were detected using the ECL system (Amersham USA, NY).

ApoAlert(TM) CPP3 and ICE colorimetric assay

Among members of the ICE family, ICE and CPP32 have different substrate specificities. ApoAlert(TM) caspase assay kits (Clontech, Inc., PaIo Alto, CA) were used in the assaying of CPP32 and ICE protease activity. Harvested cells (2 × 10^sup 6^) were centrifuged and washed twice with cold PBS. The cell pellet was re-suspended in 50 µl of chilled cell lysis buffer (1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 1 mM PMSF, 1µg ml^sup -1^ aprotinin, 1 µg ml^sup -1^ leupeptine, 2 mM DTT, 10 mM Tris-HCI, pH 8.0). After 10 minutes on ice, the lysate was centrifuged at 12 000 rpm for 3 minutes at 4°C. The supernatants were transferred to new microcentrifuge tubes. First, 50 µl of 2 × reaction buffer (10OmM HEPES, 10% sucrose, 0.1% CHAPS, pH 7.5, 1 mM PMSF, 1 µg ml^sup -1^ aprotinin, 1 µg ml^sup -1^ leupeptine, 6 mM DTT) was added to each reaction, then 5 µl of 1 mM conjugated substrate (DEVD-pNA for CPP32, YVAD-pNA for ICE) was added to each tube. After incubation at 37°C for 1 hour in a water bath, detection of protease activity was performed in a spectrophotometer at 405 nm. The negative control cells also were analyzed by assaying a culture that had not been treated with etoposide.

Plasmid transfections

The expression vector HSP-IGF-IRAS produces antisense mRNA to the first 309 bp of cDNA fragment of IGF-IR including a 30-amino acid signal peptide sequence under the control of the Drosophila HSP70 promoter20'21. A neomycin resistance gene under control of the SV40 promoter is present at the 3' end of the IGF-IR fragment. Cells near 70% confluence in 60-mm plates were transfected using CellPhect Transfection Kit (Amersham Biosciences, Piscataway, NJ, USA). After 7 days of transfection, independent colonies of transfected cells were selected by adding G418 (600 µg ml^sup -1^, Sigma, St Louis, MO). After IGF-IR antisense transfection and G418 treatment, G418-resistant colonies were isolated. Selected transfectants were maintained in medium as mentioned above with G418. The expression of IGF-IR in U-87MG/IGF-IR-antisense and KNS-42/IGF-lR-antisense cells was assessed by immunoblotting as described above.

Statistical analysis

In this study, data are expressed as the mean ±SD and were analyzed using Student's f-test. A probability value

RESULTS

Etoposide inhibited glioma cell growth

U-87MG and KNS-42 cells were exposed to differing concentrations of etoposide (10-80 µM), and an MTT assay was performed to determine the effect of etoposide on cell growth (Figure 1A,B). Etoposide, incubated with cells for 48 hours, inhibited the viability of U-87MG and KNS-42 cells in a concentration-dependent manner. More than 60% of cells died after being treated with a high dose of etoposide (80 µM). Pre-treatment of cells with IGF-I (10 nM) significantly inhibited the cytotoxic effect of etoposide and reduced the rate of viability to below 20% (Figure 1A,B).

The inhibitory effect of etoposide was time-dependent (from 1 to 4 days) in U-87MG and KNS-42 cells (Figure 2A,B). Etoposide (50 µM, roughly the IC^sub 50^ value from Figure IA,B) dramatically increased the percentage of apoptotic cells in 2 days and increased the percentage of apoptosis to 80% on day 4, the longest time period studied. IGF-I (10 nM) significantly reduced the percentage of apoptotic cells and controlled the apoptosis within 40% on day 4.

As a negative control, 0.1% DMSO had no influence on cell growth examined up to 4 days (data not shown).

Etoposide produced apoptosis (Hoechst staining)

When Hoechst staining was conducted 2 days after adding etoposide (50 µM), typical apoptotic morphology, including nuclear condensation and fragmentation, was present in 56% of U-87MG cells (Figure 2A) and 52% of KNS-42 cells (Figure 2B). The apoptotic effect of etoposide is time-dependent, and it is reversible if a low concentration of etoposide was used. In cultures that were exposed to lower concentrations of etoposide (10 or 20 μ?), removal of etoposide after 2 or 3 days of treatment interrupted the apoptotic effect of etoposide, and cells recovered and re-grew (data not shown). However, in cultures that were treated with higher doses of etoposide (50 or 80 µM), even after etoposide was removed after 2 or 3 days, cells died continuously without recovery.

Etoposide produced DNA ladders

In another series of experiments, DNA fragmentation was assessed in U-87MG and KNS-42 cells exposed to etoposide (50 µM) for 48-72 hours. Etoposide produced DNA fragmentation in U-87MG and KNS-42 cells at 48 hours (Figure 3). Pre-treatment of cells with IGF-I (1OnM) prevented DNA ladders in the presence of etoposide at 48 and 72 hours (Figure 3).

Etoposide increased wild-type p53 protein expression

The effect of etoposide (50 µM) on the wild-type p53 levels in these glioma cells during apoptosis was assessed before the addition of 50 µM of etoposide, as well as 24, 48, and 72 hours after. At the various time points, the cell lysates were analyzed using western blotting. Untreated U-87MG and KNS-42 cells had no detectable wild-type p53. Etoposide increased the expression of wild-type p53 at 48 hours and increased the levels of p53 further at 72 hours (Figure 4). Pretreatment of cells with IGF-I (10 nM) abolished the effect of etoposide on the expression of the wild-type p53 in these glioma cells (Figure 4).

Etoposide increased CPP32 but not ICE protease activity

VVe investigated whether etoposide-induced apoptosis was mediated by the activation of caspase family proteases. U-87MG and KNS-42 cells were treated with etoposide (50 μ?), and the activities of CPP32 and ICE in the cytosolic extracts were determined. Etoposide increased CPP32 but not ICE activity in U-87MG (Figure 5A) and KNS-42 (Figure 5B) cells. The CPP32 activity in U-87MG and KNS-42 cells increased at 6 hours and peaked at 8 hours after etoposide treatment. The values of the caspase activity at 8 hours after etoposide treatment were 2.18- and 2.16-fold more than the control levels in U-87MG and KNS-42 cells, respectively. Pre-treatment of cells with IGF-I (10 nM) abolished the effect of etoposide on the CPP32 activity (Figure 5A,B).

IGF-I increased bcl-2 expression

Expression of the bcl-2 protein in U-87MG and KNS-42 cells before and after treatment with IGF-I was analyzed. IGF-I (10 nM) increased the expression of bcl2 at 24 and 48 hours in U-87MG and KNS-42 cells (Figure 6). Bcl-2 was not detectable in either cell lines in the absence of IGF-I.

Expression of IGF-IR in glioma cells

The presence of the 98-kDa IGF-IR was examined in U-87MG and KNS-42 cells by western blotting. There was no detectable expression of IGF-IR in either U87MG or KNS-42 cells cultured in normal medium without IGF-I (Figure 7). The IGF-IR was highly expressed in cells either cultured in serum-free medium or in medium containing 10 nM22 IGF-I for 12 hours (Figure 7). These results show that the U-87MG and KNS-42 cells may have too few receptors in normal cultured conditions to be detected by western blotting. Serum-free medium or IGF-I can enhance the expression of IGF-IR in these glioma cells.

Effect of IGF-IR antisense on apoptosis

Western blot was used to verify the transfection of IGF-IR antisense into these glioma cells and the effect on the expression of the IGF-IR protein. The U-87MG/ IGF-IR-antisense and KNS-42/IGF-IR-antisense cells had no detectable IGF-I receptors, either in normal cell cultures or in cells treated with serum-free medium or medium containing 10 nM IGF-I (Figure 8).

Effects of IGF-IR antisense on the sensitivity of the U87MG and KNS-42 cells pre-treated with IGF-I to etoposide were examined using Hoechst 33258 staining. The U-87MG/IGF-IR-antisense and KNS-42/IGF-IR-antisense cells in the presence of IGF-I (10 nM) were treated with etoposide (50 µM). Etoposide produced a significant increase in the percentage of apoptotic cells in IGF-IR antisense-transfected cells. IGF-I failed to protect cells from apoptosis in IGF-IR antisense-transfected cells (Figure 9). Transfection of IGF-IR antisense into the human U-87MG and KNS-42 cells resulted in an enhanced effect of etoposide, although the glioma cells were pre-treated with IGF-I. The controls were not shown in Figure 9 as they were similar to those in Figure 2.

DISCUSSION

Gliomas are the most common primary brain tumors23'24. Continued progress has been made in the chemotherapeutic approach to treating patients with malignant brain tumors. Etoposide is a semi-synthetic epipodophyllotoxin derivative displaying definite antitumor activity25'26. It is a topoisomerase inhibitor and is a relatively phase-specific agent that inhibits cells in the G2 phase of the cell cycle 5~28. It induces breaks in single- and double-stranded DNA and has been shown to damage DNA by interacting with the enzyme type Il topoisomerase27'28. This enzyme normally catalyzes DNA from interconversions by introducing a transient enzyme bridged, double-strand break on one or two crossing DNA segments. We have demonstrated that the cytotoxicity of etoposide has been assigned to the induction of apoptotic cell death in glioma cell lines29. In a variety of tumors, the susceptibility of the tumor cells to apoptotic cell death following chemotherapy is a major determinant of therapeutic outcome. Etoposide has been used most often in combination chemotherapy as salvage therapy for recurrent glioma25"28. Although etoposide has shown some evidence of efficacy25'26, clinical studies with etoposide have been less encouraging than laboratory data1'2. In the present study, we have demonstrated an important role of IGF-I and IGF-IR in the protection of glioma cells from apoptosis induced by etoposide. Etoposide produced apoptosis in two glioma cell lines that were identified by DNA fragmentation, Hoechst staining, p53 expression, and CPP32 activity. IGF-I activated IGF-IR in the glioma cell lines and abolished etoposide-induced apoptosis by increasing the expression of bcl-2 and decreasing the activity of CPP32 protease. Moreover, etoposide produced apoptosis in the presence of IGF-I in IGF-IR antisense cells. These results demonstrate that IGF-I and IGF-IR reduced etoposide-induced apoptosis, and this might be related to chemo-resistance of glioma to etoposide.

Apoptosis represents a fundamental intracellular program that is regulated both positively and negatively at various levels within the signaling pathways. The susceptibility of cells to undergo apoptosis under specific receptor activation or toxic agents, such as chemotherapeutic agents or radiation, depends in part on growth factor-regulated intracellular signals that affect the progression of the apoptosis pathway22'25'30"40. The molecular characterization of gliomas has revealed a number of abnormalities, such as p53 mutation, amplification of the epidermal growth factor receptor, and overexpression of ras22' . However, little data is available on the role of IGF-I and IGF-IR on apoptosis induced by etoposide in glioma cells. In the present study, human malignant glioma cell lines were used to investigate the relationship between apoptosis (expression of wild-type p53, bcl-2, and ICE or CPP32 protease activity) induced by etoposide and the anti-apoptotic effect of IGF-I and IGF-IR. Cultured human malignant glioma cells exhibit differential time- and concentration-dependent susceptibilities to etoposide. In addition to stimulating apoptosis, chemotherapeutic agents have been shown to induce the nuclear accumulation of the p53 protein in fibroblastoid cells, as well as in epithelioid normal and immortalized cells of murine, simian, and human origin54. Accumulation occurs because of increased p53 protein stability and depends on ongoing translation34'41"43. It is not the result of enhanced gene expression34. A number of cell cycle inhibitors do not affect p53 protein accumulation, suggesting that the process may start from several points in the cell cycle34'41"43. The results that etoposide enhanced the expression of p53 and increased the activity of CPP32 protease are consistent with several recent studies indicating that DNA-damaging agents induce the accumulation of p5334'44 and enhance CPP32 protease activity45. Pre-treatment of glioma cells with IGF-I reduced the expression of p53 and CPP32 activity by etoposide and abolished apoptosis.

Part of the protective effect of IGF-I is mediated by the enhancement of bcl-2. The bcl-2 family is extremely important in the regulation of the susceptibility to apoptosis of many cell types37. Over-expression of bcl-2 or bcl-x protects cells from apoptosis °'31-36/ and loss of bcl-2 or bcl-x results in excessive apoptosis during development36'37'39. Unique among oncogenes, bcl-2 exerts its oncogenic effect via inhibition of apoptosis rather than enhancement of cell cycle progression30'36. The bcl-2 oncoprotein prolongs the survival of cells in the absence of required growth factors by blocking apoptosis even in the presence of apoptosis-inducing stimuli40. Exogenous bcl-2 has been shown to protect haemopoietic cells from apoptosis induced by growth factor withdrawal, heat shock, irradiation, and chemotherapy30'36'39'40. Loss of bcl-2 function in knockout mice results in death shortly after birth due to excessive apoptosis of lymphoid tissue and polycystic kidneys46. In the present study, the antiapoptotic effect of IGF-I was mediated probably by the enhancement of bcl-2 expression. These results are consistent with another report that IGF-I inhibits apoptosis in serum-deprived PC-12 cells by augmentation of bcl-xL expression47. Recently, it has been reported that the bcl-2 proteins are homologs of Ced-9 and regulate cell survival by controlling the activity of the ICE family proteases36'37. Our studies suggest that up-regulation of bcl-2 expression may help to decrease CPP32 activity, and may be correlated with increased cell survival. Since tumor growth can be attributed partly to decreased cellular susceptibility to apoptosis31'36'40'48, the relationship observed between increased expression of bcl-2 in cells and cell survival is further evidence that this protein can confer a survival advantage. IGF-I increased bcl-2 expression, reduced the apoptotic effect of etoposide and might lead to the poor effectiveness of etoposide in the treatment of gliomas.

In Caenorhabditis elegans, the activities of two genes, ced-3 and ced-4, are essential for apoptosis. Ced-3 shows sequence similarity to the mammalian ICErelated protease that is thought to trigger the execution phase of apoptosis35'38. ICE was described originally as a cysteine protease isolated from cells of monocytic origin49. The ICE family of protease consists of at least seven cloned enzymes22. When over-expressed in transfected cells, all of these proteases induce apoptosis. Caspase-3 (CPP32) is the most widely investigated member of the ICE family and appears to play a dominant role in apoptotic death signaling32'3 . Both ICE and CPP32 were examined in the present study in human malignant glioma cells and found that at least CPP32 is involved in etoposide-induced apoptosis. IGF-I prevented etoposide-induced apoptosis probably by decreasing the activity of CPP32. CPP32 seems more effective or more important than ICE protease in etoposide-induced apoptosis in human malignant glioma cells. Decreasing IGF-I and increasing CPP32 might need to be taken into consideration for the future design of anticancer therapies.

IGF-I, IGF-II, and insulin activate IGF-IR and play important roles in the development, growth, and survival of normal cells. IGF-IR is an important autocrine/paracrine growth factor receptor commonly expressed in malignant gliomas9. IGF-I and IGF-IR have both been found to be involved in the inhibition of apoptosis in diverse cell types8'48. Since tumor growth depends in part on the balance of cell death versus proliferation, the autocrine/paracrine pathway of IGF-I stimulation is crucial for tumor growth and thus many malignant tumors secrete IGF-I, IGF-II, and over-express IGF-IR9. The extent of IGF-IR expression in gliomas might be an important factor in the determination of the response to chemotherapy. The anti-apoptotic effect of IGF-I is mediated by its receptors' IGF-IR since antisense of IGF-IR abolished the protective effect of IGF-I. Observations explained not only a positive role of IGF-IR in the glioma growth and the protective effect of IGF-I and IGF-IR in etoposide-induced apoptosis, but also offered a new strategy, anti-IGF-IR, for future treatment.

In conclusion, IGF-I (IGF-IR) decreased etoposideinduced apoptosis in glioma cells by increasing the expression of bcl-2 and decreasing the activity of CPP32. The anti-apoptotic effect of IGF-I and IGF-IR might be related to chemo-resistance of glioma to chemotherapeutic agents.

ACKNOWLEDGEMENTS

This study was partially supported by the Department of Neurosurgery at Kobe University School of Medicine in Kobe, Japan and by the Department of Neurosurgery at the University of Mississippi Medical Center in Jackson, Mississippi, USA.

REFERENCES

1 Chamberlain MC. Recurrent supratentorial malignant gliomas in children. Long-term salvage therapy with oral etoposide. Arch Neural 1997; 54: 554-558

2 Hellman R, Neuberg DS, Wagner H, et al. A therapeutic trial of radiation therapy with vincristine, etoposide, and procarbazine (VVP) in high grade intracranial gliomas-an Eastern Cooperative Oncology Group Study (E2392). J Neurooncol 1998; 37: 55-62

3 Barry MA, Behnke CA, Eastman A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs; toxins and hyperthermia. Biochem Pharmacol 1990; 40: 2353-2362

4 Holbrook NJ, FornaceJR. Response to adversity: Molecular control of gene activation following genotoxic stress. New Biol 1991; 3: 825-833

5 Kaufmann SH. Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin and other cytotoxic anticancer drugs: A cautionary note. Cancer Res 1989; 49: 5870-5878

6 Baserga R, Sell C, Porcu P, ef al. The role of the IGF-I receptor in the growth and transformation of mammalian cells. Cell Prolif 1994; 27: 63-71

7 Leroith D, Werner H, Beitner-Johnson D, ef a/. Molecular and cellular aspect of the insulin-like growth factor-l receptor. Endocr Rev 1995; 16: 143-163

8 Harrington EA, Bennett MR, Fanidi A, ef a/. c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines. EMBO I 1994; 13: 3286-3295

9 Gammeltoft S, Ballotti R, Kowalski A, efa/. Expression of two types of receptor for insulin-like factors in human malignant glioma. Cancer Res 1988; 48: 1233-1237

10 Sandberg-Nordqvist AC, Stahlbom PA, Reinecke M, ef a/. Characterization of insulin-like growth factor I in human primary brain tumors. Cancer Res 1993; 53: 2475-2478

11 Friend KE, Khandwala HM, Flyvbjerg A, efal. Growth hormone and insulin-like growth factor-l: Effect on the growth of glioma cell lines. Growth Horm IGF Res 2001; 11: 84-91

12 Hirano H, Lopes MB, Laws ER Jr, efa/. Insulin-like growth factor-l content and pattern of expression correlates with histopathologic grade in diffusely infiltrating astrocytomas. Neuro-oncology 1991; 1: 109-119

13 Sandberg AC, Lake M, Engberg C, ef a/. The expression of insulinlike growth factor I and insulin growth Il genes in the human fetal and adult brain and in glioma. Neurose/ieff 1 988; 93: 114-119

14 Antoniades HN, Galanopoulos T, Neville-Golden J, ef al. Expression of insulin-like growth factors 1 and 2 and their receptor mRNAs in primary human astrocytomas and meningiomas: In vivo studies using in situ hybridization and immunocytochemistry. InIJ Cancer 1992; 50: 215-222

15 Click RP, Gettleman R, Patel K, ef al. Insulin and insulin-like growth factors in brain tumours: Binding and in vitro effects. Neurosurgery (Baltimore) 1989; 24: 791-797

16 Sara VR, Prisell P, Sjogren B, ef a/. Enhancement of insulin-like growth factor 2 receptors in glioblastoma. Cancer Lett 1986; 32: 229-234

17 Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assay. J lmmunol Methods 1983; 65: 55-63

18 Kobayashi T, Consoli U, Andreeff M, ef al. Activation of p21CIP1/ WAFI expression by a temperature-sensitive mutant of human p53 dose not lead to apoptosis. Oncogene 1995; 11: 2311-2316

19 Yin D, Kondo S, Takeuchi J, ef a/. Induction of apoptosis in murine ACTH-secreting pituitary adenoma cells by bromocriptine. FFBS Lett 1994; 339: 73-75

20 Craig E, McCarthy BJ, Wadsworth SC. Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster. Cell 1979; 16: 575-588

21 Resnicoff M, Sell C, Rubini M, ef al. Rat glioblastoma cells expressing an antisense RNA to the insulin-like growth factor-I(IGF-I) receptor are nontumorigenic and induce regression of wild-type tumors. Cancer Res 1994; 54: 2218-2222

22 Duan H, Chinnaiyan AM, Hudson PL, ef a/. ICE-LAP3, a novel mammalian homologue of the Caenorhabditis elegans cell death protein Ced-3 is activated during Fas- and tumor necrosis factor-induced apoptosis. ; Biol Chem 1996; 271: 1621-1625

23 Kornblith PL, Walker MD, Cassady JR. Neurologic Oncology. Philadelphia: JB Lippincott, 1987: pp. 35-48

24 Lang FF, Miller DC, Koslow M, ef a/. Pathways leading to glioblastoma multiforme: A molecular nanlysis of genetic alterations in 65 astrocytic tumors. J Neurosurg 1994; 81: 427-436

25 Chamberlain MC. Recurrent brainstem gliomas treated with oral VP-16. J Neurooncol 1993; 15: 133-142

26 Giannone L, Wolff SN. Phase Il treatment of central nervous system gliomas with high-dose etoposide and autologous bone marrow transplantation. Cancer Treat Rep 1987; 71: 759-768

27 Fleming RA, Miller AA, Stewart CF. Etoposide: An update. Clin Pharm 1989; 8: 274-293

28 Slevin ML. The clinical pharmacology of etoposide. Cancer 1991; 67: 319-329

29 Yin DL, Tamaki N, Kokunai T. Wild-type p53-dependent etoposide-induced apoptosis mediated by caspase-3 activation in human glioma cells. J Neurosurg 2000; 93: 289-297

30 Boise LH, Gonzalez-Garcia M, Postema CE. bcl-x, a bcl-2 related gene that function as a dominant regulator of apoptotic cell death. Cell 1 993; 74: 597-608

31 Chiou SK, Rao L, White E. bcl-2 blocks p53-dependent apoptosis. MoI Cell Biol 1994; 14: 2556-22563

32 Enari M, Talanian RV, Wong WW, et al. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature 1996; 380: 723-726

33 Fernandes-Alnemri T, Litwack G, Alnemri ES. CPP32, a noval human apoptotic protein with homology to Caenorhaditis eleganscell death protein Ced-3 and mammalian lnterleukin-1 β-converting enzyme. ) Biol Chem 1994; 267: 30761-30764

34 Fritsche M, Haessler C, Brandner G. Induction of nuclear accumulation of the tumor-suppressor protein p53 by DMA-damaging agents. Oncogene 1993; 8: 307-318

35 Gagliardini V, Fernandez PA, Lee RKK, ef al. Prevention of vertebrate neuronal death by the crmA gene. Science (Washington DC) 1994; 262: 826-828

36 Hockenbery DM. bcl-2 in cancer, development and apoptosis. J Cell Sd 1994; 18 (Supple.): 51-55

37 Knudson CM, Tung KSK, Tourtellotte WG. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 1995; 270: 96-99

38 Miura M, Zhu H, RotelIo R, Hartwieg ET, ef al. Induction of apoptosis in fibroblasts by IL-1 β-converting enzyme, a mammalian homolog of the C. elegants cell death gene ced-3. Cell 1993; 75: 653-660

39 Motoyama N, Wang F, Roth KA. Massive cell death of immature hamatopoietic cells and neurons in Bcl-x-deficient mice. Science 1995; 267: 1506-1510

40 Nunez G, London L, Hockenbery D, er al. Deregulated bcl-2 gene expression selectively prolongs survival of growth factor-deprived hempoietic cell lines. J lmmunol 1990; 144: 3602-3610

41 Ceraline J, Deplanque G, Duclos B, eta/. Relationships between p53 induction, cell cycle arrest and survival of normal human fibroblasts following DNA damage. Bull Cancer 1997; 84: 1007-1016

42 Chikayama S, Sugano T, Takahashi Y, et al. Nuclear accumulation of p53 protein and apoptosis induced by various anticancer agents, u.v.-irradiation and heat shock in primary normal human skin fibroblasts. lnt J Oncol 2000; 16: 1117-1124

43 Hess R, Plaumann B, Lutum AS, et al. Nuclear accumulation of p53 in response to treatment with DNA-damage agents. Toxicol Lett 1994; 72: 43-52

44 Zambetti GP, Bargonetti J, Walker K, et al. Wild-type p53 mediates positive regulation of gene expression through a specific DNA sequence element. Genes Dev 1992; 6: 1143-1152

45 Erhardt P, Cooper GM. Activation of the CPP32 apoptotic protease by distinct signaling pathway with differential sensitivity to bcl-xL. J Biol Chem 1996; 271: 17601-17604

46 Veis DJ, Sorenson CM, Shutter JR, et aL. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmental hair. Cell 1993; 75: 229-240

47 Parrizas M, Leroith D. Insulin-like growth factor-l inhibition of apoptosis is associated with increased expression of bcl-xL gene product. Endocrinology 1997; 138: 1355-1358

48 Muta K, Krantz SB. Apoptosis of human erythroid colony-forming cells is decreased by stem cell factor and insulin-like growth factor I as well as erythropoietin. J Cell Physiol 1993; 156 264-271

49 Kostura MJ, Tocci MJ, Limjuco G, et al. Identification of a monocyte specific pre-lnterleukin 1 β convertase activity. Proc Natl Acad Sd USA 1989; 86: 5227-5231

Dali Yin*,[dagger], Norihiko Tamaki[dagger], Andrew D. Parent* and John H. Zhang*

*Department of Neurosurgery, University of Mississippi Medical Center, Jackson, Mississippi, USA

[dagger] Department of Neurosurgery, Kobe University School of Medicine, Kobe, Japan

Correspondence and reprint requests to: John H. Zhang MD, PhD, Department of Neurosurgery, Louisiana State University Health Sciences Center in Shreveport, 1501 Kings Highway, PO Box 33932, Shreveport, Louisiana, USA. [johnzhang3910@yahoo.com] Accepted for publication January 2004.

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