Induction of apoptosis in rat hepatocarcinoma cells by expression of IGF-I antisense c-DNA

Journal of Hepatology 1998; 29: 807–818 Printed in Denmark ¡ All rights reserved Munksgaard ¡ Copenhagen

Copyright C European Association for the Study of the Liver 1998

Journal of Hepatology ISSN 0168-8278

Induction of apoptosis in rat hepatocarcinoma cells by expression of IGF-I antisense c-DNA Sophie Ellouk-Achard1,4, Sepideh Djenabi1, Gilberto Antonio De Oliveira1, Genevie`ve Desauty1, Huynh Thien Duc2, Mishal Zohair3, Jerzy Trojan2, Jean Roger Claude4, Alain Sarasin1 and Christiane Lafarge-Frayssinet1 1 Laboratoire de Ge´ne´tique Mole´culaire, CNRS UPR 42, 2Centre He´patobiliaire, Hoˆpital Paul-Brousse, Laboratoire de Cytome´trie, CNRS, Villejuif, and 4Laboratoire de Toxicologie (EA207), Universite´ Rene´-Descartes Paris V, Paris, France

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Background/Aims: We have developed a gene therapy strategy based on the observation that insulin-like growth factor I (IGF-I) is necessary for the acquisition and maintenance of the transformed phenotype in hepatocarcinoma. This strategy consists in transfecting the rat hepatoma cell line with an episomal vector expressing the antisense IGF-I c-DNA under the control of the metallothionein I promoter inducible by zinc, decreasing therefore the level of IGFI in these cells. The transfected clones lost their tumorigenic properties, and were able to induce, in vivo, the regression of an established tumor in syngeneic rats. To understand the loss of tumorigenic properties of these transfected clones, we have quantified, by different approaches, the number of apoptotic cells according to the level of IGF-I expression. Methods: IGF-I antisense synthesis in transfected cells was stimulated using zinc. We then characterized

and quantified apoptosis, in these transfected clones, by morphological and DNA fragmentation analyses, flow cytometry and comet assay. Results: We have demonstrated that IGF-I inhibits the development of apoptosis in parental cells, that the transfected clones are able to restore the spontaneous apoptotic programme, and that apoptosis increases massively when overexpression of IGF-I antisense is caused by zinc stimulation of the metallothionein I promoter. Conclusion: The present results allow us to conclude that the level of apoptotic pathway in liver cell lines is directly related to the amount of IGF-I deficiency.

  synthesizes and secretes insulin-like growth factor type I (IGF-I) postnatally, under growth hormone control (1). IGF-I is a polypeptide hormone that regulates a variety of biochemical pathways in many cell types (2). Initially identified as a potent physiologic mitogen, IGF-I was thought to be implicated in the regulation of cell growth and differentiation, as a consequence of its anti-apoptotic property. IGF-I needs the receptor IGF-IR to be active, and many authors have demonstrated that the couple IGFI–IGF-IR is essential to the establishment and maintenance of the transformed phenotype (3–5). Although

the liver is the major IGF-I producer in the organism, the neoplastic transformation of hepatocytes is mainly accompanied by the increase in IGF-I synthesis. The antisense IGF-I or IGF-IR strategy not only inhibits tumorigenesis but also prevents the subsequent growth of tumoral cells and, indeed, can induce the regression of parental tumors. This strategy was used successfully to cure rat glioblastoma (6–8) and rat teratocarcinoma (9), and preliminary results have been described for human melanoma (10). In our laboratory, we have recently demonstrated that this strategy was suitable to cure rat hepatocarcinoma (11). We must emphasize that the human hepatocarcinoma is the most frequent cancer of the liver, and, despite the development of various therapeutic approaches, the successful treatment of hepatocellular carcinoma remains a clinical challenge (12,13). For these reasons, much effort has

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Received 15 April; revised 2 July; accepted 3 July 1998

Correspondence: Sophie Ellouk-Achard, Laboratoire de Ge´ne´tique Mole´culaire, CNRS UPR42, 7 rue Guy Moquet, 94800 Villejuif, France. Fax: (33) 1.49.58.34.11

Key words: Apoptosis; Comet assay; DNA fragmentation; Flow cytometry; Hepatocarcinoma; Gene therapy; IGF-I; In vitro.

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been devoted to the development of gene therapy for primary liver tumors. We have established a model from a hepatoma cell line, LF, derived from a hepatocarcinoma induced in Commentry rats by the chemical carcinogen 4-dimethylaminoazobenzene (DAB). This cell line was grown in soft-agar, and a clone, LFCl2A, presenting high tumorigenic properties and synthesizing a large amount of IGF-I was isolated (14,15) and used thereafter to develop a gene therapy strategy (11). To regulate the production of IGF-I, these neoplastic cells were transfected with an episomal vector expressing IGF-I c-DNA in an antisense position under the control of the mouse metallothionein I promoter inducible by zinc. Two clonogenic (B2Cl2 and B5Cl1) lines isolated and selected by hygromycin resistance after transfection were shown to have lost their tumorigenic properties and were able to induce, in vivo, inhibition of the growth of the parental cells and/or regression of established hepatocarcinoma in syngeneic Commentry rats (11). The mechanism by which the expression of IGF-I antisense protects syngeneic Commentry rats is poorly understood. Two phenomena can be suggested to explain the molecular events associated with the inhibition of tumorigenesis in syngeneic animals. On one hand, the host immune system was demonstrated to be involved in our model, with essentially the increased expression of CMH class I on the cell surface of transfected cells when compared with parental cells (11). On the other hand, many studies have described in various in vivo and in vitro models (4,9,16,17), that antisense strategy of IGF-IR induces apoptosis (programmed cell death) of tumor cells, but no study, to our knowledge, has reported an equivalent mechanism with IGFI and hepatocarcinoma. Taking these considerations into account, our laboratory proposes that an overexpression of IGF-I antisense c-DNA can inhibit cell growth by inducing apoptosis. In fact, there is an intimate relationship between the processes of cell cycle progression and apoptosis. Programmed cell death is a normal physiological process during liver development and also in adult liver, contributing to keeping a balance between proliferation and cell death. The disturbance of this homeostasis can be induced by various stimuli such as drugs, radiation, cytokines, and also, hormones or growth factors. To survive and grow, cells continuously require growth factor receptors activated by their ligands. IGF-I is considered to be a positive regulator of cell growth during development, following hepatectomy, or in cultured hepatoma cell lines. An overexpression of IGF-I is known to inhibit spontaneous cell death by apoptosis, thus inducing cell pro-

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liferation. On the other hand, the deprivation or the absence of growth factors, or appropriate receptors, causes normal cells to enter a G0 stage of the cell cycle, resulting in slower cell growth. But, in tumor cells, which are unable to take refuge in G0, the absence of IGF-I induces massive apoptosis. Apoptosis contributes to the maintenance of tissue homeostasis. Tumor formation was formerly thought to be the result of abnormal proliferation of genetically altered cells, but it is now understood to be the result of an imbalance between cell proliferation and cell death. If cell proliferation and cell death are thought of as a ratio, then tumor mass will increase when proliferation increases or death decreases (proliferation/death ±1). Inhibition of apoptosis in malignant cells may occur by several mechanisms: overexpression of proteins such as bcl2 or c-myc, abrogation of the p53 function by mutation or deletion, or stimulation of the autocrine growth factor or its receptor (18–20). The objective of this in vitro study was to characterize apoptosis in rat hepatocarcinoma cells transfected with an episomal vector expressing IGF-I antisense cDNA. In order to correlate these in vitro results with those obtained previously in vivo (11), the experimental procedures were similar. Briefly, anti-IGF-I mRNA synthesis in transfected cells was stimulated using zinc sulphate at 40 mM for 6 h because, under these experimental conditions, when transfected cells were injected into syngeneic rats, tumoral regression and animal survival were nearly total. Under these optimal conditions, we have characterized the presence of apoptotic bodies by morphological analysis, and the formation of DNA internucleosomic fragments, and we have quantified the apoptotic cells by flow cytometry and comet assay. We have observed distinctive morphological features of apoptosis in the transfected clones, B2Cl2 and B5Cl1, in comparison with the parental cells, LFCl2A: especially vacuolization of cytoplasm, nuclear condensation and internucleosomic DNA fragmentation. This phenomenon increased massively when an overexpression of IGF-I antisense was caused by zinc stimulation of the metallothionein I promoter in transfected cells. Therefore, we suggest that intracellular growth factor deprivation, through the activation of IGF-I antisense synthesis by zinc stimulation of the metallothionein I promoter, leads to a massive apoptotic process in the cells and may be an important event in neoplastic transformation.

Materials and Methods Reagents Zinc sulphate (SO4Zn), May Gru¨nwald, Giemsa, Hœchst 33342, propidium iodide and ethidium bro-

Apoptosis and loss of IGF-I in hepatoma

mide were purchased from Sigma Chemical (La Verpille`re, France). Fluo 3 pentapotassium salts (fluo 3 and fluo 3-AM) were purchased from Molecular Probes (Eugene, OR, USA). Acridine orange was obtained from Boehringer Mannheim (Germany). All the other chemicals were of the highest purity grade commercially available. Cell lines and media LF is an established cell line, derived from a hepatocarcinoma chemically-induced in Commentry rats by 4dimethylaminoazobenzene (DAB) in our laboratory. These cells were cloned in soft-agar, and one clone, LFCl2A, was isolated and characterized (15). The LFCl2A cells were grown in minimum essential medium Eagle’s salt (MEM, Gibco Life Technologies, France) supplemented with 10% newborn calf serum (NBS, Gibco Life Technologies, France), 2 mM glutamine (Gibco Life Technologies, France), and an antibiotic mixture (streptomycin 100 mg/ml and penicillin 100 UI/ml) (Gibco Life Technologies, France). The cultured medium for the transfected clones, B2Cl2 and B5Cl1, was supplemented with hygromycin 0.2 mg/ml (Sigma Chemical, Saint Louis, MO, USA) to maintain the selection pressure of the episomal vector. The cells were seeded at the density of 105 cells/ml in 25-cc flasks, placed at 37æC in a humidified air/CO2 (95%–5%) incubator and subcultured twice a week. IGF-I antisense vector and cell transfections We used the episomal vector constructed in Joseph Ilan’s laboratory (6) for glioblastoma and teratocarcinoma therapy. This vector has been described elsewhere (11). Briefly, it contains a human hepatic c-DNA for IGF-I in an antisense orientation, placed under the control of the mouse metallothionein I promoter gene (mMT-I) inducible by zinc, the EBV-ori P origin of replication and the EBV-encoded nuclear antigen (EBNA 1), a simian virus 40 poly A as transcription terminal signal, and two genes for selection (ampicillin and hygromycin resistance genes). The transfection of the LFCl2A cells was performed using lipofectin (Gibco Life Technologies, France) according to the supplier’s instructions (6). Among about ten clones that appeared after selection, we have more extensively characterized two cell clones, B2Cl2 and B5Cl1 (11). Treatment SO4Zn (Zn) stock solution was prepared in sterile distilled water. Before stimulation with Zn, the medium was removed and replaced by a NBS-free medium for 12 h. After this conditioning time, 40 mM of Zn solu-

tion were added to the transfected cell cultures for 6 h. The parental and transfected cells used as controls, after conditioning time by a NBS-free medium, were not submitted to Zn-stimulation. After this treatment, the following studies were performed: 1. Quantification of IGF-I-expressing cells by immunocytochemical localization. The amount of IGF-I was determined by a semiquantitative immunoperoxidase technique, as described elsewhere (21). 2. Morphological analysis by May Gru¨nwald-Giemsa or Acridine Orange staining. The parental cells, LFCl2A, and the transfected cells, B2Cl2 and B5Cl1, were seeded in 9-cm2 slide flasks (Nunc, ATGC, Noisy le Grand, France) in 3 ml of MEM medium supplemented with 10% NBS. Forty-eight hours after seeding, the cells were treated as described above. After Zntreatment, the cells were fixed in methanol/acetic acid (3:1/vol:vol) and stained with May Gru¨nwald and with Giemsa 3% (MGG). The observation was made by light microscopy on 3000 cells per slide. In the second analysis, the cells were fixed in PBS/Formol 1% and stained with Acridine Orange (AO). AO, a DNA intercalating agent, is a membrane-permeable dye which stains the nucleus in green. AO was prepared as a stock solution in a phosphate buffer solution at 2 mg/ml and further diluted in a phosphate buffer at 5 mg/ml prior to use (22). 3. Analysis of DNA cleavage. Forty-eight hours after seeding in 25-cm2 flasks (Falcon, ATGC, Noisy le Grand, France), the LFCl2A, B2Cl2 and B5Cl1 cells were treated as described above. After Zn-treatment, the cells were washed in PBS (Gibco, Life Technologies, France), trypsinized, resuspended in STE buffer (NaCl: 100 mM; Tris HCl: 10 mM; EDTA: 1 mM) with SDS 10% and 200 mg/ml of proteinase K (Boehringer Mannheim, Meylan, France) and incubated overnight at 56æC. DNA was extracted with a phenol/ isoamyl alcohol mixture (Fluka, Saint Quentin Fallavier, France) and precipitated at ª20æC with ethanol and 3M sodium acetate. The DNA pellet was resuspended in the TE buffer pHΩ7.4 (Tris HCl: 10 mM; EDTA: 1 mM). Characterization of DNA cleavage was then obtained by submitting the samples to electrophoresis in 1.5% agarose gel in the TBE buffer 0.5 X (Tris base: 0.1 M; Boric acid: 0.1 M; EDTA: 0.5 mM). 4. Intracellular calcium content: [Ca2π]i. The LFCl2A, B2Cl2 and B5Cl1 cells were seeded in 3.5 cm2 slide flasks (Nunc, France) in 1 ml of complete MEM medium. Forty-eight hours after seeding, the cells were treated as described above. The [Ca2π]i in the cultured cells was investigated using fluo-3AM (23). In order to facilitate cell loading, acetoxymethylester (AM)-

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grafted probes (fluo-3AM) were used. This ester group passively crosses the plasma membrane and once inside the cell is cleaved to a cell non-permeable product by cytosolic esterases. Calibration was performed using the Tsien method (24). This protocol has been described elsewhere (25). Briefly, after Zn-treatment the cells were washed twice in MEM NBS-free without phenol red (in order to eliminate the background fluorescence due to the non-specific autofluorescence of this dye). Fluo-3AM at 10 mM was then added to the monolayer which was then incubated at 37æC for 60 min. The slides were then washed with MEM NBS-free without phenol red. Fluorimetric measurements were performed with a Laser Scanning Confocal microscope (ACAS 570, Meridian Instruments, Okenos, MI, USA). 5. Analysis of ‘‘sub-G1’’ peak. The percentage of cells in subG1 peak was determined according to the modified procedure described by Pollack & Ciancio (26). The cells were seeded in 25-cm2 flasks in 10 ml of complete MEM. Forty-eight hours after seeding, the complete medium was removed and replaced by MEM without NBS overnight. After this time, the cells were treated with Zn as described above in 10 ml of MEM without NBS. Six hours later, the cells were incubated for 1 h at 37æC in the dark in an MEM NBS-free, without phenol red, containing 10 mM of Hœchst 33342 (Ho). After this time, the cells were washed in PBS in order to eliminate the excess of the non-specific fluorescence of this dye. Then, the cells were trypsinized and resuspended for 15 min in MEM NBS-free, without phenol red containing 32 mM of propidium iodide (PI). Flow cytometry analysis was carried out with a Coulter Epics Elite Flow Cytometer (Coulter, Hiakah, FL, USA). 6. Apoptosis analysis by flow cytometry with DNA binding fluorochromes. Forty-eight hours after seeding in 25-cm2 flasks, the cells were treated as described above. After the Zn-treatment time, the cells were washed in PBS, trypsinized and resuspended in MEM NBS-free, without phenol red. Hœchst 33342 (Ho) and propidium iodide (PI) were added to the single cell suspension so that the final concentrations were 10 mM and 32 mM, respectively. 5000 cells were analyzed using a Coulter Epics Elite Flow Cytometer (Coulter, Hiakah, FL, USA) (22). 7. Alkaline, single-cell gel electrophoresis assay. The cells were seeded in 12-cm2 dishes in 2 ml of complete MEM, and 48 h after seeding the cells were treated as described above. After the Zn-treatment time, the protocol of Singh et al. (27) was followed with minor modifications (28). After minimal trypsinization, the cells were suspended in their culture medium, mixed

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with low melting point agarose (TEBU, Le Perray en Yvelines, France), maintained at 37æC in order to obtain a final concentration of 0.5% agarose. Seventy-five microliters of this suspension (3.105 cells per ml ) were pipetted onto a frosted microscope slide precoated with a layer of 1% normal melting point agarose (TEBU, Le Perray en Yvelines, France). The slides were left for 15 min over ice and immediately immersed in a lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris pH 10, sodium sarcosinate 1%, triton X-100 1%, DMSO 10%) at 4æC and left for 1 h. The slides were removed from the lysis solution and transferred to an electrophoresis box containing a fresh alkaline buffer (300 mM NaOH, 1 mM EDTA, pH 13) at room temperature. The slides were left in this buffer for 40 min to allow unwinding of the DNA. Electrophoresis was performed for 20 min at 23 V and 300 mA. After electrophoresis, the slides were rinsed three times with 400 mM Tris pH 7.5, stained with 75 ml ethidium bromide (20 mg/ml in water), and covered with a cover slip. The observation was made using an epifluorescence microscope equipped with a short wavelength arc mercury lamp HBO (Osram) and with an appropriate excitation and barrier filters at 20-fold magnification (Axioplan, Zeiss).

Results We previously demonstrated that the tumorigenic potential of LFCl2A hepatoma cells was fully inhibited by the expression of an antisense anti-IGF-I (11). These transfected cells were also able to induce an immunogenic state in the syngeneic rats which allowed them to reject parental tumoral cells (11). In order to understand these in vivo results, we carried out in vitro sudies in experimental conditions similar to those in vivo. When the B2Cl2 or B5Cl1 transfected cells were stimulated for 6 h by zinc, as described in Materials and Methods, the antisense anti-IGF-I was overexpressed via the mouse metallothionein I promoter, leading to a decrease in the level of the IGF-I proteins and an increase in cell death by apoptosis. Apoptosis was originally defined on morphological, biological and functional grounds. In this study, we considered only the morphological features because they still provide the most reliable markers of apoptosis. Various techniques have been used, such as microscopy analysis, agarose gel electrophoresis, flow cytometry and comet assay. The first techniques were used to characterize the apoptotic cells and the second to quantify this phenomenon on the transfected clones in comparison with the parental cells. Before describing our results, we will characterize the cell lines, particularly their IGF-I level.

Apoptosis and loss of IGF-I in hepatoma

Characterization of the cell clones The parental hepatoma cells, LF, were stemmed from a fast-growing transplantable hepatoma originating from a chemically (4-dimethylaminoazobenzene) induced hepatocarcinoma in Commentry rats. This cell line was cloned in soft agar, and the LFCl2A clone was selected for its powerful tumorigenic properties when reinjected into syngeneic rats and also its high ability to produce IGF-I. These cells were grown as described in Materials and Methods. LFCl2A cells presented a flattened epithelial shape growing into a mosaic pattern with numerous overlapping (loss of contact inhibition). These cells were transfected either with the vector expressing the IGF-I gene in an antisense position (giving rise to the B2Cl2 and B5Cl1 clones after hygromycine selection) or with the empty vector (in which the IGF-I gene was deleted). The growth rate, measured by the indice of multiplication, was roughly the same for the parental LFCl2A and the LFCl2A transfected with the empty vector, but the growth of B2Cl2 and B5Cl1 transfected cells decreased significantly 1.5and 1.7-fold, respectively. The expression of IGF-I in these 4 clones was measured by an immunocytochemical technique, as described in Materials and Methods. The percentage of cells expressing IGF-I was the same for the parental LFCl2A and the empty vector transfected LFCl2A; this expression was completely independent of Zn stimulation (Fig. 1). By contrast, we observed an important spontaneous decrease in the number of cells producing IGF-I in both B2Cl2 and B5Cl1 clones. This decrease became dramatically more important when these cells expressing IGF-I antisense were stimulated by Zn. In order to simplify the description of our results, only the LFCl2A parental cells without stimulation by Zn were considered for the comparison with B2Cl2 and B5Cl1 clones in the absence or presence of zinc stimulation. Apparition of apoptosis in IGF-I-deprived transfected cells Characterization of morphological features of apoptosis. The morphological analysis of the apoptotic cells in the absence or presence of zinc stimulation was carried out by phase-contrast microscopy using May Gru¨nwald-Giemsa (MGG) staining and by fluorescence microscopy using Acridine Orange (AO) labeling. Acridine orange binds to double-stranded DNA and gives green fluorescence, permitting the indentification of apoptotic cells. We observed that the parental cells, LFCl2A, in the absence of zinc stimulation (Fig. 2a), contained homogeneous DNA, without chromatin condensation, and

Fig. 1. Immunocytochemistry quantification of IGF-I-expressing cells by rat hepatocarcinoma cells transfected or not with an episomal vector expressing IGF-I antisense cDNA in the absence or presence of stimulation by zinc at 40 mM for 6 h.

an intact cytoplasm (Fig. 2b). We noted that all of these cells presented many nucleolei. Similar results were observed with LFCl2A in the presence of zinc stimulation (data not shown). The transfected cells, B2Cl2 and B5Cl1, in the absence of zinc stimulation, underwent spontaneous apoptosis. In comparison with the parental cells, LFCl2A, morphological changes occurred in both the cell nucleus and cytoplasm. Fig. 2c reveals vacuolization of cytoplasm, nuclear condensation and membrane blebbing. With AO labeling (Fig. 2d), the transfected cells presented a shrunken cytoplasm and nucleus, and a condensed chromatin which appears as spheres or as a peripheral crescent. Fig. 2e and 2f illustrate the effect of increased antisense expression on the morphological state of the transfected clones. In fact, we can see in Fig. 2f a higher proportion of cells with chromatin condensation in comparison with the untreated transfected cells (Fig. 2d). Moreover, ultrastructurally, the enhancement of apoptosis involves cell shrinkage and detachment of apoptotic cells from neighboring cells (Fig. 2e). The morphological changes of the B5Cl1 cells (results not shown) were similar to those observed with the B2Cl2 cells. Internucleosomal DNA fragmentation. These typical morphological features of apoptosis were associated with a unique change in the nuclear DNA. The cells were subjected to analysis for the presence of DNA fragmentation. The apoptotic cells produced a characteristic fragmentation pattern, the ‘‘DNA ladder’’,

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Apoptosis and loss of IGF-I in hepatoma

Fig. 2. Morphological characteristics of apoptosis in rat hepatocarcinoma cells transfected or not with an episomal vector expressing IGF-I antisense c-DNA in the absence or presence of stimulation by zinc at 40 mM for 6 h. The arrows indicate the apoptotic cells. (Magnification 200-fold). Parental cells, LFCl2A, without zinc stimulation after May-Gru¨nwald Giemsa (MGG) 2a or Acridine Orange staining (AO) 2b; Transfected cells, B2Cl2, without zinc stimulation after MGG 2c or AO staining 2d; Transfected cells, B2Cl2, after zinc stimulation, MGG 2e or AO staining 2f.

composed of approximately 180–200 base pairs, which can be separated by gel agarose electrophoresis and visualized under ultraviolet light after ethidium bromide staining. Fig. 3 shows this characteristic ‘‘DNA ladder’’ for both transfected cells, B2Cl2 and B5Cl1, in the absence of zinc stimulation (lanes 2 and 4), while no DNAfragmentation was detected for the parental cells, LFCl2A, in the absence (lane 1) or presence (data not shown) of zinc stimulation. An increase in the amount of internucleosomic DNA fragments was observed in both transfected cell lines, B2Cl2 and B5Cl1 (lanes 3 and 5), after zinc stimulation, suggesting that these cells underwent a particularly severe form of apoptosis when the intracellular level of IGF-I decreased.

These results confirmed that the increase in apoptotic rat hepatocarcinoma-transfected cells, when stimulated by zinc, as characterized by the presence of internucleosomal DNA fragmentation, was correlated with the decrease of IGF-I expression. Quantification of apoptosis events Intracellular calcium content. The cleavage of internucleosomal fragments, induced preferentially in the linker regions between nucleosomes, has often been proposed to be catalyzed by an apoptotic-specific Ca2π- Mg2π-dependent endonuclease. Therefore, the [Ca2π]i in the cells was investigated using acetoxymethylester ()-grafted probes (fluo-3) with a Confocal microscope. This ester group passively crosses the plasma membrane and once inside the cell is cleaved to a cell non-permeable product by cytosolic esterases. Intracellular Ca2π fluctuations were measured by the fluorescence level. An increase in [Ca2π]i, up to 42% and 60%, respectively, was observed with the transfected cells B2Cl2 and B5Cl1, after zinc stimulation (Table 1). No disturbance in the Ca2π homeostasis was ob-

TABLE 1 Intracellular calcium content ([Ca2π]i in nM) in rat hepatocarcinoma cells transfected, or not, with an episomal vector expressing IGF-I antisense c-DNA in the absence or presence of stimulation with zinc at 40 mM for 6 h

π2

[Ca ]i in nM

Fig. 3. Internucleosomal DNA fragmentation in rat hepatocarcinoma cells transfected or not with an episomal vector expressing IGF-I antisense c-DNA in the absence (ªZn) or presence (πZn) of zinc at 40 mM for 6 h. Agarose gel electrophoresis of DNA extracted from the culture of LFCl2A, B2Cl2 and B5Cl1 lines; reading from left to right. Laddering of DNA fragments (180 bp or multiples of this size, characteristic of apoptosis) was detected after genomic DNA extraction from both transfected cells, B2Cl2 and B5Cl1, when these cells were stimulated by zinc at 40 mM for 6 h. The molecular weight marker (jX-174-RF DNAHae III Digest, fragment sizes from 72 to 1358 bp) is shown on the right.

LFCl2A

B2Cl2

BN5Cl1

ªZn

πZn

ªZn

πZn

ªZn

πZn

121

124

116

162

132

192

TABLE 2 Flow cytometry analysis of apoptotic cells after double-staining with Hœchst 33342 (Ho 342) and propidium iodide (PI) in rat hepatocarcinoma cells transfected or not with an episomal vector expressing IGFI antisense c-DNA in the absence or presence of zinc at 40 mM for 6 h

% of viable cells % of apoptotic cells % of dead cells

LFCl2A

B2Cl2

BN5Cl1

ªZn πZn

ªZn πZn

ªZn

πZn

94.8 0.4 4.8

80.7 13.8 5.5

80.6 12.7 3.7

54.2 32.8 13.0

92.3 1.5 6.2

54.4 29.8 15.8

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Fig. 4. DNA content of apoptotic cells after double-staining with Hœchst 33342 (Ho 342) and propidium iodide (PI) in rat hepatocarcinoma cells transfected or not with an episomal vector expressing IGF-I antisense c-DNA in the absence (ªZn) or presence (πZn) of zinc at 40 mM for 6 h. The arrows indicate the «sub-G1» peak. Parental cells, LFCl2A 4a: without zinc stimulation. Transfected cells, B2Cl2 4b: without zinc stimulation; 4c: with zinc stimulation at 40 mM for 6 h. Transfected cells, B5Cl1 4d: without zinc stimulation; 4e: with zinc stimulation at 40 mM for 6 h.

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Fig. 5. Digitized comet images of individual hepatocarcinoma cells, B5Cl1, transfected with an episomal vector expressing IGF-I antisense c-DNA in the presence of zinc at 40 mM for 6 h. Panel 5a: intact nucleus. Panel 5b: ‘‘classical’’ comet; the migration of DNA fragments in the electrical field indicates the presence of single-strand breaks. Panel 5c: ‘‘atypic’’ comet characteristic of apoptosis or programmed cell death.

served with the parental cell lines, LFCl2A, under any culture conditions. This intracellular calcium change in the transfected cells, B2Cl2 and B5Cl1, can modulate various physiological functions and lead to intracellular Ca2π accumulation, triggering the apoptotic process. Quantification of apoptosis events assessed by flow cytometry. The consequences of IGF-I deprivation on apoptosis in rat hepatocarcinoma-transfected cells were also assessed by flow cytometry analysis to discriminate and quantify viable, apoptotic and necrotic cells. This technique was based on size distribution and fluorescence quantification due to staining with the DNA-binding fluorochromes Ho 342 and PI. Our results (Table 2) have shown that the transfected cells, B2Cl2 and B5Cl1, underwent a massive apoptotic process when the intracellular IGF-I content was decreased. In fact, we observed that 6 h after zinc stimulation, the percentages of cell death by apoptosis were, for B2Cl2 and B5Cl1, 2.2 and 2.6 greater than that observed with transfected cells without zinc stimulation (13.8% versus 29.8% and 12.7% versus 32.8% respectively for B2Cl2 and B5Cl1). We note that cell death by apoptosis was a minor event in the parental cells, LFCl2A (0.4 %), even after Zn-stimulation. The quantification of apoptotic cells, assessed by

flow cytometry analysis, can be correlated with the presence of a ‘‘sub-G1’’ or apoptotic peak. The ‘‘subG1’’ peak, located before G0/G1 in the DNA histogram (Fig. 4), enables us to determine the percentage of apoptotic cells as already reported (29). Our results have shown that, for both, B2Cl2- and B5Cl1-transfected cells, in the absence of Zn-stimulation, the number of cells found in the ‘‘sub-G1’’ peak was too high to be attributed only to a spontaneous process. According to the procedure used in this study, we consider that this ‘‘sub-G1’’ peak may be heavily contaminated by cells with non-specifically degraded-DNA and some debris of necrotic cells. Indeed, in an in vitro model, apoptotic cells were not phagocytosed and eventually underwent degenerative changes; the term ‘‘secondary apoptosis’’ is often applied to this change. Stimulation by zinc for 6 h increased the accumulation of events in the ‘‘subG1’’ peak corresponding to 34.2% (Fig. 4c) and 33.5% (Fig. 4e) of apoptosis found, respectively, in B2Cl2- and B5Cl1-transfected cells (arrows). By comparison, the parental cells, LFCl2A, showed no ‘‘sub-G1’’ peak in the absence of Zn-stimulation (Fig. 4a), as well as after Zn-stimulation (results not shown). Quantification of DNA damage by single-cell gel electrophoresis assay. Single-cell gel electrophoresis is a sensitive fluorescence microscopic method for detection of primary DNA damage to an individual cell. The degree of DNA damage can be quantified by measuring the displacement of the genetic material between the cell nucleus ‘‘comet head’’ and the resulting ‘‘tail’’. In the present study, only three different comet patterns observed in hepatocarcinoma cells, B5Cl1, after Zn-stimulation are described (Fig. 5). Panel 5a shows an undamaged cell with an intact nucleus, Panel 5b shows a classical comet with damaged cell and Panel 5c illustrates a comet with an extremely prominent tail without a clearly identifiable cell nucleus characteristic of apoptotic cells. As shown in Table 3, the quantification of the number of these three differ-

TABLE 3 Quantification of digitized comet images, assessed by the single-cell gel electrophoresis method, of individual parental hepatocarcinoma cells LFCl2A, or B5Cl1, transfected with an episomal vector expressing IGF-I antisense c-DNA in the absence or in the presence of zinc at 40 mM for 6 h

% of Comet Panel 5a like % of Comet Panel 5b like % of Comet Panel 5c like

LFCl2A

B5Cl1

ªZn

ªZn

πZn

93 7 0

81 6 13

63 8 23

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ent comet shapes indicates that the transfected B5Cl1 clones exhibit high levels of apoptosis in the absence of Zn (13%) as compared to parental cells LFCl2A and a higher number after Zn-stimulation (23%). It is interesting to note that these percentages of apoptotic cells were very close to those found after the flow cytometry analysis (Table 2). Similar results were obtained with B2Cl2 after Zn-stimulation (data not shown).

Discussion In this study, we demonstrate that IGF-I inhibits the development of a cell death program in a neoplastic cell line, LFCl2A. The suppression of IGF-I synthesis by transfection of parental cells with an episomal vector expressing IGF-I c-DNA antisense leads to the restoration of the apoptotic programme as a mechanism of counterbalancing cell proliferation. We observe that the proliferation of the transfected cells, B2Cl2 and B5Cl1, is 1.5- to 1.7-times lower than that of the parental cells, LFCl2A. In addition, we evaluate by different approaches how the lack of IGF-I synthesis, varying from 50% to 15% of control cells depending upon culture conditions causes massive apoptosis in transfected cell lines (Fig. 1). Morphologically, the appearance of dead cells in the culture deprived of IGF-I involves chromatin condensation and margination, cell shrinkage, blebbing and nuclear fragmentation which, in vivo, are associated with the formation of apoptotic bodies that undergo phagocytosis by macrophages or neighboring cells. The extensive DNA fragmentation observed is probably due to the effect of an endonuclease Ca2π-dependent activity in the linker region between nucleosomes that is characteristic of apoptosis (30–33). Many authors report that nuclei can compartmentalize ions, and that increases in intranuclear Ca2π can be dissociated from cytosolic increases in Ca2π (34,35). However, calcium alone is not sufficient to trigger apoptosis, which implies that additional endonuclease-activating components must be present for DNA fragmentation to take place. Another characteristic of cells undergoing apoptosis is that the integrity of the plasma membrane is preserved and most functions of the membrane remain unchanged (31). Flow cytometric methods are based on this capacity of the apoptotic cells to exclude viability dyes such as propidium iodide, in contrast with cell necrosis. With this attractive method, we have differentiated alive, apoptotic and necrotic cells. By means of DNA staining with a bis-benzimide dye (Hœchst 33342), in association with propidium iodide cell labeling, we have determined the cell cycle positions of both live and dead cell populations and the ‘‘sub G1’’ peak. Moreover, the increase in ‘‘atypic’’ comet number in B5Cl1, or in B2Cl2 (data

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not shown) after Zn-stimulation confirmed that cell death by apoptosis, for the two transfected cell lines, is directly related to the antisense IGF-I expression. All the different techniques used in this work to measure apoptosis in our various cell clones give very similar percentages of apoptotic cells that correspond to the degree of IGF-I synthesis inhibition as measured by immunohistochemistry. It is well known that survival and proliferation of almost all cells depend on the presence of growth factor. The Insulin-like Growth Factors (IGF-I and IGF-II) are critical regulators of cell growth (4,8,10,16,17,36,37) and can block the apoptosis pathway in a variety of cell lines (38) leading to a proliferation of the cell population that is normally programmed to die, and inducing subsequent transformation events that result in the uncontrolled growth of cells. Many results coming from the laboratory of Renato Baserga indicate that, when the function of IGF-I receptor is decreased or otherwise impaired, the cells undergo massive apoptosis. The work was conducted on C6 glioblastoma cells, human melanoma cell line, mouse melanoma cells as well as rhabdomyosarcoma cells. The authors conclude that IGF-IR activated by its ligand plays a very protective role in programmed cell death and that this protection is even more striking in vivo than in vitro (20). The impairment of IGF-IR was studied by antisense strategy as well as by shift frame mutation of IGF-IR gene (39). For these authors the extent of apoptosis is directly related to the number of IGF-I receptor molecules. They indicate that a decrease in the number of IGF-IR, in syngeneic animals, induced a host response that can eliminate in vivo the surviving cells (20). Futhermore, they show that rats that have been given injections with C6 glioblastoma cells expressing antisense IGF-IR not only developed tumors, but were protected from a subsequent challenge with wild-type C6 glioblastoma cells for at least 3 months, but no eradication of pre-existing tumors of the wild type was described. The experiments conducted in Joseph Ilan’s laboratory with IGF-I antisense have demonstrated that deprivation of IGF-I leads to a decrease of tumorigenic properties in the transducted cells as well as complete abrogation of pre-existing tumors in the case of glioblastoma (6,7) and teratocarcinoma (9). Apoptosis was not investigated by these authors, and the mechanism evoked was of immunological nature. We have established a hepatocarcinoma cell line, LFCl2A, that produces voluminous tumors when injected into syngeneic Commentry rats. When these cells were transfected with an episomal vector expressing IGF-I antisense, the modified LFCl2A cells, which

Apoptosis and loss of IGF-I in hepatoma

do not produce IGF-I, became less tumorigenic and when injected in rats inhibited the growth of parental tumor cells and induced regression of established tumors. These transfected hepatocarcinoma cell lines induce, in vivo, a tumor immunity mediated by CD8π T cells (11). Moreover, these transfected cells, in vitro, revealed various morphological aspects of apoptosis. We have thus hypothesized that phenotype modifications due to apoptosis could lead to recognition of the transfected cells by the immune systems, and could explain the tumor-specific immunity mediated by CD8πT cells (11,40). Indeed, we have demonstrated for the first time that our transfected cells underwent apoptosis due to the lack of IGF-I synthesis. Up to now, only IGF-IR has been considered for apoptosis studies using this type of antisense strategy (4,8,10,16,17,20). The mechanism by which this antisense strategy inhibits cell growth can be reasonably described by its negative regulation on the autocrine secretion of the growth factor, contributing to the partial autonomy and regulation of cell growth and proliferation. Various genes have been implicated in the induction or inhibition of apoptosis. The phenotypic modifications due to apoptosis may lead to the recognition of the transfected cells by the immune system, and may explain the tumor-specific immunity mediated by CD8π T cells that we have described elsewhere (11). To our knowledge, this phenomenon has not so far been described for hepatocarcinoma. We therefore suggest that the mechanism of tumor rejection proposed elsewhere (11) coexists with the apoptotic events demonstrated here. A better understanding of the relationship between the immune and apoptotic processes could contribute to a new therapeutic approach to the treatment of tumors overexpressing Insulin-like Growth Factor type I.

Acknowledgements The authors wish to thank Mr J. McCabe from the Centre Technique de Langues (CTL, Universite´ Paris V), S. De Oliveira and C. Frayssinet for critical reading of the manuscript. CAPES- «Fundac¸a˜o Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior» (Brası´lia-DF, Brazil) is greatly acknowledged for the financial support to G.A. De Oliveira. This work was supported by the ‘‘Association pour la Recherche sur la Cancer’’ (Contrat no. 9260, ARCVillejuif, France).

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