- Email: [email protected]
Amino acid transport in a human neuroblastoma cell line is regulated by the type I insulin-like growth factor receptor
Life Sciences 71 (2002) 127 – 137 www.elsevier.com/locate/lifescie
Amino acid transport in a human neuroblastoma cell line is regulated by the type I insulin-like growth factor receptor Hong-Sheng Wang, Masafumi Wasa*, Akira Okada Department of Pediatric Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Received 3 July 2001; accepted 8 January 2002
Abstract Insulin-like growth factor I (IGF-I) and IGF-II stimulate cancer cell proliferation via interaction with the type I IGF receptor (IGF-IR). We put forward the hypothesis that IGF-IR mediates cancer cell growth by regulating amino acid transport, both when sufficient nutrients are present and when key nutrients such as glutamine are in limited supply. We examined the effects of aIR3, the monoclonal antibody recognizing IGF-IR, on cell growth and amino acid transport across the cell membrane in a human neuroblastoma cell line, SK-N-SH. In the presence of aIR3 (2 Ag/ml), cell proliferation was significantly attenuated in both control (2 mM glutamine) and glutaminedeprived (0 mM glutamine) groups. Glutamine deprivation resulted in significantly increased glutamate (system XAG ), MeAIB (system A), and leucine (system L) transport, which was blocked by aIR3. Glutamine (system ASC) and MeAIB transport was significantly decreased by aIR3 in the control group. Addition of aIR3 significantly decreased DNA and protein biosynthesis in both groups. Glutamine deprivation increased the IGF-IR protein on the cell surface. Our results suggest that activation of IGF-IR promotes neuroblastoma cell proliferation by regulating trans-membrane amino acid transport. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Insulin-like growth factor; Type I IGF receptor; Neuroblastoma; Amino acid transport; Glutamine
Introduction The effects of insulin-like growth factor I (IGF-I) and IGF-II (IGFs) are mediated via interaction with the type I IGF receptor (IGF-IR) [1]. Ligand binding activates the tyrosine kinase domain, resulting in both receptor autophosphorylation and phosphorylation of cytoplasmic substrates, which subsequently
*
Corresponding author. Tel.: +81-6-6879-3753; fax: +81-6-6879-3759. E-mail address: [email protected] (M. Wasa). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 6 2 6 - 0
128
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
bind to the activated receptor [2]. Recent work has confirmed the importance of IGFs/IGF-IR interactions in cancer cell proliferation by such mechanisms as the mediation of mitogenesis, the maintenance of a transformed phenotype, and the protection of tumor cells from apoptosis [3–5]. Cancer cells require an increased supply of amino acid precursors to support key intracellular metabolic pathways. In order to support increased demands for amino acids, they must be endowed with highly efficient transport systems. The essential role of carrier-mediated amino acid uptake in malignant cells is manifested by the fact that glutamine is transported across the plasma membrane faster than in normal cells [6]. Although glutamine can serve as an important ATP source, it is also a precursor for the synthesis of proteins and nucleic acids. It has been shown for cancer cells that both cell growth rates and DNA and protein biosynthesis correlate directly with the concentration of glutamine in culture media [7]. Neuroblastomas are malignant tumors found in children that are derived from neural crest cells. Human neuroblastoma cell lines have been reported to express IGF-IR and mRNA for IGFs [8–10]. Recent studies have suggested the importance of the interaction of IGFs with IGF-IR as an autocrine or paracrine growth factor mechanism in neuroblastoma cells [11,12]. These effects are blocked by the monoclonal antibody against IGF-IR, aIR3, which inhibits the interaction of the IGF-IR with its ligands. This indicates that functional IGF-IR plays a key role in the control of the signaling pathway of IGFs [12,13]. Cancer cells survive and even grow in conditions where nutrient resources are limited. These conditions can often be observed in the poorly vascularized center of solid tumors. A precise mechanism for this capacity for survival and growth has not yet been described. We put forward the hypothesis that IGF-IR mediates cancer cell growth by regulating amino acid transport activity, both when sufficient nutrients are present and when key nutrients such as glutamine are in limited supply. We have already shown that IGF-I stimulates amino acid transport in a glutamine-deprived human neuroblastoma cell line [14]. However, no study to date has addressed the effects of the functional impairment of IGF-IR on amino acid transport. To investigate our hypothesis, we examined the effects of aIR3 on cell growth and amino acid transport in a glutamine-deprived human neuroblastoma cell line. Here, we used the well-characterized neuroblastoma cell line, SK-N-SH, which expresses specific high-affinity functional IGF-IR [10].
Materials and Methods Chemicals Radiolabeled amino acid (3H-L-glutamine, 3H-L-glutamate, 3H-L-leucine), 3H-2-(methylamino) isobutyric acid (MeAIB, a system A-specific substrate), and 3H-L-thymidine were purchased from Amersham (Arlington Heights, IL). Dulbecco’s Modified Eagle medium (DMEM) was from GIBCO/ BRL Life Technologies (Grand Island, NY) and fetal bovine serum (FBS) was from JRH Biosciences (Lenexa, KS). Tissue culture plates were obtained from Costar Corp. (Corning, NY). A monoclonal antibody against IGF-IR (aIR3) was purchased from Oncogene Science (Uniondale, NY). An isotypematched mouse IgG1 (IgG1) and MTT cell growth kit were obtained from Chemicon (Temecula, CA). The F(abV)2 fragment of fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG was purchased from Cappel (Durham, NC). Amino acids and all biochemicals were purchased from Sigma Chemical (St. Louis, MO). A Neuroblastoma cell line, SK-N-SH, was provided by Dr. Tadao Ohno (RIKEN Cell Bank, Tsukuba, Japan).
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
129
Cell culture SK-N-SH cells were cultured in DMEM supplemented with 2 mM glutamine, 10% heat-inactivated FBS, 1,000 units/ml penicillin, and 1,000 units/ml streptomycin at 37 jC under a humidified atmosphere of 5%CO2/95% air. FBS was heat-inactivated to eliminate any variable sources of IGFs or other trophic factors such as epidermal growth factor from the culture medium. The culture medium was changed every 3 days until cells were confluent, at which point they were used for experiments. Cell proliferation Cells were seeded at a density of 1 104 cells/100 Al (100 Al/well) into 96-well tissue culture plates. After 24 hours, the culture medium was removed and changed to DMEM supplemented with 10% FBS plus glutamine (2 mM or 0 mM) in the presence of either aIR3 (2 Ag/ml) or IgG1 (2 Ag/ml). Cell proliferation was measured at days 0, 1, 2, and 3 using MTT cell growth assay [15]. Cell growth in 2 mM glutamine was chosen as the control. Amino Acid Transport Cells were seeded into 24-well tissue culture plates (0.5 ml/well). After getting 90-100% cell confluence, culture medium was removed and changed to DMEM supplemented with 10% FBS plus glutamine (2 mM or 0 mM) in the presence of either aIR3 (2 Ag/ml) or IgG1 (2 Ag/ml). L-glutamine, L-glutamate, MeAIB and L-leucine transport were measured at 24 hours. The transport of radiolabeled amino acids by cell monolayers was assayed by the cluster tray method of Gazzola et al. [16]. Before the transport assays, cells were rinsed twice with warm sodium-free Krebs-Ringer Phosphate Buffer (Chol-KRP, which was made by replacing the corresponding sodium salts with choline chloride and choline phosphate) to remove extracellular sodium and amino acids. After removal of Chol-KRP, the transport assay was initiated by transferring 0.25 ml of the uptake medium to 24-well trays. The transport of radiolabeled amino acid (5 ACi 3H-amino acid/ml) was performed for 1 minute at 37 jC at 10 Amol/L unlabeled amino acid in both sodium Krebs-Ringer Phosphate (Na-KRP) and Chol-KRP buffer. The assay was terminated by discarding the uptake buffer and ringing the cells with ice-cold Chol-KRP buffer three times. The wells containing the cells were allowed to dry and were solubilized in 200 Al of 0.2 N NaOH/0.2% sodium dodecyl sulfate (SDS) solution. One hundred Al of the cell extract was neutralized with 10 Al of 2 N HCl and subjected to scintillation spectrophotometry. Protein content was measured by the bicinchoninic acid protein method [17]. The Na+-dependent transport values were obtained by subtracting the transport values in Chol-KRP from those in Na-KRP. Saturable Na+independent transport values were determined in Chol KRP by subtracting the values in the presence of excess (10 mM) unlabeled amino acid from those in its absence. Transport velocities were expressed in units of picomoles per milligram of protein per minute. DNA and protein synthesis For the determination of DNA and protein synthesis, we measured the incorporation of 3H-thymidine and 3H-leucine, respectively, into acid-insoluble material. Cells were seeded in 24-well cluster trays (0.5 ml/well). When 90-100% cell confluence was reached, the medium was removed and replaced with
130
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
DMEM supplemented with 10% FBS plus glutamine (2 mM or 0 mM) in the presence of either aIR3 (2 Ag/ml) or IgG1 (2 Ag/ml). After 24 hours, 3H-thymidine or 3H-leucine (1 ACi/ml) was added to the culture medium and the cells were incubated for 2.5 hours at 37 jC. The assay was terminated after 2.5 hours, when the cells were washed twice with phosphate-buffered saline (PBS) and fixed by washing three times with ice-cold 10% trichloroacetic acid. Thereafter, the cells were rinsed twice with 70% and 95% ethanol, respectively. They were allowed to dry and solubilized in 200 Al of 0.2 N NaOH/0.2% SDS solution. Radioactivity and protein content were measured by the same procedures described for amino acid transport measurement. Flow cytometry to detect IGF-IR Based on the cell growth curve, cells were seeded in a 6-well culture plate (2 ml/well) at different densities in order to control for density-dependent effects at each time point between control and glutamine-deprived groups. After 24 hours, culture medium was removed and changed to DMEM supplemented with 10% FBS plus glutamine (2 mM or 0 mM). After 24, 48, and 96 hours, flow cytometry was carried out to identify the IGF-IR protein on the cell surface as described previously [18]. Briefly, 1 106 cells were washed with cold PBS supplemented with 1% FBS and pelleted in microfuge tubes by centrifugation at 1,000 g at 4 jC for 3 minutes. Then, 200 Al of anti-IGF-IR mAb (400 ng) diluted with PBS was added. As control, 200 Al of irrelevant IgG1 mAb in PBS was added at the same concentration as the primary antibody. Cells were incubated for 30 minutes at 4 jC and washed three times with PBS supplemented with 1% FBS. After the final wash, 200 Al of a 1:100 dilution of FITCconjugated goat antimouse IgG1 antibody [F(abV)2 fragments] was added to each tube. Cells were incubated for additional 30 minutes at 4 jC and washed three times with PBS supplemented with 1% FBS. Then, they were resuspended in 500 Al of PBS and maintained on ice for immediate analysis by flow cytometry. Stained cells were examined on FACScan (Becton Dickinson, San Jose, CA). At least 10,000 cells were counted for each sample. Data were processed by CellQuest program (Becton Dickinson, San Jose, CA). Statistical analysis Data (mean F standard deviation) were analyzed by analysis of variance, using a commercial software program (Statview SE +, Graphics, Abacus Concepts, Berkeley, CA). A p value < 0.05 was considered statistically significant.
Results Effects of aIR3 on cell growth Cell growth decreased significantly in the absence of glutamine (0 mM glutamine), compared to the control cultures (2 mM glutamine). This effect became apparent 48 hours after glutamine deprivation. In the presence of aIR3, cell proliferation rates were significantly reduced in both the control and the glutamine-deprived groups (Fig. 1).
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
131
Fig. 1. Effects of glutamine (Gln) deprivation and aIR3 (2 Ag/ml) on cell growth in a SK-N-SH human neuroblastoma cell line. Data are presented as the mean optical density F standard deviation for quadruplicated determinations. * p < 0.01, ** p < 0.001 vs. control, # p < 0.001 vs. glutamine deprivation.
Effects of aIR3 on amino acid transport We studied the effects of aIR3 on the transport of L-glutamine, L-glutamate, MeAIB, and L-leucine in both the control (2 mM glutamine) and the glutamine-deprived (0 mM glutamine) cultures. Initial studies demonstrated that the uptake of these amino acids was linear for at least 3 minutes. Na+dependent glutamine, Na+-dependent glutamate, Na+-dependent MeAIB, and Na+-independent leucine uptake represented at least 95%, 70%, 80%, and 95% of total uptake, respectively. Therefore, a 1minute transport assay of Na+-dependent glutamine, Na+-dependent glutamate, Na+-dependent MeAIB, and Na+-independent leucine was chosen for subsequent experiments. As shown in Fig. 2, Na+dependent glutamine transport was significantly less in the glutamine-deprived culture (649 F 49 pmol/ mg protein/min) than in the control (972 F 27 pmol/mg protein/min)(p < 0.001). Na+-dependent glutamine transport decreased significantly under both conditions after the addition of aIR3 (control+ aIR3, 874 F 83, p < 0.05; glutamine deprivation + aIR3, 489 F 36 pmol/mg protein/min, p < 0.001). In contrast to glutamine transport, Na+-dependent glutamate, Na+-dependent MeAIB, and Na+independent leucine transport increased significantly in the glutamine-deprived culture (glutamate, 66 F 6; MeAIB, 77 F 9; leucine, 3540 F 419 pmol/mg protein/min) compared to the control (glutamate, 33 F 6; MeAIB, 46 F 7; leucine, 1647 F 84 pmol/mg protein/min)(p < 0.001). Addition of aIR3 significantly decreased this up-regulation of amino acid transport down to the basal levels found in the control (glutamate, 46 F 7; MeAIB, 26 F 7; leucine, 2402 F 195 pmol/mg protein/min) (p < 0.001). There was a significant decrease in MeAIB transport (25 F 4 pmol/mg protein/min, p < 0.01) in the control group treated with aIR3.
132
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
Fig. 2. Effects of glutamine (Gln) deprivation and aIR3 (2 Ag/ml) on (A) Na+-dependent glutamine, (B) Na+-dependent glutamate, (C) Na+-dependent MeAIB, and (D) Na+-independent leucine transport after 24 hours. Data are presented as mean F standard deviation for quadruplicated determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control, # p < 0.001 vs. glutamine deprivation.
Effects of aIR3 on leucine transport kinetics To determine the kinetic basis for the glutamine deprivation and aIR3-induced changes in leucine transport, the transport of leucine from 10 AM to 2 mM was determined at 24 hours in three sets of conditions: (1) control (2 mM glutamine); (2) glutamine deprivation (0 mM glutamine); and (3) glutamine deprivation + aIR3 (Fig. 3). Glutamine deprivation significantly increased the maximum transport velocity of leucine (Vmax; control, 17945 F 672; glutamine deprivation, 55998 F 5435 pmol/mg protein/ min; P < 0.001) without affecting transport affinity (Km; control, 112 F 4; glutamine deprivation, 122 F 9 AM). The addition of antibody aIR3 significantly decreased Vmax (46083 F 1978 pmol/mg/protein/min, p < 0.01) without changing Km (121 F 10 AM) in the glutamine-deprived condition. Effects of aIR3 on DNA and protein synthesis Fig. 4 shows the effects of glutamine deprivation and aIR3 on 3H-thymidine and 3H-leucine incorporation rates. The data are expressed as percent of control values. Glutamine deprivation resulted in significant decreases in both 3H-thymidine (control, 100 F 8%; glutamine deprivation, 75 F 4%, p < 0.001) and 3H-leucine (control, 100 F 8%; glutamine deprivation, 85 F 11%, p < 0.01) incorporation rates. aIR3 decreased 3H-thymidine and 3H-leucine incorporation significantly in both
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
133
Fig. 3. Effects of glutamine (Gln) deprivation and aIR3 (2 Ag/ml) on leucine transport kinetics. The figure shows Eadie-Hofstee plot of saturable Na+-independent leucine transport. Transport velocity is plotted as a function of velocity/[leucine]. Data are presented as mean F standard deviation for quadruplicated determinations. Where not shown, the error bar is contained within the symbol.
the control (3H-thymidine, 79 F 5%; 3H-leucine, 66 F 4% , P < 0.001) and the glutamine-deprived conditions (3H-thymidine, 40 F 3%; 3H-leucine, 54 F 3%, P < 0.001). Effects of glutamine deprivation on the expression of IGF-IR In order to examine whether glutamine deprivation modulates the expression of IGF-IR, flow cytometric analysis was used to directly identify the IGF-IR protein on the cell surface. As shown in Fig. 5, an increase
Fig. 4. Effects of glutamine (Gln) deprivation and aIR3 (2 Ag/ml) on (A) 3H-thymidine and (B) 3H-leucine incorporation after 24 hours. Data are expressed as percentage of control values and are presented as mean F standard deviation for quadruplicated determinations. * p < 0.01, ** p < 0.001 vs. control, # p < 0.001 vs. glutamine deprivation.
134
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
Fig. 5. Flow cytometric analysis of the expression of the type I insulin-like growth factor receptor (IGF-IR) in control and glutamine (Gln) deprivation. Data using the anti-IGF-IR mAb were plotted as cell number (ordinate) vs. relative units of fluorescence intensity (abscissa). Open histogram with solid line shows control, and open histogram with dotted line glutamine deprivation. Shaded area is isotype control. The data shown here are representative of four independent experiments.
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
135
in IGF-IR expression was observed at 48 hours (control, 43%; glutamine deprivation, 56%) and 96 hours (control, 68%; glutamine deprivation, 85%) under conditions of glutamine deprivation, compared with the control, indicating that cells deprived of glutamine express more IGF-IR protein.
Discussion Our study shows that the growth of the human neuroblastoma cell line SK-N-SH is inhibited by the anti-IGF-IR antibody aIR3, and indicates that aIR3 decreases amino acid transport activity under both normal culture conditions and under conditions of glutamine deprivation. Blocking IGF-IR with aIR3, to prevent occupancy by the IGF ligand, eliminates the ability of IGF-I to protect human neuroblastoma cells from hyper-osmotic death [19]. Chambery showed a direct link between IGF-IR activity and N-myc amplification in SK-N-SH [20]. However, although IGF-IR activity and amino acid transport are the two major events that have been identified as independently instrumental in the growth of cancer cells, the relationship between IGF-IR and amino acid transport in cancer cells has not been shown. Our results provide the first evidence of IGF-IR regulation of amino acid transport activity across the cell membrane of a human neuroblastoma cell line. Glutamine deprivation resulted in decreased glutamine transport. In contrast, SK-N-SH cells responded to glutamine deprivation by increasing the transport activities of glutamate, MeAIB, and leucine. In SK-N-SH, glutamine is predominantly taken up through system ASC (our unpublished data). Na+-dependent glutamate, Na+-dependent MeAIB, and Na+-independent leucine are transported via systems XAG-, A, and L, respectively [21]. It is unclear what intracellular signals elicited by glutamine deprivation induce the up-regulation of these amino acids, when the glutamine transporter did not exhibit adaptive regulation. Glutamine has been shown to selectively induce the gene expression of ‘‘heat shock’’ proteins [22]. Therefore, one possibility is a change in the gene expression of individual amino acid transporters. Glutamine deprivation may exert positive specific effects on the expression of amino acid transporter genes, such as systems A, XAG-, and L. It is possible that one signal involved in controlling the gene expression is associated with changes in the intracellular concentration of glutamine. We have already shown that glutamine and leucine transport increased in a glutaminedeprived human hepatoma cell line, SK-Hep, whereas glutamine transport did not change in another hepatoma cell line, Hep G2 [23]. We should be cautious in applying our findings to other tumor cells, because the cell response to glutamine deprivation differs among cell lines. aIR3 completely blocked the adaptive increase of MeAIB transport induced by glutamine deprivation, and partially blocked the up-regulation of leucine and glutamate transport. While the precise mechanism whereby IGF-IR is able to maintain amino acid transport activity is unknown, several intriguing possibilities exist. As shown in Fig. 3, aIR3 decreased the up-regulation of leucine transport by decreasing Vmax, without affecting Km, which indicates that aIR3 decreases the number of active leucine transporters in the cell membrane without affecting the transporter affinity. An increase in the number of active transporter proteins in the cell membrane usually involves an increase in the de novo protein synthesis of the carrier itself [24]. In the present study, DNA and protein synthesis was significantly decreased in the aIR3 treated group. For many cell types, the activation of the IGF-IR is the last receptormediated event before DNA synthesis and mitosis [25]. Therefore, one possible mechanism is that the decreased DNA and protein synthesis induced by aIR3 leads to decreased de novo synthesis of amino acid transport proteins. When activated by its ligands, the IGF-IR transmits a signal to its two major substrates,
136
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
insulin receptor substrate 1 and Shc, a signal which is subsequently transducted via the common signaltransducing pathway to the nucleus [2]. Signaling through the IGF-IR increases expression of CDC2 mRNA, which is a critically important control mechanism in the eukaryotic cell cycle [26]. Based on these data, another possible mechanism for the decreased transport activity effected by aIR3 is that the decrease in the number of transport carriers may be the result of decreased rates of either transcription or translation of a carrier-specific gene, leading to decelerated de novo synthesis of carrier proteins. Malignant cells require increased amounts of amino acid to support DNA and protein biosynthesis, and amino acid transport plays an important role in the proliferation of these cells [6,27]. Our observation that decreased cell growth was associated with decreased amino acid transport supports the view that an IGFs/ IGF-IR system at least partially mediates SH-N-SH neuroblastoma cell proliferation by maintaining the transport of amino acids across the cell membrane. SK-N-SH cells responded to glutamine deprivation by increasing the amount of IGF-IR protein on the cell surface (Fig. 5). This response may result in enhanced stimulation of signal transduction by IGFs, and a consequent increase in amino acid transport activities, consistent with the mechanisms discussed above. Decreased extracellular amino acid levels encountered by tumors in vivo may elicit adaptive responses similar to those shown in our in vitro study, which contribute to the maintenance of the cytoplasmic levels of amino acids essential for growth. On the other hand, aIR3 did not completely inhibit cell growth, suggesting that other mitogenic factors may also be involved. These findings throw some light on the mechanisms that regulate cancer cell growth. Both the progression of tumor growth in human rhabdomyosarcoma-bearing animals and the formation of newly established tumors were suppressed by treatment with aIR3 [26]. In addition to its role in promoting cancer growth, IGF-IR has been shown to enhance the potential for the local and distant spread of cancer cells [28]. In general, there seem to be two approaches to blocking the effects of the IGFs/IGF-IR system: one is to block IGFs synthesis/secretion, and hence reduce local and systemic IGF levels; the other is to block the interaction of IGFs with their receptors. In view of the critical role of IGF-IR signal transduction in the regulation of cellular proliferation and amino acid transport, we speculate that blocking IGF-I receptors may prove a useful and novel clinical strategy in the management of neuroblastomas. Collectively our results, as presented here, support the concept that activation of IGF-IR helps to promote neuroblastoma cell proliferation by regulating amino acid transport across the cell membrane, both when sufficient nutrients are available, and when key nutrients are in short supply, as, for example, when glutamine is not provided. This mechanism may allow cancer cells to continue to grow even in tumor tissues that are deprived of nutrients. Acknowledgement This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] Werner H, LeRoith D. The role of the insulin-like growth factor system in human cancer. Advances in Cancer Research 1996;68:183 – 222. [2] Baserga R. The insulin-like growth factor-I receptor: a key to tumor growth? Cancer Research 1995;55(2):249 – 52. [3] Dunn SE, Hardman RA, Kari FW, Barrett JC. Insulin-like growth factor I (IGF-I) alters drug sensitivity of HBL100 human breast cancer cells by inhibition of apoptosis induced by diverse anticancer drugs. Cancer Research 1997;57(13):2687 – 93.
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
137
[4] Resnicoff M, Abraham D, Yutanawiboonchai W, Rotman HL, Kajstura J, Rubin R, Zoltick P, Baserga R. The insulin-like growth factor I receptor protects tumor cells from apoptosis in vivo. Cancer Research 1995;55(11):2463 – 9. [5] Coppola D, Saunders B, Fu L, Mao WG, Nicosia SV. The insulin-like growth factor 1 receptor induces transformation and tumorigenicity of ovarian mesothelial cells and down-regulates their Fas-receptor expression. Cancer Research 1999;59(13):3264 – 70. [6] Bode BP, Kaminski DL, Souba WW, Li AP. Glutamine transport in isolated human hepatocytes and transformed liver cells. Hepatology 1995;21(2):511 – 20. [7] Wasa M, Bode BP, Abcouwer SF, Collins CL, Tanabe KK, Souba WW. Glutamine as a regulator of DNA and protein biosynthesis in human solid tumor cell lines. Annals of Surgery 1996;224(2):189 – 97. [8] Kiess W, Koepf G, Christiansen H, Blum WF. Human neuroblastoma cells use either insulin-like growth factor-I or insulin-like growth factor-II in an autocrine pathway via the IGF-I receptor: variability of IGF, IGF binding protein (IGFBP) and IGF receptor gene expression and IGF and IGFBP secretion in human neuroblastoma cells in relation to cellular proliferation. Regulatory Peptides 1997;72(1):19 – 29. [9] Ota A, Wilson GL, Spilberg O, Pruss R, LeRoith D. Functional insulin-like growth factor I receptors are expressed by neural-derived continuous cell lines. Endocrinology 1988;122(1):145 – 52. [10] Martin DM, Yee D, Carlson RO, Felfman EL. Gene expression of the insulin-like growth factors and their receptors in human neuroblasroma cell lines. Molecular Brain Research 1992;15(3 – 4):241 – 6. [11] El-Badry OM, Helman LJ, Chatten J, Steinberg SM, Evans AE, Israel MA. Insulin-like growth factor II mediated proliferation of human neuroblastoma. The Journal of Clinical Investigation 1991;87(2):648 – 57. [12] Leventhal PS, Randolph AE, Vesbit TE, Schenone A, Windebank AJ, Feldman EL. Insulin-like growth factor-II as a paracrine growth factor in human neuroblastoma cells. Experimental Cell Research 1995;221(1):179 – 86. [13] Martin DM, Singleton JR, Meghani MA, Feldman EL. IGF receptor function and regulation in autocrine human neuroblastoma cell growth. Regulatory Peptides 1993;48(1 – 2):225 – 32. [14] Wasa M, Wang HS, Tazuke Y, Okada A. Insulin-like growth factor-I stimulates amino acid transport in a glutaminedeprived human neuroblastoma cell line. Biochimica et Biophysica Acta 2001;1525(1 – 2):118 – 24. [15] Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. Journal of Immunological Methods 1989;119(2):203 – 10. [16] Gazzola GC, Dall’Asta V, Franchi-Gazzola R, White MF. The cluster tray method for rapid measurement of solute fluxes in adherent cultured cells. Analytical Biochemistry 1981;115(2):368 – 74. [17] Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Analytical Biochemistry 1985;150(1):76 – 85. [18] Li YM, Brunke DL, Dantzer R, Kelley KW. Pituitary epithelial cell implants reverse the accumulation of CD4 CD8 lymphocytes in thymus glands of aged rats. Endocrinology 1992;130(5):2703 – 9. [19] Matthews CC, Feldman EL. Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death. Journal of Cellular Physiology 1996;166(2):183 – 222. [20] Chambery D, Mohseni-Zadeh S, Galle B, Babajko S. N-myc regulation of type I insulin-like growth factor receptor in a human neuroblastoma cell line. Cancer Research 1999;59(12):2898 – 902. [21] Kilberg MS, Stevens BR, Novac DA. Recent advances in mammalian amino acid transport. Annual Review of Nutrition 1993;13:137 – 65. [22] Cai JW, Hughes CS, Shen JW, Subjeck JR. Induction of heat-shock proteins by glutamine. FEBS Letters 1991; 288(1 – 2):229 – 32. [23] Wasa M, Bode BP, Souba WW. Adaptive regulation of amino acid transport in nutrient-deprived human hepatomas. Ameican Journal of Surgery 1996;171(1):163 – 9. [24] Shotwell MA, Kilberg MS, Oxender DL. The regulation of neutral amino acid transport in mammalian cells. Biochimica et Biophysica Acta 1983;737(2):267 – 84. [25] Pietrzkowski Z, Mulholland G, Gomella L, Jameson BA, Wernicke D, Baserga R. Inhibition of growth of prostatic cancer cell lines by peptide analogues of insulin-like growth factor 1. Cancer Research 1993;53(5):1102 – 6. [26] Kalebic T, Tsokos M, Helman LJ. In vivo treatment with antibody against IGF-I receptor suppresses growth of human rhabdomyosarcoma and down-regulates p34cdc2. Cancer Research 1994;54(21):5531 – 4. [27] Collins CL, Wasa M, Souba WW, Abcouwer SF. Determinations of glutamine dependence and utilization by normal and tumor-derived breast cell lines. Journal of Cellular Physiology 1998;176(1):166 – 78. [28] Macaulay VM. Insulin-like growth factors and cancer. British Journal of Cancer 1992;65(3):311 – 20.
Amino acid transport in a human neuroblastoma cell line is regulated by the type I insulin-like growth factor receptor Hong-Sheng Wang, Masafumi Wasa*, Akira Okada Department of Pediatric Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Received 3 July 2001; accepted 8 January 2002
Abstract Insulin-like growth factor I (IGF-I) and IGF-II stimulate cancer cell proliferation via interaction with the type I IGF receptor (IGF-IR). We put forward the hypothesis that IGF-IR mediates cancer cell growth by regulating amino acid transport, both when sufficient nutrients are present and when key nutrients such as glutamine are in limited supply. We examined the effects of aIR3, the monoclonal antibody recognizing IGF-IR, on cell growth and amino acid transport across the cell membrane in a human neuroblastoma cell line, SK-N-SH. In the presence of aIR3 (2 Ag/ml), cell proliferation was significantly attenuated in both control (2 mM glutamine) and glutaminedeprived (0 mM glutamine) groups. Glutamine deprivation resulted in significantly increased glutamate (system XAG ), MeAIB (system A), and leucine (system L) transport, which was blocked by aIR3. Glutamine (system ASC) and MeAIB transport was significantly decreased by aIR3 in the control group. Addition of aIR3 significantly decreased DNA and protein biosynthesis in both groups. Glutamine deprivation increased the IGF-IR protein on the cell surface. Our results suggest that activation of IGF-IR promotes neuroblastoma cell proliferation by regulating trans-membrane amino acid transport. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Insulin-like growth factor; Type I IGF receptor; Neuroblastoma; Amino acid transport; Glutamine
Introduction The effects of insulin-like growth factor I (IGF-I) and IGF-II (IGFs) are mediated via interaction with the type I IGF receptor (IGF-IR) [1]. Ligand binding activates the tyrosine kinase domain, resulting in both receptor autophosphorylation and phosphorylation of cytoplasmic substrates, which subsequently
*
Corresponding author. Tel.: +81-6-6879-3753; fax: +81-6-6879-3759. E-mail address: [email protected] (M. Wasa). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 6 2 6 - 0
128
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
bind to the activated receptor [2]. Recent work has confirmed the importance of IGFs/IGF-IR interactions in cancer cell proliferation by such mechanisms as the mediation of mitogenesis, the maintenance of a transformed phenotype, and the protection of tumor cells from apoptosis [3–5]. Cancer cells require an increased supply of amino acid precursors to support key intracellular metabolic pathways. In order to support increased demands for amino acids, they must be endowed with highly efficient transport systems. The essential role of carrier-mediated amino acid uptake in malignant cells is manifested by the fact that glutamine is transported across the plasma membrane faster than in normal cells [6]. Although glutamine can serve as an important ATP source, it is also a precursor for the synthesis of proteins and nucleic acids. It has been shown for cancer cells that both cell growth rates and DNA and protein biosynthesis correlate directly with the concentration of glutamine in culture media [7]. Neuroblastomas are malignant tumors found in children that are derived from neural crest cells. Human neuroblastoma cell lines have been reported to express IGF-IR and mRNA for IGFs [8–10]. Recent studies have suggested the importance of the interaction of IGFs with IGF-IR as an autocrine or paracrine growth factor mechanism in neuroblastoma cells [11,12]. These effects are blocked by the monoclonal antibody against IGF-IR, aIR3, which inhibits the interaction of the IGF-IR with its ligands. This indicates that functional IGF-IR plays a key role in the control of the signaling pathway of IGFs [12,13]. Cancer cells survive and even grow in conditions where nutrient resources are limited. These conditions can often be observed in the poorly vascularized center of solid tumors. A precise mechanism for this capacity for survival and growth has not yet been described. We put forward the hypothesis that IGF-IR mediates cancer cell growth by regulating amino acid transport activity, both when sufficient nutrients are present and when key nutrients such as glutamine are in limited supply. We have already shown that IGF-I stimulates amino acid transport in a glutamine-deprived human neuroblastoma cell line [14]. However, no study to date has addressed the effects of the functional impairment of IGF-IR on amino acid transport. To investigate our hypothesis, we examined the effects of aIR3 on cell growth and amino acid transport in a glutamine-deprived human neuroblastoma cell line. Here, we used the well-characterized neuroblastoma cell line, SK-N-SH, which expresses specific high-affinity functional IGF-IR [10].
Materials and Methods Chemicals Radiolabeled amino acid (3H-L-glutamine, 3H-L-glutamate, 3H-L-leucine), 3H-2-(methylamino) isobutyric acid (MeAIB, a system A-specific substrate), and 3H-L-thymidine were purchased from Amersham (Arlington Heights, IL). Dulbecco’s Modified Eagle medium (DMEM) was from GIBCO/ BRL Life Technologies (Grand Island, NY) and fetal bovine serum (FBS) was from JRH Biosciences (Lenexa, KS). Tissue culture plates were obtained from Costar Corp. (Corning, NY). A monoclonal antibody against IGF-IR (aIR3) was purchased from Oncogene Science (Uniondale, NY). An isotypematched mouse IgG1 (IgG1) and MTT cell growth kit were obtained from Chemicon (Temecula, CA). The F(abV)2 fragment of fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG was purchased from Cappel (Durham, NC). Amino acids and all biochemicals were purchased from Sigma Chemical (St. Louis, MO). A Neuroblastoma cell line, SK-N-SH, was provided by Dr. Tadao Ohno (RIKEN Cell Bank, Tsukuba, Japan).
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
129
Cell culture SK-N-SH cells were cultured in DMEM supplemented with 2 mM glutamine, 10% heat-inactivated FBS, 1,000 units/ml penicillin, and 1,000 units/ml streptomycin at 37 jC under a humidified atmosphere of 5%CO2/95% air. FBS was heat-inactivated to eliminate any variable sources of IGFs or other trophic factors such as epidermal growth factor from the culture medium. The culture medium was changed every 3 days until cells were confluent, at which point they were used for experiments. Cell proliferation Cells were seeded at a density of 1 104 cells/100 Al (100 Al/well) into 96-well tissue culture plates. After 24 hours, the culture medium was removed and changed to DMEM supplemented with 10% FBS plus glutamine (2 mM or 0 mM) in the presence of either aIR3 (2 Ag/ml) or IgG1 (2 Ag/ml). Cell proliferation was measured at days 0, 1, 2, and 3 using MTT cell growth assay [15]. Cell growth in 2 mM glutamine was chosen as the control. Amino Acid Transport Cells were seeded into 24-well tissue culture plates (0.5 ml/well). After getting 90-100% cell confluence, culture medium was removed and changed to DMEM supplemented with 10% FBS plus glutamine (2 mM or 0 mM) in the presence of either aIR3 (2 Ag/ml) or IgG1 (2 Ag/ml). L-glutamine, L-glutamate, MeAIB and L-leucine transport were measured at 24 hours. The transport of radiolabeled amino acids by cell monolayers was assayed by the cluster tray method of Gazzola et al. [16]. Before the transport assays, cells were rinsed twice with warm sodium-free Krebs-Ringer Phosphate Buffer (Chol-KRP, which was made by replacing the corresponding sodium salts with choline chloride and choline phosphate) to remove extracellular sodium and amino acids. After removal of Chol-KRP, the transport assay was initiated by transferring 0.25 ml of the uptake medium to 24-well trays. The transport of radiolabeled amino acid (5 ACi 3H-amino acid/ml) was performed for 1 minute at 37 jC at 10 Amol/L unlabeled amino acid in both sodium Krebs-Ringer Phosphate (Na-KRP) and Chol-KRP buffer. The assay was terminated by discarding the uptake buffer and ringing the cells with ice-cold Chol-KRP buffer three times. The wells containing the cells were allowed to dry and were solubilized in 200 Al of 0.2 N NaOH/0.2% sodium dodecyl sulfate (SDS) solution. One hundred Al of the cell extract was neutralized with 10 Al of 2 N HCl and subjected to scintillation spectrophotometry. Protein content was measured by the bicinchoninic acid protein method [17]. The Na+-dependent transport values were obtained by subtracting the transport values in Chol-KRP from those in Na-KRP. Saturable Na+independent transport values were determined in Chol KRP by subtracting the values in the presence of excess (10 mM) unlabeled amino acid from those in its absence. Transport velocities were expressed in units of picomoles per milligram of protein per minute. DNA and protein synthesis For the determination of DNA and protein synthesis, we measured the incorporation of 3H-thymidine and 3H-leucine, respectively, into acid-insoluble material. Cells were seeded in 24-well cluster trays (0.5 ml/well). When 90-100% cell confluence was reached, the medium was removed and replaced with
130
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
DMEM supplemented with 10% FBS plus glutamine (2 mM or 0 mM) in the presence of either aIR3 (2 Ag/ml) or IgG1 (2 Ag/ml). After 24 hours, 3H-thymidine or 3H-leucine (1 ACi/ml) was added to the culture medium and the cells were incubated for 2.5 hours at 37 jC. The assay was terminated after 2.5 hours, when the cells were washed twice with phosphate-buffered saline (PBS) and fixed by washing three times with ice-cold 10% trichloroacetic acid. Thereafter, the cells were rinsed twice with 70% and 95% ethanol, respectively. They were allowed to dry and solubilized in 200 Al of 0.2 N NaOH/0.2% SDS solution. Radioactivity and protein content were measured by the same procedures described for amino acid transport measurement. Flow cytometry to detect IGF-IR Based on the cell growth curve, cells were seeded in a 6-well culture plate (2 ml/well) at different densities in order to control for density-dependent effects at each time point between control and glutamine-deprived groups. After 24 hours, culture medium was removed and changed to DMEM supplemented with 10% FBS plus glutamine (2 mM or 0 mM). After 24, 48, and 96 hours, flow cytometry was carried out to identify the IGF-IR protein on the cell surface as described previously [18]. Briefly, 1 106 cells were washed with cold PBS supplemented with 1% FBS and pelleted in microfuge tubes by centrifugation at 1,000 g at 4 jC for 3 minutes. Then, 200 Al of anti-IGF-IR mAb (400 ng) diluted with PBS was added. As control, 200 Al of irrelevant IgG1 mAb in PBS was added at the same concentration as the primary antibody. Cells were incubated for 30 minutes at 4 jC and washed three times with PBS supplemented with 1% FBS. After the final wash, 200 Al of a 1:100 dilution of FITCconjugated goat antimouse IgG1 antibody [F(abV)2 fragments] was added to each tube. Cells were incubated for additional 30 minutes at 4 jC and washed three times with PBS supplemented with 1% FBS. Then, they were resuspended in 500 Al of PBS and maintained on ice for immediate analysis by flow cytometry. Stained cells were examined on FACScan (Becton Dickinson, San Jose, CA). At least 10,000 cells were counted for each sample. Data were processed by CellQuest program (Becton Dickinson, San Jose, CA). Statistical analysis Data (mean F standard deviation) were analyzed by analysis of variance, using a commercial software program (Statview SE +, Graphics, Abacus Concepts, Berkeley, CA). A p value < 0.05 was considered statistically significant.
Results Effects of aIR3 on cell growth Cell growth decreased significantly in the absence of glutamine (0 mM glutamine), compared to the control cultures (2 mM glutamine). This effect became apparent 48 hours after glutamine deprivation. In the presence of aIR3, cell proliferation rates were significantly reduced in both the control and the glutamine-deprived groups (Fig. 1).
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
131
Fig. 1. Effects of glutamine (Gln) deprivation and aIR3 (2 Ag/ml) on cell growth in a SK-N-SH human neuroblastoma cell line. Data are presented as the mean optical density F standard deviation for quadruplicated determinations. * p < 0.01, ** p < 0.001 vs. control, # p < 0.001 vs. glutamine deprivation.
Effects of aIR3 on amino acid transport We studied the effects of aIR3 on the transport of L-glutamine, L-glutamate, MeAIB, and L-leucine in both the control (2 mM glutamine) and the glutamine-deprived (0 mM glutamine) cultures. Initial studies demonstrated that the uptake of these amino acids was linear for at least 3 minutes. Na+dependent glutamine, Na+-dependent glutamate, Na+-dependent MeAIB, and Na+-independent leucine uptake represented at least 95%, 70%, 80%, and 95% of total uptake, respectively. Therefore, a 1minute transport assay of Na+-dependent glutamine, Na+-dependent glutamate, Na+-dependent MeAIB, and Na+-independent leucine was chosen for subsequent experiments. As shown in Fig. 2, Na+dependent glutamine transport was significantly less in the glutamine-deprived culture (649 F 49 pmol/ mg protein/min) than in the control (972 F 27 pmol/mg protein/min)(p < 0.001). Na+-dependent glutamine transport decreased significantly under both conditions after the addition of aIR3 (control+ aIR3, 874 F 83, p < 0.05; glutamine deprivation + aIR3, 489 F 36 pmol/mg protein/min, p < 0.001). In contrast to glutamine transport, Na+-dependent glutamate, Na+-dependent MeAIB, and Na+independent leucine transport increased significantly in the glutamine-deprived culture (glutamate, 66 F 6; MeAIB, 77 F 9; leucine, 3540 F 419 pmol/mg protein/min) compared to the control (glutamate, 33 F 6; MeAIB, 46 F 7; leucine, 1647 F 84 pmol/mg protein/min)(p < 0.001). Addition of aIR3 significantly decreased this up-regulation of amino acid transport down to the basal levels found in the control (glutamate, 46 F 7; MeAIB, 26 F 7; leucine, 2402 F 195 pmol/mg protein/min) (p < 0.001). There was a significant decrease in MeAIB transport (25 F 4 pmol/mg protein/min, p < 0.01) in the control group treated with aIR3.
132
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
Fig. 2. Effects of glutamine (Gln) deprivation and aIR3 (2 Ag/ml) on (A) Na+-dependent glutamine, (B) Na+-dependent glutamate, (C) Na+-dependent MeAIB, and (D) Na+-independent leucine transport after 24 hours. Data are presented as mean F standard deviation for quadruplicated determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control, # p < 0.001 vs. glutamine deprivation.
Effects of aIR3 on leucine transport kinetics To determine the kinetic basis for the glutamine deprivation and aIR3-induced changes in leucine transport, the transport of leucine from 10 AM to 2 mM was determined at 24 hours in three sets of conditions: (1) control (2 mM glutamine); (2) glutamine deprivation (0 mM glutamine); and (3) glutamine deprivation + aIR3 (Fig. 3). Glutamine deprivation significantly increased the maximum transport velocity of leucine (Vmax; control, 17945 F 672; glutamine deprivation, 55998 F 5435 pmol/mg protein/ min; P < 0.001) without affecting transport affinity (Km; control, 112 F 4; glutamine deprivation, 122 F 9 AM). The addition of antibody aIR3 significantly decreased Vmax (46083 F 1978 pmol/mg/protein/min, p < 0.01) without changing Km (121 F 10 AM) in the glutamine-deprived condition. Effects of aIR3 on DNA and protein synthesis Fig. 4 shows the effects of glutamine deprivation and aIR3 on 3H-thymidine and 3H-leucine incorporation rates. The data are expressed as percent of control values. Glutamine deprivation resulted in significant decreases in both 3H-thymidine (control, 100 F 8%; glutamine deprivation, 75 F 4%, p < 0.001) and 3H-leucine (control, 100 F 8%; glutamine deprivation, 85 F 11%, p < 0.01) incorporation rates. aIR3 decreased 3H-thymidine and 3H-leucine incorporation significantly in both
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
133
Fig. 3. Effects of glutamine (Gln) deprivation and aIR3 (2 Ag/ml) on leucine transport kinetics. The figure shows Eadie-Hofstee plot of saturable Na+-independent leucine transport. Transport velocity is plotted as a function of velocity/[leucine]. Data are presented as mean F standard deviation for quadruplicated determinations. Where not shown, the error bar is contained within the symbol.
the control (3H-thymidine, 79 F 5%; 3H-leucine, 66 F 4% , P < 0.001) and the glutamine-deprived conditions (3H-thymidine, 40 F 3%; 3H-leucine, 54 F 3%, P < 0.001). Effects of glutamine deprivation on the expression of IGF-IR In order to examine whether glutamine deprivation modulates the expression of IGF-IR, flow cytometric analysis was used to directly identify the IGF-IR protein on the cell surface. As shown in Fig. 5, an increase
Fig. 4. Effects of glutamine (Gln) deprivation and aIR3 (2 Ag/ml) on (A) 3H-thymidine and (B) 3H-leucine incorporation after 24 hours. Data are expressed as percentage of control values and are presented as mean F standard deviation for quadruplicated determinations. * p < 0.01, ** p < 0.001 vs. control, # p < 0.001 vs. glutamine deprivation.
134
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
Fig. 5. Flow cytometric analysis of the expression of the type I insulin-like growth factor receptor (IGF-IR) in control and glutamine (Gln) deprivation. Data using the anti-IGF-IR mAb were plotted as cell number (ordinate) vs. relative units of fluorescence intensity (abscissa). Open histogram with solid line shows control, and open histogram with dotted line glutamine deprivation. Shaded area is isotype control. The data shown here are representative of four independent experiments.
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
135
in IGF-IR expression was observed at 48 hours (control, 43%; glutamine deprivation, 56%) and 96 hours (control, 68%; glutamine deprivation, 85%) under conditions of glutamine deprivation, compared with the control, indicating that cells deprived of glutamine express more IGF-IR protein.
Discussion Our study shows that the growth of the human neuroblastoma cell line SK-N-SH is inhibited by the anti-IGF-IR antibody aIR3, and indicates that aIR3 decreases amino acid transport activity under both normal culture conditions and under conditions of glutamine deprivation. Blocking IGF-IR with aIR3, to prevent occupancy by the IGF ligand, eliminates the ability of IGF-I to protect human neuroblastoma cells from hyper-osmotic death [19]. Chambery showed a direct link between IGF-IR activity and N-myc amplification in SK-N-SH [20]. However, although IGF-IR activity and amino acid transport are the two major events that have been identified as independently instrumental in the growth of cancer cells, the relationship between IGF-IR and amino acid transport in cancer cells has not been shown. Our results provide the first evidence of IGF-IR regulation of amino acid transport activity across the cell membrane of a human neuroblastoma cell line. Glutamine deprivation resulted in decreased glutamine transport. In contrast, SK-N-SH cells responded to glutamine deprivation by increasing the transport activities of glutamate, MeAIB, and leucine. In SK-N-SH, glutamine is predominantly taken up through system ASC (our unpublished data). Na+-dependent glutamate, Na+-dependent MeAIB, and Na+-independent leucine are transported via systems XAG-, A, and L, respectively [21]. It is unclear what intracellular signals elicited by glutamine deprivation induce the up-regulation of these amino acids, when the glutamine transporter did not exhibit adaptive regulation. Glutamine has been shown to selectively induce the gene expression of ‘‘heat shock’’ proteins [22]. Therefore, one possibility is a change in the gene expression of individual amino acid transporters. Glutamine deprivation may exert positive specific effects on the expression of amino acid transporter genes, such as systems A, XAG-, and L. It is possible that one signal involved in controlling the gene expression is associated with changes in the intracellular concentration of glutamine. We have already shown that glutamine and leucine transport increased in a glutaminedeprived human hepatoma cell line, SK-Hep, whereas glutamine transport did not change in another hepatoma cell line, Hep G2 [23]. We should be cautious in applying our findings to other tumor cells, because the cell response to glutamine deprivation differs among cell lines. aIR3 completely blocked the adaptive increase of MeAIB transport induced by glutamine deprivation, and partially blocked the up-regulation of leucine and glutamate transport. While the precise mechanism whereby IGF-IR is able to maintain amino acid transport activity is unknown, several intriguing possibilities exist. As shown in Fig. 3, aIR3 decreased the up-regulation of leucine transport by decreasing Vmax, without affecting Km, which indicates that aIR3 decreases the number of active leucine transporters in the cell membrane without affecting the transporter affinity. An increase in the number of active transporter proteins in the cell membrane usually involves an increase in the de novo protein synthesis of the carrier itself [24]. In the present study, DNA and protein synthesis was significantly decreased in the aIR3 treated group. For many cell types, the activation of the IGF-IR is the last receptormediated event before DNA synthesis and mitosis [25]. Therefore, one possible mechanism is that the decreased DNA and protein synthesis induced by aIR3 leads to decreased de novo synthesis of amino acid transport proteins. When activated by its ligands, the IGF-IR transmits a signal to its two major substrates,
136
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
insulin receptor substrate 1 and Shc, a signal which is subsequently transducted via the common signaltransducing pathway to the nucleus [2]. Signaling through the IGF-IR increases expression of CDC2 mRNA, which is a critically important control mechanism in the eukaryotic cell cycle [26]. Based on these data, another possible mechanism for the decreased transport activity effected by aIR3 is that the decrease in the number of transport carriers may be the result of decreased rates of either transcription or translation of a carrier-specific gene, leading to decelerated de novo synthesis of carrier proteins. Malignant cells require increased amounts of amino acid to support DNA and protein biosynthesis, and amino acid transport plays an important role in the proliferation of these cells [6,27]. Our observation that decreased cell growth was associated with decreased amino acid transport supports the view that an IGFs/ IGF-IR system at least partially mediates SH-N-SH neuroblastoma cell proliferation by maintaining the transport of amino acids across the cell membrane. SK-N-SH cells responded to glutamine deprivation by increasing the amount of IGF-IR protein on the cell surface (Fig. 5). This response may result in enhanced stimulation of signal transduction by IGFs, and a consequent increase in amino acid transport activities, consistent with the mechanisms discussed above. Decreased extracellular amino acid levels encountered by tumors in vivo may elicit adaptive responses similar to those shown in our in vitro study, which contribute to the maintenance of the cytoplasmic levels of amino acids essential for growth. On the other hand, aIR3 did not completely inhibit cell growth, suggesting that other mitogenic factors may also be involved. These findings throw some light on the mechanisms that regulate cancer cell growth. Both the progression of tumor growth in human rhabdomyosarcoma-bearing animals and the formation of newly established tumors were suppressed by treatment with aIR3 [26]. In addition to its role in promoting cancer growth, IGF-IR has been shown to enhance the potential for the local and distant spread of cancer cells [28]. In general, there seem to be two approaches to blocking the effects of the IGFs/IGF-IR system: one is to block IGFs synthesis/secretion, and hence reduce local and systemic IGF levels; the other is to block the interaction of IGFs with their receptors. In view of the critical role of IGF-IR signal transduction in the regulation of cellular proliferation and amino acid transport, we speculate that blocking IGF-I receptors may prove a useful and novel clinical strategy in the management of neuroblastomas. Collectively our results, as presented here, support the concept that activation of IGF-IR helps to promote neuroblastoma cell proliferation by regulating amino acid transport across the cell membrane, both when sufficient nutrients are available, and when key nutrients are in short supply, as, for example, when glutamine is not provided. This mechanism may allow cancer cells to continue to grow even in tumor tissues that are deprived of nutrients. Acknowledgement This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] Werner H, LeRoith D. The role of the insulin-like growth factor system in human cancer. Advances in Cancer Research 1996;68:183 – 222. [2] Baserga R. The insulin-like growth factor-I receptor: a key to tumor growth? Cancer Research 1995;55(2):249 – 52. [3] Dunn SE, Hardman RA, Kari FW, Barrett JC. Insulin-like growth factor I (IGF-I) alters drug sensitivity of HBL100 human breast cancer cells by inhibition of apoptosis induced by diverse anticancer drugs. Cancer Research 1997;57(13):2687 – 93.
H.-S. Wang et al. / Life Sciences 71 (2002) 127–137
137
[4] Resnicoff M, Abraham D, Yutanawiboonchai W, Rotman HL, Kajstura J, Rubin R, Zoltick P, Baserga R. The insulin-like growth factor I receptor protects tumor cells from apoptosis in vivo. Cancer Research 1995;55(11):2463 – 9. [5] Coppola D, Saunders B, Fu L, Mao WG, Nicosia SV. The insulin-like growth factor 1 receptor induces transformation and tumorigenicity of ovarian mesothelial cells and down-regulates their Fas-receptor expression. Cancer Research 1999;59(13):3264 – 70. [6] Bode BP, Kaminski DL, Souba WW, Li AP. Glutamine transport in isolated human hepatocytes and transformed liver cells. Hepatology 1995;21(2):511 – 20. [7] Wasa M, Bode BP, Abcouwer SF, Collins CL, Tanabe KK, Souba WW. Glutamine as a regulator of DNA and protein biosynthesis in human solid tumor cell lines. Annals of Surgery 1996;224(2):189 – 97. [8] Kiess W, Koepf G, Christiansen H, Blum WF. Human neuroblastoma cells use either insulin-like growth factor-I or insulin-like growth factor-II in an autocrine pathway via the IGF-I receptor: variability of IGF, IGF binding protein (IGFBP) and IGF receptor gene expression and IGF and IGFBP secretion in human neuroblastoma cells in relation to cellular proliferation. Regulatory Peptides 1997;72(1):19 – 29. [9] Ota A, Wilson GL, Spilberg O, Pruss R, LeRoith D. Functional insulin-like growth factor I receptors are expressed by neural-derived continuous cell lines. Endocrinology 1988;122(1):145 – 52. [10] Martin DM, Yee D, Carlson RO, Felfman EL. Gene expression of the insulin-like growth factors and their receptors in human neuroblasroma cell lines. Molecular Brain Research 1992;15(3 – 4):241 – 6. [11] El-Badry OM, Helman LJ, Chatten J, Steinberg SM, Evans AE, Israel MA. Insulin-like growth factor II mediated proliferation of human neuroblastoma. The Journal of Clinical Investigation 1991;87(2):648 – 57. [12] Leventhal PS, Randolph AE, Vesbit TE, Schenone A, Windebank AJ, Feldman EL. Insulin-like growth factor-II as a paracrine growth factor in human neuroblastoma cells. Experimental Cell Research 1995;221(1):179 – 86. [13] Martin DM, Singleton JR, Meghani MA, Feldman EL. IGF receptor function and regulation in autocrine human neuroblastoma cell growth. Regulatory Peptides 1993;48(1 – 2):225 – 32. [14] Wasa M, Wang HS, Tazuke Y, Okada A. Insulin-like growth factor-I stimulates amino acid transport in a glutaminedeprived human neuroblastoma cell line. Biochimica et Biophysica Acta 2001;1525(1 – 2):118 – 24. [15] Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. Journal of Immunological Methods 1989;119(2):203 – 10. [16] Gazzola GC, Dall’Asta V, Franchi-Gazzola R, White MF. The cluster tray method for rapid measurement of solute fluxes in adherent cultured cells. Analytical Biochemistry 1981;115(2):368 – 74. [17] Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Analytical Biochemistry 1985;150(1):76 – 85. [18] Li YM, Brunke DL, Dantzer R, Kelley KW. Pituitary epithelial cell implants reverse the accumulation of CD4 CD8 lymphocytes in thymus glands of aged rats. Endocrinology 1992;130(5):2703 – 9. [19] Matthews CC, Feldman EL. Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death. Journal of Cellular Physiology 1996;166(2):183 – 222. [20] Chambery D, Mohseni-Zadeh S, Galle B, Babajko S. N-myc regulation of type I insulin-like growth factor receptor in a human neuroblastoma cell line. Cancer Research 1999;59(12):2898 – 902. [21] Kilberg MS, Stevens BR, Novac DA. Recent advances in mammalian amino acid transport. Annual Review of Nutrition 1993;13:137 – 65. [22] Cai JW, Hughes CS, Shen JW, Subjeck JR. Induction of heat-shock proteins by glutamine. FEBS Letters 1991; 288(1 – 2):229 – 32. [23] Wasa M, Bode BP, Souba WW. Adaptive regulation of amino acid transport in nutrient-deprived human hepatomas. Ameican Journal of Surgery 1996;171(1):163 – 9. [24] Shotwell MA, Kilberg MS, Oxender DL. The regulation of neutral amino acid transport in mammalian cells. Biochimica et Biophysica Acta 1983;737(2):267 – 84. [25] Pietrzkowski Z, Mulholland G, Gomella L, Jameson BA, Wernicke D, Baserga R. Inhibition of growth of prostatic cancer cell lines by peptide analogues of insulin-like growth factor 1. Cancer Research 1993;53(5):1102 – 6. [26] Kalebic T, Tsokos M, Helman LJ. In vivo treatment with antibody against IGF-I receptor suppresses growth of human rhabdomyosarcoma and down-regulates p34cdc2. Cancer Research 1994;54(21):5531 – 4. [27] Collins CL, Wasa M, Souba WW, Abcouwer SF. Determinations of glutamine dependence and utilization by normal and tumor-derived breast cell lines. Journal of Cellular Physiology 1998;176(1):166 – 78. [28] Macaulay VM. Insulin-like growth factors and cancer. British Journal of Cancer 1992;65(3):311 – 20.