Sialylation and sulfation of lactosylceramide distinctly regulate anchorage-independent growth, apoptosis, and gene expression in3LL Lewis lung carcinoma cells

Satoshi Uemura, Kazuya Kabayama, Mariko Noguchi, Yasuyuki Igarashi and Jin-ichi Inokuchi1

Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan

Received on August 15, 2002; revised on October 2, 2002; accepted on October 21, 2002


    Abstract
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 Abstract
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 Results
 Discussion
 Materials and methods
 References
 
To investigate the significance of sialylation and sulfation of lactosylceramide in transformed cells, we established ganglioside GM3- and lactosylsulfatide (SM3)-reconstituted cells by transfecting cDNAs of GM3 synthase and cerebroside sulfotransferase into the J5 subclone of 3LL Lewis lung carcinoma cells. The J5 clone was selected for the transfection of these genes because it lacks GM3 and SM3 but accumulates lactosylceramide. The anchorage-dependent growth of both GM3- and SM3-reconstituted cells was similar. However, anchorage-independent growth (as measured by colony-forming ability in soft agar) of the SM3- reconstituted cells was almost completely lost, which supports our previous observation showing the suppression of tumorigenic potential in vivo and ß1 integrin gene expression induced by the introduction of cerebroside sulfotransferase gene (Kabayama et al. [2001] J. Biol. Chem., 276, 26777–26783). The GM3-reconstituted cells formed a significantly higher number of colonies in soft agar compared to mock-transfected cells and began to proliferate and become resistant to apoptosis when serum was depleted, indicating that endogenous GM3 is essential for maintaining these fundamental properties of malignant cells. We also found that serum-induced ERK1/2 activation was suppressed in the GM3-reconstituted cells, suggesting that anchorage-independent cell cycle initiation by endogenous GM3 is elicited through pathway(s) independent of ERK1/2 activation. The selective down-regulation of platelet-derived growth factor (PDGF)-dependent ERK1/2 activation in the GM3-reconstituted cells was due to the substantial decreases of PDGF {alpha} receptor mRNA and protein, but in the SM3-reconstituted cells PDGF {alpha} receptor expression was similar to mock cells. Thus, endogenously produced GM3 and SM3 differentially and distinctly regulate tumor-progression ability, that is, GM3 leads the transformed phenotype of J5 cells to promotion and SM3 to abrogation.

Key words: ß1 integrin / ganglioside GM3 / malignancy / PDGF {alpha} receptor / sulfatide SM3


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The relationship between gangliosides and cancer malignancy has long been a subject of focused interest (Hakomori, 1989Go, 1994Go; Hakomori and Igarashi, 1995Go). Ganglioside GM3, a major ganglioside in many mammalian cells, was found to be abundant in highly metastatic lines of B16 melanoma (Yogeeswaran et al., 1978Go; Nozue et al., 1988Go). Also, the change of ganglioside patterns has been demonstrated by the transformation with viral infection (Hakomori and Murakami, 1968Go; Mora et al., 1969Go; Yogeeswaran et al., 1972Go). For instance, GM3 has been found to be increased by transformation with oncogenic SV40 and polyoma viruses (Mora et al., 1969Go; Yogeeswaran et al., 1972Go), which could be due to simplification of sugar chains after changes in glycosyltransferase activities. Increased expression of GM3 has also been observed, in cells transformed by the v-raf proto-oncogene (Dnistrian et al., 1975Go). The working hypothesis inspired by these observations is that expression of GM3 on cancer cells might accompany increases in cancer malignancy. However, cancer cells exhibiting low GM3 expression but high malignancy also exist (Koyama et al., 1983Go; Nakakuma et al., 1984Go; Nakaishi et al., 1988aGo,bGo,cGo; Watanabe et al., 2002Go).

CMP-N-acetylneuraminate:lactosylceramide {alpha}-2,3-N-acetylneuraminyltransferase (SAT-I) (Ishii et al., 1998Go) is the first enzyme in the ganglioside biosynthetic pathways (Figure 1). The functional roles of GM3 have thus far been examined indirectly by the addition of exogenous GM3 to culture medium (Bremer et al., 1984Go, 1986Go; Stevens et al., 1989Go; Nakamura et al., 1991Go) or by the depletion of precursor glycosphingoslipids (GSLs) using inhibitors of glucosylceramide synthase (Inokuchi and Radin, 1987Go; Inokuchi et al., 1989Go, 2000Go; Kyogashima et al., 1996Go; Meuillet et al., 2000Go; Tagami et al., 2002Go). These results suggest that GM3 may be involved in cell proliferation by regulating growth factor receptor activities (Bremer et al., 1984Go, 1986Go; Meuillet et al., 2000Go; Nojiri et al., 1991Go) and in cell adhesion and motility by interacting with integrins and/or integrin-related molecule(s) (Zheng et al., 1993Go; Ono et al., 2001Go).



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Fig. 1. LacCer branching in GSL biosynthesis. Enzymes acting on LacCer: (1) GM3 synthase (SAT-I) (Ishii et al., 1998Go); (2) GA2/GM2/GD2 synthase (GalNAc-T) (Nagata et al., 1992Go); (3) CST (Honke et al., 1997Go); (4) Lc3 synthase (ß3Gn-T5) (Togayachi et al., 2001Go); (5) Gb3 synthase (Kojima et al., 2000Go); (6) iGb3 synthase (Keusch et al., 2000Go).

 
However, it has not been conclusively demonstrated that the effect of exogenously added gangliosides or pharmacologically depleted gangliosides reflects the biological function of endogenous gangliosides. Recently, we established the lactosylsulfatide (SM3)-reconsituted cells by introducing cerebroside sulfotransferase (CST) cDNA (Honke et al., 1997Go) into a subclone of 3LL Lewis lung carcinoma cells, the J5 clone, which lacks acidic GSLs but accumulates LacCer (Kabayama et al., 2001Go). The SM3-reconstituted J5 (J5/CST) cells exhibited a loss of cell adhesion to the extracellular matrix proteins laminin and fibronectin and lost their tumorigenic potential in vivo, both of which could be attributed to decreased expression of ß1 integrin gene (Kabayama et al., 2001Go).

To clarify the significance of sialylation and sulfation of LacCer (Figure 1), we have now established the GM3-reconstituted cells by transfecting the SAT-I cDNA into the J5 clone. The functional aspects of the GM3- reconstituted J5 (J5/SAT-I) cells were investigated and compared with those of J5/CST cells. We were able to demonstrate distinct functions for endogenous GM3 and SM3 in the transformed phenotypes.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Reconstitution of GM3 and SM3
The J5 clone, which lacks acidic GSLs and accumulats LacCer, was isolated previously from wild-type murine 3LL Lewis lung carcinoma cells (Inokuchi et al., 1993Go). The mouse SAT-1 (Fukumoto et al., 1999Go) was introduced into the J5 clone to generate GM3-expressing clones. Thirty transfectants were obtained; three clones, which expressed a high level of GM3, were chosen for further study (Figure 2, left panel). Analysis of the neutral GSLs of these SAT-I-transfected cells showed that most of the cellular LacCer was converted to GM3 (Figure 2). We recently established the CST-transfected cells using the same transfection procedure (Kabayama et al., 2001Go). The accumulation of SM3 in two clonal isolates is shown in Figure 2. These results indicate that SAT-I transfected cells express only GM3, whereas CST-transfected J5 cells express only SM3.



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Fig. 2. GSL analysis of SAT-I- and CST-transfected J5 cells. GSLs extracted from J5/SAT-I, J5/CST, and mock-transfected cells, corresponding to 1 mg of protein, were separated on HPTLC plates and stained with orcinol-sulfuric acid for acidic GSLs or with cupric acetate–phosphoric acid reagent for neutral sphingolipids as described in Materials and methods.

 
Comparison of anchorage-dependent and -independentcell growth
There was no difference in the anchorage-dependent growth on plastic substrate with 10% serum among J5/SAT-I and J5/CST clones and mock-transfected (mock) cells (Figure 3A). On the other hand, the GM3-reconstituted cells acquired greater ability to form colonies in soft agar than mock cells, whereas J5/CST cells were unable to grow in soft agar (Figure 3B). Anchorage-independent growth measured by colony formation in a semi-solid medium is thought to be one of the fundamental properties of malignant cells (Freedman and Shin, 1974Go; Cifone, 1982Go).



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Fig. 3. Comparison of cell growth and colony-forming ability of GM3- and SM3-reconstituted J5 cells. (A) Cell growth of the GM3- and SM3-expressing cells under normal culture conditions in the presence of 10% FCS in culture plastics (anchorage-dependent growth). Five thousand J5/SAT-I and mock cells were plated in 96-well plates, and the cell growth rate of each clone was measured every day as mentioned in Materials and methods. (B) The colony-forming ability in soft agar in the presence of 10% FBS was examined as a measure of anchorage-independent growth as described in Materials and methods. Seven days after plating, positive colonies were photographed under 40x magnification. Values represent the mean percentage of colonies having more than 50 µm in diameter. Bar=50 µm. *p < 0.005 as compared with mock control cells.

 
Endogenous SM3 selectively suppresses ß1 integrin gene expression and cell adhesion
Since the expression of ß1 integrin is decreased in J5/CST cells (Kabayama et al., 2001Go), we compared the mRNA levels of ß1 integrin between J5/SAT-I and J5/CST cells by northern blot analysis. A significant decrease of ß1 integrin mRNA was apparent in J5/CST cells (Figure 4). In contrast, there was no alteration of ß1 integrin mRNA level in J5/SAT-I cells, indicating that the action on the regulation of ß1 integrin expression was unique to SM3. J5/CST cells exhibited a marked decrease in their adhesive abilities to fibronectin-coated plastic substrates (Kabayama et al., 2001Go), yet the adhesive ability of J5/SAT-I cells to fibronectin and laminin remained comparable to mock cells (data not shown).



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Fig. 4. Decrease of ß1 integrin mRNA in SM3-reconstituted J5 cells but not in GM3-reconstituted cells. Northern blot analysis of ß1 integrin mRNA. Total RNA samples (10 µg) from J5, mock, and J5/CST or J5/SAT-I cells were electrophoresed, transblotted onto a nylon membrane, and hybridized with a digoxigenin-labeled RNA probe of ß1 integrin, as described under Materials and methods. Methylene blue staining of 18S and 28S rRNA in the same membrane is shown.

 
Endogenous GM3 enhances the transformed phenotype
Although under normal culture conditions (10% serum), anchorage-dependent growth of both J5/SAT-I and J5/CST cells was similar (Figure 3A), only J5/SAT-I cells were able to proliferate progressively in the medium containing 0.1% serum (Figure 5A). Moreover, under serum-starved condition, J5/SAT-I cells displayed resistance to apoptosis, whereas mock cells remained susceptible (Figure 5B). These results suggest that endogenous GM3 is essential for maintaining cell growth under serum depleted and anti-apoptotic properties. In contrast to GM3-reconstituted cells, SM3-reconstituted cells were not able to proliferate and exhibited higher levels of apoptosis in the 0.1% serum-containing medium (Figure 5B).



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Fig. 5. Cell growth and apoptosis in serum starved condition. (A) Five thousand J5/SAT-I, J5/CST, and mock cells were plated in 96-well plates in RPMI-1640 medium containing 0.1% FCS, and the cell growth of each clone was measured. *p < 0.001 as compared with mock control cells. (B) Cells were cultured in RPMI-1640 medium containing 0.1% FCS for 0, 1, or 2 days and subjected to a DNA fragmentation assay as described under Materials and methods.

 
Decrease in serum- and growth factor–dependentERK1/2 activation in GM3-reconstituted cells
Extracellular regulated kinase (ERK) 1/2 activity correlates well with cell proliferation, and it acts downstream of many growth factor receptors. For this reason, we compared the ERK1/2 activation in GSL-reconstituted clones. J5/SAT-I and mock cells maintained in fetal calf serum (FCS)-free medium for 24 h were subsequently stimulated with 10% FCS or lipid-depleted FCS treated with charcoal (C-FCS). ERK1/2 phosphorylation induced by FCS and lipid-depleted FCS in J5/SAT-I cells were 6- and 1.3-fold, respectively, but were 8- and 3.5-fold in mock cells (Figure 6). The activation of ERK1/2 by C-FCS lipid-depleted FCS in the GM3-reconstituted cells was remarkably low in comparison to mock cells, perhaps due to insensitivity of the cells to some polypeptide growth factors. Therefore, we examined the activation of ERK1/2 after stimulation with the polypeptide growth factors epidermal growth factor (EGF), insulin, and platelet-derived growth factor (PDGF). Subsequently, the activation of ERK1/2 in cells stimulated with the lipid growth factors sphingosin 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) was examined. As shown in Figure 7, ERK1/2 activation in J5/SAT-I cells was selectively and substantially diminished by PDGF but not the other growth factors.



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Fig. 6. Mitogen-activated protein kinase activation in GM3-reconstituted J5 cells by FCS and C-FCS. Cells were cultured in RPMI-1640 containing 0.3% FCS for 24 h. The serum-starved cells were treated with 10% FCS or 10% C-FCS for 5 min and lysed in lysis buffer. Lysates (50 µg/lane) were subjected to immunoblot analysis with anti-phospho-ERK1/2 antibody or anti-ERK1/2 antibody. The results are representative of one of three separate experiments.

 


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Fig. 7. Mitogen-activated protein kinase activation in GM3-reconstituted J5 cells by EGF, insulin, PDGF, Sph-1-P, and LPA. Cells were cultured in RPMI-1640 containing 0.3% FCS for 24 h. The serum-starved cells were then treated with the indicated amounts of EGF, insulin, PDGF, Sph-1-P, and LPA for 5 min and lysed on lysis buffer. Lysates (50 µg/lane) were subjected to immunoblot analysis with anti-phospho-ERK1/2 antibody or anti-ERK1/2 antibody.

 
Selective suppression of PDGF{alpha}R expression inGM3 reconstituted cells
Since 3LL Lewis lung carcinoma cells express PDGF {alpha} receptor (PDGF{alpha}R) (Do et al., 1992Go), its expression in J5/SAT-I cells was compared with that of mock cells. Surprisingly, there was a significant decrease in PDGF{alpha}R expression as measured by western blotting (Figure 8A, upper panel). In contrast, the expression of PDGF{alpha}R in J5/CST cells was similar to that of mock cells (Figure 8A, lower panel). To elucidate whether the decrease of PDGF{alpha}R in J5/SAT-I cells occurred at the transcriptional or posttranscriptional levels, northern analysis was performed. Very low levels of PDGF{alpha}R mRNA were detected in J5/SAT-I cells as compared to the parental or mock-transfected clones (Figure 8B). PDGFßR mRNA was undetectable in J5, mock, and J5/SAT-I cells by this technique (Figure 8C).



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Fig. 8. GM3 but not SM3 down-regulates the expression of PDGF{alpha}R. (A) Cell lysates (50 µg protein/lane) from J5, mock, and J5/SAT-I cells (top) and from J5 mock and J5/CST cells (bottom) were subjected to western blot analysis with anti-PDGF{alpha}R. (B) Northern blot analysis of PDGF{alpha}R mRNA (top). Total RNA samples (12 µg) from J5, mock, and J5/SAT-I cells were electrophoresed, transblotted onto a nylon membrane, and hybridized with a digoxigenin-labeled probe of PDGF{alpha}R. Methylene blue staining of 18S and 28S rRNA in the same membrane (bottom). (C) Northern blot analysis of PDGFßR mRNA (top) was performed using a digoxigenin-labeled probe of PDGFßR. Methylene blue staining of 18S and 28S rRNA in the same membrane (bottom).

 
Comparison of anchorage-dependent and -independent growth and PDGF{alpha}R expression between 3LL Lewislung carcinoma cell lines
We examined whether the phenomena observed in J5/SAT-I cells could also be observed in wild-type (parent) 3LL Lewis lung carcinoma cells containing high levels of GM3 (Figure 9A). There was no difference in anchorage-dependent growth between the parent 3LL and J5 cells (Figure 9B). In contrast, anchorage-independent growth was greater in the wild cells as compared to J5 cells (Figure 9C). Moreover, we confirmed that the expression of PDGF{alpha}R in the parent 3LL cells at the protein and mRNA level was decreased (Figure 9D and E), suggesting that the increased expression of endogenous GM3 could be the cause of the suppression of PDGF{alpha}R gene expression.



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Fig. 9. Comparison of anchorage-dependent and -independent growth and PDGF{alpha}R expression between 3LL Lewis lung carcinoma cell lines. (A) Total GSLs combined with the acidic and neutral lipid fractions from 3LL-P and J5 cells corresponding to 1 mg of protein were applied to an HPTLC plate, developed, and stained with cupric acetate–phosphoric acid reagent. (B) Cell growth of 3LL-P and J5 cells under normal culture conditions in the presence of 10% FCS (anchorage-dependent growth) was measured as in Figure 3A. (C) The colony-forming ability of 3LL-P and J5 in soft agar was examined as in Figure 3B. Bar=50 µm. (D) Cell lysates (50 µg protein/lane) from 3LL-P and J5 cells were subjected to immunoblot analysis with anti-PDGF{alpha}R. (E) Northern blot analysis of PDGF{alpha}R in 3LL-P and J5 cells were performed as in Figure 8B.

 

    Discussion
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 Abstract
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 Materials and methods
 References
 
LacCer is the branching point in complex GSL biosynthesis (Figure 1). GSL expression is primarily determined by cell type–specific expression of enzymes at the lumenal side of the Golgi apparatus. Because the composition and distribution of LacCer-derived GSLs are known to be greatly altered during development and oncogenic transformation (Hakomori, 1989Go, 1994Go; Hakomori and Igarashi, 1995Go), the elucidation of the biological significance of LacCer branching in various cellular functions is one of the most important issues of glycobiology. The cDNAs of six enzymes acting at LacCer branching point have recently been cloned (Ishii et al., 1998Go; Honke et al., 1997Go; Nagata et al., 1992Go; Kojima et al., 2000Go; Keusch et al., 2000Go; Togayachi et al., 2001Go), making it possible to directly examine the functions of GM3 and SM3 and other GSLs related to the cloned enzymes mentioned.

To elucidate the function of individual GSLs, we have been examining various GSL-reconstituted cells by introducing a series of GSL synthase cDNAs into deficient cells (Kabayama et al., 2001Go). Here, we established ganglioside GM3-reconstituted cells by transfecting the SAT-I cDNA into a subclone (J5) isolated without mutagenesis from wild-type 3LL Lewis lung carcinoma cells (Inokuchi et al., 1993Go). The J5 clone, which lacks acidic GSLs but accumulates LacCer, is an ideal cell line for the investigation of the biological significance of the LacCer branching point in GSL biosynthesis. In the present study, the significance of sialylation versus sulfation of LacCer was examined by comparing the properties of J5/SAT-I and J5/CST cells.

The major findings of the present study are: (1) Anchorage-independent growth was strikingly different between the GM3- and SM3-reconstituted cells, showing promotion and abrogation, respectively. There was no difference in cell growth under normal culture conditions in plastic plates; however, only J5/SAT-I cells were able to proliferate and became resistant to apoptosis under serum-starved conditions. Thus, endogenous GM3 appears to be a required molecule for maintaining the transformed phenotype.

(2) The suppression of anchorage-independent growth and cell adhesion to laminin and fibronectin in J5/CST cells could be attributed to decreased ß1 integrin gene expression as reported previously (Kabayama et al., 2001Go), whereas these properties in J5/SAT-I cells remained unchanged. (3) In J5/SAT-I cells, serum- and growth factor-dependent ERK1/2 activation was diminished due to decreased expression of PDGF{alpha}R gene, suggesting that the anchorage-independent cell cycle initiation by endogenous GM3 is elicited independently of ERK1/2 activation. (4) Because the expression of GM3 and SM3 in 3LL lung cancer cells resulted in a selective decrease in PDGF{alpha}R and ß1 integrin mRNA level, we propose the novel hypothesis that individual GSLs might regulate the gene expression of a select number of genes.

J5/SAT-I and J5/CST cells, as well as the J5 cells transfected with the empty vector alone, theoretically share an identical genetic background with the exception of the expression of the SAT-I and CST genes. Thus it is now possible to examine the functional role of an individual GSL molecule in these cells by employing the present GSL reconstitution strategy. Therefore, the selective suppression of the PDGF{alpha}R gene in the GM3-reconstituted cells and the ß1 integrin gene in the SM3-reconstituted cells can be attributed to the expression of cellular GM3 and SM3, respectively. It is well known that the composition of GSLs changes remarkably during development, differentiation and oncogenic transformation (Hakomori, 1989Go, 1994Go; Hakomori and Igarashi, 1995Go). It is generally believed that these modifications are the result of changes in expression of various genes, including those encoding growth factors, hormones, cytokines, and their receptors. In turn, these altered bioactive signaling factors transmit altered cellular signals to the nucleus, affecting the downstream gene expression of various GSL synthase genes. Conversely, we propose that individual GSLs distinctly govern gene expression, as exemplified the PDGF{alpha}R and ß1 integrin genes. We postulate that individual GSLs selectively and distinctly affect gene expression through their effects on the membrane microdomain component(s) and through the effects of the complex formed with individual GSLs on signaling molecules regulating gene expression.

Gangliosides play an important role modulating signal transduction, as evidenced by reports that exogenous gangliosides suppress auto-phosphorylation of the PDGFßR (Bremer et al., 1984Go, 1986Go), EGF receptor (Bremer et al., 1986Go; Meuillet et al., 2000Go), and insulin receptor (Nojiri et al., 1991Go; Tagami et al., 2002Go). However, no previous report has directly analyzed the interaction between endogenous GM3 and these receptor molecules. Using GM3-reconstituted cells expressing the SAT-I gene, the activation of ERK1/2 was examined as a monitor of the interaction of receptor molecules and GM3, because ERK1/2 functions downstream of multiple receptors. When the phosphorylated ERK1/2 levels were compared in J5/SAT-I cells maintained in steady-state conditions to those in mock-transfected cells, no difference was found (data not shown). This could be expected because cell proliferation under these conditions was essentially the same (Figure 3A).

Next, we examined the serum-dependent activation of ERK1/2 and found that in J5/SAT-I cells the activation was significantly reduced in comparison with that in control cells (Figure 6). Additionally, this defect in activation was apparently due to decreased PDGF{alpha}R expression (Figures 7 and 8). It has been reported that the activation of ERK1/2 mainly correlates with anchorage-dependent growth and is not involved in transformed phenotypes induced by protooncogenes (Kizaka-Kondoh and Okayama, 1993Go). Because serum-dependent ERK1/2 activation in J5/SAT-I cells was decreased without decreasing the anchorage-dependent growth (Figures 3 and 6), expression of GM3 appears to be a factor in the lowered requirement for growth factors in these and other types of cancer cells. In summary, GM3 enhances anchorage-independent growth and anti-apoptotic properties, which are the most fundamental abilities of cancer cells, through a signaling pathway independent of ERK1/2 activation.

The relationship between the up-regulation of anchorage-independent growth (Figure 3B) and the decrease in PDGF{alpha}R gene expression (Figure 8B) is an intriguing phenomenon that requires further investigation. In NIH-3T3 cells, JNK-1, which is activated by PDGF{alpha}R- dependent signaling, reportedly counteracts the enhancement of anchorage-independent growth induced by PDGFßR signaling (Yu et al., 2000Go). Thus there is a possibility that the decreased expression of PDGF{alpha}R in the GM3-reconstituted cells results in the up-regulation of the tumorigenic potential via some other mechanisms, because J5/SAT-I cells did not express PDGFßR (Figure 8C). Why is the expression of GM3 connected with the decrease in the PDGF{alpha}R gene? We are currently clarifying whether the decrease of PDGF{alpha}R mRNA in J5/SAT-I cells is due to reduced transcription or instability of the mRNA by using a nuclear runoff assay. Moreover, we have begun to monitor the whole image of gene expression control by endogenous GM3 using DNA microarray technology.

It is noteworthy that the expression of PDGF{alpha}R in the wild-type 3LL Lewis lung carcinoma cells, which possess high GM3 content and potent tumorigenic potential, is remarkably lower than that of the GM3-deficient J5 subclone (Figure 9). Therefore, it will be an important future subject to examine various cancer cells expressing GM3 as their major ganglioside to clarify the common role(s) of GM3 in malignancy.


    Materials and methods
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 Abstract
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 Materials and methods
 References
 
Reagents
For GSLs analysis, DEAE-Sephadex A-25 was purchased from Amersham Pharmacia Biotech (Buckinghamshire, England), and Sep-Pack C18 and silica gel high-performance thin-layer chromaotgraphy (HPTLC) plates were from Waters Associates (Milford, MA) and Merck, respectively. PcDNA3.1/Zeo(+), zeocin, and LipofectAMINE PLUS Reagent were from Invitrogen (Carlsbad, CA). For western blotting of anti-p-ERK1/2, mouse monoclonal IgG (sc-7383), anti-PDGF{alpha}R rabbit IgG (sc-338), and anti-insulin receptor ß subunit rabbit IgG (sc-711) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-ERK1/2 rabbit IgG was from New England BioLabs (Beverly, MA). Murine natural epidermal growth factor was from Life Technologies. Human recombinant PDGF-BB, LPA oleoyl (C18:1, [cis]-9), and insulin (bovine pancreas) were from Sigma-Aldrich (St. Louis MO). Sph-1-p was from Avanti (Alabaster, AL). Bicinchoninic acid reagent from Pierce Chemical Company (Rockford, IL) was used for protein determination. All other reagents were purchased from Sigma-Aldrich unless otherwise mentioned.

Cell lines
The J5 subclone of the murine 3LL Lewis lung carcinoma cell line has been described previously (Inokuchi et al., 1993Go). Cells were maintained in RPMI 1640 medium containing 10% (v/v) FCS, 100 U/ml penicillin, 100 ng/ml streptomycin, 9.4% (v/v) sodium bicarbonate, and 100 mM L-glutamine. Cells were cultured in a humidified, 5% CO2 atmosphere tissue culture incubator and passaged every 3 days using a 0.25% trypsin/1 mM ethylenediamine tetra-acetic acid (EDTA) solution. Transfected and mock-transfected cells were cultured in the medium containing also 300 µg/ml zeocin (Invitrogen).

Cell proliferation assay
Cell viability and proliferation was determined by using the cell proliferation reagent WST-8, a tetrazolium salt that is cleaved by mitochondrial dehydrogeneses in viable cells. Briefly, cells in RPMI 1640 containing FCS (10% or 0.1%) and 300 µg/ml zeocin at 5x104 cells/ml were plated to 96-well microtiter plates at 100 µl/well. Ten microliters of WST-8 was added to each well at the specified day of culture and then incubated for 2 h at 37°C. The absorbance at 450 nm of the formazan generated in the wells was measured with a dual-wavelength flying spot scanner (CS9300-PC, Shimadzu, Kyoto, Japan).

SAT-I gene transfection
J5 cells were transfected using LipofectAMINE PLUS reagent with the plasmid pcDNA3.1/Zeo(+)-mSAT-I (Fukumoto et al., 1999Go). Mock-transfected cells were prepared with pcDNA3.1/Zeo(+) without mSAT-I cDNA. The transfectants were selected in the same medium used for growing them in the presence of zeocin.

Lipid analysis
Cells (1x107) were collected, washed twice with phosphate buffered saline (PBS), and the lipids were extracted from the cell pellet as described (Macher and Klock, 1980Go) and fractionated (Ledeen et al., 1973Go). Briefly, the total lipid extract was dissolved in chloroform/methanol/water (30:60:8, v/v/v), passed through a DEAE-Sephadex A-25 column (0.8x4.5 cm; acetate form), and eluted with five volumes each of the same solvent (neutral lipid fraction), and chloroform/methanol/1 M aqueous Na acetate (30:60:8) (acidic lipid fraction). The solvent was evaporated to dryness, and esters were cleaved with methanolic 0.5 M NaOH for 1 h 40°C. The solution was neutralized with 1 M acetic acid in methanol and diluted with 6 ml 50 mM NaCl solution then desalted with Sep-Pack C18 reverse-phase cartridge (Waters). Neutral GSLs were separated by HPTLC using chloroform/methanol/acetic acid/formic acid/water (40:18:7.2:2.4:1.2) and detected with 3% cupric acetate–8% phosphoric acid reagent. Gangliosides were separated by HPTLC using chloroform/methanol/0.5% CaCl2 (60:40:9) and detected with orcinol-sulfuric acid reagent. The quantity of each sphingolipid was measured with a dual-wavelength flying spot scanner in the reflectance mode at 500 nm.

Colony forming assay in soft agar
Single cells were plated in 0.4% semi-solid agar in RPMI-1640 medium containing 10% fetal bovine serum (FBS). Aliquots containing 5000 cells were plated on a basal layer of 0.5% agar in growth medium in 60-mm culture dishes. The number of colonies growing in agar was determined 7 days after plating. The rate of colony formation was represented by the mean percentage of colonies exceeding 50 µm in diameter in soft agar. Always, more than 100 colonies were counted.

Cell attachment assay
One hundred microliters of fibronectin or laminin (1, 5, 10, and 25 µg/ml) in PBS were added to each well on 96-well plates, incubated overnight at room temperature, and removed. The coated wells were further incubated with 100 µl of 0.1% bovine serum albumin (BSA) in PBS at room temperature for 1 h and washed with PBS three times. Each well was incubated with 50 µl 0.01% BSA in RPMI-1640 medium at 37°C for 1 h. A 50-µl suspension of J5/CST-1, J5/CST-2, or mock cells (5x103) in 0.01% BSA-RPMI-1640 was added to the fibronectin- or laminin-coated wells and incubated for 30 min. Nonadherent cells were removed by inverting the plate, and wells were gently washed with 100 µl of serum-free RPMI 1640 medium. One hundred microliters of the same medium was added to the well followed by 10 µl of the WST-8 reagent. After incubation at 37°C for 2 h, the absorbance of formazan generated in the wells was measured. Cell attachment was calculated as the percentage ratio of the formazan generated by attached cells to that generated by the cells added to the well.

Western blot analysis
Cells were lysed in 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM NaF, 1 mM EDTA, 1 mM ethylene glycol bis(2-aminoethyl ether)-tetra acetic acid, 1% Triton, 1 mM phenylmethylsulfonyl fluoride, 75 U/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM sodium orthovanadate (lysis buffer) for 10 min at 4°C. Protein concentrations were determined with BCA Protein Assay kit. Equal amounts of protein were separated by SDS–PAGE and transferred to a polyvinylidene difluoride membrane. Membranes were blocked for 1 h with washing buffer (Tris-buffered saline containing 0.05% Tween 20) containing 5% skim milk, followed by incubation with the primary antibody in the same solution for 1 h. After three washes, the blot was incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. The antigen was detected using the Enhanced ChemiLuminescence detection system.

Northern blot analysis
Total RNA (10–12 µg) was denatured in 50% formamide, 6% formaldehyde, 20 mM 4-morpholine propane sulfonic acid (pH 7.0) at 65°C, electrophoresed in 1% agarose gels containing 6% formaldehyde, blotted onto a nylon membrane (Roche Molecular Biochemicals), and cross-linked by UV irradiation. A digoxigenin-labeled RNA probe for mouse ß1 integrin mRNA was synthesized from the AvaII-fragment of pGEM1-mouse ß1 integrin cDNA (kindly provided by Dr. R. O. Hynes) using a digoxigenin RNA labeling kit with Sp6 RNA polymerase (Roche Molecular Biochemicals) according to the manufacturer's instructions. A digoxigenin-labeled RNA probe for PDGF{alpha}R mRNA was synthesized from the EcoRV fragment of pGEM3Z-hPDGF{alpha}R cDNA (kindly provided by Dr. T. Matsui); for PDGFßR mRNA was synthesized from the DraI fragment of pGEM3Z-hPDGFßR cDNA (kindly provided by Dr. T. Matsui) using a digoxigenin RNA labeling kit. The membrane was stained with methylene blue for the detection of 18S and 28S rRNA and then hybridized with the RNA probe at 68°C. Detection was with a digoxigenin Luminescent Detection kit (Roche Molecular Biochemicals).

DNA fragmentation
The cells were washed with PBS and lysed in 10 mM Tris–HCl (pH 7.4), 5 mM EDTA, and 0.5% Triton X-100 for 10 min at 4°C. The supernatant was extracted with equal volume of phenol, phenol/chloroform (1:1, v/v), and chloroform, and then DNA was precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. The DNA was suspended in 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA and treated with 40 µg/ml RNase A for 1 h at 37°C. The concentrations of DNA were determined by the absorbance at 260 nm. A 20-µg sample of DNA was separated by agarose gel electrophoresis on a 1% gel in 40 mM Tris–HCl (pH 8.5) and 2 mM EDTA. The gel was then stained with 0.5 µg/ml ethidium bromide for 15 min, and the fragmented DNA was visualized under UV light and photographed.


    Acknowledgements
 
This work was supported by Grant-in-Aid 12033201 for Scientific Research on Priority Areas, and Grant-in-Aid 11672155 for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. This work was also supported by the Yamada Science Foundation. We wish to thank Dr. Norman S. Radin (professor emeritus, University of Michigan) and Dr. Gabor Tigyi (University of Tennessee) for valuable comments. We also thank Dr. Masaki Saito and Dr. Atsushi Ishii for providing SAT-I cDNA, Dr. Koichi Honke for CST cDNA, Dr. Richard O. Hynes for ß1 integrin cDNA, and Dr. Toshimitsu Matsui for cDNAs of PDGF{alpha}R and PDGFßR. Ganglioside and glycosphingolipid nomenclatures are in accordance with Svennerholm (1980)Go and IUPAC-IUBMB recommendations.

1 To whom correspondence should be addressed; e-mail: inokuchi{at}kinou02.pharm.hokudai.ac.jp Back


    Abbreviations
 
BSA, bovine serum albuming; C-FCS, charcoal-treated fetal calf serum; CST, cerebroside sulfotransferase; EDTA, ethylenediamine tetra-acetic acid; EGF, epidermal growth factor; ERK, extracellular regulated kinase; FBS, fetal bovine serum; FCS, fetal calf serum; GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; LPA, lysophosphatidic acid; PBS, phosphate buffered saline; PDGF, platelet-derived growth factor; PDGF{alpha}R, platelet derived growth factor {alpha} receptor; SAT-I, GM3 synthase; SM3, lactosylsulfatide; Sph-1-P, sphingosin 1-phosphate.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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