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
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Abstract |
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Key words:
ß1 integrin
/
ganglioside GM3
/
malignancy
/
PDGF receptor
/
sulfatide SM3
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Introduction |
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CMP-N-acetylneuraminate:lactosylceramide -2,3-N-acetylneuraminyltransferase (SAT-I) (Ishii et al., 1998
) 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., 1984
, 1986
; Stevens et al., 1989
; Nakamura et al., 1991
) or by the depletion of precursor glycosphingoslipids (GSLs) using inhibitors of glucosylceramide synthase (Inokuchi and Radin, 1987
; Inokuchi et al., 1989
, 2000
; Kyogashima et al., 1996
; Meuillet et al., 2000
; Tagami et al., 2002
). These results suggest that GM3 may be involved in cell proliferation by regulating growth factor receptor activities (Bremer et al., 1984
, 1986
; Meuillet et al., 2000
; Nojiri et al., 1991
) and in cell adhesion and motility by interacting with integrins and/or integrin-related molecule(s) (Zheng et al., 1993
; Ono et al., 2001
).
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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.
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Results |
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Discussion |
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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., 2001). 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., 1993
). 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., 2001), 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
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
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 PDGFR 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, 1989
, 1994
; Hakomori and Igarashi, 1995
). 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
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., 1984, 1986
), EGF receptor (Bremer et al., 1986
; Meuillet et al., 2000
), and insulin receptor (Nojiri et al., 1991
; Tagami et al., 2002
). 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 PDGFR 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, 1993
). 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 PDGFR gene expression (Figure 8B) is an intriguing phenomenon that requires further investigation. In NIH-3T3 cells, JNK-1, which is activated by PDGF
R- dependent signaling, reportedly counteracts the enhancement of anchorage-independent growth induced by PDGFßR signaling (Yu et al., 2000
). Thus there is a possibility that the decreased expression of PDGF
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
R gene? We are currently clarifying whether the decrease of PDGF
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 PDGFR 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.
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Materials and methods |
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Cell lines
The J5 subclone of the murine 3LL Lewis lung carcinoma cell line has been described previously (Inokuchi et al., 1993). 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., 1999). 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, 1980) and fractionated (Ledeen et al., 1973
). 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 acetate8% 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 TrisHCl, 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 SDSPAGE 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 (1012 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 PDGFR mRNA was synthesized from the EcoRV fragment of pGEM3Z-hPDGF
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 TrisHCl (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 TrisHCl (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 TrisHCl (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.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: inokuchi{at}kinou02.pharm.hokudai.ac.jp
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Abbreviations |
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References |
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