Lectin Binding in Meth-A Cells Polyploidized by Different Mechanisms

C 2000 The Japan Mendel Society Cytologia 65: 389-395,2000

Kohzaburo Fujikawa-Yamamoto*, Chie Ohdoi, Hiroko Yamagishi, Zhi-ping Zong and Shi-yong Wang

Division of Basic Science, Research Institute of Medical Science, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan

Accepted September 6, 2000

Summary The cell-surface hydrocarbon chains have an important role to characterize cells, however, the information for polyploid cells is little. To assess the expression of sugar chain in cells polyploidized by different mechanisms, the lectin binding was examined. Meth-A cells, a methylcholanthrene-induced mouse abdominal dropsy sarcoma cell line, were polyploidized by demecolcine and K-252a, stained with the FITC labeled lectins, wheat germ agglutinin (WGA), Ricinus corn- munis agglutinin I (RCA120),peanut agglutinin (PNA) and Ulex europeaus agglutinin I (UEA-I), and measured for their fluorescence by flow cytometry. The WGA and UEAI bindings increased proportionally to the area of cell surface, not but to the DNA content. The RCA120 and PNA bindings were significantly larger in K-252a-induced polyploid Meth-A cells than in demecolcine-induced ones. The lectin binding in the diploid cells was almost the same, regardless the presence and the absence of the 2 polyploidizing agents. The lectin binding was roughly proportional to the cell-surface area of polyploidized Meth-A cells and it was affected by the polyploidizing methods. Key words Lectin, Polyploidization, Meth-A cells, Demecolcine, K-252a.

Many membrane proteins are linked with various hydrocarbon residues which have a major influence on the chemical and physical properties of the cell membrane and can serve to modulate membrane transport, cell metabolism, growth and other functional activity of the cell (Nicolson 1976). The type and orientation of these sugar residues of membrane glycoproteins are quite vari- able and they form branching chains which extend outwardly from the lipid bilayer of the plasma membrane. The glycochains interwind with those of adjacent membrane glycoproteins and glyco- lipids to form a complex carbohydrate network on the cell surface. Though the cell-surface glycochains have an important role to determine cell character, the change of those expression have not been known fully, particularly in polyploidized mammalian cells. Meth-A cells, a methylcholanthrene-induced mouse abdominal dropsy sarcoma cell line, are easily polyploidized cells. They are polyploidized by many drugs, such as demecolcine, K-252a, staurosporine and paclitaxel (Fujikawa-Yamamotoet al. 1993, 1994, Roberts et al. 1990). In a usual culture condition, the cell population always contains a small population of large cells which were autonomously polyploidized and resulted in apoptosis (Fujikawa-Yamamoto et al. 1997). Meth-A cells are suitable for artificial polyploidizing experiments. Demecolcine (Colcemid) antagonizes tubulin polymerization and induces the disassembly of microtubules into monomers (Inoue 1981), which inhibits spindle fiber formation in M phase, resulting polyploidization of many cells. K-252a is a protein kinase inhibitor (Kase et al. 1987), and polyploidize cultured cells without mitosis (G2—G1transition) (Usui et al. 1991), though the en- zymes that are specifically inhibited by these drugs are unknown (Zollner 1993). It is of interest whether or not Meth-A cells polyploidized by different mechanisms express cell-surface glycochain with a same manner. * Corresponding author, e-mail: [email protected] 390 Kohzaburo Fujikawa-Yamamoto et al. Cytologia 65

Various plant lectins bind specifically to the glycochain and have been employed to study the cell-surface modulation as an useful indicator. In this study, the lectin binding of Meth-A cells polyploidized by demecolcine and K-252a was examined by flowcytometry (FCM).

Materials and methods

Cells Meth-A cells (methylcholanthrene-induced mouse abdominal dropsy sarcoma cell line) were maintained in a humidified atmosphere of 5% CO2 at 37•Ž as a suspension culture in Leibovitz's

L15: Ham's F10 mixture (7 : 3) supplemented with 10% fetal calf serum (M. A. Bioproducts , Md, USA), streptomycin (100 µg/ml) and penicillin (50 units/ml).

Lectins

The lectins, wheat germ agglutinin (WGA), concanavalin A (Con A), Dolichos bifluorous agglutinin (DBA), peanut agglutinin (PNA), Ricinus communis agglutinin I (RCA120), soybean agglutinin (SBA) and Ulex europeaus agglutinin I (UEA-I) were purchased as a kit (Fluorescein Lectin

Kit 1, Vector Laboratories Inc., Burlingame, CA, USA). These lectins have been labeled by FITC with the F/P (moles of fluorescein/mole lectin) ratios listed in Table 1.

Drug treatments

Exponentially growing Meth-A cells were plated in culture dishes (90 mm diameter, Nalge Nunc International, IL, USA) at a density of about 5•~105 cells/dish. Twelve h thereafter, the cells were exposed to demecolcine (270 nM, Sigma, MO, USA) or K-252a (800 nM, Funakoshi, Tokyo Japan). At various times, the cells were harvested, fixed with 20% ethanol at 4°C for 30 min and processed for FCM measurements.

Double staining of glycochain and DNA The aliquots of Meth-A cells (2 •~105 cells) were washed three times with PBS(-) (divalent- cation free phosphate buffered saline) containing 0.1% bovine serum albumin (BSAPBS) and re- suspended in 0.1 ml of the solution. The cells were incubated with FITC-labeled lectin by adding the 0.1 ml lectin solution (2 jug lectin/0.1 ml BSAPBS) into the 0.1 ml cell suspension for 30 min at room temperature (RT). Then, the cells were incubated with 0.3 ml of PBS(-) containing 0.25%

RNase (Type II-A, Sigma) for 10 min at RT. Immediately before the measurements, the cells were stained with 0.5 ml of PI (propidium iodide, 7.5•~10-5M) solution, and green and red fluorescences were examined by means of FCM. Under these staining conditions, the signal due to residual double stranded RNA is negligible and the relative intensity of the red fluorescence corresponds to the DNA content (Krishan 1975).

Flow cytometry

The fluorescence from individual cells was measured using a FACSORT (Becton Dickinson immunocytometry Systems, USA). The green and red fluorescence of individual cells irradiated with a focused laser light at a wavelength of 488 nm was detected using photomultiplier tubes. The narrow-angle scattering light, which was the scattering laser light by a cell and related to the cell size, was also detected by a photodiode. The relative intensities of green fluorescence (FL1H), red fluorescence (FL2H) and the scattering light were measured simultaneously.

Results

To examine the lectin binding in Meth-A cells growing exponentially, the fluorescence his- 2000 Lectin Binding in Polyploidized Meth-A Cells 391

Fig. 1. Lectin fluorescence histograms of Meth-A cells. Fig. 2. Changes in DNA fluorescence histograms of Exponentially growing Meth-A cells were stained by Meth-A cells after the addition of demecolcine (D), and FITC labeled WGA, ConA, RCA120, PNA, SBA, UEA-I K-252a (K). Exponentially growing Meth-A cells were ex- and DBA. The histogram of negative control is of non- posed to demecolcine (270 nM) and K-252a (800 nM) for stained Meth-A cells. The abscissa represents the relative 3 days. DNA histograms of the cells were measured 1 (-1), fluorescence intensity. 2 (-2) and 3 days (-3) after the drug addition. The histogram C is of the control. The abscissa represents the relative fluorescence intensity (upper) and the relative DNA content (lower).

Table 1. Lectin binding in Meth-A cells

tograms were measured for 7 sort of lectins by FCM (Fig. 1). The mode-fluorescence intensity was used to assess the lectin binding, because the fluorescent cells distributed almost-symmetry on loga- rithmic scale. The quantity of lectin binding were enumerated considering F/P ratio and the results were listed in Table 1. The lectin binding in Meth-A cells was roughly divided into 4 groups (1; WGA, 2; ConA and RCA120,3; SBA and PNA, 4; UEA-I and DBA), whose binding ratio was about 600 : 100 : 5 : 1. We selected WGA, RCAl20,PNA and UEA-I in the following experiments. To examine the polyploidization in Meth-A cells by demecolcine and K-252a, changes in DNA histograms were measured for Meth-A cells exposed to these drugs (Fig. 2). The peaks of 16C DNA content, revealing the polyploidized cells, appeared in the histograms at 72 h after the 392 Kohzaburo Fujikawa-Yamamoto et al. Cytologia 65 drug addition, though the slight differences were observed in the population. It was con- cluded that Meth-A cells were polyploidized by these drugs. To examine the lectin binding of polyploidized Meth-A cells, the fluorescence intensity of the S phase of polyploidized cells was measured, because the G2/M phase of hy- poploidy overlaps with the G1 phase of hyper- ploidy. Four gates, R2, R3, R4 and R5, corresponding to the S phases of diploid, tetraploid, R3 octaploid and hexadecaploid cells, respectively, were set in the forward scatter/red fluorescence cytograms and the green fluorescence histograms were measured through the gates. A representative example of measurements was shown in Fig. 3. In Fig. 3, the upper panel rep- R4 resents a forward scatter/red fluorescence cytogram (cell size/DNA content cytogram) and the lower two panels the green fluorescence histograms obtained through the R3 and R4 gates, indicated in the cytogram. As an example of measurements, the Fig. 3. The representative example of the forward scat- green fluorescence histograms of WGA conju- ter/red fluorescence cytogram (the upper panel) and the green fluorescence histograms (the lower panels). Expo- gated Meth-A cells in the S phase were shown nentially growing Meth-A cells were exposed to demecol- in Fig. 4. In Fig. 4, the left and right panel are cine (270 nM) for 48 h. The cells were double-stained with of the demecolcine and K-252a exposed Meth- FITC labeled lectins and PI. Gate-regions of R2, R3, R4 A cells, respectively. The fluorescence intensity and R5 were set in the cytogram as that the S phase cells at the mode of histograms were listed in Table of diploidy, tetraploidy, octaploidy and hexadecaploidy falling into the regions, respectively. Green fluorescence 2A with the results for other lectins. Note that histograms were measured for each region. the fluorescence signals were appropriately at- tenuated in the measurements and the values of Table 2A cannot compare to those of Table 1. The fluorescence intensity increased with the ploidy, but the detail was different by the lectins used. WGA bound with no difference between demecolcine and K-252a-induced polyploid Meth-A cells. In contrast, RCA120binding was larger in K-252a induced polyploid cells than in demecolcine-induced ones. Analogous response was observed in PNA binding. The UEA-I binding was re- semble to the WGA binding. Note that the fluorescence intensity of 2S (the S phase of diploidy) was almost the same between the exponentially growing control and drug-treated cells, suggesting that the direct effects of demecolcine or K-252a in the lectin binding is negligible small. To elucidate the differences in lectin binding of Meth-A cells polyploidized by different mechanisms, the fluorescence ratio of polyploidy to diploidy was calculated (Table 2B). The binding of WGA and UEA-I was almost the same in Meth-A cells polyploidized by demecolcine and K-252a, and the fluorescence ratio of 16S to 2S was about 3.5 to 4. In the contrast, the binding of RCAl20 and PNA was smaller in demecolcine-induced polyploidized cells than in K-252a-induced ones. The ratio of 16S/2S was 2-3 in demecolcine and 5 in K-252a. It was obvious that the lectin binding in polyploid cells was different by the sort of lectin, and by the polyploidizing methods. 2000 Lectin Binding in Polyploidized Meth-A Cells 393

Fig. 4. FITC fluorescence histograms of polyploid Meth-A cells in the S phase. Exponentially growing Meth-A cells were exposed to demecolcine (270 nM) and K-252a (800 nM) for 3 days. The cells were harvested at various times and double-stained with FITC labeled lectins and PI. The FITC fluorescence histograms were measured through the gate-regions of R2, R3, R4 and R5 indicated in Fig. 3. The left and right panels represent fluorescence histograms of diploid (R2), tetraploid (R3), octaploid (R4) and hexadecaploid (R5) Meth-A cells polyploidized by demecolcine (D) and K-252a (K) for 1 (-1), 2 (-2) and 3 (-3) days, respectively. C is of the histogram of exponentially growing Meth-A cells.

Discussion

Various lectins bind specifically to the glycoconformations. WGA binding has been attributed

primarily to a N-acetyl D-glucosamine (P-G1cNAc) residue. RCA120 and UEA-I mainly recognize mono-sugar residues, D-galactose (6-Gal)/N-acetyl D-galactosamine (f3-GalNAc) and L-fucose (a-

Fuc), respectively. PNA binds to the two-sugar conformation of GalƒÀ1-3GalNAc . The lectin binding to glycochain, however, is not so strictly specific and it occurs even intracel- lularly, particularly in permeably fixed cells. Furthermore, the amount of lectin binding tends to eas-

ily affected by the outer factors such as the release of N-acetyl neuraminic acid from the tip of glycochain. In this study, only the lectin binding to polyploidized cells will be mentioned and the ex-

pression of glycochain would not be referred. WGA, whose bindings were almost the same between demecolcine- and K-252a-induced poly-

ploid Meth-A cells, bound almost proportionally to the deductive area of cell surface of polyploid

cells, not but the DNA content. The binding ratio of 2S : 4S : 8S : 16S was 1 : 1.7 : 2 .6 : 3.7, and the cell-surface ratio of diploidy : tetraploidy : octaploidy : hexadecaploidy is 1 : 1 .6 : 2.5 : 4 assuming that the cell volume increases proportionally to the DNA content and that the state of cell-surface is

not changed in all the S phase. The UEA-I binding was similar to WGA . The binding of RCA120 and PNA was different between demecolcine- and K-252a-induced polyploid Meth-A cells, suggesting that the lectin binding to Gal/GalNAc and Gal-GalNAc was al- tered by polyploidization mechanisms. At this stand, we have no experimental evidences to explain the phenomena. The direct effect of demecolcine and K-252a to the lectin binding may be discard-

ed, because the lectin binding was almost the same in the absence (control) and presence of these 394 Kohzaburo Fujikawa-Yamamoto et al. Cytologia 65

Table 2. Lectin binding to polyploidized Meth-A cells

(A) Fluorescence intensity in the S phase of polyploid cells*

(B) Fluorescence ratio in the S phase of polyploid cells

2S, 4S, 8S and 16S represent the S phase of diploid, tetraplod, octaploid and hexadecaploid cells, respectively. Numerals in parenthesis represent the range of data.

drugs. It has been suggested that K-252a polyploidizes cells through the bypass from G2 to G1 (Abe et al. 1991), resulting multilobed-mononuclear morphology of polyploid cells (Fujikawa-Yamamoto et al. 1999). Contrarily, demecolcine polyploidizes cells through the arrest before the formation of spindle fiber in the M phase, resulting a variety of mini/multi nuclear morphology (Fujikawa-Ya- mamoto et al. 1999). It might be probable that the difference in nuclear morphology in polyploid cells resulted the different lectin binding.

Acknowledgements This study was supported in part by a grant for High-Technology Research Center Project by the Ministry of Education of Japan and Kanazawa Medical University (H1).

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