Cell surface properties of ascites sublines of the 13762 rat mammary adenocarcinoma

Experimental Cell Research 126 (1980) 417-426

CELL

SURFACE

OF THE Relationship

PROPERTIES

OF ASCITES

13762 RAT MAMMARY

qf the

Major Sialoglycoprotein

SUBLINES

ADENOCARCINOMA to Xenotransplantability

ANNE P. SHERBLOM,’ JOHN W. HUGGINS,’ ROBERT W. CHESNUT,’ ROBERT L. BUCK,’ CHARLOTTE L. OWNBY,L GERALD B. DERMER” and KERMIT L. CARRAWAY’ Departments

of ‘Biochemistty und 2Physiologicai Sciences, Oklahomu State University. OK 74074 and 3Depurtment qf Pathology, University oj’Southern Calfornia, Los Angeles, CA 90017, USA

Stillwwter,

SUMMARY The relationship between cell surface sialoglycoprotein and xenotransplantation has been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma. Two of the five sublines (MAT-C and MAT-Cl) can be transplanted into mice. These two sublines also have the greatest amounts of total, trypsin-releasable and neuraminidase-releasable sialic acid. Chemical labeling using periodate treatment followed by [3H]borohydride reduction indicates that most of the protein-bound sialic acid is associated with a single major sialoglycoprotein (or family of glycoproteins) with a low mobility on polyacrylamide gels in dodecyl sulfate (SDS). This glycoprotein, denoted ASGP-1, is also-1abelkd.b~ lactoperoxidase and iZ5i, indicating its presence at the cell surface. Metabolic labeling with r3Hlglucosamine shows that ASGP-1 is the major glycosylated protein in both xenotransplantabie 70% of the protein-bound label in each. The labeling studies indicate that the non-xenotransplantable subline does not have a substantially greater amount of ASGP-1 on its cell surface. Likewise cationized ferritin labelina and transmission electron microsconv (TEM) do not show substantially greater amounts of negatrvely charged groups distributed along the cell surfaces of MAT-Cl than of MAT-B1 cells. The results indicate that the transplantation differences between these sublines cannot be explained solely by the presence of a major sialoglycoprotein at the cell surface.

Although glycoproteins are believed to be assay [7] or proteolytic release of sialic acid important in neoplastic diseases [l, 2, 31, on glycoprotein fragments [8]. It has been that the sialoglycoprotein the nature of their contributions is largely proposed obscure. One possibility is that cell surface “masks” transplantability antigens on the glycoproteins aid the cell in escaping im- TA3-Ha cells [6, 91. An alternative explanamune surveillance [4, 51. For example, the tion for escape from immune surveillance is TA3-Ha mammary adenocarcinoma is allo- that the tumor cells shed cell surface glycoand xenotransplantable and has large proteins [4] on immune complexes [lo], amounts of sialoglycoprotein on its cell sur- which then block immune processes which face [6]. In contrast the TA3-St subline, would lead to tumor cell destruction. TA3which does not transplant across strain or Ha cells shed substantial amounts of sialospecies barriers, has little of the sialoglyco- glycoprotein into ascites fluid [ 11, 121.Kim et al. [13] have suggested that shedding is protein [6], as determined by lectin-binding

418

Sherblom et al.

important in mammary tumor metastasis. In a series of tumors selected for varied metastatic potentials, decreased cell surface glycoprotein, nucleotidase and immunogenicity were correlated with increased metastasis. It was suggested that the depletion of the cell surface glycoproteins, including the enzyme 5’-nucleotidase, resulted from shedding of these materials into the circulation [ 131. To determine whether any (or all) of these phenomena are important in various aspects of neoplastic diseases, it is necessary to obtain a clear picture of the cell surface, e.g. the molecules which contribute to its properties, their organization and their dynamics. Our studies in this area have concentrated on the metastatic 13762 rat mammary adenocarcinoma [ 141. This tumor has been converted into an ascites form from which several morphologically different sublines have been selected. In preliminary studies of two of these sublines (MAT-Bl) and (MAT-Cl) we found differences

in morphology,

5’-nucleotida8e

and sialoglycoprotein content in isolated membranes. We have now shown differences in xenotransplantation between these sublines.

MAT-Cl

can be traB8plaBted

intO

mice, but MAT-B 1 cannot. The relationship between transplantation and cell surface sialoglycoprotein has been studied by labeling and electron microscopy. Cell surface and metabolic labeling studies indicate that a single sialoglycoprotein (or class of sialoglycoproteins) dominates the cell surface of both sublines. In MAT-Cl cells, which have numerous branched microvilli extending from the cell body, the sialoglycoprotein can be observed in fibrillar structures extending from and probably between the individual microvilli. The MAT-B1 cells have a more ordinary surface morphology, less surface Exp Cell Res 126 (1980)

sialic acid and fewer fibrils. It is important to note that the differences between MATBl and MAT-Cl are quantitative rather than qualitative. There is not a substantially greater amount of the major sialoglycoprotein on the surface of the xenotransplantable subline. Thus the differences in transplantation cannot be explained by the presence of a major sialoglycoprotein, as has been postulated for the TA3 mouse mammary adenocarcinoma [6]. We suggest that the 13762 sublines are useful for investigating more subtle differences related to transplantation.

EXPERIMENTAL

PROCEDURES

Materials Lactoperoxidase, glucose oxidase, diphenylcarbamyl chloride-treated trypsin and PPG (2,5diphenyloxazole) were from Sigma (St Louis), McCoy’s 5a modifiedmediumandV%rio cholerue neuraminidase were from Gibco (GraiId Island, NY), [“H]NaBH, (specific

r;lmmr.ln\ on,4 ~n,*orr\l .1,arm . . ..m activity >1~ mL’,rrrlrl”lr, .x,1” Ll~uac.“, Wb,Cp,v-

ducts of New England Nuclear (Boston, MA), [l3H]o-glucosamine HCI (2-6 Ci/mmole) and carrier-free [12511NaI were from AmershamlSearle (Arlinaton ‘Heights, IL) and Soluene 350 and Instagel were f;om Packard (Downers Grove, IL). Ruthenium red was from Polysciences, Inc. (Warrington, PA) and cationized ferritin from Miles Biochemicals (Elkhart, IN). Cells. MAT-B and MAT-C ascites sublines of the 13762 mammary tumor were obtained from Mason Research Laboratories and were maintained as described previously [15]. MAT-B1 and MAT-Cl designate the sublines which arose from the MAT-B and MAT-C lines respectively, by routine weekly passage for about one year in our laboratory [14]. The distinguishing features of these sublines [14] have been stable for over two years since the original observations. Cells are carried by weekly transfers and as rrozen stocks. Cells used for these studies were washed 3 times by suspending in PBS (Dulbecco’s phosphate-buffered saline, pH 7.4, calcium and magnesium free) [16], and centrifuging at 120 g for 3 min. Viability was determined using Trypan blue; only preparations of cells showing viability greater than

98%wereused,

Analytical

procedures

Total cell sialic acid was determined by hydrolyzing whole cells (5-15~ lo7 cells) in 3 ml 0.1 N H/SO, at 80” for 1 h and centrifuging (2000 g for 5 min). The supernatant was applied to a 1.5 ml Dowex

Mammary ascites tumor cell sialoglycoproteins l-Xl0 (formate form) column, washed with water, eluted with 1.0 M formic acid and lyophilized. Sialic acid was assayed by a modification [ 171of the method of Warren r 181. TrypsinizaGon was performed by incubating 5x 10’ cells at 3PC with 20 pg trypsin in 2 ml buffer containing 0.8% NaCl, 5 mM KCI, 1 mM Na,HPO,, 5 mM dextrose and 10 mM HEPES. DH 7.4. The supernatant following centrifugation (2 OdOg for 5 min) was lyophilized, resuspended, hydrolyzed as described above and assayed for sialic acid. Neuraminidase treatment was performed by incubating 5~ 10’ cells in 1 ml PBS containing calcium and magnesium and 24 units Vibrio cholerae enzyme at 37°C. Samples taken at various times were centrifuged (2000 g for 5 min) and the supernatants assayed for sialic acid.

Electron microscopy Samples for TEM were fixed in 2% glutaraldehyde in 0.1 M cacodylate-O.1 M sucrose, pH 7.4. After secondary fixation with 0~0, in the same buffer they were dehydrated and embedded in Spurr resin. Silver sections were prepared using an MT-2 ultramicrotome and a diamond knife, stained with uranyl acetate and lead citrate [19] and examined in a Phillips EM 200 electron microscope at 60 kV. Cell surface staining with ruthenium red [20] or cationized ferritin [6] was performed on MAT-Cl and MAT-B1 cells, some of which had been previously treated with neuraminidase. After glutaraldehyde fixation and buffer wash, cells were post-fixed in 1% 0~0, containinp. 0.5 ma/ml ruthenium red for 3 h, dehydrated and-embedded in vestopal W. For cationized ferritin staining 1 ml suspensions containing 1x IO’ glutaraldehyde-fixed cells were added to 0.5 ml of a cationized ferritin solution to give a final concentration of 0.35 mg ferritinlml. Cells were treated for 30 min, washed several times in cacodylate buffer, post-fixed with OsO,, dehydrated and embedded in vestopal W. Sections were examined in the electron microscope without additional staining.

Cell labeling Mith periodate-borohydride Labeling with periodate-borohydride was performed according to the procedure of Gahmberg & Andersson [21]. Cells (1 x 10’) were incubated for 10 min on ice in 1 ml of PBS containing 2 mM sodium periodate; the reaction was quenched bv addition of 0.2 ml of 0.1 M glycerol in PBS, and cells were washed twice with cold PBS. Prior to reduction with r3H1NaBH, cells were washed once with a buffer containing 50 mM sodium phosphate, pH 8.0, 85 mM NaCI. The sample was suspended in 1 ml of the same buffer and incubated for 30 min at room temperature with 0.25 mCi [3H]NaBH,. Unlabeled NaBH4 (0.1 mg) was added, and the cells were washed twice in the pH 8.0 buffer and once with PBS. Plasma membranes were prepared by the Zn*+ stabilization method [ 151from 1-5~ IO9ascites cells which had been labeled in a volume of IO ml as described above.

419

[3H]Glucosamine labeling of cells Tritiated glucosamine (250 &i) was diluted with 0.5 ml of 0.9% NaCl and injected into the peritoneal cavity of a rat bearing MAT-B] or MAT-Cl ascites cells. Approx. 16 h later the rat was killed and the cells recovered and washed three times with PBS. ASGP- I, the major sialoglycoprotein, was isolated from [3H]glucosamine-labeled MAT-B] or MAT-C 1 cells by centrifugation of membrane fragments in a CsCl gradient containing 4 M guanidine HCI [22].

Lactoperoxidase-catalyzed iodination Lactoperoxidase-catalyzed iodination was performed according to the method of Hynes [23]. Carrier-free lzsl (0.1-0.5 mCi), lactoperoxidase (40 pg) and glucose oxidase (0.2 units) were added to a suspension of 1~10~ cells in 1 ml of PBS containing 1 mglml glucose. After incubation at room temperature for 10 min, 5 ml PBI (PBS with 1 mM KI) was added and the sample was agitated gently at room temperature for 5 min prior to washing 3-4 times with PBI.

Electrophoresis Labeled whole cells and plasma membranes were assayed for protein [24] and TCA (trichloroacetic acid)precipitable radioactivity [25]. The samples were solubilized by addition of an equal volume of solution containing 0.125 M Tris, pH 6.8, 3 % SDS (sodium dodecyl sulfate) and 5 % /3-mercaptoethanol and immersion in a boiling water bath. Solubilized whole cells were sonicated briefly at low frequency to disrupt viscous material, and 0.1 volume of 0.02% Pyronin Y in 50 % glycerol was added. Slab gels (13.5 cmxl6.0 cmx0.75 mm) were prepared with a linear 5-15 % acrylamide gradient using the gel buffer system of King & Laemmli [26]. A 2 cm 3.5 % acrylamide stacking gel was used. Following electrophoresis, samnles were soaked in 25% isopropanol-10% acetic acid for 30 min, stained in a solution of 25% isopropanol, 10% acetic acid (usually complete within 3&l5 min). The gel was then soaked in 2-3 changes of 10% methanol and once in 10% methanol, 1% glycerol. Gels to be processed for fluoroeranhv were soaked in 3 chances of dimethvlsulfoxyde’ (DMSO) 20 min each and 22% PPG *in DMSO for 30 min [271. After several washes in water the gels were dried, placed in contact with Kodak X-Omat R (XR-5) X-ray film which had been preflashed to increase sensitivity [28] and stored at -70°C. Kodak no-screen film NS-ST was used for ? autoradiography of slab gels. For quantitation of radioactivity, samples were electrophoresed on 5% acrylamide tube gels (5 mmx 10 cm) with 3% stacking gel using the same buffer system [26]. Slices (1.5 mm) of unstained gels were either placed in 12x75 mm polystyrene tubes and counted in a gamma counter (lz51),or solubilized with Soluene 350. mixed with Instaael and counted in a scintillation counter (3H). Agar&e gels consisted of 1% agarose in 90 mM Tris, 2.5 mM EDTA. 90 mM sodium borate, pH 8.2, cast to a height of 10 cm in 5 mm i.d. glass tubes. Solubilized samples were diluted with the Tris-borate buffer prior to electrophoresis.

420

Sherblom et al.

Table 1. Transplantation of 13762 ascites cells into mice and normal host F344 rat Animals C57BLl6j C57BLl6i C57BL/6j C57BL/6i C57BL/6j F344 Rat F344 Rat F344 Rat F344 Rat F344 Rat

Injection Mouse Mouse Mouse Mouse Mouse

Saline MAT-A MAT-B1 MAT-C MAT-C 1 Saline MAT-A MAT-B 1 MAT-C MAT-C 1

Number killed/total o/10 o/10 o/10 6110 s/10 015 4/s 415 515 515

injected with saline, MAT-A or MAT-B1 tumors. A more limited earlier study also showed the failure of MAT-B to transplant into mice. As expected, tumors were found in all of the normal host animals injected and 80-100% of the animals were dead within 90 days. These results indicate that MAT-C and MAT-Cl sublines have acquired the ability to evade transplantation rejection. Cell surface sialic acid

The transplantation results resemble previous studies in the TA3 mouse mammary adenocarcinoma, for which it was postulated that cell surface sialoglycoprotein(s) Transplantation studies plays a role in evading rejection [6, 91. Fischer 344 female rats, 90 days of age and C57BL/ Therefore the cell surface sialic acid con6j mice, 60 days of age, were injected with 1X 106 tent of the MAT sublines was investigated cells/ml in 0.2 ml of 0.9% NaCl. Animals were observed for 90 days. All animals which died showed a using enzymatic methods. progressive increase in weight relative to saline control Total cell sialic acid and the amount before death. Results are expressed as dead animals per group at 90 days. released by neuraminidase and trypsin were determined for the MAT-B1 and MAT-Cl sublines as well as for MAT-B and MAT-C RESULTS sublines (table 2). The amount of released l@ cells in saline were injected per animal. Numbers represent animals dead within 90-day period.

Transplantation

studies

The 13762 rat mammary adenocarcinoma is a dimethyl benzanthrene-induced solid tumor [29] from which were derived three distinct ascites sublines, MAT-A, MAT-B, and MAT-C. Two additional sublines, MAT-B 1 and MAT-C 1, were obtained from MAT-B and MAT-C, respectively, after passage in vivo in our laboratory [14]. As reported previously [14], the various sublines can be distinguished by differences in morphology, agglutinability, ConA receptor mobility and 5’-nucleotidase activity. Table 1 shows results obtained when four of these ascites sublines, grown in Fischer 344 rats, are transplanted into C57BL/6j mice. For MAT-C and MAT-Cl sublines 60-80% were dead within 90 days. In contrast there were no tumors or fatalities in the groups

Table 2. Analysis of total and enzymereleasable sialic acid of 13762 mammary ascites adenocarcinoma sublines (nmolesl lOa cells) Sample intact cells Neuraminidase released Trypsin released

MATB

MATBl

MATC

MATCl

230

175

460

560

140

75

265

310

45

70

150

325

Total sialic acid and sialic acid released by trypsin or neuraminidase (see Experimental Procedures) were determined usine the methods of Desuont et al. r161 and Warren [17]: Cell protein was determined by-the method of Lowry et al. [23]. No sialic acid was released in the absence of trypsin or neuraminidase. Trypsin-released and neuraminidase-released values did not increase significantly beyond 60 min of incubation. Values at 100 min of incubation was reported.

Mummary ascites tumor cell sialoglycoproteins

421

sugar was evaluated from the time course of release. The MAT-Cl cells have the largest amount of whole cell and releasable sialic acid. MAT-B 1 has the least, except by trypsin release, and MAT-C is intermediate. The trypsin results suggest that the amount of sialic acid associated with cell surface sialoglycoprotein varies in the order MAT-C 1 > MAT-C > MAT-B 1 > MAT-B. Thus there appears to be a correlation between cell surface sialic acid and xenotransplantability. Histochemical localization Previous studies by Miller et al. [6] have shown that TA3-Ha cells have sialoglycoprotein in fibrillar structures on the cell surface. Therefore the MAT-B1 and MATCl cells, which represent the extremes in cell surface sialic acid content, were examined by TEM with histochemical staining procedures. Previous SEM studies have shown pronounced morphological differences between these sublines [14]. MAT-Cl cells have extensive branched microvilli extending from the cell body, while MAT-B1 cells have a more normal morphology. MAT-B1 microvilli are unbranched and often curved. In addition the MAT-B1 cells more often exhibit ruffles, ridges and blebs than do MAT-C 1. TEM of thin sections of the MAT-Cl cells confirms the presence of numerous, branched microvilli. Close examination of such micrographs also shows the presence of fibrillar material extending from the surface of the microvilli. Sections cut perpendicular to the major axis of the microvilli show significant amounts of the extracellular Iibrillar material, apparently bridging the microvilli (fig. 1). When MAT-Cl cells are stained with ruthenium red, a heavy layer of stain is observed which permits visualization of

Fig. I. TEM of MAT-Cl cell showing fibrillar material bridging microvilli. Microfilament bundle structures can be seen in the interior of microvilli. Bar, 0.2 pm. x57000.

fibrils (fig. 2A). Neuraminidase treatment, under conditions which remove 60% of the total cellular sialic acid, reduces staining of MAT-Cl cells substantially (fig. 2B). Fibrils are still observed but they are less distinct. MAT-B1 cells also stain with ruthenium red, but show fewer fibrils (data not shown). The staining of MAT-B1 cells is not substantially reduced by a similar neuraminidase treatment. Similar results were obtained with cationized ferritin which localizes cell surface negative charges. An extensive layer offerritin is observed, particularly on the microvilli, which have ordered arrays of the ferritin exhibiting closest packing (data not shown). Neuraminidase treatment substantially reduces the staining. MAT-B 1 cells appear to have somewhat fewer catiE.rp (‘r/l Rr.\ 126 (/YXO)

422

Sherblom et al.

on cell surface components and probe possible differences between the two sublines, the cells were labeled with a variety of agents prior to electrophoresis. Lactoperoxidasel’25Z

Fig. 2. Ruthenium red staining of MAT-Cl cells. (A) Surfaces of MAT-Cl cell stained by ruthenium red. Fibrils bridging adjacent microvilli are also stained. (B) MAT-Cl cells after neuraminidase treatment. Bar, 0.2 pm. x65 000.

onized ferritin molecules on the cell surface and essentially no ordered arrays of ferritin. However, the difference between MAT-B1 and MAT-Cl is not as evident as that between TA3-St and TA3-Ha [6]. In addition treatment of MAT-B1 cells with neuraminidase does not substantially reduce ferritin staining (data not shown), suggesting the presence of negatively charged groups at the cell surface other than sialic acid. SDS polyacrylamide

gel electrophoresis

Slab gels of MAT-B 1 or MAT-Cl cells stained with Coomassie blue are not significantly different in observable polypeptides (fig. 3A, B). Thus, in order to focus Exl, Cell Kes 126 (1980)

labeling

This method specifically labels cell surface proteins and glycoproteins of intact cells [30]. Several components are labeled by lactoperoxidase-catalyzed iodination of MAT-B1 and MAT-Cl cells (fig. 3C, 0). Although the same bands appear labeled in each subline, the relative intensities of the bands are different. For instance, the two major bands in each subline are a high molecular weight band which barely enters the gel (ASGP-1) and a band of molecular weight about 150 K. The percent of cell radioactivity in the first is 9% and 26% for MAT-B1 and MAT-Cl, respectively (table 3). The relative intensity of the 150 K band is reversed, with 25 % and 9% for MAT-B1 and MAT-Cl, respectively. The reason for the differences in iodination of the bands in the different sublines is unknown. They may relate to differences in organization at the cell surface. The complexities of cell surface structures and the iodination reaction preclude a better explanation at present. Periodatelborohydride

labeling

To identify the sialoglycoproteins which might contribute to the cell surface staining patterns and the differences in total and releasable sialic acid among the sublines, the cells were labeled by the periodateborohydride procedure under conditions which label predominantly sialic acid [21]. Electrophoretic patterns of SDS-solubilized, labeled whole cells (fig. 3E, F) were quite simple. A species of apparent molecular weight >300 K (denoted ASGP-1) domi-

Mammary ascites tumor cell sialoglycoproteins

423

Fig. 3. Coomassie blue stain and

I-fK--,

AB

CD

EF

nated the profile, and two other bands of 160 K and 110 K each comprised 3-5 % of the total radioactivity. Although these other bands are not visible on the fluorogram, they are evident in the radioactive profiles of tube gels which were used for quantitation. The specific activity of the MAT-Cl cells was consistently 34 times greater than that of the MAT-B1 line, the difference being accounted for by the increased labeling of ASGP-1 (table 3). These results are the average of 6 experiments, where the standard deviation is k20 cpm/ pg protein for the specific activity and + 3 % for the contribution from ASGP- 1. 28-801819

G

H

autoradiography of SDS-solubilized MAT-B 1 and -C 1 cells. Gel electrophoresis [25] was performed on a 5-15 % polyacrylamide gradient slab with 3.5 % stacking gel, and 50 pg protein was applied to each well. Gels were stained and dried or prepared for fluorography [26] prior to drying. For each section MAT-B1 is on the left; MATCl is on the right. (A), (B) Coomassie blue stain; (C), (D) autoradiogram from [rZ51]lactoperoxidase-labeled cells; (E), (F) fluorogram of periodate-borohydride treated cells; (G), (H) fluorogram of [3H]glucosaminelabeled cells.

After plasma membrane isolation the relative amount of each labeled glycoprotein appears to be the same as in the whole cell. The specific activity of the membranes is approx. 5-fold that of the whole cell homogenate (table 3). The MAT-Cl membranes exhibit a 2-fold higher specific activity than the MAT-B1 membranes, with the ASGP-1 component dominating each. Metabolic labeling with glucosamine Because the specificity of chemical and enzymatic labeling techniques can lead to misinterpretations, MAT-B 1 and MAT-Cl cells were metabolically labeled by injec-

424

Sherblom et al.

Table 3. Incorporation of label into MAT-B1 and MAT-Cl cells Treatment

WLactoperoxidase [3H]Glucosamine HCI [3H]NaBH,/periodate Membranes prepared from [3H]NaBHdperiodate-treated

MAT-B1

MAT-Cl

Spec. act. (cpm/pg protein) 1600 3 600 130 130 170 52 cells

390

800

MAT-B1

MAT-Cl

Contribution from ASGP-1 (%) 9 26 71 86 65 82 70

83

Aliquots of cells or membranes were TCA-precipitated as described in Methods for determination of specific activity. The contribution from ASGP-1 was estimated from radioactivity profiles of 5 % polyacrylamide electrophoresis gels as follows: Background-corrected counts, were summed for all fractions except the low molecular weight dye-band region to provide “totai counts”. “Total counts” typically accounted for 80-l 10% of the TCA-precipitable radioactivity applied to the gel. Background-corrected counts were summed for the first 8-10 mm of the separating gel to provide “ASGP-1 counts”/“total counts” X 100.

tion of [3H]glucosamine into the peritoneal cavity prior to cell isolation. Incorporation of label into TCA-precipitable material appears the same for both MAT-B1 and MAT-Cl sublines (table 3). However, since growth conditions for the two sublines may be different, comparison of specific activities may not be meaningful. Electrophoresis profiles are surprisingly simple, having for each subline a single major peak corresponding to the high molecular weight sialoglycoprotein (fig. 3G, H) and representing 70-90 % of the incorporated radioactivity (table 3). Interestingly, the major sialoglycoproteins of MAT-B1 and MAT-Cl cells display slightly different electrophoretic mobilities when examined by slab gel electrophoresis (fig. 3). The major sialoglycoprotein of chemically or metabolically tritiated MAT-Cl cells migrates farther than the MAT-B1 glycoprotein; the same difference is seen in autoradiograms of Y-labeled MAT-B 1 and MAT-Cl cells. Slab gels of MAT-B1 or MAT-Cl cells stained with Coomassie blue are not significantly different in observable polypeptides (fig. 3A, B). E.rp Cell Rrs 126 11980)

Agarose gel electrophoresis Since ASGP-1 does not migrate well in 5% acrylamide gels, we also performed electrophoresis in 1% agarose gels. ASGP-1 was isolated from a crude membrane fraction of [3H]glucosamine-labeled cells as described previously [22]. The glycoprotein migrates into the gel (fig. 4C, D). MATCl ASGP-1 migrates faster and appears more homogeneous than MAT-B1 ASGP-1 both by Coomassie blue staining and radioactivity profiles. The crude membrane fraction is shown to indicate that most of the proteins have migrated to the lower half of the gel (fig. 4A, B). The radioactivity profile from the crude membrane fraction from each subline was very similar to that of the purified glycoprotein. DISCUSSION The various ascites sublines of the 13762 rat mammary adenocarcinoma differ in their ability to survive rejection upon transplantation into a foreign host. In addition these sublines show differences in morphology [14], agglutinability [14] and sialic acid content. These results are reminiscent

Mammary

ascites tumor cell sialoglycoproteins

425

which might be involved in transplantation differences, we have employed cell surface labeling techniques. Periodate-borohydride labeling indicates that most of the proteinbound cell surface sialic acid is associated with a single species of low mobility on polyacrylamide gels in SDS. This sialoglycoprotein is also labeled with lactoperoxidase and lz51. More importantly, it is the predominant species labeled metabolically by radioactive glucosamine in vivo. That this predominance and specificity of labeling is not due to non-specific aggregation or contributions from a non-tumor host product is indicated by several results. (1) The labeled glycoprotein is included in Sepharose 2B during gel filtration in 1% SDS or 4 M guanidine hydrochloride and elutes only slightly ahead of hemocyanin. (2) Fig. 4. Electrophoresis of MAT-B1 and MAT-Cl Using the Burridge technique [31] for exmembrane fragments and purified ASGP-1 on 1% amining lectin receptors, the ASGP-1 does agarose gels. Conditions of labeling with [3H]glucosamine, isolation of ASGP-1 and electrophoresis are not bind ConA, even though numerous described in Experimental Procedures. Gels were ConA receptors are observed on the gels. stained with Coomassie blue. (A) MAT-B] membrane fragments; (B) MAT-Cl membrane fragments; (C) (3) Isolated, well-washed ascites cells inMAT-B1 ASGP-1; (D) MAT-Cl ASGP-1. Radioactivity profiles for MAT-B 1 ASGP- 1 (- - -) and MAT-C 1 corporate radioactive glucosamine into ASGP-1 (-) are given above. ASGP-1 as the predominant labeled component. (4) The ASGP-1 co-isolates with plasma membranes. (5) The purified glycoof previous findings on ascites sublines of protein migrates well into the gel during the TA3 mouse mammary adenocarcinoma electrophoresis on 1% agarose. [6]. In both cases the transplantable subThe transplantation differences between line has the more highly convoluted cell the MAT sublines cannot be explained surface, the greater amount of cell surface simply by the presence or absence of a sialic acid and lower agglutinability with dominant sialoglycoprotein. More subtle ConA. The transplantability of the TA3- factors must be considered. If ASGP-1 is Ha subline has been attributed to the pres- related to the transplantation differences ence of sialoglycoprotein at the cell surface then one would expect differences between which masks histocompatibility antigens. MAT-B 1 and MAT-Cl ASGP-1. We have The sialoglycoprotein(s) has been identified demonstrated a difference in the electroby proteolytic release [8] and lectin binding phoretic behavior of MAT-B 1 and MAT-C 1 ASGP-1. Recently we have also shown by [7], which shows a virtually qualitative (400-fold) difference between TA3-St and analysis of purified ASGP-1 that MAT-Cl TA3-Ha cells. ASGP-1 contains 3-fold more sialic acid To characterize cell surface molecules than MAT-B1 ASGP-1 [22]. Thus, although Ex/J C-r// Ke.\ I26 I IYXOJ

426

Sherblom et al.

the MAT-C 1 glycoprotein migrates further on electrophoresis in SDS it appears larger than MAT-B1 ASGP-1 by both sedimentation velocity in 4 M guanidine HCl and gel filtration in SDS. Both MAT-B1 and MAT-Cl glycoproteins are large (-600 K) and rich in carbohydrate (-70%). Although the amino acid compositions are similar, there are distinct differences in the O-linked oligosaccharides [22], the most important of which involves addition of sialic acid to the oligosaccharides. Thus the sialic acid may be an important contributor to the cell surface properties of these cells. Whether it plays a direct role in xenotransplantation is under investigation, using other sublines derived from the 13762 mammary tumor. Additional investigations of the 13762 ascites sublines should provide important insights into the mechanisms by which tumor cells alter their cell surface properties through biosynthetic variations. We wish to thank Sandra McGuire, Charlene Bymaster and Margaret Thompson for technical assistance and Kathryn Kocan for assistance with transmission electron microscopy. A. P. S., R. W. S. and J. W. H. were recioients of NIH National Research Service Awards from the NCI. Research support was obtained from the NC1 (NO-l-CB-33910 and CA19985), the ACS (BC-246) a Presidential Challenge Grant from Oklahoma State Universitv. NIH Biomedical Sciences Support Grants and the Oklahoma Agricultural Experiment Station. Journal article J-3338 of the Oklahoma Agricultural Experiment Station. This research was conducted in cooperation with the USDA Agricultural Research Service, Southern Region.

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Received June 28, 1979 Revised version received November 15, 1979 Accepted November 20, 1979

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