DYNAMICS OF TOXIN AND RECEPTORS ON A LYMPHOMA CELL LINE AND ITS TOXIN-RESISTANT VARIANT USING FERRITIN-CONJUGATED, 125I-LABELED LIGAND

GARTH L. NICOLSON, JAMES R. SMITH, and ROBERT HYMAN

From the Department of Developmental and Cell , University of California, Irvine, California 92717, and the Department of Cancer Biology, The Salk Institute for Biological Studies, San Diego, California 92112

ABSTRACT

The dynamics of the toxin Ricinus communis agglutinin II (RCAH or ) on cells of a murine lymphoma line (BW5147) and a toxin-resistant variant line (BW5147Ric R. 3) that is 200 times more resistant than the parent to direct RCA n cytotoxicity were examined using ferritin-conjugated, affinity purified, lzsI-labeled RCAII (ferritin-125I-RCAn). Ferritin-125I-RCAn was indistinguishable from native RCA~ I in quantitative binding and cytotoxicity experiments. When RCAt~-sensi- tive BW5147 and -resistant BW5147Ric R. 3 cells were labeled with ferritin-125I- RCAH at various toxin concentrations (1-10 /xg/ml), no differences in toxin binding were observed. These same cells were examined by electron microscopy. At low ferritinJZSI-RCAH concentrations (1-3 /,~g/ml RCAn) where only the parental BW5147 cells were significantly more sensitive to RCAI~, toxin receptors were internalized by ferritin-125I-RCAii-induced endocytosis. In parallel experi- ments, ferritinJ25I-RCAH that bound to the resistant BW5147Rica. 3 cells remained relatively dispersed or clustered, and there was little evidence of transport into cells via endocytosis. At higher ferritin-~25I-RCAH concentrations (>7/zg/ml RCAI0 where both parental and resistant variant cells are sensitive to the cytotoxic effects of RCAH, more ferritin-conjugated toxin was bound, and subsequent endocytosis occurred to a similar degree in both cell types. Endocy- tosis of ferritin-conjugated concanavalin A was indistinguishable on RCAIr sensitive parental and resistant variant cells at all concentrations tested. The results suggest that a specific defect on the selected BW5147Ric a. 3 cells prevents RCAn entry into these cells at low toxin concentrations, rendering them more resistant to the cytotoxic effects of RCAH.

KEY WORDS toxin lectin membrane 19, 37). An -60,000 mol wt (Rici- endocytosis lymphomavariants receptor nus communis agglutinin II [RCAH or ricin]) 1 that ricin concanavalin A

1 Abbreviations used in this paper: BSA, bovine serum Certain and isolated from albumin; Con A, concanavalin A; DMEM, Dulbecco plants are extremely toxic to animals and man (2, modified minimal essential medium; DMEM-BSA,

J. CELL BIOLOGY The Rockefeller University Press 0021-9525/78/0801-056551.00 565 has been isolated and purified from the castor is altered on the variant cells to a lower apparent plant (Ricinus cornmunis) (11, 24, 25, 35, 38, molecular weight component (44). In preliminary 49) inhibits proteirr synthesis in intact cells (12- nonquantitative experiments, it appeared that the 14, 27, 38) and in cell-free synthesis resistant variant cells might not transport ferritin- systems (17, 27, 32-34, 43). The toxicity of RCAH at the same rate as the parental sensitive RCAtt or ricin has been utilized in experiments to cells (28). We now report in detail, using a fully differentially kill tumor cells in vitro (27) as well biologically active, quantitative, electron-dense as in animals (12, 41) and man (8, 50). probe consisting of ferritin-conjugated, r'SI-la- The cellular toxicity of RCAu (ricin) is generally beled-RCAH (ferritin-12SI-RCAn), that the RCAII thought to occur via the following sequence of toxin molecules bind to cell surfaces of both events: Toxin molecules bind to the cell surface resistant and sensitive cells. However, at low and induce redistribution of their receptors. Some RCAII concentrations where the maximum differ- of the aggregated toxin-receptor complexes are ential toxic effects occur, the ricin molecules only endocytosed into the cell where after a period of enter at significant levels into the sensitive cells. time a proportion of the endocytotic vesicles break down, releasing toxin molecules into the cell cy- MATERIALS AND METHODS toplasm where they irreversibly inactivate protein synthesis (18, 27, 32, 38, 40, 43, 45). To study Biochemicals and Chemicals this sequence in more detail, several somatic cell Bovine serum albumin (BSA, three times crystallized) mutants have been isolated that are more resistant was obtained from Armour Pharmaceuticals (Chicago, to the direct toxic effects of RCA~ or other Ill.). Ferritin was purchased from Immuno-Diagnostics (7, 9, 16, 17, 44, 46-48). Although many of these (Solana Beach, Calif.) and NamI (~17 Ci/mg) from toxin- and lectin-resistant mutants possess cell International Nuclear Corp. (Irvine, Calif.). L-[SH]leucine (30 Ci/mmol) was bought from New England Nuclear surface changes leading to large decreases in toxin (Boston, Mass.), and osmium tetroxide and glutaralde- or lectin binding (6, 7, 17), certain resistant hyde, EM grade, were obtained in sealed vials from variants are capable of binding substantial quanti- Polysciences, Inc., (Warrington, Pa.). Saccharides were ties of toxin or lectin molecules to their cell products of Calbiochem (San Diego, Calif.), and all surfaces (9, 16, 44). One such variant cell line inorganics were obtained from Mallinckrodt, Inc. (St. which was selected from a murine lymphoma cell Louis, Mo.). line by direct RCAa cytotoxicity is -200 times more resistant compared to its parental line in the Cell Lines and Growth absence of serum, although it has lost only -30- The BW5147 cell line is a spontaneous AKR/J lym- 40% of its total cell-binding sites for RCAa (44). phoma that has been adapted to tissue culture and grows Cell-free protein synthesis studies suggest that the in suspensions with Dulbecco modified minimal essential ribosomes of the RCA~rresistant variant line are medium (DMEM) plus 2 mM glutamine and 10% horse just as sensitive to toxin inactivation as parental serum. A thioguanine-resistant subelone (BW5147. G. 1) ribosomes, indicating that the properties of resist- is defined here as the parental BW5147 cell line (10) from which an RCA,rresistant variant line ance must reside at a site(s) other than the level of (BW5147RicR.3) was obtained by stepwise selection protein synthesis (43). In addition, although most over 1 yr against a series of RCA n concentrations surface proteins on the resistant variant cells are increasing from 5 x 10 _3 p,g/ml to 1 ~,g/ml. The similar to parental cell surface proteins as judged BW5147RicR.3 variant cell line is -200 times more by electrophoresis after lactoperoxidase-catalyzed resistant compared to the parental BW5147 line, as iodination or of mem- measured by the ability of the cells to survive a 60-min brane glycoproteins, a surface glycoprotein of exposure to RCAI~ in serum-free medium. These exper- ~80,000 mol wt on the surfaces of parental cells iments were repeated with ferritin-RCAn conjugates as follows: UV-sterilized toxin or ferritin-toxin conjugate was diluted in DMEM plus 0.1% crystalline bovine DMEM plus 0.1% bovine serum albumin; ferritin-Con serum albumin (DMEM-BSA) in place of DMEM plus A, ferritin-conjugated concanavalin A; ferritin-~25I- horse serum. The cells were washed once in DMEM- RCAn, ferritin-conjugated, lzSI-labeled RCAH; PBS 1, BSA, counted, and suspended in DMEM-BSA to 2 • 0.2 M sodium chloride-0.005 M sodium phosphate 10~ cells/ml. Ferritin-mI-RCAii or RCAII (1 ml) and buffer, pH 7.2; PBS 2, 1 M sodium chloride-0.05 M cells (1 ml) were mixed on ice and then transferred to a sodium phosphate buffer, pH 6.8; RCAtI, Ricinus com- humidified tissue culture incubator at 37~ After a 60- munis agglutinin II. min incubation with agitation every 15 min, the tubes

THE JOURNAL OF CELL BIOLOGY VOLUME 78, 1978 were placed on ice, and 2 ml of cold DMEM plus 10% Richmond, Calif.) as previously described (29). horse serum was added, and the ceils were washed twice Ferritin-conjugated concanavalin A (Ferritin-Con A) by centrifugation (600 g for 5 min at 4~ The cells was synthesized using the same procedures, except that were then resuspended in 2 ml of DMEM containing the conjugation buffer was 1 M sodium chloride-0.05 M 10% horse serum and inoculated into 35-mm dishes. sodium phosphate buffer, pH 6.8 (PBS 2), containing The dishes were sampled initially and at 48 and 72 h to 0.2 M ot-methyl-D-mannopyranoside, and the affinity determine the number of live/dead cells by trypan blue column was made with Sephadex G-75 beads (29). dye exclusion. Cell protein synthesis was measured as described Labeling Procedures previously (27), using log phase cells. Cultures treated Cells were labeled with ferritin-125I-RCA, or ferritin- with an inhibitory concentration of RCAn or ferritin-~2sI- Con A by the following procedures: BW5147 or RCA a for various times in DMEM-BSA were washed in BW5147Ric R. 3 cells were incubated for 10 rain at 22~ DMEM minus L-leucine plus 5% horse serum and with the ferritin conjugates at the appropriate concentra- incubated in the same medium at 37~ in a COs tion in PBS 1 (ferritin-lZSI-RCAu) or 1 M sodium incubator for 60 or 120 rain with added [aH]leucine (3 chloride-0.05 M sodium phosphate buffer (PBS 2) (fer- ~Ci/ml). After the incubation, the cells were washed ritin-Con A) plus 0.1% BSA. The cells were washed three times with phosphate-buffered saline and dissolved twice in PBS + 0.1% BSA and either fixed in phosphate- in 1 ml of NaOH containing 0.1% BSA. Protein was buffered 1% glutaraldehyde for 5 min at 22~ or precipitated by addition of 4-5 vol of cold 10% TCA, incubated in DMEM plus 5% horse serum for an and after a 60-min incubation at 4~ the precipitates additional 60 min at 37~ in a humidified COs incubator. were filtered through Whatman GF/C glass filters. Fil- At the end of the 60-rain incubation, the cells were fixed ters were washed twice with 5% TCA, once with 95% in phosphate-buffered 1% glutaraldehyde for 5 min at ethanol, dried, and aH was determined in a scintillation 22~ and washed in 0.1 M cacodylate buffer before counter. postfixation in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at 22~ The fixed cells were centrifuged Lectins and Ferritin- ~251- into soft agar, and the hardened pellets were processed as tissue blocks (30). The agar blocks were washed and Lectin Conjugates dehydrated in ethanol and finally propylene oxide before Concanavalin A (Con A) obtained from Calbiochem embedding in Spurr resin (Electron Microscopy Sci- was affinity purified according to the procedures of ences, Fort Washington, Pa.). Thin sections were cut on Agrawal and Goldstein (1). RCA. was affinity purified a Reichert model OMU-3 ultramicrotome C. Reichert, from Ricinus communis extracts by the method of Nicol- sold by American Optical Corp., Buffalo, N. Y. with a son and Blaustein (24). RCA n was labeled with lZSl by diamond knife (Rondiken, Honolulu), dried, and ob- the McFarlane (15) procedures as described previously served unstained in a Hitachi model HU-12 electron (25). Ferritin conjugates were synthesized and purified microscope at 75 kV. by a modification of the procedures of Nicolson and Singer (29). I~SI-RCAn was added to purified ferritin RESULTS (Immuno-Diagnostics) in a ratio of 1:5 wt/wt in 0.2 M sodium chloride-0.005 M sodium phosphate buffer, pH Properties of Ferritin- lZSl-R CA n 7.2 (PBS 1) containing 0.2 M D-galactose. The reaction Before performing the ultrastructural experi- mixture was split into two equal portions, and 10 tzl of ments, we examined the biological activities of the 1% glutaraldehyde in distilled water was added to one of the samples. 10 min later and at each successive 10- ferritin-12sI-toxin conjugates. In one experiment, min interval, 10 p.l of 1% glutaraldehyde were added to we monitored the effectiveness of ferritin-l~SI- both reaction mixtures. When the sample containing the RCA. in inhibiting the growth of the murine additional 10 /zl of glutaraldehyde displayed a slight lymphoma cell lines. Growth of the parental turbidity under strong beam of visible light, the reactions BW5147 line is completely inhibited at RCA. in both samples were terminated by addition of 0.25 vol concentrations >0.01 b*g/ml and partially in- of 1 M glycine in PBS 1. The samples were centrifuged hibited at RCAu concentrations >0.001 ~g/ml at 27,000 g for 15 min at 4~ and the small pellets were when toxin is continuously present in the cultures discarded. The supernates were pooled and centrifuged for 48-72 h. In contrast, the Ric a. 3 variant line at 160,000 g for 120 min onto a 0.5-ml cushion of shows only slight inhibition of growth at RCAa Sepharose 4B beads. After removal of the supernates containing unconjugated 125I-RCAII, the pellet of ferritin concentrations of 0.25 /.Lg/ml, and some cells and ferritin-~SI-RCAa was gently resuspended in PBS 1 survive even at 2.5 /xg/ml RCA. (44). When this and purified by affinity chromatography on a 3 x 90 cm experiment was performed with RCA~r and ferri- column of Sepharose 4B (Pharmacia Inc., Piscataway, tin-lZSI-RCA, in parallel, no difference in biologi- N. J.) or Bio-Rad A 1.5 m (Bio-rad Laboratories, cal activity (measured by cell growth) was oh-

NICOLSON ET AL. Toxin and Lectin Receptors on Lymphoma Cell Line 567 served at equivalent toxin concentrations. These rain but has no effect on protein synthesis in the effects were specific and could be blocked by BW5147Ric R. 3 variant cells." However, after 48- inclusion of 0.1 M D-gal or lactose in the incuba- 96 h, BW5147Rica.3 variant cells are growth- tion medium. Similar experiments were per- inhibited at 3 p,g/ml ferritinJZSI-RCAll "(Fig. 1). formed by pulse labeling with ~zsI-RCA, or ferri- Quantitative binding of ferritin-]ZSI-RCA[i was tin-X25I-RCA], for 10 min at 22~ or 60 min at assessed with BW5147 or BW5147Rica.3 cells. 37~ Since RCA. binds rapidly to cells (in most Although the amounts of ferritin-X25I-RCA, cases to plateau values in under 10 min [c.f. bound per cell were RCAxz dose-dependent and references 25 and 26], short incubations with usually slightly below the levels of unconjugated toxin or ferritin-toxin followed by removal of toxin, little difference was found in specific bind- excess toxin and incubation for 24-72 h gave ing of ferritin-125I-RCA,i between the sensitive essentially the same results (Fig. 1 a and b). For parental BW5147 cells and the Rica.3 variant, the ultrastructural experiments, concentrations of and sometimes the resistant Ric a. 3 variant cells ferritin-X~SI-RCA. (3-10 /zg/ml toxin) were cho- bound slightly more ferritin-~ZSI-RCA, (Fig. lc). sen that resulted in significant amounts of cell Ferritin-X25I-RCAit labeling could be blocked by surface bound ferritin-XZSI-RCA, in each experi- 0.1 M o-gal or lactose in the labeling and wash ment so that the location of a reasonable number solutions as found previously (19, 27). In other of toxin molecules could be easily determined on experiments the ability of ferritin-X2SI-RCA, to each thin-sectioned cell. At the lowest dose (3/~g/ inhibit cell protein synthesis was monitored at 60 ml), ferritin-~2SI-RCA, inhibits protein synthesis and 120 min after toxin binding to BW5147 cells. >80% in intact parental BW5147 cells within 90 Ferritin-X25I-RCA, (3 /xg/ml RCA.) almost com- pletely inhibited cell protein synthesis (>90%) b within 60 min in BW5147 cells, confirming our BW5147RIcR'3 00.01 previous results with fibroblastic cells (19, 26), .w o and there was little difference between ferritin- 0.1 conjugated and unconjugated toxin. t.O Labeling and Dynamics of //~0.01 3.O Fe rritin- x25I-R CA n _= ~ The binding and dynamics of ferritin-~25I-RCA. on RCA.-sensitive BW5147 and -resistant 0 ~ BW5147Ric R. 3 cells were determined by gamma .r c isotope counting and electron microscopy. Cells BWSI47Ric~.~ were pulsed with ferritin-125I-RCA, for 10 min at

N'~ BW5147 22~ washed, fixed with glutaraldehyde, and the radioactivity in each sample was determined (Fig. lc). Then, these same samples were postfixed, dehydrated, and embedded for electron micros- i)~*r-ltSl-nCA ~ml) copy. When BW5147 and BW5147RicR'3 cells /,5 ,o , were labeled with ferritin-~ZSI-RCA, containing 3 O.l,tO,3D, ZO.wm ,<4--: /zg/ml RCA. for 10 min, -106 ferritin-toxin mol- ecules bound specifically to either cell type. Ultra- TIME(h) structurally, the ferritin-X25I-RCAn molecules were FIGURE 1 RCA n toxicity and binding of ferritin-~Zq- found at the cell surface in a more or less dispersed RCA, to BW5147 and BW5147RicR.3 cells. (a) distribution (Fig. 2). Inclusion of 0.1 M lactose in BW5147 or (b) BW5147RicR. 3 cells were pulsed for the incubation and wash medium prevented bind- 10 min at 22~ with RCAn, washed with PBS, and then ing of ferritin-~25I-RCA. (Fig. 3). When parental plated in DMEM containing 10% horse serum. At 48, BW5147 cells were pulsed for 10 min at 22~ 72, and 96 h, cell numbers and viability were determined (see Materials and Methods). (c) Cells were incubated with ferritin-~25I-RCA, containing 1-3 /~g/ml with ferritin-XZSI-RCA,for 10 min at 22~ washed twice in PBS containing 0.1% BSA, and fixed in 1% glutaral- z Nicolson, G. L., and G. Poste. 1978. Mechanism of dehyde for 5 min (22~ Controls containing 0.1 M toxin resistance in selected lymphoma variant cell lines. lactose averaged <15% of the raw data. J. Supramol. Struct. In press.

THE JOURNAL OF CELL BIOLOGY VOLUME 78, 1978 FIGURE 2 BW5147 cells were labeled with ferritin-12SI-RCAn (3 /zg/ml RCAn) for 10 min at 22~ washed twice in PBS + 0.1% BSA, and fixed for electron microscopy. Bar, 0.2/zm. • 63,000.

FIGURE 3 Same as in Fig. 2, except that the samples contained 0.1 M lactose in the labeling and wash solutions. Bar, 0.2/zm. • 63,000.

NICOLSON ET AL. Toxin and Lectin Receptors on Lymphoma Cell Line 569 RCAn and then washed and incubated for 60 min variables. These include the specific activity and at 37~ before fixation, many of the ferritin- valence of the probe, its size, charge, binding conjugated toxin molecules did not remain at the strength, and other parameters (see references 3 cell periphery, and endocytosis of the ligand was and 23). In some studies the substitution of ferri- observed (Fig. 4). Not all of the ferritin-125I-RCAn tin-lectin reagents for native lectins did not result molecules were endocytosed after the 60-min in- in a quantitatively similar degree of labeling (4) or cubation, and the remaining surface-bound mole- ultrastructural distribution of lectin-binding sites cules appeared to be present in clusters and (3). These problems were probably attributable to patches on the cell surface. At these dose levels of the purity of the starting materials, methods of RCAn, the variant BW5147Ric R. 3 cells are more probe synthesis, and subsequent purification, the resistant to RCA~ toxic effects, and therefore the large size of the ferritin molecule (-150 ,~, experiment was repeated with the resistant cells. hydrated) and the potential problem that some Although differences in ferritin-~25I-RCAn binding saccharide-binding sites may be blocked during and location on parental and Ric R. 3 variant cells conjugation. We have used highly purified re- were not discernible immediately after the 10-min agents and very careful conjugation conditions labeling incubation, by 60 min after the ferritin- which have been continually modified over the 12SI-RCAn pulse (1-3 /~g/ml RCAn) there was years to synthesize a ferritin-125I-RCAn probe. little evidence of toxin transport via endocytosis in The probe was then purified by affinity chroma- the BW5147Rica.3 cells (Fig. 5). However, at tography to yield a potent biologically active label. higher toxin concentrations where the parental Comparison of the ferritin-125I-RCAn probe to and Ric a. 3 variant cells are equally sensitive to RCAn in a variety of experiments indicated that the cytotoxic effects of RCAH (7-10 p.g/ml the ferritin-toxin conjugate was unchanged in cell RCAn), toxin-mediated endocytosis occurred in binding and inhibition of cell growth and protein both cell types (Figs. 6 and 7 at 7/zg/ml RCA~). synthesis, important biological activities of RCAn (for reviews see references 20 and 37). Dynamics of Ferritin-Con A RCA~ Exerts its toxic effects by a sequence of The apparent defect in the Ric a. 3 cells which events that results in inhibition of cell protein prevents extensive endocytosis of ferritinJ25I- synthesis. First, the toxin binds rapidly to the cell RCAH at concentrations where the ferritin-conju- surfaces of susceptible cells (almost all cells are gated toxin enters parental BW5147 cells could susceptible [37]) through D-gal or D-galNAc-ter- have been due to a general depression in the minal membrane oligosaccharide residues (26, 38, ability to transport low concentrations of surface- 39). This binding is very specific and can be bound polyvalent ligands. Therefore, we exam- blocked or reversed by D-gal or lactose (5, 19, 26, ined BW5147 and the Ric a. 3 variant line for their 27, 38-40). Some cells fail to bind RCAH or ricin, abilities to bind and endocytose another ligand, and these are resistant to toxin-mediated cytotox- ferritin-Con A. Cells were pulsed with several icity (7, 16, 17). RCAH contains two separable concentrations of ferritin-Con A for 10 min, polypeptides (25, 42): One of these is thought to washed, and incubated for an additional 60 min at be involved in cell binding (35, 36), while the 37~ as in the ferritin-~25I-RCAH experiments. At other is thought to inactivate cell protein synthe- low concentrations of ferritin-Con A (3-10 p.g/ml sis. Only intact toxin molecules are cytotoxic in Con A), both BW5147 and Rica.3 cells bound animals and unbroken cells. similar amounts of ferritin-Con A or 125I-Con A RCAII must enter cells to act on protein synthe- (Table I) and transported the ligand into cells via sis, and its entry mechanism has been the subject endocytosis (Figs. 8 and 9). Although there ap- of several studies. The first clue to the entry of peared to be a slight enhancement in ferritin-Con RCAu or ricin into cells came from kinetic studies A transport in the parental BW5147 cells, the on inhibition of protein synthesis. Although toxin variant BW5147Ric a. 3 cells were fully capable of molecules bind rapidly to cells, a lag time of at endocytosis of this ligand. least 30-60 min occurs before onset of inhibition of protein synthesis (27, 42). This lag period DISCUSSION corresponds nicely with the time required for Quantitative ultrastructural analyses of cell sur- aggregation of surface toxin-receptor complexes, face receptor distribution and dynamics are diffi- endocytosis, and escape of some toxin molecules cult to determine and are affected by a variety of from endocytotic vesicles into the cytoplasm (19,

570 THE JOURNAL OF CELL BIOLOGY" VOLUME 78, 1978 FIGURE 4 BW5147 cells were labeled with ferritin-l~SI-RCAn (3 p,g/ml RCA~) for 10 min at 22~ washed, and incubated for 60 min at 37~ before fixation. Extensive endocytosis of the ferritin-toxin occurred. Bar, 0.2 p,m. x 63,000.

FIGURE 5 Same as in Fig. 4, except that BW5147Rica.3 variant cells were labeled with ferritin-125I - RCA. (3/zg/ml RCA,). Bar, 0.2/,Lm. • 63,000. FIOURE 6 Same as in Fig. 4, except that BW5147 cells were labeled with ferritin-12SI-RCA[~ (7 /.Lg/ml RCAII). Bar, 0.2/zm. • 63,000. FIGURE 7 Same as in Fig. 4 except that BW5147Ric a-3 cells were labeled with ferritin-125I-RCA[i (7 /~g/ml RCA]0. Bar, 0.2 ta.m. • 63,000.

572 THE JOURNAL OF CELL BIOLOGY 9VOLUME 78, 1978 TABLE I and its toxin-resistant variant line were compared for RCAII binding, transport, and cytotoxicity at Cell Line ~zsI-Con A clam Bound* low toxin concentrations where only the parental #g/m/ line is sensitive, and at higher toxin concentrations BW5147 1.0 3,300 -- 250 where both parental and resistant variant lines are 3.0 7,800 -- 400 10 20,100 +- 900 susceptible to RCAn killing. Although similar 10 + a-methyl- 1,100-+ 400 amounts of ferritin-125I-RCAu are bound to the o-mannoside parental and RicR.3 variant cells at low toxin BW5147Rica. 3 1.0 3,800 -- 300 concentrations, the ferritin-toxin molecules are 3.0 8,300 --- 500 only transported at significant levels into the sen- 10 22,100 _+ 1,100 sitive parental cells. At higher toxin concentra- 10 + a-methyl- 1,600-+ 400 tions where both parental and Ric R.3 cells are D-mannoside sensitive, ferritin-125I-RCAix molecules are bound * 4 • l0s cells were labeled with r~sI-Con A in PBS + and transported into both cell lines. The lack of 0.1% BSA for 10 min at 22~ and washed twice with endocytotic transport of ferritin-125I-RCAH in PBS + 0.1% BSA. toxin-resistant Ric a- 3 cells at low RCAxI concen- trations is not simply a generalized defect in cellular 27). The 30- to 60-min lag time can be almost endocytosis, because these cells are capable of completely eliminated by using lipid vesicles with transporting low concentrations of ferritin-Con A. encapsulated toxin capable of directly introducing The cell surface components of BW5147 and RCAB into the cytoplasm after fusion of the lipid BW5147Ric R. 3 cells have recently been analyzed vesicle membrane with the cell plasma mem- using surface labeling techniques and affinity chro- brane.2 Experiments using reticulocytes that have matography (44). Of the many cell surface com- diminished endocytotic activities also indicate that ponents identifiable by these techniques, two sur- toxin entry occurs most likely via endocytosis. face-exposed proteins of ~80,000 and 35,000 mol Refsnes et al. (42) found that ricin bound very wt are iodinatable by lactoperoxidase techniques rapidly to reticulocytes, but protein synthesis was on the parental but not on the Ric R. 3 cells. The not inhibited for at least 4 h under conditions larger of these proteins is a glycoprotein-binding where it rapidly stopped protein synthesis in Ehr- site for RCAn, while the smaller 35,000 mol wt lich ascites and HeLa cells. component could not be identified as a binding Once the toxin molecules have gained entry site for RCA~ in affinity chromatography experi- into susceptible ceils, ribosome-dependent protein ments. Ric R. 3 cells that do not possess the 80,000 synthesis is catalytically and irreversibly inacti- mol wt component have instead a component of vated (18, 32-34). Inhibition of cell-free protein lower molecular weight (-70,000) which can be synthesis was effective when ribosomes but not isolated by affinity chromatography or precipita- supernates of cell-free systems were treated with tion by anti-BW5147 in NP-40 cell membrane toxin (31), and Sperti et al. (45) found that extracts (44). separate in vitro treatment of the 40S and 60S A hypothesis for RCAn resistance in this system ribosome subunits with toxin resulted in identifi- has been proposed by Robbins et al. (44) that cation of the 60S ribosome subunit as the site of depends on the endocytotic transport of toxin toxin action. In addition, isolation and separation molecules into sensitive cells mediated by the 80K of ribosome subunits from toxin-treated cells re- component. This hypothesis presumes that the vealed that the 60S, but not the 40S, subunits toxin molecules bind to a variety of cell surface were inactivated (40). binding sites, but that only the 80K components Toxin-resistant tumor cell variants have proven are productive receptors, and that their 70K form useful in studying the various events leading to is not capable of mediating endocytotic transport toxin-mediated cellular death (see Introduction). of the toxin at low toxin concentrations. Presumed Since there are several possible mechanisms genetic mutation(s) in the RicB. 3 variant cell line whereby tumor cells can escape RCAn cytotoxic responsible for the above-described alteration effects, this could explain why toxin therapy has could be in the genes coding for the 80K compo- not proven universally effective (see review by nent, glycosyltransferases that specifically glyco- Olsnes and Pihl [37]). In the present study, an sylate this component, or in genes that code for or RCAII toxin-sensitive murine lymphoma cell line control a degradative enzyme that alters the 80K

NICOLSON ET AL. Toxin and Lectin Receptors on Lymphoma Cell Line 573 Fmul~ 8 BW5147 cells were labeled with ferritin-Con A (10 v,g/ml Con A) for 10 min at 22~ washed, and incubated for 60 min at 37~ before fixation. Bar, 0.2 ~m. x 63,000.

FmURE 9 Same as in Fig. 8, except that BW5147Ric a. 3 variant cells were labeled with ferritin-Con A (10/zg/ml Con A). Bar, 0.2/~m. x 63,000.

5'74 THE JOURNAL OF CELL BIOLOGY 9VOLUME 78, 1978 component before insertion into the cell mem- 73:526-542. brane. The data in this paper are consistent with 9. HYMAN, R., M. LACORBIERE, S. STAVAREK, and G. L. NICOLSON. 1974. Derivation of lymphoma this hypothesis but provide no direct evidence on variants with reduced sensitivity to plant lectins. J. the role of the 80K component in endocytosis of Natl. Cancer Inst. 52:963-969. ricin. The experiments presented here suggest that 10. HYMAN, R., and V. STALLINGS. 1974. Complemen- either these or other changes in the variant could tation patterns of Thy-1 variants and evidence that disrupt the normal transmembrane communica- antigen loss variants "pre-exist" in the parental tion (21, 22) that is required to initiate a specific population. J. Nad. Cancer Inst. 52:429-436. endocytotic process resulting in toxin sensitivity. 11. ISHmURO, M., T. TAKAHASHI, G. FUNATSU., K. HAYASHI, and M. FUNATSU. 1964. Purification of We thank V. Stallings, B. Nork, and A. Brodginski for ricin. J. Biochem. (Tokyo). 55:587-592. assistance. 12. LIN, J-Y., W-Y. KAO, K-Y. TSERNG, C-C. CHEW, These studies were supported by a contract (CB- and T-C. TUNG. 1970. Effect of crystalline on 33879) from the Tumor Immunology Program of the U. the biosynthesis of protein, RCA, and DNA in S. National Cancer Institute (to G. L. Nicolson) and experimental tumors. Cancer Res. 30:2431-2433. grants from the U. S. National Science Foundation 13. LIN, J-Y., K. LIE, C-C. CHEW, and T-C. TUNG. (PCM76-18528) (to G. L. Nicolson) and U. S. Public 1971. Effect of crystalline ricin on the biosynthesis Health Service (Ca-13287) (to R. Hyman). Dr. Hyman of protein, RNA, and DNA in experimental tumor is a Scholar of the Leukemia Society of America. cells. Cancer Res. 31:921-924. 14. LIN, J-Y., C-C. PAD, S-T. Ju, and T-C. TUNG. Received for publication 16 May 1977, and in revised 1972. 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