Eur. J. Biochem. 72, 275-281 (1977)

Properties of a Baby-Hamster-Kidney Cell Line with Increased Resistance to 2-Deoxy-~-glucose Anthony MEAGER, Roderick NAIRN, and R. Colin I1I'C;IIES National Institute for Medical Research. Mill Hill, London (Received September 2 / 0 c t o h e r 14, 1976)

A cultured cell line with increased resistance to 2-deoxy-D-glucose was obtained from cloned baby hamster kidney fibroblasts, BHK 21jCl3, after repeated exposure to high concentrations of 2-deoxyglucose. The increased resistance could not be attributed to a decreased permeability of deoxysugar. The resistant cell line incorporated radioactive 2-deoxy-~-glucoseinto glycoproteins at a similar rate as parental BHK 21/C13 cells. Incorporation of radioactive glucosamine, galactose and to lesser extent mannose into cellular glycoproteins was inhibited by 2-deoxyglucose to similar extents in the resistant cells and parental BHK 21/C13 cells. Changes induced by 2-deoxyglucose in cell surface carbohydrate structure of the resistant cells were detected by altered sensitivities to toxic plant lectins and by surface labelling as described for parental cells in the preceding paper. It is suggested that the toxicity of 2-deoxy-~-glucoseto normal fibroblasts is not mediated through effects on glycosylation of cellular glycoproteins.

The glucose and mannose analogue, 2-deoxy-~glucose is taken up by cells by the same transport system as D-glucose [1,2]. Furthermore, it has been shown that the deoxysugar is phosphorylated and converted to the activated sugar nucleotides, UDP-2deoxy-D-glucoseand GDP-2-deoxy-~-glucose in several cell types, including hamster fibroblasts 13- 51. Labeled 2-deoxy-~-glucoseis incorporated into glycoproteins and glycolipids by a variety of cells [6- 81 and has been shown in some systems to block the biosynthesis of the carbohydrate chains of glycoproteins 19- 141. Since the effect of 2-deoxy-~-glucoseon viral glycoprotein biosynthesis is counteracted by mannose [8,15] it has been suggested [8,14] that it acts mainly as an antimetabolic of mannose. Failure to make normal glycoproteins is believed to occur by the addition of 2-deoxy-~-glucosein place of mannose in the nascent carbohydrate chains during assembly. In addition to these interesting effects on glycosylation processes, 2-deoxy-~-glucosein sufficiently high concentrations over extended periods of time inhibits cell growth 116- 191 as well as viral replication [8,12, 14,151. In the latter case the effect of 2-deoxy-~glucose was concluded to be directly due to inhibition Ahhrevzations. BHK cells, baby hamster kidney cell h e ; 2dGR cells, BHK cells resistant to 2-deoxy-D-glucose.

of chain elongation of the carbohydrate chains of viral envelope glycoproteins, such that the poorly glycosylated polypeptides were unable to integrate and form fully assembled viral particles [ 8 ] . Other effects of 2-deoxy-D-glucose have also been implicated in the growth-retarding properties of the deoxysugar. At relatively low conceiit rations, this antimetabolite has been shown to interfere with levels of adenine nucleotides and to decrease the energy charge of cells [20,21]. Furthermore, 2-deoxy-~-glucoseappears to inhibit the interconversions of glucose 6-phosphate into fructose 6-phosphate and glucono 6-phosphate respectively 15,161 in the first steps of the glycolytic and shunt pathways. There are consequently a number of possible targets for the growth inhibitory activity of 2-deoxy-~-glucose. We were interested particularly in the question of whether perturbation of glycosylation reactions involving cellular glycoproteins is a major contributing factor to 2-deoxy-~-glucosetoxicity. In order to approach this problem baby hamster kidney cells BHK 21/C 13 were exposed to increasing concentrations of the antimetabolite and a resistant culture was isolated and cloned. We have examined the effects of 2-deoxyD-glUcOSe on various properties relating to the glycoprotein constitution of these cells and compare these properties with parental cells sensitive to deoxysugar.

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216

MATERIALS AND METHODS Cell Culture Baby hamster kidney fibroblasts, cell line BHK 21/C 13, was obtained from Flow Laboratories (Irvine, Scotland) and grown either in supplement Glasgow modified minimal essential medium (Glasgow medium) or a similar minimal essential medium containing 6mM glucose in place of 25mM glucose [22]. Details of culture technique, harvesting and passage of cells, and colony growth are given in the preceding paper P21.

Selection of Cells Resistant to 2-Deoxyglucose Toxicity

Our selection technique was designed to isolate cells resistant to 2-deoxyglucose on a temperaturesensitive basis. However, as discussed below, tests carried out at 33 "C or 39 "C show that the 2-deoxyglucose-resistant cells isolated were resistant to similar extents at either temperature. Cells were grown in normal Glasgow medium at 33 "C on 60-mm culture dishes and treated at 2 x 10' cells per dish with the (0.5 mg/ mutagen, methyl-N-nitro-N-nitrosoguanidine ml of culture medium). After 24 h at 33 "C the medium was removed and replaced with fresh medium containing no mutagen. The cells (approximately 28 % of the total) surviving the mutagen treatment were grown for 4 days at 33 "C to establish mutant phenotypes and then the culture dishes were transferred to 39 "C. At this time the medium was changed to one containing 30 mM 2-deoxyglucose and incubation continued at 39 "C for 6 days. After this treatment surviving cells were allowed to grow out at 33 "C for 1 day in medium minus 2-deoxyglucose before adding 50 mM 2-deoxyglucose. After a further 6 days at 39 "C the medium was removed, fresh medium minus 2-deoxyglucose added and surviving cells grown out at 33 "C to confluency (approximately 1O7 cells/dish). These cells were subsequently treated with 100 mM and 150 mM 2-deoxyglucose in a similar fashion. After this selection process only a few colonies of resistant cells were visible microscopically on the culture dishes after incubation at 33 "C.Large well-separated colonies ( > 100 cells) were removed by trypsin treatment under sterile glass cylinders and transferred to culture dishes containing fresh medium containing 10 mM 2-deoxyglucose and incubated at 39 "C. Cells from one clone of these 2-deoxyglucose-resistant cells were re-selected at low cell density (103/culture dish) in Ham's FIO growth medium containing 20 mM 2-deoxyglucose. After 10 days at 39 "C about four reasonably sized colonies were selected and these were separately transferred after cylinder-cloning to new culture vessels containing fresh Ham's FIO medium without 2-deoxyglucose. When sufficient numbers of cells were avail-

able stocks were made, and the cells were tested for colony-forming ability in increasing concentrations of 2-deoxyglucose. The cell line, designated 2 dGR,showing the highest colony-forming ability in the presence of 2-deoxyglucose was chosen for further study. It was routinely grown in the usual Glasgow modified minimal essential medium supplemented with 10 mM 2-deoxy-~-glucose. Other Methods

To determine the rate of uptake of 2-deoxy-~glucose by 2dGRcells, subconfluent monolayer cultures of these cells growing on 35-mm diameter dishes at 36 "C as well as similar cultures of parental BHK 21/C 13 cells were washed once with phosphatebuffered saline and then incubated at 36 "C with 1 ml of 2-deoxy-~-[l-~H]glucose (2 pCi/ml, 25 Ci/mmol, Radiochemical Centre, Amersham, Bucks) in phosphate-buffered saline. At times up to 50 min duplicate dishes were removed from the 36 "C incubator. The monolayers were washed with warm phosphatebuffered saline, dissolved in 1 M sodium hydroxide (1 ml) and aliquots removed for radioactive counting and determination of protein content as described in the preceding paper [22]. Methods used for measurement of the rates of incorporation of radioactive sugars into cells in the presence or absence of 2-deoxyglucose, surface labelling procedures and determination of the sensitivity of cells to toxic lectins are fully described in the preceding paper [22]. Brief descriptions are given here in the figure legends. RESULTS Growth Properties of 2-Deoxy-~-g~ucose-Resistant Cells

The growth of normal BHK cells in medium containing 25 mM glucose is prevented by concentrations greater than about 20 mM 2-deoxyglucose [22]. By contrast the resistant clone, 2dGR, in mass culture grew without lag when seeded at approximately 10' cells per culture dish in medium containing up to 30 mM 2-deoxyglucose, the highest concentration tested (Fig. 1). However, the growth rates in medium containing the antimetabolite were slower than found with cells grown in the absence of 2-deoxyglucose and the cell densities of monolayer cultures at saturation decreased with increasing concentration of 2-deoxyglucose as follows: no 2-deoxyglucose, 3.5 x lo5 cells/ cm2; 10mM, 1.5 x 10' cells/cm2; 20mM, 1.1 x lo5cells/ cm2; 30 mM, 0.8 x 10' cells/cm2. The 2dGR cell line also exhibited increased resistance to 2-deoxyglucose at very low cell densities. As indicated in Fig. 2, when approximately lo3 cells

277

A. Meager, R. Nairn, and R. C. Hughes

c

/

!-

o

I

20

40

60

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a0 100 120 Time ( h )

I

I

140 160

40

I

iao

30

Fig. 1. Growth cu~ve.sof 2dGK cells in medium containing 10 mM 2-deo.uyglucose ( o ) ,20 mM 2-deo.~yglucose( 0 )or 30 mM 2-deoxyglucose (Hi.Growth in the absence of 2-deoxyglucose is also shown (0)

20

10 5

'0

were seeded per culture dish in Ham's FIO medium containing increasing concentrations of 2-deoxyglucose the numbers of visible colonies formed after several days at any concentration of 2-deoxyglucose was always substantially higher for 2dGR cells compared with parental BHK cells. The absolute plating efficiencies of the parental cells and the 2dGR cells in the absence of 2-deoxyglucose, i.e. numbers of colonies forming expressed relative to the numbers of cells plated (usually lo3 per plate) were very similar and approximately 20- 30 %. Therefore, the results shown in Fig.2 indicate that the 2dGR cells were at least 10- 20 times more resistant to the deoxysugar. The increased resistance of 2dGRcells to the toxic effects of 2-deoxyglucose as revealed either by colony formation or by mass culturing was unstable (Table 1). If 2dGR cells were serially passaged in the absence of 2-deoxyglucose for several months the sensitivity returned to the parental pattern. Resistance was maintained for long periods (at least six months), however, if 2dGR cells were grown continuously in the presence of high (10 mM) concentrations of 2deoxyglucose. Morphologically 2dGR were similar to normal BHK cells but were larger (410 pg cell protein/cell compared to 250 pg cell protein/cell for normal BHK cells) and grew to slightly less (10 - 30 %) dense monolayers at confluency in the absence of 2-deoxyglucose.

Fig. 2. E f f c t of2-deoxyglucose on the colony-forming ubility of B H K cells. Dose response curves are drawn with relative plating efficiency (i.e. the number of colonies developed in the presence of 2-deoxyglucose compared with the number of colonies developed in the absence of 2-deoxyglucose expressed as a percentage) as ordinate against 2-deoxyglucose concentrations. Normal wild-type BHK 2dGRcells, (0) 21/C13 cells, (0);

Table 1 Resistance of clonal isolates of 2dGR cells to 2-deoxyglucose and reversion to sensitivity after growth in the absence of 2-deoxyglucose Resistant cell line 2dGR immediately after isolation as described in Materials and Methods was grown in medium containing 10 mM 2-deoxyglucose and separate clonal isolates were tested for colonyforming ability in the absence and presence of 10 mM 2-deoxyglucose respectively. The relative plating efficiency is the percentage ratios of colonies formed. Each clonal isolate was grown continuously in 2-deoxyglucose-freemedium for 3 months (10- 12 passages) and retested for plating efficiency 2-deoxyglucose. Results are also shown for the maintenance of resistance of clone 4 after growth for a similar period in 2-deoxyglucose. n.d. = not determined Cell isolates

Culture medium supplemented with 10 mM 2-deoxyglucose

Relative plating efficiency

Clone 1

Yes no Yes no Yes no Yes no yes a

106 14- 17 123 n.d. 109 18-19 73 8-9 > 70

Clone 2 Clone 3 Clone 4

Metabolic Effects of 2-Deoxyglucose on 2dGR Cells

In the preceding paper [22] we showed that 2deoxyglucose interferes with normal biosynthesis of cellular glycoproteins in BHK cells. Thus, incorporation of radioactivity from labelled sugar precursors into acid-precipitable fractions was rapidly inhibited by the antimetabolite. The cells bound less ricin, a lectin which seeks galactose or N-acetylgalactosamine, and became more resistant to ricin cytotoxicity. Furthermore, glycoproteins made by normal cells in

10 15 2 0 25 30 35 40 45 Final concn of 2-deoxyglucose (mM)

~~~

~

Retested resistance after 3 - 12 months continuous passage in medium containing 10 mM 2-deoxyglucose. a

the presence of the inhibitor migrated more quickly during gel electrophoresis, indicating a lower apparent molecular weight. This has been attributed [22] to a defect in glycosylation and hence a smaller carbo-

2-Deoxyglucose-Resistant BHK Cells

278

0 Time (min)

4

8 12 Time ( h )

I6

20

Fig. 3. Uptake of2-deoxygluco.se by BHK cells. (A) Subconfluent monolayer cultures of normal BHK 2ljC 13 cells (0)or resistant 2dGRcells (0) were incubated at 36 "C with 2-deo~y-[l-~H]gIucose (2 pCi) in phosphate-buffered saline (1 ml). At times duplicate plates were removed from the incubator. The monolayers were washed with phosphate-buffered saline, dissolved in alkali and the extent of radioactivity associated with the cells was measured. (B) Subconfluent monolayer cultures o f normal (0)or resistant ( 0 )cells were incubated at 36 "C with 2-deo~y-[l-~H]glucose (2 pCi) in glucose-free Glasgow-medium. At times duplicate plates were removed from the incubator. The cells were washed with phosphate-buffered saline followed by phosphotungstic acid/perchloric acid mixture and finally with ethanol. Incorporation of radioactivity into the acid-washed cells was measured as in (A)

hydrate content than glycoproteins made in the absence of 2-deoxyglucose. In the following series of experiments, 2dGR cells were exposed to 2-deoxyglucose and compared with normal BHK 21/C13 cells in their response to the inhibitor as reported in detail in the accompanying paper [22]. The most obvious mechanism of acquiring resistance to 2-deoxyglucose is by an inability of resistant cells to take up the sugar. However, the rate of uptake of 3H-labelled 2-deoxyglucose by 2dGRcells and incorporation into acid-precipitable glycoproteins was the same as normal BHK cells (Fig. 3). Similarly, 2-deoxyglucose prevents incorporation of radioactivity from 3H-labelled glucosamine and galactose into a total acid-precipitable fraction of 2dGR cells (Fig. 4). The extent of inhibition is similar to that reported [22] for wild-type BHK 21/C 13 cells. Mannose incorporation into macromolecules was less affected by 2deoxyglucose in both 2dGRcells (Fig.4C) and normal BHK cells [22]. Under exactly comparable conditions, for example pre-treatment of cells for 2 h with 5 mM 2-deoxyglucose at 39 "C before addition of isotopically labelled mannose, it consistently appears that qualitatively the effects of 2-deoxyglucose on mannose incorporation are similar in 2dGR cells and normal BHK cells. We conclude therefore that glycosylation events are equally affected by 2-deoxyglucose in the resistant and sensitive strains of BHK cells. Supportive evidence for modulation of the structure of surface glycoproteins of 2dGR cells by 2-deoxyglucose treatment is shown in Fig.5. 2dGR cells (Fig.5) are as sensitive to ricin and concanavalin A cytotoxicity 123,241 as normal BHK cells [22]. Like the latter, 2dGR cells become progressively more re-

sistant to the toxic effects of ricin by prolonged pretreatment of cells with 2-deoxyglucose. Conversely, 2dGR cells became increasingly more sensitive to concanavalin A, a mannose-seeking lectin, after 2deoxyglucose treatment and again behaved similarly to wild-type BHK cells in this property. There is a suggestion in Fig.5 that lOmM concentration of 2deoxyglucose may be required to induce a 3-fold increase inresistance to ricin on 2dGR cells while a lesser concentration (1 mM) is sufficient to induce this degree of resistance in normal BHK cells 1221.

Suvface Labelling of 2dCR Cells When normal BHK cells were grown to confluency in the presence of 1 mM or 10 mM 2-deoxyglucose and labelled by lactoperoxidase-catalysed iodination, several changes were found in the 1251-labellingprofile following dodecylsulphate polyacrylamide gel electrophoresis of the iodinated protein components 1221, which other evidence discussed previously [22] has shown to be largely glycoprotein in BHK cells (R. Nairn, unpublished results) as in other cells. There was a small shift of labelled species to lower-molecularweight values, consistent with a reduction in glycosylation of several glycoproteins. Replicate cultures of resistant 2dGR cells (2 x 10' cells/60-mm culture dish) which had been maintained i,n Glasgow medium plus 10 mM 2-deoxyglucose were set up for iodination. One set of cultures was allowed to grow out in 10 mM 2-deoxyglucose-containing medium while the other grew out over 4- 5 days in the absence of 2-deoxyglucose. The cells were then iodinated. The gel iodination profiles are shown in

A. Meager, R. Nairn, and R. C. Hughes

279

Lectin final concentratlon ( p g l m l )

Fig.5. Toxiciry of ricin and concanavalin A towards 2dGR cells. (A) Ricin (Ricinus communis toxin) was added at the stated final concentrations to 60 mm diameter tissue culture dishes to which approximately lo3 cells in Ham's FIO medium had been added 24 h previously. Incubation was continued at 39 "C until visible colonies appeared. These were counted and expressed as percentages of the colonies appearing under the same condition in absence of ricin. (B) Concanavalin A toxicity towards 2dGRcells was measured exactly as described in (A). (0)Culture medium without 2-deoxyglucose; (0)1 mM 2-deoxyglucose; (n)10 mM 2-deoxyglucose

Lh-uLu-

'0

2 4 6 8 10 12 Time of incubation i h )

Fig. 4. Incorporation of radioactive sugars into 2dGKcellglycoproteins. Monolayer cultures growing on 35-mm tissue culture dishes at 39 'C were incubated in medium (final glucose concentration 6 mM) supplemented in some sets of didhes (A in A) with 5 mM 2-deoxyglucose. After growth for 2 days to near confluency the 2-deoxyglucose-containing medium was replaced with medium containing no deoxysugar (0 in A, B and C) and 2-deoxyglucose to a final concentration of 5 mM was added to other dishes (0 in A, B and C). Incubation at 39 "C was continued for 2 h. Then the following radioactive precursors were added separately (0.05 ml per dish, 5 pCi/dish); (B) D-[l-3H]galactose ( 5 Ci/mmol), (A) D [ ~ - ~ H ] glucosamine hydrochloride (12 Ci/mmol) or (C) D-[l-3H]mannose (2 Ci/mmol). At times thereafter duplicate dishes were removed from incubation. The cells were washed successively with phosphatebuffered saline, a perchloric acid/phosphotungstic acid mixture and cold ethanol. Each washed cell fraction was then dissolved in alkali for radioactive counting. Full details of the experimental procedure are given in the preceding paper [22]

Fig.6B and C respectively, and can be compared with the iodination profile of normal BHK cells grown in the absence of 2-deoxyglucose shown in Fig.6A. Clearly there are striking differences between the iodination profiles of 2dGR cells and normal BHK cells. Of particular interest are the apparent loss of labelling of a fraction of nominal molecular weight 250000 in 2dGR cells (Fig,6C), the overall low level of '251-labelling (Fig. 6C) and the enhanced mobility

of glycoprotein species when 2dGR cells grown in the presence of 2-deoxyglucose are iodinated (Fig. 6 B). Without 2-deoxyglucose, the iodination pattern of 2dGR cell surface components was broadly similar to normal BHK cells except for the absence of the highmolecular-weight glycoprotein mentioned above. It was mentioned earlier that 2dGRcells retain their resistance to 2-deoxyglucose for long periods provided this sugar (10 mM or greater) was present in the growth medium. If, however, 2dGRcells are serially passaged in the absence of 2-deoxyglucose for several weeks, the resistance is lost and a 'revertant' cell line, 2dG'"', obtained. The cell surface labelling profiles of 2dGR and 2dG'"' cells were examined and compared to those of normal BHK cells. Consistently we have found that the iodination profile of the revertant cells approximated quite closely to the iodination profile of normal BHK cells (Fig. 6E). In particular the iodinated glycoprotein species of nominal molecular weight 250 000 reappeared, the overall extent of iodination increased and the mobility of the iodinated bands approached that of iodinated bands in normal BHK cells. If revertant 2dG'"' cells were plated out in medium containing 10 mM 2-deoxyglucose and grown to confluency before iodination then the iodination profile (Fig. 6D) again became essentially similar to that of 2dGR cells (Fig. 6B) maintained in 2-deoxyglucose-containing medium.

2-Deoxyglucose-Resistant BHK Cells

280

0

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1.5

O

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i

.i

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4 6 8 10 Distance o f m i gr at i on ( c m )

1 2 1 4

Fig. 6 . Comparison of the surfhce components of' normal BHK cells and 2dGR cells labelled by lactoperoxiduse-catalysedradioiodinntion. Cells were grown at 39 "C on 60-mm culture dishes and when confluent were labelled with and bdctoperoxidase as described previously [22]. After labelling, the monolayers of viable intact cells were washed extensively to remove labelling reagents, dissolved in 1 %sodium dodecylsulphate, 1 % 2-mercaptoethanol and analysed by slab gel electrophoresis on 7.5 % polyacrylamide. Parallel tracks of the resolving gel were separately cut into 2-mm slices and counted for ''1 radioactivity in a gamma spectrometer. (A) Normal BHK 21/C13 cells grown in medium without 2-deoxyglucose and then labelled. (B) 2dGR cells grown in the continual presence of 10 mM 2-deoxyglucose and then labelled. (C) 2dGR cells grown for 5 days in the absence of 2-deoxyglucose before babelling; these cells showed the full degree of resistance to 2-deoxygiucose of 2dGRcells grown continuously in 10 mM 2-deoxyglucose. (D) 'Reverted' 2dGRcells, grown for three months continuously in the absence of 2-deoxyglucose and showing wild-type sensitivity to 2-deoxyglucose, were exposed to 10 mM 2-deoxyglucose for 2 days before labelling. (E) 'Reverted' 2dGR cells, defined as in (D), labelled after growth in the absence of 2-deoxyglucose. The arrows indicated approximate molecular weights of several peaks of radioactivity found in normal BHK cells. These were determined using standard proteins of known molecular weights run separately. A bromophenol blue marker ran to the point indicated

DISCUSSION Our results demonstrate that induction of resistance in BHK cells towards 2-deoxy-~-glucose does not

affect the inhibition of glycosylation reactions brought about by exposure ofcells to the deoxysugar. Similarly, some interference to cell surface organization and composition is still apparent in 2-deoxyglucose-resistant 2 dGR cells exposed to 2-deoxyglucose as shown by the increased resistance of treated 2dGR cells to ricin cytotoxicity and by surface labelling techniques. Therefore, such alterations in cellular glycoproteins cannot in themselves play an essential role in the cytotoxicity of 2-deoxy-~-glucose. While the mechanism for maintaining resistance to 2-deoxyglucose in 2dGR cells is not understood, a further comparison of the metabolic responses of these cells and parental, sensitive cells to the sugar may point to the basic cause(s) of 2-deoxyglucose toxicity. One outstanding question is the genetic basis for the phenotypic changes apparent in 2dCR cells. It is not known if these changes result from genetic mutation rather than adaptation and phenotypic variation within a stable genotype. We suggest that the former is likely on the following grounds : 2-deoxyglucoseresistant cells were isolated only from mutagenized BHK cells and no resistant colonies could be obtained from a non-mutagenized population ; the colonies arise at a low frequency m7); clonal isolates of the 2dGR population show similar degrees of resistance to the antimetabolite (Table 1). The decline in resistance of 2dGRcells found in cultures maintained without 2-deoxyglucose is not necessarily inconsistent with our view. For example, a high reversion rate of the mutation controlling dehydrofolate reductase production in methotrexate-resistant leukemia has been ascribed to genetic instability [25]. The study of the effects of 2-deoxyglucose on cell surface glycoproteins with regard to their iodination by lactoperoxidase as reported in this and the preceding paper [22] has provided interesting results. Particularly noteworthy was the much decreased extent of labelling of surface glycoproteins by lactoperoxidase-catalysed iodination after normal BHK cells had been cultured in medium containing 2-deoxyglucose [22]. It is relevant to this finding that Melchers [13] briefly reports that the expression of surface-bound immunoglobulin in mouse myeloma cells as revealed by immunofluorescent antibody staining is not affected appreciably by 2deoxyglucose although secretion of the soluble immunoglobulin molecules is markedly inhibited. Our results by contrast suggest that in BHK cells the antimetabolite may disturb normal expression of surface glycoproteins although the mechanism by which it does so is, of course, unexplained. This could reflect either decreased biosynthesis and processing of glycoproteins destined for the cell membrane, or poor insertion and orientation of glycoproteins in the plasma membrane resulting in reduced exposure of available tyrosine residues for iodination. Increased turnover of glycoproteins from the cell surface may

A. Meager, R. Nairn, and R. C. Hughes

also be involved, since it is known that if glycoproteins are not fully glycosylated they become more susceptible to breakdown by proteolytic enzymes [14]. In the case of the 2dGR cell line, cells resistant to the toxicity of 2-deoxyglucose selected by repeated exposure to high levels of 2-deoxyglucose, the extent of 1251-labellingof surface glycoproteins appears to be reduced if the cells are grown in medium with or without 2-deoxyglucose. However, 2dGR cells grown for extended times in the absence of 2-deoxyglucose return to the normal level of "'1-surface labelling. The change back to a normal level of iodination as in the untreated BHK cells, is not immediate, although a partial return to full glycosylating activity, as evidenced by the more normal electrophoretic mobilities of surface glycoproteins, is apparent relatively quickly, within 2-4 days of culturing in the absence of 2deoxyglucose. Only after several weeks propagation in the absence of 2-deoxyglucose, when the resistance of 2dGRcells to 2-deoxyglucose is essentially lost and 'reverted' cells (2dG"') have taken over the culture, is anything like the usual gel iodination profile of normal BHK cell surface glycoproteins restored however. The level of iodination of surface components then approaches that of normal BHK cells under comparable conditions and more interestingly the glycoprotein band of high molecular weight (250 000) reappears on the gels. Hynes [26,27] has described the loss of a highmolecular-weight surface glycoprotein species, capable of iodination, from hamster NIL cells after transformation by RNA tumour viruses. Such a change also occurs in BHK cells transformed by polyoma virus [28] and by type 5 adenovirus in some but not all instances [29]. Loss of this glycoprotein, however, is apparent in 2dGR cells as reported in the present paper and in several ricin-resistant BHK cell lines (R. Nairn and R. C. Hughes, unpublished results). The validity, therefore, of the loss of this high-molecularweight glycoprotein to uniquely indicate the virally transformed state may be questioned.

281

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We thank the Medical Research Council for a Research Studentship (RN). AM was a Beit Memorial Fellow.

A. Meager, R. Nairn, and R. C. Hughes, M. R. C. National Institute for Medical Research, The Ridgeway, Mill Hill, London, Great Britain, NW7 1AA