354 T. Marshall and A. L. Latner Electrophoresis 1983, 4, 354-358

Thomas Marshall and Albert L. Latner

High resolution electrophoresis of extracts of a baby hamster kidney cell line before and after transformation by polyoma virus Microbiological Chemistry Research

Laboratory, The University, Newcastle-upon Tyne

The polypeptide patterns obtained following denaturation and high resolution two- dimensional electrophoresis of proteins from baby hamster kidney cells before and after transformation by Polyoma virus have been compared by silver staining incor- porating methylamine. The polypeptides were resolved either by non-equilibrium pH-gradient electrophoresis or isoelectric focusing in the first dimension. Two dif- ferent approaches to isoelectric focusing were employed in order to achieve satisfac- tory resolution over a wide range of isoelectric point. Both cell lines gave remarkably similar two-dimensional patterns of spot distribution which allowed changes to be detected in the relative abundance of a number of polypeptides. None of the polypeptides detected were specific to either of the two cell lines.

1 Introduction

The detection of proteins associated with neoplastic transfor- mation would be important for the clinical diagnosis of cancer and possibly its treatment. The demonstration of cancer- specific proteins would further facilitate the raising of cancer- specific monoclonal antibodies. Such an antibody to the so- called Cal antigen has already been prepared and its diagnostic usefulness demonstrated [ 1-31. The detection of possible specific proteins in cancer tissue is complicated by difficulties associated with the collection and preparation of homogeneous samples and contamination with serum pro- teins. These problems can be overcome by the use of estab- lished tissue culture cell lines. In this respect a human cell line compared to the same line which has been rendered malignant would be ideal. Some such observations have been made on human amniotic and other cell lines 141. Nude mice were used as the recipients for tumour production from the transformed amniotic cells. Unfortunately, the only real test of malignancy is the demonstration of invasion and or metastasis in the species of animal from which the cell lines have been derived. Similar criticism is applicable to the work with human cell lines [41, in which labelling was done with [35S1methionine or [32Plorthophosphate. Such studies could not be done with human beings and it is therefore necessary to revert to an animal model. For this purpose baby hamster kidney (BHK) cells are suitable. These cells rarely produce metastases when injected subcutaneously into hamsters but are transformed by polyoma virus to yield cells (PyY) which produce a high incidence of invasion and metastasis after injection. Both cell lines can be readily grown in tissue culture.

High resolution two-dimensional electrophoresis [51 provides an excellent means of comparing complex protein mixtures following denaturation of the proteins to their constituent polypeptides. This approach has been used in conjunction with autoradiography to compare the polypeptides of BHK and polyoma transformed BHK (PyY) cell lines following tissue culture in a growth medium containing [35Slmethionine

Correspondence: Professor A. L. Latner, Microbiological Chemistry Re- search Laboratory,The University,Newcastle uponTyneNE 1 7RU, UK

Abbreviations: BHK: Baby hamster kidney cells; PyY: Polyoma tran- sformed BHK; C: Clone; IEF: Isoelectric focusing; NEPHGE: Non- equilibrium pH-gradient electrophoresis; Tris: Tns(hydroxymethy1) ami- nomethane; SDS: Sodium dodecyl sulphate

0 Verlag Chemie GmbH, D-6940 Weinheirn, 1983

[6,71. The polypeptide patterns so obtained, however, are not indicative of the original protein distribution. Proteins with a low rate of turnover or a low methionine content may incorporate insufficient [35S1methionine to permit detection. Proteins with a higher turnover rate vary in this ability. It follows therefore that the pattern obtained with auto- radiography must differ considerably from that which would have been obtained from the proteins originally present.

Highly sensitive silver stains for the detection ofprotein have a sensitivity equivalent to that achieved using autoradiography IS]. We have recently described the use of methylamine in the preparation of the silver stain [91 which is suitable for thin and the relatively thick (3 mm) polyacrylamide gradient gels used in our simplified technique of high resolution two-dimensional electrophoresis [ 101. The use of silver staining, in contrast to that employing radioactive labelling, should give a better in- dication of the polypeptide constituents obtained from pro- teins originally present in both normal and transformed cells. Whilst the staining properties may vary from polypeptide to polypeptide, it is quite legitimate to compare and contrast in different extracts the amount of stain taken up by polypeptide spots present in identical positions in the second dimension

2 Materials and methods

2.1 Chemicals

Ampholines of pH ranges 3.5-10, 4-6, 5-7, 7-9 and 9-1 were supplied by LKB Instruments Ltd., Croydon, Surrey, UK. Acrylamide (specially purified for electrophoresis), NN’ methylenebisacrylamide, agarose (electrophoresis grade), glycerol, glycine, 2-mercaptoethanol and sodium dodecyl sulphate were purchased from BDH Chemicals, Poole, Dorset, UK. Tris (hydroxymethyl) aminomethane and molec- ular weight markers for sodium dodecyl sulphate (SDS) elec- trophoresis (Dalton Mark VI) were purchasedfrom the Sigma Chemical Co., Poole, Dorset, UK.

2.2 Cell culture

Established tissue culture cell lines, BHK 2 1/C 13 and PyY (Polyoma transformed BHK 2 1/C 13) were purchased from Flow Laboratories, Irvine, Scotland. The cells were grown as

0173-0835/83/05 10-0354802.50/0

Electrophoresis 1983, 4, 354-358 Polypeptide patterns normal and transformed baby hamster kidney cells 355

monolayers in glass Winchester bottles using the technique described for another cell line [91. Growth took place in Dulbecco’s modification of Eagle’s medium (Flow Lab- oratories) containing 10% v/v calf serum (Flow Lab- oratories). The proteins of each cell line were extracted and prepared for high resolution two-dimensional electrophoresis as previously described 191. The protein content ofthe extracts was determined by a modified Lowry estimation [ 1 11.

2.3 High resolution two-dimensional electrophoresis

The polypeptides were resolved by isoelectric focusing (IEF) or non-equilibrium pH-gradient electrophoresis (NEPHGE) in the first dimension and SDS polyacrylamide gel electro- phoresis in the second dimension [5,121. The actual simplified procedures we employed were similar to those described pre- viously [9, 101.

2.3.1 First dimension

IEF was carried out in 4 % w/v polyacrylamide gels contain- ing 9 M urea and 2 % w/v Ampholine. The Ampholine was a mixture of the pH ranges 3.5-10,5-7 and 9-1 1 (9:l:l vlv). The gels were prepared as previously described [lo] but assembled in the IEF apparatus with extensively degassed 0.02 M sodium hydroxide in the lower cathode reservoir and 0.01 M phosphoric acid in the upper anode reservoir. This reversal of the positions of the anode and cathode reservoirs, with loading of the sample (50 pg protein) at the anode end of the IEF gel, gave better resolution of the polypeptides present in most of that half ofthe gel proximal to the anode as well as in the anode side of the half proximal to the cathode. Electrofo- cusing was continued for 16 h at 330 V with a 90 K n resistor in series [ 101. This technique did not give good resolution of those polypeptide spots which were either very basic or very acidic. Many ofthe more basic polypeptides failed toremain in the IEF gel. Presumably, their isoelectric points were higher than the upper limit of the pH gradient generated during IEF and so passed out of the gel cylinder.

Polypeptides with the more basic isoelectric points were separated by NEPHGE [ 121, which resulted in their retention within the gel cylinder. The samples contained 50 pg of pro- tein. The procedure was similar to that described above except that the gels contained equal volumes of the Ampholine pH ranges 7-9 and 9-1 1. It should be pointed out that the latter range is supplied at half the concentration of the other ranges. This resulted in a final Ampholine concentration of 1.5 % w/v. Electrophoresis was continued for only 5 h [91.

Polypeptides with the more acidic isoelectric points were resolved by IEF in gels containing 2 % w/v Ampholine which was amixtureofthepHranges4-6and3.5-10(4:1 v/v).They were assembled in the IEF apparatus as previously described [lo]. In this case the 0.01 M phosphoric acid was in the lower anode reservoir and extensively degassed 0.02 M sodium hydroxide in the upper cathode reservoir. The samples, con- taining 50 pg of protein, were loaded at the cathode end of the gel cylinder prior to IEF.

2.3.2 Second dimension

SDS polyacrylamide gel electrophoresis was performed as previously described 19,101 except that mercaptoethanol was

omitted from the equilibration solution 0 (10 % w/v glycerol, 5 % v/v mercaptoethanol, 2.3 % w/v SDS in 0.0625 M Tris- HCl, pH 6.8) recommended by O’Farrell[51 for the equilibra- tion of the first-dimension gels prior to SDS electrophoresis. This omission did not alter the polypeptide patterns but eliminated the streaky artefacts previously detected with the silver stain [91. The polyacrylamide gradient gels (75 x 75 x 3 mm) were prepared with a 4- 10 % w/v polyacrylamide gra- dient over the upper 1.0 cm and alinear 10-20 % w/vgradient over the remaining 6.5 cm [81. A 1 % w/v solution of agarose in the equilibration solution devoid of mercaptoethanol was used to cement the equilibrated first-dimension gels to the top of the polyacrylamide gradient gel. Molecular weight markers for SDS gel electrophoresis (range 12 000-66 000) were ap- plied to the top of the electrophoresis gel as previously de- scribed [ 101. Electrophoresis was performed in 0.025 M Tris base, 0.192 M glycine containing 0.1 % w/v SDS, at 20 mA/ gel, until the dye front (Bromophenol Blue) reached the bot- tom of the gel 101.

2.4 Silver stain

The polypeptide patterns were detected using the silver stain incorporating methylamine [ 101. The gels were soaked over- night at room temperature in a solution containing 50% methanol and 10 % acetic acid and then stained using a proce- dure similar to that previously described [ 101. Briefly, the gels were washed for 10, 20 and 30 min, respectively, in three changes of an aqueous solution containing 5 % methanol and 7 % acetic acid (200 ml/gel) at 60 *C and then treated with a fresh solution containing 4 % w/v paraformaldehyde and 1.43 % w/v sodium cacodylate, pH 7.3, for 15 min (100 ml/ gel) at 60 “C [lo]. The gels were then washed for 10,20 and 30 min in three changes of distilled water (200 ml/gel) at 60 “C, in a fourth change at room temperature for 10 min, and then gently shaken in freshly prepared diamine solution (50 ml/gel) for 10 min at room temperature. Thediamine solu- tion was prepared by adding 10 ml of amixture of 5 volumes of 0.36 % sodium hydroxide and one volume of 25/30 % wlv methylamine solution to 4.0 ml of 20 % w/v silver nitrate. A further volume (- 2 ml) ofthe alkaline mixture was then slow- ly added until the brown precipitate disappeared. The volume was now adjusted to 100 ml with distilled water. This gave a more consistent diamine preparation than slowly adding all of the required NaOH/methylamine mixture [91. Following a brief 3 min rinse in distilled water, the gels were transferred to a freshly prepared aqueous solution (100 ml/gel) containing 0.005 % w/v citric acid and 0.19 % w/v formaldehyde [ 1 11. This solution was subsequently changed at 5 min intervals over the following 20 min. The stained proteins first appeared as brown black spots against a clear background. Further development intensified the protein-stained spots against an intense amber background. The gels were subsequently destained using the photographic reducer described by Switzer et al. 181. The proportion of the mixed solutions ‘A’ and ‘B’ was, however, modified (3 volumes ‘A’ + 1 volume ‘B’ + 3 volumes HzO) to give a greater control over the destaining procedure. The gels were shaken in Kodak Hypo clearing agent (Kodak Ltd., Manchester, UK) for 30 min and then washed thoroughly in tap water [91.

Each pair of gels, corresponding to those shown in each of the figures, were stained and destained alongside each other in the

356 T. Marshall and A. L. Latner Electrophoresis 1983, 4, 354-358

same bath. It was therefore possible to assure that corre- sponding molecular weight markers in each of the gels were stained to the same extent. This allowed meaningful com- parison of the intensity of staining of corresponding peptide spots. Moreover, the polypeptides in each pair of gels had pre- viously been simultaneously subjected to IEF or NEPHGE, in each case in the same apparatus. The subsequent gel electrophoreses were also carried out at the same time in the same electrophoresis tank.

3 Results

The two-dimensional patterns of the polypeptides derived from proteins extracted from BHK and PyY cell lines are shown in Fig. 1-3. The patterns of distribution were highly reproducible and those shown are typical of the results ob- tained from five completely separate extractions of protein from different cultures of each cell line. In each of the paired observations, the patterns of distribution, as opposed to con- centration, were identical. For the purposes of comparison, spots were located by their positions in relation to the first- dimension gel and to the molecular weight markers in the sec- ond dimension. This facilitated even more exact identification by observing the relation of the spots in question to neighbour- ing spots.

The intensely stained zones at the bottom of the gels in Fig. 3 were due to incomplete removal of basic Ampholines. It could be argued that they masked possible polypeptide differences. These zones did not appear when the initial overnight soak of the gels in 50 % methanol, 10 % acetic acid was carried out at 6 0 "C. The basic polypeptide spots so revealed were identical in a paired observation. Polypeptide spots which were con- sistently more prominent in BHK are numbered 1-46, whereas those consistently more prominent in PyY are let- tered a-t. Other differences are apparent in the figures but these were not detected in all of the preparations analysed.

None of the polypeptides detected were specific to either ofthe two cell lines. Polypeptides with the more acidic isoelectric points are denoted by the lowest numbers and letters (Fig. 2). Those with the more basicisoelectric points are denoted by the highest numbers and letters (Fig. 3).

Fig. 1 demonstrates the patterns obtained following IEF ofthe polypeptides in a pH gradient generated from the Ampholine mixture of the widest pH range (3.5-1 1; predominantly pH 3.5-10). It is evident that this pHgradientisinsufficient togive satisfactory resolution of those polypeptides with the more basic or more acidic isoelectric points. The more acidic polypeptides were adequately resolved (Fig. 2) by reversing the polarity of the first-dimension procedure and resolving the polypeptides in a pH gradient generated from Ampholine of the lower pH range (predominantly pH 4-6). Adequate resolution of the more basic polypeptides (Fig. 3) was achiev- ed by maintaining the original polarity of electrophoresis but obtaining resolution in the first dimension using NEPHGE in the presence of Ampholine of high pH range (pH 7-1 1). The resulting two-dimensional patterns shown in the three figures cover the complete range ofpolypeptide isoelectric points. The two arrows above each of the patterns shown in Fig. 1 are at either side of the region most clearly defined when the Ampho- line mixture ofwidest pH range was used in the first dimension. The anode and cathode sections outside this region corres- pond respectively to the anode side of the arrow in Fig. 2 and to the cathode side of that in Fig. 3. The molecular weight markers employed were bovine albumin, ovalbumin, pepsin, trypsinogen, lactoglobulin and lysozyme. Comparison with the two-dimensional patterns obtained with human serum showed that, under our experimental conditions, anomalous apparent molecular weight indications of 44 000, 18 000, 13 000 and 12 000 were given by pepsin, trypsinogen, lac- toglobulin and lysozyme respectively. Those for bovine albu- min and ovalbumin were correct. The lines in the figures show- ing apparent molecular weight correspond to our own exper- imental findings. Since pepsin was so close to ovalbumin, its

Figure I . Two-dimensional high resolution electrophoretogram of polypeptides of intermediate isoelectric points stained with silver methylamine. In this and subsequent figures, (A) designates BHK extract and (B) PyY extract; numerals indicate polypeptide spots consistently more prominent in BHK and lower case letters those consistently more prominent in PyY; the numbers between (A) and (B) indicate apparent molecular weight x 10-3.

Electrophoresis 1983, 4, 354-358 Polypeptide patterns normal and transformed baby hamster kidney cells 35 7

Figure 2. Two-dimensional high resolution electrophoretogram of the acidic polypeptides stained with silver methylamine.

corresponding line is not shown. Although all of the poly- peptide differences detected were quantitative in nature rather than specific to one cell line, some were prominent in one line and virtually absent from the other. Such polypeptides include a and b, which were prominent in PyY, and spots 4,25 and 32, which were prominent in BHK.

4 Discussion

The use of three different pH mixtures of Ampholines, in con- junction with either IEF or NEPHGE, in the first dimension resulted in three overlapping two-dimensional polypeptide

patterns. The composite of these three patterns represents a virtually complete separation of the polypeptides over a wide range ofisoelectric point. It is only on the basis of such adegree of separation that avalid comparison can be made between the polypeptide constituents of the two cell lines.

There are many more spots of a basic nature in Fig. 3 than are apparent in the cathode region when the broad range Ampho- line gel was used (Fig. 1). This is because NEPHGE retains all the more basic polypeptides [ 121 whereas the IEF procedure used to obtain Fig. 1 appears to result in the passage ofthose polypeptides through the length of the gel cylinder and out of its cathode end into the electrode solution. It is also evident

Figure 3. Two-dimensional high resolution electrophoretogram of the basic polypeptides stained with silver methylamine. In this case the first-dimension procedure was NEPHGE.

358 T. Marshall and A. L. Latner Electrophoresis 1983, 4, 354-358

that, during the latter procedure, many of the more acidic polypeptides failed to enter the IEF gel. Many more were detected following use of the predominantly pH 4-6 Ampho- line gel (Fig. 2). This can be attributed to the pH gradients generated by the two different Ampholine mixtures after IEF. The technique of gel slicing was used to measure the pH gradients generated. This indicated that the anode end of the broad-range Ampholine gel (Fig. 1) was approximately at pH 5.0, whereas that of theother gel(Fig. 2)was at approximately pH 4.3. Consequently, polypeptide spots with isoelectric points between these two values would appear in the second dimension only when the latter IEF gel formed the first dimen- sion. The difference between the two levels of anode pH cor- responded to approximately one-third of the length of the more acid IEF gel. This was the part of the gel giving rise to many additional spots in the second dimension.

Silver staining using methylamine with overstaining and destaining [91 proved to be a highly sensitive means of detec- ting the two-dimensional polypeptide patterns. The destaining procedure was greatly improved by the modification of the proportions in the mixture containing the two destaining solu- tions “A” and “B” previously described [Sl. This gave rapid and effective removal of the non-specific background stain without excessive loss of polypeptide staining and allowed the destaining procedure to be more finely controlled. The clarity of the two-dimensional patterns, following silver staining, was also greatly improved by the omission of mercaptoethanol from the solution used to equilibrate first-dimension gels prior to SDS electrophoresis [91. This did not alter the pattern ofthe polypeptide spots.

It is unlikely that the differences in intensity detected in the polypeptide patterns of the two cell lines were due to the ex- pression of viral protein, since many differences were found. Furthermore, apart from differences in intensity, the distribu- tion patterns of the spots from the two cell lines were identical. This indicates that the same polypeptides were present in each cell extract. This is very interesting in relation to the question of the possible production of a specific protein or proteins by a cancer cell line. When considering the large differences in the intensity of some ofthe PyY polypeptide spots relative to those of BHK it is evident that, at an appropriate dilution, the BHK polypeptides would not be detected and so give rise to the illu- sion of specificity. This concentration effect might easily ac- count for the apparent specificity of a few polypeptide spots found in carcinoma of the colon [ 131 and, as the authors point out, for those in childhood leukemia [ 141. Our own findings do not point to the presence of specific polypeptides and confirm, with greater certainty, those of Bravo and Celis 141. If cancer of the bladder does result from a point mutation of the protein- coding sequence of an oncogene [ 15,161, theonlydifferencein the protein produced would be the substitution of a valine

residue for a single glycine residue. Such a small change in mo- lecular weight, without a change in charge, as well as the single amino acid changes resulting from other tumour oncogenes, would result in a difference not detectable by high resolution two-dimensional electrophoresis.

Our findings do not indicate whether any of the concentration differences we have found are related to membrane structure. This question could be answered by membrane preparation. Trypsin treatment of the whole cells prior to extraction might also possibly clarify the situation. Such work is currently in progress. Finally, our observations indicate that any in- vestigation involving two-dimensional electrophoresis of tissue extracts or body fluids should, where necessary, include three separations in the first-dimension gel designed to demon- strate the more basic and acidic polypeptides in addition to those of intermediate PI. Failure to do this would lower the value of the investigation and even lead to incorrect conclu- sions.

We wish to thank Moira Gambie for technicalassistance. This work was supported by a grant to one of us (A.L.L.) from the North of England Cancer Research Campaign.

Received February 7, 1983

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