Glucose 6-phosphate dehydrogenase isoenzymes in cultured human cell lines: Separation by isoelectric focusing

ANALYTICAL BIOCHEMISTRY 101, 78-87 (1980)

Glucose 6-Phosphate Dehydrogenase lsoenzymes in Cultured Human Cell Lines: Separation by Isoelectric Focusing

LEON HUNTER

Cell Culture Laboratory, University of California, School of Public Health, Naval Biosciences Laboratory, Oakland, California 94625

Received May 21, 1979

Isoelectric focusing in acrylamide gel slabs has been used to separate glucose 6-phosphate dehydrogenase isoenzymes in human cell lines. The pattern of four to six bands has been found to vary both in band position (isoelectric point) and in relative intensity. These differences can be used to characterize and distinguish different cell lines, both from different donors and from different tissues from the same donor. The method should provide a much-needed supplement to the limited number of techniques currently available for monitoring changes in cell cultures.

The separation of glucose 6-phosphate dehydrogenase (G6PD)’ isoenzymes by conventional gel electrophoresis produces rela- tively simple banding patterns which never- theless have proved extremely useful in cell characterization. For example, human cell lines give one of two single band types, the so-called “A” or “B” isoenzymes. The fact that the ubiquitous HeLa line possesses the A type and that the latter is substantially less prevalent in human populations than B has provided useful supporting evidence for the presence or absence of HeLa contami- nation in a variety of suspect cell cultures (1,2).

However the deceptive simplicity of this A/B classification is seen to be suspect when one carries out electrophoresis under carefully controlled, reproducible conditions. Then it becomes evident that the B isoenzyme at least is not a single entity but rather is seen to shift slightly but repro- ducibly in position and bandwidth around the so-called B position with different cell lines. The reason for this variability be-

r Abbreviations used: G6PD, glucose 6-phosphate dehydrogenase; IEF, isoelectric focusing.

comes evident when cell homogenates are examined by isoelectric focusing (IEF) as described in the present paper.

IEF is a powerful technique that separates proteins in a pH gradient on the basis of their isoelectric points. In the most com- monly used method samples are focused in individual tubes of acrylamide gel which are subsequently stained appropriately to locate the proteins of interest. This technique has been used by Turner and Ceder- baum (3) to demonstrate the complexity of G6PD isoenzymes. While it has the ad- vantage of minimizing band distortions it is difficult to make a close comparison of and detect subtle differences in related samples. Such advantages are possible when samples are run side by side in a single gel slab. Kaloustian et al. (4) have described slab electrofocusing experiments for G6PD in human, rat, and rabbit erythrocytes. Un- fortunately this technique has its own problems not the least of which is a tendency toward pH gradient drift and the resulting occurrence of protein band instabilities and distortions. In a recent paper (5) I suggested one method of minimizing such cathodic drift problems. In the present paper I would

0003-2697/80/010078-10$02.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.

78

HUMAN GLUCOSE 6-PHOSPHATE DEHYDROGENASE ISOENZYME SEPARATION 79

like to report some of the successful results of applying this technique to the separation of G6PD isoenzymes in human cell lines.

MATERIALS AND METHODS

Materials

Cell lines. Cells were grown as monolayers in 75ml flasks and incubated at 37°C in 20 ml medium (Dulbecco’s modification of Fsgle’s minimum essential medium, Gibco, Grand Island, N. Y.) supplemented with 10% fetal bovine serum. No antibiotics were used. Confluent monolayers were trypsinized and the cells collected by cen- trifugation (225s for 10 min). The cell pellets were stored at -70°C. Whole cell homogenates were prepared by freeze- thawing (three times) a 1:3 dilution of the pellets in distilled water and storing at -70°C until required. Homogenates could be stored for extended periods with virtually no change in the G6PD band pattern. A tabu- lation of the cell lines used in this study is given in Table 1.

Reagents. Acrylamide and N,N’-methy- lenebisacrylamide were supplied by East- man Kodak (Rochester, N. Y.), riboflavin by Sigma Chemical Company (St. Louis, MO.). pHisolyte ampholytes (Brinkmann Instruments, Westbay, N. Y.) were used. G6PD stain components (NADP, phenazine methosulfate, nitroblue and MTT tetrazolium, glucose 6-phosphate) were obtained from Sigma Chemical Company.

Equipment. A modified (see below) Ortec (Oak Ridge, Tenn.) 4200 electrophoresis system was used for electrofocusing. The constant-voltage/constant-current power supply was a Buchler (Fort Lee, N. J.) Model 3-l 155.

Methods

(I) Isoelectric focusing. The basic aim was to use the simplest possible procedure and gel formulation in order to eliminate or at least minimize outside influences on

the enzyme. A typical gel contained mono- mers (acrylamide (5.88% w/v), N,N’-methy- lenebisacrylamide (0.12% w/v)), riboflavin catalyst (1 x 10e5% w/v), and pH gradient- forming ampholytes (2% w/v). All ingredi- ents except the catalyst were dissolved in distilled water and degassed. The catalyst in distilled water (0.004% w-v) was added and the mixture poured into the Ortec glass cell in sufficient quantity to form in one operation the 3-mm-thick gel slab and the 12 sample wells, the latter formed by a Teflon insert. Polymerization was initiated by irradiation with a uv or fluorescent light source.

Sample mixtures consisting of the cell homogenate (5-62 ~1, depending on enzyme activity), NADP (0.03 pmol in 5 ~1 H,O), 80% aqueous sucrose (12.5 pl), and water (to make 80 ~1 total volume) were incubated at 37°C for 60 min. They were carefully transferred to the sample wells and capped with a gel monomer mix to form a 3- to S-mm layer above the wells. Capping formulation was similar to that above except that con- centration was usually raised to 7% to en- sure a firmer, tighter seal at the top of the gel. The cap was layered with degassed distilled water and light polymerized. During this final polymerization it was helpful to screen the samples from the uv light with aluminum foil.

The isoelectric focusing technique has been previously described (5). Briefly, electrofocusing was conducted in an Ortec electrophoresis chamber. The anolyte in the lower chamber was dilute sulfuric acid (0.05-o. 1 N). The conventional Ortec upper electrolyte arrangement was dispensed with. Instead, after removing the water cap a thin layer (l-2 mm) of cotton (for example, Telfa nonadherent strip) was placed on the exposed upper surface of the gel and wetted with a 2% aqueous solution of the same ampholytes used in the slab. The electrical circuit was completed with a platinum wire cathode, mounted along the bottom edge of a weighted Teflon block, in contact with,

TABLE 1

SUMMARY OF~SOELECTRICALLY FOCUSEDCELL LINES

Donor

Designation Passage No.” Age Sex Tissue source Pathology Morphology

Hs88 T 12 Hs350 T 11 Hs392 SK 13 Hs414 T 12 Hs439 T 13 Hs456 BT 11 Hs4.56 SK 7 Hs479 T 10 Hs484 T 13 Hs578 T 40 Hs.578 T 53 Hs578 T 54 Hs578 Bst 8 Hs588 T 8 Hs597 T 8 Hs600 T 6

Hs674 T 17 Hs674 Sk 10 Hs746 T 18 Hs746 T 20 Hs750 T 12 Hs767 B 1 25 Hs888 Sk 5 Hs913 T(E) 14 Murphy 25 MBA9812 18 Co1016 5

DET.562 54 ME180 73 P,J 11 P,HR 7 S W527’ 8 SW527e 26

HeLa lines (I) Known strains

CCL2 118 HeLaS3K 7 HeLaS3 16 RT-4 5 HEp2 360 HEp2 414 HEp2 419 HEp2 502 HEp2 507

(II) HeLa contaminant Gil 10 + 12 HBT, 38 2563 6 MA160 110 CLL74 9

25 55 46 13 67 80 80 53 83 74 74 74 74 29 - -

63 63 74 74 74 45 20 51 - 67 59

- - - - - -

-

*

F F M M M F F F F F F F F F Fb Mb

M M M M M M M M Mb M F

- F M M - -

Connect. tissue-arm Breast Skin-Arm/Shoulder Lymph node Testis Skin Skin Breast Urinary bladder Breast Breast Breast Breast Cervix Skin

-

Rectum Skin Connect. tissue-leg Connect. tissue-Ieg Lung Bladder Skin Lung Bone Lung Metastasis from leg

tumor Pleural fluid Cervix Lymph node Lymph node

-

F Cervix

*

Cancerous Cancerous Normal Cancerous Cancerous Abnormal Norma! Cancerous Cancerous Cancerous Cancerous Cancerous Normal Cancerous Cancerous Cancerous

(melanoma) Cancerous

Normal Cancerous Cancerous Cancerous Normal Normal Cancerous Cancerous Cancerous Cancerous

Fibroblast - -

Mixed - - -

Fibroblast -

Epithelial Mixed Mixed Fibroblast

- - -

Mixed -

Transformed Transformed Epithelial Epithelial Fibroblast

- Fibroblast Epithelial Epithelial

Cancerous - Cancerous Epithelial Cancerous Lymphoid Cancerous Lymphoid

- Epithelial - Epithelial

Cancerous

‘I In our laboratory. b By karyology in tumor cells (and related skin cells, not part of this study). c The origin of this cell line (tissue and donor) is not verified.

80

HUMAN GLUCOSE &PHOSPHATE DEHYDROGENASE ISOENZYME SEPARATION 81

SW527

p8 ~26 p8 ~26

QI,,,;* * ,’ - .’

Hs7467

pl8 ~20 ~16 p20

- Lll “‘- -

HcLaS3K

P?

Us484T

PI3

i. .,i\ i b.2 L.._J

E IEF E IEF E IEF E IEF

FIG. 1. Comparison of electrophoretic and isoelectric focused G6PD band patterns from human cell lines. IEF conditions vary with different cell lines; electrophoresis conditions as in Fig. 5.

and running the full length of, the cotton distilled H,O), MTTlnitroblue tetrazolium strip. The complete apparatus was installed mixture (6) (6 mg of each in 1.5 ml distilled in a refrigerator to prevent overheating. H,O), and distilled water to give a total of Focusing was carried out at a constant 40 ml. Staining (at 37°C in subdued light) voltage in the range SO-200 V and took generally took 30-90 min at which time the a minimum of 12 h (generally overnight). slab was washed free of excess staining Some runs were extended to 2 days. mixture with repeated water changes.

(II) Measurement ofpH gradient. The gel slab was removed from the Ortec cell and the pH gradient measured by one of two methods: (a) Sections (1 cm) of a 3- to S-mm- wide vertical strip, cut from the center of the slab, were soaked in 5 ml H,O for 2-3 h under nitrogen and the pH of the resulting solution was measured with conventional electrodes; or (b) direct pH measurements were made on the intact gel slab at l-cm intervals using a microelectrode (MI-410 microcombination probe, Microelectrodes Inc., Londonderry, N. H.).

RESULTS AND DISCUSSION

Comparison of Electrophoretic and ZEF Results

(ZZZ) Gt5PD stain. A conventional G6PD- specific blue formazan staining mixture was formulated from 0.2 M Tris buffer, pH 8.0 (20 ml), NADP (7.5 mg in 1.5 ml distilled H,O), 0.1 M MgCl, (0.4 ml), glucose 6- phosphate (38 mg in 2 ml distilled H,O), phenazine methosulfate (1.5 mg in 0.4 ml

Hitherto electrophoresis has been used to classify human G6PD into one of two types on the basis of band mobility in starch or acrylamide gels, the “fast” A type or the “slow” B type (plus of course the AB com- posite that results from random X-chromo- some suppression in a heterozygous fe- male). Although the variable mobility of the B isoenzyme from different cell lines has led to the introduction of a number of B sub- classes this A/B classification is still widely used.

A selection of electrophoretic and IEF band patterns from a variety of cell lines is shown in Fig. 1. It is clear that while each

LEON HUNTER

&8

8.8

FIG. 2. IEF band patterns for G6PD isoenzymes in human cell lines. Conditions: 6% gel; 2% pHisolyte 2- 11; 100 V for 18 h; pH measured on water extract from excized (1 cm) gel sections.

cell line produces the expected single electrophoretic A or B band it can be shown by IEF to contain at least four to six separate G6PD bands, confirming the observations of Turner and Cederbaum (3) and Kaloustian et at. (4). Some of the implications of these results, with regard to the fundamental nature of the enzyme complex but primarily from the practical aspect of cell characterization, will be discussed below.

Comparison of Relative Band Intensity Patterns

Figure 2 shows the IEF patterns from a representative selection of human cell lines. The general picture is a pattern of from three to six fairly evenly spaced bands in similar if not identical positions in the pH gradient from sample to sample. However it is evident that there is a wide variation in the relative intensity of the bands within a pattern. Thus even although all these cell lines with the exception of HeLaS3K (fifth from the left) are B types they could in most cases be readily distinguished from one

another on the basis of these G6PD band patterns (especially with densitometric scanning).

Of critical importance to the utilization of this technique for cell characterization is the stability of the banding patterns. We have been able to compare patterns of a number of lines at varying passage levels with the following results.

(I) With long-established lines cultivated in a well-defined, unchanging manner the band pattern is remarkably stable. Figure 3 shows a series of HEp2 samples ranging from passage 360 to passage 507. The band patterns remain essentially unchanged over this extended series of cultures.

(II) With long-established lines that have branched and been subjected, sometimes unwittingly, to widely different growing conditions, differences and similarities can be detected. A prime example of course is HeLa which has frequently displaced quite different cell lines and continued to be cultivated under conditions appropriate to these other lines. A comparison of a selection of HeLa or suspect HeLa lines is shown in Fig. 4. A common feature of these sam-


p.360

ate@

p.414

v..,-.-

p.419 p.502 p. 507

0 be .a - __

FIG. 3. IEF comparison of G6PD band patterns at different passage levels for cell line H.Ep2. Conditions: 6% gel; 3% pHisolyte 2-11; 100 V for 17.5 h.

ples is the presence of band 4 (counting from duced a major band 4. However despite the top) as a major band. HeLa is an A-type this similarity it is evident that these HeLa line. No B-type cell line to date has pro- lines can be divided into groups on the basis

GII Hf3T3 2563 Mm0 cLL74 b!eLas3 HCLOSW

.-. . .; -*

‘.

FIG. 4. IEF comparison of HeLa or HeLa contaminant G6PD band patterns. Conditions: 6% gel; 2% pHisolyte mix containing 75% pH 6-8 and 25% pH 2-l 1; 100 V for 17.5 h.

84 LEON HUNTER

of their relative band intensity patterns, namely, 2563 and CLL74, whose bands 2,3, and 4 are of almost equal intensity, and Gil and HeLaS3K, in which band 4 is substantially more intense than band 2 or 3. (HBT3, MA160, and HeLaS3 appear to form an intermediate group with band 2 and 3 intensities slightly less than that of band 4.) Further differences can be detected in the pattern of the minor bands below band 4, and in the presence and nature of (minor) band 1, e.g., present as a very faint doublet (barely visible in photo) in HeLaS3K. In summary it appears that the prolonged separation of these various HeLa strains has lead to divergences in their G6PD band patterns which can be picked up by our IEF technique even when they are all classed as type A by conventional electrophoresis.

(III) Shorter-term cultures seem to be consistent at least when nearby passages are compared (see Fig. 1, Hs746T). How- ever, differences may show up with continued culture (see Fig. 1, SW527). While such differences might seem to be un- desirable from the point of view of cell characterization they point to the fact that changes in cell population composition and/ or individual cell characteristics are taking place and that therefore it is misleading to assume, even if only tacitly (as in many studies utilizing cell lines), that a static cell line is the norm. The G6PD patterns re- vealed by IEF (and presumably those of other enzymes) would appear to constitute an important monitoring system for cell line stability.

IEF Comparison of A- and B-Type Cell Lines

Electrophoretic A and B classification of human G6PD isoenzymes is based on slight differences in mobility. It was of interest to see whether characteristic differences persisted after the more effective separation achieved by IEF. Figure 5 (upper half) shows typical electrophoresis bands in a

ISOELECTRIC FOCUSSING

FIG. 5. Comparison of A-type (left side) and B-type (right side) G6PD cell lines by electrophoresis and isoelectric focusing. Electrophoresis conditions: 5% persulfate-catalyzed gel containing 0.1 M Tris-HCI buffer, pH 8.5; cathode buffer, 0.38 M Tris-glycine, pH 8.5; anode buffer, 0.1 M Tris-HCI, pH 8.5; 325 V for 1 h (approx). Isoelectric focusing conditions: 6% gel; 1% pHisolyte 6-8, 1% pHisolyte 5-7: 100 V for 17 h.

representative selection of A (left) and B (right) cell lines. Figure 5 (lower half) shows the results of electrofocusing the same samples. All the samples have similar or closely related series of bands. In some cases the bands appear to be displaced slightly relative to other samples. This is difficult to detect with certainty unless the samples are run side by side on the same gel slab. How- ever, this does not seem to be a characteristic difference between A and B cell types.


QH (ffQQfOX.1

1 ?.o

- 6.8

- 6.6

6.4

- 62

- 6.0

” 5.6

FIG. 6. IEF comparison of G6PD band patterns from related tumor and normal cell lines. Hs578 T is derived from a breast carcinosarcoma, Hs578 Bst from normal tissue from the same subject. Conditions: 6% gel; 3% pHisolyte mix containing 75% pH 6-8 and 25% pH Z- 11; 100 V for 19 h. Direct pH measurement by microelectrode.

(See below for further discussion.) The common factor that seems to distinguish A from B is the presence of band 4 (counting from the top) as a major band. In some cases (for example in some HeLa lines) it is rhe major band while in other A lines it may be only one of several strong bands. How- ever, in no case have B-type cells been found to possess other than a very minor band 4. The resulting shift in the “center of gravity” of the band patterns and hence their isoelectric points is in accord with the greater electrophoretic mobility of the so-called A isoenzyme since the components of the latter would have a lower over- all isoelectric point and hence a greater negative charge under the usual electrophoretic conditions. (Similarly the variation in band intensity patterns, and therefore the relative amounts of the enzyme variants, within a cell type (say B), is presumably

the reason for the slight shifting in the electrophoretic band position in different cell lines.)

Comparison of Normal and Tumor Cell Lines

So far we have discussed only unrelated cell lines. It is of very considerable interest to compare band patterns in related lines from different tissue sources in the same subject. Our results show that differences are present and can be quite clearly detected. One example, shown in Fig. 6, is the normal breast cell line Hs578 Bst and the breast tumor cell line Hs578 T, at three different passages, from the same subject. The tumor samples are all similar in having a band 3 that is much weaker than the other two major bands (1 and 2) (differences in band 3 intensities among the tumor samples are probably due to variations in sample concentrations). On the other hand all three bands in the normal cell line are nearly equal in intensity. Furthermore all three bands in the latter sample show a slight but distinct displacement. This subtle difference is not unique to the present samples but in fact can serve as a further point of distinction between both related and unrelated samples. Several examples are shown in Fig. 7. ME180 and the sample to the left, CCL2, are both displaced relative to the adjacent sample Hs456 SK. Also, in another, related pair the tumor line Hs674 T (a carcinoma of the rectum) is displaced relative to the normal skin line Hs647 Sk (in addition to distinct differences in relative band intensities).

These differences in related tumor and normal cell lines are of great interest and presumably reflect metabolic differences in the cells. It is important to note that related lines do not always show such differences. However it is often difficult to assess the tumorogenicity of an established cell line even when it has originated from a genuine tumor. Thus it is quite possible that the

86 LEON HUNTER

F3HR P3J Hs350T Hs473T Hs597T HsGOOT CCL2 MEtF30 Hs456Sk Hs456BT Hs674T Us674SX P7 Pfl PII PI0 P8 ~6 PI16 P73 P7 PII Pi7 PI0

- 6.4

- 6.2

- 6.0

- 5.8

- 5.6

- 5.4

- 5.2 - 5.0

FIG. 7. Representative G6PD isoenzyme band patterns from human cell lines by IEF. See Table 1 for cell line derivations and text for discussion. Conditions: 6% gel; 3% pHisolyte mix containing 75% pH 6-8 and 25% pH 2-11; 100 V for 19 h. Direct pH measurement by microelectrode.

surviving cells in a so-called ‘tumor’ cell line are as ‘normal’ as the related ‘normal’ line. In this regard the two series discussed above (Hs674 and HsS78) were selected on the basis of their well characterized tumoro- genie properties (7-9) which clearly distinguished them from their ‘normal’ partner. At the present time further work is under- way in this interesting area with related lines from tumors and a variety of body tissues.

Unusual Banding Patterns

Most of the cell lines examined by our IEF technique have shown the same kind of regularly spaced band patterns. However, in a few cases a completely different pattern of closely spaced bands at a lower pH has been observed. Two examples are shown on the left side of the gel slab in Fig. 7. Two or three other cell lines that have shown this unusual pattern have, perhaps significantly, also been derived like P3J and P3HR from lymphoid tissue, both neoplastic (EB3)

and “normal” (NC37). Whether this pattern is unique to lymphoid cells, however, can- not be stated with certainty until more cell lines have been examined. It is significant that a similar pattern of “hyperanodic,” closely spaced bands has been obtained from leukemic white cell extracts by Kahn and his coworkers (10) who have carried out extensive investigations of G6PD components in human blood cells.

The above data illustrate the wide diver- sity with regard to position and relative intensity of G6PD banding patterns from human cell lines and are primarily intended to demonstrate the usefulness of the technique in providing an additional, much- needed tool for characterizing and dif- ferentiating between the many cell lines used in biological and medical research. Beyond this practical aspect, however, are the many fundamental questions that the presence of so many bands in such a variety of intensity variations raises with regard to their nature and their structural inter- relationships. It is obvious that the situa-


tion is considerably more complex than predicted by the simple existence of “A” and “B” isoenzymes. The derivation of these complex band patterns has been discussed inconclusively by Turner and Ceder- baum (3) and by Kaloustian et al. (4). As mentioned above Kahn and his coworkers (10,ll) have investigated G6PD band patterns in a variety of human blood cells, both normal and pathological. Their work has developed useful explanations (12) for the interconversion of one band pattern system to another, involving limited pro- teolysis at the C-terminal end of the G6PD subunit chain. However, the derivation of the individual bands within a pattern is still uncertain at this time.

Possibilities include post-translational changes with additional protein and/or car- bohydrate complexing, subunit rearrange- ments, or modification by proteolytic enzymes. With regard to the latter, tests with trypsin (unpublished data) have shown that all our G6PD bands are completely elimi- nated with the appearance of a new enzy- matically active band at a substantially higher isoelectric point (pH 6.9-7.3). The possibility of artifactual modification during electrofocusing must be kept in mind but certainly seems minimal in view of the mild- ness of our IEF procedure. The obvious first step in the elucidation of this problem is the isolation of purified individual band components for further study. Unfortu- nately to date this has been frustrated by the difficulty of recovering more than a trace of active material from the acrylamide gel slab and the fact that no other, more amenable technique can approach the sepa-

rations achieved by IEF. Work in this area is continuing.

ACKNOWLEDGMENTS

The author would like to express his gratitude for the invaluable assistance of the members of the Cell Culture Laboratory in providing the wide variety of cell lines used in this project. This work was supported by the National Cancer Institute under the auspices of the Office of Naval Research and the Regents of the University of California.

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Nelson-Rees, W. A., and Flandermeyer, R. R. (1976) Science 191, %-98.

Nelson-Rees, W. A., Flandermeyer, R. R., and Hawthorne, P. K. (1974) Science 184, 1093.

Turner, B. J., and Cederbaum, S. D. (1975) in Isozymes I: Molecular Structure (Market& C. L., ed.), pp. 653-660, Academic Press, New York.

4. Kaloustian, V. M. D., Idriss-Daouk, S. H., Hallal, R. T., and Awdeh, Z. L. (1974) Biochem. Genet. 12, 51-58.

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Hunter, L. (1978) Anal. Biochem. 89, 279-283. Melnick, R. 1. (1971)Prog. Histochem. Cytochem.

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Gershwin, M. E., Ikeda, R. M., Kawakami, T. Cl., and Owens, R. B. (1977) J. Nat. Cancer Inst. 58, 1455- 1461.

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Documents

Glucose 6-phosphate dehydrogenase isoenzymes in cultured human cell lines: Separation by isoelectric focusing