Phenotypic conversion of TK-deficient cells following electroporation of functional TK enzyme

EXPERIMENTAL

CELL RESEARCH

198,36-42

(1992)

Phenotypic Conversion of TK-Deficient Cells following Electroporation of Functional TK Enzyme SUE F. DAGHER, SUSANE. CONRAD, ELIZABETH A. WERNER, AND RONALD J. PA?TERSON’ Graduate Program in Genetics and Department of Microbiology, Michigan State University, East Lansing, Michigan 48824-l 101

rescent dextrans [ 71into cultured cells using electroporation. In one case, a relatively crude extract from normal human cells was introduced into xeroderma pigmentosum cells in order to correct the DNA repair deficiency characteristic of this disease [6]. It is not known what factor(s) in the crude extract was responsible for correcting the mutant phenotype, nor is it easily discernible what percentage of electroporated xeroderma pigmentosum cells were able to perform DNA repair. In this report, we show phenotypic conversion of mutant Rat-3 (thymidine kinase-deficient; [8] ) cells following electroporation of functional TK enzyme. TK has many advantages as a model enzyme for these studies; (i) stable TK- cell lines with low background levels of activity exist, (ii) cell extracts can be assayed for TK activity, (iii) incorporation of radiolabeled thymidine into DNA can be quantitated when TK is present in the cytosol, (iv) in situ autoradiography can be used to quantitate the percentage of cells containing functional enzyme, and (v) enzymatically active recombinant TK (rTK) has been expressed in bacteria and partially purified and is available in large quantities.

The ability to phenotypically rescue a mutant (Rat-3, thymidine kinase-deficient) cell line by electroporation of functional TK enzyme has been investigated. Extracts of electroporated cells showed a 35-fold increase in TK enzyme levels under conditions where >90% of the cells remained viable. The electroporated enzyme was intracellular, as demonstrated by the fact that cells were able to utilize exogenous [‘Hlthymidine for DNA synthesis. By in situ autoradiography, 92% of electroporated cells contained functional enzyme and incorporated [‘Hlthymidine into DNA. Thus, this technique can efficiently provide a missing metabolic function to cultured mammalian Cells. 0 1992 Academic Press. 1~.

INTRODUCTION

The introduction into mammalian cells of biologically active macromolecules has broad applicability, particularly for studying their impact on cellular metabolism. A number of techniques for doing this have been developed (including transfection, liposome-mediated fusion, microinjection and electroporation), each with its own advantages and disadvantages [ 11.Electroporation has the advantage that it is rapid, and large numbers of cells can be simultaneously exposed to an electric field. Electroporation is a balance between the ability to build up a breakdown potential across a membrane, creating “pores” large enough to allow the entry of macromolecules, and the ability of the membrane to reseal before excessive leakage from the cell’s interior takes place. Under appropriate conditions, a high percentage of cells remain viable and efficiently take up macromolecules. Electroporation has been most frequently used to introduce exogenous DNA into a wide variety of cells as an alternative to other transfection techniques [2]. Theoretically, electroporation should also be an effective technique for introducing proteins and other macromolecules into cells. In fact, several reports have shown uptake of antibodies [ 31,enzymes [4-61, and fluo’ To whom reprint 8957.

requests

should be addressed.

0014-4827/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

MATERIALS

AND METHODS

Cells. Rat-l (TK+; [9]) and Rat-3 (TK-) cells were maintained in Dulbecco’s modified Eagle’s medium (DME) supplemented with 10% calf serum. Prior to electroporation, confluent monolayers were arrested by serum starvation (0.1% serum) for 24 h and serum stimulated by the addition of fresh medium containing 10% calf serum for 8 h. Ekctroporation. Various buffers (21 mA4 Hepes, pH 8.0, phosphate-buffered saline [PBS], Opti-MEM [GIBCO, Grant Island, NY], and MEM [GIBCO]) with and without sucrose and/or calf serum were tested for use in electroporation. Opti-MEM supplemented with 0.3 M sucrose and 10% calf serum consistently yielded high cell viability and efficient uptake and was used as the standard electroporation buffer (EB). Eight hours after serum stimulation, cells were trypsinized, washed one time in PBS and one time in DME with serum, and resuspended at a density of 2-9 X 10’ cells/ml in EB. Fluorescent-dextran (FITC-dextran), rTK enzyme (see below), or buffer was added immediately before electroporation to 0.4 ml of cell suspension in a 0.4-cm (0.4-cm electrode gap) disposable Bio-Rad cuvette. The cuvette was chilled and then exposed to a single pulse (field strength 825 V/cm, capacitance 250 pF, which gave a pulse duration of 13.5-15.5 ma) using a Bio-Rad gene pulser. Immediately follow-

Fax: (517)353-

36

CONVERSION

OF TK-

PHENOTYPE

ing the pulse, the cuvette was placed on ice for lo-15 min, then the cells were washed twice in EB. Electroporated cells were plated on coverslips (fluorescent microscopy), microscope slides (in situ autoradiography), or tissue culture plates in DME plus 10% calf serum. Cell viability was determined by trypan blue exclusion and fluorescein diacetate fluorescence [lo] immediately after electroporation, and cell counts were determined at various times after plating. Electroporation efficiency was determined by fluorescent microscopy or assaying for TK enzymatic activity. Production of recombinant TK. A truncated form of human TK protein was produced in Escherichiu coli in the following way. A 1125bp SmaI-BamHl fragment from within the human TK cDNA [ll, 121 was cloned into the E. coli expression vector pCPll0 (a gift of Nikos Panayotatos; [13]). This vector contains a lactose inducible (lac uv5) promoter, a ribosome binding site from bacteriophage T7, and an AUG translation initiation codon. pCPll0 DNA was digested with Sal1 and the ends were filled in with Klenow polymerase. The SmaIBamHl TK fragment was then inserted into the vector. The resulting construct (pCPllO-TK) encodes a TK protein that has the first 16 amino acids of the TK enzyme deleted and 3 amino acids (Met-SerArg) inserted from the vector. We have previously shown that a similar truncated protein is enzymatically active in mammalian cells [ 121. E. coli W3110 (iq, L8) containing pCPllO-TK were grown to late log phase in LB and induced with 1% lactose for 4 h. Cells were pelleted, resuspended in buffer A (10 m&4 Tris, pH 7.0,1.5 n&f MgCl, ,3.0 m&f L-mercaptoethanol, 0.1 m&f phenylmethylsulfonyl fluoride [PMSF], 250 mA4 sucrose), and disrupted in a French press at 18,000 psi. Membranes were pelleted by centrifugation at 20,000 rpm for 90 min in an SW27 rotor (Beckman, Fullerton, CA). The supernatant was dialyzed against buffer A, and the TK protein was partially purified by carboxymethyl-Sepharose (CM-Sepharose) chromatography using buffer B (10 m&f Tris, pH 8.0,1.5 n&f MgCl,, 3.0 mM 2-mercaptoethanol, 100 mM KCl, 0.1 mM PMSF, 250 mM sucrose) for elution as previously described [14]. Fractions were assayed for TK both by enzyme assays [15-171 and by SDS-polyacrylamide gel electrophoresis, and those fractions containing the highest TK activity were pooled and concentrated by Amicon YMlO filtration. The rTK is enxymatically active and stable for at least 6 months when stored in elution buffer at -70°C. Purity of the enzyme was determined by laser densitometry of Coomassie blue-stained gels. Assays for TK enzymatic actiuity. The enzymatic activity of rTK assayed either during purification from bacteria or in lysates of electroporated cells is given in units. One unit is the amount of enzyme required to convert 1 pmol of thymidine to 1 pmol of dTMP in 30 min at 37°C under standard reaction conditions [15-171. TK activity in cell lysates of electroporated cells was assayed as described previously [15-171. cells were plated on glass In situ autoradiography. Electroporated slides in DME plus 10% calf serum. One hour after plating, [3H]thymidine (5 pCi/ml, 10 ml/plate containing three slides) was added for 2 h. The slides were washed three times in cold PBS and fixed by immersion one time in cold 5% trichloroacetic acid and one time in 100% ethanol. Slides were dried, then coated with Kodak NTB-2 NUclear Track emulsion, and exposed for 6 days.

RESULTS

Optimal electroporation conditions have been shown to vary from cell line to cell line and to depend on a number of factors including buffer, voltage, capacitance, pulse time, and growth state of the cell. Since we wanted to monitor the capacity of an introduced enzyme to rescue a deficient phenotype, it was crucial to achieve high cell viability and a high percentage of cells contain-

BY ENZYME

ELECTROPORATION

37

ing the introduced enzyme. We therefore tested a number of previously published conditions [3-61 and modifications thereof. The uptake of macromolecules was measured using FITC-dextran and cell viability was determined by trypan blue exclusion, fluorescein diacetate fluorescence, or cell counts up to 3 h after electroporation. In our experiments, electroporation in PBS or Hepes buffer, alone or supplemented with 0.3 M sucrose, resulted in low cell viability (<70%), low uptake (<60%), or both. Chakrabarti et al. [3] previously showed efficient (approaching 100%) uptake of antibodies while maintaining cell viability of 80-90% using Opti-MEM plus serum. We therefore tested two tissue culture media, MEM and Opti-MEM, supplemented with 10% calf serum. Both resulted in >90% cell viability, but only 30-40% of the cells took up FITC-dextran. The reasons for the disparity in these results are unknown but may be due to the use of different cell lines or electroporation apparati. When sucrose was added to either MEM/calf serum or Opti-MEM/calf serum, high cell viability (~85%) was maintained, and uptake was increased two- to threefold to >90%. The difference between MEM and Opti-MEM was slight, with OptiMEM yielding a higher cell viability. We thus chose Opti-MEM, 10% calf serum, and 0.3 A4 sucrose as an electroporation buffer. We also optimized the voltage and capacitance used for electroporation. When Rat-3 cells were exposed to an electrical field less than 825 V/cm, viability approached 100% but less than 70% of the cells showed uptake of FITC-dextran. Conversely, voltages greater than 825 V/cm showed excellent FITC-dextran uptake (>90% of the cells), but viability was less than 70%. Additionally, high voltages resulted in marked expansion of cells with numerous “blebs” at the cell periphery. Optimal uptake and minimal cell death occurred at 825 V/cm, capacitance 250 PF, and pulse time 13.5-15.5 ms. Exposure of Rat-3 cells to an electrical field under these conditions resulted in the efficient transfer of FITC-dextran, as demonstrated by the uniformly fluorescent cytoplasms and dark nuclei seen in Fig. 1D. In five independent experiments, an average of 90% of electroporated cells took up FITC-dextran while maintaining cell viabilities of 82-96%. The cells were nonfluorescent when electroporated in the absence of FITCdextran (Fig. 1E) or when FITC-dextran was added 15 min after the electrical pulse (Fig. 1F). Figures lA, lB, and 1C show the same fields of cells viewed by phase contrast microscopy. Having established the conditions for efficient uptake of FITC-dextran, we investigated the electroporation of functional TK enzyme into TK- cells. TK enzyme was expressed in E. coli as described under Materials and Methods. rTK enzyme was purified to near homogeneity by sequential chromatography on CM-Sepharose and thymidine-Sepharose as previously described [ 141.

DAGHER

ET AL.

FIG. 1. Electroporation of FITC-dextran into Rat-3 cells. Rapidly growing Rat-3 cells were trypsinized and washed once in PBS and once (1.2 mg) was added immediately before in DME + 10% calf serum. Cells were resuspended at a density of 1 X lo7 cells/ml in EB. FITC-dextran electroporation. Conditions for electroporation are as described under Materials and Methods. The presence of FITC-dextran in the cells was examined by fluorescent microscopy. A and D, electroporation in the presence of FITC-dextran; B and E, electroporation in the absence of FITC-dextran; C and F, FITC-dextran added 15 min following electroporation. A-C, phase contrast microscopy; D-F, fluorescent microscopy.

As shown in Fig. 2, the enzyme is highly purified after elution from CM-Sepharose and essentially homogeneous after elution from thymidine-Sepharose. Since

the capacity of the thymidine-Sepharose column was limited, and we required large quantities of TK for electroporation experiments, the partially purified enzyme

CONVERSION

OF TK-

PHENOTYPE

123456M 69 46

30 rTKj 21 14

FIG. 2. Purity of rTK isolated from E. coli. rTK in lysates prepared from pCPllO-TK was purified by sequential chromatography on CM-Sepbarose and tbymidine-Sepharose as described in the text. Lanes 1 and 2, fractions from the CM-Sepharose column; lanes 3-6, fractions from the thymidine-Sepharose column; lane M, size markers from top to bottom are 69, 46, 30, 21, and 14 kDa. Arrow indicates rTK.

obtained by CM-Sepharose chromatography was used for these studies. Pooled and concentrated fractions from CM-Sepharose contained 12.8 mg total protein/ ml with a specific activity of 3.9 units of TK/mg protein. The purity of the rTK was approximately 70%. To ensure that exposure to an electric field had no effect on enzyme function, the activity of various concentrations of rTK was compared before and after electroporation under our standard conditions in the absence of cells. The activities of electroporated and nonelectroporated rTK were within 520% of each other (data not shown). The half-life of TK protein reportedly varies during the cell cycle, with the enzyme being stable throughout most of the cycle but rapidly degraded soon after mitosis [la]. To be certain that the protein was introduced into cells at a time when it would be relatively stable, the following protocol was used. Cells were grown to confluence and serum starved for 24 h to synchronize them in G,. At t = 0, fresh medium plus 10% serum was added, and cells were harvested for electroporation 8 h later. At this point, the majority of cells are in late G,/early S phase (data not shown) where the TK enzyme is expected to be stable. To determine the level of electroporated rTK activity detectable in cell lysates, increasing concentrations of rTK were electroporated into a constant number of Rat-3 TK cells. Electroporated cells were plated at 37°C for 3 h, and lysates were then prepared and assayed for enzyme activity. As shown in Table 1, the amount of activity in extracts was directly proportional to the amount of enzyme electroporated. All further electroporation experiments were conducted with 5 units (1.3 mg protein) of rTK per l-4 X lo7 cells in a final volume of 0.4 ml.

BY ENZYME

39

ELECTROPORATION

The amount of rTK electroporated per cell was calculated for all experiments. Approximately 0.1-l% of the rTK, which represents 0.1-0.6 pglcell, was transferred. This amount varied with both the number of cells and the amount of rTK electroporated. To assess the stability of electroporated rTK, cells were plated after electroporation and TK activity in lysates was assayed at 2-h intervals for 10 h. Cell number and viability did not vary over the duration of these experiments (data not shown). The average TK half-life was 7.3 h (Fig. 3). Whether this half-life would vary in cells electroporated at different stages of the cell cycle is unknown. The data presented above suggest that TK is localized in the cell interior following electroporation. A possible objection to this interpretation is that the TK activity detected in cell extracts following electroporation is due to enzyme adhering to the plasma membrane rather than to intracellular rTK. Since Rat-3 cells cannot phosphorylate thymidine, and since TMP is not transported across the plasma membrane, exogenous [3H]thymidine will be incorporated into cellular DNA only if rTK is intracellular following electroporation. Cells electroporated under our standard conditions should efficiently incorporate [3H]thymidine into cellular DNA, since they are electroporated in late G,/early S phase. Rat-3 cells were electroporated 8 h after serum stimulation, the cells were plated, incubated for 1 h at 37°C and then labeled for 2 h with [3H]thymidine. Incorporation of [3H]thymidine into cellular DNA was assayed by measuring TCA-precipitable counts. Lysates from identical cultures electroporated in parallel were assayed in vitro for TK activity after the 3-h incubation. Two control experiments were performed: (i) electro-

TABLE

1

Dose Response of ITK Electroporated into Rat-3 Cells TK electroporated (units)” None 0.05 0.5 2.5 5.0

TK activity * (X10’)

Fold increase in TK activity ’

2.1 5.1 8.0 44.9 74.5

2.3 3.8 21.4 35.5

a Units electroporated into 7 X 10’ cells. b Cell lysates were prepared 3 h following electroporation and assayed for TK activity as described previously 115-171. Protein concentrations in the extracts were determined by the Bradford assay 1191 using a standard curve of y globulin. Activity is expressed as units of TK per microgram protein in lysate. ’ TK activity in lysates of cells electroporated in the presence of rTK divided by the activity in lysates of cells electroporated in the absence of rTK.

40

DAGHER

ET AL. DISCUSSION

101 2

I 4

I I I I 6 6 10 12 TIME (h post slectmpomtion)

I 14

I

FIG. 3. Stability of rTK electroporated into Rat-3 TK- cells. In three independent experiments, Rat-3 cells were electroporated 8 h after serum stimulation. Cell lysates were prepared 4 h after electroporation and at 2-h intervals thereafter, and TK activity (units/lo6 cells) was determined. Activity at the 2-h intervals is expressed as a percentage of the activity at 4 h after electroporation. The average half-life of electroporated rTK was 7.3 h (line); the actual half-lives in the three experiments were 6.2 (O), 6.8 (Cl), and 9.4 (A) h.

poration of cells in buffer alone and (ii) electroporation of cells in buffer followed by the addition of rTK enzyme after membrane resealing (15 min). As shown in Table 2, cells electroporated in the presence of rTK incorporated 23 times more [3H]thymidine into cellular DNA than control cells. In the parallel experiment, TK activity in lysates of cells electroporated in the presence of rTK was 39 times greater than in cells electroporated in the absence of rTK. These data indicate that most of the electroporated rTK is in an intracellular compartment where phosphorylation of thymidine occurs. To quantitate the percentage of cells containing functional rTK, in situ autoradiography was used. Rat-l (TK+) cells electroporated in the absence of rTK were compared to Rat-3 (TK-) cells electroporated in the absence and presence of rTK. Figure 4 shows representative autoradiograms. Rat-3 (Fig. 4A) and Rat-l (Fig. 4B) cells which incorporated [3H]thymidine into DNA show nuclear localized silver grains. Rat-3 cells electroporated in the absence of rTK showed no [3H]thymidine incorporation (not shown) nor did cells where rTK was added 15 min after electroporation (Fig. 4C). Approximately 57% of Rat-l cells and 47% of Rat-3 cells incorporated [3H]thymidine during the labeling period. Assuming that the percentage of cells in S phase is the same for the two cell lines, we conclude that approximately 82% of the electroporated cells contained functional enzyme.

A number of techniques exist for introducing protein into eukaryotic cells (for review see Ref. [l] ); however, these have various disadvantages. Microinjection, for example, is very time consuming and technically difficult and only a few hundred cells can be injected at a time. Red cell ghosts or liposome-mediated fusion require preparation and loading of vesicles and careful titration to ensure adequate transfer without toxicity. Electroporation offers unique advantages for protein introduction over these other techniques. Its greatest appeal is its relative simplicity. Large populations of cells can be simultaneously and efficiently electroporated (greater than 80% of cells show uptake with 8090% cell viability) to give sufficient quantities of material for biochemical analysis after intracellular residence. Electroporation also provides a high degree of control and reproducibility compared to liposome-mediated transfer or microinjection. A negative aspect is the efficiency of transfer. In our studies, less than 1% of the rTK was transferred to the cellular interior. Lowering the protein concentration of the electroporation buffer (i.e., reducing the percentage of calf serum) might increase rTK uptake but could cause a decrease in cell viability. We have not varied the serum concentration, since the amount of rTK transferred and the cell viability resulted in phenotypic conversion of a high percentage of cells and readily detectable TK activity in lysates. Electroporation of proteins offers an alternative to some of the standard techniques used to investigate protein structure/function relationships. Currently, cDNA cloning coupled with in vitro mutagenesis is widely used. Mutant proteins produced in E. coli or

TABLE Functional

Assays

Electroporation conditions Electroporation minus rTK Electroporation minus rTK, rTK added after 15 min Electroporation plus rTK during electroporation

2

of Electroporated TK activity” (X107)

rTK

Enzyme [3H]Thymidine incorporation *

2.4 (1)’

34 (1)

4.6 (1.9)

29 (0.85)

94.3 (39.2)

789 (23.2)

o Cell lysates were prepared 3 h following electroporation and assayed for TK activity. Activity is described in the legend to Table 1. * Electroporated cells were allowed to attach to plates for 1 b, and then [3H]thymidine (5 pCilml,5 ml per plate) was added for an additional 2 h. Incorporation of thymidine is expressed as TCA-precipitable cpmlrg total cellular protein. c Values in parentheses indicate stimulation relative to control (electroporation minus rTK).

CONVERSION

OF TK-

PHENOTYPE

BY ENZYME

ELECTROPORATION

41

other expression systems have been studied in vitro in either crude or purified systems. Since the activity and/ or regulation of many enzymes and regulatory molecules may be modified by interactions with other cellular components, this approach may not produce a true picture of the functions and domains of the original protein. The alternative of introducing numerous mutated genes into mammalian cells is time- and labor-intensive, especially if stable transformants are required. In addition, appropriate expression systems are not always available. Introducing proteins into cells by electroporation overcomes some of these difficulties and allows one to study a protein in its “native” environment. In cells deficient for a given protein, electroporation of the missing protein could be used to characterize post-translational modifications and structural and functional stability. Even in cells synthesizing a protein, electroporation of labeled or in vitro-modified molecules could permit half-life studies without the necessity of using inhibitors to block protein synthesis. Due to the stability of the electroporated protein (a half-life of 7-8 h in our system), this technique could be used to examine the effect of the introduced protein on the cell’s metabolism. Proteins altered by site-specific mutagenesis could be introduced into cells to assess regions within the protein responsible for biologic function, subcellular compartmentalization, or post-translational modification. The functional properties of molecules such as transcription or differentiation factors could be evaluated following electroporation into appropriate cells. Finally, a transient null phenotype might be obtained by transferring specific antibodies or inhibitors which normally are unable to enter the cell. In spite of its advantages, a number of problems potentially exist with this technique. Recombinant proteins introduced into cells might not be appropriately modified, might not localize to the correct intracellular compartment, or might be rapidly degraded or inactivated in some cell systems. In many cases, however, electroporation offers significant advantages over assaying proteins in vitro and will open new areas of investigation. We have demonstrated at least one of the potential uses of electroporation for protein transfer, the rescue of a defective phenotype using a recombinant enzyme. We were able to detect TK enzymatic activity in cell lysates and show that the electroporated rTK catalyzed incorporation of exogenous [3H]thymidine into cellular DNA in a cell deficient for this activity. The general utility of this technique for the study of other eukaryotic enzymes or proteins awaits further investigation. FIG. 4. In situ autoradiogramsof electroporatedcells. Rat-l and Rat-3 cells were electroporated 8 h after serum stimulation. (A) Rat-3 cells electroporated in the presence of 5 units of rTK, (B) Rat-l cells electroporated in the absence of rTK, (C) Rat-3 cells electroporated in buffer, rTK added 15 min following electroporation.

The authors thank Drs. Walt Esselman, Pat Oriel, and Rich Schwartz for their discussions and critical reading of this manuscript. This work was supported by NIH Grant CA37144 to S.E.C. and funds from the College of Human Medicine at Michigan State University.

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