Radioactive labeling of DNA in cells in culture

ANALYTICAL

BIOCHEMKTRY

79, 95- 103 (1977)

Radioactive Labeling of DNA in Cells in Culture LAWRENCE D. GROUSE AND BRUCE K. SCHRIER Behavioral

Biology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Received April 6, 1976; accepted December 10, 1976

We have compared and evaluated various methods for recovery and deproteinization of DNA from cells in culture and for obtaining labeled DNA at high specific activities with [3H]thymidine. The kinetics of labeling cells in culture with [3H]thymidine were examined using the Chang human liver cell line. The extent of labeling was limited by the presence of the radioactive nuclides in the intracellular pool, and the limitation was associated with cessation of cell multiplication after approximately one doubling. Incorporation of [3H]thymidine was found to be most cost-efficient at low cell densities and at a precursor concentration of 3 $X/ml in the presence of 54uorodeoxyuridine; however, higher precursor concentrations led to greater DNA specific activity. The extent of label incorporation varied with different vertebrate cell lines tested.

Many techniques have been employed to obtain radioactively labeled DNA and RNA from vertebrates. These techniques fall into three groups. (A) Labeling in vivo by injection of radioactive precursors in the animal (1) has the advantage that the nucleic acid subsequently isolated should be “normal” in every characteristic, except for the presence of the label and the chemical events attendant to its disintegration. The major disadvantage of the in vivo technique is the low specific activity of the nucleic acids so obtained. (B) Labeling of nucleic acids in vitro by reaction with labeled chemicals (2-4) and by enzymatic reactions [reverse transcriptase (5), polynucleotide kinase (6), or DNA polymerase (7)] can produce DNA of very high specific acitivty. Disadvantages of these methods include the necessity for rigorous purity of the material to be chemically labeled, the need for further purification of labeled DNA after labeling (8), and the uncertainties of fidelity of copying present in enzymatically synthesized DNA probes (9). (C) Labeling of DNA by cells in culture (10) has the potential for synthesis of high specific activity DNA under more physiologic conditions. For our studies involving unique sequence hybridization (DNA/DNA and DNA/RNA), we decided to employ the cell culture technique for obtaining labeled nucleic acids. Despite the availability of several procedures reported in the literature for labeling nucleic acid, often these methods are not evaluated in detail. We felt the need to determine optimal conditions for the labeling of DNA in cells in culture. This paper presents some of these evaluations and the methods for obtaining maximal labeling. 95 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.

ISSN ooO3-2697

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Cell culture techniques. Cells were grown in 75cm2 Falcon flasks in 15 ml of either Dulbecco’s Modified Eagles Medium of high glucose formulation (DMEM), or Minimal Eagles Medium (MEM) (both from GIBCO) with 10% fetal bovine serum (GIBCO). All cells, except the Xenopus A-6line, were grown at 365°C; A-6 cells were.grown at 26°C. The LLC-MK2 rhesus monkey, WI-38 human diploid,Xenopus laevis A-6, and Chang human liver cell lines were obtained from the American Type Culture Collection Cell Repository (CCL Nos. 7.1, 75, 102, and 13, respectively), The CRFK cat cell line was provided by Contract E-73-2001-NOL within the special Virus-Cancer Program (NIH, PHS) through the courtesy of Dr. Walter Nelson-Rees; mouse “L” cell (LM derivative, deficient in thymidine kinase activity) was provided by Dr. Frank Ruddle; rat muscle L8 and NS-20 mouse neuroblastoma lines were obtained through courtesy of Dr. Marshall Nirenberg. Primary cultures of chick fibroblasts were provided by Dr. Clifford N. Christian. To determine the number of cells in a flask, cells were removed by 0.05% trypsin treatment and were counted in a hemocytometer. The inhibitor, 5’-fluorodeoxyuridine (FdU) was a gift of Dr. W. E. Scott of HoffmanLaRoche. Preparation of 3H-labeled DNA. Except as noted in the individual experiments, cells at approximately one-half confluency were labeled with [3H]thymidine (New England Nuclear, 3 @i/ml, 50.8 Ci/mmol) for 3-5 days, and DNA was prepared by a modified Marmur method (11). This method involves cell lysis in 1% sodium dodecyl sulfate (Sigma), digestion with 100 pg/ml of preincubated pronase (Calbiochem) at 37°C for 4 hr, phenol-chloroform extraction, and winding out on a glass rod at an ethanol-water interface. An advantage of the modified Marmur method is the effective deproteinization (96%) during the initial pronase incubation and phenol-chloroform extraction. DNA recovery, however, averaged only 50%. Chloroform, phenol, or phenol-chloroform extraction alone resulted in 61, 42, and 55% deproteinization and 78, 94, and 72% DNA recovery, respectively. Complete recovery of the DNA during winding-out was only accomplished at DNA concentrations of 350 pg/ml and greater. A marked decrease in the recovery of DNA resulted from winding-out at lower concentrations. TCA Precipitation. Acid-insoluble counts were determined by precipitation in 10% trichloroacetic acid, using 500 pg of crystalline bovine serum albumin (BSA, Sigma) as carrier, and filtration on GF/C glass fiber filters. Filters were counted in toluene-Liquifluor scintillant using a Packard Tri-Carb scintillation counter at 28% efficiency. Fluorimetric determination of DNA. Assays of SDS lysates of cultured cells for DNA were performed by a modification of the method of Kissane

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and Robins (12). Briefly, a portion of the detergent extract containing an estimated 5-10 pg of DNA was treated with one-tenth volume of 50% TCA at room temperature for 30 min. The pellet obtained from this material at 6780g was suspended in 1 ml of 5% TCA, and this suspension was recentrifuged. The second pellet was resuspended in 1 ml of 0.1 N KOAc in ETOH at 4°C for 30 min, centrifuged, dissolved in 100~1 of 1 N NH40H and distributed in portions containing an estimated 0.5-2 pg of DNA which were frozen and lyophilized. To the dry material in each tube was added 30 ~1 of 2 M 3,5diaminobenzoic acid-2HCl (decolorized by Norit A) in water. The tightly capped tubes were then incubated at 60°C for 30 min; 1 ml of 0.6 N HCIO, was added to each and was mixed immediately before reading fluorescence at 504 nm (activation at 416 nm) in a AmincoBowman spectrophotofluorometer. The DNA standard was a water solution of calf thymus DNA (Sigma). RESULTS Uptake of [3H]Thymidine

by Cells in Culture

To discover general rules concerning the optimal labeling of DNA using E3H]thymidine as precursor, we monitored the uptake of labeled precursor into Chang liver cells, a commonly used human cell line. Figure 1 illustrates various parameters of the labeling of Chang cells at several different concentrations of precursor with and without FdU (and uridine), as described previously with cultured cells (13). In all cases, a fraction of the label appeared in the cells over a 2-day period (Fig. lA), with little additional uptake thereafter. Uptake of radioactivity was greatest in cells given 3 pCi/ml and FdU; in the other flasks, uptake increased with increasing precursor concentration, but this increase was always less than proportional to the concentration change. The effects on DNA accumulation (Fig. 1B) in the flasks, at least by Day 3, were the same at all thymidine concentrations in the absence of FdU. In all flasks, there was a burst of DNA synthesis (approximately a doubling) which peaked on the first day and decreased thereafter, although a small late rise was recorded in flasks labeled with 1 &i/ml. A different response was found in the presence of FdU; DNA accumulation continued to the second day and was at a higher level on Day 3 than it was in any of the other treatment regimens. After 3-4 days, in all regimens, a small percentage (0.5- 1.5%) of the counts present in the medium were acid-precipitable, suggesting that cell death and lysis were occurring. The number of thymidine molecules taken up by the cells labeled at 3 @i/ml without FdU during the first day was calculated to be only about 5% of the available thymidine locations in the newly synthesized DNA and corresponded to only about 9% of the total labeled precursor in the medium. Thus, the remaining thymidine must have been synthesized de

GROUSE AND SCHRIER

INCORPORATION

0 1 2 DAYS OF CULTURE

3

4

5

6

FIG. 1. Effect of [3H]thymidine concentration on uptake and specific activity. Replicate flasks were inoculated with 0.39 x lo8 Chang cells in MEM- 10% FCS on Day 0 minus 1. On Day 0, rH]thymidine (50.8 Ci/mmol) was added at the following concentrations: 1 &i/ml (solid circles); 3 &i/ml (triangles); 3 $Xml with 20 &ml of FdU and 50 pg/ml of uridine (inverted triangles); 6 @/ml (open circles); and 10 &i/ml (squares). Each datum represents the average of analyses of the contents of two or three flasks. In most instances, variability among replicates was within the diameter of the data points. Panel A: At the times indicated, 25-4 portions of a S.O-ml (1% SDS) lysate of the cells from each flask were added to 10 ml of Aquasol for radioactivity determination. Panel B: DNA content of the cell lysate was determined as described in Materials and Methods. Brackets show the range of values for selected significant data points. Panel C: At the times indicated, 25-4 portions of the 5.0-ml (1% SDS) cellular lysate were precipitated in 10% TCA with 500 pg of bovine serum albumin as carrier, filtered on GFK glass fiber filters, and counted in a toluene-based scintillant. Panel D: Specitic activities were calculated from the data used to plot Panels B and C.

nova by the cells rather than being taken from the medium. Certainly, lack of thymidine could not have been responsible for cessation of labeled thymidine incorporation. The addition of fresh medium with [3H]thymidine (3 &i/ml, 50.8 Ci/mmol) to cells labeled for 3 days led to no further uptake of precursor; thus, the cessation of uptake appeared to be a property of the labeled cells rather than of the medium. The inhibitory effect of [3H]thymidine on cell growth was shown to occur through the action of labeled material taken up and utilized by the cells, since inclusion in the medium of excess unlabeled

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thymidine competed with the uptake of [3H]thymidine, and cell division was not inhibited. Under the labeling conditions listed in Fig. 1, the cells incorporated (into acid-precipitable material) similar percentages of the intracellular pools of thymidine (Fig. 1, A and C). Thus, incorporation was also less than proportionate to the increased concentration of labeled precursor; FdU seemed to have its effects on getting the thymidine into the cells rather than on stimulating the incorporation of those molecules which were already in the intracellular pools. The specific activities of the DNA recovered from these cells (Fig. 1D) were highest at 6 or 10 &i/ml; there was no advantage of the higher concentration on Day 3. The specific activity after 3 &i/ml and FdU was somewhat less than that with 6 ~Cilml, but 1.6 times more DNA was recovered in these cultures treated with the anti-metabolite. From these data, it was clear that, at least for Chang human liver cells in this culture medium, the use of moderate concentrations of labeled precursor and FdU was the most economical labeling method. The theoretical maximum specific activity, if all the thymidine taken up from medium containing 10 @i/ml was incorporated into DNA, approaches 1 x lo6 cpm/pg of DNA (28% counting efficiency). If, in fact, incorporation were limited to only 25-33% of thymidine brought into the intracellular pools, since FdU enhanced thymidine uptake, an apparent practical limit of 300,000-400,000 cpm/kg of DNA might be eclipsed by the use of this nucleoside. From these data, it is reasonable to conclude that the regimen including 3 @i/ml of rH]thymidine and FdU was the most effective of those tested here for obtaining high specific activity DNA in good return. These data do not speak to the question of whether the same increment in DNA recovery and precursor uptake and incorporation, obtained with FdU at [3H]thymidine concentrations of 3 ,&i/ml, could be also realized at 10 &i/ml or higher concentrations of precursor. We have not performed that experiment, but would be surprised if the anti-metabolite did not stimulate a significant increase in those circumstances as well. It should be noted that, although cells growing in 3 @i/ml of rH]thymidine tend to lose their adherence to the substrate necessitating recovery of floating cells from the medium, no such precaution was necessary for cells treated with FdU, as they retained their adherence to the flasks. Thus, the use of FdU not only facilitated recovery of the DNA, but, with equivalent recovery per flask, it also increased the specific activity of DNA over that attained by simple incubation with labeled precursor. Labeling of DNA was more extensive at low than at high cell density. We labeled half of a group of flasks of Chang cells with 3 pCi/ml of [3H]thymidine 1 day after plating them with a low cell density. Uptake of label from the medium into the cells over the next 4 days is shown in the left-hand curve of Fig. 2. When the labeled cells were harvested on Day 4, each flask contained an average of 2.8 x lo6 total cells, an increase of

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FIG. 2. Uptake of [3H]thymidine at low and high cell densities. Twenty flasks were inoculated with 0.8 x 106 Chang cells/flask in MEM- 10% FCS on Day 0 minus 1. Ten flasks received 3 &i/ml of [3H]thymidine on Day 0, and the remaining flasks grew without labeled precursor until Day 6, at which time they received fresh medium and 3 &i/ml of [3H]thymidine. Each point represents an assay of tritium in the medium, as described in the legend to Fig. 1, averaged from 10 flasks.

2 x lo6 cells in each flask; and 80% of the tritium contained in those cells was acid-precipitable. This resulted in an average labeling of approximately 2.6 pCi/cell. Cells in the remaining flasks grew without tritiated precursor until Day 6, at which time they had nearly reached confluency. Uptake of [3H]thymidine by these higher density cells (right-hand curve of Fig. 2) also reached a maximum after 2 days of labeling. From Days 6 to 10, in the presence of tritium, an increase of cell number by 2 x lo6 cells per flask (to 6.2 x lo6 cells/flask) was recorded. Only 40% of the tritium counts in these cells at Day 10 was acid-precipitable. Therefore, the average tritium content for all cells in the flask was only 0.87 pCi/cell. Labeling at high density under these conditions produced 2.2 times more DNA containing three-fourths as many total counts with less than 25% as great specific activity as did labeling at lower density. If each cell in the lower density flasks contained 8 pg of DNA, it also would have contained approximately 8 dpm of tritium, and the specific activity of this DNA would have been about 280,000 cpm/pg at 28% efficiency. Considerable variation was observed in the efficiency of labeling different cell lines. This variability is apparent in Table 1, where the specific activities, obtained in purified DNA with nine cell lines from seven species labeled with 3 &i/ml of [3H]thymidine and no FdU, are compared. The result with L cells, this strain of which is lacking in thymidine kinase activity, shows that little labeling occurred in the absence of that enzyme.

RADIOACTIVE

LABELING TABLE

SPECIFIC

ACTIVITIES

1

DNA FROM VARIOUS

OF PURIFIED

Species

L Cells (TK-) Neuroblastoma NS20 LLC-MK, Primary fibroblast CRFK WI-38 A-6 Kidney Chang liver L8 Muscle

Mouse Mouse Rhesus monkey Chick Cat Human laevis

Human Rat

n DNA was purified, as described in Materials lysates of 25 or 50 flasks of cells grown for 3-5 of rH]thymidine (50.8 Ci/mmol). The cells were x 10” ceils/flask), and the rH]thymidine was added these studies, cells were grown in either DMEM 10% FCS.

CELL

LINES

DNA specific activity (cpm/pg of DNA)

Cell line”

Xenopus

101

OF DNA

950 51,800 53,600 71,100 76,600 119,000 152,000 203,000

214,000 and Methods, from combined SDS days in medium containing 3 &i/ml inoculated at low densities (0.5- 1.0 either immediately or 24 hr later. For or MEM, both of which contained

The other cell lines gave few clues to the reason for a greater than fourfold variation in labeling efficiency. Differences in radiosensitivity may have been responsible for this variability, since the data for two human lines indicate that species differences alone were not an adequate explanation. Although labeling with FdU and [3H]thymidine was not attempted with each cell line, in additional experiments with the A-6Xenopus cell line, a similar increase in the specific activity of DNA was obtained in A-6 as was shown for Chang cell DNA (Fig. 1D). Chang cell DNA prepared from cells labeled for 3 days at 3 &i/ml was greater than 98% acid soluble after treatment with DNase. Analysis of this same Chang cell DNA, using the method of Dingman (14), revealed sedimentation coefficients of 23.3s and 15.5s in neutral and alkaline sucrose gradients, respectively. This indicated that little nicking had occurred, even though the DNA had been stored at -20°C for 18 months prior to this analysis. Use of gentler methods for isolation of very high molecular weight DNA, such as enzymic deproteinization alone (15), would be appropriate if contamination by RNA and proteolytic enzyme were acceptable: however, for molecular hybridization in which further fragmentation of the DNA will be performed, the observed molecular weight and nicking of DNA prepared by the modified Marmur method is satisfactory. DISCUSSION

Several factors influence the labeling of DNA in tissue culture. The time course of uptake of radioactive precursor into cells and the radiation-

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induced inhibition of cell division seem to be the most important factors in Chang cells. The variability of these factors in different cell lines probably explains differential labeling under apparently identical labeling conditions. The increase in the specific activity of DNA, as observed with the FdU treatment, was most likely due to the increased uptake and incorporation of PH]thymidine into DNA because of decreased cellular synthesis of thymidylic acid (16). FdU at high doses is known to arrest cells in S phase due to complete thymidine depletion (16). In the present series of experiments, arrest of cell division was not observed at the concentration of FdU used, and the retardation of cell division caused by the radiation effect predominated. Labeling of DNA is a very time-dependent phenomenon, and this can be shown in two ways. First, when a high concentration of label was used over a short period of exposure, the specific activity of the resulting DNA was doubled over that obtained using a lower concentration of label for several days; this suggested that the toxic effects of radiation were not expressed until the cell cycle following labeling. This idea is supported by the observation that cell number and DNA content doubled in the first 1 or 2 days in the presence of the [3H]thymidine, but either no further increase, or a slight decrease in cell number occurred thereafter. Second, the use of FdU allowed an increase in the labeling of DNA above a level which was normally toxic, and this labeling also occurred only during the period in which the cell number doubled. Burki and Okada (17) have reviewed the radiation-induced killing of cultured mammalian cells by tritiated thymidine. From their data it is clear that the survival of cells decreases rapidly at concentrations of [3H]thymidine in excess of 1 &i/ml. For cells labeled at a [3H]thymidine concentration of less than 1 &i/ml, essentially 100% cell survival was found. Thus, survival was related to the number of tritium disintegrations per genome, and, though lower concentrations of tritiated precursor were associated with greater survival and cell division, diminished specific activity of cell DNA resulted. Burki et al. (18) found that differences among cell lines in the degree of inactivation by [3H]thymidine incorporated into DNA were similar to the differences in radiosensitivity seen when the cell lines were exposed, in the frozen state, to external radiation in the form of y- or X-rays. The nature of the radiation-induced lesions in the experiments reported here has not been characterized, although thymidine dimer formation, chromosome aberrations, and chain breakage are likely candidates ( 19,20). In conclusion, as a result of these studies, it is clear that: (a) The ability to obtain high specific activity tritiated DNA in cells in culture is limited by a radiosensitive process within the cells, a process which has different sensitivity in different cell lines. (b) These limits may be abridged by the use of FdU and/or large amounts of radioactive precursor during the period of

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initial cell doubling. (c) Small increments in DNA specific activity can be obtained by the use of increased concentrations of tritiated precursor over a few days. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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