the journal of b~o~.ocical chemistry prrnted m u.s.a ... · as a model cell culture system for...

THE JOURNAL OF B~O~.OCICAL CHEMISTRY Vol. 2%. No. 2, Issue of January 25, pp. 309-318, 1979 Prrnted m U.S.A.

Polyoma Virus and Cyclic AMP-mediated Control of Dihydrofolate Reductase mRNA Abundance in Methotrexate-resistant Mouse Fibroblasts*

(Received for publication, June 26, 1978)

Rodney E. Kellems,$ Vera B. Morhenn,$j Eva A. Pfendt,@ Frederick W. Alt,f and Robert T. Schimke

From the Departments of Biological Sciences and §Dermatology, Stanford University, Stanford, California 94305

As a model cell culture system for studying polyoma- mediated control of host gene expression, we isolated methotrexate-resistant 3T6 cells in which one of the virus-induced enzymes, dihydrofolate reductase, is a major cellular protein. In highly methotrexate-resistant cell lines dihydrofolate reductase synthesis accounts for over 10% that of soluble protein, corresponding to an increase of approximately loo-fold over the level in parental cells. This increase in dihydrofolate reductase synthesis is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. We have used these cells to show that infection with polyoma virus results in a 4- to &fold increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in dihydrofolate reductase mRNA abundance. The increase in dihydrofolate reductase synthesis begins 15 to 20 h after infection and continues to increase until cell lysis. These observations represent the first direct evidence that viral infection of eukaryotic cells results in the increased synthesis of a specific cellular enzyme and an increase in the abundance of a specific cellular mRNA.

In order to gain additional insight into the control of dihydrofolate reductase synthesis we examined other parameters affecting dihydrofolate reductase synthesis. We found that the addition of fresh serum to stationary phase cells results in a a-fold stimulation of dihydrofolate reductase synthesis, beginning 10 to 12 h after serum addition. Serum stimulation of dihydrofolate reductase synthesis is completely inhibited by the presence of dibutyryl cyclic AMP as well as by theophylline or prostaglandin El, compounds which cause an increase in intracellular cyclic AMP levels. In fact, the presence of dibutyryl cyclic AMP and theophylline results in a 2- to 3-fold decrease in the rate of dihydrofolate reductase synthesis and the abundance of dihydrofolate reductase mRNA. However, in contrast to the effect on serum stimulation, dibutyryl cyclic AMP and theophylline do not inhibit polyoma virus induction of dihydrofolate reductase synthesis or dihydrofolate reductase mRNA levels. These observations suggest that dihydrofolate reductase gene expression is controlled by at least two regulatory pathways: one involving

* This work was supported by grants from the American Cancer Society (NP148) and the National Cancer Institute (CA 16318). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.&C. Section 1734 solely to indicate this fact.

+ Present address, Department of Biochemistry, Baylor College of Medicine. Texas Medical Center, Houston. Tex. 77030.

1 Present address, Center for Cancer Research E17-517, Massachu- setts Institute of Technology, 77 Massachusetts Ave., Cambridge, Mass. 02139.

serum that is blocked by high levels of cyclic AMP and another involving polyoma induction that is not inhibited by cyclic AMP.

Polyoma and SV40 are small DNA tumor viruses that grow lytically in cells derived from their natural hosts (mice and monkeys, respectively) and cause tumors in a variety of rodent species. These viruses can be conveniently propagated in vitro by the use of appropriate lines of cultured cells. As a result the molecular biology of polyoma and SV40 gene expression has been the subject of intensive study for a number of years and we now have a great deal of information concerning the organization and regulation of these viral genomes (for recent reviews see Refs. 1 to 3). In contrast, very little is known about the biochemical mechanisms by which viral gene products affect host gene expression.

One of the major biochemical events resulting from polyoma infection of mouse fibroblasts is a substantial increase in the activities of numerous cellular enzymes related to DNA synthesis (4-7; for early, but still largely relevant reviews see Refs. 8 and 9). The precise biochemical basis for the increase in host enzyme activities has not been defined. In particular it has not been shown directly that these enzyme inductions result from an increase in specific enzyme synthesis and a corresponding increase in the abundance of specific cellular mRNAs. In order to examine these biochemical events in detail we have focused our attention on one particular polyoma-induced host enzyme, dihydrofolate reductase (10). For these studies we isolated methotrexate-resistant mouse fibroblasts in which dihydrofolate reductase synthesis accounts for over 10% that of soluble protein, representing an increase of approximately loo-fold over the level in parental cells. As with other methotrexate-resistant cells we have examined (ll), this increase in dihydrofolate reductase synthesis is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. The use of these cells has enabled us to show that polyoma infection results in a substantial increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in the abundance of dihydrofolate reductase mRNA. Additional parameters affecting dihydrofolate reductase synthesis, including cell growth phase, fresh serum, and cyclic nucleotides, were also examined.

EXPERIMENTAL PROCEDURES

Cells and Viruses-Permanent lines of mouse embryo fibroblasts (Swiss 3T6, a generous gift of Dr. Paul Berg and Marianne Die&man, Stanford University) were grown in Dulbecco’s modified Eagle’s medium (DME, Microbiological Associates), containing 5% dialyzed fetal calf serum (Grand Island Biological Co.), and were incubated at

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310 Control of Host Gene Expression by Polyoma Virus

37°C in a humidified atmosphere 02:CO2 (9O:lO). Cells were routinely cultured in the presence of 50 units/ml of penicillin and 50 pg/ml of streptomycin. Periodic testing for mycoplasma (the mycoplasma culture test and the uridine to uracil incorporation test, Irvine Scientific) indicated that cultures were free of contamination. Highly methotrexate-resistant 3T6 cells were selected in a stepwise fashion for the ability to grow in increasing concentrations of the drug. Cells main- tained in the presence of 400 PM methotrexate are referred to as 3T6- Rl and those in 5 mM methotrexate as 3T6-R2. Cloned sublines were indistinguishable from the original populations and are also referred to as 3T6-Rl and 3T6-R2.

An inoculum of polyoma virus, large plaque type, was kindly provided by Dr. Walter Eckart (The Salk Institute, San Diego, Calif.). Large quantities of virus were grown in 3T6 cells as described by Estes et al. (12), modified for polyoma. Supernatants were prepared from extracts of infected cells by centrifugation at 1000 rpm for 10 min. Virus titers in these extracts were quantitated as plaque-forming units as described previously (13).

Serum Stimulation Procedure-Cells were seeded at approximately 1.6 x lo4 cells/cm’ in Lux tissue culture dishes and cultured without medium change for at least 7 days. (The single exception to this procedure is the experiment described in the legend to Fig. 6 in which case preconfluent cells were used.) At least 3 days after the cells reached confluency the conditioned medium was replaced with fresh culture medium (including 10% dialyzed fetal calf serum) either with or without various drugs (see text). Control cultures were main- tained in the conditioned (i.e. used) medium. Control experiments determined that neither the removal and readdition of conditioned media nor the addition of fresh DME medium’ (without fetal calf serum) had any effect on either thymidine incorporation or dihydrofolate reductase synthesis.

Viral Infection Procedure-For initial experiments cells were prepared exactly as described under “Serum Stimulation Procedure.” Later we found that lytic infection was more reproducible if virus was inoculated on preconfluent monolayers. Thereafter, we seeded cells at 1.6 x lo4 cells/cm* and infected with virus after 3 days. At this time, the conditioned medium was removed and saved. The cells were washed with DME medium and infected with aliquots of virus extract, diluted when necessary in a solution of 1% bovine serum albumin in DME medium. Control cultures were mock-infected with 1% bovine serum albumin in DME medium for 2 h at 37°C. Following incubation, the virus was removed and replaced with the original conditioned cell culture medium. Visual lysis of infected cells was usually apparent by the 3rd day. In control experiments we determined that a cell extract prepared from noninfected cells does not stimulate dihydrofolate reductase synthesis or thymidine incorporation. Minor variations of this basic procedure are pointed out in the text where appropriate.

Measurement of Dihydrofolate Reductase Synthesis-The relative rate of dihydrofolate reductase synthesis was determined by direct immunoprecipitation of the enzyme from extracts of pulse-labeled cells as described previously (10). Incorporation into dihydrofolate reductase is expressed as a percentage of total incorporation in the high speed supernatant fraction of cell extracts.

Measurement of Z’hymidine Incorporation-Cells were labeled for 15 min with [“Hlthymidine (2 @J/ml New England Nuclear) in Dulbecco’s modified Eagle’s medium, rinsed twice with phosphate- buffered saline, and lysed by addition of a solution of 0.1% sodium dodecyl sulfate (14). A portion of the cell extract was used to determine the protein concentration (15) and another aliquot was used to measure the incorporation of [“Hlthymidine (expressed as counts per min incorporated per mg of protein).

Quantitation of Dihydrofolate Reductase mRNA Sequences- Total cytoplasmic RNA was prepared as described previously (16) and either used immediately or stored at -80°C. The relative abundance of dihydrofolate reductase mRNA sequences in various RNA preparations was determined from the kinetics of hybridization with trace amounts of [JH]cDNA complementary to dihydrofolate reductase mRNA (dihydrofolate reductase-cDNA). Conditions are as described previously (17) except that reactions were boiled for 10 min immediately prior to incubation at 68°C. The amount of cDNA hybridized was measured by resistance to S1 nuclease digestion (17).

Quantitation of Dihydrofolate Reductase Gene Sequences-DNA was extracted from cell nuclei and processed as described (17). The DNA was denatured and allowed to reanneal in the presence of trace quantities of radioactive dihydrofolate reductase-cDNA. The abun-

’ The abbreviation used is: DME medium, Dulbecco’s modified Eagle’s medium.

dance of dihydrofolate reductase structural genes was determined from the kinetics by which the cDNA was rendered double-stranded (17).

Preparation of cDNA Complementary to Dihydrofolate Reductase mRNA-Dihydrofolate reductase-cDNA was purified by using the two-step purification procedure that we have recently described (17).

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electropho- resis-High speed supernatant extracts of pulse-labeled cells were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 3.5% stacking gel and 12.5% separating gel (18). The distribution of radioactivity throughout the gel was determined as described previously by Palmiter et al. (19).

RESULTS

Isolation and Characterization of Methotrexate-resistant 3T6 Cells-The cellular enzymes induced as a result of polyoma infection are usually present in very small quantities making biochemical analysis of their regulation a difficult task. To alleviate this difficulty we isolated methotrexate- resistant 3T6 cells (an established line of mouse fibroblasts) in which one of the virus-induced enzymes, dihydrofolate reductase, constitutes a major cellular protein. The selection for methotrexate-resistant 3T6 cells was carried out in a stepwise fashion by selecting cells for the ability to grow in increasing concentrations of methotrexate, eventually obtain- ing highly resistant cell lines. The relative rate of dihydrofolate reductase synthesis in several highly resistant cell lines was measured by direct immunoprecipitation of the enzyme from extracts of pulse-labeled cells (Table I). Incorporation into dihydrofolate reductase is expressed as a percentage of total incorporation in the high speed supernatant fraction of cell extracts (11). Based on our earlier data (11) the relative rate of dihydrofolate reductase synthesis in cells selected for growth in 5 mM methotrexate (10%) represents an increase of approximately lOO- to 200-fold over that of parental cells. Cells selected for growth in 400 ,uM and 5 mM methotrexate were cloned and will be referred to as 3T6-Rl and 3T6-R2, respectively.

The data in Table I indicate that dihydrofolate reductase accounts for a large percentage of the radioactivity incorporated into the high speed supernatant protein of 3T6-R2 cells (5 mM methotrexate). To see if radioactivity is incorporated into any other resistant cell-soluble protein at such an increased rate, a high speed supernatant cell extract was prepared from a mixed population of pulse-labeled 3T6 and 3T6- R2 cells and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As shown in Fig. 1, the pattern of incorporation into the parental 3T6 cells and the methotrexate-resistant 3T6-R2 cells is virtually identical except for the presence of a major peak of radioactivity in 3T6-R2 cells which appears in a region corresponding to a molecular weight of approximately 22,000. We have shown in other experiments that this major 22,000 molecular weight peak co-migrates with pure dihydrofolate reductase.’ Therefore, dihydrofolate reductase is a major protein species in the 3T6-R2 cells and represents the only difference between 3T6-R2 and the parental 3T6 cell protein that is readily detectable by this type of analysis.

In order to determine whether the increased rate of dihydrofolate reductase synthesis in the methotrexate-resistant 3T6 cells is accompanied by a corresponding increase in the level of dihydrofolate reductase mRNA sequences we hybridized cytoplasmic RNA from either the parental (3T6) or resistant (3T6-Rl and 3T6-R2) cells with cDNA complementary to dihydrofolate reductase mRNA (hereinafter referred to as dihydrofolate reductase-cDNA). The kinetics and the extent of these reactions (Fig. 2A) indicate that dihydrofolate

’ Unpublished observation.


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Control of Host Gene Expression by Polyoma Virus

TABLE I

Dihydrofolate reductase synthesis in methotrexate-resistant 3T6 cells

Methotrexate-resistant 3T6 cells were selected in a stepwise fashion for the ability to grow in the concentrations of drug indicated. The relative rate of dihydrofolate reductase synthesis in these cell lines was measured in log phase and stationary phase cultures of cells as described previously (11). The values represent the average of multiple determinations of two independent experiments.

Level of methotrexate resistance

50 PM 100 PM 400pM

5InM

Dihydrofolate reductase synthesis

Log phase Stationary phase

5%. total 1.3 0.7 1.8 0.6 3.3 1.6

10.4 5.3

SLICE NUMBER FIG. 1. Analysis of pulse-labeled protein from parental (3T6) and

methotrexate-resistant (3T6-R2) cells. Logarithmically growing cultures of 3T6 and 3T6-R2 cells were pulse-labeled for 1 h with [‘4C]leucine (5 $X/ml) and [3H]leucine (10 pCi/ml), respectively, in leucine-free minimal essential medium (Eagle’s) containing 10% dialyzed fetal calf serum. A high speed supernatant extract was prepared (11) from a mixture of parental and methotrexate-resistant cells and was fractionated by electrophoresis through sodium dodecyl sulfate- polyacrylamide gels (see “Experimental Procedures”). Panel B indicates the distribution of incorporation into protein from methotrexate-resistant (3T6-R2, M) and parental (3T6, 0- - -0) cells. Panel A shows the ratio of “H to 14C radioactivity (i.e. resistant to parental) for each corresponding gel slice of B. Pure dihydrofolate reductase (DHFR) migrates to a position indicated by the arrow. Migration is from left to right.

reductase mRNA sequences are present at a much greater abundance in RNA from methotrexate-resistant cells as compared to similar RNA preparations from the parental cells. The quantity of parental 3T6 cell RNA used in these reactions (21 pg) was able to hybridize only 20% of the input cDNA whereas &fold less RNA from 3T6-Rl cells (3.5 pg) was sufficient to hybridize virtually all the input cDNA. The extents of these reactions indicate that there is at least a 30- fold increase in the level of dihydrofolate reductase mRNA in the methotrexate-resistant 3T6-Rl cells. Judging from the reaction kinetics shown in Fig. 2A, dihydrofolate reductase mRNA sequences are an additional severalfold more abundant in RNA from 3T6-R2 cells.

80 - 3T6-R2

311

FIG. 2. Hybridization kinetics of dihydrofolate reductase-cDNA with RNA (A) and DNA (B) from parental (3T6) and methotrexate- resistant (3T6-Rl and 3T6-R2) cells. A, Hybridization of dihydrofolate reductase-cDNA (30 pg, 300 cpm) to cytoplasmic RNA from 3T6 cells (A-A, 21 pg of RNA per reaction), 3T6-Rl cells (t--l, 3.5 pg of RNA per reaction), and 3T6-R2 cells (M, 17 pg of RNA per reaction). Cytoplasmic RNA was prepared as described by Kel- lems et al. (16). For details concerning reaction conditions and the procedure used to measure the per cent of cDNA hybridized (i.e. resistance to S, nuclease digestion), consult Alt et al. (17). The data are corrected for an endogenous SI nuclease resistance of 3% that characterized the cDNA used in these reactions. B, association kinetics of dihydrofolate reductase-cDNA (50 pg, 500 cpm) with DNA from 3T6 cells (A-A, 1.5 mg per reaction), 3T6-Rl cells (H, 0.5 mg per reaction), and 3T6-R2 cells (W, 0.5 mg per reaction). For each reaction the DNA was melted by heating to 102°C for 10 min, then immediately placed at 68°C and allowed to reanneal in the presence of cDNA. Reactions were terminated at the indicated Cot values by fractionating single- and double-stranded DNA by hydrox- ylapatite chromatography. The per cent of cDNA double-stranded is corrected for a value of 2% which represents the double-stranded behavior of the cDNA under these conditions, when incubated in the absence of driver DNA. The reassociation kinetics of the driver DNA from the three cell lines were indistinguishable and are summarized by the dashed line. For complete details see Alt et al. (17).

The abundance of dihydrofolate reductase structural gene sequences in parental and methotrexate-resistant 3T6 cells was determined by reannealing total cellular DNA in the presence of trace quantities of dihydrofolate reductase-cDNA. The kinetics with which dihydrofolate reductase-cDNA was hybridized (Fig. 2B) indicate that the dihydrofolate reductase structural gene is approximately 35 and 100 times more abundant in DNA from 3T6-Rl and 3T6-R2 cells, respectively, than in DNA from the parental 3T6 cells. These observations serve to characterize the basis for methotrexate resistance in these cell lines and to extend our earlier finding (17) that selective multiplication of the dihydrofolate reductase structural gene accounts for overproduction of dihydrofolate reductase in methotrexate-resistant lines of mouse cells.

Increased Dihydrofolate Reductase Synthesis and Dihy- drofolate Reductase mRNA Levels Following Polyoma In- fection-Frearson et al. (10) originally observed that dihydrofolate reductase activity was one of numerous cellular enzyme activities that increased severalfold or more following polyoma infection of primary mouse kidney cells. To learn more about


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this induction process we determined the effect of polyoma infection on dihydrofolate reductase synthesis in methotrexate-resistant 3T6 cells. As shown in Fig. 3A, 72 h after polyoma infection, dihydrofolate reductase synthesis is greater than 16% in infected cells, compared to a value of less than 4% in control, mock-infected, cells. An examination of the time course of polyoma induction in 3T6-Rl cells (Fig. 3B) shows that the rate of dihydrofolate reductase synthesis in infected cells begins to increase over control cells approximately 15 to 20 h after infection and continues to increase until cell lysis (70 to 80 h). The relative increase in dihydrofolate reductase synthesis that we observe after polyoma infection (4- to 5- fold, Fig. 3, A and B) is in good agreement with the relative increase in dihydrofolate reductase activity reported by Frear- son et al. (10).

We also examined the effect of the multiplicity of infection on both the time course and the extent of the polyoma induction of dihydrofolate reductase synthesis. As shown in Fig. 3B, the time course of the induction of dihydrofolate reductase synthesis is not significantly affected by the multiplicity of infection. However, the magnitude of the induction of dihydrofolate reductase synthesis does increase as a function of the multiplicity of infection up to 100 plaque-forming units/cell. Beyond this dihydrofolate reductase synthesis is only marginally increased by additional increases in the quantity of virus used (200 and 500 plaque-forming units/cell, data not shown).

Dihydrofolate reductase is a major protein in 3T6-R2 cells (see Fig. 1B) and as shown in Fig. 3 its rate of synthesis increases nearly 5-fold following polyoma infection. To see if any other major change in the synthesis of soluble protein occurs in these cells as a result of polyoma infection we compared the pattern of radioactivity incorporated into soluble protein from infected and control 3T6-R2 cells. Fig. 4 shows that the increased incorporation of radioactive leucine into dihydrofolate reductase represents the major difference in the pattern of incorporation between infected and control cells. The radioactivity profiles of Fig. 1 differ somewhat from those shown in Fig. 4 because the former were obtained from a log phase culture of cells and the latter from stationary phase cultures. The data in Fig. 4 are not intended to indicate that dihydrofolate reductase is the only cellular enzyme whose synthesis increases following polyoma infection. The synthesis

6-

0020 Oo- 80

TIME AFTER VIRAL INFECTION (hours)

FIG. 3. The effect of polyoma infection on the relative rate of dihydrofolate reductase synthesis. Quiescent cultures of 3T6-R2 cells (A) or 3T6-Rl cells (II) were infected with polyoma virus as described under “Experimental Procedures.” At various times after infection, the relative rate of dihydrofolate reductase synthesis was determined by direct immunoprecipitation of the enzyme from extracts of pulse- labeled cells (11). The numbers within the figure refer to different multiplicities of infection (plaque-forming units per cell) used in each viral infection. The values for dihydrofolate reductase synthesis in control, mock-infected, cells are indicated by O---O.

Y

r‘ m

60 I

_ INFECTED/ UNINFECTED

40 -

(4 I

01 I I I I I

-8 -i

SLICE NUMBER

FIG. 4. The effect of polyoma infection on the incorporation of leucine into cellular protein. Quiescent cultures of 3T6-R2 cells were infected with polyoma virus (100 plaque-forming units/cell), as described under “Experimental Procedures.” Uninfected cells were treated in the same way except that no virus was used. After 72 h, infected and uninfected cells were pulse-labeled with r3H]- and [‘?]leucine, respectively, in leucine-free minimal essential medium (Eagle’s) containing 10% dialyzed fetal calf serum. Infected and uninfected cells were mixed, a high speed supernatant cell extract was prepared, and the protein was fractionated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (11). Panel B indicates the distribution of incorporation into protein from infected (U) and uninfected (M) cells. Panel A shows the ratio of “H to 14C (i.e. infected to uninfected) radioactivity for the corresponding gel slice shown in Panel B.

of numerous other cellular enzymes is undoubtedly stimulated along with dihydrofolate reductase, but since these enzymes are present in such small quantities an increase in their rate of synthesis is not detectable by this method of analysis.

To determine whether the polyoma-mediated increase in dihydrofolate reductase synthesis is due to a corresponding increase in dihydrofolate reductase mRNA levels we hybridized cytoplasmic RNA from polyoma-infected and control 3T6-R2 cells to dihydrofolate reductase-cDNA. These RNA excess reactions (see Fig. 5) were terminated intentionally before no more than 30% of the cDNA was hybridized. Under these conditions the percentage of cDNA hybridized is proportional to the concentration of the reacting RNA species (20). As shown in Fig. 5, the percentage of cDNA hybridized was approximately linearly related to the Rot value of the reaction when using RNA from either infected or control cells. The slopes of the curves relating per cent of cDNA hybridized to Rot indicate that dihydrofolate reductase mRNA sequences are 4- to 5-fold more abundant in cytoplasmic RNA from polyoma-infected cells than from control cells. Therefore, the increase in dihydrofolate reductase synthesis following polyoma infection (Fig. 3) is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA.

The Role of Fresh Serum and Cyclic Nucleotides in the Control of Dihydrofolate Reductase Synthesis-The relative rate of dihydrofolate reductase synthesis in stationary phase


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Control of Host Gene Expression by Polyoma Virus 313

24

16

12

8

1 I 1 I I

+POLYOMA A

/

/

A

0.4 0.8 1.2 1.6 2.0

Rot FIG. 5. The effect of polyoma infection on dihydrofolate reductase

mRNA abundance. Preconfluent cultures of 3T&R2 cells were infected with polyoma virus (100 plaque-forming units/cell) as described under “Experimental Procedures.” Uninfected control cells were treated in an identical manner except that no virus was used. Cells (1 to 2 g wet weight) were harvested after 72 h and total cytoplasmic RNA was prepared as described previously (16). A series of hybridization reactions containing 5 pg of RNA and 50 pg (500 cpm) of dihydrofolate reductase-cDNA each were boiled for 10 min and then placed at 68°C. Reactions were terminated at the indicated Rot values and the per cent of cDNA hybridized was measured by resistance to SI nuclease hydrolysis. Details concerning hybridization conditions and the S, nuclease assay have been given (17).

cultures is 2-fold less than the rate of synthesis in log phase cells (Table I). As shown in Fig. 6A, the addition of fresh culture medium (including 10% dialyzed fetal calf serum) to such stationary phase (i.e. quiescent) cells results in a stimulation of dihydrofolate reductase synthesis beginning 10 to 12 h after treatment. Maximum stimulation (2-fold) is achieved 20 to 25 h following addition of fresh medium, after which time dihydrofolate reductase synthesis declines to the initial low values. Fig. 6B shows that thymidine incorporation is also stimulated and that the time course of this stimulation par- allels the increase in dihydrofolate reductase synthesis. Con- trol experiments indicate that neither the removal and readdition of conditioned medium nor the addition of medium without serum will stimulate dihydrofolate reductase synthesis or thymidine incorporation. Since dialyzed serum was used for all experiments it is likely that macramolecular serum components are responsible for the induction process. Addi- tion of fresh serum to polyoma-infected cells (multiplicity of infection equals 100) does not cause a significant additional stimulation of dihydrofolate reductase synthesis.’

Serum effects have been correlated with changes in cellular cyclic AMP levels. For example several reports indicate that intracellular cyclic AMP levels decline substantially following serum stimulation of quiescent cells (21-24). In addition, Kram et al. (25) have shown that serum stimulation of various cellular functions, including thymidine incorporation, is blocked by the presence of high concentrations of dibutyryl cyclic AMP and theophylline. In view of these observations

i (4 ’ I I I

+DBcAMP

g

I I I I

(B)

F -\ \ +SERUM +DBcAMP

? 0 I ‘s* ----*B--N 0 10 20 30 40

TIME AFTER SERUM ADDITION (hours) FIG. 6. The effect of fresh serum and dibutyryl cyclic AMP and

theophylline on dihydrofolate reductase synthesis and thymidine incorporation. A, dihydrofolate reductase synthesis. Conditioned medium was removed from preconfluent cultures of 3T6-Rl cells and replaced with an equal volume of fresh cell culture medium (including 10% dialyzed fetal calf serum) either with or without 0.2 mM dibutyryl cyclic AMP and 1 InM theophylline (together indicated as +DBcAMP). The original conditioned medium was returned to a portion of the cells which served as controls. At various times after treatment dihydrofolate reductase (DHFR) synthesis was measured by immunoprecipitation from extracts of pulse-labeled cells (11). Key to symbols: fresh medium + 0.2 mM dibutyryl cyclic AMP + 1 mM

theophylline, A- - -A; fresh medium, A-A; control cells, u. B, [3H]thymidine incorporation. Preconfluent cultures of 3T6-Rl cells were treated as above and pulse-labeled with 2 &i/ml of r3H]thymidine at the indicated times. Thymidine incorporation was measured as described under “Experimental Procedures” and expressed as counts per min incorporated per mg of protein. The level of thymidine incorporation in the control cells is much higher in this experiment than for those shown in Tables II and IV because preconfluent cells were used in this experiment whereas quiescent cells were used for the experiments described in Tables II and IV. For a key to the symbols see above.

we wanted to know if these compounds would prevent serum stimulation of dihydrofolate reductase synthesis. When fresh serum is added to quiescent cells in the presence of dibutyryl cyclic AMP and theophylline serum stimulation of dihydrofolate reductase synthesis and thymidine incorporation is blocked (Fig. 6, A and B). Dibutyryl cyclic AMP and theophylline actually cause a substantial decline in both dihydrofolate reductase synthesis and thymidine incorporation relative to control cells (Fig. 6). Dibutyryl cyclic AMP and theophylline completely block serum stimulation of dihydrofolate reductase synthesis and thymidine incorporation even when added as much as 10 h after the addition of serum (data not shown).


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TABLE II

The role of cyclic AMP in the control of dihydrofolate reductase synthesis and thymidine incorporation

Conditioned media was removed from quiescent cultures of 3T6- Rl and 3T6-R2 cells and was replaced with an equal volume of fresh DME medium containing 10% dialyzed fetal calf serum (FCS) and supplemented, when indicated, with dibutyryl cyclic AMP, dibutyryl cyclic GMP, prostaglandin El, or theophylline. After 24 h dihydrofolate reductase synthesis and thymidine incorporation were measured as described under “Experimental Procedures.” Untreated quiescent cells served as controls. The data represent the average of multiple measurements taken on two independent experiments. In some cases thymidine incorporation was not measured (indicated by -).

Experimental procedure

Dihydrofol- Thymidine ate reduc-

tase synthe- lnc;;fp- sis

‘H cpm/mg B total protein x

lo- 3 A. 3T6-Rl Cells

1. Control 2. FCS 3. FCS + 0.2 mM dibutyryl cyclic AMP

+ 1 mM theophylline 4. FCS + 1 mM dibutyryl cyclic AMP 5. FCS + 5 IIIM dibutyryl cyclic AMP 6. FCS + 1 mM dibutyryl cyclic GMP 7. FCS + 5 mM dibutyryl cyclic GMP 8. FCS + 10 pg/ml prostaglandin E1 9. FCS + 50 pg/ml prostaglandin El

B. 3T6-R2 Cells 1. Control 2. FCS 3. FCS + 0.2 mM dibutyryl cyclic AMP

+ 1 mM theophylline 4. FCS + 0.2 mM dibutyryl cyclic AMP 5. FCS + 1 mM theouhvlline

1.3 11 2.5 100 1.3 6

1.2 1.2 2.8 3.7 1.9 1.2

5.4 12.0

3.5

7.4 4.4

-

-

-

To ensure that the effects of dibutyryl cyclic AMP and theophylline are indeed cyclic AMP related, we examined the effects of theophylline (alone) or prostaglandin El (alone) on the serum stimulation of dihydrofolate reductase synthesis. These compounds cause an increase in intracellular cyclic AMP levels, either by inhibiting phosphodiesterase (theophylline, 26) or stimulating adenylate cyclase (prostaglandin E1, 27-29). As shown in Table II, either theophylline (1 mM)

or prostaglandin E1 (50 pg/ml) will block serum stimulation of dihydrofolate reductase synthesis in methotrexate-resistant 3T6 cells. Kram et al. (25) have previously observed that prostaglandin E1 blocks serum stimulation of thymidine incorporation. Dibutyryl cyclic AMP alone, at relatively high concentrations (1 and 5 mM), will block serum stimulation of dihydrofolate reductase synthesis and thymidine incorporation (Table II). However, similarly high concentrations of dibutyryl cyclic GMP do not prevent serum stimulation of dihydrofolate reductase synthesis or thymidine incorporation (Table II). In fact, the presence of high concentrations of dibutyryl cyclic GMP causes a significant increase in both dihydrofolate reductase synthesis and thymidine incorporation. Taken together, the data presented in Table II allow us to conclude that the effects of dibutyryl cyclic AMP on dihydrofolate reductase synthesis are cyclic AMP-related, and are not due to a nonspecific effect of the high cyclic nucleotide concentration used.

The Effect of Dibutyryl Cyclic AMP and Theophylline on Polyoma Virus Induction of Dihydrofolate Reductase Syn- thesis and Dihydrofolate Reductase mRNA Levels-Since polyoma and serum both result in stimulation of dihydrofolate reductase synthesis (and thymidine incorporation), it is possible that closely related control mechanisms are involved in

each case. To explore this possibility and to compare serum and polyoma induction of dihydrofolate reductase synthesis, we determined the effect of dibutyryl cyclic AMP and theophylline on the polyoma induction process. As shown in Fig. 7A, dibutyryl cyclic AMP and theophylline have virtually no effect on the polyoma induction of dihydrofolate reductase synthesis. This is in marked contrast to the fact that these compounds completely block serum stimulation of dihydrofolate reductase synthesis (Fig. 6), even when added as much as 10 h after fresh serum (data not shown). Fig. 7A also shows that the presence of dibutyryl cyclic AMP and theophylline results in a decrease in dihydrofolate reductase synthesis in uninfected cells.

We have also measured thymidine incorporation as a func-

I +POLYOMA

UNINFECTED

1

(B; ” I

+POLbOMA +DBcAMP P- -m----- 73

I /’

t

i

9 /

jr / , +POLYOMA

TIME AFTER INFECTION (hours) FIG. 7. The effect of dibutyryl cyclic AMP and theophylline on

polyoma virus stimulation of dihydrofolate reductase synthesis. Quiescent cultures of 3T6-Rl cells were exposed to polyoma virus (25 plaque-forming units/cell) for 2 h as described under “Experimental Procedures.” Immediately following removal of virus, the original volume of conditioned medium, either with or without 0.2 mM dibutyryl cyclic AMP and 1 mM theophylline (together indicated as +DBcAMP) was added to the cells. Uninfected cells were treated in the same manner except that no virus was used. Dihydrofolate reductase synthesis (A) and [3H]thymidine incorporation (B) were measured (see “Experimental Procedures”) at zero time and at 50 and 60 h following treatment. Key to symbols: polyoma, A---A; polyoma + 0.2 mM dibutyryl cyclic AMP and 1 mM theophylline, a- - -a; uninfected, w uninfected + 0.2 mM dibutyryl cyclic AMP and 1 mM theophylline, 0- - -0.


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TABLE III Polyoma and dibutyryl cyclic AMP-mediated control of

dihydrofolate reductase synthesis in 3T6-R2 cells

Preconfluent 3T6-R2 cells were infected for 2 h with polyoma virus (100 plaque-forming units/cell) as described under “Experimental Procedures.” Immediately following removal of virus, the original volume of conditioned medium, either with or without 0.2 mM dibutyryl cyclic AMP and 1 mM theophylline, was added to the cells. Uninfected cells were treated in an identical fashion except that no virus was used. After 72 h, a small portion of the cells was used to measure dihydrofolate reductase synthesis (see “Experimental Pro- cedures”) and the remainder (approximately 1 ml of packed cells in each case) was used as a source of cytoplasmic RNA for the experiment described in Fig. 8. The values for dihydrofolate reductase synthesis represent the average of two independent determinations.

Dihydrofol-

Experimental procedure ate reductase synthe-

sis

1. Uninfected 2. Uninfected + dibutyryl cyclic AMP + theophylline 3. Polyoma 4. Polyoma + dibutyryl cyclic AMP + theophylline

% total 5.1 2.3

16.8 17.5

25

20

15

IO

5

UNINFECTED

1 2 3 4 5

Rot FIG. 8. Polyoma virus- and dibutyryl cyclic AMP-mediated control

of dihydrofolate reductase mRNA abundance. Preconfluent 3T6-R2 cells was treated as described in the legend to Table III. After 72 h, a small portion of cells were used to measure dihydrofolate reductase synthesis (see Table III) and the remainder (approximately 1 ml of packed cells in each case) was harvested and total cytoplasmic RNA was prepared (16). The relative abundance of dihydrofolate reductase mRNA sequences in each RNA preparation was measured by hybridization to dihydrofolate reductase-cDNA (5 pg RNA and 50 pg, i.e. 500 cpm, cDNA per reaction) as described in the legend to Fig. 5. Key to symbols: polyoma, A-A; polyoma + 0.2 mM dibutyryl cyclic AMP and 1 mM theophylline, n---n, uninfected, w uninfected + 0.2 mrvr dibutyryl cyclic AMP and 1 mM theophylline, 0- - -0. The presence of dibutyryl cyclic AMP and theophylline is indicated as +DBcAMP.

tion of polyoma infection in the presence or absence of dibutyryl cyclic AMP and theophylline. The effect of polyoma infection on thymidine incorporation (Fig. 7B) roughly par- allels that of dihydrofolate reductase synthesis in that 1) polyoma induction of thymidine incorporation is not blocked by dibutyryl cyclic AMP and theophylline and 2) thymidine incorporation in control cells is depressed as a result of expo-

sure to dibutyryl cyclic AMP and theophylline. A curious feature of the data in Fig. 7B is that dibutyryl cyclic AMP and theophylline actually cause a substantial increase in thymidine incorporation in polyoma-infected cells. This effect has been observed in each of several cases when this experiment was repeated. The biochemical basis for this increase in thymidine incorporation is not known.

To investigate the relationship between intracellular cyclic AMP levels and polyoma-mediated induction of dihydrofolate reductase mRNA, we carried out a lytic infection of 3T6-R2 cells either in the presence or absence of dibutyryl cyclic AMP and theophylline. Mock-infected control cells were treated similarly. A small portion of the cells was used for the measurement of dihydrofolate reductase synthesis, and the remainder for the preparation of cytoplasmic RNA. The data regarding dihydrofolate reductase synthesis are presented in Table III and indicate that dibutyryl cyclic AMP and theophylline have no detectable effect on viral induction of dihydrofolate reductase synthesis, although, as expected, a decrease in dihydrofolate reductase synthesis in uninfected cells

TABLE IV

The effect of inhibitors of DNA synthesis on dihydrofolate reductase synthesis in 3T6-RI cells

A, Conditioned medium was removed from quiescent 3T6-Rl cells and replaced with an equal volume of fresh DME medium containing 10% dialyzed fetal calf serum (FCS) and supplemented as indicated, with either 8-methoxypsoralen, cytosine arabinoside, or hydroxyurea. Cells exposed to 8methoxypsoralen were irradiated at zero time with 1.6 J/cm2 UV light (360 nm). Subsequent to irradiation the medium was removed and fresh cell culture medium, without 8methoxypsor alen, was added. After 24 h, dihydrofolate reductase synthesis and thymidine incorporation were measured as described under “Experi- mental Procedures.” Untreated quiescent cells served as controls. The data represent the average of multiple determinations of two independent experiments. B, cuitures of 3T6-Rl cells were exposed to polyoma virus (100 plaque-forming units/cell) for 2 h as described under “Experimental Procedures.” Immediately following removal of virus, the original volume of conditioned medium, either with or without cytosine arabinoside, was added. The cells used in Part B.3. were exposed to 8-methoxypsoralen and ultraviolet light (as described above) just prior to viral infection. Dihydrofolate reductase synthesis and thymidine incorporation were determined at 50 and 75 h after viral infection. See “Experimental Procedures” for details. The data represent the average of multiple determinations performed on two indenendent exueriments.

% total

A. Serum stimulation 1. Control 2. FCS 3. FCS + 0.5 pg/ml8-methoxypsoralen 4. FCS + 25 pg/ml cytosine arabinoside

FCS + 100 pg/ml cytosine arabinoside 5. FCS + 2.5 mM hydroxyurea

B. Polyoma stimulation 1. Control (50 h)

Control (75 h) 2. Polyoma infected (50 h)

Polyoma infected (75 h) 3. Polyoma infected + 0.5 pg/ml

8-methoxypsoralen (50 h) Polyoma infected + 0.5 pg/ml

8-methoxypsoralen (75 h) 4. Polyoma infected + 100 pg/ml cytosine

arabinoside (50 h) Polyoma infected + 100 pg/ml cytosine

arabinoside (75 h)

1.2 2.5 2.6 2.6 2.5 2.5

0.9 0.9 1.5 2.8 1.6

2.9

1.8

2.0

Experimental procedure

DiWro- Thymidine folate reductase incorpora-

synthesis tion

‘H cpm/mg protein X

IO-"

2 110

8 0.9 0.5 1.0

2.5 0.5 6.4 7.0 2.5

1.0

0.2

0.2


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does result from this treatment. The relative abundance of dihydrofolate reductase mRNA sequences in the various RNA preparations was measured by hybridization to dihydrofolate reductase-cDNA. The results (Fig. 8) indicate that 1) the increase in dihydrofolate reductase mRNA levels resulting from polyoma infection is not significantly affected by dibutyryl cyclic AMP and theophylline, 2) exposure of uninfected cells to dibutyryl cyclic AMP and theophylline results in a 3- fold decrease in dihydrofolate reductase mRNA levels, and 3) the relative abundance of dihydrofolate reductase mRNA sequences is proportional to the relative rate of dihydrofolate reductase synthesis in each case.

The Stimulation of Dihydrofolate Reductase Synthesis by Polyoma Virus and Serum Is Not Tightly Coupled to Thy- midine Incorporation-Polyoma infection or serum stimulation of quiescent cells was carried out in the presence of a variety of drugs that are known to block DNA synthesis and thymidine incorporation. The effect on dihydrofolate reductase synthesis was determined. As shown in Table IV, hydroxyurea (30, 31), cytosine arabinoside, (32, 33), or a-methoxypsoralen plus exposure to ultraviolet light (34, 35) were effective in blocking serum stimulation of thymidine incorporation but had no detectable effect on the stimulation of dihydrofolate reductase synthesis. Likewise, cytosine arabinoside and 8-methoxypsoralen (and ultraviolet light) were effective in inhibiting polyoma-mediated stimulation of thymidine incorporation but had no significant effect on dihydrofolate reductase synthesis. In summary, the data in Table IV indicate that dihydrofolate reductase synthesis can be stimulated by either polyoma virus or fresh serum under conditions where thymidine incorporation and presumably DNA synthesis (see references just cited) have been blocked. These data are in agreement with the studies of Frearson et al. (10) showing that inhibitors of DNA synthesis did not affect polyoma or SV40 induction of dihydrofolate reductase activity in primary cell lines.

DISCUSSION

Numerous cellular enzymes, normally present in ex- tremely small quantities, are induced as a result of polyoma infection. The biochemical basis for polyoma-mediated regulation of cellular gene expression is unknown. To facilitate a biochemical analysis of this polyoma induction process, we isolated methotrexate-resistant 3T6 cells in which one of the virus-induced enzymes, dihydrofolate reductase, is a major cellular protein. In one highly resistant cell line, 3T6-R2, dihydrofolate reductase synthesis is increased approximately loo-fold over parental cells due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. These cells are permissive for polyoma infection. Because methotrexate-resistant cells contain such a large quantity of dihydrofolate reductase enzyme and mRNA, it has been relatively easy to show that infection with polyoma virus results in a 4- to 5-fold increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in the level of dihydrofolate reductase mRNA. The increase in dihydrofolate reductase synthesis begins 15 to 20 h after infection and continues to increase-until cell lysis (70 to 80 h). This represents the first direct evidence that viral infection of eukaryotic cells results in the increased synthesis of a specific cellular enzyme and an increase in the abundance of the specific mRNA coding for the enzyme.

To gain additional insight into polyoma-mediated control of dihydrofolate reductase synthesis we examined other parameters for their effect on dihydrofolate reductase synthesis. We found that the addition of fresh serum to quiescent cells

results in a 2-fold stimulation of dihydrofolate reductase synthesis. The increase in dihydrofolate reductase synthesis begins 10 to 12 h after addition of serum, reaches a maximum at 20 to 25 h, and thereafter declines. Serum stimulation of thymidine incorporation follows a similar time course. Dibu- tyryl cyclic AMP, as well as theophylline or prostaglandin E,, which cause an increase in intracellular cyclic AMP levels, completely block serum stimulation of dihydrofolate reductase synthesis and thymidine incorporation. However, polyoma virus induction of dihydrofolate reductase synthesis and dihydrofolate reductase mRNA levels is unaffected by these compounds. These observations suggest that control of dihydrofolate reductase gene expression is regulated by at least two regulatory pathways; one involving serum components that is blocked by elevated levels of cyclic AMP and another involving polyoma that is insensitive to cyclic AMP levels. Alternatively, polyoma may exert its effect on dihydrofolate reductase gene expression at a point beyond the involvement of cyclic nucleotides.

The effect of dibutyryl cyclic AMP on dihydrofolate reductase gene expression deserves additional comment. Although this cyclic nucleotide has no effect on the polyoma induction of dihydrofolate reductase synthesis and dihydrofolate reductase mRNA abundance as discussed above, we did observe that when quiescent cells were exposed to dibutyryl cyclic AMP and theophylline (in the absence of virus) the rate of dihydrofolate reductase synthesis and the level of dihydrofolate reductase mRNA decreased severalfold. The drop in dihydrofolate reductase mRNA levels in cells exposed to dibutyryl cyclic AMP is the first reported example in which this cyclic nucleotide is shown to cause a decrease in the abundance of a specific cellular mRNA. In at least two other cases dibutyryl cyclic AMP was shown to cause an increase in the mRNA activities specifying two hepatic enzymes, phosphoenolpyruvate carboxykinase (36) and tyrosine aminotrans- ferase (37). These curiously opposite effects of the cyclic nucleotide are, in fact, consistent with two currently accepted models concerning the physiological activity of cyclic AMP. One physiological role of cyclic AMP is to serve as a second messenger for numerous polypeptide hormones in specialized target tissues (38). It is most likely this, or a related function, which accounts for the dibutyryl cyclic AMP induction of phosphoenolpyruvate carboxykinase and tyrosine amino- transferase mRNA activities. A second physiological response to cyclic AMP is related to the control of cell proliferation (reviewed in Refs. 26 and 39). Exposure to dibutyryl cyclic AMP, or to agents which cause an increase in intracellular cyclic AMP levels, inhibits cell growth by arresting cells in the G1, or G2 phase of the cell cycle (never in the S phase) (40). This effect may explain the cyclic AMP-mediated drop in dihydrofolate reductase protein synthesis and mRNA abundance, since dihydrofolate reductase is a growth phase and, therefore, possibly a cell cycle-regulated enzyme.

It is possible that all of the parameters we have examined (polyoma, serum, growth phase, cyclic nucleotides) mediate their control of dihydrofolate reductase synthesis by interaction with cell cycle control programs. For example, serum stimulation of quiescent cells has been used to achieve par- tially synchronous populations of cultured animal cells (41-43). The induction of dihydrofolate reductase synthesis following serum stimulation is closely correlated in time with the stimulation of thymidine incorporation (and presumably DNA synthesis, see references just cited) and may therefore result from the activation of a cellular program required for S phase. Careful studies regarding the cell cycle-dependent regulation of dihydrofolate reductase synthesis are required to


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explore this possibility further. In this regard, the use of inhibitors of DNA synthesis has enabled us to show that neither serum- nor polyoma-mediated stimulation of dihydrofolate reductase synthesis is dependent on an increase in thymidine incorporation and presumably DNA synthesis. This is in contrast to the control of histone synthesis which is tightly coupled to DNA synthesis (48, 49).

The pattern of cellular gene expression that accompanies the appearance of T-antigen in polyoma- or SV40-infected cells also resembles that normally associated with the S phase of the cell cycle (i.e. increased DNA, histone, and polyamine synthesis and increased activities of known S phase enzymes; see Tooze (9) for further discussion). Therefore, polyoma infection may act as a mitogenic stimulus that activates a cell cycle program required for S phase. If polyoma-induced changes in dihydrofolate reductase gene expression result from viral interaction with cell cycle control mechanisms then closer examination of the polyoma induction process may lead to a better understanding of cell cycle regulation and the control of cell proliferation.

Many investigations concerning the regulation of gene expression in eukaryotic cells are directed at understanding the factors controlling the synthesis of specialized products of highly differentiated cells. A major advantage afforded by these systems is that the gene products studied are synthe- sized in large quantities making biochemical analysis relatively easy. Furthermore, gene expression in such systems is regulated in response to well defined stimuli such as steroid or polypeptide hormones. The cellular enzymes induced following papovavirus infection belong to a group sometimes referred to as household, or ubiquitous, enzymes (44) that are present in most cell types regardless of the state of cellular differentiation. The intracellular levels of these enzymes are generally very low and, as a result, studies concerning their regulation have usually relied only on measuring enzyme activities in cell extracts. By exploring the biochemical parameters underlying the regulation of this important group of cellular enzymes, we may uncover control mechanisms somewhat different from those mediating hormonal regulation of gene expression. Drug-resistant cell lines that overproduce specific cellular enzymes normally present in small quantities (45-47) will be valuable tools for use in studying the control of enzyme synthesis in animal cells. We believe that, when cells are selected for resistance to specific inhibitors of essential cellular enzymes, the mechanism of drug resistance will frequently involve overproduction of the target enzyme. Based on our observations with methotrexate-resistant cells, it is likely that such overproduction will result from selective multiplication of those genes coding for the target enzyme. Furthermore, we have found that although methotrexate-resistant cells contain highly amplified copies of the dihydrofolate reductase structural gene, control of dihydrofolate gene expression can still occur and is similar to that in parental cells (10, 11, 16, 50,51). The use of methotrexate-resistant 3T6 cells to examine the biochemical basis of polyoma-, growth phase-, cyclic nucleotide-, and cell cycle-mediated control of dihydrofolate reductase gene expression should increase our understanding of these important regulatory parameters and their relationship to one another.

Acknowledgments-We appreciate the helpful advice and thought- ful discussion provided bv Grav Grouse, Randv Kaufman. Jack Nun- berg, and Marvin Wickens while these’studies were in progress. We are also grateful to Luean Anthony and John Wilson for critical evaluation of the manuscript.

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R E Kellems, V B Morhenn, E A Pfendt, F W Alt and R T SchimkemRNA abundance in methotrexate-resistant mouse fibroblasts.

Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase

1979, 254:309-318.J. Biol. Chem.

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