In situ assay for ornithine decarboxylase in tissue culture

ANALYTICAL

BIOCHEMISTRY

104,452-456

(1980~

In Situ Assay for Ornithine

Decarboxylase

in Tissue

Culture

M. K. PATTERSON, JR.. AND M. D. MAXWELL Eioturdic~al

Dit%ior~.

77~

Sunrrrrl

Rohrrt.~

Nob/r

Forrndatiotr.

Irrc~.. Ardmorr.

Oklrrirr~t~cr

73-W/

Received October 29. 1979 An in .vifrlmethod for assaying ornithine decarboxylase of cells in tissue culture is described. Values obtained were higher than those derived from homogenates and sonicates. An added advantage of the irr sit/r assay was the use of the cultures following assay to determine cell nuclei and protein.

Current interest in the relationship of polyamine biosynthesis and growth has led to the development of assay methods for ornithine decarboxylase (EC 4.1.1.17: ODC’). Most have been modifications of the early methods described by Pegg and Williams-Ashman (1) or Russell and Snyder (2), who used I-[‘“Clornithine as a substrate. Released ‘“CO, was trapped and counted. Assays in general have been performed on homogenates or on cytosol fractions derived from centrifugation of homogenized or sonicated cell preparations. Recently the assay has been applied to wholecell suspensions of spleen and lymph node (3) and malignant ascites or fibrosarcoma (4). It was found that ODC activity was always “significantly reduced” when extracts of disrupted cells rather than whole cells were assayed. The current study was undertaken to determine if attached cells grown as monolayers in tissue culture could be assayed for ODC in .sirrr, thus avoiding potential enzyme losses and the time involved in manipulating cells to obtain cell fractions. ’ Abbreviations used: ODC, ornithine decarboxylase: pCMP. p-chloromercuriphenylsulfonic acid: PLP. pyridoxal 5’.phosphate; DTT, dithiothreitol: FBS, fetal bovine serum; TCA, trichloroacetic acid: cAMP. cyclic AMP. 0003.2697/80/080452-05$02.00/O Copyrrght ‘I 1980 by Academic Press, Inc. All right< of reproductmn rn any form reerved

452

MATERIALS

AND METHODS

Reagents. The following reagents and suppliers were used: L-[ I-“Clornithine, Aquasol. New England Nuclear: p-chloromercuriphenylsulfonic acid monosodium salt (pCMP). pyridoxal 5’-phosphate (PLP). DL-dithiothreitol (DTT), semicarbazide, Tris.HCI buffer, putrescine dihydrochloride. amino-oxyacetic acid, rotenone, and I.-ornithine, Sigma Chemical Company; ethanolamine, Eastman; citric acid, sodium hydroxide, Mallinckrodt; ethylene glycol monomethyl ether, Fisher Scientific; trichloroacetic acid (TCA). Sargent- Welch: cu-difluoromethyl ornithine. Centre de Recherche Merrell International. Strasbourg, France; a-methyl ornithine, Calbiochem. Tissue culture medium, McCoy’s 5a. was prepared in this laboratory according to the published formulary (5). Fetal bovine serum (FBS) was obtained from Reheis. Tissue culture c.~lls. Human diploid lung cells (WI-38. CCL 75) were purchased from the American Type Culture Collection at “passage” 19. Viral transformed WI-38 cells (VA13A) were supplied by DR. V. J. Cristofalo, The Wistar Institute, Philadelphia. and the BHK/C13 cells by Dr. Fred Grinnell, The University of Texas Southwestern Medical School, Dallas.

ORNITHINE TABLE Otwt-rtitI*iE

DW~RBOX~ CELL

I

LASE Ac I IVITY PRLPAKA-IIONS

ODC

tnmol

CO,!mg

2.01

z

Homogenate Supernatant Pellet Sonicate Supernatant Pellet

0.96 0.80

-t 0.03 e 0.04 Trace 2 0.05 2 0.03 Trace

0.87 0.75

15 VARI~LIS

protein/h)” BHK!Cl3

WI-38” 10 Situ

DECARBOXYLASE

0.19"l11)"

(8, (4) (8) (4)

8.53 5.92 6.13 0.27 6.45 6.13 0.13

+ i z -+ f i2

I.29 0.3 0.58 0.03 0.36 0.59 0.03

(8) (7) (4) (4) (7) (4) (4)

” For comparative purposes all values were normalized by maintaining equivalent sample sires and using protein values from the irl .rirlr cultures. ” WI-38 cells between “passages” 97 and 37. IO h serum stimulation; BHKiCl3 values 4 h after serum stimulation. Values are means i SE. ” Number in parentheses is number of assays. Homogenate and sonicate values were obtained from IO pooled cultures, each from which replicate assays were prepared.

Cell inocula were prepared (6), introduced into plastic T-25 flasks, and grown on McCoy’s 5a medium supplemented with 10% FBS. Activation of ODC was achieved by l8- to 24-h serum starvation in McCoy’s 5a medium minus serum followed by replacement with McCoy’s 5a medium supplemented with 10% FBS (time 0). Ettzyttte c~~sc~.v.For the in sitrt assay the tissue culture medium was removed, the cell layer washed three times with saline or a balanced salt solution, and the flask drained briefly by inversion. Control flasks (inactivated enzyme) were prepared at this point by exposing the flasks to 620 W (2.5 GHz) microwave irradiation for 30 s (7). With the culture flasks upright the following assay reagents were added, either singly or for routine purposes as a mixture, by pipetting such that they did not wash over the cell layer: 0.2 ml. 0.5 mM PLP: 0.2 ml, 25 mM DTT; 0.4 ml, L-[‘Vlornithine prepared by mixing the isotope with 1 mM carrier r.-ornithine to give 250-300 dpmi

ASSAY

453

nmol: and 1.2 ml of 0.1 M Tris HCl buffer, pH 7. I. For reagent controls the PLP was replaced by 0.2 ml of 50 mM semicarbazide and the DTT by 0.2 ml of 1 mM pCMP. A 1 x l-cm square of Whatman 3MM chromatography paper, folded in half, was immersed in ethanolamine:ethylene glycol monomethyl ether (2: 1) and blotted briefly on a paper towel. The saturated paper was placed into the well of a reaction flask stopper assembly (Kontes glass). The assembly was used to stopper the flask by folding the rubber cap over the flask opening lip. The reaction was initiated by laying the flask horizontally and incubating at 37°C. Standard incubation in this study was 2 h. although this time can vary depending upon the initial activity of the isotope and the sensitivity of the isotope detection. The reaction was terminated by injecting I.0 ml of I M citric acid through the rubber stopper. Incubation was continued for 3045 min to allow release and trapping of the ‘TO,. The flasks were unstoppered and the saturated paper squares transferred to Biovials (Beckman) containing 2 ml Aquasol and 0.1 ml H,O. The vials were agitated and allowed to stand several hours before counting to establish equilibrium. An added advantage to this assay is that the material remaining in the flask can be used for direct assay of protein or for nuclei counting. For the protein measurement the cell layer. after the ODC assay was completed, was scraped and a l.O-ml aliquot treated with 0.2 ml of 60% TCA. The solution was chilled for 30-60 min and centrifuged. and the precipitate drained. The precipitate was dissolved in 1.0 ml of 0.5 N NaOH and the protein determined on an aliquot by the Lowry procedure (8). For nuclei counts the cell layer was scraped and an aliquot of the cell suspension mixed with crystal violet solution (0.1%’ in 0.1 hf citric acid) at a 1: I or greater dilution. Following aspiration several times in a Pasteur pipet.

454

PATTERSON

the nuclei were counted in a hemocytometer. Homogenate and sonicate preparations were made by adding to each T-25 flask of washed monolayers 1.2 ml of a homogenizing buffer prepared by mixing 50 ml of 0.1 M Tris.HCl buffer, pH 7.1, and 10 ml solution containing 10 mM Tris.HCl, pH 7.1, 0.1 M EDTA, 50 pm PLP, and 50 pm DTT, and scraping the cell layer. The resultant cell suspensions were pooled (e.g., from 10 T-25 flasks) and either sonicated, using four 50-W pulses for 5 s each, or homogenized in a Potter-Elvehjem tissue grinder. Fractions were prepared by refrigerated centrifugation at 45001: for 15 min. Replicate ODC assays of these preparations were on aliquots equivalent to one T-25 culture. RESULTS To establish the validity of the in situ assay and its relationship to published procedures, comparative assays were carried out on two cell lines using the in situ assay and the methods described for disrupted cell preparations. The results are shown in Table 1. All homogenate and sonicate assays were made on samples equivalent to one T-25 flask, i.e., 1.2 ml. Protein measurement indicated up to a 30% loss during cell harvest and disruption. The ODC activities in Table 1 were therefore normalized on the basis of sample equivalence and the assumption that no protein losses resulted in handling. In situ ODC values assayed 45 to 130% higher than homogenates or sonicates. Good recovery (relative to homogenate or sonicate) of activity in the supernatants was found, with only traces of activity detected in the pellets. To determine if the lower values obtained from the disrupted cell preparations were due to losses incurred during the incubation period, a time curve was established. Figure 1 shows that both the irz situ and homogenate assays were linear through 2 h incubation.

AND

MAXWELL

O-

3.0 HOURS

INCUBATION

FIG. 1. Comparison of WI-38 VA13A ODC values derived from in siru assay (0) and homogenate (0) at various incubation times. Assay conditions were described under Material and Methods. Points on curves are means -t SE (vertical bars) from three assays.

Although L-ornithine, labeled only in the carboxyl carbon, was used as a substrate, the specificity of the decarboxylase was determined. Table 2 shows that PLP was required for maximum enzyme activity and that ODC inhibitors, a-methyl ornithine and difluoromethyl ornithine. significantly reduced enzyme activity. The reaction product, putrescine, inhibited the reaction with a logarithmic fashion. Moreover, the reaction proceeded under anaerobic conditions (9) and in the presence of 0.01 mM amino-oxyacetate or 0.1 mM rotenone (10) where other pathways that could decarboxylate ornithine are inactive. Following 18-h serum starvation the enzyme was activated 2- to 3-fold in WI-38 cells and 8- to 17-fold in BHKICl3 cells after addition of serum-supplemented medium. Addition of I .O mM dibutyryl cyclic AMP and 1.O mM theophylline resulted in a 24-fold increase in BHK/C13 cells (data not shown). Serum activation was also used to demonstrate application of the assay. Figure 2 shows the activation curve of ODC in WI-38 cells and its transformed counterpart, WI-38 VA13A, following repletion of serum to 18-h serum-starved cultures. WI-38 cells showed a 2-fold increase in enzyme activity

ORNITHINE

DECARBOXYLASE TABLE

ORNIT

HINE

DECARBOXVLASE

A~TIVI

2

11 ASSAYED

In

Sir/r

ODC

‘I WI-38 cells between “passage” stimulation. ” Values are mean i- SE. ’ Number in parentheses is number ” Cultures continued on 5a medium

CO,/mg

k f_ -+ +

0.19” (Ill’ 0.09 (6) 0.03 (6) 0.10 (71

40.7 12.4 71.1

I.27 1.94 1.18 0.37 2.62

2 2 2 -t f

0.11 0.13 0.02 0.02 0.06

60.0 96.5 58.7 18.4 130

(71 (4) (41 (4) (7)

37. 10 h after

of assays. without serum

80

E \ .g 6.0 x

IO AFTER STIMULATON

FIG. 2. ODC values of WI-38 (01 and WI-38 VAl3A (0) cells using ilr situ method following serum starvation and subsequent addition of complete growth medium. Values after the addition of 10 mM Lasparagine are also shown (B). Assay conditions were described under Material and Methods. Points on curves are means k SE (vertical bars) from four experiments for the WI-38 cells and three for the WI-38 VAl3A with at least three replicate tubes for each point.

serum

as negative

CONDITIONS

protein/h) ‘1; Control

BHK/Cl3

2.01 0.82 0.3 1.45

37 and

V.~RIOUS

‘; Control

at 8 h, while the WI-38 VAl3A showed a 5-fold increase at 6 h. Addition of 10 mM L-asparagine resulted in a 4-fold activation in WI-38 cells.

HOURS

UNDER

(nmol

WI-38 Control Minus serum” Minus pyridoxal 5phosphate Plus 0.5 ITIM a-methyl ornithine Plus 0.01 mM difluoromethyl ornithine Plus I .O mM putrescine Plus 10.0 mM putrescine Plus 100 mM putrescine N, atmosphere Microwave irradiation (30 s)

455

ASSAY

stimulation:

8.53 i- 1.19 (8) 1.75 + 0.06 (2) 0.93 (1) 4.50 + 0.13 (2)

20.5 10.9 52.7

2.48 t 0.36 (2)

79.0

9.57 f 0.34 (2) 0.09 i 0.01 (3)

II?. 1.0

BHKIC13

values

4 h after

controls.

DISCUSSION

An in situ method for assaying ornithine decarboxylase in tissue cultured cells has been shown to offer several advantages over “conventional” methods. First, the true enzyme activity may best be demonstrated by the method, since the values obtained using the in situ method were higher than those found in the “disrupted cell” procedures. The lower values obtained in the latter procedures appear to reflect losses incurred during cell harvest and preparation and not during incubation. Second, the time element involved is reduced considerably in the in situ method. since cell harvest and preparation is eliminated. Third, following assay the cell nuclei can be enumerated and the protein measured on the same cell population used in the assay. ODC in the cell lines tested responded to either serum depletion-repletion, CAMP. or L-asparagine in a manner and magnitude similar to that previously reported (9. I I - 131. Confirmation that ODC

456

PATTERSON

AND

was the enzyme responding was shown by inhibition of the enzyme activity with DLa-methyl ornithine ( 14, IS), DL-a-difluoromethyl ornithine (16). and putrescine (17), the product of the reaction. Inactivation of enzyme activity occurred following microwave irradiation of the cultures for 30 s (7). Moreover. the expected pyridoxal Sphosphate dependency (1.18,19), the lack of dependency on atmospheric oxygen (9). and the absence of ornithine aminotransferase activity (IO) were demonstrated. Thus, ODC behaves in the it? situ assay as expected from previous reports using the “conventional” assay system for the enzyme.

9.

Vaage. 39,

5. Patterson. (1978) 6.

E..

and

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