J. Cell Sci. 68, 305-319 (1984) 305Printed in Great Britain © The Company of Biologists Limited 1984

ARGININE SYNTHESIS BY HEPATOMAS IN VITRO

II. ISOLATION AND CHARACTERIZATION OF MORRISHEPATOMA VARIANTS UNABLE TO CONVERT ORNITHINE TOARGININE, AND MODULATION OF UREA-CYCLE ENZYMES BYDEXAMETHASONE AND CYCLIC-AMP

S. J. GOSSSir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, U.K.

SUMMARY

'77orn', a derivative of the Morris rat hepatoma 7777, stably expresses high levels of ornithinetranscarbamoylase (OTC) and carbamoylphosphate synthetase I (CPS-I), and is able to growindefinitely in ornithine-medium (medium with ornithine in place of arginine). Variants that havelost this ability are isolated from 77orn by a 'suicide' selective technique dependent on the cellularincorporation of [3H]ornithine. These variants, which have reduced levels of CPS-I, or of bothCPS-I and OTC, are shown to have developed multiple hormonal requirements; their enzymedeficiencies can be reversed by use of an appropriately supplemented medium. In particular,CPS-I is inducible by dexamethasone and dibutyryl-cyclic-AMP in combination. Cholera toxin canbe used instead of cyclic-AMP, but then butyrate is additionally required if the induction is to bemaintained in the long term. The use of these agents in excess can depress OTC. Several otherhepatomas, and also explanted foetal rat liver cells, have similar requirements for CPS-I expression.It is argued that multiple hormonal requirements for CPS-I production are normal in liver cells invitro, and that hormone-independent hepatomas should be regarded as abnormal. The implicationsof this for the somatic cell genetic investigation of differentiation are briefly discussed.

INTRODUCTION

The preceding paper (Goss, 1984) describes the growth of various rat hepatomasin vitro in a medium containing ornithine in place of arginine. This medium selectsfor the growth of well-differentiated hepatomas possessing both ornithine trans-carbamoylase (OTC) and carbamoylphosphate synthetase I (CPS-I), the liver-specific urea-cycle enzymes required for the conversion of ornithine to citrulline. Thesubsequent steps of the urea-cycle, leading to the synthesis of arginine, are catalysedby enzymes found in most tissues of the body and in most cultured cell lines. Variantsthat had lost the ability to grow in ornithine-medium were isolated from the hepatoma0i, a derivative of the widely used line H4-II-E (Pitot, Peraino, Morse & Potter,1964). Two classes of these orn~ variants were identified. In both it was the tissue-specific conversion of ornithine to citrulline that had failed; there were unstablevariants whose ability to grow in ornithine-medium could be restored by the inclusionof 50 nM-dexamethasone in the medium, and there were relatively stable variants thathad suffered a general loss of liver-specific traits. It was argued that neither class ofvariants was suitable for genetic tests aimed at investigating the control of the produc-tion of urea-cycle enzymes; accordingly, it seemed worthwhile our attempting toisolate further, different, variants.

306 5. J. Goss

This paper describes the isolation of orn~ variants from an orn+ derivative of theMorris hepatoma, 7777. It was hoped that the use of a hepatoma not related to 0iwould improve our chances of finding new types of variant. Furthermore, a newselection method was adopted in place of BrdUrd (bromodeoxyuridine)/light treat-ment. Cells were grown for several hours in the presence of [3H]ornithine. orn+ cellsthen synthesized radiolabelled arginine, incorporated it into their proteins, andsubsequently were lethally damaged by self-irradiation. Similar selection by 'suicide'following 3H-labelled amino acid incorporation has been used successfully by manyauthors (e.g. Thompson, Stanners & Siminovitch, 1975; Dantig, Slayman & Adel-berg, 1982). There might be some advantage in selecting against liver-specific traitswithout the use of BrdUrd, since BrdUrd has often been shown to interfere with theexpression of tissue-specific genes (Bischoff & Holtzer, 1970; Davidson & Kaufman,1977). Finally, to avoid the isolation of more dexamethasone-requiring variants,50 nM-dexamethasone was included in the medium used in these experiments. It waspossible in this way to isolate a series of variants with differing hormonal requirementsfor the maintenance of OTC and CPS-I. These requirements were found to resemblethose of normal liver. The orn~ 'variants' then appear to approximate more closely tothe 'wild-type' than do the so-called 'minimal deviation hepatomas' that possess theseenzymes in the absence of hormonal supplements.

MATERIALS AND METHODS

Cells and cell cultureThis paper is primarily concerned with the Morris hepatoma, 7777 (Morris & Wagner, 1968).

Other cells used are the Chinese hamster fibroblast line, wg3-H (Westerveld, Visser, Meera Khan& Bootsma, 1971), and the rat hepatoma, HTC (Thompson, Tomkins & Curran, 1966). Referenceis made to Fu5-TG1 and to 0i and its variants. These derivatives of the H4-II-E rat hepatoma aredescribed in the preceding paper (Goss, 1984). That paper also describes the basic media (arginine-medium, citrulline-medium, ornithine-medium, and medium without tyrosine) that are usedthroughout the present work. The addition of hormones and other supplements to these media isdescribed in Results. All cells were maintained as monolayer cultures by standard techniques.

Enzyme assays

The assays for ornithine transcarbamoylase (OTC, EC 2.1.3.3) and carbamoyl phosphatesynthetase I (CPS-I, EC 2.7.2.5) are described in the preceding paper (Goss, 1984). The resultsgiven below are from single assays, it having been established that duplicate assays on any onesample generally agreed to within 10%. Assays on replicate cultures of 7777 derivatives agreedsurprisingly well with each other (see CPS-I assays on 77varl in Table 1). This uniformity clearlycontributes to the production of smooth dose-response relationships between enzyme activity andthe dose of inducing agents (see Tables 2, 3 and 5).

Isolation oforn ~ cells by a pH]ornithine suicide techniqueThis method has been employed extensively on 77orn cells and occasionally on 0i cells. In some

instances the cells were first mutagenized (details are given in Results). The cells were brought intoa single-cell suspension by trypsinization at 37 °C for 10—15min in a mixture of phosphate-bufferedsaline (PBS), Na2EDTA (0-25g/1), trypsin ('1:250 trypsin', Difco, Detroit, 0-125%, w/v) andglucose (lg/l)- They were collected by centrifugation, pipetted gently to resuspend them inmedium, and plated in 75 cm2 plastic tissue-culture flasks (lCr cells/flask, in arginine-medium with50 nM-dexamethasone). After an overnight incubation at 37 °C, these cultures were washed, and the

Hormonal regulation of OTC and CPS-I 307

medium was replaced with ornithine-medium containing [3H]ornithine (L-[2, 3, -3H]ornithine,20Ci/mmol, 10/iCi/ml, from New England Nuclear) in place of the normal, non-radioactive,ornithine. L-proline (0-35 min) and L-glutamic acid (1 mM) were added to this medium in order toquench the incorporation of labelled proline and glutamate (label can be transferred from ornithineto these amino acids by the action of ornithine-<5-amino transferase; see Results). The cultures wereincubated at 37 °C for 5 h, to allow orn+ cells to synthesize proteins labelled with [3H]arginine. Afterthis labelling period, the radioactive medium was removed, the cells were gently washed with PBS,and then they were incubated for a further 45 min in non-radioactive ornithine medium. During thistime, the unincorporated [3H]ornithine was lost from the cells. Finally, the cells were harvested andfrozen by standard techniques in medium with 10% serum and 10% dimethylsulphoxide. Thefrozen samples were stored over liquid nitrogen for up to 5 weeks, to allow the labelled cells toaccumulate damage. After thawing, the survivors were grown up in arginine-medium with 50 nM-dexamethasone. A variation of this technique, in which the cells were treated before the labellingperiod with theophylline and cholera toxin, is described in Results.

Preliminary investigations of the labelling of cells in medium containing radioactive ornithinewere done using confluent coverslip cultures. These cultures were incubated for 5 h in labellingmedium like that described above, but containing [14C]ornithine (L-[l-14C]ornithine, 57mCi/mmol, 0-5/^Ci/ml, from Amersham) instead of [3H]ornithine. At the end of the labelling period,the coverslips were transferred to ethanol to fix the cells, and then to 5 % trichloroacetic acid toextract the unincorporated label (extraction for 20 min at 4 °C). Finally, the coverslips were washedwith water, dried and put in scintillation vials for scintillation counting in Unisolve E (Koch LightLaboratories, U.K.).

RESULTS

Horn, an orn+ derivative of 7777

The Morris hepatoma 7777 has a low specific activity of CPS-I and therefore cannotgrow in ornithine-medium (i.e. 7777 is orn~). However, when plated in ornithine-medium, 7777 readily gives rise to clones that have more CPS-I and are stably orn+

(Goss, 1984). Several hundred clones arising from one such plating were pooled,designated 77orn, and used as starting material for the experiments described in thispaper. The growth of 77orn in ornithine-medium is facilitated by the addition ofdexamethasone (50 nM) to the medium, though this supplement is not essential.(Dexamethasone further increases CPS-I in these cells; cf. Goss (1984).) 77ornretains high levels of both OTC and CPS-I even when it is grown in non-selectivemedium: cells kept growing for 5 weeks in arginine-medium plus 50 nM-dexamethasone were assayed at intervals; OTC ranged between 80 and 185, and CPS-I between 4-5 and 8-6nmol/min per mg. 77orn can also grow in medium withouttyrosine. This ability is correlated with the presence of another liver-specific enzyme,phenylalanine hydroxylase (Choo, Cotton &Danks, 1976; Haggerty, Young &Buese,1975).

Killing of77orn by incorporation of radioactivity from [3H] ornithine

If the incorporation of radio-label from ornithine is to be used successfully to selectagainst cells able to convert ornithine to arginine, then that conversion must be themajor pathway by which the cells incorporate ornithine. Widman, Golden & Chasin(1979) have remarked that [14C]ornithine can be incorporated by other routes. TheH4-II-E derivative they studied lacked OTC and so did not convert ornithine to

308 5. jf. Goss

arginine, nevertheless there was significant incorporation of label from ornithine intoneutral and acidic amino acids. This labelling, which probably involved mainlyproline and glutamate, would have proceeded via ornithine-<5-aminotransferase(OAT), an enzyme known to be present at high levels in rat liver and kidney (Peraino& Pitot, 1963; Herzfeld & Knox, 1968). Fortunately, 77orn was found to have a lowlevel of OAT, comparable to that in fibroblasts. In contrast, the H4-II-E derivativesavailable in this laboratory have considerably more OAT. (OAT, sp. act. nmol/minper mg: published result for human skin fibroblasts, 2 units (O'Donnell, Sandman &Martin, 1977). Our results: 77orn in arginine-medium+50nM-dexamethasone, 6-5units; wg3-H Chinese hamster fibroblasts in arginine-medium, 3 units; Fu5-TG1, anH4-II-E derivative, in arginine-medium, 90 units.) In preliminary trials of the[3H] ornithine selection technique, 77orn and wg3-H were compared for their incor-poration of label from ornithine and for the extent of the ensuing cell death. wg3-His a useful control since Chinese hamster fibroblasts lack not only OTC and CPS-I butalso argininosuccinate synthetase (Sun, Sun, Tennant & Hsie, 1979; Carritt, Gold-farb, Hooper & Slack, 1977), and so there can be no question of their convertingornithine to arginine. On the other hand, wg3-H should utilize ornithine via OAT ata rate similar to that obtaining in 77orn. In the presence of 0-35 mM-proline and 1 DIM-glutamate, the incorporation of label from [14C]ornithine into trichloroacetic acid-insoluble material was found to be 100 times faster in confluent coverslips of 77ornthan in similar confluent coverslip cultures of wg3-H (see Materials and Methods fordetails). When 77orn and wg3-H were subjected to the standard selection procedureinvolving growth in medium with [3H]ornithine followed by a period of storage inliquid nitrogen, it was found that the survival of 77orn was reduced to about five cellsin 105 after just 7 days' storage. (After 23 days' storage there was no further killing:when 5 X 105 77orn cells were plated out, 26 clones arose.) In contrast, there was nomeasurable killing of the hamster cells even after storage for 5 weeks. The rapid killingof most of the 77orn cells indicates that these cells must fairly uniformly incorporatelarge amounts of radio-label. The selection procedure achieves a massive killing oforn+ cells whilst leaving orn~ cells unscathed.

77varl, an orn~ derivative of Horn

77orn was put through a round of [3H]ornithine killing without prior mutagenesis.The treated cells were recovered after storage for 3 weeks and clones grew up with afrequency of about 10~5. Seven clones were isolated and were tested for growth inornithine-medium+50nM-dexamethasone. Of the seven, four grew immediately, andthe other three showed very extensive cell death, but yielded orn+ sub-clones with afrequency of about 10~4. The three orn~ clones were further tested and were foundto grow well in citrulline-medium. It follows that the defect in these clones lies in thesynthesis of citrulline from ornithine. This was confirmed by enzyme assay: the threeclones have normal levels of OTC, but their CPS-I is greatly reduced. Subsequentwork on these clones showed that they were essentially identical. The results obtainedfrom just one of them, 77varl, are considered below. Table 1 shows the preliminaryOTC and CPS-I assays on this clone.

Hormonal regulation of OTC and CPS-I 309

Table 1. Specific activities of OTC and CPS-I in 77orn and 77varl

Cell

77orn77varl77varl

Medium

Arg-med + 50 nM-dexArg-med + 50 nM-dexCit-med + 50 nM-dex

Sp. act.

OTC

120 (186)107Not done

(nmol/min per mg)

CPS-I

4-5 (8-6)0-23 ±0-05*0-3

77orn and 77varl were grown to confluency in the media indicated and assayed for OTC andCPS-I. Results in parentheses are assays on independent, equivalent cultures, med, medium; dex,dexamethasone; cit, citrulline.

•The CPS-I activity in 77varl was highly repeatable in independent cultures; the mean andstandard deviation of four determinations are given. Otherwise, CPS-I activities are rounded to thenearest 0-1 unit, and OTC activities to the nearest whole unit.

CPS-I is induced in 77varl by dibutyryl-cAMP and by cholera toxin

77varl is able to grow in medium without tyrosine, and so it clearly has not suffereda total loss of liver-specific traits (in contrast to variant Arg-6 of 0i; Goss, 1984). Itseemed possible that, like variants Arg-2 or Arg-20 of 0i (Goss, 1984), 77varl hadsome hormonal requirement for the production of CPS-I. In view of the general roleof cyclic-AMP (cAMP) in the coordination of liver metabolism, the effects of addingdibutyryl-cAMP (db-cAMP) to the medium were observed. A dose-dependent induc-tion of CPS-I was apparent by 40 h. This action of db-cAMP was facilitated by thefurther addition of the phosphodiesterase inhibitor, theophylline, to the medium.The slow onset of the induction of CPS-I (monitored over 48 h) is consistent with theinvolvement of new synthesis of protein (Table 2).

Since the continued presence of db-cAMP in the medium was found to besomewhat toxic, alternative attempts were made to raise the intracellular level ofcAMP by supplementing the medium with cholera toxin (Gill, 1977). That choleratoxin can induce CPS-I in 77varl is shown in Table 2. Unfortunately, the choleratoxin-supplemented media of Table 2 also proved too toxic to be suitable for long-term experiments. Various media were tried without theophylline, and with lesscholera toxin and less dexamethasone. Such media were indeed less toxic, but theydid not maintain the expression of CPS-I.

Both dexamethasone and cholera toxin are required for CPS-I induction

In the course of the investigations described in the preceding section, 77orn wasgrown for 3 weeks in arginine-medium without dexamethasone and theophylline, butwith 4 x 10~"M cholera toxin. The specific activity of CPS-I in these culturesremained less than 1 nmol/min per mg, the threshold believed necessary to supportgrowth in ornithine-medium (Goss, 1984). At the end of this period, the cells weresplit into a series of parallel cultures in the same medium but with a range of con-centrations of dexamethasone. One week later, at the end of the second passage in

310 S.jf.Goss

these new conditions, the cells were assayed for CPS-I and OTC. The results in Table3, taken together with the results in Table 2, show that, though neither dexametha-sone nor cholera toxin alone causes any significant induction of CPS-I, together theycan cause an induction of at least 20-fold. It also appears that dexamethasone andcholera toxin act together to cause a reduction of OTC.

Table 2. Induction of CPS-I in 77varl by dibutyryl-cyclic-AMP and by cholera toxin

MediumiTheophylline

————++++

+++

Theophylline

supplementsA

^

db-cAMP(ITIM)

_

0-030 10-3_0-030-10-3

0-30-10-1

Cholera toxin

Exposure time(h)

40404040404040

12448

CPS-I(nmol/min per mg)

0-2310-050-20-81-50-41-42-42-7

0-210-582-6

+ 4 X 1 0 " " M 48 2-0

+ 4 X 1 0 - ' ° M 48 3-1

+ 4x 10~9 M 48 Cells died

CPS-I was assayed in 77varl cultures near to confluency. The cultures had previously beenmaintained for the specified times in arginine-medum + 50 nM-dexamethasone with supplements asindicated. Theophylline was used at 0-5 miu.

Table 3. Effect of dexamethasone on CPS-I and OTC in 77varl grown in the presenceof cholera toxin

Sp. act. (nmol/min per mg)

Dexamethasone CPS-I OTC(mu)

0 0-4 661-85 2-8 635-56 3-9 41

16-7 4-5 3050 5-2 20

77varl cells were grown for 1 week in arginine-medium + cholera toxin (4 X 1 0 ~ " M ) withdexamethasone supplements as indicated. The cells were assayed at confluency and the resultsexpressed according to the conventions of Table 1.

For further details see the text.

Hormonal regulation of OTC and CPS-I 311

Selection of further orn~ variants of 77orn

In an attempt to isolate further variants, different from 77varl, 77orn cells weremutagenized overnight with ethylmethane sulphonate (400 or 680^ig/ml in experi-ments H and I, respectively). These treatments killed 70—90 % of the cells and raisedthe frequency of cells resistant to 1 |ig/ml 6-thioguanine from less than 10~6 in controlpopulations to 1-2(X 10~s). Following mutagenesis the cells were allowed to recoverin arginine-medium + 50 nM-dexamethasone for 3 days, and then cholera toxin(4 X 10~10M) and theophylline (0-5 ITIM) were added to the medium for a further 2days. After this the cells were subjected to [3H]ornithine selection and stored for 4weeks; the survival rate was 7 X 10~5. In all, 27 clones were isolated from the twoexperiments, H and I. There was no obvious difference between the two sets ofclones; their properties are described below.

Properties of H and I clones

The 27 clones were grown up in arginine-medium and then tested for their abilityto use either citrulline or ornithine as a precursor of arginine. The media used in allthese tests contained dexamethasone at 50 nM. All the clones grew well in citrulline-medium, but 26 out of the 27 failed to grow in ornithine-medium. Twelve of theseorn~ clones yielded orn+ sub-clones with a frequency of 10~4 to 10~5, and the otherorn~ clones showed no growth at all in ornithine-medium. A total of 17 orn" cloneswere tested for their ability to grow in medium without tyrosine (again with 50 nM-dexamethasone). Of these clones, 13 grew immediately, two grew after a brief delayand the remaining two died at the first test. However, after a further 2 weeks' growthin arginine-medium, this last pair of clones was retested and found to grow immediate-ly without tyrosine. The H and I orn~ variants have thus lost the ability to convertornithine to citrulline, and yet they have retained the ability to synthesize tyrosine;in this respect they resemble 77varl. However, in contrast to 77varl, most were foundto have reduced levels of OTC: of 22 assayed, 10 had less than lOnmol/min per mg,and only seven had more than 20 nmol/min per mg. CPS-I was assayed in 17 clones:it was in many cases undetectable, and otherwise greatly reduced (to about 0-1 nmol/min per mg). To test their responsiveness to cAMP, 10 variants were grown for 2 daysin arginine-medium with 50 nM-dexamethasone, 0-5 mM-theophylline and 0-1 ntM-db-cAMP. Only three of the 10 showed CPS-I induction resembling that seen in77varl. In summary, H and I orn~ variants commonly have low levels of OTC, andtheir CPS-I is in every case greatly reduced. Unlike 77varl, few of these variantsrecover their CPS-I in response to the standard dexamethasone+db-cAMP treat-ment. Two orn~ clones, H15 and 16, were chosen for further study. Neither has evergiven rise to orn+ derivatives when plated from standard arginine-medium toornithine-medium (both media with 50nM-dexamethasone).

Induction of OTC and CPS-I in the variants HI5 and 16

In a long series of experiments, most of which were conducted blindly on a trial-and-error basis, a new medium was devised that caused the restoration of CPS-I and

312 S.J.Goss

the partial restoration of OTC in both H15 and 16. The main steps in these experi-ments are shown in Table 4. It will be seen that after growth for about 1 month inarginine-medium + 50nM-dexamethasone, both cells had no detectable CPS-I andvery little OTC. Treatment of the cells with db-cAMP and theophylline had no effectin the short term, but did cause the appearance of a low level of CPS-I after 17 days.A large rise in OTC occurred in both variants after the withdrawal of dexamethasone(a depression of OTC by dexamethasone has already been noted in other circum-stances; see above). Some evidence was found that the inclusion of ornithine (5 miu)and high levels of TV-carbamoylglutamate 10 mM) in the medium caused increases inboth CPS-I and OTC, though these effects were small and difficult to reproduce. Incontrast, reducing the serum supplement from 10% to 1% regularly increasedCPS-I and often also increased OTC. These various conditions were combined in anew medium, medium 1 of Table 4. If this medium was further supplemented withcholera toxin (10~I 2M) and the potent phosphodiesterase inhibitor, isobutylmethyl-xanthine (IBMX, 30/ZM), there was a rapid induction of CPS-I in both variants(Table 4).

Adaptation of the variants to cholera toxin; the effects ofbutyrate

Cholera toxin was inhibitory to cell growth, even at 10~12M. The cells grew slowlywith a marked tendency to multinucleation leading to cell death. The use of choleratoxin at 10~13 M was tried, and it was found in the first instance to give an adequateinduction of CPS-I. However, with time, both H15 and 16 seemed to adapt to thistreatment and CPS-I activity became progressively reduced. This effect was more

Table 4. Restoration of OTC and CPS-I in the variants H15 and 16

Sp. act. (nmol/min per mg)

H15 16Growth conditions CPS-I/OTC CPS-I/OTC

Arginine-medium + 50 nM-dex 1st assay, day 1 0-1/17 0-1/72nd assay, day 14 Nil/4 Nil/13rd assay, day 24 Nil/6 Nil/1

Arg-med + 50 nM-dex + for 2 days Nil/n.d. Nil/n.d.

db-cAMP and theophylline for 17 days 0-5/6 0-3/nil

Arg-med - dex 1 month Nil/33 Nil/48

Medium 1 3 weeks 0-4/144 0-3/22Medium 1 + cholera toxin 4 days 9-1/28 1-2/14H15 and 16 cells were grown in various media for the times indicated. CPS-I and OTC assays were

done -on the cells at confluency. db-cAMP and theophylline were used at 0-1 mM and 0-5 min,respectively. Cholera toxin was used at 10~I 2M and isobutylmethylxanthine (IBMX) at 30/iM.Medium 1 is as arginine-medium, but contains only 1 % foetal calf serum, lOmM-A'-carbamoyl-glutamate (in place of TV-acetylglutamate), 5 mM-ornithine (in addition to the arginine), anddexamethasone (1-85 nM). n.d., not done; nil, <0-l unit CPS-I, or <0-2 unit OTC.

Hormonal regulation of OTC and CPS-I 313

Table 5. Adaptation of HIS cells to prolonged cholera toxin treatment; the effect ofbutyrate

Medium

Medium 1Medium 1 + IBMX +

cholera toxin, 10"13

Medium 2Medium 2 + butyrate,Medium 2 + butyrate,Medium 2 + butyrate,

M

0-0-1-

2mM5mMOmM

Exposure time(days)

214

101931

6, 126, 126, 126, 12

Sp. act. (nmol/min

CPS-I

0-43-33-81-00-9

0-4, 0-21-0,0-91-6, 1-72-6, 3-8

per mg)

OTC

14484683943

43, 5538,4630,3817, 14

H15 cells were treated as shown in the table and assayed for OTC and CPS-I. Medium 1 is definedin the legend to Table 4. IBMX was used at 30//M. Medium 2 is as medium 1, but has 5-56nM-dexamethasone, 5 X 10"13M-cholera toxin, and 30/iM-IBMX.

noticeable in H15, for which the results are set out in Table 5. Slightly raising thedexamethasone and cholera toxin supplements (medium 2) was of no assistance, butadding butyrate to the medium induced CPS-I in a dose-dependent fashion (Table5). In control experiments butyrate was found to have no effect on CPS-I in theabsence of cholera toxin (data not shown). Butyrate is probably acting to inducecholera toxin receptors; it is known to have this effect in several cell lines (Fishman& Atikkan, 1979). Butyrate (at 0-5 mM) also had the unexpected effect of improvingcell growth by preventing multinucleation. (Some multinucleation had still beenoccurring despite the use of cholera toxin at only 10~13M.)

Our subsequent experience has led to our adopting a growth medium ('inducingmedium') that consists of standard arginine-medium plus: ornithine (Snra), N-carbamoylglutamate (10 mM, in place of N-acetylglutamate), dexamethasone(5-56 niw), cholera toxin (5 X 1(T1 3M), IBMX (30 fwi), butyrate (0-5 mM) and foetalcalf serum 5%. H15 and 16 cells grow well and apparently indefinitely in thismedium. They continue to grow if they are transferred from this medium to anequivalent medium prepared without arginine and with dialysed serum; i.e. inducingmedium renders both H15 and 16 orn+. Inducing medium unfortunately does notsupport the growth of any of our hepatomas in low-density culture, even if arginineis included.

Induction of CPS-I in other hepatomasIt will be clear from the experiments described above that H15 and 16 require an

extraordinarily complex medium if they are to express significant levels of CPS-I andOTC. It is of interest to know whether these variants are extremely abnormal, or

314 S.jf.Goss

whether their requirements might be of some general importance. Several other orn~hepatomas were tested to see if they showed any response to inducing medium orclosely related media. These tests gave a number of positive results. The original 7777line, which has little CPS-I activity, shows CPS-I induction in inducing medium andis rendered orn+. The same is true of a 0i variant, Ad-1. This variant will not bedescribed here in any detail; suffice it to say that it was derived from 0i after ultravioletmutagenesis and selection with [3H]ornithine. Ad-1 closely resembles H-15, and soit belongs to a class of 0i variants distinct from those previously isolated by theBrdUrd/light technique (Goss, 1984).

Similar tests were also done on the hepatoma HTC. This hepatoma is orn~, it lacksdetectable OTC and has little CPS-I (<0-5 nmol/min per mg in arginine-medium + 50 nM-dexamethasone). Contrary to the reports of Naylor, Busby & Klebe(1976), we find HTC grows well in citrulline-medium. Inducing medium had noeffect on HTC, but CPS-I could be induced up to 10-fold if 0 1 mM-db-cAMP wasused in place of cholera toxin (HTC cells are apparently insensitive to cholera toxin).No treatment has yet been found that induces OTC in HTC cells. CPS-I can also beinduced in these cells simply by using arginine-medium with 1 %, instead of 10%,serum. This tendency of low concentrations of serum to mimick the effect of cAMPin inducing CPS-I has been seen above in the case of H15 and 16 (but there the effectwas smaller). It is possible that insulin (or some insulin-like factor) in the serum isacting to reduce intracellular cAMP.

Inducing medium and related media have also been tried on the 'stable' 0i variant,Arg-6D. This cell line differs from those described above in showing no response toany of the media. Tests are currently under way on partial revertants of Arg-6 thathave regained their ability to grow in medium with tyrosine. (Arg-6 and Arg-6D aredescribed in the preceding paper; Goss (1984).)

In conclusion, a number of hepatomas have been found whose requirements forCPS-I expression are similar, if not necessarily identical, to those of H15 and 16. Inthe following section this study is extended to include normal foetal liver cells.

Effect of inducing medium on foetal hepatocytes in vitro

Cultures of foetal hepatocytes were prepared essentially according to the methodof Leffert & Paul (1972). The cultures were initially set up in arginine-mediumwithout dexamethasone in 25 cm2 tissue-culture flasks. After 2 days, when confluentmonolayers had formed, each was split to a pair of flasks, one with arginine-containingmedium and one with ornithine in place of arginine. Paired flasks were prepared inthis way using either standard medium without hormones, or inducing medium. Theappearance of the cultures after twice weekly feeding for 6 weeks is shown in Fig. 1.

Fig. 1. Effect of inducing medium on foetal hepatocytes in vitro. Foetal rat hepatocyteswere cultured in plastic tissue-culture flasks (see the text for details). Phase-contrastmicrographs were taken of live 6-week-old cultures. Media were as follows: A, arginine-medium without dexamethasone; B, ornithine-medium without dexamethasone: C, induc-ing medium; D, inducing medium without arginine. In B and D ornithine is the onlyprecursor of arginine provided to the cells. X100.

Hormonal regulation of OTC and CPS-I 315

Fig. l

316 S.Jf.Goss

In ornithine-medium without hormones the cells mostly died; in the other media thecells survived well, but there was little growth. Typical hepatocyte morphology waspreserved in both cultures in inducing medium (i.e. both with and without arginine)but was rapidly lost in the culture in arginine-medium without hormones. Thearginine-containing cultures were assayed for OTC and CPS-I (at 6 weeks). OTC wasnot detectable in either culture. CPS-I was not detectable in the culture withouthormones, but was present at 2-7 nmol/min per mg in the culture in inducingmedium. In summary, inducing medium allows both the maintenance of hepatocytemorphology and the continued presence of CPS-I in cultured foetal liver cells. In-ducing medium enables foetal liver cultures to survive for at least 6 weeks in theabsence of arginine.

DISCUSSION

The experiments described in this paper were designed to produce orn~ varianthepatomas that differed from those already isolated from 0i (Goss, 1984). To this end,a different starting cell (77orn) was used, and a new selective procedure was adopted.The success of this approach appears to be attributable largely to the new selectiveprocedure, since, when this procedure was used on 0i cells, a variant (Ad-1) wasproduced that resembled the 77orn variant, H15, and was different from the 0ivariants previously isolated by the BrdUrd/light technique.

The orn~ variants described in this paper have revealed the importance of thehormonal composition of tissue-culture medium in maintaining the expression ofOTC and CPS-I in cells in vitro. That the requirements of the variants H15 and 16may be of general significance is suggested by the finding that the inducing mediumdesigned to restore OTC and CPS-I to these variants is also able to maintain bothCPS-I and hepatocyte morphology in explanted foetal rat liver. Furthermore, thereare similar requirements for CPS-I expression in 7777, Ad-1 and HTC. That it wasnot possible to preserve OTC levels in explanted liver shows that our present con-ditions of culture are still not optimal for normal hepatocyte function. This couldlikewise explain our inability to restore OTC to HTC cells.

The main active components of inducing medium have obvious natural counter-parts. It is reasonable to assume that the dexamethasone is acting as would a naturalglucocorticoid, and that the cholera toxin is mimicking the effect of some hormone(for instance, glucagon) that normally acts by elevating intracellular cAMP levels.Hepatomas are commonly unresponsive to glucagon, and cholera toxin can then beused in its stead (Wimalasena etal. 1981). Other workers have found that a combina-tion of dexamethasone and glucagon induces CPS-I in short-term cultures oi adult rathepatocytes. Some reports show rather small inductive effects (Lin, Snodgrass &Rabier, 1982; Gebhardt & Mecke, 1979), but another finds that both hormones areessential for the synthesis of CPS-I in such cultures (Rozen & Shore, 1982). Thissecond finding clearly agrees well with our experience.

It would appear that the variants H15 and 16 have hormonal requirements resemb-ling those seen in normal liver in vitro. It then follows that cells like 77orn and the

Hormonal regulation of OTC and CPS-I 317

H4-II-E derivatives, Fu5, Fu5-TG1 and0i (Goss, 1984), must be regarded as abnor-mal, since they express CPS-I in the absence of hormonal stimulation. We are atpresent investigating whether this abnormality involves enzymes other than CPS-I.H4-II-E derivatives are often described as 'minimal deviation hepatomas', but thiswould seem inappropriate if their continued expression of hepatic traits were to someextent due to an aberrant independence from normal hormonal requirements. Thiscomplication could influence the outcome of investigations into differentiation usingthese cells. It is interesting in this context to note that certain hybrids made with H4-II-E derivatives show little or no induction of tyrosine amino transferase (TAT) inresponse to either dexamethasone or cAMP alone, but nevertheless they show sig-nificant TAT induction if both agents are used together (Weiss & Chaplain, 1971;Leichtling et al. 1978). This finding suggests that the development of multiple hor-monal requirements might play some role in the 'extinction' of tissue-specific func-tions in hybrid cells.

Whatever the general significance of the various hormonal requirements of thehepatomas described in this paper, one conclusion is certain: careful attention mustbe paid to medium formulation if true genetic mutants affecting the production ofurea-cycle enzymes are to be isolated. At present we have no evidence that our variantsare indeed mutants. The relative ease with which they were obtained suggests we aredetecting a variation that occurs more frequently than mutation. This suggestion issupported by the lack of variants stably deficient in just one enzyme; in particular, thecomplete lack of variants unable to use citrulline indicates that the generation of truemutants is most probably obscured by the lability of expression of tissue-specificgenes. The stepwise isolation and characterization of increasingly fastidious hormone-requiring variants might facilitate the design of a medium that can suppress thislability. The difficulty in this approach lies in the complex interactions of hormones,and in the opposing effects that any one treatment might have on different enzymes.For example, it has been shown above that conditions inducing CPS-I tend to depressOTC. Other problems lie in the slow adaptation of cells to prolonged inducing treat-ments (Table 5), and in the tendency of inducing treatments to inhibit cell growth.Current work in this laboratory is aimed at producing still more variants, in the hopethat the use of more appropriate growth conditions will permit the isolation of truemutants suitable for the analysis of differentiation by the techniques of somatic cellgenetics.

The author is most grateful to Mrs Heather Smith for her reliable and energetic technical assis-tance, and for her enthusiasm and persistence (a valuable contribution at times when progress wasslow). She also made useful criticisms of the early drafts of this and the preceding paper. The workwas supported by the Cancer Research Campaign. The author also thanks Professor H. Harris andthe staff of the Dunn School for providing the support and encouragement necessary for this work.

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{Received 30 January 1984-Accepted 2 February 1984)