Effects of conditioned medium on nucleoside uptake, cell cycle progression and apparent DNA repair

Cells in culture can ‘condition’ the medium in which they grow, changing its capacity to promote further cell growth. Prolonged conditioning has generally a nonspecific inhibitory effect; this may involve nutrient depletion (Holley, Armour & Baldwin, 1978a), excretion of ammonia and lactic acid (Holley, Armour & Baldwin, 19786), or release of growth-inhibitory macromolecules, which have been partially characterized (Whittenberger & Glaser, 1977; Natraj & Datta, 1978; Holley et al. 1980; Strobel-Stevens & Lacey, 1981; Voss, Steck, Calamia & Wang, 1982). Cells grown to confluence and kept in conditioned medium can be held conveniently in a quiescent, non-proliferating state.

Studies involving the regulation of cell growth control employ the release from quiescence by serum or other factors. But quiescence is also useful as a period during which DNA repair can be studied without the complications of cell proliferation. Quiescent cells can recover from damage that would be lethal to proliferating cells, if the lesions only become lethal in 5 phase and can be repaired in quiescence. This quiescent repair of potentially lethal damage has been frequently investigated in confluent populations kept in the medium they have conditioned, to avoid the mitogenic stimulus of medium with fresh serum (e.g. Little, 1970; Hahn, Bagshaw, Evans & Gordon, 1973; Weichselbaum, Nove & Little, 1978; Simons, 1979; Chan & Little, 1979; Konze-Thomas, Levinson, Maher & McCormick, 1979; Iliakis & Pohlit, 1979; Pohlit & Heyden, 1981; Malcolm, Tomkinson & Little, 1982). Conditioned medium has also been reported to improve the survival of proliferating Chang liver cells after X-irradiation (Little, 1970, 1973) and Chinese hamster V-79 cells after ultraviolet (u.v.) irradiation (Nakano, Yamagami & Takaki, 1979), again by apparently suppressing replication and allowing more time for the repair of potentially lethal damage.

But there is a possible difficulty in measuring DNA repair in the presence of conditioned medium. Some of the commonest methods for detecting DNA repair require the repairing cells to take up nucleosides from the medium; sometimes radioactively labelled thymidine, which is then incorporated by repair synthesis into DNA where it can be detected in non-S-phase cells by autoradiography (Cleaver & Thomas, 1981) or in non-replicated DNA by the bromodeoxyuridine/caesium chloride gradient method (Smith, Cooper & Hanawalt, 1981). Alternatively, a very sensitive repair assay requires the repairing cells to take up l-/J-D-arabinofuranosyl cytosine (cytosine arabinoside; araC) which is converted to araCTP, an inhibitor of DNA repair synthesis but not of the previous excision repair step of DNA strand incision; this causes DNA strand breaks to accumulate at repair sites, and these are easily detected (Collins & Johnson, 1981). However, we have observed that medium conditioned by quiescent fibroblasts of Microtus agrestis, the field vole, inhibits the uptake of both thymidine and araC by those cells, and can seriously interfere with repair assays (Johnson, Collins, Downes & Squires, 1982; Downes, Johnson & Collins, 1982).

We present here evidence that this effect of conditioned medium on nucleoside uptake is widespread but not uniform, and must be seriously considered in studies of repair and proliferation.

Cells were grown in plastic Petri dishes at 37°C in a 5% COz atmosphere in Earle’s minimal essential medium, supplemented with non-essential amino acids, penicillin and streptomycin, and with various levels of serum: 12% heat-inactivated foetal bovine serum (FBS) for early-passage Chinese hamster untransformed fibroblasts; 10 % heat-inactivated FBS for mouse myeloma ascites cells (strain NS-1, provided from peritoneal culture by Dr D. W. Taylor, Department of Pathology, Cambridge), mouse embryo fibroblasts (provided by Dr G.C. Elliott) and Microtus agrestis established fibroblasts (strain MA Cantab); 10% non-heat-inactivated FBS for human embryo diploid fibroblasts (strain BCL-D1); and 2% heat-inactivated FBS plus 3% newborn calf serum for HeLa and CHO-K1 cells.

Quiescent cultures of Microtus fibroblasts were obtained as previously described (Downes et al. 1982); cultures were grown to confluence and then given step-down medium with 1 % foetal bovine serum for 3 days. Quiescent butyrate-detransformed Chinese hamster ovary (CHO) cells were obtained by 3 days growth with 5 mM-sodium butyrate till DNA synthesis (as measured by incorporation of [³H]thymidine into trichloroacetic acid-insoluble material) had ceased, and the cells had become elongated and fibroblast-like (Storrie, Puck & Wenger, 1978). CHO cell cultures in exponential growth were allowed to condition their media for 24 h following a 1:3 split of subconfluent cultures. Other cultures were allowed to condition their media for up to 7 days. Conditioned media were briefly centrifuged to remove debris, and warmed to 37 °C before being added to dishes of cells that had been rinsed with warm Dulbecco’s phosphate-buffered saline (PBS) after their media had been removed.

Uptake of ³H-labelled nucleosides was measured by a modification of the method of Rogers et al, (1980). Cells in monolayer culture in 30mm dishes were labelled with ³H-labelled cytosine arabinoside or other nucleosides at 37 °C. For zero-time control experiments, ³H-labelled medium was put into the dishes and immediately removed with a Pasteur pipette; otherwise, dishes were incubated at 37 °C for various times, and then rapidly transferred to ice-cooled metal trays, washed four times with about 4ml/dish ice-cold PBS, and then extracted three times with 0·7 ml/dish of 50% ethanol. The pooled extracts from each dish were added to 10ml Tritosol (Fricke, 1975) for scintillation counting. Numbers of cells per dish were estimated by Coulter counting of trypsinized parallel cultures. The rate of uptake was sometimes calculated by a least-squares fit to the uptake data for incorporation into the soluble pool.

Some ethanol extracts from [³H]araC-labelled cells grown in 90 mm dishes were prepared similarly, with proportionally larger volumes. They were lyophilized, and each residue dissolved in 50μl water. Samples (20 μ l) were loaded onto 5 cm × 20 cm polyethyleneimine-cellulose thin-layer plates (Machery-Nagl), and subjected to ascending chromatography in a 1:4 mixture of 3M-LiC1/1M-acetic acid for about 2h. Mobilities of araC, araCMP and araCTP standards were observed under ultraviolet (u.v.) light. Dried thin-layer plates were cut into 5 mm strips, and put in vials for scintillation counting.

Quiescent cultures of Microtus fibroblasts in 30 mm dishes, after 3 days in confluence in 1 % serum, were stimulated either by fresh medium containing 10 % foetal bovine serum, or by medium conditioned by quiescent Microtus cells, but supplemented with 10% FBS and 2 mM-glutamine. S phase progression was monitored by cumulative incorporation of [³H] thymidine as previously described (Downes et al. 1982). In some dishes, cells were held in mitosis by 6 h periods of treatment with 0· lμ g/ml Colcemid, added at various times after stimulation; mitotic indices were measured in cytocentrifuge preparations of trypsinized cells, stained with crystal violet.

u.v.-dependent DNA incision was measured as described by Collins & Johnson (1981), using hydroxyurea plus araC, or aphidicolin, which is also a potent inhibitor of u.v.-induced DNA synthesis (Collins, Squires & Johnson, 1982), to inhibit the post-incision stages of excision repair and so to accumulate DNA strand breaks at excision repair sites. Cells so treated were lysed in 0· 15 M-NaCl, 0· 1 M-NaOH, 0· 01 M-EDTA, causing the DNA to unwind around the strand breaks; the DNA was then sheared, and single- and double-stranded fractions were separated by hydroxyapatite chromatography. In this system, the fraction of DNA converted to the singlestranded form by alkaline lysis is a function of the abundance of DNA strand breaks, and so of the abundance of sites at which excision repair has been inhibited.

[5-³H]araC (15-5 Ci/mmol), [methyl-³H]thymidine (41-44 Ci/mmol), [1′, 2‱-³H]bromodeoxy-uridine (41 Ci/mmol), [8-³H]deoxyguanosine (5 Ci/mmol), [G-³H]deoxyadenosine (12Ci/mmol), and [5-³H]deoxycytidine (2 Ci/mmol), were obtained from Amersham International. Unlabelled chemicals were obtained from Sigma, except for aphidicolin (a gift from I.C.I.) and tetrahydrouridine (from Calbiochem). Culture media, Colcemid and sera were obtained from Gibco Europe Ltd.

Different cell lines may condition their media so as to inhibit, more or less, the uptake of both thymidine and araC, or araC but not thymidine, or neither. Figs 1-3 show some examples of this variability. Affcroius-conditioned medium partly inhibits the uptake of both thymidine and araC by Microtus cells; residual uptake is greatest at early times after addition of conditioned medium, but continues at a linear rate from 10-30 min (Fig. 1). A quite different effect is shown with CHO-conditioned medium acting on BCL-D1 cells (Fig. 2); here, araC uptake shows a very slight initial rise and is then completely inhibited, while thymidine uptake is unaffected. (CHO cells themselves respond similarly to CHO-conditioned medium, though the inhibition of araC uptake is not so extreme; see below, Table 2.) But though BCL-D1 cells can respond to inhibitory conditioning factors, they do not release them; BCL-D1 conditioned medium has no effect (Fig. 3).

We have tried a limited number of permutations of responses of cell lines to conditioning factors, mostly using the very sensitive BCL-D1 line as responder, and measuring uptake over the period 10-20 min after addition of conditioned medium, when the effect is fully established. The approach is not recommended for detailed studies of uptake mechanisms, but is sufficient to indicate whether or not conditioning factors are likely to interfere with other experiments. We have concentrated on inhibition of araC uptake, which appears to be vyidely distributed; we realise that varieties of conditioning effects other than those reported here are quite possible, and that some conditioned media may well block the uptake of other nucleosides but not of araC. A summary of various results is shown in Table 1. It is apparent that the susceptibility of BCL-D1 cells to inhibitory factors is not much affected by their growth state; cells from passage 18 to passage 30, and from 1 to 15 days after subculturing, all respond to CHO-conditioned medium. But the ability to condition medium with inhibitory factors is unevenly distributed among cell lines: Microtus, CHO, early-passage Chinese hamster and mouse normal fibroblasts, and late-passage (transformed) mouse fibroblasts can condition medium; HeLa, BCL-D1 and mouse ascites cells cannot, nor does fresh mouse serum have any effect.

And though, as Table 1 shows, the conditioning effect is usually not connected with the growth state or transformation of the conditioning line, there is one exception. Table 2 demonstrates the effect of treatment with sodium butyrate on CHO cells; the quiescent, morphologically detransformed CHO can still respond to normal CHO-conditioned medium, but they have lost the ability to condition it themselves.

As would be expected from the effect on araC uptake, medium conditioned by CHO cells has a considerable effect in reducing the apparent rate of repair of u.v. damage in human diploid cells, as measured by the accumulation of DNA breaks in the presence of hydroxyurea and araC (Fig. 4A). But an alternative assay, using the terpenoid antibiotic aphidicolin to accumulate strand breaks, shows no such effect (Fig. 4B). We conclude that the real rate of u.v. excision repair is not affected by CHO-conditioned medium. This is analogous to the failure of A/zcro/ws-conditioned medium to inhibit apparent excision repair in Microtus cells when hydroxyurea alone, or hydroxyurea and very large amounts of araC, are used (Downes et al. 1982).

Microtus-conditioned medium not only keeps confluent cells in quiescence (Collins & Johnson, 1979), it prevents them from responding to fresh serum. This is shown in Fig. 5; the addition to confluent cultures of 10% fresh foetal calf serum in fresh medium produces a wave of cell proliferation, which is greatly reduced in cells given fresh serum and conditioned medium. (The reduced [³H]thymidine incorporation into DNA might be due to reduction of [³H]thymidine uptake; the reduced mitotic accumulation cannot be.)

That the difference in uptake between cells in fresh and conditioned medium is due to inhibition of the latter rather than stimulation by the former is shown in Fig. 6. The addition of 5 % fresh serum to CHO-conditioned medium does not remove its inhibitory effect on araC uptake in BCL-D1 cells (cf. Fig. 2); conversely, cells in PBS take up araC as in complete fresh medium.

The inhibition of araC uptake takes effect at the stage of araC phosphorylation (Fig.7). When the alcohol-soluble extracts from Microtus cells, given [³H]araC in the presence of fresh or A/zcroZus-conditioned medium, are analysed by thin-layer chromatography it is evident that the difference in uptake is due to a reduction in araCTP and araCMP formation in the cells given conditioned medium. (araCDP levels are too low to detect.)

We have examined the specificity of nucleoside uptake inhibition by CHO-conditioned medium (Table 3). Of the nucleosides tested, only araC and deoxycytidine are affected. (The uptake rate for the thymidine analogue 5-bromo-deoxyuridine, incidentally, is far less than that for thymidine. Mattern (1980) has shown that human diploid cells preferentially incorporate thymidine, rather than bromodeoxyüridine, into their DNA; this discrimination seems to be exerted at the uptake stage.) Microtws-conditioned medium has a broader range of uptake inhibition (Fig. 1), which we have not investigated fully.

araC and deoxycytidine are both substrates for the degradative enzyme cytidine deaminase (Camiener, 1968). But Table 3 also shows that tetrahydrouridine, a potent inhibitor of cytidine deaminase (Cohen, 1977), does not relieve the specific inhibition of araC uptake by CHO-conditioned medium.

The variation in the capacity of cells to condition their media so as to block nucleoside uptake is as unexpected, and as yet inexplicable, as the effect itself. The inhibition by conditioned media is quite separate from the stimulatory effect of fresh serum on nucleoside transport, which initially operates only on ribonucleosides and does not affect araC (Rozengurt, Stein & Wigglesworth, 1977). The inhibition by conditioned medium is not overcome by fresh serum, but can be removed simply by placing the cells in buffered saline solution. Three mechanisms for this inhibitory effect seem possible. Inhibitory conditioned media may contain factors that inhibit the uptake enzymes, or compete with added nucleosides in the uptake process, or degrade nucleosides faster than they can be taken up.

Nucleoside uptake in cultured cells is generally believed to be a two-step process: nucleosides are transported across the plasma membrane by facilitated diffusion, then phosphorylated to nucleotides to which the membrane is impermeable and which become the predominant form within a few minutes of the start of uptake. The transport system is fairly non-specific, being unable to distinguish between deoxyribonucleosides (or arabinosides), and rapidly reaches equilibrium with the external medium, generally in less than 15 s; there are several base-specific kinases, whose action is generally rate-limiting and which are responsible for the control of uptake (see Plagemann & Wohlhueter (1980) for a review).

Inhibition of the non-specific transport system is ruled out, at least for CHO-type conditioned medium. But since araC and deoxycytidine uptake both rely on deoxycytidine kinase for the phosphorylation step (Plagemann, Marz & Wohlhueter, 1977), the common effect on araC and deoxycytidine could be exerted at the kinase level. Certainly, Microtus-conditioned medium seems to block araC phosphorylation (Fig. 7) (the rather large amount of unphosphorylated araC seen here, unaffected by conditioned medium, is probably due to the difficulty of washing a large dish of Microtus cells thoroughly without detaching the thick confluent layer).

Competition for the uptake system by components of conditioned medium is possible. Conditioned medium from 3T3 mouse cells has been shown to inhibit uridine uptake (Pariser & Cunningham, 1971), and this may be due to the excretion of uridine into the medium, which occurs as cells enter quiescence and reduce their RNA content (Uziel & Selkirk, 1980). Whether deoxyribonucleosides are also excreted, and how CHO cells would excrete only deoxycytidine, we do not know. Such a competitive mechanism could explain the finding that sufficiently high levels of araC -around 10 mM -can overcome the apparent inhibition of formation of u.v.-induced DNA breaks byMicrotws-conditioned medium (Downeset al. 1982); this is not easily reconcilable with a kinase inhibition.

The third possible mechanism, stimulation of degradation of nucleosides by conditioned medium, would also be consistent with ineffectiveness at high araC concentrations. It is consistent with most of the other data; for degradation of araC or deoxycytidine by cytidine deaminase would yield uracil arabinoside or deoxyuridine, which on phosphorylation would be rapidly methylated to thymine arabinoside, or thymidine phosphates. The tritiated araC and deoxycytidine used here was labelled only at the 5’ position, and this label would be lost on methylation, which would give an apparent inhibition of uptake and of nucleoside phosphorylation; and uracil or thymine arabinosides are very poor inhibitors of DNA synthesis (Müller et al. 1978; Müller & Zahn, 1979) and may not cause DNA strand breaks to accumulate. But if such a degradative pathway exists, it is unexpectedly insensitive to tetrahydrouridine.

Whatever the mechanism by which the inhibition of uptake works, its existence is of considerable importance in DNA-repair studies. It is not yet possible to predict whether, or when, a given cell line will condition its medium so as to block nucleoside uptake (it may be only a coincidence that all the lines that have yet been shown to do so are of rodent origin, not human). But if a cell line has a Microtus-type effect, any of the popular, simple assays for DNA repair will be seriously misleading in the presence of conditioned medium. It should be noted that we have not established how long conditioning takes -more than 30 min but less than 24 h. Quite short exposures may well be effective.

Whether the inhibition of nucleoside uptake is related to the growth-inhibitory properties of conditioned medium remains to be seen. Stimulation of quiescent cells into proliferation usually involves a stimulation of ribonucleoside, and later of deoxyribonucleoside, uptake. It has been shown by Schor & Rozengurt (1973) that serum stimulation of DNA synthesis in quiescent mouse 3T3 cells can be enhanced by nucleosides in the medium; conceivably, suppression of nucleoside uptake by conditioned medium may directly maintain quiescence. In any case, any measurements of proliferation involving [³H] thymidine incorporation may be sensitive to Microtus-type conditioning. It would be advisable to test for inhibition of nucleoside uptake in any experiment where cells are given nucleosides in the presence of anything but fresh medium. And since araC is a commonly used antitumour drug (Cohen, 1977) the possibility should be considered that its uptake into target cells may be regulated by diffusible factors.

We are grateful to the Cancer Research Campaign of which R.T. J. is a Research Fellow, for continued support, and to R. G. W. Northfield for skilled technical assistance.