Human diploid fibroblasts with alterations in ribonucleotide reductase activity, deoxyribonucleotide pools and in vitro lifespan

Mechanisms o f Ageing and Development, 26 (1984) 37-49

37

Elsevier Scientific Publishers Ireland Ltd.

HUMAN DIPLOID FIBROBLASTS WITH ALTERATIONS IN RIBONUCLEOTIDE REDUCTASE ACTIVITY, DEOXYRIBONUCLEOTIDE POOLS AND I N VITRO LIFESPAN

JOHN E. DICK and JIM A. WRIGHT* Departments of Microbiology and Biochemistry, University o f Manitoba, and Manitoba Institute of Cell Biology, 100 Olivia Street, Winnipeg, Manitoba, R3E OV9 (Canada)

(Received September 7th, 1983) (Revision received January 11th, 1984)

SUMMARY Three drug resistant human diploid fibroblast clones were isolated which contained elevated levels o f ribonucleotide reductase activity when compared to wild type fibroblasts. The drug resistant cells do not appear to possess an enzyme with altered affinity for hydroxyurea. The increase in enzyme activity can entirely account for cellular drug resistance. In keeping with the observed changes in reductase activity in drug resistant fibroblasts, deoxyribonucleotide pools were also found to be altered. Most significantly, there was a 1.8-fold expansion of the dCTP pool size, suggesting that elevation in intracellular dCTP concentrations plays an important role in cellular resistance. Furthermore, the drug resistant fibroblasts exhibited substantial reductions in their replicative abilities, suggesting that the regulation of ribonucleotide reductase and the accompanying deoxyribonucleotide pools in human diploid cells is involved in aspects of cellular senescence. K e y words: Ribonucleotide reductase; Deoxyribonucleotides; Hydroxyurea resistance;

Senescence ; Genetics INTRODUCTION In mammalian cells, a key control point in the replication of DNA occurs in the reduction of ribonucleoside diphosphates to the deoxyribonucleotide precursors of DNA [ 1 - 3 ] . This reaction is catalyzed by the highly regulated ribonucleotide reductase [1 ], and alterations in this key activity have wide ranging consequences for the cell [3]. For example, the enzyme appears to play an important role in tumor transformation [4,5], certain immunodeficiency diseases in man [6], alterations in spontaneous mutation rates of cultured cells [7,8], and in certain aspects of cellular differentiation [9]. Furthermore, *To whom correspondence should be addressed.

38 structural features of the enzyme are unusual [10-13], and during DNA synthesis the reductase appears to be physically and functionally associated with other enzymes of DNA synthesis, in a multienzyme complex which channels deoxyribonucleotides to the replication forks [14,15]. From biological, regulatory, and enzyme structural points of view, ribonucleotide reductase is one of the most complex enzymes in the cell [3]. In a previous communication, we showed that ribonucleotide reductase activity declines significantly during cellular senescence [16]. This observation, along with earlier findings of a mutator gene associated with the mammalian reductase [7,8], suggested a possible mechanism of cellular aging which involves changes in reductase activity and the accompanying deoxyribonucleotide pools during cellular senescence. To test this hypothesis further, we have used hydroxyurea as a selective agent in culture to isolate, for the first time, human diploid fibroblasts with changes in ribonucleotide reductase activity. Our studies with these fibroblasts provide further evidence for a link between ribonucleotide reduction, deoxyribonucleotide pools, and changes in cellular senescence. MATERIALS AND METHODS

Cells and growth conditions The fetal lung fibroblast strain, HSC172, kindly provided by Dr. Buchwald, University of Toronto, was derived from a 12-week-old female fetus [17]. In agreement with other studies (e.g. ref. 17), we have observed that the karyotype of these cells is normal 46, XX. Cells were routinely maintained on 100 mm plastic tissue culture plates (Lux Scientific Corp.) in a-minimal essential medium (t~-MEM; Flow Laboratories) supplemented with penicillin (100 units/ml), streptomycin sulfate (68 /ag/ml) and 15% fetal bovine serum (Gibco Laboratories) preselected for high plating efficiency characteristics. The cells were subcultured according to a rigid protocol using 1:4 split ratio, counting two mean population doublings (MPD) each time [19-21]. Purified trypsin (Sigma Chemical Co.) at 0.1% in phosphate-buffered saline (PBS) was used to remove cells from the surface of plates. The effect of hydroxyurea on the colony forming ability of HSC172 cells was determined by incubating varying numbers of cells in 100 mm tissue culture plates containing growth medium with and without drug. After about 14 days, the number of surviving colonies with more than 15 ceils was counted. Relative colony forming ability is defined as the ability to form colonies in the presence of drug divided by this ability in the absence of drug. Assay for ribonucleotide reductase activity in intact cells We have described an assay for ribonucleotide reductase using permeabilized human diploid fibroblasts [16,22]. The important features of the assay are as follows. Exponentially growing HSC172 cells were plated at a density of 2 × 106 cells per 150 mm plate and incubated at 37°C for approximately 40 h. Cells were removed with 0.1% trypsin (Sigma), centrifuged, resuspended in growth medium and counted using a particle counter

39 (Coulter Electronics). The cell pellet was resuspended at 5 × 106 cells/ml in permeabilizing buffer consisting of 1.0% Tween 80 (J.T. Baker Chemical Co.), 0.25 M sucrose, 0.05 M N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer, pH 7.2, and 2 mM dithiothreitol (DTT). The cells were incubated at 30°C for 75 min, after which they were centrifuged and resuspended in fresh permeabilizing buffer as described above. The cell suspension was then dispensed at 200 tzl/tube into reaction tubes containing 100/al of reaction mixture. The final assay for CDP reduction consisted of 0.05 M Hepes pH 7.2, 6 mM DDT, 8 mM MgC12, 4 mM ATP, 0.4 mM 14C-labelled CDP (5000 cpm/ nmol, Amersham Corporation), 0.67% Tween 80, 0.167 M sucrose, and 1 × 106 to 3 Z 106 cells. A 2 × 7 mm magnetic stirring bar was placed in each assay tube, and the reaction tubes were incubated at 370C for 30 min in a water bath on top of a magnetic stirrer (Belco Glass Co.) operating at high speed. The reactions were terminated by boiling for 4 min, after which 2 mg/assay of Crotalus atrox venom in 0.1 M Hepes buffer, pH 6.8, containing 10 mM MgCI2 were added to each tube. Cells were incubated for a further 2 h at 37°C. The reaction was terminated by boiling and the deoxycytidine formed was measured as described by Steeper and Steuart [23]. The deoxycytidine was eluted from a Dowex-1 borate ion-exchange resin (Bio-Rad Laboratories) with 5 ml of water. Radioactivity was determined by adding Scintiverse II cocktail (Fisher) to the 5 ml of water containing deoxycytidine. Enzyme activity was expressed as nmoles of product formed per h per 3 × 106 cells. Selection experiments Low MPD cells (MPD between 10 and 32) were plated at 8 × 10 s cells per 100 mm tissue culture plate and grown for 18-24 h, at which time the growth medium was removed and replaced with medium containing 100 mM hydroxyurea. Cells (a total of 2 × 106 cells/experiment) were cultured in the presence of drug for 6 0 - 7 5 h and then removed with trypsin solution and subcultured at 1 : 2 or 1 : 4 dilution. The cells were refed with growth medium without hydroxyurea and incubated at 37°C. This removal of cells with trypsin was necessary since a large fraction of cells incapable of cell division would not lift off the plates after drug treatment, and would hinder the formation of colonies by cells capable of proliferation. Three to four weeks later colonies were observed on some of the plates against a light background of non-dividing cells which may be acting as a feeder layer for proliferating cells. The largest colonies of about 1 cm in diameter were removed using glass cloning cylinder or were picked with a sterile Pasteur pipette and transferred to 60 mm tissue culture plates for incubation. When these plates contained confluent cultures, the cells were frozen in liquid nitrogen and tested for drug sensitivity. In some experiments, colonies appearing after drug treatment were not picked; instead the culture was grown to a partial monolayer and then subjected to one or more additional rounds of drug treatment as described above, before isolating a colony for testing. When cultures were treated with ethyl methane sulfonate (EMS; Eastman) prior to selection for drug resistance, low MPD HSC172 cells were added at 4 X 105 cells per 100 mm plate and cultured for 18-24 h. EMS was then added to these cells at a final

40 concentration between 300 and 400/ag/ml and incubated for about 24 h. This treatment resulted in a reduction in colony forming ability of 50-90%. The viable cells were then allowed to grow to confluence, subcultured, and used in the selection procedure described above.

Determination o f nucleo tide poo ls The levels of intracellular deoxyribonucleotide pools in wild type and drug resistant cells were measured by high-performance liquid chromatography (HPLC) using a modification of the procedures of Garrett and Santi [24] and Hartwick and Brown [25]. The details of these modifications will be described elsewhere (Creasey and Wright). Briefly, cells were plated at a density of 2 × 106per 150 mm tissue culture plate and grown for about 40 h. Wild type and drug resistant cells were harvested and extractions of each were made at the same time at 4°C [26]. The cells were removed with trypsin (0.2%), counted, and centrifuged in as short a time as possible. The cell pellets were resuspended at 5 × 107 cetls/ml in PBS and a small aliquot of [3H] thymidine was added to estimate the dilution factor. This resuspension was then transferred to glass test tubes with sufficient 70% perchloric acid to achieve a final concentration of 0.5 M, and the tubes were placed on ice for 30 min. Diethyl pyrocarbamate (100/A/ml) and tri-N-octylamine in Freon (2 vols. to 1 vol. of extract) were added to the extract and vortexed for 2 min. The tubes were then centrifuged at low speed for 2 min, and the supernatant was collected and immediately frozen at --65°C. The separation and identification of the deoxyribonucleotides first required the destruction of ribonucleotides with periodate and methylamine [24]. The extracts were then separated by HPLC on an anion-exchange resin [27]. A Beckman model 332 gradient liquid chromatograph consisting of two model 110A pumps coordinated by a model 420 system controller programmer was used. The nucleotides were detected with a Beckman model 160 detector at 254 nm, with maximum sensitivity of 0.001 AUFS (absorbance units full scale). A Partisil 10/25 SAX (Whatman) strong anion-exchange column (25 cm X 4.6 mm) preceded by a guard column (7.0 cm × 2.0 mm) was used in the separation process. Good resolution of the four deoxyribonucleotides was achieved using isocratic elution with 0.25 M KH2PO4 (Fisher, HPLC grade) and 0.25 M KC1 (Fisher, ACS grade), pH 5.0 at a flow rate of 2 ml/min. All buffers were filtered through a 0.45 /am membrane filter and degassed prior to use. Identification was by retention times as compared to known standards. The peaks were quantitated on the basis of peak weight. RESULTS

Selection o f drug resistant lines Hydroxyurea resistant Chinese hamster ovary and mouse L cells can be isolated at a frequency of about 10-5 by adding 5 × l0 s cells to 100 mm culture plates containing growth medium supplemented with drug [ 2 8 - 3 0 ] . Attempts to use a similar procedure

41 to isolate human diploid fibroblasts resistant to hydroxyurea were not successful, since killing of these cells by hydroxyurea was cell density dependent even at low cell concentrations. When cells were plated at less than 1000 cells per 100 mm culture plate in the presence of hydroxyurea, colonies formed in approximately two weeks. However, at greater cell concentrations distinct colonies were not observed; instead, nearly all the cells remained attached to the plate and proliferated to form a monolayer culture. Cultures could be maintained in this state for up to several months, with little detachment o f cells from the plate surface. This ability of human diploid fibroblasts to remain quiescent for months has been documented [18]. Clearly, if rare drug resistant clones were to be isolated, a selection scheme would have to be used which involved large numbers o f cells and reduced attachment of non-proliferating cells. This was achieved by exposing the fibroblasts to 100 mM hydroxyurea for a relatively short period o f time and then plating the cells in the absence of drug, as described in Materials and Methods. As shown in Fig. la, this procedure leads to several logs of killing as determined by colony forming ability; however, the slope of the killing curve is rather steep so that drug concentration, length of exposure, and cell density are critical for colony development and data reproducibility. Furthermore, we have previously reported a correlation between cellular senescence and increased sensitivity to hydroxyurea [16]. This phenomenon is evident in Fig. 1, where it can be seen that cell cultures at higher MPD are significantly more sensitive to drug than lower MPD cultures. As shown in Fig. lb, three fibroblast clones were isolated which exhibited drug resistance properties as determined by colony forming ability in the presence of various concentrations of hydroxyurea. For comparison, the colony forming abilities of two 1.0 >

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Fig. 1. (a) Relative colony forming ability of HSC172 MPD 40 (D) and QCI-1 (o) after exposure to 100 mM hydroxyurea for various lengths of time. (b) Relative colony forming ability of HSC172 MPD 10 (+) and MPD 77 (o), QCI-1 (*), WCI-2 (o), and MCI-1 (o) in the presence of various concentrations of hydroxyurea. Similar results with each culture have been obtained in three separate experiments.

42 drug sensitive cultures at low and high MPD (10 and 77) are also shown. Details of the selection procedure used to isolate these drug resistant clones are described in Table I. The clones WCI-2 and MCI-1 showed a Dlo value o f about 1.4 mM hydroxyurea which is 2-fold higher than the Dr0 for the low MPD wild type culture and 3-4-fold greater than the high MPD culture. Clone QCI-1 was less resistant than WCI-2 and MCI-1 but exhibited drug resistance properties when compared to wild type fibroblast cultures. In addition, Fig. la shows that QCI-1 is more resistant than wild type cells when resistance is determined using short exposure to high concentrations of drug, as was carried out in selection experiments. Senescence o f hydroxyurea resistant cultures It is well documented that normal human diploid fibroblasts exh~it limited proliferative abilities in culture [18,22,31]. This reduced capacity for cell division has been

TABLE I LIFESPAN OF WILD TYPE AND MUTANT CLONES Steps in isolation o f mutants

MCI-1 Start Mutagenesis Selection Regrowth Selection Regrowth Selection Cloning

MPD during selection

17_+2

71

18_+2

76

20_+3

57

4 3 14 9

32 15 11 26

QCI-1 Start Mutagenesis Selection Cloning

Cumulative total

15

39 WCI-2 Start Selection Cloning

MPD after cloning

10 4 9 14 27

Wild-type HSC172 *An identical result has been reported by Gupta using HSC172 [ 191

95 -+5*

43 studied extensively as a model for aging at the ceUular level. Table I shows that the three drug resistant clones and the wild type human diploid fibroblasts also exhibit a limited replicative lifetime in vitro. The number of MPD that accumulated during selection of drug resistant cultures was determined by actual cell counts except in the cloning steps where the number of cells per colony was estimated from colony size. These values closely matched the estimated number of MPD accumulated during isolation of mutant cell lines in other studies [32]. Interestingly, these calculations indicated that there was a significant decrease in the lifespan of the three drug resistant clones as compared to the wild type fibroblasts. It is important to note that a great deal of heterogeneity of lifespan exists among the individual cells of a population, with low MPD populations showing a distinctly bimodal distribution of doubling potentials [21,33]. The individual cells in the high doubling potential mode have lifespans similar to the mass populations, and generally are capable of forming large colonies [33]. The mutant selection scheme outlined above inherently selects for cells with high growth potential since only large colonies (>1 cm in diameter) were isolated from drug selection plates.

Ribonucleotide reductase activity Previous studies with permanent cell lines have shown that hydroxyurea resistant cells contain alterations in the activity of ribonucleotide reductase, a key rate-limiting step in DNA synthesis [3,29,30]. Since drug resistant human diploid fibroblasts senesce in culture at earlier MPD (Table I) only a limited amount of material was available for enzyme studies. However, the intact cell assay system developed for analyzing ribonucleotide reductase activity in permeabilized human diploid fibroblasts [16,22] allowed us to investigate the reductase in resistant and wild type ceils. We have shown that ribonucleotide reductase levels change during the lifespan of human diploid fibroblasts [16]. In order to compare enzyme activity in drug resistant and wild type cells accurately it was necessary to match these cells with respect to MPD prior to senescence. Wild type HSC172 cells, under our culture conditions, senesced after 95 -+ 5 MPD whereas the drug resistant cultures senesced within 20 MPD after isolation (Table I). Therefore, wild type cells that were between MPD 75 and 90 were used for comparative purposes in enzyme experiments. Figure 2a shows that the three drug resistant cultures contain elevated levels of ribonucleotide reductase activity when compared to wild type cells. The increased levels of enzyme activity, between 2- and 3.5-fold, correlate very closely to cellular resistance t o hydroxyurea (Fig. lb). To test whether or not drug resistant cells possess an enzyme with altered sensitivity to hydroxyurea, the activity of ribonucleotide reductase was examined in the presence of 0.5 mM hydroxyurea, a concentration which reduces the wild type activity by approximately 50%. There appears to be only slight differences in enzyme sensitivity to drug among the various cultures, suggesting that drug resistance'in these human diploid fibroblasts is due to an overproduction of normal enzyme activity (Fig. 2b).

44

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Fig. 2. (a) R e l a t i v e CDP reductase activity in QCI-1, WC1-2 and MCI-1 compared to age matched wild type H S C 1 7 2 cells. Results are from an average of eight determinations for QCI-1 and WC1-2, and three determinations for MCI-1. (b) CDP reductase activity remaining in the presence of 0.5 m M hydroxyurea. Results are from an average of at least three determinations for each culture.

Deoxyribonucleotide pools Alterations in ribonucleotide reductase activity due to the effects of inhibitors or mutations affecting enzyme regulation can lead to perturbations in intracellular deoxyribonucleotide concentrations [ 3 4 - 3 7 ] . The effects of adding hydroxyurea to cultured cells remain somewhat inconclusive as various studies using different analytical methods show a variety of pool changes; however, there seems to be general agreement that pyrimidine pools are rapidly depleted [35,38]. Several studies have suggested that the level of the dCTP pool correlates more closely than the three other deoxyribonucleotide pools, to DNA synthesis [39], cell proliferation [40] and the mutagenic nature of altered ribonucleotide reductase regulation [37]. Therefore, we examined the deoxyribouucleotide pool sizes in wild type and drug resistant human diploid fibroblasts. Figure 3 shows that there is a significant (1.8-fold) expansion of the dCTP pool and slight increases in dTTP and dATP concentrations. In addition, there was a large decline in the dGTP pool, although it should be noted that the level of dGTP was very low in wild type cells as well, almost at the limit of detection (<10 pmol per 2 × 106 cells).

45 2.0 O O x (t)

1.5 I,Z Z U,.I

(9 z

1.0

TTP

dCTP

dATP

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Fig. 3. Relative deoxyribonucleotide pool sizes in WCI-2and wild type cells. Two separate experiments were performed and similar results were obtained. DISCUSSION We describe for the first time a procedure for isolating hydroxyurea resistant human diploid fibroblasts. Over the course of about two years, sixty selection experiments have been carried out, each with 2 × l06 cells. From a total of 1.2 × 108 cells 150 colonies which developed after drug treatment were retested for hydroxyurea resistance, and three were found to exhibit stable drug resistance properties. This indicates that the frequency of hydroxyurea resistant cells in these human diploid fibroblasts is very low (about 2.5 × 10 -8) when compared to permanent cell lines such as mouse L and Chinese hamster ovary cells [29,30,41], but resembles frequencies reported for other drug resistant markers in human diploid fibroblasts [ 17,42]. Although we have not determined if EMS increases the mutation frequency for hydroxyurea resistance, it should be noted that the mutation frequency in untreated cultures may be lower than our estimate since two of the clones, MCI-I and QCI-1, were isolated from EMS treated populations. Also, in some selection experiments, cell cultures were treated several times with hydroxyurea, whereas in others the ceils were exposed to a single drug treatment (Table I). Therefore, the mutation frequency of 2.5 X 10-a should be considered as only a rough estimate. Studies of ribonucleotide reductase activity in hydroxyurea resistant permanent rodent cell lines have detected three drug resistant classes [3,29,30]. Class one drug resistant ceils contain an enzyme less sensitive than wild type to drug inhibition; class two cell lines overproduce enzyme activity with a wild type sensitivity to drug; and class three cells overproduce enzyme activity less sensitive than wild type to drug inhibition. The three drug resistant human diploid fibroblast clones showed elevated levels of ribonucleotide reductase activity similar to class two and three rodent cell lines. Although detailed drug inhibition studies were not carried out, it appears that the human drug resistant cells contain enzyme activity with wild type sensitivity to hydroxyurea, suggest-

46 ing that they belong to the class two category of drug resistance. In support of this view, the 2- to 3.5-fold increase in enzyme activity can totally account for resistance to hydroxyurea at the cellular level. In keeping with the observed changes in ribonucleotide reductase activity in drug resistant fibroblasts, it was found that the deoxyribonucleotide pools, the products of the reductase reaction, were also altered. Although several changes were noticed, including a decline in dGTP concentration, the most significant change was a 1.8-fold expansion of the dCTP pool size. These findings suggest that the elevated dCTP pool in resistant cells may play a special role in cellular resistance, and support previous observations that the levels of CDP reductase are particularly important for resistance mechanisms involving hydroxyurea and other drugs with a similar mode of action [43]. Ribonucleotide reductase is responsible for providing a continuous and balanced supply of the deoxyribonucleotide precursors essential for DNA synthesis. The fidelity of DNA replication is influenced by the relative concentrations of the deoxyribonucleotide pools, and contributes to the characteristic mutation rate of the cell. The importance of this point is emphasized in the recent findings of a mutator gene associated with mammalian ribonucleotide reductase, which results in increased rates of spontaneous mutation due to an imbalance of endogenous deoxyribonucleoside triphosphate pools [7,8]. In a previous study, we showed that there are significant changes in ribonucleotide reductase levels during senescence of normal human diploid fibroblasts [16]. In view of the hypothesis that randomly accumulating mutations incapacitate somatic cells and are involved in aging [44,45], modifications in ribonucleotide reductase activity (along with accompanying changes in deoxyribonucleotide pools) could be a mechanism whereby the reductase participates in an aging process [16]. This study supports the above hypothesis but does not eliminate the possibility that ribonucleotide reductase may be an important protein involved in a programmed mechanism of aging. This latter theory predicts that several key proteins govern senescence in much the same way as they are involved in cellular differentiation [46]. It is appropriate to note that ribonucleotide reductase appears to play a role in aspects of cellular differentiation [9] and it has been suggested that one of the subunits of the reductase may belong to a small group of proteins that are directly involved in regulating cell cycle progression [12]. Our results provide further evidence for a link between altered ribonucleotide reduction, changes in deoxyribonucleotide pools, and modifications in cellular senescence, since estimates of the in vitro lifespans of the three drug resistant human fibroblast clones showed that they experienced substantial reductions, in the order of 20-40%, in their proliferative abilities when compared to cultures of normal human diploid fibroblasts. It is worth noting that human mutants selected for resistance to other agents such as ouabain [46], 8-azaguanine [47], c~-amanitin [17], diphtheria toxin [48] and ouabain and 6-thioguanine [49] do not appear to exhibit alterations in their lifespan. These observations support the idea [16] that ribonucleotide reduction has a unique role to play in the senescence of cells in culture. Whether this mechanism involves a general change in the balance of endogenous deoxyribonucleotide pools or is due to more specific pool alterations is unknown. In this regard, it would be worth investigating further the

47 changes in dCTP, as it has been suggested that the level of this pool correlates more closely to DNA synthesis than any of the other three deoxyribonucleotides [39], and it has recently been reported that dCTP has an affinity for DNA,v-polymerase [50], which along with ribonucleotide reductase, appears to be a component of a multienzyme complex involved in the synthesis of DNA [14,15]. Furthermore, perturbations in dCTP concentrations are important in mutagenesis mechanisms [8,37]. ACKNOWLEDGEMENTS Research funds were provided by the Natural Sciences and Engineering Research Council of Canada and the National Cancer Institute of Canada to J.A. Wright. J.E. Dick acknowledges receipt of graduate scholarships from the Natural Sciences and Engineering Research Council of Canada and the Manitoba Health Research Council. We also thank David C. Creasey for helpful discussions and aid in setting up the HPLC protocol. J.A. Wright is a recipient of a Terry Fox Cancer Research Scientist Award.

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