Isolation of variant cells with defective metabolic cooperation (mec−) from polyoma virus transformed Syrian hamster cells

ISOLATION METABOLIC VIRUS E. II.

OF VARIANT COOPERATION

TRANSFORMED WRIGHT,’

CELLS (MEC‘) SYRIAN

P. S. G. GGLDFARB

WITH

DEFECTIVE

FROM HAMSTER

POLYOMA CEI>LS

and J. H. SUBAK-SHARPE2

SUMMARY A selection system has been designed to select for variant cells having a reduced ability in metabolic co-operation (intercellular nuclcotide transfer). In this system. cells resistant to pyrimidine deoxyribonucleoside analogues or purine analogues (recipients) are repeatedly grown in dense culture with excess sensitive cells (donors) in the presence of the relevant analogue. When co-operation occurs. donor cells take up the analogue and nucleotidc derivatives are transtcrrcd to recipients. As a consequence, both donors and co-operating recipients die. Recipients which fail to co-operate survive. Using 5-bromodeoxyuridine (BUdR) as the selective agent. cells have been obtained with rcduccd ability to co-operate. as measured both hy autoradiography and a DNA separation method. ‘These cells, termed met -. are smaller. more epithelioid and have half the chromosome number of their met- parent’s mode. Using the purine analogue 8-azaadenine (MA). as the selective agent, the selection system did not produce cells with reduced ability for metabolic co-operation.

Intercellular communication involving direct molecular transfer can be studied by the use of mutant mammalian cell lines lacking certain enzyme activities of the purine and pyrimidine salvage pathways. These mutants are unable to incorporate exogenously supplied preformed purines and pyrimidine deoxyribonucleosides into nucleic acid, but do so in mixed culture when in direct or indirect contact with wild type cells. This observation was first made by Subak-Sharpe et al. [I] who called the phenomenon metabolic co-operation. The change in metabolic state of the mutant ceils * Present address: The Royal Free Hospital, Medicine. 8 Hunter Street, London WCIE\; z To whom reprint requests should be sent. 5

761X10

School of IBP. UK.

resulting from interaction with the wild type, is readily detected using autoradiographic techniques, thus providing a system for the study of this type of intercellular communication. Metabolic co-operation has been demonstrated between wild type cells and several different salvage pathway mutants lacking at least one of the following enzyme activities: hypoxanthine-guanine phosphoribosyl transferasc (HGPRT-) [ 1. 2], adenine phosphoribosyl transferase (APRT-) [3], thymidine kinase (TK-) f4]. or deoxycytidine kinase (dCK-) [5]. The requirement for cell contact in metabolic co-operation was demonstrated by two types of experiments [6]: (1) co-operation does not occur when co-cultures are grown at den-

64

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Wright, Goldfarb

1

and Subak-Sharpe

10

Id

IO>

IO 1 , 0 1

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Figs l-2. Abscissa: analogue cone. @g/ml); ordinate: cell yield. Fig. I. Total cell yields of PyY/HGPRT-/dCK-/TKcells grown for 7 days in the presence of varying concentrations of BUdR (W-W); CAR (0-U); 6-TG (O-O); or 8-AA (O-O), expressed as percentage of control with no analogue added. lo5 cells were seeded

in medium plus analogue and grown at 37°C. Each point represents the mean of two estimates. Fig. 2. Total cell yields of PyY/APRT- cells grown for 5 days in the presence of varying concentrations of 8-AA (0-O) or BUdR (W--M), expressed as percentage of control with no analogue added. Conditions asinfig. 1.

sities below that allowing the frequent formation of cell contacts, and (2) it cannot be induced by growing recipient mutant cells in medium conditioned by wild type cells, There is strong evidence [4,6,7, 81 that the material transferred between donors and recipients during co-operation are small molecules, probably phosphorylated nucleosides, which feed into the purine or pyrimidine biosynthetic pathways beyond the step blocked in the mutant, recipient cells. It has been shown [9] that preformed nucleotides are normally unable to enter cells through the plasma membrane, unless the phosphate moiety is lost. The co-operative ability of cells may therefore depend on locally modified membrane properties at the regions of cellular contact allowing nucleotide transfer. Of the several structurally specialized sites which have been identified in electron micrographs of junctional membranes, the gap junction is thought to mediate ionic coupling between cells [lo, 111.Observations by Gilula et al. [ 121,using and freeze-etching electrophysiological techniques, showed that metabolic cooperation in fibroblasts is associated with

both the presence of gap junctions and ionic coupling between interacting cells. Cocultures of Don (Chinese hamster) cells and their HGPRT- variants showed co-operation, ionic coupling and the presence of gap junctions between the two cell types. However, co-cultures of Don and A9 cells (an HGPRT- variant of the mouse L cell line) showed neither metabolic co-operation nor ionic coupling, and gap junctions between the cells could not be detected. The failure of mouse L cells to interact both metabolically [4, 6, 131 and ionically [14, 151 has been shown previously. Critical examination of the mechanism mediating metabolic co-operation requires a comparison of closely related cells differing, if possible, only in their ability to cooperate: in less closely related cells the presence of species specific or strain differences between mutant and wild type may mask or confuse the differences concerned with ability to co-operate. It is therefore essential to obtain metabolic co-operation of positive or negative (met+ and met-) cells originating from the same cell line. In this paper we describe two selection sys-

Exp Cell Res IO3 (I 976)

terns designed to obtain met- cells from a parental met’ population and examine some properties of the selected cells resulting from them. MATERIALS

AND METHODS

The variant cell lines used were derived from the polyoma virus transformed BHKC 13 cell line PyY, by Subak-Sharpe [lh]. The PyYIHGPR’r-/dCK-/TK.. line (in previous publications designated PyY/TG/ C?rR/BUdR) had been successively selected for resistance to Gthioguanine, cytosine arahinoside and S-bromodeoxyuridine (BUdR) (see fig. I), and was subsequently shown to be deficient in HGPRT activity (hypoxanthine guanine phosphoribosyl transferase), dCK activity (deoxycytidine kinasc) and TK activity (thymidine kinasc). The PyY/APRTline (previously PyY/AA/AAR) had been selected for resistance to 8-azaddenine @-AA) (see fig. 2) and tubercidin and shown to be deficient in APRT activity (adenine phosphoribosyl transferase). Fig. I also shows the sensitivity of the PyY/HGPRT/dCK-/TKline to 8-AA and fig. 2 also shows the sensitivity of the PyY/APRTline to BUdR. LMTKcells were derived from I~929 cells by Kit et al. [17] and lack TK activity. These were kindly provided by Mrs J. Macnah (Institute of Virology, Glasgow). Cell lines and selection cultures were grown in “Pyrex” glass (babv feeding) bottles in Eagle’s medium (Glasgow modification) supplemented with 10% v/v fetal calf serum under an atmosphere of 5% CO? in air at 37°C. Cells were taken off the glass by treating monolayers with 0.05% trypsin in Ca-.Mg-free isotonic phosphate-buffered saline (PBS) containing 0.6 mM EDTA. After washing and resuspension in growth medium. cells were seeded into bottles for stock cultures or on to I3 mm diameter glass coverslips in Petri dishes for microscopy or autoradiography. or into 50 mm diameter dishes (Sunclon or Biocult Ltd) for labelling of DNA.

Metabolic co-operation was detected by autoradiograph? of co-cultures of donor and recipient cells grown for some hours in the presence of a tritiated purine base or pyrimidine deoxyrihonucleosidc. The donor: recipient ratio was I : 20 and the culture density for seeding 4x I@’ cells/SO mm diameter dish. Tritiatcd compounds to a final concentration of 2 &i/ml were added after the cells had spread. usually 34 h following seeding. The radiochemicals were obtained from .4mersham at the following specific activities: [ U-“Cladcnine 287 mCi/mmol, [8-“Hladenine. 25 Ci/ mmol: (G-:‘H]hypoxanthinc. I Ci/mmol; [6-:‘H]TdR. 26 Cilmmol; [S-‘H]deoxycytidine (CdR). 21.2 Ci/ mmol. Covet-slips. bearing cells. were rcmovcd after suit-

able incubation periods. washed twice in PBS at 4°C. drained, and fixed in methanol: acetic acid (3: I by volume) vapour for l-2 min. They were then placed in fixative for a further 3-4 min followed by extraction with 10% TCA at 4°C (2~ IO min). Coverslips were washed overnight in running tap water, then air-dried and mounted on microscope slides for autoradiography. Slides were dipped into Ilford nuclear research emulsion K2 diluted I : 1 by volume with distilled ~~UtOI’ddiOgrdphS were exposed for suitable water. periods (6-13 days) at 4°C and after developing with IIford contrast FF developer. they were stained with Giemsa-May-Grunwald for 2-3 min. washed and airdried. Metabolic co-operation for pyrimidine deoxyribonuclcotides will only be detected provided that the recipients have passed through S phase allowing them to incorporate labellcd. transferred nucleotides into their DNA [S]. Thus co-cultures labelled with [“H]TdR or [3H]CdR were incubated for extended periods (2448 h) to allow a large proportion of the recipient cells to pass through S phase. ‘To avoid depletion of TdR in the medium, cold TdR was added to a final concentration of IO (i M to the [:‘H]TdR lahelled cultures. Co-cultures labelled with tritiated purines were grown for shorter times (5-10 h) since detection of co-operation for purine ribonucleotides is not ceil-cycle dependent. In order to distinguish donors unambtguously from recipients over these short labelling times. donors were strongly prelabelled with the tritiated purine for IX h before the co-culture. When j3H]adenine was used for Iahelling. cold hypoxanthine was added to lo-’ M to reduce the incorporation 31‘ any pH]hypoxanthine or [“Hlinosine formed by deamination of [:‘H]adenosine.

Fig. 3. Selection systems for obtaining met- cells from meet celi populations. (a) Selection I. Donor cells (TK-) co-operate with recipient cells (TK ) in the presence of exogenous BUdR at 1 mglml. Both are killed following the incorporation of BUdR into their DNA and subsequent exposure to blue light. BtiJR enters the recipients by metabolic co-operation. Meccells present in the ‘TK- cell population should survive since their inability to co-operate prevents BUdR incorporation into their DN.4; (h) selection II. Donor cells. (APRT’), co-operate with recipient ceils (APRT ) in the presence of exogenous 8-.4A at 100 pg/ ml. Both are killed following the toxic action of the drug, this reaching recipients by metaho!ic co-operation. Any metcells in the APRT cell population should survive.

66

Wright, Goldfarb and Subak-Sharpe

Co-operation was quantified by counting autoradiographic grains over recipients in contact with heavilylabelled donors. These grain counts were then compared with counts made of grains over recipients not in contact with donors on the same coverslip. Autoradiographs were exposed for periods which gave low grain counts in recipients, since counts greater than 50 grains/cell were difficult to make. In all comparisons, grain counts were made on autoradiographs exposed for identical times and treated similarly whenever possible. Histograms of grain counts were plotted with the counts classified into intervals of width five. In most cases a sample size of 200 cells was used. The results were tested for statistically significant differences using the median test [23].

Isopycnic centrifugation of cell DNA Following removal of the culture medium, labelled cells were washed with PBS and solubilized in 4 ml of 2 % SDS in standard saline citrate at pH 7.4. Pronase (Sigma, pre-boiled for 10min) was added to a final concentration of 1 mg/ml and after an overnight incubation at 37°C 5.5 g of CsCl (Analar) was added to each solution. The SDS precipitate which formed was removed by low speed centrifugation, and the resulting CsCl solutions placed in 10 ml MSE polypropylene tubes. The refraitive index of each solution was adjusted to 1.401-2 with a saturated solution of CsCl and each tube was then topped up with liquid paraftin, capped and spun at 40 000 mm (average g of 1I5 000) for 72 h at 2eC in an MSE* 65 ‘cent&g; using a 10X 10 ml titanium angle rotor. Three drop fractions were collected from the bottom, on paper discs (Whatman No. 1) 2.5 cm diameter, which were dried at room temperature. Every tenth fraction was taken for refractive index measurements. The discs from each gradient were together extracted twice in 800 ml 10% TCA for 10 min at 4°C and then washed in absolute ethanol, followed by a single wash in ether. Air-dried discs were placed in scintillation vials with 10 ml of NE233 scintillant and counted using a Philips Liquid Scintillation analyser. Where cultures had been labelled with C3H]adenine, discs from the fractionated gradients were treated individually with 0.1 ml of 2.5 N NaOH, and incubated for 3-4 h at 37°C in a humid atmosphere, to hydrolyse RNA which had been labelled during the experiment. After this treatment the discs were TCA extracted and counted as above.

Karyology Colcemid (Ciba) was added to semiconfluent, exponentially growing cultures in 20 oz bottles, to a final concentration of 0.25 pg/ml, for l-2 h. Cells in mitosis were then shaken -from the monolayer into the medium. removed. and resusuended in 5 ml of hvootonic medium consisting of 2 vol distilled HZ0 and 1 vol Eagle’s medium. After 5-10 min at 37”C, the swollen cells were resuspended in glacial acetic acid/methanol 1 : 3 (v/v) at 4°C. The fixative was changed twice after 1 h, by spinning down and resuspending the cells. Exp Cell Res 103 (1976)

Cells were finally resuspended in 0.5 ml of fixative and 1 drop of this suspension was dropped on a cooled, damp glass cover&p. The air-dried coverslips were then stained for 5 min with Giemsa diluted l/lOth with 0.1 M phosphate buffer pH 6.8 and mounted.

Preparation of cells for scanning electron microscopy Cultures of cells growing on 13 mm diameter glass coverslips were washed with PBS and the cells fixed in 2% glutaraldehyde for 30 min. The coverslips were washed three times in PBS, drained, and nlaced in osmium tetroxide vapour for 15 min. They were washed in three changes of double-distilled water and then placed in isopentane cooled by liquid nitrogen, in order to cool the cells rapidly. The coverslip cultures were then freeze dried using an Edwards ‘Speedivac’ apparatus. Cells were coated with gold-palladium and examined in a Cambridge scanning electron microscope.

RESULTS The selection for met- cells The principles of the two selection systems used are shown in fig. 3. The parental cells to be selected for loss of met+ were, in selection I, PyY/HGPRT-/dCK-/TK-/ (met+) and in selection II, PyY/APRT-/ (me&). The basis for both systems is that these analogue resistant met+ cells are placed in a situation where they die if they co-operate. Metabolic co-operation is not expected to differentiate between nucleotides and their toxic analogues. Thus met+ recipients should be killed. Since the wildtype donor cells are themselves killed by the analogue, the sole survivors of such selections should be met- mutant recipient cells. In selection I, co-operation occurs between TK+ donors (PyYIAPRT-) and TKrecipients (PyY/HGPRT-/dCK-/TK-) in the presence of the thymidine analogue BUdR, which, once it is converted to BUdRMP can be incorporated into the DNA of both donors and recipients. BUdRcontaining DNA is sensitised to photoinduced breakage [IS] and thus the applica-

Isolation of met- cells 10’

c

Fig. 4. Abscissa:

time (days); ordinate: cell no. Growth of PyY/HGPRT-/dCK-/TKme& cells (A-A); met- IA (O-O); met- IG (O----O); and met- IN (A-A) cells in medium at 37°C. lo5 cells were seeded into.50 mm diameter dishes and incubated for 5 davs. Each dav 2 dishes/cell line were removed and cell-yields estimated. Each point represents the mean yield from two dishes.

tion of blue light after a suitable co-culture period kills not only TK+ donors but also me& recipient cells. Only TK-, met- cells present in the population should survive. In selection II PyY/APRT- cells are recipients with PyY/HGPRT-/dCK-/TK(APRT’) cells as donors, in the presence of the adenine analogue &AA. Met+ APRTrecipients cannot metabolise 8-AA since they are APRT-, but provided that they cooperate with the APRT+ donors receiving 8-AA nucleotides, they incorporate the toxic analogue. In this system only APRT-, met- cells should survive. In both selection systems, 9x lo5 donors were mixed in suspension with 1X lo5 recipients, seeded and allowed to spread. Either 1 mg/ml BUdR (selection I) or 100 pg/ml 8-AA (selection II) was added and

the co-cultures incubated for 2-3 days. Selection I co-cultures were then exposed to blue light from a Philips Actinic blue (05) 65-80 W lamp for 30 min. The medium in both systems was then replaced with fres medium plus analogue and incubation continued for 2-3 days. Surviving cells were removed and counted. IX lo5 survivors (or cells grown up from the survivors) were mixed with 9~ 1Ojfresh donor cells for each subsequent round of selection. In both cases, co-cultures were seeded at high density (1 x lo6 cells/bottle) to maximise the immediate opportunity for co-operation, and the donor : recipient ratio was 9 : 1 to ensure that recipient/recipient contacts would only occur rarely. The first few passages of selection I yielded few survivors (about 3 %) an was necessary to sub-culture these to obtain enough cells for the subsequent selection step. Later passages yielded increasingly more survivors (-30%). After 46 successive selection passages the survivors were cloned in soft agar in the presence of 1 mg/ml BUdR, clones being designated met- IA, B, C, etc. Unlike selection I,

analogue cont. (@g/ml); ordinate: cell yield. Total cell yields of met- IA cells grown for- 7 days in the presence of varying concentrations of BUdR (M-W); CAR (CL-O); and 6-TG (O-O), expressed as percentage of control with no analogue added. Conditions as in fig. 1.

Fig. 5. Abscissa:

.&xp Cell Res 103 (1976)

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Wright, Goldfarb and Subak-Sharpe

Figs 6, 7. Abscissn: grains/nucleus; ordinate: frequency. Metabolic co-operation between PyY/APRTdonors and (a) PvYIHGPRT-/dCK-ITKmet+ recipients; or (6) ‘me;- IA recipients. Grains over recipients (top) in contact; (bottom) not in contact with donors.

Co-cultures of donors and recipients (1: 20) were labelled with fig. 6) 2 &i/ml E3H]TdR for 24 h in the presence of 10m6M TdR; Ifis. 7) 2 &i/ml [$H]CdR for 24 h. On comparison of (top) and (bottom) for fig. 6b x:=O.42;p>O.2. For fig. 7b x:= 1.65;p=O.2.

large numbers of survivors were obtained from early passages of selection II (--4050%) indicating failure of the system to kill most of the APRT- recipients. Survivors were cloned in soft agar in the presence of 100 ~glml g-AA after 36 passages, clones being termed met- IIA, B , C, etc.

The generation times are respectively 15.5h and -24 h. This increase in generation time of the met- cells compared with me?, remained stable on subsequent passage of the met- I clones in the absence of selective pressure.

Properties of presumptive metcells Selection I. Cloned cells were examined and compared with the parental met+ cells, PyY/HGPRT-/dCK-/TK-, with respect to growth rate, analogue resistance, metabolic co-operation, morphology and chromosome number.

Analogue resistance The resistance of the parental me& cells to the three analogues is shown in fig. 1. Met- I cells were tested for the presence of these three markers to check that neither contamination of the selection cultures, e.g. by LMTK- cells, nor reversion of the dCKand HGPRT- markers had occurred. The analogue resistance of met- IA which shows characteristic results (as did the other three clones tested) is shown in fig. 5, indicating that met- I cells are similar to

Growth rates Fig. 4 shows growth curves of met+ PyY/ HGPRT-/dCK-/TKand 3 met- I clones. Exp Cell Res 103 (1976)

Figs 8, 9. Ahsci.s.sn: grains/cell; ordinure: frequency. Metabolic co-operation between c/ig. X) PyY/APRTdonors and (u) @Y/HGPRT. IdCK-ITK met- recipients; or (b) met’ IA recipients; fig. 9) (~1)PyY/ HGPRT-/dCK‘/TK-. mecf donors; or (6) met- IA donors and PyY/APKT- recipients. Grains over rccipicnts (fop) in contact: (bosom) not in contact with donors,

Co-cultures were labelled with 2 pCi/ml vig. CI [“Hlhypoxanthinc; (lig. 9) [“H]adeninc for 7 h, in the presence of 10 * M hypoxanthine. Donors were pre labelled with 2 @/ml (/is. X) [“Hlhypoxanthine fig. 9) [3H]adcnine for I8 h prior to co-culture. On comparison of (u) and (h) for fig. 8 (top) x!= l77.4:p~O.OOl. For fig. 9 (top) x:= 182.8:1;~0.001.

met- cells in this respect, and ruling out contamination.

with donors were tested for difference usin! the median test and in both cases no signifi cant difference was found between them a the 5% level. (The control met” distribu tions similarly tested were highly signifi cant.) These results indicate that thl selected cells differ from the parental met. cells and there is no significant co-operatior for pyrimidine deoxyribonucleotides !I met- I cells, under the experimental condi tions used. Fig. 8 shows distributions of grain count from met-. and met- IA cells either in con tact or not in contact with donors, co cultured in the presence of 2 /*Ci/ml [%I: hypoxanthine for 7 h. Co-operation is see to occur in both cell types; however, this i reduced in the met - IA cells. Compariso of the distributions from met ’ and met TI cells in contact with donors using th

Met’ and met- PyY/HGPRT -/dCK-./T’Kcell lines wet-c compared as recipients of [“H]TdR, [3H]CdR and [3H]hypoxanthine labelled nucleotides from PyY/APRTdonors and as donors of [3H]adenine labelled nucleotides to PyY/APRT- recipients. Fig. 6 shows distribution of autoradiographic grain counts from met- control and met- IA cells either in contact (to/>) or not in contact (horror77) with donors, the cocu!tures being labelled with 2 pCi/ml [3H]TdR for 24 h. Similar distributions from co-cultures labelled with 2 &i/ml [“H]CdR are shown in fig. 7. Distributions from met - I cells in contact and not in contact

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Fig. 10. Autoradiographs of metabolic co-operation between: (u-c) PyY/APRTdonors and PyYi HGPRT-/dCK-/TKmet+ recipients labelled with (u-c) 2 &i/ml. (a) [“H]TdR for 24 h with 10m6M TdR; (b) r3H]CdR for 24 h; (c) [3H]hypoxanthine for 7 h; and (d) PyY/HGPRT-/dCK-/TKme& donors and PyY/

APRT- recipients labelled with 2 &i/ml r3H]adenine for 7 h with 10e4M hypoxanthine. As shown, metabolic co-operation takes place between donor and recipient cells in direct contact. Indirect transfer is also observed in these co-cultures, but this is not illustrated.

median test shows that they are significantly different. A similar situation is seen in co-cultures labelled with C3H]adenine, the donors being the met- IA or met+ cells. Fig. 9 shows the distributions obtained when co-cultures were pulsed with [3H]adenine at 2 &i/ml for 7 h. Again a reduction in co-operation is seen with the met- IA cells. Figs 10 (a-d) and 11 (a-d) show autoradiographs of co-

operation in met+ and met- IA cells respectively. These results indicate that the selected met- I cells show a general reduction in ability to co-operate: there is apparent absence of pyrimidine deoxyribonucleotide transfer, while some purine ribonucleotide transfer is retained. Tests with other met- I clones showed essentially similar results. Met- IA cells were also cultured for 31

E.rp CrllR~s

103 (1976)

Fig. II. Autoradiographs of metabolic co-operation between; a-c, PyY/APRT- donors and met- IA recipients, labelled with (a-c) 2 #Z/ml. (a) [‘H]TdR for 24 h with 1O-6M TdR; (h) r3H]CdR for 24 h; (c) r3H]-

hypoxanthine for 7 h; (d) met IA donors and PyY/ APRT- recipients, labelled with 2 &i/ml [3H]adenine for 7 h with IO+ hypoxanthine.

passages in the absence of selective pressure and pass 31 cells showed the same characteristics with respect to co-operation as early passage cells. An independent method of detecting metabolic co-operation for TdR nucleotides was devised in order to confirm the autoradiographic results obtained with met+ and met- I cells. The incorporation of transferred TdR nucleotides into recipient DNA can be detected only if donor and recipient DNA are separated. This can be done by utilising the separation of dense BUdR-substituted ‘TK- cell DNA from light TK- cell DNA by isopycnic centrifugation in neutral

cesium chloride gradients [lg, 191.When a co-culture of TK’ (PyY/APRT‘-) and TK(PyY/HGPRT-/dCK-/TK-) cells is grown in the presence of BUdR, [14C]TdR and [“Hladenine, the TK+ donor cells take up BUdR and [*IC]TdR and give rise to [‘“Cjlabelled heavy-heavy (HH) and heavy-light (HL) duplexes. TK- recipient cells incorporate only the [“Hladenine and give rise to [3H]labelled light-light (LL) duplexes. The presence of 14Ccounts in the 3H LL peak will indicate that [“C]TdR nucleotides have been transferred from donors to recipients by metabolic co-operation and have been incorporated into recipient DN,4. Transfer

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Wright, Goldfarb and Subak-Sharpe

r a

12

13

Figs 12, 13. Abscissa: fraction no.; ordinate: (left) 0, cpm %X lo-‘; (right) A, cpm 3H~ 10-a. CsCl isopycnic gradient profiles of DNA from cocultures of PyY/APRTdonors with (a) PyYI HGPRT-/dCK-/TKme&; (b) met- IA; (c) LMTKrecipients. Co-cultures with a final cell density of fig. 12) 2x 106cells/50 mm dish; fig. 13) 5x IO5cells/90 mm

dish, were labelled with 1 &i/ml [I*C]TdR, 8 pg/mJ BUdR and 2 &i/ml [3H]adenine for 24 h. Extracted DNA was spun on neutral CsCl gradients in a 10X 10 titanium angle rotor in an MSE 65 centrifuge at 20°C at 40000 rpm for 72 h. The arrows over peaks are densities in g/ml.

Exp Cell Res 103 (1976)

Psolation of met- cells

73

Figs 14, 15. Stereoscan electron micrograph of fig. 14) PyY/HGPRT-/dCK--/TKmet- cells; (/Ix. 15) met- 1.4 cells.

of BUdR-nucleotides from donor to recipient under these conditions is suggested by the small shift of the LL peak (from between 1.699-1.702 to 1.708 g/ml) but is insufficient to cause a major density shift. The labelling time of the co-culture must be long enough to ensure that no TK’, (‘*C. donor) DNA remains in the LL peak. Some SH counts in the HH and HL peaks would indicate that adenine nucleotides have been transferred in the opposite direction by metabolic co-operation and incorporated into DNA. Most, of course, would be expected to go into RNA. PyY/APRT-/(TK’) donors were cocultured with PyY HGPRT-/dCK-ITK-I (APRT+) recipients (met- or met- or LMTK- in the ratio 1 : 1 at high density (2X 10” cells/50 mm diameter dish) for 24 h in the presence of 1 &i/ml [14C]TdR, 8 pg/ ml BUdR and 2 &i/ml [“Hladenine. DNA was then extracted and separated on neutral CsCl gradients. Fig. 12(a-c) shows the protiles obtained when the recipients were met’ , met- or LMTK- (negative control), respectively. Donor (APRT-, TK+‘) DNA is

identified as the “C-1abelled HH and HL peaks at densities 1.749 and 1.726-9 g/ml. respectively. Recipient (APRT- . TK ) DNA is represented as the “H-labelled LL peak at the density of 1.699 g/ml. TdR nucleotides ( “C-labe11ed) transferred from donors to recipients by metabolic co-operation which are incorporated into their DNA appear as a “‘C peak coincident with the “H-labelled LL peak, see fig. 12~. This “C peak appears to be absent in profiles in which either LMTK- or met- I cells are the recipients, indicating lack of detectable transfer of TdR nucleotides to these cells. Similar co-cultures were made at low density. 5~ 105 cells/90 mm diameter dish, where cells could not make contact. Profiles from the gradients on which DNA from these were separated are shown in fig. 13(u--c). In all cases there is no :.‘C peak coincident with the LL DNA, indicating that the co-operative transfer found in fig. 12a is, in fact, dependent on cell contact formation. The above results confirm that met- I cells are defective in ability to cooperate for TdR nucleotides. In these ex-

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Wright, Goldfarb and Subak-Shapre

6-

a

4a0. 40

Lllh-nn so

16 1

I 60

Ill 70

I7 80

b

C

a 0 30

PO

40

50

60

70

L

JO

J 40

50

L!R-u 60

Fig.

16. Abscissa: chromosome no.; ordinate: frequency. Frequency distributions of chromosome number in

(a) PyY/HGPRT-/dCK-/TKmet+ cells; (b) met- IA cells; (c) met- IN cells. Modal numbers are 98, 49 and 50, respectively.

periments tritium label transfer in the opposite direction and subsequent incorporation into DNA, could not be demonstrated.

will also be seen that met- I cells are considerably smaller than the parental cells. (Average cell diameter, measured by Coulter counter, of cells grown to high density were met+ 16.1 and met- 14.2 pm.)

Morphology During selection I, it was noticed that survivors appeared to be smaller and more epithelioid in appearance compared with the parental met+ cells. Met- I clones were extremely rounded and failed to flatten on the substrate. Figs 14 and 1.5show scanning electron micrographs of met+ and met- I cells, respectively. Met- I cells are seen to have irregular blebbed surfaces with no microvilli visible. This pronounced change in morphology persisted through many passages, the met- IA clone at pass 31 being similar to early passage met- IA cells. It Exp Cell Rrs 103 (1976)

Chromosome number Frequency distributions of the chromosome number in met+ and met- I cells are shown in fig. 16. The me& PyYIHGPRT-IdCK-/ TK- cells have a modal chromosome number of 98, whereas met- I clones A and N have modal numbers of 49 and 50, respectively. A small proportion of the met+ cells were found to have a chromosome number of around 50. It is therefore possible that either the met- clones are derived from these cells with about 50 chromosomes, or

b

co-cultures were labelled with [:lH]adenine for 8 h. The close similarity between the distributions from the two cell types indicates that there is no change in ability to cooperate in presumptive met- II cells and that this selection has not succeeded in producing mec- cells after 46 successive passages. DISCUSSION

Two selective systems have been designed to obtain cells with reduced capability for metabolic co-operation, met cells. As selective agent, one system employed the TdR analogue BUdR, while the other system used the adenine analogue, g-AA. The cells resulting from the selection I;‘ig. 17. Ah.scis.ru: grains/cell: dim/e: frequency. Metabolic co-operation between PyY/HGPRT / in which BUdR was used, were found dCK./TK- met+ donors and (a) PyY/APRT- met’ recipients; (b) met- II recipients. to show virtual abolition of pyrimidine deCo-cultures were labclled with 2 &i/ml [:1H]adenine oxyribonucleotide co-operation: not only for 8 h, in the presence of 10 4 M hypoxanthine. Donors were prclabelled with 2 &i/ml r3Hladenine for TdR but also CdR nucleotides were no 18 h prior to’co-culture. Grains over iecfpients (ray) longer detectably transferred. Moreover. in contact: (hotfom) not in contact with donors. the transfer of TdR nucleotides to these cells from competent donor cells cou!d not be detected using two different meththere has been a loss of chromosomes from ods; autoradiography and analysis of the selected cells during the met selec- DNA by isopycnic centrifugation. When cooperation for purine ribonucleotides was tion. tested in these same cells, they showed only a reduction in co-operation for both hypoxanthine and adenine nucleotides. Thus the Selection II. The presumptive met- II cells ability to act as recipients of purine ribowere unchanged in morphology, chromo- nucleotides by metabolic co-operation resome number and resistance to 8-AA when mains present in the cells. In addition. these compared with the met’ PyYIAPRT- pa- met- 1 cells show morphological changes, rental cells. Both cell types had modal chro- being smaller, more epithelioid and having mosome numbers of 38. These cells were an extremely blebbed surface, compared tested for ability to act as recipients of [3H]- with their met+ parents. The met phenotype could be exp!ained adenine labelled nucleotides from PyY/ HGPRT-/dCK-/TKmecf donors. Fig. 17 in several possible ways: (I) The met compares one representative clone with the selections could have become contaminated met’ APRT- parent. In these experiments, by LMTK- cells which were also cultured

76

Wright, Goldfarb and Subak-Sharpe

in the laboratory. This is excluded because [22], and Slack (personal communication) LMTK- cells have a typical morphology has isolated a variant of the Chinese unlike the met- I morphology which is hamster cell line B 14 FAF which is resistant characteristically rounded and blebbed, and to 300 pug/ml 8-AA, yet has unchanged ability to incorporate [3H]adenine. This by the presence of the three characteristic enzyme deficiencies HGPRT-, dCK- and suggests that certain cells may be able to TK- in the met- I cells. discriminate between adenine and 8-AA at (2) It is possible that we have obtained some point in the salvage pathway. The isolation of met- cells from a parencells which have preferentially lost the capacity for pyrimidine deoxyribonucleotide tal me& line as described here, enables us co-operation. If this were the case, it would to investigate factors involved in metabolic imply that the mechanism for metabolic co- co-operation. In particular, investigation of whether the defect in the met- cells is due operation is through a multi-component system in which capability of transfer of dif- to an intracellular metabolic defect or to ferent molecules is separable in some way. alterations in components of intracellular (3) The met- cells could have an altered junctions, for example, gap junctions, can pyrimidine deoxyribonucleotide metabo- now be examined and this investigation is lism. This would imply a quantitative or reported in a separate paper by Wright qualitative change in the cell’s anabolic or et al. [20]. catabolic pathways or their compartmen- It is a pleasure to acknowledge the invaluable techtalisation. nical assistance of Mrs Martha Macnamara, who unpursued the successive rounds of met- cell (4) The explanation may lie in the re- tiringly selection. We thank Dr C. Slack for helpful criticism throughduced capability of the met- cell of forming out this work. This work was done while E. D. W. was functional gap junctions or in a quantitative the recipient of an MRC award. P. S. G. G. is supconstraint on their number. At critical num- ported by a grant No. SP1341 from the Cancer Rebers this, in combination with other factors search Campaign. such as pool sizes, could result in the observed differential reduction in metabolic REFERENCES co-operation. These possibilities are examined in a sub- 1. Subak-Sharpe, J H, Biirk, R R & Pitts, J D, Heredity 21 (1966) 342. sequent paper [20]. 2. Subak-Sharpe, J H, Ciba foundation symp homeoThe second selective system in which the static regulators (1969) 276. 3. Btirk, R R, Pitts, J D & Subak-Sharpe, J H, Exp selective agent was 8-AA, failed to produce cell res 53 (1968) 297. met- cells. This suggests that either insuf4. Pitts, J D, Proc third lepetite colloquium (1972) ficient transfer of toxic nucleotide ana- 5. 277. Wright, E D. Unpublished results. logues occurred to kill the recipient cells, or 6. Cox. R P. Krauss. M R. Balis. M E & Dancis. J. Prod natl acad sci ‘US 67 (1970) 1573. that the recipients have adapted to survive 7. Goldfarb, P S G, Slack, C, Subak-Sharpe, J H & the transfer of analogue nucleotides in some Wright, E D, Transport at the cellular level, 28th symposium of the society for experimental way other than by a reduction in ability to biology, p. 463. Cambridge University Press, co-operate. The initial high survival rate of Cambridge (1974). 8. Cox, R P Krauss, M R, Balis, M E & Dancis, J, these recipients is consistent with the forExp cell res 74 (1972) 251. mer explanation. 9. Leibman, K C & Heidelberger, C, J biol them 216 (1955) 823. Very little is known about the mode of 10. Furshpan, E J & Potter, D D, Curr topics dev biol action of 8-AA. It has low affinity for APRT 3 (1968) 95. Exp Cell Res 103 (1976)

I I. Goodenough. D A & Revel, J P, J cell biol 45 (1970) 272. 12. Gilula, N B, Reeves, 0 R & Steinbach, A, Nature 235 (1972) 262. 13. Widmer-Favre, C. J cell sci I I (1972) 261. 14. Goshima, K. Exp cell res 65 (1971) Ihl. 1.5. Nelson. P G & Peacock. J H. J gen physiol 62 (1973) 2s. 16. Subak-Sharpe, H, Exp cell res 38 (1965) 106. 17. Kit, S, Dubbs, D R, Piekarski, 1~J & Hsu, T C, Exp cell res 3 1 (1963) 297. 18. Djordjevic, D & Szybalski. W, J exp med II2 (1960) 509.

19. Meselson, M. Stahl, F W & Vinograd. J. Proc natl acad sci US 43 (1957) 581. 20. Wright, E D, Slack. C, Goldfarb. P S G & Subak-Sharpe, J H, Exp cell res 103(1976) 79. 21. Gadd, R E A & Henderson. J F. Can j biochem 48 (1970) 295. 22. Lindgren, B W, Statistical theory. Collier-Macmillan Ltd, I>ondon (1968).

Received April 13. 1976 Accepted June I, 1976

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