Communication between normal and enzyme deficient cells in tissue culture

Copyright 0 1972 by Academic Press, Inc. All rixkts of reproduction in any form reserwd

Experimental

COMMUNICATION DEFICIENT R. P. COX, MARJORIE

Cell Research 74 (1972) 251-268

BETWEEN NORMAL

AND ENZYME

CELLS IN TISSUE CULTURE R. KRAUSS, M. E. BALIS and J. DANCIS

Departments of Medicine, Pharmacology, and Pediatrics and the Stella and Charles Guttman Laboratory for Human Pharmacology and Pharmacogenetics, New York University School of Medicine, New York, N.Y. 10016 and the Sloan-Kettering Institute for Cancer Research, New York, N. Y. 10021, USA

SUMMARY Correction of certain mutant phenotypes by intimate contact with normal cells, i.e. ‘metabolic cooperation’, is an easily studied form of cell communication. Metabolic cooperation between normal cells and mutant cells deficient in hypoxanthine-guanine or adenine phosphoribosyl transferase (HGPRTase and APRTase resoectivelv) aonears to be the result of transfer of the enzyme prohuct, nucleotide or nucleotide cierivat& f;orn normal to mutant cells. This process shows selectivity in that mutant derivatives of mouse L cells are unable to function as recipients of HGPRTase or APRTase products, while hamster and human fibroblasts with these enzyme deficiencies, exhibit correction of the mutant phenotype, when in contact with normal donor cells. There is also selectivity with respect to substances transferred, since other mutant phenotypes, i.e. G-6 PD deficiency, are not corrected by contact with normal cells. Species specificities do not appear to influence metabolic cooperation, therefore heterospecific cell mixtures provide an opportunity to cytologically distinguish cells and study individual cell interactions. Transfer of nucleotide from normal to mutant cells is less dependent on energy production than is the incorporation of radioactive purines into cellular material. The nucleotide translocation mechanism is not susceptible to sulfhydryl blocking agents.

Cell to cell communication may be involved in a variety of biological processes including induction of the immune response [l]; cell recognition during the sorting out of embryonic cells [2, 3, 41; contact inhibition of both cell locomotion [5, 61 and cell division 17, 81; electrical coupling between cells [9, lo]; and metabolic cooperation-that is the correction of certain mutant phenotypes in cell culture by contact with normal cells [l I-151. Although the mechanisms involved in cell communication in these various phenomena may not be identical, an understanding of any one of the processes may provide insight into the others.

Metabolic cooperation between tissue culture fibroblasts derived from patients with Lesch-Nyhan disease (congenital hyperuricosuria) and normal cells provides a relatively easily analysed model for studying one kind of cell communication. Lesch-Nyhan fibroblasts are deficient in IMP, pyrophosphate phosphoribosyl-transferase activity (EC 2.4. 2.8) alternatively named hypoxanthinejguanine phosphoribosyltransferase (HGPRTase) [16, 171. Normal human cells (HGPRTase- ) incorporate radioactive hypoxanthine or guanine into intracellular nucleotides demonstrable at the cellular level by radioautographic methods, while skin fibroblasts Exptl Cell Res 74 iI

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grown from patients with Lesch-Nyhan disease(HGPRTase-) show a marked reduction in incorporation of these nucleotides under similar conditions. However, Lesch-Nyhan cells grown in close contact with normal human fibroblasts become labeled. This phenomenon, called metabolic cooperation, requires cell to cell contact and is a form of cell communication. Several hypotheses have been advanced to explain this phenomenon [12, 151. It has been suggested that HGPRTase+ cells may provide HGPRTase- cells with a substance(s) that corrects the defect and enables mutant cells to synthesize a functional HGPRTase. This substance might be episomal DNA, informational RNA or a regulatory molecule that stabilizes or activates a mutant enzyme. Another possibility is that HGPRTase+ cells may provide HGPRTasecells with preformed enzyme and thereby confer the capacity to metabolize guanine and hypoxanthine. Recent experiments from our laboratory and from Pitts’ provide evidence for a third possibility, that is normal cells synthesize the radioactive nucleotide which is transferred to the mutant cells as nucleotide or as a product of the nucleotide [ 15, 181.This conclusion is based on experiments which showed that incorporation of labeled hypoxanthine into mutant cells ceasedpromptly after separating HGPRTasecells from normal. The experiments were carried out under conditions in which HGPRTase is stable for many hours [15]. In the present study intercellular communication was investigated using several different enzymatic markers: HGPRTase, adenine phosphoribosyltransferase (APRTase EC 2.4.2.7) and glucose-6 phosphate dehydrogenase (G-6 PD EC 1.1.1.4.9). Cell lines and strains deficient in certain of these enzymes were derived from humans, hamsters and mice. Certain of the non-human cell Exptl Cell Res 74 (1972)

lines have a relatively distinctive nuclear and, in some cases, cytoplasmic morphology so that in most instances they can be distinguished from human cells. Therefore, the capacity of human or animal cells to donate or receive ‘correcting factors’ by cell contact could be determined by observing individual cells. The ability to distinguish cytologically donor from recipient cells permitted a study of the conditions necessary for metabolic cooperation and the effect of metabolic inhibitors and other substances on the capacity of cells to transfer or receive. Moreover, the specificity of cell communication was evaluated by studying various intra- and interspecies cell mixtures, normal and neoplastic cells, and mutants with different enzymatic deficiencies. MATERIALS AND METHODS Cells Table 1 shows the various cell lines and strains used in the present study, the species and tissue of origin, cellular morphology, enzymatic markers, saturation density in confluent cultures and laboratories from which the cultures were obtained. Cells were derived from humans, hamsters and mice. The human cells were diploid fibroblasts derived from 14 normal subjects, 3 children with Lesch-Nyhan disease (HGPRTase-), and one child with non-spherocytic hemolytic anemia secondary to a marked deficiency of G-6 PD activity [19]. Human heteroploid cell lines of neoplastic origin were represented by two clonal derivatives of HeLa cells: one having a modal number of 65 chromosomes andthe other, 71 chromosomes. Hamster cells were the established baby Syrian hamster kidney cell line BHK 21 clone 13 which has a diploid male karvotvpe 1201.Thev tend to grow as elongated bipolar ceiis exhibiting -a parallel &ientation. TG-2 is a variant of BHK 21 C 13, selected for resistance to thioguanine (SO-100 rug/ml) and has a marked deficiency of HGPRTase activity (table 2) [21]. PyY/APP is a polyoma virus transformant of BHK21 C 13 selected for resistance to 8 azoadenine (40 ,ug/ml) and lacks APRTase activity (table 2) [22]. This cell is stellate although in confluent cultures it becomes elongated and grows randomly rather than preserving an oriented layer of cells. Two mouse lines of independent origin and their variants were studied. 3T3 was derived from 17- to 19-day-old embryonic Swiss mice under conditions of culture which preserve a very high degree of contact inhibition of growth [23]. When cultures of 3T3

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Table 1

Cell line

Origin

Human skin fibroblasts Lesch-Nyhan fibroblasts G-6PDfibroblasts

Skin biopsy from normal subjects Skin biopsy from patients Skin biopsy from patients with non-spherocytic anemia Syrian baby hamster kidney BHK 21 cells resistant to thioguanine Polyoma virus transformed BHK21 resistant to 2,6 diamino purine Swiss mouse embryo

BHK21-Cl3 TG-2

PyY/APP-

3T3M

3T3 M-CI-

3T3 Py Littlefield’s L cell

A9 MCR2 Clone ID HeLa,, HeLa,,

Cellular rnorpholO!sY

Enzyme marker’

Saturation density (cells ,< 105/cm2) Source b

Spindle-shaped fibroblasts Spindle-shaped fibroblasts Spindle-shaped fibroblasts

None

0.5-2

HGPRTase-

1-2

13-15, 17

G-6PD-

1-2

19

Elongated bipolar cells Elongated bipolar cells

None

8-10

Littlefield

20

HGPRTase-

8-10

Littlefield

21

Elongated bipolar cells or stellate cells in sparse culture

APRTase-

> 10

Subak-Sharpe

22

0.5-0.8

Green

23

5-7

Basilic0

24

APRTase-

b 10

Littlefield

25, 26

HGPRTaseand APRTaseAPRTase-

> 10

Littlefield

26

> 10

Gartler

TK-

> 10

Green

27

None

8-10

Scharf

28

None

5-7

Rounded bipolar None fibroblasts tend to become cuboida1 when confluent None Bipolar fibroblasts tend to be spindle-shaped None Stellate fibroblasts

3T3 cell which lost contact inhibition Polyoma virus transformant of 3T3 C,H mouse sub- Thick bipolar cutaneous tissue fibroblasts (NCTC clone L929) L cell resistant to Thick bipolar 8 azaguanine fibroblasts

L cell resistant to Thick bipolar 2,3 diaminopurine fibroblasts Thick bipolar L cell fibroblasts HeLa cell-clone Grow as round with 65 chrome- bipolar cells somes HeLa cell clone Cuboidal cells with 71 chromo- as closely packed somes cell clusters

Reference

2-4

28

a Enzyme markers HGPRTase-, deficient in hypoxanthine-guanine phosphoribosyl transferase activity, APRTase-, deficient in adenine phosphoribosyl transferase activity, G-6 PD-, deficient in glucose 6 phosphate dehydrogenase activity, Tk-, deficient in thymidine kinase activity. b Source is the laboratory from which the cells were obtained. Exptl Cell Res 74 (1972)

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2. Specific activities of HGPRTase and APRTase in cultured cells Table

Spec. act. Cell line

HGPRTase

Human cells Normal skin fibroblasts Lesch-Nyhan (3) HeLa,, HeLa,,

2.0 to 2.8 < 0.05 14.3 5.8

Hamster lines BHK21 Cl3 TG-2 PyY APP-

5.7 < 0.05 3.8

Mouse lines 3T3 Py 3T3 L cell (Clone 1D) L cell (Littlefield) A9 MCR2

3.0 3.4 3.5 11.9 < 0.05 7.1

APRTase

2.0 to 2.4 3.1 to 1.3

18.7 22.6 < 0.05

3.2 3 < 0:05 < 0.05 < 0.05

Spec. act. are nmoles AMP or IMP formed/min/mg protein. These values were determined by taking aliquots at 4 time points during assay, determining the amount of product formed in each and calculating the activity from the slope of the product formed vs time.

are passed infrequently and at high population density, they exhibit reduced contact inhibition and form multilayers in culture. 3T3CI- is a 3T3 cell which has reduced contact inhibition. A polyoma virus transformant of 3T3 (PY3T3) has a greater reduction of contact inhibition of growth [24]. The second mouse line and its variants are derivatives of L cells (NCTC clone 929 L) which originated from the subcutaneous tissue of a C3H mouse [25, 261. The variants of this line are morphologically indistinguishable from one another occurring as thick bipolar fibroblasts, and they have been selected for certain enzyme deficiencies which provide biochemical markers [26]. Table 2 shows the specific activity of HGPRTase and APRTase in the various cell lines and strains. The enzvme activities found indicate that the cell lines which are resistant to purine analogs are in fact enzyme deficient and not transport mutants. An unexpected finding was that A9 mouse cells, an L cell variant, are not only deficient in HGPRTase but also lack APRTase activity. When the parent L cell line from which A9 was derived was examined (Littlefield’s L cell), it was found to have very low levels of APRTase (table 2). Other variants of L cells, for example, Clone 1D [27], had high levels of both HGPRTase and APRTase activity. Exptl Cell Res 74 (1972)

Media and cultures Cells were grown in Waymouth’s medium [29] containing 10 % fetal calf serum and antibiotics (penicillin 50 units, streptomycin 50 pg, and kanamycin 30 ,ug/ ml). Cultures were carried in flat-bottomed flint glass bottles employing nutrient media and sera as nreviouslv described 1151. Cells were harvested by detaching confluent cell monolayers from glass surfaces with 0.04 % trvnsin (GIBCo, Grand Island, N.Y.) and 0.02 % disodium EDTA in Puck’s saline A: Cell suspensions were diluted in complete medium, counted in a hemocytometer and either inoculated into other bottles or into Leighton tubes containing 6 x 30 mm coverslips for radioautographic studies. Approx. 150 000 cells in 2 ml of medium were inoculated into each Leighton tube having an 11 x 37 mm window and the cultures were grown at 37°C in an atmosphere of 10 % CO, in balanced air. For certain studies that compared the metabolic cooperation in cell monolayers and in recently separated cell suspensions, cultures were grown in glass Petri dishes (100 mm diameter) containing two 6 x 30 mm coverslips. In these experiments the coverslips were removed from the dishes and placed in individual Leighton tubes prior to harvesting the confluent cell monolayers. These coverslips were therefore representative of the cell monolayer prior to trypsinizing. The cell suspensions prepared from the dishes were diluted in complete medium and the cells were incubated in 5 ml siliconized Erlenmeyer flasks in a gyratory shaker in a water bath at 36°C for 2 h.

Radioautographic studies Radioactive purines were chromatographed prior to use. 3H-adenine (New England Nuclear Corp., Boston) was sufficiently pure to use as received, but %Hhypoxanthine (New England Nuclear) required further purification on Dowex 50. Radioautographic studies were carried out by adding the isotope to Waymouth’s medium containing 15 % fetal calf serum. The final concentration of label varied, depending on the cell line or cell mixture being studied. The concentrations used and the duration of incubation are described in the legends to the tables and figures. In general, sufficient 3H-purine was used to produce heavy labeling (30 grains or more per nucleus) in about 90 % of the HGPRTase+ cells and less than 10 grains/nucleus in the HGPRTaselines and strains. In certain experiments mutant cell lines deficient in either HGPRTase or APRTase were incubated with both substrates. This provided a control of cell viability, metabolic activity, and availability of phosphoribosylpyrosphosphate (PRPP). __ since-both enzymes carry out analogous reactions. .’ Labeled cultures grown on coverslips were prepared for radioautography by washing them 4 times with Hanks buffered salt solution. Cover slips were then left in methanol overnight. They were attached with Permount to 75 x 25 mm microscope slides. Initially, slides were washed in running tap water for 1 h, but in later experiments they were washed for 6-7 h. The longer washing removed more of the background radioactivity. Slides were dipped in Kodak NTB3

Cell communication liquid emulsion and after one week exposure, the radioautographs were developed and stained with May-Griinwald-Giemsa [30]. Variation in the intensity of staining of the same cell line in different experiments was achieved by varying the duration of decolorization. Suspensions of cells were incubated with radioactive substrate for 2 h in a 37°C gyratory water bath. The cells were collected by centrifuging for 2.5 min at 700 rpm and washed twice with fresh medium without serum and once with Hanks saline. Siliconized tubes and pipettes were used throughout. The cell pellets were fixed overnight with ethanol/ acetic acid 3 : 1. Slides were prepared from the cell pellet and radioautographs were made as described above. In all radioautographic studies of heterospecific cell mixtures, duplicate or triplicate coverslips were prepared. At least 200 cells on each coverslip were examined and photographs were made of representative areas.

Assay of APRTase and HGPRTase activity Enzyme assays were carried out with 20 000 g supernates prepared from suspensions of cells disrupted by repeated freezing and thawing in 10 vol of 0.01 M Tris pH 7.2 with 0.001 M mercaptoethanol. The supernate was used for enzyme assays and protein was measured by the method of Lowry et al. [31]. HGPRTase and APRTase activity was assayed by the method of Rubin et al. [32], -with either- 14C-8_ hvnoxanthine or 14C-S-adenine as substrates. Specific-activities are expressed as nmoles AMP or IMP formed/min/mg protein. These values were measured by taking aliquots of the reaction mixture at four time intervals during the incubation, determining the amount of product formed in each, and calculating the activity from the slope of the product formed versus time. All samples were also assayed by chromatography using two different solvents [5% Na,HPO, in isoamyl alcohol, and butanol/acetic acid/water (120 : 30 : SO)]. When nucleoside formation, indicative of nucleotidase or phosphatase activity was detected, the radioactivities of nucleoside and nucleotide were totaled.

Cytochemical method for G-6 PD For identification of cellular phenotypes, cells and cell mixtures were grown for 48 h in four-chambered Lab-Teck tissue c&me slides. The activity of G-6 PD was examined by the method described by Siniscalco [19]. Each chamber was washed with Hanks solution and dried rapidly in an air stream. About 0.5 ml of a freshly prepared reaction mixture containing one Dart of 0.2 M vhosphate buffer pH 7.4; one part glucose-6-phosphate solution (60 mgjml of disodium salt); one part triphosphorpyridine nucleotide solution (10 mg/ml of sodium salt 99 % purity); two parts of nitro-blue-tetrazolium (1 mg/ml, grade III); one part phenazine methosulfate (0.02 mg/ml) and a final concentration of 0.1 % potassium azide. The slides were incubated at 37°C and 100% humi-

25.5

dity for 20 to 30 min. The reaction mixture was decanted, the plastic chambers removed, and the slides drained. Slides were then fixed in form01 vapors foi 5 min, washed in Hanks solution, counterstained with 0.1 56neutral red for 10 min, washed again and dried. G-6 PD+ cells show large dark granules of orecipitated formazan, indicating enzyme activity. There is slight diffusion of formazan extracellularly which appears as small fine particles. The effects of formazan diffusion from G-6 PD+ cells to G-6 PD deficient cells was investigated by growing G-6 PD+ cells on one coverslip and G-6 PD- cells on another. The coverslips were rinsed in Hanks solution and one coverslip was suspended on clay pillars 0.5 mm above the other, and the capillary space filled with the reaction mixture. After incubation for 30 min at 37 C. the coverslips were separated, rinsed and handled as described above. The coverslip containing G-6 PDf cells was heavily stained and the cells showed the enzyme specific large dark granules. The G-h PD- cells and the extracellular spaces on the other coverslip contained slight fine particles of formazan which had diffused from the G-6 PD+ cell monolayer This fine precipitate was easily distinguished from the large granules found in G-6 PD ‘- cells.

RESULTS Cell specificity in metabolic cooperation Intraspecific cell mixtures: Intraspecific cell communication was first explored with hamster and mouse cell lines. HGPRTaseTG-2 hamster cells were grown to confluence with normal hamster BHK21 cells. Under these conditions, nearly all cells incorporated the label, suggesting metabolic cooperation. In contrast, a derivative of the mouse L cell, A9, deficient in HGPRTase, failed to show cooperation with HGPRTasef mouse embryo cell 3T3 (fig. la). Since BHK21 and its derivative TG-2 resemble each other morphologically, cooperation between them must be determined on a statistical basis, but the two mouse cell lines, A9 and 3T3 look different, and communication between individual cells can be studied. 3T3 cells are larger and tend to spread on the glass surface where they assume a more cuboidal shape, and their nuclei contain several small nucleoli. The cytoplasm of A9 stains more darkly with MayGrtinwald-Giemsa stain than 3T3, and A9 Exptl Cell Res 74 (1972)

256 R. P. Cox et al. Table 3. Metabolic cooperation in intra and interspecific cell mixtures

Cell Human skin fibroblasts BHK21 Cl3 3T3M 3T3 ClPy 3T3 Clone 1D Littlefield L cell HeLa,, HeLa,,

Lesch-Nyhan (HGPRTase-)

TG-2 (HGPRTase-)

A9 (HGPRTaseand APRTase-)

+ + + + f -

+ + +

-

+

-

Clone Py Y/APP(APRTase-)

MCR 2 (APRTase-)

&K-)

FL?

+ + +

-

-

-

-

-

-

-

See Materials and Methods for experimental details.

cells tend to grow as thick bipolar fibroblasts (fig. 1). A9 cells are also APRTase-. Therefore, the correction of this phenotype by APRTase+ 3T3 cells was investigated with 3Hadenine as substrate. Once again metabolic cooperation did not occur (fig. 1b). A second L cell derivative, MCR2, also deficient in APRTase, failed to exhibit metabolic cooperation following growth with APRTase+ L cells (clone 1D) or mouse embryo cells 3T3 (table 3). Previous studies have demonstrated metabolic cooperation between HGPRTase deficient hamster and HGPRTase deficient human cells and normal fibroblasts of the same species [l l-l 51. Similarly, an APRTase deficient polyoma transformant of BHK21 showed correction of the mutant phenotype by contact with APRTase+ hamster cells

[33]. Recently, Pitts has reported that a HGPRTase- L cell does not show metabolic cooperation with normal L cells [18]. Our finding that A9 cells are unable to undergo metabolic cooperation for either HGPRTase or APRTase markers may be a characteristic either of the mutant recipient or of the donor cell. To study the phenomenon further, heterospecific cell mixtures were carried out between strains known to function as donors or recipients and certain L cells and their mutant derivatives. Morphological differences between cells of different species permits the identification of donor and recipient cells on an individual basis, eliminating the need for the statistical type of analysis of metabolic cooperation presented in previous experiments [ 14, 151. Heterospecific

cell mixtures.

Fig. I. Radioautographs of cocultured HGPRTase-, APRTase-, L cells (strain A9) and embryonic mouse cells (3T3). A9 cells are small with darkly stained cytoplasm and nuclei containing one or two large nucleoli. 3T3 cells are larger and paler with several small nucleoli. Label is not transferred from 3T3 to A9, indicating lack of metabolic cooperation. (n) Cultures were incubated at 37°C for 3 h with 35 ,&i/ml 3H-hypoxanthine; (b) cultures were incubated at 37°C for lt h with 0.5 ,&i/ml 3H-adenine. Fig. 2. Radioautographs of HGPRTase- human fibroblasts grown with (a) mouse embryo cells (3T3) and with (b) mouse L cells (clone 1D). Human fibroblasts marked by an arrow, are lightly stained while both mouse lines are darker. (a) 3T3 cells donate label to mutant human cells. (b) L cells do not correct mutant phenotype. Cultures were incubated at 37°C for 2 h with SH-hypoxanthine; ‘(a) 5 ,&i/ml; (6) 35 &i/ml. Exptl Cell Res 74 (1972)

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17 - 521809

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R. P. Cox et al.

Fig. 3. Radioautographs of HGPRTase- L cells (strain A9) grown (a) with normal human fibroblasts and (b)

with hamster BHK21 cells. A9 are small darkly stained cells, while both the human and hamster fibroblasts are large and lightly stained. A9 cells remain unlabeled despite contact with HGPRTase+ fibroblasts. Cultures were incubated at 37°C for 2 h with SH-hypoxanthine; (a) 25 ,&i/ml; (6) 20 &i/ml.

Heterospecific cell mixtures were used to determine whether L cells and other established mouse lines could participate as donors in metabolic cooperation. 3T3 cells and L cells (clone ID) were mixed and grown to confluency with HGPRTase- human fibroblasts which are known to be efficient recipients in metabolic cooperation with normal human cells. Both of these mouse cell lines have sufficiently different morphology and nucleolar staining properties to be distinguishable in most cases from human fibroblasts. Fig. 2a shows that 3T3 cells are capable of correcting the LeschNyhan phenotype, while the L cells are not (fig. 2b). This finding raises the possibility Exptl Cell Res 74 (1972)

that the L cell surface is not able to interact with the surface of other cells in a way that would lead to effective communication between them. To extend this observation, an L cell mutant, A9, which is deficient in both HGPRTase and APRTase, was mixed with cells of other species known to be donors for metabolic cooperation. Fig. 3a shows a radioautograph of a mixture of human skin fibroblasts and A9 cells pulsed with tritiated hypoxanthine. Human fibroblasts are heavily labeled, while the A9 cells, which are in contact with the human cells, show little or no incorporation of the purine. Fig. 3 b is a similar radioautograph of BHK21 hamster cells grown with

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Fig. 4. Radioautographs of APRTase- hamster cells Py Y/APP grown with (a) normal human fibroblasts: (b) mouse embryo cells (3T3); (c) mouse L cells (Clone 1D). The hamster cells are stellate shaped, dark staining with a prominent nucleolus, while the human and 3T3 cells are larger and lighter staining. The L cells are very dark staining rounded bipolar fibroblasts. Both (a) human fibroblasts and (6) 3T3 cells donate label to APRTase hamster cells, but (c) L cells do not correct the mutant phenotype. Cultures were incubated at 37°C for 2 h with 3H-adenine 3.0 ,&i/ml.

A9. The hamster cells are well-labeled, but the A9 L cells which are in apparent contact with BHK cells remain unlabeled. These findings indicate that a mutant L cell derivative is unable to receive correcting factors from other cells which are known to be effective donors for metabolic cooperation, suggesting that these cells lack the properties necessary to establish effective intercellular interactions. Heterospecific correction of APRTasemutants has not been previously investigated. Therefore, an APRTase- hamster cell (Py Y/APP) was mixed either with human fibroblasts (fig. 4a), or 3T3 mouse embryo cells (fig. 46) or L cells (fig. 4~). These celis are morphologically distinguishable. When grown to confluency and pulsed with 3Hadenine, heterospecific correction of the hamster APRTase- mutation was observed with human fibroblasts and mouse 3T3 cells but not with L cells (fig. 4).

Moreover, an L cell variant MCR2 deficient in APRTase was unable to undergo metabolic cooperation with either human fibroblasts or 3T3 cells. These findings reemphasize the inability of L cells and their variants to participate in this form of cell communication. Table 3 summarizes studies of metabolic cooperation between HGPRTase- human, hamster, and mouse cells; APRTase- hamster and mouse cells; and various non-mutant cells which are potential donor strains. Species specificities do not appear to influence this form of metabolic cooperation. Effect of contact inhibition and cell morphology on metabolic cooperation Surface interactions between potential donor and recipient cells are likely to be important prerequisites for metabolic cooperation. Therefore, studies were carried out comparing the donor capacity of 3T3 cells, a line Exptl Cell Res 74 (1972)

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with high contact inhibition, with that of certain derivatives of 3T3 cells that have reduced contact properties. Reduced contact inhibition may be the result of alteration in cell interactions and, in the case of polyoma virus transformants, there are also known changes in the glycoprotein constituents of cell surfaces [34]. However, as seen in table 3, when 3T3 CI- or 3T3 Py cells, two lines with reduced contact inhibition, were grown to confluency with human HGPRTase- cells, both variants functioned as donors in metabolic cooperation as efficiently as 3T3 cells with high contact inhibition. It would appear that contact inhibition of growth is not related to function as a donor in metabolic cooperation. Certain human tumor cell lines, for example HeLa cells, exhibit differences in their cellular morphology and in their binding to glass surfaces. HeLa,, (table 1) is a clonal line grown for many years in suspension culture and readapted to monolayer culture 4 years ago. This line grows in monolayer culture as rounded bipolar cells. When mixtures of HeLa,, cells and human Lesch-Nyhan fibroblasts were grown to confluency and pulsed with 3H-hypoxanthine, HeLa,, cells incorporated label, while the HGPRTase- fibroblasts remained unlabeled (fig. 5a), indicating a lack of metabolic cooperation. On the other hand, another clonal line of HeLa cells, HeLa,,, when grown with HGPRTase- human fibroblasts, showed metabolic cooperation (fig. 5b). The findings

that one HeLa line donates in metabolic cooperation, while another cannot, reemphasized the selectivity and specificity of cell communication. Perhaps alterations in cell membranes, responsible for cell shape and mode of attachment, play a role in the intercellular associations formed. These interactions may be important in determining cell communication. Nature of substance transferred from normal to mutant cells

Previous studies of metabolic cooperation between normal human fibroblasts and HGPRTase- human cells were most compatible with the transfer of a product of the enzyme HGPRTase, a nucleotide or nucleotide derivative rather than the transfer of preformed enzyme or informational macromolecules leading to the synthesis of the enzyme [15]. Therefore, the nature of heterospecific metabolic cooperation and the mechanism for correcting APRTase- marker warranted study to determine if a similar process, transfer of a product of the enzyme APRTase, is responsible for metabolic cooperation in this cell system. Morphological differences, particularly in the staining characteristics of the cells and the nucleolar pattern of human and hamster fibroblasts, permit identification of a cell as a human or hamster. Fig. 6a shows metabolic cooperation in monolayer cultures of Py Y/APP hamster APRTase- fibroblasts and normal human fibroblasts when they are incubated

Fip. 5. Radioautoaraahs of HGPRTase- human fibroblasts grown with (a) HeLass (b) HeLa,, cell lines. Human L&ch-Nyhan cell; are more lightly stained and have two or three nucleoli. (a) BeLa,, cells grow as thick binolar fibroblasts with large nucleoli and are more darkly stained than human cells; (b), HeLa,, are cuboidal cells with large nucleoli. I%GPRTase- human fibroblasts are fed by HeLa,, (6) but not by HeLa, (a). Cultures were incubated for 3 h at 37°C with 3H-hypoxanthine; (a), 50 ,&i/ml; (b) 35 pCi/ml. Fig, 6 (a), Radioautograph of a monolayer culture of normal human fibroblasts and APRTase- hamster cells (py Y/APP) showing metabolic cooperation. Human cells are more lightly stained while hamster cells are darker and have a large nucleus; (b) radioautograph of cell suspension prepared from confluent monolayers of (a) showing prompt reversion to mutant phenotype in hamster cells. In (b), a single heavily labeled human cell is shown with several unlabeled hamster fibroblasts. Confluent cultures were incubated for 3 h at 37°C with *H-adenine 3.0 &i/ml. Suspension cultures were incubated for 2 h with 12.5 &i/ml 3H-adenine. Exptl Cell Res 74 (1972)

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R. P. Cox et al.

with 3H-adenine. Upon separating the cells by trypsinization the mutant hamster cells promptly revert to the APRTase- phenotype (fig. 6b). These results suggest that correction of the APRTase- marker by contact with normal cells is also due to transfer of enzyme product. Cell contact required for metabolic cooperation

Previous studies strongly suggest that cell contact is required for metabolic cooperation. When cells of heterozygotes for the Lesch-Nyhan mutation or mixtures of HGPRTase+ and HGPRTase- cells are separated from one another they exhibit either the normal or mutant phenotype. However, as cultures become confluent all cells become labeled [14]. Further evidence that cell contact is required for metabolic cooperation is shown by the following experiments. (1) Transfer of ‘conditioned medium’ from confluent normal cultures to HGPRTasecell monolayers did not increase nuclear labeling of mutant cells when they were pulsed with 3H-hypoxanthine. (2) Confluent cultures of HGPRTase- cells on coverslips incubated side by side with HGPRTase+ cells in radioactive medium failed to increase labeling of mutant cells; nor was metabolic cooperation observed when one coverslip was suspended 0.5 mm above the other and the capillary space filled with radioactive medium. (3) Addition of bull semen 5’ nucleotidase and yeast RNase to confluent mixtures of HGPRTase+ BHK cells and HGPRTase- human cells at the time cultures were pulsed with 3H-hypoxanthine did not reduce transfer and incorporation of label in mutant cells. Since nucleotides or their derivatives appear to be the substances transferred from normal to mutant cells during metabolic cooperation, the addition of these enzymes to medium should prevent Exptl Cell Res 74 (1972)

or reduce labeling of mutant cells if the correcting factors were even transiently extracellular. This evidence strongly suggests that the transfer process involves intimate cell contact and does not occur through the medium. Nature of label in donor and recipient cells as detected by enzymatic digestion

The ability to distinguish cytologically donor cells from recipients in heterospecific cell mixtures permits comparison of the nature of incorporated label in mutant and recipient cells by the susceptibility of the label to enzymatic digestion in fixed preparations. The nature of the label in both donor and recipient cells can be approximated by studying the effect of hydrolytic enzymes, on the assumption that extensive cleavage of the labeled macromolecule would release 3Hpurine and markedly reduce the grains in subsequent radioautographs. As shown in fig. 7a the amount and distribution of grains from incorporated 3H-hypoxanthine is similar in HGPRTase+ and HGPRTase- cells following metabolic cooperation. Fig. 7b, c show the effects of incubating labeled cells, fixed in methanol, with either RNase (7 b) or DNase (7~) as compared with replicate cells not treated with enzymes. Incubation of fixed coverslip preparations in solutions containing RNase markedly decreased the grain count in both cytoplasm and nucleus of cells which had undergone metabolic cooperation (fig. 7b). The basophilic staining characteristic of May-Griinwald-Giemsa stains is also markedly decreased in these cells confirming the loss of nuclear and cytoplasmic RNA in the treated cells. On the other hand, incubation of similar preparations with solutions of DNase caused only moderate diminution in the labeling of nuclei (fig. 7~). The loss of labeling from both nuclei and cytoplasm observed following

Fig. 7. Comparison of RNase and DNase treatment with respect to the release of incorporated 3H-purine from fixed preparations of cocultured HGPRTasef hamster donor and HGPRTase- human recipient cells follo\ving metabolic cooperation. Cultures had been incubated with 20 pCi 3H-hypoxanthine for 3 h at 37’C followed by fixation in methyl alcohol overnight. (a) Control-no enzyme treatment; (b) RNase: 1.7 jrg/ml for 30 min; (c) DNase: 2 rceiml for 30 min. The enzvme were in 0.04 M Tris-0.9 “,, saline containing 4 mM MgCl, _ preparations _. at pH 7.2.’ I’ Fig. 8. Effect of sulfhydryl reactive substance and metabolic inhibitors on metabolic cooperation between cocultured HGPRTasef hamster cells and HGPRTase- human fibroblasts. (a) Control-no additions. Hamster cells are dark staining smaller cells which are feeding the paler and larger human cells; (b) p-chloromercuribenzoate (PCMB) treated: Cultures were incubated for 30 min with 0.1 mM PCMB and then were pulsed with 3Hhypoxanthine in the presence of PCMB; (c) dinitrophenol (DNP) treated: Cultures were incubated for 30 min with 5.0 mM DNP and then were pulsed with 3H-hypoxanthine in the presence of DNP; (d) potassium aride (5 mM) and sodium fluoride (2 mM) treated. Cultures were incubated for 30 min with inhibitors and then were pulsed with 3H-hypoxanthine in the presence of inhibitors. All cultures were incubated at 37°C for 3 h with 20 &i/ml of 3H-hypoxanthine. In this experiment cultures of HGPRTase- human fibroblasts exhibit between 0 and 5 grains/cell nucleus following a similar incubation with label.

264 R. P. Cox et al. digestion with RNase, and from nuclei following DNase, were similar for the donor HGPRTase+ hamster cells and for the recipient HGPRTase- human cells, indicating that radioactive hypoxanthine or its transferred derivative was probably incorporated into similar macromolecules in both species.

Effects of suljhydryl-blocking agents and metabolic inhibitors on metabolic co-operation Sulfhydryl-containing proteins have been implicated in the translocation mechanism of several transport systems [35, 361.Therefore, the effects on metabolic cooperation of sulfhydryl-blocking agents, were studied. Confluent mixtures of HGPRTasef hamster and HGPRTase- human cells were pulsed with SH-hypoxanthine in the absence and presence of 0.1 mM p-chloromercuribenzoate (PCMB) or 1.O mM N-ethylmaleimide (NEM). These concentrations of sulfhydryl blocking agents have previously been shown to inhibit cation transport in mammalian cell cultures [36]. NEM was toxic to these cultures and many cells detached from the monolayer. PCMB caused no apparent cytological effects and only slightly reduced the incorporation of 3H-hypoxanthine into HGPRTase+ hamster cells (fig. 8b). Moreover, PCMB did not significantly affect the amount of label transferred to HGPRTasehuman cells by contact with HGPRTase+ hamster cells. An uncoupler of oxidative phosphorylation, dinitrophenol (DNP), reduced the incorporation of 3H-hypoxanthine into HGPRTase+ cells. In the presence of 5 mM DNP transfer of label to mutant cells still occurred despite the reduced labeling of donor cells (fig. 8~). Two potent inhibitors of both glycolysis and oxidative metabolism, Exptl

Cell Res 74 (1972)

potassium azide (5 mM) and sodium fluoride (2 mM), markedly decreased the labeling of HGPRTase+ donor cells but did not appear to significantly interfere with the transfer of label to mutant cells (fig. 8d). These results suggest that energy is required for incorporation of radioactive purines into cellular material, but transfer of label from normal to mutant cells may be less dependent on energy production. Specificity and selectivity of metabolic cooperation The specificity and selectivity of transfer were further studied using another mutation, glucose-6-phosphate dehydrogenase deficiency (G-6 PD-), which like the phosphoribosyltransferase mutants described above can be recognized at the cellular level. The phenotype is detected histochemically through the use of the enzymatic reduction of NADP to NADPH in the presence of the substrate (glucose-6-phosphate) coupled to the reduction of tetrazolium salts [19]. Insoluble deposits of formazan appear within G-6 PD+ cells but not in G-6 PD- cells if potassium azide is also included in the incubation mixture, as recommended by Piomelli, to inhibit reduction of NADP by glycolysis [37]. In populations of G-6 PD+ cells cultured alone, nearly all cells show formazan precipitation (fig. 9a) while G-6 PD- fibroblasts do not stain (fig. 9 b). Fig 9 c shows a representative field in which G-6 PD+ normal human fibroblasts are cocultured with G-6 PDhuman fibroblasts in a ratio of 1 normal per 100 mutant cells. As seenin fig. 9c, formazan precipitation is present in one cell while G-6 PD- cells which are in intimate contact with the G-6 PD+ fibroblast do not contain the specific precipitate. These findings suggest that metabolic cooperation does not occur with respect to the G-6 PD- phenotype.

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Fig. 9. Histochemical detection of G-6 PD. (a) G-6 PD+ normal fibroblasts. The intracellular dark staining large

granular material is precipitated formazan and indicates enzyme activity. There is slight diffusion of formazan extracellularly which appears as small fine indistinct particles surrounding the cells; (b) G-6 PD- deficient l‘ibroblasts from patients with nonspherocytic hemolytic anemia; (c) cocultured G-6 PD- and G-6 PDI- cells at a ratio of 100 to 1. A G-6 PD+ cell is heavily stained showing the large granules indicating enzyme activity. There is no detectable metabolic cooperation since the G-6 PD- fibroblasts that are in intimate contact remain unstained. A slight nonspecific diffusion of formazan which appears as small indistinct particles is observed around the G-6 PD+ cell. Histochemical method for G-6 PD is that recommended by Siniscalco et al. [19] with the addition 0.1 % potassium azide as proposed by Piomelli [37].

DISCUSSION The results of the present study indicate that metabolic cooperation between cells growing in tissue culture requires cell to cell contact and appears highly selective and specific. Specificity is exhibited in the nature of substances transferred and by the finding that not all cells are able to undergo metabolic cooperation. Derivatives of L cells are unable to function as recipients of HGPRTase or APRTase products, while hamster and human cells with these enzyme deficiencies exhibit correction of the mutant phenotype when in intimate association with certain normal donors. Similarly, L cell derivatives cannot function as donors while the established mouse 3T3 cell, mouse embryo cells, hamster fibroblasts and human strains can. The reason certain cells act as recipients or

donors while other cells cannot give or receive, is not known. Recently Gilula and his associates have shown that mixtures of norma1 and HGPRTase- Chinese hamster cells which undergo metabolic cooperation are also ionically coupled and have gap junctions [38]. However, mixtures of HGPRTasemouse L cells with normal hamster fibroblasts do not exhibit metabolic correction nor are they ionically coupled and they also lack gap junctions between hamster and L cells. These findings provide evidence for the irnportance of a specific cellular structure, the gap or low resistance junctions, in mediating both metabolic cooperation and ionic coupling [38]. There is also selectivity with respect to substances transferred. Correction of the APRTase- and HGPRTase- phenotypes observed with hamster and human cells apparExptl Cell Res 74 (1972)

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ently is the result of transfer of the nucleotide or a nucleotide derivative from normal to mutant cell. Subak-Sharpe has recently shown that metabolic cooperation can also occur with hamster cells that are deficient in either deoxycytidine kinase or thymidine kinase (personal communication). Thus to date, metabolic cooperation has been observed only in cells deficient in one of the enzymes of nucleotide biosynthesis. On the other hand, the phenotype of (G-6 PD-) cells is not corrected by contact with normal cells (G-6 PD+) suggesting that the enzyme (G-6 PD) or an informational macromolecule leading to its synthesis in mutant cells is not extensively transferred. The present study emphasizes the importance of intimate cell contacts as a prerequisite for metabolic cooperation. Inclusion in the medium of enzymes which hydrolyse nucleotides and their derivatives failed to affect transfer of label even though nucleotides or their derivatives appear to be the substances transferred. Recently Ashkenazi & Gartler [39] have reported that incubation of freshly trypsinized Lesch-Nyhan cells with homogenates of normal cells confers the ability to transiently incorporate small amounts of 3H-hypoxanthine. Under these circumstances, the label is primarily cytoplasmic rather than the nuclear and nucleolar pattern seen with metabolic cooperation. This difference in cytological localization of incorporated label suggests that a mechanism different from metabolic cooperation is involved. It is possible that recently trypsinized mutant HGPRTase- cells ingest particles containing the enzyme HGPRTase from normal cell sonicates and therefore temporarily are able to incorporate 3H-hypoxanthine. The processes responsible for transfer of nucleotide from donor to recipient cells apparently do not involve sulfhydryl groups, since treatment of cultures with PCMB does Exptl Cell Res 74 (1972)

not affect metabolic cooperation. Energy does not appear to be required for transfer of label from donor to mutant, since treatment of cells with either DNP or a combination of azide and fluoride which markedly decreases labeling of donor cells permits a proportion of label to be transferred to the mutant cells. Heterospecific cell mixtures provide an advantage for studying metabolic cooperation, since morphological differences between cells of different species permit individual cells to be identified and eliminate the need for statistical evaluation. Stoker previously demonstrated that freshly isolated mouse embryo cells were able to correct the phenotype of HGPRTase- hamster cells [40]. The mouse cells were marked by ingested carbon or carmine granules to distinguish them from the mutant hamster cells. This method of distinguishing cells is somewhat hampered by the transfer of carbon from marked to unmarked cells and by the toxicity of certain ingested particles. Recently Michalke & Lowenstein have used heterospecific cell mixtures in which the cells are morphologically different to study electrical coupling [41]. Their results also emphasize the value of cytologically distinguishing cell types in studies on cell communication. Cell-cell interactions among mammalian somatic cell populations which require cell contact may involve several mechanisms. Transfer of substances from one cell to another is being recognized with increasing frequency. At a recent symposium on cell-cell interactions [42]. Cruikshank reported that melanocytes can transfer pigment material to keratinocytes when they form an intimate association, and Mann described a similar interaction between mast cells and eosinophiles with a transfer of granular material to eosinophiles. Kolodny has presented evidence for transfer of RNA between mouse

Cell communication

cells in culture and such transfer also requires intimate cell contact [43]. Electrical coupling between cells has been studied by Lowenstein and by Potter and their associates [9, 10, 411.Although the special membrane contacts, or low resistance gap junctions, which are believed to be of importance in the electrical coupling of cells were discovered in the nervous system, it has become apparent that they exist between many cell types both in vivo and in cultures. These specialized membrane junctions or electrical synapses are probably the site of ion transfer and are responsible for electrical transmission. Recent evidence suggests that gap junctions also may be important structures for the passage of nucleotides from normal to mutant cells during metabolic cooperation [38]. Further support for this hypothesis is the evidence that fluorescein ‘(mol. wt 330) passed into adjacent cells when injected electrophoretically into BHK21 fibroblasts in culture with a microelectrode [lo]. Fluorescein often was detected two or three cells away, demonstrating spread between cells not impaled by the electrode. The spread of dye was never observed in the absence of cell contact. Short-range interactions between cells may play a role in contact inhibition of cell division and cell locomotion. The exchange of metabolites and other small molecuIes when a cell in culture establishescontact with other cells may stabilize the metabolic activity of susceptible cells as each cell tends to acquire an average concentration of substances exchanged. In essence, this exchange on contact could profoundly affect the metabolic activity of susceptible cells. The correction of mutant phenotypes by cell contact with normal cells, as observed with Lesch-Nyhan cells in cuhure, may be of importance in vivo for maintaining normal function in mutant cells of heterozygotes

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for this sex-linked recessive gene [ 13, 14, 15, 441. Moreover, the exchange of molecules between cells may play a more general role in embryonic differentiation and in modulation of gene expression. Failure of cell--cell communication could be of importance in abnormalities of development and perhaps in neoplasia. The authors aupreciate the advice of Dr M. Siniscalco and Dr.% Piomelli on methods for studying G-6 PD. This investigation was supported by USPHS grants AM 14528, CA-08748, and HD 04526.

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34. Defendi, V & Gasic, G, J cellular camp physio162 (1963) 23. 35. Fox, C F & Kennedy, E P, Proc natl acad sci US 54 (1965) 891. 36. Cox, R P, Mol pharm 4 (1968) 510. 37. Piomelli, S. In preparation (1972). 38. Gilula, N B, Reeves, 0 R & Steinbach, A, Nature 235 (1972) 262. 39. Ashkenazi, Y E & Gartler, S M, Exptl cell res 64 (1971) 9. 40. Stoker, M G P, J cell sci 2 (1967) 293. 41. Michalke, W & Lowenstein, W R, Nature 232 (1971) 121. 42. Editorial, Interactions in populations, Nature 228 (1970) 15. 43. Kolodny, G M, Exptl cell res 65 (1971) 313. 44. Fujimoto, WY, Subak-Sharpe, J H & Seegmiller, J E, Proc natl acad sci US 68 (1971) 1516. Received December 21, 1971 Revised version received March 22, 1972