metabolic co-operation between biochemically marked mammalian cells in tissue culture

12
CLASSIC PAPER Metabolic co-operation between biochemically marked mammalian cells in tissue culture{ H. Subak-Sharpe, R. R. Bu ¨ rk and J. D. Pitts Reviewed by Marcela Kudelova* Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic Accepted: 27 September 2001 INTRODUCTION Adjacent cells must be sealed together to prevent passage of fluids through the cell layer and to give the tissue strength. These impermeable seals are termed tight junctions and desmosomes (various adhering junctions). Gap junctions do not form a seal and so allow the free passage of small molecules between a cell and its neighbour. The particles in the gap are actually tiny channels that directly link the cytoplasms of the two cells [1,2]. A vivid example of such cell-to-cell transfer is the phenomenon of metabolic coupling or metabolic co-operation, where cells are capable of transfer- ring to neighbouring cells molecules that the recipients are unable to synthesise. For example, adenosine mono-, di- or tripho- sphate can pass through gap junctions. Hypo- xanthine can serve as a precursor of DNA. It is converted first to inosine 5k-phosphate, in a reaction catalysed by the enzyme hypoxanthine- guanine phosphoribosyltransferase (HPRT) and then to dATP, the immediate precursor of DNA. If cells lacking HPRT are cocultured with cells that have this enzyme, and labelled hypoxanthine is added to the medium, then radioactivity is frequently found in the nuclear DNA of enzyme deficient cells. The more than 30-year-old publication by Subak-Sharpe et al. is a classic example of the application of this phenomenon that they referred to as ‘metabolic co-operation between cells’. The authors used cells of a genetic variant of the hamster fibroblast line BHK 21 which lack inosinic pyrophosphorylase activity (IPP x cells) and there- fore were unable to incorporate radioactive hypoxanthine into the nuclear DNA. However, when in direct or indirect contact with IPP + cells, IPP x cells also incorporated the isotope. In discussing the significance of their work, their foreseeable application of this phenomenon in the gene-based chemotherapy of cancer is wor- thy of mention. Metabolic co-operation between biochemically marked mammalian cells in tissue culture H. SUBAK-SHARPE, R. R. BU ¨ RK AND J. D. PITTS* M.R.C. Experimental Virus Research Unit, Institute of Virology, University of Glasgow, Scotland Reprinted from J. Cell Sci. 4, 353–367 (1969) Summary Cells of a genetic variant of the hamster fibroblast line BHK 21 which lack inosinic pyrophosphorylase activity (IPP x cells) and therefore cannot normally incorporate [ 3 H]hypoxanthine were grown in mixed culture with cells of BHK 21 sublines which have inosinic pyrophosphorylase activity (IPP + cells). If not in contact with IPP + cells, IPP x cells do not incorporate added [ 3 H]hypoxanthine into nucleic acid. IPP + cells always do incorporate [ 3 H]hypoxanthine and IPP x cells when in direct or indirect contact with IPP + cells also incorporate the isotope. Cell to cell contact appears to be essential for this gain of a metabolic function by IPP x cells. The possible molecular basis and general implications of the phenomenon are discussed. *Corresponding to: Dr M. Kudelova, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 842 45 Bratislava, Slovak Republic. {Reproduced from J. Cell Sci. 4, 353–367 (1969) with kind permission of the publishers. *Present address: Institute of Biochemistry, University of Glasgow, Scotland. Reviews in Medical Virology Rev. Med. Virol. 2002; 12: 69–80. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rmv.342 Copyright # 2002 John Wiley & Sons, Ltd.

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Page 1: Metabolic co-operation between biochemically marked mammalian cells in tissue culture

C L A S S I C

P A P E R

Metabolic co-operation between biochemicallymarked mammalian cells in tissue culture{H. Subak-Sharpe, R. R. Burk and J. D. Pitts

Reviewed by Marcela Kudelova*Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic

Accepted: 27 September 2001

INTRODUCTIONAdjacent cells must be sealed together to preventpassage of fluids through the cell layer and to givethe tissue strength. These impermeable seals aretermed tight junctions and desmosomes (variousadhering junctions). Gap junctions do not forma seal and so allow the free passage of smallmolecules between a cell and its neighbour. Theparticles in the gap are actually tiny channels thatdirectly link the cytoplasms of the two cells [1,2].A vivid example of such cell-to-cell transfer is thephenomenon of metabolic coupling or metabolicco-operation, where cells are capable of transfer-ring to neighbouring cells molecules that therecipients are unable to synthesise.

For example, adenosine mono-, di- or tripho-sphate can pass through gap junctions. Hypo-xanthine can serve as a precursor of DNA. Itis converted first to inosine 5k-phosphate, in areaction catalysed by the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) andthen to dATP, the immediate precursor of DNA.If cells lacking HPRT are cocultured with cellsthat have this enzyme, and labelled hypoxanthineis added to the medium, then radioactivity isfrequently found in the nuclear DNA of enzymedeficient cells.

The more than 30-year-old publication bySubak-Sharpe et al. is a classic example of theapplication of this phenomenon that they referredto as ‘metabolic co-operation between cells’. Theauthors used cells of a genetic variant of thehamster fibroblast line BHK 21 which lack inosinic

pyrophosphorylase activity (IPPx cells) and there-fore were unable to incorporate radioactivehypoxanthine into the nuclear DNA. However,when in direct or indirect contact with IPP+

cells, IPPx cells also incorporated the isotope. Indiscussing the significance of their work, theirforeseeable application of this phenomenon inthe gene-based chemotherapy of cancer is wor-thy of mention.

Metabolic co-operation betweenbiochemically marked mammaliancells in tissue cultureH. SUBAK-SHARPE, R. R. BURK ANDJ. D. PITTS*M.R.C. Experimental Virus Research Unit, Institute of Virology,

University of Glasgow, Scotland

Reprinted from J. Cell Sci. 4, 353–367 (1969)

Summary

Cells of a genetic variant of the hamster fibroblast line BHK 21

which lack inosinic pyrophosphorylase activity (IPPx cells)

and therefore cannot normally incorporate [3H]hypoxanthine

were grown in mixed culture with cells of BHK 21 sublines

which have inosinic pyrophosphorylase activity (IPP+ cells). If

not in contact with IPP+ cells, IPPx cells do not incorporate

added [3H]hypoxanthine into nucleic acid. IPP+ cells always

do incorporate [3H]hypoxanthine and IPPx cells when in direct

or indirect contact with IPP+ cells also incorporate the isotope.

Cell to cell contact appears to be essential for this gain of a

metabolic function by IPPx cells.

The possible molecular basis and general implications of the

phenomenon are discussed.

*Corresponding to: Dr M. Kudelova, Institute of Virology, SlovakAcademy of Sciences, Dubravska cesta 9, 842 45 Bratislava, SlovakRepublic.

{Reproduced from J. Cell Sci. 4, 353–367 (1969) with kindpermission of the publishers.

*Present address: Institute of Biochemistry, University ofGlasgow, Scotland.

Reviews in Medical Virology

Rev. Med. Virol. 2002; 12: 69–80.Published online in Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/rmv.342

Copyright # 2002 John Wiley & Sons, Ltd.

Page 2: Metabolic co-operation between biochemically marked mammalian cells in tissue culture

Introduction

Variants of a cultured mammalian cell line (HEp 2) with

reduced inosinic pyrophosphorylase (IMP: pyrophosphate

phosphoribosyl transferase E.C. 2. 4. 2. 8) activity were first

reported in 1961 by Brockman, Kelley, Stutts & Copeland. In

1962 Brockman, Roosa, Law & Stutts described analogous

variants of the mouse cell line P-388, and in the same year

Szybalski, Szybalska & Ragni reported the isolation of a variant

of the Detroit 98 (normal human sternal bone marrow) cell line

which lacked inosinic pyrophosphorylase activity. Similar

variants of mouse L cells (Littlefield, 1963) and hamster BHK

21 cells (Subak-Sharpe, 1965) have subsequently been isolated

in other laboratories. Later in 1962 Szybalska & Szybalski

reported experiments which indicated that cells which lacked

inosinic pyrophosphorylase activity could be transformed

by DNA extracted from mammalian cells which possessed

this enzymic activity. These genetically transformed cells

formed stable clones with apparently normal enzyme activity.

Stimulated by this report we have been investigating a

radioautographic approach which might allow us to detect

genetic transformation with the BHK 21 cell system which

is studied in this Institute. In preliminary reconstruction

experiments designed to detect hypoxanthine incorporation

by rare cells with inosinic pyrophosphorylase activity among

many which lack it, we observed a phenomenon which will be

referred to as ‘metabolic co-operation between cells’. The

consistent finding is that genetically deficient cells incorporate

tritiated hypoxanthine into their nucleic acid when they are in

contact with cells known to have inosinic pyrophosphorylase

activity.

Materials and methods

The cells used originate from the hamster fibroblast line BHK

21 clone C 13 described by Macpherson & Stoker (1962). The

derivation of the control parent cell lines, the ‘normal’ C 13 and

the polyoma virus-transformed Py Y, has been given by Stoker

& Macpherson (1964) and that of the biochemical variant

Py Y/TG 1 by Subak-Sharpe (1965). Py Y/TG 1 cells cannot

convert hypoxanthine, guanine or their ribosides to the

respective nucleotides inosinic or guanylic acid. Thus Py

Y/TG 1 do not incorporate hypoxanthine into RNA or DNA.

These cells have been shown to lack detectable inosinic

pyrophosphorylase activity by incorporation experiments

with cells in culture (Subak-Sharpe, 1965), by in vitro enzyme

Figures 1 and 2. Incorporation of [3H]hypoxanthine into nucleic acid during 48 h of growth by Py Y/TG 1 cells (IPPx) (Fig. 1); and Py Y

cells (IPP+) (Fig. 2). Magnification approximately r800. All cells were in pure culture. The radioautographic technique is described in

the text. Film was developed after 42 days.

70 Classic Paper

Copyright # 2002 John Wiley & Sons, Ltd. Rev. Med. Virol. 2002; 12: 69–80.

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assays (Hay & Subak-Sharpe, unpublished), and now by

radioautography. Py Y/TG 1 will be referred to as IPPx.

Inosinic pyrophosphorylase can be readily demonstrated in

both the C 13 and Py Y cell lines in vivo, in vitro, and also by

radioautography. As both cell lines behave similarly in the

experiments to be described, they will not be distinguished and

only referred to as IPP+.

The medium used was ETC (8 parts modified Eagles, 1 part

tryptose phosphate broth, 1 part calf serum). Incorporation of

[3H]hypoxanthine by IPP+ varied with different batches of

tryptose phosphate broth. Therefore a single batch of tryptose

phosphate broth and one of calf serum, known to result in good

incorporation, were used for these experiments.

Preparation for radioautographyCells were grown to confluent monolayers, removed from the

glass by short exposure to 0.05% trypsin plus 1 mM EDTA at room

temperature, resuspended in ETC, and counted. (These were

single-cell suspensions—on no occasion were more than 5% of

the cells in clumps.) Then 105 cells were seeded into 60-mm glass

Petri dishes containing from 3 to 6 13-mm glass coverslips under

5 ml of ETC. The cells were incubated in a gassed incubator at

37uC. At the appropriate time 10 mc of [3H]hypoxanthine (specific

activity 462 mc/mM, Radiochemical Centre, Amersham) were

added. After further incubation coverslips were fixed and

processed for radioauto-graphy as described below.

In some experiments IPP+ cells were prelabelled with

[3H]hypoxanthine by preincubation for 48 h in the presence

of 0.5–1.0 mc/ml.

RadioautographyCells grown on clean coverslips were washed in phosphate-

buffered saline (PBS (a) of Dulbecco & Vogt, 1957) and fixed in

glacial acetic acid/ethanol (3/1). Acid-soluble material was

removed by washing in 10% trichloroacetic acid, followed by

two further washes in water—each operation for 10 min at 0uC;

the coverslips were then dipped in ethanol and allowed to dry

at room temperature.

The coverslips, mounted with DePeX (Gurr) on gelatine/

chrome alum treated slides (0.5% gelatine, 0.05% chrome alum,

0.5% formaldehyde (40% solution), 0.1% Kodak Photoflo), were

covered with Kodak AR 10 stripping film. After exposure for 7

or more days at 4uC in a dry, light-tight box, the film was

developed at 20uC in Kodak D 19 b for 5 min, rinsed in water

and fixed in Amfix (May and Baker) for twice clearing time.

The slides were washed in running water for 5 min and then

dried and stained with Giemsa.

Figure 2.

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Copyright # 2002 John Wiley & Sons, Ltd. Rev. Med. Virol. 2002; 12: 69–80.

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Results

Figures 1 and 2 show radioautographs of Py Y/TG 1 (IPPx) and

Py Y (IPP+) cells respectively after 48 h growth in the presence

of [3H]hypoxanthine. IPPx cells show no observable incorpora-

tion above background. All IPP+ cells incorporate label heavily

into their nucleic acid, though there is some quantitative

variation. As these cells are not synchronized and probably

vary in their metabolic activity this is to be expected.

In mixed culture when no contact is made or appears to have

taken place between IPP+ and IPPx cells, each behaves

independently of the other (Fig. 3): IPPx cells typically do

not incorporate [3H]hypoxanthine into their nucleic acid while

IPP+ cells become heavily labelled. ‘Heavily labelled’ means

well over the threshold of countability—virtually black.

The situation is different wherever IPPx cells are in contact

with IPP+ cells: the IPP+ cells in the presence of a great excess

of IPPx cells stand out clearly, as they incorporate label heavily

(Fig. 4). Usually the IPP+ cells are seen to be surrounded by

more lightly labelled cells, which are in direct or indirect

contact with the heavily labelled IPP+ cells (Figs. 5–7). (There is

clear discontinuity between heavily and lightly labelled: in the

latter case the silver grains are usually easily countable.) For

the time being these lightly labelled cells will be referred to as

IPPx. In pure cultures of IPP+ cells although there is variation

in the amount of label incorporated, IPP+ cells that could be

mistaken for the clones of lightly labelled IPPx type of cell are

never observed.

An illustration of the phenomenon and the clue to its nature

is given by the following experiment. IPPx and IPP+ cells were

seeded to give a mixture of 300 : 1 or 3000 : 1 respectively at 106,

5r105, 2r105 and 105 cells per plate, incubated and coverslips

fixed after exposure to [3H]hypoxanthine from 0–12, 12–24,

24–36 and 36–48 h respectively. After radioautography, heavily

incorporating clones of 1, 2, 4, 8 or more cells stood out clearly.

Cells in direct or indirect contact with these heavily labelled

cells were lightly labelled, but lightly labelled cells were not

found elsewhere. The mean doubling time of all these BHK-

derivative cells is about 9–12 h which allows positive identifi-

cation of the heavily labelled cells as IPP+. Figures 5–7

illustrate typical clones of 1, 4 and about 16 IPP+ cells in

contact with IPPx cells. For the labelling periods 0–12, 12–24

and 24–36 h in another representative experiment Table I

gives the relative distribution of clones containing various

numbers of heavily labelled cells and various numbers of

lightly labelled cells in direct or indirect contact with these.

With exposure to [3H]hypoxanthine during the first 12-h

Figure 3. Incorporation of [3H]hypoxanthine into nucleic acid. Py Y/TG 1 (IPPx) and (IPP+) cells in mixed culture but not in contact:

the mixed cells were plated at high dilution and pulsed 24–36 h (IPP+ were Py Y). Magnification approximately r1000. Developed after

42 days.

72 Classic Paper

Copyright # 2002 John Wiley & Sons, Ltd. Rev. Med. Virol. 2002; 12: 69–80.

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period mainly groups of 1, 2 or 4 heavily labelled cells were

observed. After the second and third pulse periods groups

containing progressively larger numbers of heavily labelled

cells were found. This is just what is expected if the heavily

labelled cells are directly derived by cell division (at the normal

rate) from the initially single IPP+ cells.

On the other hand the lightly labelled (IPPx) cells per group

occurred on average in much larger numbers than expected, if

these IPPx cells are derived by normal cell division from the

initially single IPP+ cells. With labelling from 0–12 h, clones

with as many as 10, 12, 13 and even 20 were observed and in

the 12- to 24-h labelled sample some clones contained over 40

(see Table 1). The IPPx cells can therefore not derive from IPP+

cells by normal division.

As the number of heavily labelled (presumptive IPP+) cells

available for contact increased through cell division the

number of lightly labelled IPPx cells per group also increased.

The number of IPPx cells for a given number of IPP+ cells in a

group varied considerably, and was relatable to the cell density

in that particular area of the coverslip. Contact with a heavily

labelled cell appeared to be a pre-requisite for the occurrence of

IPPx cells and cells growing isolated from the heavily labelled

cells did not show any labelling—they had normal IPPx

phenotype. These results clearly indicate that the IPPx cells

must be originally IPPx cells which have gained the ability to

incorporate [3H]hypoxanthine through direct or indirect con-

tact with IPP+ cells, but as IPPx cells which are ‘metabolic co-

operators’. Similarly the heavily labelled cells will be referred

to as IPP+.

Other experiments, which will be given in detail in a future

communication, utilized the technique of prelabelling one of

the cell lines by allowing them to engorge carbon or carmine

(Stoker, 1964). Individual cells could then be identified even in

the mixture. The results were in complete agreement with the

above conclusions.

Extensive observations have been made in many mixed cell

experiments. They indicate that metabolic co-operation in this

BHK 21 system needs cell-to-cell contact between IPPx and

IPP+ cells. Though it is difficult to rule out completely, there is

no evidence that the phenomenon occurs without such contact.

It might be argued that the observations are due to increased

permeability of the cell membranes or to changed RNA

Figure 4. IPP+ cells growing in the presence of a great excess of IPPx cells. A mixture of 1 IPP+: 300 IPPx was plated at high dilution

and pulsed with [3H]hypoxanthine from 24–36 h. A clone of 16 IPP+ cells can be seen in the centre, 15 cells on one side and 1 on the

other of a gap in the cell sheet. Note how the individual cells have separated due to movement after cell division. Magnification

approximately r30. Developed after 42 days.

Classic Paper 73

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metabolism caused by the trypsin/versene treatment. This is

not so, for in experiments where cells were collected by shaking

off the glass, good metabolic co-operation was observed.

At times, mycoplasma have been detected in these cell lines.

Could this explain the phenomenon? The transfer of myco-

plasma from donor to recipient is unlikely to furnish the

explanation of metabolic co-operation, for the phenomenon

occurred in the same manner whether or not mycoplasma

could be detected, using the method of Fogh & Fogh (1964)

with increased sensitivity through modifications by W. House

(to be published). Metabolic co-operation has also been

repeatedly observed by Stoker (1967a) in experiments using

as donors primary mouse embryo cells, which have never been

found to be contaminated with mycoplasma at this Institute.

The following 5 points summarize the observations which

have been made consistently in mixed culture experiments.

IPPx cells not in contact with IPP+ cells do not incorporate

label into their nucleic acid (e.g. Fig. 3). (There are very rare

exceptions, whose position always suggests that they have been

in such cell-to-cell contact previously and have become recently

separated due to cell movement.) IPPx cells in microscopically

observable contact with IPP+ cells almost always incorporate

label; these will be called primary co-operators (e.g. Fig. 5). In

addition, IPPx cells in contact with primary co-operators

usually incorporate label; such cells will be referred to as

secondary co-operators, and IPPx cells in contact with these

may also incorporate some label (e.g. Fig. 7). A gradient of

incorporation from the initiating IPP+ cell can frequently be

discerned. Effective co-operation is established in less than 12 h

and does not need division of the IPP+ cells as is shown by

Fig. 5 and Table 1. The distribution of IPP+ cells in clones at the

later times indicates that, in addition to cell division, sometimes

quite extensive cell movement occurs (e.g. Fig. 7); this can make

interpretation of results progressively more difficult. Of course

cell division and movement not only affect IPP+ cells but also

co-operator and other IPPx cells.

Discussion

Metabolic co-operation as observed in [3H]hypoxanthine

incorporation experiments necessarily manifests a donor-

recipient relationship between IPP+ and IPPx cells. However,

we consider the co-operation to be reciprocal, for primary

co-operators show both recipient (from IPP+ cells) and donor

(to secondary co-operators) properties. This can be better

Figures 5, 6 and 7. Incorporation of [3H]hypoxanthine into nucleic acid by Py Y/TG 1 (IPPx) and Py Y cells grown in mixed culture in

contact: pulsed 0–12 h (Fig. 5), pulsed 12–24 h (Fig. 6), and pulsed 36–48 h (Fig. 7, on next page). Note heavily labelled and lightly

labelled cells in contact and unlabelled cells either not in contact or far distal. Magnification approximately r800. Developed after

42 days.

74 Classic Paper

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Figure 6.

Figure 7.

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Page 8: Metabolic co-operation between biochemically marked mammalian cells in tissue culture

demonstrated by use of an independent second marker (Burk,

Pitts & Subak-Sharpe, in preparation). Thus metabolic co-

operation, which is defined as the process whereby the

metabolism of cells in contact is modified (perhaps controlled)

by exchange of material, appears to be a general phenomenon.

Cell contact despite cell movement is sometimes strikingly

illustrated by long cytoplasmic connecting ‘bridges’ between

co-operating cells. Figure 8 particularly focuses on a cytoplas-

mic ‘bridge’ between a primary and a secondary co-operator.

Figure 8 also demonstrates that the labelled nucleic acid is

widely dispersed through-out the cytoplasm and nucleus of

co-operator cells.

Subak-Sharpe, Burk & Pitts (1966) described metabolic co-

operation in outline in a preliminary report, and Stoker (1967a)

confirmed the findings, extending the investigations to primary

mouse cells. Some published work on organ cultures may

concern the same phenomenon. Loewenstein & Kanno (1964)

have investigated the membrane permeability of epithelial cell

junctions in the Drosophila salivary gland, using intracellular

microelectrodes. They found that small ions as well as sodium

fluorescein (mol. wt. 376) can move rather freely from cell to

cell without leaking to the exterior and conclude that the organ

rather than the single cell appears to be the unit of ion-

environment. In a further paper Kanno & Loewenstein (1966)

Table I. IPPx cells (Py Y/TG 1) and IPP+ cells (C 13 or Py Y) were mixed 300 : 1 and distributed at 5r105,

2r105 and 105 cells per 50 mm plate containing coverslips. [H3]hypoxanthine was added to the plates at

0–12 h, 12–24 h and 24–36 h respectively, and the coverslips fixed for radioautography after each 12-h pulse.

Processed radioautographs were examined for groups containing one or more heavily labelled cells and the

lightly labelled cells associated with each such group were scored. 4 coverslips from 2 experiments were

examined for each mixture.

No. of heavily

labelled cells

in the group

0–12 h* 12–24 h* 24–36 h*

No. of groups

observed

Mean no. of

lightly labelled

cells/group

No. of groups

observed

Mean no. of

lightly labelled

cells/group

No. of groups

observed

Mean no. of

lightly labelled

cells/group

IPP+=C13

1 13 4 (0–10){ 3 5 (4–5) 0 —

2 17 5 (0–12) 8 5 (1–11) 4 5 (3–8)

3 1 13 (—) 1 50+ (—) 0 —

4 4 10 (2–20) 12 13+ (0–40+) 5 8 (3–14)

5 1 2 (—) 2 13 (3–23) 1 23 (—)

6 — — 5 10 (7–17) 1 10 (—)

7 — — 0 — 1 8 (—)

8 — — 4 4 (2–6) 8 15+ (6–40+)

9–15 — — — — 10 13+ (3–40+)

16 — — — — 3 27+ (12–50+)

IPP+=Py Y

1 6 2 (0–5) 1 5 (—) 0 —

2 16 3 (0–7) 7 6 (0–20) 2 10 (9–12)

3 1 10 (—) 4 7 (5–10) 2 16 (12–20)

4 9 6 (0–10) 10 18 (5–28) 1 10 (—)

5 — — 3 21 (9–31) 2 6 (2–10)

6 — — 4 8 (5–13) 0 —

7 — — 0 — 1 17 (—)

8 — — 7 13 (3–25) 7 14 (0–26)

9–15 — — 3 30 (20–41) 14 8+ (0–30+)

16 — — — — 5 38+ (16–50+)

*Period of [3H]hypoxanthine pulse.{Numbers in parenthesis give the range of numbers of co-operators per group.

76 Classic Paper

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investigated the size of molecules able to move from cell to cell

in this system, and obtained evidence that molecules with the

immunological specificity of serum albumin (mol. wt. 69 000)

could be transferred.

Analogous observations have been made by Potter, Furshpan

& Lennox (1966) using squid embryos. These writers from

occasional observations of dye (Niagara Sky Blue 6?B, mol. wt.

993) leakage to cells neighbouring upon the cells into which

microelectrodes were introduced also suggest that intercellular

connexions permit the passage of such molecules. Moreover

they have found electrical coupling between BHK cells and

between polyoma or SV 40 virus-transformed BHK cells. In

other experiments with micro-electrodes using rat liver and rat

liver cancers in situ, Loewenstein & Kanno (1966) showed that

intercellular ion communication characteristically occurs

between normal liver cells, but that liver cancer cells have no

detectable electrical intercellular communication. Their find-

ing appears only at first sight to be at variance with our

observations and those of Potter et al., for Loewenstein &

Kanno were observing the behaviour of invasive tumour cells,

whereas the cells we and Potter et al. have studied were either

‘normal’ C 13 cells or polyomainduced non-invasive tumour

cells (Stoker, 1967b).

It should be noted that metabolic co-operation has been

demonstrated between freshly isolated (Stoker, 1967a) or

‘normal’ C 13 and polyoma-transformed cells as well as

between polyoma-transformed cells of different lines.

What is the molecular basis of metabolic co-operation

between mammalian cells in tissue culture? It cannot be

permeability to hypoxanthine, because Subak-Sharpe (1965)

has shown that the deficiency of the IPPx cells is not due to

their impermeability to hypoxanthine and related compounds.

There remain five categories of molecular species, transfer of

any of which could result in the experimental observations.

They are: (1) nucleotides formed from [3H]hypoxanthine in

IPP+ cells; (2) labelled polynucleotides formed in IPP+ cells;

(3) informational polynucleotides, e.g. messenger RNA or

episomal DNA coding for inosinic pyrophosphorylase;

(4) protein, e.g. the enzyme inosinic pyrophosphorylase; and

(5) a regulator substance for inosinic pyrophosphorylase.

With regard to point (1), the generally recognized role of

nucleotides in regulatory mechanisms (Davidson, 1966) should

be borne in mind. Transfer of polynucleotides as covered by

point (2), such as that of ribosomal RNA, may also affect cell

metabolism.

Point (3), (4) or (5) would imply that a cell in contact with a

different cell may be capable—at least for some time—of a

metabolic function for which it lacks genetic information. Thus,

Figure 8. Incorporation of [3H]hypoxanthine into nucleic acid by Py Y/TG 1 (IPPx) and C 13 (IPP+) cells grown in mixed culture in

contact and pulsed 36–48 h. Magnification approximately r1850 to illustrate a cytoplasmic connecting ‘bridge’.

Classic Paper 77

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in a monolayer, an individual cell’s metabolic capability would

not be totally limited by the cell’s own genotype, but rather by

the total gene pool of all the cells with which it is directly or

indirectly in contact.

Metabolic co-operation demands consideration in the design

of any experimental procedure for the selection of genetic

variants of mammalian cells in tissue culture. Where cells are

plated at high concentrations and contact between cells is

frequent, selection would only favour changed clls whose

genotype is not masked by metabolic co-operation. But where

cells are plated in low concentration, their phenotype is likely

to reflect their genotype; moreover it may be advantageous if

selective conditions are not applied until some time after

plating, so as to allow exhaustion of molecules obtained during

previous co-operative contact.

If metabolic co-operation takes place in the living organism

and if it applies to a wide variety of metabolic functions and

to malignant and normal cells to the same extent then it is

highly pertinent to the problem of cancer (e.g. Stoker, 1967c).

Unfortunately the phenomenon implies that some tumour cells

may be camouflaged, at least temporarily, by the metabolic

potentialities of surrounding normal cells, or alternatively that

tumour-cell characteristics may become manifest in surround-

ing normal cells, causing them to respond to chemotherapy as

tumour cells.

The relevance of the phenomenon to the problem of contact

inhibition is obvious, but this aspect has already been

discussed by Stoker (1967c).

We are currently interested in elucidating the molecular basis

of metabolic co-operation and the generality of the phenom-

enon with respect to other biochemical variants and with

respect to other cells, particularly invasive tumour cells.

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The authors thank Professor M. G. P. Stoker for his helpfulencouragement and constructive criticism. The technicalassistance of Miss I. Keenan is gratefully acknowledged.

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COMMENTARYThe ‘metabolic co-operation between cells’, nowalso known as gap-junctional intercellular com-munication (GJIC), plays an important role intumour suicide gene therapy. Its principle residesin the introduction into cancer cells of a gene(suicide gene) capable of converting a non-toxicprodrug into a cytotoxic drug responsible forinhibition of cellular DNA synthesis. Althoughthe suicide gene does not enter every cell of thetumour, its toxic metabolite can also affectadjacent cells by GJIC. This so-called bystandereffect significantly contributes to killing of tumourcells. One such suicide gene, the thymidine kinase(TK) from herpes simplex virus 1 (HSV-1), incombination with the prodrug ganciclovir (GCV)has been extensively and successfully used insome animal models exhibiting a strong bystandereffect [3,4,5]. Promising early results of the HSV-1-TK/GCV enzyme/prodrug system (a tumourregressed although only 10% of its cells expressedthe suicide enzyme) led to clinical trials of therapyfor the treatment of brain and ovarian tumours inpatients with end-stage cancer, leukaemia andAIDS [6,7]. However, the use of HSV-TK is limitedby the toxicity of the nucleoside analogues.

TK is a multifunctional enzyme that possessesthree separate cellular enzyme activities phos-phorylating thymidine (dT), deoxycytidine (dC)and thymidylate (dTMP). Unlike its cellularcounterpart human TK, HSV-1 TK is able tophosphorylate purine as well as pyrimidineanalogues. Natural mutability and tolerance tomodification by site directed mutagenesis ofHSV-1 TK provides an opportunity to designnovel antiviral drugs and to use lower, less toxicdoses of drugs in gene therapy of cancer.Thus, introduction of mutations into HSV-1 TKenhances the specificity of phosphorylation ofGCV and 3k-azido-3kdeoxythymidine (AZT, zido-vudine) in tumour cell killing assays [8,9]. Thecytotoxicity of other enzyme/prodrug systemshas also been investigated. A strong bystandereffect was observed in human breast cancercells as well as 9L rat gliosarcoma cells, whichwere effectively killed by (E)-5-(2-bromovinyl)-2k-deoxyuridine (BVDU) in the presence of cellsexpressing varicella-zoster virus-TK (VZV-TK).However, GCV lacked activity in this system [10].

Cazaux et al. [11] compared the TK activityof three a-herpesviruses (HSV-1, VZV, equine

herpesvirus-4 (EHV-4)) and that of two c-herpesviruses (Epstein-Barr virus (EBV) and her-pesvirus saimiri 2 (HVS-2)). They tested the abilityof these TKs to phosphorylate different nucleosideanalogues such as AZT, stavudine (d4T, 3k-deoxy-2k,3k-didehydrothymidine), didanosine (ddI)and 5-FUdR (5k-fluoro-2k-deoxyuridine). Whencomparing c-herpesvirus TKs with the a-herpesvirus TKs, the range of activation of allthe analogues was found to be superior in theformer. Murine herpesvirus (MHV) strains 68and 72, originally isolated from the bank voleClethrionomys glareolus [12], were found to beclosely related to c-herpesviruses. To determineif any MHV-TK/prodrug system could be effec-tive and less toxic than the system previouslydescribed, the sensitivity of MHV-72-TK todifferent nucleoside analogues was investigated[13]. The results showed that 5-FUdR wasextremely cytotoxic and effectively killed cellsexpressing MHV-72-TK. The IC50 value of 2.1 mM

was 16 times lower than that required to kill cellsexpressing HSV-1-TK. To test whether the bystan-der effect between two heterologous cell typescould be mediated by the MHV-72-TK/5-FUdRsystem in vitro, cells expressing MHV-72-TK wereco-cultured with the tumour fibroblastoid cell lineNAD. The bystander effect was observed whenonly 1% of MHV-72-TK-expressing cells enhancedmouse tumour cell killing, so decreasing theirsurvival to 25.6% in the presence of 5-FUdR(10.8 mM). In parallel, the same concentration of5-FUdR only marginally inhibited tumour cellgrowth in the absence of exogenous TK activity.

Strategies to optimise the HSV-TK for gene-basechemotherapy of cancer were recently highlightedby A. Karlsson [14]. Several approaches have beendeveloped to enhance the extent of tumour cellkilling by increasing the capacity of GJIC withpharmaceutical agents or by transducing a vectorcarrying a tumour repressor gene (Cx26) into thecancer cells [15]. To boost in situ cytotoxicity,vectors containing suicide genes have been com-plemented with vectors delivering a variety ofcytokines [16]. Thus the finding of Subak-Sharpeet al. opened up new horizons for understandingthe molecular basis of cell-to-cell communicationsand for the development of rational approaches toits utilisation. Great progress has been made overthe past 30 years in devising the applications ofmetabolic co-operation between cells so it is clear

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that the profound hypothesis in this paper led tonew thinking about the treatment of cancer andthe field of gene therapy.

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11. Cazaux CH, Tiraby M, Loubiere L, et al.Phosphorylation and cytotoxicity of therapeu-tic nucleoside analogues: a comparison of aand c herpesvirus thymidine kinase suicidegenes. Cancer Gene Ther 1998; 5: 83–91.

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13. Raslova H, Matis J, Rezuchova I, et al. Thebystander effect mediated by the new murinegammaherpesvirus 72 – thymidine kinase/5kfluoro-2k-deoxyuridine (MHV72-TK/5-FUdR)system in vitro. Antiviral Chem Chemother 2000;11: 273–282.

14. Karlsson A. Optimized herpes simplex virusthymidine kinase for gene-base chemotherapyof cancer. Int Antiviral News 2001; 9: 73.

15. Mesnil M, Yamasaki H. Bystander effect inherpes simplex virus-thymidine kinase/ganci-clovir cancer gene therapy: role of gap-junctional intercellular communication. CancerRes 2000; 60: 3983–3999.

16. Campain JA, Matassa AA, Feiger PL, et al.Lipid and adenoviral-mediated gene transferinto AIDS-Kaposi’s sarcoma cell lines. CancerGene Ther 1998; 5: 131–143.

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