metabolic co-operation between biochemically marked mammalian cells in tissue culture
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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.
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.
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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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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.
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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.
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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.
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Figure 6.
Figure 7.
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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.
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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’.
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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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