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The relationship between the growth characteristics of somatic cell hybrids and their levels of cAMP and activities of adenylate cyclase and cAMP phosphodiesterase
Experimental
THE RELATIONSHIP OF SOMATIC
CELL
Cell Research 99 ( 1976) 63-71
BETWEEN HYBRIDS
AND ACTIVITIES
THE GROWTH AND
THEIR
OF ADENYLATE
CHARACTERISTICS LEVELS
OF CAMP
CYCLASE
AND CAMP PHOSPHODIESTERASE M. .I. TISDALE and B. J. PHILLIPS Department of Biochemistry, St Thomas’s Hospital Medicd School. Lorzdon SE1 7EH, und Ludwig Institutefor Cancer Research, Chester Beatty Research Institute, London SW3 6JB. UK
SUMMARY The relationship between malignancy, responsiveness to density dependent inhibition of growth and intracellular levels of adenosine 3’S’-monophosphate (CAMP) has been investigated using two mouse cell lines, A9 (tibroblastic) and TLXS (transplantable lymphoma), and two lkes of hybrids, A9HITLX and A9H/TLX (turnout), formed between them. The highly malignant line TLXS. which does not exhibit density-dependent inhibition of growth, has a low intracellular level of CAMP in vitro, which remains constant, irrespective of cell density. In contrast. the less malignant lines A9. A9H/TLX and A9H/TLX (turnour), which do show density dependence of growth, have higher levels of CAMP, which increase at confluency. The activities of the enzymes adenylate cyclase and CAMP phosphodiesterase in these cell lines have been measured at both high and low cell density. When assayed at 0.2 mM ATP both the basal and fluoride-stimulated cyclase activity in A9, A9H/TLX and A9H/TLX (tumour) are higher in confluent cultures. In TLXS cells, there is no increase in fluoride-stimulated cyclase activity at high cell density. The observed increase in cyclase activity at high density in A9 and the hybrids is not so pronounced when a substrate concentration of 2 mM is used for the assay. The activity of CAMP phosphodiesterase seems to follow the intracellular CAMP level. Thus, for A9 cells, the activity of both the high and low affinity forms of the enzyme is I .7 times higher in cells at high density compared with those at low density. The multiple forms of the CAMP phosphodiesterase from all four cell lines have been resolved using Sepharose 6B gel filtration. An attempt has been made to correlate the presence of different forms of the enzyme with malignancy and density dependence of growth.
Normal cells growing in tissue culture exhibit “density dependent” [1] or “contact” [2] inhibition of ceil growth; i.e. the cells stop dividing when they reach confluency. Transformed cells, however, do not show this type of growth control [3]. Since growth stimulation can be inhibited by CAMP [4, 51 and elevated levels are associated with a decreased rate of cell growth [6, 7, 81 it has been suggested that this nucleotide is the intracellular mediator of the cessation of growth at confluency. 5 -761802
The level of CAMP has been shown to rise at confluency [9, lo] and the levels are higher in contact-inhibited cultures than in non-confluent, rapidly growing cultures [I 11. Treatment with CAMP analogues, however, does not restore contact inhibition of growth to transformed cells [ 12. 131. The intracellular level of CAMP is lower in tumour and transformed cells than in normal cells [ 12, 14, 151and these cells are also more sensitive to growth inhibition by agents which stimulate increases in intrat lprl
(‘d/
xc\
ov IlY’hl
64
Tisdale and Phillips
cellular CAMP [16]. Using somatic cell hybrids, an apparent correlation has previously been obtained between the malignancy of a cell population and the intracellular level of CAMP [15]. Thus, it would appear that both the malignancy of a cell type and its responsiveness to growth control are related to SOme feature Of CAMP metabolism. The steady-state concentration of CAMP in any cell type is determined by leakage to the outside and by the activities of the adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1. l), which catalyses the synthesis of CAMP from ATP, and the CAMP phosphodiesterase (3’,5’cAMP 5’-nucleotidohydrolase, EC 3.1.4.17) which cleaves CAMP to 5’-AMP. The high level of CAMP previously observed in a hybrid line of low malignancy [15] was shown not to be due to a decreased activity of the phosphodiesterase, since the activity of this enzyme actually increased with the rise in the basal level of CAMP. Previous reports have shown CAMP to increase the phosphodiesterase activity of a number of cell types [17, 181. The present report investigates the intracellular level of CAMP and the activity of adenylate cyclase in A9 and TLX5 cells and in hybrids between them, both in the logarithmic and confluent phases of growth. The activity of CAMP phosphodiesterase has been reported to be doubled in cultures of 3T3 fibroblasts on contact inhibition [18]. Therefore, the kinetics of hydrolysis of CAMP have also been investigated as a function of cell density, as have the multiple forms of the enzyme. MATERIALS
AND METHODS
[8-3H]cAMP, ammonium salt (27.5 Ci/mmole), was purchased from the Radiochemical Centre, Amersham. PCS solubiliser was obtained from Hopkin & Exptl Cell Res 99 (1976)
Williams, Essex, and 5’.ribonucleotide phosphohydrolase from Ophiophagus hannah and Sepharose 6B from Sigma Chemical Co., St Louis. MO, USA. Adenosinr 5’-triphosphate, disodium salt, phosphocreatine kinase and phosphocreatine were purchased from the Boehringer Corporation, London. Theophylline and CAMP were obtained from British Drug Houses Ltd., Poole, Dorset.
Cell culture All the cell lines were grown in Dulbecco’s modified Eagle’s medium plus IO% fetal calf serum, gassed with 10% CO, in air. The A9 cells used in this study were kindly supplied by Professor H. Harris. The A9 line was originally isolated by Littlefield [24] as a clone of Earle’s L cells. A9 cells are resistant to I-azaguanine, and lack the enzyme hypoxanthine-guanine phosphoribosyl transferase. Although originally derived from normal mouse tissue and described in a number of papers [44, 45, 461 as being of low malignancy (all these papers quoting about 12% takes for a poorly specified number of cells in newborn irradiated mice), A9 has been reported as giving takes in nude mice [47] and as reported below, these cells are highly tumorigenic in immune-deprived mice. The derivation of the TLXS line has been previously described [15]. A9 and TLXS cells were fused, using inactivated Sendai virus, by the method of Davidson [42]. Hybrids were isolated by thoroughly washing off the unfused TLXS cells after fusion and inhibition of the growth of A9 cells by the use of HAT selective medium [43]. Hybrid colonies were grown separately and cloned before use. The karyotypes of the various clones which were isolated will be described in a forthcoming publication (B. Phillips, in preparation). The hybrid clone used in this study was designated A9H/TLX D and had a chromosome complement which was almost exactly the sum of the complements of the two parent cells. A tumour line, A9H/TLX D (turnout), was derived from this hybrid by serial transplantation (four passages) in immune-deprived mice (see below), followed by trypsinisation of the tumour and re-cloning of the hybrid cells. The karyotype of these cells was unaltered.
Test for malignancy Cells were injected subcutaneously into the inguinal region of thymectomised CBA mice, which had been X-irradiated and injected with syngenic bone marrow cells. Mice were examined daily and tumours and normal tissues were examined histologically in samples of
thetumour-bearingmice,
Density dependence studies Cells of the four lines under study were seeded in 6 cm plastic Petri dishes at 100cells/mm2 in 5 ml of medium. At daily intervals, four dishes from each series were trypsinised and cell counts were made. The medium was unchanged throughout the experiment. When maximum density had been reached in each case, the medium was removed from some of the dishes and tested for its ability to support the growth of cells freshly seeded at 100 cells/mmz.
Growth rate, malignancy and CAMP metabolism Table 1. Results of injection of cells into immune-deprived CBA mice
Cell type A9 TLXS A9H/TLX A9HITLX (tumour) (4th passage and I month culture)
No. injected
Day of Turnour” death takes (range)
104 104 IO’
IO/l0 IO/IO IO/IO
35-62 1t-13 65-90
104
IO/IO
40-a
o Total number of animals tumoursltotal number injected.
with
progressive
Adenylate cyclase assay Cells were seeded in 9 cm plastic dishes, containing 15 ml of medium, 3 days bkfore the assay was performed. To obtain confluent cultures of A9 and hybrid cells, I 000 cells/mm2 were seeded, and 4 000 cells~mm2 for TLXS, which grew to a much higher density. For log phase cultures 200 cells/mm2 were seeded in each case. The medium was not changed before assay. Cells were removed from the culture dishes with a rubber policeman, sedimented by centrifugation at low speed and washed with 0.9% NaCI. The cell pellets were then homogenized in ice-cold 50 mM Tris-HCI buffer, pH 7.4, containing 25 mM KCI and 5 mM MgCI, in a Dounce homogenizer, and the homogenate was centrifuged at 600 g for 10 min at 4°C. The precipitate was finally resuspended in a volume of the Tris-HCI buffer to give a protein concentration of about 0.5 mglml. The standard adenylate cyclase assay system was similar to that reported by Albano et al. [19]. The reaction was initiated by adding 100 /LI of the membrane suspension to 300 PI of 50 mM Tris-HCI, pH 7.4, containing final concentrations of ATP (2 mM or 0.2 mM), 3 mM MgCI,, IO mM NaCl, 10 mM KCI, 6 mM theophylline, 400 pg phosphocreatine kinase and 20 mM phosphocreatine at 35°C. Reaction mixtures were incubated for various time periods up to 15 min and the incubation was terminated by placing the tubes in a boiling water bath for 3 min, followed by cooling, and centrifugation at 1250 g for 10 min. The supernatant fluids were suitably diluted before determination of their content of CAMP using a kit purchased from the Radiochemical Centre, Amersham. Radioactivity was determined in PCS solubiliser using a Tracer Lab liquid scintillation spectrometer. Protein was determined by the method of Lowry et al. [20] using bovine serum albumin as a standard.
hZ
was determined using the Radiochemical Centre assal kit. A standard curve was performed for each determination.
Phosphodiesterase assay The enzymic activity of column eluates was determined using the two step assay of Thompson & Appleman [2l]. 5’-[3H]AMP formed by the phosphodies(erase was converted to [YH]adenosine by the action of a nucleotidase from Ophiophagus hannuh. L!nreacted substrate was removed by Amberlite CG 400 Type I, 100-200 mesh ion exchange resin, and after centrifugation the [3H]adenosine in the supernatant fluid was determined by liquid scintillation counting. For the determination of the kinetics of hydrolysis of CAMP by A9 cells, a portion of a sonicated cell suspension was incubated with [8JH]cAMP for a time interval which gave less than 10% hydrolysis of the substrate. Separation of CAMP from 5’-AMP was achieved by a thin layer chromatographic procedure previously described [15].
Chromatograph) Gel filtration was carried out on a Sepharose 6B column with dimensions of 35x 1.5 cm. The flow rate for the best seoaration was about 20 ml/h. Fraction volumes were I ml. The buffer conditions were 50 mM Tris HCI. OH I - and IO mM 2. 7.5. containing 10 mM M&l, mercaptoethanol. Total sonicated cell suspensions were applied in buffer (I ml). Column calibrations were run under the same conditions using myoglobin. chymotrypsinogen, ovalbumin, bovine albumin, human y-globulin and apoferritin. The column void volume was determined using Dextran 2000.
RESULTS
Malignancy As shown in table 1, all four cell types gave 100% incidence of tumours when lo4 cells were injected into deprived mice. The properties of these tumours were quite different, however. TLXS tumours were very fast growing, killing the host after only 12 days, and invading almost all of the host organs. This tumour normally grows as an ascites which spreads rapidly throughout the body. The tumours derived from A9 and hybrid cells were more slow growing, the CAMP assay A9HITLX cells being exceptionally slow High and low density cultures were prepared as for the to produce tumours. The rate of growth of assay of adenylate cyclase. CAMP was determined as previously described [ 151. these tumours increased almost to that of The ether extracted, lyophilized TCA supernatant was A9 after four transplant passages, and the puritied on a column of Dowex 5OW-X8 200-400 mesh in the H+ form. and the amount of CAMP in the eluate cells derived from the transplanted tumours ExptlCrllRes
YY 11976)
66
Tisdale and Phillips not affecting the growth of the TLXS cells and that cell division was continuing until the medium no longer supported the cells. A9, A9H/TLX and A9H/TLX (tumour), in contrast to TLXS, grew rather more slowly and reached a plateau phase at much lower densities. Cells of these lines remained viable at maximum density for
Fig. 1. Abscissa: time (days); ordinate: log cell density A9 (cells/mm*). Growth curves for TLXS (X-X), (O-O), A9H/TLX (O-O) and A9H/TLX (turnour) (A-A). (Each point is the mean of four separate determinations.)
(i.e. A9H/TLX(tumour)) retained this rate of growth when re-implanted after some months in culture. The A9 and hybrid cell turnout-s showed very little tendency to invade or metastasize but formed large, well circumscribed masses in the inguinal region. A9 and A9H/TLX (tumour) showed some local invasion and regional lymph node involvement shortly before the death of the host. Density dependent growth inhibition Growth curves in vitro, for the four cell lines are shown in fig. 1. These curves were constructed from the data from four separate experiments. It can be seen that TLX5 cells grew most rapidly and reached a very much higher density than any of the other lines. They did not exhibit a plateau phase but started to die soon after reaching maximum density. When medium was removed from cells at maximum density and transferred to cultures of cells at low density, it failed to support more than one cell division. Thus, it seemed that cell density was Exptl Cell Res 99 (1976)
Fig. 2. Abscissa: V/S nmoles/min/mg protein (PM); ordinate: V nmoles/min/mg protein. Hofstee plots of the CAMP phosphodiesterase activities of sonicated suspensions of (a) confluent and (b) logarithmic cultures of A9 cells. The 0.2 ml final reaction mixtures contained 100 mM Tris-HCI (pH 8. I), 10 mM MgSO,, 3H-labelled CAMP (200000 cpm, 0.2-l 000 PM) and 9-100 pg total cell protein. At appropriate intervals of incubation at 35”C, 50 ~1 samples were removed and analysed as in Methods. The initial velocities were estimated from the linear portions of the degradation curves. The slope is the negative value of the apparent K,,,, and the intercept on the ordinate is the value of the apparent V,,,.
Grolz,th rate, mnlignclncy trnd cAMP mrtuholism
67
Adenylute cycluse trctivit! The activity of membrane-bound adenylate cyclase from TLXS and A9 and the two Low density” High density” Cell type hybrids is shown in table 3. Enzyme activity 85f2.9b 55+_1.85 A9 was measured at 0.2 mM ATP in the pres24k2.2 18kl.3 TLXS ence of the activator NaF for cells at either 160f7.2b 75k2.5 A9H/TLX 43+ I .4 71+2.3* AVH/TLX (turnour) high or low population densities. In all cases adenylate cyclase activity is linear (I CAMP concentration expressed as pmole/mg protein +S.E.M. with time for at least 10 min. For all cell h Difference between cAMP levels at high and low types the basal cyclase activity in cultures density significant (~~0.05) according to Student’s f-test. in the logarithmic phase of growth is too low to be measured accurately and so basal levels are not included in the table. In rhe several weeks. The differences between the presence of NaF, basal cyclase activity is densities reached by these three lines were increased between 7- and 14-fold over the small but A9H/TLX (tumour) attained a basal level for each cell type. Except for slightly higher density than A9 which in the density unresponsive cell line TL,XS. turn was higher than A9H/TLX. Medium fluoride-stimulated enzyme activity is xigremoved from cultures several days after nificantly higher in confluent cultures. For maximum density had been achieved was TLXS, the fluoride-stimulated enzyme acfound to support at least one further com- tivity does not vary significantly with cell plete round of culture growth, from 100 density. The fluoride-stimulated enzyme cells/mm2 to maximum density. Thus these activity parallels the intracellular CAMP three lines can be regarded as showing a and the malignancy of these cells. Thus strong density dependence of growth. A9H/TLX which has the highest intracellular level of CAMP has also the highest Influence of cell population density fluoride-stimulated cyclase activity, while on cAMP levels TLXS which has the lowest intracellular The intracellular levels of CAMP in the cell level of CAMP has the lowest adenylate lines A9, TLXS and the two hybrids A9HI cyclase activity. TLX and A9H/TLX (tumour) for cells at both high and low population density is Table 3. Adenylate cyclase activity, detershown in table 2. The highly malignant mined at 0.2 mM ATP in the presence of TLXS cells have a low basal level of CAMP 10 mM NaF --which does not change significantly from Adenylate cyclase activity low to high cell density. In contrast A9, (pmole/min/mg protein) +S.E.M. A9HITLX and A9H/TLX (tumour) have higher levels of CAMP during the period of Cell type Low density High density __ logarithmic growth than does TLXS. Fur51.7+3.2” 38.9k2.6 -ther, these levels are appreciably elevated A9 22.5t1.7 28.6Ll.8 when the cells approach confluency. The TLX5 121.0+4.2” 96.Ok3.8 A9H/TLX 112.0+3.9” 67.Ok3.2 least malignant hybrid, ABHITLX, has the A9H/TLX (tumour) highest level of CAMP both at low and high a Differences between activities at high and low density significant (ptO.05) according to Student’s t-test. cell population density.
Table 2. cAMP concentration density
and ceil
68
T&dale and Phillips
Table 4. Adenylate cyclase activity, determined at 2 mM ATP, in either the presence or absence of 10 mM NaF Adenylate cyclase activity (pmole/min/mg protein) +S.E.M. Low density
High density
Cell type
- NaF
+NaF
-NaF
+NaF
A9 TLXS A9H/TLX A9H/TLX (turnour)
24k1.4 25k1.2 45k2.4 32kl.8
99k5.5 51k2.2 132L6.5 12lk6.2
2lkl.O 29k1.6 42k2.2 29k1.5
13Ok 6.5” 46+ 2.0 339rf 15.0” 106t 5.4
(1 Value differs significantly @<0.05) from corresponding low density value according to Student’s f-test.
parent K, value, whilst the intercept on the ordinate gives the value of the apparent V max. These cells appear to contain two enzymatic activities as reported for a large number of other cell types [23] with apparent K, values for CAMP of 200 PM and 2.5 PM. There is a 1.7-fold increase in both the high and low affinity forms of the CAMP phosphodiesterase when these cells become confluent. The apparent K, values, however, are unchanged (fig. 2). Since the intracellular concentration of CAMP rises almost 2-fold at confluency, CAMP itself could be the inducer of the increased enzyme activity, as suggested in a number of reports [17, 181. In a number of tissues, CAMP phosphodiesterase has been shown to exist in multiple forms [2 1,231 which can be separated either by electrophoresis [25] or by gel chromatography [21, 261. To investigate the possibility of an alteration in such forms of the enzyme with contact inhibition of cell growth, total cell sonicated suspensions of A9, TLXS and the two hybrids were subCyclic nucleotide phosphodiesterase jected to Sepharose 6B gel filtration, and The rate of hydrolysis of CAMP by the enzyme activity in the eluent was deterphosphodiesterase from A9 cells at both mined at a substrate concentration of 5 PM, high and low density is expressed by means which was considered to measure mainly of a Hofstee plot [22] in fig. 2. In this plot the high affinity form. The results of the the slope is the negative value of the ap- CAMP phosphodiesterase elution patterns To detect if any change in enzyme activity observed is due to a change in affinity for the substrate, cyclase levels for cells growing at high and low density and in the presence or absence of NaF have also been measured at 2 mM ATP. The results are shown in table 4. At this substrate concentration the basal cyclase activity is similar in both logarithmically growing and in confluent cultures. Also for TLXS and A9H/TLX (tumour) the fluoride-stimulated enzyme activity does not vary appreciably with cell density. For A9 and A9H/TLX, however, the fluoride-stimulated activity is higher in confluent cultures. The fact that major differences in the adenylate cyclase activity of these four cell types were only observed at low ATP concentrations suggests that the affinity of the enzyme for ATP is not the same for all cell lines. Preliminary experiments suggest that while the two hybrids have a K, ATP of about 0.20.3 mM, that of the two parent lines is much higher (ca 2 mM).
trpfl
Cdl
RPS YY t/976)
Growth rate, malignancy
Table 5. Forms
of CAMP phosphodiesterase in TLXS and A9 cells and hybrids A9HITLX and A9HITLX (tumour) Cell line Apparent mol. wt
A9
TLXS
>loooooo 5ooooo 450 000 360000 3ooooo 250 000 200 000 150ooo 100000
+ + + + ~ + + -+
f + + + + + -
A9H/ TLX
A9H/TLX (turnour)
+ + + + + + +
+ + + + f +
Symbols: +, presence: -, absence
of the four cell types is shown in table 5. Material eluting at the void volume of the column with apparent molecular weight 1000 000 probably represents membrane bound enzyme. For all cell types the elution profiles at both high and low cell densities were similar. The parent cells A9 and TLXS exhibit seven and six separable forms of the enzyme, respectively. Of these all are present in either one or other of the hybrids except the forms with apparent molecular weight 500000 and 360000. One new form of the enzyme with an apparent molecular weight of 300000 is found in both of the hybrids but in neither of the parents. A form with an apparent molecular weight of 250000 is only found in the less malignant parent A9 and the less malignant hybrid A9H/TLX. DISCUSSION The relationship between the malignancy of a cell type and the intracellular level of CAMP has previously been investigated using somatic cell hybrids [15]. Although the hybrids used in the present study are different from the ones used previously, the
and CAMP metabolism
69
same trend of decreasing CAMP levels with increasing malignancy is still observed. For the highly malignant non-contact inhibited lymphoma cell line, TLXS, the basal level of CAMP is low, and further, remains at this level irrespective of cell density. However, the hybrid A9H/TLX. produced by the fusion of TLXS with the mouse fibrohlast line A9, shows a low degree of malignancy and four times as much CAMP as found in TLXS. Also this level is doubled when these cells become confluent. For A9 and the transplanted hybrid tumour cell line A9H/TLX (tumour) both of which show density dependent inhibition of growth, there is similarly an approximately two-fold rise in CAMP levels at confluency. The association of adenylate cyclase with the plasma membrane would place it in an ideal position to relay the message of “density dependent” or “contact” inhibition. However, the characteristics of the adenylate cyclase from a range of tumours appear confused. While in some tumour and transformed cells the activity of the cyclase is lower than that of the tissue of origin [27, 28, 291, in other cell types the opposite situation is found [30, 3 1. 321. It is difficult, therefore, to reconcile the lowered intracellular level of CAMP in some tumour cells with changes in activity of adenylate cyclase. The variation in cyclase activity with cell density for the cell types which we have investigated is similar to that found by Anderson et al. [33, 341. Since the K,, ATP for a number of cell types is close to 0.2 mM [28, 33, 341 cyclase activity was measured at 0.2 mM ATP. At this concentration both the basal and NaF-stimulated adenylate cyclase activity increase as a function of cell density for those cell lines (A9, A9H/ TLX (tumour) and A9H/TLX) showing density-dependent inhibition of growth. In Expd(‘r/i KC,\vu 11g76)
70
Tisdale and Phillips
contrast, for the non-contact inhibited cell line TLXS neither the basal nor the fluoridestimulated enzyme activity varies appreciably between cells at low or high density. When measured at 2 mM ATP there is little variation in cyclase activity with cell population density for any cell type. This is similar to the effect of Rous sarcoma virus transformation of chick embryo fibroblasts on adenylate cyclase activity when a decrease in enzyme activity was only observed at low concentrations of ATP [28]. This was due to an increase in the apparent K, ATP for the transformed cells. The mechanism by which NaF causes a stimulation of adenylate cyclase activity is unknown, although Constantopoulos & Najar [35] have suggested that it results from a nucleophilic displacement of a phosphate group from an inhibited phosphorylated enzyme or an activation of an endogenous membrane phosphatase [36]. This latter possibility seems to be less probable with the cell types considered in this investigation since the activity of membrane phosphoprotein phosphatase increases with increasing malignancy [37]. This enzyme was shown to be part of an ATPase system. Thus TLXS cells which have a low cyclase activity have a high endogenous phosphatase activity, whilst A9H/TLX cells, which have the highest cyclase activity, have the lowest phosphatase activity. Kasarov & Friedman [38] have shown a four- to five-fold greater activity of Na+-K+ activated ATPase in transformed fibroblasts than occurs in normal non-transformed cell lines. They consider the inability of transformed cells to raise their CAMP levels to be attributable to competition between ATPase and adenylate cyclase for the substrate ATP. This may be important in malignant cells if the K, ATP of adenylate cyclase is increased, as is sugExptl Cd Res 99 (1976)
gested by the present experiments for A9 and TLXS, especially since these cell types have a higher ATPase activity [36]. Russel & Pastan [39] have suggested that a membrane-associated CAMP phosphodiesterase exerts a substantial effect on the cellular CAMP level. Accordingly the CAMP phosphodiesterase in all four cell lines has been studied for cells at both high and low density. This enzyme exists as several different molecular entities [23] and can be fractionated into forms of differing molecular weight by gel filtration on Sepharose 6B. No qualitative difference in the forms of the enzyme was observed for cells growing at either high or low population density, irrespective of whether they showed density-dependent inhibition of cell growth (A9 and hybrids) or not (TLXS). The inheritance pattern of the various forms of the enzyme in the two hybrids appears complicated. While most of the forms of the enzyme found in the hybrids are found in either one or other of the parents, one form of the enzyme with an apparent molecular weight of 300 000 is found in both of the hybrids but in neither of the parents. This could be due to a degree of heterogeneity in the two parent lines at the time of fusion, while the hybrids were derived from the fusion of only one of each of the parent cells. When the density-dependent A9 cells become confluent both high and low affinity forms of the CAMP phosphodiesterase increase in activity approximately two-fold without a concurrent change in apparent affinity value. This is similar to the results of D’Armiento et al. [18] with 3T3-4 cells. They suggested that the level of CAMP phosphodiesterase activity is regulated by the intracellular concentration of CAMP. Our own results [IS] would support this conclusion since there appears to be a direct
Growth
relationship between basal CAMP levels and the activity of both the high and the low affinity forms of the phosphodiesterase. Thus, for A9, TLXS and the two hybrids A9HITLX and A9H/TLX (turnout-) there appears to be an inverse correlation between the intracellular CAMP and malignancy. The increased basal level of CAMP found in the low-malignancy cell types is not due to a decreased activity of the CAMP phosphodiesterase, but to an increased adenylate cyclase activity. Neoplastic cells show a change in the surface glycolipids [40], and these may serve as receptors for substances modulating adenylate cyclase activity [41]. The possibility therefore exists that deficient membrane interactions coupled with a lower cyclase activity in neoplastic cells leads directly to the lowered basal CAMP levels. This would also explain the inability of such cells to respond to density dependent inhibition of growth if CAMP was a mediator of such an event. The authors wish to thank Professor L. Young for his interest. M. J. Tisdale wishes to acknowledge the receipt of a research grant from the Cancer Research Campaign.
REFERENCES 1. Stoker, M G P & Rubin, H, Nature 215 (1967) 171. 2. Abercrombie. M & Ambrose, E J, Cancer res 22 (1962) 525. 3. Dulbecco, R, Science 166(1969) 962. 4. Bombik, B M & Burger, M M, Exp cell res 80 (1973) 8X. 5. Burgkr,M M, Bombik, B M, Breckenridge, B McL & Sheppard, J R, Nature new biol239 (1972) 161. 6. Taylor-Papadimitriou, J, Intj cancer 13 (1974)404. 7. Heidrick, M L & Ryan, W L, Cancer res 30 (1970) 376. 8. Cho-Chung, Y S & Gullino, P M, Science 183 (1974) 87. 9. Otten, J, Johnson, G S & Pastan, I, Biochem biophys res commun 44 (1971) 1192. 10. Bannai,S & Sheppard, J R, Nature 250 (1974) 62. Il. Gtten, I, Johnson, G S & Pastan, I, J biol them 247 (1972) 7082. 12. Carchman, R A, Johnson,G S & Pastan, I, Cell 1 (1974) 59.
rate, malignancy
cd
CAMP metabolism
7L
13. Smets. L A, Nature new biol 239 (1972) 123. 14. Rein, A, Carchman, R A. Johnson, G S & Pastan. I. Biochem biophys res commun 52 (1973) 899. 15. Tisdale, M J & Phillips. B J, Exp cell t-esXXI 19741 111. 16. Abell, C W, Kamp. C W & Johnson, I, 1). Cancel res 30 (1970) 717. 17. Schwartz. J P. Morris. N R & Breckenridge. B McL. J biol them 248 (1973) 2699. 18. D’Armiento, M, Johnson, G S & Paatan. I. Proc natl acad sci US 69 (1972) 459. 19. Albano, J D M, Maudsley. D V. Brown, B I. & Barnes. G D, Biochem sot trans I ( 1973)477 20. Lowry, 0 H. Rosebrough. N J. Farr. A I & Randall, R J, J biol them 193 (195I) 265. 21. Thompson, W J & Appleman. M M. Biocheml\tr! 10(1971)311. 22. Hofstee, B H J. Science 116 (1952) 329. 23. Appleman, M M. Thompson, W J & Russel, I‘ K. Adv cyclic nucleotide res 3 (1973) 6s. 24. Littlefield. J W. Proc natl acad sci C!S 50 t 1963) 568. 25. Mann. E & Christiansen. R 0. Science 173 (1971) 540. 26. Schroder, J & Rickenberg, H V. Biochim biophy\ acta 302 (1973) 50. 27. Burk, RR, Nature 219 (1968) 1272. 28. Anderson, W B. Johnson, G S & Pastan. I. Proc natl acad sci US 70 (1973) 1055. 29. Anderson, W B. Lovelace, E & Pastan, I. Biochem biophys res commun 52 (1973) 1293. 30. Kemp. R G & Duquesnoy, R J, Science IX3 I 1974) 218. 31. Boyd, H. Louis, C J & Martin, T J. Cancer res 34 (1974) 1720. 32. Schorr, I & Russel, A. Biochim biophy\ acta 364 (1974) 173. 33. Anderson. W B, Russell, T R, Carchman. R ‘4 & Pastan. I. Proc natl acad sci US 70 (1973) 3802. 34. Anderson. W B. Gallo, M & Pastan. I. J trio1 them 249 (1974) 7041. 3s. Constantopoulos, A & Najjar, V A. Biochem biophys res commun 53 (1973) 794. 36. Layne, P, Constantopoulos, A, Judge, J F X, Rauner. R & Najjar, ‘V A. Biochem hiophys rex commun 53 (1973) 800. 37. Tisdale. M J & Phillips. B J. Biochem biophys res commun. In press. 38. Kasarov. LB & Friedman. H. Cancer res 34 (1974) 1862. 39. Russell, T R & Pastan. I. J biol them 248 (1973) 5835. 40. Emmelot, P, Eur j cancer 9 (1973) 319. 41. Bennett, V, O’Reefe, E & Cuatrecasass, P. Proc natl acad sci US 72 (1975) 33. 42. Davidson, R, Exp cell res 55 (1%9) 424. 43. Littletield, J W, Exp cell res 41 (1966) 190. 44. Harris, H, Proc roy sot ser B 179 (1971) 1. 45. Klein, G, Bregula, U, Wiener, F & Harris, H. J cell sci 8 (1971) 659. 46. Wiener, F, Cochran, A, Klein, G & Harris. H. J natl cancer inst 48 (1972) 465. 47. Freedman, V H & Seung-il Shin, Cell 3 (1974) 35.5. Received June 16, 1975 Accepted October 21, 1975 Exprl Ceil Rrs 99 11976)
THE RELATIONSHIP OF SOMATIC
CELL
Cell Research 99 ( 1976) 63-71
BETWEEN HYBRIDS
AND ACTIVITIES
THE GROWTH AND
THEIR
OF ADENYLATE
CHARACTERISTICS LEVELS
OF CAMP
CYCLASE
AND CAMP PHOSPHODIESTERASE M. .I. TISDALE and B. J. PHILLIPS Department of Biochemistry, St Thomas’s Hospital Medicd School. Lorzdon SE1 7EH, und Ludwig Institutefor Cancer Research, Chester Beatty Research Institute, London SW3 6JB. UK
SUMMARY The relationship between malignancy, responsiveness to density dependent inhibition of growth and intracellular levels of adenosine 3’S’-monophosphate (CAMP) has been investigated using two mouse cell lines, A9 (tibroblastic) and TLXS (transplantable lymphoma), and two lkes of hybrids, A9HITLX and A9H/TLX (turnout), formed between them. The highly malignant line TLXS. which does not exhibit density-dependent inhibition of growth, has a low intracellular level of CAMP in vitro, which remains constant, irrespective of cell density. In contrast. the less malignant lines A9. A9H/TLX and A9H/TLX (turnour), which do show density dependence of growth, have higher levels of CAMP, which increase at confluency. The activities of the enzymes adenylate cyclase and CAMP phosphodiesterase in these cell lines have been measured at both high and low cell density. When assayed at 0.2 mM ATP both the basal and fluoride-stimulated cyclase activity in A9, A9H/TLX and A9H/TLX (tumour) are higher in confluent cultures. In TLXS cells, there is no increase in fluoride-stimulated cyclase activity at high cell density. The observed increase in cyclase activity at high density in A9 and the hybrids is not so pronounced when a substrate concentration of 2 mM is used for the assay. The activity of CAMP phosphodiesterase seems to follow the intracellular CAMP level. Thus, for A9 cells, the activity of both the high and low affinity forms of the enzyme is I .7 times higher in cells at high density compared with those at low density. The multiple forms of the CAMP phosphodiesterase from all four cell lines have been resolved using Sepharose 6B gel filtration. An attempt has been made to correlate the presence of different forms of the enzyme with malignancy and density dependence of growth.
Normal cells growing in tissue culture exhibit “density dependent” [1] or “contact” [2] inhibition of ceil growth; i.e. the cells stop dividing when they reach confluency. Transformed cells, however, do not show this type of growth control [3]. Since growth stimulation can be inhibited by CAMP [4, 51 and elevated levels are associated with a decreased rate of cell growth [6, 7, 81 it has been suggested that this nucleotide is the intracellular mediator of the cessation of growth at confluency. 5 -761802
The level of CAMP has been shown to rise at confluency [9, lo] and the levels are higher in contact-inhibited cultures than in non-confluent, rapidly growing cultures [I 11. Treatment with CAMP analogues, however, does not restore contact inhibition of growth to transformed cells [ 12. 131. The intracellular level of CAMP is lower in tumour and transformed cells than in normal cells [ 12, 14, 151and these cells are also more sensitive to growth inhibition by agents which stimulate increases in intrat lprl
(‘d/
xc\
ov IlY’hl
64
Tisdale and Phillips
cellular CAMP [16]. Using somatic cell hybrids, an apparent correlation has previously been obtained between the malignancy of a cell population and the intracellular level of CAMP [15]. Thus, it would appear that both the malignancy of a cell type and its responsiveness to growth control are related to SOme feature Of CAMP metabolism. The steady-state concentration of CAMP in any cell type is determined by leakage to the outside and by the activities of the adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1. l), which catalyses the synthesis of CAMP from ATP, and the CAMP phosphodiesterase (3’,5’cAMP 5’-nucleotidohydrolase, EC 3.1.4.17) which cleaves CAMP to 5’-AMP. The high level of CAMP previously observed in a hybrid line of low malignancy [15] was shown not to be due to a decreased activity of the phosphodiesterase, since the activity of this enzyme actually increased with the rise in the basal level of CAMP. Previous reports have shown CAMP to increase the phosphodiesterase activity of a number of cell types [17, 181. The present report investigates the intracellular level of CAMP and the activity of adenylate cyclase in A9 and TLX5 cells and in hybrids between them, both in the logarithmic and confluent phases of growth. The activity of CAMP phosphodiesterase has been reported to be doubled in cultures of 3T3 fibroblasts on contact inhibition [18]. Therefore, the kinetics of hydrolysis of CAMP have also been investigated as a function of cell density, as have the multiple forms of the enzyme. MATERIALS
AND METHODS
[8-3H]cAMP, ammonium salt (27.5 Ci/mmole), was purchased from the Radiochemical Centre, Amersham. PCS solubiliser was obtained from Hopkin & Exptl Cell Res 99 (1976)
Williams, Essex, and 5’.ribonucleotide phosphohydrolase from Ophiophagus hannah and Sepharose 6B from Sigma Chemical Co., St Louis. MO, USA. Adenosinr 5’-triphosphate, disodium salt, phosphocreatine kinase and phosphocreatine were purchased from the Boehringer Corporation, London. Theophylline and CAMP were obtained from British Drug Houses Ltd., Poole, Dorset.
Cell culture All the cell lines were grown in Dulbecco’s modified Eagle’s medium plus IO% fetal calf serum, gassed with 10% CO, in air. The A9 cells used in this study were kindly supplied by Professor H. Harris. The A9 line was originally isolated by Littlefield [24] as a clone of Earle’s L cells. A9 cells are resistant to I-azaguanine, and lack the enzyme hypoxanthine-guanine phosphoribosyl transferase. Although originally derived from normal mouse tissue and described in a number of papers [44, 45, 461 as being of low malignancy (all these papers quoting about 12% takes for a poorly specified number of cells in newborn irradiated mice), A9 has been reported as giving takes in nude mice [47] and as reported below, these cells are highly tumorigenic in immune-deprived mice. The derivation of the TLXS line has been previously described [15]. A9 and TLXS cells were fused, using inactivated Sendai virus, by the method of Davidson [42]. Hybrids were isolated by thoroughly washing off the unfused TLXS cells after fusion and inhibition of the growth of A9 cells by the use of HAT selective medium [43]. Hybrid colonies were grown separately and cloned before use. The karyotypes of the various clones which were isolated will be described in a forthcoming publication (B. Phillips, in preparation). The hybrid clone used in this study was designated A9H/TLX D and had a chromosome complement which was almost exactly the sum of the complements of the two parent cells. A tumour line, A9H/TLX D (turnout), was derived from this hybrid by serial transplantation (four passages) in immune-deprived mice (see below), followed by trypsinisation of the tumour and re-cloning of the hybrid cells. The karyotype of these cells was unaltered.
Test for malignancy Cells were injected subcutaneously into the inguinal region of thymectomised CBA mice, which had been X-irradiated and injected with syngenic bone marrow cells. Mice were examined daily and tumours and normal tissues were examined histologically in samples of
thetumour-bearingmice,
Density dependence studies Cells of the four lines under study were seeded in 6 cm plastic Petri dishes at 100cells/mm2 in 5 ml of medium. At daily intervals, four dishes from each series were trypsinised and cell counts were made. The medium was unchanged throughout the experiment. When maximum density had been reached in each case, the medium was removed from some of the dishes and tested for its ability to support the growth of cells freshly seeded at 100 cells/mmz.
Growth rate, malignancy and CAMP metabolism Table 1. Results of injection of cells into immune-deprived CBA mice
Cell type A9 TLXS A9H/TLX A9HITLX (tumour) (4th passage and I month culture)
No. injected
Day of Turnour” death takes (range)
104 104 IO’
IO/l0 IO/IO IO/IO
35-62 1t-13 65-90
104
IO/IO
40-a
o Total number of animals tumoursltotal number injected.
with
progressive
Adenylate cyclase assay Cells were seeded in 9 cm plastic dishes, containing 15 ml of medium, 3 days bkfore the assay was performed. To obtain confluent cultures of A9 and hybrid cells, I 000 cells/mm2 were seeded, and 4 000 cells~mm2 for TLXS, which grew to a much higher density. For log phase cultures 200 cells/mm2 were seeded in each case. The medium was not changed before assay. Cells were removed from the culture dishes with a rubber policeman, sedimented by centrifugation at low speed and washed with 0.9% NaCI. The cell pellets were then homogenized in ice-cold 50 mM Tris-HCI buffer, pH 7.4, containing 25 mM KCI and 5 mM MgCI, in a Dounce homogenizer, and the homogenate was centrifuged at 600 g for 10 min at 4°C. The precipitate was finally resuspended in a volume of the Tris-HCI buffer to give a protein concentration of about 0.5 mglml. The standard adenylate cyclase assay system was similar to that reported by Albano et al. [19]. The reaction was initiated by adding 100 /LI of the membrane suspension to 300 PI of 50 mM Tris-HCI, pH 7.4, containing final concentrations of ATP (2 mM or 0.2 mM), 3 mM MgCI,, IO mM NaCl, 10 mM KCI, 6 mM theophylline, 400 pg phosphocreatine kinase and 20 mM phosphocreatine at 35°C. Reaction mixtures were incubated for various time periods up to 15 min and the incubation was terminated by placing the tubes in a boiling water bath for 3 min, followed by cooling, and centrifugation at 1250 g for 10 min. The supernatant fluids were suitably diluted before determination of their content of CAMP using a kit purchased from the Radiochemical Centre, Amersham. Radioactivity was determined in PCS solubiliser using a Tracer Lab liquid scintillation spectrometer. Protein was determined by the method of Lowry et al. [20] using bovine serum albumin as a standard.
hZ
was determined using the Radiochemical Centre assal kit. A standard curve was performed for each determination.
Phosphodiesterase assay The enzymic activity of column eluates was determined using the two step assay of Thompson & Appleman [2l]. 5’-[3H]AMP formed by the phosphodies(erase was converted to [YH]adenosine by the action of a nucleotidase from Ophiophagus hannuh. L!nreacted substrate was removed by Amberlite CG 400 Type I, 100-200 mesh ion exchange resin, and after centrifugation the [3H]adenosine in the supernatant fluid was determined by liquid scintillation counting. For the determination of the kinetics of hydrolysis of CAMP by A9 cells, a portion of a sonicated cell suspension was incubated with [8JH]cAMP for a time interval which gave less than 10% hydrolysis of the substrate. Separation of CAMP from 5’-AMP was achieved by a thin layer chromatographic procedure previously described [15].
Chromatograph) Gel filtration was carried out on a Sepharose 6B column with dimensions of 35x 1.5 cm. The flow rate for the best seoaration was about 20 ml/h. Fraction volumes were I ml. The buffer conditions were 50 mM Tris HCI. OH I - and IO mM 2. 7.5. containing 10 mM M&l, mercaptoethanol. Total sonicated cell suspensions were applied in buffer (I ml). Column calibrations were run under the same conditions using myoglobin. chymotrypsinogen, ovalbumin, bovine albumin, human y-globulin and apoferritin. The column void volume was determined using Dextran 2000.
RESULTS
Malignancy As shown in table 1, all four cell types gave 100% incidence of tumours when lo4 cells were injected into deprived mice. The properties of these tumours were quite different, however. TLXS tumours were very fast growing, killing the host after only 12 days, and invading almost all of the host organs. This tumour normally grows as an ascites which spreads rapidly throughout the body. The tumours derived from A9 and hybrid cells were more slow growing, the CAMP assay A9HITLX cells being exceptionally slow High and low density cultures were prepared as for the to produce tumours. The rate of growth of assay of adenylate cyclase. CAMP was determined as previously described [ 151. these tumours increased almost to that of The ether extracted, lyophilized TCA supernatant was A9 after four transplant passages, and the puritied on a column of Dowex 5OW-X8 200-400 mesh in the H+ form. and the amount of CAMP in the eluate cells derived from the transplanted tumours ExptlCrllRes
YY 11976)
66
Tisdale and Phillips not affecting the growth of the TLXS cells and that cell division was continuing until the medium no longer supported the cells. A9, A9H/TLX and A9H/TLX (tumour), in contrast to TLXS, grew rather more slowly and reached a plateau phase at much lower densities. Cells of these lines remained viable at maximum density for
Fig. 1. Abscissa: time (days); ordinate: log cell density A9 (cells/mm*). Growth curves for TLXS (X-X), (O-O), A9H/TLX (O-O) and A9H/TLX (turnour) (A-A). (Each point is the mean of four separate determinations.)
(i.e. A9H/TLX(tumour)) retained this rate of growth when re-implanted after some months in culture. The A9 and hybrid cell turnout-s showed very little tendency to invade or metastasize but formed large, well circumscribed masses in the inguinal region. A9 and A9H/TLX (tumour) showed some local invasion and regional lymph node involvement shortly before the death of the host. Density dependent growth inhibition Growth curves in vitro, for the four cell lines are shown in fig. 1. These curves were constructed from the data from four separate experiments. It can be seen that TLX5 cells grew most rapidly and reached a very much higher density than any of the other lines. They did not exhibit a plateau phase but started to die soon after reaching maximum density. When medium was removed from cells at maximum density and transferred to cultures of cells at low density, it failed to support more than one cell division. Thus, it seemed that cell density was Exptl Cell Res 99 (1976)
Fig. 2. Abscissa: V/S nmoles/min/mg protein (PM); ordinate: V nmoles/min/mg protein. Hofstee plots of the CAMP phosphodiesterase activities of sonicated suspensions of (a) confluent and (b) logarithmic cultures of A9 cells. The 0.2 ml final reaction mixtures contained 100 mM Tris-HCI (pH 8. I), 10 mM MgSO,, 3H-labelled CAMP (200000 cpm, 0.2-l 000 PM) and 9-100 pg total cell protein. At appropriate intervals of incubation at 35”C, 50 ~1 samples were removed and analysed as in Methods. The initial velocities were estimated from the linear portions of the degradation curves. The slope is the negative value of the apparent K,,,, and the intercept on the ordinate is the value of the apparent V,,,.
Grolz,th rate, mnlignclncy trnd cAMP mrtuholism
67
Adenylute cycluse trctivit! The activity of membrane-bound adenylate cyclase from TLXS and A9 and the two Low density” High density” Cell type hybrids is shown in table 3. Enzyme activity 85f2.9b 55+_1.85 A9 was measured at 0.2 mM ATP in the pres24k2.2 18kl.3 TLXS ence of the activator NaF for cells at either 160f7.2b 75k2.5 A9H/TLX 43+ I .4 71+2.3* AVH/TLX (turnour) high or low population densities. In all cases adenylate cyclase activity is linear (I CAMP concentration expressed as pmole/mg protein +S.E.M. with time for at least 10 min. For all cell h Difference between cAMP levels at high and low types the basal cyclase activity in cultures density significant (~~0.05) according to Student’s f-test. in the logarithmic phase of growth is too low to be measured accurately and so basal levels are not included in the table. In rhe several weeks. The differences between the presence of NaF, basal cyclase activity is densities reached by these three lines were increased between 7- and 14-fold over the small but A9H/TLX (tumour) attained a basal level for each cell type. Except for slightly higher density than A9 which in the density unresponsive cell line TL,XS. turn was higher than A9H/TLX. Medium fluoride-stimulated enzyme activity is xigremoved from cultures several days after nificantly higher in confluent cultures. For maximum density had been achieved was TLXS, the fluoride-stimulated enzyme acfound to support at least one further com- tivity does not vary significantly with cell plete round of culture growth, from 100 density. The fluoride-stimulated enzyme cells/mm2 to maximum density. Thus these activity parallels the intracellular CAMP three lines can be regarded as showing a and the malignancy of these cells. Thus strong density dependence of growth. A9H/TLX which has the highest intracellular level of CAMP has also the highest Influence of cell population density fluoride-stimulated cyclase activity, while on cAMP levels TLXS which has the lowest intracellular The intracellular levels of CAMP in the cell level of CAMP has the lowest adenylate lines A9, TLXS and the two hybrids A9HI cyclase activity. TLX and A9H/TLX (tumour) for cells at both high and low population density is Table 3. Adenylate cyclase activity, detershown in table 2. The highly malignant mined at 0.2 mM ATP in the presence of TLXS cells have a low basal level of CAMP 10 mM NaF --which does not change significantly from Adenylate cyclase activity low to high cell density. In contrast A9, (pmole/min/mg protein) +S.E.M. A9HITLX and A9H/TLX (tumour) have higher levels of CAMP during the period of Cell type Low density High density __ logarithmic growth than does TLXS. Fur51.7+3.2” 38.9k2.6 -ther, these levels are appreciably elevated A9 22.5t1.7 28.6Ll.8 when the cells approach confluency. The TLX5 121.0+4.2” 96.Ok3.8 A9H/TLX 112.0+3.9” 67.Ok3.2 least malignant hybrid, ABHITLX, has the A9H/TLX (tumour) highest level of CAMP both at low and high a Differences between activities at high and low density significant (ptO.05) according to Student’s t-test. cell population density.
Table 2. cAMP concentration density
and ceil
68
T&dale and Phillips
Table 4. Adenylate cyclase activity, determined at 2 mM ATP, in either the presence or absence of 10 mM NaF Adenylate cyclase activity (pmole/min/mg protein) +S.E.M. Low density
High density
Cell type
- NaF
+NaF
-NaF
+NaF
A9 TLXS A9H/TLX A9H/TLX (turnour)
24k1.4 25k1.2 45k2.4 32kl.8
99k5.5 51k2.2 132L6.5 12lk6.2
2lkl.O 29k1.6 42k2.2 29k1.5
13Ok 6.5” 46+ 2.0 339rf 15.0” 106t 5.4
(1 Value differs significantly @<0.05) from corresponding low density value according to Student’s f-test.
parent K, value, whilst the intercept on the ordinate gives the value of the apparent V max. These cells appear to contain two enzymatic activities as reported for a large number of other cell types [23] with apparent K, values for CAMP of 200 PM and 2.5 PM. There is a 1.7-fold increase in both the high and low affinity forms of the CAMP phosphodiesterase when these cells become confluent. The apparent K, values, however, are unchanged (fig. 2). Since the intracellular concentration of CAMP rises almost 2-fold at confluency, CAMP itself could be the inducer of the increased enzyme activity, as suggested in a number of reports [17, 181. In a number of tissues, CAMP phosphodiesterase has been shown to exist in multiple forms [2 1,231 which can be separated either by electrophoresis [25] or by gel chromatography [21, 261. To investigate the possibility of an alteration in such forms of the enzyme with contact inhibition of cell growth, total cell sonicated suspensions of A9, TLXS and the two hybrids were subCyclic nucleotide phosphodiesterase jected to Sepharose 6B gel filtration, and The rate of hydrolysis of CAMP by the enzyme activity in the eluent was deterphosphodiesterase from A9 cells at both mined at a substrate concentration of 5 PM, high and low density is expressed by means which was considered to measure mainly of a Hofstee plot [22] in fig. 2. In this plot the high affinity form. The results of the the slope is the negative value of the ap- CAMP phosphodiesterase elution patterns To detect if any change in enzyme activity observed is due to a change in affinity for the substrate, cyclase levels for cells growing at high and low density and in the presence or absence of NaF have also been measured at 2 mM ATP. The results are shown in table 4. At this substrate concentration the basal cyclase activity is similar in both logarithmically growing and in confluent cultures. Also for TLXS and A9H/TLX (tumour) the fluoride-stimulated enzyme activity does not vary appreciably with cell density. For A9 and A9H/TLX, however, the fluoride-stimulated activity is higher in confluent cultures. The fact that major differences in the adenylate cyclase activity of these four cell types were only observed at low ATP concentrations suggests that the affinity of the enzyme for ATP is not the same for all cell lines. Preliminary experiments suggest that while the two hybrids have a K, ATP of about 0.20.3 mM, that of the two parent lines is much higher (ca 2 mM).
trpfl
Cdl
RPS YY t/976)
Growth rate, malignancy
Table 5. Forms
of CAMP phosphodiesterase in TLXS and A9 cells and hybrids A9HITLX and A9HITLX (tumour) Cell line Apparent mol. wt
A9
TLXS
>loooooo 5ooooo 450 000 360000 3ooooo 250 000 200 000 150ooo 100000
+ + + + ~ + + -+
f + + + + + -
A9H/ TLX
A9H/TLX (turnour)
+ + + + + + +
+ + + + f +
Symbols: +, presence: -, absence
of the four cell types is shown in table 5. Material eluting at the void volume of the column with apparent molecular weight 1000 000 probably represents membrane bound enzyme. For all cell types the elution profiles at both high and low cell densities were similar. The parent cells A9 and TLXS exhibit seven and six separable forms of the enzyme, respectively. Of these all are present in either one or other of the hybrids except the forms with apparent molecular weight 500000 and 360000. One new form of the enzyme with an apparent molecular weight of 300000 is found in both of the hybrids but in neither of the parents. A form with an apparent molecular weight of 250000 is only found in the less malignant parent A9 and the less malignant hybrid A9H/TLX. DISCUSSION The relationship between the malignancy of a cell type and the intracellular level of CAMP has previously been investigated using somatic cell hybrids [15]. Although the hybrids used in the present study are different from the ones used previously, the
and CAMP metabolism
69
same trend of decreasing CAMP levels with increasing malignancy is still observed. For the highly malignant non-contact inhibited lymphoma cell line, TLXS, the basal level of CAMP is low, and further, remains at this level irrespective of cell density. However, the hybrid A9H/TLX. produced by the fusion of TLXS with the mouse fibrohlast line A9, shows a low degree of malignancy and four times as much CAMP as found in TLXS. Also this level is doubled when these cells become confluent. For A9 and the transplanted hybrid tumour cell line A9H/TLX (tumour) both of which show density dependent inhibition of growth, there is similarly an approximately two-fold rise in CAMP levels at confluency. The association of adenylate cyclase with the plasma membrane would place it in an ideal position to relay the message of “density dependent” or “contact” inhibition. However, the characteristics of the adenylate cyclase from a range of tumours appear confused. While in some tumour and transformed cells the activity of the cyclase is lower than that of the tissue of origin [27, 28, 291, in other cell types the opposite situation is found [30, 3 1. 321. It is difficult, therefore, to reconcile the lowered intracellular level of CAMP in some tumour cells with changes in activity of adenylate cyclase. The variation in cyclase activity with cell density for the cell types which we have investigated is similar to that found by Anderson et al. [33, 341. Since the K,, ATP for a number of cell types is close to 0.2 mM [28, 33, 341 cyclase activity was measured at 0.2 mM ATP. At this concentration both the basal and NaF-stimulated adenylate cyclase activity increase as a function of cell density for those cell lines (A9, A9H/ TLX (tumour) and A9H/TLX) showing density-dependent inhibition of growth. In Expd(‘r/i KC,\vu 11g76)
70
Tisdale and Phillips
contrast, for the non-contact inhibited cell line TLXS neither the basal nor the fluoridestimulated enzyme activity varies appreciably between cells at low or high density. When measured at 2 mM ATP there is little variation in cyclase activity with cell population density for any cell type. This is similar to the effect of Rous sarcoma virus transformation of chick embryo fibroblasts on adenylate cyclase activity when a decrease in enzyme activity was only observed at low concentrations of ATP [28]. This was due to an increase in the apparent K, ATP for the transformed cells. The mechanism by which NaF causes a stimulation of adenylate cyclase activity is unknown, although Constantopoulos & Najar [35] have suggested that it results from a nucleophilic displacement of a phosphate group from an inhibited phosphorylated enzyme or an activation of an endogenous membrane phosphatase [36]. This latter possibility seems to be less probable with the cell types considered in this investigation since the activity of membrane phosphoprotein phosphatase increases with increasing malignancy [37]. This enzyme was shown to be part of an ATPase system. Thus TLXS cells which have a low cyclase activity have a high endogenous phosphatase activity, whilst A9H/TLX cells, which have the highest cyclase activity, have the lowest phosphatase activity. Kasarov & Friedman [38] have shown a four- to five-fold greater activity of Na+-K+ activated ATPase in transformed fibroblasts than occurs in normal non-transformed cell lines. They consider the inability of transformed cells to raise their CAMP levels to be attributable to competition between ATPase and adenylate cyclase for the substrate ATP. This may be important in malignant cells if the K, ATP of adenylate cyclase is increased, as is sugExptl Cd Res 99 (1976)
gested by the present experiments for A9 and TLXS, especially since these cell types have a higher ATPase activity [36]. Russel & Pastan [39] have suggested that a membrane-associated CAMP phosphodiesterase exerts a substantial effect on the cellular CAMP level. Accordingly the CAMP phosphodiesterase in all four cell lines has been studied for cells at both high and low density. This enzyme exists as several different molecular entities [23] and can be fractionated into forms of differing molecular weight by gel filtration on Sepharose 6B. No qualitative difference in the forms of the enzyme was observed for cells growing at either high or low population density, irrespective of whether they showed density-dependent inhibition of cell growth (A9 and hybrids) or not (TLXS). The inheritance pattern of the various forms of the enzyme in the two hybrids appears complicated. While most of the forms of the enzyme found in the hybrids are found in either one or other of the parents, one form of the enzyme with an apparent molecular weight of 300 000 is found in both of the hybrids but in neither of the parents. This could be due to a degree of heterogeneity in the two parent lines at the time of fusion, while the hybrids were derived from the fusion of only one of each of the parent cells. When the density-dependent A9 cells become confluent both high and low affinity forms of the CAMP phosphodiesterase increase in activity approximately two-fold without a concurrent change in apparent affinity value. This is similar to the results of D’Armiento et al. [18] with 3T3-4 cells. They suggested that the level of CAMP phosphodiesterase activity is regulated by the intracellular concentration of CAMP. Our own results [IS] would support this conclusion since there appears to be a direct
Growth
relationship between basal CAMP levels and the activity of both the high and the low affinity forms of the phosphodiesterase. Thus, for A9, TLXS and the two hybrids A9HITLX and A9H/TLX (turnout-) there appears to be an inverse correlation between the intracellular CAMP and malignancy. The increased basal level of CAMP found in the low-malignancy cell types is not due to a decreased activity of the CAMP phosphodiesterase, but to an increased adenylate cyclase activity. Neoplastic cells show a change in the surface glycolipids [40], and these may serve as receptors for substances modulating adenylate cyclase activity [41]. The possibility therefore exists that deficient membrane interactions coupled with a lower cyclase activity in neoplastic cells leads directly to the lowered basal CAMP levels. This would also explain the inability of such cells to respond to density dependent inhibition of growth if CAMP was a mediator of such an event. The authors wish to thank Professor L. Young for his interest. M. J. Tisdale wishes to acknowledge the receipt of a research grant from the Cancer Research Campaign.
REFERENCES 1. Stoker, M G P & Rubin, H, Nature 215 (1967) 171. 2. Abercrombie. M & Ambrose, E J, Cancer res 22 (1962) 525. 3. Dulbecco, R, Science 166(1969) 962. 4. Bombik, B M & Burger, M M, Exp cell res 80 (1973) 8X. 5. Burgkr,M M, Bombik, B M, Breckenridge, B McL & Sheppard, J R, Nature new biol239 (1972) 161. 6. Taylor-Papadimitriou, J, Intj cancer 13 (1974)404. 7. Heidrick, M L & Ryan, W L, Cancer res 30 (1970) 376. 8. Cho-Chung, Y S & Gullino, P M, Science 183 (1974) 87. 9. Otten, J, Johnson, G S & Pastan, I, Biochem biophys res commun 44 (1971) 1192. 10. Bannai,S & Sheppard, J R, Nature 250 (1974) 62. Il. Gtten, I, Johnson, G S & Pastan, I, J biol them 247 (1972) 7082. 12. Carchman, R A, Johnson,G S & Pastan, I, Cell 1 (1974) 59.
rate, malignancy
cd
CAMP metabolism
7L
13. Smets. L A, Nature new biol 239 (1972) 123. 14. Rein, A, Carchman, R A. Johnson, G S & Pastan. I. Biochem biophys res commun 52 (1973) 899. 15. Tisdale, M J & Phillips. B J, Exp cell t-esXXI 19741 111. 16. Abell, C W, Kamp. C W & Johnson, I, 1). Cancel res 30 (1970) 717. 17. Schwartz. J P. Morris. N R & Breckenridge. B McL. J biol them 248 (1973) 2699. 18. D’Armiento, M, Johnson, G S & Paatan. I. Proc natl acad sci US 69 (1972) 459. 19. Albano, J D M, Maudsley. D V. Brown, B I. & Barnes. G D, Biochem sot trans I ( 1973)477 20. Lowry, 0 H. Rosebrough. N J. Farr. A I & Randall, R J, J biol them 193 (195I) 265. 21. Thompson, W J & Appleman. M M. Biocheml\tr! 10(1971)311. 22. Hofstee, B H J. Science 116 (1952) 329. 23. Appleman, M M. Thompson, W J & Russel, I‘ K. Adv cyclic nucleotide res 3 (1973) 6s. 24. Littlefield. J W. Proc natl acad sci C!S 50 t 1963) 568. 25. Mann. E & Christiansen. R 0. Science 173 (1971) 540. 26. Schroder, J & Rickenberg, H V. Biochim biophy\ acta 302 (1973) 50. 27. Burk, RR, Nature 219 (1968) 1272. 28. Anderson, W B. Johnson, G S & Pastan. I. Proc natl acad sci US 70 (1973) 1055. 29. Anderson, W B. Lovelace, E & Pastan, I. Biochem biophys res commun 52 (1973) 1293. 30. Kemp. R G & Duquesnoy, R J, Science IX3 I 1974) 218. 31. Boyd, H. Louis, C J & Martin, T J. Cancer res 34 (1974) 1720. 32. Schorr, I & Russel, A. Biochim biophy\ acta 364 (1974) 173. 33. Anderson. W B, Russell, T R, Carchman. R ‘4 & Pastan. I. Proc natl acad sci US 70 (1973) 3802. 34. Anderson. W B. Gallo, M & Pastan. I. J trio1 them 249 (1974) 7041. 3s. Constantopoulos, A & Najjar, V A. Biochem biophys res commun 53 (1973) 794. 36. Layne, P, Constantopoulos, A, Judge, J F X, Rauner. R & Najjar, ‘V A. Biochem hiophys rex commun 53 (1973) 800. 37. Tisdale. M J & Phillips. B J. Biochem biophys res commun. In press. 38. Kasarov. LB & Friedman. H. Cancer res 34 (1974) 1862. 39. Russell, T R & Pastan. I. J biol them 248 (1973) 5835. 40. Emmelot, P, Eur j cancer 9 (1973) 319. 41. Bennett, V, O’Reefe, E & Cuatrecasass, P. Proc natl acad sci US 72 (1975) 33. 42. Davidson, R, Exp cell res 55 (1%9) 424. 43. Littletield, J W, Exp cell res 41 (1966) 190. 44. Harris, H, Proc roy sot ser B 179 (1971) 1. 45. Klein, G, Bregula, U, Wiener, F & Harris, H. J cell sci 8 (1971) 659. 46. Wiener, F, Cochran, A, Klein, G & Harris. H. J natl cancer inst 48 (1972) 465. 47. Freedman, V H & Seung-il Shin, Cell 3 (1974) 35.5. Received June 16, 1975 Accepted October 21, 1975 Exprl Ceil Rrs 99 11976)