Znt. J. Cancer: 20, 560-563 (1977)

ADENYLATE CYCLASE ACTIVITY AND THE cAMP LEVEL ARE NOT DIRECTLY CORRELATED WITH TRANSFORMATION BY AVIAN SARCOMA VIRUSES

M. YOSHIDA, Y. IKAWA, M. OWADA Laboratory of Viral Oncology, Cancer Institute, Toshima-ku, Tokyo; and Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

and K. TOYOSHIMA Department of Tumor Viruses,

The adenylate cyclase activity was measured in chick embryo fibroblasts (CEF) infected with temperature-sensitive mutants ( t s ) of avian sarcoma virus (ASV). When CEF transformed with a ( ts ) mutant at 36" C were incubated at the non-permiss- ive temperature (41" C), recovery from the low adenylate cyclase activity detectable in the trans- formed state was slower than the disappearance of signs of morphological transformation. After a downward shift of the temperature the activity decreased and this change was also slower than the alteration of cell morphology. The affinity of the enzyme system for ATP also changed after, and not during, morphological alteration. N o significant difference was observed between the cAMP levels in ASV-transformed and non-infected CEF. These findings are consistent with the idea that adenylate cyclase is not involved in cell transformation and that the change in its activity is secondary t o cell transformation,

Since the role of cAMP in cell proliferation was first suggested (Biirk, 1968) there have been many reports on changes in the cAMP level accompanying neoplastic transformation (Ryan and Heidrick, 1968 ; Heidrick and Ryan, 1971; Otten et a/ . , 1971, 1972; Sheppard, 1972). Otten et al. (1972) first reported a decrease in the cAMP level in chick embryo fibro- blasts (CEF) as an early event in cell transformation by the Bryan strain of Rous sarcoma virus (BH- RSV). This reduction of the cAMP level was deduced by Anderson et al. (1973) to be due to decreased activity of adenylate cyclase in the transformed cells. We previously reported that when ts mutants of RSV were used, reduction of adenylate cyclase activity was related to a transformed phenotype of the cells and that the kinetic parameters of adenylate cyclase activity in transformed cells appeared to be specific to the strain of avian sarcoma virus (ASV) used (Yoshida et al., 1975, 1976). These previous observa- tions suggested that reductions of adenylate cyclase activity and cAMP level might be essential for expression of transformed phenotypes. Therefore, we analysed the changes in adenylate cyclase activity of CEF infected with the ts mutant of ASV after temperature shifts. The results reported in this paper suggest that the change in adenylate cyclase activity is not due to a primary effect of the temperature- sensitive gene of ASV used.

MATERIAL AND METHODS

Cells used for assay of adenylate cyclase and cAMP content were secondary cultures of C/E or C/E chicken embryo fibroblasts maintained in Parker's medium 199 supplemented with 10% TPB, 5 % calf serum and 0.5% chicken serum as described pre- viously (Yoshida et al., 1975). Virus infection was carried out in primary culture, and the number of seeded cells was carefully controlled so that the monolayers were semi-confluent when used for assay. The PH of the culture was also kept as constant as possible until just before the assay. Every culture was re-fed with the fresh medium 22-24 h before the assay.

LA 334 is a temperature-sensitive ( ts) mutant isolated from B77 strain of ASV and has ts lesions in both replication and transformation (Owada and Toyoshima, 1973). LA23, LA27, LA31 and LA34 are f s mutants of class T isolated from the Prague strain of Rous sarcoma virus (Wyke, 1973; Wyke et al., 1974) and were kindly supplied by Dr. P. K. Vogt of the University of Southern California, USA.

Activity of adenylate cyclase was assayed as described previously (Yoshida et al., 1975). Princi- pally, [32P]cAMP formation from [d2P]ATP was detected in the presence of theophylline using the total cell homogenate as the source of membrane- bound adenylate cyclase, and the activity was expressed as pmoles of cAMP formed during 10 min incubation per total mg of protein.

Extraction and assay of cAMP were carried out according to Gilman (Gilman, 1970). Briefly, culture fluid was carefully drained from the cultures as quickly as possible then, without washing with buffered solution, 4-5 ml of cold 5 % TCA were added to the cell layer in a 100-mm plate. The plates were maintained for 3-5 h at 4" C, after which the cells were scraped into TCA, homogenized in a Potter-type homogenizer with a tightly-fitting pestle and centrifuged for 20 min at 3,500 rpm and 4" C.

Received: May 25, 1977 and in revised form July 25, 1977.

ADENYLATE CYCLASE AND ASV-TRANSFORMATION 56 1

The supernatant was then removed carefully, the top 5-mm and bottom 5-mm layers being excluded. After extraction of TCA with ether, the cAMP solution was lyophilized and used for assay of cAMP without further purification. The protein binding assay of cAMP was carried out in the presence of inhibitor protein as in Gilman's pro- cedure. Recovery of cAMP was followed by adding t3H]cAMP to the plate with TCA.

0 6 12 I8 24 0 6 12 18 24

111+ A F T E R TEIIPEPATUFE S H I F T [ h r . )

FIGURE 1 Effect of temperature shift of LA334-infected CEF on

activity (A) and Km value for ATP(B) of adenylate cyclase system. CEF were infected with LA 334 at high multiplicity (at least 0.1) in primary culture and grown at 36" C for 5 or 6 days. The transformed cells were transferred into four sets of 100-mm plates at a density of 3-3.5 x lo6 cells per plate; two sets were maintained at 36" C and the other two at 41°C for 44-48h before the temperature shift experiment (0 time). One of the two sets maintained at 41" C was shifted down to 36" C [v) at 0 time and the other was kept at 41" C (0). One of the other sets main- tained at 36" C was shifted up to 41",C (A) and the remaining set was left at 36" C (0). Each point represents the mean of two measurements. (+) indicates more than 60% cell transformation judged by morphology and (-) indicates normal cell morphology; ( 5 ) represents morphology closely but not completely resembling that of normal cells.

The sensitivity of apparent cAMP in the cell extract to cAMP phosphodiesterase was assayed as follows. The cell extract was divided into two portions and the cAMP content of one portion assayed directly. The other portion was digested with 0.2 mg of beef heart cAMP phosphodiesterase (Boehringer Mannheim GmbH, Waldhof, West Germany) in the presence of 50 mM Tris-HCI (PH 8.0) and 10 mM MgC12 at 30" C for 60 min.; then lO0pg of acid- washed bovine serum albumin were added as carrier and protein was precipitated with cold 10% TCA; the mixture was centrifuged and TCA in the super-

0 G O 6 0 6 0 6 T I N E AFTER TEMPERATURE SHIFT (hr)

FIGURE 2 Effect of temperature shift of CEF infected with ts

mutants of PR-RSV on adenylate cyclase activity. Cells infected with fs mutants were grown and maintained at 36" C (0) or 41" C (0). The cells were shifted up from 36" C to 41" C (A) at 0 time. (A) LA23; (B) LA27; (c) LA31 ; (D) LA34. The other conditions were the same as described in Figure 1.

natant was removed by extraction with ether, then the content of cAMP was measured as described above.

RESULTS AND DISCUSSION

As reported previously (Yoshida et al., 1975), chick embryo fibroblasts infected with LA 334, a ts mutant of B 77, had lower adenylate cyclase activity than uninfected cells at 36" C, the permissive temperature for the expression of transforming phenotypes, whereas at the non-permissive tempera- ture of 41" C, their enzyme activity was as high as that in normal cells. The changes in activity were reversible on temperature shift of the culture and were accompained with change in the apparent Km value of the adenylate cyclase system for APT: the value was higher in LA334-infected cells at 36°C than in non-infected cells, but was normal at 41" C.

To test whether these changes were early events in cell transformation, we measured the kinetics of the changes after temperature shift of the cultures. When LA334-infected cells that had been maintained at 41" C for 44-48 h were shifted down to 36 "C, they showed distinct morphological transformation within a few hours. During this period, however, the adenylate cyclase activity decreased by less than 20% of the total decrease observed after incubation of the cells for 20 h at 36" C (Fig. 1 ~ ) . Very similar delays in increase of adenylate cyclase activity were also

562 YOSHIDA ET AL.

observed after upward shift of temperature in CEF infected with LA23, LA27, LA31 and LA34 respect- ively, each of which has a different ts-site in its sarcoma gene(s) (Fig. 2) (Wyke, 1973; Wyke, et al., 1974).

The change in Km value of the adenylate cyclase system for ATP was also slower than the appearance or disappearance of morphological transformation (Fig. 16).

In our system the rates of change in adenylate cyclase activity were much slower than those reported by Anderson (Anderson et al., 1973) for ts 5 of the Bryan strain of RSV. This difference seemed to be due to the difference in the ts mutants used.

The observed rates of change of adenylate cyclase activity were similar to, or slower than, those of other transformation phenotypes of the cells infected with ts ASV-mutants, such as sugar transport (Kawai and Hanafusa, 1971), agglutinability by plant lectins (Burger and Martin, 1972) and membrane polypep- tide composition (Isaka et al., 1975). However, the techniques for measuring these changes have different sensitivities, making it difficult to compare the respective results directly. This delay in the changes of adenylate cyclase relative to morphological trans- formation might be explained as a reflection of changes in the cell membrane.

TABLE I

CYCLLC AMP CONTENT OF NON-INFECTED AND TRANSFORMED CEF

CAMP concentration (pmol/mg protein)' Average (PmoU mg

I I1 Ill IV v pro- tein)

Cells infected Experiment No. with

-(control) 11.6 11.4 13.6 12.8 14.3 12.7 B77 12.4 9.3 12.6 14.7 12.3

RSV(R AV-60) 10.8 RSV ' (RAV-1) 13.6

' Each value is the mean of three measurements. - a Bryan strain of Rous sarcoma virus.

It is possible that a slight decrease of adenylate cyclase activity might reduce the cAMP level sufficiently to induce cell transformation, assuming that cAMP is essential for transformation. To test this, the cAMP level in transformed cells was measured by Gilman's method (Gilman, 1970) and compared with that of non-transformed cells. Table I shows that there was no significant difference between the cAMP levels in normal and transformed cells. The observed amount of cAMP seems to reflect the actual level of cAMP because the apparent amount of cAMP increased linearly with the amount of cell

0 0.1 0.2 0.3

EXTRACT CORRESPONDING TO P R O T E I N (mq)

FIGURE 3 Apparent cAMP content as a function of the amount of

cell extract expressed in terms of protein. Non-infected (0) and B77-transformed ( 0 ) cells were grown at 39" C and cAMP was extracted and estimated by protein binding assay, as described by Gilman (1970).

extract; more than 90% of the total was sensitive to cAMP phosphodiesterase (Fig. 3). Furthermore, in another control experiment, the extracts did not contain appreciable amounts of substances interfer- ing with the assay of cAMP when the cell extract was divided into two similar series with various amounts, and a known amount of standard cAMP was added to one of the series. The cAMP content of the tubes containing added cAMP appeared to be the sum of the cAMP in the cell extract and added CAMP. The finding that the cAMP levels in non-infected and transformed CEF were similar shows that decrease in adenylate cyclase activity did not induce decrease in the total level of CAMP.

The results in Table I are not consistent with those of Otten et al. (1972), possibly owing to differences in conditions such as cell density and PH or nutrients in the medium. On the other hand, they are consistent with the report of Hovi et al. (1974) that the cAMP levels in normal and transformed CEF were similar. Primary cultures of CEF differ from non-transformed cell lines of mammalian fibroblasts in that they do not show strict inhibition of cell growth by cell-to-cell contact. If the cAMP level is related to growth of the cells (Heidrick and Ryan, 1971 ; Otten et al., 1971), but not to transformation, then no difference between the levels in normal and transformed CEF would necessarily be expected, especially in our system where semi-confluent cultures were used. It might be argued that only a transient change in the cAMP level might be sufficient to initiate or terminate the transformation. This possibility cannot be completely ruled out, but it seems unlikely because continuous expression of the sarcoma gene(s) of ASV is required to maintain the cells in the transformed state.

ADENYLATE CYCLASE AND ASV-TRANSFORMATION 563

It is concluded that the changes in adenylate cyclase do not have an essential role in transforma- tion or in reduction of the cAMP level in transformed chick cells.

cyclase activity are transformation-dependent and specific for the viral strain. However, both the delay in the changes relative to the appearance or dis- appearance of morphological transformation of ts ASV-mutant-infected cells and also the finding that the cAMP levels in normal and transformedcells were not different suggest that the changes in adenylate

ACGNOWLEDGEMENTS

This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan,

L’ACTIVITE ADENYLATE CYCLASE ET LE NIVEAU DE cAMP NE SONT PAS EN CORReLATION DIRECTE AVEC LA TRANSFORMATION

PAR LE VIRUS DU SARCOME AVIAIRE

L’activitk adtnylate cyclase a Bt6 mesur6e dans des fibroblastes d’embryon de poulet (CEF) infect& par des mutants thermosensibles (ts) du virus du sarcome aviaire (ASV). Lorsque des CEF transformb par un mutant ts A 36’ C ont 6t6 incubb ti la temperature non permissive (41’ C), la reprise de l’activite adenylate cyclase, qui etait faible 1’6tat transforme, a Btt plus lente que la disparition des signes de transformation morphologique. A la suite d’un abaissement de la temperature, I’activite a diminut et ce changement a lui aussi tte plus lent que l’alttration de la morphologie cellulaire. L’affinitt du systbme enzy- matique pour I’ATP a egalement change apres, et non pendant, I’alttration morphologique. Aucune difference significative n’a ete constatee entre les niveaux de cAMP dans les CEF transform& par 1’ASV et dans les CEF non infectts. Ces observations concordent avec I’idQ que I’adenylate cyclase n’intervient pas dans la transformation cellulaire et que la modification de son activite ne joue pas un rBle essentiel dans cette transformation.

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