436
Preliminary notes
7. Barsh, G S, Greenberg, D B & Cunningham, D D, J cell physiol 92 (1977) 115. 8. Greenberg, D B, Barsh, G S, Ho, T-S & Cunningham, D D, J cell physiol 90 (1977) 193. 9. Rozengurt, E, Legg, A & Pettican, P, Proc natl acad sci US 76 (1979) 1284. 10. Dicker, P & Rozengurt, E. Nature 287 (1980) 607. II. - Ibid 276 (1978) 723. 12. Friedkin, M, Legg, A & Rozengurt, E, Proc natl acad sci US 76 (1979) 3909. 13. Rozengurt, E, Legg, A, Strang, G & CourtenayLuck, N, Proc natl acad sci US 78 (1981) 4392. 14. Bourne, H R & Rozengurt, E, Proc natl acad sci US 73 (1976) 4555. 15. Rozengurt, h, Curr topics cell regul 17 (1980) 59. 16. Todaro, G J & Green, H, J cell biol 17 (1963) 299. 17. Mierzejewski, K & Rozengurt, E, Exp cell res 106 (1977) 394. 18. .$hod& M, Nature 256 (1975) 578. 19. Fiske. C H & Subbarow. Y. J biol them 66 (1975) 375. 20. Rothblat. G H. Arboaast, L Y, Omellette, L & Howard,’ B V, In vitro-12 (1976) 554. 21. Pastan, I H & Willingham, M C, Ann rev physiol 43 (1981) 239. 22. Brown, M S & Goldstein, J L, Science 191 (1976) 150. 23. Goldstein, J L & Brown, M S, Ann rev biochem 46 (1977) 897. Received November 18, 1981 Accepted January 19, 1982
Copyrght @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0014.48?7/82/06043h-W$O2.00/0
pp60”“-Dependent increase in adenosinestimulated CAMP formation R. F. HOWARD,‘* H. GLOSSMAN;’ R. R. FRIIS’ and J. R. SHEPPARD,3** ‘Rudolf-Buchheim-lnstitutfiir Pharmakologie, Zlnstitutftir Virologie, Fachbereich Humanmedizin der Justus-Liebig-Universitiit Giessen, D-6300 Giessen, Germany and 3Dight Institute for Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA Summary. Responsiveness to extracellular signals may participate in the control of cell division. This hypothesis was examined by comparing the adenosine responsiveness of Rous sarcoma virus (RSV)-transformed cells and untransformed cells. The results show that RSV-transformed chicken embryo cells (CEC) are more responsive to adenosine than Rous associated virus (RAV)-infected or normal CEC. Experiments using temperature-sensitive, transformation-defective (ts-td) RSV-CEC crown under oermissive and res&cti;e tempera&s exhibit the same differences as RSV- and RAV-infected cells, respectively. Moreover, the ts-td virus-infected cells exExp Cell Res 13Y (19.32)
hibit an enhanced adenosine responsiveness within 1 h following a shift from the restrictive to the permissive temperature. These data indicate that increased adenosine responsiveness is an early event in RSV-induced transformation of CEC. Biological communication via membrane receptor-mediated mechanisms is altered in a wide variety of pathological and physiological conditions. In studies using cell cultures, cellular response to hormones
has been reported to change as a function of genetic mutation (e.g., hereditary disorders) [ 1, 21, extended exposure to hormones [3, 41, the cell cycle phase [5], and different growth states (e.g., neoplastic transformation) [6-81. Adenosine (Ado) is a molecule endogenous to all living cells which exhibits hormonal properties [9] through a receptor-dependent mechanism that regulates adenylate cyclase activity [IO-121. We report here that Rous sarcoma virus (RSV)transformed chicken embryo cells (CEC) respond to Ado more than untransformed control cultures. Methods CEC cultures were obtained from blO-day-old embryonated specific-pathogen-free eggs as previously described [ 131.Newly passaged cells were infected with cloned Rous associated virus (RAV) type 5 (subgroup A), wild-type Prague strain of RSV subgroup A (PrA) or RSV mutants at an m.o.i.=0.1-0.5. To measure hormone-elevated CAMP levels, 2-day-old cultures of cells were washed twice with a ohosphatebuffered saline solution supplemented with-0.02? mM MgCI, and 0.1 mM of the phosphodiesterase inhibitor Ro 20-1724(buffer A), and incubated at 37°C for the indicated times with 10 uM ClAdo in buffer A. The CAMP content of a perchloric acid cellular extract was analysed by radioimmunoassay [ 14, 151and protein by the method of Lowry et al. [16]. These procedures have been described in detail elsewhere [8].
Results and Discussion Chicken embryo cells (CEC) transformed by RSV, as well as CEC-infected, but not * Present address: Scripps Clinic, 10666 N. Torrey Pines Rd, La Jolla, CA 92037, USA. ** To whom offprint requests should be addressed. Printed
in Sweden
Preliminary
notes
437
35c
I 35c
II
:
3%
I 35c
ok+ IO
minutes
of rxubaiion
20 with
I O 2
32 IOyM
chloradenosine
Fig. 2. Z-Chloradenosine stimulation, of RSV-transformed and RAV-infected CEC. Each time point is the mean of duplicate plates (variation
transformed, by RAV physiologically respond to the nucleoside Ado or its nonmetabolized analog, 2-chloroadenosine (ClAdo), by synthesizing increased amounts of CAMP. RSV-transformed cells, however, accumulate much greater levels of CAMP over a 30 min period, in response to ClAdo, than do the RAV-infected cells (fig. 1). Cells infected with the temperaturesensitive, transformation-defective (ts-td) RSV mutants G1201, 203, 251, and 253 [17] also respond to ClAdo with the increased synthesis of intracellular CAMP (fig. 2). This synthesis and accumulation of cyclic nucleotide is greater at the temperature permissive for transformation (35°C) than at the restrictive temperature (42°C). The response of the ts-td mutants at 35°C reflects the increased ClAdo response of the wildtype RSV-transformed compared with the RAV-infected cells. Interestingly, the rela-
m,n
of
I5 10
ncubotlon
Fig. 2. 2-Chloradenosine stimulation of ts-td mutantinfected CEC. Sister cultures were maintained for 24 h at either 35°C (permissive) or 42°C (restrictive), and incubations were performed as described in Methods, except that warm tables adjusted to 42°C (in a 40.5”C warm room) and 35°C were employed. The bar graph indicates the CAMP values determined without CIAdo, and after 2, 5, and 10 min of incubation. Each value is the mean of duplicate plates assayed in duplicate. These data are representative of at least three experiments for each RSV mutant.
tively high level of response seen with GI251-infected cells at 42°C correlates well with an unusual feature of this mutant. Growth regulation of cells infected by the G1251 RSV mutant is not restricted at 42°C although all other parameters of transformed behavior are blocked [ 181. These data suggest that expression of pp60”“, the RSV src gene product, is associated with dramatically increased responsiveness to extracellular signals such as Ado. Thus, we sought to determine the kinetics of the enhanced Ado responsiveness and when this change occurred in the transformation process. G1203 and 251-infected cells were grown at 42°C (the restrictive temperature) and shifted to 35°C. After intervals of l-24 h, these cells were incubated with ClAdo E.rp Cdl Res 13Y lIY82)
438
Preliminary
notes
them from normal cells is an altered regulation by environmental signals. The membrane receptor-regulated adenylate cyclase enzyme is a well characterized system by which normal cells communicate and react to environmental changes. Previous studies [FL221 have indicated that the in vitro basal adenylate cyclase activity in RSVtransformed CEC was lower than normal, growth-regulated CEC. We have confirmed those data using membranes and homotime after 42”-35”dums%ift (ham) genates in adenylate cyclase assays [S]. Fig. 3. Kinetics of the transformation-dependent inMore importantly, however, we have crease in ClAdo responsiveness. Cells infected with G1203 or 251 were grown at 42°C and shifted to 35°C shown here that intact CEC transformed by on the second day after passage. ClAdo responsiveRSV respond to Ado with an excessive ness was determined at 42°C (0 h) and at 35°C at each production of CAMP. This observation with time after downshift (1, 2, 4, 24 h) by incubation with 10 I.LM ClAdo for 10 min. The mean accumulation of ClAdo is especially significant, since all CAMP at 0, 1, 2, 4, and 24 h is shown. These data are representative of three experiments. O-O, GI251-in- viable cells possess the capability of releasfected CEC; 0-Q GI203-infected CEC. ing Ado as a hormone for interaction with their own and neighboring cellular receptors [ 121. In cell growth experiments conducted (10 PM) for 10 min. The intracellular CAMP was then assayed as indicated in the caption during the past decade, CAMP has been reto fig. 1. The CAMP accumulated in re- ported to exert both positive [23-251 and sponse to ClAdo at the various times after negative [26, 271 control. Perhaps the cell temperature downshift is illustrated in fig. cycle-dependent changes in CAMP metabo3. A 2- to 3-fold increase in the ClAdo re- lism or different cell types examined may sponsiveness is observed in the first 4 h account for these paradoxical data. The following the temperature downshift. Most studies presented in this report, demonof this change occurs within 1 h. Thus, an strating an excessive production of CAMP increase in ClAdo responsiveness is ob- in response to adenosine, suggest that a positive effect of CAMP, e.g., the CAMPserved quite early in the transformation process. Much later (fig. 3, 24 h) a further dependent phosphorylation of pp60”” [28], increase in ClAdo responsiveness could be may be involved in the expression of the detected. The ClAdo responsiveness of transformed phenotype. wild-type RSV-transformed and RAV-inThe authors wish to thank Sandra M. H. Howard for fected CEC was also examined at 42°C and excellent technical and artistic assistance. This research was supported by grants from Volks24 h after a shift to 35°C. We found no wagen-Werk and Sonderforschungsbereich 47 of the temperature-dependent differences in the Deutsche Forschungsgemeinschaft. R. F. H. and .I. R. S. were supported by the Alexander von HumClAdo responses of cells infected with boldt-Foundation and the US NIH. either wild-type RSV or RAV (data not References shown). 1. Drezner, M K & Burch, W M, J clin invest 62 A primary characteristic of hyperplastic (1978) 1221. 2. Farfel, 2, Brickman, A S, Kaslow, H R, Brothers, and malignant cells which distinguishes Exp Cdl Res 139 (/9X2)
Preliminary
Received November 23, 1981 Revised version received February 22, 1982 Accepted February 24, 1982
439
Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/82/060939-05SO2.CQ/O
V M & Boume, H R, New eng j med 303 (1980)
231. -_
3. Mickey, J V, Tate, R M & Lefkowitz, R J, J biol them 250 (1975) 5727. 4. Perkins, J P, Moore, M M, Kalisker, A & Su, Y-F, J cyclic nucl res 5 (1975) 641. 5. Howard, R F & Sheppard, J R, J cell biol90 (1981) 169. 6. Sheppard, J R, Gormus, R & Moldow, C F, Nature 269 (1977) 693. 7. DeRubertis, F R, Chayoth, R & Field, J B, J clin invest 57 (1976) 641. 8. Howard, R F; Friis, R R, Sheppard, J R & Glossmann, H, J cell phys. In press. 9. Sattin. A & Rall, T, Mol pharmacol6 (1970) 13. 10. Schultz, J &Daly, J W, J biol them 248 (1973) 843. 11. Premont, J, Perez, M & Bockaert, J, Mol pharmacol 13 (1977) 662. 12. Londos, C, Wolfe, J & Cooper, D M F, Physiological and regulatory functions of adenosine and adenine analogs -(ed H Barr & G I Drummond) p. 271. Raven Press, New York (1979). 13. Friis, RR, Mason, W C, Chen, Y C & Halpern, M S, Virology 64 (1975) 49. 14. Harper, F J & Brooker, G J, Cyclic nucl res 1 (1975) 207. 15. Struck, C J, Ahnert, G, Glossmann, H & Schaeg, W, Arch pharm 298 (1977) 67. 16. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall. R J. J biol them 193(1951) 265. 17. Becker, ‘D, Kurth, R, Critchley, D, Friis, R R & Bauer H, J viral 21 (1977) 1042. 18. Weber, M J & Friis, R R, Cell 16 (1979) 25. 19. Anderson, W B, Johnson, G S & Pastan, I, Proc natl acad sci US 70 (1973) 1055. 20. Anderson, W B, Lovelace, E & Pastan, I, Biochem biophys res commun 52 (1973) 1293. 21. Gidwitz, S, Weber, M J & Storm, D R, J biol them 251 (1976) 7950. 22. Yoshida, M, Owada, M & Toyoshima, K, Virology 63 (1975) 68. 23. Whittield, J F, MacManus, J P& Gillian, D J, J cell physiol 76 (1970) 65. 24. Wang, T, Sheppard, J R & Foker, J, Science 201 (1978) 155. 25. Green, H, Cell 15 (1978) 801. 26. Sheppard, J R, Nature new bio1236 (1972) 14. 27. Pastan, I H, Johnson, G S & Anderson, W B, Ann rev biochem 44 (1975) 491. 28. Collett, M S, Erikson, E & Erickson, R L, J virol 29 (1979) 770.
notes
Subcuttivation of human hair follicle keratinocytes P. J. J. M. WETERINGS,’ A. J. M. VERMORKEN’ * and H. BLOEMENDAL,’ ‘Research Unit
for Cellular Differentiation
ZDepartment ofBiochemistry, Nijmegen, The Netherlands
and Transformation, and University of Nijmegen,
A method is described for the subcultivation of human hair follicle keratinocytes. Primary cultures of these cells were grown on bovine eye lens capsules. Fragments of the colonies could successfully be transplanted onto new capsules. After two subcultivation steps, the keratinocytes remain dipIoid and still exhibit the same pattern of protein biosynthesis as primary cultures. The cultured hair follicle cells may be useful in investigations on genetically determined sensitivity towards carcinogens.
Summary.
Among differentiated cell types, epithelial cells have proved particularly difficult to establish in culture. This fact has seriously hampered some studies in cancer research, since most human tumors (carcinomas) arise in epithelial tissue. In recent years several new techniques have been developed that greatly lengthen the lifetime of human epithelial cells in vitro. In most cases, the cells are cultivated on collagen or in the presence of irradiated mesenchymal cells with or without the support of a collagenous matrix or on irradiated dermis, mostly from pig skin (see e.g. [l-4]). Recently we developed a method to obtain primary cultures of pure epithelial cells, originating from human hair follicles [5]. This technique has a number of advantages. Firstly, plucked scalp hair follicles contain no fibroblasts. Therefore, pure epithelial cell cultures are obtained without laborious manipulations needed to purify cells of epidermal origin. Secondly, because the lens * To whom offprint requests should be addressed: Research Unit for Cellular Differentiation and Transformation, University of Nijmegen, Geert Grooteplein Noord 21, 6500 HB Nijmegen, The Netherlands.
Printed
I” Sweden
E.rp Cd
Res 139 (19612)