5. Carroll, E J & Epel, D, Exptl cell res 90 (1975) lacks the structural material supplied by the 429. granules . Few microvilli project from the 6. Endo, Y, Exptl cell res 25 (1961) 518. 7. Bryan, J, J cell biol 44 (1970) 635. cell surface. The eggs do not develop nor8. Ito, S, Revel, J P & Goodenough, D A, Biol bull mally and only a low percentage cleave. If 133 (1967) 471. 9. Epel, D, Weaver, A M, Muchmore, A V & eggs are fertilized in sea water containing 0.01 Schimke, R T, Science 163 (1969) 294. M a-methyl-D-mannoside after having been 10. Anderson, E, J cell biol 37 (1968) 514. treated with ConA, fertilization and developReceived October 4, 1974 ment are normal. These effects of ConA on cortical granule dispersion were not found in eggs of the sea urchins Strongylocentrotus purpuratus and Purine and pyrimidine modification of growth Lytechinus pictus. The cancellation of these and surface properties of baby hamster kidney cells (BHK21 /C 13) effects on D. excenfricus eggs by the competing sugar (a-methyl-o-mannoside) shows that J. C. TAYLOR,r D. W. HlLL and M. ROGOLSKY,2 in Dendraster eggs, ConA must be crosslinkDepartment of Microbiology, University of Utah, Coling the granule material by binding to lege of Medicine, Salt Lake City, I/t 84112, USA mannose-like residues. In the normal process Summary. Hypoxanthine or adenosine and thymidine of fertilization membrane formation, the added to soft agar medium stimulated the growth of non-transformed BHK2liC13 cells. Increases in concontents of the granules may be processed canavalin A agglutinability and synthesis of a plasby enzymes secreted by the granules before minogen activator also occurred. Thymidine was refor colony formation in agar but not for incondensation on the vitelline layer. In sea quired creased lectin agglutinability or activator activity. urchin eggs, a p-1,3-glucanase of unknown function is released from the cortical granules Clarke & Smith observed stimulatory effects . Although Dendraster eggs do not contain of exogenously added purines on the growth glucanase, they may contain another carboof normal BHK21 cells in monolayer culhydrase which may alter the granule contents tures [I]. Montagnier  also described purine thus preparing them for polymerization on stimulation of the growth of transformed the vitelline surface . Isolation of the (but not normal) BHK21 cells in agar suspenspheres shown in fig. 3 may provide a method sion. The reason for the growth stimulation for obtaining the granule contents before is unknown. enzymatic alteration. Since cortical granules In the present report we extend these preare Golgi-produced secretory vesicles [lo] vious studies [l, 21 with the observation that it is possible that plant lectins such as Con A thymidine, added with hypoxanthine or ademay be of use in isolating the contents of nosine can induce the growth of non-transsecretory vesicles from other cells. formed BHK21/C13 cells in semisolid medium. Agglutinability of cells with conWe thank Dr David Epel for his hospitality during canavalin A (ConA) is also increased. Conour stay in his laboratory. This work was supported by NIH Grant, Number 1 ROl HD 08645-01. currently with the increased lectin agglutinability, we have noted an increase in the References production of a fibrinolytic factor by the I. Runnstrom, J, Adv morphogenesis 5 (1966) 291. Cl3 cells. This cell factor has been shown to 2. Schuel, H, Wilson, W L, Chen, K & Lorand, L, Dev biol 34 (1973) 175. 3. Longo, F J & Schuel, H, Dev biol 34 (1973) 187. 4. Vacquier, V D, Tegner, M J & Epel, D, Exptl cell res 80 (1973) 111.
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1 Present address: Department of Microbiology, Scripps Clinic and Research Foundation, La Jolla, Ca 92037, USA.
Table 1. The effect of the addition of hypoxanthine and thymidine or adenosineon ConA agglutinability, growth in agar suspensionand plasminogen activator (PA) activity in BHKZI cells Cell line and treatment BHK2l/Cl3 (None) BHK21/HPb (None) BHK21/Cl3 Hypoxanthine & thymidineC BHK2l/Cl3 Adenosine & thymidine’
PA activity of cell strain in agar suspension culture
Formation of colonies in agar suspension culture
by ConA &g/ml)
a 0, 0%; 1 t-, 30%; 2+, 50%; and 3 +, 70% of the maximum number (4+) of cells agglutinated . * Represents high passage sublines of the BHK21/Cl3 cell line. (Ten sublines were examined. Data presents cumulative results from all high passage sublines.) ’ For ConA agglutinability, cells were grown in culture dishes with 7.14 x lO-5 M of hypoxanthine (Nutritional Biochemicals) and 3.12 x lO-5 M of thymidine (Sigma). d For agar suspension culture, hypoxanthine was added to final concentrations of 0.15-3.16 x lo-” M and thvmidine to final concentrations of 0.08-1.46 x lO-4 M. e Partial hydrolysis of casein was evident around some of the single cells in suspension. f In monolayer culture, adenosine (Nutritional Biochemicals) was added to a final concentration of 3.7 x 10e5 M and thymidine to a final concentration of 3.12 x lO-5 M. For agar suspension culture cells were grown in the presence of 1.59 x IO-” M of adenosine and 1.46 % lo-* M of thymidine. NT, not tested.
activate plasminogen as does urokinase  and, therefore, has been designated in the present report as a plasminogen activator (PA). Materials and Methods Cl3 cells were obtained from the American Type Culture Collection in the 52nd passage from tissue of origin. Cells were grown in Eagles minimum essential medium with 10 % calf serum, non-essential amino acids, penicillin, and streptomycin. Repeated tests have shown them to be free of mycoplasma contamination. High passage Cl3 cells were propagated in the same medium as the low passage cells but with 5 96 calf serum . Cells to be tested for the formation of colonies in soft agar were suspended in agar as described by Macpherson  and grown for 10 days at 37°C in an atmosphere of approx. 5 % CO,. The soft agar was then overlayed with casein-fibrin solutions to detect PA activity as follows: After incubation for 10 days a 1.3 % human fibrinogen solution was diluted 1:3 with a sterile 5 % skim milk solution, added as an overlay to the agar and clotted with thrombin (bovine, Parke-Davis). Skim milk and fibrinogen solutions
were prepared as described previously . The plates were incubated overninht at 37°C and observed for liquefaction of fibrin -and/or hydrolysis of casein. BHK2l/Cl3 cells to be tested for agglutination with ConA (Calbiochem) were removed If;om surfaces of culture vessels with a 0.02 % disodium versenate solution, centrifuged, and resuspended in an appropriate volume of calcium and magnesium-deficient saline (PD). One-tenth ml of ConA at different concentrations was added to 0.2 ml of the cell suspension (l-3 x lo6 cells/ml) in 96well flat-bottom culture trays (Linbro Chemical Co.) and incubated at room temperature for 20 min. The tray was then inverted and shaken to remove the fluid contents of the wells. After 2 min, 0.2 ml of PD solution was added to each well and the cells examined microscopically for agglutination .
When suspended in agar according to the method of Macpherson  Cl3 cells did not grow (table 1). High passage sublines of the Cl3 cells , on the other hand, grew readily in agar suspension. All those cells which Exprl
Cdl Res 90 (1975)
grew in suspension also produced PA in the semisolid medium and were found to be agglutinable by ConA (table 1). Cl3 cells, however, did not demonstrate any of these characteristics (table 1). An attempt was made to see if the traits of the high passage cells described above could be transferred to Cl3 cells. DNA was purified from those strains which could grow in suspension after the method of Berns & Thomas . Procedures used were the same except that sodium lauryl sulfate concentration was reduced to 0.025 Y0 to lyse the cells. The majority of the Cl3 cells grew and formed colonies when incubated in the presence of the DNA (data not shown). Growth did not occur, however, when DNA was removed by washing prior to suspending the cells in soft agar. This suggested that individual components of the DNA preparation had stimulated the growth of the cells. Subsequently, we found that hypoxanthine and thymidine or adenosine and thymidine could completely replace the effect of the DNA. As seen in table 1, growth in suspension occurred as well as production of PA and increased agglutinability by ConA. These effects were reversible. Cl3 cells grown in suspension in the presence of hypoxanthine and thymidine were isolated. After growing the cells in monolayer culture for a week in the absence of hypoxanthine and thymidine they were again suspended in soft agar without hypoxanthine and thymidine. These cells did not grow and produced little detectable PA. ConA agglutinability had also returned to normal. PA activity (stimulated by purine and pyrimidine addition) not only was detected when the Cl3 cells were assayed following colony formation but also was observed in single cells in suspension by 24-h after addition of hypoxanthine and thymidine (data not shown). Cell division did not begin in these Exprl
Cell Res 90 (1975)
experiments until 48-h after purine and pyrimidine supplementation. It was of interest to note that ConA agglutinability had also increased by 24-h. Clarke & Smith [l] observed that Cl3 cells suspended in agar in the presence of hypoxanthine alone could not grow. We have also observed this phenomenon. Thymidine, therefore, is a prerequisite for the growth of normal cells in agar. It is not required, however, for the increase in ConA agglutinability of the cells grown in monolayer culture. Hypoxanthine or adenosine alone will cause the cells to become agglutinable by ConA as well as to synthesize PA (data not shown). Discussion
The role of PA in the growth of cells in agar is not known at the present time. A positive correlation, however, has been consistently observed between increased PA activity and increased ConA agglutinability (table 1). We have also isolated high passage BHK21 cells which are deficient in PA activity. These cells are not agglutinable by ConA, and do not form colonies in agar. Moreover, a high passage cell line able to grow in the presence of 5.7 x 1O-4 M 8azahypoxanthine and 1.94 x 1O-4 M 5-bromodeoxyuridine was isolated which also demonstrated the characteristics of the low passage Cl3 cells seen in table 1 (unpublished observations). Although the implication of the above observations is that PA is involved in surface modifications, no direct demonstration has yet been made of either its role in surface changes or growth stimulation. Indirect evidence has been obtained, however, suggesting that PA interacts with extracellular factors (e.g., plasminogen) to stimulate growth of cells in agar. Ossowski et al. , have noted that removal of plasminogen from the serum used to grow cells in soft agar caused a three-fold decrease in the plating
Pr cuminary notes efficiency of transformed cells. PA produced by BHK21/C13 cells  and transformed cells  can activate plasminogen, with the subsequent formation of plasmin, a potent, trypsin-like enzyme. Although the PA produced by Cl3 cells while proliferating in agar in the presence of purines and pyrimidines could be the growth promoting substance hypothesized by Clarke & Smith [l] and Montagnier  much work needs to be done to establish this point. This work described in the present report, however, will be able to serve as the basis for future experimental approaches to the problem of growth regulation in cell culture. This work was partially supported by a Predoctoral Fellowship from NIGMS to J. C. T. (5-FOl GM 46-
276) and by NIH Grant A108494 and NSF Grant
GK-29382’ References 1. Clarke, G D & Smith, C, J cell physiol 81 (1973) 125. 2. Montagnier, L, Ciba foundation symposium growth control in cell cultures (ed G E W Wolstenholme & J Knight) p. 33. Churchill, London (1971). 3. Torres, A R, Snyder, R W & Hill, D W, J natl cancer inst (1974). In press. 4. Taylor, J C, Hill, D W & Rogolsky, M, Exptl cell res 73 (1972) 422. 5. MacPherson, I, Fundamental techniques in virology (ed K Habel & N P Salzman) p. 214. Academic Press, New York (1969). 6. Poste, G, Exptl cell res 73 (1972) 319. 7. Berns, K I & Thomas, C A, J mol biol 1I (1965) 476.
8. Ossowski, L, Quigley, J P, Kellerman, G M & Reich, E, J exptl med 138 (1973) 1056. Received November 4, 1974
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