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Utilization of serum proteins by normal and poliovirus infected monkey kidney cells
J. G. Bachfold and L. P. Gebhardf
432
The total thickness of these structures is not great enough to resolve with the light microscope. A functional significance cannot be assigned to the pentalaminar bodies with the knowledge available at present. However, a fact which may be of significance is that all of these bodies so far observed were in cells active in either phagocytosis or pinocytosis. Since our observations are necessarily limited it may be that cells not carrying out these functions also possess these structures, although we have not seen them in several other kinds of tissue cells which were carrying out different kinds of activities. REFERENCES 1. DALTON, A. J., Anaf. Rec. 121, 281 (1955). 2. KUFF, E. L. and DALTON, A. J., InternaZI. J. Ultrastructure 3. PALADE, G. E., J. Ezpfl. Med. 95, 285 (1952).
UTILIZATION
OF SERUM
PROTEINS
INFECTED
MONKEY
POLIOVIRUS
J. G. BACHTOLD Department
of Bacteriology,
College
of Medicine,
Research 1, 1 (1957).
BY NORMAL KIDNEY
AND
CELLS’
and L. P. GEBHARDT University
of Utah, Salt Lake City, Utah, U.S.A.
Received August 19, 1957
T HE necessity for adding complex substances to tissue culture systems to promote fibroblastic outgrowth was first reported by Carrel [a]. He observed that the addition Later of embryonic tissue extracts accelerated the growth of chicken fibroblasts. Simms and Sanders [9] reported the successful use of serum ultrafiltrates as an additive to tissue culture fluid for certain systems. Sanford et al. [8] concluded that, although serum ultrafiltrate as well as serum residue supported cell proliferation, neither was as effective as whole serum. Allgoewer ef al. [l] also reported that the addition of whole human serum to the medium was necessary to promote epidermal outgrowth of human skin in vitro, while gamma globulin as an additive showed no growth promoting activity. Kent and Gey [5] found that cultured cells of neoplastic origin utilized serum proteins. It was the purpose of our investigation to determine quantitatively by means of electrophoretic analysis the patterns of serum utilization during the course of epithelial-like outgrowth of monkey kidney cells in vitro prior to and after infection with poliovirus. 1 Aided by a grant from The National Foundation for Infantile Paralysis. Experimental
Cell Research 13
Serum protein utilization
by kidney cells
433
Methods and Material&-Flat bottomed 8 ounce “prescription” bottles were seeded with 50 ml of a 1:200 suspension of trypsinized monkey kidney cells in a growth medium essentially the same as that described by ~Ielni~k 16). Instead of the recommended 2 per cent calf serum, IO per cent was used to increase the accuracy of the electrophoretic assay. The addition of this relatively large amount of serum did not interfere with epithelial-like outgrowth. The serum employed in all of these studies
I 0 Fig. I.-Fraction Protein N in mg/ml vs time before and after infection. Albumen-globulin ratios vs time before and after infection.
I I I I
my*
3 4 5 of ceil growth
6
2 I I I i
0 I 2 3 days offer virus
Fig. 2.-Total Protein N in mg/ml vs time before and after infection.
was obtained from a single calf and was inactivated for 30 min. at 56°C before use. The virus employed was the Mahoney strain of poliovirus containing 1 x 10’ PFL! (plaque-forming units) per ml, as determined by the plaque technique of Dulbecco [3]. Two milliliters of this virus fluid in 50 ml of the medium was used to infect the cells. Electrophoretic studies were performed at 1°C in a Perkin-Elmer apparatus equipped with the Philpot-Svensen optics. A 2 ml Tiselius cell was used in the standard open system. The solutions were equilibrated by dialysis for 14 hours under constant agitation at 4°C with diethyl barbiturate buffer at pH 8.5 to 8.6 and at an ionic strength of 0.1. All solutions were centrifuged at 105,000 x g for one hour to remove particulate matter prior to dialysis and prior to Kjeldahl nitrogen determinations. The variations which took place in the serum protein as a result of cell growth were measured by comparison with the supernatant of a centrifuged sample from the cell suspension as used to seed the bottles. Changes which took place as a result of virus activity were measured by comparison with standard medium containing filtered virus fluid. For electrophoretic assays and for nitrogen determinations 8 ml samples were withdrawn from the respective bottle cultures without replacement of the fluid. Kesults and Discussion.-The data in Fig. 1 which represent the average of 15 values show that during the first 3 days when epithelial like outgrowth was most
J. G. Bachfold and L. P. Gebhardf intense a decrease in albumin as well as in the globulins occurred. The increased albumin-globulin ratio indicates, however, a greater utilization of the globulins than of albumin. At this point the monolayers were well established but not confluent. During the fourth day an apparent increase from the 3 day level was observed for albumin as well as for alpha globulin. This increase was attributed to lysis of unattached cells and the subsequent liberation of soluble cellular proteins of high
Fig. 3.-Electrophoretic patterns of different samples. A, Standard medium containing 10 per cent calf-serum; B, standard medium after exposure to cells for one hour; C, standard medium after 6 days of cell growth; D, standard medium containing filtered virus; E, standard medium 3 days after infection.
mobilities which migrated with albumin and alpha globulin. The presence of a significant number of non-viable cells in trypsinized monkey kidney cell preparations has recently been emphasized by Rappaport [7]. It was difficult to determine whether utilization of the serum proteins occurred from the fourth to the sixth day since significant amounts of proteins from lysed cells were present in the solution. After the sixth day the fluid was decanted, the cell layer washed twice with the standard medium, and then infected with poliovirus. During the first day after infection a sharp drop in albumin as well as the globulins was observed. This corresponds to the period when an accelerated metabolic activity is apparent in the infected cells as observed by HydCn [4]. There was a gradual increase in the serum proteins for the following 2 days. This increase was again attributed to the liberation of soluble cellular proteins from virus-induced cell destruction. When total protein nitrogen values were plotted as a function of time a rapid decrease during the first 3 days of epithelial-like outgrowth became evident (Fig. 2). Calculations made from the electrophoretic data showed that of the total decrease in protein nitrogen, 49.7 per cent was due to a loss in the globulins and 44 per cent due to the loss in albumin. The possible utilization of the serum proteins during the second 3 days was offset by the release of cellular proteins. Within the first day after infection a sharp drop in protein nitrogen was observed. Of the total decrease 33.5 per cent was due to the loss of albumin and 27 per cent due to the loss in globulins. This represents a much more rapid utilization of the serum proteins compared to that during normal cell growth. A subsequent gradual increase was again attributed to lysis of cells. As shown in Fig. 3 a difference was noted between the electrophoretic patterns of medium which had contained cells for approximately one hour prior to centrifugation and those of the medium only. In the medium without cells an alpha-2 globulin appeared consistently, whereas in the medium exposed to cells and then centrifuged no alpha-2 globulin separated. It Experimental
Cell Research 13
Serum protein utilization
by kidney cells
435
appears that the cells actively participate in the removal of the alpha-2 globulin. No boundary anomalies were observed in the electrophoretic patterns during the period of cell growth and after infection. Furthermore, the total amount of protein was higher in the sample exposed to cells for 1 hour than in the medium only. Although no fluid change was made during the course of cell growth, a continuous monolayer was formed. The cells retained their susceptibility to the virus since the supernatant fluid after infection and lysis of the cells contained 1 y 10’ PFU per ml. The total protein nitrogen of the control medium without cells did not change during 6 days of incubation. Likewise no significant changes in the mobilities of the serum components either before or after infection were observed. From the results obtained, several points emerge: all electrophoretically separable serum proteins were utilized by outgrowing epithelial like cells under the experimental conditions. It appeared that approximately 50 per cent of the globulin was utilized compared to 44 per cent of the albumin. Since a continuous monolayer of polygonal cells was obtained in six days, evidently a routine fluid change after 3 days of cell growth is not necessary to insure a confluent monolayer if a sufficient amount of serum is initially added to the medium. The yield of virus from cells grown, under such conditions is identical to the one obtained in bottle cultures where the nutrient fluid was changed after 3 days of cell growth. Summary.-The quantitative utilization of serum protein by epithelial-like cells in monkey kidney cell cultures in vitro has been studied. It was found that all electrophoretically separable serum components were utilized by the cells during the most active growth phase, namely the first 3 days. During the second three days of growth very little protein was utilized. After infection of the cells serum protein utilization was restricted to the first day, namely the period of virus multiplication. A continuous monolayer of epithelial like cells was obtained in 6 days without the 3 day routine fluid change. Since approximately half of the 10 per cent added calf serum was utilized it appears that the addition of 5 per cent calf serum would eliminate the necessity of a fluid change. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
ALLGOEWER,M.,POMERAT, C.M.and BLOCKER, T. G., Ann. Surg. 135,923(1952). CARREL, A., J. Expfl. Med. 17, 14 (1913). DULBECCO, R., Proc. N&l. Acad. Sci. 38, 747 (1952). HYDBN, H., Cold Spring Harbor Symposia Quanf. Biot. 12, 104 (1947). KENT, H. N. and GEY, G. O., Proc. Sot. Exptf. Biot. Med. 94, 205 (1957). MELNICK, J. L., Ann. N.Y. Acad. Sci. 61, 754 (1955). RAPPAPORT, C., Bull. World. Health. Organization 14, 147 (1956). SANFORD, K. K., WALTA, H. K., SHANNON, J. E., EARLE, W. R. and EVAXS, V. J., J. Natt. Cancer Inst. 13, 121 (1952). 9. SIMMS, H. S. and SANDERS, M., Arch. Pathol. 33, 619 (1942).
Experimental
Cell Research 13
432
The total thickness of these structures is not great enough to resolve with the light microscope. A functional significance cannot be assigned to the pentalaminar bodies with the knowledge available at present. However, a fact which may be of significance is that all of these bodies so far observed were in cells active in either phagocytosis or pinocytosis. Since our observations are necessarily limited it may be that cells not carrying out these functions also possess these structures, although we have not seen them in several other kinds of tissue cells which were carrying out different kinds of activities. REFERENCES 1. DALTON, A. J., Anaf. Rec. 121, 281 (1955). 2. KUFF, E. L. and DALTON, A. J., InternaZI. J. Ultrastructure 3. PALADE, G. E., J. Ezpfl. Med. 95, 285 (1952).
UTILIZATION
OF SERUM
PROTEINS
INFECTED
MONKEY
POLIOVIRUS
J. G. BACHTOLD Department
of Bacteriology,
College
of Medicine,
Research 1, 1 (1957).
BY NORMAL KIDNEY
AND
CELLS’
and L. P. GEBHARDT University
of Utah, Salt Lake City, Utah, U.S.A.
Received August 19, 1957
T HE necessity for adding complex substances to tissue culture systems to promote fibroblastic outgrowth was first reported by Carrel [a]. He observed that the addition Later of embryonic tissue extracts accelerated the growth of chicken fibroblasts. Simms and Sanders [9] reported the successful use of serum ultrafiltrates as an additive to tissue culture fluid for certain systems. Sanford et al. [8] concluded that, although serum ultrafiltrate as well as serum residue supported cell proliferation, neither was as effective as whole serum. Allgoewer ef al. [l] also reported that the addition of whole human serum to the medium was necessary to promote epidermal outgrowth of human skin in vitro, while gamma globulin as an additive showed no growth promoting activity. Kent and Gey [5] found that cultured cells of neoplastic origin utilized serum proteins. It was the purpose of our investigation to determine quantitatively by means of electrophoretic analysis the patterns of serum utilization during the course of epithelial-like outgrowth of monkey kidney cells in vitro prior to and after infection with poliovirus. 1 Aided by a grant from The National Foundation for Infantile Paralysis. Experimental
Cell Research 13
Serum protein utilization
by kidney cells
433
Methods and Material&-Flat bottomed 8 ounce “prescription” bottles were seeded with 50 ml of a 1:200 suspension of trypsinized monkey kidney cells in a growth medium essentially the same as that described by ~Ielni~k 16). Instead of the recommended 2 per cent calf serum, IO per cent was used to increase the accuracy of the electrophoretic assay. The addition of this relatively large amount of serum did not interfere with epithelial-like outgrowth. The serum employed in all of these studies
I 0 Fig. I.-Fraction Protein N in mg/ml vs time before and after infection. Albumen-globulin ratios vs time before and after infection.
I I I I
my*
3 4 5 of ceil growth
6
2 I I I i
0 I 2 3 days offer virus
Fig. 2.-Total Protein N in mg/ml vs time before and after infection.
was obtained from a single calf and was inactivated for 30 min. at 56°C before use. The virus employed was the Mahoney strain of poliovirus containing 1 x 10’ PFL! (plaque-forming units) per ml, as determined by the plaque technique of Dulbecco [3]. Two milliliters of this virus fluid in 50 ml of the medium was used to infect the cells. Electrophoretic studies were performed at 1°C in a Perkin-Elmer apparatus equipped with the Philpot-Svensen optics. A 2 ml Tiselius cell was used in the standard open system. The solutions were equilibrated by dialysis for 14 hours under constant agitation at 4°C with diethyl barbiturate buffer at pH 8.5 to 8.6 and at an ionic strength of 0.1. All solutions were centrifuged at 105,000 x g for one hour to remove particulate matter prior to dialysis and prior to Kjeldahl nitrogen determinations. The variations which took place in the serum protein as a result of cell growth were measured by comparison with the supernatant of a centrifuged sample from the cell suspension as used to seed the bottles. Changes which took place as a result of virus activity were measured by comparison with standard medium containing filtered virus fluid. For electrophoretic assays and for nitrogen determinations 8 ml samples were withdrawn from the respective bottle cultures without replacement of the fluid. Kesults and Discussion.-The data in Fig. 1 which represent the average of 15 values show that during the first 3 days when epithelial like outgrowth was most
J. G. Bachfold and L. P. Gebhardf intense a decrease in albumin as well as in the globulins occurred. The increased albumin-globulin ratio indicates, however, a greater utilization of the globulins than of albumin. At this point the monolayers were well established but not confluent. During the fourth day an apparent increase from the 3 day level was observed for albumin as well as for alpha globulin. This increase was attributed to lysis of unattached cells and the subsequent liberation of soluble cellular proteins of high
Fig. 3.-Electrophoretic patterns of different samples. A, Standard medium containing 10 per cent calf-serum; B, standard medium after exposure to cells for one hour; C, standard medium after 6 days of cell growth; D, standard medium containing filtered virus; E, standard medium 3 days after infection.
mobilities which migrated with albumin and alpha globulin. The presence of a significant number of non-viable cells in trypsinized monkey kidney cell preparations has recently been emphasized by Rappaport [7]. It was difficult to determine whether utilization of the serum proteins occurred from the fourth to the sixth day since significant amounts of proteins from lysed cells were present in the solution. After the sixth day the fluid was decanted, the cell layer washed twice with the standard medium, and then infected with poliovirus. During the first day after infection a sharp drop in albumin as well as the globulins was observed. This corresponds to the period when an accelerated metabolic activity is apparent in the infected cells as observed by HydCn [4]. There was a gradual increase in the serum proteins for the following 2 days. This increase was again attributed to the liberation of soluble cellular proteins from virus-induced cell destruction. When total protein nitrogen values were plotted as a function of time a rapid decrease during the first 3 days of epithelial-like outgrowth became evident (Fig. 2). Calculations made from the electrophoretic data showed that of the total decrease in protein nitrogen, 49.7 per cent was due to a loss in the globulins and 44 per cent due to the loss in albumin. The possible utilization of the serum proteins during the second 3 days was offset by the release of cellular proteins. Within the first day after infection a sharp drop in protein nitrogen was observed. Of the total decrease 33.5 per cent was due to the loss of albumin and 27 per cent due to the loss in globulins. This represents a much more rapid utilization of the serum proteins compared to that during normal cell growth. A subsequent gradual increase was again attributed to lysis of cells. As shown in Fig. 3 a difference was noted between the electrophoretic patterns of medium which had contained cells for approximately one hour prior to centrifugation and those of the medium only. In the medium without cells an alpha-2 globulin appeared consistently, whereas in the medium exposed to cells and then centrifuged no alpha-2 globulin separated. It Experimental
Cell Research 13
Serum protein utilization
by kidney cells
435
appears that the cells actively participate in the removal of the alpha-2 globulin. No boundary anomalies were observed in the electrophoretic patterns during the period of cell growth and after infection. Furthermore, the total amount of protein was higher in the sample exposed to cells for 1 hour than in the medium only. Although no fluid change was made during the course of cell growth, a continuous monolayer was formed. The cells retained their susceptibility to the virus since the supernatant fluid after infection and lysis of the cells contained 1 y 10’ PFU per ml. The total protein nitrogen of the control medium without cells did not change during 6 days of incubation. Likewise no significant changes in the mobilities of the serum components either before or after infection were observed. From the results obtained, several points emerge: all electrophoretically separable serum proteins were utilized by outgrowing epithelial like cells under the experimental conditions. It appeared that approximately 50 per cent of the globulin was utilized compared to 44 per cent of the albumin. Since a continuous monolayer of polygonal cells was obtained in six days, evidently a routine fluid change after 3 days of cell growth is not necessary to insure a confluent monolayer if a sufficient amount of serum is initially added to the medium. The yield of virus from cells grown, under such conditions is identical to the one obtained in bottle cultures where the nutrient fluid was changed after 3 days of cell growth. Summary.-The quantitative utilization of serum protein by epithelial-like cells in monkey kidney cell cultures in vitro has been studied. It was found that all electrophoretically separable serum components were utilized by the cells during the most active growth phase, namely the first 3 days. During the second three days of growth very little protein was utilized. After infection of the cells serum protein utilization was restricted to the first day, namely the period of virus multiplication. A continuous monolayer of epithelial like cells was obtained in 6 days without the 3 day routine fluid change. Since approximately half of the 10 per cent added calf serum was utilized it appears that the addition of 5 per cent calf serum would eliminate the necessity of a fluid change. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
ALLGOEWER,M.,POMERAT, C.M.and BLOCKER, T. G., Ann. Surg. 135,923(1952). CARREL, A., J. Expfl. Med. 17, 14 (1913). DULBECCO, R., Proc. N&l. Acad. Sci. 38, 747 (1952). HYDBN, H., Cold Spring Harbor Symposia Quanf. Biot. 12, 104 (1947). KENT, H. N. and GEY, G. O., Proc. Sot. Exptf. Biot. Med. 94, 205 (1957). MELNICK, J. L., Ann. N.Y. Acad. Sci. 61, 754 (1955). RAPPAPORT, C., Bull. World. Health. Organization 14, 147 (1956). SANFORD, K. K., WALTA, H. K., SHANNON, J. E., EARLE, W. R. and EVAXS, V. J., J. Natt. Cancer Inst. 13, 121 (1952). 9. SIMMS, H. S. and SANDERS, M., Arch. Pathol. 33, 619 (1942).
Experimental
Cell Research 13