Printedin Sweden Copyright @)1975by Academic Press,Inc. All rights of reproduction in anyform reserved
Experimental Cell Research 96 (1975) 58-62
INITIATION OF STATIONARY BY A POLYPEPTIDE CONTAINING
OF DNA HUMAN
FRACTION
SYNTHESIS GLIA-LIKE
CELLS
FROM HUMAN
SOMATOMEDIN
PLASMA
ACTIVITY
B. WESTERMARK, A.. WASTESON and K. U T H N E The Wallenberg Laboratory and Institute o f Medical and Physiological Chemistry, University o f Uppsala, S-75 247 Uppsala, Sweden and Department o f Clinical Chemistry, Veterinary College, S-10501 Stockholm, Sweden
SUMMARY A polypeptide fraction, mol.wt 4 000-7 000, isolated from human plasma, released the cell cycle block of serum-deprived or density-inhibited human glial cells. The factor responsible for this effect was not insulin. The same polypeptide fraction was previously shown to enhance sulphation and DNA synthesis of embryonic chick cartilage. Separation by isoelectric focusing implied that the factor responsible for the glial cell stimulation was not identical to that active in cartilage.
Most animal cells in culture require serum for their survival and multiplication. Exponentially growing cells cease to proliferate when serum is removed. The proliferation restraint that occurs in dense cultures of normal cells (density-dependent inhibition of growth) is released by the addition of fresh serum (see [11] for review). The basis for the effect of serum on cell multiplication is unknown. Temin and collaborators have isolated polypeptides from calf serum [6] and from conditioned medium from rat liver cell cultures [1] that stimulate cell multiplication. These polypeptides also displayed somatomedin- or insulin-like properties, implying similarities within this group of substances [1, 6]. In the present investigation we have studied the multiplication stimulatory effect on human glia-like cells of a polypeptide Exptl Cell Res 96 (1975)
fraction isolated from human plasma. This fraction was previously shown to contain somatomedin (sulphation factor) activity, as indicated by its stimulating effect on polysaccharide biosynthesis in chick cartilage [12], human fibroblasts [14] or human glia-like cells [14]. MATERIALS AND METHODS Cell lines Human glia-like lines were initiated from brain biopsies as described previously [7, 8]. These cells have been characterized with regard to origin, morphology, growth pattern, contact inhibition, density-dependent inhibition (cf [8, 15, 16]). The cell lines were used during their rapid growth phase (phase II according to Hayflick) [5]. The cells were grown in Eagle's MEM supplemented with 10% baby calf serum and antibiotics (50 p.g/ml of streptomycin, 100 U/ml of penicillin, 1.25 /zg/ml of amphotericin B). Falcon plastic Petri dishes ( 0 50 ram) were employed. Cultures were incubated at 37~ in humidified air containing 5 % CO2.
Stimulation o f D N A synthesis by polypeptides
59
The cells were then dissolved in 1 ml/dish of 0.3 M sodium hydroxide. An aliquot of the lysate was transferred to scintillation vials and a dioxan-based cocktail containing Cab-O-Sil was added. The samples were counted at 10~ in an Intertechnique liquid scintillator. The radioactivity contained in the acid-insoluble material was taken as a measure of DNA synthesis.
300
200
Polypeptide fraction 100 0.5
I
2
3OO
0
200
100
A polypeptide fraction (mol.wt 4000-7000) was isolated from human plasma as described [12], An acid ethanol extract of Cohn fraction IV was subjected to gel chromatography on Sephadex G-75. Effluent fractions corresponding to the Kar values 0.6--0.8 were pooled and lyophilized. The somatomedin activity of the preparation was 0.08 U//zg of protein, estimated by its stimulatory activity on the incorporation of [35S]O4 into embryonic chick cartilage [3]. One U is defined as the somatomedin activity of I ml of a pooled reference serum [3]. The insulin content of the fraction was 0.1/zU//xg of protein [2].
. . . . 0 s 10 15
25
50
Fig. 1. Abscissa: (top) serum conc. (%); (bottom) tzg/ ml of PPF; ordinate: cpm (mean of duplicate cultures) as % of control (250 cpm). Stimulation of DNA synthesis of serum-deprived sparse cells by (top) calf serum and (bottom) PPF.
A s s e s s m e n t o f D N A synthesis in cell cultures after exposure to test samples Serum-deprived sparse cultures. Pooled cells were plated at low density, about 10000 cells/cmz, in Eagle's MEM 10% serum and grown for 24 h. Medium was then changed to 5 ml/dish of serumfree Ham's F-12 [4] containing 15 mM Hepes | (Sigma), pH 7.3. After another 24 h incubation, medium was again changed with F-12 Hepes and test samples were added to duplicate dishes in a small volume (20-100/~1/5 ml medium). Dense, stationary cultures. Pooled glial cells were seeded into dishes and incubated with biweekly medium renewal. Eagle's MEM with 10% calf serum was employed. After 2 weeks the cell cultures had reached a stationary phase [8]. The substances to be tested were then added to the old incubation medium 3 days after a medium change. Measurement of DNA synthesis. After incubation with test samples for 24 h of either sparse or dense cultures, [aH]thymidine was added (0.02 ~Ci/ml, final conc., spec. act. 2 Ci/mM). By this procedure, the isotope was present during the peak of DNA synthesis [17]. After incubation for another 24 h period, the dishes were washed twice with phosphate-buffered saline and extracted 3• rain with 5 ml of 5% trichloroacetic acid at +4~ After one wash in 5 ml of PBS, the cultures were incubated at room temperature for 5 min in ethanol-ether (3 : 1) and air-dried.
Isoelectric focusing Further separation of the lyophilized polypeptide fraction was performed by isoelectric focusing [9]. Portions of 100 mg were dissolved in 6 M urea-1% Ampholine (LKB, Stockholm, Sweden) and subjected to isoelectric focusing in beds of Sephadex G-75 (40• cm) containing 6 M urea and 1% Ampholine (pH 3-10) at +5~ for 24 h at 10 V/cm. At the end of the run, the bed was cut into I cm bands which were eluted with distilled water. Fractions were tested for growth-promoting activity on sparse cultures at a final conc. of I0 /zg of dry weight/ml.
Insulin A mixture of crystalline bovine and porcine insulin (Insulin Special | 40 U/ml was bought from Vitrum (Stockholm, Sweden). Crystalline human insulin was a gift from Novo Industri (Copenhagen, Denmark). It was directly dissolved in the growth medium at its final concentration.
RESULTS
Effect o f serum and polypeptide fraction (PPF) on serum-deprived sparse cells The
incorporation
of [3H]TdR
into acid-
insoluble material of serum-deprived after
exposure
to
calf
serum
and
cells poly-
p e p t i d e f r a c t i o n at v a r i o u s c o n c e n t r a t i o n s is r e c o r d e d curve
in fig.
obtained
1. T h e
dose-response
with cultures
exposed
to
Exptl CellRes 96 (1975)
60
Westermark, Wasteson and Uthne 200~
200
100 100
9
,
,
I
2
,,,,
/"
1135 I04
163 1(]2
1{~1,4
300
200
O
I0 20 100
o ~ To F5
2'5
sb
Fig. 2. Abscissa: (top) serum conc. (%); (bottom) tzg/ ml of PPF; ordinate: cpm (mean of duplicate cultures) as % of control (1200 cpm). Stimulation of DNA synthesis of density-inhibited cells by (top) calf serum and (bottom) PPF.
8,
,0.3
7, ,0.2
6 5 4
50
10"0
Fig. 4. Abscissa." (top) conc, of a mixture of bovine and porcine insulin (U/ml); (bottom) conc. of human insulin (p,U/ml); ordinate: cpm (mean of duplicate cultures) as % of control (340 cpm). Stimulation of sparse, serum-deprived cells by insulin.
calf serum followed an expected course with a rapid increase at low concentration and a plateau phase at higher concentrations of serum. The serum-deprived glial cells were also markedly stimulated by the polypeptide fraction. The corresponding dose-response curve had essentially the same course as that obtained when serum was used as a stimulator.
Effect o f serum and polypeptide fraction when added to the old medium o f dense cultures 150 Stationary, density-inhibited ceils 3 days after medium change were employed. The 10 20 30 samples to be tested were added in a low Fig. 3. Abscissa: fraction no.; ordinate: (top) Az6o volume to the spent growth medium, The (right) (0---0); pH (left) (O--O); (bottom) cpm plot of [3H]TdR incorporation vs the (mean of duplicate cultures) as % of control (275 cpm). respective concentrations appears in fig. 2. lsoelectric focusing of polypeptide fraction. Eluted fractions were analysed for absorbance at 260 nm, The incorporation of [3H]TdR was markedpH and thymidine incorporation in sparse cultures ly stimulated by serum and polypeptide of glial cells. Exptl Cell Res 96 (1975)
Stimulation of DNA synthesis by polypeptides
61
nificant stimulation occurred in the range of insulin concentrations studied.
0
100
165 16~ 163 T62 161.s Fig. 5. Abscissa: conc. of a mixture of bovine and porcine insulin (U/ml); ordinate: cpm (mean of dupli-
cate cultures) as % of control (1350 cpm). Effect of bovine and porcine insulin on densityinhibited cells.
fraction, respectively. It should be noted that in both cases, the dose-response curves were linear in the range of serum concentrations tested in contrast to those obtained with sparse cells (fig. 1). The level of maximal stimulation was not reached. It is also evident that, with serum as a standard, the specific stimulatory activity of PPF was higher on density-inhibited cells than on sparse, serum-deprived cells.
Isoelectric focusing of polypeptide fraction An attempt was made to purify the PPF by isoelectric focusing (fig. 3). As can be seen, growth-promoting activity was recovered in an acidic pH range (4.5-5.9).
Effect o f insulin Sparse cells. The effect of insulin on the DNA synthesis of sparse glial cells appears in fig. 4. The highest concentrations of bovine-porcine insulin was slightly stimulatory. Moderate doses of either bovine-porcine or human insulin failed to cause any stimulation. Dense cultures. The effect of mixtures of porcine and bovine insulin on densityinhibited cells is depicted in fig. 5. No sig-
DISCUSSION It appears that the polypeptide fraction from human plasma displayed not only somatomedin-like activity in different kinds of cells [12, 14] but was also a potent initiator of DNA synthesis in cells of human astrocytic origin. The fraction produced a more pronounced effect when it was used along with the spent growth medium on density-inhibited cells than when tested in a serum-free medium on sparse cells (figs 1, 2). This finding may indicate an interaction between separate stimulating factors present in serum. In some cell systems, insulin may act as a stimulator of DNA synthesis [10]. However, the possibility that the contaminating insulin was responsible for the observed enhancement on DNA synthesis in the present experiments, seems to be ruled out by the low response to insulin of sparse cells and the virtual absence of any response of density-inhibited cells. It should also be pointed out that the dosage levels of insulin at which glia-like cells were stimulated by far exceeded the low concentration of insulin in PPF. Further separation of the PPF by isoelectric focusing revealed an acidic nature of the active component. This finding discriminates the present glial factor from the cartilage sulphation factor which has an isoelectric point near neutral pH [13]. Thus, the glial factor described may be another representative of the group of cellstimulating polypeptides [1 1]. This work was supported by grants from the Swedish Medical Research Council (13X-2309, 13X-4486) and the Swedish Cancer Society (55-B73-09XC and 689B75-01X). Exptl Cell Res 96 (1975)
62
Westermark, Wasteson and Uthne REFERENCES
1. Dulak, N C & Temin, H M, J cell physiol 81 (1973) 153. 2. Hales, C N & Randle, P J, Biochemj 88 (1963) 137. 3. Hall, K, Acta endocrinol, suppl. 163 (1972). 4. Ham, R G, Proc natl acad sci US 53 (1965) 288. 5. Hayflick, L, Exp cell res 37 (1965) 614. 6. Pierson, R W & Temin, H M, J cell physiol 79 (1972) 319. 7. Pont6n, J & Maclntyre, E, Acta pathol microbiol Scand 74 (1968) 465. 8. Pont6n, J, Westermark, B & Hugosson, R, Exp cell res 58 (1969) 393. 9. Radola, B J, Biochim biophys acta 194 (1969) 335. 10. Temin, H M, J cell physiol 69 (1967) 377. 11. Temin, H M, Pierson, R W & Dulak, N C, Growth, nutrition and metabolism of cells in
Exptl Cell Res 96 (1975)
12. 13. 14. 15. 16. 17.
culture (ed H Rothblat & V J Cristofalo) vol. 1, p. 49. Academic Press, New York & London (1972). Uthne, K, Acta endocrinol, suppl. 175 (1973). Van Wyk, J J, Hall, K, Van den Brande, J L & Weaver, R P, J clin endocrinol metab 32 (1971) 389. Wasteson, /~, Uthne, K & Westermark, B, Bioc h e m j 136 (1973) 1069. Westermark, B, Exp cell res 69 (1971) 259. - - Abstracts of Uppsala dissertations in medicine, Acta Univ Upsaliensis, no. 164 (1973). Westermark, B & Wasteson, A, Adv metab disorders (ed R Luft & K Hall) vol. 8, p. 85. Academic Press, New York (1975).
Received February 17, 1975 Revised version received May 12, 1975
Experimental Cell Research 96 (1975) 58-62
INITIATION OF STATIONARY BY A POLYPEPTIDE CONTAINING
OF DNA HUMAN
FRACTION
SYNTHESIS GLIA-LIKE
CELLS
FROM HUMAN
SOMATOMEDIN
PLASMA
ACTIVITY
B. WESTERMARK, A.. WASTESON and K. U T H N E The Wallenberg Laboratory and Institute o f Medical and Physiological Chemistry, University o f Uppsala, S-75 247 Uppsala, Sweden and Department o f Clinical Chemistry, Veterinary College, S-10501 Stockholm, Sweden
SUMMARY A polypeptide fraction, mol.wt 4 000-7 000, isolated from human plasma, released the cell cycle block of serum-deprived or density-inhibited human glial cells. The factor responsible for this effect was not insulin. The same polypeptide fraction was previously shown to enhance sulphation and DNA synthesis of embryonic chick cartilage. Separation by isoelectric focusing implied that the factor responsible for the glial cell stimulation was not identical to that active in cartilage.
Most animal cells in culture require serum for their survival and multiplication. Exponentially growing cells cease to proliferate when serum is removed. The proliferation restraint that occurs in dense cultures of normal cells (density-dependent inhibition of growth) is released by the addition of fresh serum (see [11] for review). The basis for the effect of serum on cell multiplication is unknown. Temin and collaborators have isolated polypeptides from calf serum [6] and from conditioned medium from rat liver cell cultures [1] that stimulate cell multiplication. These polypeptides also displayed somatomedin- or insulin-like properties, implying similarities within this group of substances [1, 6]. In the present investigation we have studied the multiplication stimulatory effect on human glia-like cells of a polypeptide Exptl Cell Res 96 (1975)
fraction isolated from human plasma. This fraction was previously shown to contain somatomedin (sulphation factor) activity, as indicated by its stimulating effect on polysaccharide biosynthesis in chick cartilage [12], human fibroblasts [14] or human glia-like cells [14]. MATERIALS AND METHODS Cell lines Human glia-like lines were initiated from brain biopsies as described previously [7, 8]. These cells have been characterized with regard to origin, morphology, growth pattern, contact inhibition, density-dependent inhibition (cf [8, 15, 16]). The cell lines were used during their rapid growth phase (phase II according to Hayflick) [5]. The cells were grown in Eagle's MEM supplemented with 10% baby calf serum and antibiotics (50 p.g/ml of streptomycin, 100 U/ml of penicillin, 1.25 /zg/ml of amphotericin B). Falcon plastic Petri dishes ( 0 50 ram) were employed. Cultures were incubated at 37~ in humidified air containing 5 % CO2.
Stimulation o f D N A synthesis by polypeptides
59
The cells were then dissolved in 1 ml/dish of 0.3 M sodium hydroxide. An aliquot of the lysate was transferred to scintillation vials and a dioxan-based cocktail containing Cab-O-Sil was added. The samples were counted at 10~ in an Intertechnique liquid scintillator. The radioactivity contained in the acid-insoluble material was taken as a measure of DNA synthesis.
300
200
Polypeptide fraction 100 0.5
I
2
3OO
0
200
100
A polypeptide fraction (mol.wt 4000-7000) was isolated from human plasma as described [12], An acid ethanol extract of Cohn fraction IV was subjected to gel chromatography on Sephadex G-75. Effluent fractions corresponding to the Kar values 0.6--0.8 were pooled and lyophilized. The somatomedin activity of the preparation was 0.08 U//zg of protein, estimated by its stimulatory activity on the incorporation of [35S]O4 into embryonic chick cartilage [3]. One U is defined as the somatomedin activity of I ml of a pooled reference serum [3]. The insulin content of the fraction was 0.1/zU//xg of protein [2].
. . . . 0 s 10 15
25
50
Fig. 1. Abscissa: (top) serum conc. (%); (bottom) tzg/ ml of PPF; ordinate: cpm (mean of duplicate cultures) as % of control (250 cpm). Stimulation of DNA synthesis of serum-deprived sparse cells by (top) calf serum and (bottom) PPF.
A s s e s s m e n t o f D N A synthesis in cell cultures after exposure to test samples Serum-deprived sparse cultures. Pooled cells were plated at low density, about 10000 cells/cmz, in Eagle's MEM 10% serum and grown for 24 h. Medium was then changed to 5 ml/dish of serumfree Ham's F-12 [4] containing 15 mM Hepes | (Sigma), pH 7.3. After another 24 h incubation, medium was again changed with F-12 Hepes and test samples were added to duplicate dishes in a small volume (20-100/~1/5 ml medium). Dense, stationary cultures. Pooled glial cells were seeded into dishes and incubated with biweekly medium renewal. Eagle's MEM with 10% calf serum was employed. After 2 weeks the cell cultures had reached a stationary phase [8]. The substances to be tested were then added to the old incubation medium 3 days after a medium change. Measurement of DNA synthesis. After incubation with test samples for 24 h of either sparse or dense cultures, [aH]thymidine was added (0.02 ~Ci/ml, final conc., spec. act. 2 Ci/mM). By this procedure, the isotope was present during the peak of DNA synthesis [17]. After incubation for another 24 h period, the dishes were washed twice with phosphate-buffered saline and extracted 3• rain with 5 ml of 5% trichloroacetic acid at +4~ After one wash in 5 ml of PBS, the cultures were incubated at room temperature for 5 min in ethanol-ether (3 : 1) and air-dried.
Isoelectric focusing Further separation of the lyophilized polypeptide fraction was performed by isoelectric focusing [9]. Portions of 100 mg were dissolved in 6 M urea-1% Ampholine (LKB, Stockholm, Sweden) and subjected to isoelectric focusing in beds of Sephadex G-75 (40• cm) containing 6 M urea and 1% Ampholine (pH 3-10) at +5~ for 24 h at 10 V/cm. At the end of the run, the bed was cut into I cm bands which were eluted with distilled water. Fractions were tested for growth-promoting activity on sparse cultures at a final conc. of I0 /zg of dry weight/ml.
Insulin A mixture of crystalline bovine and porcine insulin (Insulin Special | 40 U/ml was bought from Vitrum (Stockholm, Sweden). Crystalline human insulin was a gift from Novo Industri (Copenhagen, Denmark). It was directly dissolved in the growth medium at its final concentration.
RESULTS
Effect o f serum and polypeptide fraction (PPF) on serum-deprived sparse cells The
incorporation
of [3H]TdR
into acid-
insoluble material of serum-deprived after
exposure
to
calf
serum
and
cells poly-
p e p t i d e f r a c t i o n at v a r i o u s c o n c e n t r a t i o n s is r e c o r d e d curve
in fig.
obtained
1. T h e
dose-response
with cultures
exposed
to
Exptl CellRes 96 (1975)
60
Westermark, Wasteson and Uthne 200~
200
100 100
9
,
,
I
2
,,,,
/"
1135 I04
163 1(]2
1{~1,4
300
200
O
I0 20 100
o ~ To F5
2'5
sb
Fig. 2. Abscissa: (top) serum conc. (%); (bottom) tzg/ ml of PPF; ordinate: cpm (mean of duplicate cultures) as % of control (1200 cpm). Stimulation of DNA synthesis of density-inhibited cells by (top) calf serum and (bottom) PPF.
8,
,0.3
7, ,0.2
6 5 4
50
10"0
Fig. 4. Abscissa." (top) conc, of a mixture of bovine and porcine insulin (U/ml); (bottom) conc. of human insulin (p,U/ml); ordinate: cpm (mean of duplicate cultures) as % of control (340 cpm). Stimulation of sparse, serum-deprived cells by insulin.
calf serum followed an expected course with a rapid increase at low concentration and a plateau phase at higher concentrations of serum. The serum-deprived glial cells were also markedly stimulated by the polypeptide fraction. The corresponding dose-response curve had essentially the same course as that obtained when serum was used as a stimulator.
Effect o f serum and polypeptide fraction when added to the old medium o f dense cultures 150 Stationary, density-inhibited ceils 3 days after medium change were employed. The 10 20 30 samples to be tested were added in a low Fig. 3. Abscissa: fraction no.; ordinate: (top) Az6o volume to the spent growth medium, The (right) (0---0); pH (left) (O--O); (bottom) cpm plot of [3H]TdR incorporation vs the (mean of duplicate cultures) as % of control (275 cpm). respective concentrations appears in fig. 2. lsoelectric focusing of polypeptide fraction. Eluted fractions were analysed for absorbance at 260 nm, The incorporation of [3H]TdR was markedpH and thymidine incorporation in sparse cultures ly stimulated by serum and polypeptide of glial cells. Exptl Cell Res 96 (1975)
Stimulation of DNA synthesis by polypeptides
61
nificant stimulation occurred in the range of insulin concentrations studied.
0
100
165 16~ 163 T62 161.s Fig. 5. Abscissa: conc. of a mixture of bovine and porcine insulin (U/ml); ordinate: cpm (mean of dupli-
cate cultures) as % of control (1350 cpm). Effect of bovine and porcine insulin on densityinhibited cells.
fraction, respectively. It should be noted that in both cases, the dose-response curves were linear in the range of serum concentrations tested in contrast to those obtained with sparse cells (fig. 1). The level of maximal stimulation was not reached. It is also evident that, with serum as a standard, the specific stimulatory activity of PPF was higher on density-inhibited cells than on sparse, serum-deprived cells.
Isoelectric focusing of polypeptide fraction An attempt was made to purify the PPF by isoelectric focusing (fig. 3). As can be seen, growth-promoting activity was recovered in an acidic pH range (4.5-5.9).
Effect o f insulin Sparse cells. The effect of insulin on the DNA synthesis of sparse glial cells appears in fig. 4. The highest concentrations of bovine-porcine insulin was slightly stimulatory. Moderate doses of either bovine-porcine or human insulin failed to cause any stimulation. Dense cultures. The effect of mixtures of porcine and bovine insulin on densityinhibited cells is depicted in fig. 5. No sig-
DISCUSSION It appears that the polypeptide fraction from human plasma displayed not only somatomedin-like activity in different kinds of cells [12, 14] but was also a potent initiator of DNA synthesis in cells of human astrocytic origin. The fraction produced a more pronounced effect when it was used along with the spent growth medium on density-inhibited cells than when tested in a serum-free medium on sparse cells (figs 1, 2). This finding may indicate an interaction between separate stimulating factors present in serum. In some cell systems, insulin may act as a stimulator of DNA synthesis [10]. However, the possibility that the contaminating insulin was responsible for the observed enhancement on DNA synthesis in the present experiments, seems to be ruled out by the low response to insulin of sparse cells and the virtual absence of any response of density-inhibited cells. It should also be pointed out that the dosage levels of insulin at which glia-like cells were stimulated by far exceeded the low concentration of insulin in PPF. Further separation of the PPF by isoelectric focusing revealed an acidic nature of the active component. This finding discriminates the present glial factor from the cartilage sulphation factor which has an isoelectric point near neutral pH [13]. Thus, the glial factor described may be another representative of the group of cellstimulating polypeptides [1 1]. This work was supported by grants from the Swedish Medical Research Council (13X-2309, 13X-4486) and the Swedish Cancer Society (55-B73-09XC and 689B75-01X). Exptl Cell Res 96 (1975)
62
Westermark, Wasteson and Uthne REFERENCES
1. Dulak, N C & Temin, H M, J cell physiol 81 (1973) 153. 2. Hales, C N & Randle, P J, Biochemj 88 (1963) 137. 3. Hall, K, Acta endocrinol, suppl. 163 (1972). 4. Ham, R G, Proc natl acad sci US 53 (1965) 288. 5. Hayflick, L, Exp cell res 37 (1965) 614. 6. Pierson, R W & Temin, H M, J cell physiol 79 (1972) 319. 7. Pont6n, J & Maclntyre, E, Acta pathol microbiol Scand 74 (1968) 465. 8. Pont6n, J, Westermark, B & Hugosson, R, Exp cell res 58 (1969) 393. 9. Radola, B J, Biochim biophys acta 194 (1969) 335. 10. Temin, H M, J cell physiol 69 (1967) 377. 11. Temin, H M, Pierson, R W & Dulak, N C, Growth, nutrition and metabolism of cells in
Exptl Cell Res 96 (1975)
12. 13. 14. 15. 16. 17.
culture (ed H Rothblat & V J Cristofalo) vol. 1, p. 49. Academic Press, New York & London (1972). Uthne, K, Acta endocrinol, suppl. 175 (1973). Van Wyk, J J, Hall, K, Van den Brande, J L & Weaver, R P, J clin endocrinol metab 32 (1971) 389. Wasteson, /~, Uthne, K & Westermark, B, Bioc h e m j 136 (1973) 1069. Westermark, B, Exp cell res 69 (1971) 259. - - Abstracts of Uppsala dissertations in medicine, Acta Univ Upsaliensis, no. 164 (1973). Westermark, B & Wasteson, A, Adv metab disorders (ed R Luft & K Hall) vol. 8, p. 85. Academic Press, New York (1975).
Received February 17, 1975 Revised version received May 12, 1975
