Epidermal Growth Factor and the Control of Proliferation of Balb 3T3 and Benzo[alpyrene-transformed Balb 3T3 Cells KENNETH D. BROWN AND ROBERT W. HOLLEY Molecular Biology Laboratory, The Salk Institute, Post Office Box 1809, Sun Diego, CaZifornia 921 12
ABSTRACT Benzo[alpyrene-transformed Balb 3T3 cells (BP3T3) exhibit “normal” growth controls at low concentrations of serum. Epidermal growth factor (EGF) stimulates DNA synthesis and cell division in both Balb 3T3 and BP3T3 cells a t physiological concentrations. The growth response of BP3T3 cells to EGF is qualitatively the same as t h a t of 3T3 cells, however, the transformed cells have a lower quantitative requirement. Both 3T3 and BP3T3 cells show a density-dependent response to EGF, but the shift in the dose response curve for BP3T3 cells a t high cell density is smaller than that seen for 3T3 cells. One cause of the restricted growth of 3T3 cells at high cell density compared with BP3T3 cells is the increased concentration of growth factor needed for stimulation of 3T3 cells at higher cell densities. A lower rate of depletion of other growth factory by BP3T3 cells may also explain the smaller effect of cell density on the EGF response of these cells. The growth characteristics observed in benzo[alpyrene-transformed 3T3 cells (BP3T3) are superficially the same as the growth characteristics of virally transformed, SV3T3 cells. Both cell types have a low serum requirement. Both grow to high cell density in culture medium with 10% calf serum, both form colonies in Methocel, and both are tumorigenic (Holley et al., ’76).However, in contrast to the behavior of SV3T3 cells, the initiation of DNA synthesis in BP3T3 cells is still dependent on a serum factor. If BP3T3 cells are grown in medium containing a low concentration of serum, the cells become quiescent with growth arrested in the G, or Go phase of the cell cycle. The addition of serum or purified growth factor to such quiescent cells leads to the initiation of DNA synthesis and the resumption of growth (Holley et al., ’76). Thus BP3T3 cells exhibit “normal” growth controls a t low concentrations of serum. Since BP3T3 cells retain a requirement for a growth factor required by normal 3T3 cells, these transformed, tumorigenic cells can be used to explore the possible causes of a decrease in the quantitative requirement for a growth factor. For this purpose, we have chosen to use the epidermal growth factor (EGF) of &hen (‘62). EGF is present in serum J. CELL. PHYSIOL. (1979)100: 139-146.
(Byyny e t al., ’74) and is active as a growth factor for 3T3 and BP3T3 cells. EGF can be prepared in pure form and is well characterized physically and chemically (Taylor et al., ’72; Savage et al., ’73; Holladay e t al., ’76). This paper describes the effect of EGF on cell growth. A subsequent paper will describe the molecular interaction of the labeled growth factor with 3T3 and BP3T3 cells. MATERIALS AND METHODS
Cell cultures The original culture of benzo[alpyrenetransformed Balb 3T3 cells (BP3T3) was obtained from Doctor Joseph DiPaolo. Several clones of BP3T3 cells were derived from this stock. The clone used in the experiments described in this paper (BP3T3 clone 7) was the same as that used in a previous study (Holley et al., ’76). Normal Balb 3T3 cells were obtained from Doctor Marguerite Vogt. Cells were grown in Dulbecco modified Eagle’s medium (DME) (Morton, ’70) with 0.45%glucose Received Nov. 6, ’78. Accepted Feb. 13, ’79. I Abbreviations: BP3T3, benzolalpyene-transformedBalb 3T3 cells; SV3T3, Simian-virus-40-transformed Swiss 3T3 cells; EGF, epidermal growth factor. Present address: Tumor Cell Laboratory, ICRF Laboratories, P. 0. Box 123, Lincoln’s Inn Fields, London WC2A 3PX. England.
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and with 10%calf serum in a VO, incubator. Frozen stock cells were thawed a t approximately 10-week intervals.
Epidermal growth factor (EGF) and antiserum to EGF EGF was purified as described by Savage and Cohen ('72) from the submaxillary glands of adult male, Swiss Webster mice that had been injected daily (subcutaneously) with testosterone propionate (1 mg per animal) for nine days. Rabbits were immunized with unconjugated EGF emulsified in incomplete Freund's adjuvant (DIFCO).Each animal was injected at six sites (2 each subcutaneously, subscapularly, intramusculary) with a total of 250 Fg of antigen. The injections were repeated a t 4-week intervals. After the second injection the rabbits were bled weekly from an ear vein and the antiserum was stored a t - 20°C. The antiserum was partially purified by ammonium sulfate fractionation and ion exchange chromatography (Fahey, '67).
and Kiernan, ' 7 4 , except that nonradioactive thymidine was omitted from the labeling medium and the labeling period was 0 to 24 hours after the addition of EGF.
Measurement of cell growth Cells were seeded at low density (3,00050,000cells per 5-cm dish) in 5 ml of DME plus 10% calf serum. Sixteen hours later the medium was changed to DME containing serum and EGF a t the required concentration. The medium was renewed as described for the individual experiments. RESULTS
The initiation of DNA synthesis by EGF in 3T3 and BP3T3 cells The effect of EGF on the initiation of DNA synthesis was investigated by adding increasing concentrations of EGF to quiescent cells and measuring the incorporation of [3H1-thymidine from 16 to 24 hours later. EGF was found to be a potent stimulator of DNA synthesis in both 3T3 and BP3T3 cells. The reAssay for the initiation o f DNA synthesis sponse of 3T3 cells was markedly dependent in quiescent cells on the cell population density. For sparse culOne-hundred microliter aliquots of various tures (
ABSTRACT Benzo[alpyrene-transformed Balb 3T3 cells (BP3T3) exhibit “normal” growth controls at low concentrations of serum. Epidermal growth factor (EGF) stimulates DNA synthesis and cell division in both Balb 3T3 and BP3T3 cells a t physiological concentrations. The growth response of BP3T3 cells to EGF is qualitatively the same as t h a t of 3T3 cells, however, the transformed cells have a lower quantitative requirement. Both 3T3 and BP3T3 cells show a density-dependent response to EGF, but the shift in the dose response curve for BP3T3 cells a t high cell density is smaller than that seen for 3T3 cells. One cause of the restricted growth of 3T3 cells at high cell density compared with BP3T3 cells is the increased concentration of growth factor needed for stimulation of 3T3 cells at higher cell densities. A lower rate of depletion of other growth factory by BP3T3 cells may also explain the smaller effect of cell density on the EGF response of these cells. The growth characteristics observed in benzo[alpyrene-transformed 3T3 cells (BP3T3) are superficially the same as the growth characteristics of virally transformed, SV3T3 cells. Both cell types have a low serum requirement. Both grow to high cell density in culture medium with 10% calf serum, both form colonies in Methocel, and both are tumorigenic (Holley et al., ’76).However, in contrast to the behavior of SV3T3 cells, the initiation of DNA synthesis in BP3T3 cells is still dependent on a serum factor. If BP3T3 cells are grown in medium containing a low concentration of serum, the cells become quiescent with growth arrested in the G, or Go phase of the cell cycle. The addition of serum or purified growth factor to such quiescent cells leads to the initiation of DNA synthesis and the resumption of growth (Holley et al., ’76). Thus BP3T3 cells exhibit “normal” growth controls a t low concentrations of serum. Since BP3T3 cells retain a requirement for a growth factor required by normal 3T3 cells, these transformed, tumorigenic cells can be used to explore the possible causes of a decrease in the quantitative requirement for a growth factor. For this purpose, we have chosen to use the epidermal growth factor (EGF) of &hen (‘62). EGF is present in serum J. CELL. PHYSIOL. (1979)100: 139-146.
(Byyny e t al., ’74) and is active as a growth factor for 3T3 and BP3T3 cells. EGF can be prepared in pure form and is well characterized physically and chemically (Taylor et al., ’72; Savage et al., ’73; Holladay e t al., ’76). This paper describes the effect of EGF on cell growth. A subsequent paper will describe the molecular interaction of the labeled growth factor with 3T3 and BP3T3 cells. MATERIALS AND METHODS
Cell cultures The original culture of benzo[alpyrenetransformed Balb 3T3 cells (BP3T3) was obtained from Doctor Joseph DiPaolo. Several clones of BP3T3 cells were derived from this stock. The clone used in the experiments described in this paper (BP3T3 clone 7) was the same as that used in a previous study (Holley et al., ’76). Normal Balb 3T3 cells were obtained from Doctor Marguerite Vogt. Cells were grown in Dulbecco modified Eagle’s medium (DME) (Morton, ’70) with 0.45%glucose Received Nov. 6, ’78. Accepted Feb. 13, ’79. I Abbreviations: BP3T3, benzolalpyene-transformedBalb 3T3 cells; SV3T3, Simian-virus-40-transformed Swiss 3T3 cells; EGF, epidermal growth factor. Present address: Tumor Cell Laboratory, ICRF Laboratories, P. 0. Box 123, Lincoln’s Inn Fields, London WC2A 3PX. England.
139
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KENNETH D. BROWN AND ROBERT W. HOLLEY
and with 10%calf serum in a VO, incubator. Frozen stock cells were thawed a t approximately 10-week intervals.
Epidermal growth factor (EGF) and antiserum to EGF EGF was purified as described by Savage and Cohen ('72) from the submaxillary glands of adult male, Swiss Webster mice that had been injected daily (subcutaneously) with testosterone propionate (1 mg per animal) for nine days. Rabbits were immunized with unconjugated EGF emulsified in incomplete Freund's adjuvant (DIFCO).Each animal was injected at six sites (2 each subcutaneously, subscapularly, intramusculary) with a total of 250 Fg of antigen. The injections were repeated a t 4-week intervals. After the second injection the rabbits were bled weekly from an ear vein and the antiserum was stored a t - 20°C. The antiserum was partially purified by ammonium sulfate fractionation and ion exchange chromatography (Fahey, '67).
and Kiernan, ' 7 4 , except that nonradioactive thymidine was omitted from the labeling medium and the labeling period was 0 to 24 hours after the addition of EGF.
Measurement of cell growth Cells were seeded at low density (3,00050,000cells per 5-cm dish) in 5 ml of DME plus 10% calf serum. Sixteen hours later the medium was changed to DME containing serum and EGF a t the required concentration. The medium was renewed as described for the individual experiments. RESULTS
The initiation of DNA synthesis by EGF in 3T3 and BP3T3 cells The effect of EGF on the initiation of DNA synthesis was investigated by adding increasing concentrations of EGF to quiescent cells and measuring the incorporation of [3H1-thymidine from 16 to 24 hours later. EGF was found to be a potent stimulator of DNA synthesis in both 3T3 and BP3T3 cells. The reAssay for the initiation o f DNA synthesis sponse of 3T3 cells was markedly dependent in quiescent cells on the cell population density. For sparse culOne-hundred microliter aliquots of various tures (
