Polyamine Influences on the Prolactin Stimulation of Phosphoprotein Synthesis in Hydroxyurea Synchronized MCF-7 Human Mammary Epithelial Cells
Summary The actions of prolactin on the rate of synthesis of an isoelectrically precipitable (pH 4.6) phosphoprotein fraction of the MCF-7 human mammary epithelial cell line were determined in cells synchronized at the G i : S interphase of the cell cycle employing hydroxyurea in a serum-free defined medium. Cells not allowed to enter the S-phase of DNA replication, by maintaining hydroxyurea in the incubation medium, exhibited an increased rate of [ H ] leucine incorporation into the isoelectrically precipitable phosphoprotein fraction when exposed to prolactin and 1 — 5 mM spermidine. Cells released from the hydroxyurea induced synchrony exhibited an increased rate of [ 3H] leucine incorporation in response to prolactin when ornithine, putrescine, or spermidine were present. The polyamine spermine was ineffective in allowing prolactin's action on phosphoprotein synthesis. In synchronized cells released from the hydroxyurea block, prolactin was shown to effect an increased rate of phosphoprotein synthesis at the posttranscriptional G1 stage of the cell cycle. All prolactin responses were attained with physiological concentrations of the hormone. During and subsequent to the synchrony period with hydroxyurea, the presence or absence of insulin was found to be useful for the "staging" of the cell cycle to maintain cell synchrony and obtain prolactin effects on phosphoprotein synthesis. Key words MCF-7 Human Mammary Epithelial Cells — Hydroxyurea Synchronization — Polyamines — Prolactin — Phosphoprotein Synthesis
Introduction The effects of insulin, hydrocortisone, spermidine, and prolactin on the rate of [3H] leucine uptake and incorporation into phosphoproteins in MCF-7 cells have been reported (Linebaugh and Rillema 1984). These studies showed that the polyamine spermidine allows prolactin to manifest its
Horm.metab.Res.23(1991)414-422 © GeorgThieme Verlag Stuttgart-New York
effects on macromolecular synthesis in MCF-7 cells in a serum-free, defined medium. Spermidine, in addition to insulin and hydrocortisone, is essential for prolactin to manifest a stimulation of the rate of [ 3H] uridine incorporation into RNA, [ 3H] leucine incorporation into total cellular protein, and [ 3H] leucine incorporation into an isoelectrically precipitable phosphoprotein fraction. The prolactin responses were observed at physiological concentrations of the hormone. Specificity was established by demonstrating that bovine growth hormone had no effects on RNA or phosphoprotein synthesis in the MCF-7 cells. In more recent studies we have characterized the optimal conditions for synchrony and arrest of MCF-7 cells at the G 1 : S interphase of the cell cycle employing hydroxyurea alone, or in combination with insulin (Linebaugh and Rillema 1987). Employing the information acquired in earlier studies, the present investigations were performed to more closely define the stage of the cell cycle in which prolactin is expressing its action on [3H] leucine incorporation into phosphoproteins. In addition, the information concerning polyamine specificty and cell cycle progression through the S-phase and the resultant effects these factors have on the action of prolactin is further explored. Materials and Methods Materials Materials used in these studies were from the following sources: bovine insulin (lot 9SH35AE, 26.1 USP units/mg), penicillin and streptomycin from Eli Lilly (Indianapolis, IN); hydrocortisone from Pfizer Laboratories (New York, NY); spermidine trihydrochloride, ornithine hydrochloride, putrescine dihydrochloride, spermine tetrahydrochloride, and hydroxyurea from Sigma (St. Louis, MO); ovine prolactin (NIADDK-oPRL-16) from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases; Bacto-trypsin from Difco Labs (Detroit, MI); Eagle's minimal essential medium (Earle's salts), medium 199 with Earle's salts (EM199), Hank's balanced salts solution (HBSS), nonessential amino acids, L-glutamine, and newborn bovine serum from K. C. Biological (Lenexa, KS); tissue culture plates and flasks from Corning Glass (Corning, NY); [methyl-3H] thymidine (20.0 Ci/mmol), and L-[4,5(n)-3H] leucine (60.0 Ci/mmol) from New England Nuclear (Boston, MA). The MCF-7 human mammary epithelial cell line was obtained from the Michigan Cancer Foundation (Detroit, MI) and maintained in continuous culture.
Received: 8 Dec. 1989
Accepted: 11 March 1991 after revision
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B. E. Linebaugh and J. A. Rillema Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan, U. S. A.
Prolactin Stimulation of MCF-7 Cell Phosphoprotein
Horm. metab. Res. 23 (1991)
415
Table 1 Effects of insulin, hydrocortisone and spermidine on the uptake and incorporation of [3H] leucine into phosphoprotein in MCF-7 cells retained or released from theG1:S interphase hydroxyurea block. G1:S Block
Incorporation dpm [3H ] Leu/u.g DNA/60 min
M199 I IH ISpd IHSpd
Retained Retained Retained Retained Retained
30.8 ±2.7 53.5 ±3.0 66.4 ±6.0 72.6 ±2.0 82.5 ±2.6
250.9 ±13.2 273.9 ±8.8 369.5 ±15.0 347.7 ±5.9 427.8 ±10.7
157.4 ±7.8 171.4±5.2 165.8 ±5.5
M199 I IH ISpd IHSpd
Released Released Released Released Released
33.1 ±1.4 60.7 ±2.9 77.3 ±2.9 66.0 ±3.7 78.3 ±3.4
235.1 ±12.2
165.4 ±4.9 193.1 ±7.8 197.3 ±1.3 189.8 ±4.7 181.9 ±2.2
Uptake (acid soluble) dpm [3H] Leu/u.g DNA/60 min
295.2 ±11.6 321.8 ±9.8 342.6 ±16.5 412.2±6.1
DNA u.g/plate
156.8 ±1.9
160.9 ±4.3
Cell culture The MCF-7 cells were cultured in Eagle's minimal essential medium (E-MEM) supplemented with 2X nonessential amino acids, L-glutamine (292 mg/1), 10% newborn bovine serum (inactivated), penicillin (100 U/ml), streptomycin (100 ug/ml), and insulin (10~ M). Cultures in log growth were harvested with 0.25% trypsin in phosphate-buffered saline (pH = 7.4). The cells were seeded at a uniform density of 0.5-1 x 106/60 mm plate, unless otherwise indicated, in supplemented Eagle's minimal essential medium (Earle's salts) and incubated at 37 °C in a humidified atmosphere of 5% CC2/95% air. This medium was aspirated and replaced on day 2. On day 4, the medium was removed, the cells washed with 5 ml/plate Hank's balanced salts solution (37 °C, 30 min) and then incubated in 5 ml/plate serum and hormone-free medium 199 (EM 199) supplemented with 2X nonessential amino acids, L-glutamine (292 mg/ml), penicillin (100 U/ml) and streptomycin (100 |xg/ml) as previously described (Linebaugh and Rillema 1977). In initial studies, this period of time referred to as the "depletion period" was varied as was the concentration of hydroxyurea to determine the optimal conditions for synchrony and arrest at the G1: S interphase (Linebaugh and Rillema 1987).
Assay for rate of DNA synthesis Experiments concerned with the rate of DNA synthesis were carried out by first aspirating the 5 ml medium present during the 16 h depletion period. The cells were then washed with 5 ml supplemented serum and hormone-free EM 199 and refed with EM 199 containing 10- 6 M insulin (I), 10- 6 M hydrocortisone (H), 5 mM spermidine (Spd) and 1 ug/ml prolactin (PRL), as indicated. Incubation was continued (37 °C) for the specified time periods with [3H] thymidine (1 uCi/ml) present in EM 199 (3 ml/plate) for the last 60 min before termination of the incubations. After the 60 min pulse, the medium was aspirated, the cells washed with 5 ml/plate HBSS ( 0 4 °C) and 1.0 ml/plate 0.5N perchloric acid (PCA) was added. The cells were scraped from the plates, homogenized in 2.0 ml PCA, centrifuged (l000xg) for 5 min, and the radioactivity in 1.0 ml aliquots of the supernatants measured. The 0.5N PCA-insoluble fraction was washed with 5 ml 0.5N PCA (0-4 °C); the DNA was then extracted by resuspending the pellet in 3.0 ml 0.5N PCA, heating at 90 °C for 30 min, and centrifuging (l000xg) for 5 min. The solubilized DNA in a 1.0 ml aliquot was quantitated by the diphenylamine reaction procedure of Burton (1956). The specific activity of 3H in the DNA was cal-
culated after determining the radioactivity in a 1.0 ml aliquot of the DNA extract.
Assay for the rate of phosphoprotein syntheis Experiments concerned with hormonal influences on phosphoprotein synthesis were assessed by measuring the rate of cellular [3H]leucine uptake and incorporation into a pH 4.6 precipitable fraction of the cell (Juergens, Stockdale, Topper and Ettas 1965). After a terminal 60 min pulse-labelling period with [3H] leucine, the cells were washed with 5 ml/plate Hank's balanced salt solution (0— 4 °C), overlayed with 1.0 ml homogenization buffer [0.15M KC1/0.004M NaH2PO4-H 2O/0.01M imidazole (pH = 6.7)] and rapidly frozen (— 20 °C) in situ. The plates were thawed, cells scraped from the surface, the plates washed with 3.0 ml homogenization buffer, and the combined 4.0 ml divided into aliquots for the determination of 1) total DNA, 2)[3H] leucine present in the acid-soluble fraction, and 3) [3H]leucine incorporatioon into the phosphoprotein fraction. A 2.0 ml aliquot of the 4.0 ml total cell lysate was removed and mixed with 5.0 ml 0.5N PCA for determination of acid-soluble [ 3H] leucine uptake and total DNA content. Methods employed to measure DNA content and the acid-soluble radiolabel uptake are similar to the [3H ] thymidine incorporation experiments. The remaining 2 ml of the 4.0 ml lysate was centrifuged (l00xg, 5 min) and 1.0 ml aliquots of the supernates were removed for isoelectric precipitation as previously detailed (Linebaugh and Rillema 1984). The 3H in the pH 4.6 phosphoprotein precipitate was expressed as dpm of [3H]leucine incorporated into phosphoprotein per ug cellular DNA per 60 min. Numbers in the Tables and values in the Figure are mean ± SE of 5 observations. Statistical comparisons were made with Student's t-test when two mean differences were compared. Differences were considered significant with p < 0.05.
Results Effect of hydroxyurea block on cellular metabolism Table 1 depicts the 16 hour effects of insulin, hydrocortisone, and/or spermidine on the rates of [ 3H] leucine uptake and incorporation into phosphoproteins in MCF-7 cells that are released or retained at the G\ :S inter-
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MCF-7 cells were synchronized by exposure to 1 mM hydroxyurea in medium 199 for 16 h. Cultures were then refed with medium 199 containing 1 mM hydroxyurea(G1:S interphase block retained) or washed free of the hydroxyurea (G1 :S interphase block released) and refed with medium containing 10~6M insulin (I), 10~6M hydrocortisone (H), and 5 mM spermidine (Spd) as indicated. Incubation was continued (37 °C) for 16 h with a terminal inclusive pulse with [3H] leucine (1 uCi/ml medium 199, 3 ml/plate). Analysis of pH] leucine uptake and incorporation into phosphoprotein was performed as detailed in Materials and Methods. n = 5. a Greater than M199 (p < 0.05). "Greater than I (p < 0.05.
416 Horm. metab. Res. 23 (1991)
B. E. Linebaugh and J. A. Rillema
phase of the cell cycle with hydroxyurea. Shown are the concentrations of hydrocortisone and spermidine, when added with insulin, that were found to be optimal for enhancing the rate of [3H]leucine incorporation into phosphoproteins. The
following relevant observations were noted: a) insulin stimulates the rate of [3H]leucine uptake and the rate of incorporation of [3H]leucine into the phosphoprotein fraction, b) hydrocortisone and spermidine individually enhance insulin's action on phosphoprotein synthesis, and c) when hydrocortisone and spermidine are tested together, they have no greater effect on the insulin response than when each is combined individually with insulin. The efficacy of the hydroxyurea block is apparent from the unaltered DNA content of the cultures when the hydroxyurea block is retained. When the block was released, the DNA content was increased with all experimental combinations that included insulin. Prolactin effect on phosphoprotein
Fig. 1 Prolactin stimulation of the rate of [3H] leucine incorporation into phosphoprotein in MCF-7 cells blocked at the G1 :S interphase by hydroxyurea. MCF-7 cells were incubated in medium 199 containing 1 mM hydroxyurea for 16 h. At 0 h the medium 199 (containing 1 mM hydroxyurea) was replaced with medium 199 containing 1 mM hydroxyurea plus: 10- 6 M insulin (I), 10- 6 M hydrocortisone (H), 5 mM spermidine (Spd), and 1 (ug/ml prolactin (PRL) as indicated. Incubation was continued (37 °C) for 3,6,12,18 and 24 h with a terminal inclusive 60 min pulse with [3H] leucine in medium 199 (0.5 uCi/ml, 3 ml/plate). Analysis of [3H] leucine incorporation into phosphoprotein was performed as detailed in Materials and Methods. n = 5. - IHSpd; — IHSpd PRL.
In earlier studies we reported that spermidine is a required media constituent for prolactin to express an effect on the rate of [ 3H] leucine incorporation into phosphoproteins in MCF-7 cells (Linebaugh and Rillema 1984); these experiments were carried out with cells that were synchronized by serum and hormone depletion. One goal of the present studies was to determine if prolactin will stimulate 3H] leucine incorporation into phosphoproteins in MCF-7 cells that a) are maintained at the G1 :S interphase by treatment with hydroxyurea and b) are cultured with insulin, hydrocortisone and spermidine. Figure 1 shows the time-course for the prolactin response under these conditions. Prolactin significantly elevates the rate of [3H]leucine incorporation at 6 and 12 hours, compared to controls containing insulin, hydrocortisone and spermidine. The prolactin stimulation is no longer apparent at 24 hours after the 0 h medium change.
Table 2 Polyamine effects on the rate of [3H] leucine uptake and incorporation into phosphoprotein in hydroxyurea synchronized cells: G1 :S block retained vs released.
G 1:S Block M199
IH
IH Orn
IH Put
IH Spd
IHSpn
Incorporation dpm [3H] Leu/ug DNA/60 min
Uptake (acid sol) dpm [3H1 Leu/u.g DNA/60 min
Retained
Released
Retained
79.5 ±7.4
144.9a ±8.6
124.0 ±4.7
267.3a ±8.4
1427
1899
±21
±29
178.3 ±5.4
300.7 a ±6.5
1769
2339
±52
±82
170.1 ±6.5
287.6 a ±9.3
1998
2733
±54
±55
225.0 ±8.4
269.7 a ±10.9
2367
2821
±63
±41
178.7 ±3.1
174.4 ±15.0
2283
2471
±89
±11
DNA u.g/plate
Released
Retained
Released
1424
1867
±81
±84
41.5 ±0.7
41.1 ±1.1
67.1 ±2.0
78.3a ±0.7
63.5 ±1.6
71.8a ±1.3
55.5 ±1.1
65.9a ±0.7
49.8 ±1.6
68.0a ±0.8
48.2 ±1.3
65.6a ±1.6
MCF-7 cells were exposed to 1 mM hydroxyurea for 16 h. The hydroxyureaG1:S block was either retained or released at Oh and the cells refed with medium 199 containing 10- 6 M insulin (I), 10- 6 M hydrocortisone (H), 5 mM ornithine (Orn), 5 mM putrescine (Put), 5 mM spermidine (Spd), and 5 mM spermine (Spn) as indicated. Incubation was continued for 16 h with a terminal 60 min pulse with [3H] leucine (2 uCi/ml medium 199,3 ml/plate). Analysis of [3H] leucine uptake and incorporation into phosphoprotein was performed as described in Materials and Methods. n = 5. aGreater than G1 :S retained (p < 0.05).
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synthesis
Horm. metab. Res. 23 (1991) 417
Prolactin Stimulation ofMCF-7 Cell Phosphoprotein
Table 3 Polyamine specificity on the influence of prolactin's stimulation of [3H]leucine uptake and incorporation into MCF-7 cell phosphoprotein: comparison of hydroxyurea G1 :S interphase block release vs maintenance. Incorporation dpm [3H] Leu/ug DNA/60 min
Uptake (Acid Soluble) dpm [3H] Leu/ug DNA/60 min
DNA ug/plate
Hydroxyurea G1:S Interphase Block Released IHSpd IH Spd PRL
91.0 ±4.6 120.2 ±6.3
234.6 ±24.9
175.2 ±2.7 153.1 ±12.6
IHOrn IH Orn PRL
94.1 ±3.4 108.7 ±4.6
212.8 ±6.4 253.5 ±15.5
173.1 ±3.6 149.3 ±4.9
IH Put IH Put PRL
81.4 ±3.9 105.8 ±3.8
211.5 ± 11.8
183.8 ±6.3
259.0 ±8.8
152.9 ±2.2
IHSpn IHSpnPRL
57.5 ±3.6 55.9 ±1.7
206.9 ±7.3 203.7 ±10.5
177.9 ±6.3 177.6 ±3.2
117.8±7.1 122.2 ±5.2
188.8 ±7.4
IHSpd IH Spd PRL
113.1 ±3.7 133.3 ±6.8
333.1 ±15.0 341.1 ±16.9
IHOrn IH Orn PRL
156.0 ±7.6 146.6 ±12.2
413.3 + 34.4
99.5 ±4.6
358.7 ±23.8
107.5 ±6.9
IHPut IH Put PRL
137.7 ±8.5 130.8 ±4.3
364.9 ±11.7 378.5 ±12.3
106.6 ±5.3 112.2 ±4.2
IH Spn IH Spn PRL
76.9 ±4.6 82.0 ±3.6
316.0 ±28.1 324.8 ±12.8
123.4 ±7.0 115.5 + 6.1
MCF-7 cells were exposed to 1 mM hydroxyurea for 16 h. TheG1:S interphase block was either released or retained and the cells reincubated for 16 h with medium 199 containing 10- 6 M hydrocortisone (H), 5 mM polyamines spermidine (Spd), ornithine (Orn), putrescine (Put) and spermine (Spn), Prolactin (PRL) was present at 1 ug/ml. A terminal 60 minute pulse employing [3H] leucine at 0.5 uCi/ml medium 199,3 ml/plate was used to label the cells. Analysis of [3H ] leucine incorporation and uptake was performed as described in Materials and Methods. n = 5. a Greater than control (p < 0.05).
pared to cells treated only with insulin and hydrocortisone. Spermidine was the most effective. When the G1: S interphase block is released, cells incubated with 5 mM polyamines added to the medium (except for spermine), exhibited a rate of Table 2 shows the effects of ornithine, [3H] leucine incorporation into phosphoprotein equal to or putrescine, spermidine and spermine on the rates of [ 3H] greater than that in insulin and hydrocortisone treated cells. In leucine uptake and incorporation into MCF-7 cell phospho- this instance, spermine was ineffective. protein in cultures synchronized with 1 mM hydroxyurea and subsequently retained or released from the G1: S interphase Polyamine specificity: block. The data show that in all instances, except for cells ininfluence on prolactin stimulation cubated with spermine, release from the hydroxyurea interof phosphoprotein synthesis phase block results in a greater rate of [3H] leucine incorporation into phosphoproteins when compared to the counterpart The specificity of the polyamine influence on cultures maintained at the Gi: S interphase with hydroxyurea. the prolactin stimulation of [3H]leucine uptake and incorporaIn addition, the total DNA accumulated in cells released from tion into phosphoproteins was determined in cells synchroGi: S interphase block was increased over the comparable nized with hydroxyurea (Table 3). The data indicate that when cells retained at the G1: S interphase. These effects, attribu- the G1: S interphase block is released, prolactin stimulates the table to insulin, are consistent with those presented in Table 1 rate of [3H]leucine incorporation when the cells were cultured and also those reported earlier (Linebaugh and Rillema 1987). with putrescine, ornithine, or spermidine but not with sperThe combined effects of insulin, hydrocortisone and sper- mine. When spermine was present, the basal [3H]leucine inmidine, to increase the rate of [3H]leucine incorporation into corporation rate was lower than when the other polymines phosphoprotein in cells retained at the G1: S interphase with were present. This agrees with the data presented in Table 2. hydroxyurea, are also consistent with the results shown in When cells were retained at the G1: S interphase by maintainTable 1. When the Gi :S interphase block is retained, all the ing 1 mM hydroxyurea in the culture medium, prolactin stimupolyamines (5 mM) caused an added increase in the rate of lated the rate of [3H]leucine incorporation into the phospho[ H] leucine incorporation into phosphoproteins, when com- protein fraction only when spermidine was present during the
Effect of retention or release of G1:S interphase block on phosphoprotein synthesis: specificity ofpolyamine influence
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Hydroxyurea G1 :S Interphase Block Rets
Horm. metab. Res. 23 (1991)
B. E. Linebaugh and J. A. Rillema
Table 4 Time-course of prolactin action on [3H] leucine uptake and incorporatiion into phosphoprotein in synchronized MCF-7 cells at varying culture densities. Time (h) 0
12
18
Plating Density # Cells x 106/plate
Incorporation dpm [3H] leucine/ng DNA/60 min
Uptake (Acid Soluble) dpm [3H] leucine/ug DNA/60 min
DNA ug/plate
158.2 ±17.1 166.8 ±5.6 184.6 ±13.8
2221 ±265.3 1940 ±24.0 3626 ±462.8
75.1 ±7.3 58.9 ±3.7 14.6 ±1.3
IH Orn IH Orn PRL
186.2 ±5.7 189.0 ±7.6
1412 ±56.5 1425 ±40.3
140.6 ±4.3
0.5
IH Orn IH Orn PRL
232.3 ±11.0 226.5 ±15.0
1568 ±104.1 1471 ±83.8
87.4 ±3.9 85.6 ±4.9
0.1
IH Orn IH Orn PRL
214.3 ±5.9 200.1 ± 16.8
2192 ±159.7 2024 ±64.8
26.4 ±2.1 26.4 ±0.7
IHOrn IH Orn PRL
127.7 ±8.8 103.1 ±5.9
1716±81.7 1796 ±62.7
150.6 ±7.7 147.4 ±2.9
0.5
IHOrn IH Orn PRL
263.3 ±23.5 239.9 ±16.2
2218 ±212.5 2253 ±139.3
85.5 ±7.8 83.1 ±5.4
0.1
IHOrn IH Orn PRL
233.3 ±10.7 258.9 ±14.0
3007 ±199.0 3618 ±374.8
28.1 ±1.2 22.2 ±1.9
IHOrn IH Orn PRL
118.2 ±9.6 125.3 ±3.9
2269 ±52.3 2224 ±78.0
132.6 ±5.3
0.5
IHOrn IH Orn PRL
268.0 ±12.0 311.4 ±14.6
2452± 181.3 2585 ±63.2
88.9 ±6.3 81.9±1.7
0.1
IHOrn IH Orn PRL
349.3 ±21.0 496.9 ±49.5
3493 ±144.5 3587 ±426.7
25.7 ±2.1 25.7 ±2.1
1 0.5 0.1
135.7 ±2.1
139.7 ±0.9
MCF-7 cells plated at varying density were synchronized with 1 mM hydroxyurea, released from the G1: S interphase at Oh, refed with medium 199 containing 10 M insulin (I), 10 M hydrocortisone (H), 5 mM ornithine (Orn) and 1 ug/ml prolactin (PRL), reincubated (37 °C) for 6,12 and 18 h, and pulsed with [3H] leucine (2 u,Ci/ml, 3 ml medium 199/plate) for the terminal 60 min. Analysis of [3H leucine uptake and incorporation into phosphoprotein was performed as explained in Materials and Methods. n = 5. a Greater than I, H, Orn (p < 0.05).
16 h incubation. Ornithine and putrescine were ineffective as positive effectors of prolactin's action under the condition of G1: S interphase maintenance with 1 mM hydroxyurea. Effect of cell density on prolactin course of cellular phosphoprotein
timesynthesis
Table 4 presents the time-course of prolactin's action on [ 3H] leucine incorporation into phosphoproteins in MCF-7 cells initially plated at varying specific cell densities. The hydroxyurea was washed out of the cultures at Oh, and the cells refed with 1 0 _ 6 M insulin, 1 0 - 6 M hydrocortisone, 5 mM ornithine, ± prolactin (1 ug/ml). The net DNA accumulation (ug/plate) at Oh reflects the initial plating density variations after four days in complete growth medium (minimal essential medium with 10% serum) and in those synchronized at the G1: S interphase with exposure to 1 mM hydroxyurea for 16 h. The net amount of DNA nearly doubles 6 — 12 h after the block is released and the synchronized cells complete one S-phase of DNA replication {Linebaugh and Rillema 1987). Only at 18 h and at the lower initial plating densities (0.1 and 0.5 x 10 cells/60 mm dish) did prolactin treated cells (IHOrnPrl) exhibit an enhanced rate of [ 3H] leucine incorporation into phosphoprotein compared to control cells (IHOrn).
The effects of the polyamines ornithine and spermidine, with and without prolactin, on the rate of [3H] thymidine uptake and incorporation into DNA were also evaluated in MCF-7 cells seeded at varying initial plating densities. The results (not presented) indicated that neither the polyamines nor prolactin had any effect on the rate of [ 3H] thymidine uptake, [ 3H] thymidine incorporated into DNA, or total DNA accumulated when assayed every 3 h for 24 h. Effect of prolactin concentration on stimulation of phosphoprotein synthesis Table 5 shows the effect of various prolactin concentrations on the stimulation of the rate of [3H] leucine incorporation into phosphoproteins in synchronized MCF-7 cells seeded at a dilute initial plating density (0.5 x 106 cells/60 mm dish). The total DNA accumulated at the end of the incubation period was appropriate for the cell density initially plated and comparable to earlier experiments (Table 4). Prolactin is effective at concentrations as low as 10 pg/ml and appears to be most effective at the more dilute concentrations. At prolactin concentrations of 10 (ug/ml or greater, leucine incorporation into the phosphoprotein fraction was inhibited (data not included). Explanations for this observation may in-
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418
Horm. metab. Res. 23 (1991) 419
Prolactin Stimulation ofMCF-7 Cell Phosphoprotein
Prolactin Conc. 0 1 100 10 1 100 10
ug/ml ng/ml ng/ml ng/ml pg/ml pg/ml
Incorporation dpm[ 3 H] leucine/ ug DNA/60 min 102.4±3.3 125.7 ±6.1 127.4 ±5.6 136.3 ±4.6 153.5 ±7.4 159.8 ±9.8 165.7 ±2.9
clude the presence of contaminent(s) in the prolactin preparation; however, there exists no evidence of such that we are aware of.
Uptake (Acid Sol) dpm [3H]leucine/ ug DNA/60 min 1145± 10 1343±56 1410±32 1529 ±37 1608 ±40 1645 ±87 1545 ±42
a a a a a a
a a a a a a
MCF-7 cells (plated at 0.5 x 10 /cells/60 mm plate) were exposed to 1 mM hydroxyurea for 16 h. TheG1:S hydroxyurea block was removed by washing with medium 199 (5 ml/plate) and the cells refed with medium 199 containing 10 _6 M insulin , 10 - 6 M hydrocortisone, 5 mM spermidine and prolactin at the concentrations indicated. Incubation was continued (37 °C) for 18 h with a 60 min terminal pulse with [3H] leucine (2 uCi/ml, 3 ml medium 199/plate). Analysis of [3H] leucine uptake and incorporation into phosphoprotein was performed as detailed in Materials and Methods. n = 5. a Greater than 0 prolactin (p < 0.05).
Insulin influence on prolactin action in hydroxyurea synchronized cells The experiments in Table 6 were designed to determine the influence of insulin on the prolactin action in hydroxyurea synchronized MCF-7 cells. The cells (seeded at a uniform low density of 0.5 x 10 760 mm dish) were synchronized by exposure to 1 mM hydroxyurea during the 16 h synchrony (depletion) period. In some cases the cells were also exposed to 10- 6 M insulin during the depletion period. At Oh all cells were released from the G1: S interphase block and cultured for 18 h in the presence of hormones and polyamines as indicated. The accumulated DNA (ug/plate) of all groups is indicative of the low initial plating density. In cells exposed to 10- 6 M insulin and 1 mM hydroxyurea during the 16 h synchrony period and having no fresh insulin added after the G1:S block release (Oh), the low amount of accumulated DNA and the narrow range difference among treatment
Table 6 The influence of insulin on the prolactin stimulation of [ 3H] leucine uptake and incorporation into phosphoprotein of hydroxyurea synchronized MCF-7 cells: A) Insulin presence vs absence during 16 h hydroxyurea synchrony period (ii vs i) B) Insulin presence vs absence afterG1:S hydroxyurea block release (ii vs iii). Medium 199+ (18 h incubation)
IH IHSpd IH Spd PRL IH Orn IH Orn PRL IHPut IH Put PRL
IH IHSpd IH Spd PRL IH Orn IH Orn PRL IH Put IH Put PRL
-H -HSpd - H Spd PRL -HOrn - H Orn PRL -HPut -HPutPRL
Incorporation dpm [3H] leucine/ug DNA/60 min
Uptake (Acid Soluble) dpm [3H] leucine/ug DNA/60 min
i Hydroxyurea (1 mM) during 16 h Synchrony Period 34.4 ±1.8 889 ±53 42.1 ±2.4 1089 ±56 50.8 ±1.8 1222 ±49 56.6 ±2.4 a 1280 ±50 53.4±2.7 1281 ±67 65.8 ±3.0 a 1355 ±53
50.9 ±3.0 60.9±1.5
a
1199 ±39 1199±28
Insulin (10- 6 M) and Hydroxyurea (1 mM) during 16 h Synchrony Period 42.8 ±2.9 1024±81 50.1 ±1.8 1391 ±87 1247 ±64 48.4 ±0.6 1201 ±84 57.5 ±3.4
41.6 ± 1.0 58.3 ±1.3 40.6 ±1.0 49.5 ±1.1
1174 ±46 1299 ±20 1048 ±68 1225 ±69
Insulin (10 M) and Hydroxyurea (1 mM) during 16 h Synchrony Period. No Fresh Insulin Added after Hydroxyurea Block Release (Oh) 44.8 ±2.8 1660 ±119 43.7 ±1.0 1588 ± 4 2 47.3 ±2.2 1458 ± 5 0 64.6 ±2.1 1522 ±56 1509 ± 6 2 44.3 ±1.8 1532 ± 4 9 64.0 ±2.4 1375 ± 4 6 43.4 ±1.7 1343 ±53 63.1 ±1.9
DNA ug/plate
64.4 ±2.5 58.2 ±2.9 50.2 ±1.6 51.1 ±1.9 47.9 ±2.5 52.6 ±1.5 50.7 ±1.5 53.6 ±1.0
52.1 ±3.1 49.9 ±1.9 54.9 ±2.5 57.4 ±3.7 60.0 ±0.9 54.3 ±1.9 60.5 ±3.5 55.0 ±2.4
37.5 ±2.3 42.3 ±1.8 38.6 ±1.9 42.2 ±1.0 41.1 ± 1.8 42.3 ±0.7 41.5 ± 1.2
40.6 ±0.9
MCF-7 cells were synchronized atG1:S interphase by 16 h exposure to 1 mM hydroxyurea with insulin (10-6M) absent or present as indicated. At Oh cells were washed (releasingG1:S interphase hydroxyurea block) refed with medium 199+ 10 M insulin (I), 10~6 M hydrocortisone (H), 5 mM polyamines ornithine (Orn), putrescine (Put), and spermidine (Spd), with/without 0.1 jig/ml prolactin (PRL). Incubation was continued for 18 h with a terminal 60 min inclusive pulse with 1 uCi/ml[ 3 H] leucine (3 ml/plate). Analysis of [3H] leucine uptake and incorporation into phosphoprotein was performed as detailed in Materials and Methods, n = a Greater than control group lacking prolactin (p < 0.05).
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Table 5 Stimulation of [3H]leucine uptake and incorporation into phosphoprotein of hydroxyurea synchronized MCF-7 cells by dilute prolactin concentrations.
Horm. metab. Res. 23 (1991) groups, is indicative of the maintenance of a great degree of synchrony and also the lack of cell entry into the insulin induced second S-phase of DNA synthesis (Linebaugh and Rillema 1987). When no fresh insulin is added in the medium (Table 6, iii) after the hydroxyurea induced g1: S block is released, the increased rate of [ 3H] leucine incorporation into phosphoprotein in response to prolactin is proportionately greater than when insulin is present (Table 6, ii) in the final 18 h culture period. Discussion That the polyamine biosynthetic pathway is associated with cell division is apparent from the observations that a) the polyamines are involved in the regulation of cell growth (Janne, Poso and Raina 1978; Tabor and Tabor 1976), and b) there is an increased rate of polyamine-synthesis associated with the action of growth promoting factors such as pituitary growth hormone (Russell 1980), prolactin (Richards, Beer, Bourgeault, Chen and Gout 1982), TPA (Jetten, Ganong, Vanderbark, Shirley and Bell 1985), and estrogen (Cohen, O'Malley and Stastny 1970; Russell and Taylor 1971). In a study on the role of polyamines in Hela Cell DNA replication, Gallo, Koza and Herbst (1986) studied the role of the polyamines in S-phase DNA synthesis. Employing cells that were depleted of polyamines by inhibition with the ornithine decarboxylase inhibitor a-difluomethylornithine (DFMO), it was shown that DNA replication was inhibited. The inhibitory effect of D F M O was reversed by the provision of spermidine at least 10 h before the initiation of the S-phase of the cell cycle. Moreover, failure of polyamine supplements, added later in the synchronization schedule, to initiate DNA synthesis at the same time as control cells, suggested a polyamine function in the "staging" of the cell cycle. The DFMO induced deficiency of polyamines delayed the traversal of G1 cells through Sphase, but this delay is also avoided by the addition of exogenous polyamine (spermidine) at least 10 h before S-phase initiation. The actions of several mitogenic agents on the stimulation of the proliferation of human bronchial epithelial cells (Willey, Laveck, McClendon and Lechner 1985) were also shown to involve the activation of ornithine decarboxylase (ODC); these studies were carried out with a serum-free medium. In these studies it was demonstrated that the activation of ODC, the G1 specific, rate-limiting enzyme of polyamine synthesis, (Heby, Gray, Lindl, Morton and Wilson 1976), is necessary for the stimulation of cell division. The activity of L-omithine decarboxylase in Chinese Hamster ovary cells growing in a defined serum-free medium was found to be dependent on variations in exogenous ornithine concentrations (Sertich, Glass, Fuller and Gerner 1986). These variations lead to changes in ODC activity and intracellular putrescine and spermidine concentrations with ODC activity increasing most dramatically when the concentrations of end-products were minimal. The ODC activity was found to vary as a function of cell density. In contrast to the changes occurring in ODC activity, both putrescine and spermidine-dependent S-adenosyl-L-methionine decarboxylase (SAMD) activities were unchanged in cells growing in a defined medium. The levels of intracellular putrescine did not increase in cells maintained in defined medium even though
B. E. Linebaugh and J. A. Rillema ODC activities were much higher than in cells growing in serum supplemented medium. Intracellular spermidine was significantly lower in cells growing in defined medium compared to those maintained in serum supplemented medium, thus confirming the earlier findings of Melvin and Keir (1977). Intracellular spermine levels on the other hand, were unaffected by medium supplements. One of the major differences between defined medium and medium supplemented with serum is that the defined medium lacks ornithine. Although variable between serum lots the concentration of ornithine in serum has been estimated to be about 5 x 1 0 - 6 M (Baetz, Hubbert and Graham 1975). In studies employing defined media (Sertich, Glass, Fuller and Gerner 1986; Linebaugh and Rillema 1984) the cells can only generate ornithine from arginine, which is a component of medium 199. Apparently the cells cultured in defined media are incapable of ornithine synthesis in sufficient quantity to maintain adequate levels of ornithine, putrescine, and spermidine. It is also possible that the polyamines are released from the cells into the culture medium at a rate that does not allow the intracellular accumulation of significant quantities of the polyamines. In any event, it is essential to add the polyamines or ornithine to defined media in order for prolactin to stimulate phosphoprotein synthesis in the MCF-7 cells. The fact that 1—5 mM polyamines is a reasonable concentration to add to the culture medium is inferred from earlier studies in which the intracellular spermidine concentration of mouse mammary cells was determined to be 1—5 mM (Rillema, Linebaugh and Mulder 1977). In recent studies on the insulin stimulation of DNA synthesis in mouse lens epithelial cells, 3T3 cells (Reid and Reid 1987) and MCF-7 cells (Linebaugh and Rillema 1987), it was shown that insulin must be continuously present in the medium bathing the cells in order to stimulate progress from G1/G 0 to the S-phase of the cell cycle. Previous to Sphase, the effect of insulin and the presumed signal produced in the cell is lost when insulin is removed; this causes cells to reset their cycle and upon addition of insulin, the cells must pass through the entire G1 phase of the cell cycle before reaching the S-phase (Reid and Reid 1987). When insulin was removed from the bathing medium and the MCF-7 cells were ready to enter the S-phase in synchrony (blocked by hydroxyurea at the G1: S interphase), the cells progress through one Sphase of DNA synthesis. They remain at a posttranscriptional stage of the cell cycle, where differentiative actions of insulin were shown to occur (Linebaugh and Rillema 1987). At approximately the same posttranscriptional period in the cell cycle, prolactin was shown to increase the rate of synthesis phosphoproteins in the MCF-7 cell (Linebaugh and Rillema 1984). In an attempt to study the mechanism(s) of prolactin's actions(s), efforts have been made to standardize the variable conditions of cell density, synchrony time periods, synchrony medium constituents, incubation times after G1: S interphase block release at Oh, and constituents of the defined incubation medium. Results of these experiments indicate that when hydroxyurea synchronized MCF-7 cells are released from the G1: S interphase block and refed with medium containing insulin, hydrocortisone, prolactin, and one of the polyamines (ornithine, putrescine, or spermidine), prolactin
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Prolactin Stimulation of MCF-7 Cell Phosphoprotein
When hydroxyurea synchronized MCF-7 cells are not released from the G 1 : S interphase block prolactin was shown to be effective in stimulating the rate of [3H]leucine incorporation into phosphoprotein only when the polyamine spermidine was present. Under these conditions neither ornithine nor other polyamines allowed the prolactin response. In the G 1 : S blocked cells the time-course of the prolactin stimulation of [3H]leucine incorporation into phosphoprotein involves an initially detectable increase at 6 h, and a maximum increase by 12 h; at 24 h the effect is no longer apparent. In cells maintained at the G 1 : S interphase, the prolactin effects are earlier, last longer, and the magnitude of response is greater than in cells allowed to proceed through the cell cycle. The specificity of the polyamines as effectors of prolactin's action was demonstrated by showing that ornithine, putrescine, and spermidine allowed prolactin to effect an increased rate of synthesis of phosphoproteins in cells in which the hydroxyurea block was released. Spermine, was shown not to be a positive effector for prolactin's action on phosphoprotein synthesis. At concentrations of 1—5 mM, spermine significantly reduced the basal rate of [ 3 H ] leucine incorporation into phosphoproteins. The effect of MCF-7 culture density on the prolactin stimulation of the rate of [3H]leucine incorporation was determined with ornithine added to the bathing medium. The activation of ODC (G1 specific rate-limiting enzyme of polyamine synthesis) and the resultant changes in intracellular putrescine and spermidine levels have been found to vary as a function of cell density in other cell systems (Sertich et al. 1986). The function of MCF-7 cell density on ODC activation appears to also be a post-transcriptional event that deserves more study. The effect of prolactin on phosphoprotein synthesis was studied in cells in which insulin was present (Table 6, ii) or absent (Table 6, i) during the synchrony culture period and/or during the culture period subsequent to hydroxyurea synchronization. Results of these experiments (Table 6) indicated that cells exposed to insulin (10-6 M) during the synchrony period (ii), but not after the hydroxyurea G1: S interphase block was released (iii), exhibited a prolactin stimulation of phosphoprotein synthesis 18 hr after the block was released. This corresponds with the post-transcriptional G1 stage of the cell cycle. Evidently the insulin bound to the cell membranes, its signal generated, or both are sufficient to carry the cell through the S-phase into G1; this apparently allows prolactin to express its action when any of the polyamines spermidine, ornithine, or putrescine are provided. The maintenance of cell synchrony and lack of entry into a second Sphase of D N A synthesis is apparent (Table 6) when insulin is not present (iii) in the incubation medium after the hydroxyurea block is released. The results of these studies indicate that the polyamines and the "staging" of the cell cycle are essential variables that must be controlled in detecting prolactin's
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stimulation of phosphoprotein synthesis in MCF-7 cells. Prolactin does not appear to effect changes in the rate of phosphoprotein synthesis during the S-phase of the cell cycle. The changes that are effected are periodic and most likely occur during the posttranscriptional G1 period of the cell cycle. Also, spermidine appears to be the specific polyamine component essential for prolactin to effect its action on the rate of phosphoprotein synthesis. Insulin is necessary for the growth of MCF-7 cells, specifically for the traversal of the G1 period of the cell cycle. Insulin along with hydrocortisone, and the polyamine spermidine were previously shown to be essential for prolactin to express its action on phosphoprotein synthesis in the MCF-7 cells (Linebaugh and Rillema 1984). The use of insulin during the synchrony period and its removal at the same time that the hydroxyurea induced G1: S interphase block is released, has been shown in these studies to be a useful device for "staging" of the cell cycle, maintaining cell synchrony, and obtaining a prolactin effect on phosphoprotein synthesis. Acknowledgements This investigation was supported by grant number HD 06571 from the NICHHD. References Baetz, A. L., W. T. Hubbert, C. K. Graham: Developmental changes of free amino acids in bovine fetal fluids with gestational age and the interrelationships between the amino acid concentrations in the fluid compartments. J. Reprod. Fertil. 44:437-477 (1975) Burton, K. A.: A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62:315—323 (1956) Cohen, S., B. W. O'Malley, M. Stastny: Estrogenic induction of ornithine decarboxylase in vivo and in vitro. Science 170: 336—338 (1970) Gallo, C. J., R. A. Koza, E. J. Herbst: Polyamines and Hela-cell DNA replication. Biochem. J. 238:37-42(1986) Heby, O., J. W. Gray, P. A. Lindl, L. J. Marton, C. B. Wilson: Changes in L-ornithine decarboxylase activity during the cell cycle. Biochem. Biophys. res. Commun. 71:99-105 (1976) Janne, J., H. Poso, A. Raina: Polyamines in rapid growth and cancer. Biochim.Biophys.Acta473-241-293(1978) Jetten, A. M., B. R. Ganong, G. R. Vanderbark, J. E. Shirley, R. M. Bell: Role of protein kinase C in diacylglycerol-mediated induction of ornithine decarboxylase and reduction of epidermal growth factor binding. Proc. Nat. Acad. Sci. U. S. A. 82:1941-1945 (1985) Juergens, W. G., F. E. Stockdale, Y. J. Topper, J, J. Elias: Hormone-dependent differentiation of mammary gland in vitro. Proc. Nat. Acad.Sci.U. S. A.54629-634(1965) Linebaugh, B. E., J. A. Rillema: Hydrocortisone enhancement of insulin's action on macromolecular synthesis in MCF-7 cells. Mol. Cell. Endocrinol. 7:335-343 (1977) Linebaugh, B. E., J. A. Rillema: Polyamine requirement for prolactin action on macromolecular synthesis in the MCF-7 human mammary epithelial cell line. Biochim. Biophys. Acta 804: 348-355 (1984) Linebaugh, B. E., J. A. Rillema: Actions of insulin on MCF-7 cells that are synchronized with hydroxyurea. Mol. Cell. Endocrinol. 52: 227-233(1987) Mehin, M. A., H. M. Keir: Release of polyamines from growing and serum-deprived BHK-21/C-13 cells. Biochem. Soc. Lond. Trans. 5:711-712(1977) Reid, T. W., W. A. Reid: The labile nature of the insulin signal(s) for the stimulation of DNA synthesis in mouse lens epithelial and 3T3 cells. J. Biol. Chem. 262:229-233 (1987)
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stimulates the rate of [ 3H] leucine incorporation into the isoelectrically precipitable phosphoprotein fraction. This prolactin effect becomes significant between 12—24 h, with an optimal response occurring between 16 - 1 8 h.
Horm. metab. Res. 23 (1991)
Horm. metab. Res. 23 (1991)
B. E. Linebaugh and J. A. Rillema
Richards, J. F., C. T. Beer, C. Bourgeault, K. Chen, P. W. Gout: Bio- Willey, J. C, M. A. Laveck, I. A. McClendon, J. F. Lechner: Relationchemical response of lymphoma cells to mitogenic stimulation by ship of ornithine decarboxylase activity and cAMP metabolism to prolactin. Mol. Cell. Endocrinol. 265:41-49 (1982) proliferation of normal human bronchial epithelial cells. J. Cell. Physiol. 124:207-212(1985) Rillema, J. A., B. E. Linebaugh, J. A. Mulder: Regulation of casein synthesis by polyamines in mammary gland explants of mice. Endocrinology 100: 529-536 (1977) Russell, D. H.: Ornithine decarboxylase as a biological and pharmacological tool. Pharmacology 20:117-129 (1980) Requests for reprints should be addressed to: Russell, D. H., R. L. Taylor: Polyamine synthesis and accumulation in Dr. James A. Rillema the castrated rat uterus after estradiol-17B stimulation. Endocrinology 88; 1397-1403(1971) Department of Physiology Sertich, G. J., J. R. Glass, D. J. M. Fuller, E. W. Gerner. Altered poly-Wayne State University amine metabolism in Chinese hamster cells growing in a defined School of Medicine medium. J. Cell. Physiol. 127:114-120(1986) Tabor, C. W., H. Tabor: 1,4-Diaminobutane (putrescine), spermidine, 540 E. Canfield Detroit, MI 48201 (U. S. A.) and spermine. Ann. Rev. Biochem. 45:285-306 (1976)
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