Thyrotropin-releasing hormone inhibits GH4 pituitary cell proliferation by blocking entry into S phase.

0013-7227/90/1261-0472$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society

Vol. 126, No. 1 Printed in U.S.A.

Thyrotropin-Releasing Hormone Inhibits GH4 Pituitary Cell Proliferation by Blocking Entry into S Phase* JOHN S. RAMSDELL Division of Molecular and Cellular Endocrinology, Department of Anatomy and Cell Biology, Medical University of South Carolina, Charleston, South Carolina 29425

160,000 sites/cell, suggesting no spare receptors for TRH on

ABSTRACT. TRH inhibits the proliferation of GH4 rat pituitary cells. We have characterized TRH inhibition of cell proliferation by four approaches: cell number, [3H]thymidine incorporation per culture, bromodeoxyuridine (BrdUrd) incorporation per cell, and cell cycle distribution. TRH decreases GH4 cell number within 18 h of treatment, and this inhibition is maintained for up to 96 h. TRH inhibits [3H]thymidine incorporation into GH4cell cultures as early as 12 h, and the inhibition of [3H] thymidine incorporation correlates, after a 6-h lag, with decreased GH4 cell number. TRH inhibition of [3H] thymidine incorporation is concentration dependent and saturable, with half-maximal inhibition (IC50) of 2 nM. TRH inhibition of [3H] thymidine incorporation is receptor number dependent up to

GH4C! cells. The precise action of TRH on GH4cell proliferation

was examined by flow cytometry of fluorescein isothiocyanateanti-BrdUrd- and propidium iodide-DNA stained cells. TRH inhibits the number of cells that incorporate BrdUrd and not the amount of BrdUrd incorporated per cell. Dual analysis indicates that the decreased anti-BrdUrd staining is largely restricted to cells in the early S phase. This action of TRH is prolonged (>32 h) and results in a parallel increase in the number of cells in G2-M and Gx. These findings indicate that TRH inhibits GH4cell proliferation at least in part by inhibiting the number of cells entering the S phase. (Endocrinology 126: 472-479, 1990)

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TRH is a well characterized agonist for several responses in the GH3 rat pituitary cell line and the GH4 clonal variant (5). A rapid response to TRH is the acute release of previously synthesized hormone (PRL and GH). Prolonged responses to TRH include enhanced PRL synthesis, reduced GH synthesis, and increased cell substratum adhesion. A potential fourth prolonged response elicited by TRH is inhibition of GH4 cell proliferation. TRH has been reported to have no effect (6) or cause small inhibition (4) or substantial inhibition (7, 8) of GH4 cell proliferation. Because TRH has little or no effect on total cellular protein, TRH is often assumed to have no effect on GH4 cell growth. However, more detailed study indicates that TRH increases GH4 cell size (3), protein per cell (7, 8), and DNA content per cell (7, 8) when it has been found to decrease cell number. The work by Hayashi and Sato (9) on the derivation of serumfree medium, indicated that a low concentration (1 nM) of TRH (together with several other growth factors and hormones) is a growth-promoting factor for GH3 cells. However, subsequent studies indicate that in serum-free medium supplemented with insulin-like growth factor-II (IGF-II), TRH (at 10 nM or higher concentrations) effectively inhibits GH3 cell proliferation (8). This report investigates the action of TRH on GH4 cell proliferation by four approaches: cell number, [3H]thymidine incorporation in cell cultures, bromodeoxyuridine (BrdUrd)

ELL proliferation is normally under the balanced control of growth stimulatory and growth inhibitory factors. A large number of growth stimulatory factors have been investigated; however, only a few growth inhibitory factors (tumor growth factor-/?, tumor necrosis factor, and interferon-/5) have been identified for most cell types (1, 2). GH4 cells are unique in that several traditionally growth stimulatory factors [epidermal growth factor (EGF) and fibroblast growth factor] inhibit cell proliferation (3). The significance of this has not been determined; however, it may be related to the observations that mammotrophs are primarily under inhibitory regulation (4). TRH has also been reported to inhibit GH4 cell proliferation; however, this response is not observed by all investigators and has not been thoroughly characterized. Because TRH is one of the better characterized first messengers in terms of signal transduction, we chose to investigate this action of TRH to advance our understanding of the mechanisms that inhibit pituitary cell proliferation. Received August 24, 1989.[cmAddress all correspondence and requests for reprints to: John S. Ramsdell, Ph.D., Division of Molecular and Cellular Endocrinology, Department of Anatomy and Cell Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425-2203. * This work was supported by the Medical University of South Carolina Biomedical Support Grant of 1987-1988 and American Cancer Society Grant 1N-175.

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TRH INHIBITS GH4 CELL PROLIFERATION

incorporation in individual cells, and cell cycle distribution. Materials and Methods Materials TRH was purchased from Peninsula Laboratories (Belmont, CA). [3H]Thymidine (6.7 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Mouse anti-BrdUrd was purchased from Bectin Dickinson (Mountain View, CA), and fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin G (IgG) F(ab') was purchased from Sigma Chemical Co. (St. Louis, MO). FITC-conjugated mouse antiBrdUrd was purchased from Boehringer Mannheim (Indianapolis, IN). Ham's F-10, fetal bovine serum, and horse serum were obtained from Flow Laboratories (McLean, VA). Plastic culture dishes and flasks were obtained from Costar (Cambridge, MA). All other compounds, unless indicated, were purchased from Sigma Chemical Co. Methods Cell culture. Stock GH4 cultures were maintained in Ham's F10 Nutrient Mixture supplemented with 15% horse and 2.5% fetal bovine serum (F10+) in the absence of antibiotics for a maximum of 10 passages. The isolation of GH4 variants and characterization of TRH receptor number and several TRHenhanced biological responses and intracellular signals has been previously described (10, 11). Original passages of these variants were used for these studies. Cell number determinations. Cells are plated at 0.5 x 105/16mm2well in 0.3 ml F10+. For long treatments with TRH, cells were treated with TRH and were incubated for 24-96 h. For short treatments with TRH, cells were allowed to attach to the wells for 12-24 h and were then treated with TRH. Previous studies have shown the bioactivity of TRH to be stable for at least 24 h under these conditions (12). The cells were detached from the dish by the addition of 10 p\ EDTA (0.1 M) and incubated for 10 min at 37 C. Cells were resuspended in wells and counted with a hemocytometer. Each value represents the mean of nine grids. pH]Thymidine incorporation assay. Cells were plated at 0.5 x 107l6-mm2well in 0.3 ml F10+. For long treatments with TRH cells were treated with TRH and incubated for 24-96 h. The cultures were treated with 2.5 /iCi/ml [3H]thymidine for the final 4 h of the incubation. For short treatments with TRH, cells were allowed to attach to the wells for 12-24 h and then were treated with 2.5 /iCi/ml [3H]thymidine in the presence of TRH. The cells were detached from the dish by the addition of 10 ti\ EDTA (0.1 M) and incubated for 10 min at 37 C. Cells were collected, wells were washed with PBS, and the combined suspension was centrifuged at 10,000 x g for 5 min. The supernatant was removed, and the pellet was extracted with 10% trichloroacetic acid for 30 min at 4 C. Acid-insoluble material was separated by filtration (Whatman GFA filters, Clifton, NJ), and trapped radioactivity was quantified by liquid scintillation spectroscopy. Cell number was quantified in parallel experiments using a hemocytometer. The [3H]thymidine

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incorporation assays are a measure of both active DNA synthesis and (at times after a cell doubling, 24-36 h) decreased cell number. BrdUrd/DNA staining. Cells were stained for BrdUrd and DNA based on previously described methods and modifications (13, 14). Cells were plated at 5 X 105/100-mm2 dish in 8 ml F10+ and allowed to initiate log phase growth. At 24 h cells were treated in the absence or presence of 100 nM TRH for the indicated times. For the final 60 min of treatment the dishes were labeled with 15 pM BrdUrd. The cultures were washed twice with ice-cold PBS-0.2% EDTA containing 5 mM thymidine and incubated for 10 min with this medium at 4 C. The cells were removed by gentle trituration, 106 cells were centrifuged at 1000 x g for 5 min, and the pellet was resuspended in 70% ethanol for 15 min at 4 C. The fixed cells were centrifuged at 1000 x g for 3 min and resuspended in 2 ml 2.5 M HCl-0.5% Triton X-100 for 30 min at 23 C. The cell suspension was centrifuged, washed, and resuspended in 5 ml PBS-0.5% Tween-20 three times and centrifuged through a cushion of bovine calf serum, and the pellet was resuspended in 20 (A mouse anti-BrdUrd for 30 min at 23 C. The cell suspension was centrifuged, washed three times, and resuspended in 5 ml PBS-0.5% Tween-20, and the pellet was resuspended in 30 i*\ FITC-conjugated goat antimouse anti-IgG F(ab') for 30 min at 23 C. The cell suspension was centrifuged, washed three times, and resuspended in 1 ng/va\ propidium iodide (PI) for 15 min at 23 C. For some experiments FITC-conjugated anti-BrdUrd was substituted for the anti-BrdUrd and FITC-conjugated goat antimouse anti-IgG F(ab') with similar results. Flow cytofluorometry. Cells were analyzed on a Coulter Epics 753 (Coulter Electronics, Hialeah, FL). Cells were excited at 488 nm with a 5-watt argon laser to excite both the PI and FITC fluorescence. The emitted light passes through a 635band pass filter to reach the first detector for PI. The emitted light passes through a 525-band pass filter to reach the second detector for FITC. Data were recorded and stored in a multiparameter data acquisition and display system. Data analysis. Specific BrdUrd fluorescence was determined by subtracting anti-BrdUrd FITC fluorescence in cells not labeled with BrdUrd from that in those labeled with BrdUrd. This was performed for each of 256 channels of increasing FITC fluorescence, as described by Khochbin et al. (15). The data are then presented as the number of cells per fluorescent channel (yaxis) us. increasing fluoroscent intensity per channel (x-axis), as shown in Ref. 13. Cell cycle distribution was determined in dual PI-BrdUrd DNA-stained cells based on a previously described method (15). The maximum of Gx and G2-M peaks was determined automatically by the computer program. In all cases, the G2-M peak was twice the fluorescence intensity of the Gi peak, even though samples did vary slightly in the given PI fluoroscent intensity in each peak. The distribution of cells between the Gi and G2-M peaks was approximated visually by three-dimensional representation of BrdUrd/DNA bivarate histograms. The upper extent of Gx and G2-M peaks was defined as the extent of nonspecific FITC-anti-BrdUrd fluorescence. Cells within these coordinates were quantified by a computer program. In some experiments a clear separation without any


Endo • 1990 Voll26»Nol


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overlap between Gx and G2-M distribution was observed. In other experiments a small number of cells with nonspecific FITC anti-BrdUrd fluorescence spanned the two peaks. Comparison of calculations between these staining patterns indicates that these are S phase cells which did not readily stain with the FITC-anti-BrdUrd (possibly due to heterogeneity in the DNA denaturation step). Accordingly, the number of cells in the S phase were calculated as the sum of specific FITCanti-BrdUrd fluorescence and those nonspecific FITC-antiBrdUrd fluoroscent cells between a normal distribution of the d and G2-M peaks. The combination of these two groups of S phase cells did not make any qualitative difference on the effect of TRH on cell cycle distribution.

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incorporation TRH action on asynchronous GH4 cells in exponential growth was examined at prolonged times (several completed cycles) and early times (before a complete cell cycle). Cell proliferation was determined by cell counts and [3H]thymidine incorporation. GH4C1 cells treated at the time of plating with 100 nM TRH numbered less than vehicle-treated cells at each time point between 2496 h (Fig. 1, top). When cells were labeled for 4 h with [3H]thymidine, TRH-treated cells incorporated less radiolabel (Fig. 1, bottom). To determine the earliest onset of TRH action and if TRH has a similar effect on cells after they attach to the substratum, cells were allowed 12 h to attach to the dishes before treatment with TRH. TRH decreased GH4Ci cell number at 18 and 24 h of treatment (Fig. 2, top). When replicate cultures were incubated with [3H]thymidine for the entire period of TRH treatment, thymidine incorporation was inhibited within 12 h. The next experiments examined the concentration and receptor dependencies for TRH inhibition of [3H]thymidine incorporation. Receptor dependency for TRH thymidine incorporation

inhibition of PH]

Dose-response curves and a panel of TRH receptor variants were used to assess TRH-receptor interactions that inhibit [3H]thymidine incorporation. TRH-inhibited [3H]thymidine incorporation was monophasic and saturable at 100 nM, with half-maximal inhibition (IC50) at 2 nM (Fig. 3). The receptor number dependency for TRH inhibition of [3H]thymidine incorporation was examined in a panel of previously characterized GH4 cell variants with increasing numbers of TRH receptors (0.19, 1.1, 1.6, and 2.6 X 105 sites/cell). TRH caused a progressively larger inhibition of [3H] thymidine incorporation in variants with increasing TRH receptor number up to 1.6 x 105 sites/cell (the number of sites found

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FlG. 1. Prolonged TRH inhibition of cell number and [3H]thymidine incorporation. GH4C1 cells were plated at 0.5 x 106/16-mm2 well in 0.3 ml F10+ in the presence or absence of 100 nM TRH. For thymidine incorporation, cultures were treated with 2.5 fiCi/ral [33H]thymidine for the final 4 h. At the designated times, the cells were either counted to determine cell number or acid precipitated to determine thymidine incorporation. Values given are the mean ± SE for quadruplicate samples and are fit to exponential curves. [3H]Thymidine incorporation within these time points was repeated in at least three independent experiments. TRH inhibition of [3H] thymidine incorporation at 24 h (from 4513 ± 98 to 3708 ± 83) may not be evident due to the scale of the graph. VEH, Vehicle.

on GH 4 d cells; Fig. 4). The variant with 2.6 X 105 TRH receptor sites/cell did not show further TRH inhibition of [3H]thymidine incorporation. The next experiments examined the action of TRH on individual cells. TRH inhibition of BrdUrd incorporation TRH inhibition of DNA synthesis in individual cells was examined by flow cytometry of GH4 cells labeled with the thymidine analog BrdUrd. TRH decreased the number of cells with specific anti-BrdUrd fluorescence without affecting the distribution of fluorescence intensity (amount of BrdUrd incorporated per cell; Fig. 5, see legend for analysis). This effect of TRH was evident as early as 6 h and was maintained for up to 32 h. The percent inhibition of the total number of cells specifically BrdUrd labeled (35% at 18 h) was comparable to that found for TRH inhibition of [3H]thymidine incorporation per cultures. Likewise, TRH inhibition of BrdUrd per cell and [3H] thymidine incorporation per culture were prolonged actions of TRH (up to 32 h).The next


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FIG. 3. Concentration dependency for TRH inhibition of [3H]thymidine incorporation. GH4Ci cells were plated at 0.5 X 106/16-mm2 well in 0.3 ml F10+ in the presence of increasing concentrations (10 u -10 6 M) of TRH for 72 h. Cultures were treated with 2.5 /xCi/ml [3H] thymidine for the final 4 h, and the cells were acid precipitated to determine thymidine incorporation. Values given are the mean ± SE for quadruplicate samples from a single experiment.

0

10 Treatment (h)

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FlG. 2. TRH inhibition of cell number and [3H]thymidine incorporation within a single cell cycle. GH4Ci cells were plated at 0.5 x 105/16mm2 well in 0.3 ml F10+ and incubated for 24 h. Cultures were then treated without or with 100 nM TRH for the indicated times. For thymidine incorporation, cultures were treated with 2.5 /iCi/ml [3H] thymidine for the duration of the TRH treatment. At the designated times, the cells were either counted to determine cell number or acid precipitated to determine thymidine incorporation. Values given are the mean ± SE for quadruplicate samples. TRH inhibition of cell number and [3H]thymidine incorporation within these time points was repeated in at least three independent experiments. The TRH-treated data did not fit well to an exponential curve, probably as a result of the intitial action of TRH, and for this reason are presented without curve fitting. VEH, Vehicle.

experiments examined the action of TRH on the distribution of GH4 cells in the different stages of the cell cycle. TRH effects on GH4 cell cycle distribution The effect of TRH on GH4 cell cycle distribution was examined by flow cytometry using cells stained for both DNA content and newly synthesized DNA. This assay uses PI to measure DNA content and FITC-anti-BrdUrd to measure newly incorporated BrdUrd. The results are presented as three-dimensional maps turned 150° toward the reader to emphasize S phase (Fig. 6, see legend for analysis). Control cells not BrdUrd labeled (Fig. 6, left panel) have a very large peak with low PI fluorescence (red) intensity (x-axis) that contains cells with a 2n DNA content (primarily in the Gi phase). A second population of cells to the left of the Gi peak has twice the intensity

80,000

240,000

160,000

TRH Receptor Number

(sites/cell)

FIG. 4. Receptor dependency for TRH inhibition of [3H]thymidine incorporation. GH4Ci cells (solid symbols) and variants (xl7, p l l - 1 , yl3-3, and yl3-5; open symbols) with different TRH receptor numbers were plated at 0.5 x 106/16-mm2well in 0.3 ml F10+ in the presence or absence of 100 nM TRH for 72 h. Cultures were treated with 2.5 ixCi/ ml [3H]thymidine for the final 4 h, and the cells were acid precipitated to determine thymidine incorporation. Each data point is the mean difference between treated and nontreated values for quadruplicate pairs of samples derived from five independent experiments (O, D, A, V, 0) using each variant three times.

of PI fluoresence (4n) and consists primarily of cells in the G2-M phase. Cells in the S phase have an intermediate DNA content because they are actively synthesizing DNA and, therefore, span the Gi and G2-M peaks of PI fluoresence. Because of the overlap of S phase cells with those in Gx and G2-M, cell cycle distribution is best



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— 80 24 h) TRH has little or no effect on total cellular protein. This might be interpreted as meaning that TRH has no effect GH4 cell proliferation. When the action of TRH on GH4 (or GH3) cell proliferation is examined directly, TRH has been found to cause no inhibition (6), a small inhibition (10%) (3), or a substantial inhibition (>40%) (7, 8) of GH4 cell number. The basis for these different observations is not known, but may result from different cell density, serum, or GH3 clones. Under the conditions described in this report we consistently find that TRH inhibits GH4 cell proliferation, as measured by any of four different methods: cell counts, [3H]thymidine incorporation per culture, BrdUrd incorporation per cell, and cell cycle distribution. A clear definition of TRH action on GH4 cell growth is of importance for in vitro studies of prolonged (>12 h) TRH treatment and particularly to assess those studies that indicate that TRH decreases the amount of particular gene products. TRH has three actions on GH4 cell growth that are seemingly paradoxical with inhibition of cell proliferation. First, TRH increases cell volume (3). Second, TRH increases DNA content per cell (7, 8). Third, TRH increases protein per cell (7, 8). These findings, based on 2- to 7-day treatments, indicate that TRH increases the number of large cells with high protein and DNA contents. This is consistent with a higher percentage of cells in the later stages of the cell cycle (16). The data presented in this report indicate that TRH reduces the number of cells in early S phase, with an accompanying accumulation of late S and G2-M phase cells as well as Gi phase cells. However, since TRH action on cell cycle



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FIG. 6. Flow cytometric analysis of cell cycle analysis of TRH-treated GHid cells. GH4Ci cells in exponential growth were treated with or without 100 nM TRH for 6 h and labeled with 15 fiM BrdUrd for the final hour. A replicate group for each experiment received no BrdUrd and was used a control to determine nonspecific BrdUrd staining. Cells were stained with FITC-anti-BrdUrd plus PI, and 10,000 cells for each histogram were analyzed by flow cytometry. Data are shown as three-dimensional maps of 256 channels of increasing (from right to left in each panel) red (PI) fluorescence on the x-axis, 256 channels of increasing (from back to front) green (anti-BrdUrd) fluorescence on the z-axis, and increasing cell number for each red-green coordinate on the y-axis. Gi phase is the very large peak closest to the x,z ordinate. G2-M phase is a smaller accumulation of cells to the left of Gi. S phase cells are between Gl and G2-M, extending out toward the reader in cells treated with BrdUrd. Early S phase are those S phase cells with low red (PI) fluroscence intensity, extending outward from the Gx peak. In this experiment TRH given at 6 h decreased the percentage of S phase cells from 19.5% to 12.8% and increased the percentage of G2-M and Gi from 7.4% to 10% and from 73% to 77%, respectively. TRH given at 18 h caused a similar effect (not shown). This experiment was repeated two additional times at several time points with similar results. One such experiment is presented in Fig. 7.

distribution was not examined beyond 32 h, additional studies are necessary to determine if TRH continues to increase the percentage of cells in the later stages of the cell cycle. A fourth paradoxical action of TRH is that it was originally identified as a growth stimulatory factor for GH3 cells in defined medium formulations (9). This may result from the concentration (1 nM) chosen for this study rather than the serum-free us. serum-supplemented medium. TRH has been confirmed as having a small growth stimulatory action at 1 nM, but a growth inhibitory action at higher concentrations (10 nM) in both serum-free and serum-supplemented medium (8). Similar differences in the concentration dependencies have been observed for other TRH responses in GH4 cells, including enhanced PRL release and synthesis and acute enhancement and chronic inhibition of uridine uptake (17, 18). TRH inhibition of [3H]thymidine incorporation is concentration dependent and saturable, with half-maximal inhibition (IC50) at 2 nM. However, TRH was found to have no stimulatory effect on [3H]thymidine incorporation between 0.01-1.0 nM, as described for its action on GH3 cell number. Because the IC50 for TRH is less than its Kd (20 nM) for the receptor (12), different concentration dependencies could result from different receptor occupancy requirements. Receptor dependency for TRH inhibition of GH4cell proliferation was assessed

using a panel of GH4 cell variants with decreased TRH receptor number. TRH inhibited [3H]thymidine incorporation in all variants in proportion to the number of TRH receptor sites per cell (up to 160,000 sites/cell) indicating tight receptor-response coupling and an absence of spare receptors for TRH inhibition of thymidine incorporation in GH4Ci cells. This finding also indicates that a single class of TRH receptors mediates inhibition of [3H]thymidine incorporation, as previously described for other TRH-mediated responses in these cells (10,11). One possible mechanism by which TRH may inhibit thymidine incorporation and cell proliferation is inhibition of thymidine kinase activity. Martin and co-workers (18) previously found that TRH causes an acute accumulation and a chronic inhibition of [3H]uridine uptake and incorporation into GH4 cells. This action of TRH was restricted to pyrimidine nucleosides and was the result of uridine phosphorylation, possibly by an action on uridine kinase (19). Likewise, one possible means by which TRH inhibits [3H]thymidine incorporation may be direct inhibition of thymidine kinase activity. However, because thymidine kinase activity increases dramatically as cells enter the S phase (20), TRH may alternatively inhibit [3H]thymidine incorporation by reducing the number of cells entering the S phase. To discriminate between these possibilities, flow cytometry was used to examine the effect of TRH on the incorpo-


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a decreased entry into the S phase. Entry into the S phase is a commonly regulated event in the cell cycle (21, 22). Growth stimulatory factors promote entry into the S phase for up to 2 h before DNA synthesis (the restriction point), after which time progression through the cell cycle is committed (23). During this time the activity of enzymes for DNA replication (i.e. thymidine kinase and thymidylate synthase) is enhanced, possibly through the formation of a multienzyme complex with other enzymes involved in DNA transcription (24). If TRH solely inhibited entry into the S phase, a parallel accumulation of cells in Gi should occur with decreases in the other stages of the cell cycle. However, TRH inhibition of the percentage of early S phase cells does not result in an accumulation of cells exclusively in Gi.

E 3

At 6 h, the primary accumulation of cells occurs in late

hours of TRH treatment

FIG. 7. Cell cycle distribution of TRH-treated G H A cells. Cells were treated and stained as described in Fig. 6, and cell distribution in d , G2-M, and S phases was determined as described in Materials and Methods. This experiment was repeated three times. In each case, the number of S phase cells was decreased and the number of G2-M cells increased at all times; the number of Gi cells increased by 12 h of TRH treatment.

ration of a thymidine analog (BrdUrd) into individual cells. TRH decreases the number of cells that incorporate BrdUrd without affecting the amount of BrdUrd labeling per cell. If TRH were to specifically inhibit thymidine kinase activity, cells in the S phase would have a decreased amount of BrdUrd incorporation but not be decreased in number. This is because thymidine kinase provides only a salvage pathway for deoxythymidine triphosphate (DTTP) synthesis (deoxythymidine monophosphate (dTMP) is synthesized de novo by thymidylate synthase). Because TRH decreases the number of cells that incorporate BrdUrd at times (6-12 h) before a decrease in total cell number, TRH is not cytotoxic. Accordingly, TRH inhibition of the number cells actively synthesizing DNA should be paralleled by increases in the number of cells in other stages of the cell cycle. The mechanisms by which TRH inhibits the number of cells actively synthesizing DNA were examined by determining its action on GH4 cell cycle distribution. TRH caused a dramatic decrease in the number of cells in the S phase within 6 h. This action of TRH is seen primarily in the early S phase, which is consistent with

S phase and G2-M. The accumulation of cells in G2-M leads to a transient decrease in Gi, however in all experiments the percentage of cells in both Gx and G2-M phases is increased by 12 h. An analogous finding has been reported for EGF inhibition of A431 human carcinoma cell proliferation (25). This action of EGF, like TRH action on GH4 cells, clearly decreases the entry of cells into the S phase, yet leads to an accumulation of both G2-M and Gi phase cells. The accumulation of EGF treated A431 cells in both G2-M and Gi phases appears to result from two independent blocks in the cell cycle. Evidence for this hypothesis was provided by blocking cells from passing through mitosis with vinblastine, and then demonstrating that EGF still decreased the percentage of cells in the S phase (blocking entry from Gi to S phase) and increased the percentage of cells in late S and/or G2 (blocking entry of G2to mitosis). The action of TRH on cell cycle distribution is consistent with this hypothesis. According to the model derived for EGF action of A431 cells, TRH may have two actions on cell cycle progression that could inhibit GH4 cell proliferation. One is to decrease the number of cells entering the S phase (observed as a decrease in early S and increase in Gi), and the second may be to inhibit the entry of cells into mitosis (observed as an increase in G2-M and transient decrease in Gx). Cell proliferation is normally under the control of both growth stimulatory and growth inhibitory factors. Therefore, TRH probably inhibits the action of certain growth stimulatory factors. Studies with GHi cells and GH3 clonal variants (GC and GH4Ci) indicate that proliferation is highly dependent on thyroid hormone (T3) (2628) and this action occurs during the first 4-6 h of the Gi phase (22). The dependence on T 3 may result in part from autocrine or serum-derived factors (28-30). However, the identity of such a factor remains unknown, because no traditional growth stimulatory factors will substitute for the action of T 3 to promote GH4 cell growth


TRH INHIBITS GH4 CELL PROLIFERATION in serum-free medium (30). Yajima and Saito (8) found that IGF-II (multiplication-stimulating activity), which together with IGF-I comprises 90% of the mitogenic activity of serum (31), is necessary for TRH to inhibit GH3 cell proliferation in serum-free medium. GH3 cells express receptors for IGF-I and IGF-II (32), and each of these factors is secreted by pituitary cells (33, 34). Because TRH inhibits GH3 cell proliferation in IGF-IIcontaining medium, even in the absence of T3, TRH most likely selectively inhibits the growth-promoting activity of IGF-II, rather than T 3 or a T3-stimulated autocrine factor. In summary, TRH inhibits GH4 cell proliferation at least in part by blocking cells from entering the S phase. Inhibitory mechanisms that control entry into the S phase may play an important role in lactotrophs, which are known to proliferate during different physiological and pathological conditions (35, 36).

Acknowledgments I thank Ms. Christine Carr and Joanne Koffskey for assistance with the flow cytometry and Drs. F. R. Bookfor and W. C. Gorospe for helpful discussions.

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Phosphorylation of BMK1 induces prostatic carcinoma cell proliferation by promoting entry into the S phase of the cell cycle.

Mutations in the PCNA-binding site of CDKN1C inhibit cell proliferation by impairing the entry into S phase.

Voltage-dependent calcium channels regulate GH4 pituitary cell proliferation at two stages of the cell cycle.

Expression of 1N3R-Tau isoform inhibits cell proliferation by inducing S phase arrest in N2a cells.

Plumericin inhibits proliferation of vascular smooth muscle cells by blocking STAT3 signaling via S-glutathionylation.

S Phase Cell Cycle Arrest.

Transient heat shock in mid-G1-phase of the C6 glioma cell cycle impairs entry into S-phase.

Transforming growth factor-alpha and -beta are potent and effective inhibitors of GH4 pituitary tumor cell proliferation.

Dehydroepiandrosterone inhibits cell proliferation and improves viability by regulating S phase and mitochondrial permeability in primary rat Leydig cells.

Tenovin-6 inhibits proliferation and survival of diffuse large B-cell lymphoma cells by blocking autophagy.

Activation of the murine thyrotropin beta-subunit promoter by GH4 rat pituitary cell-free extracts.

Immunosuppressive activity of pogostone on T cells: Blocking proliferation via S phase arrest.

OVEREXPRESSION OF BOTH CLOCK AND BMAL1 INHIBITS ENTRY TO S PHASE IN HUMAN COLON CANCER CELLS.

Regulatory T cells inhibit CD34+ cell differentiation into NK cells by blocking their proliferation.

p27 levels.

MicroRNA-200b inhibits pituitary tumor cell proliferation and invasion by targeting PKCα.

The Ubp15 deubiquitinase promotes timely entry into S phase in Saccharomyces cerevisiae.

Accelerated entry of aortic smooth muscle cells from spontaneously hypertensive rats into the S phase of the cell cycle.

The Drosophila plutonium and pan gu genes regulate entry into S phase at fertilization.

Metformin inhibits proliferation and growth hormone secretion of GH3 pituitary adenoma cells.

Continuous protein synthesis is required to maintain the probability of entry into S phase.

Lipopolysaccharide-binding alkylpolyamine DS-96 inhibits Chlamydia trachomatis infection by blocking attachment and entry.

GADD45β induction by S-adenosylmethionine inhibits hepatocellular carcinoma cell proliferation during acute ischemia-hypoxia.

Growth hormone-releasing factor stimulates calcium entry in the GH3 pituitary cell line.