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Endocrinology Vol. 139, No. 4 2155-2162
Copyright © 1998 by The Endocrine Society

ARTICLES

Thymosin Fraction 5 Inhibits the Proliferation of the Rat Neuroendocrine MMQ Pituitary Adenoma and C6 Glioma Cell Lines in Vitro1

Bryan L. Spangelo, Derald D. Farrimond, Mahesh Thapa, Charles M. Bulathsinghala, Kay-Lynn Bowman, Alaleh Sareh, Francis M. Hughes, Jr., Allan L. Goldstein and Mahnaz Badamchian

Department of Chemistry (B.L.S., D.D.F., M.T., C.M.B., K.B., A.S.), University of Nevada Las Vegas, Las Vegas, Nevada 89154; Laboratory of Signal Transduction (F.M.H.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and the Department of Biochemistry and Molecular Biology (A.L.G., M.B.), The George Washington University School of Medicine, Washington D.C. 20037

Address all correspondence and requests for reprints to: Bryan L. Spangelo, Department of Chemistry, University of Nevada Las Vegas, 4505 Maryland Parkway, Las Vegas, Nevada 89154-4003. E-mail: spangelb{at}nevada.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cytokines such as interleukin-1 (IL-1) and IL-6 stimulate the hypothalamic-pituitary-adrenal (HPA) axis. In addition, these proteins affect pituitary cell proliferation in vitro. Thymosin fraction 5 (TF5) is a partially purified preparation of the bovine thymus that enhances immune system functioning. Because TF5 similarly stimulates the HPA axis, we examined the effects of this preparation on neuroendocrine tumor cell proliferation. Cells of the PRL-secreting rat anterior pituitary adenoma, MMQ (5–50 x 103 cells/well), were exposed to vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or TF5 (100–500 µg/ml) for up to 96 h and the proliferation of MMQ cells monitored using the MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). TF5-mediated inhibition of cell proliferation was dependent on both TF5 concentration and the initial MMQ cell number. Minimal reductions in optical densities resulted from exposure to 100 µg/ml TF5, whereas the highest concentration of this preparation (i.e. 500 µg/ml) completely blocked MMQ cell division. The concentration-dependent effects of TF5 were particularly striking at initial plating densities of 25 and 50 x 103 MMQ cells/well; in contrast, all concentrations of TF5 completely inhibited MMQ cell growth at 5 and 10 x 103 cells/well. The antiproliferative actions of TF5 on MMQ cells were demonstrable within 24 h and remained for up to 96 h as determined by the MTT assay and actual cell counts. Because the highest densities of MMQ cells were partially refractive to the antiproliferative effects of TF5, we examined the effects of PRL (1–1000 nM) and MMQ cell conditioned medium (50%) on TF5 inhibition of MMQ adenoma proliferation. The TF5 concentration-dependent inhibition of MMQ cell growth was largely reversed by the 50% conditioned medium, whereas PRL slightly potentiated the antiproliferative actions of TF5. The proliferation of the rat C6 glioma cell line (10–30 x 103 cells/well) demonstrated greater sensitivity to TF5: concentrations as low as 10 µg/ml TF5 inhibited C6 cell proliferation (P < 0.01), and near-maximal inhibition was noted at 200 µg/ml TF5. Significant reductions in MMQ and C6 cell viabilities accompanied decreases in cell number and morphological analysis indicated these cells were dying by apoptosis. The peptides thymosin {alpha}1 (T{alpha}1), thymosin ß4 (Tß4), MB35, and MB40 had no effect on either MMQ or C6 cell proliferation, indicating that these TF5 components are not the principle active peptides. Therefore, TF5 was further separated into 60 fractions by preparative reverse phase HPLC. HPLC fractions 17, 25, 26, and 27 significantly suppressed MMQ cell proliferation (P < 0.01) to the same extent as TF5; other HPLC fractions had no effect. These data demonstrate a new biological property of TF5: the inhibition of cell proliferation and the induction of apoptosis in neuroendocrine tumor cells. The proliferation effects were time and concentration dependent and could be partially reversed by an activity present in the MMQ cell conditioned medium. Thus, TF5 and cytokines have opposite effects on adenoma cells because IL-2 and IL-6 stimulate GH3 cell proliferation. We propose that circulating thymic peptides may act to prevent pituitary adenoma and glioma tumor formation, an action opposed by autocrine growth factors secreted by these tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A FOCAL POINT of neuroendocrine-immune system interactions is the regulation of the hypothalamic-pituitary axis by cytokine proteins and thymic peptides (reviewed in Refs. 1–3). Interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-{alpha} (TNF{alpha}) each stimulate the hypothalamic-pituitary-adrenal (HPA) axis. For example, IL-1ß increases corticosterone secretion in rats via the enhanced release of the hypothalamic neuropeptide CRH (3). Similarly, the bovine thymic hormone preparation thymosin fraction 5 (TF5) activates the HPA axis in vivo (4, 5) via a possible direct action on hypothalamic CRH release (6). Goya et al. (7) also demonstrated that TF5 increases ACTH release from mouse corticotroph AtT20 cells in vitro. In contrast, a constituent peptide of TF5, thymosin {alpha}1 (T{alpha}1), suppresses plasma ACTH, TSH, and PRL levels following its intracerebroventricular injection in rats (8). T{alpha}1 also inhibits CRH, TRH, and SRIF release from explanted medial basal hypothalami in vitro (9). Previously, we reported that TF5 and a basic 35 residue peptide purified from TF5 (i.e., MB-35) stimulate rat anterior pituitary PRL and GH release in vitro (10, 11, 12).

In addition to the regulation of neuroendocrine peptide secretion, certain cytokines also affect the proliferation of pituitary cells in vitro. The cytokines IL-2 and IL-6 stimulate rat pituitary adenoma GH3 cell proliferation but inhibit anterior pituitary cell division (13). Similarly, IL-6 increases the proliferation of MtT/E cells, a rat pituitary adenoma cell line (14). Although IL-1 has no direct effect on the rate of GH3 cell division, this cytokine inhibits rat anterior pituitary cell division as well as the amount of [3H]-thymidine incorporation into these cells (15). Because cytokines and thymic peptides have similar effects on the HPA axis, we hypothesized that TF5 would also affect the proliferation rates of neuroendocrine cells. Thus, we have examined the effects of TF5 and certain of its constituent peptides on the proliferation of the rat anterior pituitary adenoma MMQ (16) and the rat C6 glioma (17) cell lines. Thymosin inhibited both MMQ and C6 cell proliferation in a concentration-, time-, and cell number-dependent manner. In MMQ cells, these effects were apparently mediated by increased apoptosis. We suggest that thymic hormone immune surveillance mechanisms may affect neuroendocrine tumor formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Rat MMQ pituitary adenoma cell culture
The MMQ pituitary adenoma cell line (16) was maintained in a humidified atmosphere of 5% CO2-95% air at 37 C in RPMI-1640 (10 ml/T-25 cm2 flask) supplemented with 2.5% FCS, 7.5% horse serum, and antibiotics (7.5 µg/ml streptomycin, 15 µg/ml gentamycin, 19 µg/ml penicillin, 0.6 µg/ml fungizone; Gibco, Grand Island, NY). MMQ cells were subcultured every 3–4 days. In initial experiments, 500 µg/ml TF5 was added to the T-25 cm2 flasks and aliquots removed for cell counting each day for 4 days. In subsequent experiments, MMQ cells were rinsed two times with serum-free RPMI-1640 and cultured in 96-well plates (25 x 103 cells/0.2 ml/well; Intermountain Scientific, Bountiful, UT) or in 12-well plates (200 x 103 cells/3 ml/well) for the determination of cell viability and proliferation. The MMQ cells were treated with either vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum and antibiotics) or TF5, T{alpha}1, Tß4, MB-35 or MB-40 for 24–96 h (4 wells/treatment group). In addition, differing numbers of MMQ cells (5, 10, 25 and 50 x 103 cells/well) cultured in 96-well plates were exposed to TF5 for 96 h. In certain experiments, MMQ cells (25 x 103 cells/well) were exposed to varying concentrations of rat PRL (National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases), or to their own conditioned medium (10%–50%), both in the absence and presence of TF5.

The extent of cellular proliferation in the 96-well plates was determined using the MTT cell viability and proliferation assay (18). Following the designated incubation interval, 20 µl of MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma Chemical Co., St. Louis, MO) were added to each well in the 96-well plates. After 4 h, 150 µl of medium was removed from each well and replaced with an equal volume of 0.04 M HCl/isopropanol. Following an overnight incubation in darkness, the dissolved MTT crystals were quantitated. Optical densities were obtained using a test wavelength of 570 nm and a reference wavelength of 630 nm (Dynatech MR5000 microelisa spectrophotometer, Chantilly, VA).

Rat C6 glioma cell culture
We maintained the C6 glioma cell line (17; American Type Culture Collection, Rockville, MD) in a humidified atmosphere of 5% CO2-95% air at 37 C in RPMI-1640 supplemented with 2.5% FCS, 7.5% horse serum, and antibiotics (10 ml in a T-25 cm2 flask). C6 glioma cells were also subcultured every 3–4 days. For an experiment, confluent monolayers of C6 cells were released from the tissue culture flask using 0.25% trypsin (Gibco) and rinsed two times with serum-free RPMI-1640. Cells were cultured in 96-well plates (20 x 103/0.2 ml/well) or in 12-well plates (160 x 103/3 ml/well) for the determination of cell viability and proliferation. Following an overnight attachment period, the 96-well cultured cells were rinsed twice with serum-free RPMI-1640 and then treated with either vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum and antibiotics) or TF5, T{alpha}1, Tß4, MB-35 or MB-40 for 24–72 h (4 wells/treatment group). In addition, differing numbers of cultured C6 cells (10, 15, 20, 25, and 30 x 103 cells/well) were exposed to TF5 for 72 h. Following the designated incubation interval, 20 µl of MTT were added to each well in the 96-well plates as an indicator for cellular proliferation rates.

Cell viability and morphology
MMQ and C6 cells cultured in 12-well plates were exposed to maximally effective concentrations of TF5 (e.g. 500 µg/ml) for 72 h. Following trituration (MMQ) or detachment with trypsin and trituration (C6), we quantitated both cell number and viability using standard trypan blue and hemocytometer techniques. For morphological analysis, cells were resuspended in 95% ethanol and dried onto glass slides. Slides were stained with hematoxylin and eosin and photographed with standard light microscopic techniques.

Reverse-phase HPLC separation of TF5
Preparative reverse-phase HPLC of TF5 was performed as described (11, 12). TF5 was applied (1.5 g) to a {delta}-prep HPLC system equipped with a model 481 variable wavelength detector with a semipreparative flow cell (280 nM) and a 300 x 50 mm {delta}-pak 300 Å 15 µM C18 column (Waters Chromatography Division of Millipore Corp., Milford, MA). Eluent A was 0.02 M ammonium acetate (pH 6.8) and eluent B was acetonitrile. A 60-min linear gradient from 0–80% B was run at a flow rate of 80 ml/min. TF5 was dissolved in the initial solvent A, applied to the column through a port in the solvent delivery system, and 1-min fractions were collected. A representative absorbance profile of the HPLC separation of TF5 is shown in Fig. 1Go. Actual protein concentrations were determined using the Lowry assay (19). Aliquots from each of 60 fractions (5 µl/well) were added to cultured MMQ cells (25 x 103 cells) for 4 days, and the extent of proliferation was determined using the MTT assay. TF5 was used as a positive control (100–500 µg/ml).



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  		Figure 1. Reverse-phase HPLC separation of 1.5 g of TF5 on a 300 x 50 mm {delta}-pak 300 Å 15 µm C18 column. Eluent A was 0.02 M ammonium acetate (pH 6.8), and eluent B was acetonitrile. A 60-min linear gradient from 0 to 80% B was performed at a flow rate of 80 ml/minute and 1 min fractions were collected. Detection was at 280 nm.

 
Statistical analysis
ANOVA and the Bonferroni analysis for multiple comparisons were used for statistical evaluation of the data. A P value of <= 0.05 was considered as significant. Unless noted otherwise, data are expressed as the mean ± SE of groups consisting of four observations. Each experiment was performed independently at least three times and, unless noted otherwise, representative results are reported.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TF5 inhibits MMQ adenoma cell proliferation in vitro
The MMQ cell line is a rat pituitary adenoma that secretes PRL in vivo and in vitro (16). In preliminary studies, we found that the proliferation of MMQ cells was suppressed by TF5. As shown in Fig. 2Go, treatment of MMQ cells with 500 µg/ml TF5 during a 4-day culture period resulted in decreased numbers of these cells. A clear reduction in total viable cell number was noted within 24 h of exposure to TF5 (Fig. 2Go). This suppression remained throughout the 4-day treatment period.



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  		Figure 2. Time course of TF5 inhibition of rat MMQ pituitary adenoma cell number in vitro. MMQ cells were subcultured (1.0 x 106 cells/25 cm2 flask) in culture medium (RPMI-1640 containing 2.5% FCS, 7.5% horse serum and antibiotics) or culture medium supplemented with 500 µg/ml TF5. Cells were removed from the flasks every 24 h and quantified using standard microscopy. TF5 significantly reduced the accumulation of viable MMQ cells on each day (P < 0.01). The data are expressed as the mean ± SEM of four separate experiments.

 
We subsequently evaluated MMQ cell proliferation in the presence of TF5 using the MTT cell viability and proliferation assay (18). As shown in Fig. 3Go, TF5 inhibition of the MMQ cells was dependent on both MMQ cell number and the concentration of this thymic preparation. Thus, following a 96-h incubation, all concentrations of TF5 effectively blocked MMQ cell proliferation at initial plating densities of 5 and 10 x 103 MMQ cells/well. Higher concentrations of MMQ cells (25 and 50 x 103 MMQ cells/well) allowed pronounced concentration-dependent effects of TF5. Thus, 100 µg/ml only partially reduced the proliferation of 25 and 50 x 103 MMQ cells/well. In addition, higher concentrations of TF5 (300–500 µg/ml) were required to substantially suppress MMQ cell proliferation at these greater cell densities. Consistent with actual cell numbers (Fig. 2Go), TF5 inhibited MMQ cell proliferation within a 24-h treatment period as monitored by the MTT assay (Fig. 4Go). These effects were dependent on TF5 concentration and remained for 96 h, although the lower concentrations of TF5 (e.g. 100 and 200 µg/ml) were increasingly less effective with time. Based on Figs. 3Go and 4Go, we estimate that the ED50 for the TF5-mediated inhibition of 25 x 103 MMQ cells during a 96-h incubation period is 200 µg/ml.



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  		Figure 3. TF5 inhibits rat MMQ pituitary adenoma cell proliferation in vitro: effects of TF5 concentration and cell number. Cultured MMQ cells (5–50 x 103 cells/well) were exposed to either vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or different concentrations of TF5 (100–500 µg/ml) for 96 h. At 5–25 x 103 MMQ cells, TF5 significantly inhibited cell proliferation at all concentrations tested (P < 0.01). At 50 x 103 MMQ cells, only 300–500 µg/ml TF5 significantly suppressed cell proliferation (P < 0.01). The data are expressed as the mean ± SEM of groups consisting of four observations.

 


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  		Figure 4. Time course of TF5 inhibition of MMQ pituitary adenoma cell proliferation in vitro: MTT reaction. Cultured MMQ cells (25 x 103 cells/well) were exposed to either vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or different concentrations of TF5 (100–500 µg/ml) for 24–96 h. All concentrations of TF5 significantly inhibited MMQ cell proliferation at each time point (P < 0.01). The data are expressed as the mean ± SEM of groups consisting of four observations.

 
Because the suppression of MMQ cell proliferation by TF5 could be partially reversed by either 1) increasing the initial culture density of MMQ cells (Fig. 3Go), or 2) increasing the culture period to 96 h (Fig. 4Go), we hypothesized that the MMQ cells produced an activity capable of inhibiting the antiproliferative actions of TF5. We therefore examined the effects of 50% MMQ cell conditioned medium on TF5-mediated inhibition of MMQ cell growth. Because these cells release PRL (16), we also exposed MMQ cells to 1 µM PRL in the absence and presence of TF5. As shown in Fig. 5Go, TF5 inhibited MMQ cell proliferation in a concentration-dependent manner during a 96-h treatment period. Although MMQ conditioned medium did not significantly affect basal proliferation of these cells, the TF5 inhibition of MMQ cell proliferation was nonetheless either completely (e.g. 100–300 µg/ml) or largely (e.g. 400 and 500 µg/ml) reversed by this conditioned medium. In addition, concentrations of MMQ cell conditioned medium as low as 10% produced a partial reversal of TF5-mediated inhibition of cell growth (data not shown). These effects were probably not due to PRL because this polypeptide induced a modest potentiation of TF5-mediated suppression of MMQ cells. Lower concentrations of PRL (i.e. 1–500 nM) either slightly suppressed or had no affect on MMQ cell proliferation (data not shown). Thus, MMQ cells release an activity that mitigates the antiproliferative effects of TF5.



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  		Figure 5. TF5 inhibits MMQ pituitary adenoma cell proliferation in vitro: effects of MMQ cell conditioned medium and PRL. Cultured MMQ cells (25 x 103 cells/well) were exposed to either vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum and antibiotics) or different concentrations of TF5 (100–500 µg/ml) for 96 h. In addition, MMQ cells were exposed to either 50% MMQ conditioned medium or 1 µM PRL in the absence or presence of each concentration of TF5. TF5 significantly suppressed MMQ cell proliferation (vehicle vs. 100 µg/ml, P < 0.05; 200–500 µg/ml, P < 0.01). MMQ cell conditioned medium did not affect MMQ cell basal proliferation but did significantly reverse the TF5 inhibition of cell growth (200–500 µg/ml TF5 vs. 200–500 µg/ml TF5 + 50% MMQ conditioned medium, P < 0.01). Rat PRL (1 µM) marginally reduced basal MMQ cell proliferation (P < 0.01) and slightly potentiated TF5 suppression of cell growth (200 and 300 µg/ml TF5 vs. 200 and 300 µg/ml TF5 + 1 µM PRL, P < 0.01). The data are expressed as the mean ± SEM of groups consisting of four observations.

 
TF5 inhibits C6 glioma cell proliferation in vitro
Consistent with glial cells in situ, the rat C6 glioma cell line synthesizes the S100 protein as well as the glial fibrillary acidic protein (GFAP). In culture these cells have a stellate (i.e. astrocytic) appearance (17). In preliminary experiments, 50 and 200 µg/ml TF5 completely inhibited C6 cell proliferation (20 x 103 cells/well) within 48 h (data not shown). As for the MMQ cell line, TF5 inhibition of the proliferation of C6 cells was dependent on TF5 concentration and the initial C6 cell plating density (Fig. 6Go). Thus, as little as 10 µg/ml TF5 blocked the proliferation of 10 and 15 x 103 cells/well following a 72-h incubation. However, 10 µg/ml TF5 had little effect on 25 and 30 x 103 cultured C6 cells. These higher densities of C6 cells were increasingly refractive to TF5, requiring 200 µg/ml for substantial suppression of cell growth. Nonetheless, C6 cells were apparently more sensitive to TF5 compared with MMQ cells. At maximal cell densities (i.e. 25 x 103), we estimate an ED50 of 50 µg/ml TF5 for inhibition of C6 cell growth.



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  		Figure 6. TF5 inhibits C6 glioma cell proliferation in vitro: effects of TF5 concentration and cell number. Cultured C6 cells (10–30 x 103 cells/well) were exposed to either vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or different concentrations of TF5 (10–200 µg/ml) for 72 h. At 10–25 x 103 MMQ cells, TF5 significantly inhibited cell proliferation at all concentrations tested (P < 0.01). At 30 x 103 MMQ cells, only 25–200 µg/ml TF5 significantly suppressed cell proliferation (P < 0.01). The data are expressed as the mean ± SEM of groups consisting of four observations.

 
TF5 reduces MMQ and C6 cell viabilities and induces apoptosis in vitro
In Table 1Go we show the effects of TF5 on C6 glioma and MMQ pituitary adenoma cell numbers and viability following 72-h incubations. For both cell lines, 500 µg/ml TF5 significantly suppressed viable cell numbers and total cell viabilities (P<0.01). Thus, TF5 inhibited the proliferation of viable, living cells and at the same time reduced the viability of the total cell population. To ascertain the mode of cell death, MMQ cells treated with 500 µg/ml TF5 or vehicle for 72 h were analyzed by light microscopy. As shown in Fig. 7Go, vehicle-treated cells were rounded and stained diffusely with hematoxylin, indicating uncondensed chromatin and healthy cells. In contrast, cells treated with 500 µg/ml TF5 showed clearly condensed chromatin, a significant loss of cell size and blebbing of the plasma membrane, all distinct characteristics of apoptosis or programmed cell death. Thus, in addition to suppressing cell proliferation, TF5 also apparently stimulates apoptosis in these cells.


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  		Table 1. Effects of TF5 on C6 glioma and MMQ pituitary adenoma cell numbers and viability following 72-h incubations

 


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  		Figure 7. TF5 induces apoptotic morphology in MMQ cells. Cultured MMQ cells (1 x 106 cells/flask) were exposed to either vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or TF5 (500 µg/ml) for 72 h. Following culture, cells were resuspended in 95% ethanol, dried and stained with hematoxylin and eosin. The data are representative of five separate experiments.

 
HPLC fractionation of TF5
We tested all currently available purified and synthetic thymosin peptides on C6 and MMQ cell growth. T{alpha}1, Tß4, MB-35 and MB-40 (all 1 µM) had no effect on either C6 glioma cell proliferation following a 72-h incubation (Fig. 8Go) or MMQ cells during a 96-h incubation (data not shown). Because these TF5 peptide constituents had no effect on C6 and MMQ cells, we analyzed the effects of HPLC-separated TF5 (Fig. 1Go) on MMQ cell proliferation. Of the 60 fractions examined, only fractions 17, 25, 26, and 27 consistently inhibited MMQ cell growth (Fig. 9Go). Compared with fractions 25–27, fraction 17 maximally inhibited MMQ cell proliferation at the lowest final protein concentration (i.e., 60 µg/ml).



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  		Figure 8. Effect of purified thymosin peptides on C6 glioma cell proliferation in vitro. Cultured C6 cells (160 x 103 cells/well) were exposed to either vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or TF5 (500 µg/ml), T{alpha}1, Tß4, MB-35 or MB-40 (all 1 µM) for 72 h. Only 500 µg/ml TF5 significantly suppressed cell proliferation (P < 0.01). The data are expressed as the mean ± SEM of groups consisting of four observations.

 


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  		Figure 9. Effect of HPLC-purified TF5 fractions on MMQ cell proliferation. Cultured MMQ cells (25 x 103 cells/well) were exposed to either vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics), 500 µg/ml TF5, or HPLC fractions of TF5 (1–60) for 4 days. Compared with vehicle (0.712 ± 0.0142), HPLC fractions 17, 25, 26, and 27 consistently inhibited (P < 0.01) MMQ cell proliferation in vitro. For comparison, treatment of MMQ cells with TF5 (500 µg/ml) resulted in a MTT assay result of 0.035 ± 0.0012. The data are expressed as the mean of three separate experiments. For clarity, the SEM are not shown, but in all cases were less than 10% of the mean. The MTT optical density units are given as filled diamonds (continuous line) and final protein concentrations of the individual fractions are displayed as open triangles (broken line).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The bovine thymic preparation TF5 is an immunologically active group of small peptides that additionally affects neuroendocrine function. This preparation stimulates various aspects of T cell-mediated immunity including increased mixed lymphocyte responses and increased responses to T cell mitogens (2, 20, 21). Injections of TF5 potentiated the ability of an interleukin cocktail administered in vivo to enhance the recovery of function in vitro of lymphocytes obtained from glucocorticoid-treated mice (22). This preparation also tended to normalize thymic weight and thymocyte subpopulation abnormalities associated with severe uremia (23). Several peptides have been isolated from TF5, including T{alpha}1 and Tß4. T{alpha}1 enhances natural killer cell activity in tumor bearing mice (24) and is an effective agent in combination therapy for the treatment of lung carcinoma in mice (25). TF5 and T{alpha}1 both increase the production of a B cell growth factor [BCGF-12kDa] and IL-2 in human peripheral blood mononuclear cells (26). We have previously reported that TF5 also increases IL-6 production in mitogen-activated rat spleen cell cultures (21).

In addition to these effects on the immune system, TF5 stimulates neuroendocrine hormone production (2). Thus, this preparation enhanced PRL and GH release from rat anterior pituitary cells following a 30 min incubation (10). Subsequent studies revealed that a 35-amino acid basic peptide, termed MB35, is the active component in TF5 that stimulates PRL and GH release from rat anterior pituitary cells in vitro (11, 12). Injections of TF5 lead to increased levels of glucocorticoids in rodents (4) and primates (5) via the probable stimulation of hypothalamic CRH release (6). Because other immune system products such as IL-1 and IL-6 also stimulate pituitary hormone release (reviewed in Ref.1) and affect the proliferation of pituitary cells (13, 14, 15), we hypothesized that TF5 may regulate the proliferation of pituitary adenoma and glioma cells.

Despite the fact that TF5 generally stimulates lymphocyte proliferation (20, 21), this preparation suppressed both actual cell counts as well as MTT assay optical density units in MMQ pituitary adenoma and C6 glioma cell cultures. These effects were rapid and time-dependent: within 24 h MMQ cell numbers and MTT-derived optical density units were significantly depressed in TF5-treated cultures compared with vehicle-treated control cells. Using high concentrations of TF5 (e.g. 300–500 µg/ml), the suppression of MMQ cell proliferation remained for up to 96 h. Depending on the initial cell density, concentrations as low as 100 µg/ml significantly inhibited MMQ cell growth. In contrast, C6 glioma cells were apparently more sensitive than MMQ cells to the antiproliferative effects of TF5: as little as 10 µg/ml of this preparation significantly reduced C6 cell growth. These differences are reflected in the estimated ED50 of TF5 for the inhibition of MMQ and C6 cell proliferation (i.e. 200 and 50 µg/ml TF5, respectively).

Interestingly, the effects of TF5 on both MMQ and C6 cell proliferation were partially reversed when the initial cell densities were increased. For example, 100 and 200 µg/ml TF5 did not significantly inhibit the proliferation of 50 x 103 MMQ cells after 4 days. Low concentrations of TF5 (e.g. 10 µg/ml) were similarly less effective at higher plating densities of C6 glioma cells (i.e. 30 x 103 cells/well). Because the effects of low concentrations of TF5 were also increasingly less dramatic with time, we proposed that an anti-TF5 activity accumulates in these neuroendocrine tumor cell cultures. We found that 50% conditioned medium obtained from MMQ cell cultures largely reversed the antiproliferative actions of TF5. Although PRL is a mitogenic hormone affecting a variety of cell types including Nb2 lymphoma cells (27) and is released by MMQ cells (16), this hormone did not reverse the effects of TF5. In fact, PRL induced a modest potentiation of the TF5-mediated inhibition of MMQ cell proliferation. Thus, the MMQ cells apparently release an unidentified activity that antagonizes TF5 suppression of cell growth.

The antiproliferative activity in TF5 is undoubtedly due to one or more peptide components because salting out of the thymic peptides using ammonium sulfate is used during the purification procedure for TF5. In addition, TF5 is essentially free of nucleic and fatty acids (20). Peptides previously isolated from TF5 (i.e. T{alpha}1, Tß4, MB35, MB40) had no demonstrable effect on either MMQ or C6 cell proliferation. Despite previous reports documenting the effects of cytokines on pituitary cell proliferation (13, 14, 15), neither IL-1ß nor IL-6 had any effect on MMQ cell proliferation (data not shown). Thus, TF5 was further purified using HPLC: only 4 of 60 total HPLC fractions exhibited significant antiproliferative activity. Of these, fraction 17 maximally inhibited MMQ cell growth at the lowest protein concentration. It is noteworthy that none of the previously purified thymosin peptides elute in either fractions 17, 25, 26, or 27. Because fraction 17 was the most potent and was separated from the protein peak (which was centered at fraction 25), we are using fraction 17 as starting material for the further purification of this growth inhibiting activity.

In addition to its growth inhibitory effects, we have also shown that TF5 stimulates a significant loss of cellular viability in the MMQ and C6 cultures. Morphological analysis revealed that this loss may be due to the induction of the apoptotic death process in the MMQ cells. Recent studies have shown that growth inhibition and induction of apoptosis are separable phenomena and may be mediated by different pathways (28). A constituent of TF5, Tß10, has been reported to affect the apoptotic program. Thus, overexpression of Tß10 in transfected NIH 3T3 fibroblasts predisposes these cells to enter apoptosis (29). Although the role of Tß10 in the present study is unclear, the results suggest that certain thymosin peptides may function as tumor suppressors. We suggest that circulating thymic peptides may engage in an immune surveillance program to inhibit the proliferation of neoplastic cells. For example, immunoreactive T{alpha}1 is detectable in the circulation (20) and nude mice bearing non-small cell lung carcinoma tumors have a reduction of 75% in tumor volume following T{alpha}1 injections (30). In the case of a developing pituitary adenoma, a circulating thymic peptide may gain access to the sinusoids of this tissue via the typical vascular feedback pathways. However, a malignant glioma requires that a tumor-suppressing thymic peptide cross the blood-brain barrier. Although no thymic peptide has been reported to gain access to the CNS from the circulation, certain cytokines (e.g., IL-1{alpha}, TNF{alpha}) apparently possess specific, saturable transport systems at the blood-brain barrier (31, 32). Alternatively, thymosin peptides may be produced within neuroendocrine tissues (33, 34, 35), providing an autocrine or paracrine mechanism for regulation of cell growth in the pituitary and CNS. Thus, a novel thymosin peptide may inhibit C6 glioma and MMQ cell proliferation by the induction of apoptosis. Future work will be directed toward the isolation and characterization of this anti-tumorigenic thymic activity as well as its mechanism of action.


   Footnotes
 
1 This work was supported by grants to B.L.S. (NIH DK-42059) and M.B. (Alpha-1 Biomedicals, Inc.), and NIH intramural funds provided to F.M.H. Back

Received August 25, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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