Pituitary Tumor Cell Proliferation Research Articles

Loss of 15-hydroxyprostaglandin dehydrogenase indicates a tumor suppressor role in pituitary adenomas

15-hydroxyprostaglandin dehydrogenase (15-PGDH) may function as a tumor suppressor that antagonizes the action of the cyclooxygenase-2 (COX-2) oncogene in several types of tumors. However, it is unknown if it has a role in the pituitary. Recently, our group found that 15-PGDH expression was low in prolactin (PRL) secreting adenomas (prolactinomas) and growth hormone (GH) secreting adenomas (GHomas) using fiber-optic BeadArray technology. In this study, we examined the relative expression of 15-PGDH and COX-2 mRNA in clinical specimens and examined the effects of 15-PGDH on GH3 rat pituitary tumor cell proliferation, apoptosis and hormone secretion. 15-PGDH expression was lower and COX-2 expression was higher in prolactinomas and GHomas compared with normal controls. Overexpressed 15-PGDH inhibited tumor cell proliferation and induced apoptosis. It had a significant suppressive effect on mRNA levels and on the secretion of PRL and GH in GH3 cells. The inhibition of cell proliferation was accompanied by the decreased expression of cox-2, matrix metalloproteinase-9 (MMP-9) and B cell leukemia/lymphoma-2 (Bcl-2). These data are suggestive of a previously unrecognized pathway in pituitary tumorigenesis, and this novel observation may shed light on therapeutic strategies for pituitary tumors.

Function of a novel SOX5 splicing isoform expressed in mouse brain during sleep

MiR-132, miR-15a and miR-16 have been implicated in the pathogenesis of many types of cancer, including pituitary tumors. However, the molecular mechanism of these miRNAs in pituitary tumor growth and metastasis is still unclear. Here, we showed that miR-132 and miR-15a/16 were less expressed in pituitary tumor cell lines, as well as in invasive pituitary tumor tissues, compared to non-invasive tumor tissues. We described that overexpression of miR-132 and miR-15a/16 resulted in the suppression of pituitary tumor cell proliferation, migration and invasion, respectively, and also inhibits the expression of proteins involved in Epithelial to Mesenchymal Transition (EMT). Then, we show that these miRNAs synergistically target Sox5 in pituitary tumor. Moreover, we found that Sox5 overexpression partially rescued miR-132, miR-15a and miR-16-mediated inhibition of cell migration, invasion and cell growth. Finally, we confirmed that Sox5 was upregulated in invasive pituitary tumor tissues, compared to non-invasion tissues. In conclusion, our data indicate that miR-132 and miR-15a/16 act as tumor suppressor genes in pituitary tumor by directly targetting Sox5, and imply that these miRNAs have potential as therapeutic targets for invasive pituitary tumor.

GH3B6 Pituitary Tumor Cell Proliferation is Mediated by PKCα and PKCε via ERK 1/2-dependent Pathway

Background: In this report, we explored the role of PKCα and PKCε as mediators of phorbol 12-myristate13-acetate (PMA)-induced proliferation in pituitary tumor GH3B6 cells, and determined if the ERK1/2 and Akt pathways were activated. Methods: The GH3B6 cell proliferation was estimated by BrdU incorporation and the cell cycle progression by flow cytometric cell cycle analysis. We determined the expression of PKCα and PKCε in membrane and cytosolic fractions by western blotting. The subcellular redistribution of both PKC isozymes was analyzed by confocal microscopy. Results: Incubation with PMA for 15 min stimulated PKCα and PKCε activation, which was correlated with the phosphorylation of ERK1/2 but not Akt. The activation of both these PKC isozymes was closely associated with the stimulation of proliferation and the cell cycle progression induced by PMA in GH3B6 cells, an effect that was blocked by the inhibitors of PKCα (Gö6976) and PKCε (εV1-2). In addition, the pretreatment with the inhibitor of ERK1/2 (PD98059) prevented the mitogenic activity induced by treatment with PMA for 15 min. Conclusion: We demonstrated that the activation of PKCα and PKCε by phorbol ester in tumor pituitary GH3B6 cells led to cell proliferation and cell cycle progression, effects that involved ERK1/2 activation.

Folate receptor α regulates cell proliferation in mouse gonadotroph αT3-1 cells

We have previously found that the mRNA and protein levels of the folate receptor alpha (FRα) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FRα has not fully been determined. We investigated the effect of FRα over-expression in the mouse gonadotroph αT3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FRα were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FRα promotes cell proliferation. These effects were abrogated in the same αT3-1 cells when transfected with a mutant FRα cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FRα over-expressing cells. In summary, our data suggests that FRα regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

Nongenomic actions of estradiol compared with estrone and estriol in pituitary tumor cell signaling and proliferation

Physiological estrogens, including estrone (E(1)), estradiol (E(2)), and estriol (E(3)), fluctuate with life stage, suggesting specific roles for them in biological and disease processes. We compared their nongenomic signaling and functional actions in GH3/B6/F10 rat pituitary tumor cells. All hormones caused prolactin release at 1 min; the lowest effective concentrations were 10(-11) M E(2), 10(-10) M E(1), and 10(-7) M E(3). All estrogens increased the oscillation frequency of calcium (Ca) spikes, with the same time delay (approximately 200 s) at all levels (10(-15) to 10(-9) M). At some concentrations, E(1) and E(3) provoked more Ca-responding cells than E(2). The amplitude and volume of Ca peaks were elevated by all hormones at > or = 10(-15) M. All hormones caused cell proliferation, with the lowest effective concentrations of E(2) (10(-15) M) > E(1) (10(-12) M) > E(3) (10(-10) M); E(2) caused higher maximal cell numbers at most concentrations. All estrogens caused oscillating extracellular-regulated kinase (ERK) activations, with relative potencies of E(1) and E(2) > E(3). All estrogens were ineffective in activation of ERKs or causing proliferation in a subline expressing low levels of membrane estrogen receptor-alpha. Dose-response patterns were frequently nonmonotonic. Therefore, the hormones E(1) and E(3), which have been designated "weak" estrogens in genomic actions, are strong estrogens in the nongenomic signaling pathways and functional responses in the pituitary.

Curcumin (Diferuloylmethane) Inhibits Cell Proliferation, Induces Apoptosis, and Decreases Hormone Levels and Secretion in Pituitary Tumor Cells

Prolactinomas are the most prevalent functional pituitary adenomas. Dopamine D2 receptor (D2R) agonists, such as bromocriptine are the first line of therapy; however, drug intolerance/resistance to D2R agonists exists. Apart from D2R agonists, there is no established medical therapy for prolactinomas; therefore, identifying novel therapeutics is warranted. Curcumin, a commonly used food additive in South Asian cooking, inhibits proliferation of several tumor cell lines; however, its effect on pituitary tumor cell proliferation has not been determined. Our objectives were to: 1) determine whether curcumin inhibits proliferation of pituitary tumor cell lines; 2) identify the signaling intermediaries that mediate the effect of curcumin; 3) examine whether curcumin inhibited pituitary hormone production and release; and 4) examine whether curcumin could enhance the growth-inhibitory effect of bromocriptine. Using rat lactotroph cell lines, GH3 and MMQ cells, we report that curcumin had a robust dose and time-dependent inhibitory effect on GH3 and MMQ cell proliferation. Inhibitory effects of curcumin persisted, even on removal of curcumin, and curcumin also blocked colony formation ability of pituitary tumor cells. The growth-inhibitory effect of curcumin was accompanied by decreased expression of cyclin D3 and ser 780 phosphorylation of retinoblastoma protein. In addition, curcumin also induced apoptosis in both GH3 and MMQ cells. Furthermore, curcumin suppresses intracellular levels and release of both prolactin and GH. Finally, we show that low concentrations of curcumin enhanced the growth-inhibitory effect of bromocriptine on MMQ cell proliferation. Taken together we demonstrate that curcumin inhibits pituitary tumor cell proliferation, induces apoptosis, and decreases hormone production and release, and thus, we propose developing curcumin as a novel therapeutic tool in the management of prolactinomas.

HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells

Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the β-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms.

Expression of galectin-3 correlates with apoptosis in pituitary adenoma cells

To investigate the expressions of Galectin-3 (Gal-3), Bcl-2 and Bax in human pituitary adenomas, and to explore the interrelation among them. RT-PCR and immunohistochemistry were applied to detect the mRNA and protein expressions of Gal-3, Bcl-2 and Bax in surgically excised human pituitary adenoma tissues, including invasive and non-invasive pituitary adenomas, and the correlation analysis was performed. The Gal-3 and Bcl-2 expressions in the invasive pituitary group were significantly higher than those in the non-invasive group, and the expression of Bax had no significant difference between the two groups. Pearsonos correlation analyses showed that the Gal-3 expression was positively correlated with Bcl-2, but was not correlated with Bax, which was inversely correlated with expression of Bcl-2. Gal-3 may function through a cell death inhibition pathway involving Bcl-2 to enhance cell proliferation, which result in the invasive growth of pituitary adenoma. These results indicate that Gal-3 has an important role in pituitary tumor cell proliferation and may serves as a possible therapeutic target in treatment of pituitary tumors.

α1-Adrenergic Receptor Antagonists: Novel Therapy for Pituitary Adenomas

Pituitary tumors are common and cause considerable morbidity due to local invasion and altered hormone secretion. Doxazosin (dox), a selective alpha(1)-adrenergic receptor antagonist, used to treat hypertension, also inhibits prostate cancer cell proliferation. We examined the effects of dox on murine and human pituitary tumor cell proliferation in vitro and in vivo. dox treatment inhibited proliferation of murine pituitary tumor cells, induced G(0)-G(1) cell cycle arrest, and reduced phosphorylated retinoblastoma levels. In addition, increased annexin-fluorescein isothiocyanate immunoreactivity and cleaved caspase-3 levels, in keeping with dox-mediated apoptosis, were observed in the human and murine pituitary tumor cells, and dox administration to mice, harboring corticotroph tumors, decreased tumor growth and reduced plasma ACTH levels. dox-mediated antiproliferative and proapoptotic actions were not confined to alpha-adrenergic receptor-expressing pituitary tumor cells and were unaffected by cotreatment with the alpha-adrenergic receptor blocker, phenoxybenzamine. dox treatment led to reduced phosphorylated inhibitory kappaB (IkappaB)-alpha expression, and nuclear factor-kappaB transcription and decreased basal and TNFalpha-induced proopiomelanocortin transcriptional activation. These results demonstrate that the selective alpha(1)-adrenergic receptor antagonist dox inhibits pituitary tumor cell growth in vitro and in vivo by mechanisms that are in part independent of its alpha-adrenergic receptor-blocking actions and involve down-regulation of nuclear factor-kappaB signaling. dox is proposed as a possible novel medical therapy for pituitary tumors.

Tamoxifen-induced growth arrest and apoptosis in pituitary tumor cells in vitro via a protein kinase C-independent pathway

Protein kinase C (PKC), a kinase family involved in cell signal transduction, is overexpressed in most pituitary adenoma cells. We studied the effect of tamoxifen, an estrogen receptor antagonist and also a protein kinase inhibitor, on pituitary tumor cell proliferation and the induction of apoptosis; and we compared its effects with those of another PKC inhibitor, staurosporine. Tamoxifen induced growth arrest and apoptosis in a mouse pituitary adenoma cell line, AtT20, and in low-passage human primary pituitary tumor cell cultures. Staurosporine also inhibited pituitary tumor cell growth. PKC activity in AtT20 cells was inhibited by staurosporine and by prolonged treatment with phorbol myristate acetate, which down-regulates PKC activity, but not by tamoxifen, at the dosages used to induce apoptosis. Our findings suggest that tamoxifen induces apoptosis in AtT20 cells independent of a classical PKC isozyme pathway.

Cyclosporin A, but not FK506, increases arachidonic acid release and inhibits proliferation of pituitary corticotrope tumor cells

The selective immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) are used in the prevention of allogenic transplant rejection and in the therapy of chronic autoimmune inflammatory pathologies. Chronic treatment with CsA leads to secondary functional and trophic alterations of multiple organs and cell systems among which endocrine ones, through insofar uncharacterized mechanisms. With the recent use of FK506 there have been reports of an improved therapeutic efficacy and a reduction of side-effects, as compared to CsA. An intriguing hypothesis is that toxic damage could be due to a systemic CsA activation of arachidonic acid (AA) metabolism, through pathways as yet only partially characterized. The side-effects of both drugs have been poorly studied on cells from tissues other than blood or kidney. We have thus proceeded to study their action on AA release in corticotropic AtT-20/D16-16 cells. The results obtained are as follows: 1) during incubation times ≥12 h, basal AA release is increased by CsA, but not FK506; the acute effect (10 min) of melittin, a PLA 2 activator, is significantly potentiated starting from a 30 min pretreatment with CsA but not FK506; manoalide, a PLA 2 inhibitor, antagonizes the melittin potentiation of AA release by CsA whereas the inhibition of the melittin stimulus by glucocorticoids is antagonized both by CsA and FK506. 2) during longer (>2 d) incubation times, cell growth is inhibited by CsA but not FK506. These results indicate a role for CsA, not apparent for FK506, in the activation of PLA 2 and in the inhibition of cell growth. They also suggest that CsA does not have a direct (i.e. not mediated by the immune system) therapeutic effect in inflammatory processes.

Effects of long term treatment with corticotropin releasing factor on corticotropic tumor cells in vitro

hCRF inhibits proliferation of corticotropic tumor cells cultivated in serum-reduced medium via interaction with CRF-receptors. This effect was attenuated by the specific antagonist hCRF (9-41), but not by a variety of substances which are inhibitors of cAMP production or cAMP-dependent kinases, suggesting that the effect was not mediated via cAMP. The growth inhibiting effect of hCRH was developed after 4 h incubation, a longer hCRF treatment did not change the effect observed after 4 h. Simultaneously, after hCRF treatment for 4 days the cells were insensitive for ACTH release by hCRF stimulation despite of an increase in the number of secretory granules. The results show that the inhibition of proliferation of pituitary tumor cells by hCRF seems to be a rapid receptor-mediated process connected with morphological changes, but not mediated via activation of adenylate cyclase.

Tumor-specific expression of alternatively spliced estrogen receptor messenger ribonucleic acid variants in human pituitary adenomas.

The well documented mitogenic and hormone regulatory effects of estrogen (E2) on pituitary cells are mediated via its nuclear receptor (ER), a cellular homolog of v-erbA oncogene. ER isoforms generated by alternative exon splicing, termed ER variants delta 2ER to delta 7ER, have been identified in breast cancer and have been postulated to have important pathogenetic and clinical implications in tumorigenesis and/or development of hormone resistance. Because pituitary tumors, particularly prolactinomas, are known to be E2-dependent, we investigated alternatively spliced ER variant messenger ribonucleic acid expression in 40 human pituitary tumors of various phenotypes and normal pituitary tissues, using reverse transcription-PCR and Southern blot analyses. Nine of 11 prolactinomas readily expressed multiple ER variants (delta 2ER, delta 4ER, 5ER, and delta 7ER), whereas 6 of 11 tumors showed faint expression of delta 3ER. Four of 7 glycoprotein hormone-producing tumors that synthesized FSH beta expressed delta 2ER, delta 5ER, and delta 7ER. In 9 GH- and 10 ACTH-secreting tumors examined, the expression of normal and variant ER was restricted to tumors that also exhibited scattered PRL immunoreactivity. Variant and normal ER were not found in three null cell tumors (oncocytomas) that showed negative immunoreactivity for all pituitary hormones or their subunits. In contrast, only delta 4ER and delta 7ER were uniformly detected in normal pituitaries. delta 6ER was not detected in any normal or neoplastic pituitary specimen studied. We conclude that multiple alternatively spliced ER variants are coexpressed with normal ER in a tumor phenotype-specific manner. In addition, ER variants delta 2ER and delta 5ER were found to be tumor specific. Future functional studies will be required to determine whether coexpression of multiple ER variants along with normal ER confers a pathophysiological role in pituitary hormone regulation and/or tumor cell proliferation.

Somatostatin-14 and its analog octreotide exert a cytostatic effect on GH3 rat pituitary tumor cell proliferation via a transient G0/G1 cell cycle block.

Somatostatin (SRIF) and its analogs exert potent inhibitory effects on hormonal hypersecretion. In addition, they have been demonstrated to inhibit the proliferation of various cell lines as well as the growth of some endocrine tumors in vivo. To evaluate the action of SRIF and its analog octreotide on the proliferation and cell cycle kinetics of endocrine cells, we investigated their effect on GH3 rat pituitary tumor cells, a GH-producing cell line. Using flow cytometric DNA analysis with propidium iodide staining, we found that octreotide inhibits the proliferation of synchronized GH3 cells, achieving a maximal reduction, compared to controls, of 19.4 +/- 5.3% and 22.4 +/- 5.1% with 100 ng/ml and 1000 ng/ml octreotide, respectively (P < 0.05). This effect was demonstrated to be due to a block in progression from the G0/G1 phase to the S phase of the cell cycle. This was most evident after 24 h of exposure to 100 ng/ml octreotide, at which time there was a 7.1 +/- 1.4% increase in cells in G0/G1 (P < 0.01) and a 6.6 +/- 1.3% decrease in cells in S phase (P < 0.01). However, unless octreotide was replenished, this effect was transient and overcome by 36-48 h. No apoptosis was seen, and trypan blue studies confirmed that cell death by necrosis did not occur. A single exposure to native SRIF-14 had little effect, but a G0/G1 cell cycle block and inhibition of proliferation were seen if SRIF was regularly replenished. We conclude that SRIF and octreotide exert a cytostatic effect on GH3 cells by causing a partial G0/G1 cell cycle block. These findings suggest that the actions of SRIF and octreotide occur through signal transduction pathways that act predominantly on downstream regulators.

Somatostatin-14 and its analog octreotide exert a cytostatic effect on GH3 rat pituitary tumor cell proliferation via a transient G0/G1 cell cycle block

Somatostatin-14 and its analog octreotide exert a cytostatic effect on GH3 rat pituitary tumor cell proliferation via a transient G0/G1 cell cycle block

Inhibition of rat pituitary tumor cell proliferation by benzodiazepines in vitro.

The effect of various benzodiazepines (peripheral-type receptor ligands: Ro 5-4864, PK 11135; central-type receptor ligands: clonazepam, Ro 15-1788, Ro 15-4513; mixed type: diazepam) on the proliferation of estrogen-induced rat pituitary prolactin-secreting tumor cells was studied in vitro. [3H]thymidine incorporation into DNA was used as an index of cell proliferation. It was found that tested peripheral- and mixed-type benzodiazepine receptor ligands significantly suppressed the pituitary cell proliferation in a dose-dependent manner (10(-4)-10(-8) M). The inhibitory effect of Ro 5-4864 was reversed by 5 x 10(-3) M calcium chloride. On the other hand, central-type benzodiazepine receptor ligands suppressed tumor cell proliferation only at the highest concentration studied (10(-4) M). Our results indicate that benzodiazepines might exert an antiproliferative action on pituitary tumor cell growth, and that this effect seems to be a calcium-dependent process.

Regulation of prothymosin alpha mRNA levels in rat pituitary tumor cells.

Prothymosin alpha (PTA) mRNA and histone H4 (H4) mRNA levels were studied in various experimental conditions that affected GH1 pituitary tumor cell proliferation. Cell proliferation and progression through the cell cycle was assessed by counting cells, 3H-thymidine incorporation and flow cytometry. PTA mRNA levels were decreased in a time-dependent fashion following serum deprivation; when the cells were induced to grow by serum refeeding, PTA mRNA expression was greatly stimulated. Interestingly, after caprylic acid treatment (2.5 mM for 24 h) that arrested cells in the G0/G1 phase of the cell cycle, PTA mRNA and H4 mRNA levels were almost undetectable; conversely, following caprylic acid withdrawal, PTA mRNA and H4 mRNA expression were greatly stimulated. Furthermore, cells cultured in T3-deprived serum, which was found to decrease GH1 cell proliferation, had low levels of PTA and H4 mRNAs. This effect was reversed by the addition of nanomolar concentrations of T3 to the culture. On the other hand, IGF-1 addition to the culture did not substantially modify PTA mRNA levels. The present data clearly indicate that PTA mRNA expression is tied to the proliferating activity of GH1 cells and, thus, could be used as a marker of the action that various agents have on GH1 cell proliferation.

Dissociation of antiproliferative and antihormonal effects of the somatostatin analog octreotide on 7315b pituitary tumor cells.

The somatostatin (SS) analog octreotide has been successfully used in the treatment of (neuro)endocrine tumors. The mechanism of action of the tumor (growth) inhibitory action by octreotide is not fully understood. We have investigated the effect of octreotide on 7315b rat pituitary tumor cell growth, PRL release, and intracellular PRL concentrations in vitro. When cultured in medium with 10% fetal calf serum, the number of high affinity SS receptors increased with increasing culture time. On days 7, 14, and 21 of culture, the number of SS receptors amounted to 978 +/- 217, 3588 +/- 705, and 5865 +/- 3332 fmol/mg protein, respectively, whereas they were not measurable on day 0. From days 0-7, 7-14, and 14-21 of culture, octreotide (1 pM to 1 microM) inhibited PRL release and the intracellular PRL concentration, with IC50 values in the nanomolar range. However, no inhibition of cell growth was observed by these octreotide concentrations from day 0-7 of culture, while octreotide inhibited cell growth in a dose-dependent fashion from days 7-14 and 14-21 of culture (maximal inhibition by 25% and 26%, respectively). In a series of nine consecutive experiments we found a significant positive correlation between the percent inhibition of cell growth induced by 1 microM octreotide and the number of SS receptors on 7315b cells (r = 0.7865; P = 0.012). Inhibition of PRL release did not correlate with SS receptor numbers. Octreotide (1 microM) inhibited forskolin (0.5 microM)-stimulated cell growth and intracellular PRL concentrations, while in the presence of a high concentration of forskolin (10 microM), octreotide had no effect on forskolin-stimulated cell growth and intracellular PRL concentrations. In addition, its PRL release inhibitory effect was significantly lower in forskolin-stimulated cultures. Pretreatment of the cells with pertussis toxin (10 micrograms/liter) completely prevented the inhibition of cell growth by octreotide and diminished the inhibitory effect of octreotide on PRL release. Finally, 1 microM octreotide significantly inhibited forskolin-stimulated cAMP production (by 29% and 53% on days 7 and 14 of culture, respectively). We conclude that 1) octreotide inhibits 7315b rat pituitary tumor cell proliferation via a pertussis toxin-sensitive GTP-binding protein- and adenylate cyclase-dependent mechanism; and 2) the number of SS receptors on 7315b pituitary tumor cells may determine whether octreotide exerts a direct antiproliferative effect, whereas its antihormonal effect occurs in the presence of relatively low numbers of SS receptors. This suggests a dissociation of the antiproliferative and antihormonal effects induced by octreotide.

Bryostatins selectively regulate protein kinase C-mediated effects on GH4 cell proliferation

The phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate [TPA) or phorbol 12-myristate 13-acetate), directly activates the calcium- and phospholipid-dependent protein kinase C (protein kinase C), which, in turn, generates a number of cellular responses. The bryostatins, a family of macrocyclic lactones isolated from marine bryozoans, also bind to and active protein kinase C. However, they differ from TPA in the selectivity of their responses in that they behave either as agonists or antagonists of protein kinase C actions. We used several bryostatins and TPA to examine the role of protein kinase C in the regulation of GH4C1 rat pituitary tumor cell proliferation. TPA inhibited [3H]thymidine incorporation in GH4 cells in a stereoselective and concentration-dependent manner. Examination of cell cycle distribution by flow cytometry revealed that TPA decreased the percentage of cells in S-phase and proportionally increased the percentage of G1-phase cells. Bryostatin 1 alone did not affect cell proliferation, but prevented the TPA inhibition of cell proliferation. Bryostatin 1 treatment from 30 min to 6 h after TPA treatment also prevented the growth-inhibitory action of TPA, suggesting that prolonged stimulation of protein kinase C is necessary for growth inhibition. Both bryostatin 1 and TPA down-regulated protein kinase C, indicating that down regulation of the enzyme cannot account for the growth inhibitory action of TPA. Bryostatin 2, which differs from bryostatin 1 by a hydroxyl substitution for the acetyl group at the C-7 carbon of the macrocyclic lactone ring (R1), inhibited cell proliferation and did not reduce the growth-inhibitory action of TPA. Bryostatins 3 and 8 (each of which has an ester group in the R1 position, yet contains other structural modifications) are antagonists for TPA inhibition of GH4 cell proliferation like bryostatin 1. We next examined the effect of bryostatins 3 and 8 on cell-substratum adhesion, a cellular response observed after GH4 cells are treated with growth-inhibitory agents. Bryostatin 8 (like bryostatin 1) did not enhance cell-substratum adhesion and blocked the action of TPA. In contrast, bryostatin 3 enhanced cell-substratum adhesion. Because bryostatin 3 blocked TPA inhibition of cell proliferation, yet did not block TPA-enhanced cell-substratum adhesion, these responses are not interdependent. We next examined the effect of bryostatin on other growth-inhibitory agents for GH4 cells. Bryostatin 8 blocks the effect of TPA on [3H]thymidine incorporation and the entry of G1 cells into S-phase, but does not block the growth-inhibitory action of thyrotropin-releasing hormone or epidermal growth factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Transforming Growth Factor-a and -0 Are Potent and Effective Inhibitors of GH4 Pituitary Tumor Cell Proliferation*

The mechanisms that restrict cell proliferation play an important regulatory role in differentiation and tumorigenesis. The growth of PRL-secreting cells of the anterior pituitary is known to be highly estrogen dependent; however, estrogen may act indirectly via growth regulatory polypeptides. We have used the GH4C1 rat pituitary cell line to investigate the action of two classes of growth regulatory polypeptides, transforming growth factor-alpha (TGF alpha) and TGF beta. TGF alpha and TGF beta each inhibit GH4 cell proliferation, as measured by cell number and [3H]thymidine incorporation, and given together arrest GH4 cell proliferation. The growth inhibitory action of TGF alpha is concentration dependent (IC50 = 100 pM) and saturable. Activin-A, a TGF beta-related polypeptide, also inhibits proliferation, but is less effective than TGF beta. TGF alpha and TGF beta each alter GH4 cell cycle distribution by decreasing in the percentage of S phase cells (74% and 34%, respectively) and increasing proportionally G0-G1 phase cells. The growth inhibitory action of TGF alpha differs from that of TGF beta in that TGF alpha also causes a temporary accumulation of cells in G2-M phases. We next initiated experiments to evaluate the role of protein kinase-C in the growth inhibitory actions of TGF alpha and TGF beta. The alpha- and beta-isoforms of protein kinase-C were down-regulated by pretreatment with 12-O-tetradecanoylphorbol-13-acetate, yet TGF alpha and TGF beta still substantially inhibited GH4 cell proliferation. We next compared the actions of TGF alpha and TGF beta on two other well characterized prolonged GH4 responses. TGF alpha and TGF beta each increased GH4 cell adhesion, but differed in their effects on PRL production. This indicates that TGF alpha and TGF beta activate different signaling pathways in GH4 cells. Activin-A acted like TGF beta by enhancing cell-substratum adhesion and inhibiting PRL production, consistent with an interaction at a common receptor site. Taken together these results identify biological functions for TGF alpha, TGF beta, and activin-A on PRL cells and open the possibility that they may represent the direct in vivo mediators of estrogen action to regulate the growth of PRL cells in the anterior pituitary gland.