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Molecular Endocrinology, doi:10.1210/me.2005-0001
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Molecular Endocrinology 20 (9): 2010-2019
Copyright © 2006 by The Endocrine Society

Autocrine/Paracrine Regulation of Breast Cancer Cell Proliferation by Growth Hormone Releasing Hormone via Ras, Raf, and Mitogen-Activated Protein Kinase

G. Siriwardana, A. Bradford, D. Coy and P. Zeitler

Departments of Pediatrics (G.S.) and Obstetrics and Gynecology (A.B., P.Z.), University of Colorado Health Sciences Center, Denver, Colorado 80262; and Department of Medicine (D.C.), Tulane University School of Medicine, New Orleans, Louisiana 70146

Address all correspondence and requests for reprints to: Dr. Philip S. Zeitler, Children’s Hospital/University of Colorado, Division of Endocrinology, 1056 19th Avenue, Denver, Colorado 80218. E-mail: zeitler.philip{at}tchden.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although GHRH has previously been shown to regulate proliferation of breast cancer cells and prevent apoptosis, the intracellular pathways mediating this effect have not been clarified. Exogenous GHRH stimulated a dose-dependent proliferative response within 24 h in MDA-231, as well as in T47D cells and in MCF-7 cells transfected with the GHRH receptor. The proliferation of MDA-MB-231 (MDA-231) cells was associated with an increase in tritiated thymidine uptake. In addition, phosphorylation of MAPK was rapidly stimulated by GHRH. The phosphorylation of MAPK by GHRH was prevented by transfection of the cells with dominant-negative Ras or Raf or by pretreatment of cells with Raf kinase 1 inhibitor. The inhibition of Ras and Raf, as well as the inhibition of MAPK phosphorylation by PD98059, also prevented GHRH-induced cell proliferation. Finally, pretreatment of cells with the somatostatin analog, BIM23014, also prevented GHRH-induced MAPK phosphorylation and cell proliferation. These results indicate that GHRH stimulates dose-dependent cell proliferation of MDA-231 breast cancer cells through a pathway that requires Ras, Raf, and MAPK phosphorylation. The results also provide support for a possible autocrine/paracrine antagonism between GHRH and somatostatin in the regulation of MDA-231 cell population maintenance. Taken together, the studies provide further insight into the possible role of GHRH as a growth factor in breast cancer.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GHRH, SYNTHESIZED IN THE hypothalamus and secreted into the pituitary portal system, is primarily responsible for stimulation of the synthesis and release of GH. However, it is also an important trophic factor promoting development and proliferation of pituitary somatotrophs (1, 2, 3, 4, 5, 6, 7, 8). GHRH is also expressed in a limited set of other tissues, including lymphocytes, placenta, gut, kidney, thymus, testis, and breast (9, 10, 11, 12, 13, 14, 15). The role of extrahypothalamic GHRH is not known, although mitogenic activity has been reported in lymphocytes and testicular germ cells (16, 17, 18).

GHRH is also expressed in tumors of the central nervous system, lungs, intestine, breast, prostate, ovary, and endometrium (15, 19, 20, 21, 22, 23, 24). In addition, several splice variants of the GHRH receptor (GHRHr) have been isolated from rat pituitary, and human prostate, breast, ovarian, and small-cell lung cancer tissues (25, 26). Splice variants of the GHRHr in 3T3 fibroblasts specifically activate cell proliferation responses to GHRH analogs (27). Conversely, antagonists of GHRH have antitumorigenic activity in many human cell lines, including those of gastrointestinal tract, renal, prostrate, ovary, and breast (28, 29, 30, 31, 32, 33). In MDA-231 breast cancer cells, we have previously reported that antagonism of endogenous GHRH leads to apoptosis in MDA-231 breast cancer cells (34).

Information on the mechanism of mitogenic stimulation by GHRH is scant. In GH4 rat pituitary cells, we have demonstrated that stimulation of cell proliferation by GHRH is mediated by MAPK phosphorylation, and both cell proliferation and MAPK phosphorylation are inhibited by the somatostatin analog BIM23014 (35). These studies also suggested that activation of MAPK in somatotrophs is mediated by Raf and Ras proteins. Transfection of Chinese hamster ovary (CHO) cells with the GHRHr led to activation of MAPK via p21ras (Ras) (36). Furthermore, in MCF-7 and T47D breast cancer cells, BIM23014 had antitumorigenic activity (37), associated with inhibition of MAPK phosphorylation. However, little is known about the details of GHRH action as a mitogenic factor or about the specific biology of GHRH in breast cancer cells.

In this study, we demonstrate that GHRH stimulates MDA-MB-231 breast cancer cell (MDA-231 cells) proliferation in a dose- and receptor-dependent manner, that activation of MAPK is required for this mitogenic activity, that the signaling to MAPK by GHRH occurs through Ras and Raf proteins, and that this effect of GHRH is attenuated by BIM23014.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
As shown in Fig. 1Go, MDA-231 cells exhibited reduced proliferation in the presence of heat-inactivated fetal calf serum (FCS), suggesting the presence of a heat-labile serum growth factor. Addition of GHRH to heat-inactivated FCS-containing medium was sufficient to fully reconstitute proliferation of MDA-231 cells. However, addition of GHRH to untreated FCS did not further stimulate cell proliferation. This effect of GHRH on MDA-231 cell numbers in DMEM without FCS was dose dependent (Fig. 2Go), with a linear increase in cell numbers in response to logarithmically increasing doses of added GHRH. The effect of GHRH on MDA-231 cell proliferation in serum-free DMEM was highly labile over time. When GHRH (1 µM) was incubated in serum-free DMEM for varying lengths of time before addition to plated cells, the proliferative response diminished after 6-h preincubation (data not shown).


Figure 1
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  		Fig. 1. Effect of GHRH on Proliferation of MDA-231 Cells

MDA-231 cells were plated in 96-well tissue culture plates as described. Sixteen hours later, the medium was replaced with DMEM with 10% freshly thawed, or heat-deactivated FCS (30 min at 55 C) and with or without GHRH at 1 µM. The cells were counted 24 h after the treatments. The data are from a representative set of two experiments. **, P < 0.01.

 

Figure 2
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  		Fig. 2. Dose-Dependent Effect of GHRH on MDA-231 Cell Proliferation

MDA-231 cells were plated in 96-well tissue culture plates as described. The medium was replaced with DMEM and was treated with either the carrier for the GHRH or GHRH ranging from 62 nM to 1 µM. The cells were counted 24 h after the treatments. The data are from a representative set from two experiments. Values represent the mean ± SEM; n = 8 replicates at each time point for each experiment.

 
To examine the effect of GHRH on DNA synthesis, cells grown overnight as indicated were treated with GHRH (1 µM), in new serum-free medium, followed 2 h later by addition of tritiated thymidine (Fig. 3Go). Consistent with studies in pituitary GH4 cells (35), treatment of MDA-231 breast cancer cells with GHRH, resulted in a 40–50% increase in thymidine uptake relative to control over 2–6 h. Taken together, these results indicate that GHRH stimulates proliferation of MDA-231 cells.


Figure 3
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  		Fig. 3. Effect of GHRH on Thymidine Incorporation by MDA-231 Cells

Cells were plated at 20,000 cells per well in 96-well plates in 100 µl DMEM with 10% FCS and were grown overnight. Treatment with GHRH (1 µM) or vehicle alone was made 16 h after plating, in newly replaced DMEM with no FCS. Two hours after the addition of GHRH, 6 µl tritiated thymidine was added to each well. At 2, 4, and 6 h after the addition of thymidine, the wells were washed thrice with 150 µl PBS, added with 25 µl 10% trichloroacetic acid for 5 min followed by 100 µl of 0.1 M sodium hydroxide and 27.5 µl of 0.1 M hydrochloric acid. The entire content of the wells was transferred to scintillation counter tubes, and the activity was counted for 10 min/treatment. Eight wells per treatment were made during these experiments. Results from three representative experiments are shown. **, P < 0.01

 
To demonstrate directly endogenous production of GHRH by MDA-231 cells, we incubated cells with [35S]methionine and immunoprecipitated newly formed radiolabeled GHRH using an anti-GHRH antibody. As shown in Fig. 4Go, MDA-231 cells synthesize GHRH in a time-dependent fashion. Relatively little of the newly formed GHRH was isolated from the cell growth medium, although, as noted, GHRH is highly labile in serum-containing media and may have been degraded.


Figure 4
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  		Fig. 4. Endogenous Production of GHRH by MDA-231 Breast Cancer Cells

MDA-231 cells were grown in 3.5-cm dishes to 80% confluency in 3 ml DMEM/10% FCS. The medium was replaced with 3 ml methionine and cysteine-free DMEM for 2 h. Then, the medium was again changed to 0.6 ml methionine and cysteine-free DMEM with 50 µCi 35S (5 µl/plate), and the cells were incubated for 3 or 16 h. At 3 or 16 h, the growth medium was collected and treated with protease inhibitors immediately. The cells were harvested with 0.5 ml cold PBS and centrifuged for 5 min at 1000 rpm to obtain the cell pellet. The cells were lysed using 0.5 ml RIPA buffer. The cell lysate and the growth medium were subjected to immunoprecipitation with anti-GHRH or nonimmune immunoglobulin (IGG). The isolated product was resolved in a 10–20% tricine gradient gel (Bio-Rad), and the gel was dried and autoradiographed. The band that corresponds to the migration pattern of GHRH is shown.

 
We have previously demonstrated that MDA-231 and T47D cells both express mRNA for the GHRHr (34). To test the ability of GHRH to cause proliferation in another cell line expressing GHRHr, we treated T47D cells with GHRH in the absence of serum and the cells were counted. As shown in Fig. 5Go, the results of three independent experiments with different starting cell numbers, demonstrate a 20–30% increase in T47D cell number 16 h after treatment with GHRH in vitro.


Figure 5
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  		Fig. 5. Effect of GHRH on T47D Cell Proliferation

T47-D breast cancer cells were plated in 96-well tissue culture plates as described. The medium was replaced with DMEM and was treated with either the carrier for the GHRH or GHRH at 1 µM. The cells were counted 24 h after the treatments. The data are from a representative set from two experiments. Values represent the mean ± SEM; n = 8 replicates at each time point for each experiment. Values represent the mean ± SEM; n = 8 replicates. **, P < 0.01; *, P < 0.05.

 
Unlike MDA-231 and T47D cells, MCF-7 cells do not express GHRHr mRNA. However, as shown in Fig. 6Go, Western blot analysis demonstrates that MCF7 cells contain a greater amount of GHRH peptide than either MDA-231 or T47-D cells. We were, therefore, interested in determining whether GHRH produced endogenously by MCF7 cells can promote cell proliferation when the cells are transfected with the GHRHr. MCF7 cells were plated and counted 16 and 40 h after transfection with GHRHr or vector alone. As shown in Fig. 7Go, there was a 50–110% increase in cell proliferation between 16 and 40 h posttransfection in cells transfected with GHRHr compared with cells transfected with vector alone.


Figure 6
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  		Fig. 6. Relative of GHRH by Breast Cancer Cell Lines

MDA-231, T47-D, and MCF-7 cells were plated in DMEM/10% FCS in 3.5-cm tissue culture dishes. Beginning on the 16th hour, cells were harvested at 20-min intervals for 40 min using 200 µl Laemmli SDS sample buffer. Twenty-microliter samples from the lysate were resolved in 16.5% Tris tricine gel, and the proteins were transferred to Immobilon membranes and probed for human GHRH using anti-human GHRH.

 

Figure 7
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  		Fig. 7. Effect of GHRH on MCF-7 Cell Proliferation after Transfection with GHRHr

MCF-7 cells were transfected with pcDNA or pcDNA containing rat GHRHr, and were plated at 20,000 cells per well for overnight growth in DMEM/10% FCS. No exogenous GHRH was added. The cells were counted 16 and 40 h after the transfection. Values represent the mean ± SEM; n = 8 replicates. **, P < 0.01.

 
GHRH stimulation of proliferation in pituitary cells requires phosphorylation and activation of MAPK (35). To examine the role of MAPK in GHRH-induced breast cancer cell proliferation, MDA-231 cells grown overnight were washed and incubated in serum-free DMEM for 1.5 h, followed by addition of GHRH (0.1 nM to 1 µM). SDS-PAGE resolved extracts were probed with a phosphospecific MAPK antibody. As seen in Fig. 8Go, GHRH stimulated robust and rapid phosphorylation of MAPK in a time- and dose-dependent manner. No significant changes in total MAPK were noted.


Figure 8
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  		Fig. 8. Stimulation of MAPK Phosphorylation by GHRH

MDA-231 cells were grown in DMEM with 10% FCS for 16 h. At this time, the medium was replaced with DMEM with no FCS for 1.5 h and treated with different doses of GHRH ranging from 0.1 nM to 1 µM for 2.5 or 5 min and harvested in SDS sample buffer. Lysate (40 µl) was subjected to 10% SDS-PAGE, and proteins were transferred to Immobilon membranes and probed for phospho-MAPK. The blot was stripped and probed for MAPK.

 
To determine the signaling pathway involved in the stimulation of MAPK phosphorylation by GHRH, we examined the effect of specific inhibitors of Raf1 kinase (5-iodo-3[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-indolinone) and MAPK (PD98059) (2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one) (38, 39) on cell proliferation. As shown in Fig. 9Go, pretreatment of MDA-231 cells with either the Raf1 kinase or MAPK inhibitor prevented GHRH-induced cell proliferation. To further examine these pathways, MDA-231 cells were transiently transfected with constructs encoding dominant-negative forms of Ras (pZCR17N) or Raf (pRSV-Raf-C4) (40). Forty hours posttransfection, cells were serum starved for 1.5 h and subsequently treated with GHRH as described. As seen in Fig. 10AGo, dominant-negative Ras and dominant-negative Raf constructs blocked GHRH stimulation of MDA-231 cell proliferation. Furthermore, expression of dominant-negative Ras or Raf effectively eliminated the phosphorylation of MAPK caused by GHRH, although there was a small increase in basal MAPK phosphorylation in the Ras or Raf inhibited cells (Fig. 10BGo). Taken together, the inhibitor and dominant-negative experiments indicate that stimulation of MDA-231 cell proliferation by GHRH is mediated by the Ras/Raf/MAPK pathway.


Figure 9
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  		Fig. 9. Inhibition of GHRH-Induced MDA-231 Cell Proliferation by Inhibitors of Ras and Raf

MDA-231 cells were plated in 96-well tissue culture plates as described. The medium was replaced with serum-free DMEM 16 h later, and the cells were pretreated for 20 min with PD98059 (20 µM), Raf1 kinase inhibitor (50 nM), or vehicle followed by treatment with GHRH (1 µM). The cells were counted 24 h later. The data are from a representative set of three experiments. Values represent the mean ± SEM; n = 8 replicates. **, P < 0.01.

 

Figure 10
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  		Fig. 10. Inhibition of MDA-231 Cell Proliferation and MAPK Phosphorylation by Dominant-Negative Ras and Raf

A, MDA-231 cells were transfected with pRSV, pZCR17N, or pRSV-Raf-C4 and were plated at 16,000 cells per well for overnight growth. The medium was replaced with serum-free DMEM 16 h after transfection, and the cells were treated with GHRH (1 µM) or vehicle. The cells were counted 24 h after the GHRH treatment. The data are from a representative set from two experiments that produced similar results. Values represent the mean ± SEM; n = 8 replicates. B, MDA-231 cells were transfected with 1 µg pRSV (lanes 1 and 2), pZCR17N (lanes 3 and 4), or pRSV-Raf-C4 (lanes 5 and 6) were grown in DMEM with 10% FCS for 40 h. The medium was replaced with DMEM with no FCS for 1.5 h, and cells were treated with GHRH (10 nM) for 5 min and harvested in SDS sample buffer. Lysate (40 µl) was subjected to 10% SDS-PAGE, and proteins were transferred to Immobilon membranes and probed for phospho-MAPK. The blot was stripped and was probed for MAPK. Blot was again stripped and probed for actin. **, P < 0.01.

 
Phosphorylation of MAPK by GHRH is inhibited in GH4 pituitary somatotroph cells by the presence of somatostatin (35). We used the long-acting somatostatin analog, BIM23014, to determine whether somatostatin also inhibits GHRH-induced phosphorylation in MDA-231 cells. As seen in Fig. 11AGo, pretreatment of MDA-231 cells with BIM23014, prevented the phosphorylation of MAPK in response to GHRH. Furthermore, pretreatment with BIM23014 prevented GHRH-induced cell proliferation (Fig. 11BGo). Thus, analogous to its role in pituitary cells, somatostatin antagonizes GHRH-dependent MAPK phosphorylation and cell proliferation in breast cancer cells.


Figure 11
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  		Fig. 11. Inhibition of MDA-231 Cell Proliferation and MAPK Phosphorylation by the Somatostatin Analog BIM23014

A, MDA-231 cells were grown in DMEM with 10% FCS for 16 h. At this time, the medium was replaced with DMEM with no FCS, kept for 1.5 h, and treated with PBS or 10 nM BIM23014. Fifteen minutes later, the cells were treated with GHRH at 10 nM, and the cells were harvested 2.5 and 5 min later. The lysate (40 µl) was subjected to 10% SDS-PAGE, and proteins were transferred to Immobilon membranes and probed for phospho-MAPK. The blot was stripped and probed for MAPK. B, MDA-231 cells were plated in 96-well tissue culture plates as described. The medium was replaced with serum-free DMEM 16 h later, and the cells were pretreated with BIM23014 20 min before the addition of GHRH (1 µM). The cells were counted 24 h after the treatments. The data are from a representative set of three experiments. Values represent the mean ± SEM; n = 8 replicates. *, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The results reported here indicate that GHRH promotes a dose- and receptor-dependent increase in cell proliferation, DNA synthesis, and MAPK phosphorylation in breast cancer cells. Furthermore, the effects of GHRH on cell proliferation and MAPK phosphorylation appear to be mediated by the Ras and Raf pathway, because inhibition of these pathways in diverse ways prevented the effects of GHRH. These experiments also indicate the potential for an autocrine/paracrine coupling of GHRH and somatostatin in regulation of breast cancer cell proliferation. Taken together, the results indicate that GHRH could play an important functional role in the development, growth, and progression of human breast tumors

We have previously reported that antagonism of endogenous GHRH leads to dose-dependent apoptosis in MDA-231 cells (34). In addition, there have been multiple reports demonstrating that antagonism of GHRH has antiproliferative effects on a variety of human tumor types (28, 29, 30, 31, 32, 33). These reports have suggested that endogenous GHRH may be acting as an autocrine/paracrine mitogenic growth factor for various tumor cell types. However, although GHRH has been shown to be mitogenic for pituitary GH4 cells in vitro (6, 35), evidence of direct mitogenic activity of GHRH in cancer cells has been lacking before this report.

It has been argued that the mitogenic actions of GHRH are mediated through related receptors, such as those for vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide (41, 42), because of the inability to identify GHRH receptors in all responsive tissues. MCF7 cells produce endogenous GHRH but do not express the GHRHr and are nonresponsive to GHRH. However, when these cells were transfected with vectors containing the full-length GHRH receptor construct, cell proliferation was stimulated in response to endogenous GHRH, indicating that the GHRHr is capable of transducing a mitogenic signal in response to GHRH. The failure to identify GHRH receptors in other responsive tumor cell lines may indicate that multiple receptors can mediate GHRH responsiveness or may reflect technical problems in identifying low-level receptor expression.

Despite numerous reports on the antitumorigenic effect of GHRH antagonists in human cancer, little is known regarding the mechanisms of action of GHRH in cancer cells. Phosphorylation of MAPK after GHRH treatment has been reported previously (35, 36). Pombo et al. (36) used CHO cells transfected with the GHRHr, whereas we (35) used GH4 and HeLa cells. The results reported here confirm that GHRH stimulates phosphorylation of MAPK in MDA-231 cells, and extend these observations to indicate that this stimulation occurs through a Raf- and Ras-dependent pathway. Furthermore, the results indicate that activation of this pathway is required for promotion of proliferation.

Although the phosphorylation of MAPK in MDA-231 cells in response to GHRH fits with previous demonstrations in other cell lines, the involvement of Raf kinase during the signal transduction is in contrast with the observations of Pombo et al. (36) who demonstrated that Ras, but not Raf, is involved in signaling to MAPK from the GHRHr. However, these experiments were carried out in CHO cells transfected with a variant of the GHRHr. Several splice variants of GHRHr have been described in humans (27) and animal models (26) that differ in terms of signaling through the cAMP and other pathways. Therefore, the differences in the involvement of Raf and Ras reported here and in the CHO cells may be due to cell line-related differences or due to differences in the specifics of signaling of various GHRH variants receptors. We have observed the participation of Raf and Ras in the stimulation of MAPK phosphorylation by GHRH in GH4 cells similar to that seen in the MDA-231 cells reported here (our unpublished observations).

The inhibition of both Raf1 and MAPK prevented proliferation of MDA-231 cells in response to GHRH. This observation suggests that the effects of GHRH on cell proliferation demonstrated in previous studies are mediated by the Ras/Raf/MAPK pathway. However, Raf and MAPK are central components of many cellular signaling pathways. Therefore, the effect of inhibition of Raf and MAPK on proliferation of MDA-231 cells could reflect effects due to other mitogenic agents present in the system, rather than a specific effect on GHRH-mediated proliferation. However, there were no effects of Raf/MAPK inhibitors on rates of proliferation in the absence of GHRH. These results argue against the presence of a growth factor that activates Raf/MAPK independent of GHRH. Furthermore, the cells were maintained in serum-free conditions after the addition of GHRH and hence were not exposed to additional exogenous growth factors.

We have previously reported that the somatostatin analog, BIM23014, prevents GHRH-induced phosphorylation of MAPK in GH4 rat pituitary cells (35). We now expand this observation to report a similar antagonism between somatostatin and GHRH in MDA-231 cells. Thus, the pretreatment of MDA-231 cells with BIM23014 prevented the phosphorylation of MAPK as well as cell proliferation. Because it has been reported that somatostatin prevents MCF-7 cell proliferation (37), the antiproliferative effect of BIM23014 in MDA-231 and MCF 7 cells may reflect the prevention of signaling to MAPK. Furthermore, the apparent mutual antagonism between GHRH and somatostatin raises the possibility that these two gut-brain peptides form a regulatory unit for cell growth of cancer cells, similar that that seen in the hypothalamus and normal extrahypothalamic tissues (37, 43, 44)

In summary, GHRH stimulates dose-dependent cell proliferation of MDA-231 and T47D breast cancer cells, and MCF7 cells transfected with the GHRH receptor b in the absence of other exogenous growth factors. Furthermore, antagonism of GHRH reduces cell proliferation (28, 29, 30, 31, 32, 33, 34). Activation of MAPK is required for GHRH-induced proliferation in MDA-231 cells, and this activation occurs via Raf and Ras oncoproteins. These observations, along with the evidence for endogenous GHRH secretion, suggest that endogenous autocrine/paracrine secretion of GHRH from breast cancer cells may participate in the promotion of proliferation and prevention of apoptosis. Finally, these results provide support for a possible autocrine/paracrine antagonism between GHRH and somatostatin in the regulation of MDA-231 cell populations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
MDA-231 cells, originally obtained from American Type Culture Collection (Manassas, VA), were grown in DMEM with 10% FCS at 37 C and 5% CO2. Cells for treatment were plated at 60,000 cells/cm2 and incubated at 37 C for 16 h. Cultures for cell count were plated in 96-well tissue culture dishes in 100 µl of growth medium. Cell cultures for Western blotting were plated at 60,000 cells/cm2 in 3.5-cm diameter tissue culture dishes, using 3 ml growth medium. As appropriate, treatments were added to newly replaced DMEM with no FCS. FCS was heat inactivated by heating to 55 C for 30 min.

Plasmid Constructs and Transfections
Dominant-negative Ras (pZCR17N) and dominant-negative Raf (pRSV-Raf-C4) vector constructs have been described elsewhere (40). Rat GHRHr has been described previously (25). pCDNA was from Invitrogen (Carlsbad, CA). Transient transfection was performed by electroporation (40). Briefly, media were changed 4 h before each transfection with DMEM/10% FCS, and cells were harvested at 80% confluency and electroporated in DMEM/10% FCS using 1 µg plasmid/0.2 million cells in a 200-µl volume. After electroporation, cells were plated in DMEM/10% FCS and maintained until treatments/harvesting.

Reagents
Human GHRH (Sigma Chemical Co., St. Louis, MO) and PRL 2460 (H-Tyr-D-Arg-Asp-Ala-Ile-Phe-Thr-Aib-Ala-Tyr-Arg-Lys-Val-Leu-Ala-Ala-Leu-Ala-Ala-Arg-Lys-Ala-Leu-Aib-Ala-Ala-Nle-Ala-NH2) were dissolved at 250 and 1 µM respectively, in a solution of 1% insulin-free BSA and 0.2% acetic acid and stored at –80 C. The Raf1 kinase inhibitor (5-iodo-3[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-indolinone) (38) and the MAPK inhibitor PD98059 (2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one) (39) (Calbiochem-Novabiochem Corp., San Diego, CA) were dissolved in dimethyl sulfoxide to 100 µM and 20 mM, respectively, and stored at –20 C. BIM23014 was dissolved in PBS to a stock solution of 10 mM (courtesy of Dr. John Tentler, Department of Endocrinology, University of Colorado Health Sciences Center). MAPK and phospho-MAPK antibodies were from New England Biolabs (Beverly, MA). Ras, Raf, and actin D antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and antibody against GHRH was from Peninsula Laboratories, Inc. (Belmont, CA).

Cell Counts
For counting, 20,000 cells per well were plated in 96-well plates in 100 µl DMEM with 10% FCS and incubated overnight. Treatments were applied 16 h after plating, in newly replaced DMEM with no FCS unless indicated otherwise. The growth medium was discarded 24 h after the treatment and 200 µl PBS with 2 mM EDTA was added to detach the cells. Twenty-microliter samples were placed in a hemocytometer and cells in four 0.1-µl volumes were counted visually. Counts represent eight wells per treatment.

Tritiated Thymidine Uptake
Cells were plated at 20,000 cells per well in 96-well plates in 100 µl DMEM with 10% FCS and grown overnight. GHRH (1 µM) or vehicle was added 16 h after plating, in fresh DMEM with no FCS. Two hours after the addition of GHRH, 6 µl [3H]thymidine was added to each well. At 2, 4, and 6 h after the addition of thymidine, the wells were washed three times with 150 µl PBS, followed by addition of 25 µl 10% trichloroacetic acid for 5 min, 100 µl 0.1 M NaOH, and 27.5 µl 0.1 M HCl (34). The entire content of each well was transferred to a scintillation vial, and the activity was counted for 10 min/sample. Counts represent eight wells per treatment.

35S Incorporation and Immunoprecipitation Procedure
MDA-231 cells were grown in 3.5-cm dishes to 80% confluency in 3 ml DMEM/10% FCS. The medium was replaced with 3 ml methionine- and cysteine-free DMEM for 2 h. Medium was then changed to 0.6 ml methionine- and cysteine-free DMEM with 20 µCi (5 µl) [35S]methionine per plate, and the cells were incubated. At 3 or 16 h, the growth medium was collected and treated with protease inhibitors immediately. The cells were harvested using 0.5 ml cold PBS and centrifuged for 2 min at 1000 rpm to obtain the cell pellet. The cells were lysed using 0.5 ml radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors for 30 min on ice, with a brief vortexing every 10 min. Cells were then centrifuged at 14,000 rpm for 15 min. The cell lysate and the collected growth medium were each treated with 10 µl protein G-Sepharose beads and incubated for 1 h. These were centrifuged for 10 sec at 14,000 rpm, and the supernatant was saved. One hundred microliters of the supernatant was added to 300 µl RIPA buffer with protease inhibitors (control). The rest of the cell lysate supernatant and 400 µl of the cell growth medium supernatant were incubated with 4 µl anti-GHRH antibody at 4 C in a rotator. After 4 h of incubation, 20 µl protein G-Sepharose was added to each tube and incubated for 2 h in a rotator at 4 C. The beads were then separated by a 10-sec centrifuging at 2000 rpm and washed four times with 500 µl RIPA buffer. After washing, the beads were added to 25 µl of 2x SDS gel loading buffer and boiled, and proteins were resolved in a gradient gel. The gel was dried and visualized by autoradiography.

Western Blotting
Cells were grown in 3.5-cm tissue culture dishes in 3 ml DMEM with 10% FCS for 16 h and were at 80% confluency at the time of treatment. After treatments, cells were exposed to 200 µl of hot Laemmli SDS sample buffer and homogenized. Proteins were resolved by subjecting 40-µl samples to SDS-PAGE, except for the GHRH peptide where the samples were run in a 16% Tris tricine gel (Bio-Rad, Hercules, CA). Proteins were transferred to Immobilon-P (Millipore, Bedford, MA) membranes in 192 mM glycine, 25 mM Tris, and 20% methanol at 400 mA for 1.5 h. Filters were blocked in Tris-buffered saline with 0.2% Tween 20/5% nonfat milk and probed with the specific antibodies. The proteins of interest were detected using the enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) procedure. The density of bands was quantified using the AlphaImager (Alpha Innotech, San Leandro, CA).


   ACKNOWLEDGMENTS
 
We acknowledge the advice and support of Drs. Arthur Gutierrez-Hartman and William Wood.


   FOOTNOTES
 
This work was supported by an Individual Investigator Award from the Howard Hughes Medical Institute at the University of Colorado, a basic science research grant from Pharmacia and Upjohn, and a grant from the Breast Cancer Research Program of the Department of Defense (DAMD17-00-1-0212).

First Published Online April 13, 2006

Abbreviations: CHO, Chinese hamster ovary; FCS, fetal calf serum; GHRHr, GHRH receptor; RIPA, radioimmunoprecipitation assay.

Received for publication January 4, 2005. Accepted for publication April 4, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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