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Endocrine, Polypeptide and Cancer Institute (K.S., A.V.S., P.A., K.G., F.H., A.F., J.L.V., G.H.), Veterans Affairs Medical Center, New Orleans, Louisiana 70112; and Department of Medicine (K.S., A.V.S., A.F., J.L.V., G.H.), Tulane University School of Medicine, New Orleans, Louisiana 70112
Address all correspondence and requests for reprints to: Dr. Andrew V. Schally, Endocrine, Polypeptide, and Cancer Institute, VA Medical Center, 1601 Perdido Street, New Orleans, Louisiana 70112-1262.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the search for new therapeutic approaches for IGF-I-dependent cancers, we recently developed GHRH antagonists (9). Tumor inhibitory effects of GHRH antagonists have been demonstrated in a wide range of experimental malignancies (9), including osteosarcomas (10); lung (11, 12), prostate (13, 14, 15), kidney (16), breast (17), pancreatic (18), and colon cancers (19); and glioblastomas (20). Several classes of GHRH analogs have been synthesized in our laboratory in an endeavor to develop antagonists with increased receptor-binding affinity, biological activity, and metabolic stability (9, 21). The evaluation of the tumor inhibitory effects of GHRH antagonists revealed that their mechanism of action is complex and not restricted solely to the blockade of the pituitary GH/hepatic IGF-I axis (9). The reduction of serum GH and IGF-I levels by prolonged administration of GHRH antagonists might explain the inhibitory effect of these antagonists on some IGF-I-dependent tumors such as osteosarcomas, lung, renal, and prostate cancers (10, 11, 13, 14, 16). However, in the course of inhibition of mammary, prostatic, pancreatic, and colorectal cancers and glioblastomas by the GHRH antagonists, the intratumoral concentrations of IGF-I and/or IGF-II are decreased, and the serum GH and IGF-I levels are only slightly affected or unchanged (15, 17, 18, 19, 20). This indicates that in some cancers the local action of the antagonists on tumor cells may be more important for the inhibitory process than systemic effects on the GHRH-GH-IGF-I axis. Whether the local target of GHRH antagonists is IGF-I or IGF-II appears to depend on the type of tumor. GHRH antagonists can also inhibit tumor growth by mechanisms that are IGF independent (12)—that is, by blocking the actions of locally produced autocrine/paracrine GHRH (12, 22).
Recent work indicates that GH also may be an autocrine growth substance in mammary tumors (23, 24). These findings raise the question of whether GHRH antagonists could affect GH synthesis at extrapituitary sites and thus interfere with the local production and effects of GH in cancers.
In the present study, we investigated the effects of GHRH antagonists on growth of E-independent MXT mouse mammary cancers. In addition to following the changes in tumor volume, we also analyzed cancer growth characteristics by histological methods such as silver staining of nucleolar organizer regions (AgNORs), immunohistochemical detection of proliferating cell nuclear antigen (PCNA), and assaying apoptotic cells. We likewise performed in vitro experiments to elucidate the mechanism of action of the antagonists. Our studies reveal that in MXT breast cancers, GHRH antagonists can inhibit tumor growth by suppressing the local production and effects of GH. This represents a new, previously unknown mechanism of tumor inhibitory action of GHRH antagonists. Our findings are reported herein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All chemicals were purchased from Sigma (St. Louis, MO) unless stated otherwise.
Animals and tumors
Female B6D2F1 mice
were obtained from the National Cancer Institute, Frederick Cancer
Research Facility (Frederick, MD). MXT(3.2)ovex mammary carcinoma was
originally acquired from Dr. A. E. Bogden
(Biomeasure, Hopkinton, MA) and was regularly transplanted
in our laboratory. The maintenance of animals, the transplantation
method of cancers, and the calculation of tumor volume were reported
previously (26). All experiments were performed according
to institutional ethical guidelines on animal care.
In vivo experimental protocol
One day after transplantation of tumors, the mice were randomly
divided into groups, and the treatment was started. The GHRH
antagonists were administered by single daily sc injections at a dose
of 20 µg/d or continuously from Alzet osmotic pumps (model
2002) (ALZA, Palo Alto, CA) implanted sc and releasing 20
µg/d. The groups were as follows: (1) control, vehicle only; (2)
MZ-5–156, injections; (3) JV-1–36, injections; (4) JV-1–38,
injections; (5) JV-1–36, osmotic pump; and (6) JV-1–38, osmotic pump.
The experiment was terminated on day 18. Body weights and tumors were
measured regularly, and tumor volume was determined. Tumor growth
reduction was calculated according to the formula: tumor growth
reduction % = 100 - 100 x (T-t)/(C-c), where t
= mean initial volume of treated tumors, T = mean final volume of
treated tumors, c = mean initial volume of control tumors, and
C = mean final volume of control tumors. The mice were killed by
exsanguination under Metofane (Malinkrodt Vet., Mundelein,
IL) anesthesia. The tumors were cleaned and weighed. Tumor samples were
taken for histology, receptor analysis, RIA, and molecular biology
studies.
Histological procedures
Tumor samples were fixed in 10% buffered formalin. The
specimens were embedded in Paraplast (Oxford Labware, St.
Louis, MO) and sections were stained with hematoxylin-eosin. The
mitotic and apoptotic cells were counted in 10 standard high-power
microscopic fields containing, on average, 500 cells, and their numbers
per 1000 cells were accepted as the mitotic and apoptotic indices,
respectively. For the demonstration of the nucleolar organizer region
in tumor cell nuclei, the AgNOR method was used as described
(26). The number of AgNOR granules is an indicator of cell
proliferation (26). The silver-stained black dots in 50
cells of each tumor were counted, and the AgNOR number per cell was
calculated.
Immunohistochemical detection of PCNA
The sections of paraffin-embedded tumor tissue on silanated
glass slides underwent standard processing with additional 2 x 5
min microwave treatment in 0.01 M sodium citrate buffer, pH
6.0. Primary antibody for PCNA (Ab-1) (Calbiochem,
Cambridge, MA) 1:500 was used for 1 h followed by biotinylated
antimouse IgG 1:300 for 1 h and ExtrAvidine-peroxidase 1:100 for
30 min. Peroxidase was detected with Sigma Fast
diaminobenzidine tablets. All incubations were carried out at room
temperature. The nuclei containing PCNA were counted and the percentage
of positive nuclei was determined.
Receptor assay
The IGF-I receptor assay on membranes of MXT cancers was
described previously (11, 12, 13). The LIGAND PC computerized
curve-fitting program of Munson and Rodbard (11, 12, 13) was
used to evaluate the types of receptor binding, the maximal binding
capacity of the receptor and the dissociation constant
(Kd) values.
RIAs for GH, IGF-I, and IGF-II in mouse MXT breast cancers
The tumor tissue was homogenized in 2 M acetic acid
supplemented with protease inhibitors. The homogenate was centrifuged
at 10,000 g for 30 min. The supernatants were lyophilized and
reconstituted in RIA buffer. Mouse GH was determined using materials
provided by Dr. A. F. Parlow (NIDDK National Hormone and Pituitary
Program, Torrance, CA): mGH reference preparation AFP 10783B, mGH
antigen AFP 10783B, and antirat GH-RIA-5/AFP-411S.
The method used for determination of IGF-I and IGF-II in tumor samples
was described (14). Briefly, 100 mg of tumor tissue was
homogenized in 0.5 M homogenization buffer containing
protease inhibitors. Tumor homogenates were extracted by a modified
acid-ethanol cryoprecipitation method to eliminate the interference by
IGF-binding proteins and 300 µl of homogenate was separated for
protein determination using the Bio-Rad Laboratories, Inc.
(Hercules, CA) protein assay kit. In the cell culture medium, IGF-I and
IGF-II were measured directly without extraction. Rat IGF-I
(Diagnostics Systems Laboratories, Inc., [DSL], Webster,
TX) was iodinated by the chloramine T method. The standard was in the
range of 2–2,000 pg/tube. Goat anti-rIGF-I (DSL) was used at the final
dilution of 1:20,833. Recombinant IGF-II (Bachem,
Torrance, CA) was iodinated by the Lactoperoxidase method, the standard
was set up in the range of 2–1,000 pg/tube. Mouse antibody generated
against rIGF-II (Amano Enzyme, Troy, VA) (10 µg/ml) was
used at the final dilution of 1:14,205. Inter- and intra-assay
variation was less than 15% and less than 10%, respectively. The
results were evaluated by using a computer-controlled APEX automatic
counter (Micromedic, Huntsville, AL).
mRNA extraction and RT-PCR analyses
Total RNA was extracted from MXT mouse mammary tumors using the
Micro RNA extraction kit (Stratagene, La Jolla, CA)
according to the manufacturer’s instructions. Resuspended RNA was
quantified spectrophotometrically at 260 nm. One microgram total RNA
was reverse transcribed into single-strand cDNA using Moloney murine
leukemia reverse transcriptase according to manufacturer’s
instructions (Perkin-Elmer Corp., Norwalk, CT). Following
an initial 1.5-min denaturation step at 95 C, the reaction mixture was
subjected to 35–40 cycles of PCR amplification using specific primers
for mouse ß-actin (35 cycles), mouse/human IGF-I (35 cycles), mouse
GH (40 cycles), and mouse GH receptors (GHRs) (40 cycles). Primer
sequences for mouse ß-actin (27), IGF-I
(28), GH, and GHR (29) were:
5'-GTCACCCACACTGTGCCCATCT-3' (ß-actin sense),
5'-ACAGAGTACTTGCGCTCAGGAG-3' (ß-actin antisense),
5'-ACATCTCCCATCTCTCTGGATTTCCTTTTGC-3' (IGF-I sense) and
5'-CCCTCTACTTGCGTTCTTCAAATGTACTTCC-3' (IGF-I
antisense),
5'-CAGCCTGATGTTTGGTACCTCGGA-3' (GH
sense), 5'-GCGGCGACACTTCATGACCCGCA-3' (GH antisense),
5'-AGTTGGAGGAGGTGAACACCAT-3' (GHR sense), and
5'-GGCACAAGAGATCAGCTTCCAT-3' (GHR antisense). Each cycle consisted of a
15-sec denaturation step at 95 C and a 30-sec annealing step at 60
C. The last cycle was followed by a 7-min elongation at 72 C using a
GeneAmp PCR system 2400 cycler (Perkin-Elmer Corp.).
Negative controls were run in parallel by performing the above
reactions without the addition of reverse transcriptase as a test for
the presence of contaminating genomic DNA in the RNA preparations from
these tumors. Ten microliters of PCR-amplified product was resolved by
electrophoresis on a 1.8% agarose gel that was then stained with
ethidium bromide and visualized under UV light. PCR product bands of
the expected sizes (542 bp for mouse ß-actin, 514 bp for IGF-I,
253 bp for GH, and 330 bp for GHR) were then analyzed using a model
GS-700 imaging densitometer (Bio-Rad Laboratories, Inc.).
In vitro studies
The E-independent MXT breast cancer cell line was kindly
provided by Dr. Gunter Bernhard (University Regensburg, Germany). This
cell line was maintained in Roswell Park Memorial Institute 1640 (RPMI
1640) medium with 10% FBS, 4 mM L-glutamine,
100 U/ml penicillin G sodium, 100 U/ml Streptomycin sulfate, and 0.25
µg/ml Amphotericin. The effects of the addition of
IGF-I, IGF-II, GH, and JV-1–38 alone and in combinations were
evaluated by the crystal violet proliferation assay as described
previously (20). The results were calculated as percent
T/C where T = optical density of treated cultures and C =
optical density of untreated cultures. JV-1–38 was selected for these
studies because in previous in vitro work, it appeared to be
the most potent among the analogs tested (9, 15, 21).
For the measurement of the IGF-I, IGF-II, and GH levels in media, 12-well plates were seeded with 1500 cells/well and quadruplicate wells were treated with: (1) culture medium only; (2) serum-free medium (SFM) (RPMI 1640 + insulin-transferrin-selenium + 5% Fetuin); (3) JV-1–38 10-5 M in culture medium; and (4) JV-1–38 10-5 M in SFM. Aliquots of the respective incubation media were removed at 24, 48, 72, and 96 h of treatment and assayed for IGF-I, IGF-II, and GH along with samples of media without cells or compounds.
The incorporation of [methyl-3H]thymidine into DNA of MXT cells was determined as follows. MXT cells were seeded into duplicate microplates in culture medium. After the cells reached the confluence, the medium was removed and replaced with medium containing 5% FBS and the test compounds. Control wells received medium only. After 19 h of culture, one set of plates was pulsed with 0.25 µCi/well of [methyl-3H]thymidine (specific activity 25 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ) in a total volume of 175 µl/well for 5 h. The cells were fixed with ice-cold 10% trichloroacetic acid, washed twice with 4 C trichloroacetic acid and solubilized overnight in 0.2 N NaOH at 37 C. Radioactivity was determined by liquid scintillation counting (analytical model 6880, Searle, Des Plaines, IL). The results were expressed as 100 x T/C, where T = average dpm of test cultures and C = average dpm of control cultures.
For the analysis of cyclin B2 in MXT cells, nine 75-cm2 flasks were seeded with 2 x 106 cells each in standard culture medium. Triplicate flasks were treated with 10-7 M mouse GH, 10-6 M JV-1–38, and control medium. After 24, 48 and 96 h, one flask of each group was harvested using Trypsin 0.05% EDTA 0.53 mM (Life Technologies, Inc., Rockville, MD) and washed with PBS.
Standard immunoblotting procedure
The protein concentration of whole MXT cell lysates was
determined by Bio-Rad Laboratories, Inc. protein assay,
and samples containing equal amounts of protein were loaded onto 7.5%
SDS/polyacrylamide gel. After electrophoretic separation, the proteins
were electrotransferred to nitrocellulose membranes and incubated as
follows: blocking overnight at 4 C with 5% BSA, anti-cyclin B2 (N-20)
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) 1:1000
for 1 h, and then peroxidase-conjugated antigoat serum 1:10,000
for 1 h. The amounts loaded were confirmed by assaying actin with
antiactin (I-19) (Santa Cruz Biotechnology, Inc.) 1:1000
for 1 h. The proteins were detected using a super signal
chemiluminescent detection system (Pierce Chemical Co.,
Rockford, IL). The bands were analyzed with an imaging densitometer
(model GS-700, Bio-Rad Laboratories, Inc.) and the values
related to actin densities.
Statistical methods
The SigmaStat software (Jandel, San Rafael,
CA) was used for the statistical analysis of data. Tumor volume changes
were evaluated by two-way repeated-measure ANOVA and other data by
one-way ANOVA, and the groups were compared with Dunnett’s multiple
comparison test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table 1). The efficacy of administration from
the osmotic pump was comparable to that from the injection.
Histological investigation of proliferation characteristics showed that
the mitotic index was unchanged, but AgNOR numbers were significantly
decreased by the treatment with GHRH antagonists. PCNA levels were
lowered in the tumors treated with JV-1–38 or JV-1–36. AgNOR scores
correlated well with PCNA indices (r = 0.5, P =
0.005). The number of apoptotic cells was increased in the tumors
treated with JV-1–38 (Table 1).
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. The levels of IGF-I were decreased in
all the tumors treated with GHRH antagonists, but significant
differences from controls were obtained only in the group receiving
JV-1–38 by osmotic pumps (P = 0.032). IGF-II levels
were not changed by the therapy. GH was present in the control tumors
and in those treated with MZ-5–156. However, after the therapy with
JV-1–36 and JV-1–38, GH levels in tumors were virtually undetectable
(Table 2).
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and Table 2
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and Fig. 2).
In vitro studies on effects of IGF-I, IGF-II, mouse GH, and
GHRH antagonist JV-1–38 in MXT-breast cancer cell line
Addition of IGF-I to the medium slightly enhanced proliferation of
MXT cells at 73 h, but IGF-II had no significant effect. In
contrast, mGH caused a major increase in cell number after 43 and
73 h. JV-1–38 at 3 x 10-6M
significantly inhibited proliferation of MXT cells at both time points
(Fig. 3). The GHRH antagonist
JV-1–38 also decreased the promoting effect of IGF-I and GH on
proliferation, and conversely, GH but not IGF-I abolished the
inhibitory effect of JV-1–38 (Fig. 3).
3H-thymidine incorporation in MXT cells was
increased by IGF-I and GH after 24 h (Fig. 4). JV-1–38 inhibited the stimulatory
effect of IGF-I and GH on 3H-thymidine-incorporation.
Immunoblotting analysis showed that cyclin B2, which characteristically
appears in late G2 phase of cell cycle, accumulated in
MXT cells 24 and 48 h after addition of GH or JV-1–38 to the
medium. After 96 h, cyclin B2 in GH-treated cells was decreased to
control levels but remained high in cells treated with JV-1–38 (Fig. 5). Cyclin B2 concentrations in the cells
exposed to JV-1–38 were significantly higher than in controls
(P = 0.036, with repeated-measure ANOVA).
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). In SFM, the initial levels of IGF-I
and IGF-II were lower than in serum-containing medium, and IGF-I
concentration increased with the time of culture showing that the cells
produce this growth factor (Fig. 6B). There was only a very low
concentration of GH in the SFM, close to the detection limit. The
levels of GH and IGF-II did not change with the time of culture.
Addition of 10-5 M JV-1–38 had no significant
effect on the concentration of IGF-I, IGF-II, or GH in SFM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The central role of GH in the development and differentiation of the mammary glands became apparent when both GH and its receptors were discovered in mammary tissues of various animal species (23, 24). GH receptors have been detected in rodent, bovine, and human mammary epithelial cells (23, 24, 30) and can mediate direct effects of this hormone. It is now also known that GH is produced at several extrapituitary sites including the brain, blood vessels, and the immune system (31). Furthermore, GH and mRNA for GH were detected in the mammary glands of dogs, cats, and humans (32, 33, 34). In addition to a role of GH in the development of normal mammary tissue, experimental observations accumulated in the past few years also suggest its involvement in mammary carcinogenesis. Thus, GH production was demonstrated in mammary tumors of dogs (32), and it was also revealed that transgenic mice, which overexpress GH, develop mammary carcinomas more frequently (35, 36).
In a like manner to animal tumors, the expression of the GH receptor gene was shown in human breast cancer specimens and breast cancer cell lines (37). It was also reported that GH stimulates the growth of breast cancer cells (38) and that this effect can be inhibited by a GH receptor antagonist (39). Some researchers postulate the existence of a local mammary GH-IGF-I axis (34) and the involvement of GH in mammary carcinogenesis by autocrine/paracrine mechanisms (24). This hypothesis is supported by a very recent study demonstrating the expression of GH receptors in a large number of human breast cancer samples (40). In spite of the growing information about the role of GH in mammary tumor growth, the pathway responsible for GH-stimulated mitogenesis has not yet been determined (24). Some effects of GH can be indirect and mediated by locally produced IGF-I. However, GH may also have a direct effect on tumor cells through GH receptors or through PRL receptors (23). There are several common mediators and possibilities for cross-talk in the intracellular signaling pathways of the receptors for GH and PRL (41). In view of the accumulation of information on the role of the GH-IGF-I system in the development of certain cancers, some investigators emphasize the need for further studies on a possible carcinogenic effect of GH therapy (42) or even an examination of an eventual link between dietary consumption of IGF-I from the milk of cows injected with bGH and breast cancer risk (43, 44).
The present study shows that GHRH antagonists inhibit growth of E-independent MXT mouse mammary cancers. This mouse carcinoma is a good and reliable model of breast cancer as the tumors grow invasively, contain little necrosis and metastasize to regional lymph nodes. Thus, although this tumor is not of human origin, it is more similar in many characteristics to breast cancers in patients than the xenografts of human cancer lines in nude mice. MXT cancers also express IGF-I receptors, indicating a possible role of the GHRH-GH-IGF-I axis in the progression of this tumor. Our study demonstrated that GH and IGF-I concentrations and mRNA expression for GH and IGF-I are decreased in the tumor tissue after treatment with GHRH antagonists, and the mechanisms of inhibitory action of GHRH antagonists in MXT mammary cancers could be based on these phenomena. Previously, we showed that GHRH antagonists decreased IGF-I and/or IGF-II levels in various human experimental tumors (9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20). However, this is the first demonstration that in an experimental setting, GHRH antagonists can also decrease the production of GH in a tumor.
Considering the complex role of the GHRH-GH-IGF-I axis in the growth of breast cancers, GHRH antagonists could interfere with this system in several ways. It is not presently clear whether GHRH antagonists suppress the production and actions of IGF-I directly or through the inhibition of GH. Some findings support the view that these antagonists can probably inhibit the synthesis and action of both GH and IGF-I in tumors. Thus, GHRH antagonists decrease the synthesis of both IGF-I or IGF-II and growth in a series of tumors in which the GH production was never detected (9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20). Likewise, the proliferation of MXT cells was inhibited in vitro by JV-1–38, although we could not detect GH secretion by the cells. Apparently, GHRH antagonists can use multiple mechanisms of action even in one type of tumor.
Our in vitro experiments demonstrate that the proliferation of MXT cells is enhanced not only by IGF-I but also even more powerfully by GH. Antagonist JV-1–38 significantly inhibited the autonomous growth of the cells as well as the proliferation stimulated by IGF-I or GH.
The receptors for GH and IGF-I have several common intracellular pathways. From insulin receptor substrate-1/2 to Ras-MAPKs, or PI3K and PKC, there are many mediators that become involved after activation of receptors of either IGF-I or GH (45, 46, 47). Both IGF-I and GH can protect the cells from apoptosis by acting through their own receptors and activating PI3K (45, 48).
In our in vitro study, JV-1–38 significantly inhibited the autonomous or stimulated proliferation of MXT cells. The GHRH antagonist alone had no significant effect on 3H-thymidine-incorporation into autonomously growing cells, but decreased the incorporation stimulated by IGF-I or GH. The finding that JV-1–38 decreased cell number without inhibiting 3H-thymidine-incorporation indicates that the cells pass through the S phase but do not undergo mitosis. The addition of GH or JV-1–38 to the medium resulted in an early increase of cyclin B2 concentrations in MXT cells. Cyclin B2 returned to control levels after 96 h in the GH-treated cells but remained high in the cells exposed to JV-1–38. B-type cyclin-cdc2 kinase complexes are essential for cell cycle progression from G2 to mitosis. Cyclin B is not present in the G1 phase but accumulates in G2 and disappears during mitosis (49). Elevated cyclin B2 levels suggest that some MXT cells treated with either GH or JV-1–38 enter the G2 phase of the cycle. Subsequently the cells exposed to GH undergo mitosis, as shown by the increase in cell numbers. In contrast, JV-1–38 reduced the cell number and preserved high cyclin B2 levels in the cells, indicating that the cell cycle was arrested for a longer time at the G2-M point. It is known that apoptosis is triggered when cells remain arrested at G2-M for a critical period (50). The arrest in G2 is a common response to DNA damage (50). GHRH antagonists are obviously not DNA-damaging agents, and their effect is more comparable to that of growth factor withdrawal, which is a stress for cells growing in vitro and which can result in apoptosis. In our experiments, we did not measure cyclin B/cdc2 activity, and used cyclin B2 detection only to follow the amount of cells in G2 phase of cycle.
These in vitro results are in accord with the in vivo studies in which the GHRH antagonists decreased cell proliferation and increased apoptosis in MXT tumors. These effects were linked to a decrease in local production of GH and IGF-I.
In conclusion, GHRH antagonists can inhibit growth of mammary cancers by several mechanisms. Because GHRH antagonists appear to interfere at each level of the GHRH-GH-IGF axis, this class of compounds could be applied to the treatment of a variety of tumors that depend on endocrine-paracrine-autocrine stimulation by GHRH, GH, or IGFs. Moreover, the GHRH antagonists block the growth promoting action of IGFs and also the direct effects of GH on tumors. This action of GHRH antagonists results in an arrest at the G2-M checkpoint of cell cycle and cell death. Considering that various findings have been recently reported about the role of GH in mammary carcinogenesis, the therapies that antagonize GH activity might be important. In addition, GHRH antagonists, by preventing the antiapoptotic effects of IGFs and GH, may also enhance the efficacy of cytotoxic compounds in combination therapies.
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Acknowledgments |
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Footnotes |
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Abbreviations: AgNOR, Silver staining of nucleolar organizer region; GHR, GH receptor; PCNA, proliferating cell nuclear antigen; SFM, serum-free medium.
Received April 27, 2001.
Accepted for publication June 6, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 Z. Szereday, A. V. Schally, J. L. Varga, C. A. Kanashiro, F. Hebert, P. Armatis, K. Groot, K. Szepeshazi, G. Halmos, and R. Busto Antagonists of Growth Hormone-Releasing Hormone Inhibit the Proliferation of Experimental Non-Small Cell Lung Carcinoma Cancer Res., November 15, 2003; 63(22): 7913 - 7919. [Abstract] [Full Text] [PDF]
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G. Halmos, A. V. Schally, T. Czompoly, M. Krupa, J. L. Varga, and Z. Rekasi Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in Human Prostate Cancer J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4707 - 4714. [Abstract] [Full Text] [PDF]
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H. Kiaris, A. V. Schally, R. Busto, G. Halmos, S. Artavanis-Tsakonas, and J. L. Varga Expression of a splice variant of the receptor for GHRH in 3T3 fibroblasts activates cell proliferation responses to GHRH analogs PNAS, January 1, 2002; (2002) 12590999. [Abstract] [Full Text] [PDF]
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H. Kiaris, A. V. Schally, R. Busto, G. Halmos, S. Artavanis-Tsakonas, and J. L. Varga Expression of a splice variant of the receptor for GHRH in 3T3 fibroblasts activates cell proliferation responses to GHRH analogs PNAS, January 8, 2002; 99(1): 196 - 200. [Abstract] [Full Text] [PDF]
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