Differential Expression of Parathyroid Hormone–Related Protein in Adrenocortical Tumors: Autocrine/Paracrine Effects on the Growth and Signaling Pathways in H295R Cells

The parathyroid hormone–related protein (PTHrP) is present in many common malignancies, such as breast and prostate cancer, and in normal and malignant endocrine tissues, including pancreatic islet cells and endocrine tumors such as thyroid and ovarian tumors (1-3). Several investigators have shown that PTHrP acts as an autocrine or paracrine growth factor in malignancy, independently of its hypercalcemic effects (4, 5). PTHrP is an oncoprotein that seems to be implicated in tumor proliferation and differentiation. It is produced by tumors commonly associated with hypercalcemia, as well as nonneoplastic tissue and several endocrine glands and tumors (6). The human PTHrP gene is composed of nine exons. The products of exons 5 and 6 are present in all PTHrP transcripts, and encode the pre–pro-region and the majority of mature peptides (7). The NH₂ terminus of PTHrP acts through a receptor that is common to PTH and PTHrP. The activated receptor initiates both the adenylate cyclase/protein kinase A (PKA) and the cytosolic calcium/inositol phosphate/protein kinase C pathways (8, 9). PTH and PTHrP belong to the vasoactive intestinal peptide-secretin-glucagon family of peptides, some of which, like vasoactive intestinal peptide and pituitary adenylate cyclase–activating peptide, modulate the secretory activity of the adrenal cortex in a paracrine manner. PTHrP immunoreactivity has been detected in the adult human adrenal cortex and in the adrenal cortex and medulla of human fetuses at ages 8 to 40 weeks (10, 11) PTH-binding sites have been located in the rat adrenal cortex by autoradiography, and PTH/PTHrP receptor mRNA has been found in the rat adrenal gland (12, 13). PTHrP was also detected in a case of adrenal cortical carcinoma associated with hypercalcemia (13, 14), in which the serum calcium concentration returned to normal after removal of the tumor. A secretagogue of PTHrP has been found in human normal adrenocortical cells (15), and in chicken adrenocortical cells in primary culture (16). PTHrP, like PTH, enhances the secretion of steroid hormones by human adrenocortical cells via a signaling mechanism involving the activation of both the adenylate cyclase/PKA and PLC/protein kinase C cascades (15). The function and clinical importance of PTHrP are poorly understood, and little is known about its role in adrenocortical carcinoma (ACC) and adrenocortical adenoma (ACA).

Although PTHrP has been detected in the normal adrenal cortex, in adrenocortical tumors (ACT) and in ACC (6), no information is available on the relationship between PTHrP synthesis and the tumor phenotype. ACC is a rare tumor with a poor prognosis. The incidence of ACC has been estimated at 0.5 to 2 per million per year in adults. Symptoms may result from steroid oversecretion and/or tumor growth and metastases. The estimated 5-year survival rate is less than 30%, demonstrating the poor prognosis for this rare cancer (17, 18). However, the pathophysiology of ACTs is not well documented because very few genetic alterations have been identified in these tumors (19, 20). Nevertheless, recent progress has shed some light on the biology of ACTs, and discrete genetic markers are closely associated with the malignant phenotype (21-23).

This study investigates the synthesis of PTHrP in benign and malignant ACTs, and evaluates its physiologic role in the ACC cell line H295R (24, 25). We used quantitative real-time PCR to assay PTHrP gene expression, and measured the PTHrP protein and its processing in tumor tissues by immunohistochemistry and Western blotting. We studied the synthesis of PTHrP and the PTHrP receptor and cell signaling pathways using H295R cells. Lastly, we investigated the function of PTHrP(1-34) by measuring its effect on the proliferation, cell cycle progression, and apoptosis of H295R cells using an antagonist of PTHrP, PTHrP(7-34), and a specific anti-PTHrP NH₂ terminus antibody. Our findings suggest that active PTHrP gene expression plays a role in ACC development and may be implicated in tumor malignancy. The effects of PTHrP on cell growth and apoptosis in vitro may well involve the cyclic AMP (cAMP)/PKA and PLC/protein kinase C pathways via activation of the PTHrP receptor.

All the patients included in the study underwent surgery for a sporadic ACT. None of the 25 patients had any anadrenocortical tumor–predisposing syndromes (Beckwith-Wiedemann, Carney complex, McCune-Albright, multiple endocrine neoplasia type 1, or Li-Fraumeni syndrome). The clinical data, hormonal status, and tumor stage (McFarlane classification) shown in Table 1 were assessed as previously described (19-21, 26). The study was approved by an institutional review board (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale, Hôpital Cochin, Paris), and patients gave their informed consent. After surgery, all patients were examined twice a year for 2 years, and annually thereafter. Each evaluation for ACC included hormone evaluation, chest X-ray, and computerized tomography scans of the abdomen and thorax. The patients were followed until their death, their last examination, or the end of the follow-up period. The minimum and the maximum follow-up periods were 3 and 119 months.

Total RNA was extracted by homogenizing 50 to 100 mg of frozen tissue or 5 × 10⁶ H295R cells with RNABLe reagent (Laboratoires Eurobio), followed by two separate elution steps in a final volume of 30 μL by the RNAeasy Minikit (Qiagen) according to the manufacturer's instructions. RNA was measured using a NanoDrop ND-1000 spectrophotometer (Labtech). Reverse transcription was done in triplicate on each sample using 2 μg of total RNA, according to the Superscript III priming (Invitrogen) manufacturer's protocol. Negative controls were prepared under the same conditions, but without reverse transcriptase. Completed reverse transcription reactions were brought to a final volume of 20 μL by adding RNase-free/DNase-free double-distilled water.

The cDNA strand was synthesized from 2 μg of total RNA using the Superscript II reverse transcriptase enzyme protocol (Invitrogen). PCR was used to measure the synthesis of the cDNAs encoding PTHrP (285 bp), S14 (140 bp), and the PTH/PTHrP receptor (486 bp; refs. 1, 7, 9).

Quantitative PCR amplification of PTHrP and β₂-microglobulin cDNAs was done with previously published primers (27). For the PTHrP common region: forward primer (5′-GTCTCAGCCGCCGCCTCAA-3′) and reverse primer (5′-GGAAGAATCGTCGCCGTAAA-3′) corresponding to exons 5/6, and having a 93-bp amplicon. For β₂-microglobulin (114 bp): forward primer (5′-GATGAGTATGCCTGCCGTGTG-3′) and reverse primer (5′-CAATCCAAATGCGGCATCT-3′) were used for normalization as described by Wellmann et al. (28). The plasmids of cDNA calibrators were generous gifts from Dr. T.J. Rosol (Department of Veterinary Biosciences, College of Veterinary Medicine, and Division of Human Cancer Genetics, Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, OH). Assays were done in duplicate in two separate PCR assays. The SYBR Green reaction mixture (10 μL; Qiagen) contained 2 μL of sample (diluted 1:10) or calibrator cDNA, 1.2 μL of 25 mmol/L MgCl₂, 0.5 μL of each primer (10 pmol/μL) for PTHrP or β₂-microglobulin, 1 μL of DNA Master, and 4.2 μL of water. The mixture was run in a LightCycler apparatus (Roche Diagnostics) using QuantiTect SYBR Green PCR mixture (Qiagen), according to the manufacturer's protocol. The Hot Start Taq DNA polymerase was activated at 95°C for 10 min, and 40 cycles were then run as follows: 95°C for 15 s, 58°C for 7 s (PTHrP) or 57°C for 7 s (β₂-microglobulin), and 72°C for 15 s for extension synthesis. Total RNA from breast cancer cell lines was used as negative and positive control for PTHrP mRNA amplification (9). A melting curve analysis was routinely done (55-95°C) to verify the specificity of the PCR products (27, 28). Calibration curves were log-linear over the quantification range with correlation coefficients (r) of ≥−1 to (r) ≥−0.99, and slopes from −3.567 (β₂-microglobulin) to −3.331 [PTHrP (1-139), common region], respectively. These indicated comparable PCR amplification efficiencies. The resulting cDNAs produced single bands on agarose gels of the expected size for β₂-microglobulin (114 bp) and for PTHrP (1-139; common region = 93 bp; data not shown). Data were normalized using the ratio of the PTHrP cDNA to that of β₂-microglobulin cDNA to correct for differences in the amounts of RNA in samples.

The human ACC cell line H295R was derived from a female patient aged 48 years who presented with weight loss, acne, facial hirsutism, a large ACC tumor (2,002 g), high steroid secretions (glucocorticoids, androgens, and mineralocorticoids), and metastases. H295R is a pluripotential cell line that produces steroids (24). Cells were cultured in 12-well plates (250 × 10³ cells/well), or in 96-well plates (10 × 10³ cells/well) for 3 days in complete medium, and then for 48 h in serum-free, ITS-free medium (depleted medium; ref. 25). Cells were then incubated with PTHrP(1-34), (Asn¹⁰, Leu¹¹, D-Trp¹²)-PTHrP(7-34) antagonist, or anti-PTHrP antibody. The medium was changed and fresh drugs added every 48 h. To determine the effects of antiproteases, H295R cells were cultured for 48 h, then incubated for 20 min, 6 h or 24 h in fresh medium containing the proteasome inhibitor, MG-132 (25 μmol/L; Sigma), or vehicle (DMSO, Me₂SO, final concentration 0.2%).

Frozen tissue samples were ground in liquid nitrogen, and the proteins extracted (1). The protein concentration was measured using the Bio-Rad assay, and equal amounts were loaded onto 15% SDS gels for electrophoresis. The separated proteins were electrophoretically transferred to Hybond-ECL nitrocellulose membranes (Amersham Biosciences; ref. 1). These membranes were then incubated overnight at 4°C with the appropriate primary antibody: rabbit polyclonal (Biovalley) and mouse monoclonal (IDS) anti-PTHrP antibody (1-34) against PTHrP(1-34) (1/100), or mouse monoclonal anti-PTH/PTHr-P receptor antibody (1/100; Santa Cruz Biotechnology). An anti–β-actin antibody (1/5000) was used for standardization. The antigen-antibody complexes were visualized using appropriate secondary antibodies (Santa Cruz Biotechnology), and the chemiluminescence detection system ECL kit (Amersham). Negative controls were obtained by incubating the anti-PTHrP antibody for 4 h with excess antigen (PTHrP 1-34 peptide; 100 μg/mL) at 4°C. The signals were digitized with the GeneTool analysis system ver. 0.1 (1).

The PepTag nonradioactive Protein Kinase Assay Kit (Promega) was used to measure the activity of PKA, as recommended by the manufacturer. The reaction buffer (supplied with the PepTag kit) contained 1 μmol/L of cAMP to ensure activation of PKA. Bovine PKA (Promega) was used as a control for kinase activity, and protein kinase inhibitor (Sigma) was used as a specific PKA inhibitor. The PepTag A1 peptide substrate was subjected to electrophoresis for 20 min on 1% agarose gels, and the separated bands were photographed using a phosphoimager. The intensities of the bands were analyzed with Gene tools software.

Cells grown on glass coverslips were washed with Hanks' HEPES buffer (pH 7.4), and loaded with 1 μmol/L of Fura-2/AM for 40 min in the same buffer at room temperature. The Fura-2/AM fluorescence response was then used to measure the intracellular calcium concentration ([Ca²⁺]_i; ref. 9).

This assay, based on the conversion of the yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Promega) to purple formazan crystals by metabolically active cells, provides a quantitative estimate of viable cells. H295R cells were seeded at 10 × 10³ cells/well in 96-well plates and cultured for 3 days and then in serum-free and growth factor–free medium for 48 h. They were then incubated for 1, 2, 5, or 7 days with the test substances at the concentrations indicated in the figures. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagents were used according to the manufacturer's recommendations. The absorbance at 570 nm was measured in a microplate reader.

The cells were seeded in 12-well plates, and their proliferation was measured as above. Cells were removed with trypsin, rinsed with PBS and fixed in 500 μL of 70% ethanol. They were then centrifuged, rinsed in PBS, suspended in 50 μg/mL of propidium iodide plus 100 μg/mL RNase A, and analyzed by flow cytometry with a FACscan (EPICS XL). Data were analyzed with Multicycle software. All experiments were done thrice. The histogram was constructed with at least 10,000 cells.

Apoptotic cells were quantified using the Annexin V-FITC Apoptosis detection kit according to the manufacturer's instructions (Roche). The cells were analyzed using a Coulter Epics Altra flow cytometer.

The tumor samples were fixed in formalin, embedded in paraffin and 4-μm sections were cut. The wax was removed, and the sections rehydrated. They were then heated for 10 min in 0.01 mol/L of citrate buffer (pH 6) to retrieve fixation-concealed antigens. The H295R cells were plated out in a Labtek chamber (Nunc, Dominique Dutsher), and fixed by immersion in 3% paraformaldehyde for 10 min. Endogenous peroxidase activity in tissue sections and cells was blocked by incubation in 3% H₂O₂ for 30 min. The sections and cells were incubated with anti-PTHrP(1-34) antibody (1/250), or anti-PTHrP receptor antibody (1/250) overnight in PBS containing 1% BSA. The control sections and cells for PTHrP and PTH-R1 immunostaining were incubated with buffer alone or anti-species IgG. The rabbit vectastain or mouse Elite Kit were used as the secondary antibody for 1 h, and the samples were then incubated with the antibody complex (Vectastain kit Clinisciences) for 30 min. Samples were washed as above, treated with 3,3′-diaminobenzidine tablets (Sigma) for 15 min and rinsed in distilled water. All samples were counterstained with hematoxylin, and mounted in Vecta mount (Vector Clinisciences).

Statistical analyses were done using the StatView 5.0 program (SAS Institute). Data were analyzed using ANOVA and regression analysis and Fisher's projected least significant difference to compare means. Data are expressed as means ± SD. Significance was set at P < 0.05.

The pathologic variables of the 25 ACTs are summarized in Table 1. The patients with ACC (n = 12) were ages 15 to 73 years. The patients with ACA (n = 13) were ages 27 to 71 years. Five ACA (40%) were nonsecreting, and eight ACA (60%) produced excess adrenal steroids.

Eight patients (66%) had localized tumors (McFarlane stage I and II). Four patients (34%) had disseminated tumors at diagnosis (McFarlane stages III-IV), whereas six patients (50%) had distant metastases. Eleven patients (96%) had a Weiss score of 2, with obviously malignant tumors (stages II-IV). The patients with ACA included 13 patients ages 27 to 71 years. This group was mostly (90%) female. Five patients (40%) were steroid-negative, and four patients (60%) presented an adrenal steroid excess. Seven patients (90%) were stage I (McFarlane) with a Weiss score of 0 to 1.

The PTHrP/β₂-microglobulin ratio was significantly higher in the ACC samples (0.008 ± 0.014) than in the ACA samples (0.001 ± 0.001, P < 0.006; Fig. 1A). The concentrations of PTHrP mRNA were positively correlated with the markers of malignancy: McFarlane stage (r² = 0.225, P < 0.0001) and Weiss score (r² = 0.175, P < 0.004). The concentrations of PTHrP mRNA in ACC were also correlated with metastases (P < 0.05).

We used immunohistochemistry to identify the cells containing PTHrP in six ACC samples and six ACA samples, normal adrenal tissue, and H295R human adrenocortical cancer cells. The ACC sections contained dense staining and numerous foci of positive cells (Fig. 2A). The ACA sections contained few positive cells, and these were located mainly in the endothelial layer within the interstitial tissue delimiting the nodules. Normal tissue sections also contained a few cells with PTHrP in their cytoplasm/nucleus (Fig. 2A). The positive cells were mainly in the interstitial zone of endothelial cells and in vacuolated cells in the zona fasciculata. H295R cells stained positively for PTHrP in the cytoplasm or nucleus (Fig. 2B).

Western blotting of tissue lysates confirmed the presence of PTHrP in all tumors and in H295R cells. The band patterns were characteristic, with bands at 25, 27, and 35 kDa and at 50 and 70 kDa, respectively (data not shown), suggesting that each tumor may specifically regulate its PTHrP protein products. The same pattern was obtained for H295R lysates, as excepted for the 70 kDa band (Fig. 2Ca). The specificity of the immunoreactive bands of pro-PTHrP (25 kDa) and pre–pro-PTHrP (27 kDa), and the 35 and 50 kDa bands produced by the endopeptidase cleavage of the PTHrP, was confirmed by incubating the blots with anti-PTHrP antibody that had been incubated with blocking peptide (PTHrP 1-34; data not shown). However, the 17 kDa band corresponding to PTHrP(1-34) appeared only in lysates of H295R cells (Fig. 2Ca). We checked that PTHrP was indeed degraded by proteasomes. Blots of equal amounts of H295R proteins treated with the proteasome inhibitor (MG132) showed more intense protein bands, and particularly that of PTHrP(1-34) (17 kDa), than did blots of untreated protein (Fig. 2C). The specificity of these immunoreactive bands was confirmed by incubating the blot with anti-PTHrP antibody that has been previously saturated with blocking peptide PTHrP(1-34) (Fig. 2Cb).

The amplified cDNA from ACCs and adenomas had similar intensities, and a single 489-bp band was revealed by agarose gel analysis. PTHrP-R1 mRNA was also found in H295R cells (Fig. 2D). Western blots showed a 74 kDa band, corresponding to the PTH/PTHrP receptor, in the lysates of all the tumors and H295R cells. However, the concentrations of PTHrP-R1 in the nonsecreting adrenocortical adenomas and the secreting adrenocortical adenomas were not significantly different (Fig. 2E).

The basal intracellular calcium concentration ([Ca²⁺]_i) in confluent H295R cells was 110 ± 10 nmol/L (mean ± SD, n = 6). The NH₂-terminal PTHrP(1-34) (10 nmol/L) triggered a transient increase in intracellular calcium concentration ([Ca²⁺]_i) that decreased rapidly after 15 seconds, but remained above the basal level (plateau phase, 23 ± 5%; mean ± SD, n = 4; Fig. 3Aa). The PLC inhibitor U-73122 inhibited the transient peak when added 60 seconds before PTHrP, but had no effect on the plateau phase (Fig. 3Ab). U-73343 (0.5-5 μmol/L), an inactive analogue of U-73122, had no effect (data not shown). The calcium channel inhibitor, verapamil (1 μmol/L), inhibited part of the transient peak when added 60 seconds before PTHrP; it also abolished the plateau phase (Fig. 3Ac). Thus, PTHrP increased the [Ca²⁺]_i via two mechanisms, one involving a calcium influx from the extracellular milieu (verapamil), and the other, a mobilization of calcium from the endoplasmic reticulum (U-73122).

The kinase activity in cells treated with 10 to 100 nmol/L of PTHrP for 15 minutes was higher than that in untreated cells (Fig. 3Ba). Exposure of protein lysates of both controls and PTHrP-treated cells to cAMP significantly (P ≤ 0001) increased their kinase activity (Fig. 3Bb). Incubation with protein kinase inhibitor inhibited the enhancement of PKA activity (data not shown).

PTHrP(1-34) stimulated the growth of H295R cells in serum-free and growth factor–free medium in a dose-dependent fashion (Fig. 4A). PTHrP(1-34) increased cell proliferation from day 2 to day 5. The PTHrP-induced proliferation was neutralized by the receptor antagonist PTHrP(7-34) (100 nmol/L) and anti-PTHrP antibody (0.5 μg/mL) when added with PTHrP(1-34) (100 nmol/L) for 48 hours (Fig. 4B).

The H295R cells were incubated with a potent, specific PTH/PTHrP receptor antagonist, PTHrP(7-34) (Asn¹⁰, Leu¹¹, D-Trp¹²), or an anti-PTHrP(1-34) antibody recognizing all the NH₂-terminal–containing PTHrP peptides to further show that endogenous PTHrP is also required to maintain increased cell proliferation. Cells were analyzed for apoptosis by Annexin V incorporation. Incubation with PTHrP reduced the percentage of apoptotic cells (Fig. 5B) compared with control-depleted medium (Fig. 5A). Incubation with PTHrP(7-34) (1 μmol/L) or anti-PTHrP antibody (1-2.5 μg/mL) induced apoptosis on day 5 (Fig. 5C and D). Incubation of cells with PTHrP(1-34) (100 nmol/L) plus either the PTHrP(7-34) or anti-PTHrP antibody reduced the percentage of apoptotic cells (Fig. 5C and D).

Flow cytometry analyses corroborated the observation that PTHrP increased cell proliferation. There was no change in the cell cycle distribution after 48 hours of treatment (data not shown). The number of cells in S phase was increased on day 5 of treatment with PTHrP(1-34), whereas the number of cells in G₁ phase was decreased (Fig. 5E), followed by an increase in the number of cells in G₀-G₁ on day 7 (PTHrP, 65.30 ± 0.01; controls, 57.14 ± 0.57; P < 0.01; Fig. 5F). Thus, the proliferative profile of the cells incubated with PTHrP (65.30 ± 0.01) was similar to that of cells cultured in normal medium (75.05 ± 1.2; Fig. 5F). PTHrP-R1 antagonist and anti-PTHrP antibody also decreased the number of cells in G₁ phase and increased the number of cells in S phase at day 5 (Fig. 5D). However, in contrast to PThrP(1-34) treatment, cells incubated with PTHrP(7-34) or anti-PTHrP antibody for 7 days had entered apoptosis, as indicated by the sustained accumulation of cells in S phase (PTHrP 7-34, 32.40 ± 0.03, P < 0.001; antibody, 32.07 ± 0.7, P < 0.01 as compared with controls, 25.80 ± 0.49). Similarly, the percentage of cells in G₂ phase was increased (PTHrP 7-34, 24.21 ± 2.30; controls, 17.04 ± 1.04; P < 0.001), whereas the percentage of cells in G₁ phase was markedly decreased (PTHrP 7-34, 43.41 ± 2.06; antibody, 50.05 ± 0.5; controls, 57.14 ± 0.57; P < 0.001). Adding PTHrP(1-34) to either PTHrP(7-34) or antibody-treated cells caused them to enter the G₁ phase on days 5 and 7 (Fig. 5E and F). A similar cell cycle profile was obtained when H295R cells were triggered to apoptosis by 5 ng/mL of transforming growth factor (data not shown).

This study examines the relationship between the concentration of PTHrP mRNA and clinical-pathologic variables of human ACC and adrenal adenoma by quantitative real-time PCR. All the tumors contained the PTHrP 93-bp amplicon (common exon VI), but there were significantly more in the ACCs and the H295R cells than in the adenomas. The highest PTHrP mRNA concentration was correlated with the usual criteria of aggressive malignancy and elevated steroid secretion. And differences in the immunolocalization of PTHrP enabled us to distinguish between ACC, ACA, and normal tissue. ACC tumors contained many cells with a dense, focal PTHrP staining, whereas the PTHrP in ACT adenoma was mainly in endothelial cells within the interstitial tissue delimiting the nodules. Normal tissue contained few PTHrP-positive cells, and these were endothelial cells and vacuolated cells in the zona fasciculata. The intense PTHrP staining observed in invasive and metastatic tumors, both pituitary and thyroid tumors, also varies with the type of tumor (5). Western blots show PTHrP processing by these tumors and H295R cells. The observed multiple immunoreactive bands correspond to pro- and pre–pro-PTHrP (25-27 kDa), and to higher molecular weights in all the tissue extracts and H295R cells. However, only the H295R cell line possesses the 17 kDa band, corresponding to the PTHrP(1-34) peptide. The amino acid sequence of pro-PTHrP, a precursor of PTHrP, can serve as a substrate for the prohormone convertase furine (29). The PTHrP(1-34), detected in H295R cells, but not in adrenal tumors, is probably due to ubiquitination and to the proteasome-dependent degradation of the peptide (29, 30). PTHrP(1-34) (17 kDa) and other immunoreactive proteins accumulated in H295R cells incubated with a proteasome inhibitor. The different patterns of intensities within the tumors may be due to different processing of PTHrP by the tumors, as reported for pancreatic adenocarcinoma (31). PTHrP contains many basic amino acid motifs that allow extensive posttranslational processing before secretion; these products have short half-lives, as do many regulatory proteins, including oncoproteins.

The PTH/PTHrP receptor mRNA and protein were found in all the tumors and in the H295R cells, suggesting that PTHrP acts as an autocrine/paracrine factor, influencing proliferation and differentiation. The signaling pathway activated by PTHrP depends on the cell type. PTHrP has no effect on PKA activity in Walker 256 tumor cells or in human mammary invasive cells, but it activates the phospholipase C pathway (8, 32). Low (10 pmol/L-10 nmol/L) concentrations of PTHrP(1-34) increased the concentration of intracellular calcium in H295R, whereas higher concentrations (100 nmol/L-1 μmol/L) activated the cAMP/PKA signaling pathway. This suggests that the concentration of PTHrP is an important discriminating factor for using a particular route in these H295R human adrenocortical cancer cells. The aldosterone and cortisol responses to PTHrP are totally or partially blocked by inhibitors of adenylyl cyclase or phospholipase C, when they are added alone or together to dispersed human adrenocortical cells in primary culture (15). Thus, there may be a positive association between PTHrP and steroid secretion in human adrenocortical cancers and in H295R cells, revealing another crucial role of the peptide in ACTs.

One of the main actions of PTHrP on tumors is to increase cell multiplication (8, 32). PTHrP(1-34) stimulates the proliferation of H295R cells in medium lacking both serum and growth factors in a dose-dependent fashion. The specific, competitive antagonist of the PTH/PTHrP receptor blocks the PTHrP-induced increase in cell proliferation, showing that PTHrP promotes tumor cell survival by interacting with the PTH/PTHrP-R1. Specific anti–NH₂-terminal PTHrP antibodies also neutralize endogenous PTHrP-induced cell proliferation. This corroborates data showing that reducing PTHrP concentrations with neutralizing antibodies in vivo and in vitro or with PTHrP siRNA or shRNA in vitro reduces cell invasion, tumor size, and metastases (33-39). Treating H295R cells with (1-2.5 μg/mL) anti-PTHrP antibody enhances their apoptosis by day 5, revealing that the cells produce endogenous PTHrP. Adding PTHrP reverses the inhibitory effect of the antibody by reducing the percentage of apoptotic cells. This suggests that PTHrP has an antiapoptotic effect on H295R cells, in agreement with its effect on cell proliferation.

Increasing cell growth and division with PTHrP(1-34) could well be associated with increases in the percentages of cells in the S and G₂-M cell cycle phases. PTHrP did indeed activate the cell cycle in H295R cells and released the G₁-S checkpoint, resulting in a redistribution of cells in the S and G₂-M phases. Flow cytometry analyses showed that cells incubated with antibodies against PTHrP increased the percentage of cells in S phase, and decreased the percentage of cells in G₁ phase, whereas the PTH/PTHrP-R1 antagonist decreased the percentage of cells in G₁ phase and increased the percentage in the S-G₂ phase. The anti-PTHrP antibody induced apoptosis after 5 days, probably after a premitotic block (40, 41), whereas PTHrP counteracted the effect of the anti-PTHrP antibody causing cells to enter the G₁ phase after day 5. The blockade of cells in the S or G₂ phase may be linked to cell cycle–regulating genes. Specific PTHrP siRNA affects the CDC2 and CDC25B cell cycle–regulating genes implicated in the progression from S-G₂ phase to the mitotic phase (34). PTHrP was shown to protect cells from apoptosis by treating a human medulloblastoma with antisense PTHrP (42). PTHrP also regulates the synthesis of the antiapoptotic protein Bcl2 in HEK293 cells, and inhibits the apoptosis induced in chondrogenic cells and HEK 293 cells by TNFα (43, 44). These results indicate that PTHrP participates in the growth of H295R cells, and by extension, is involved in tumor growth.

In conclusion, the concentration of PTHrP mRNA may be used to discriminate between ACCs and adenomas. The high activity of the PTHrP gene may play a role in the development of ACCs and could be implicated in steroid hormone dysregulation and tumor metastasis. The experiments on H295R cells show that PTHrP is a key regulator of important biological activities, such as proliferation, cell cycle dysregulation, and apoptosis. PTHrP uses two signaling pathways, intracellular cAMP/PKA and calcium/PLC in H295R cells that can be involved in the mitogenic effects of PTHrP because they are largely dependent on mitogen-activated protein kinase, whose activity can be modulated by both PKA and protein kinase C (45, 46). Further studies are needed to characterize the cascade of intracellular proteins activated by PTHrP in ACT cells leading to the activation of mitogen-activated protein kinase that in turn activates early genes involved in cell proliferation and differentiation (46, 47).

No potential conflicts of interest were disclosed.

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