RWGT2 and HARA cells express predominately EGFR and ErbB2 receptors and produce EGF-related ligand mRNA for AREG, TGF-α and HB-EGF. (A) Parathyroid hormone-related protein/GAPDH mRNA ratios in RWGT2 and HARA cell cultures measured by QRT-PCR. The relative ratios of PTHrP mRNA to GAPDH mRNA levels were expressed as mean±s.e.m. of four cultures from a single experiment. ^*P=0.003. (B) Relative epidermal growth factor family receptor expression as detected by western blots of cell extracts from RWGT2, HARA and the breast cancer cell line MCF-7. The right side of the figure indicates the specific erbB receptor antibodies. Protein extracts were precipitated using conA as indicated by ppt ConA on lower panel of the figure. (C, D) AREG, TGF-α, and HB-EGF/GAPDH mRNA ratios in RWGT2 and HARA cultures were measured by QRT-PCR. The relative ratios of EGF-like ligand mRNA to GAPDH mRNA level were expressed as mean±s.e.m. of four cultures from a single experiment. Note that the y axis for AREG mRNA is 100-fold greater than the y axis for TGF-α and HB-EGF mRNA. (E) Phosphorylation of the EGFR was measured in extracts from cells grown in basal medium. The band labelled with phosphotyrosine antibody reactivity was increased with EGF treatment (EGF). The general phosphotyrosine antibody 4G10 and EGFR antibodies used for protein detection are indicated on the right. These experiments were repeated four times with cells derived from independent passages with similar results.

Mitogen-activated protein kinase signalling mediates EGFR-induced activation of PTHrP gene expression. (A) Epidermal growth factor receptor and ERK phosphorylation was reduced by EGFR TKI treatment in both RWGT2 and HARA cells. Upper bands are from a Western blot of conA-Sepharose precipitated proteins probed first with the generic phosphotyrosine monoclonal antibody 4G10, then stripped and reprobed with an EGFR antibody. Lower bands are from a Western blot of 30 μg of protein probed first with a phospho-ERK 1/2 antibody, then stripped and reprobed with an antibody to ERK 1/2 and finally stripped and reprobed with an antibody to β-actin (not shown). C (control) indicates cells treated with DMSO vehicle for 6 h. EGFRI indicates cells treated with PD (1 μM) for 6 h. This experiment was repeated twice. (B) A MEK inhibitor significantly reduced basal PTHrP/GAPDH mRNA ratios in both cell lines. RWGT2 and HARA cells were treated with 1 μM DMSO (C), PD (EGFRI, 1 μM) or PD98059 (MEKI, 10 μM) for 6 h. The mRNA was harvested from four independent cultures and analysed. Two-tailed Student's t-test. ^*P<0.05 relative to C (control). For the RWGT2 experiments: MEKI, P=0.045; EGFRI, P=0.03; for HARA experiments: MEKI, P=0.04; EGFRI, P=0.04. (C, D) A MEK inhibitor blunted EGFR ligand-induced increases in PTHrP/GAPDH mRNA ratios in both cell lines. Cells were preincubated with 1 μM DMSO (C), PD (EGFRI, 1 μM) or PD98059 (MEKI, 10 μM) for 1 h and then stimulated with 100 ng ml⁻¹ EGF (+EGF) or 1 μg ml⁻¹ AREG (+AREG) for 6 h. B represents untreated cells in all experiments. Panels B–D represent four replicates of samples with all experiments repeated twice. Two-tailed Student's t-test. ^*P<0.05 relative to C (control). (C) EGF EGFRI, P=0.01; MEKI, P=0.05; AREG EGFRI, P=0.02; MEKI, P=0.053 not significant. (D) EGF EGFRI, P=0.001; MEKI, P=0.046; AREG EGFRI, P=0.049; MEKI, P=0.03. (E) Basal reporter gene activity from a PTHrP-P3 construct was reduced by a dominant-negative Raf construct in both the RWGT2 and HARA cells. RLU represents relative luciferase unit as defined in the Materials and methods. Co-transfection of the PTHrP reporter gene with the empty vector is indicated by pcDNA3, a dominant-negative Ras construct (−ras) a dominant-negative Raf construct (−raf). Values in all panels represent the mean of four samples from individual cultures±s.e.m. These experiments were repeated three times with cells derived from independent passages with similar results. Two-tailed Student's t-test. ^*P<0.05 relative to C (control). RWGT2: ras, P=0.019; raf, P=0.033; HARA: ras P=0.02.

Gefitinib reduced plasma total calcium concentrations and urinary cAMP creatinine ratios in nude mice with RWGT2 xenografts and hypercalcaemia. (A) Hypercalcaemic mice with RWGT2 xenografts (n=9) were treated daily for 3 days with gefitinib (200 mg kg⁻¹, per os (p.o.)). The first treatment occurred at the onset of hypercalcaemia. Treatment resulted in a statistically significant decrease in total plasma calcium concentrations compared to pretreatment values at all time points (6, 24, 52 and 78 h) and compared to untreated mice (n=8). Repeated measures one-way ANOVA, mean±s.e.m., ^*P<0.001. Normal nude mice (n=5) were treated daily for 3 days with gefitinib (200 mg kg⁻¹, p.o.) or untreated (n=5). Gefitinib did not decrease total plasma calcium concentrations compared to pretreatment values at all time points and compared to untreated mice. Repeated measures one-way ANOVA, mean±s.e.m. ^*P=0.001 relative to treated; ^§P=0.001 relative to baseline values. (B) Spontaneously voided urines were collected at the indicated times after the first gefitinib treatment (200 mg kg⁻¹). Cyclic AMP and creatinine concentrations were measured. The cAMP and creatinine ratio was expressed as nM mg⁻¹. Mice received additional doses of gefitinib at 24 and 48 h. Values at each time point represent the mean of two measurements from eight tumour-bearing mice from both the gefitinib-treated and untreated groups, as well as, 16 nontumour-bearing mice at baseline. Cyclic AMP levels were decreased in the gefitinib-treated mice when compared to untreated mice at all time points. Repeated measures one-way ANOVA, Mean±s.e.m., ^*P=0.02 relative to treated; ^§P=0.004 relative to baseline values. In all figures, the baseline and pretreatment groups are represented by the columns labelled base and pretreat, respectively. (C) Plasma PTHrP concentrations were measured using plasma samples collected before tumour injection (base) and at 6 and 78 h after gefitinib or placebo treatment. Experiments included nine gefitinib-treated mice and eight untreated mice. Baseline PTHrP values were from the same 17 mice. In the gefitinib-treated mice (black column), there was a mild reduction at 6 h followed by a marked reduction (50%) in plasma PTHrP concentrations when compared to untreated mice (white columns) at the respective time points. Untreated and gefitinib-treated mice with RWGT2 xenografts had PTHrP concentrations that were significantly greater than baseline at both 6 and 78 h. ^*P=0.02 relative to treated; ^§P=0.006 relative to baseline values. (D) Gefitinib treatment reduced PTHrP mRNA levels in RWGT2 tumours. RNA was extracted from tumours that were removed 78 h after hypercalcaemia was identified. The ratio of PTHrP to GAPDH mRNA was assayed by QRT-PCR. The ratio of PTHrP to GAPDH mRNA in the gefitinib-treated tumours (n=6) was decreased 60% as compared to untreated (n=6). The QRT-PCR was repeated twice with similar results. P=0.014.

Gefitinib reduced plasma total calcium and PTHrP concentrations in nude mice with HARA xenografts and hypercalcaemia. (A) Hypercalcaemic mice with HARA xenografts (n=5) were treated daily for 3 days with gefitinib (200 mg kg⁻¹, p.o.). The first treatment occurred at the onset of hypercalcaemia. Treatment resulted in a statistically significant decrease in total plasma calcium concentrations compared to pretreatment values at 78 h and compared to untreated mice (n=5). Repeated measures one-way ANOVA, mean±s.e.m., ^*P=0.042 relative to treated; ^§P=0.001 relative to baseline. (B) Plasma PTHrP concentrations were measured using plasma samples collected before HARA cell injections (baseline) and at 78 h after gefitinib or placebo treatment. Experiments included five gefitinib-treated mice and five untreated mice. Baseline PTHrP values were from the same 10 mice. In the gefitinib-treated mice (black column), there was a marked reduction (60%) in plasma PTHrP concentrations when compared to untreated mice (white columns). Untreated and gefitinib-treated mice with HARA xenografts had PTHrP concentrations that were significantly greater than baseline at 78 h. ^*P=0.047 relative to treated; ^§P=0.001 relative to baseline. (C) Gefitinib treatment reduced PTHrP mRNA levels in RWGT2 tumours. RNA was extracted from tumours that were removed 78 h after hypercalcaemia was identified. cDNA was produced with the MultiScribe Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA) and QRT-PCR performed. PTHrP to GAPDH mRNA was assayed by QRT-PCR. The ratio of PTHrP to GAPDH mRNA in the gefitinib-treated tumours (n=3) was decreased 90% as compared to untreated (n=4). The QRT-PCR was repeated twice with similar results. ^*P=0.02.

Parathyroid hormone-related protein mRNA expression is reduced by EGFR inhibitors and increased by the addition of exogenous EGFR ligands. (A) RWGT2 cells were treated with 1 μM PD for 6 h, a neutralising polyclonal antibody to AREG (10 μg ml⁻¹), a ligand blocking EGFR antibody (αEGFR, 10 μg ml⁻¹) or PBS (C) for 24 h. Neutralising antibodies to growth factors are represented by the symbol (α). The mRNA was harvested from four independent cultures and analysed by QRT-PCR. PD, P=0.03; αAREG, P=0.05. (B) HARA cells were treated with 1 μM PD for 6 h or a neutralising polyclonal antibody to AREG (10 μg ml⁻¹) or PBS (C) for 24 h. The mRNA was harvested from four independent cultures and analysed by QRT-PCR. PD, P=0.017; αAREG, P=0.034. (C) RWGT2 cells were incubated with PBS (C), 100 ng ml⁻¹ of EGF or 1 mg ml⁻¹ of AREG for 2, 4 and 6 h. The mRNA was harvested from four independent cultures and analysed by QRT-PCR. Two-hour EGF, P=0.02; 4 h EGF, P=0.002; 4 h AREG, P=0.002; 6 h EGF, P=0.004; 6 h AREG, P=0.001. (D) HARA cells were incubated with PBS (C), 100 ng ml⁻¹ of EGF or 1 mg ml⁻¹ of AREG for 2, 4 and 6 h. The mRNA was harvested from four independent cultures and analysed by QRT-PCR. Four-hour AREG, P=0.02; 6 h EGF, P=0.02; 6 h AREG P=0.013. Values in all panels represent the mean of four samples from individual cultures±s.e.m. These experiments were repeated three times with cells derived from independent passages with similar results, ^*P<0.05 relative to C (control). Two-tailed Student's t-test.

Comparison of RWGT2 and HARA models of hypercalcaemia. (A) Kaplan–Meir analysis of time to the development of hypercalcaemia. Fifty percent of mice developed HHM 40 days after injection of HARA cells compared to the RWGT2 xenograft mice that developed HHM 60 days after injection of cells. P=0.01. RWGT2 tumours (n=18) and HARA tumours (n=10). (B) Average tumour volume at time of hypercalcaemia. When hypercalcaemia was diagnosed, the RWGT2 (n=18) tumours were nearly twice as large as the HARA tumours (n=10) ^*P=0.04 HARA compared to RWGT2. Two-tailed Student's t-test. (C) Comparison of plasma PTHrP concentrations in hypercalcaemic untreated mice with RWGT2 and HARA xenografts. Plasma PTHrP concentrations were determined 78 h after the development of HHM in both xenograft models. Average plasma PTHrP concentrations in the untreated mice were 80% higher (22 vs 12 pM) in the HARA (n=5) as compared to RWGT2-bearing mice (n=8). (D) Comparison of PTHrP mRNA expression between cells grown in vitro and in vivo. RNA was extracted from tumours that were removed 78 h after hypercalcaemia was identified. The ratio of PTHrP to GAPDH mRNA was assayed by QRT-PCR. The ratio of PTHrP to GAPDH mRNA in HARA tumours (HARA-T) was increased 100-fold compared to HARA cells (HARA-C) grown in vitro. The ratio of PTHrP to GAPDH mRNA in HARA tumours was sixfold higher than RWGT2 tumours (RWGT2-T). Values in all panels represent the mean of four samples from individual cultures or tumours±s.e.m. ^*P=0.027 HARA tumours relative to HARA cells; ^§P<0.05 HARA tumours relative to RWGT2 tumours. The QRT-PCR was repeated twice with similar results. Two-tailed Student's t-test. (E) Phosphorylation of the EGFR was measured in protein extracts from RWGT2 and HARA tumours by Western blotting. The general phosphotyrosine antibody 4G10 and polyclonal antibody for phosphorylated Tyr 992 was used to probe 1 mg of extracted tumour protein precipitated with conA-Sepharose. Blots were stripped and reprobed with EGFR antibodies. Four tumours from each cell line were evaluated.

Gefitinib treatment reduced RWGT2 tumour EGFR and MAPK phosphorylation at 78 h but not tumour volume. (A) Comparison of EGFR and MAPK phosphorylation as assessed in untreated and gefitinib-treated RWGT2 tumours by Western blots. Tumours from untreated RWGT2 mice had greater phosphorylation of both EGFR phosphotyrosine residue 1068 and ERK1/2 when compared to tumour lysates from RWGT2 gefitinib-treated mice. Furthermore, all lysates had equivalent EGFR and ERK1/2 expression. Equal amounts of total protein (10 μg) were separated by electrophoresis using 4–20% tris-glycine sodium dodecyl sulphate-polyacrylamide gels, transferred onto nitrocellulose membranes, and analysed by immunoblotting using rabbit polyclonal antibodies to phosphotyrosine 1068 and phospho ERK1/2. Blots were stripped and reprobed with rabbit polyclonal antibodies against EGFR and ERK1/2. The blots were stripped a final time and reprobed with a polyclonal antibody to β-actin. Experiments were repeated three times, and the data from a representative blot are shown. (B) Tumour volumes were measured serially as described in ‘Materials and methods.' No reduction in tumour volume was present (pretreatment, 48 and 78 h) between gefitinib-treated and untreated mice at any time point. Each bar represents the mean tumour volume of eight or nine mice; bars, s.e.m. ^*P=0.048 vs treated at the pretreatment time point even though the animals were randomised. (C) Gefitinib treatment of mice with RWGT2 xenografts did not cause an increase in apoptosis. Representative samples at 78 h from gefitinib-treated and untreated tumours. H&E staining revealed similar numbers of apoptotic cells represented as shrunken cells with eosinophilic cytoplasm and karyorrhectic or pyknotic nuclei in both the treated and untreated mice (black, arrows); bar 100 μm. Fluorescent TUNEL staining for apoptosis (black, arrows) revealed similar numbers of apoptotic cells throughout the tumour parenchyma. × 400. (D) Comparison of numbers of apoptotic cells in gefitinib-treated and untreated RWGT2 tumours at 78 h. Ten × 100 fields from a fluorescent TUNEL slide for each mouse were counted to assess the number of apoptotic cells per field. The mean apoptotic cell number per × 100 field was represented as the black bar for gefitinib-treated and white bar for the untreated. No significant differences in mean apoptotic cells in tumours were present between the gefitinib-treated vs untreated mice.