Cell cycle actions of parathyroid hormone-related protein in non-small cell lung carcinoma

Abstract

Parathyroid hormone-related protein (PTHrP), a paraneoplastic protein expressed by two-thirds of human non-small cell lung cancers, has been reported to slow progression of lung carcinomas in mouse models and to lengthen survival of patients with lung cancer. This study investigated the effects of ectopic expression of PTHrP on proliferation and cell cycle progression of two human lung adenocarcinoma cell lines that are normally PTHrP negative. Stable transfection with PTHrP decreased H1944 cell DNA synthesis, measured by thymidine incorporation, bromodeoxyuridine uptake, and MTT proliferation assay. A substantial fraction of PTHrP-positive cells was arrested in or slowly progressing through G1. Cyclin D2 and cyclin A2 protein levels were 60-70% lower in PTHrP-expressing cells compared with control cells (P < 0.05, N = 3 independent clones per group), while expression of p27(Kip1), a cyclin-dependent kinase inhibitor, was increased by 35 +/- 9% (mean +/- SE, P < 0.05) in the presence of PTHrP. Expression of other cyclins, including cyclins D1 and D3, and cyclin-dependent kinases was unaffected by PTHrP. PTHrP did not alter the phosphorylation state of Rb, but decreased cyclin-dependent kinase (CDK) 2-cyclin A2 complex formation. Ectopic expression of PTHrP stimulated ERK phosphorylation. In MV522 cells, PTHrP had similar effects on DNA synthesis, cyclin A2 expression, pRb levels, CDK2-cyclin A2 association, and ERK activation. In summary, PTHrP appears to slow progression of lung cancer cells into S phase, possibly by decreasing activation of CDK2. Slower cancer cell proliferation could contribute to slower tumor progression and increased survival of patients with PTHrP-positive lung cancer.

Fig. 1.

Parathyroid hormone-related protein (PTHrP) and type I parathyroid hormone/PTHrP receptor (PTH1R) expression. A: immunoassay with anti-PTHrP 1-34 antibody detected PTHrP in G418-resistant colonies of human H1944 lung adenocarcinoma cells stably transfected with PTHrP 1-87, but not in cells transfected with empty vector. The PTHrP-transfected clones made, on average, four times as much PTHrP in 24 h as BEN squamous lung carcinoma cells (see text), which are considered to produce high levels of PTHrP (2). Data in the graphs represent means ± SE for 3–5 replicate wells. B: RT-PCR (top) and immunoblots (bottom) revealed that H1944 cells expressed the receptor for PTHrP, PTH1R. Protein and RNA from BEN squamous lung cancer cells, A549 adenocarcinoma cells, H1395 lung adenocarcinoma cells, and H460 lung large-cell carcinoma cells were used as positive controls for PTH1R.

Fig. 2.

PTHrP effects on DNA synthesis and proliferation. A: stable clones of PTHrP-positive and PTHrP-negative H1944 cells were assayed for rate of DNA synthesis by incubation with [³H]thymidine. The PTHrP-positive cells incorporated thymidine into DNA at 43 ± 7% of the rate demonstrated by PTHrP-negative clones. **P < 0.01. N = 5 PTHrP clones and 4 vector-transfected clones. B: cells were incubated with 10 μM bromodeoxyuridine (BrdU) for 2 or 24 h and analyzed for BrdU content by flow cytometry. PTHrP expression reduced the percentage of cells that were BrdU positive by at least one-half at both time points. Data represent results from 3 clones each for the control and experimental groups. *P < 0.05 vs. vector. C: growth was assessed by MTT assay in PTHrP-producing and nonproducing H1944 cells plated in 96-well plates at 4,000 cells/well. The PTHrP-positive cells grew at ∼50% of the rate of the vector-transfected cells. The data represent results from 5 vector clones and 4 PTHrP clones. *P < 0.05 and **P < 0.01 vs. vector.

Fig. 3.

Effects of PTHrP on cell cycle distribution, progression, and cell size by flow cytometry. A: cell cycle distributions for H1944 cells at 50% confluency were similar between PTHrP-expressing clones and vector clones. Plots show representative distributions, while the graphs below represent average results from 5 PTHrP-positive clones and 3 PTHrP-negative clones (P = not significant). B: bivariate plots of 24-h BrdU vs. DNA content found a high percentage of BrdU-negative cells in the PTHrP-expressing clones. In contrast, nearly all of the vector-transfected cells had taken up BrdU by 24 h. The bivariate plots show representative results from the control and experimental groups, while the graphs show averages from 3 clones per treatment. *P < 0.05 vs. vector control. C: forward scatter, a flow cytometry marker of cell size, was significantly greater for PTHrP-negative cells compared with PTHrP-transfected cells. Bar graphs represent mean data from 3 PTHrP-negative clones and 4 PTHrP-positive clones. **P < 0.01.

Fig. 4.

Effects of PTHrP on cell cycle proteins. PTHrP transfection decreased levels of cyclin D2 and cyclin A2 (A), while increasing expression of the p27^Kip1 cyclin-dependent kinase (CDK) inhibitor (B). The CDKs, cyclin B1, cyclin D1, cyclin D3, and cyclin E2, were unaffected by PTHrP. Representative immunoblots appear on the left, and bar graphs of relative density, normalized by the densities of the actin bands, on the right. Data represent means ± SE from 3 independent clones per experimental group. *P < 0.05, **P < 0.01 vs. vector control. Blots here and in other figures were performed multiple times with reproducible results.

Fig. 5.

PTHrP alters association of cyclin A2 with CDK2. Cell lysates from PTHrP-positive and -negative H1944 cells were immunoprecipitated (IP) with a rabbit polyclonal antibody for CDK2, then immunoblotted (IB) for cyclin A2 or with mouse monoclonal CDK2 antibody. The PTHrP-positive cells contained significantly less cyclin A2 associated with CDK2 than did the vector controls. *P < 0.05. N = 3 clones/group.

Fig. 6.

Interaction of p27 with H1944 cell proliferation. A: PTHrP-positive H1944 cells contained 67% less p27 after treatment with anti-p27 lentiviral small interfering RNA (siRNA) particles compared with nonsilencing particles. The p27 knockdown resulted in a 60% increase in Rb phosphorylation and a 15% increase in thymidine incorporation over 24 h (B) compared with control cells treated with nonsilencing siRNA. *P < 0.05.

Fig. 7.

Effects of PTHrP on signaling pathways. A: PTHrP-positive H1944 clones demonstrated increased levels of extracellular signal-regulated kinase (ERK) phosphorylation (p-ERK) compared with the PTHrP-negative vector controls, but Src activation was unaffected. The bar graphs show average values from 3 independent clones for each group. **P < 0.01 vs. vector. B: wild-type H1944 cells were treated with 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), a cell-permeant, long-acting cAMP mimetic. The compound increased ERK phosphorylation in dose-dependent fashion acutely, but the effect was gone by 60 min of treatment. **P < 0.01 vs. vector. n = 3 wells/time point. C: treatment with exogenous PTHrP 1-34 stimulated an acute increase in cAMP levels in wild-type H1944 cells. *P < 0.05 vs. vehicle treatment. n = 3 wells/group. However, PTHrP-producing cells did not differ in cAMP content from vector-transfected cells (n = 3 clones per group tested in duplicate). Units are pmol/μg cell protein.

Fig. 8.

PTHrP effects in MV522 lung adenocarcinoma cells. A: 24-h thymidine uptake was reduced by 25% in PTHrP-producing MV522 cells compared with vector controls. B: cyclin A2, cyclin D1, cyclin D2, and cyclin D3 levels were reduced in PTHrP-producing clones compared with controls. C: less cyclin A2 was associated with CDK2 in PTHrP-positive cells than in the PTHrP-null line. D: PTHrP-expressing cells demonstrated greater levels of ERK phosphorylation than did the controls. *P < 0.05. **P < 0.01.