Growth inhibition of non-small cell lung cancer cells by AP-1 blockade using a cJun dominant-negative mutant

Abstract

cJun, a major constituent of AP-1 transcription factor transducing multiple mitogen growth signals, is frequently overexpressed in non-small cell lung cancers (NSCLCs). The purpose of this study is to determine the effects of AP-1 blockade on the growth of NSCLC cells using a cJun dominant-negative mutant, TAM67. Transiently transfected TAM67 inhibited AP-1 transcriptional activity in NSCLC cell lines, NCI-H1299 (H1299), A549 and NCI-H520 (H520). The colony-forming efficiency of H1299 and A549 was reduced by TAM67, while that of H520 was not. To elucidate the effects of TAM67 on the growth of H1299, we established H1299 clone cells that expressed TAM67 under the control of a doxycycline-inducible promoter. In the H1299 clone cells, the induced TAM67 inhibited anchorage-dependent growth by promoting G1 cell-cycle block, but not by apoptosis. The induced TAM67 decreased the expression of a cell-cycle regulatory protein, cyclin A. TAM67 also inhibited anchorage-independent growth of these cells. Furthermore, TAM67 reduced growth of established xenograft tumours from these cells in nude mice. These results suggest that AP-1 plays an essential role in the growth of at least some of NSCLC cells.

Figure 1

Inhibition of AP-1 transcriptional activity by TAM67 in NSCLC cells. H1299, A549 and H520 cells were cotransfected with 0.1 μg of TRE2-luciferase plasmid and 8 ng of renilla-luciferase plasmid and either 0.2 μg of pCMV-TAM67 or pCMV control plasmid. Transfected cells were lysed 36 h after transfection and luciferase activity was measured. To increase AP-1 activity, cells were treated with 0.1 nM TPA for 6 h before harvesting. The luciferase activity was shown relative to the basal activity in H520 cotransfected with pCMV plasmid without TPA, which was set to 1. Each value represents the mean±s.d. (n=3). ^*P<0.01.

Figure 2

Colony-forming efficiency of TAM67-transfected NSCLC cells. H1299, A549 and H520 cells were cotransfected with 0.3 μg of pSV2neo containing a neomycin-resistant gene and either 2 μg of pCMV-TAM67 or pCMV. After 2 weeks of selection in geneticin, resistant colonies were stained with crystal violet. The number of colonies was shown as the mean±s.d. (n=3). ^*P<0.01.

Figure 3

Induction of TAM67 using Tet-on system in H1299 cells and its effect on AP-1 activity. (A) TAM67 expression in H1299 Tet-on TAM67 clone cells (TAM67 #8 and TAM67 #34). The cells were cultured in the absence or presence of doxycycline for 48 h, and the extracted cellular protein was examined for the TAM67 expression by western blot analysis. H1299 Tet-on GFP clone cells (GFP #1 and GFP #3) were used as controls. (B) Inhibition of AP-1 transcriptional activity in H1299 Tet-on TAM67 clone cells. The cells were cultured in the absence or presence of doxycycline for a week and were cotransfected with 0.1 μg of TRE2-luciferase plasmid and 8 ng of renilla-luciferase plasmid. Transfected cells were lysed 36 h after transfection and luciferase activity was measured. To increase AP-1 activity, cells were treated with 0.1 nM TPA for 6 h before harvesting. Each value represents the mean±s.d. (n=3). ^*P<0.01.

Figure 4

DNA-binding activities of different AP-1 subunits in H1299 cells. H1299 Tet-on clone cells were cultured in the absence or presence of doxycycline for a week and then the nuclear extracts were prepared. DNA-binding activity of each AP-1 family member was measured using TransAM AP-1 kit as described in Materials and Methods. (A) DNA-binding activity of cJun. The wild-type (Wt) or mutated (Mut) AP-1 consensus oligonucleotide was used as a competitor for cJun binding. Each value represents the mean±s.d. (n=3). (B) DNA-binding activities of cJun, JunB, JunD, cFos, FosB, Fra-1 and Fra-2 (data of p-cJun were replotted from Figure 4A). Nuclear extract of K562 cells stimulated by TPA was used as positive control for cJun, JunB, JunD, cFos, FosB or Fra-1 and that of WI-38 cells stimulated by TPA for Fra-2. Each value represents the mean±s.d. (n=3). ^*P<0.01.

Figure 5

Cell growth assay, cell-cycle analysis and apoptosis assay. (A) Inhibition of anchorage-dependent growth by the induction of TAM67. H1299 Tet-on clone cells were cultured in the absence or presence of doxycycline for a week and then cell growth was measured using an MTT assay. Each value represents the mean±s.d. (n=4). (B) Effects of the induction of TAM67 on cell cycle. H1299 Tet-on clone cells were incubated with 10% FBS in the absence or presence of doxycyline for 4 days. The percentage of cells in each phase was measured by a FACS flow cytometer and analysed using ModFit LT software. Each value represents the mean±s.d. (n=5). ^*P<0.01. (C) Apoptosis assay under the induction of TAM67. H1299 Tet-on clone cells were cultured in the absence or presence of doxycycline for 4 days and then apoptosis assay was performed using a flow cytometric analysis as described in Materials and Methods.

Figure 6

Effects of the induction of TAM67 on the expression of cell-cycle regulatory proteins. H1299 Tet-on clone cells were incubated with 10% FBS in the absence or presence of doxycyline for 4 days. The extracted protein (20 μg) was analysed for cyclin A, cyclin E and p27 expression by western blot analysis as described in Materials and Methods. To normalise the total protein quantity in each sample, the band intensities of cyclin A, cyclin E and p27 were divided by those of actin in the same sample. The expression levels were normalised to those in the absence of doxycycline. Each value represents the mean±s.d. (n=5).

Figure 7

Inhibition of anchorage-independent growth under the induction of TAM67. H1299 Tet-on clone cells were cultured in soft agarose in the absence or presence of doxycycline for 2 weeks. Each value represents the mean±s.d. (n=3). ^*P<0.01.

Figure 8

Tumour growth inhibition under the induction of TAM67 in nude mice. H1299 Tet-on clone cells were injected subcutaneously into BALB/c nude mice. After tumours developed and reached the size of 30 mm³, the mice were randomised to receive doxycycline-containing or doxycycline-free water. (A) Induction of TAM67 expression in established tumours from TAM67 #8 cells. Mice were killed 7 days after the randomisation and local skin tumours were removed. The extracted tumour protein (20 μg) was analysed for TAM67 expression by western blot analysis. (B) The tumour sizes were measured at intervals of 5 days and tumour volumes were estimated as described in Materials and Methods. Each value represents the mean±s.d. (n=5). ^*P<0.01.