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

Shimizu Y, Kinoshita I, Kikuchi J, Yamazaki K, Nishimura M, Birrer MJ, Dosaka-Akita H - Br. J. Cancer (2008)

Bottom Line: The colony-forming efficiency of H1299 and A549 was reduced by TAM67, while that of H520 was not.The induced TAM67 decreased the expression of a cell-cycle regulatory protein, cyclin A.Furthermore, TAM67 reduced growth of established xenograft tumours from these cells in nude mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan.

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.

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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.
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fig5: 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.

Mentions: We investigated the effects of TAM67 on anchorage-dependent growth using an MTT assay. The growth of TAM67 #8 and TAM67 #34 was suppressed in the presence of doxycycline, but not in GFP #1 or GFP #3 (Figure 5A). We next performed flow cytometric analysis to determine whether TAM67 affects the cell cycle using TAM67 #8 and GFP #3. The induction of TAM67 by doxycycline significantly reduced the percentage of cells in S phase, and increased that in G0/G1 phase in TAM67 #8, while the induction of GFP did not show such effects in GFP #3 (Figure 5B). We further examined whether TAM67 induces apoptosis using TUNEL assay in TAM67 #8. The fraction of DNA fragmentation in the presence of doxycycline remained low at the same level as that in the absence of doxycycline (Figure 5C). These results suggest that TAM67 inhibits cell growth by promoting G1 cell-cycle block in H1299, but not by apoptosis.


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

Shimizu Y, Kinoshita I, Kikuchi J, Yamazaki K, Nishimura M, Birrer MJ, Dosaka-Akita H - Br. J. Cancer (2008)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2266861&req=5

fig5: 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.
Mentions: We investigated the effects of TAM67 on anchorage-dependent growth using an MTT assay. The growth of TAM67 #8 and TAM67 #34 was suppressed in the presence of doxycycline, but not in GFP #1 or GFP #3 (Figure 5A). We next performed flow cytometric analysis to determine whether TAM67 affects the cell cycle using TAM67 #8 and GFP #3. The induction of TAM67 by doxycycline significantly reduced the percentage of cells in S phase, and increased that in G0/G1 phase in TAM67 #8, while the induction of GFP did not show such effects in GFP #3 (Figure 5B). We further examined whether TAM67 induces apoptosis using TUNEL assay in TAM67 #8. The fraction of DNA fragmentation in the presence of doxycycline remained low at the same level as that in the absence of doxycycline (Figure 5C). These results suggest that TAM67 inhibits cell growth by promoting G1 cell-cycle block in H1299, but not by apoptosis.

Bottom Line: The colony-forming efficiency of H1299 and A549 was reduced by TAM67, while that of H520 was not.The induced TAM67 decreased the expression of a cell-cycle regulatory protein, cyclin A.Furthermore, TAM67 reduced growth of established xenograft tumours from these cells in nude mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan.

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.

Show MeSH
Related in: MedlinePlus