Limits...
LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Petrilli A, Copik A, Posadas M, Chang LS, Welling DB, Giovannini M, Fernández-Valle C - Oncogene (2013)

Bottom Line: We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs.Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs.Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL, USA.

ABSTRACT
Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

Show MeSH

Related in: MedlinePlus

Inhibition of LIMK interferes with cell cycle progression of Nf2ΔEx2 MSCs. (a)Nf2ΔEx2 MSCs were grown in a 6-well format and treated with 5 µM BMS-5 for 7 hours prior to labeling. Cells were analyzed by flow cytometry. Propidium iodide labeling profile of the diploid cell population analyzed with the ModFit program. Tabulation of the distribution of cell cycle phases of 3 independent experiments (mean± SEM, n=3). (b)Nf2ΔEx2 MSCs were treated with 5 µM BMS-5 or DMSO control overnight prior to 3 hr BrdU labeling and analysis by flow cytometry. Distribution of BrdU- and 7-AAD-labelled cells analyzed with FlowJo software. These distribution plots are representative of 4 independent experiments (n=4). (c) Graph of the distribution of the cell cycle phases of all the experiments as mean ± SEM, *P<0.05 determined by two-way ANOVA and Bonferroni multiple comparisons post-test. (d) Overlay of histograms of 7-AAD content comparing the vehicle and BMS-5 treated cells shown in (b).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4016185&req=5

Figure 6: Inhibition of LIMK interferes with cell cycle progression of Nf2ΔEx2 MSCs. (a)Nf2ΔEx2 MSCs were grown in a 6-well format and treated with 5 µM BMS-5 for 7 hours prior to labeling. Cells were analyzed by flow cytometry. Propidium iodide labeling profile of the diploid cell population analyzed with the ModFit program. Tabulation of the distribution of cell cycle phases of 3 independent experiments (mean± SEM, n=3). (b)Nf2ΔEx2 MSCs were treated with 5 µM BMS-5 or DMSO control overnight prior to 3 hr BrdU labeling and analysis by flow cytometry. Distribution of BrdU- and 7-AAD-labelled cells analyzed with FlowJo software. These distribution plots are representative of 4 independent experiments (n=4). (c) Graph of the distribution of the cell cycle phases of all the experiments as mean ± SEM, *P<0.05 determined by two-way ANOVA and Bonferroni multiple comparisons post-test. (d) Overlay of histograms of 7-AAD content comparing the vehicle and BMS-5 treated cells shown in (b).

Mentions: To test the possibility that the decrease in proliferation was a consequence of cell-cycle inhibition, we performed flow cytometry analysis of cells stained with propidium iodine (PI). BMS-5 interfered with Nf2ΔEx2 MSCs cell-cycle progression as evidenced by an increase in the percentage of diploid cells trapped in the G2/M phase (Figure 6 a). We further analyzed the distribution of cells among the cell-cycle phases using a BrdU/7AAD assay. There was a significant increase in the number of cells accumulating in the G2/M phase in samples treated with 5 µM BMS-5 as compared to vehicle control (20% vs. 42%). This was accompanied by a decrease in the number of BMS-5 treated cells in G1 as compared to control (41% vs. 26%). This is consistent with the results obtained by analysis of PI staining (Figure 6 b–c). Overlay of the 7AAD-area histograms clearly demonstrated a significant increase in the G2/M population in BMS-5 compared to DMSO treated cells (Figure 6 d). Furthermore, the increase in the G2/M population was accompanied by an increase in the number of cells with higher DNA content consistent with a polyploidy or multinucleated cell population. There was also a smaller but substantial change in number of cells detected in the S-phase of cell cycle in samples treated with BMS-5 as compared to DMSO control. Lastly, the population of cells in the S phase decreased with increasing BMS-5 concentration (Supplementary Figure S2). These results taken together suggest that inhibition of LIMK activity in Nf2ΔEx2 MSCs interferes with progression of the G2/M phase of the cell cycle, thereby decreasing the number of viable Nf2ΔEx2 MSCs.


LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Petrilli A, Copik A, Posadas M, Chang LS, Welling DB, Giovannini M, Fernández-Valle C - Oncogene (2013)

Inhibition of LIMK interferes with cell cycle progression of Nf2ΔEx2 MSCs. (a)Nf2ΔEx2 MSCs were grown in a 6-well format and treated with 5 µM BMS-5 for 7 hours prior to labeling. Cells were analyzed by flow cytometry. Propidium iodide labeling profile of the diploid cell population analyzed with the ModFit program. Tabulation of the distribution of cell cycle phases of 3 independent experiments (mean± SEM, n=3). (b)Nf2ΔEx2 MSCs were treated with 5 µM BMS-5 or DMSO control overnight prior to 3 hr BrdU labeling and analysis by flow cytometry. Distribution of BrdU- and 7-AAD-labelled cells analyzed with FlowJo software. These distribution plots are representative of 4 independent experiments (n=4). (c) Graph of the distribution of the cell cycle phases of all the experiments as mean ± SEM, *P<0.05 determined by two-way ANOVA and Bonferroni multiple comparisons post-test. (d) Overlay of histograms of 7-AAD content comparing the vehicle and BMS-5 treated cells shown in (b).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4016185&req=5

Figure 6: Inhibition of LIMK interferes with cell cycle progression of Nf2ΔEx2 MSCs. (a)Nf2ΔEx2 MSCs were grown in a 6-well format and treated with 5 µM BMS-5 for 7 hours prior to labeling. Cells were analyzed by flow cytometry. Propidium iodide labeling profile of the diploid cell population analyzed with the ModFit program. Tabulation of the distribution of cell cycle phases of 3 independent experiments (mean± SEM, n=3). (b)Nf2ΔEx2 MSCs were treated with 5 µM BMS-5 or DMSO control overnight prior to 3 hr BrdU labeling and analysis by flow cytometry. Distribution of BrdU- and 7-AAD-labelled cells analyzed with FlowJo software. These distribution plots are representative of 4 independent experiments (n=4). (c) Graph of the distribution of the cell cycle phases of all the experiments as mean ± SEM, *P<0.05 determined by two-way ANOVA and Bonferroni multiple comparisons post-test. (d) Overlay of histograms of 7-AAD content comparing the vehicle and BMS-5 treated cells shown in (b).
Mentions: To test the possibility that the decrease in proliferation was a consequence of cell-cycle inhibition, we performed flow cytometry analysis of cells stained with propidium iodine (PI). BMS-5 interfered with Nf2ΔEx2 MSCs cell-cycle progression as evidenced by an increase in the percentage of diploid cells trapped in the G2/M phase (Figure 6 a). We further analyzed the distribution of cells among the cell-cycle phases using a BrdU/7AAD assay. There was a significant increase in the number of cells accumulating in the G2/M phase in samples treated with 5 µM BMS-5 as compared to vehicle control (20% vs. 42%). This was accompanied by a decrease in the number of BMS-5 treated cells in G1 as compared to control (41% vs. 26%). This is consistent with the results obtained by analysis of PI staining (Figure 6 b–c). Overlay of the 7AAD-area histograms clearly demonstrated a significant increase in the G2/M population in BMS-5 compared to DMSO treated cells (Figure 6 d). Furthermore, the increase in the G2/M population was accompanied by an increase in the number of cells with higher DNA content consistent with a polyploidy or multinucleated cell population. There was also a smaller but substantial change in number of cells detected in the S-phase of cell cycle in samples treated with BMS-5 as compared to DMSO control. Lastly, the population of cells in the S phase decreased with increasing BMS-5 concentration (Supplementary Figure S2). These results taken together suggest that inhibition of LIMK activity in Nf2ΔEx2 MSCs interferes with progression of the G2/M phase of the cell cycle, thereby decreasing the number of viable Nf2ΔEx2 MSCs.

Bottom Line: We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs.Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs.Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL, USA.

ABSTRACT
Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

Show MeSH
Related in: MedlinePlus