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Analogues of Y27632 increase gap junction communication and suppress the formation of transformed NIH3T3 colonies.

Hampson L, He XT, Oliver AW, Hadfield JA, Kemp T, Butler J, McGown A, Kitchener HC, Hampson IN - Br. J. Cancer (2009)

Bottom Line: Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras.In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4).Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.

View Article: PubMed Central - PubMed

Affiliation: University of Manchester School of Cancer Studies and Imaging Science, Gynaecological Oncology Laboratories, St Mary's Hospital, Manchester M13 OJH, UK.

ABSTRACT

Background: Constitutive activation of RhoA-dependent RhoA kinase (ROCK) signalling is known to promote cellular transformation and the ROCK inhibitor Y-27632 has the ability to suppress focus formation of RhoA transformed NIH3T3 cells.

Methods: Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras. In vitro kinase inhibitor profiling, co-culture of transformed cells with non-transformed cells and a novel Lucifer yellow/PKH67 dye transfer method were used to investigate their mode of action.

Results: Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn. No toxicity was observed against either transformed or non-transformed cells and the effect was dependent on co-culture of these two cell types. In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4). Dye transfer analysis showed they increased gap junction intercellular communication (GJIC) between transformed and non-transformed cells.

Conclusions: These data are the first to suggest that transient blockade of specific kinases can induce a persistent inhibition of non-contact inhibited transformed colony formation and can also remove pre-formed colonies. These effects could potentially be mediated by the observed increase in GJIC between transformed and non-transformed cells. Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.

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YA1 and Y27632 irreversibly suppress the formation of GEF16 transformed NIH3T3 colonies whereas, unlike Y27632, YA1 eliminates pre-existing transformed colonies. (A) Polyclonal GEF16 transfected cells were seeded at 2.0 × 105 cells per 30 mm dish and incubated overnight. A measure of 10 μM YA1 or Y27632 was then added to each of these for 2, 4, 6, 8 and 10 days, respectively, where upon the cells were detached with trypsin and re-plated at a density of 2.0 × 105 cells. After a further 10 days culture in the absence of inhibitors the cells were stained with Toluidine blue. (B) Polyclonal GEF16 and vector transfected cells were seeded at 2.0 × 105 cells per 30 mm dish and incubated for 10 days after which 10 or 20 μM of either, YA1, Y27632 or DMSO control was added. These were incubated for 3 or 6 days then stained with Toluidine blue.
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fig4: YA1 and Y27632 irreversibly suppress the formation of GEF16 transformed NIH3T3 colonies whereas, unlike Y27632, YA1 eliminates pre-existing transformed colonies. (A) Polyclonal GEF16 transfected cells were seeded at 2.0 × 105 cells per 30 mm dish and incubated overnight. A measure of 10 μM YA1 or Y27632 was then added to each of these for 2, 4, 6, 8 and 10 days, respectively, where upon the cells were detached with trypsin and re-plated at a density of 2.0 × 105 cells. After a further 10 days culture in the absence of inhibitors the cells were stained with Toluidine blue. (B) Polyclonal GEF16 and vector transfected cells were seeded at 2.0 × 105 cells per 30 mm dish and incubated for 10 days after which 10 or 20 μM of either, YA1, Y27632 or DMSO control was added. These were incubated for 3 or 6 days then stained with Toluidine blue.

Mentions: Freshly plated GEF16 polyclonal cells were treated with 10 μM of YA1 for 2, 4, 6, 8 and 10 days, respectively, after which the compound was removed from the culture media and the cells maintained in normal media for a further 10-day chase period. This shows a progressive decrease in the number of transformed foci associated with increased time of exposure to the compound (Figure 3A) and indicates that YA1 not only inhibits the formation of transformed foci but, on withdrawal, also prevents transformed foci from reforming. Significantly, there is no detectable difference in growth rates of sub-confluent GEF16 polyclonal cells treated with either inhibitor YA1 or DMSO control (Figure 3B) (P>0.05), and flow cytometry shows no evidence of alterations in cell cycle or the accumulation of an apoptotic sub-G1 population (Figure 3C). In Figure 3A the number of cells per dish increases with each successive inhibitor-treatment time interval before withdrawal of the compound. To remove this variable and to ensure that each 10-day chase period starts with the same number of inhibitor-treated cells, GEF16 polyclonal cells were treated with either inhibitor YA1 or Y27632 for 2, 4, 6, 8 and 10 days. After this, cells were detached, re-seeded at 2 × 105 per well in 6-well plates and then maintained in the absence of inhibitors for a 10-day chase period. This shows suppression of transformed focus formation by either YA1 or Y27632 treatment, which correlates with the duration of exposure (Figure 4A). Collectively these observations are the first to show that transient treatment with Y27632 or YA1 permanently suppresses transformed colony formation and does not involve cell killing.


Analogues of Y27632 increase gap junction communication and suppress the formation of transformed NIH3T3 colonies.

Hampson L, He XT, Oliver AW, Hadfield JA, Kemp T, Butler J, McGown A, Kitchener HC, Hampson IN - Br. J. Cancer (2009)

YA1 and Y27632 irreversibly suppress the formation of GEF16 transformed NIH3T3 colonies whereas, unlike Y27632, YA1 eliminates pre-existing transformed colonies. (A) Polyclonal GEF16 transfected cells were seeded at 2.0 × 105 cells per 30 mm dish and incubated overnight. A measure of 10 μM YA1 or Y27632 was then added to each of these for 2, 4, 6, 8 and 10 days, respectively, where upon the cells were detached with trypsin and re-plated at a density of 2.0 × 105 cells. After a further 10 days culture in the absence of inhibitors the cells were stained with Toluidine blue. (B) Polyclonal GEF16 and vector transfected cells were seeded at 2.0 × 105 cells per 30 mm dish and incubated for 10 days after which 10 or 20 μM of either, YA1, Y27632 or DMSO control was added. These were incubated for 3 or 6 days then stained with Toluidine blue.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2736836&req=5

fig4: YA1 and Y27632 irreversibly suppress the formation of GEF16 transformed NIH3T3 colonies whereas, unlike Y27632, YA1 eliminates pre-existing transformed colonies. (A) Polyclonal GEF16 transfected cells were seeded at 2.0 × 105 cells per 30 mm dish and incubated overnight. A measure of 10 μM YA1 or Y27632 was then added to each of these for 2, 4, 6, 8 and 10 days, respectively, where upon the cells were detached with trypsin and re-plated at a density of 2.0 × 105 cells. After a further 10 days culture in the absence of inhibitors the cells were stained with Toluidine blue. (B) Polyclonal GEF16 and vector transfected cells were seeded at 2.0 × 105 cells per 30 mm dish and incubated for 10 days after which 10 or 20 μM of either, YA1, Y27632 or DMSO control was added. These were incubated for 3 or 6 days then stained with Toluidine blue.
Mentions: Freshly plated GEF16 polyclonal cells were treated with 10 μM of YA1 for 2, 4, 6, 8 and 10 days, respectively, after which the compound was removed from the culture media and the cells maintained in normal media for a further 10-day chase period. This shows a progressive decrease in the number of transformed foci associated with increased time of exposure to the compound (Figure 3A) and indicates that YA1 not only inhibits the formation of transformed foci but, on withdrawal, also prevents transformed foci from reforming. Significantly, there is no detectable difference in growth rates of sub-confluent GEF16 polyclonal cells treated with either inhibitor YA1 or DMSO control (Figure 3B) (P>0.05), and flow cytometry shows no evidence of alterations in cell cycle or the accumulation of an apoptotic sub-G1 population (Figure 3C). In Figure 3A the number of cells per dish increases with each successive inhibitor-treatment time interval before withdrawal of the compound. To remove this variable and to ensure that each 10-day chase period starts with the same number of inhibitor-treated cells, GEF16 polyclonal cells were treated with either inhibitor YA1 or Y27632 for 2, 4, 6, 8 and 10 days. After this, cells were detached, re-seeded at 2 × 105 per well in 6-well plates and then maintained in the absence of inhibitors for a 10-day chase period. This shows suppression of transformed focus formation by either YA1 or Y27632 treatment, which correlates with the duration of exposure (Figure 4A). Collectively these observations are the first to show that transient treatment with Y27632 or YA1 permanently suppresses transformed colony formation and does not involve cell killing.

Bottom Line: Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras.In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4).Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.

View Article: PubMed Central - PubMed

Affiliation: University of Manchester School of Cancer Studies and Imaging Science, Gynaecological Oncology Laboratories, St Mary's Hospital, Manchester M13 OJH, UK.

ABSTRACT

Background: Constitutive activation of RhoA-dependent RhoA kinase (ROCK) signalling is known to promote cellular transformation and the ROCK inhibitor Y-27632 has the ability to suppress focus formation of RhoA transformed NIH3T3 cells.

Methods: Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras. In vitro kinase inhibitor profiling, co-culture of transformed cells with non-transformed cells and a novel Lucifer yellow/PKH67 dye transfer method were used to investigate their mode of action.

Results: Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn. No toxicity was observed against either transformed or non-transformed cells and the effect was dependent on co-culture of these two cell types. In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4). Dye transfer analysis showed they increased gap junction intercellular communication (GJIC) between transformed and non-transformed cells.

Conclusions: These data are the first to suggest that transient blockade of specific kinases can induce a persistent inhibition of non-contact inhibited transformed colony formation and can also remove pre-formed colonies. These effects could potentially be mediated by the observed increase in GJIC between transformed and non-transformed cells. Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.

Show MeSH
Related in: MedlinePlus