Limits...
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Results: Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn.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.

Show MeSH

Related in: MedlinePlus

The growth suppressive effects of YA1 on single transformed colony-derived GEF16 NIH3T3 cells and polyclonal Ras transformed NIH3T3 cells are more pronounced when these are co-cultured with non-transformed cells. (A) Single transformed colonies were picked from 10-day cultures of GEF16 polyclonal NIH3T3 cells and expanded. These cells were then seeded at 2.0 × 105 cells per 30 mm dish and treated with 10 μM of YA1 or DMSO control either immediately or after 10 days in culture. Duplicate wells were collected for flow cytometry. (B) A total of 2 × 105 cells per well were plated consisting of increasing numbers of non-transformed vector cells co-cultured with decreasing numbers of single transformed colony-derived GEF16 cells. These were treated with 10 μM of either YA1 or DMSO for 10 days. (C) The same co-culture experiment described in (B) was performed substituting polyclonal Ras transformed NIH3T3 cells for GEF16 transformed cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2736836&req=5

fig5: The growth suppressive effects of YA1 on single transformed colony-derived GEF16 NIH3T3 cells and polyclonal Ras transformed NIH3T3 cells are more pronounced when these are co-cultured with non-transformed cells. (A) Single transformed colonies were picked from 10-day cultures of GEF16 polyclonal NIH3T3 cells and expanded. These cells were then seeded at 2.0 × 105 cells per 30 mm dish and treated with 10 μM of YA1 or DMSO control either immediately or after 10 days in culture. Duplicate wells were collected for flow cytometry. (B) A total of 2 × 105 cells per well were plated consisting of increasing numbers of non-transformed vector cells co-cultured with decreasing numbers of single transformed colony-derived GEF16 cells. These were treated with 10 μM of either YA1 or DMSO for 10 days. (C) The same co-culture experiment described in (B) was performed substituting polyclonal Ras transformed NIH3T3 cells for GEF16 transformed cells.

Mentions: Single transformed colonies were picked from GEF16 polyclonal NIH3T3 cells and expanded (Figure 5A). These were plated and inhibitor YA1 added either immediately or after 10 days when the cells were post-confluent. It can be seen that this has a modest effect when added to low density cultures whereby these do not achieve the same saturation density as control cells. However, addition of YA1 to post-confluent cultures has no discernible effect when compared with controls. Flow cytometry shows YA1 has no effect on the cell cycle and no apoptotic sub-G1 population is seen in cultures treated either pre- or post-confluence.


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)

The growth suppressive effects of YA1 on single transformed colony-derived GEF16 NIH3T3 cells and polyclonal Ras transformed NIH3T3 cells are more pronounced when these are co-cultured with non-transformed cells. (A) Single transformed colonies were picked from 10-day cultures of GEF16 polyclonal NIH3T3 cells and expanded. These cells were then seeded at 2.0 × 105 cells per 30 mm dish and treated with 10 μM of YA1 or DMSO control either immediately or after 10 days in culture. Duplicate wells were collected for flow cytometry. (B) A total of 2 × 105 cells per well were plated consisting of increasing numbers of non-transformed vector cells co-cultured with decreasing numbers of single transformed colony-derived GEF16 cells. These were treated with 10 μM of either YA1 or DMSO for 10 days. (C) The same co-culture experiment described in (B) was performed substituting polyclonal Ras transformed NIH3T3 cells for GEF16 transformed cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2736836&req=5

fig5: The growth suppressive effects of YA1 on single transformed colony-derived GEF16 NIH3T3 cells and polyclonal Ras transformed NIH3T3 cells are more pronounced when these are co-cultured with non-transformed cells. (A) Single transformed colonies were picked from 10-day cultures of GEF16 polyclonal NIH3T3 cells and expanded. These cells were then seeded at 2.0 × 105 cells per 30 mm dish and treated with 10 μM of YA1 or DMSO control either immediately or after 10 days in culture. Duplicate wells were collected for flow cytometry. (B) A total of 2 × 105 cells per well were plated consisting of increasing numbers of non-transformed vector cells co-cultured with decreasing numbers of single transformed colony-derived GEF16 cells. These were treated with 10 μM of either YA1 or DMSO for 10 days. (C) The same co-culture experiment described in (B) was performed substituting polyclonal Ras transformed NIH3T3 cells for GEF16 transformed cells.
Mentions: Single transformed colonies were picked from GEF16 polyclonal NIH3T3 cells and expanded (Figure 5A). These were plated and inhibitor YA1 added either immediately or after 10 days when the cells were post-confluent. It can be seen that this has a modest effect when added to low density cultures whereby these do not achieve the same saturation density as control cells. However, addition of YA1 to post-confluent cultures has no discernible effect when compared with controls. Flow cytometry shows YA1 has no effect on the cell cycle and no apoptotic sub-G1 population is seen in cultures treated either pre- or post-confluence.

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