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Results: Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn.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

YA1 stimulates GJIC between transformed and non-transformed cells. (A) Cells derived from a single GEF16 transformed colony were electroporated with LY and co-cultured with non-transformed cells that had been labelled earlier with PKH67. Either YA1 (10 μM) or DMSO control was added to duplicate cultures and these were collected at T=0, 1.5, 4.5 and 7.5 h for analysis by flow cytometry. The numbers of cells displaying each type of fluorescence is shown. (B) The ratio of double LY/PKH67-labelled cells expressed as percentage of the total LY population.
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fig6: YA1 stimulates GJIC between transformed and non-transformed cells. (A) Cells derived from a single GEF16 transformed colony were electroporated with LY and co-cultured with non-transformed cells that had been labelled earlier with PKH67. Either YA1 (10 μM) or DMSO control was added to duplicate cultures and these were collected at T=0, 1.5, 4.5 and 7.5 h for analysis by flow cytometry. The numbers of cells displaying each type of fluorescence is shown. (B) The ratio of double LY/PKH67-labelled cells expressed as percentage of the total LY population.

Mentions: Figure 6A shows the effect of either YA1 or DMSO control on the extent of dye transfer from co-cultured donor LY-labelled GEF16 single colony transformed cells to recipient PKH67 stained non-transformed cells. It can be seen from Figure 6B that YA1-treated cultures have approximately three times the number of double LY/PKH67-labelled cells when compared with DMSO control (P<0.05). This indicates an increase in the transfer of dye from LY to PKH67 stained cells, which is consistent with an inhibitor-induced increase in GJIC.


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 stimulates GJIC between transformed and non-transformed cells. (A) Cells derived from a single GEF16 transformed colony were electroporated with LY and co-cultured with non-transformed cells that had been labelled earlier with PKH67. Either YA1 (10 μM) or DMSO control was added to duplicate cultures and these were collected at T=0, 1.5, 4.5 and 7.5 h for analysis by flow cytometry. The numbers of cells displaying each type of fluorescence is shown. (B) The ratio of double LY/PKH67-labelled cells expressed as percentage of the total LY population.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: YA1 stimulates GJIC between transformed and non-transformed cells. (A) Cells derived from a single GEF16 transformed colony were electroporated with LY and co-cultured with non-transformed cells that had been labelled earlier with PKH67. Either YA1 (10 μM) or DMSO control was added to duplicate cultures and these were collected at T=0, 1.5, 4.5 and 7.5 h for analysis by flow cytometry. The numbers of cells displaying each type of fluorescence is shown. (B) The ratio of double LY/PKH67-labelled cells expressed as percentage of the total LY population.
Mentions: Figure 6A shows the effect of either YA1 or DMSO control on the extent of dye transfer from co-cultured donor LY-labelled GEF16 single colony transformed cells to recipient PKH67 stained non-transformed cells. It can be seen from Figure 6B that YA1-treated cultures have approximately three times the number of double LY/PKH67-labelled cells when compared with DMSO control (P<0.05). This indicates an increase in the transfer of dye from LY to PKH67 stained cells, which is consistent with an inhibitor-induced increase in GJIC.

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