2002 Fiscal Year Final Research Report Summary
The essential role of Rho family G protein in tumor metastasis and invasion.
| Project/Area Number |
13670557
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Gastroenterology
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| Research Institution | Keio University |
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Principal Investigator |
AZUMA Toshifumi Keio University, School of Medicine, Assistant professor, 医学部, 専任講師 (00222612)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIDA Jiro Tokyo Dentalcollege, Detalmedicine professor, 医学部, 教授 (50198470)
ISHII Hiromasa Keio University, School of Medicine, professor, 医学部, 教授 (20051500)
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| Project Period (FY) |
2001 – 2002
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| Keywords | Cancer / Metastasis / Rho GTPase |
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| Research Abstract |
We examined total Rac1 protein levels in each cell line and found no significant difference. All cell lines express similar level. We also examined active Rac1 levels using PBD pull down assay. We found that some cell lines kept significant amount of active Rac1 even under unstimulated condition. LoVo, DLD1 are kept significantly high level of active Rac1 compared to those of T84, HT29N2, HCT-15. ] We also examined total expression level of Cdc42 among these cell lines. Total amount of Cdc42 expression in each cell line was similar as Rac1. We then investigated active Cdc42 level using PBD pull down assay. Unlike Rac1 active Cdc42 level in each cell lines was quite similar from cell line to cell line. RhoA was reported to be the key protein for cell motility and liver cancer cell invasion. We then examined total RhoA expression level in each cell line. We found no outstanding difference in each cell lines, but we saw some cell lines express more than other cell lines. HCT 15 expresses
… More
most and LoVo have least amount of RhoA.
We then investigated this high active Rac1 level in some cell lines may be caused by Rac1 gene mutation as Ras gene mutation, which caused loss of GTPase function. We sequenced Rac1 gene in each cell lines and found no mutations. Then we investigated whether this active Rac1 level may correlate to cell motility. We used Matrigel invasion assay to see any differences in cell invasiveness. We found LoVo and DLD1 showed significantly higher invasiveness and T84 and HT29N2 did lowest motility. This correlation we saw could suggest that active Rac1 level is the most important factor for colon cancer. We then examined to see how important RhoA signaling. LPA is known to be the simulator of RhoA. We treated each cell line with LPA and we found some cell lines responded to this stimuli but other cell lined did not. We found that the motility of LoVo and DLD1 was inhibited after LPA stimulation. We also found that HCT15 and HT29N2 cells were responded to LPA stimulation. We also examined active RhoA levels in LoVo and DLD1 after LPA stimulation ; we found that RhoA was activated by LPA. We also examined RhoGDI levels of these cell lines but we found no significant differences. Interestingly LPA stimulation activates Rac1 inHT29N2 and HCT 15 cell lines. This activation was diminished after Rock inhibitor Y27632 treatment indicating that RhoA activation end up with Rac1 activation and this signal transduction was key for cell lines with low active Rac1. On the other hand cells with high active Rac1 lacks some signaling cascade to transfer RhoA activation signal to Rac1 activation. This is the reason why these cell lies did not show any Rac1 activation after RhoA simulation even though RhoA was activated. Less
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Research Products
(5 results)
