RhoA inactivation inhibits cell migration but does not mediate the effects of polyamine depletion

Ramesh M. Ray, Anami Patel, Mary Jane Viar, Shirley A. McCormack, Yi Zheng, Gabor Tigyi, Leonard R. Johnson

Research output: Contribution to journalArticle

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Abstract

Background & Aims: Inhibition of RhoA activity and depletion of polyamines inhibits cell migration and causes changes in the actin cytoskeleton. In this article we have examined the effect of polyamine depletion on RhoA and evaluated these effects on cell migration. Methods: Polyamines were depleted in intestinal epithelial cell (IEC)-6 cells by incubating them for 4 days with 5 mmol/L α-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase, the first rate-limiting enzyme in the synthesis of polyamines. IEC-6 cells were then transfected with vectors containing HA tags and constitutively active (HA-V14) or dominant-negative (HA-N19) RhoA with pcDNA3 (vector). Results: DFMO caused a significant decrease in Rho levels in the cytoplasm and membranes of IEC-6 cells. This decrease was caused by an approximate 50% inhibition of RhoA protein synthesis. Neither the half-life of RhoA nor the level of RhoA messenger RNA (mRNA) was affected. HA-V14-RhoA cells migrated much more rapidly than vector-transfected cells, and HA-N19-RhoA cells exhibited almost no motility. The migration of HA-V14-RhoA cells, however, was inhibited markedly by polyamine depletion. Polyamine depletion did not affect the activity of RhoA in HA-V14-RhoA cells, but inhibited it dramatically in the vector-transfected cells. In the presence of DFMO, the HA-V14-RhoA cells lost stress fibers and gained the appearance of HA-N19-RhoA cells or wild-type cells treated with DFMO. Conclusions: First, polyamines are essential for the activity and synthesis and, therefore, normal levels of RhoA protein. Second, RhoA does not mediate the inhibitory effects of polyamine depletion on cell migration.

Original languageEnglish (US)
Pages (from-to)196-205
Number of pages10
JournalGastroenterology
Volume123
Issue number1
DOIs
StatePublished - Jan 1 2002

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Polyamines
Cell Movement
Eflornithine
rhoA GTP-Binding Protein
Epithelial Cells
Stress Fibers
Ornithine Decarboxylase
Actin Cytoskeleton
Half-Life
Cytoplasm

All Science Journal Classification (ASJC) codes

  • Gastroenterology

Cite this

Ray, R. M., Patel, A., Viar, M. J., McCormack, S. A., Zheng, Y., Tigyi, G., & Johnson, L. R. (2002). RhoA inactivation inhibits cell migration but does not mediate the effects of polyamine depletion. Gastroenterology, 123(1), 196-205. https://doi.org/10.1053/gast.2002.34216

RhoA inactivation inhibits cell migration but does not mediate the effects of polyamine depletion. / Ray, Ramesh M.; Patel, Anami; Viar, Mary Jane; McCormack, Shirley A.; Zheng, Yi; Tigyi, Gabor; Johnson, Leonard R.

In: Gastroenterology, Vol. 123, No. 1, 01.01.2002, p. 196-205.

Research output: Contribution to journalArticle

Ray, RM, Patel, A, Viar, MJ, McCormack, SA, Zheng, Y, Tigyi, G & Johnson, LR 2002, 'RhoA inactivation inhibits cell migration but does not mediate the effects of polyamine depletion', Gastroenterology, vol. 123, no. 1, pp. 196-205. https://doi.org/10.1053/gast.2002.34216
Ray, Ramesh M. ; Patel, Anami ; Viar, Mary Jane ; McCormack, Shirley A. ; Zheng, Yi ; Tigyi, Gabor ; Johnson, Leonard R. / RhoA inactivation inhibits cell migration but does not mediate the effects of polyamine depletion. In: Gastroenterology. 2002 ; Vol. 123, No. 1. pp. 196-205.
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abstract = "Background & Aims: Inhibition of RhoA activity and depletion of polyamines inhibits cell migration and causes changes in the actin cytoskeleton. In this article we have examined the effect of polyamine depletion on RhoA and evaluated these effects on cell migration. Methods: Polyamines were depleted in intestinal epithelial cell (IEC)-6 cells by incubating them for 4 days with 5 mmol/L α-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase, the first rate-limiting enzyme in the synthesis of polyamines. IEC-6 cells were then transfected with vectors containing HA tags and constitutively active (HA-V14) or dominant-negative (HA-N19) RhoA with pcDNA3 (vector). Results: DFMO caused a significant decrease in Rho levels in the cytoplasm and membranes of IEC-6 cells. This decrease was caused by an approximate 50{\%} inhibition of RhoA protein synthesis. Neither the half-life of RhoA nor the level of RhoA messenger RNA (mRNA) was affected. HA-V14-RhoA cells migrated much more rapidly than vector-transfected cells, and HA-N19-RhoA cells exhibited almost no motility. The migration of HA-V14-RhoA cells, however, was inhibited markedly by polyamine depletion. Polyamine depletion did not affect the activity of RhoA in HA-V14-RhoA cells, but inhibited it dramatically in the vector-transfected cells. In the presence of DFMO, the HA-V14-RhoA cells lost stress fibers and gained the appearance of HA-N19-RhoA cells or wild-type cells treated with DFMO. Conclusions: First, polyamines are essential for the activity and synthesis and, therefore, normal levels of RhoA protein. Second, RhoA does not mediate the inhibitory effects of polyamine depletion on cell migration.",
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AU - Patel, Anami

AU - Viar, Mary Jane

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AU - Zheng, Yi

AU - Tigyi, Gabor

AU - Johnson, Leonard R.

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AB - Background & Aims: Inhibition of RhoA activity and depletion of polyamines inhibits cell migration and causes changes in the actin cytoskeleton. In this article we have examined the effect of polyamine depletion on RhoA and evaluated these effects on cell migration. Methods: Polyamines were depleted in intestinal epithelial cell (IEC)-6 cells by incubating them for 4 days with 5 mmol/L α-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase, the first rate-limiting enzyme in the synthesis of polyamines. IEC-6 cells were then transfected with vectors containing HA tags and constitutively active (HA-V14) or dominant-negative (HA-N19) RhoA with pcDNA3 (vector). Results: DFMO caused a significant decrease in Rho levels in the cytoplasm and membranes of IEC-6 cells. This decrease was caused by an approximate 50% inhibition of RhoA protein synthesis. Neither the half-life of RhoA nor the level of RhoA messenger RNA (mRNA) was affected. HA-V14-RhoA cells migrated much more rapidly than vector-transfected cells, and HA-N19-RhoA cells exhibited almost no motility. The migration of HA-V14-RhoA cells, however, was inhibited markedly by polyamine depletion. Polyamine depletion did not affect the activity of RhoA in HA-V14-RhoA cells, but inhibited it dramatically in the vector-transfected cells. In the presence of DFMO, the HA-V14-RhoA cells lost stress fibers and gained the appearance of HA-N19-RhoA cells or wild-type cells treated with DFMO. Conclusions: First, polyamines are essential for the activity and synthesis and, therefore, normal levels of RhoA protein. Second, RhoA does not mediate the inhibitory effects of polyamine depletion on cell migration.

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