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Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin.

Li D, Mrsny RJ - J. Cell Biol. (2000)

Bottom Line: Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell-cell contact growth control.Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs.Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Research and Development, Genentech Inc., South San Francisco, California 94080, USA.

ABSTRACT
Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell-cell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1-induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.

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Expression of occludin suppressed anchorage-independent growth of Pa-4ΔRaf-1:ER cells in soft agarose. (A) Representative photographs of dimethylthiazol-diphenyltetrazolium bromide (MTT)-stained dishes. Pa-4-vec (panels a and b), Pa-4ΔRaf-1:ER (panels c and d), and Pa-4ΔRaf-1:ER-occludin (panels e and f) cells were plated at 10,000 cells/35-mm dish in semisolid media containing 0.35% low melting temperature agarose in the absence (panels a, c, and e) or presence (panels b, d, and f) of 1 mM estradiol. The cells were stained with 0.05 mg/ml MTT after 15 d. (B) The average number of colonies obtained from three 35-mm dishes/group ± SD is represented graphically. Colonies >0.3 mm in diameter were counted. Similar results were seen in seven of eight Pa-4ΔRaf-1:ER-occludin clones.
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Figure 6: Expression of occludin suppressed anchorage-independent growth of Pa-4ΔRaf-1:ER cells in soft agarose. (A) Representative photographs of dimethylthiazol-diphenyltetrazolium bromide (MTT)-stained dishes. Pa-4-vec (panels a and b), Pa-4ΔRaf-1:ER (panels c and d), and Pa-4ΔRaf-1:ER-occludin (panels e and f) cells were plated at 10,000 cells/35-mm dish in semisolid media containing 0.35% low melting temperature agarose in the absence (panels a, c, and e) or presence (panels b, d, and f) of 1 mM estradiol. The cells were stained with 0.05 mg/ml MTT after 15 d. (B) The average number of colonies obtained from three 35-mm dishes/group ± SD is represented graphically. Colonies >0.3 mm in diameter were counted. Similar results were seen in seven of eight Pa-4ΔRaf-1:ER-occludin clones.

Mentions: Cancer cells grow aggressively and invasively. In a cell culture system, this is represented by their ability to grow anchorage-independently in soft agarose. Although the basal activity of ΔRaf-1:ER fusion kinase was sufficient to disrupt TJ function and suppress occludin expression in Pa-4 cells, the kinase activity of ΔRaf-1:ER can be further induced in the presence of added estradiol. Further induction of Raf-1 activity can greatly increase the ability of Pa-4ΔRaf-1:ER cells to form colonies in soft agarose plates (Li et al. 1997). We examined the effect of occludin reintroduction on the growth characteristics of Raf-1–activated Pa-4 cells. We found that expression of occludin in Pa-4ΔRaf-1:ER cells significantly decreased their ability to grow in soft agarose in the absence or presence of estradiol (Fig. 6). Thus, there is a possibility that upregulation of occludin expression could be a potential approach to treat Raf-1–induced epithelial cancers.


Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin.

Li D, Mrsny RJ - J. Cell Biol. (2000)

Expression of occludin suppressed anchorage-independent growth of Pa-4ΔRaf-1:ER cells in soft agarose. (A) Representative photographs of dimethylthiazol-diphenyltetrazolium bromide (MTT)-stained dishes. Pa-4-vec (panels a and b), Pa-4ΔRaf-1:ER (panels c and d), and Pa-4ΔRaf-1:ER-occludin (panels e and f) cells were plated at 10,000 cells/35-mm dish in semisolid media containing 0.35% low melting temperature agarose in the absence (panels a, c, and e) or presence (panels b, d, and f) of 1 mM estradiol. The cells were stained with 0.05 mg/ml MTT after 15 d. (B) The average number of colonies obtained from three 35-mm dishes/group ± SD is represented graphically. Colonies >0.3 mm in diameter were counted. Similar results were seen in seven of eight Pa-4ΔRaf-1:ER-occludin clones.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169369&req=5

Figure 6: Expression of occludin suppressed anchorage-independent growth of Pa-4ΔRaf-1:ER cells in soft agarose. (A) Representative photographs of dimethylthiazol-diphenyltetrazolium bromide (MTT)-stained dishes. Pa-4-vec (panels a and b), Pa-4ΔRaf-1:ER (panels c and d), and Pa-4ΔRaf-1:ER-occludin (panels e and f) cells were plated at 10,000 cells/35-mm dish in semisolid media containing 0.35% low melting temperature agarose in the absence (panels a, c, and e) or presence (panels b, d, and f) of 1 mM estradiol. The cells were stained with 0.05 mg/ml MTT after 15 d. (B) The average number of colonies obtained from three 35-mm dishes/group ± SD is represented graphically. Colonies >0.3 mm in diameter were counted. Similar results were seen in seven of eight Pa-4ΔRaf-1:ER-occludin clones.
Mentions: Cancer cells grow aggressively and invasively. In a cell culture system, this is represented by their ability to grow anchorage-independently in soft agarose. Although the basal activity of ΔRaf-1:ER fusion kinase was sufficient to disrupt TJ function and suppress occludin expression in Pa-4 cells, the kinase activity of ΔRaf-1:ER can be further induced in the presence of added estradiol. Further induction of Raf-1 activity can greatly increase the ability of Pa-4ΔRaf-1:ER cells to form colonies in soft agarose plates (Li et al. 1997). We examined the effect of occludin reintroduction on the growth characteristics of Raf-1–activated Pa-4 cells. We found that expression of occludin in Pa-4ΔRaf-1:ER cells significantly decreased their ability to grow in soft agarose in the absence or presence of estradiol (Fig. 6). Thus, there is a possibility that upregulation of occludin expression could be a potential approach to treat Raf-1–induced epithelial cancers.

Bottom Line: Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell-cell contact growth control.Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs.Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Research and Development, Genentech Inc., South San Francisco, California 94080, USA.

ABSTRACT
Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell-cell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1-induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.

Show MeSH
Related in: MedlinePlus