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Differential partitioning of Galphai1 with the cellular microtubules: a possible mechanism of development of Taxol resistance in human ovarian carcinoma cells.

Parekh HK, Adikari M, Vennapusa B - J Mol Signal (2006)

Bottom Line: Recently, changes in the dynamic instability of the microtubules have also been associated with development of taxol resistance in a lung cancer cell line.Interestingly, increased association of the Galphai1 protein and the microtubules in the taxol-resistant cells compared to the parental 2008 cells was observed, both prior to and after treatment of these cells with taxol.Based on the opposing effects of taxol and the Galphai1 protein on the microtubule dynamic instability (taxol suppresses microtubule dynamic instability whilst the Galphai1 protein inhibits the suppression) our results indicate the operation of a novel pathway that would enable the cells to escape the cytotoxic effects of taxol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Temple University School of Medicine, 3400 N, Broad Street, Philadelphia, PA 19140, USA. hemant.parekh@temple.edu

ABSTRACT

Background: Taxol binds to the cellular microtubules and suppresses their dynamic instability. Development of tumor cell resistance to taxol is typically associated with increased expression of the drug efflux pump P-glycoprotein and/or alterations in the microtubules. Recently, changes in the dynamic instability of the microtubules have also been associated with development of taxol resistance in a lung cancer cell line. We have established a 250-fold taxol-resistant human ovarian carcinoma subline (2008/13/4) that does not display the typical alterations associated with development of drug resistance.

Results: Utilizing the mRNA differential display technique, we observed increased expression of an alpha subunit of the guanine nucleotide-binding protein, Galphai1, in the taxol-resistant human ovarian carcinoma cell lines compared to the parental 2008 cells. Several isoforms of the alpha-subunit of the G protein have been identified and the Galphai (inhibitory) are so named because they inhibit the activity of adenylate cyclase leading to inactivation of the cAMP-dependent protein kinase A (PKA) pathway. In addition, Galphai1 is also known to bind to microtubules and activates their GTPase activity and thus induces depolymerization of the microtubules. In the present study we demonstrate that the intracellular level of cAMP and the PKA activity were higher in the taxol-resistant 2008/13/4 and the 2008/17/4 cells despite the increased expression of Galphai1 in these cells. Moreover, Galphai1 was found to be localized not on the cell membrane, but in intracellular compartments in both the taxol-sensitive and -resistant human ovarian carcinoma cells. Interestingly, increased association of the Galphai1 protein and the microtubules in the taxol-resistant cells compared to the parental 2008 cells was observed, both prior to and after treatment of these cells with taxol.

Conclusion: Based on the opposing effects of taxol and the Galphai1 protein on the microtubule dynamic instability (taxol suppresses microtubule dynamic instability whilst the Galphai1 protein inhibits the suppression) our results indicate the operation of a novel pathway that would enable the cells to escape the cytotoxic effects of taxol.

No MeSH data available.


Related in: MedlinePlus

Expression of Gαi1 in the taxol-sensitive and -resistant cells. Whole cell lysate was prepared from each of the cell line by scraping into a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton-X-100, 0.5% (v/v) Nonidet P40, 2.5 mM Na pyrophosphate, 1 mM NaOV, 50 mM NaF and 1× protease inhibitor cocktail and incubated on ice for 15 minutes. The lysate was then centrifuged at 13,000 × g for 20 min and the supernatant was transferred to a fresh tube and stored at -80°C until use. Proteins (25 μg/lane) were separated on a SDS-PAG and transferred to a PVDF membrane. Western blotting analysis was performed using rabbit polyclonal antibody against Gαi1 and enhanced chemiluminescence reagents. Expression of α-tubulin was evaluated in the same lysates to ensure equal protein concentrations in each sample. For Northern blotting analysis, total RNA (20 μg) extracted from each cell was separated and transferred to Nylon membrane. Full-length Gαi1 cDNA was used as probe. The ethidium bromide stained RNA gel is shown in the bottom right-hand corner to ensure equal RNA loading.
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Figure 1: Expression of Gαi1 in the taxol-sensitive and -resistant cells. Whole cell lysate was prepared from each of the cell line by scraping into a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton-X-100, 0.5% (v/v) Nonidet P40, 2.5 mM Na pyrophosphate, 1 mM NaOV, 50 mM NaF and 1× protease inhibitor cocktail and incubated on ice for 15 minutes. The lysate was then centrifuged at 13,000 × g for 20 min and the supernatant was transferred to a fresh tube and stored at -80°C until use. Proteins (25 μg/lane) were separated on a SDS-PAG and transferred to a PVDF membrane. Western blotting analysis was performed using rabbit polyclonal antibody against Gαi1 and enhanced chemiluminescence reagents. Expression of α-tubulin was evaluated in the same lysates to ensure equal protein concentrations in each sample. For Northern blotting analysis, total RNA (20 μg) extracted from each cell was separated and transferred to Nylon membrane. Full-length Gαi1 cDNA was used as probe. The ethidium bromide stained RNA gel is shown in the bottom right-hand corner to ensure equal RNA loading.

Mentions: To identify genes whose expression is either induced or reduced in the taxol-resistant cells, we systematically compared mRNA display patterns between parental (2008) and the taxol-resistant (2008/13/4 and 2008/17/4) cells. We choose only those bands that showed a consistent differential expression in the duplicate samples from the 2008/13/4 cells and/or 2008/17/4 cells compared to the 2008 cells. Furthermore, we chose only those bands that exhibited at least 3-fold change in densitometric intensity. We thus identified the upregulation of the alpha-i1 subunit of the guanine nucleotide binding protein mRNA (Gαi1; data not shown) in the 2008/13/4 and 2008/17/4 cells compared to the 2008 cells. The increased expression of the Gαi1 in the taxol resistant cells was confirmed by Northern and Western blotting analysis. As shown in Figure 1, a 15-fold and 6-fold higher expression of Gαi1 (protein and mRNA, respectively) was observed in the 2008/13/4 and 2008/17/4 cells, respectively, compared to the 2008 cells.


Differential partitioning of Galphai1 with the cellular microtubules: a possible mechanism of development of Taxol resistance in human ovarian carcinoma cells.

Parekh HK, Adikari M, Vennapusa B - J Mol Signal (2006)

Expression of Gαi1 in the taxol-sensitive and -resistant cells. Whole cell lysate was prepared from each of the cell line by scraping into a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton-X-100, 0.5% (v/v) Nonidet P40, 2.5 mM Na pyrophosphate, 1 mM NaOV, 50 mM NaF and 1× protease inhibitor cocktail and incubated on ice for 15 minutes. The lysate was then centrifuged at 13,000 × g for 20 min and the supernatant was transferred to a fresh tube and stored at -80°C until use. Proteins (25 μg/lane) were separated on a SDS-PAG and transferred to a PVDF membrane. Western blotting analysis was performed using rabbit polyclonal antibody against Gαi1 and enhanced chemiluminescence reagents. Expression of α-tubulin was evaluated in the same lysates to ensure equal protein concentrations in each sample. For Northern blotting analysis, total RNA (20 μg) extracted from each cell was separated and transferred to Nylon membrane. Full-length Gαi1 cDNA was used as probe. The ethidium bromide stained RNA gel is shown in the bottom right-hand corner to ensure equal RNA loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1761139&req=5

Figure 1: Expression of Gαi1 in the taxol-sensitive and -resistant cells. Whole cell lysate was prepared from each of the cell line by scraping into a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton-X-100, 0.5% (v/v) Nonidet P40, 2.5 mM Na pyrophosphate, 1 mM NaOV, 50 mM NaF and 1× protease inhibitor cocktail and incubated on ice for 15 minutes. The lysate was then centrifuged at 13,000 × g for 20 min and the supernatant was transferred to a fresh tube and stored at -80°C until use. Proteins (25 μg/lane) were separated on a SDS-PAG and transferred to a PVDF membrane. Western blotting analysis was performed using rabbit polyclonal antibody against Gαi1 and enhanced chemiluminescence reagents. Expression of α-tubulin was evaluated in the same lysates to ensure equal protein concentrations in each sample. For Northern blotting analysis, total RNA (20 μg) extracted from each cell was separated and transferred to Nylon membrane. Full-length Gαi1 cDNA was used as probe. The ethidium bromide stained RNA gel is shown in the bottom right-hand corner to ensure equal RNA loading.
Mentions: To identify genes whose expression is either induced or reduced in the taxol-resistant cells, we systematically compared mRNA display patterns between parental (2008) and the taxol-resistant (2008/13/4 and 2008/17/4) cells. We choose only those bands that showed a consistent differential expression in the duplicate samples from the 2008/13/4 cells and/or 2008/17/4 cells compared to the 2008 cells. Furthermore, we chose only those bands that exhibited at least 3-fold change in densitometric intensity. We thus identified the upregulation of the alpha-i1 subunit of the guanine nucleotide binding protein mRNA (Gαi1; data not shown) in the 2008/13/4 and 2008/17/4 cells compared to the 2008 cells. The increased expression of the Gαi1 in the taxol resistant cells was confirmed by Northern and Western blotting analysis. As shown in Figure 1, a 15-fold and 6-fold higher expression of Gαi1 (protein and mRNA, respectively) was observed in the 2008/13/4 and 2008/17/4 cells, respectively, compared to the 2008 cells.

Bottom Line: Recently, changes in the dynamic instability of the microtubules have also been associated with development of taxol resistance in a lung cancer cell line.Interestingly, increased association of the Galphai1 protein and the microtubules in the taxol-resistant cells compared to the parental 2008 cells was observed, both prior to and after treatment of these cells with taxol.Based on the opposing effects of taxol and the Galphai1 protein on the microtubule dynamic instability (taxol suppresses microtubule dynamic instability whilst the Galphai1 protein inhibits the suppression) our results indicate the operation of a novel pathway that would enable the cells to escape the cytotoxic effects of taxol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Temple University School of Medicine, 3400 N, Broad Street, Philadelphia, PA 19140, USA. hemant.parekh@temple.edu

ABSTRACT

Background: Taxol binds to the cellular microtubules and suppresses their dynamic instability. Development of tumor cell resistance to taxol is typically associated with increased expression of the drug efflux pump P-glycoprotein and/or alterations in the microtubules. Recently, changes in the dynamic instability of the microtubules have also been associated with development of taxol resistance in a lung cancer cell line. We have established a 250-fold taxol-resistant human ovarian carcinoma subline (2008/13/4) that does not display the typical alterations associated with development of drug resistance.

Results: Utilizing the mRNA differential display technique, we observed increased expression of an alpha subunit of the guanine nucleotide-binding protein, Galphai1, in the taxol-resistant human ovarian carcinoma cell lines compared to the parental 2008 cells. Several isoforms of the alpha-subunit of the G protein have been identified and the Galphai (inhibitory) are so named because they inhibit the activity of adenylate cyclase leading to inactivation of the cAMP-dependent protein kinase A (PKA) pathway. In addition, Galphai1 is also known to bind to microtubules and activates their GTPase activity and thus induces depolymerization of the microtubules. In the present study we demonstrate that the intracellular level of cAMP and the PKA activity were higher in the taxol-resistant 2008/13/4 and the 2008/17/4 cells despite the increased expression of Galphai1 in these cells. Moreover, Galphai1 was found to be localized not on the cell membrane, but in intracellular compartments in both the taxol-sensitive and -resistant human ovarian carcinoma cells. Interestingly, increased association of the Galphai1 protein and the microtubules in the taxol-resistant cells compared to the parental 2008 cells was observed, both prior to and after treatment of these cells with taxol.

Conclusion: Based on the opposing effects of taxol and the Galphai1 protein on the microtubule dynamic instability (taxol suppresses microtubule dynamic instability whilst the Galphai1 protein inhibits the suppression) our results indicate the operation of a novel pathway that would enable the cells to escape the cytotoxic effects of taxol.

No MeSH data available.


Related in: MedlinePlus