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Hsp90 and Hsp40/Erdj3 are required for the expression and anti-apoptotic function of KSHV K1.

Wen KW, Damania B - Oncogene (2010)

Bottom Line: Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family.We report that small-interfering RNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90, dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones.Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC(50) values of 50 nM and below.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family. It is the etiological agent of three different human cancers, Kaposi sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman disease. The far left end of the KSHV genome encodes a unique transmembrane glycoprotein called K1. K1 possesses the ability to transform rodent fibroblasts and block apoptosis. K1 has also been shown to activate the PI3K/Akt/mTOR pathway in different cells. Using tandem affinity purification, we identified heat shock protein 90beta (Hsp90beta) and endoplasmic reticulum-associated Hsp40 (Erdj3/DnaJB11), as cellular binding partners of K1. Interactions of K1 with Hsp90beta and Hsp40 were confirmed by co-immunoprecipitation in both directions. Furthermore, K1 also interacted with the Hsp90alpha isoform. We report that small-interfering RNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90, dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones. In addition, both Hsp90 and Hsp40/Erdj3 were essential for K1's anti-apoptotic function. Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC(50) values of 50 nM and below.

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The Hsp90 inhibitors, 17-AAG and 17-DMAG, inhibit the proliferation of KSHV-positive PEL cell linesA. BCP-1, JSC-1, and BCBL-1 were incubated with 50 nM of 17-AAG or 17-DMAG and subjected to a MTS assay over a period of four days. The absorption at 490 nm in the presence of 50 nM of 17-AAG or 17-DMAG, or equivalent volume of vehicle control (DMSO) is shown on the vertical axis, and time in days after addition of the drugs is shown on the horizontal axis. Error bars represent standard deviation from the mean. B. IC50 values of 17-AAG on BCP-1, JSC-1, and BCBL-1 were determined after 72 h incubation with 17-AAG. A range of concentrations of 17-AAG was used: 0, 1, 10, 100, 1000, or 10000 nM. A nonlinear fit, sigmoidal curve was generated by plotting percent growth inhibition against the log concentration of 17-AAG. Error bars represent SEM. C. IC50 values of 17-DMAG on BCP-1, JSC-1, and BCBL-1 were determined by MTS assays after 72 h incubation with 17-DMAG.
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Figure 7: The Hsp90 inhibitors, 17-AAG and 17-DMAG, inhibit the proliferation of KSHV-positive PEL cell linesA. BCP-1, JSC-1, and BCBL-1 were incubated with 50 nM of 17-AAG or 17-DMAG and subjected to a MTS assay over a period of four days. The absorption at 490 nm in the presence of 50 nM of 17-AAG or 17-DMAG, or equivalent volume of vehicle control (DMSO) is shown on the vertical axis, and time in days after addition of the drugs is shown on the horizontal axis. Error bars represent standard deviation from the mean. B. IC50 values of 17-AAG on BCP-1, JSC-1, and BCBL-1 were determined after 72 h incubation with 17-AAG. A range of concentrations of 17-AAG was used: 0, 1, 10, 100, 1000, or 10000 nM. A nonlinear fit, sigmoidal curve was generated by plotting percent growth inhibition against the log concentration of 17-AAG. Error bars represent SEM. C. IC50 values of 17-DMAG on BCP-1, JSC-1, and BCBL-1 were determined by MTS assays after 72 h incubation with 17-DMAG.

Mentions: Since Hsp90 and Hsp40 regulate the protein expression and anti-apoptotic function of K1, we wanted to determine if Hsp90 inhibitors can block proliferation of KSHV-positive PEL cells. PEL cells have previously been shown to express K1 transcripts and protein at low levels (Bowser et al., 2002; Lee et al., 2003). We treated PEL cells with DMSO (vehicle control) or 50nM of the GA-derived Hsp90 inhibitors, 17-AAG and 17-DMAG, that are currently in clinical trials (Goetz et al., 2005; Ivy & Schoenfeldt, 2004) for 0, 24, 48, 72, and 96 hours. We found that both inhibitors suppressed the growth of KSHV-harboring PEL cell lines, BCP-1, BCBL-1, and JSC-1, in a MTS-based cell proliferation assay (Fig. 7A). We also determined the IC50 of 17-AAG and 17-DMAG in BCP-1, BCBL-1, and JSC-1 cell lines 72h post-treatment. The IC50 of 17-AAG and 17-DMAG were ~50nM and ~10nM, respectively, in all three cell lines (Fig. 7B and 7C). These IC50 values are consistent with other tumor cell lines that are dependent on Hsp90 (Hollingshead et al., 2005). Hence, 17-AAG and 17-DMAG can effectively inhibit KSHV-infected primary effusion lymphoma cell lines in vitro. We also investigated the effects of 17-DMAG on 293-K1 cells compared to 293-Vec cells. 1×106 293-K1 and 293-Vec cells were incubated with 17-DMAG and a Trypan blue exclusion assay was performed at 24, 48 and 72 hours post-treatment. We found that at all three timepoints tested, there were more live cells present in the 293-K1 cells compared to the 293-Vec cells. This suggests that K1 confers a survival advantage to 293 cells upon Hsp90 inhibition (Supplemental Fig. 6) and that higher concentrations of drug are required to inhibit K1-expressing 293 cells compared to 293 cells that do not express the K1 protein.


Hsp90 and Hsp40/Erdj3 are required for the expression and anti-apoptotic function of KSHV K1.

Wen KW, Damania B - Oncogene (2010)

The Hsp90 inhibitors, 17-AAG and 17-DMAG, inhibit the proliferation of KSHV-positive PEL cell linesA. BCP-1, JSC-1, and BCBL-1 were incubated with 50 nM of 17-AAG or 17-DMAG and subjected to a MTS assay over a period of four days. The absorption at 490 nm in the presence of 50 nM of 17-AAG or 17-DMAG, or equivalent volume of vehicle control (DMSO) is shown on the vertical axis, and time in days after addition of the drugs is shown on the horizontal axis. Error bars represent standard deviation from the mean. B. IC50 values of 17-AAG on BCP-1, JSC-1, and BCBL-1 were determined after 72 h incubation with 17-AAG. A range of concentrations of 17-AAG was used: 0, 1, 10, 100, 1000, or 10000 nM. A nonlinear fit, sigmoidal curve was generated by plotting percent growth inhibition against the log concentration of 17-AAG. Error bars represent SEM. C. IC50 values of 17-DMAG on BCP-1, JSC-1, and BCBL-1 were determined by MTS assays after 72 h incubation with 17-DMAG.
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Figure 7: The Hsp90 inhibitors, 17-AAG and 17-DMAG, inhibit the proliferation of KSHV-positive PEL cell linesA. BCP-1, JSC-1, and BCBL-1 were incubated with 50 nM of 17-AAG or 17-DMAG and subjected to a MTS assay over a period of four days. The absorption at 490 nm in the presence of 50 nM of 17-AAG or 17-DMAG, or equivalent volume of vehicle control (DMSO) is shown on the vertical axis, and time in days after addition of the drugs is shown on the horizontal axis. Error bars represent standard deviation from the mean. B. IC50 values of 17-AAG on BCP-1, JSC-1, and BCBL-1 were determined after 72 h incubation with 17-AAG. A range of concentrations of 17-AAG was used: 0, 1, 10, 100, 1000, or 10000 nM. A nonlinear fit, sigmoidal curve was generated by plotting percent growth inhibition against the log concentration of 17-AAG. Error bars represent SEM. C. IC50 values of 17-DMAG on BCP-1, JSC-1, and BCBL-1 were determined by MTS assays after 72 h incubation with 17-DMAG.
Mentions: Since Hsp90 and Hsp40 regulate the protein expression and anti-apoptotic function of K1, we wanted to determine if Hsp90 inhibitors can block proliferation of KSHV-positive PEL cells. PEL cells have previously been shown to express K1 transcripts and protein at low levels (Bowser et al., 2002; Lee et al., 2003). We treated PEL cells with DMSO (vehicle control) or 50nM of the GA-derived Hsp90 inhibitors, 17-AAG and 17-DMAG, that are currently in clinical trials (Goetz et al., 2005; Ivy & Schoenfeldt, 2004) for 0, 24, 48, 72, and 96 hours. We found that both inhibitors suppressed the growth of KSHV-harboring PEL cell lines, BCP-1, BCBL-1, and JSC-1, in a MTS-based cell proliferation assay (Fig. 7A). We also determined the IC50 of 17-AAG and 17-DMAG in BCP-1, BCBL-1, and JSC-1 cell lines 72h post-treatment. The IC50 of 17-AAG and 17-DMAG were ~50nM and ~10nM, respectively, in all three cell lines (Fig. 7B and 7C). These IC50 values are consistent with other tumor cell lines that are dependent on Hsp90 (Hollingshead et al., 2005). Hence, 17-AAG and 17-DMAG can effectively inhibit KSHV-infected primary effusion lymphoma cell lines in vitro. We also investigated the effects of 17-DMAG on 293-K1 cells compared to 293-Vec cells. 1×106 293-K1 and 293-Vec cells were incubated with 17-DMAG and a Trypan blue exclusion assay was performed at 24, 48 and 72 hours post-treatment. We found that at all three timepoints tested, there were more live cells present in the 293-K1 cells compared to the 293-Vec cells. This suggests that K1 confers a survival advantage to 293 cells upon Hsp90 inhibition (Supplemental Fig. 6) and that higher concentrations of drug are required to inhibit K1-expressing 293 cells compared to 293 cells that do not express the K1 protein.

Bottom Line: Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family.We report that small-interfering RNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90, dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones.Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC(50) values of 50 nM and below.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family. It is the etiological agent of three different human cancers, Kaposi sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman disease. The far left end of the KSHV genome encodes a unique transmembrane glycoprotein called K1. K1 possesses the ability to transform rodent fibroblasts and block apoptosis. K1 has also been shown to activate the PI3K/Akt/mTOR pathway in different cells. Using tandem affinity purification, we identified heat shock protein 90beta (Hsp90beta) and endoplasmic reticulum-associated Hsp40 (Erdj3/DnaJB11), as cellular binding partners of K1. Interactions of K1 with Hsp90beta and Hsp40 were confirmed by co-immunoprecipitation in both directions. Furthermore, K1 also interacted with the Hsp90alpha isoform. We report that small-interfering RNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90, dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones. In addition, both Hsp90 and Hsp40/Erdj3 were essential for K1's anti-apoptotic function. Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC(50) values of 50 nM and below.

Show MeSH
Related in: MedlinePlus