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Knock-down of PRAME increases retinoic acid signaling and cytotoxic drug sensitivity of Hodgkin lymphoma cells.

Kewitz S, Staege MS - PLoS ONE (2013)

Bottom Line: DNA microarray analysis of HL cells after PRAME knock-down indicated regulation of several genes including down-regulation of known anti-apoptotic factors.Increased retinoic acid signaling in these cells was revealed by increased expression of the retinoic acid metabolizing cytochrome P450 (CYP26B1), a transcriptional target of retinoic acid signaling.Our data suggest that PRAME inhibits retinoic acid signaling in HL cells and that the knock-down of PRAME might be an interesting option for the development of new therapy strategies for patients with chemo-resistant HL.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Martin-Luther-University Halle-Wittenberg, Halle, Germany.

ABSTRACT
The prognosis for patients with Hodgkin lymphoma (HL) has improved in recent decades. On the other hand, not all patients can be cured with the currently established therapy regimes and this therapy is associated with several adverse late effects. Therefore it is necessary to develop new therapy strategies. After treatment of L-540 HL cells with 5'-azacytidine (5AC), we observed increased expression of the preferentially expressed antigen in melanoma (PRAME). In addition, we detected an increased resistance of 5AC-treated cells against cytotoxic drugs. We analyzed the influence of PRAME on cell survival of HL cells by knocking down PRAME in the chemotherapy resistant cell line L-428, a cell line that express PRAME at a high level. After knock-down of PRAME using vector based RNA interference we observed increased sensitivity for cisplatin, etoposide and retinoic acid. DNA microarray analysis of HL cells after PRAME knock-down indicated regulation of several genes including down-regulation of known anti-apoptotic factors. Increased retinoic acid signaling in these cells was revealed by increased expression of the retinoic acid metabolizing cytochrome P450 (CYP26B1), a transcriptional target of retinoic acid signaling. Our data suggest that PRAME inhibits retinoic acid signaling in HL cells and that the knock-down of PRAME might be an interesting option for the development of new therapy strategies for patients with chemo-resistant HL.

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Knock-down of PRAME and increased sensitivity for retinoic acid.A) Expression of PRAME was analyzed in HL cell line L-428 by qRT-PCR. The control cells were transfected with the empty vector, whereas the other cells were transfected with a vector allowing suppression of PRAME by RNA interference. Presented are means and standard errors from six/eleven determinations. For comparative analysis, the mean of L-428 cells with the empty vector was set as 1. B) Cells of the HL cell line L-428 with empty vector, L-428 cells after knock-down of PRAME, and the HL cell line L-540 were incubated with 2.5×10−4 M ATRA or DMSO. After four days the viability was assessed by propidium iodide staining. The number of living cells in the samples with DMSO was set as 100%. Presented are means and standard errors from triplicate determinations. Asterisks indicate significance (p<0.05; Students t test).
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pone-0055897-g003: Knock-down of PRAME and increased sensitivity for retinoic acid.A) Expression of PRAME was analyzed in HL cell line L-428 by qRT-PCR. The control cells were transfected with the empty vector, whereas the other cells were transfected with a vector allowing suppression of PRAME by RNA interference. Presented are means and standard errors from six/eleven determinations. For comparative analysis, the mean of L-428 cells with the empty vector was set as 1. B) Cells of the HL cell line L-428 with empty vector, L-428 cells after knock-down of PRAME, and the HL cell line L-540 were incubated with 2.5×10−4 M ATRA or DMSO. After four days the viability was assessed by propidium iodide staining. The number of living cells in the samples with DMSO was set as 100%. Presented are means and standard errors from triplicate determinations. Asterisks indicate significance (p<0.05; Students t test).

Mentions: It is known from other tumor models that PRAME inhibits the retinoic acid (RA) receptor [18]. In order to investigate the influence of PRAME on RA signaling in HL cells, we transfected cells of the HL cell line L-428 with a vector allowing knock-down of PRAME expression. L-428 cells express high amounts of PRAME and are resistant against RA (Figure S4). Suppression of PRAME was tested by qRT-PCR (Figure 3). Expression of PRAME in the transfected cells was 66% lower than in the control cells. The viability of the EmGFP positive cells was not decreased in PRAME knock-down cells (Figure S5). We incubated cells with low PRAME expression and control cells for 4 days with 2.5×10−4 M all-trans RA. Thereafter, viability was assessed by propidium iodide staining. As seen in Figure 3 and Figure S5, cells transfected with the control vector showed a viability of 90% whereas the cells with PRAME knock-down had a viability of only 61.45%. As a control we also incubated L-540 cells with ATRA. These cells express only low levels of PRAME (Figure S4) and showed the lowest viability (Figure 3).


Knock-down of PRAME increases retinoic acid signaling and cytotoxic drug sensitivity of Hodgkin lymphoma cells.

Kewitz S, Staege MS - PLoS ONE (2013)

Knock-down of PRAME and increased sensitivity for retinoic acid.A) Expression of PRAME was analyzed in HL cell line L-428 by qRT-PCR. The control cells were transfected with the empty vector, whereas the other cells were transfected with a vector allowing suppression of PRAME by RNA interference. Presented are means and standard errors from six/eleven determinations. For comparative analysis, the mean of L-428 cells with the empty vector was set as 1. B) Cells of the HL cell line L-428 with empty vector, L-428 cells after knock-down of PRAME, and the HL cell line L-540 were incubated with 2.5×10−4 M ATRA or DMSO. After four days the viability was assessed by propidium iodide staining. The number of living cells in the samples with DMSO was set as 100%. Presented are means and standard errors from triplicate determinations. Asterisks indicate significance (p<0.05; Students t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569423&req=5

pone-0055897-g003: Knock-down of PRAME and increased sensitivity for retinoic acid.A) Expression of PRAME was analyzed in HL cell line L-428 by qRT-PCR. The control cells were transfected with the empty vector, whereas the other cells were transfected with a vector allowing suppression of PRAME by RNA interference. Presented are means and standard errors from six/eleven determinations. For comparative analysis, the mean of L-428 cells with the empty vector was set as 1. B) Cells of the HL cell line L-428 with empty vector, L-428 cells after knock-down of PRAME, and the HL cell line L-540 were incubated with 2.5×10−4 M ATRA or DMSO. After four days the viability was assessed by propidium iodide staining. The number of living cells in the samples with DMSO was set as 100%. Presented are means and standard errors from triplicate determinations. Asterisks indicate significance (p<0.05; Students t test).
Mentions: It is known from other tumor models that PRAME inhibits the retinoic acid (RA) receptor [18]. In order to investigate the influence of PRAME on RA signaling in HL cells, we transfected cells of the HL cell line L-428 with a vector allowing knock-down of PRAME expression. L-428 cells express high amounts of PRAME and are resistant against RA (Figure S4). Suppression of PRAME was tested by qRT-PCR (Figure 3). Expression of PRAME in the transfected cells was 66% lower than in the control cells. The viability of the EmGFP positive cells was not decreased in PRAME knock-down cells (Figure S5). We incubated cells with low PRAME expression and control cells for 4 days with 2.5×10−4 M all-trans RA. Thereafter, viability was assessed by propidium iodide staining. As seen in Figure 3 and Figure S5, cells transfected with the control vector showed a viability of 90% whereas the cells with PRAME knock-down had a viability of only 61.45%. As a control we also incubated L-540 cells with ATRA. These cells express only low levels of PRAME (Figure S4) and showed the lowest viability (Figure 3).

Bottom Line: DNA microarray analysis of HL cells after PRAME knock-down indicated regulation of several genes including down-regulation of known anti-apoptotic factors.Increased retinoic acid signaling in these cells was revealed by increased expression of the retinoic acid metabolizing cytochrome P450 (CYP26B1), a transcriptional target of retinoic acid signaling.Our data suggest that PRAME inhibits retinoic acid signaling in HL cells and that the knock-down of PRAME might be an interesting option for the development of new therapy strategies for patients with chemo-resistant HL.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Martin-Luther-University Halle-Wittenberg, Halle, Germany.

ABSTRACT
The prognosis for patients with Hodgkin lymphoma (HL) has improved in recent decades. On the other hand, not all patients can be cured with the currently established therapy regimes and this therapy is associated with several adverse late effects. Therefore it is necessary to develop new therapy strategies. After treatment of L-540 HL cells with 5'-azacytidine (5AC), we observed increased expression of the preferentially expressed antigen in melanoma (PRAME). In addition, we detected an increased resistance of 5AC-treated cells against cytotoxic drugs. We analyzed the influence of PRAME on cell survival of HL cells by knocking down PRAME in the chemotherapy resistant cell line L-428, a cell line that express PRAME at a high level. After knock-down of PRAME using vector based RNA interference we observed increased sensitivity for cisplatin, etoposide and retinoic acid. DNA microarray analysis of HL cells after PRAME knock-down indicated regulation of several genes including down-regulation of known anti-apoptotic factors. Increased retinoic acid signaling in these cells was revealed by increased expression of the retinoic acid metabolizing cytochrome P450 (CYP26B1), a transcriptional target of retinoic acid signaling. Our data suggest that PRAME inhibits retinoic acid signaling in HL cells and that the knock-down of PRAME might be an interesting option for the development of new therapy strategies for patients with chemo-resistant HL.

Show MeSH
Related in: MedlinePlus