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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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Increased sensitivity for cisplatin and roscovitin after incubation of HL cells with 5′-azacytidine.Cells of the HL cell line L-540 were incubated with 5′-azacytidine or medium. Thereafter, cells were treated with 25 µg/mL cisplatin or 60 µM roscovitin. The viability was assessed by propidium iodide staining. The number of living cells in the samples without drugs 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-g002: Increased sensitivity for cisplatin and roscovitin after incubation of HL cells with 5′-azacytidine.Cells of the HL cell line L-540 were incubated with 5′-azacytidine or medium. Thereafter, cells were treated with 25 µg/mL cisplatin or 60 µM roscovitin. The viability was assessed by propidium iodide staining. The number of living cells in the samples without drugs was set as 100%. Presented are means and standard errors from triplicate determinations. Asterisks indicate significance (p<0.05; Students t test).

Mentions: In our previous work we observed differences in the expression of PRAME between chemo-resistant and -sensitive HL cells. We asked whether the low expression of PRAME in HL cell lines L-540 might be epigenetically regulated. Therefore, we analyzed the methylation status of PRAME in HL cell lines. We isolated DNA from HL cell lines and treated them with bisulfite. Bisulfite converts cytosine into uracil but methylated cytosines are not converted. After the conversion reaction, a methylation specific PCR was performed with specific primers for non-methylated PRAME [27]. As control we performed PCR with primers for an ALU repetitive sequence (not modified by bisulfite) [27]. A representative result is shown in Figure 1. HL cell lines L-428, L-1236, HDLM-2 and KM-H2 showed stronger signals for non-methylated PRAME than the cell line L-540. After incubation of L-540 cells with 5′-azacytidine (5AC) for two weeks we observed stronger signals for non-methylated PRAME (Figure 1). In parallel, expression of PRAME increased with time of 5AC treatment ([19] and Figure S1). We tested whether increased PRAME expression has an influence on the resistance against cytostatic drugs. We incubated the 5AC-treated cells for 24 hours with cisplatin or roscovitin, respectively. The results of these experiments are shown in Figure 2 and Figure S2. After 7 days of incubation with 5AC the expression of PRAME was 14 times higher than in the control cells; at the same time, more cells survived after treatment with cisplatin or roscovitin. We observed that the sensitivity against cytostatic drugs decreased in parallel with the increased expression of PRAME. No such decrease of sensitivity was observed when we treated constitutively PRAME expressing HDLM-2 cells with 5AC (Figure S3).


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

Kewitz S, Staege MS - PLoS ONE (2013)

Increased sensitivity for cisplatin and roscovitin after incubation of HL cells with 5′-azacytidine.Cells of the HL cell line L-540 were incubated with 5′-azacytidine or medium. Thereafter, cells were treated with 25 µg/mL cisplatin or 60 µM roscovitin. The viability was assessed by propidium iodide staining. The number of living cells in the samples without drugs 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-g002: Increased sensitivity for cisplatin and roscovitin after incubation of HL cells with 5′-azacytidine.Cells of the HL cell line L-540 were incubated with 5′-azacytidine or medium. Thereafter, cells were treated with 25 µg/mL cisplatin or 60 µM roscovitin. The viability was assessed by propidium iodide staining. The number of living cells in the samples without drugs was set as 100%. Presented are means and standard errors from triplicate determinations. Asterisks indicate significance (p<0.05; Students t test).
Mentions: In our previous work we observed differences in the expression of PRAME between chemo-resistant and -sensitive HL cells. We asked whether the low expression of PRAME in HL cell lines L-540 might be epigenetically regulated. Therefore, we analyzed the methylation status of PRAME in HL cell lines. We isolated DNA from HL cell lines and treated them with bisulfite. Bisulfite converts cytosine into uracil but methylated cytosines are not converted. After the conversion reaction, a methylation specific PCR was performed with specific primers for non-methylated PRAME [27]. As control we performed PCR with primers for an ALU repetitive sequence (not modified by bisulfite) [27]. A representative result is shown in Figure 1. HL cell lines L-428, L-1236, HDLM-2 and KM-H2 showed stronger signals for non-methylated PRAME than the cell line L-540. After incubation of L-540 cells with 5′-azacytidine (5AC) for two weeks we observed stronger signals for non-methylated PRAME (Figure 1). In parallel, expression of PRAME increased with time of 5AC treatment ([19] and Figure S1). We tested whether increased PRAME expression has an influence on the resistance against cytostatic drugs. We incubated the 5AC-treated cells for 24 hours with cisplatin or roscovitin, respectively. The results of these experiments are shown in Figure 2 and Figure S2. After 7 days of incubation with 5AC the expression of PRAME was 14 times higher than in the control cells; at the same time, more cells survived after treatment with cisplatin or roscovitin. We observed that the sensitivity against cytostatic drugs decreased in parallel with the increased expression of PRAME. No such decrease of sensitivity was observed when we treated constitutively PRAME expressing HDLM-2 cells with 5AC (Figure S3).

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