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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 decreases expression of anti-apoptotic genes.Gene Expression in cells of the HL cell line L-428 after PRAME knock-down or transfection with control vector was analyzed by DNA microarray analysis. A) Presented are RMA normalized signal intensities from representative probe sets (CD40∶205153_s_at, PRAME: 204086_at, EGR2∶205249_at, BCL2∶203685_at, BCL2L1∶215037_s_at IL13RA1∶210904_s_at, DHRS2∶206463_s_at). Numbers indicate fold changes. B) Validation of differentially expressed genes by qRT-PCR. Presented are the relative expression values for four PRAME knock-down samples from three independent time points and three control samples. ACTB was used as housekeeping control and for each gene the expression in the sample with highest expression was set as one.
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pone-0055897-g006: Knock-down of PRAME decreases expression of anti-apoptotic genes.Gene Expression in cells of the HL cell line L-428 after PRAME knock-down or transfection with control vector was analyzed by DNA microarray analysis. A) Presented are RMA normalized signal intensities from representative probe sets (CD40∶205153_s_at, PRAME: 204086_at, EGR2∶205249_at, BCL2∶203685_at, BCL2L1∶215037_s_at IL13RA1∶210904_s_at, DHRS2∶206463_s_at). Numbers indicate fold changes. B) Validation of differentially expressed genes by qRT-PCR. Presented are the relative expression values for four PRAME knock-down samples from three independent time points and three control samples. ACTB was used as housekeeping control and for each gene the expression in the sample with highest expression was set as one.

Mentions: We asked which genes might be responsible for the increased sensitivity of L-428 cells after knock-down of PRAME. In order to identify potential candidate genes we performed a microarray analysis and compared the gene expression profile of L-428 cells that had been transfected with PRAME knock-down vector with the gene expression profile of cells that had been transfected with empty control vector. We found several genes that were differentially expressed in PRAME knock-down cells. Down regulated genes include several genes with known anti-apoptotic function, e.g. CD40, B cell leukemia/lymphoma 2 (BCL2), BCL2 like 1 or the interleukin 13 receptor alpha 1. From the genes shown in Figure 6, Affymetrix MAS5 detection p values of probesets for CD40 (205153_s_at and 215346_at), BCL2 (203684_s_at), and early growth response 2 (EGR2, 205249_at) increased after knock-down of PRAME. MAS5 Detection calls for CD40 (215346_at) and BCL2 changed from present to marginal (CD40) or marginal to absent (BCL2), respectively. Quantitative PCR proved down-regulation of these genes in PRAME knock-down cells (Figure 6). A significant correlation was found between expression of PRAME and CD40 (p<0.01) and IL13RA1 (p<0.05), respectively.


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 decreases expression of anti-apoptotic genes.Gene Expression in cells of the HL cell line L-428 after PRAME knock-down or transfection with control vector was analyzed by DNA microarray analysis. A) Presented are RMA normalized signal intensities from representative probe sets (CD40∶205153_s_at, PRAME: 204086_at, EGR2∶205249_at, BCL2∶203685_at, BCL2L1∶215037_s_at IL13RA1∶210904_s_at, DHRS2∶206463_s_at). Numbers indicate fold changes. B) Validation of differentially expressed genes by qRT-PCR. Presented are the relative expression values for four PRAME knock-down samples from three independent time points and three control samples. ACTB was used as housekeeping control and for each gene the expression in the sample with highest expression was set as one.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569423&req=5

pone-0055897-g006: Knock-down of PRAME decreases expression of anti-apoptotic genes.Gene Expression in cells of the HL cell line L-428 after PRAME knock-down or transfection with control vector was analyzed by DNA microarray analysis. A) Presented are RMA normalized signal intensities from representative probe sets (CD40∶205153_s_at, PRAME: 204086_at, EGR2∶205249_at, BCL2∶203685_at, BCL2L1∶215037_s_at IL13RA1∶210904_s_at, DHRS2∶206463_s_at). Numbers indicate fold changes. B) Validation of differentially expressed genes by qRT-PCR. Presented are the relative expression values for four PRAME knock-down samples from three independent time points and three control samples. ACTB was used as housekeeping control and for each gene the expression in the sample with highest expression was set as one.
Mentions: We asked which genes might be responsible for the increased sensitivity of L-428 cells after knock-down of PRAME. In order to identify potential candidate genes we performed a microarray analysis and compared the gene expression profile of L-428 cells that had been transfected with PRAME knock-down vector with the gene expression profile of cells that had been transfected with empty control vector. We found several genes that were differentially expressed in PRAME knock-down cells. Down regulated genes include several genes with known anti-apoptotic function, e.g. CD40, B cell leukemia/lymphoma 2 (BCL2), BCL2 like 1 or the interleukin 13 receptor alpha 1. From the genes shown in Figure 6, Affymetrix MAS5 detection p values of probesets for CD40 (205153_s_at and 215346_at), BCL2 (203684_s_at), and early growth response 2 (EGR2, 205249_at) increased after knock-down of PRAME. MAS5 Detection calls for CD40 (215346_at) and BCL2 changed from present to marginal (CD40) or marginal to absent (BCL2), respectively. Quantitative PCR proved down-regulation of these genes in PRAME knock-down cells (Figure 6). A significant correlation was found between expression of PRAME and CD40 (p<0.01) and IL13RA1 (p<0.05), respectively.

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