Expression of Dual-Specificity Phosphatase 5 Pseudogene 1 (DUSP5P1) in Tumor Cells

<div><p>Sequencing of individual clones from a newly established cDNA library from the chemoresistant Hodgkin's lymphoma cell line L-1236 led to the isolation of a cDNA clone corresponding to a short sequence from chromosome 1. Reverse transcriptase-polymerase chain reaction indicated high expression of this sequence in Hodgkin's lymphoma derived cell lines but not in normal blood cells. Further characterization of this sequence and the surrounding genomic DNA revealed that this sequence is part of a human endogenous retrovirus locus. The sequence of this endogenous retrovirus is interrupted by a pseudogene of the dual specificity phosphatase 5 (DUSP5). Reverse transcriptase-polymerase chain reaction revealed high expression of this pseudogene (DUSP5P1) in HL cell lines but not in normal blood cells or Epstein-Barr virus-immortalized B cells. Cells from other tumor types (Burkitt's lymphoma, leukemia, neuroblastoma, Ewing sarcoma) also showed a higher DUSP5P1/DUSP5 ratio than normal cells. Furthermore, we observed that higher expression of DUSP5 in relation to DUSP5P1 correlated with the expression of the pro-apoptotic factor B cell leukemia/lymphoma 2-like 11 (BCL2L11) in peripheral blood cells and HL cells. Knock-down of DUSP5 in HL cells resulted in down-regulation of BCL2L11. Thus, the DUSP5/DUSP5P1 system could be responsible for regulation of BCL2L11 leading to inhibition of apoptosis in these tumor cells.</p></div

Transgenic over-expression of the short transcript from the cDNA library has no effect on expression of BCL2L11.

<p>The vector from the cDNA library with the insert from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089577#pone-0089577-g001" target="_blank">Figure 1</a> was used for tranfection of L-428 cells. RNA was isolated 48 h after transfection. Quantitative RT-PCR was used for determination of expression of the transgene (ERVK_1q42.13), BCL2L11, DUSP5P1, and DUSP5, respectively. For calculation of relative expression values, GAPDH was used as housekeeping control and the mean Δct value from control cells was set as 1. Presented are means and standard deviations from 3 independent experiments. With exception of ERVK_1q42.13 (p<10⁻⁶), all other differences are statistically not significant.</p

Expression of ERVK_1q42.13 in HL cell lines.

<p>(A) Presented are results from a RT-PCR with primers with specificity for actin beta (ACTB) and ERVK_1q42.13. cDNA from HL cell lines and normal PBMC was used as template for PCR. NTC: no template control. (B) Presented are results from a RT-PCR with primers with specificity for ERVK_1q42.13. cDNA from HL cell line L-1236 and normal PBMC was used as template for PCR. cDNA was synthesized by using reverse transcriptase (RT:+) from three different vendors (see Material and methods). In addition, RNA was used without reverse transcription (RT:-). NTC: no template control. The black dividing line indicates removal of five irrelevant lanes from the original image. (C) Presented are results from a RT-PCR with primers with specificity for ERVK_1q42.13. cDNA from HL cell line KM-H2 and three different lymphoblastoid cell lines (LCL) was used as template for PCR. NTC: no template control.</p

Identification of ERV expression in HL cell lines.

<p>(A) 13 individual vectors from a cDNA library from HL cell line L-1236 were digested with <i>Not</i>I, analyzed by gel electrophoresis, and sequenced as described in Material and Methods. The sequence of the insert from the shortest vector (arrow) is presented. The vector sequence is printed in italics; the insert sequence that is also present in the human genome is presented in bold type. Binding sites for the primers used in panel B are underlined. (B) Presented are results from a RT-PCR with primers (ERVK_1q42.13_library) with specificity for the sequence from panel A. cDNA from HL cell lines and normal PBMC was used as template for PCR. NTC: no template control.</p

Expression of DUSP5P1 and DUSP5 in HL cell lines.

<p>(A) Presented are results from a RT-PCR with primers with specificity for DUSP5 and DUSP5P1. cDNA from HL cell lines and normal PBMC was used as template for PCR. NTC: no template control. (B) Presented are results from a quantitative RT-PCR (means and standard deviations from 3 experiments) with primers with specificity for DUSP5 and DUSP5P1. cDNA from HL cell lines and normal PBMC was used as template for PCR. For calculation of relative expression values, GAPDH was used as housekeeping control and the mean Δct value was set as 1. In addition to the results from individual cell lines, the mean values from HL cell lines and PBMC are presented. Asterisk indicate statistical significance (p<0.05; Student's t-test).</p

Schematic presentation of human chromosome 1 band 1q42.13 and the DUSP5P1 locus.

<p>(A) The sequence of the vector shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089577#pone-0089577-g001" target="_blank">Figure 1</a> is indicated by an orange rectangle. This sequence is part of a larger ERV sequence (colored red) on chromosome 1 at band 1q42.13 which was identified as described in Material and Methods. This larger sequence is split into several parts. Only the 4 parts with highest homology to an ERV in chromosome 4 are shown. Part one and two of this ERV are separated by a pseudogene of DUSP5 (DUSP5P1). Locations of PCR Products with primers with specificity for DUSP5P1 (green), the cloned cDNA (ERVK_1q42.13_library; yellow) and the ERV segments 2 and 3 (ERVK_1q42.13; blue) are indicated by horizontal bars. (B) Sequence alignment of the newly identified ERV and an ERV from chromosome 4 (MER65I, RetroSearch ERV-ID: 16168). The position of the DUSP5P1 insertion in ERVK_1q42.13 is indicated. Color code: red: A; green: C; yellow: G; blue: T. Data visualization was performed with GeneDoc (<a href="http://www.psc.edu/biomed/genedoc" target="_blank">http://www.psc.edu/biomed/genedoc</a>).</p

Down-regulation of BCL2L11 after knock-down of DUSP5 in HL cells.

<p>Vector based knock-down of DUSP5 in L-428 cells was performed by using the BLOCK-iT POL II miR RNAi expression vector kit as described in Materials and Methods. Gene expression in cells after knock-down of DUSP5 and control cells was analyzed by microarray analysis and quantitative RT-PCR. (A) Results from the microarray analysis. Presented are signal intensities from probe sets corresponding to DUSP5 and BCL2L11 in L-428 cells after knock-down of DUSP5 or after transfection with control vector. (B) Results from RT-PCR analysis. Presented are means and standard deviations from triplicate determinations. For calculation of relative expression values, GAPDH was used as housekeeping control and the median Δct value from control cells was set as 1. Asterisk indicate statistical significance (p<0.05; Student's t-test).</p

The ratio of DUSP5 and DUSP5P1 discriminates between malignant and non-malignant cells.

<p>Quantitative RT-PCR was used for determination of expression of DUSP5 and DUSP5P1 in cell lines and normal PBMC. (A) For calculation of relative expression values, GAPDH was used as housekeeping control and the mean Δct value was set as 1. Presented are the DUSP5P1/DUSP5 ratios in the indicated samples (from left to right: 10 PBMC, 7 EBV immortalized B cell lines (LCL), 5 HL cell lines (L-1236, L-428, L-540, KM-H2, HDLM-2), hematopoietic (hem.) tumor cell lines (Daudi, Raji, Jurkat, THP-1, 697, cALL2, NALM-6, Kasumi, K562, HL-60, U937), 4 neuroblastoma cell lines (SiMa, Kelly, SH-Sy5y, CHP-134), 4 Ewing sarcoma cell lines (SK-N-MC, A673, RD-ES, TC71). (B) Absolute copy numbers of DUSP5 and DUSP5P1 were calculated based on the vector titrations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089577#pone.0089577.s001" target="_blank">Figure S1</a>). Presented are the DUSP5/DUSP5P1 copy numbers ratios (means and standard deviations) in the indicated samples (same samples as in panel A). (C) Absolute copy numbers of DUSP5 and DUSP5P1 were calculated based on the vector titrations. Presented are the DUSP5/DUSP5P1 copy numbers ratios (means and standard deviations) of in the indicated sample groups. With the exception of the difference between hematopoietic and non-hematopoietic tumor cells, all other differences are statistically significant (non-malignant vs malignant: p<10⁻¹⁰; non-malignant vs. HL: p<10⁻⁸; Student's t-test;).</p

Homology modeling of a putative DUSP5P1 derived polypeptide.

<p>Presented is the result from an <i>in silico</i> homology modeling experiment using the substrate binding domain from DUSP6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089577#pone.0089577-Farooq1" target="_blank">[29]</a> as template. The putative DUSP5P1 peptide (red) was predicted on the basis of a promoter scan followed by translation of all possible reading frames. This peptide (MLRKEAAAGW MVLGCRPYLA FTALSVPGSL NINLYSLVCA SPGRLWGQRA TCCQMPRSTL LLQEGSILAA VMVLN) is derived from the first open reading frame after the predicted transcription start site. In addition, the structure of the homologue region of DUSP5 (green) was predicted by using DUSP6 (white) as template. For better visibility, only the sequences from DUSP6 and DUSP5 corresponding to the predicted DUSP5P1 peptide are shown. Amino acids important for substrate binding by DUSP6 and the corresponding amino acids from DUSP5 and DUSP5P1 are highlighted.</p

Correlation of DUSP5 and BCL2L11 expression in HL cells and PBMC.

<p>Quantitative RT-PCR was used for determination of expression of DUSP5, DUSP5P1, and BCL2L11 in normal PBMCs (squares; N = 5) and HL cell lines (circles; N = 5). For calculation of relative expression values, GAPDH was used as housekeeping control and the mean Δct value from 5 PBMC samples from healthy donors was set as 1. For cell line L-540 no expression of BCL2L11 was detected.</p