DEK protein level is a biomarker of CD138positive normal and malignant plasma cells

Abstract

Overexpression of DEK oncogene is associated with increased proliferation of carcinoma cells and it is observed in several solid tumors due to the amplification of the 6p22.3 chromosomal region where DEK locates. Although the same chromosomal amplification occurs in multiple myeloma (MM), a plasma cell neoplasm, whether the expression and the copy number of the DEK gene are affected in MM remains elusive. We show that despite the increased copy number in CD138positive MM cells (4 out of 41 MM samples), DEK mRNA expression was down-regulated compared with that in CD138negative bone marrow (BM) cells of the same patients (P<0.0001). DEK protein was not detectable by immunohistochemistry (IHC) in CD138positive normal plasma cells or in malignant plasma cells of MM patients (n = 56) whereas it was widely expressed in normal and neoplastic B-cells. Stable knockdown or overexpression of DEK in CD138positive MM cell lines did not affect the proliferation and viability of the cells profoundly in the presence or absence of chemotherapeutic agent melphalan whereas knockdown of DEK moderately but significantly increased the expression level of CD138 (p<0.01). Decreased DEK expression in plasma cells suggests a potential role of this gene in plasma cell development and lack of detectable DEK protein by IHC could be used as a biomarker for normal and malignant plasma cells.

Doxorubicin induces prolonged DNA damage signal in cells overexpressing DEK isoform-2

“…Since both retroviral-MIG (does not allow antibiotic selection) and lentiviral-shRNA constructs contain GFP, we first generated DEK1 or DEK2 overexpressing cells. Then, after sorting GFP + cells by FACS, we suppressed DEK expression in these HS-27A-DEK2 or HS-27A-DEK1 cells by DEK-specific lentiviral sh-RNA (shDEK) [ 19 ] and selected the cells with puromycin to generate HS-27A-shDEK+DEK2 or HS-27A-shDEK+DEK1 cells. We couldn’t achieve DEK1 overexpression in the presence of shDEK ( S2 Fig ) since the DEK-specific sh-RNA showed more efficient knockdown of DEK1 , but we were able to overexpress DEK2, and we generated HS-27A-shDEK+DEK2 cells that have about 70% reduction in the endogenous DEK1 expression ( Fig 4A and 4B ).…”

“…Stable overexpression of epitope-tagged DEK isoforms (HS-27A-DEK1-GFP, HS-27A-DEK2-GFP) or the knockdown by sh-RNA (Origene, Rockville, MD, USA, TL313507) of endogenous DEK isoforms in HS-27A cells (HS-27A-shDEK) performed as described before using the corresponding VSVg pseudotyped MIG retrovirus or sh-RNA lentivirus [19]. HS-27A-DEK1-GFP, HS-27A-DEK2-GFP, and the control cells transduced with an empty MIG (HS-27A-GFP) were sorted by BD FACSJazz TM (BD Bioscience, Franklin Lakes, New Jersey, U.S.), and over 90% GFP-positive cells were obtained (S1 Fig) . HS-27A-sh-negative and HS-27A-shDEK cells were selected with 1.25μg/ml puromycin for 10 days.…”

Doxorubicin induces prolonged DNA damage signal in cells overexpressing DEK isoform-2

“…Since the HS-27A-DEK2 cells were more prone to doxorubicin-induced cell death, we analyzed if the response to DNA damage differs between the DEK1 and DEK2 overexpressing cells by assessing the γH2AX signal (phosphorylated histone H2AX, which labels the damaged DNA areas on the chromatin) using immunofluorescence staining. We stably suppressed DEK expression in HS-27A-DEK2 or HS-27A-DEK1 cells by DEK specific lentiviral sh-RNA (shDEK) [19] aiming to generate the HS-27A-shDEK+DEK2 or HS-27A-shDEK+DEK1 cells. We couldn’t achieve DEK1 overexpression (data not shown) in the presence of shDEK since the DEK-specific sh-RNA showed more efficient knockdown of DEK1 , but we were able to overexpress DEK2, and we generated HS-27A-shDEK+DEK2 cells that have about 70% reduction in the endogenous DEK1 expression (Figures 4A and 4B).…”

“…Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (for 293T, HeLa) or RPMI-1640 medium (for HS-27A) containing 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA, 15140122) at 37°C with 5% CO 2 atmosphere.Stable overexpression of epitope-tagged DEK isoforms (HS-27A-DEK1-GFP, HS-27A-DEK2-GFP) or the knockdown by sh-RNA (Origene, Rockville, MD, USA, TL313507) of endogenous DEK isoforms in HS-27A cells (HS-27A-shDEK) performed as described before using the corresponding VSVg pseudotyped MIG retrovirus or sh-RNA lentivirus[19]. HS-27A-DEK1-GFP, HS-27A-DEK2-GFP, and the control cells transduced with an empty MIG (HS-27A-GFP) were sorted by BD FACSJazz TM (BD Bioscience, Franklin Lakes, New Jersey, U.S.), and over 90% of GFP-positive cells were obtained (data not shown).…”

DEK Is a Potential Biomarker Associated with Malignant Phenotype in Gastric Cancer Tissues and Plasma

“…DEK is a well-known proto-oncogene found in a range of nuclear proteins involved in chromatin remodeling, transcriptional repression or activation, DNA damage repair, cell proliferation, and suppression of apoptotic pathways [8]. DEK overexpression has been documented in various human malignant tissues, including GC, pancreatic ductal adenocarcinoma, oral squamous cell carcinoma (OSCC), hepatocellular carcinoma (HCC), chronic lymphocytic leukemia (CLL), lung cancer, cervical cancer, breast cancer, melanoma, head-and-neck cancer, bladder cancer, retinoblastoma, T-cell large granular lymphocytic leukemia, colon cancer, prostate cancer, acute myeloid leukemia (AML), and malignant glioma melanoma [8,9,10,11,12,13,14,15,16,17,18,19,20,21]. Moreover, DEK has been detected extracellularly in the urine of patients with bladder cancer and shown to be released by activated macrophages in the hemopoietic system [11].…”