Pro-apoptotic and antiproliferative activity of human KCNRG, a putative tumor suppressor in 13q14 region

Abstract

Deletion of 13q14.3 and a candidate gene KCNRG (potassium channel regulating gene) is the most frequent chromosomal abnormality in B-cell chronic lymphocytic leukemia and is a common finding in multiple myeloma (MM). KCNRG protein may interfere with the normal assembly of the K+ channel proteins causing the suppression of Kv currents. We aimed to examine possible role of KCNRG haploinsufficiency in chronic lymphocytic leukemia (CLL) and MM cells. We performed detailed genomic analysis of the KCNRG locus; studied effects of the stable overexpression of KCNRG isoforms in RPMI-8226, HL-60, and LnCaP cells; and evaluated relative expression of its transcripts in various human lymphomas. Three MM cell lines and 35 CLL PBL samples were screened for KCNRG mutations. KCNRG exerts growth suppressive and pro-apoptotic effects in HL-60, LnCaP, and RPMI-8226 cells. Direct sequencing of KCNRG exons revealed point mutation delT in RPMI-8226 cell line. Levels of major isoform of KCNRG mRNA are lower in DLBL lymphomas compared to normal PBL samples, while levels of its minor mRNA are decreased across the broad range of the lymphoma types. The haploinsufficiency of KCNRG might be relevant to the progression of CLL and MM at least in a subset of patients.

Fig. 1

Genomic organization of RFP2/KCNRG gene locus. Schemes represent the structure of the mRNA isoforms of the human RFP2 and KCNRG genes and the hybrid mRNA isoform. Open reading frame of RFP2 is represented by white arrow. Open reading frames of KCNRG are represented by black arrows. Hybrid mRNA RFP2/KCNRG is not translated. Promoter of RFP2 marked as PR, promoter of KCNRG marked as PK

Fig. 2

An alignment of KCNRG with other proteins of KCTD family. Degree of shading indicates different degree of conservation for a given amino acid position: invariant positions are darkest, other conserved positions are shaded lighter, and non-conserved positions are not shaded. Total length of T1 domain, its position, and full length of the proteins are summarized in the table adjacent to the aligned protein sequences. Truncated versions of T1 domain are marked by star

Fig. 3

Expression of KCNRG inhibits proliferation and enhances apoptosis. Axis Y reflects chemoluminescence readings (ChemU) in respective assays. a–c Results BrdU enzyme-linked immunosorbent assay to quantify proliferation in HL-60 (a), RPMI-8226 (b), and LnCaP (c) cells. d–f Results of the caspase assays to quantify apoptosis in HL-60 (d), RPMI-8226 (e), and LnCaP (f) cells

Fig. 4

Propidium iodide FACS analysis of the cell cycle in the vector control and KCNRG-L overexpressing HL-60 cells. a, b Scatter plot and frequency histogram reflecting cell sizes and DNA content in HL-60 cells transfected with empty vector (vector control). c, d Scatter plot and frequency histogram reflecting cell sizes and DNA content in HL-60 cells transfected with KCNRG-L overexpressing plasmid. e Overlay of images (a) and (c). f, g Detailed frequency histograms displaying DNA content of vector control HL-60 cells (f) and KCNRG-L overexpressing HL-60 cells (g) located by their size in each of four quadrants

Fig. 5

Images of HL-60 cells. a–d HL-60/control. e–h HL-60/KCNRG-L. i–l RPMI-8226/control. m–p RPMI-8226/KCNRG-L. Cells stained with lipohilic probe Dioc 18 (a, e, i, m), Nucleic acid probe DAPI (b, f, j, n), and F-actin probe rhodamine phalloidin (c, g, k, o). Overlay of the individual images (d, h, l, p). Original magnification ×100

Fig. 6

Quantification of cell death in HL-60/control, HL-60/KCNRG-L, and HL-60/KCNRG-S by staining with Annexin V/7-AAD. a–c Scatter plots and frequency histograms reflecting cell sizes and staining with Annexin V/7-AAD in HL-60 cells vector control (a), HL-60 cells transfected with KCNRG-L overexpressing plasmid (b), and HL-60 cells transfected with KCNRG-L overexpressing plasmid (c). d Overlay of images (a) and (b). e Overlay of images (a) and (c). f Bar graph reflecting relative changes in the number of cells stained with Annexin V/7-AAD after transfection with KCNRG isoforms. Q1 reflects necrosis, Q2 reflects late apoptosis, Q3 reflects healthy cell population not affected by apoptosis or necrosis, and Q4 reflects early apoptosis

Fig. 7

Real-time polymerase chain reaction (PCR) profiling of KCNRG mRNAs in human lymphoma samples. N Normal PBL, FL follicular lymphoma, DLBL diffuse large B-cell lymphoma, SLL small lymphocytic lymphoma, HL Hodgkin lymphoma, MZBL marginal zone B-cell lymphoma, MCL mantle cell lymphoma, TL T-cell lymphoma. a, b Reverse transcription (RT)-PCR profiling using primer pair KCNRG-SL that amplifies both isoforms of KCNRG mRNA. c, d RT-PCR profiling using primer pair KCNRG-S that amplifies only mRNA isoform KCNRG-S. Statistically significant changes denoted by star symbol. Expression levels of KCNRG-L/S and KCNRG-S isoforms are reflected at Y axis in artificial expression units (AU) after normalization to β-actin mRNA levels