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KCNRG as a Tumor Suppressor Gene for CLL and Multiple Myeloma
Aybike Birerdinc, Elizabeth Nohelty, Andrey Marakhonov, Ganiraju Manyam, Ivan Panov, Stephanie Coon, Eugene Nikitin, Mikhail Skoblov, Vikas Chandhoke, Ancha Baranova

This is a collaborative project between

Molecular and Microbiology Department, College of Science,George Mason University, Fairfax, VA

Research Center for Medical Genetics, RAMS, Moscow, Russia

Hematology Research Center of Russia, Moscow, Russia

This project was performed in frame of NIH R1R15CA113331-01 “KCNRG gene as candidate tumor suppressor for CLL and MM” (2005-2009), RFFI 07-04-00379-a, 07-04-12232-ofi, and 04-04-08154-ofi.

B-cell chronic lymphocyte leukemia (B-CLL) accounts for ~30% of all leukemias in the Western world and has so far been treated with variable success (Byrd et al., 2004). KCNRG (chromosome 13q14.3) is thought to be a tumor suppressor candidate gene involved in the development of B-cell chronic lymphocytic leukemia due to its significant homology and functional similarity to potassium channel regulating proteins (Ivanov et al., 2003). In addition it has been found that KCNRG has two isoforms, both of which incorporate the potassium channel like segment, and differ in the end regions. The aim of this study is to determine whether the loss of KCNRG does indeed cause tumorigecinity and to elucidate a putative role for this gene in B-CLL disease development and progression.

Since KCNRG has been shown to have a negative mutation status in B-CLL samples, a novel approach for study based on haploinsufficiency is suggested (Baranova 2004). Hence, the experimental design consists of a series of overexpression studies by using cell lines natively expressing KCNRG, cell lines that can be induced to express KCNRG and cell lines that do not natively express KCNRG to determine the role of KCNRG in apoptosis, differentiation, invasiveness, and effects of overexpression of KCNRG on gene expression profiles. The KCNRG gene has been cloned into pcDNA 3.1 myc/his vector, and the cell lines: RPMI-8668, HL60, and LNCaP have been transfected with this clone using Fugene 6 (Roche Sciences). The transfected cells are grown in 400ug/ml of Geneticin supplemented RPMI media to select for stable transfectants.

We showed that KCNRG exerts growth suppressive and pro-apoptotic in the cellular models relevant to CLL and MM and described delT mutation in the KCNRG core promoter initiator element in MM cell line RPMI-8226. Real-time PCR profiling of KCNRG mRNAs revealed that levels of the major isoform of KCNRG mRNA in DLBL lymphomas are lower compared to normal PBL samples, while levels of its minor mRNA are decreased across the broad range of the lymphoma types. We conclude that the loss of KCNRG might be relevant to the progression of these hematological malignancies at least in a subset of the patients with these disorders.

Thiis study was based on our previous studies published in: FEBS Lett. 2003 Mar 27;539(1-3):156-60

Figure 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. ORF of RFP2 is represented by white arrow. ORFs 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.

Figure 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, 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 (*).

Figure 5. Images of HL-60 and RPMI-8226 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.

Supplementary Figure 1 and 2.

Suppl. Fig.1. Comparative analysis of the RFP2/KCNRG locus in human, rat and mouse genomes. MAR/SARs are denoted by blue squares. Mouse RFP2/KCNRG locus does not possess any MAR/SAR elements.
Suppl. Fig. 2. A phylogenetic tree of prealigned T1 domains of KCNRG-like proteins.