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