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Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G 1 in non-New Zealand Black B cell and New Zealand Black-derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G 1 accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G 1 /S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3′ untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/ 16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease. [Mol Cancer Ther 2009;8(9):2684-92]

SummaryMouse models are valuable tools in the study of human chronic lymphocytic leukaemia (CLL). The New Zealand Black (NZB) strain is a naturally occurring model of late-onset CLL characterized by B-cell hyperproliferation and autoimmunity early in life, followed by progression to CLL. Other genetically engineered models of CLL that have been developed include (NZB · NZW) F1 mice engineered to express IL5, mice expressing human TCL1A, and mice overexpressing both BCL2 and a tumour necrosis factor receptor-associated factor. The applicability to human CLL varies with each model, suggesting that CLL is a multifactorial disease. Our work with the de novo NZB model has revealed many similarities to the human situation, particularly familial CLL. In NZB, the malignant clones express CD5, zap-70, and have chromosomal instability and germline Ig sequence. We also identified a point mutation in the 3¢-flanking sequence of Mirn16-1, which resulted in decreased levels of the microRNA, miR-16 in lymphoid tissue. Exogenous restoration of miR-16 to an NZB malignant B-1 cell line resulted in cell cycle alterations, suggesting that the altered expression of Mirn15a/16-1 is an important molecular lesion in CLL. Future studies utilizing the NZB mouse could ascertain the role of environmental triggers, such as low dose radiation and organic chemicals in the augmentation of a pre-existing propensity to develop CLL.Keywords: mouse models of chronic lymphocytic leukaemia, New Zealand Black, microRNA.The development of chronic lymphocytic leukaemia (CLL) in humans is believed to be polygenetic, exhibiting the highest incidence of familial leukaemia, yet emerging from many factors (Ishibe et al, 2001;Caporaso et al, 2004;Sellick et al, 2006). Molecular cytogenetic abnormalities associated with CLL have been reported (Stilgenbauer & Dohner, 2004), and include chromosome duplications (Juliusson et al, 1990;Molica et al, 1995;Dohner et al, 2000) and deletions, particularly 13q14 deletions (Juliusson et al, 1990;Dohner et al, 2000). Mouse models with known genetic backgrounds allow for the development of CLL murine models, which serve as valuable tools, not only in understanding disease mechanisms, but also in evaluating dietary/environmental triggers and efficacy of novel therapies. Mouse models of CLLThis article presents a comparison of several mouse models of CLL (Table I). With the exception of the New Zealand Black (NZB) model, all the models are induced by the expression of exogenous genes. The NZB mouse model of CLL, in contrast, is a de novo model that has been studied extensively as a model to investigate both autoimmune diseases, such as systemic lupus erythematosus (SLE), as well as B-cell lymphoproliferative disorders (Raveche et al, 1981;Manohar et al, 1982;Stall et al, 1988;Theofilopoulos, 1996). Autoreactivity has also been associated with CLL (Lugassy et al, 1992;Barcellini et al, 2006), and the NZB model displays mild autoimmunity that is associated with B-cell hyperactivity, resulting in autoimmune haemolytic anaemia ...

Similar to human CLL, the de novo NZB mouse model has a genetically determined age-associated increase in malignant B-1 clones and decreased expression of microRNAs miR-15a and miR-16 in B-1 cells. In the present study, lentiviral vectors were employed in vivo to restore miR-15a/16, and both the short-term single injection and long-term multiple injection effects of this delivery were observed in NZB. Control lentivirus without the mir-15a/16 sequence was used for comparison. We found that in vivo lentiviral delivery of mir-15a/16 increased miR-15a/16 expression in cells that were transduced (detected by GFP expression) and sera when compared to control lentivirus treatment. More importantly, mice treated with the miR- expressing lentivirus had decreased disease. The lentivirus had little systemic toxicity while preferentially targeting B-1 cells. Short-term effects on B-1 cells were direct effects and only malignant B-1 cells transduced with miR-15a/16 lentivirus had decreased viability. In contrast, long-term studies suggested both direct and indirect effects resulting from miR-15a/16 lentivirus treatment. A decrease in B-1 cells was found in both the transduced and non-transduced populations. Our data support the potential use of systemic lentiviral delivery of miR-15a/16 to ameliorate disease manifestations of CLL.