The Role of Ink4a-Arf Locus in Control of Hematopoietic Stem Cell Proliferation

GENE REGULATION: TARGETS 633. Dose-Dependent Gene Silencing by Lentivirus-Mediated RNA Interference (RNAi) in bcr-abl Positive Cells Michaela Scherr, Karin Battmer, Arnold Ganser, Matthias Eder. 1 Hematology and Oncology, Hannover Medical School, Hannover, Germany. RNA interference (RNAi) describes a process of double-stranded RNA-dependent post-transcriptional gene silencing. This gene silencing is mediated by 21-23 nt small interfering RNAs (siRNAs) which induce sequence-specific mRNA degradation. Fusiontranscripts encoding oncogenic proteins may represent potential targets for a tumor-specific RNAi-approach. Recently, siRNAs have been shown to specifically inhibit expression of the bcr-abl oncogene essential for chronic myeloid and bcr-abl positive acute lymphoblastic leukemia (CML, ALL) in hematopoietic cell lines. Since RNAi triggered by a single siRNA application is only transient in mammalian cells potential anti-tumor effects are limited in the models studied so far. Gene transfer of transcription cassettes for suitable RNAi-triggers, however, can induce long-term gene silencing even in primary hematopoietic cells. Using lentiviral gene transfer to transcribe different shRNAs targeting the bcr-abl oncogene and to express red fluorescence protein (RFP) as a quantitative reporter to track cellular transduction we demonstrate that stable but not transient RNAi can efficiently deplete bcr-abl+ human K562 and murine TonB cells. Importantly, killing of bcr-abl+ cells depends on the dose of lentivirus used for transduction and correlates with the RFP-expression level of transduced cells. Furthermore, we show that lentiviral transduction of homogenous cell lines results in heterogenous cell populations in terms of lentiviral integrations, RFP- and siRNA expression, target gene silencing, and functional phenotype. Whereas transduced K562 cells with high RFPexpression are eradicated, a limited number of cells with lower RFPexpression and smaller number of lentiviral integrations may outgrow after longer periods of cell culture. Interestingly, these cells still show some reduction in bcr-abl mRNA levels, aberrant proliferation kinetics, and higher sensitivity to the Bcr-Abl kinase inhibitor STI571 as compared to controls. Furthermore, lentiviral gene transfer of anti bcr-abl shRNA into primary CML cells can inhibit colony formation of transduced CD34+ progenitor cells by about 50%. These data demonstrate that lentivirus-mediated RNAi can induce high efficient and stable gene silencing depending on the number of lentiviral integrations in cell lines and in primary cells. Selection processes of gene modified cells, however, have implications for any potential therapeutic application and for functional genomic analysis based on gene transfer strategies to induce stable RNAi in mammalian cells.

634. The Role of Ink4a-Arf Locus in Control of Hematopoietic Stem Cell Proliferation Lilia Stepanova,1 Brian P. Sorrentino.1 1 Hematology/Oncology, St.Jude Children’s Research Hospital, Memphis, TN. The mechanisms that control hematopoietic stem cell (HSC) proliferation and self-renewal are incompletely understood, but are relevant for developing gene therapy strategies that target HSCs. Recent evidence shows that loss of Bmi-1, a polycomb transcriptional repressor of the Ink4a-Arf locus, results in progressive loss of HSCs in adult mice with subsequent failure of hematopoiesis. The Ink4a-Arf locus encodes two tumor suppressor proteins, p16Ink4a and p19Arf. Induced transcription of these genes results in cell cycle arrest in several model systems. Based on these facts, we have tested the hypothesis that these Ink4a-Arf gene products may play a role in limiting HSC self renewal during conditions of enhanced proliferation. We first tested whether p19Arf was induced in murine S240

bone marrow cells that were cultured for 10-12 days in IL3, IL6, SCF, and 15% fetal calf serum, hypothesizing that the well described loss of HSCs under these conditions could be due to induction of p19Arf. Using cells from mice that had a GFP reporter gene inserted into the first exon of the p19Arf gene, we found that the p19Arf/GFP allele was significantly induced in the bulk cell population after culture, but not expressed in lineage negative, c-kit+, Sca1+ cells derived from uncultured bone marrow. Competitive repopulation studies were then performed using bone marrow from either p19Arf null mice, or from mice with a homozygous deletion that disrupt both p19Arf and p16Ink4a (Ink4a-Arf -/-). In both cases, there were no defects in repopulating activity when using either freshly isolated bone marrow cells, or cells grown in our 12 day cultures. These data indicate that the loss of HSCs with in vitro culture does not require either p19Arf or p16Ink4a. We next asked whether these genes play a role in the self-renewal of HSCs in vivo using serial bone marrow transplantation assay. Bone marrow cells from either strain or from wildtype controls were serially transplanted into lethally irradiated recipients in doses ranging from 1X105 to 1X106 cells per mouse. We are currently following the fourth group of transplant recipients. In the mice transplanted with bone marrow cells from Ink4a-Arf -/donors, a survival advantage was noted relative to mice transplanted with strain-matched, wildtype controls. For instance, in secondary recipients receiving 1X105 bone marrow cells, 100% survival was noted in mice transplanted with Ink4a-Arf -/- cells after 10 weeks compared to 20% survival in mice receiving the same dose of wildtype cells. A similar survival advantage was also noted in groups given the lowest cell doses within the 3rd and 4th transplants. In parallel experiments using p19Arf null mice, there was no difference in survival relative to wildtype controls. Measurement of blood counts from recipients in all cohorts clearly showed that the deaths were due to hematopoietic failure. These results show that either both p16Ink4a and p19Arf can inhibit HSC self-renewal in a serial transplant setting, or that only p16Ink4a is necessary. Because animals transplanted with Ink4a-Arf -/- cells do die with hematopoietic failure, albeit later than wildtype controls, we conclude that mechanisms not associated with the Ink4a genes also play a role in limiting HSC self-renewal in vivo.

635. Macrophage Colony Stimulating Factor Expression in Retrovirally Transfected Cells Is Dependent upon the Adherence Status of the Target Cells JianGang Zhang,1,2 Qinghong Dan,1,2 Timothy C. Fong,3 Christopher C. Williams,1,2 Maria D. Avina,1,2 Mehrdokht Tarbiyat-Boldaji,1,2 Sakineh Khalaghizadeh,1,2 Michael Irwin,1,2 Amy Nyugen,1,2 H. Terry Wepsic,1,2 Martin R. Jadus.1,2 1 Diagnostic and Molecular Medicine Healthcare Group, Veterans Affairs Medical Center, Long Beach, Long Beach, CA; 2 Department of Pathology, University of California, Irvine, Irvine, CA; 3Chiron Corporation, Chiron Corporation, Emeryville, CA. Numerous cell types including both normal and cancerous cells retrovirally transduced with either the membrane or the secreted forms of macrophage colony stimulating factor (mM-CSF and sMCSF, respectively) using LXSN-based vectors showed a variable expression of the transgene. Expression of either form of M-CSF correlated with the cells’ adhesion to culture dishes. Transduced adherent cells produced more sM-CSF by ELISA and mM-CSF by flow cytometry; whereas, the non-adherent cells synthesized little M-CSF. Use of a granulocyte-macrophage colony stimulating factor (GM-CSF) LXSN-based retrovirus failed to show this dichotomy. Real-time polymerase chain reaction analysis using the ∆∆CT method showed that adherent cells produced more M-CSF-specific mRNA than the non-adherent cells, while maintaining similar mRNA levels for the β-actin and neomycin resistance genes. Examination of the Molecular Therapy Volume 9, Supplement 1, Ma y 2004

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