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Functional Cloning of SPIN-2, a Nuclear Anti-Apoptotic Protein with Roles in Cell Cycle Progression BS Fletcher1, C Dragstedt1, L Notterpek2 and GP Nolan3

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Functional Cloning of SPIN-2, a Nuclear Anti-Apoptotic Protein with Roles in Cell Cycle Progression BS Fletcher1, C Dragstedt1, L Notterpek2 and GP Nolan3 Leukemia (2002) 16, 1507–1518 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Functional cloning of SPIN-2, a nuclear anti-apoptotic protein with roles in cell cycle progression BS Fletcher1, C Dragstedt1, L Notterpek2 and GP Nolan3 1Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, FL, USA; 2Department of Neuroscience, University of Florida, College of Medicine, Gainesville, FL, USA;and 3Departments of Molecular Pharmacology and Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. The balance between hematopoietic cell viability and apoptosis plasm.8,9 Apoptosis then proceeds by the association of cyto- is regulated by exogenous growth factors, however, the mol- chrome c with the mammalian apaf-1 protein, which results ecular mechanisms by which these trophic factors exert their effects remain obscure. A functional retroviral cDNA library- in the activation of caspase 9 and other members in the 10 based screen was employed to identify genes that prevent caspase cascade. growth factor withdrawal-mediated apoptosis in the myeloid In hematopoietic tissues, in addition to controlling cell progenitor cell 32Dcl3. This approach identified three classes number and differentiation, apoptosis likely plays a role in of genes: those with known roles in apoptosis (bcl-X and orni- L leukemogenesis, as transformed cells must be able to over- thine decarboxylase); genes previously identified but not linked directly to apoptotic signaling (O-linked N-acetylglucosamine come pro-apoptotic cellular signals. The pro-survival pheno- transferase); and a previously uncharacterized gene we termed type may be due to the up-regulation of intact anti-apoptotic SPIN-2. In 32Dcl3 cells, expression of exogenous SPIN-2 pro- genes or to the expression of mutant proteins with similar vides 25% protection from apoptosis following growth factor functions. An example of this is seen in follicular B cell lym- ෂ withdrawal compared to controls which show 1–2% survival. phomas in which chromosomal translocations (ie t(8:14) and SPIN-2 overexpression slows cell growth rates and increases the percentage of cells in G /M (32% vs control cells at 12%). t(8:22)) result in the transcriptional upregulation of the anti- 2 11,12 Immunolocalization studies indicate that myc-epitope tagged apoptotic gene product bcl-2. Bcl-2 overexpression has SPIN-2 proteins, which retain their anti-apoptotic function, also been demonstrated in patients with acute myelogenous reside in the nucleus, whereas a C-terminal deletion mutant leukemia and was shown to correlate with poor prognosis.13,14 that loses its anti-apoptotic activity is located in the cytoplasm. Several members of the bcl-2 family have been identified that These studies suggest that SPIN-2 is a novel nuclear protein 15 16 that functions to regulate cell cycle progression and this have anti-apoptotic properties, including bcl-XL, A1 and 17 activity is related to the inhibition of apoptosis following the mcl-1. A second family of proteins that can inhibit apoptosis removal of essential growth factors. by blocking activation of the caspase cascade are the IAP pro- Leukemia (2002) 16, 1507–1517. doi:10.1038/sj.leu.2402557 teins (inhibitors of apoptosis proteins), such as survivin, XIAP, Keywords: apoptosis; growth factor withdrawal; retroviral cDNA cIAP1 and cIAP2.18,19 Finally, several recently described pro- libraries; spindilin teins have been demonstrated to affect apoptosis in hemato- poietic cells including cFLIP and Toso.20,21 While expression of anti-apoptotic bcl-2 family members can prolong survival Introduction following growth factor withdrawal, few other proteins besides transforming oncogenes (ie bcr/abl) are able to Programmed cell death, or apoptosis, has emerged as an promote survival or growth in these paradigms. essential mechanism by which an organism controls cell num- To identify novel proteins with potential anti-apoptotic ber and fate. In hematopoietic systems, apoptosis is highly regulated as it can function to restrict cell number, eliminate activity, a functional genetic screen was devised utilizing auto-reactive immune cells, and control differentiation. With- retroviral cDNA libraries. We report on the characterization drawal of essential cytokines that function as growth factors of SPIN-2, a novel nuclear gene that can protect the factor- has been shown to initiate the apoptotic cascade in both dependent hematopoietic progenitor cell lines 32Dcl3 and lymphoid and myeloid lineages.1,2 Cytokines, such as NSF/N1-H7 from apoptosis following interleukin (IL)-3 with- interleukins and growth factors, play roles in regulating cell drawal. The anti-apoptotic property of SPIN-2 appears specific number and promoting differentiation of specific hemato- for growth factor withdrawal-induced apoptosis as over- poietic lineages. expression in 32Dcl3 cells, or the lymphoid cell line Jurkat, Following growth factor withdrawal one of the earliest failed to protect against cytotoxic drug (doxorubicin)-induced physiological changes is a time-dependent increase in phos- apoptosis, or Fas-mediated apoptosis, respectively. Over- phatidylserine externalization.3 Subsequently, there is a expression of SPIN-2 in 32Dcl3 cells slowed cell cycle pro- 4,5 decrease in mitochondrial membrane potential and a cal- gression and increased the percentage of cells in G2/M phase cium influx-dependent redistribution of mitochondria.6 of the cell cycle. This effect of SPIN-2 on cell cycle pro- Additional changes to the mitochondria include interactions gression appears to be linked to its anti-apoptotic activity, as with the protein bax, a pro-apoptotic bcl-2 family member, mutants that are unable to promote G2/M slowing are no that translocates from the cytoplasm and associates with mito- longer able to prevent apoptosis following growth factor with- chondrial bound bcl-2.7 Ultimately, these cellular changes drawal. Thus, it is possible to define genes that have specific result in mitochondrial release of cytochrome c into the cyto- anti-apoptotic activities under defined circumstance that dis- tinguish the actions of these genes from those of more broadly acting anti-apoptotic genes. It is likely that an understanding Correspondence: BS Fletcher, Department of Pharmacology and of such genes will help refine our knowledge of the many Therapeutics, Box 100267, University of Florida, College of Medicine, mechanisms by which leukemias become resistant to Gainesville, FL 32610-0267, USA; Fax: 352 392–9696 pro-apoptotic signaling. Received 20 November 2001; accepted 20 March 2002 SPIN-2 effects on cell cycle and apoptosis BS Fletcher et al 1508 Materials and methods derivative of pBabeMN-lacZ in which the lacZ gene has been removed and the polylinker has been modified to create a SalI Cell culture site upstream of the NotI site.21,22 Following ligation, the cDNA was transformed into chemically competent UltraMAX 32Dcl3 cells (a gift of Dr Alan Friedman, Johns Hopkins Uni- DH5␣ cells (Gibco BRL). Transformants were amplified on versity, Baltimore, MD, USA) were grown in Iscove’s modified solid LB ampicillin plates and plasmid DNA was purified. A Dulbecco’s media (IMDM) with 1.5 g sodium bicarbonate per sampling of 20 random clones revealed that Ͼ95% of the liter. Additional supplements included 10% fetal bovine clones contained inserts with an average insert size of 2.0 kb. serum (FBS), 1 ng/ml murine IL-3 (R & D Systems, Minnea- Overall complexity of the library was estimated by counting polis, MN, USA) and penicillin/streptomycin/glutamine the number of independent colonies from a dilution of each (Pen/Strept/Glut) (Gibco BRL, Rockville, MD, USA). The bacterial transformation that was plated and harvested. The amphotropic and ecotropic retroviral packaging cell lines total number of colonies that were pooled together to generate (Phoenix A and E, developed by Garry Nolan, Stanford the library was ෂ350 000 independent clones. University) and NIH3T3 cells were grown in high glucose Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% FBS and Pen/Strept/Glut. The NSF/N1-H7 cell line Retroviral cDNA library screening (a gift of Dr Deng, University of Florida) was grown in RPMI- 1640 supplemented with 10% FBS, Pen/Strept/Glut and The plasmid cDNA library was transiently transfected using 1 ng/ml murine IL-3. calcium phosphate into the amphotropic packaging cell line Phoenix A that produces infectious retrovirus with titers approaching ෂ5 × 106 infectious units/ml.21 Briefly, the pack- Apoptosis assays aging cell line was plated at 5 × 106 cells per 10 cm plate (or 2 × 106 cells/6 cm plate), 18 h prior to transfection. Plasmid To demonstrate apoptotic cell death, cells were either grown DNA (20 ␮g/10 cm plate or 8 ␮g/6 cm plate) was added by in the presence (+IL-3) or absence of IL-3 (−IL-3) for 24 and standard Hepes calcium phosphate transfection in the pres- 48 h. One million cells from each condition were collected ence of chloroquine 50 ␮M. Eight hours after the addition of by centrifugation and washed once in PBS. The cell pellets the precipitate, the media was replaced with 10 ml (3 ml/6 cm were resuspended in 100 ␮l of labeling solution (containing plate) of fresh DMEM with 10% FBS. Sixteen hours later, the Hepes buffer with calcium, annexin-V-FLUOS and propidium medium was replaced again and the cells were placed at 32°C iodide (PI)) per manufacturer’s recommendations (Roche, Indi- for ෂ30 h. Retroviral supernatant was then collected and fil- anapolis, IN, USA). After a 15 min room temperature incu- tered through a 0.45 ␮m syringe filter to prevent transfer of bation, 0.3 ml of additional Hepes buffer containing PI was the packaging cell to the target culture. added to each sample followed by fluorescence activated cell Target 32Dcl3 cells were spin-infected with the recombi- sorting (FACS) analysis (FACScan; Becton Dickinson, Immun- nant retroviral library supernatant or specific cDNA super- ocytometry Systems, San Jose, CA, USA).
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