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Functional cloning of SPIN-2, a nuclear anti-apoptotic protein with roles in cell cycle progression
B S Fletcher1, C Dragstedt, L Notterpek
1Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, FL 32610-0267, USA.
Abstract:
The balance between hematopoietic cell viability and apoptosis is regulated by exogenous growth factors, however, the molecular mechanisms by which these trophic factors exert their effects remain obscure. A functional retroviral cDNA library-based screen was employed to identify genes that prevent growth factor withdrawal-mediated apoptosis in the myeloid progenitor cell 32Dcl3. This approach identified three classes of genes: those with known roles in apoptosis (bcl-X(L) and ornithine decarboxylase); genes previously identified but not linked directly to apoptotic signaling (O-linked N-acetylglucosamine transferase); and a previously uncharacterized gene we termed SPIN-2. In 32Dcl3 cells, expression of exogenous SPIN-2 provides 25% protection from apoptosis following growth factor withdrawal compared to controls which show approximately 1-2% survival. SPIN-2 overexpression slows cell growth rates and increases the percentage of cells in G(2)/M (32% vs control cells at 12%). Immunolocalization studies indicate that myc-epitope tagged SPIN-2 proteins, which retain their anti-apoptotic function, reside in the nucleus, whereas a C-terminal deletion mutant that loses its anti-apoptotic activity is located in the cytoplasm. These studies suggest that SPIN-2 is a novel nuclear protein that functions to regulate cell cycle progression and this activity is related to the inhibition of apoptosis following the removal of essential growth factors.
Insights
Researchers identified SPIN-2, a novel nuclear protein that protects myeloid progenitor cells from apoptosis after growth factor withdrawal. SPIN-2 regulates cell cycle progression, offering significant protection against programmed cell death.
Area of Science:
- Cell Biology
- Molecular Biology
- Hematopoiesis
Background:
- Growth factors are crucial for hematopoietic cell survival, regulating the balance between viability and apoptosis.
- The precise molecular mechanisms underlying growth factor-mediated cell survival are not fully understood.
- Understanding these mechanisms is key to controlling cell proliferation and preventing cell death.
Purpose of the Study:
- To identify novel genes involved in preventing apoptosis induced by growth factor withdrawal in myeloid progenitor cells.
- To characterize the function and localization of a newly discovered gene, SPIN-2.
- To elucidate the role of SPIN-2 in cell cycle regulation and its relationship to anti-apoptotic activity.
Main Methods:
- A functional retroviral cDNA library screen was used to identify apoptosis-protective genes in 32Dcl3 myeloid progenitor cells.
- Overexpression of candidate genes, including SPIN-2, was performed to assess their anti-apoptotic effects.
- Cell cycle analysis and immunolocalization studies were conducted to determine SPIN-2's cellular location and impact on cell cycle progression.
Main Results:
- The screen identified known apoptosis regulators (bcl-X(L), ornithine decarboxylase) and a novel gene, SPIN-2.
- Overexpression of SPIN-2 conferred significant protection (25% survival) against growth factor withdrawal-induced apoptosis in 32Dcl3 cells.
- SPIN-2 localized to the nucleus, slowed cell growth, and increased the percentage of cells in G(2)/M phase, with nuclear localization essential for its anti-apoptotic function.
Conclusions:
- SPIN-2 is a novel nuclear protein that inhibits apoptosis following growth factor withdrawal.
- SPIN-2 plays a role in regulating cell cycle progression, which is linked to its anti-apoptotic activity.
- These findings provide new insights into the molecular pathways governing hematopoietic cell survival and proliferation.