An Intron-Retaining Splice Variant of Human Cyclin A2, Expressed in Adult Differentiated Tissues, Induces a G1/S Cell Cycle Arrest In Vitro

BACKGROUND: Human cyclin A2 is a key regulator of S phase progression and entry into mitosis. Alternative splice variants of the G1 and mitotic cyclins have been shown to interfere with full-length cyclin functions to modulate cell cycle progression and are therefore likely to play a role in differentiation or oncogenesis. The alternative splicing of human cyclin A2 has not yet been studied. METHODOLOGY/PRINCIPAL FINDINGS: Sequence-specific primers were designed to amplify various exon-intron regions of cyclin A2 mRNA in cell lines and human tissues. Intron retaining PCR products were cloned and sequenced and then overexpressed in HeLa cells. The subcellular localization of the splice variants was studied using confocal and time-lapse microscopy, and their impact on the cell cycle by flow cytometry, immunoblotting and histone H1 kinase activity. We found a splice variant of cyclin A2 mRNA called A2V6 that partly retains Intron 6. The gene expression pattern of A2V6 mRNA in human tissues was noticeably different from that of wild-type cyclin A2 (A2WT) mRNA. It was lower in proliferating fetal tissues and stronger in some differentiated adult tissues, especially, heart. In transfected HeLa cells, A2V6 localized exclusively in the cytoplasm whereas A2WT accumulated in the nucleus. We show that A2V6 induced a clear G1/S cell cycle arrest associated with a p21 and p27 upregulation and an inhibition of retinoblastoma protein phosphorylation. Like A2WT, A2V6 bound CDK2, but the A2V6/CDK2 complex did not phosphorylate histone H1. CONCLUSION/SIGNIFICANCE: This study has revealed that some highly differentiated human tissues express an intron-retaining cyclin A2 mRNA that induced a G1/S block in vitro. Contrary to full-length cyclin A2, which regulates cell proliferation, the A2V6 splice variant might play a role in regulating nondividing cell states such as terminal differentiation or senescence

The modified absolute-value factorization norm for trust-region minimization

A trust-region method for unconstrained minimization, using a trust-region norm based upon a modified absolute-value factorization of the model Hessian, is proposed. It is shown that the resulting trust-region subproblem may be solved using a single factorization. In the convex case, the method reduces to a backtracking Newton linesearch procedure. The resulting software package is available as HSL VF06 within the Harwell Subroutine Library. Numerical evidence shows that the approach is effective in the nonconvex case. 1 Department for Computation and Information, Rutherford Appleton Laboratory, Chilton, Oxfordshire, OX11 0QX, England, EU Email : [email protected] 2 Current reports available by anonymous ftp from joyous-gard.cc.rl.ac.uk (internet 130.246.9.91) in the directory "pub/reports". 3 Department of Electrical and Computer Engineering, Northwestern University, Evanston, IL 60208-3118, USA Email : [email protected]. This author was supported by National Science Foundation..