Regulation of Glioblastoma Progression by Cord Blood Stem Cells Is Mediated by Downregulation of Cyclin D1

et al. (2011) Regulation of Glioblastoma Progression by Cord Blood Stem Cells Is Mediated by Downregulation of Cyclin D1. PLoS ONE 6(3): e18017. doi:10.1371/journal.pone.0018017 Regulation of Glioblastoma Progression by Cord Blood Stem Cells Is Mediated by Downregulation of Cyclin D1 Kiran Kumar Velpula 0 Venkata Ramesh Dasari 0 Andrew J. Tsung 0 Christopher S. Gondi 0 Jeffrey D. 0 Klopfenstein 0 Sanjeeva Mohanam 0 Jasti S. Rao 0 Maciej Lesniak, The University of Chicago, United States of America 0 1 Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America, 2 Department of Neurosurgery, University of Illinois College of Medicine at Peoria , Peoria, Illinois , United States of America Background: The normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, often is considered a rate-limiting step during cell cycle progression through the G1 phase. Methodology/Principal Findings: In the present study, human umbilical cord blood stem cells (hUCBSC) in co-cultures with glioblastoma cells (U251 and 5310) not only induced G0-G1 phase arrest, but also reduced the number of cells at S and G2-M phases of cell cycle. Cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC as revealed by Western and FACS analyses. Inhibition of cyclin D1 activity by hUCBSC treatment is sufficient to abolish the expression levels of Cdk 4, Cdk 6, cyclin B1, b-Catenin levels. Our immuno precipitation experiments present evidence that, treatment of glioma cells with hUCBSC leads to the arrest of cell-cycle progression through inactivation of both cyclin D1/ Cdk 4 and cyclin D1/Cdk 6 complexes. It is observed that hUCBSC, when co-cultured with glioma cells, caused an increased G0-G1 phase despite the reduction of G0-G1 regulatory proteins cyclin D1 and Cdk 4. We found that this reduction of G0-G1 regulatory proteins, cyclin D1 and Cdk 4 may be in part compensated by the expression of cyclin E1, when co-cultured with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated, decreased expression of cyclin D1 in the presence of hUCBSC. Conclusions/Significance: This paper elucidates a model to regulate glioma cell cycle progression in which hUCBSC acts to control cyclin D1 induction and in concert its partner kinases, Cdk 4 and Cdk 6 by mediating cell cycle arrest at G0-G1 phase. - Funding: The project was supported by Award Number NS057529 (J.S.R.) from the National Institute of Neurological Disorders and Stroke (NINDS). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health (NIH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Glioblastoma multiforme (GBM) is the most virulent and fatal form of brain cancer. Currently, no ideal treatment exists for glioblastoma multiforme, and patients generally survive less than one year [1,2]. Although there have been many achievements in surgery, radiotherapy and chemotherapy, glioblastoma continues to have a very poor prognosis [35]. Since finding an effective mechanism to deliver therapeutic agents to the targeted site of the tumor has proved problematic, the therapeutic potential of novel gene therapies has been greatly diminished [6]. Several researchers have studied the effect of neuronal stem cells with regard to the tropism and growth reduction of tumors, but their clinical application is restricted by their potential immunologic incompatibility [710]. Because of these limitations, most studies have focused on evaluating murine neural stem cells. The inherent problems with neural stem cells create the need for evaluation of other types of stem cells that are more readily available and clinically pertinent and may be used as vehicles for delivering therapeutic agents to brain tumors. Human umbilical cord blood (hUCB), a rich source of hematopoietic and mesenchymal stem cells provides an alternative source of stem cells. Of the various progenitor cells that exist in cord blood, mesenchymal stem cells in particular are attractive for clinical use because of their easy isolation, availability, and expansion in cultures [11,12]. In addition, it has been established that hUCB-derived stem cells (hUCBSC) exhibit higher proliferation and expansion potential than their adult bone marrow counterparts [13,14]. Recent investigations on mesenchymal stem cells have revealed their participation in tumor growth and metastasis, partially due to their immunosuppressive and proangiogenic properties [15]. Lu et al. reported that mesenchymal stem cells could upregulate the expression of p21, a cell cycle inhibitor, at the transcriptional level, thereby facilitating G0-G1 phase arrest and leading to apoptotic death of the tumor cells [16]. Normal progression of the cell cycle requires sequential expression of cyclins. Cyclins activate cyclin-dependent kinases (Cdk), which comprise a family of serine/threonine protein kinases, to phosphorylate target proteins required for cell-cycle progression [17]. Rapid induction of cyclin D1 and its associated binding with Cdk 4/Cdk 6, in the presence or absence of mitogenic signals, is considered to be a rate-limiting step, and is essential for a cell to pass through G1 phase during cell cycle progression. The loss of regulatory control of the cell cycle, which leads to unrestrained cell proliferation, is a hallmark of cancer. Cyclin D1 is overexpressed in breast, liver, lung and brain cancers [1820]. Suppression of cyclin D1 gene expression is an indication of cell differentiation [2123]. Dormant cells, when mitogen stimulated, enter the cell division cycle by activating cyclin D1, along with the dependant cyclin kinases Cdk 4 and Cdk 6, by phosphorylating retinoblastoma protein to release E2F transcription factors [24]. In the present study, we sought to provide insight into the functional and regulatory characteristics of cyclin D1, to determine its potential as a therapeutic target for glioblastoma, and to evaluate whether the expression of cyclin D1 is associated with clinical and pathological features in glioblastoma. Here, we show that human umbilical cord blood stem cells (hUCBSC) cocultured with U251 and 5310 cells resulted in G0-G1 phase arrest, probably by down regulating the expression of cyclin D1 and its associated kinases Cdk 4 and Cdk 6. hUCBSC arrest cell cycle of glioma cell lines U251 and 5310 at G0-G1 phase The authenticity of hUCBSC was characterized by the expression levels of the specific markers CD29 (...truncated)