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CGGBP1 Regulates Cell Cycle in Cancer Cells Umashankar Singh1*, Pernilla Roswall2, Lene Uhrbom1 and Bengt Westermark1

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CGGBP1 Regulates Cell Cycle in Cancer Cells Umashankar Singh1*, Pernilla Roswall2, Lene Uhrbom1 and Bengt Westermark1 Singh et al. BMC Molecular Biology 2011, 12:28 http://www.biomedcentral.com/1471-2199/12/28 RESEARCH ARTICLE Open Access CGGBP1 regulates cell cycle in cancer cells Umashankar Singh1*, Pernilla Roswall2, Lene Uhrbom1 and Bengt Westermark1 Abstract Background: CGGBP1 is a CGG-triplet repeat binding protein, which affects transcription from CGG-triplet-rich promoters such as the FMR1 gene and the ribosomal RNA gene clusters. Earlier, we reported some previously unknown functions of CGGBP1 in gene expression during heat shock stress response. Recently we had found CGGBP1 to be a cell cycle regulatory midbody protein required for normal cytokinetic abscission in normal human fibroblasts, which have all the cell cycle regulatory mechanisms intact. Results: In this study we explored the role of CGGBP1 in the cell cycle in various cancer cell lines. CGGBP1 depletion by RNA interference in tumor-derived cells caused an increase in the cell population at G0/G1 phase and reduced the number of cells in the S phase. CGGBP1 depletion also increased the expression of cell cycle regulatory genes CDKN1A and GAS1, associated with reductions in histone H3 lysine 9 trimethylation in their promoters. By combining RNA interference and genetic mutations, we found that the role of CGGBP1 in cell cycle involves multiple mechanisms, as single deficiencies of CDKN1A, GAS1 as well as TP53, INK4A or ARF failed to rescue the G0/G1 arrest caused by CGGBP1 depletion. Conclusions: Our results show that CGGBP1 expression is important for cell cycle progression through multiple parallel mechanisms including the regulation of CDKN1A and GAS1 levels. Background well as intracellular environment. Physical or chemical CGGBP1 was identified as a CGG triplet repeat binding stress to the cells, inability to respond to mitogenic sig- protein in vitro [1]. Ever since, different studies have nals, trans-mitotic inheritance of polyploidy, DNA focused on its ability to bind to CGG triplet repeats and damage response or loss of function of critical cell cycle exert transcriptional repression. Previously, we found regulatory genes [5-9] exemplify some such conditions that CGGBP1 participates in heat shock stress response that can cause a cell cycle block. The kind of effects by regulating HSF1 expression through heat-sensitive these conditions can have on the cell cycle progression interactions with NFIX and HMGN1 [2,3]. In normal could however vary from one cell type to the other human fibroblasts, which are expected to have all the depending on their genetic and epigenetic profiles. checkpoints and DNA repair capabilities intact, we Under normal conditions, cell cycle arrest in the G0/ recently reported functions of CGGBP1 in cell cycle reg- G1 phase is associated with the phenomena of quies- ulation at the abscission and consequential prevention of cence, when cells do not receive enough mitogenic sti- tetraploidy [4]. In cancer cells however, which often mulation in terms of growth factors, and senescence, have various abnormalities in the cell cycle regulatory when cells are terminally differentiated and enter a mechanisms, function of CGGBP1 is unknown and is of post-mitotic state [10]. Altered expression of critical obvious interest since loss of cell cycle regulation is an genes, due to genetic and epigenetic disturbances, can event central to tumorigenesis. also cause cell cycle disturbances [11-13]. The ability of Cell proliferation is tightly regulated by different cells to undergo cell cycle arrest is paramount to the mechanisms, which can halt it at an appropriate stage of health of any multicellular organism and a complex net- cell cycle in response to abnormalities in extracellular as work of proteins has evolved to execute it. The progres- sion of cell cycle from G1 to S phase is regulated by a well-studied series of events. The cyclin dependent * Correspondence: [email protected] 1Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, kinases CDK4 and CDK6 must interact with Cyclin D Uppsala University, 751 85 Uppsala, Sweden to become active and phosphorylate Rb [14-17]. This Full list of author information is available at the end of the article © 2011 Singh et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Singh et al. BMC Molecular Biology 2011, 12:28 Page 2 of 9 http://www.biomedcentral.com/1471-2199/12/28 phosphorylation of Rb releases it from the transcrip- siRNA was efficient as immunofluorescence showed a tional inhibitory complex with E2F, which then drives strong reduction in protein levels 96 h after transfection the expression of many genes including Cyclin E. Cyclin as compared to control siRNA transfected cells (Figure E complexes with CDK2 to drive entry into S phase. 1, A and 1B). The cells were transfected at 50% conflu- The very first step of this cascade, interactions between ence and after 96 h, while control siRNA transfected CDK4/6 and Cyclin D is inhibited by INK4A and ARF, cultures grew to confluence, CGGBP1 siRNA transfected as they compete with Cyclin D for binding to CDK4/6 cultures did not show increase in cell density with no [18-20]. Another protein, CDKN1A is a multifaceted visible difference in the proportion of dead cells, sug- regulator of the cell cycle. It inhibits Cyclin E-CDK2 as gesting that the CGGBP1 transfected cells underwent a well as Cyclin D-CDK4 interactions and can arrest cell cell cycle arrest. Expression of the proliferation marker cycle at G1 or early S phase in response to DNA Ki67 was also reduced in the CGGBP1 siRNA trans- damage [18-20]. Furthermore, CDKN1A expression is fected cells (Figure 1, A and 1B). Flow cytometric mea- controlled by TP53, a strong tumor suppressor gene surement of DNA content per nucleus showed that in activated by DNA damage response, which frequently the CGGBP1 depleted cultures the G0/G1 population exhibiting loss of function in many cancers. The muta- was increased while the population of S phase was tions in some or many of these cell cycle regulatory strongly decreased (Table 1 and Figure 1, C and 1D). genes such as TP53, CDKN1A, INK4A and ARF often The depletion of CGGBP1 by using the previously underlie the aberrant control of cell cycle and the ability described UTR siRNA against CGGBP1 [4] also gener- of cancer cells to escape the cell cycle block at G0/G1 ated a similar cell cycle arrest in G1/G0 phase with a phase in response to the stimuli, which would normally concomitant decrease in the S phase in U-2987 MG cause a G0/G1 arrest. The growth arrest specific gene cells stably transfected with an empty expression vector GAS1 is known to cause G0/G1 arrest in normal as well (Additional File 1). However, in U-2987 MG cells stably as transformed cells [21,22] and the mechanisms of the expressing CGGBP1 from a UTR-devoid transcript, the regulation of its levels are less well understood. UTR siRNA failed to produce the G0/G1 arrest (Addi- In this study, we combined CGGBP1 knockdown by tional File 1), showing the specificity of siRNA response. siRNA with different genetic mutations of various cell These results showed that in the U-2987 MG cells, cycle regulatory genes to rigorously test how CGGBP1 CGGBP1 is required for normal cell cycle progression regulates cell cycle in cancer cells. For this we have used well-established human cancer cell lines and also gener- ated new murine glioblastoma cell lines. We report that in all the cell lines examined, CGGBP1 deficiency pro- duced a cell cycle block at G0/G1 phase. Expression analysis of candidate genes showed that CGGBP1 regu- lates expression of CDKN1A and GAS1 genes. Mutation of CDKN1A and siRNA-mediated depletion of GAS1 could not rescue the CGGBP1 deficiency-induced cell cycle block at G0/G1 phase, suggesting that CGGBP1 controls cell cycle progression through the G1 phase through multiple parallel mechanisms. Our results show new functions for CGGBP1 in cell cycle, in regulation of expression of CDKN1A and GAS1, and provide insights into how CGGBP1 depletion overrides the redundancies of checkpoint escape mechanisms present in different cancer cells. Results CGGBP1 depletion by siRNA increases G0/G1 phase and reduces S phase cell populations Figure 1 CGGBP1 siRNA causes decrease in CGGBP1 expression We have previously established a protocol of siRNA- and Ki67 positivity of the nuclei in U-2987 MG cells and blocks mediated knockdown of CGGBP1 at both mRNA and cell cycle at G0/G1 phase. A: Control siRNA, B: CGGBP1 siRNA, C: protein levels [3,4]. In this study, we used the same pro- Flow cytometric pattern of U-2987 MG cells treated with control tocol of siRNA mediated knockdown of CGGBP1, as siRNA, D: Flow cytometric pattern of U-2987 MG cells treated with described earlier [3,4], to deplete CGGBP1 in the CGGBP1 siRNA. The strong decrease in the S-phase population in D as compared to C is visible in the plots. See table 1 for details. human glioma cell line U-2987 MG. Knockdown by Singh et al. BMC Molecular Biology 2011, 12:28 Page 3 of 9 http://www.biomedcentral.com/1471-2199/12/28 Table 1 Effect of CGGBP1 depletion in different cell types on the percentage of cells in different stages of cell cycle as measured by flow cytometric analysis of DNA content per nucleus Cells siRNA G1/G0 (% of total) S (% of total) G2/M (% of total) U-2987
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