The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.     

Heterogenic Final Cell Cycle by Chicken Retinal Lim1 Horizontal Progenitor Cells Leads to Heteroploid Cells with a Remaining Replicated Genome

Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs) exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal) mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.     

Bone morphogenetic protein 4 (BMP4) signaling in retinoblastoma cells

Bone morphogenetic proteins (BMPs) - expressed in the developing retina - are known to be involved in the regulation of cell proliferation and apoptosis in several tumor entities. The objective of this study was to determine the role of the BMP4 pathway in retinoblastoma cells, which are absent in a functional retinoblastoma (RB1) gene. BMP receptors were detected in all retinoblastoma cell lines investigated. A correct transmission of BMP signaling via the Smad1/5/8 pathway could be demonstrated in WERI-Rb1 retinoblastoma cells and application of recombinant human BMP4 resulted in an increase in apoptosis, which to a large extend is caspase independent. Cell proliferation was not affected by BMP4 signaling, although the pRb-related proteins p107 and p130, contributing to the regulation of the same genes, are still expressed. WERI-Rb1 cells exhibit elevated endogenous levels of p21(CIP1) and p53, but we did not detect any increase in p53, p21(CIP1)or p27(KIP1) expression levels. Id proteins became, however, strongly up-regulated upon exogenous BMP4 treatment. Thus, RB1 loss in WERI-Rb1 cells is obviously not compensated for by pRb-independent (e.g. p53-dependent) cell cycle control mechanisms, preventing an anti-proliferative response to BMP4, which normally induces cell cycle arrest.     

DNA双链断裂损伤反应及它的医学意义

DNA损伤应激反应是维持基因组稳定性的基石.细胞在长期进化中形成了由损伤监视、周期调控、损伤修复、凋亡诱导等在内的自稳平衡机制.一方面,借助感应、识别并启动精细而复杂的修复机制修复损伤;另一方面,通过DNA损伤应激活化的细胞周期检查点机制,延迟或阻断细胞周期进程,为损伤修复提供时间,使细胞能安全进入新一轮细胞周期;损伤无法修复时则诱导细胞凋亡.DNA双链断裂(double strand breaks,DSBs)是真核基因组后果最严重的损伤类型之一,其修复不利,同肿瘤等人类疾病的发生发展密切相关.新进展揭示:DSBs损伤反应信号分子ATM-Chk2-p53、H2AX等的组成性活化,是肿瘤形成早期所激活的细胞内可诱导的抗癌屏障,其信号网络的精确、精细调控在基因组稳定性维持中发挥重要作用.此外,HIV病毒整合进入宿主细胞基因组的过程也依赖于宿主细胞中ATM介导的DSBs损伤反应信号转导;ATM特异性的小分子抑制剂在抗HIV感染中显示重要的功能意义.文中重点讨论调控DSBs损伤应激反应信号网络的主要研究进展,及其在肿瘤发生、发展及抗HIV感染中的新医学意义.     

Behind the wheel and under the hood: Functions of cyclin-dependent kinases in response to DNA damage

Cell division and the response to genotoxic stress are intimately connected in eukaryotes, for example, by checkpoint pathways that signal the presence of DNA damage or its ongoing repair to the cell cycle machinery, leading to reversible arrest or apoptosis. Recent studies reveal another connection: the cyclin-dependent kinases (CDKs) that govern both DNA synthesis (S) phase and mitosis directly coordinate DNA repair processes with progression through the cell cycle. In both mammalian cells and yeast, the two major modes of double strand break (DSB) repair – homologous recombination (HR) and non-homologous end joining (NHEJ) – are reciprocally regulated during the cell cycle. In yeast, the cell cycle kinase Cdk1 directly promotes DSB repair by HR during the G2 phase. In mammalian cells, loss of Cdk2, which is active throughout S and G2 phases, results in defective DNA damage repair and checkpoint signaling. Here we provide an overview of data that implicate CDKs in the regulation of DNA damage responses in yeast and metazoans. In yeast, CDK activity is required at multiple points in the HR pathway; the precise roles of CDKs in mammalian HR have yet to be determined. Finally, we consider how the two different, and in some cases opposing, roles of CDKs – as targets of negative regulation by checkpoint signaling and as positive effectors of repair pathway selection and function – could be balanced to produce a coordinated and effective response to DNA damage.     

Crosstalk between Phosphoinositide 3-kinase/Akt signaling pathway with DNA damage response and oxidative stress in cancer

The phosphatidylinositol 3-kinases (PI3K)/Akt signaling pathway is one of the well-characterized and most important signaling pathways activated in response to DNA damage. This review discusses the most recent discoveries on the involvement of PI3K/Akt signaling pathway in cancer development, as well as stimulation of some important signaling networks involved in the maintenance of cellular homeostasis upon DNA damage, with an exploration of how PI3K/Akt signaling pathway contributes to the regulation of modulators and effectors underlying DNA damage response, the intricate, protein-based signal transduction network, which decides between cell cycle arrest, DNA repair, and apoptosis, the elimination of irreparably damaged cells to maintain homeostasis. The review continues by looking at the interplay between cell cycle checkpoints, checking the repair of damage inflicted to the DNA before entering DNA replication to facilitate DNA synthesis, and PI3K/Akt signaling pathway. We then investigate the challenges the cells overcome to ameliorate damages induced by oxidative activities, for example, the recruitment of many pathways and factors to maintain integrity and hemostasis. Finally, the review provides a discussion of how cells use the PI3K/Akt signaling pathway to regulate the balance between these networks.     

Topoisomerase IIα mediates E2F-1-induced chemosensitivity and is a target for p53-mediated transcriptional repression

Mutations of the retinoblastoma tumor suppressor, pRb, or its cyclin-cyclin-dependent kinase (CDK) regulatory kinases or CDK inhibitors, allows unrestrained E2F activity, leading to unregulated cell cycle progression. However, overexpression of E2F-1 also sensitizes cells to apoptosis, suggesting that targeting this pathway may be of therapeutic benefit. Enforced expression of E2F-1 in interleukin-3-dependent myeloid cells led to preferential sensitivity to the topoisomerase II inhibitor, etoposide, which was independent of p53 accumulation. Pretreatment of the E2F-1-expressing cells with ICRF-193, a second topoisomerase II inhibitor that does not cause DNA damage, protected these cells against etoposide-induced apoptosis. However, ICRF-193 cooperated with other DNA-damaging agents to induce apoptosis. Enforced expression of E2F-1 led to accumulation of p53 protein. An E2F-1 mutant that is defective in inducing cell cycle progression also induced p53, suggesting that p53 was responding directly to E2F, and not to secondary events caused by inappropriate cell cycle progression (i.e., DNA damage). Thus, topoisomerase II inhibition and DNA damage cooperate to selectively induce apoptosis in cells that have mutations in the pRb pathway.     

The nucleotide excision repair pathway is required for UV-C-induced apoptosis in Caenorhabditis elegans

Ultraviolet (UV) radiation is a mutagen of major clinical importance in humans. UV-induced damage activates multiple signaling pathways, which initiate DNA repair, cell cycle arrest and apoptosis. To better understand these pathways, we studied the responses to UV-C light (254 nm) of germ cells in Caenorhabditis elegans. We found that UV activates the same cellular responses in worms as in mammalian cells. Both UV-induced apoptosis and cell cycle arrest were completely dependent on the p53 homolog CEP-1, the checkpoint proteins HUS-1 and CLK-2, and the checkpoint kinases CHK-2 and ATL-1 (the C. elegans homolog of ataxia telangiectasia and Rad3-related); ATM-1 (ataxia telangiectasia mutated-1) was also required, but only at low irradiation doses. Importantly, mutation of genes encoding nucleotide excision repair pathway components severely disrupted both apoptosis and cell cycle arrest, suggesting that these genes not only participate in repair, but also signal the presence of damage to downstream components of the UV response pathway that we delineate here. Our study suggests that whereas DNA damage response pathways are conserved in metazoans in their general outline, there is significant evolution in the relative importance of individual checkpoint genes in the response to specific types of DNA damage.     

Role of human Cds1 (Chk2) kinase in DNA damage checkpoint and its regulation by p53.

In response to DNA damage, mammalian cells adopt checkpoint regulation, by phosphorylation and stabilization of p53, to delay cell cycle progression. However, most cancer cells that lack functional p53 retain an unknown checkpoint mechanism(s) by which cells are arrested at the G(2)/M phase. Here we demonstrate that a human homolog of Cds1/Rad53 kinase (hCds1) is rapidly phosphorylated and activated in response to DNA damage not only in normal cells but in cancer cells lacking functional p53. A survey of various cancer cell lines revealed that the expression level of hCds1 mRNA is inversely related to the presence of functional p53. In addition, transfection of normal human fibroblasts with SV40 T antigen or human papilloma viruses E6 or E7 causes a marked induction of hCds1 mRNA, and the introduction of functional p53 into SV40 T antigen- and E6-, but not E7-, transfected cells decreases the hCds1 level, suggesting that p53 negatively regulates the expression of hCds1. In cells without functional ataxia telangiectasia mutated (ATM) protein, phosphorylation and activation of hCds1 were observed in response to DNA damage induced by UV but not by ionizing irradiation. These results suggest that hCds1 is activated through an ATM-dependent as well as -independent pathway and that it may complement the function of p53 in DNA damage checkpoints in mammalian cells.     

Two faces of p53: aging and tumor suppression

The p53 tumor suppressor protein, often termed guardian of the genome, integrates diverse physiological signals in mammalian cells. In response to stress signals, perhaps the best studied of which is the response to DNA damage, p53 becomes functionally active and triggers either a transient cell cycle arrest, cell death (apoptosis) or permanent cell cycle arrest (cellular senescence). Both apoptosis and cellular senescence are potent tumor suppressor mechanisms that irreversibly prevent damaged cells from undergoing neoplastic transformation. However, both processes can also deplete renewable tissues of proliferation-competent progenitor or stem cells. Such depletion, in turn, can compromise the structure and function of tissues, which is a hallmark of aging. Moreover, whereas apoptotic cells are by definition eliminated from tissues, senescent cells can persist, acquire altered functions, and thus alter tissue microenvironments in ways that can promote both cancer and aging phenotypes. Recent evidence suggests that increased p53 activity can, at least under some circumstances, promote organismal aging. Here, we discuss the role of p53 as a key regulator of the DNA damage responses, and discuss how p53 integrates the outcome of the DNA damage response to optimally balance tumor suppression and longevity.     

The long noncoding RNA Vax2os1 controls the cell cycle progression of photoreceptor progenitors in the mouse retina

Long noncoding RNAs (lncRNAs) are emerging as regulators of many basic cellular pathways. Several lncRNAs are selectively expressed in the developing retina, although little is known about their functional role in this tissue. Vax2os1 is a retina-specific lncRNA whose expression is restricted to the mouse ventral retina. Here we demonstrate that spatiotemporal misexpression of Vax2os1 determines cell cycle alterations in photoreceptor progenitor cells. In particular, the overexpression of Vax2os1 in the developing early postnatal mouse retina causes an impaired cell cycle progression of photoreceptor progenitors toward their final committed fate and a consequent delay of their differentiation processes. At later developmental stages, this perturbation is accompanied by an increase of apoptotic events in the photoreceptor cell layer, in comparison with control retinas, without affecting the proper cell layering in the adult retina. Similar results are observed in mouse photoreceptor-derived 661W cells in which Vax2os1 overexpression results in an impairment of the cell cycle progression rate and cell differentiation. Based on these results, we conclude that Vax2os1 is involved in the control of cell cycle progression of photoreceptor progenitor cells in the ventral retina. Therefore, we propose Vax2os1 as the first example of lncRNA that acts as a cell cycle regulator in the mammalian retina during development.     

Regulation of Nucleotide Excision Repair by Nuclear Lamin B1

The nuclear lamins play important roles in the structural organization and function of the metazoan cell nucleus. Recent studies on B-type lamins identified a requirement for lamin B1 (LB1) in the regulation of cell proliferation in normal diploid cells. In order to further investigate the function of LB1 in proliferation, we disrupted its normal expression in U-2 OS human osteosarcoma and other tumor cell lines. Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis. The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation. Several proteins involved in the detection and repair of UV damage by the nucleotide excision repair (NER) pathway are down-regulated in LB1 silenced cells including DDB1, CSB and PCNA. We propose that LB1 regulates the DNA damage response to UV irradiation by modulating the expression of specific genes and activating persistent DNA damage signaling. Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.     

乳腺癌易感蛋白1在DNA损伤修复中的作用

人类乳腺癌易感基因1(breast cancer susceptibility gene 1,BRCA1)首先是在乳腺癌家族中发现的,是具有遗传倾向的乳腺癌和卵巢癌易感基因,其基因的突变与家族性乳腺癌及卵巢癌的发生有密切联系。BRCA1是一种抑癌基因,其基因产物可以参与维持基因组稳定性的多条细胞信号通路,例如DNA损伤诱导的细胞周期调控、DNA损伤修复、基因转录调节、细胞凋亡、泛素化等重要的细胞活动。本文就近几年来BRCA1在DNA损伤修复中的作用的研究进展作一综述,包括DNA损伤诱导的细胞周期检查点的激活和DNA损伤修复两方面。