Paradoxical roles of cyclin D1 in DNA stability

Maintenance of DNA integrity is vital for all of the living organisms. Consequence of DNA damaging ranges from, introducing harmless synonymous mutations, to causing disease-associated mutations, genome instability, and cell death. A cell cycle protein cyclin D1 is an established cancer-driving protein. However, contribution of cyclin D1 to cancer formation and cancer survival is not entirely known. In cancer tissues, overexpression of cyclin D1 is associated with both cancer genome instability, and resistance to DNA-damaging cancer drugs. Emerging evidence indicated that cyclin D1 may play novel direct roles in regulating DNA repair. Here we provide an insight how cyclin D1 expression may contribute to DNA repair and chromosome instability, and how these functions may facilitate cancer formation, and drug resistance.     

Oxidative stress-induced cyclin D1 depletion and its role in cell cycle processing

Background

Cyclin D1 is immediately down-regulated in response to reactive oxygen species (ROS) and implicated in the induction of cell cycle arrest in G2 phase by an unknown mechanism. Either treatment with a protease inhibitor alone or expression of protease-resistant cyclin D1 T286A resulted in only a partial relief from the ROS-induced cell cycle arrest, indicating the presence of an additional control mechanism.

Methods

Cells were exposed to hydrogen peroxide (H₂O₂), and analyzed to assess the changes in cyclin D1 level and its effects on cell cycle processing by kinase assay, de novo synthesis, gene silencing, and polysomal analysis, etc.

Results

Exposure of cells to excessive H₂O₂ induced ubiquitin-dependent proteasomal degradation of cyclin D1, which was subsequently followed by translational repression. This dual control mechanism was found to contribute to the induction of cell cycle arrest in G2 phase under oxidative stress. Silencing of an eIF2α kinase PERK significantly retarded cyclin D1 depletion, and contributed largely to rescuing cells from G2 arrest. Also the cyclin D1 level was found to be correlated with Chk1 activity.

Conlclusions

In addition to an immediate removal of the pre-existing cyclin D1 under oxidative stress, the following translational repression appear to be required for ensuring full depletion of cyclin D1 and cell cycle arrest. Oxidative stress-induced cyclin D1 depletion is linked to the regulation of G2/M transit via the Chk1–Cdc2 DNA damage checkpoint pathway.

General significance

The control of cyclin D1 is a gate keeping program to protect cells from severe oxidative damages.     

Estrogen-induced cyclin D1 and D3 gene expressions during mouse uterine cell proliferation in vivo: Differential induction mechanism of cyclin D1 and D3

D-type cyclins are involved in the regulation of the G₁/S transition of the cell cycle in various cell types cultured in vitro. Little is, however, known about the expression pattern and functional role of D-type cyclins in physiological processes in vivo. In this report, we studied whether the expression of murine D-type cyclins correlates with the states of mouse uterine cell proliferation in vivo. Time-course changes in cyclin D1 and D3 mRNA levels in the uterine tissues of immature mice primed with 17β-estradiol (E₂) were examined by Northern blot hybridization. c-fos and thymidine kinase (TK) mRNA levels were also examined as markers for the transition from G₀ to G₁ and the onset of S phase, respectively. Cyclin D1 and D3 mRNAs were induced 2.5-fold between c-fos and TK mRNA peaks. The E₂-induced cyclin D1 and D3 gene expressions were blocked by antiestrogens tamoxifen and ICI 182,780. We also investigated the effects of cycloheximide (CHX), a protein synthesis inhibitor, on cyclin D1 and D3 gene expressions. When CHX was treated alone, cyclin D3, but not cyclin D1, mRNA was immediately superinduced. The E₂-induced cyclin D3 gene expression was shifted by approximately 6 h when CHX was pretreated 1 hr before E₂ administration. Interestingly, the ³H-thymidine incorporation experiment showed that the mouse uterine cell cycle progression also shifted by 6 hr with pretreatment of CHX. The overall results suggest that both cyclin D1 and D3 mRNAs are constitutively expressed in uterine tissues and induced by E₂ at G₁ phase of the mouse uterine cell cycle. However, the superinducibility and temporal shift of cyclin D3 by CHX suggest that there is a different regulatory mechanism underlying cyclin D1 and D3 gene expressions in the mouse uterine cell cycle progression. Mol. Reprod. Dev. 46:450–458, 1997. © 1997 Wiley-Liss, Inc.     

Genetic replacement of cyclin D1 function in mouse development by cyclin D2

D cyclins (D1, D2, and D3) are components of the core cell cycle machinery in mammalian cells. It is unclear whether each of the D cyclins performs unique, tissue-specific functions or the three proteins have virtually identical functions and differ mainly in their pattern of expression. We previously generated mice lacking cyclin D1, and we observed that these animals displayed hypoplastic retinas and underdeveloped mammary glands and a presented developmental neurological abnormality. We now asked whether the specific requirement for cyclin D1 in these tissues reflected a unique pattern of D cyclin expression or the presence of specialized functions for cyclin D1 in cyclin D1-dependent compartments. We generated a knock-in strain of mice expressing cyclin D2 in place of D1. Cyclin D2 was able to drive nearly normal development of retinas and mammary glands, and it partially replaced cyclin D1's function in neurological development. We conclude that the differences between these two D cyclins lie mostly in the tissue-specific pattern of their expression. However, we propose that subtle differences between the two D cyclins do exist and they may allow D cyclins to function in a highly optimized fashion. We reason that the acquisition of multiple D cyclins may allow mammalian cells to drive optimal proliferation of a diverse array of cell types.     

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.     

Timing the changes of cyclin E cell content in G1 in exponentially growing cells

We present a method for measuring the content of immunocytochemically detected proteins in individual cells progressing through G(1) phase and its application in the analysis of cyclin E levels. The sequence of G(1) events is tracked under unaltered cycling conditions, in a cell line in the phase of balanced growth in vitro, to avoid the pitfalls of synchronization. Cells were pulse-labeled with BrdUrd and analyzed sequentially by multiparameter flow cytometry, focusing on the subpopulation of labeled cells progressively entering G(1). We use the time-from-birth ("age") of individual cells to track their position inside G(1). Using the average content of cyclin E in the whole population of G(1) cells as the internal reference for each sample, we analyzed the time course of the frequency histograms of cyclin E content within BrdUrd-labeled G(1) cells by exploiting the properties of the age distributions of asynchronous populations. This way we could calculate the average cyclin E content of cells in each age cohort. Cyclin E values were low until age 3 h, after which they rose gradually, reaching six times the value of newborn cells at the end of G(1).     

Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr286 in squamous cell carcinoma

Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G0/G1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3beta (GSK-3beta). Depletion of endogenous GSK-3beta by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3beta and found that DIF-1 dephosphorylated GSK-3beta on Ser9 and induced the nuclear translocation of GSK-3beta, suggesting that DIF-1 activated GSK-3beta. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3beta-mediated phosphorylation of Thr286.     

Ca(2+)-promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest

CDK1-cyclin B1 is a universal cell cycle kinase required for mitotic/meiotic cell cycle entry and its activity needs to decline for mitotic/meiotic exit. During their maturation, mouse oocytes proceed through meiosis I and arrest at second meiotic metaphase with high CDK1-cyclin B1 activity. Meiotic arrest is achieved by the action of a cytostatic factor (CSF), which reduces cyclin B1 degradation. Meiotic arrest is broken by a Ca2+ signal from the sperm that accelerates it. Here we visualised degradation of cyclin B1::GFP in oocytes and found that its degradation rate was the same for both meiotic divisions. Ca2+ was the necessary and sufficient trigger for cyclin B1 destruction during meiosis II; but it played no role during meiosis I and furthermore could not accelerate cyclin B1 destruction during this time. The ability of Ca2+ to trigger cyclin B1 destruction developed in oocytes following a restabilisation of cyclin B1 levels at about 12 h of culture. This was independent of actual first polar body extrusion. Thus, in metaphase I arrested oocytes, Ca2+ would induce cyclin B1 destruction and the first polar body would be extruded. In contrast to some reports in lower species, we found no evidence that oocyte activation was associated with an increase in 26S proteasome activity. We therefore conclude that Ca2+ mediates cyclin B1 degradation by increasing the activity of an E3 ubiquitin ligase. However, this stimulation occurs only in the presence of the ubiquitin ligase inhibitor CSF. We propose a model in which Ca2+ directly stimulates destruction of CSF during mammalian fertilisation.     

Identification of connexin 50 and 57 mRNA in A-type horizontal cells of the rabbit retina

Horizontal cells (HCs) mediate negative feedback to photoreceptors. In the mammalian retina, there are two types of HCs, which are extensively coupled to neighboring cells through homologous gap junctions. The permeability and therefore the strength of feedback can be regulated by light intensity, dopamine and many other factors. However, the component(s) of the most prominent gap junctions, those between A-type HCs in the rabbit retina, is still unknown. In this study, we compared the sequences of many types of mammalian connexins, obtained partial sequences of rabbit connexin 50 and 57. Using specific primers designed against the rabbit sequences, we identified mRNAs of connexin 50 and/or 57 in visually selected single A-type HC using multiplex RT-PCR.     

Gene structure and chromosomal localization of mouse cyclin G2 (Ccng2)

Cyclins are essential activators of cyclin-dependent kinases (Cdk) which, in turn, play pivotal roles in controlling transition through cell-cycle checkpoints. Cyclin G2 is a recently discovered second member of the G-type cyclins. The two members of the G-type cyclins, cyclin G1 and cyclin G2, share high structural similarity but their function remains to be defined. Here we characterize the structure of the mouse cyclin G2 gene by first cloning and sequencing the full-length mouse cyclin G2 cDNA. The cyclin G2 cDNA was used to isolate the cyclin G2 gene from a BAC library and to establish that the gene was transcribed from eight exons spanning a total of 8604 bp. The cyclin G2 gene was mapped by fluorescence in situ hybridization (FISH) to mouse chromosome 5E3.3.–F1.3. This region is syntenic to a region on human chromosome 4. The expression of cyclins G1 and G2 was examined in various tissues, but no correlation between expression patterns of the two genes was observed. However, during hepatic ontogenesis the cyclin G2 expression level decreased with age, whereas cyclin G1 expression increased. Transient expression of cyclin G2-green fluorescent protein (GFP) fusion protein in NIH3T3 cells showed that cyclin G2 is essentially a cytoplasmic protein, in contrast to the largely nuclear localization of cyclin G1. Our data suggest that, despite the close structural similarity between mouse cyclins G1 and G2, these proteins most likely perform distinct functions.     

Mitotic polyploidization of mouse heart myocytes during the first postnatal week

Summary Polyploidization of myocytes in the cardiac ventricle of mice occurs predominantly during the first week of the postnatal life. Using isolated cells it was shown that about 70 % of the cardiomyocytes become binuclear at this time (2c×2), while 10 % are mononuclear but contain 4c of DNA, where c was the haploid level of DNA. About 2 % of the cell population were represented by 4c×2 or 8c cells.Cytophotometry of Feulgen-stained DNA in ¹⁴C-thymidine-labeled nuclei has shown that the cells that enter the mitotic cycle are mainly diploid. After mitosis (30 h or more after thymidine application) the label was found predominantly in 2c×2 and 4c cell types. Comparison of the curves presenting dynamics of labeled mitoses and the accumulation of labeled binuclear cells reveals that binuclear 2c×2 cells are formed by acytokinetic mitosis. The formation of 4c mononuclear cells is accomplished via other types of mitotic arrest; this may be due, for example, to a block in the pro-or metaphase.Only very rare cases of cytotomy were detected and the number of newly formed 2c cells was very low. It is concluded that cell multiplication is practically arrested at this period of life, and growth of the ventricular mass is due to polyploidization of virtually all cycling cells, while their number remains unchanged. Mechanisms and functional significance of cardiomyocyte polyploidization are discussed.     

Cloning and characterization of a novel gene SRG-L expressed in late stages of mouse spermatogenic cells

Spermatogenesis is a continuum of spermatogoniarenewal and proliferation, meiosis, and spermiogenesisin mammalian. First the stem cells (primitive type A sper-matogonia) divide to preleptotene primary spermato-cytes, which develop to leptotene primary spermatocytes,zygotene primary spermatocytes, and pachytene anddiplotene primary spermatocytes in sequence. Then thediplotene primary spermatocytes go through two meioticdivisions, and produce round spermatids. Subsequently,round spermatids enter…     

Overexpression of cyclin D1 inhibits TNF-induced growth arrest

Although activated macrophages destroy cancer cells more effectively than normal cells, the facility to escape activated macrophages is a characteristic of tumor cells. One of the mechanisms responsible for the specific killing of tumor cells by macrophages is the production of the cytokine tumor necrosis factor alpha (TNF). Therefore, resistance to TNF may provide such cancer cells a selective advantage against host elimination. In the present work we explore the possibility that cyclin D1 overrides the cytostatic effect of TNF. We show that TNF induces p21(waf1) protein in malignant melanoma A375 cells and its binding to CDK2/4 and 6 proteins, and thereby inhibiting the activity of these complexes. This inhibition leads the cells to a G1 arrest. Overexpression of cyclin D1 in these cells makes them insensitive to TNF treatment with the recovery of CDK activity, however, is unable to overcome the inhibitory action of etoposide blocking the cells on G2/M. The bypass of TNF-induced G1 arrest seems to be related to the increase in the stability of cyclin D bound CDK complexes, increasing the total amount of CDK2/4 and 6 complexes and leading to a functional down titration of the p21(waf1) molecules. In these conditions the TNF-induced increase of p21(waf1) is not sufficient to inhibit the high amount of cyclin D-bound complexes. This hypothesis is supported by the fact that a reduction in the levels of p21(waf1) protein, induced by the expression of a mRNA antisense against p21(waf1), is also able to bypass of TNF-induced arrest. Our results confirm that p21(waf1) has an essential role in TNF-induced arrest and that the deregulation of cyclin D1 may be one of the mechanisms to escape physiological signals to restrict tumoral growth.