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G1 phase progression: Cycling on cue
Abstract
Charles J. Sherr Howard Hughes Medical Institute Department of Tumor Cell Biology St. Jude Children’s Research Hospital 332 North Lauderdale Memphis, Tennessee 38104 Recent advances in our understanding of the cell division cycle are now tying the functions of Gl phase regulators to diverse processes involving signal transduction, differ- entiation, senescence, apoptosis, and malignant transfor- mation. What determines the rate of Gl phase progression, and how do cells integrate mitogenic and antiproliferative signals with the cell cycle machinery? Lessons From Budding Yeast In Saccharomyces cerevisiae, a single 34 kDa cyclin- dependent kinase (cdk) (p34cDCZB/cdc2, also known as cdkl) serves as a master controller of the cell cycle, assembling sequentially into active holoenzyme complexes with Gl, S phase, or mitotic cyclins temporally to direct distinct transitions (reviewed by Nasmyth, 1993; Reed, 1992). In the presence of appropriate nutrients, Gl cells that reach a critical size initiate DNA replication, form buds, and dupli- cate their spindle bodies in preparation for subsequent division. Gl cyclins (Clnl, Cln2, and Cln3) are required for these processes (Richardson et al., 1989) (see Figure I), and their overexpression contracts Gl phase and de- creases cell size. Cln3-Cdc28 is present throughout Gl, and its kinase activity appears necessary for the subse- quent transcriptional activation of the CLN7 and CLN2 genes (Tyers et al., 1993). In turn, the induced Clnl and Cln2 proteins associate with Cdc28, whose kinase activity further stimulates CLN7 and CLN2 transcription. CLN7 and CLN2 gene expression is controlled by a heterodimeric transcription factor composed of Swi4 and Swi6, and Cln- CdcPm Schwab and Nasmyth, 1993). The kinase activities of Clb-Cdc28 complexes are held in check by an inhibitory protein (p40sfc’) (Mendenhall, 1993) that accumulates early in Gl and is degraded shortly before S phase (Schwab et al., 1994). Phosphorylation of ~40~‘~’ by Clnl,Cln2-Cdc28 might trigger its ubiquitin- mediated degradation, thereby enabling the Cln-regulated kinases to control S phase entry indirectly. Haploid Gl phase cells can also undergo cell cycle ar- rest and mate to form diploids. Conjugation is provoked by pheromones (a and a factors), secreted by cells of oppo- site mating types, that trigger a receptor-mediated sig- naling pathway (serpentine receptor-heterotrimeric G
... The S/G2 transition is facilitated by cyclin A-CDK2-mediated phosphorylation of DNA replication machinery components [16,17]. Finally, B-type cyclins and CDK1 orchestrate the G2/M phase transition [7,18,19]. Disruptions in cell cycle control mechanisms are a hallmark of cancer, including lung adenocarcinoma. Targeting these pathways offers a promising therapeutic strategy [8,11,12,17] (Figure 1). ...
... The S/G2 transition is facilitated by cyclin A-CDK diated phosphorylation of DNA replication machinery components [16,17]. Fina type cyclins and CDK1 orchestrate the G2/M phase transition [7,18,19]. Disruptions cycle control mechanisms are a hallmark of cancer, including lung adenocarcinom geting these pathways offers a promising therapeutic strategy [8,11,12,17] (Figure sides cell biological factors, external mechanical stimulation, such as local nanomech forces prevalent within the soft matter environment [20] of the tumor extracellular m is regulated by matrix stiffness and modulates PD-L1 expression through the act of the Yes-associated proteins to contribute to tumor cell proliferation [21]. Dysregulation of signaling pathways, in EGFR, PI3K/AKT/mTOR, RAS-MAPK, NTRK/ROS1, and cell cycling has been implicated cancer development and progression. ...
Lung adenocarcinoma (LUAD) is the most prevalent and aggressive subtype of lung cancer, exhibiting a dismal prognosis with a five-year survival rate below 5%. DEAD-box RNA helicase 18 (DDX18, gene symbol DDX18), a crucial regulator of RNA metabolism, has been implicated in various cellular processes, including cell cycle control and tumorigenesis. However, its role in LUAD pathogenesis remains elusive. This study demonstrates the significant upregulation of DDX18 in LUAD tissues and its association with poor patient survival (from public databases). Functional in vivo and in vitro assays revealed that DDX18 knockdown potently suppresses LUAD progression. RNA sequencing and chromatin immunoprecipitation experiments identified cyclin-dependent kinase 4 (CDK4), a cell cycle regulator, as a direct transcriptional target of DDX18. Notably, DDX18 depletion induced G1 cell cycle arrest, while its overexpression promoted cell cycle progression even in normal lung cells. Interestingly, while the oncogenic protein c-Myc bound to the DDX18 promoter, it did not influence its expression. Collectively, these findings establish DDX18 as a potential oncogene in LUAD, functioning through the CDK4-mediated cell cycle pathway. DDX18 may represent a promising therapeutic target for LUAD intervention.
... Although cells require nutrients and lipids for a successful cell division 1 , the role of lipids in the cell-cycle decision process is unknown. Traditionally, cell-cycle decision research has focused primarily on the role of mitogens and DNA damage [2][3][4] . ...
... Our study set out to determine if, similar to the DNA damage checkpoint, there is a lipid checkpoint that senses whether sufficient lipids are available for cells to progress through the cell cycle and when during the cell cycle such a lipid checkpoint engages. Regulatory signaling mechanisms are considered cell-cycle checkpoints if they (1) stop cell-cycle progression, (2) are rapidly induced, and (3) are reversed once optimal conditions are reached or the stress is resolved 17,18 . ...
Lipid synthesis increases during the cell cycle to ensure sufficient membrane mass, but how insufficient synthesis restricts cell-cycle entry is not understood. Here, we identify a lipid checkpoint in G1 phase of the mammalian cell cycle by using live single-cell imaging, lipidome, and transcriptome analysis of a non-transformed cell. We show that synthesis of fatty acids in G1 not only increases lipid mass but extensively shifts the lipid composition to unsaturated phospholipids and neutral lipids. Strikingly, acute lowering of lipid synthesis rapidly activates the PERK/ATF4 endoplasmic reticulum (ER) stress pathway that blocks cell-cycle entry by increasing p21 levels, decreasing Cyclin D levels, and suppressing Retinoblastoma protein phosphorylation. Together, our study identifies a rapid anticipatory ER lipid checkpoint in G1 that prevents cells from starting the cell cycle as long as lipid synthesis is low, thereby preventing mitotic defects, which are triggered by low lipid synthesis much later in mitosis.
... The cell cycle is the most fundamental molecular process of all life forms, orchestrating consecutive phases of cell growth (G1), DNA replication (S), DNA proofreading (G2), and cell division (mitosis, M) (Harper and Brooks, 2005). In mammalian cells, cell cycle progression is driven by cyclin-dependent kinases (CDKs) and their specific activators cyclins, while specialised molecular checkpoints ensure correct completion of each phase (Agarwal et al., 1995;Girard et al., 1991;Harper and Brooks, 2005;Lukas et al., 2004;Ohtsubo et al., 1995;Sherr, 1993Sherr, , 1994Sherr andRoberts, 1999, 2004;Vermeulen et al., 2003;Walker and Maller, 1991). If a problem occurs during replication, such as DNA damage, checkpoint activation serves to pause the cell cycle and repair the damage or, if not possible, to induce apoptosis (Agarwal et al., 1995;Awasthi et al., 2015;Lukas et al., 2004;Maréchal and Zou, 2013). ...
... The cell cycle is the most fundamental molecular process of all life forms, orchestrating consecutive phases of cell growth (G1), DNA replication (S), DNA proofreading (G2), and cell division (mitosis, M) (Harper and Brooks, 2005). In mammalian cells, cell cycle progression is driven by cyclin-dependent kinases (CDKs) and their specific activators cyclins, while specialised molecular checkpoints ensure correct completion of each phase (Agarwal et al., 1995;Girard et al., 1991;Harper and Brooks, 2005;Lukas et al., 2004;Ohtsubo et al., 1995;Sherr, 1993Sherr, , 1994Sherr andRoberts, 1999, 2004;Vermeulen et al., 2003;Walker and Maller, 1991). If a problem occurs during replication, such as DNA damage, checkpoint activation serves to pause the cell cycle and repair the damage or, if not possible, to induce apoptosis (Agarwal et al., 1995;Awasthi et al., 2015;Lukas et al., 2004;Maréchal and Zou, 2013). ...
Modulation of the host cell cycle is a common strategy used by viruses to create a pro-replicative environment. To facilitate viral genome replication, vaccinia virus (VACV) has been reported to alter cell cycle regulation and trigger the host cell DNA damage response. However, the cellular factors and viral effectors that mediate these changes remain unknown. Here, we set out to investigate the effect of VACV infection on cell proliferation and host cell cycle progression. Using a subset of VACV mutants we characterize the stage of infection required for inhibition of cell proliferation and define the viral effectors required to dysregulate the host cell cycle. Consistent with previous studies, we show that VACV inhibits, and subsequently shifts the host cell cycle. We demonstrate that these two phenomena are independent of one another, with viral early genes being responsible for cell cycle inhibition, and post-replicative viral gene(s) responsible for the cell cycle shift. Extending previous findings, we show that the viral kinase F10 is required to activate the DNA damage checkpoint and that the viral B1/B12 (pseudo) kinases mediate degradation of checkpoint effectors p53 and p21 during infection. We conclude that VACV modulates host cell proliferation and host cell cycle progression through temporal expression of multiple VACV effector proteins.
... The IR spectra secured the molecular structures of compounds 5a-d, revealing the absence of stretching absorption peaks of C-H sp 3 and nitrile functions (ESI, Fig. S4, S8, S11, and S17 †). The characteristic absorption peaks for NH 2 signals at d 9. 11, 9.48 (coumarin-H4), 10. 16,10.67 (thioamide NH), and 11. 52,13.04 ppm (amidic NH), respectively. ...
A series of novel coumarin–thiazoles was designed and synthesized as a possible CDK2 inhibitor with anticancer activity with low toxicity. The design relied on having hydrazine thiazole or its open-form thioamide to form H-bonds with the ATP binding site while coumarin maintained the crucial hydrophobic interactions for proper fitting. The biological evaluation revealed that the hydroxycoumarin-thiazole derivative 6c demonstrated the best inhibition with HepG2 and HCT116 IC50 2.6 and 3.5 μM, respectively. Similarly, its open thioamide chain congener 5c exhibited potent inhibition on MCF-7 and HepG2 with IC50 of 4.5 and 5.4 μM, respectively. Molecular docking simulations supported the assumption of inhibiting CDK2 by preserving the crucial interaction pattern with the hinge ATP site and the surrounding hydrophobic (HPO) side chains. Furthermore, molecular dynamics simulations of 5c and 6c established satisfactory stability and affinity within the CDK2 active site.
... suggesting novel gene functions within socket-building cells. Within subclusters i to k, we identified the 336 expression of cyclin genes CycE and CycD3, both being key factors in determining the transition from G1 337 to S, which supports the endocycling state of scale-building cells in these subclusters (Audibert et al., 338 2005;Lilly & Spradling, 1996;Richardson et al., 1995;Sherr, 1994). Subclusters m and n arranged in 339 ...
The success of butterflies and moths is tightly linked to the origin of scales within the group. A long-standing hypothesis postulates that scales are homologous to the well-described mechanosensory bristles found in the fruit fly Drosophila melanogaster , where both derive from an epithelial precursor specified by lateral inhibition that then undergoes multiple rounds of division. Previous histological examination and candidate gene approaches identified parallels in genes involved in scale and bristle development. Here, we provide definitive developmental and transcriptomic evidence that the differentiation of lepidopteran scales derives from the canonical cell lineage, known as the Sensory Organ Precursor (SOP). Live imaging in moth and butterfly pupae shows that SOP cells undergo two rounds of asymmetric divisions that first abrogate the neurogenic lineage, and then lead to a differentiated scale precursor and its associated socket cell. Single-nucleus RNA sequencing across a time-series of early pupal development revealed differential gene expression patterns that mirror canonical lineage development, including Notch-Delta signalling components, cell adhesion molecules, cell cycling factors, and terminal cell differentiation markers, suggesting a shared origin of the SOP developmental program. Additionally, we recovered a novel gene, the POU-domain transcription factor pdm3 , involved in the proper differentiation of butterfly wing scales. Altogether, these data open up avenues for understanding scale type specification and development, and illustrate how single-cell transcriptomics provide a powerful platform for understanding the evolution of cell types.
... During quiescence, non-phosphorylated Rb directly binds and inhibits E2F transcription factors, which are crucial regulators of cellcycle genes, thereby restraining cell-cycle entry 4 . In the canonical model of cell-cycle entry, mitogenic signaling increases cyclin D expression to activate CDK4/6 5 (Fig. 1a left). In turn, active CDK4/6 phosphorylates and inactivates Rb, resulting in E2F activation for cellcycle entry [6][7][8][9][10] . ...
Cyclin-dependent kinases 4 and 6 (CDK4/6) are critical for initiating cell proliferation by inactivating the retinoblastoma (Rb) protein. However, mammalian cells can bypass CDK4/6 for Rb inactivation. Here we show a non-canonical pathway for Rb inactivation and its interplay with external signals. We find that the non-phosphorylated Rb protein in quiescent cells is intrinsically unstable, offering an alternative mechanism for initiating E2F activity. Nevertheless, this pathway incompletely induces Rb-protein loss, resulting in minimal E2F activity. To trigger cell proliferation, upregulation of mitogenic signaling is required for stabilizing c-Myc, thereby augmenting E2F activity. Concurrently, stress signaling promotes Cip/Kip levels, competitively regulating cell proliferation with mitogenic signaling. In cancer, driver mutations elevate c-Myc levels, facilitating adaptation to CDK4/6 inhibitors. Differentiated cells, despite Rb-protein loss, maintain quiescence through the modulation of c-Myc and Cip/Kip levels. Our findings provide mechanistic insights into an alternative model of cell-cycle entry and the maintenance of quiescence.
... Este es un punto importante de revisión, ya que si las células presentan DNA dañado deben ser arrestadas en esta fase para que no se sintetice DNA dañado. La carga genética en humanos es diploide 3,4 . ...
Resumen El ciclo celular es un proceso en el cual una célula crece y se divide para crear una copia de sí misma, permitiendo crecer y reemplazar las células a medida que se desgastan. En los animales, el ciclo de una célula normal toma alrededor de 24 horas de principio a fin para los diferentes tipos de células, aunque algunas, como las de la piel o las tumorales, están constantemente pasando por este ciclo, mientras que otras pueden dividirse rara vez, o no hacerlo. La secuencia de eventos que se producen cuando se estimula una célula para crecer y dividirse constituye el ciclo celular. Inicia con células en reposo (fase G0), las cuales tienen que ser estimuladas por factores de crecimiento con el fin de entrar en el ciclo celular, lo que comienza con el primer período de crecimiento (fase G1) en el que se prepara para un período de síntesis de ADN (fase S). Hacia el final de G1, hay un punto de restricción (R), en que se repara el ADN en caso de estar dañado. De no ser así, sigue adelante el ciclo. Una vez que se han duplicado sus cromosomas, la célula entra a un segundo período de crecimiento (fase G2), cuando se prepara para dividirse en dos células hijas durante el período de la mitosis (fase M). Esta fase M se divide en una serie de pasos discretos que comienzan con la profase y luego pasan a través de la metafase, anafase, telofase y, finalmente, el proceso de la citocinesis, que divide la célula en dos iguales. AbstRAct The cell cycle is a process in which a cell grows and divides to create a copy of itself, allowing the cells to grow and replace as they wear out. In animals, the cycle of a normal cell takes about 24 hours from the beginning to the end for most cell types, although some, such as skin cells, or tumor cells, are constantly undergoing this cycle, while others can divide rarely or do not divide at all. The sequence of events that occur when a cell is stimulated to grow and divide is the cell cycle. It starts with resting cells (G0) that have to be stimulated by growth factors to enter the cell cycle, which begins with the first period of growth (G1) to be prepared for a period of DNA synthesis (S-phase). Towards the end of G1, there is a restriction point (R), in which DNA should be repaired if damaged, if not the cycle goes on. Once the cell has doubled its chromosomes, the cell goes into a second period of growth (G2), as it prepares to divide into two daughter cells during the mitosis (M phase). This phase M is separated into a series of discrete steps beginning with prophase and then pass through the metaphase, anaphase, telophase and, finally, the process of cytokinesis, then the cell divides into two equal.
... Its monomeric form needs to be activated by binding to cyclin E/A or phosphorylating its catalytic segment to initiate its kinase activity [34]. When CDK2 binds to cyclin A, it facilitates the S-phase advancement [35], while binding to cyclin E promotes the retinoblastoma protein phosphorylation to access the G1/S-phase [36]. CDK2 possesses four possible binding sites, one of which appears only during activation, which is accompanied by structural conformational alteration [37]. ...
... Both CDK4 and CDK6 organize in active enzymatic complexes together with cyclin D1, D2, or D3 [111] preferentially involved in Rb family members phosphorylation, thus favoring E2F release. Specifically, this transcription factor participates in G1 withdrawal and DNA duplication in mammalian cells. ...
The adult heart is made up of cardiomyocytes (CMs) that maintain pump function but are unable to divide and form new myocytes in response to myocardial injury. In contrast, the developmental cardiac tissue is made up of proliferative CMs that regenerate injured myocardium. In mammals, CMs during development are diploid and mononucleated. In response to cardiac maturation, CMs undergo polyploidization and binucleation associated with CM functional changes. The transition from mononucleation to binucleation coincides with unique metabolic changes and shift in energy generation. Recent studies provide evidence that metabolic reprogramming promotes CM cell cycle reentry and changes in ploidy and nucleation state in the heart that together enhances cardiac structure and function after injury. This review summarizes current literature regarding changes in CM ploidy and nucleation during development, maturation and in response to cardiac injury. Importantly, how metabolism affects CM fate transition between mononucleation and binucleation and its impact on cell cycle progression, proliferation and ability to regenerate the heart will be discussed.
... In the present study, it was found that cyclin B1 and cyclin E expression levels were upregulated in HepG2 cells following LSS knockdown. It is known that cyclin E promotes the transition from the G1 phase to the S phase, and that cyclin B1 undergoes dynamic changes throughout the cell cycle; a marked increase in the levels of these markers is observed when the cells enter the S phase (20)(21)(22). In the present study, following LSS loss of function in HepG2 cells, the expression of cyclin B1 and cyclin E increased, regulating the cell cycle in S phase, and leading to S phase arrest and the inhibition of cell proliferation. ...
Cholesterol is critical for tumor cells to maintain their membrane components, cell morphology and activity functions. The inhibition of the cholesterol pathway may be an efficient strategy with which to limit tumor growth and the metastatic process. In the present study, lanosterol synthase (LSS) was knocked down by transfecting LSS short hairpin RNA into HepG2 cells, and cell growth, apoptosis and migratory potential were then detected by Cell Counting Kit-8 cell proliferation assay, flow cytometric analysis and wound healing assay, respectively. In addition, proteins associated with the regulation of the aforementioned cell biological behaviors were analyzed by western blot analysis. The activity of the Src/MAPK signaling pathway was measured by western blotting to elucidate the possible signal transduction mechanisms. LSS knockdown in the HepG2 liver cancer cell line inhibited cell proliferation, with cell cycle arrest at the S phase; it also decreased cell migratory ability and increased apoptosis. The expression proteins involved in the regulation of cell cycle, cell apoptosis and migration was altered by LSS knockdown in HepG2 cells. Furthermore, a decreased Src/MAPK activity was observed in the HepG2 cells subjected to LSS knockdown. LSS loss of function decreased the malignant phenotypes of HepG2 cells by deactivating the Src/MAPK signaling pathway and regulating expression of genes involved in cell cycle regulation, cell apoptosis and migration.
... p21 and p27 belong to the kinase inhibitor protein (KIP) family. Further, the progression of the cell cycle is controlled by the cyclin-dependent kinases (CDKs) family and inhibited by CDK (CDK4 or INK4) and KIP inhibitors [15][16][17]. Therefore, we explored the effect of o-GQD on cell cycle arrest in the ER+ breast cancer cell line, MCF-7. ...
Graphene quantum dots (GQDs), nanomaterials derived from graphene and carbon dots, are highly stable, soluble, and have exceptional optical properties. Further, they have low toxicity and are excellent vehicles for carrying drugs or fluorescein dyes. Specific forms of GQDs can induce apoptosis and could be used to treat cancers. In this study, three forms of GQDs (GQD (nitrogen:carbon = 1:3), ortho-GQD, and meta-GQD) were screened and tested for their potential to inhibit breast cancer cell (MCF-7, BT-474, MDA-MB-231, and T-47D) growth. All three GQDs decreased cell viability after 72 h of treatment and specifically affected breast cancer cell proliferation. An assay for the expression of apoptotic proteins revealed that p21 and p27 were up-regulated (1.41-fold and 4.75-fold) after treatment. In particular, ortho-GQD-treated cells showed G2/M phase arrest. The GQDs specifically induced apoptosis in estrogen receptor-positive breast cancer cell lines. These results indicate that these GQDs induce apoptosis and G2/M cell cycle arrest in specific breast cancer subtypes and could potentially be used for treating breast cancers.
... An uncontrolled cell cycle that is observed in cancer cells is e caused by dysregulated growth signals, and as a consequence may affect other cellular characteristics such as cell survival [24]. As the G1 phase is a crucial phase during the cell cycle [30], unrestricted G1 to S phase transition in cancer cells will lead to accelerated cell maturation and differentiation [31]. ...
Breast carcinoma is the most prevalent cancer in women globally, with complex genetic and molecular mechanisms that underlie its development and progression. Several challenges such as metastasis and drug resistance limit the prognosis of breast cancer, and hence a constant search for better treatment regimes, including novel molecular therapeutic targets is necessary. Complement component 1, q subcomponent binding protein (C1QBP), a promising molecular target, has been implicated in breast carcinogenesis. In this study, the role of C1QBP in breast cancer progression, in particular cancer cell growth, was determined in triple negative MDA-MB-231 breast cancer cells. Depletion of C1QBP decreased cell proliferation, whereas the opposite effect was observed when C1QBP was overexpressed in MDA-MB-231 cells. Furthermore, gene expression profiling and pathway analysis in C1QBP depleted cells revealed that C1QBP regulates several signaling pathways crucial for cell growth and survival. Taken together, these findings provide a deeper comprehension of the role of C1QBP in triple negative breast cancer, and could possibly pave the way for future advancement of C1QBP-targeted breast cancer therapy.
... CDK4 and CDK6 belong to cycling-dependent kinase (CDK) complexes and promote G1 to G1/S phase conversion via retinoblastoma tumor suppressor protein (RB) phosphorylation. [7,8] RB has been reported to be a key negative regulator of the G1/S phase transition by interacting with and repressing E2F transcription factors. [9,10] When RB is phosphorylated by CDK, it becomes inactive rather than promote the transition. ...
Objective:
Revealing microfibrillar-associated protein 4 (MFAP4)'s function and its clinical significance in hepatocellular carcinoma (HCC).
Methods:
Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to evaluate MFAP4 mRNA and protein expression in paired HCC and paracarcinoma tissues, respectively. MFAP4 serum concentration was detected using enzyme-linked immunosorbent assays in healthy people (n = 30), cirrhosis (n = 15) and HCC patients (n = 80). MFAP4 protein expression was detected in two tissue microarrays (n = 60 and n = 90). Plasmids were transfected into human HCC cell line Bel-7402, and MFAP4 function was determined in vitro in cell experiments. Furthermore, tumorigenicity studies in nude mice served to assess the function of MFAP4 for HCC.
Results:
Both MFAP4 mRNA and protein expression were significantly downregulated in HCC tissue compared with paracarcinoma tissue (P < 0.05). Decreased MFAP4 expression in paracarcinoma tissue was associated with poor postoperative survival in HCC patients (P = 0.027). MFAP4 was also downregulated in HCC sera compared with healthy people (P < 0.05). In vitro, MFAP4 upregulation in Bel-7402 cells induced S phase arrest, promoted apoptosis, and inhibited migration and invasion. Western blotting indicated MFAP4 overexpression increased CDK4, CDK6, pRB, P27, and BCL-XS expression. Tumorigenicity study showed that the upregulation of MFAP4 inhibited the proliferation of Bel-7402 cells in nude mice.
Conclusions:
MFAP4 expression was significantly lower both in sera and tissue of HCC patients. MFAP4 can serve as molecular marker for HCC diagnosis and prognosis. Additionally, MFAP4 acted as an important HCC tumor suppressor by inducing S phase arrest, and promoting apoptosis, cell migration, and invasion.
... The second group of inhibitors consists of p27 Kip1 and p21 Cip1 (56). They only interact with cyclin-CDK complexes, not with CDKs alone. ...
The role of proteasomes in T cell activation, proliferation, and apoptosis was investigated using a proteasome-specific inhibitor lactacystin (LAC). Inhibition of the proteasome activity by LAC repressed the mitogen-induced T cell proliferation. The proteasome activity was definitively required for the T cells to progress from the G0 to S phase. It was necessary to optimize the progress from the G1/S boundary to the G2/M phase, but not for the progress from the G2/M phase to the next G1 phase. Probably as a result of a blockage of cell cycle progress, the cycling, but not the resting, T cells underwent apoptosis when treated with LAC. Mechanistically, we have found that cyclin-dependent kinase-2 (CDK2) and the cyclin E-associated kinase (largely CDK2), but not CDK4, in the G1 phase were strongly inhibited by LAC. This could be an important mechanism for the proteasome to regulate the cell cycle. The degradation of cyclin E in the late G1 and early S phases was dependent on the proteasome, although it was unlikely that this accounted for the observed inhibition of T cell proliferation. There was a reduced decay of p27Kip1 in the late G1 phase when the proteasome activity was suppressed, and this might be a contributing mechanism for the observed inhibition of CDK2 activity. Interestingly, p21Cip1 was up-regulated during the G1 phase, and the up-regulation was inhibited by LAC. Our study shows that the proteasome plays pivotal roles in regulating T cell activation and proliferation, and its effect is probably exerted through multiple mechanisms.
... CDK-2, a member of the CDKs, is activated by the formation of a complex with a cyclin and is required for G1 phase progression and entry into S phase [57][58][59] . The role of CDK-2 is investigated in the cellular differentiation of AML cells 60 , CDK-2 was specifically degraded upon the therapeutic differentiation of AML cells, and this depletion overcame the myeloid differentiation blockade of AML cells. ...
A series of benzo[h]chromenes, benzo[f]chromenes, and benzo[h]chromeno[2,3-d]pyrimidines were prepared. All the newly synthesised compounds were selected by National Cancer Institute for single-dose testing against 60 cell lines. Benzo[h]chromenes 5a and 6a showed promising anti-cancer activity and selected for the five-dose testing. Compounds 5a and 6a suppressed cell growth in HL-60 by the induction of cell cycle arrest, which was confirmed using flow cytometry and Annexin V-FITC/PI assays showed at the G1/S phase by regulating the expression of CDK-2/CyclinD1, triggering cell apoptosis by activating both the extrinsic (Fas/Caspase 8) and intrinsic (Bcl-2/Caspase 3) apoptosis pathways, which were determined by the western blot. Benzo[h]chromenes 5a and 6a decreased the protein expression levels of Bcl-2, CDK-2, and CyclinD1 and increased the expression of caspase 3, caspase 8, and Fas. In silico molecular analysis of compounds 5a and 6a in CDK-2 and Bcl-2 was performed.
... Expression of D-type cyclins is induced by extracellular mitogens and promotes G1 phase progression by activating cyclin-dependent kinases (CDK) 4/6 32 . Growing evidence has also indicated that both D1 and D3 cyclins can act as transcriptional regulators controlling developmental gene expression [33][34][35][36][37] . ...
Mammalian Müller glia express transcription factors and cell cycle regulators essential for the function of retinal progenitors, indicating the latent neurogenic capacity; however, the role of these regulators remains unclear. To gain insights into the role of these regulators in Müller glia, we analyzed expression of transcription factors (Pax6, Vsx2 and Nfia) and cell cycle regulators (cyclin D1 and D3) in rodent Müller glia, focusing on their age- and cell cycle-related expression patterns. Expression of Pax6, Vsx2, Nfia and cyclin D3, but not cyclin D1, increased in Müller glia during development. Photoreceptor injury induced cell cycle-associated increase of Vsx2 and cyclin D1, but not Pax6, Nfia, and cyclin D3. In dissociated cultures, cell cycle-associated increase of Pax6 and Vsx2 was observed in Müller glia from P10 mice but not from P21 mice. Nfia levels were highly correlated with EdU incorporation suggesting their activation during S phase progression. Cyclin D1 and D3 were transiently upregulated in G1 phase but downregulated after S phase entry. Our findings revealed previously unknown links between cell cycle progression and regulator protein expression, which likely affect the cell fate decision of proliferating Müller glia.
... 82 Furthermore, the genes associated with the progression of the G1 phase and the transition from the G1 to S phase have been found to be involved in various physio-pathological processes. 83 Therefore, 16 and 57 can be developed as chemotherapeutic anticancer drugs targeting the inhibition of cell cycle progression at the G1 phase. ...
Covering: up to the end of 2021Bacterial polycyclic xanthone natural products (BPXNPs) are a growing family of natural xanthones featuring a pentangular architecture with various modifications to the tricyclic xanthone chromophore. Their structural diversities and various activities have fueled biosynthetic and chemical synthetic studies. Moreover, their more potent activities than the clinically used drugs make them potential candidates for the treatment of diseases. Future unraveling of structure activity relationships (SARs) will provide new options for the (bio)-synthesis of drug analogues with higher activities. This review summarizes the isolation, structural elucidation and biological activities and more importantly, the recent strategies for the microbial biosynthesis and chemical synthesis of BPXNPs. Regarding their biosynthesis, we discuss the recent progress in enzymes that synthesize tricyclic xanthone, the protein candidates for structural moieties (methylene dioxygen bridge and nitrogen heterocycle), tailoring enzymes for methylation and halogenation. The chemical synthesis part summarizes the recent methodology for the division synthesis and coupling construction of achiral molecular skeletons. Ultimately, perspectives on the biosynthetic study of BPXNPs are discussed.
... In addition to the first discovered protein, cdc2, other CDK family members and their natural binding partners, the cyclins, were uncovered and demonstrated to regulate cell cycle checkpoints such as G1-S and G2-M [14]. These checkpoints turned out to be critically important for normal cell proliferation and gained significant attention for their role in cancer [22][23][24]. ...
Glioma stem cells (GSCs) are thought to drive growth and therapy resistance in glioblastoma (GBM) by “hijacking” at least a subset of signaling pathways active in normal neural stem cells (NSCs). Though the origins of GSCs still remain elusive, uncovering the mechanisms of self-renewing division and cell differentiation in normal NSCs has shed light on their dysfunction in GSCs. However, the distinction between self-renewing division pathways utilized by NSC and GSC becomes critical when considering options for therapeutically targeting signaling pathways that are specifically active or altered in GSCs. It is well-established that cyclin-dependent kinases (CDKs) regulate the cell cycle, yet more recent studies have shown that CDKs also play important roles in the regulation of neuronal survival, metabolism, differentiation, and self-renewal. The intimate relationship between cell cycle regulation and the cellular programs that determine self-renewing division versus cell differentiation is only beginning to be understood, yet seems to suggest potential differential vulnerabilities in GSCs. In this timely review, we focus on the role of CDKs in regulating the self-renewal properties of normal NSCs and GSCs, highlighting novel opportunities to therapeutically target self-renewing signaling pathways specifically in GBM.
... The mechanism through which iron enhances Cyclin D1 is not known yet. Studies proved that increased Cyclin D1 that assembles with cdk4 or cdk6 to promote cells from G1 to S phase plays a major role in tumorigenesis [56,57]. In their studies they showed that iron depletion via iron chelators inhibited tumor growth by targeting cyclin D1 through ubiquitinindependent proteasomal degradation [58]. ...
Iron is the most abundant metal in the human body. No independent life forms on earth can survive without iron. However, excess iron is closely associated with carcinogenesis by increasing oxidative stress via its catalytic activity to generate hydroxyl radicals. Therefore, it is speculated that iron might play a dual role in cells, by both stimulating cell growth and causing cell death. Dietary iron is absorbed by the intestinal enterocytes in the form of ferrous ion which forms cLIP. Excess iron stored in the form of Ferritin serves as a reservoir under iron depletion conditions. Ferroptosis, is an iron-dependent non-mutational form of cell death process and is suppressed by iron-binding compounds such as deferoxamine. Blocking transferrin-mediated iron import or recycling of iron-containing storage proteins (i.e., ferritin) also attenuates ferroptosis, consistent with the iron-dependent nature of this process. Unsurprisingly, ferroptosis also plays a role in the development of cancer and maybe a beneficial strategy for an-ticancer treatment. Different lines of evidence suggest that ferroptosis plays a crucial role in the suppression of tumorigenesis. In this review, we have discussed the pros and cons of iron accumulation, utilization and, its role in cell proliferation, ferroptosis and pathophysiology of cancer.
... Inactivation of Rb triggers the activation of E2F transcription factors, which in turn regulate the expression of genes whose products drive cell cycle progression. Among the E2F-responsive genes are also CCNE and CCNA, which combine with and activate CDK2 to facilitate S phase entry and progression (reviewed in Sherr, 1994Sherr, , 1996. The CCN/CDK complexes are in turn inhibited by a family of CDKs inhibitors, such as p27 kip1 (CDKN1B) (reviewed in Besson et al., 2008). ...
The aims of the present study were to investigate the expression of the classic estrogen receptors ESR1 and ESR2, the splicing variant ESR1-36 and GPER in human testicular embryonal carcinoma NT2/D1 cells, and the effects of the activation of the ESR1 and ESR2 on cell proliferation. Immunostaining of ESR1, ESR2, and GPER were predominantly found in the nuclei, and less abundant in the cytoplasm. ESR1-36 isoform was predominantly expressed in the perinuclear region and cytoplasm, and some weakly immunostained in the nuclei. In nonstimulated NT2/D1 cells (control), proteins of the cell cycle CCND1, CCND2, CCNE1 and CDKN1B are present. Activation of ESR1 and ESR2 increases, respectively, CCND2 and CCNE1 expression, but not CCND1. Activation of ESR2 also mediates upregulation of the cell cycle inhibitor CDKN1B. This protein co-immunoprecipitated with CCND2. Also, E2 induces an increase in the number and viability of the NT2/D1 cells. These effects are blocked by simultaneous pretreatment with ESR1-and ESR2-selective antagonists, confirming that both estrogen receptors regulate NT2/D1 cell proliferation. In addition, E2 increases SRC phosphorylation, and SRC mediates cell proliferation. Our study provides novel insights into the signatures and molecular mechanisms of estrogen receptor in NT2/D1 cells.
... The cell cycle in most eukaryotic cells is a complex process, including a series of coordinated events, namely, preparation for cell growth (G1 phase), the replication of genetic material (S phase), the segregation of duplicated chromosomes (G2 phase) and cell division (M phase), which is regulated by cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors [23]. The three D type cyclins (CCND1, CCND2 and CCND3) that modulate the cell cycle by engaging and activating CDKs, such as CDK4 and CDK6 and CDK-cyclin D complexes, are essential for G1 entrance [24]. Cyclin A combines with CDK1 in the late G2 and early M phase, and its protein levels stay constant throughout the cell cycle. ...
The Yangtze River Delta white goat is a rare goat species capable of producing high-quality brush hair. Dual specificity protein phosphatase 1 (DUSP1) may play a role in the formation of high-quality brush hair, as evidenced by our previous research. We investigated the potential mechanisms that regulate the proliferation and apoptosis of goat hair follicle stem cells. We particularly focused on the relationship between DUSP1 and miR-101, which directly targets DUSP1, predicted and screened through bioinformatics websites. Then, fluorescence assays, flow cytometry, RT-qPCR, and Western blotting were used to investigate the effects of miR-101 on the proliferation and apoptosis of hair follicle stem cells. We found that miR-101 overexpression significantly decreased (p < 0.01) apoptosis and promoted the proliferation of hair follicle stem cells. Furthermore, the overexpression of miR-101 increased (p < 0.05) the mRNA and protein expression levels of the proliferation-related gene (PCNA) and anti-apoptotic gene (Bcl-2), and it decreased (p < 0.05) the mRNA and protein expression levels of the apoptotic gene (Bax). In conclusion, miR-101 can promote the proliferation of and inhibit the apoptosis of hair follicle stem cells by targeting DUSP1, which provides a theoretical basis for further elucidating the molecular mechanism that regulates the production of high-quality brush hair of Yangtze River Delta white goats.
... To further explore these findings, the levels of cyclin D1, p27, and p21, as well as the phosphorylation of their downstream protein Rb, were determined using Western blotting after treatment with 5, 10, and 20 μM amlodipine for 48 h. Cyclin D-cyclin dependent kinase (CDK) 4/6 complex phosphorylates Rb to facilitate cell cycle progression from G1 to S phase (Sherr, 1994;Shapiro, 2006). As shown in Figure 1E, the expression of cyclin D1 and level of p-Rb were decreased, while the expression levels of p27 and p21 (CDK inhibitors) were increased in a dosedependent manner, compared with the non-treated group. ...
Amlodipine is a Ca2+ channel blocker commonly used to cardiovascular diseases such as hypertension and angina; however, its anticancer effects in lung cancer A549 cells remain unknown. In the present study, we explored the antitumor effects and molecular mechanisms underlying the action of amlodipine in non-small cell lung cancer (NSCLC) A549 cells in vitro and in vivo. We observed that amlodipine suppressed the proliferation of A549 lung cancer cells by arresting the tumor cell cycle. Mechanistically, our results revealed that amlodipine could attenuate the phosphoinositide 3 kinase (PI3K)/Akt and Raf/MEK/extracellular signal-regulated kinase (ERK) pathways through epidermal growth factor receptor (EGFR) and modulated cell cycle-related proteins such as cyclin D1, p-Rb, p27, and p21. Subsequently, amlodipine combined with gefitinib could synergistically inhibit cell proliferation by arresting the cell cycle. Moreover, amlodipine combined with gefitinib effectively attenuated the growth of A549 lung cancer xenografts when compared with monotherapy, affording an excellent therapeutic effect. Collectively, our results indicate that amlodipine alone or combined with the novel anticancer drug gefitinib might be a potential therapeutic strategy for NSCLC patients with wild-type EGFR.
... When cyclin D binds to CDK4/6 [17,18], the activated cyclin D/CDK4/6 complex, in turn, activates retinoblastoma (RB) protein [19]. Cyclin E binds to CDK2 following activation of cyclin D/CDK; when the cyclin E/CDK2 complex is activated, it promotes the G1 phase to the S phase [20,21]. After the cell enters the S phase, cyclin E is decomposed. ...
Background:
Desmodium gangeticum (L.)DC., which belongs to the Leguminosae family, has been used in Taiwan and other subtropical countries as an external medicine to remove blood stasis, activate blood circulation, and reduce inflammation. It has been reported to have antioxidant effects and improve inflammatory responses in rats stimulated by pro-inflammatory agents and induced gastric ulcers in experimental animals over the past few decades. This plant has also been used to treat parasitic infections, but there are no reports regarding its effects on lung cancer. Therefore, this study attempted to investigate its water crude extract (in abbreviation DG) on lung cancer cells.
Methods:
A549 human lung cancer cells were tested for survival using MTT, trypan blue, and propidium iodide. The effects of various concentrations of the crude extract of D. gangeticum (DG) (0.125~1 mg/ml) on the cell cycle and apoptosis of A549 cells were analyzed by flow cytometry and Western blotting methods.
Results:
DG can inhibit the growth of A549 human lung cancer cells in a concentration- and time-dependent manner. DG arrested A549 cells in the G1 phase by increasing the proteins expression of p21, p27, cyclin D1, and cyclin E. Additionally, DG decreased the expression of cyclin A, B1, and Cdc 2 (CDK1) proteins.
Conclusions:
DG demonstrated the anti-lung cancer activity by arresting the cell cycle in G1 via increasing the p21, p27, cyclin D1, cyclin E, and decreasing Cdc2, cyclin A, and B1 proteins expression in A549 human lung cancer cells.
... Hence, we supposed that MCGP could also promote liver regeneration through the activation of the Nrf2 signaling pathway. PCNA and HNF4A were reported to be critical on regulating liver regeneration (Sherr 1994;Lee et al., 2020). CSF1R and its ligand colony stimulating factor 1 (CSF1) regulate the proliferation, differentiation, and function of macrophages, including Kupffer cells (Santamaria-Barria et al., 2019). ...
Excessive stimulation of hepatotoxins and drugs often lead to acute liver injury, while treatment strategies for acute liver injury have been limited. Methyl 6-O-cinnamoyl-α-d-glucopyranoside (MCGP) is a structure modified compound from cinnamic acid, a key chemical found in plants with significant antioxidant, anti-inflammatory, and antidiabetic effects. In this study, we investigated the effects and underlying mechanisms of MCGP on acetaminophen (APAP)- or carbon tetrachloride (CCl4)-induced acute liver injury. As a result, MCGP inhibited cell death and apoptosis induced by APAP or CCl4, and suppressed the reactive oxygen species (ROS) generation stimulated by H2O2 in liver AML12 cells. In vivo, MCGP alleviated APAP/CCl4-induced hepatic necrosis and resumed abnormal aminotransferase activities and liver antioxidase activities. In addition, MCGP depressed APAP- or CCl4-induced oxidative stress through the suppression of CYP2E1 and activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. MCGP also enhanced the number of PCNA-positive hepatocytes, increased hepatic PCNA and Bcl-XL, and decreased BAX expression in APAP-/CCl4-intoxicated mice. Furthermore, MCGP activated the GSDMD-N/cleaved caspase 1 pathway. In summary, MCGP might act as a potential therapeutic drug against drug-induced and chemical-induced acute liver injuries, and its underlying mechanisms might engage on the pressing of oxidative stress, refraining of hepatocyte apoptosis, and facilitating of liver regeneration.
... Overexpression of E2F results in the cyclin D1 inhibition, kinase activity, and the kinase inhibitor 2A (CDKN2A, p16) gene product induction and overexpression [120][121][122]. With an association with Bax and Bak, E6 also interferes with the ongoing apoptotic processes [123,124]. ...
Cervical cancer is an aggressive type of cancer affecting women worldwide. Many affected individuals rely on smear tests for the diagnosis, surgery, chemotherapy, or radiation for their treatment. However, due to a broad set of undesired results and side-effects associated with the existing protocols, the search for better diagnostic and therapeutic interventions is a never-ending pursuit. In the purview, the bio-concentration of trace elements (copper, selenium, zinc, iron, arsenic, manganese, and cadmium) is seen to fluctuate during the occurrence of cervical cancer and its progression from pre-cancerous to metastatic nature. Thus, during the occurrence of cervical cancer, the detection of trace elements and their supplementation will prove to be highly advantageous in developing diagnostic tools and therapeutics, respectively. This review provides a detailed overview of cervical cancer, its encouragement by human papillomavirus infections, the mechanism of pathology, and resistance. Majorly, the review emphasizes the less explored role of trace elements, their contribution to the growth and inhibition of cervical cancer. Numerous clinical trials have been listed, thereby providing a comprehensive reference to the exploration of trace elements in the management of cervical cancer.
... CCND1 is located on the long arm of chromosome 11 at 11q13.3 and encodes cyclin D1 protein [3]. Cyclin D1 is involved in cell cycle progression by inducing G1-S transition through activation of cyclin-dependent kinases, Cdk4 and Cdk6 [4,5]. Cyclin D1 may also impact steroid hormone receptors, activating the oestrogen receptor (ER) [6,7], and inhibiting the androgen receptor in breast epithelium [8]. ...
CCND1 is located on 11q13. Increased CCND1 copy number (CN) in breast cancer (BC) is associated with high histopathological grade, high proliferation, and Luminal B subtype. In this study of CCND1 in primary BCs and corresponding axillary lymph node metastases (LNM),we examine associations between CCND1 CN in primary BCs and proliferation status, molecular subtype, and prognosis. Furthermore, we studied associations between CCND1 CN and CNs of FGFR1 and ZNF703, both of which are located on 8p12. Fluorescence in situ hybridization probes for CCND1 and chromosome 11 centromere were used on tissue microarrays comprising 526 BCs and 123 LNM. We assessed associations between CCND1 CN and tumour characteristics using Pearson’s χ² test, and estimated cumulative risks of death from BC and hazard ratios in analysis of prognosis. We found CCND1 CN ≥ 4 < 6 in 45 (8.6%) tumours, and ≥ 6 in 42 (8.0%). CCND1 CN (≥ 6) was seen in all molecular subtypes, most frequently in Luminal B (HER2⁻) (20/126; 16%). Increased CCND1 CN was associated with high histopathological grade, high Ki-67, and high mitotic count, but not prognosis. CCND1 CN ≥ 6 was accompanied by CN increase of FGFR1 in 6/40 cases (15.0%) and ZNF703 in 5/38 cases (13.2%). Three cases showed CN increase of all three genes. High CCND1 CN was most frequent in Luminal B (HER2⁻) tumours. Good correlation between CCND1 CNs in BCs and LNM was observed. Despite associations between high CCND1 CN and aggressive tumour characteristics, the prognostic impact of CCND1 CN remains unresolved.
... The mechanism through which iron enhances Cyclin D1 is not known yet. Studies proved that increased Cyclin D1 that assembles with cdk4 or cdk6 to promote cells from G1 to S phase plays a major role in tumorigenesis [56,57]. In their studies they showed that iron depletion via iron chelators inhibited tumor growth by targeting cyclin D1 through ubiquitin-independent proteasomal degradation [58]. ...
Iron is the most abundant metal in the human body. No independent life forms on earth can survive without iron. However, excess iron is closely associated with carcinogenesis by increasing oxidative stress via its catalytic activity to generate hydroxyl radicals. Therefore, it is speculated that iron might play a dual role in cells, by both stimulating cell growth and causing cell death. Dietary iron is absorbed by the intestinal enterocytes in the form of ferrous ion which forms cLIP. Excess iron stored in the form of Ferritin serves as a reservoir under iron depletion conditions. Ferroptosis, is an iron-dependent non-mutational form of cell death process and is suppressed by iron-binding compounds such as deferoxamine. Blocking transferrin-mediated iron import or recycling of iron-containing storage proteins (i.e., ferritin) also attenuates ferroptosis, consistent with the iron-dependent nature of this process. Unsurprisingly, ferroptosis also plays a role in the development of cancer and maybe a beneficial strategy for anticancer treatment. Different lines of evidence suggest that ferroptosis plays a crucial role in the suppression of tumorigenesis. In this review, we have discussed the pros and cons of iron accumulation, utilization and, its role in cell proliferation, ferroptosis and pathophysiology of cancer.
... INTRODUCTION D-type cyclins play a pivotal role in regulating the G1-S phase transition of the cell cycle as regulatory subunits of Cyclin-Dependent Kinases 4 and 6 (CDK4/6) which phosphorylate tumor suppressor retinoblastoma protein (Rb) [1][2][3]. Phosphorylated Rb dissociates from the E2f transcription factor permitting expression of genes necessary for S phase progression. CCND1 transcription depends on Ras-MAP kinase signaling [4,5] and likewise protein accumulation is also regulated via Ras-dependent mechanisms that control the rapid, ubiquitin-mediated protein degradation at the G1/S phase boundary [6]. ...
Cyclin D1 is a regulatory subunit of -Cyclin Dependent Kinases 4 and 6 (CDK4/6) and regulates progression from G1 to S phase of the cell cycle. Dysregulated cyclin D1-CDK4/6 contributes to abnormal cell proliferation and tumor development. Phosphorylation of threonine 286 of cyclin D1 is necessary for ubiquitin-dependent degradation. Non-phosphorylatable cyclin D1 mutants are stabilized and concentrated in the nucleus, contributing to genomic instability and tumor development. Studies investigating the tumor-promoting functions of cyclin D1 mutants have focused on the use of artificial promoters to drive the expression which unfortunately may not accurately reflect tumorigenic functions of mutant cyclin D1 in cancer development. We have generated a conditional knock-in mouse model where cyclin D1T286A is expressed under the control of its endogenous promoter following Cre-dependent excision of a lox-stop-lox sequence. Acute expression of cyclin D1T286A following tamoxifen-inducible Cre recombinase triggers inflammation, lymphocyte abnormality and ultimately mesenteric tumors in the intestine. Tissue-specific expression of cyclin D1T286A in the uterus and endometrium cooperates with Pten loss to drive endometrial hyperplasia and cancer. Mechanistically, cyclin D1T286A mutant activates NF-κB signaling, augments inflammation, and contributes to tumor development. These results indicate that mutation of cyclin D1 at threonine 286 has a critical role in regulating inflammation and tumor development.
Background:
Cyclin-dependent kinase (CDK) 7 is aberrantly overexpressed in many types of cancer and is an attractive target for cancer therapy due to its dual role in transcription and cell cycle progression. Moreover, CDK7 can directly modulate the activities of estrogen receptor (ER), which is a major driver in breast cancer. Breast cancer cells have exhibited high sensitivity to CDK7 inhibition in pre-clinical studies.
Methods:
In this review, we provide a comprehensive summary of the latest insights into CDK7 biology and recent advancements in CDK7 inhibitor development for breast cancer treatment. We also discuss the current application of CDK7 inhibitors in different molecular types of breast cancer to provide potential strategies for the treatment of breast cancer.
Results:
Significant progress has been made in the development of selective CDK7 inhibitors, which show efficacy in both triple-negative breast cancer (TNBC) and hormone receptor-positive breast cancer (HR+). Moreover, combined with other agents, CDK7 inhibitors may provide synergistic effects for endocrine therapy and chemotherapy. Thus, high-quality studies for developing potent CDK7 inhibitors and investigating their applications in breast cancer therapy are rapidly emerging.
Conclusion:
CDK7 inhibitors have emerged as a promising therapeutic strategy and have demonstrated significant anti-cancer activity in different subtypes of breast cancer, especially those that have been resistant to current therapies.
Ovarian clear-cell cancer is a rare subtype of epithelial ovarian cancer with unique clinical and biological features. Despite optimal cytoreductive surgery and platinum-based chemotherapy being the standard of care, most patients experience drug resistance and a poor prognosis. Therefore, novel therapeutic approaches have been developed, including immune checkpoint blockade, angiogenesis-targeted therapy, ARID1A synthetic lethal interactions, targeting hepatocyte nuclear factor 1β, and ferroptosis. Refining predictive biomarkers can lead to more personalized medicine, identifying patients who would benefit from chemotherapy, targeted therapy, or immunotherapy. Collaboration between academic research groups is crucial for developing prognostic outcomes and conducting clinical trials to advance treatment for ovarian clear-cell cancer. Immediate progress is essential, and research efforts should prioritize the development of more effective therapeutic strategies to benefit all patients.
Purpose Esophageal squamous cell carcinoma (ESCC) is a highly fatal malignant tumor of the digestive tract. Although the relationship between the bacterial genus Prevotella and ESCC has attracted attention recently, its specific role and molecular mechanisms remain unclear.
Methods In this study, we employed 16S rDNA sequencing technology to analyze the differences in Prevotella in 25 cases of esophageal squamous cell carcinoma (ESCC) and adjacent tissues (3 cm from the lesion). Subsequently, we co-cultured Prevotella with ESCC cells Eca109 and TE- 1, and through CCK8 assays, colony formation assays, and scratch assays, we investigated the influence of Prevotella on the proliferation and migration abilities of ESCC cells. Utilizing qT-PCR and protein immunoblot assays to examine the expression of proteins related to epithelial-mesenchymal transition (EMT) and NF-κB.
Results The relative abundance of Prevotella was markedly elevated in ESCC tissues as compared to adjacent non-tumor tissues. Upon infection, we observed a significant enhancement in the proliferation and migration capacities of ESCC cells. Besides, the stimulation by Prevotella led to a significant upregulation in the expression of essential EMT-associated proteins, including N-cadherin, Matrix metalloproteinase-9 (MMP9), and Vimentin, in ESCC cells, with a concurrent downregulation of E-cadherin. Quantitative PCR and Western blot analysis further indicated that the mRNA levels of NF-κB and c-Myc and the protein expression of Bcl-xl and NF-κB were significant upregulation in ESCC cells by stimulating with Prevotella.
Conclusion Our research unveiled the pivotal role of Prevotella in the genesis and advancement of ESCC, proposing its influence on ESCC proliferation, migration, and EMT process via the NF-κB pathway, Our results offered a novel avenue for delving into the microbial mechanisms underpinning esophageal cancer.
Objective:
Acetaminophen (APAP) is one of the world's popular and safe painkillers, and overdose can cause severe liver damage and even acute liver failure. The effect and mechanism of the xanthohumol on acetaminophen-induced hepatotoxicity remains unclear.
Methods:
The hepatoprotective effects of xanthohumol were studied using APAP-induced HepG2 cells and acute liver injury of mouse, seperately.
Results:
In vitro, xanthohumol inhibited H2O2- and acetaminophen-induced cytotoxicity and oxidative stress. Xanthohumol up-regulated the expression of Nrf2. Further mechanistic studies showed that xanthohumol triggered Nrf2 activation via the AMPK/Akt/GSK3β pathway to exert a cytoprotective effect. In vivo, xanthohumol significantly ameliorated acetaminophen-induced mortality, the elevation of ALT and AST, GSH depletion, MDA formation and histopathological changes. Xanthohumol effectively suppressed the phosphorylation and mitochondrial translocation of JNK, mitochondrial translocation of Bax, the activation o cytochrome c, AIF secretion and Caspase-3. In vivo, xanthohumol increased Nrf2 nuclear transcription and AMPK, Akt and GSK3β phosphorylation in vivo. In addition, whether xanthohumol protected against acetaminophen-induced liver injury in Nrf2 knockout mice has not been illustated.
Conclusion:
Thus, xanthohumol exerted a hepatoprotective effect by inhibiting oxidative stress and mitochondrial dysfunction through the AMPK/Akt/GSK3β/Nrf2 antioxidant pathway.
CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.
Plant growth and development rely heavily on cyclins, which compose an important class of cell division regulators. D-type cyclins (CYCDs) are responsible for the rate-limiting step of G1 cells. In the plant kingdom, despite the importance of CYCDs in herbaceous plants, there is little knowledge of these proteins in perennial woody plants. Here, a nucleus-localized cyclin gene, PsnCYCD1;1, was cloned from Populus. PsnCYCD1;1 was highly expressed in tissues with active cell division, especially the leaf buds, and could be induced by sucrose and phytohormones. Moreover, overexpression of PsnCYCD1;1 in poplar could stimulate cell division, resulting in the generation of small cells and causing severe morphological changes in the vascular bundles, resulting in "S"-shaped tortuous stems and curled leaves. Furthermore, transcriptomic analysis revealed that endogenous genes related to cell division and vascular cambium development were significantly upregulated in the transgenic plants. In addition, PsnCDKA1, PsnICK3 and PsnICK5 were identified as interacting proteins of PsnCYCD1;1 according to yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Our study demonstrates that PsnCYCD1;1 accelerates plant cell division and participates in secondary growth of vascular bundles in poplar.
Today, treatment options for cancer patients typically include surgery, radiation therapy, immunotherapy, and chemotherapy. While these therapies have saved lives and reduced pain and suffering, cancer still takes millions of lives every year around the world. Researchers are now developing advanced therapeutic strategies such as immunotherapy, targeted therapy, and combination nanotechnology for drug delivery. In addition, the identification of new biomarkers will potentiate early-stage diagnosis. Molecular Targets and Cancer presents information about cancer diagnosis and therapy in a simple way. It covers several aspects of the topic with updated information on par with medical board levels. The book features contributions from experts and includes an overview of cancer from basic biology and pathology, classifications, surveillance, prevention, diagnosis, types of cancer, treatment and prognosis. The first part of this book introduces the reader to cancer epidemiology, genetic alterations in cancer, exogenous and endogenous factors in carcinogenesis, roles for growth factors in cancer progression, cell signaling in cancer, transcription factors in cancer, and cancer genetics and epigenetics. This comprehensive guide is a valuable resource for oncologists, researchers, and all medical professionals who work in cancer care and research.
Organ fibrosis represents a dysregulated, maladaptive wound repair response that results in progressive disruption of normal tissue architecture leading to detrimental deterioration in physiological function, and significant morbidity/mortality. Fibrosis is thought to contribute to nearly 50% of all deaths in the Western world with current treatment modalities effective in slowing disease progression but not effective in restoring organ function or reversing fibrotic changes. When physiological wound repair is complete, myofibroblasts are programmed to undergo cell death and self-clearance, however, in fibrosis there is a characteristic absence of myofibroblast apoptosis. It has been shown that in fibrosis, myofibroblasts adopt an apoptotic-resistant, highly proliferative phenotype leading to persistent myofibroblast activation and perpetuation of the fibrotic disease process. Recently, this pathological adaptation has been linked to dysregulated expression of tumour suppressor gene p53. In this review, we discuss p53 dysregulation and apoptotic failure in myofibroblasts and demonstrate its consistent link to fibrotic disease development in all types of organ fibrosis. An enhanced understanding of the role of p53 dysregulation and myofibroblast apoptosis may aid in future novel therapeutic and/or diagnostic strategies in organ fibrosis.
The prenylated flavonoid icaritin (ICT, 1), a new drug for treating advanced hepatocellular carcinoma (HCC), was selected as a template to develop more potent inhibitors. An initial semisynthetic modification of ICT was performed to obtain a structure-activity relationship (SAR), which indicated that the cytotoxicity is enhanced by OH-3 rhamnosylation and that OH-7 is an important modification site. Based on the results of the SAR study, 46 N-containing ICT derivatives were synthesized and evaluated as the anti-HCC inhibitors. The results showed that most of the derivatives produced inhibited three HCC cell lines used (Hep3B, HepG2 and SMMC-7721). The modification strategy was validated by 3D-QSAR, which provided information for the further design and optimization of ICT. The most potent compound, 11c, exhibited IC50 values of 7.6 and 3.1 μM against HepG2 and SMMC-7721 cells, respectively, which were more potent than those of ICT and sorafenib, respectively. Further mechanistic studies indicated that 11c caused arrest at the G0/G1 phase in the cell cycle and induced cell apoptosis in HepG2 and SMMC-7721 cells.
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder that can lead to severe joint damage and is often associated with a high morbidity and disability. Disease-modifying anti-rheumatic drugs (DMARDs) are the mainstay of treatment in RA. DMARDs not only relieve the clinical signs and symptoms of RA but also inhibit the radiographic progression of disease and reduce the effects of chronic systemic inflammation. Since the introduction of biologic DMARDs in the late 1990s, the therapeutic range of options for the management of RA has significantly expanded. However, patients’ response to these agents is not uniform with considerable variability in both efficacy and toxicity. There are no reliable means of predicting an individual patient’s response to a given DMARD prior to initiation of therapy. In this chapter, the current published literature on the pharmacogenetics of traditional DMARDS and the newer biologic DMARDs in RA is highlighted. Pharmacogenetics may help individualize drug therapy in patients with RA by providing reliable biomarkers to predict medication toxicity and efficacy.Key wordsPharmacogeneticsPolymorphismsRheumatoid arthritisMethotrexateAzathioprineSulfasalazineTumor necrosis factor antagonistsRituximabTocilizumab
Drug addiction is a serious relapsing disease that has high costs to society and to the individual addicts. Treatment of these addictions is still in its nascency, with only a few examples of successful therapies. Therapeutic response depends upon genetic, biological, social, and environmental components. A role for genetic makeup in the response to treatment has been shown for several addiction pharmacotherapies with response to treatment based on individual genetic makeup. In this chapter, we will discuss the role of genetics in pharmacotherapies, specifically for cocaine, alcohol, and opioid dependences. The continued elucidation of the role of genetics should aid in the development of new treatments and increase the efficacy of existing treatments.Key wordsGeneAlcoholCocaineOpioidAddictionDependenceAbuseDrugTherapyPolymorphism
Nuclear receptor pregnane X receptor (PXR) can induce significant liver enlargement through hepatocyte hypertrophy and proliferation. A previous report showed that during the process of PXR-induced liver enlargement, hepatocyte hypertrophy occurs around the central vein (CV) area while hepatocyte proliferation occurs around the portal vein (PV) area. However, the features of this spatial change remain unclear. Therefore, this study aims to explore the features of the spatial changes in hepatocytes in PXR-induced liver enlargement. PXR-induced spatial changes in hepatocyte hypertrophy and proliferation were confirmed in C57BL/6 mice. The liver was perfused with digitonin to destroy the hepatocytes around the CV or PV areas, and then the regional expression of proteins related to hepatocyte hypertrophy and proliferation was further measured. The results showed that the expression of PXR downstream proteins, such as cytochrome P450 (CYP) 3A11, CYP2B10, P-glycoprotein (P-gp) and organ anion transporting polypeptide 2 (OATP2) was upregulated around the CV area, while the expression of proliferation-related proteins such as cyclin B1 (CCNB1), cyclin D1 (CCND1) and serine/threonine NIMA-related kinase 2 (NEK2) was upregulated around the PV area. At the same time, the expression of cyclin-dependent kinase inhibitors such as retinoblastoma-like protein 2 (RBL2), cyclin-dependent kinase inhibitor 1B (CDKN1B) and CDKN1A was downregulated around the PV area. This study demonstrated that the spatial change in PXR-induced hepatocyte hypertrophy and proliferation is associated with the regional expression of PXR downstream targets and proliferation-related proteins and the regional distribution of triglycerides (TGs). These findings provide new insight into the understanding of PXR-induced hepatomegaly.
The cyclins and cyclin-dependent kinases (CDKs) are two large protein families that function in a wide variety of cellular contexts. Most notably, the cyclin-CDKs govern and orchestrate progression through the eukaryotic cell cycle. In a sequential fashion, different cyclin-CDK complexes execute the signaling, transcriptional, and enzymatic events that effect transitions through and between cell cycle phases. As such, cyclin-CDKs are intricately regulated through complex formation, phosphorylation, stability, subcellular localization, and association with CDK inhibitors. Given their connection to cellular proliferation, cyclin-CDKs are poised at the nexus of oncogenes and tumor suppressors and have a unique role in the pathogenesis of cancer and developmental disorders.
Synchronization of ovary development is the premise for realizing large-scale seed breeding of small- and medium-sized shrimps. To explore the genetic mechanism that determines ovarian development in the ridgetail white prawn Exopalaemon carinicauda, we successfully cloned the full-length cDNA sequence of the cdk2 gene of E. carinicauda. The open reading frame (ORF) of cdk2 is 918 bp, which encodes 305 amino acids with a conserved cyclin binding motif S_TKc. The phylogenetic analysis showed that cdk2 has the highest homology with the cdk2 of the decapods, Macrobrachium japonicus and M. rosenbergii, which are clustered together. The results of qRT-PCR showed that the expression of cdk2 gradually increased in embryos from fertilized eggs, reaching the highest level in the anterior nodal larvae. The expression level of cdk2 decreased significantly after the anterior nodal larva stage (P < 0.05). Cdk2 was expressed in nine tissues (eye stalk, stomach, heart, gill, intestine, muscle, ovary, hepatopancreas, and ventral cord), with the highest expression in the ovary, at a level that was significantly different from the other tissues (P < 0.05). In the ovary, the expression level of this gene was highest in developmental stage Ⅲ and lowest in stage Ⅱ, which was significantly different from the other stages (P < 0.05). RNAi results showed that reducing the cdk2 expression level could significantly delay ovarian development. The results of in situ hybridization further revealed that cdk2 plays an important role in the proliferation of oocytes, and the synthesis and transportation of exogenous yolk material.
p>The aim of this study was to investigate pathological tissue from cases of enteropathy- associated T cell lymphoma (EATL), previously shown to over-express p53 protein, for the presence of p53 mutations with the view of correlating over-expression of the protein with the presence of genetic alterations. Single-stranded conformation polymorphism analysis was used to screen a series of formalin-fixed, paraffin-embedded EATL tissue samples and histologically uninvolved tissue Irom the same patients for the presence of p53 mutations in exons 5-8. DNA sequence analysis was performed on cases showing PCR fragments with mobility shifts to confirm the presence of mutations. Immunohistochemistry was used to assess expression of p53 and related cell cycle regulatory proteins in the same cases. Mobility shifts were detected in 10/29 (34.5%) tumour (T) samples and in 3/20 (15%) samples of adjacent (A) uninvolved tissue. Only 2 samples (1 T and I A sample) showing mobility shifts showed no over-expression of p53. DNA sequence analysis identified the presence of mutations in 3/29 (10%) T samples and in 2/20 (10%) A samples. Additional silent mutations were also detected in 2 T samples from different cases. The DNA sequencing results suggest that p53 over-expression is associated with p53 mutations in a small proportion of EATL cases. An alternative mechanism may be responsible for the stabilisation of p53 protein in the remaining cases. The detection of multiple mutations in samples 6om some individual patients may indicate that mutations arise in more than one T cell clone within enteropathic bowel.</p
Bromodeoxyuridine/5-bromo-2′-deoxyuridine (BrdU) is a nucleoside analog of thymidine and its incorporation into DNA during replication within S-phase of the cell cycle is used to quantify cell proliferation. Quantification of incorporated BrdU is considered the most direct measure of cell proliferation, and here we describe BrdU incorporation into cultured vascular smooth muscle cells (VSMCs) and endothelial cells in vitro. Incorporation of fluorescent-labeled ethynyldeoxyuridine/5-ethynyl-2′-deoxyuridine (EdU) is a novel alternative to BrdU assays and presents significant advantages. This method of detection of EdU based on a simple “click” chemical reaction, which covalently bonds EdU to a fluorescent dye is also outlined in this chapter with a protocol for quantitative analysis of EdU incorporation using a Fiji-based macro. We also describe how proliferation can be assessed by quantification of classical proliferative markers such as phopsho-Ser807/811 retinoblastoma (Rb), proliferating cell nuclear antigen (PCNA) and cyclin D1 by Western blotting. As these markers are involved in different aspects of the cell cycle regulation, examining their expression levels can not only reveal the relative population of proliferating cells but can also improve our understanding of the mechanism of action of a given treatment or intervention. The scratch wound assay is a simple and cost-effective technique to quantify cell migration. A protocol which involves creating a wound in a cell cultured monolayer and measuring the distance migrated by the cells after a predefined time period is also described. Gap creation can also be achieved via physical cell exclusion where cells are seeded in distinct reservoirs of a cell culture insert which reveal a gap upon removal. Cell migration may then be quantified by monitoring the rate of gap closure. The presence of cleaved caspase-3 is a marker of programmed cell death (apoptosis). To detect cleaved caspase-3 in vitro, immunocytochemistry and fluorescence can be performed as outlined in this chapter.
Alachlor is a widely used herbicide for the cultivation of various grains employed as food for cattle. The mechanisms leading to the toxic effects of alachlor on epithelial cells of the bovine mammary gland are not well known. Thus, this study was conducted to clarify the toxicological effects of alachlor on the immortalized epithelial cell line of the bovine mammary gland (MAC-T) cells. After treatment, many factors related to cell viability, proliferation, and cellular homeostasis were evaluated. Alachlor arrested cell cycle progression by blocking the expression of cyclin and cyclin-dependent kinases, and induced the breakdown of Ca²⁺ homeostasis. The cytosolic and mitochondrial levels of Ca²⁺ were also abnormally increased after the treatment of cells with alachlor, ultimately leading to the depolarization of mitochondrial membrane potential in MAC-T cells. The signaling cascade was found to be dysregulated by the abnormal phosphorylation of signaling molecules involved in PI3K/AKT (AKT, p70S6K, and S6) and MAPK/JNK (JNK and c-Jun) pathways. In these mechanisms, exposure to alachlor led to a reduction in the viability and proliferation of MAC-T cells. Altogether, the toxic effects of alachlor can lead to abnormal conditions in epithelial cells of the bovine mammary gland, which might hinder these cells from performing their main role, such as producing milk.
G1 cyclins control the G1 to S phase transition in the budding yeast, Saccharomyces cerevisiae. Cyclin E was discovered in
the course of a screen for human complementary DNAs that rescue a deficiency of G1 cyclin function in budding yeast. The amounts
of both the cyclin E protein and an associated protein kinase activity fluctuated periodically through the human cell cycle;
both were maximal in late G1 and early S phases. Cyclin E-associated kinase activity was correlated with the appearance of
complexes containing cyclin E and the cyclin-dependent kinase Cdk2. Thus, the cyclin E-Cdk2 complex may constitute a human
G1-S phase-specific regulatory protein kinase.
Budding yeast strains have three CLN genes, which have limited cyclin homology. At least one of the three is required for cell cycle START. Four B cyclins are known in yeast; two have been shown to function in mitosis. We have discovered a fifth B-cyclin gene, called CLB5, which when cloned on a CEN plasmid can rescue strains deleted for all three CLN genes. CLB5 transcript abundance peaks in G1, coincident with the CLN2 transcript but earlier than the CLB2 transcript. CLB5 deletion does not cause lethality, either alone or in combination with other CLN or CLB deletions. However, strains deleted for CLB5 require more time to complete S phase, suggesting that CLB5 promotes some step in DNA synthesis. CLB5 is the only yeast cyclin whose deletion lengthens S phase. CLB5 may also have some role in promoting the G1/S transition, because cln1 cln2 strains require both CLN3 and CLB5 for viability on glycerol media and cln1,2,3- strains require CLB5 for rescue by the Drosophila melanogaster cdc2 gene. In conjunction with cln1,2,3- rescue by CLB5 overexpression and the coincident transcriptional regulation of CLB5 and CLN2, these observations are suggestive of partial functional redundancy between CLB5 and CLN genes.
Peripheral blood T lymphocytes require two sequential mitogenic signals to reenter the cell cycle from their natural, quiescent state. One signal is provided by stimulation of the T-cell antigen receptor, and this induces the synthesis of both cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through G1. Antigen receptor stimulation alone, however, is insufficient to promote activation of G1 cyclin-Cdk2 complexes. This is because quiescent lymphocytes contain an inhibitor of Cdk2 that binds directly to this kinase and prevents its activation by cyclins. The second mitogenic signal, which can be provided by the cytokine interleukin 2, leads to inactivation of this inhibitor, thereby allowing Cdk2 activation and progression into S phase. Enrichment of the Cdk2 inhibitor from G1 lymphocytes by cyclin-CDK affinity chromatography indicates that it may be p27Kip1. These observations show how sequentially acting mitogenic signals can combine to promote activation of cell cycle proteins and thereby cause cell proliferation to start. CDK inhibitors have been shown previously to be induced by signals that negatively regulate cell proliferation. Our new observations show that similar proteins are down-regulated by positively acting signals, such as interleukin 2. This finding suggests that both positive and negative growth signals converge on common targets which are regulators of G1 cyclin-CDK complexes. Inactivation of G1 cyclin-CDK inhibitors by mitogenic growth factors may be one biochemical pathway underlying cell cycle commitment at the restriction point in G1.
The p53 tumour-suppressor protein controls the expression of a gene encoding the p21 cyclin-dependent protein kinase (CDK) regulator. Levels of p21 protein are increased in senescent cells and p21 overexpression blocks the growth of tumour cells. In normal human cells, but not in many tumour cells, p21 exists in a quaternary complex with a cyclin, a CDK, and the proliferating-cell nuclear antigen (PCNA). p21 controls CDK activity, thereby affecting cell-cycle control, whereas PCNA functions in both DNA replication and repair. Here we use simian virus 40 DNA replication in vitro to show than p21 directly inhibits PCNA-dependent DNA replication in the absence of a cyclin/CDK. Furthermore, p21 blocks the ability of PCNA to activate DNA polymerase delta, the principal replicative DNA polymerase. This regulation results from a direct interaction between p21 and PCNA. Thus, during p53-mediated suppression of cell proliferation, p21 and PCNA may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA.
Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.
The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.
In mammalian cells, DNA damage increases the levels of the nuclear tumour-suppressor p53, resulting in elevated synthesis of p21, an inhibitor of cyclin-dependent kinases (CDK). p21 may also directly block DNA replication by inhibiting the proliferating-cell nuclear antigen (PCNA), an essential DNA replication protein. However, PCNA is also required for nucleotide-excision repair of DNA, an intrinsic part of the cellular response to ultraviolet irradiation. Using an in vitro system, we now show that p21 does not block PCNA-dependent nucleotide-excision repair, in contrast to its inhibition of simian virus 40 DNA replication. Furthermore, the short gap-filling DNA synthesis by PCNA-dependent DNA polymerases delta and epsilon is less sensitive to inhibition by p21 than is long primer-extension synthesis. The ability of p21 to inhibit the role of PCNA in DNA replication but not in DNA repair rationalizes in vivo data showing that genetic damage leads to inactivation of chromosomal replication while allowing damage-responsive repair.
The retinoblastoma gene product (pRB) constrains cell proliferation by preventing cell-cycle progression from the G1 to S phase. Its growth-inhibitory effects appear to be reversed by hyperphosphorylation occurring during G1. This process is thought to involve G1 cyclins and cyclin-dependent kinases (cdks). Here we report that the cell cycle-dependent phosphorylation of mammalian pRB is faithfully reproduced when it is expressed in Saccharomyces cerevisiae. As is the case in mammalian cells, this phosphorylation requires an intact oncoprotein-binding domain and is inhibited by a negative growth factor, in this case a mating pheromone. Expression of pRB in cln (-) mutants indicates that specific combinations of endogenous G1 cyclins, Cln3 and either Cln1 or Cln2 are required for pRB hyperphosphorylation in yeast. Moreover, expression of mammalian G1 cyclins in cln (-) yeast cells indicates that the functions of Cln2 and Cln3 in pRB hyperphosphorylation can be complemented by human cyclin E and cyclin D1, respectively. These observations suggest a functional heterogeneity among G1 cyclin-cdk complexes and indicate a need for the involvement of multiple G1 cyclins in promoting pRB hyperphosphorylation and resulting cell-cycle progression.
In this study we have surveyed by immunoblotting the protein levels of Cyclin D1, D2, D3 and their catalytic partners, Cdk4 and Cdk6 in normal and transformed human cells. We found that all these proteins were differentially expressed in diploid cells derived from different tissues, in contrast to Cyclin E, Cyclin A and Cdk2 which are ubiquitously expressed. D-type Cyclins were never dramatically overexpressed and often very poorly expressed in tumor cell lines when compared to the levels in their normal counterparts. In contrast, Cdk4 was expressed at high levels in several tumor cell lines and Cdk6 was ectopically expressed in two sarcoma lines, suggesting a possible involvement of these two Cdks in oncogenesis. Interestingly, low levels of Cyclin D1 and D3 proteins always correlated with functional inactivation of the retinoblastoma gene product (pRb). In cells displaying active pRb, Cyclin D1 was found associated with Cdk4 regardless of whether the p53 gene was wild-type or mutant. Microinjection during G1 of Cyclin D1 anti-sense cDNA or anti-Cyclin D1 antibody in these cells arrested the cell cycle in G1. In cells lacking pRb function, Cyclin D1 was dissociated from Cdk4. Microinjection during G1 of Cyclin D1 antisense cDNA or anti-Cyclin D1 antibody in these cells did not affect G1 progression. These results show that (i) in the absence of pRb, Cyclin D1 is expressed at low levels, is dissociated from Cdk4 and becomes dispensable in G1; (ii) Cyclin D1 needs to be associated with its catalytic subunit, Cdk4, to function as a positive regulator of G1 progression.
D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins D1, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone H1 or casein. Virtually all cyclin D1-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin D1-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin D1-cdk4 complexes. Thus, cyclin D1-associated kinase activity was not detected during the G0-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin D1 alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.
The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.
Transforming growth factor beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Cyclins E and A in association
with Cdk2 have been shown to play a role in the G1-to-S phase transition in mammalian cells. We have studied the effects of
TGF-beta-mediated growth arrest on G1/S cyclins E and A. Inhibition of cyclin A-associated kinase by TGF-beta is primarily
due to a decrease in cyclin A mRNA and protein. By contrast, while TGF-beta inhibits accumulation of cyclin E mRNA, the reduction
in cyclin E protein is minimal. Instead, we find that the activation of cyclin E-associated kinase that normally accompanies
the G1-to-S phase transition is inhibited. A novel inhibitor of cyclin-Cdk complexes was detected in TGF-beta-treated cell
lysates. Inhibition is mediated by a heat-stable protein that targets both Cdk2 and Cdc2 kinases. In G0-arrested cells, a
similar inhibitor of Cdk2 kinase was detected. These data suggest the existence of an inhibitor of cyclin-dependent kinases
induced under different conditions to mediate antiproliferative responses.
Cell-cell contact and TGF-beta can arrest the cell cycle in G1. Mv1Lu mink epithelial cells arrested by either mechanism are incapable of assembling active complexes containing the G1 cyclin, cyclin E, and its catalytic subunit, Cdk2. These growth inhibitory signals block Cdk2 activation by raising the threshold level of cyclin E necessary to activate Cdk2. In arrested cells the threshold is set higher than physiological cyclin E levels and is determined by an inhibitor that binds to cyclin E-Cdk2 complexes. A 27-kD protein that binds to and prevents the activation of cyclin E-Cdk2 complexes can be purified from arrested cells but not from proliferating cells, using cyclin E-Cdk2 affinity chromatography. p27 is present in proliferating cells, but it is sequestered and unavailable to interact with cyclin E-Cdk2 complexes. Cyclin D2-Cdk4 complexes bind competitively to and down-regulate the activity of p27 and may thereby act in a pathway that reverses Cdk2 inhibition and enables G1 progression.
The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.
The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.
In mammalian cells inhibition of the cdc2 function results in arrest in the G2-phase of the cell cycle. Several cdc2-related gene products have been identified recently and it has been hypothesized that they control earlier cell cycle events. Here we have studied the relationship between activation of one of these cdc2 homologs, the cdk2 protein kinase, and the progression through the cell cycle in cultured human fibroblasts. We found that cdk2 was activated and specifically localized to the nucleus during S phase and G2. Microinjection of affinity-purified anti-cdk2 antibodies but not of affinity-purified anti-cdc2 antibodies, during G1, inhibited entry into S phase. The specificity of these effects was demonstrated by the fact that a plasmid-driven cdk2 overexpression counteracted the inhibition. These results demonstrate that the cdk2 protein kinase is involved in the activation of DNA synthesis.
Human cyclin D1 has been associated with a wide variety of proliferative diseases but its biochemical role is unknown. In diploid fibroblasts we find that cyclin D1 is complexed with many other cellular proteins. Among them are protein kinase catalytic subunits CDK2, CDK4 (previously called PSK-J3), and CDK5 (also called PSSALRE). In addition, polypeptides of 21 kd and 36 kd are identified in association with cyclin D1. We show that the 36 kd protein is the proliferating cell nuclear antigen, PCNA. Cyclin D3 also associates with multiple protein kinases, p21 and PCNA. It is proposed that there exists a quaternary complex of D cyclin, CDK, PCNA, and p21 and that many combinatorial variations (cyclin D1, D3, CDK2, 4, and 5) may assemble in vivo. These findings link a human putative G1 cyclin that is associated with oncogenesis with a well-characterized DNA replication and repair factor.
Human cyclin E, originally identified on the basis of its ability to function as a G1 cyclin in budding yeast, associated
with a cell cycle-regulated protein kinase in human cells. The cyclin E-associated kinase activity peaked during G1, before
the appearance of cyclin A, and was diminished during exit from the cell cycle after differentiation or serum withdrawal.
The major cyclin E-associated kinase in human cells was Cdk2 (cyclin-dependent kinase 2). The abundance of the cyclin E protein
and the cyclin E-Cdk2 complex was maximal in G1 cells. These results provide further evidence that in all eukaryotes assembly
of a cyclin-Cdk complex is an important step in the biochemical pathway that controls cell proliferation during G1.
The cellular transcription factor E2F, previously identified as a component of early adenovirus transcription, has now been
shown to be important in cell proliferation control. E2F appears to be a functional target for the action of the tumor suppressor
protein Rb that is encoded by the retinoblastoma susceptibility gene. The disruption of this E2F-Rb interaction, as well as
a complex involving E2F in association with the cell cycle-regulated cyclin A-cdk2 kinase complex, may be a common mechanism
of action for the oncoproteins encoded by the DNA tumor viruses.
Murine D type cyclins associate with a catalytic subunit (p34PSK-J3) with properties distinct from known cyclin-dependent kinases (cdks). Mouse p34PSK-J3 shows less than 50% amino acid identity to p34cdc2, p33cdk2, and p36cdk3, lacks a PSTAIRE motif, and does not bind to p13suc1. Cyclin D1-p34PSK-J3 complexes accumulate in macrophages during G1 and decline in S phase, whereas complexes involving cyclins D2 and D3 form in proliferating T cells. Although histone H1 kinase activity is not detected in cyclin D or PSK-J3 immunoprecipitates, cyclin D-p34PSK-J3 complexes assembled in vitro stably bind and phosphorylate the retinoblastoma gene product (pRb) and an Rb-like protein (p107) but do not interact with pRb mutants that are functionally inactive. Thus, p34PSK-J3 is a cyclin D-regulated catalytic subunit that acts as an Rb (but not H1) kinase.
Cyclins were discovered in marine invertebrates based on their dramatic cell cycle periodicity. Recently, the products of three genes associated with cell cycle progression in S. cerevisiae were found to share limited homology with cyclins. Mutational elimination of the CLN1, CLN2, and DAF1/WHI1 products leads to cell cycle arrest independent of cell type, while expression of any one of the genes allows cell proliferation. Using strains where CLN1 was expressed conditionally, the essential function of Cln proteins was found to be limited to the G1 phase. Furthermore, the ability of the Cln proteins to carry out this function was found to decay rapidly upon cessation of Cln biosynthesis. The data are consistent with the hypothesis that Cln proteins activate the Cdc28 protein kinase, shown to be essential for the G1 to S phase transition in S. cerevisiae. Because of the apparent functional redundancy of these genes, DAF1/WHI1 has been renamed CLN3.
Cells prepare for S phase during the G1 phase of the cell cycle. Cell biological methods have provided knowledge of cycle kinetics and of substages of G1 that are determined by extracellular signals. Through the use of biochemical and molecular biological techniques to study effects of growth factors, oncogenes, and inhibitors, intracellular events during G1 that lead to DNA synthesis are rapidly being discovered. Many cells in vivo are in a quiescent state (G0), with unduplicated DNA. Cells can be activated to reenter the cycle during G1. Similarly, cells in culture can be shifted between G0 and G1. These switches in and out of G1 are the main determinants of post-embryonic cell proliferation rate and are defectively controlled in cancer cells.
For many species of pathogenic bacteria, invasion and survival within animal cells is central to establishing a successful host-parasite relationship. Localization within host cells protects the microorganism from host defences, or permits it to cross epithelial barriers and subsequently become systemically distributed. The precise mechanisms that permit entry of bacteria into host tissues are unclear, therefore we have been studying the invasion of epithelial cells by Yersinia pseudotuberculosis. As a first step towards identifying the factors required for this process, we report here the identification of a single genetic locus from this organism that is sufficient to convert the innocuous Escherichia coli K-12 strain into an organism capable of invading cultured animal cells.
Extracellular signals can affect the rate of proliferation and the state of differentiation of eukaryotic cells. Signal transduction pathways have evolved to detect these signals at the plasma membrane, transmit them through the cytoplasm and into the nucleus, and thereby generate the appropriate changes in metabolism and transcription. Much attention has been focused recently on regulatory pathways of this sort that lead to activation of a family of protein kinases known as the mitogen- or messenger-activated, or extracellular signal-regulated protein kinases (MAPKs or ERKs) because this particular class of enzyme is highly conserved among eukaryotes, as is documented here and in the accompanying reviews in this issue. The mating pheromone response pathway in a unicellular microbe, the budding yeast Saccharomyces cerevisiae, is perhaps the best understood multicomponent signaling pathway known in any eukaryotic organism, especially at the genetic level. Furthermore, structural homologs and functional analogs of the components of the yeast pheromone response pathway are recapitulated in the signaling systems present in multicellular eukaryotes. This article emphasizes recent findings and common molecular themes for understanding the organization and regulation of MAPK-dependent signaling cascades that have emerged from biochemical and genetic analysis of the mating pheromone response pathway in yeast.
In normal human fibroblast cells, the primary cell cycle regulators, the cyclin-dependent kinases (CDKs), exist predominantly in multiple quaternary complexes, each consisting of a CDK, a cyclin, proliferating cell nuclear antigen (PCNA) and p21. p21 encodes a universal inhibitor of cyclin-dependent kinases. Here we show that the level of p21 mRNA and the interaction of p21 protein with cyclin-CDK enzymes are regulated during the cell cycle. When normal human fibroblast IMR90 cells were released from serum starvation, p21 mRNA reached its highest level immediately following serum stimulation, began to decrease at the G1/S boundary, fell to its lowest level during S phase, and accumulated again as cells exited from S phase. p21 protein associates with each cyclin-CDK complex in a cell cycle dependent manner. Cyclin A-CDK2-p21-PCNA and Cyclin B1-CDC2-p21-PCNA complexes are assembled in early S and G2 phase, respectively, indicating that p21 and/or PCNA regulates the enzymatic activity of each kinase at the time of their functioning. Cyclin D1-CDK4-p21-PCNA complexes, on the other hand, persist throughout the cell cycle, suggesting that cyclin D1-CDK4 quaternary complexes may play a role in monitoring an event(s) that may occur at any time, rather than at a specific stage of the cell cycle. The level of p21 mRNA in early passage Li-Fraumeni cells that are heterozygous for p53 mutation remained similar to that in normal fibroblasts, but was undetectable in immortalized Li-Fraumeni cells homozygous for mutant p53. This finding provides a plausible molecular explanation for the loss of genetic stability associated with cells homozygous, but not heterozygous, for p53 mutation.
Cyclic AMP (cAMP) blocks the mitogenic effects of colony-stimulating factor 1 (CSF-1) in macrophages, inducing cell cycle arrest in mid-G1 phase. Complexes between cyclin D1 and cyclin-dependent kinase 4 (cdk4) assemble in growth arrested cells, but cdk4 is not phosphorylated in vivo by the cdk-activating kinase (CAK) and remains inactive. Although undetectable in lysates of cAMP-treated cells, active CAK is recovered after antibody precipitation, indicating that it is not the direct target of inhibition. Levels of the cdk inhibitor p27Klp1 increase in cAMP-treated cells, and its immunodepletion from inhibitory lysates restores CAK-mediated cdk4 activation. Kip1 does not bind to CAK, but its association with cyclin D-cdk4 prevents CAK from phosphorylating and activating the holoenzyme.
When yeast cells reach a critical size, they initiate bud formation, spindle pole body duplication, and DNA replication almost simultaneously. All three events depend on activation of Cdc28 protein kinase by the G1 cyclins Cln1, -2, and -3. We show that DNA replication also requires activation of Cdc28 by B-type (Clb) cyclins. A sextuple clb1-6 mutant arrests as multibudded G1 cells that resemble cells lacking the Cdc34 ubiquitin-conjugating enzyme. cdc34 mutants cannot enter S phase because they fail to destroy p40SIC1, which is a potent inhibitor of Clb but not Cln forms of the Cdc28 kinase. In wild-type cells, p40SIC1 protein appears at the end of mitosis and disappears shortly before S phase. Proteolysis of a cyclin-specific inhibitor of Cdc28 is therefore an essential aspect of the G1 to S phase transition.
In normal fibroblasts CDKs exist predominantly in p21/PCNA/cyclin/CDK quaternary complexes, whereas in p53-deficient cells, p21 expression is depressed and the kinases are reduced to a cyclin/CDK binary state. p21 is a universal cyclin kinase inhibitor, but we show here that p21-containing complexes exist in both catalytically active and inactive forms. This finding challenges the current view that active cyclin kinases function only in the binary state and reveals the subtlety with which tumor-suppressor proteins modulate the cell cycle.
Using a yeast interaction screen to search for proteins that interact with cyclin D1-Cdk4, we identified a 27 kDa mouse protein related to the p21 cyclin-Cdk inhibitor. p27 interacts strongly with D-type cyclins and Cdk4 in vitro and more weakly with cyclin E and Cdk2. In mouse fibroblasts, p27 is associated predominantly with cyclin D1-Cdk4. Recombinant p27 is a potent inhibitor of cyclin D1-Cdk4 and cyclin A-Cdk2 protein kinase activity and a weaker inhibitor of cyclin B1-Cdc2. Overexpression of p27 in Saos-2 cells causes G1 arrest. p27 protein levels do not change as serum-stimulated quiescent mouse fibroblasts progress through the cell cycle. p27 is identical to p27Kip1, a cyclin-Cdk inhibitor present in TGF beta-treated cells. p27 has the hallmarks of a negative regulator of G1 progression and may mediate TGF beta-induced G1 arrest.
We cloned p27Kip1, a cyclin-dependent kinase inhibitor implicated in G1 phase arrest by TGF beta and cell-cell contact. p27Kip1 associates with cyclin E-Cdk2 complexes in vivo and in vitro, prevents their activation, and inhibits previously activated complexes, and p27Kip1 overexpression obstructs cell entry into S phase. p27Kip1 potently inhibits Rb phosphorylation by cyclin E-Cdk2, cyclin A-Cdk2, and cyclin D2-Cdk4. p27Kip1 is highly conserved and broadly expressed in human tissues, and its mRNA levels are similar in proliferating and quiescent cells. p27Kip1 has a region of sequence similarity to p21Cip1/WAF1, the Cdk inhibitor whose transcription is stimulated by p53. A p27Kip1 peptide corresponding to this region retains Cdk inhibitory activity. We suggest that cell contact, TGF beta, and p53 all restrain cell proliferation through related Cdk inhibitors.
E2F is a cellular transcription factor that is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene (RB1) and the pRb-like p107 and p130 proteins. Analysis of mutations within both adenovirus E1A and pRb, which affected their ability to regulate cellular proliferation and alter E2F activity, suggested that E2F may play a role in cell cycle progression. Microinjection of a GST-E2F-1 fusion protein into quiescent Balb/c 3T3 cells induced DNA synthesis whereas co-injection of GST-E2F-1 and GST-E2F(95-191) protein, encoding only the DNA binding domain of E2F-1, blocked the induction of S-phase. While E1A likely targets multiple cellular pathways, co-injection of the GST-E2F(95-191) dominant inhibitory protein with 12S E1A protein blocked E1A-mediated induction of DNA synthesis, suggesting that the E2F-dependent pathway is dominant. Analysis of the interval required for microinjected quiescent cells to enter S-phase indicated that E2F-1 acted faster than either E1A or serum.
Cell cycle arrest of Saccharomyces cerevisiae in G1 by the antimitogen alpha-factor is mediated by activation of a signal
transduction pathway that results in inhibition of the cyclin-dependent kinase Cdc28-Cln. The Far1 protein is required for
cell cycle arrest and associates with the Cdc28-Cln complex. The kinase activity of Cdc28-Cln was directly inhibited by Far1
both in vivo and in vitro, thus demonstrating that Far1 acts at the final step in the alpha-factor response pathway by inhibiting
a G1 cyclin-dependent kinase.
Phosphorylation by the CDK-activating kinase (CAK) is a required step in the activation of cyclin-dependent kinases. We have purified CAK from mammalian cells; the enzyme comprises two major polypeptides of 42 and 37 kDa. Protein sequencing indicates that the 42 kDa subunit is the mammalian homolog of MO15, a protein kinase known to be a component of CAK in amphibians and echinoderms. Cloning of a cDNA encoding the 37 kDa subunit identifies it as a novel cyclin (cyclin H). We have reconstituted CAK in vitro with the MO15 catalytic subunit and cyclin H, demonstrating that MO15 is a cyclin-dependent kinase (CDK7). Like other CDKs, MO15/CDK7 contains a conserved threonine required for full activity; mutation of this residue severely reduces CAK activity. The CAK holoenzyme activates complexes of CDK2 and CDC2 with various cyclins and also phosphorylates CDK2, but not CDC2, in the absence of cyclin. Thus, CAK is a CDK-cyclin complex implicated in the control of multiple cell cycle transitions.
Transforming growth factor-beta (TGF-beta) inhibits cell proliferation by inducing a G1-phase cell cycle arrest. Normal progression through G1 is promoted by the activity of the cyclin-dependent protein kinases CDK4 and CDK6 (ref. 2), which are inhibited by the protein p16INK4. We have isolated a new member of the p16INK4 family, p15INK4B. p15 expression is induced approximately 30-fold in human keratinocytes by treatment with TGF-beta, suggesting that p15 may act as an effector of TGF-beta-mediated cell cycle arrest. The gene encoding p15 is located on chromosome 9 adjacent to the p16 gene at a frequent site of chromosomal abnormality in human tumours (9p21).
In normal human diploid fibroblasts, cyclins of the A, B, and D classes each associate with cyclin-dependent kinases (CDKs), proliferating cell nuclear antigen (PCNA), and p21, thereby forming multiple independent quaternary complexes. Upon transformation of diploid fibroblasts with the DNA tumor virus SV40, or its transforming tumor antigen (T), the cyclin D/p21/CDK/PCNA complexes are disrupted. In transformed cells, CDK4 totally dissociates from cyclin D, PCNA, and p21 and, instead, associates exclusively with a polypeptide of 16 kD (p16). Quaternary complexes containing cyclins A or B1 and p21/CDK/PCNA also undergo subunit rearrangement in transformed cells. Both PCNA and p21 are no longer associated with CDC2-cyclin B1 binary complexes. Cyclin A complexes no longer contain p21, and a new 19-kD polypeptide (p19) is found in association with cyclin A. The pattern of subunit rearrangement of cyclin-CDK complexes in SV40-transformed cells is also shared in those containing adeno- or papilloma viral oncoproteins. Rearrangement also occurs in p53-deficient cells derived from Li-Fraumeni patients that carry no known DNA tumor virus. These findings suggest a mechanism by which oncogenic proteins alter the cell cycle of transformed cells.
A family of vertebrate cdc2-related kinases has been identified, and these kinases are candidates for roles in cell cycle
regulation. Here, we show that the human PLSTIRE gene product is a novel cyclin-dependent kinase, cdk6. The cdk6 kinase is
associated with cyclins D1, D2, and D3 in lysates of human cells and is activated by coexpression with D-type cyclins in Sf9
insect cells. Furthermore, we demonstrate that endogenous cdk6 from human cell extracts is an active kinase which can phosphorylate
pRB, the product of the retinoblastoma tumor suppressor gene. The activation of cdk6 kinase occurs during mid-G1 in phytohemagglutinin-stimulated
T cells, well prior to the activation of cdk2 kinase. This timing suggests that cdk6, and by analogy its homolog cdk4, links
growth factor stimulation with the onset of cell cycle progression.
Conditional overexpression of human cyclins B1, D1, and E was accomplished by using a synthetic cDNA expression system based on the Escherichia coli tetracycline repressor. After induction of these cyclins in asynchronous Rat-1 fibroblasts, a decrease in the length of the G1 interval was observed for cyclins D1 and E, consistent with an acceleration of the G1/S phase transition. We observed, in addition, a compensatory lengthening of S phase and G2 so that the mean cell cycle length in populations constitutively expressing these cyclins was unchanged relative to those of their uninduced counterparts. We found that expression of cyclin B1 had no effect on cell cycle dynamics, despite elevated levels of cyclin B-associated histone H1 kinase activity. Induction of cyclins D1 and E also accelerated entry into S phase for synchronized cultures emerging from quiescence. However, whereas cyclin E exerted a greater effect than cyclin D1 in asynchronous cycling cells, cyclin D1 conferred a greater effect upon stimulation from quiescence, suggesting a specific role for cyclin D1 in the G0-to-G1 transition. Overexpression of cyclins did not prevent cells from entering into quiescence upon serum starvation, although a slight delay in attainment of quiescence was observed for cells expressing either cyclin D1 or cyclin E. These results suggest that cyclins D1 and E are rate-limiting activators of the G1-to-S phase transition and that cyclin D1 might play a specialized role in facilitating emergence from quiescence.
Most cells of the dorsal epidermis exit from the mitotic cycle after division 16 in Drosophila embryogenesis. This exit is dependent on the down-regulation of Drosophila cyclin E (DmcycE) during the final mitotic cycle. Ectopic expression of DmcycE after the final mitosis induces entry into S phase and reaccumulation of G2 cyclins and results in progression through a complete additional cell cycle. Conversely, analyses in DmcycE mutant embryos indicate that cyclin E is required for progression through S phase of the mitotic cycle. Moreover, endoreplication, which occurs in late wild-type embryos in the same pattern as DmcycE expression, is not observed in the mutant embryos. Therefore, Drosophila cyclin E, which forms a complex with the Dmcdc2c kinase, controls progression through S phase and its down-regulation limits embryonic proliferation.
The tumor-suppressor protein p53 appears to function at the G1 phase of the cell cycle as a checkpoint in response to DNA damage. Mutations in the p53 gene lead to an increased rate of genomic instability and tumorigenesis. The E2F-1 transcription factor is a protein partner of the retinoblastoma-susceptibility gene product, RB. E2F-1 appears to function as a positive regulator or signal for entry into S phase. To explore possible interactions of p53 and E2F-1 in the cell cycle, a human E2F-1 expression plasmid was introduced into a murine cell line containing a temperature-sensitive p53 allele which produces a p53 protein in the wild-type conformation at 32 degrees C and the mutant form at 37.5 degrees C. Coexpression of the wild-type p53 protein and E2F-1 in these cells resulted in a rapid loss of cell viability through a process of apoptosis. Thus, the cell cycle utilizes an interacting or communicative pathway between RB-E2F-1 and p53.
Cyclins D1, D2 and D3 are thought to function in the G1 phase of the cell division cycle by regulating the activity of cyclin-dependent protein kinases. All three D-type cyclins can be shown to associate with two specific kinases, cdk4 and cdk6, providing at least six possible combinations. To establish whether different cell types require different subsets of these complexes and whether they are altered in tumours where D-cyclin expression is perturbed, we surveyed a series of tumour cell lines and compared them where possible to non-tumorigenic counterparts. Although complexes involving cdk4 or cdk6 were readily observed in many of the cell lines, no complexes were detectable in human cells harbouring DNA tumour virus oncoproteins or in which the retinblastoma gene product (pRb) is mutated or missing. These data suggest that as well as being a potential substrate for D-cyclin-kinases, functional pRb contributes to the formation or stability of the complexes, at least in human cells.
The ability of p53 to activate transcription from specific sequences suggests that genes induced by p53 may mediate its biological role as a tumor suppressor. Using a subtractive hybridization approach, we identified a gene, named WAF1, whose induction was associated with wild-type but not mutant p53 gene expression in a human brain tumor cell line. The WAF1 gene was localized to chromosome 6p21.2, and its sequence, structure, and activation by p53 was conserved in rodents. Introduction of WAF1 cDNA suppressed the growth of human brain, lung, and colon tumor cells in culture. Using a yeast enhancer trap, a p53-binding site was identified 2.4 kb upstream of WAF1 coding sequences. The WAF1 promoter, including this p53-binding site, conferred p53-dependent inducibility upon a heterologous reporter gene. These studies define a gene whose expression is directly induced by p53 and that could be an important mediator of p53-dependent tumor growth suppression.
The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.
The major events of the cell division cycle are triggered by periodic changes in the activity of cyclin-dependent protein kinases (CDKs). In mammals, the members of the CDK family include CDK2 and CDC2, which are thought to be involved in the control of DNA replication and mitosis, respectively. The protein kinase activity of these enzymes is controlled by a complex array of mechanisms. Activation of the CDK catalytic subunit requires association with a positive regulatory subunit (cyclin) and phosphorylation (at Thr 160 in CDK2). This activated complex can be inhibited by additional phosphorylation at Thr 14 and Tyr 15. Here we report the identification of a new mechanism for the regulation of CDK2 activity. We find that CDK2/cyclin complexes in mouse fibroblasts associate tightly with a 20K protein (CAP20). Complexes containing CAP20 were isolated from cell lysates and found to have negligible kinase activity, indicating that CAP20 association in vivo may inhibit CDK2 activity. We purified CAP20 from 3T3 cells and found that low concentrations of the protein completely inhibit the kinase activity of CDK2 in vitro. Thus CAP20 represents a new negative regulatory subunit that inhibits the activity of CDK2/cyclin complexes in mammalian cells.
The division cycle of eukaryotic cells is regulated by a family of protein kinases known as the cyclin-dependent kinases (CDKs). The sequential activation of individual members of this family and their consequent phosphorylation of critical substrates promotes orderly progression through the cell cycle. The complexes formed by CDK4 and the D-type cyclins have been strongly implicated in the control of cell proliferation during the G1 phase. CDK4 exists, in part, as a multi-protein complex with a D-type cyclin, proliferating cell nuclear antigen and a protein, p21 (refs 7-9). CDK4 associates separately with a protein of M(r) 16K, particularly in cells lacking a functional retinoblastoma protein. Here we report the isolation of a human p16 complementary DNA and demonstrate that p16 binds to CDK4 and inhibits the catalytic activity of the CDK4/cyclin D enzymes. p16 seems to act in a regulatory feedback circuit with CDK4, D-type cyclins and retinoblastoma protein.
Deregulation of cell proliferation is a hallmark of neoplastic transformation. Alteration in growth control pathways must translate into changes in the cell-cycle regulatory machinery, but the mechanism by which this occurs is largely unknown. Compared with normal human fibroblasts, cells transformed with a variety of viral oncoproteins show striking changes in the subunit composition of the cyclin-dependent kinases (CDKs). In normal cells, CDKs exist predominantly in multiple quaternary complexes, each containing a CDK, cyclin, proliferating cell nuclear antigen and the p21 protein. However, in many transformed cells, proliferating cell nuclear antigen and p21 are lost from these multiprotein enzymes. Here we have investigated the significance of this phenomenon by molecular cloning of p21 and in vitro reconstitution of the quaternary cell-cycle kinase complexes. We find that p21 inhibits the activity of each member of the cyclin/CDK family. Furthermore, overexpression of p21 inhibits the proliferation of mammalian cells. Our results indicate that p21 may be a universal inhibitor of cyclin kinases.
The key cell-cycle regulator Cdc2 belongs to a family of cyclin-dependent kinases in higher eukaryotes. Dominant-negative
mutations were used to address the requirement for kinases of this family in progression through the human cell cycle. A dominant-negative
Cdc2 mutant arrested cells at the G2 to M phase transition, whereas mutants of the cyclin-dependent kinases Cdk2 and Cdk3
caused a G1 block. The mutant phenotypes were specifically rescued by the corresponding wild-type kinases. These data reveal
that Cdk3, in addition to Cdc2 and Cdk2, executes a distinct and essential function in the mammalian cell cycle.