sirolimus and palbociclib

Phosphorylation of translational repressor eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) controls the initiation of cap-dependent translation, a type of protein synthesis that is frequently upregulated in human diseases such as cancer. Because of its critical cellular function, it is not surprising that multiple kinases can post-translationally modify 4E-BP1 to drive aberrant cap-dependent translation. We recently reported a site-selective chemoproteomic method for uncovering kinase-substrate interactions, and using this approach, we discovered the cyclin-dependent kinase (CDK)4 as a new 4E-BP1 kinase. Herein, we describe our extension of this work and reveal the role of CDK4 in modulating 4E-BP1 activity in the transition from mitosis to G1, thereby demonstrating a novel role for this kinase in cell cycle regulation.

Topics: Adaptor Proteins, Signal Transducing; Cell Cycle Proteins; Cyclin-Dependent Kinase 4; G1 Phase; HeLa Cells; Humans; Mitosis; Phosphorylation; Piperazines; Protein Biosynthesis; Pyridines; Sirolimus; TOR Serine-Threonine Kinases

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.

Topics: Animals; Butadienes; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Chromatin; Chromatin Assembly and Disassembly; Core Binding Factor Alpha 3 Subunit; Cyclin-Dependent Kinase 4; Drosophila melanogaster; Epithelial Cells; Gene Expression Regulation; HEK293 Cells; Histone-Lysine N-Methyltransferase; Humans; Imidazoles; MAP Kinase Kinase 1; MAP Kinase Kinase 4; Myeloid-Lymphoid Leukemia Protein; Nitriles; p38 Mitogen-Activated Protein Kinases; Piperazines; Polycomb-Group Proteins; Protein Kinase Inhibitors; Pyridines; ras Proteins; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease among aging individuals, affecting greatly the quality of their life. However, the pathogenesis of Parkinson's disease is still incompletely understood to date. Increasing experimental evidence suggests that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Since cell cycle dysfunction is not restricted to neurons, we investigated this issue in peripheral cells from patients suffering from sporadic PD and age-matched control individuals. Here, we describe increased cell cycle activity in immortalized lymphocytes from PD patients that is associated to enhanced activity of the cyclin D3/CDK6 complex, resulting in higher phosphorylation of the pRb family protein and thus, in a G1/S regulatory failure. Decreased degradation of cyclin D3, together with increased p21 degradation, as well as elevated levels of CDK6 mRNA and protein were found in PD lymphoblasts. Inhibitors of cyclin D3/CDK6 activity like sodium butyrate, PD-332991, and rapamycin were able to restore the response of PD cells to serum stimulation. We conclude that lymphoblasts from PD patients are a suitable model to investigate cell biochemical aspects of this disease. It is suggested that cyclin D3/CDK6-associated kinase activity could be potentially a novel therapeutic target for the treatment of PD.

Topics: Aged; Aged, 80 and over; Butyric Acid; Case-Control Studies; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Proliferation; Cyclin-Dependent Kinases; DNA-Binding Proteins; G1 Phase; Humans; Lymphocytes; Middle Aged; Parkinson Disease; Phosphorylation; Piperazines; Proteolysis; Pyridines; RNA, Messenger; S Phase; Sirolimus; Subcellular Fractions; Time Factors

When the cell cycle becomes arrested, MTOR (mechanistic Target of Rapamycin) converts reversible arrest into senescence (geroconversion). Hyperexpression of cyclin D1 is a universal marker of senescence along with hypertrophy, beta-Gal staining and loss of replicative/regenerative potential (RP), namely, the ability to restart proliferation when the cell cycle is released. Inhibition of MTOR decelerates geroconversion, although only partially decreases cyclin D1. Here we show that in p21- and p16-induced senescence, inhibitors of mitogen-activated/extracellular signal-regulated kinase (MEK) (U0126, PD184352 and siRNA) completely prevented cyclin D1 accumulation, making it undetectable. We also used MEL10 cells in which MEK inhibitors do not inhibit MTOR. In such cells, U0126 by itself induced senescence that was remarkably cyclin D1 negative. In contrast, inhibition of cyclin-dependent kinase (CDK) 4/6 by PD0332991 caused cyclin D1-positive senescence in MEL10 cells. Both types of senescence were suppressed by rapamycin, converting it into reversible arrest. We confirmed that the inhibitor of CDK4/6 caused cyclin D1 positive senescence in normal RPE cells, whereas U0126 prevented cyclin D1 expression. Elimination of cyclin D1 by siRNA did not prevent other markers of senescence that are consistent with the lack of its effect on MTOR. Our data confirmed that a mere inhibition of the cell cycle was sufficient to cause senescence, providing MTOR was active, and inhibition of MEK partially inhibited MTOR in a cell-type-dependent manner. Second, hallmarks of senescence may be dissociated, and hyperelevated cyclin D1, a marker of hyperactivation of senescent cells, did not necessarily determine other markers of senescence. Third, inhibition of MEK was sufficient to eliminate cyclin D1, regardless of MTOR.

Topics: Antibiotics, Antineoplastic; Benzamides; Butadienes; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; Cellular Senescence; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Inhibitors; Humans; MAP Kinase Kinase 1; Neoplasm Proteins; Nitriles; Piperazines; Pyridines; RNA Interference; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases

CDKN1A (p21) and CDKN2A (p16) inhibit CDK4/6, initiating senescence. According to our view on senescence, the role of p21 and p16 is to cause cell cycle arrest, whereas MTOR (mechanistic target of rapamycin) drives geroconversion to senescence. Recently we demonstrated that one of the markers of p21- and p16-initiated senescence is MEK-dependent hyper-elevation of cyclin D1. We noticed that a synthetic inhibitor of CDK 4/6 (PD0332991) also induced cyclin D1-positive senescence. We demonstrated that PD0332991 and p21 caused almost identical senescence phenotypes. p21, p16, and PD0332991 do not inhibit MTOR, and rapamycin decelerates geroconversion caused by all 3 molecules. Like p21, PD0332991 initiated senescence at any concentration that inhibited cell proliferation. This confirms the notion that a mere arrest in the presence of active MTOR may lead to senescence.

Topics: Butadienes; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Humans; Nitriles; Piperazines; Pyridines; Sirolimus; TOR Serine-Threonine Kinases