Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.     

Raf-Induced Cell Cycle Progression in Human TF-1 Hematopoietic Cells

Ras/Raf/MEK/ERK is a crucial pathway regulating cell cycle progression, apoptosis, and drug resistance. The Ras oncogene is frequently mutated in human cancer, which can result in the activation of the downstream Raf/MEK/ERK cascade leading to cell cycle progression in the absence of a growth stimulus. Raf-induced proliferation has been observed in hematopoietic cells. However, the mechanisms by which Raf affects cell cycle progression are not well described. To investigate the importance of Raf/MEK/ERK signaling in human hematopoietic cell growth, the effects of three different Raf genes, A-Raf, B-Raf and Raf-1, on cell cycle progression and regulatory gene expression were examined in TF-1 cells transformed to grow in response to b-estradiol-regulated DRaf:ER genes. Raf activation increased the expression of cyclin A, cyclin D, cyclin E, and p21Cip1, which are associated with G1 progression. Activated DRaf-1:ER and DA-Raf:ER but not DB-Raf:ER increased Cdk2 and Cdk4 kinase activity. The regulatory role of p16Ink4a, a potent Cdk4 kinase inhibitor, on the kinase activity of Cdk2 and Cdk4 was also examined. Raf induced p16Ink4a suppressor but this did not eliminate Cdk4 kinase activity. These results indicate that human hematopoietic cells transformed to grow in response to activated Raf can be used to elucidate the mechanisms by which various cell cycle regulatory molecules effect cell cycle progression. Furthermore, the differences that the various Raf isoforms have on Cdk4 activity and other cell cycle regulatory molecules can be determined in these cells.

Key Words:

Cell cycle, Raf, p21Cip1, p27Kip1, Cyclins, Cdks, Hematopoietic cells     

Differential regulation of Cdc2 and Cdk2 by RINGO and cyclins.

Cyclin-dependent kinases (Cdks) are key regulators of the eukaryotic cell division cycle. Cdk1 (Cdc2) and Cdk2 should be bound to regulatory subunits named cyclins as well as phosphorylated on a conserved Thr located in the T-loop for full enzymatic activity. Cdc2- and Cdk2-cyclin complexes can be inactivated by phosphorylation on the catalytic cleft-located Thr-14 and Tyr-15 residues or by association with inhibitory subunits such as p21(Cip1). We have recently identified a novel Cdc2 regulator named RINGO that plays an important role in the meiotic cell cycle of Xenopus oocytes. RINGO can bind and activate Cdc2 but has no sequence homology to cyclins. Here we report that, in contrast with Cdc2- cyclin complexes, the phosphorylation of Thr-161 is not required for full activation of Cdc2 by RINGO. We also show that RINGO can directly stimulate the kinase activity of Cdk2 independently of Thr-160 phosphorylation. Moreover, RINGO-bound Cdc2 and Cdk2 are both less susceptible to inhibition by p21(Cip1), whereas the Thr-14/Tyr-15 kinase Myt1 can negatively regulate the activity of Cdc2-RINGO with reduced efficiency. Our results indicate that Cdk-RINGO complexes may be active under conditions in which cyclin-bound Cdks are inhibited and can therefore play different regulatory roles.     

p53 dephosphorylation and p21Cip1/Waf1 translocation correlate with caspase-3 activation in TGF-β1-induced apoptosis of HuH-7 cells

The p53 tumor suppressor gene product plays an important role in the regulation of apoptosis. Transforming growth factor beta1 (TGF-beta1)-induced apoptosis in hepatic cells is associated with reduced expression of the retinoblastoma protein (pRb) and subsequent E2F-1-activated expression of apoptosis-related genes. In this study, we explored the potential role of p53 in TGF-beta1-induced apoptosis. HuH-7 human hepatoma cells were either synchronized in G1, S and G2/M phases, or treated with 1 nM TGF-beta1. The results indicated that greater than 90% of the TGF-beta1-treated cells were arrested in G1 phase of the cell cycle. This was associated with enhanced p53 dephosphorylation and p21(Cip1/Waf1) expression, which coincided with decreased Cdk2, Cdk4, and cyclin E expression, compared with synchronized G1 cells. In addition, p53 dephosphorylation coincided with caspase-3 activation, and translocation of p21(Cip1/Waf1) and p27(Kip1) into the cytoplasm, all of which were suppressed by caspase inhibition of TGF-beta1-induced apoptosis. Finally, phosphatase inhibition and pRb overexpression partially inhibited p53-mediated apoptosis. In conclusion, the results demonstrated that TGF-beta1-induced p53 dephosphorylation is associated with caspase-3 activation, and cytosolic translocation of p21(Cip1/Waf1) and p27(Kip1), resulting in decreased expression of Cdks and cyclins. Further, p53 appears to mediate TGF-beta1-induced apoptosis downstream of the pRb/E2F-1 pathway.     

Interplay between the p53 tumor suppressor protein family and Cdk5: novel therapeutic approaches for the treatment of neurodegenerative diseases using selective Cdk inhibitors

Cyclin-dependent kinases (Cdks) play a key role in orchestrating the coordination of cell cycle progression in proliferating cells. The escape from the proper control of the cell cycle by the upregulation of cyclins or aberrant activation of Cdks leads to malignant transformation. In quiescent cells and/or terminally differentiated cells, the expression pattern and activity of Cdks is altered. In postmitotic neurons, expression of mitotic kinases is downregulated, whereas Cdk5 expression becomes upregulated. Similarly to other Cdks, free Cdk5 displays no enzymatic activity and requires complex formation with a specific regulatory subunit. Two activators of Cdk5 have been identified. p35 and its isoform p39 bind to, and thereby activate, Cdk5. Unlike mitotic kinases, Cdk5 does not require activating phosphorylation within the T-loop. Because p35 is a short-lived protein, the p35/Cdk5 complexes are unstable. The stability of the p35 protein is regulated by its Cdk5-mediated phosphorylation of p35. Activated p35/Cdk5 kinase phosphorylates numerous physiological targets. The proper phosphorylation of the most important substrates, such as tau protein and neurofilament H, is essential for the correct regulation of the cytoskeletal organization, thereby regulating cell adhesion, motility, and synaptic plasticity. Moreover, Cdk5 regulates the activity of the p53 tumor suppressor via phosphorylation. p53 is upregulated in multiple neuronal death paradigms, including hypoxia, ischemia, and excitotoxicity, and plays a key role in the induction of apoptosis. On the other hand, an abnormally high expression and elevated activity of Cdk5 was observed in neurodegenerative diseases, suggesting the application of Cdk inhibitors for their therapy. Considering the action of some Cdk inhibitors on the expression and activity of the p53 protein, their therapeutic efficacy must be carefully evaluated.     

Interplay between the p53 tumor suppressor protein family and Cdk5

Cyclin-dependent kinases (Cdks) play a key role in orchestrating the coordination of cell cycle progression in proliferating cells. The escape from the proper, control of the cell cycle by the upregulation of cyclins or aberrant activation of Cdks leads to malignant transformation. In quiescent cells and/or terminally differentiated cells, the expression pattern and activity of Cdks is altered. In postmitotic neurons, expression of mitotic kinases is downregulated, whereas Cdk5 expression becomes upregulated. Similarly to other Cdks, free Cdk5 displays no enzymatic activity and requires complex formation with a specific regulatory subunit. Two activators of Cdk5 have been identified. p35 and its isoform p39 bind to, and thereby activate, Cdk5. Unlike mitotic kinases, Cdk5 does not require activating phosphorylation within the T-loop. Because p35 is a short-lived protein, the p35/Cdk5 complexes are unstable. The stability of the p35 protein is regulated by its Cdk5-mediated phosphorylation of p35. Activated p35/Cdk5 kinase phosphorylates numerous physiological targets. The proper phosphorylation of the most important substrates, such as τ protein and neurofilament H, is essential for the correct regulation of the cytoskeletal organization, thereby regulating cell adhesion, motility, and synaptic plasticity. Moreover, Cdk5 regulates the activity of the p53 tumor suppressor via phosphorylation. p53 is upregulated in multiple neuronal death paradigms, including hypoxia, ischemia, and excitotoxicity, and plays a key role in the induction of apoptosis. On the other hand, an abnormally high expression and elevated activity of Cdk5 was observed in neurodegenerative diseases, suggesting the application of Cdk inhibitors for their therapy. Considering the action of some Cdk inhibitors on the expression and activity of the p53 protein, their therapeutic efficacy must be carefully evaluated.     

Interplay Between the p53 Tumor Suppressor Protein Family and Cdk5: Novel Therapeutic Approaches for the Treatment of Neurodegenerative Diseases Using Selective Cdk Inhibitors.

Cyclin-dependent kinases (Cdks) play a key role in orchestrating the coordination of cell cycle progression in proliferating cells. The escape from the proper control of the cell cycle by the upregulation of cyclins or aberrant activation of Cdks leads to malignant transformation. In quiescent cells and/or terminally differentiated cells, the expression pattern and activity of Cdks is altered. In postmitotic neurons, expression of mitotic kinases is downregulated, whereas Cdk5 expression becomes upregulated. Similarly to other Cdks, free Cdk5 displays no enzymatic activity and requires complex formation with a specific regulatory subunit. Two activators of Cdk5 have been identified. p35 and its isoform p39 bind to, and thereby activate, Cdk5. Unlike mitotic kinases, Cdk5 does not require activating phosphorylation within the T-loop. Because p35 is a short-lived protein, the p35/Cdk5 complexes are unstable. The stability of the p35 protein is regulated by its Cdk5-mediated phosphorylation of p35. Activated p35/Cdk5 kinase phosphorylates numerous physiological targets. The proper phosphorylation of the most important substrates, such as tau protein and neurofilament H, is essential for the correct regulation of the cytoskeletal organization, thereby regulating cell adhesion, motility, and synaptic plasticity. Moreover, Cdk5 regulates the activity of the p53 tumor suppressor via phosphorylation. p53 is upregulated in multiple neuronal death paradigms, including hypoxia, ischemia, and excitotoxicity, and plays a key role in the induction of apoptosis. On the other hand, an abnormally high expression and elevated activity of Cdk5 was observed in neurodegenerative diseases, suggesting the application of Cdk inhibitors for their therapy. Considering the action of some Cdk inhibitors on the expression and activity of the p53 protein, their therapeutic efficacy must be carefully evaluated.     

Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells.

Lactoferrin inhibits cell proliferation and suppresses tumor growth in vivo. However, the molecular mechanisms underlying these effects remain unknown. In this in vitro study, we demonstrate that treatment of breast carcinoma cells MDA-MB-231 with human lactoferrin induces growth arrest at the G1 to S transition of the cell cycle. This G1 arrest is associated with a dramatic decrease in the protein levels of Cdk2 and cyclin E correlated with an inhibition of the Cdk2 kinase activity. Cdk4 activity is also significantly decreased in the treated cells and is accompanied by an increased expression of the Cdk inhibitor p21(CIP1). Furthermore, we show that lactoferrin maintains the cell cycle progression regulator retinoblastoma protein pRb in a hypophosphorylated form. Additional experiments with synchronized cells by serum depletion confirm the anti-proliferative activity of human lactoferrin. These effects of lactoferrin occur through a p53-independent mechanism both in MDA-MB-231 cells and other epithelial cell lines such as HBL-100, MCF-7, and HT-29. These findings demonstrate that lactoferrin induces growth arrest by modulating the expression and the activity of key G1 regulatory proteins.     

Molecular basis for the specificity of p27 toward cyclin-dependent kinases that regulate cell division

The cyclin-dependent kinase inhibitors (CKIs) bind to and directly regulate the catalytic activity of cyclin-dependent kinase (Cdk)/cyclin complexes involved in cell cycle control and do not regulate other, closely related Cdks. We showed previously that the CKI, p27, binds to Cdk2/cyclin A though a sequential mechanism that involves folding-on-binding. The first step in the kinetic mechanism is interaction of a small, highly dynamic domain of p27 (domain 1) with the cyclin subunit of the Cdk2/cyclin A complex, followed by much slower binding of a more lengthy and less flexible domain (domain 2) to Cdk2. The second step requires folding of domain 2 into the kinase inhibitory conformation. Rapid binding of p27 domain 1 to cyclin A tethers the inhibitor to the binary Cdk2/cyclin A complex, which reduces the entropic barrier associated with slow binding of domain 2 to the catalytic subunit. We show here that p27/cyclin interactions are an important determinant of p27 specificity towards cell cycle Cdks. We used surface plasmon resonance, limited proteolysis, mass spectrometry, and NMR spectroscopy to study the interaction of p27 with Cdk2/cyclin A, and with another Cdk complex, Cdk5/p25, that is involved in neurodegeneration. Importantly, Cdk5/p35 (the parent complex of Cdk5/p25) is not regulated by p27 in neurons. Our results show that p27 binds to Cdk5 and Cdk2 with similar, slow kinetics. However, p27 fails to interact with p25 within the Cdk5/p25 complex, which we believe prevents formation of a kinetically trapped, inhibited p27/Cdk5/p25 complex in vivo. The helical topology of p25 is very similar to that of cyclin A. However, p25 lacks the MRAIL sequence in one helix that, in the cell cycle cyclins, mediates specific interactions with domain 1 of p21 and p27. Our results strongly suggest that p21 and p27, related Cdk inhibitors, select their cell cycle regulatory Cdk targets by binding specifically to the cyclin subunit of these Cdk/cyclin complexes as a first step in a sequential, folding-on-binding mechanism.     

Dna damage-induced G(1) arrest in hematopoietic cells is overridden following phosphatidylinositol 3-kinase-dependent activation of cyclin-dependent kinase 2

Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G(1) checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that gamma-irradiated murine myeloid 32D cells arrest in G(1) with active cyclin D-cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21(Cip1) and p27(Kip1), yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G(1) arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.     

Cdk2 acts upstream of mitochondrial permeability transition during paclitaxel-induced apoptosis

Sequential activation of cyclin-dependent kinases (Cdks) controls mammalian cell cycle. Here we demonstrate that the upregulation of cyclin-dependent kinase 2 (Cdk2) activity coincides with the loss of mitochondrial membrane potential (MMP) in paclitaxel-induced apoptosis. Ectopic expression of the dominant negative Cdk2 (Cdk2-dn) and a specific Cdk2 inhibitor, p21^WAF1/CIP1, effectively suppresses the loss of MMP, the release of cytochrome c, and subsequent activation of caspase-3 in paclitaxel-treated cells. Whereas forced activation of Cdk2 by overexpression of cyclin A dramatically promotes these events. We further show that Cdk2 activation status does not interfere with a procedure that lies downstream of cytochrome c release induced by Bax protein. These findings suggest that Cdk2 kinase can regulate apoptosis at earlier stages than mitochondrial permeability transition and cytochrome c release.     

The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G(1) when exposed to 95% oxygen. Instead, cells arrested in S and G(2). Stable expression of p53 restored induction of p21 and G(1) arrest without affecting mRNA expression of the other Cip or INK4 G(1) kinase inhibitors. To confirm the role of p21 in G(1) arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G(1) arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G(1) during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G(1) arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.     

Non-malignant and tumor-derived cells differ in their requirement for p27Kip1 in transforming growth factor-beta-mediated G1 arrest

Transforming growth factor beta (TGF-beta) induces G(1) arrest in susceptible cells by multiple mechanisms that inhibit the G(1) cyclin-dependent kinases (Cdks), including Cdk2, Cdk4, and Cdk6. TGF-beta treatment of early passage finite lifespan human mammary epithelial cells (HMECs) led to an accumulation of p27(Kip1) in cyclin E1-Cdk2 complexes and kinase inhibition. The requirement for p27 in the G(1) arrest by TGF-beta was assessed by transfection of antisense p27 (ASp27) oligonucleotides into TGF-beta-treated HMECs. Despite a reduction in total and cyclin E-Cdk2 bound p27 after ASp27 transfection, HMECs remained arrested in the G(1) phase. Maintenance of the G(1) arrest was accompanied by increased association of the Cdk inhibitor p21(WAF-1/Cip-1) and the retinoblastoma family member p130(Rb2) in cyclin E1-Cdk2 complexes along with kinase inhibition. In contrast to the findings in HMECs, p27 was essential for G(1) arrest by TGF-beta in two tumor-derived lines. ASp27 transfection into two TGF-beta-responsive, cancer-derived lines was not associated with increased compensatory binding of p21 and p130 to cyclin E1-Cdk2, and these cell lines failed to maintain G(1) arrest despite the continued presence of TGF-beta. Progressive cell cycle deregulation leading to impaired checkpoint controls during malignant tumor progression may alter the role of p27 from a redundant to an essential inhibitor of G(1)-to-S phase progression.     

Calcitonin receptor-mediated growth suppression of HEK-293 cells is accompanied by induction of p21WAF1/CIP1 and G2/M arrest.

We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.     

The role of the LH subdomain in the function of the Cip/Kip cyclin-dependent kinase regulators

The Cip/Kip protein family, which includes p27, p21, and p57, modulates the activity of cyclin-dependent kinases (Cdks). A domain within these proteins, termed the kinase inhibitory domain (KID), is necessary and sufficient for Cdk inhibition. The KID consists of a cyclin-binding subdomain (termed D1) and a Cdk-binding subdomain (termed D2) joined by a 22-residue linker subdomain (termed LH). Before binding the Cdks, D1 and D2 are largely unstructured and the LH subdomain exhibits nascent helical characteristics. Curiously, although the sequence of the linker subdomain is not highly conserved within the family, its nascent helical structure is conserved. In this study, we explored the role of this structural conservation in interactions with cyclin-dependent kinase 2 (Cdk2) and cyclin A. We constructed chimeric p27-KID molecules in which the p27 LH subdomain was replaced with the corresponding segments of either p21 or p57. The chimeric molecules bind and inhibit Cdk2 in a manner similar to wild-type p27-KID. However, the extent of enthalpy/entropy compensation associated with these interactions was dramatically different, indicating different extents of LH subdomain folding upon binding. Our results indicate that the different LH subdomains, despite their sequence and thermodynamic differences, play similar roles in binding and inhibiting Cdk2/cyclin A.