Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation

Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.     

Distinct versus redundant properties among members of the INK4 family of cyclin-dependent kinase inhibitors

p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d) comprise a family of cyclin-dependent kinase inhibitors and tumor suppressors. We report that the INK4 proteins share the ability to arrest cells in G1, and interact with CDK4 or CDK6 with similar avidity. In contrast, only p18 and particularly p19 are phosphorylated in vivo, and each of the human INK4 proteins shows unique expression patterns dependent on cell and tissue type, and differentiation stage. Thus, the INK4 proteins harbor redundant as well as non-overlapping properties, suggesting distinct regulatory modes, and diverse roles for the individual INK4 family members in cell cycle control, cellular differentiation, and multistep oncogenesis.     

The multiple roles of the cyclin-dependent kinase inhibitory protein p57(KIP2) in cerebral cortical neurogenesis

The members of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitory proteins (CKIs), including p57(KIP2), p27(KIP1), and p21(CIP1), block the progression of the cell cycle by binding and inhibiting cyclin/CDK complexes of the G1 phase. In addition to this well-characterized function, p57(KIP2) and p27(KIP1) have been shown to participate in an increasing number of other important cellular processes including cell fate and differentiation, cell motility and migration, and cell death/survival, both in peripheral and central nervous systems. Increasing evidence over the past few years has characterized the functions of the newest CIP/KIP member p57(KIP2) in orchestrating cell proliferation, differentiation, and migration during neurogenesis. Here, we focus our discussion on the multiple roles played by p57(KIP2) during cortical development, making comparisons to p27(KIP1) as well as the INK4 family of CKIs.     

Limited overlapping roles of P15(INK4b) and P18(INK4c) cell cycle inhibitors in proliferation and tumorigenesis

Entry of quiescent cells into the cell cycle is driven by the cyclin D-dependent kinases Cdk4 and Cdk6. These kinases are negatively regulated by the INK4 cell cycle inhibitors. We report the generation of mice defective in P15(INK4b) and P18(INK4c). Ablation of these genes, either alone or in combination, does not abrogate cell contact inhibition or senescence of mouse embryo fibroblasts in culture. However, loss of P15(INK4b), but not of P18(INK4c), confers proliferative advantage to these cells and makes them more sensitive to transformation by H-ras oncogenes. In vivo, ablation of P15(INK4b) and P18(INK4c) genes results in lymphoproliferative disorders and tumor formation. Mice lacking P18(INK4c) have deregulated epithelial cell growth leading to the formation of cysts, mostly in the cortical region of the kidneys and the mammary epithelium. Loss of both P15(INK4b) and P18(INK4c) does not result in significantly distinct phenotypic manifestations except for the appearance of cysts in additional tissues. These results indicate that P15(INK4b) and P18(IKN4c) are tumor suppressor proteins that act in different cellular lineages and/or pathways with limited compensatory roles.     

Cellular and molecular evidence for malignancy-inhibitory functions of p15RS

The gene for the INK4 family Cdk inhibitor p15^INK4B is frequently deleted or inactivated in multiple types of human cancers, indicating that p15 is a tumor suppressor. p15RS is a ubiquitously expressed nuclear protein that is positively regulated by p15 and, in turn, inhibits the expression of cyclin D and cyclin E. To determine whether p15RS has malignancy inhibitory functions in addition to its inhibitory effects on cell cycle entry, we ectopically expressed p15RS in metastatic melanoma A375 cells, in which p15 gene is deleted and p15RS expression is dramatically downregulated, and examined the effect on various malignant phenotypes. Here, we report that while the p15RS expression had little effect on cell growth in monolayer cultures, it dramatically inhibited anchorage-independent cell growth in soft agar, a hallmark for malignancy. p15RS expression also inhibited cell migration and invasion, which are key determinants of metastasis. At molecular levels, p15RS expression specifically downregulates the expression of cathepsin B and MMP-9 at RNA levels, which are known to promote cell invasion through degrading extracellular matrix proteins. These results indicate that p15RS has malignancy inhibitory functions independent of cell cycle inhibition and provide novel insights on the role of p15 in tumor inhibition.     

Alterations in the cell cycle and in the protein level of cyclin D1, p21CIP1, and p16INK4a after exposure to 50 Hz MF in human cells

The effect of exposure to 50 Hz, 1 mT magnetic fields (MF) on the cell cycle in general, on the DNA synthesis in S-phase, and on the G1-phase regulating proteins Cdk4, cyclin D1, p16INK4a, and p21CIP1 was investigated in human amniotic fluid cells. The BrdU-incorporation assay revealed a significant diminution of S-phase cells in MF-exposed cultures. The protein level of Cdk4 did not change, but MF induced a decreased expression of cyclin D1 after 24 h and 30 h exposures. The level of p16INK4a increased at 1 h and 12 h after exposure, whereas the expression of p21CIP1 was enhanced at 6 h and 12 h after exposure. Reduced levels of both Cdk inhibitors were observed at longer exposure times (24 h, 30 h). Our results suggest an inhibitory effect of MF on the G1-phase induced by altered expression of p16INK4a and p21CIP1.     

Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function

Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G(1)/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16(INK4a) to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16(INK4a) inhibited G(1)/S transition induced in MCF-7 cells by 17-beta-estradiol (E(2)) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G(1) and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21(Cip1) and p27(Kip1) was decreased, however, in both control and p16(INK4a)-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E(2) in control and p16(INK4a)-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16(INK4a). Inhibition of Cdc25A activity in p16(INK4a)-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E(2)-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16(INK4a)-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21(Cip1) and p27(Kip1).     

Requirements for cell cycle arrest by p16INK4a

Analysis of tumor-derived mutations has led to the suggestion that p16INK4a, cyclin D1, cdk4, and the retinoblastoma protein (pRB) are components of a regulatory pathway that is inactivated in most tumor cells. Cell cycle arrest induced by p16INK4a, an inhibitor of cyclin D-dependent kinases, requires pRB, and it has been proposed that this G1 arrest is mediated by pRB-E2F repressor complexes. By comparing the properties of primary mouse embryonic fibroblasts specifically lacking pRB-family members, we find that pRB is insufficient for a p16INK4a-induced arrest. In addition to pRB, a second function provided by either p107 or p130, two pRB-related proteins, is required for p16INK4a to block DNA synthesis. We infer that p16INK4a-induced arrest is not mediated exclusively by pRB, but depends on the nonredundant functions of at least two pRB-family members.     

Genetic evidence for functional dependency of p18Ink4c on Cdk4

The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and induces the growth-suppressive function of Rb family proteins. Mutations in the Cdk4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice, suggesting that INK4 is a major regulator of CDK4. Mice lacking the Cdk4 gene exhibit various defects in many organs associated with hypocellularity, whereas loss of the p18(Ink4c) gene results in widespread hyperplasia and organomegaly. To genetically test the notion that the function of INK4 is dependent on CDK4, we generated p18; Cdk4 double-mutant mice and examined the organs and tissues which developed abnormalities when either gene is deleted. We show here that, in all organs we have examined, including pituitary, testis, pancreas, kidney, and adrenal gland, hyperproliferative phenotypes associated with p18 loss were canceled. The double-mutant mice exhibited phenotypes very close to or indistinguishable from that of Cdk4 single-mutant mice. Mice lacking p27(Kip1) develop widespread hyperplasia and organomegaly similar to those developed by p18-deficient mice. The p27; Cdk4 double-mutant mice, however, displayed phenotypes intermediate between those of p27 and Cdk4 single-mutant mice. These results provide genetic evidence that in mice p18(Ink4c) and p27(Kip1) mediate the transduction of different cell growth and proliferation signals to CDK4 and that p18(Ink4c) is functionally dependent on CDK4.     

Cooperation of p27(Kip1) and p18(INK4c) in progestin-mediated cell cycle arrest in T-47D breast cancer cells

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G(1) cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27(Kip1) among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27(Kip1) and few were bound to p21(Cip1). In vitro, recombinant His(6)-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His(6)-p27 in vitro or p27(Kip1) in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18(INK4c) and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18(INK4c) led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27(Kip1) and p18(INK4c) cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor beta and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.     

Cyclin D-CDK subunit arrangement is dependent on the availability of competing INK4 and p21 class inhibitors

The D-type cyclins and their major kinase partners CDK4 and CDK6 regulate G0-G1-S progression by contributing to the phosphorylation and inactivation of the retinoblastoma gene product, pRB. Assembly of active cyclin D-CDK complexes in response to mitogenic signals is negatively regulated by INK4 family members. Here we show that although all four INK4 proteins associate with CDK4 and CDK6 in vitro, only p16(INK4a) can form stable, binary complexes with both CDK4 and CDK6 in proliferating cells. The other INK4 family members form stable complexes with CDK6 but associate only transiently with CDK4. Conversely, CDK4 stably associates with both p21(CIP1) and p27(KIP1) in cyclin-containing complexes, suggesting that CDK4 is in equilibrium between INK4 and p21(CIP1)- or p27(KIP1)-bound states. In agreement with this hypothesis, overexpression of p21(CIP1) in 293 cells, where CDK4 is bound to p16(INK4a), stimulates the formation of ternary cyclin D-CDK4-p21(CIP1) complexes. These data suggest that members of the p21 family of proteins promote the association of D-type cyclins with CDKs by counteracting the effects of INK4 molecules.     

Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product.

D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, regulate events in the G1 phase of the cell cycle and may contribute to the phosphorylation of the retinoblastoma gene product (Rb). However, in cells in which the function of Rb has been compromised, either by naturally arising mutations or through binding to proteins encoded by DNA tumour viruses, Cdk4 and Cdk6 are not associated with D cyclins. Instead, both kinases form binary complexes with a stable 16 kDa protein (p16) encoded by the putative tumour suppressor gene INK4/MTS1 on human chromosome 9p21. Here we show an inverse correlation between Rb status and the expression of p16. Since Rb-negative cells express high levels of p16, we suggest that in these cells p16 competes with D cyclins for binding to Cdk4 and Cdk6 and prevents formation of active complexes. In line with these predictions, DNA tumour virus oncoproteins do not disrupt cyclin D1-Cdk4 complexes in cells lacking p16.     

Mechanism of cell cycle entry mediated by the intrinsically disordered protein p27(Kip1)

p27(Kip1) (p27), a prototypical intrinsically disordered protein (IDP), regulates eukaryotic cell division through interactions with cyclin-dependent kinase (Cdk)/cyclin complexes. The activity, stability, and subcellular localization of p27 are regulated by phosphorylation. We illustrate how p27 integrates regulatory signals from several non-receptor tyrosine kinases (NRTKs) to activate Cdk4 and initiate cell cycle entry. Unmodified p27 potently inhibits Cdk/cyclin complexes, including Cdk4/cyclin D (IC(50), 1 nM). Some NRTKs (e.g., Abl) phosphorylate p27 on Tyr 88, which facilitates a second modification on Tyr 74 by another NRTK (e.g., Src). Importantly, this second modification causes partial reactivation of Cdk4 within ternary complexes containing doubly Tyr phosphorylated p27. Partial activation of Cdk4 initiates entry into the cell division cycle. Therefore, p27's disordered features enable NRTKs to sequentially promote a phosphorylation cascade that controls cell fate. Beyond cell cycle control, these results illustrate general concepts regarding why IDPs are well-suited for roles in signaling and regulation in biological systems.     

Cdk4 activation is dependent on the subunit rearrangement in the complexes

Although several factors have been implicated in the regulation of Cdk4 activity, little is known regarding the contributions of cyclin-dependent kinase inhibitors (CKIs) in Cdk4 activation in the mid G1 phase. Using a mouse macrophage cell line (Bac1.2F5), we found that most of Cdk4 bound to p15 when cells were in a quiescent state. Following CSF-1 stimulation, Cdk4 bound to cyclin D1 and then to p21, concomitant with the dissociation of p15 from the complexes. The activation of Cdk4 correlated well with p21 binding to the complexes, and the majority of active Cdk4 complexes contained p21. During regeneration of mouse liver after partial hepatectomy, Cdk4 activity coincided precisely with ternary complex formation of cyclin D1/Cdk4/p21. Using the baculovirus expression system, we succeeded in reconstituting a capacity for Cdk4 activation in insect cells, forming an active cyclin D1/Cdk4/p21 ternary complex. Taken together, it is suggested that p21 and cyclin D1 act cooperatively as activators of Cdk4 through the release of CKIs of the INK4 family.     

Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors

The cyclin-dependent kinases (Cdks) have a central role in coordinating the eukaryotic cell division cycle, and also serve to integrate diverse growth-regulatory signals. Cdks are controlled through several different processes involving the binding of activating cyclin subunits, of inhibitory Cip or INK4 subunits, and phosphorylation. Crystallographic studies of Cdks in four different complexes, reviewed here, have revealed the mechanisms by which these regulatory processes control the Cdk switches. All of these mechanisms involve conformational changes in and around the catalytic cleft of the kinase, indicating that Cdks have evolved an intrinsic conformational flexibility. This flexibility is central to their ability to switch states in response to a diverse range of growth-regulatory signals.     

Incomplete folding upon binding mediates Cdk4/cyclin D complex activation by tyrosine phosphorylation of inhibitor p27 protein

p27(Kip1) (p27), an intrinsically disordered protein, regulates the various Cdk/cyclin complexes that control cell cycle progression. The kinase inhibitory domain of p27 contains a cyclin-binding subdomain (D1), a Cdk-binding subdomain (D2), and a linker helix subdomain that connects D1 and D2. Here, we report that, despite extensive sequence conservation between Cdk4/cyclin D1 (hereafter Cdk4/cyclin D) and Cdk2/cyclin A, the thermodynamic details describing how the individual p27 subdomains contribute to equally high affinity binding to these two Cdk/cyclin complexes are strikingly different. Differences in enthalpy/entropy compensation revealed that the D2 subdomain of p27 folds incompletely when binding Cdk4/cyclin D versus Cdk2/cyclin A. Incomplete binding-induced folding exposes tyrosine 88 of p27 for phosphorylation by the nonreceptor tyrosine kinase Abl. Importantly, tyrosine phosphorylation (of p27) relieves Cdk inhibition by p27, enabling cell cycle entry. Furthermore, the interaction between a conserved hydrophobic patch on cyclin D and subdomain D1 is much weaker than that with cyclin A; consequently, a construct containing subdomains D1 and LH (p27-D1LH) does not inhibit substrate binding to Cdk4/cyclin D as it does to Cdk2/cyclin A. Our results provide a mechanism by which Cdk4 (within the p27/Cdk4/cyclin D complex) is poised to be activated by extrinsic mitogenic signals that impinge upon p27 at the earliest stage of cell division. More broadly, our results further illustrate the regulatory versatility of intrinsically disordered proteins.