Tumor suppressor gene p16/INK4A/CDKN2A‐dependent regulation into and out of the cell cycle in a spontaneous canine model of breast cancer

p16/INK4A/CDKN2A is an important tumor suppressor gene that arrests cell cycle in G1 phase inhibiting binding of CDK4/6 with cyclin D1, leaving the Rb tumor suppressor protein unphosphorylated and E2F bound and inactive. We hypothesized that p16 has a role in exit from cell cycle that becomes defective in cancer cells. Well characterized p16‐defective canine mammary cancer cell lines (CMT28, CMT27, and CMT12), derived stably p16‐transfected CMT cell clones (CMT27A, CMT27H, CMT28A, and CMT28F), and normal canine fibroblasts (NCF), were used to investigate expression of p16 after serum starvation into quiescence followed by re‐feeding to induce cell cycle re‐entry. The parental CMT cell lines used lack p16 expression either at the mRNA or protein expression levels, while p27 and other p16‐associated proteins, including CDK4, CDK6, cyclin D1, and Rb, were expressed. We have successfully demonstrated cell cycle arrest and relatively synchronous cell cycle re‐entry in parental CMT12, CMT28 and NCF cells as well as p16 transfected CMT27A, CMT27H, CMT28A, and CMT28F cells and confirmed this by ³H‐thymidine incorporation and flow cytometric analysis of cell cycle phase distribution. p16‐transfected CMT27A and CMT27H cells exited cell cycle post‐serum‐starvation in contrast to parental CMT27 cells. NCF, CMT27A, and CMT28F cells expressed upregulated levels of p27 and p16 mRNA, post‐serum starvation, as cells exited cell cycle and entered quiescence. Because quiescence and differentiation are associated with increased levels of p27, our data demonstrating that p16 was upregulated along with p27 during quiescence, suggests a potential role for p16 in maintaining these non‐proliferative states. J. Cell. Biochem. 114: 1355–1363, 2013. © 2012 Wiley Periodicals, Inc.     

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 cyclin-dependent kinase 11 interacts with 14-3-3 proteins

Cyclin-dependent kinase 11 isoforms (CDK11) are members of the p34(cdc2) superfamily. They have been shown to play a role in RNA processing and apoptosis. In the present study, we investigate whether CDK11 interacts with 14-3-3 proteins. Our study shows that the putative 14-3-3 binding site (113-RHRSHS-118) within the N-terminal domain of CDK11(p110) is functional. Endogenous CDK11(p110) binds directly to 14-3-3 proteins and phosphorylation of the serine 118 within the RHRSHS motif seems to be required for the binding. Besides, CDK11(p110) is capable of interacting with several different isoforms of 14-3-3 proteins both in vitro and in vivo. The interaction of 14-3-3 gamma with CDK11(p110) occurs throughout the entire cell cycle and reaches maximum at the G2/M phase. Interestingly, 14-3-3 gamma shows strong interaction with N-terminal portion of caspase-cleaved CDK11(p110) (CDK11(p60)) product at 48 h after Fas treatment, which correlates with the maximal cleavage level of CDK11(p110) and the maximum activation level of CDK11 kinase activity during apoptosis. Collectively, these results suggest that CDK11 kinases could be regulated by interaction with 14-3-3 proteins during cell cycle and apoptosis.     

Role of PI3K/AKT/mTOR signaling in the cell cycle progression of human prostate cancer

Prostate cancer is one of the most common cancers among men. Recent studies demonstrated that PI3K signaling is an important intracellular mediator which is involved in multiple cellular functions including proliferation, differentiation, anti-apoptosis, tumorigenesis, and angiogenesis. In the present study, we demonstrate that the inhibition of PI3K activity by LY294002, inhibited prostate cancer cell proliferation and induced the G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins including cyclin D1, CDK4, and Rb phosphorylation at Ser780, Ser795, and Ser807/811, whereas expression of CDK6 and beta-actin was not affected by LY294002. The expression of cyclin kinase inhibitor, p21(CIP1/WAF1), was induced by LY294002, while levels of p16(INK4) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation and p70(S6K), but not MAPK. PI3K regulates cell cycle through AKT, mTOR to p70(S6K). The mTOR inhibitor rapamycin has similar inhibitory effects on G(1) cell cycle progression and expression of cyclin D1, CDK4, and Rb phosphorylation. These results suggest that PI3K mediates G(1) cell cycle progression and cyclin expression through the activation of AKT/mTOR/p70(S6K) signaling pathway in the prostate cancer cells.     

2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.     

Induction of p18INK4c and its predominant association with CDK4 and CDK6 during myogenic differentiation.

Terminal cell differentiation involves permanent withdrawal from the cell division cycle. The inhibitors of cyclin-dependent kinases (CDKs) are potential molecules functioning to couple cell cycle arrest and cell differentiation. In murine C2C12 myoblast cells, G1 CDK enzymes (CDK2, CDK4, and CDK6) associate with four CDK inhibitors: p18INK4c, p19INK4d, p21, and p27Kip1. During induced myogenesis, p21 and its associated CDK proteins underwent an initial increase followed by a decrease as cells became terminally differentiated. The level of p27 protein gradually increased, but the amount of total associated CDK proteins remained unchanged. p19 protein decreased gradually during differentiation, as did its associated CDK4 protein. In contrast, p18 protein increased 50-fold, from negligible levels in proliferating myoblasts to clearly detectable levels within 8-12 h of myogenic induction. This initial rise was followed by a precipitous increase between 12 and 24 h postinduction, with p18 protein finally accumulating to its highest level in terminally differentiated cells. Induction of p18 correlated with increased and sequential complex formation--first increasing association with CDK6 and then with CDK4 over the course of myogenic differentiation. All of the CDK6 and half of the CDK4 were complexed with p18 in terminally differentiated C2C12 cells as well as in adult mouse muscle tissue. Finally, kinase activity of CDK2 and CDK4 decreases as C2C12 cells differentiate, whereas the CDK6 kinase activity is low in both proliferating myoblasts and differentiated myotubes. Our results indicate that p18 may play a critical role in causing and/or maintaining permanent cell cycle arrest associated with mature muscle formation.     

Direct binding of the N-terminus of HTLV-1 tax oncoprotein to cyclin-dependent kinase 4 is a dominant path to stimulate the kinase activity

The involvement of Tax oncoprotein in the INK4-CDK4/6-Rb pathway has been regarded as a key factor for immortalization and transformation of human T-cell leukemia virus 1 (HTLV-1) infected cells. In both p16 -/- and +/+ cells, expression of Tax has been correlated with an increase in CDK4 activity, which subsequently increases the phosphorylation of Rb and drives the infected cells into cell cycle progression. In relation to these effects, Tax has been shown to interact with two components of the INK4-CDK4/6-Rb pathway, p16 and cyclin D(s). While Tax competes with CDK4 for p16 binding, thus suppressing p16 inhibition of CDK4, Tax also binds to cyclin D(s) with concomitant increases in both CDK4 activity and the phosphorylation of cyclin D(s). Here we show that both Tax and residues 1-40 of the N-terminus of Tax, Tax40N, bind to and activate CDK4 in vitro. In the presence of INK4 proteins, binding of Tax and Tax40N to CDK4 counteracts against the inhibition of p16 and p18 and acts as the major path to regulate Tax-mediated activation of CDK4. We also report that Tax40N retains the transactivation ability. These results of in vitro studies demonstrate a potentially novel, p16-independent route to regulate CDK4 activity by the Tax oncoprotein in HTLV-1 infected cells.     

Parathyroid hormone and the regulation of cell cycle in colon adenocarcinoma cells

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In this study, we investigated the role of PTH in the regulation of the cell cycle in human colon adenocarcinoma Caco-2 cells. Flow cytometry analysis revealed that PTH (10^− 8 M, 12-24 h) treatment increases the number of cells in the G0/G1 phase and diminishes the number in both phases S and G2/M. In addition, analysis by Western blot showed that the hormone increases the expression of the inhibitory protein p27Kip1 and diminishes the expression of cyclin D1, cyclin D3 and CDK6. However, the amounts of CDK4, p21Cip1, p15INK4B and p16INK4A were not different in the absence or presence of PTH. Inhibitors of PKC (Ro-318220, bisindolylmaleimide and chelerythine), but not JNK (SP600125) and PP2A (okadaic acid and calyculin A), reversed PTH response in Caco-2 cells. Taken together, our results suggest that PTH induces G0/G1 phase arrest of Caco-2 intestinal cells and changes the expression of proteins involved in cell cycle regulation via the PKC signaling pathway.     

Induced expression of p16(INK4a) inhibits both CDK4- and CDK2-associated kinase activity by reassortment of cyclin-CDK-inhibitor complexes

To investigate the mode of action of the p16(INK4a) tumor suppressor protein, we have established U2-OS cells in which the expression of p16(INK4a) can be regulated by addition or removal of isopropyl-beta-D-thiogalactopyranoside. As expected, induction of p16(INK4a) results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16(INK4a) also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27(KIP1). Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16(INK4a), this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16(INK4a). Sequestration of CDK4 by p16(INK4a) allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16(INK4a), p27(KIP1) appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27(KIP1) and p21(CIP1). Significantly, p16(INK4a) itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.     

Association of the cyclin-dependent kinases and 14-3-3 sigma negatively regulates cell cycle progression

14-3-3 sigma, implicated in cell cycle arrest by p53, was cloned by expression cloning through cyclin-dependent kinase 2 (CDK2) association. 14-3-3 sigma shares cyclin-CDK2 binding motifs with different cell cycle regulators, including p107, p130, p21(CIP1), p27(KIP1), and p57(KIP2), and is associated with cyclin.CDK complexes in vitro and in vivo. Overexpression of 14-3-3 sigma obstructs cell cycle entry by inhibiting cyclin-CDK activity in many breast cancer cell lines. Overexpression of 14-3-3 sigma can also inhibit cell proliferation and prevent anchorage-independent growth of these cell lines. These findings define 14-3-3 sigma as a negative regulator of the cell cycle progression and suggest that it has an important function in preventing breast tumor cell growth.     

INK4d-deficient mice are fertile despite testicular atrophy

The INK4 family of cyclin-dependent kinase (CDK) inhibitors includes four 15- to 19-kDa polypeptides (p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d)) that bind to CDK4 and CDK6. By disrupting cyclin D-dependent holoenzymes, INK4 proteins prevent phosphorylation of the retinoblastoma protein and block entry into the DNA-synthetic phase of the cell division cycle. The founding family member, p16(INK4a), is a potent tumor suppressor in humans, whereas involvement, if any, of other INK4 proteins in tumor surveillance is less well documented. INK4c and INK4d are expressed during mouse embryogenesis in stereotypic tissue-specific patterns and are also detected, together with INK4b, in tissues of young mice. INK4a is expressed neither before birth nor at readily appreciable levels in young animals, but its increased expression later in life suggests that it plays some checkpoint function in response to cell stress, genotoxic damage, or aging per se. We used targeted gene disruption to generate mice lacking INK4d. These animals developed into adulthood, had a normal life span, and did not spontaneously develop tumors. Tumors did not arise at increased frequency in animals neonatally exposed to ionizing radiation or the carcinogen dimethylbenzanthrene. Mouse embryo fibroblasts, bone marrow-derived macrophages, and lymphoid T and B cells isolated from these animals proliferated normally and displayed typical lineage-specific differentiation markers. Males exhibited marked testicular atrophy associated with increased apoptosis of germ cells, although they remained fertile. The absence of tumors in INK4d-deficient animals demonstrates that, unlike INK4a, INK4d is not a tumor suppressor but is instead involved in spermatogenesis.     

An important role of CDK inhibitor p18(INK4c) in modulating antigen receptor-mediated T cell proliferation.

The inhibitors of cyclin-dependent kinase (CDK) 4 (INK4) bind CDK4/6 to prevent their association with D-cyclins and G(1) cell cycle initiation and progression. We report here that among the seven CDK inhibitors, p18(INK4c) played an important role in modulating TCR-mediated T cell proliferation. Loss of p18(INK4c) in T cells led to hyperproliferation in response to CD3 stimulation. p18(INK4c)-null mice developed lymphoproliferative disorder and T cell lymphomas. Expression of IL-2, IL-2R-alpha, and the major G(1) cell cycle regulatory proteins was not altered in p18-null T cells. Both FK506 and rapamycin efficiently inhibited proliferation of p18-null T cells. In activated T cells, p18(INK4c) remained constant, and preferentially associated with and inhibited CDK6 but not CDK4. We propose that p18(INK4c) sets an inhibitory threshold in T cells and one function of CD28 costimulation is to counteract the p18(INK4c) inhibitory activity on CDK6-cyclin D complexes. The p18(INK4c) protein may provide a novel target to modulate T cell immunity.     

Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors

Cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.     

Molecular Docking of Known Carcinogen 4- (Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with Cyclin Dependent Kinases towards Its Potential Role in Cell Cycle Perturbation

Cell cycle is maintained almost all the times and is controlled by various regulatory proteins and their complexes (Cdk+Cyclin) in different phases of interphase (G₁, S and G₂) and mitosis of cell cycle. A number of mechanisms have been proposed for the initiation and progression of carcinogenesis by abruption in cell cycle process. One of the important features of cancer/carcinogenesis is functional loss of these cell cycle regulatory proteins particularly in CDKs and cyclins. We hypothesize that there is a direct involvement of these cell cycle regulatory proteins not only at the genetic level but also proteins level, during the initiation of carcinogenesis. Therefore, it becomes significant to determine inconsistency in the functioning of regulatory proteins due to interaction with carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Hence, we investigated the interaction efficiency of NNK, against cell cycle regulatory proteins. We found a different value of ΔG (free energy of binding) among the studied proteins ranging between -3.29 to -7.25 kcal/mol was observed. To validate the results, we considered Human Oxy-Hemoglobin at 1.25 Å Resolution, [PDB_ID:1HHO] as a +ve control, (binding energy -6.06 kcal/mol). Finally, the CDK8 (PDB_ID:3RGF) and CDK2 (PDB_ID:3DDP) regulatory proteins showing significantly strong molecular interaction with NNK -7.25 kcal/mol, -6.19 kcal/mol respectively were analyzed in details. In this study we predicted that CDK8 protein fails to form functional complex with its complementary partner cyclin C in presence of NNK. Consequently, inconsistency of functioning in regulatory proteins might lead to the abruption in cell cycle progression; contribute to the loss of cell cycle control and subsequently increasing the possibility of carcinogenesis.