Inhibition Of Cdk2 Kinase Activity Research Articles

Indisulam synergizes with palbociclib to induce senescence through inhibition of CDK2 kinase activity.

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

The cytotoxic and mechanistic effects of aaptamine on hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common form of cancer and the third most frequent cause of cancer-associated mortality worldwide. We isolated aaptamine from the marine sponge Aaptos, and synthesized derivatives of this compound. Aaptamine and synthetic derivatives displayed various biological activities. This represents the first account of studies on the effects of aaptamine and its derivatives in hepatocarcinogenesis. In this study, Cell Counting Kit (CCK8) was used to evaluate the anti-proliferative effect of aaptamine on HCC in vitro. Additionally, a subcutaneous xenograft model was used to determine if aaptamine could inhibit hepatocellular carcinoma in vivo. We also used RT-PCR and Western blot to analyze the mechanisms behind these effects. Our results showed that aaptamine has anti-proliferation effects on the cell lines LM3 and HepG2. Aaptamine also suppressed the colony-formation ability of HCC cells. We found that aaptamine treatment led to cell cycle arrest in HCC cells, reduced the expression of SOX9 and CDK2. Significant anti-tumor effects were observed in aaptamine-administered tumor-bearing mice as compared to controls. However, structural changes made to aaptamine yielded two derivatives for which all the effects listed above were considerably reduced as compared to the original compound aaptamine. In conclusion, aaptamine is demonstrated for the first time to inhibit liver cancer progression. The aaptamine-induced cell cycle arrest was associated with the increased binding of p21 to Cdk2-cyclin D/E complexes, inhibition of Cdk2 kinase activity in HCC cells. Furthermore, aaptamine appears to be a promising and efficient treatment of liver cancer HCC-LM3 in vivo. We have also uncovered structural changes that might affect the biological activity. The work provides a promising drug candidate for HCC treatment.

Inhibition of Cdk2 kinase activity selectively targets the CD44⁺/CD24⁻/Low stem-like subpopulation and restores chemosensitivity of SUM149PT triple-negative breast cancer cells.

Inflammatory breast cancer (IBC) is an angioinvasive and most aggressive type of advanced breast cancer characterized by rapid proliferation, chemoresistance, early metastatic development and poor prognosis. IBC tumors display a triple-negative breast cancer (TNBC) phenotype characterized by centrosome amplification, high grade of chromosomal instability (CIN) and low levels of expression of estrogen receptor α (ERα), progesterone receptor (PR) and HER-2 tyrosine kinase receptor. Since the TNBC cells lack these receptors necessary to promote tumor growth, common treatments such as endocrine therapy and molecular targeting of HER-2 receptor are ineffective for this subtype of breast cancer. To date, not a single targeted therapy has been approved for non-inflammatory and inflammatory TNBC tumors and combination of conventional cytotoxic chemotherapeutic agents remains the standard therapy. IBC tumors generally display activation of epithelial to mesenchymal transition (EMT) that is functionally linked to a CD44+/CD24−/Low stem-like phenotype. Development of EMT and consequent activation of stemness programming is responsible for invasion, tumor self-renewal and drug resistance leading to breast cancer progression, distant metastases and poor prognosis. In this study, we employed the luminal ER+ MCF-7 and the IBC SUM149PT breast cancer cell lines to establish the extent to which high grade of CIN and chemoresistance were mechanistically linked to the enrichment of CD44+/CD24low/− CSCs. Here, we demonstrate that SUM149PT cells displayed higher CIN than MCF-7 cells characterized by higher percentage of structural and numerical chromosomal aberrations. Moreover, centrosome amplification, cyclin E overexpression and phosphorylation of retinoblastoma (Rb) were restricted to the stem-like CD44+/CD24−/Low subpopulation isolated from SUM149PT cells. Significantly, CD44+/CD24−/Low CSCs displayed resistance to conventional chemotherapy but higher sensitivity to SU9516, a specific cyclin-dependent kinase 2 (Cdk2) inhibitor, demonstrating that aberrant activation of cyclin E/Cdk2 oncogenic signaling is essential for the maintenance and expansion of CD44+/CD24−/Low CSC subpopulation in IBC. In conclusion, our findings propose a novel therapeutic approach to restore chemosensitivity and delay recurrence of IBC tumors based on the combination of conventional chemotherapy with small molecule inhibitors of the Cdk2 cell cycle kinase.

Chemical-genetic analysis of cyclin dependent kinase 2 function reveals an important role in cellular transformation by multiple oncogenic pathways

A family of conserved serine/threonine kinases known as cyclin-dependent kinases (CDKs) drives orderly cell cycle progression in mammalian cells. Prior studies have suggested that CDK2 regulates S-phase entry and progression, and frequently shows increased activity in a wide spectrum of human tumors. Genetic KO/knockdown approaches, however, have suggested that lack of CDK2 protein does not prevent cellular proliferation, both during somatic development in mice as well as in human cancer cell lines. Here, we use an alternative, chemical-genetic approach to achieve specific inhibition of CDK2 kinase activity in cells. We directly compare small-molecule inhibition of CDK2 kinase activity with siRNA knockdown and show that small-molecule inhibition results in marked defects in proliferation of nontransformed cells, whereas siRNA knockdown does not, highlighting the differences between these two approaches. In addition, CDK2 inhibition drastically diminishes anchorage-independent growth of human cancer cells and cells transformed with various oncogenes. Our results establish that CDK2 activity is necessary for normal mammalian cell cycle progression and suggest that it might be a useful therapeutic target for treating cancer.

HDAC1 Cooperates with C/EBPα in the Inhibition of Liver Proliferation in Old Mice

Epigenetic control of liver proliferation involves cooperation between transcription factors and chromatin-remodeling proteins. In this work, we found that the levels of HDAC1 (histone deacetylase 1) are increased in quiescent livers of old mice. The elevation of HDAC1 in liver is mediated by the RNA-binding protein CUGBP1. We found that the age-associated CUGBP1-eIF2 complex binds to the 5' region of HDAC1 mRNA and increases translation of HDAC1 in the liver. Further analyses showed that CUGBP1 also increases expression of HDAC1 in cultured cells, in the livers of CUGBP1 transgenic mice, and in the livers of mice injected with cyclin D3, which enhances the formation of the CUGBP1-eIF2 complex. In livers of old mice, HDAC1 interacts with the transcription factor C/EBPalpha and is recruited by this protein to E2F-dependent promoters as a component of high M(r) C/EBPalpha-Brm complexes. The recruitment of HDAC1 to c-Myc and FoxM1B promoters leads to deacetylation of histone H3 at Lys-9 on these E2F-dependent promoters. We show that HDAC1 is an important mediator of growth-inhibitory activity of C/EBPalpha and that small interfering RNA-mediated inhibition of HDAC1 reduces the ability of C/EBPalpha to inhibit cell proliferation. In addition, we have found that both elevation of HDAC1 and interaction of C/EBPalpha with HDAC1 are controlled by cyclin D3-dependent mechanisms. Treatment of old mice with growth hormone, which reduces cyclin D3 levels, leads to the reduction of the CUGBP1-eIF2 complex, normalization of HDAC1 levels, and inhibition of interactions of HDAC1 with C/EBPalpha-Brm complexes. Thus, our data demonstrate that translational elevation of HDAC1 in livers of old mice is involved in the assembly of high M(r) protein-protein complexes that inhibit liver proliferation.

Indole-3-Carbinol (I3C) Inhibits Cyclin-dependent Kinase-2 Function in Human Breast Cancer Cells by Regulating the Size Distribution, Associated Cyclin E Forms, and Subcellular Localization of the CDK2 Protein Complex

Indole-3-carbinol (I3C), a dietary compound found in cruciferous vegetables, induces a robust inhibition of CDK2 specific kinase activity as part of a G1 cell cycle arrest of human breast cancer cells. Treatment with I3C causes a significant shift in the size distribution of the CDK2 protein complex from an enzymatically active 90 kDa complex to a larger 200 kDa complex with significantly reduced kinase activity. Co-immunoprecipitations revealed an increased association of both a 50 kDa cyclin E and a 75 kDa cyclin E immunoreactive protein with the CDK2 protein complex under I3C-treated conditions, whereas the 90 kDa CDK2 protein complexes detected in proliferating control cells contain the lower molecular mass forms of cyclin E. I3C treatment caused no change in the level of CDK2 inhibitors (p21, p27) or in the inhibitory phosphorylation states of CDK2. The effects of I3C are specific for this indole and not a consequence of the cell cycle arrest because treatment of MCF-7 breast cancer cells with either the I3C dimerization product DIM or the anti-estrogen tamoxifen induced a G1 cell cycle arrest with no changes in the associated cyclin E or subcellular localization of the CDK2 protein complex. Taken together, our results have uncovered a unique effect of I3C on cell cycle control in which the inhibition of CDK2 kinase activity is accompanied by selective alterations in cyclin E composition, size distribution, and subcellular localization of the CDK2 protein complex.

CAMP-mediated Inhibition of DNA Replication and S Phase Progression: Involvement of Rb, p21Cip1, and PCNA

cAMP exerts an antiproliferative effect on a number of cell types including lymphocytes. This effect of cAMP is proposed to be mediated by its ability to inhibit G1/S transition. In this report, we provide evidence for a new mechanism whereby cAMP might inhibit cellular proliferation. We show that elevation of intracellular levels of cAMP inhibits DNA replication and arrests the cells in S phase. The cAMP-induced inhibition of DNA synthesis was associated with the increased binding of p21Cip1 to Cdk2-cyclin complexes, inhibition of Cdk2 kinase activity, dephosphorylation of Rb, and dissociation of PCNA from chromatin in S phase cells. The ability of cAMP to inhibit DNA replication and trigger release of PCNA from chromatin required Rb and p21Cip1 proteins, since both processes were only marginally affected by increased levels of cAMP in Rb-/- and p21Cip1-/- 3T3 fibroblasts. Importantly, the implications of cAMP-induced inhibition of DNA synthesis in cancer treatment was demonstrated by the ability of cAMP to reduce apoptosis induced by S phase-specific cytotoxic drugs. Taken together, these results demonstrate a novel role for cAMP in regulation of DNA synthesis and support a model in which activation of cAMP-dependent signaling protects cells from the effect of S phase-specific antitumor agents.

Transforming growth factor-beta induces Cdk2 relocalization to the cytoplasm coincident with dephosphorylation of retinoblastoma tumor suppressor protein.

The transforming growth factor-beta (TGF-beta) signaling pathway functions to prevent tumorigenesis, and loss of sensitivity to TGF-beta-mediated cell cycle arrest is nearly ubiquitous among human cancers. Our previous studies demonstrated that rapamycin potentiates TGF-beta-induced cell cycle arrest in nontransformed epithelial cells and partially restores TGF-beta-induced growth arrest of some human cancer cell lines. Growth arrest correlated with increased binding of p21 and p27 to cyclin-dependent kinase-2 (Cdk2), and inhibition of Cdk2 kinase activity. However, it was unclear how TGF-beta caused increased binding of p21 and p27 to Cdk2. Cell fractionation and immunofluorescence microscopy experiments were performed to examine the effect of TGF-beta on the intracellular localization of Cdk2, p21, and p27. Kinase assays were performed on cytoplasmic and nuclear extracts to determine how TGF-beta altered Cdk2 activity in both subcellular compartments. In breast epithelial cells treatment with TGF-beta induced a decrease in nuclear Cdk2 concentrations and relocalization of Cdk2 to the cytoplasm. Cdk2 relocalization to the cytoplasm correlated with dephosphorylation of nuclear retinoblastoma tumor suppressor protein and decreased nuclear Cdk2 activity. In these epithelial cell lines, p21 and p27 were localized primarily in the cytoplasm. Decreases in nuclear Cdk2 concentrations correlated with increased binding of Cdk2 to cytoplasmic p21 and p27. Cooperative growth arrest induced by treatment with TGF-beta + rapamycin causes inhibition of nuclear Cdk2 activity through multiple mechanisms, including Cdk2 relocalization to the cytoplasm, increased p27 and p21 binding to Cdk2, and increased phosphorylation of nuclear Cdk2 on its inhibitory site, Tyr15.

Bcl-2 activates a programme of premature senescence in human carcinoma cells.

The apoptosis regulator Bcl-2 has been shown to modulate cell-cycle progression, favouring a quiescent state over a proliferative state, in both normal and tumour cells. We show here that constitutive expression of Bcl-2 in human carcinoma cells results in a cell-cycle arrest that within a few days can become irreversible. Arrested cells acquire a senescent-like phenotype, which consists of several characteristic morphological alterations and increased activity of senescence-associated beta-galactosidase. The induction of the premature senescence programme is mediated by inhibition of Cdk2 kinase activity, and p27(KIP1) is required to maintain the senescent phenotype. We propose that the ability to activate an endogenous premature senescence programme allows Bcl-2 to suppress tumour growth. These results suggest that the down-regulation of Bcl-2 expression, which has been observed during the development and progression of human carcinoma, is related to the ability of Bcl-2 to severely hamper the growth of carcinoma cells and to induce a permanent cell-cycle arrest, with the features of senescence.

Mechanisms of Cell Cycle Arrest in Response to TGF-β in Progestin-Dependent and -Independent Growth of Mammary Tumors

TGF-β1 modulation of cell cycle components was assessed in an experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary tumors in Balb/c mice. TGF-β1 inhibited both MPA-induced proliferation of progestin-dependent C4HD epithelial cells and proliferation of the progestin-independent variant cell type C4HI, arresting cells in G1 phase of the cell cycle. Progestin-independent 60 epithelial cells evidenced reduced response to TGF-β1 antiproliferative effects. TGF-β1 inhibition of cyclins D1 and A expression and up-regulation of p21CIP1 levels were the common findings in all three cell types. In addition, a significant content reduction of cyclin D1/cdk4 and cyclin A/cdk2 complexes was found after TGF-β1 inhibition of MPA-dependent and -independent proliferation. TGF-β1 inhibited cyclin D2 expression and up-regulated p27KIP1 levels only when acting as inhibitor of MPA-induced proliferation of C4HD cells. Regulation of these two cell cycle components resulted in decreased cyclin D2/cdk2 complex and in increased p27KIP1 association with cdk2 in C4HD cells treated with TGF-β1. These two molecular mechanisms, unobserved in progestin-independent growth of C4HI or 60 cells, were associated with a significantly higher degree of inhibition of cdk2 kinase activity in C4HD cells compared to that found in TGF-β-treated C4HI or 60 cells. Reduced sensitivity of 60 cells to the growth-inhibitory effects of TGF-β1 correlated with significantly lower levels of p15INK4B, p21CIP1, and p27KIP1 expressed in these cells, compared to the levels present in C4HD or C4HI cells, and correlated as well with lack of expression of p16INK4. Thus, common targets were found to exist in TGF-β1 inhibitory action on breast cancer cells, but regulation of specific targets was found when TGF-β1-inhibited proliferation driven by the progesterone receptor.

Cell cycle exit during terminal erythroid differentiation is associated with accumulation of p27Kip1 and inactivation of cdk2 kinase

Progression through the mammalian cell cycle is regulated by cyclins, cyclin- dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs). The function of these proteins in the irreversible growth arrest associated with terminally differentiated cells is largely unknown. The function of Cip/Kip proteins p21Cip1and p27Kip1 during erythropoietin-induced terminal differentiation of primary erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus was investigated. Both p21Cip1 and p27Kip1 proteins were induced during erythroid differentiation, but only p27Kip1 associated with the principal G1CDKs—cdk4, cdk6, and cdk2. The kinetics of binding of p27Kip1 to CDK complexes was distinct in that p27Kip1 associated primarily with cdk4 (and, to a lesser extent, cdk6) early in differentiation, followed by subsequent association with cdk2. Binding of p27Kip1 to cdk4 had no apparent inhibitory effect on cdk4 kinase activity, whereas inhibition of cdk2 kinase activity was associated with p27Kip1binding, accumulation of hypo-phosphorylated retinoblastoma protein, and G1 growth arrest. Inhibition of cdk4 kinase activity late in differentiation resulted from events other than p27Kip1 binding or loss of cyclin D from the complex. The data demonstrate that p27Kip1 differentially regulates the activity of cdk4 and cdk2 during terminal erythroid differentiation and suggests a switching mechanism whereby cdk4 functions to sequester p27Kip1 until a specified time in differentiation when cdk2 kinase activity is targeted by p27Kip1 to elicit G1 growth arrest. Further, the data imply that p21Cip1 may have a function independent of growth arrest during erythroid differentiation.

Cell cycle exit during terminal erythroid differentiation is associated with accumulation of p27Kip1 and inactivation of cdk2 kinase

AbstractProgression through the mammalian cell cycle is regulated by cyclins, cyclin- dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs). The function of these proteins in the irreversible growth arrest associated with terminally differentiated cells is largely unknown. The function of Cip/Kip proteins p21Cip1and p27Kip1 during erythropoietin-induced terminal differentiation of primary erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus was investigated. Both p21Cip1 and p27Kip1 proteins were induced during erythroid differentiation, but only p27Kip1 associated with the principal G1CDKs—cdk4, cdk6, and cdk2. The kinetics of binding of p27Kip1 to CDK complexes was distinct in that p27Kip1 associated primarily with cdk4 (and, to a lesser extent, cdk6) early in differentiation, followed by subsequent association with cdk2. Binding of p27Kip1 to cdk4 had no apparent inhibitory effect on cdk4 kinase activity, whereas inhibition of cdk2 kinase activity was associated with p27Kip1binding, accumulation of hypo-phosphorylated retinoblastoma protein, and G1 growth arrest. Inhibition of cdk4 kinase activity late in differentiation resulted from events other than p27Kip1 binding or loss of cyclin D from the complex. The data demonstrate that p27Kip1 differentially regulates the activity of cdk4 and cdk2 during terminal erythroid differentiation and suggests a switching mechanism whereby cdk4 functions to sequester p27Kip1 until a specified time in differentiation when cdk2 kinase activity is targeted by p27Kip1 to elicit G1 growth arrest. Further, the data imply that p21Cip1 may have a function independent of growth arrest during erythroid differentiation.

Disruption of the Actin Cytoskeleton Leads to Inhibition of Mitogen-Induced Cyclin E Expression, Cdk2 Phosphorylation, and Nuclear Accumulation of the Retinoblastoma Protein-Related p107 Protein

The actin cytoskeleton has been found to be required for mitogen-stimulated cells to passage through the cell cycle checkpoint. Here we show that selective disruption of the actin cytoskeleton by dihydrocytochalasin B (H2CB) blocked the mitogenic effect in normal Swiss 3T3 cells, leading to cell cycle arrest at mid to late G1 phase. Cells treated with H2CB remain tightly attached to the substratum and respond to mitogen-induced MAP kinase activation. Upon cytoskeleton disruption, however, growth factors fail to induce hyperphosphorylation of the retinoblastoma protein (pRb) and the pRb-related p107. While cyclin D1 induction and cdk4-associated kinase activity are not affected, induction of cyclin E expression and activation of cyclin E–cdk2 complexes are greatly inhibited in growth-stimulated cells treated with H2CB. The inhibition of cyclin E expression appears to be mediated at least in part at the RNA level and the inhibition of cdk2 kinase activity is also attributed to the decrease in cdk2 phosphorylation and proper subcellular localization. The expression patterns of cdk inhibitors p21 and p27 are similar in both untreated and H2CB-treated cells upon serum stimulation. In addition, the changes in subcellular localization of pRb and p107 appear to be linked to their phosphorylation states and disruption of normal actin structure affects nuclear migration of p107 during G1-to-S progression. Taken together, our results suggest that the actin cytoskeleton-dependent G1 arrest is linked to the cyclin–cdk pathway. We hypothesize that normal actin structure may be important for proper localization of certain G1 regulators, consequently modulating specific cyclin and kinase expression.

Herpes Simplex Virus Type 1 Infection Imposes a G1/S Block in Asynchronously Growing Cells and Prevents G1 Entry in Quiescent Cells

Herpes simplex virus type 1 (HSV-1) infection disrupted cell cycle regulation in at least two ways. First, infection of quiescent human embryonic lung cells simultaneously with readdition of serum caused inhibition of cyclin D/cyclin-dependent kinase (CDK) 4,6-specific and cyclin E/CDK2-specific phosphorylation of the retinoblastoma protein pRb. The inhibition of cyclin D/CDK4,6 kinase activity corresponded to a loss of cyclin D1 protein and a failure of CDK4 and CDK6 to translocate to the nucleus. Failure to detect cyclin E/CDK2 kinase activity was accompanied by a loss of cyclin E protein and a failure of CDK2 to translocate to the nucleus. Levels of pocket protein p130 persisted, whereas p107 did not accumulate. As a result of these effects on cyclin kinase, G0-infected cells failed to reenter the cell cycle. The second type of HSV-induced cell cycle dysregulation was observed in asynchronously dividing cell cultures. A rapid inhibition of preexisting cyclin E/CDK2 and cyclin A/CDK2 activities was observed in human embryonic lung cells, as well as two other human cell lines: C33 and U2OS. HSV-1 immediate-early gene expression was necessary for the inhibition of CDK2 kinase activity. Cyclin and CDK subunit protein levels, intracellular localization, and complex stability were unaffected by infection. In addition, levels of cyclin-dependent kinase inhibitors, p27 and p21, were not affected by HSV-1. Previous experiments demonstrated that in asynchronous infected cells, hypophosphorylated pRb and pocket protein–E2F complexes accumulated, and cellular DNA synthesis was rapidly inhibited. Coupled with the present results, this indicates that HSV-1 has evolved mechanisms for preventing cells in G1 from proceeding through the restriction point and for cells in S from completing a round of DNA replication.

Induction of differentiation accompanies inhibition of Cdk2 in a non-small cell lung cancer cell line.

Induction of differentiation in a variety of model systems is accompanied by cell cycle exit and inhibition of Cdk2 kinase activity. We asked whether inhibition of Cdk2 activity is sufficient to allow differentiation to occur in a non-small cell lung cancer cell line. Treatment of NCI-H358 with flavopiridol, an inhibitor of multiple Cdk's, resulted in growth arrest and induction of mucinous differentiation. The onset of differentiation coincided temporally with loss of Cdk2 kinase activity. Western analysis revealed that flavopiridol treatment resulted in depletion of both cyclin E and D1, suggesting that loss of the regulatory subunits is at least partially responsible for the loss of Cdk kinase activity. Similarly, roscovitine, an inhibitor of Cdk's 1, 2, and 5, but not Cdk4, also induced differentiation in NCI-H358, although the resulting pattern of expression of cell cycle regulatory genes differed from the pattern obtained with flavopiridol. Furthermore, stable expression of an antisense Cdk2 construct in NCI-H358 also resulted in the appearance of a marker of mucinous differentiation. These results show that the inhibition of activity of cyclin dependent kinases, particularly Cdk2, by multiple different mechanisms is accompanied by differentiation. Thus, induction of differentiation is one potential mechanism of action for agents that down-regulate Cdk activity.

A link between cell cycle and cell death: Bax and Bcl-2 modulate Cdk2 activation during thymocyte apoptosis.

Resting thymocytes undergoing apoptosis in response to specific stimuli degrade the cdk inhibitor p27(Kip1) and upregulate Cdk2 kinase activity. Inhibition of Cdk2 kinase activity efficiently blocks cell death via certain apoptosis pathways whereas overexpression of Cdk2 accelerates such cell death, suggesting its involvement in the signal transduction pathways activated by certain apoptotic stimuli. We found that Cdk2 activation during thymocyte apoptosis can be regulated by p53, Bax and Bcl-2. The highly elevated Cdk2 kinase activity in the apoptosing thymocytes is not associated with its canonical cyclins, cyclin E and cyclin A, and requires de novo synthesis of proteins for activation to take place. We therefore propose Cdk2 activation to be a crucial event in distinct pathways of apoptosis and the point at which the cell cycle and cell death pathways interact.

The Cyclin-Dependent Kinase Inhibitors Olomoucine and Roscovitine Arrest Human Fibroblasts in G1 Phase by Specific Inhibition of CDK2 Kinase Activity

The specificity and the temporal location of cell cycle arrest induced by the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine were investigated in normal human fibroblasts. Effects on the cell cycle were compared with those induced by the kinase inhibitor staurosporine, which arrests normal cells in early G1 phase by acting upstream of CDK2. Consistent with theirin vitroactivity, olomoucine and roscovitine, but not the related compound iso-olomoucine, induced a dose-dependent arrest in G1 phase. Following removal of CDK inhibitors, cells resumed cycle progression entering S phase with a kinetics faster than staurosporine-treated samples. Cellular levels of PCNA, cyclin D1, and cyclin E were not affected by the CDK inhibitors. In contrast, staurosporine significantly reduced the levels of these proteins, as determined by immunocytometry and Western blot analysis. Cyclin A was detectable only in some cells remaining in the G2 + M compartment of samples treated with CDK inhibitors, but not in samples treated with staurosporine. Significant reduction in the hyperphosphorylated forms of retinoblastoma protein was found in samples treated with CDK inhibitors, while only hypophosphorylated forms were observed in staurosporine-treated samples. Concomitantly, CDK2, but not CDK4, activity immunoprecipitated from cells treated with olomoucine or roscovitine was markedly inhibited. These results suggest that in normal cells, CDK2 kinase activity is the specific target of olomoucine and roscovitine.

Mechanism of antitumor action of PKC activator, gnidimacrin.

Daphnane-type diterpene gnidimacrin isolated from the Chinese plant Stellera chamaejasme L. is an antitumor agent that activates protein kinase C (PKC). The mechanism of antitumor action of gnidimacrin and the possible involvement of PKC were examined using sensitive K562 and refractory HLE cells. Gnidimacrin did bind to K562 cells 3 times more than to HLE cells. Immunoblot analyses revealed pronounced PKC betaII expression in gnidimacrin sensitive cell lines including K562 cells, while refractory HLE cells strongly expressed PKC alpha, but not PKC betaII. In a 24-hr exposure of K562 cells to gnidimacrin, G1 phase arrest and inhibition of cdk2 kinase activity was found at growth-inhibitory concentration (0.0005 microg/ml). Complete inhibition of cdk2 activity and maximum G1 phase arrest were observed at 0.005 microg/ml, however, these biological effects were reduced at 0.05 microg/ml (260 times the 50% inhibitory concentration). Cellular PKC after a 24-hr exposure was examined by immunoblot analysis and specific binding of [3H]phorbol-12,13-dibutyrate as a ligand of PKC. Expression and the amount of functional PKC of K562 cells were not changed at 0.002 microg/ml, but down-regulated to less than 1/10th of the control at 0.05 microg/ml. The reduction of biological effects at 0.05 microg/ml is most likely due to PKC down-regulation. Our results suggest that PKC (particularly betaII) is one of the major determinants of the ability of cells to respond to gnidimacrin and that the antitumor action might be associated with cell-cycle regulation through suppression of cdk2 activity.

Identification of cdk2 binding sites on the p27Kip1 cyclin-dependent kinase inhibitor.

A cdk2 binding domain on p27Kip1 located within the sequence of amino acids 53-85 was further characterized by generating a series of point mutations within amino acid residues 62-75. Two regions, FDF (residues 62-64) and GXY (residues 72 and 74), were identified within the beta hairpin region of p27Kip1. Mutations within these regions essentially completely inhibited the binding to in vitro translated cdk2 and cdk2/cyclin E complexes formed in vitro or in vivo. The p27Kip1 GST-fusion protein of the point mutation that replaces phenylalanine at residue 64 to alanine (F64A) showed approximately twofold less inhibition of cdk2 kinase activity. The cellular response to the introduction of the F64A mutant form of p27Kip1 was compared to that of p27Kip1 wild type by transfecting HeLa cells with constructs of full length sense and antisense coding sequences. Overexpression of the F64A mutant form of p27Kip1 bound significantly lower levels of cdk2 as compared to wild type and did not affect the cdk2 related kinase activity of the transfected HeLa cells. Overexpression of wild type p27Kip1 resulted in a reduction of the level of cdk2 kinase activity and effectively suppressed the growth of the transfected HeLa cells.

Inhibition of cdk2 kinase activity by methylselenocysteine in synchronized mouse mammary epithelial tumor cells.

Methylselenocysteine (MSC), an organic selenium compound has significant anticarcinogenic activity against mammary tumorigenesis. Previous experiments have demonstrated that MSC and inorganic selenite inhibit mammary cell (TM6 cell line) growth through different pathways. The present investigation demonstrated that MSC arrested cells in S phase during the TM6 cell cycle, which was followed by cells entering apoptosis at 48 h. Methylselenocysteine specifically affected the cdk2 kinase activity of the TM6 cells (54% reduction) at 16 h after release from growth arrest. The cdk4 kinase activity did not change during the cell cycle, confirming that cells had passed the G1 checkpoint and had entered S phase. The amount of cyclin E associated with cdk2 was increased by MSC by the 12 h time point, thereby facilitating entry of cells into S phase. Afterwards, cyclin E and cyclin A associated with cdk2 did not change for the remainder of the cell cycle. The data demonstrate that inhibition of mammary cell growth by MSC is mediated by alterations in progression of cells through S phase. The decrease in cdk2 kinase activity is coincident with prolonged arrest in S phase. One consequence of prolonged arrest may be apoptosis.