The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
Oxidative stress is a contributing factor in a number of chronic diseases, including cancer, atherosclerosis, and neurodegenerative diseases. Lipid peroxidation that occurs during periods of oxidative stress results in the formation of lipid electrophiles, which can modify a multitude of proteins in the cell. 4-Hydroxy-2-nonenal (HNE) is one of the most well-studied lipid electrophiles and has previously been shown to arrest cells at the G1/S transition. Recently, proteomic data have shown that HNE is capable of covalently modifying CDK2, the kinase responsible for the G1/S transition. Here, we identify the sites adducted by HNE using recombinant CDK2 and show that HNE treatment suppresses the kinase activity of the enzyme. We further identify sites of adduction in HNE-treated intact human colorectal carcinoma cells (RKO) and show that HNE-dependent modification in cells is long-lived, disrupts CDK2 function, and correlates with a delay of progression of the cells into S-phase. We propose that adduction of CDK2 by HNE directly alters its activity, contributing to the cell cycle delay.
Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.
Several lines of evidence suggest that tumor cells show elevated activity of the NF-kappaB transcription factor, a phenomenon often resulting from constitutive activity of IkappaB kinase beta (IKKbeta). However, others have found that loss of NF-kappaB activity or IKKbeta is tumor promoting. The role of NF-kappaB in tumor progression is therefore controversial and varies with tumor type. We sought to more extensively investigate the role IKKbeta in melanoma tumor development by specifically disrupting Ikkb in melanocytes in an established mouse model of spontaneous melanoma, whereby HRasV12 is expressed in a melanocyte-specific, doxycycline-inducible manner in mice null for the gene encoding the tumor suppressor inhibitor cyclin-dependent kinase 4/alternative reading frame (Ink4a/Arf). Our results show that Ink4a/Arf-/- mice with melanocyte-specific deletion of Ikkb were protected from HRasV12-initiated melanoma only when p53 was expressed. This protection was accompanied by cell cycle arrest, with reduced cyclin-dependent kinase 2 (Cdk2), Cdk4, Aurora kinase A, and Aurora kinase B expression. Increased p53-mediated apoptosis was also observed, with decreased expression of the antiapoptotic proteins Bcl2 and survivin. Enhanced stabilization of p53 involved increased phosphorylation at Ser15 and reduced phosphorylation of double minute 2 (Mdm2) at Ser166. Together, our findings provide genetic and mechanistic evidence that mutant HRas initiation of tumorigenesis requires Ikkbeta-mediated NF-kappaB activity.
The basal-like subtype of breast cancer is associated with invasiveness, high rates of postsurgical recurrence, and poor prognosis. Aside from inactivation of the BRCA1 tumor-suppressor gene, little is known concerning the mechanisms that cause basal breast cancer or the mechanisms responsible for its invasiveness. Here, we show that the heterogeneous mouse mammary tumor virus-cyclin D1-Cdk2 (MMTV-D1K2) transgenic mouse mammary tumors contain regions of spindle-shaped cells expressing both luminal and myoepithelial markers. Cell lines cultured from these tumors exhibit the same luminal/myoepithelial mixed-lineage phenotype that is associated with human basal-like breast cancer and express a number of myoepithelial markers including cytokeratin 14, P-cadherin, alpha smooth muscle actin, and nestin. The MMTV-D1K2 tumor-derived cell lines form highly invasive tumors when injected into mouse mammary glands. Invasion is associated with E-cadherin localization to the cytoplasm or loss of E-cadherin expression. Cytoplasmic E-cadherin correlates with lack of colony formation in vitro and beta-catenin and p120(ctn) localization to the cytoplasm. The data suggest that the invasiveness of these cell lines results from a combination of factors including mislocalization of E-cadherin, beta-catenin, and p120(ctn) to the cytoplasm. Nestin expression and E-cadherin mislocalization were also observed in human basal-like breast cancer cell lines, suggesting that these results are relevant to human tumors. Together, these results suggest that abnormal Cdk2 activation may contribute to the formation of basal-like breast cancers.
p57(Kip2) has been considered a candidate tumor suppressor gene because of its location in the genome, biochemical activities, and imprinting status. However, little is known about the role of p57(Kip2) in tumorigenesis and cancer progression. Here, we show that the expression of p57(Kip2) is significantly decreased in human prostate cancer, and the overexpression of p57(Kip2) in prostate cancer cells significantly suppressed cell proliferation and reduced invasive ability. In addition, overexpression of p57(Kip2) in LNCaP cells inhibited tumor formation in nude mice, resulting in well-differentiated squamous tumors rather than adenocarcinoma. Furthermore, the prostates of p57(Kip2) knockout mice developed prostatic intraepithelial neoplasia and adenocarcinoma. Remarkably, this mouse prostate cancer is pathologically identical to human prostate adenocarcinoma. Therefore, these results strongly suggest that p57(Kip2) is an important gene in prostate cancer tumorigenesis, and the p57(Kip2) pathway may be a potential target for prostate cancer prevention and therapy.
Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyte antigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleated cells. The identification of novel, tumor-specific epitopes is a critical step in the development of immunotherapeutics for breast cancer. To directly identify peptide epitopes unique to cancerous cells, secreted human class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed in breast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, nontumorigenic cell line MCF10A. Stable transfectants were seeded into bioreactors for production of > 25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy. Comparative analysis of HLA-A*0201 peptides revealed 5 previously uncharacterized epitopes uniquely presented on breast cancer cells. These peptides were derived from intracellular proteins with either well-defined or putative roles in breast cancer development and progression: Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC1), Kinetochore Associated 2 (KNTC2 or HEC1), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6). Cellular recognition of the MIF, KNTC2, EXOSC6, and Cdk2 peptides by circulating CD8+ cells was demonstrated by tetramer staining and IFN-gamma ELISPOT. The identification and characterization of peptides unique to the class I of breast cancer cells provide putative targets for the development of immune diagnostic tools and therapeutics.
The ATR-ATRIP kinase complex regulates cellular responses to DNA damage and replication stress. Mass spectrometry was used to identify phosphorylation sites on ATR and ATRIP to understand how the kinase complex is regulated by post-translational modifications. Two novel phosphorylation sites on ATRIP were identified, S224 and S239. Phosphopeptide-specific antibodies to S224 indicate that it is phosphorylated in a cell cycle-dependent manner. S224 matches a consensus site for cyclin-dependent kinase (CDK) phosphorylation and is phosphorylated by CDK2-cyclin A in vitro. S224 phosphorylation in cells is sensitive to CDK2 inhibitors. Mutation of S224 to alanine causes a defect in the ATR-ATRIP-dependent maintenance of the G(2)-M checkpoint to ionizing and UV radiation. Thus, ATRIP is a CDK2 substrate, and CDK2-dependent phosphorylation of S224 regulates the ability of ATR-ATRIP to promote cell cycle arrest in response to DNA damage.
Cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes are present at high frequency in human breast cancer cell lines, but the significance of this observation is unknown. This report shows that expression of a cyclin D1-Cdk2 fusion protein under the control of the mouse mammary tumor virus (MMTV) promoter results in mammary gland hyperplasia and fibrosis, and mammary tumors. Cell lines isolated from MMTV-cyclin D1-Cdk2 (MMTV-D1K2) tumors exhibit Rb and p130 hyperphosphorylation and up-regulation of the protein products of E2F-dependent genes. These results suggest that cyclin D1/Cdk2 complexes may mediate some of the transforming effects that result from cyclin D1 overexpression in human breast cancers. MMTV-D1K2 cancer cells express the hepatocyte growth factor (HGF) receptor, c-Met. MMTV-D1K2 cancer cells also secrete transforming growth factor beta (TGFbeta), but are relatively resistant to TGFbeta antiproliferative effects. Fibroblasts derived from MMTV-D1K2 tumors secrete factors that stimulate the proliferation of MMTV-D1K2 cancer cells, stimulate c-Met tyrosine phosphorylation, and stimulate the phosphorylation of the downstream signaling intermediates p70(s6k) and Akt on activating sites. Together, these results suggest that deregulation of the Cdk/Rb/E2F axis reprograms mammary epithelial cells to initiate a paracrine loop with tumor-associated fibroblasts involving TGFbeta and HGF, resulting in desmoplasia. The MMTV-D1K2 mice should provide a useful model system for the development of therapeutic approaches to block the stromal desmoplastic reaction that likely plays an important role in the progression of multiple types of human tumors.
BACKGROUND - Kaposi sarcoma-associated human herpesvirus (KSHV) encodes K-cyclin, a homologue of D-type cellular cyclins, which binds cyclin-dependent kinases to phosphorylate various substrates. K-cyclin/cdk phosphorylates a subset of substrates normally targeted by cyclins D, E, and A. We used cells naturally infected with KSHV to further characterize the biochemical features of K-cyclin.
METHODS - We used immunoprecipitation with K-cyclin antibodies to examine the association of K-cyclin with cdk2, cdk6, p21Cip1, and p27Kip1 proteins in BC3 cells. We separated populations of BC3 cells enriched in cells in G1, S, or G2/M phases by elutriation and measured K-cyclin protein and the kinase activity of K-cyclin/cdk6 complexes. The half-life of K-cyclin and cyclin D2 proteins was determined by blocking protein synthesis with cycloheximide and measuring proteins in cell lysates by western blot analysis. We fused the entire K-cyclin sequence to the carboxyl-terminal sequence of cellular cyclin D that contains the PEST degradation sequence to produce K-cyclin/D2 and transfected K-cyclin/D2 into K-cyclin-negative cells to investigate the effect of the PEST sequence on K-cyclin's stability.
RESULTS - Viral K-cyclin interacted with cyclin-dependent kinases cdk2, cdk4, and cdk6 and with the cyclin/cdk inhibitory proteins p21Cip1 and p27Kip1 in BC3 cell lysates. Unlike D-type cyclins, whose expression is cell cycle dependent, the level of K-cyclin was stable throughout the cell cycle, and the kinase associated with the K-cyclin/cdk6 complex was constitutively active. The half-life of K-cyclin (6.9 hours) was much longer than that of cellular cyclin D2 (0.6 hour) and that of K-cyclin/D2 (0.5 hour), probably because K-cyclin lacks the PEST degradation sequence present in D-type cyclins.
CONCLUSION - The constitutive activation of K-cyclin/cdk complexes in KSHV-infected cells appears to result from the extended half-life of K-cyclin and may explain its role in Kaposi sarcoma.
Mdm2 and MdmX function as cellular regulators of the p53 tumor suppressor protein. Intriguingly, the activities of these proteins are interdependent; MdmX stabilizes Mdm2, enabling its activities towards p53, but it also requires Mdm2 for its nuclear localization. Here we demonstrate that via its phosphorylation by CDK2/Cdc2p34, MdmX regulates nuclear export of Mdm2. Cdc2p34 phosphorylates MdmX on Ser 96 in vitro. Mutation within this site (MdmX(S96A)) impairs, whereas phosphomimic substitution (MdmX(S96D)) increases the cytoplasmic localization of MdmX, suggesting that CDK2/Cdc2p34 phosphorylation is required for export of MdmX from the nucleus. Consequently, cells that express MdmX(S96A) retain Mdm2 in their nuclei, suggesting that export of Mdm2 to the cytoplasm is MdmX-dependent. Similarly, treatment of cells with the pharmacological inhibitor of CDK2/Cdc2p34 or with a dominant-negative Cdc2 results in nuclear localization of MdmX and Mdm2 and decreases the level of Mdm2 expression. Since Cdc2p34 is active in nonstressed conditions, our finding provides a novel insight into the signaling cascade involved in the regulation of MdmX localization and for regulation of Mdm2 localization and stability.