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1.
Oncogene ; 34(15): 1928-37, 2015 Apr 09.
Article in English | MEDLINE | ID: mdl-24882580

ABSTRACT

Lysyl oxidase (LOX) is a multifunctional protein required for normal collagen and elastin biosynthesis and maturation. In addition, LOX has complex roles in cancer in which the lysyl oxidase propeptide (LOX-PP) domain of secreted pro-LOX has tumor-suppressor activity, while the active enzyme promotes metastasis. In prostate cancer cell lines, recombinant LOX-PP (rLOX-PP) inhibits the growth of PC3 cells in vitro by mechanisms that were not characterized, while in DU145 cells rLOX-PP targeted fibroblast growth factor signaling. Because rLOX-PP can enhance effects of a genotoxic chemotherapeutic on breast cancer cell apoptosis, we reasoned that rLOX-PP could target DNA repair pathways typically elevated in cancer. Here we demonstrate for the first time that rLOX-PP inhibits prostate xenograft growth in vivo and that activating phosphorylations of the key DNA repair molecules ataxia-telangiectasia mutated (ATM) and checkpoint kinase 2 (CHK2) are inhibited by rLOX-PP expression in vivo. In addition, in vitro studies showed that rLOX-PP inhibits radiation-induced activating phosphorylations of ATM and CHK2 and that exogenously added rLOX-PP protein can localize to the nucleus in both DU145 and PC3 cells. rLOX-PP pull-down studies resulted in detection of a protein complex with the nuclear DNA repair regulator MRE11 in both cell lines, and rLOX-PP localized to radiation-induced nuclear DNA repair foci. Finally, rLOX-PP was shown to sensitize both DU145 and PC3 cells to radiation-induced cell death determined in colony-formation assays. These data provide evidence that rLOX-PP has a nuclear mechanism of action in which it directly interacts with DNA repair proteins to sensitize prostate cancer cells to the effects of ionizing radiation.


Subject(s)
DNA Repair , Prostatic Neoplasms/genetics , Prostatic Neoplasms/therapy , Protein-Lysine 6-Oxidase/biosynthesis , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Damage , Humans , Male , Mice , Mice, Nude , Phosphorylation , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein-Lysine 6-Oxidase/genetics , Xenograft Model Antitumor Assays
2.
Oncogene ; 29(2): 201-13, 2010 Jan 14.
Article in English | MEDLINE | ID: mdl-19838210

ABSTRACT

Approximately 80% of breast cancers express the estrogen receptor-alpha (ERalpha) and are treated with anti-estrogens. Resistance to these agents is a major cause of mortality. We have shown that estrogen inhibits Notch, whereas anti-estrogens or estrogen withdrawal activate Notch signaling. Combined inhibition of Notch and estrogen signaling has synergistic effects in ERalpha-positive breast cancer models. However, the mechanisms whereby Notch-1 promotes the growth of ERalpha-positive breast cancer cells are unknown. Here, we demonstrate that Notch-1 increases the transcription of ERalpha-responsive genes in the presence or absence of estrogen via a novel chromatin crosstalk mechanism. Our data support a model in which Notch-1 can activate the transcription of ERalpha-target genes via IKKalpha-dependent cooperative chromatin recruitment of Notch-CSL-MAML1 transcriptional complexes (NTC) and ERalpha, which promotes the recruitment of p300. CSL binding elements frequently occur in close proximity to estrogen-responsive elements (EREs) in the human and mouse genomes. Our observations suggest that a hitherto unknown Notch-1/ERalpha chromatin crosstalk mediates Notch signaling effects in ERalpha-positive breast cancer cells and contributes to regulate the transcriptional functions of ERalpha itself.


Subject(s)
Breast Neoplasms/genetics , Estrogen Receptor alpha/physiology , I-kappa B Kinase/genetics , Receptor, Notch1/physiology , Animals , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , E1A-Associated p300 Protein/genetics , E1A-Associated p300 Protein/metabolism , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , I-kappa B Kinase/metabolism , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Oligopeptides/pharmacology , Promoter Regions, Genetic/genetics , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tamoxifen/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Xenograft Model Antitumor Assays
3.
Oncogene ; 28(38): 3390-400, 2009 Sep 24.
Article in English | MEDLINE | ID: mdl-19597471

ABSTRACT

Enhanced RAS signaling and decreased androgen dependence of prostate cancer cells accompany poor clinical outcomes. Elevated autocrine fibroblast growth factors 2 (FGF-2) signaling promotes prostate cancer cell growth and survival. Expression of lysyl oxidase (LOX) inhibits RAS transforming activity. LOX is secreted as 50 kDa pro-LOX protein and then undergoes extracellular proteolytic processing to form approximately 30 kDa LOX enzyme and approximately 18 kDa propeptide (LOX-PP). We have previously shown that LOX-PP inhibits breast cancer cell transformation and tumor formation, but mechanisms of action of LOX-PP have not been fully elucidated. Here we report that LOX expression is reduced in prostate cancer cell lines and that recombinant LOX-PP protein inhibits serum-stimulated DNA synthesis and MEK/ERK and PI3K/AKT pathways in DU 145 and PC-3 androgen-independent cell lines. In DU 145 cells, treatment with a pharmacologic FGF-receptor inhibitor or a neutralizing anti-FGFR1 antibody mimicked LOX-PP inhibition of serum-stimulated DNA synthesis. FGF-2-stimulated DNA synthesis, ERK1/2, AKT and FRS2alpha activation were found all to be inhibited by LOX-PP in DU 145 cells. LOX-PP reduced specific binding of FGF-2 to DU 145 cells, suggesting that LOX-PP targets FGF signaling at the receptor. Interestingly, PC-3 cells did not respond to FGF-2, consistent with previous reports. We conclude that LOX-PP inhibits proliferation of DU 145 cells by interfering with FGFR(s) binding and signaling, and that LOX-PP has other mechanisms of action in PC-3 cells.


Subject(s)
Enzyme Precursors/pharmacology , Fibroblast Growth Factor 2/antagonists & inhibitors , Prostatic Neoplasms/prevention & control , Protein-Lysine 6-Oxidase/pharmacology , Signal Transduction , Cell Line, Tumor , Cell Proliferation , DNA/biosynthesis , Fibroblast Growth Factor 2/metabolism , Humans , Male , Prostate/enzymology , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Receptors, Fibroblast Growth Factor/metabolism , Recombinant Proteins/pharmacology
4.
Cell Mol Life Sci ; 66(11-12): 1850-7, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19387549

ABSTRACT

CK2 is a highly conserved tetrameric serine/ threonine kinase present in all eukaryotic organisms. It is constitutively active, and appears to be regulated by level of expression and activity, and subcellular localization. In turn, it has been postulated to control the function of many proteins through changes in phosphorylation that affect protein stability, protein-protein interactions, and subcellular localization. Through these mechanisms, CK2 regulates many fundamental cellular properties. An enzyme that carries out such a master regulatory function is likely to be important in organismic development and in cancer. We have shown that overexpression of CK2 catalytic subunits is capable of promoting tumorigenesis, and that loss of CK2 catalytic subunits in development can be lethal. Through studies in cells, mice, and frogs, we and others have identified the Wnt and NF-kappaB pathways as two key signal transduction pathways that are regulated by CK2 activity, in embryonic development and in cancer. These results suggest that inhibiting CK2 could be useful in treating cancer, but dangerous to developing organisms.


Subject(s)
Casein Kinase II/physiology , Cell Transformation, Neoplastic/metabolism , Embryonic Development/physiology , NF-kappa B/physiology , Signal Transduction/physiology , Wnt Proteins/physiology , Animals , Humans , Phosphorylation
5.
Oncogene ; 27(44): 5833-44, 2008 Oct 02.
Article in English | MEDLINE | ID: mdl-18560356

ABSTRACT

Notch-1 inhibits apoptosis in some transformed cells through incompletely understood mechanisms. Notch-1 can increase nuclear factor-kappa B (NF-kappaB) activity through a variety of mechanisms. Overexpression of cleaved Notch-1 in T-cell acute lymphoblastic leukemia cells activates NF-kappaB via interaction with the I kappa B kinase (IKK) signalosome. Concomitant activation of the Notch and NF-kappaB pathways has been described in a large series of cervical cancer specimens. Here, we show that wild-type, spontaneously expressed Notch-1 stimulates NF-kappaB activity in CaSki cervical cancer cells by associating with the IKK signalosome through IKKalpha. A significant fraction of tumor necrosis factor (TNF)-alpha-stimulated IkappaB kinase activity in CaSki cells is Notch-1-dependent. In addition, Notch-1 is found in the nucleus in association with IKKalpha at IKKalpha-stimulated promoters and is required for association of IKKalpha with these promoters under basal and TNF-alpha-stimulated conditions. Notch-1-IKKalpha complexes are found in normal human keratinocytes as well, suggesting that IKK regulation is a physiological function of Notch-1. Both Notch-1 and IKKalpha knockdown sensitize CaSki cells to cisplatin-induced apoptosis to equivalent extents. Our data indicate that Notch-1 regulates NF-kappaB in cervical cancer cells at least in part via cytoplasmic and nuclear IKK-mediated pathways.


Subject(s)
I-kappa B Kinase/metabolism , Receptor, Notch1/metabolism , Uterine Cervical Neoplasms/metabolism , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line, Tumor , Chromatin/metabolism , Cisplatin/pharmacology , Female , Humans , I-kappa B Kinase/genetics , NF-kappa B/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins/metabolism , Receptor, Notch1/genetics , Receptor, Notch2/metabolism , Receptor, Notch4 , Receptors, Notch/metabolism
6.
J Immunol ; 167(9): 4919-25, 2001 Nov 01.
Article in English | MEDLINE | ID: mdl-11673497

ABSTRACT

Rapid IkappaBalpha turnover has been implicated in the high basal NF-kappaB activity in WEHI 231 B immature IgM(+) B cells. Here we show that treatment of WEHI 231 cells with apigenin, a selective inhibitor of the protein kinase CK2, decreased the rate of IkappaBalpha turnover and nuclear levels of NF-kappaB. Turnover of IkappaBalpha in these cells is mediated in part by the protease calpain. Since both CK2 and calpain target the proline-glutamic acid-serine-threonine (PEST) domain, we investigated the role of CK2 in the degradation of IkappaBalpha by calpain using an in vitro phosphorylation/degradation assay. CK2 phosphorylation enhanced mu-calpain-mediated degradation of wild-type IkappaBalpha, but not of mutant 3CIkappaBalpha, with S283A, T291A, and T299A mutations in phosphorylation sites within the PEST domain. Roles for CK2 and calpain in IkappaBalpha turnover were similarly shown in CH31 immature and CH12 mature IgM(+) B cells, but not in A20 and M12 IgG(+) B cells. These findings demonstrate for the first time that CK2 phosphorylation of serine/threonine residues in the PEST domain promotes calpain-mediated degradation of IkappaBalpha and thereby increases basal NF-kappaB levels in IgM(+) B cells.


Subject(s)
Calpain/physiology , DNA-Binding Proteins/metabolism , I-kappa B Proteins , Protein Serine-Threonine Kinases/physiology , Apigenin , Casein Kinase II , Cells, Cultured , Flavonoids/pharmacology , Humans , NF-KappaB Inhibitor alpha , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors
7.
J Cell Biochem ; 82(3): 387-98, 2001.
Article in English | MEDLINE | ID: mdl-11500915

ABSTRACT

Epidemiological evidence suggests tea (Camellia sinensis L.) has chemopreventive effects against various tumors. Green tea contains many polyphenols, including epigallocatechin-3 gallate (EGCG), which possess anti-oxidant qualities. Reduction of chemically induced mammary gland carcinogenesis by green tea in a carcinogen-induced rat model has been suggested previously, but the results reported were not statistically significant. Here we have tested the effects of green tea on mammary tumorigenesis using the 7,12-dimethylbenz(a)anthracene (DMBA) Sprague-Dawley (S-D) rat model. We report that green tea significantly increased mean latency to first tumor, and reduced tumor burden and number of invasive tumors per tumor-bearing animal; although, it did not affect tumor number in the female rats. Furthermore, we show that proliferation and/or viability of cultured Hs578T and MDA-MB-231 estrogen receptor-negative breast cancer cell lines was reduced by EGCG treatment. Similar negative effects on proliferation were observed with the DMBA-transformed D3-1 cell line. Growth inhibition of Hs578T cells correlated with induction of p27(Kip1) cyclin-dependent kinase inhibitor (CKI) expression. Hs578T cells expressing elevated levels of p27(Kip1) protein due to stable ectopic expression displayed increased G1 arrest. Thus, green tea had significant chemopreventive effects on carcinogen-induced mammary tumorigenesis in female S-D rats. In culture, inhibition of human breast cancer cell proliferation by EGCG was mediated in part via induction of the p27(Kip1) CKI.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinogens/pharmacology , Flavonoids , Mammary Neoplasms, Animal/chemically induced , Mammary Neoplasms, Animal/pathology , Plant Extracts/pharmacology , Tea , Tumor Suppressor Proteins , 9,10-Dimethyl-1,2-benzanthracene/antagonists & inhibitors , 9,10-Dimethyl-1,2-benzanthracene/pharmacology , Animals , Antineoplastic Agents/therapeutic use , Carcinogens/antagonists & inhibitors , Catechin/analogs & derivatives , Catechin/pharmacology , Cell Cycle Proteins/metabolism , Cell Death/drug effects , Cell Division/drug effects , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/antagonists & inhibitors , Female , Flow Cytometry , Mammary Neoplasms, Animal/enzymology , Phenols/metabolism , Plant Extracts/therapeutic use , Polymers/metabolism , Probability , Rats , Tumor Cells, Cultured
8.
Hepatology ; 34(1): 32-41, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11431731

ABSTRACT

NF-kappaB regulates liver cell death during development, regeneration, and neoplastic transformation. For example, we showed that oncogenic Ras- or Raf-mediated transformation of rat liver epithelial cells (RLEs) led to altered NF-kappaB regulation through IKK complex activation, which rendered these cells more resistant to TGF-beta1-induced apoptosis. Thus, based on these findings, we sought to determine whether NF-kappaB could also be involved in tumor growth of liver cells in vivo. Hepatocellular carcinomas (HCCs) derived from bitransgenic mice harboring TGF-alpha and c-myc transgenes targeted specifically to the liver were compared with HCCs from c-myc single transgenic mice. Tumors from bitransgenic mice are characterized by a higher frequency of appearance, lower apoptotic index, and a higher rate of cell proliferation. Here we show that NF-kappaB is activated in HCCs of double TGF-alpha/c-myc transgenic mice, but not of c-myc single transgenic mice, suggesting that TGF-alpha mediates induction of NF-kappaB. Activation of the IKK complex was observed in the HCCs of double TGF-alpha/c-myc transgenic mice, implicating this pathway in NF-kappaB induction. Lastly, activation of the Akt/protein kinase B (PKB), which has recently been implicated in NF-kappaB activation by PDGF, TNF-alpha, and Ras, was also observed. Importantly, human HCC cell lines similarly displayed NF-kappaB activation. Thus, these studies elucidate an anti-apoptotic mechanism by a TGF-alpha-Akt/PKB-IKK pathway, which likely contributes to survival and proliferation, thereby accelerating c-myc-induced liver neoplastic development in vivo.


Subject(s)
Carrier Proteins/physiology , Liver Neoplasms, Experimental/metabolism , NF-kappa B/biosynthesis , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins/physiology , Transforming Growth Factor alpha/genetics , Animals , Apoptosis , Cell Division , Enzyme Activation , Gene Expression , Intracellular Signaling Peptides and Proteins , Liver/metabolism , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Transgenic , NF-kappa B/metabolism , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-myc/physiology , RNA-Binding Proteins , Transforming Growth Factor alpha/physiology
9.
Oncogene ; 20(25): 3247-57, 2001 May 31.
Article in English | MEDLINE | ID: mdl-11423974

ABSTRACT

Protein kinase CK2 is a ubiquitous and evolutionarily conserved serine/threonine kinase that is upregulated in many human cancers and can serve as an oncogene in lymphocytes. Recently, we have demonstrated that CK2 potentiates Wnt/beta-catenin signaling in mammary epithelial cells. To determine whether CK2 overexpression contributes to mammary tumorigenesis, we have performed comparative studies of human and rat breast cancer specimens and we have engineered transgenic mice with dysregulated expression of CK2alpha in the mammary gland. We find that CK2 is highly expressed in human breast tumor specimens and in carcinogen-induced rat mammary tumors. Overexpression of CK2alpha in the mammary gland of transgenic mice, under control of the MMTV-LTR, causes hyperplasia and dysplasia of the female mammary gland. Thirty per cent of the female MMTV-CK2alpha transgenic mice develop mammary adenocarcinomas at a median of 23 months of age, often associated with Wnt pathway activation, as evidenced by upregulation of beta-catenin protein. NF-kappaB activation and upregulation of c-Myc also occur frequently. Thus, in mice, rats, and humans, dysregulated expression of CK2 is associated with and is capable of contributing to mammary tumorigenesis. Targeted inhibition of CK2 could be useful in the treatment of breast cancer.


Subject(s)
Adenocarcinoma/etiology , Breast Neoplasms/etiology , Cell Transformation, Neoplastic , Mammary Neoplasms, Animal/etiology , Protein Serine-Threonine Kinases/biosynthesis , Trans-Activators , Zebrafish Proteins , Age of Onset , Animals , Casein Kinase II , Cytoskeletal Proteins/metabolism , Female , Fibrocystic Breast Disease , Humans , Hyperplasia , Lactation , Mammary Glands, Animal/pathology , Mammary Neoplasms, Animal/chemically induced , Mammary Tumor Virus, Mouse/genetics , Mice , Mice, Transgenic , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction , Wnt Proteins , beta Catenin
10.
Oncogene ; 20(11): 1287-99, 2001 Mar 15.
Article in English | MEDLINE | ID: mdl-11313873

ABSTRACT

The Nuclear Factor (NF)-kappaB family of transcription factors controls expression of genes which promote cell growth, survival, and neoplastic transformation. Recently we demonstrated aberrant constitutive activation of NF-kappaB in primary human and rat breast cancer specimens and in cell lines. Overexpression of the epidermal growth factor receptor (EGFR) family member Her-2/neu, seen in approximately 30% of breast cancers, is associated with poor prognosis. Previously, Her-2/neu has been shown to signal via a phosphatidylinositol 3 (PI3)-kinase to Akt/protein kinase B (PKB) pathway. Since this signaling pathway was recently shown to activate NF-kappaB, here we have tested the hypothesis that Her-2/neu can activate NF-kappaB in breast cancer. Overexpression of Her-2/neu and EGFR-4 in Ba/F3 cells led to constitutive PI3- and Akt kinase activities, and induction of classical NF-kappaB (p50/p65). Similarly, a tumor cell line and tumors derived from MMTV-Her-2/neu transgenic mice displayed elevated levels of classical NF-kappaB. Engagement of Her-2/neu receptor downregulated the level of NF-kappaB. NF-kappaB binding and activity in the cultured cells was reduced upon inhibition of the PI3- to Akt kinase signaling pathway via ectopic expression of kinase inactive mutants, incubation with wortmannin, or expression of the tumor suppressor phosphatase PTEN. Inhibitors of calpain, but not the proteasome, blocked IkappaB-alpha degradation. Inhibition of Akt did not affect IKK activity. These results indicate that Her-2/neu activates NF-kappaB via a PI3- to Akt kinase signaling pathway that can be inhibited via the tumor suppressor PTEN, and is mediated by calpain rather than the IkappaB kinase complex.


Subject(s)
Breast Neoplasms/metabolism , Genes, Tumor Suppressor , I-kappa B Proteins , Phosphoric Monoester Hydrolases/metabolism , Protein Serine-Threonine Kinases , Receptor, ErbB-2/metabolism , Signal Transduction , Tumor Suppressor Proteins , Animals , Calpain/metabolism , DNA-Binding Proteins/metabolism , Female , Humans , Mammary Neoplasms, Animal/metabolism , Mice , Mice, Transgenic , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Tumor Cells, Cultured
11.
Oncogene ; 20(14): 1688-702, 2001 Mar 29.
Article in English | MEDLINE | ID: mdl-11313917

ABSTRACT

Upon engagement of the B Cell Receptor (BCR) of WEHI 231 immature B cells, a drop in c-Myc expression is followed by activation of the cyclin-dependent kinase inhibitor (CKI) p27(Kip1), which induces growth arrest and apoptosis. Here, we report inverse patterns of p27 and c-Myc protein expression follow BCR engagement. We present evidence demonstrating, for the first time, that the p27(Kip1) gene is a target of transcriptional repression by c-Myc. Specifically, the changes in p27 protein levels correlated with changes in p27 mRNA levels, and gene transcription. Induction of p27 promoter activity followed BCR engagement of WEHI 231 cells, and this induction could be repressed upon co-transfection of a c-Myc expression vector. Inhibition of the TATA-less p27 promoter by c-Myc was also observed in Jurkat T cells, vascular smooth muscle, and Hs578T breast cancer cells, extending the observation beyond immune cells. Consistent with a putative Inr element CCAGACC (where +1 is underlined) at the start site of transcription in the p27 promoter, deletion of Myc homology box II reduced the extent of repression. Furthermore, enhanced repression was observed upon transfection of the c-Myc 'super-repressor', with mutation of Phe115 to Leu. The sequences mediating transcriptional activity and c-Myc repression were mapped to bp -20 to +20 of the p27 gene. Finally, binding of Max was shown to facilitate c-Myc binding and repression of p27 promoter activity. Overall, these studies identify the p27 CKI gene as a new target whereby c-Myc can control cell proliferation, survival and neoplastic transformation.


Subject(s)
Cell Cycle Proteins , Microtubule-Associated Proteins/genetics , Proto-Oncogene Proteins c-myc/physiology , Repressor Proteins/physiology , Transcription Factors , Tumor Suppressor Proteins , Animals , Antibodies, Anti-Idiotypic/immunology , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Basic-Leucine Zipper Transcription Factors , Cell Transformation, Neoplastic , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p27 , DNA-Binding Proteins/metabolism , G1 Phase , Humans , Muscle, Smooth, Vascular/metabolism , Promoter Regions, Genetic , RNA, Messenger/analysis , Receptors, Antigen, B-Cell/physiology
12.
Cancer Res ; 61(9): 3810-8, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11325857

ABSTRACT

Nuclear factor-kappaB (NF-kappaB)/Rel transcription factors regulate genes that control cell proliferation, survival, and transformation. In normal breast epithelial cells, NF-kappaB/Rel proteins are mainly sequestered in the cytoplasm bound to one of the specific inhibitory IkappaB proteins, whereas in breast cancers they are activated aberrantly. Human breast tumor cell lines, carcinogen-transformed mammary epithelial cells, and the majority of primary human or rodent breast tumor tissue samples express constitutively high levels of nuclear NF-kappaB/REL: To begin to understand the mechanism of this aberrant NF-kappaB/Rel expression, in this study we measured the activity of the major kinases implicated in regulation of IkappaB stability, namely IKKalpha, IKKbeta, and protein kinase, CK2 (formerly casein kinase II). Hs578T, D3-1, and BP-1 breast cancer cell lines displayed higher levels of IKKalpha, IKKbeta, and CK2 activity than untransformed MCF-10F mammary epithelial cells. Inhibition of IKK activity upon expression of dominant negative kinases or of CK2 activity by treatment with selective inhibitors decreased NF-kappaB/Rel activity in breast cancer cells. Inactivation of the IkappaB kinase complex in Hs578T cells via expression of a dominant negative IKKgamma/NF-kappaB essential modulator/IKK-associated protein 1 reduced soft agar colony growth. Thus, the aberrant expression of CK2 or IKK kinases promotes increased nuclear levels of NF-kappaB/Rel and transformation of breast cancer cells. Furthermore, primary human breast cancer specimens that displayed aberrant constitutive expression of NF-kappaB/Rel were found to exhibit increased CK2 and/or IKK kinase activity. These observations suggest these kinases play a similar role in an intracellular signaling pathway that leads to the elevated NF-kappaB/Rel levels seen in primary human mammary tumors and, therefore, represent potential therapeutic targets in the treatment of patients with breast cancer.


Subject(s)
Breast Neoplasms/enzymology , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Breast Neoplasms/pathology , Casein Kinase II , Cell Adhesion/physiology , Cell Division/physiology , Cell Survival/physiology , Cell Transformation, Neoplastic/metabolism , Enzyme Activation , Humans , I-kappa B Kinase , NF-kappa B/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Tumor Cells, Cultured
13.
Endocrinology ; 142(1): 147-56, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11145577

ABSTRACT

Dependence of murine pro-B Ba/F3 cells on interleukin-3 can be substituted by GH when cells are stably transfected with the GH receptor (GHR) complementary DNA. Recently, we demonstrated that Ba/F3 cells produce GH, which is responsible for the survival of cells expressing the GHR. This GH effect involves the activation of nuclear factor-kappaB (NF-kappaB). Here, we examined the signaling pathways mediating proliferation of growth factor-deprived Ba/F3 GHR cells. Exogenous GH stimulation of Ba/F3 GHR cells induced cyclins E and A and the cyclin-dependent kinase inhibitor p21(waf1/cip1) and repressed cyclin-dependent kinase inhibitor p27(kip1). The presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor Ly 294002 abolished proliferation induced by GH, arresting Ba/F3 GHR cells at the G(1)/S boundary, but did not promote apoptosis. Thus, the proliferative effect of GH is closely related to PI 3-kinase activation, whereas PI 3-kinase is not essential for GH-induced cell survival. Addition of Ly 294002 resulted in a moderate decrease in NF-kappaB activation by GH, suggesting a possible link between PI 3-kinase and NF-kappaB signaling by GH. Expression of c-myc was also induced by GH in Ba/F3 GHR cells, and inactivation of either PI 3-kinase or NF-kappaB reduced this induction. Overexpression of the dominant negative repressor mutant c-Myc-RX resulted in an inhibition of the GH proliferative effect, suggesting the involvement of c-myc in GH-induced proliferation. Taken together, these results suggest that the effects of GH on cell survival and proliferation are mediated through two different signaling pathways, NF-kappaB and PI 3-kinase, respectively; although cross-talk between them has not been excluded. NF-kappaB, which has been shown to be responsible for the antiapoptotic effect of GH, could also participate in GH-induced proliferation, as c-myc expression is promoted by PI 3-kinase, in an NF-kappaB-dependent and -independent manner.


Subject(s)
Apoptosis/drug effects , Cell Cycle/drug effects , Growth Hormone/pharmacology , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Somatotropin/physiology , Signal Transduction/physiology , Animals , Cell Division/drug effects , Cell Line , Cell Survival/drug effects , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , G1 Phase , Genes, Reporter , Genes, myc , Luciferases/genetics , Mice , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Rats , Receptors, Somatotropin/drug effects , Receptors, Somatotropin/genetics , S Phase , Signal Transduction/drug effects , Transfection
14.
Mol Cell Biochem ; 227(1-2): 153-65, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11827167

ABSTRACT

Breast cancer is a major cause of cancer death in women, and the genetic abnormalities leading to the common sporadic forms of the disease are still under active investigation. CK2 has been reported to be upregulated in human breast cancer, which these studies confirm; CK2 is also upregulated in rat carcinogen-induced breast tumors. Transgenic mice overexpressing CK2alpha in the mammary gland develop mammary hyperplasia, dysplasia, and eventually adenocarcinomas, demonstrating that dysregulated expression of CK2 can contribute to transformation of the mammary epithelium. These mammary tumors have evidence of activation of the Wnt and NFkappaB pathways and upregulation of c-Myc. CK2 is capable of phosphorylating the key signaling molecule in the Wnt pathway, the transcriptional cofactor beta-catenin, and regulating its turnover. CK2 is known to phosphorylate IkappaB and thereby regulate basal NFkappaB levels; in the mammary cell lines and tumors, CK2 activity correlates with NFkappaB levels and inhibition of CK2 downregulates NFkappaB. Thus, CK2 may promote breast cancer through dysregulation of key pathways of transcriptional control in the mammary epithelium, and inhibition of CK2 has a potential role in the treatment of breast and other cancers.


Subject(s)
Breast Neoplasms/metabolism , Mammary Neoplasms, Animal/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , Zebrafish Proteins , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Animals , Apigenin , Blotting, Western , Casein Kinase II , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Down-Regulation , Flavonoids/pharmacology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Genes, Reporter , Humans , Immunohistochemistry , Mice , Mice, Transgenic , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasms, Experimental , Phosphorylation , Precipitin Tests , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Rats , Signal Transduction , Time Factors , Transcription, Genetic , Up-Regulation , Wnt Proteins
15.
Oncogene ; 19(48): 5498-506, 2000 Nov 16.
Article in English | MEDLINE | ID: mdl-11114727

ABSTRACT

NF-kappaB/Rel transcription factors regulate many genes involved in control of cellular proliferation, neoplastic transformation, and apoptosis, including the c-myc oncogene. Recently, we have observed that levels of NF-kappaB and aryl hydrocarbon receptor (AhR), which mediates malignant transformation by environmental carcinogens, are highly elevated and appear constitutively active in breast cancer cells. Rel factors have been found to functionally interact with other transcription factors. Here we demonstrate a physical and functional association between the RelA subunit of NF-kappaB and AhR resulting in the activation of c-myc gene transcription in breast cancer cells. RelA and AhR proteins were co-immunoprecipitated from cytoplasmic and nuclear extracts of non-malignant MCF-10F breast epithelial and malignant Hs578T breast cancer cells. In transient co-transfection, RelA and AhR gene products demonstrated cooperation in transactivation of the c-myc promoter, which was dependent on the NF-kappaB elements, and in induction of endogenous c-Myc protein levels. A novel AhR/RelA-containing NF-kappaB element binding complex was identified by electrophoretic mobility shift analysis of nuclear extracts from RelA and AhR co-transfected Hs578T cells. Thus, the RelA and AhR proteins functionally cooperate to bind to NF-kappaB elements and induce c-myc gene expression. These findings suggest a novel signaling mechanism whereby the Ah receptor can stimulate proliferation and tumorigenesis of mammary cells.


Subject(s)
Breast/physiology , Genes, myc/genetics , NF-kappa B/physiology , Receptors, Aryl Hydrocarbon/physiology , Transcriptional Activation/physiology , Binding Sites , Breast/cytology , Breast/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Division/genetics , Cell Line , Cell Transformation, Neoplastic/genetics , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/physiology , Gene Expression Regulation/physiology , Humans , NF-kappa B/genetics , NF-kappa B/metabolism , Precipitin Tests , Promoter Regions, Genetic/genetics , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Transcription Factor RelA , Transfection , Tumor Cells, Cultured
16.
Breast Cancer Res Treat ; 63(2): 117-31, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11097088

ABSTRACT

Exposure to ubiquitous environmental chemicals, such as polycyclic aromatic hydrocarbons (PAH), may contribute to human breast cancer. In animals, PAH induce tumors in part by activating the aryl hydrocarbon receptor (AhR)/transcription factor. Historically, investigations into AhR-regulated carcinogenesis have focused on AhR-dependent transcriptional regulation of cytochrome P450 (CYP) enzymes which oxidize PAH to mutagenic intermediates. However, recent studies suggest that the AhR directly regulates cell growth. Given the postulated role of the AhR in carcinogenesis, we predicted that: (1) tissue predisposed to PAH tumorigenesis would express the AhR and (2) aberrant AhR and/or AhR-regulated gene expression would accompany malignant transformation. To test these hypotheses, AhR and CYP1 protein and/or mRNA levels were evaluated in rat mammary tumors induced with 7, 12-dimethylbenz[a]anthracene (DMBA), a prototypic PAH and AhR ligand. Results indicate modest AhR expression in normal mammary myoepithelial and ductal epithelial cells. In contrast, high AhR levels were detected in DMBA-induced tumors. Nuclear AhR localization in tumors suggested constitutive AhR activation. In situ hybridization and quantitative RT-PCR assays indicated high AhR mRNA levels in neoplastic epithelial cells. While both AhR-regulated CYP1A1 and CYP1B1 mRNAs were induced in breast tissue within 6 h of DMBA gavage, only CYP1B1 mRNA remained elevated in tumors. These results: (1) help explain targeting of breast tissue by carcinogenic PAH, (2) imply that AhR and CYP1B1 hyper-expression represent molecular biomarkers for, at least, PAH-induced mammary cell transformation, and (3) suggest mechanisms through which the AhR may contribute to carcinogenesis well after exogenous AhR ligands have been eliminated.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 Enzyme System/genetics , Mammary Neoplasms, Experimental/metabolism , RNA, Messenger/analysis , Receptors, Aryl Hydrocarbon/analysis , 9,10-Dimethyl-1,2-benzanthracene , Animals , Cytochrome P-450 CYP1B1 , Female , Mammary Glands, Animal/metabolism , Mammary Neoplasms, Experimental/chemically induced , Rats , Rats, Sprague-Dawley
17.
J Cell Biochem ; 80(1): 146-55, 2000 Sep 18.
Article in English | MEDLINE | ID: mdl-11029762

ABSTRACT

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell-layer associated pepsin-resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils.


Subject(s)
Collagen/metabolism , Muscle, Smooth, Vascular/metabolism , Animals , Cattle , Cells, Cultured , Collagen/ultrastructure , Extracellular Matrix/metabolism , Extracellular Matrix/ultrastructure , Immunohistochemistry , Microscopy, Electron/methods , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/ultrastructure
18.
Mol Cell Biol ; 20(15): 5381-91, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10891479

ABSTRACT

NF-kappaB/Rel factors have been implicated in the regulation of liver cell death during development, after partial hepatectomy, and in hepatocytes in culture. Rat liver epithelial cells (RLEs) display many biochemical and ultrastructural characteristics of oval cells, which are multipotent cells that can differentiate into mature hepatocytes. While untransformed RLEs undergo growth arrest and apoptosis in response to transforming growth factor beta1 (TGF-beta1) treatment, oncogenic Ras- or Raf-transformed RLEs are insensitive to TGF-beta1-mediated growth arrest. Here we have tested the hypothesis that Ras- or Raf-transformed RLEs have altered NF-kappaB regulation, leading to this resistance to TGF-beta1. We show that classical NF-kappaB is aberrantly activated in Ras- or Raf-transformed RLEs, due to increased phosphorylation and degradation of IkappaB-alpha protein. Inhibition of NF-kappaB activity with a dominant negative form of IkappaB-alpha restored TGF-beta1-mediated cell killing of transformed RLEs. IKK activity mediates this hyperphosphorylation of IkappaB-alpha protein. As judged by kinase assays and transfection of dominant negative IKK-1 and IKK-2 expression vectors, NF-kappaB activation by Ras appeared to be mediated by both IKK-1 and IKK-2, while Raf-induced NF-kappaB activation was mediated by IKK-2. NF-kappaB activation in the Ras-transformed cells was mediated by both the Raf and phosphatidylinositol 3-kinase pathways, while in the Raf-transformed cells, NF-kappaB induction was mediated by the mitogen-activated protein kinase cascade. Last, inhibition of either IKK-1 or IKK-2 reduced focus-forming activity in Ras-transformed RLEs. Overall, these studies elucidate a mechanism that contributes to the process of transformation of liver cells by oncogene Ras and Raf through the IkappaB kinase complex leading to constitutive activation of NF-kappaB.


Subject(s)
Cell Transformation, Neoplastic/genetics , Genes, ras , I-kappa B Proteins/metabolism , Liver/pathology , Retroviridae Proteins, Oncogenic/genetics , Animals , Apoptosis/drug effects , Cell Division/drug effects , Cell Line, Transformed , Epithelium/drug effects , Epithelium/metabolism , Epithelium/pathology , I-kappa B Proteins/antagonists & inhibitors , I-kappa B Proteins/genetics , Liver/drug effects , NF-kappa B/metabolism , Oncogene Proteins v-raf , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Rats , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology
19.
Blood ; 96(3): 1013-20, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10910917

ABSTRACT

A-myb is a member of the myb family of transcription factors, which regulates proliferation, differentiation, and apoptosis of hematopoietic cells. A-Myb expression is normally restricted to the proliferating B-cell centroblasts and transgenic mice overexpressing A-myb displayed enhanced hyperplasia of the lymph nodes. Because A-Myb is highly expressed in several subtypes of human B-cell neoplasias, we sought to determine whether the A-myb gene promoted proliferation and survival of B lymphocytes, using the WEHI 231 and CH33 murine B-cell lymphomas as models. Here, we show that ectopic expression of A-myb rescues WEHI 231 and CH33 cells from growth arrest and apoptosis induced by anti-IgM treatment. Previously, we demonstrated an essential role of the c-myc gene in promoting cell survival of WEHI 231 cells in response to a variety of apoptotic stimuli. Furthermore, we and others have shown that the c-myc gene is potently transactivated by A-Myb in several cell types. Thus, we sought to determine whether c-Myc would mediate the A-Myb antiapoptotic effect in B cells. Here we show that ectopic expression of A-myb leads to maintenance of c-myc expression, and that expression of antisense c-myc RNA ablates A-Myb-mediated survival signals. Thus, these findings strongly implicate the A-myb gene in the regulation of B-cell survival and confirm the c-myc gene as one of the downstream targets of A-myb in these cells. Overall, our observation suggests that A-myb expression may be relevant to the pathology of human B-cell neoplasias.


Subject(s)
Apoptosis/genetics , Genes, myb , Genes, myc , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/pathology , Receptors, Fc/genetics , Animals , Apoptosis/immunology , Gene Expression Regulation, Neoplastic , Humans , Mice , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-myb , Receptors, Fc/immunology , Trans-Activators/genetics , Transcription, Genetic
20.
Mol Endocrinol ; 14(5): 650-61, 2000 May.
Article in English | MEDLINE | ID: mdl-10809229

ABSTRACT

The pro-B Ba/F3 cell line requires interleukin-3 and serum for growth, and their removal results in cell apoptosis. Ba/F3 cells transfected with the GH receptor (GHR) cDNA become able to proliferate in response to GH. To investigate the role of GH in the control of apoptosis, Ba/F3 cells expressing either the wild-type rat GHR (Ba/F3 GHR) or a mutated rat GHR (Ba/F3 ILV/T) were used. We show that Ba/F3 GHR cells, but not parental Ba/F3 or Ba/F3 ILV/T cells, were able to survive in the absence of growth factor. Furthermore, an autocrine/paracrine mode of GH action was suggested by the demonstration that Ba/F3 cells produce GH, and that addition of GH antagonists (B2036 and G120K) promotes apoptosis of Ba/F3 GHR cells. Consistent with survival, the levels of both antiapoptotic proteins Bcl-2 and Bag-1 were maintained in Ba/F3 GHR cells, but not in parental Ba/F3 cells upon growth factor deprivation. Constitutive activation of the transcription factor nuclear factor-kappaB (NF-kappaB), which has been shown to promote cell survival, was sustained in Ba/F3 GHR cells, whereas no NF-kappaB activation was detected in parental Ba/F3 cells in the absence of growth factor. Furthermore, addition of GH induced NF-kappaB DNA binding activity in Ba/F3 GHR cells. Overexpression of the mutated IkappaB alpha (A32/36) protein, known to inhibit NF-kappaB activity, resulted in death of growth factor-deprived Ba/F3 GHR cells, and addition of GH was no longer able to rescue these cells from apoptosis. Together, our results provide evidence for a new GH-mediated pathway that initiates a survival signal through activation of the transcription factor NF-kappaB and sustained levels of the antiapoptotic proteins Bcl-2 and Bag-1.


Subject(s)
Apoptosis/drug effects , Growth Hormone/pharmacology , Hematopoietic Stem Cells/drug effects , I-kappa B Proteins , Interleukin-3/pharmacology , NF-kappa B/physiology , Receptors, Somatotropin/drug effects , Transcription, Genetic/drug effects , Animals , Autocrine Communication , Carrier Proteins/biosynthesis , Cattle , Cell Cycle/drug effects , Cell Line , Culture Media/pharmacology , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Depression, Chemical , Dimerization , Genes, bcl-2 , Growth Hormone/antagonists & inhibitors , Hematopoietic Stem Cells/cytology , Mice , NF-KappaB Inhibitor alpha , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Rats , Receptors, Somatotropin/genetics , Receptors, Somatotropin/metabolism , Transcription Factors , Transfection , bcl-X Protein
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