Elevated ornithine decarboxylase levels activate ataxia telangiectasia mutated-DNA damage signaling in normal keratinocytes

We examined the effect of increased expression of ornithine decarboxylase (ODC), a key rate-limiting enzyme in polyamine biosynthesis, on cell survival in primary cultures of keratinocytes isolated from the skin of K6/ODC transgenic mice (Ker/ODC) and their normal littermates (Ker/Norm). Although elevated levels of ODC and polyamines stimulate proliferation of keratinocytes, Ker/ODC undergo apoptotic cell death within days of primary culture unlike Ker/Norm that continue to proliferate. Phosphorylation of ataxia telangiectasia mutated (ATM) and its substrate p53 are significantly induced both in Ker/ODC and in K6/ODC transgenic skin. Chromatin immunoprecipitation analyses show that the increased level of p53 in Ker/ODC is accompanied by increased recruitment of p53 to the Bax proximal promoter. ATM activation is polyamine dependent because alpha-difluoromethylornithine, a specific inhibitor of ODC activity, blocks its phosphorylation. Ker/ODC also displays increased generation of H(2)O(2), acrolein-lysine conjugates, and protein oxidation products as well as polyamine-dependent DNA damage, as measured by the comet assay and the expression of the phosphorylated form of the histone variant gamma H2AX. Both reactive oxygen species generation and apoptotic cell death of Ker/ODC may, at least in part, be due to induction of a polyamine catabolic pathway that generates both H(2)O(2) and cytotoxic aldehydes, because spermine oxidase (SMO) levels are induced in Ker/ODC. In addition, treatment with MDL 72,527, an inhibitor of SMO, blocks the production of H(2)O(2) and increases the survival of Ker/ODC. These results show a novel activation of the ATM-DNA damage signaling pathway in response to increased ODC activity in nontumorigenic keratinocytes.     

Ras transformation of RIE-1 cells activates cap-independent translation of ornithine decarboxylase: regulation by the Raf/MEK/ERK and phosphatidylinositol 3-kinase pathways

Ornithine decarboxylase (ODC) is the first and generally rate-limiting enzyme in polyamine biosynthesis. Deregulation of ODC is critical for oncogenic growth, and ODC is a target of Ras. These experiments examine translational regulation of ODC in RIE-1 cells, comparing untransformed cells with those transformed by an activated Ras12V mutant. Analysis of the ODC 5' untranslated region (5'UTR) revealed four splice variants with the presence or absence of two intronic sequences. All four 5'UTR species were found in both cell lines; however, variants containing intronic sequences were more abundant in Ras-transformed cells. All splice variants support internal ribosome entry site (IRES)-mediated translation, and IRES activity is markedly elevated in cells transformed by Ras. Inhibition of Ras effector targets indicated that the ODC IRES element is regulated by the phosphorylation status of the translation factor eIF4E. Dephosphorylation of eIF4E by inhibition of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) or the eIF4E kinase Mnk1/2 increases ODC IRES activity in both cell lines. When both the Raf/MEK/ERK and phosphatidylinositol 3-kinase/mammalian target of rapamycin pathways are inhibited in normal cells, ODC IRES activity is very low and cells arrest in G(1). When these pathways are inhibited in Ras-transformed cells, cell cycle arrest does not occur and ODC IRES activity increases, helping to maintain high ODC activity.     

Elevated levels of ornithine decarboxylase and polyamines in the kidneys of estradiol-treated male hamsters

The effects of 17 ß-estradiol (E₂) on the levels ofornithlne decarboxytase (ODC), S-adenosylmethionine decarboxylase(SAMDC), and of the polyamines putrescine, spermidine and sperminein the kidneys of castrated male hamsters were determined. Thei.p. injection of E₂ into male hamsters led to renal ODC levelsthree times above the control levels 6–12 h after treatment.Similarly, the renal ODC levels in hamsters treated with chronicdoses of E₂ for 60–180 days were 13–1.9 times thecorresponding enzyme levels in control, sham-operated animals.With a series of estrogen analogues, there was a direct correlationbetween the rise in renal ODC in vitro and the binding to renalestradiol receptor sites in vitro. The hamster kidney levelsof the polyamines putrescine, spermidine and spermine all declinedduring the 180-day experimental period. A single i.p. injectionof E led to a 70% increase in renal SAMDC activity 12 h aftertreatment. However, the administration of E for 180–270days was without effect on the normal age-dependent declinein SAMDC levels noted in kidneys. These results indicate that,like other carcinogens and promoters, E₂ increases the levelsof ODC and of polyamines in its target tissue and that the risein ODC is mediated by a specific estradiol-binding protein.     

Blocking the Raf/MEK/ERK pathway sensitizes acute myelogenous leukemia cells to lovastatin-induced apoptosis

The statin family of drugs are well-established inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and are used clinically in the control of hypercholesterolemia. Recent evidence, from ourselves and others, shows that statins can also trigger tumor-specific apoptosis by blocking protein geranylgeranylation. We and others have proposed that statins disrupt localization and function of geranylgeranylated proteins responsible for activating signal transduction pathways essential for the growth and/or survival of transformed cells. To explore this further, we have investigated whether the mitogen-activated protein kinase (MAPK) signaling cascades play a role in regulating statin-induced apoptosis. Cells derived from acute myelogenous leukemia (AML) are used as our model system. We show that p38 and c-Jun NH2-terminal kinase/stress-activated kinase MAPK pathways are not altered during lovastatin-induced apoptosis. By contrast, exposure of primary and established AML cells to statins results in significant disruption of basal extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Addition of geranylgeranyl PPi reverses statin-induced loss of ERK1/2 phosphorylation and apoptosis. By establishing and evaluating the inducible Raf-1:ER system in AML cells, we show that constitutive activation of the Raf/MAPK kinase (MEK)/ERK pathway significantly represses but does not completely block lovastatin-induced apoptosis. Our results strongly suggest statins trigger apoptosis by regulating several signaling pathways, including the Raf/MEK/ERK pathway. Indeed, down-regulation of the Raf/MEK/ERK pathway potentiates statin-induced apoptosis because exposure to the MEK1 inhibitor PD98059 sensitizes AML cells to low, physiologically achievable concentrations of lovastatin. Our study suggests that lovastatin, alone or in combination with a MEK1 inhibitor, may represent a new and immediately available therapeutic approach to combat tumors with activated ERK1/2, such as AML.     

REVERSION OF MALIGNANT PHENOTYPES OF HUMAN LUNG SQUAMOUS CARCINOMA CELLS BY ORNITHINE DECARBOXYLASE ANTISENSE RNA

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Estrogen-dependent regulation of ornithine decarboxylase in breast cancer cells through activation of nongenomic cAMP-dependent pathways

17beta-estradiol (E2) induces ornithine decarboxylase (ODC) activity in several E2-responsive tissues/cells, and this study investigated the mechanism of hormone-induced transactivation in MCF-7 human breast cancer cells. E2-induced reporter gene (luciferase) activity in MCF-7 cells transfected with a construct (pODC1) containing the -164 to +29 region of the human ODC gene promoter linked to bacterial luciferase. This promoter sequence contains GC-rich Sp1 binding sites, CAAT, LSF, cAMP response element (CRE), and TATA motifs. Deletion and mutational analysis of the ODC promoter showed that both CAAT and LSF sites were required for hormone-induced transactivation. Gel mobility shift and DNA footprinting assays indicated that NFYA and LSF bound the CAAT and LSF motifs, respectively, and GAL4-NFYA/GAL4-LSF chimeras were also activated by E2, 8-bromo-cAMP, and protein kinase A (PKA) expression plasmid. However, E2-induced transactivation of GAL4-NFYA and GAL4-LSF was blocked by the PKA inhibitor SQ22356 indicating that the mechanism of ODC induction by E2 involves upregulation of cAMP/PKA through nongenomic pathways of estrogen action.     

Interleukin-6, osteopontin and Raf/MEK/ERK signaling modulate the sensitivity of human myeloma cells to alkylphosphocholines

Alkylphosphocholines are highly active against multiple myeloma (MM) cells in vitro and are devoid of myelotoxicity. Little is known about the determinants of MM cell responsiveness or resistance to these drugs. In this study we investigated the effects of disease-relevant cytokines, such as interleukin-6 (IL-6) and osteopontin (OPN), on the in vitro antimyeloma activity of erufosine and perifosine. The role of the Raf/MEK/ERK pathway was also studied. Exogenous IL-6 reduced the cytotoxicity of erufosine against OPM-2 cells and, to a smaller extent, against U-266 cells. This was accompanied by inhibition of apoptosis in OPM-2 cells. The efficacy of perifosine was similarly affected, but to a greater extent. IL-6 slightly enhanced the sensitivity of RPMI-8226 cells to erufosine, thus emphasizing the heterogeneity of MM. Induced overexpression of OPN isoforms made OPM-2 cells less sensitive to erufosine. In all cases of IL-6- or OPN-induced resistance, the effective concentrations of erufosine were still within the clinically achievable range. Like other alkylphosphocholines, erufosine enhanced Raf/MEK/ERK signaling in MM cells but in some cases this contributed to cytotoxicity.     

Endogenous tumor growth factor indicated by increased ornithine decarboxylase activity in malignant cells treated with host serum ascites fluid.

Ornithine decarboxylase (ODC) production was used as an indicator of mitotic activity in neoplastic cells removed from murine hosts at progressive stages of growth. Cells from three ascites cancers and one fibrosarcoma were tested and showed declining ODC production with progressive growth. The cells were incubated with serum or malignant effusion fluid taken from the murine hosts at progressive stages of growth. For 2 to 3 weeks after tumor implantation, sera and, in particular, ascites fluids increasingly stimulated ODC production in cells at all stages of growth. With advancing disease, without the malignant growth having reached a stationary phase, the collected fluids decreasingly stimulated ODC production in the cells. The stimulating factor(s) in host serum and malignant effusion fluid were not tumor specific in the one combination tested.     

Inositol hexaphosphate reduces 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase independent of protein kinase C isoform expression in keratinocytes

High-fiber diets have been shown to have beneficial effects on preventing tumorigenesis. Inositol hexaphosphate (InsP₆ or phytic acid) which is a fiber-associated component of cereals and legumes has been demonstrated to inhibit cell proliferation and enhance cell differentiation, indicating its potential for chemopreventive roles. In this study, we investigated the effect of InsP₆ on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, an essential event in tumor promotion in HEL-30 cells, a murine keratinocyte cell line and SENCAR mouse skin. ODC activity was significantly reduced by 0.5 mM InsP₆ in keratinocytes (P<0.01). Furthermore, when mouse skin was treated with 10 mM InsP₆, ODC induction was significantly inhibited (P<0.05). In addition, the expression of TPA-induced c-myc mRNA was significantly inhibited by the same InsP₆ treatments in HEL-30 cells and CD-1 mouse skin (P<0.01). No changes in protein kinase C (PKC) isoform expression and phorbol dibutyrate binding due to InsP₆ treatment were found in HEL-30 cells. These results indicate that InsP₆ reduces TPA-induced ODC activity independent of PKC isoform expression.     

Elevated levels of angiostatin in effusions from patients with malignant disease

The aim of this study was to determine the presence of angiostatin in ascitic and pleural effusions from cancer patients, as well as of metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA), both involved in angiostatin generation in in vitro models. Ascitic fluids, pleural exudates, and sera from 21 cancer patients were analyzed for the presence of angiostatin by western blot, whereas gelatinases MMP-2 and MMP-9, and uPA were evaluated by zymography. Our study revealed elevated levels of angiostatin in effusions of cancer patients, contrasting with mostly intermediate levels in less than half of their sera, and undetectable levels in normal sera. Despite the observation of enhanced levels of HMW-uPA and MMP-2 in malignant effusions from cancer patients, their analysis in individual samples showed no association between angiostatin presence and the enzymes, suggesting that the latter would not play an unimportant role, if any, in in vivo generation of angiostatin.     

Oncogenic KRAS sensitizes premalignant,but not malignant cells,to Noxa-dependent apoptosis through the activation of the MEK/ERK pathway

KRAS is mutated in about 20-25% of all human cancers and especially in pancreatic, lung and colorectal tumors. Oncogenic KRAS stimulates several pro-survival pathways, but it also triggers the trans-activation of pro-apoptotic genes. In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels. Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner. In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression. We therefore speculated that other survival pathways are counteracting the pro-apoptotic effect of mutated KRAS and found that the inhibition of AKT restores sensitivity to treatment, especially in presence of oncogenic KRAS. In conclusion, our work suggests that the pharmacological inhibition of the pathways triggered by mutated KRAS could also switch off its oncogene-activated pro-apoptotic stimulation. On the contrary, the combination of chemotherapy to inhibitors of specific pro-survival pathways, such as the one controlled by AKT, could enhance treatment efficacy by exploiting the pro-death stimulation derived by oncogene activation.     

Elevated ornithine decarboxylase activity, polyamines and cell proliferation in neoplastic and vacuolated liver cells of winter flounder (Pleuronectes americanus)

Liver neoplasms, including hepatocellular and cholangiocellulartumors, commonly occur in winter flounder (Pleuronectes americanus)caught from some chemically contaminated areas such as BostonHarbor. Hydropically vacuolated cells, very often associatedwith neoplasia in winter flounder liver, appear to representthe first cellular abnormality in animals that later developfrank neoplasms. The proliferative capacity of hydropicallyvacuolated cells was studied by analyzing both ornithine decarboxylase(ODC) activity and bromodeoxyuridine (BrdU) labeling indices.Liver of winter flounder with vacuolated cellular lesions hadODC activity more than 5- to 12-fold greater than that in liverthat lacked such vacuolation, whether caught from Boston Harboror Georges Bank. Large focal areas of hydropically vacuolatedcells dissected from severely affected livers had ODC activityas high or higher than surrounding parenchymal tissue. Significantelevations in hepatic polyamine levels and ratios of putrescine/spermidinewere also present in all Boston Harbor animals studied, especiallythose exhibiting vacuolated cellular lesions, as compared toGeorges Bank fish. BrdU labeling techniques indicate that hydropicallyvacuolated cells, along with perivacuolar small basophilic cellsand neoplastic cholangiocytes, appear to have the capacity tosynthesize DNA and undergo mitosis. The frequent associationof hydropically vacuolated cells with hepatic neoplasia, alongwith high ODC activity and DNA synthesis capability, suggestthat the vacuolated cells and/or perivacuolar basophilic cellsmay be integral to the development of some neoplastic phenotypesin winter flounder liver.     

Elevated ornithine decarboxylase activity, polyamines and cell proliferation in neoplastic and vacuolated liver cells of winter flounder (Pleuronectes americanus)

The publishers would like to apologise for publishing the incorrectfigure lc in this paper. The correct figure and legend is shownbelow.     

A network of genetic events sufficient to convert normal human cells to a tumorigenic state

Although great progress has been made at identifying and characterizing individual genes involved in cancer, less is known about how the combination of such genes collaborate to form tumors in humans. To this end, we sought to genetically recreate tumorigenesis in normal human cells using genes altered in human cancer. We now show that expression of mammalian proteins that inactivate the tumor suppressors Rb and p53 in conjunction with the oncoproteins Ras and Myc and the telomerase subunit hTERT is sufficient to drive a number of normal human somatic cells to a tumorigenic fate. This provides a blueprint of the events that lead to human cancer, allowing different cancers to be genetically modeled from normal human cells.     

Expression of B cell activation antigens on normal and malignant B cells

In an attempt to relate the functional events of B cell activation with changes in cell surface molecules, we have used a panel of monoclonal antibodies directed against cell surface antigens expressed on activated but not resting B cells, to determine a sequence of activation antigen expression following anti-immunoglobulin stimulation. Within the first 24 hr of culture with anti-Ig, resting splenic B cells were induced to express B5 and interleukin-2 receptor (IL-2R) and subsequently express T9 and BB1 by 48 hr. Maximum antigen expression was seen by day 3 with the majority of cells expressing B5, IL-2R, T9, and BB1, and fewer numbers of cells expressing Blast-1 and Blast-2. By day 6, the expression of these antigens significantly decreased. Dual fluorochrome staining of anti-Ig activated B cells demonstrated heterogeneity of activation antigen expression, suggesting the existence of subpopulations of activated B cells. In an attempt to relate the non-Hodgkin's lymphomas (NHLs) to this sequence of activation, 69 tumor samples from patients with B cell NHLs were then examined for expression of these activation antigens. Histologically defined subgroups of B cell NHLs demonstrated differential expression of activation antigens with B5, BB1, and T9 exhibiting the widest distribution, whereas IL-2R, Blast-1, and Blast-2 demonstrated more limited expression. The finding that no B cell malignancy phenotypically resembles the small resting B lymphocyte coupled with the observation that virtually all B cell NHLs examined expressed activation antigens suggests that these tumors may be the neoplastic counterparts of subpopulations of activated B lymphocytes.     

Modulation of Raf/MEK/ERK kinase activity does not affect the chemoresistance profile of advanced prostate cancer cells

The Raf/MEK/ERK signaling cascade has been extensively studied for its roles in growth and differentiation of a variety of cell types. Confliciting evidence exists regarding the function of classical MAPK signaling with regards to the development of chemotherapeutic drug resistance; some reports describe an pro-survival role, whereas others have suggested that activation of Raf/MEK/ERK is essential for drug-induced death. To elucidate the importance of MAPK signaling in the development of advanced prostate cancer drug resistance, DU145 and PC3 prostate cells were stably-infected/transfected with constitutively-activated mutants of both Raf-1 and B-Raf. Results from MTT analyses suggested that activation of either Raf-1 or B-Raf is inconsequential in prostate cancer chemoresistance. To confirm these findings, the MAPK signal transduction cascade was activated with EGF and response to doxorubicin or paclitaxel was measured in the presence/absence of the MEK-specific inhibitor, U0126. These results showed that inhibition of signals transduced by the MAPK pathway are insufficient to affect the chemoresistance profile of advanced prostate cancer cells. Together, these data demonstrate that the response of prostatic tumors to the chemotherapeutic compounds doxorubicin and paclitaxel is independent of Raf/MEK/ERK signaling.     

Role of asbestos and active oxygen species in activation and expression of ornithine decarboxylase in hamster tracheal epithelial cells.

Induction of ornithine decarboxylase (ODC) enzyme activity occurs after exposure of hamster tracheal epithelial (HTE) cells to asbestos and the soluble tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Since active oxygen species are implicated as mediators of asbestos-induced biological responses studies here were designed to examine whether active oxygen species generated by asbestos or oxidants caused increased ODC activity. In confluent HTE cells, significant blockage of chrysotile or crocidolite asbestos-stimulated ODC activity occurred with simultaneous addition of catalase, but not superoxide dismutase, to medium. The addition of xanthine plus xanthine oxidase caused a dose-dependent increase in ODC activity, which was inhibited significantly after addition of catalase or mannitol, indicating that H2O2 was the principal oxidant produced in that reaction. Addition of phenazine methosulfate, a redox reagent used to generate superoxide, resulted in significant elevation of ODC, which was inhibited by addition of superoxide dismutase but not catalase. Hydrogen peroxide added to culture medium also caused a potent increase in ODC activity inhabitable by catalase. Hypochlorous acid caused increases in ODC activity, although the magnitude of this response was less than that observed with other oxidants. Therefore, although all active oxygen species examined triggered ODC, less reduced species (O2- and H2O2) were more proficient than OH or a halogenated oxidant. All oxidants, except HOCl, caused a significant increase in [3H] thymidine incorporation at 24 or 48 h after their addition to HTE cells. In comparative studies, exposure of HTE cells to either asbestos or xanthine plus xanthine oxide increased the level of ODC mRNAs proportionate to oxidant concentration and the extent of enzyme induction. Thus, data indicate that H2O2 plays a major role in asbestos-stimulated ODC induction and proliferation of epithelial cells of the respiratory tract by altering the regulation of a gene critical to proliferation.     

Inhibition of the Raf/MEK/ERK pathway up-regulates expression of the coxsackievirus and adenovirus receptor in cancer cells

Recombinant adenoviruses are presently being tested clinically as a new strategy for the treatment of cancer. An important determining factor for the successful entry of such adenoviruses into target cells is expression of the coxsackievirus and adenovirus receptor (CAR) at the cell surface. Recent observations suggest that expression of this receptor, which physiologically participates in formation of cell-cell adhesions, is frequently reduced in highly malignant cancer cells. This raises the possibility that those tumors representing the greatest therapeutic challenge might be the least susceptible to infection with therapeutic adenoviruses. We explored the role of the Raf-MEK-ERK pathway on CAR expression in a panel of cancer cells because this pathway is frequently up-regulated in cancer cells and is known to down-regulate cell-cell adhesion molecules. We found that disruption of signaling through the Raf-MEK-ERK pathway by inhibition of MEK up-regulated CAR expression, which was accompanied by increased representation of the protein at the cell surface. After Raf-MEK-ERK inhibition, adenovirus entry into cells was increased and cell killing by replication competent adenoviruses was enhanced in a CAR-dependent manner. Conversely, induction of Raf-1 resulted in reduction and disruption of CAR expression at the cell surface. We conclude that loss of CAR expression in cancer cells is, at least in part, mediated through the Raf-MEK-ERK signal transduction pathway and that pharmacological restoration of CAR at the cell surface could improve adenovirus-based treatments of cancer.