Autoradiographic localization of ornithine decarboxylase in mouse kidney by use of radiolabeled α-difluoromethylornithine

Summary Ornithine decarboxylase, a key enzyme in polyamine biosynthesis and cell growth, has been localized in mouse kidney by autoradiography after administration of radiolabeled -difluoromethylornithine. This drug is an enzyme-activated irreversible inhibitor of ornithine decarboxylase and forms a covalent bond with the enzyme. It was found that ornithine decarboxylase is present in all cell types studied but that the highest content occurs in the proximal convoluted tubules followed by the distal convoluted tubules and the collecting tubules. The majority of the enzyme is located in the cytoplasm but about 10–15% is present in the nuclei (often associated with nucleolus-like components) of the cells of the proximal and distal convoluted tubules. The labeled ornithine decarboxylase was lost rapidly from both nucleus and cytoplasm of all the cell types examined, and labeling by radioactive -difluoromethylornithine was greatly reduced if the mice were pretreated for 5 h with cycloheximide to block protein synthesis. These results indicate that ornithine decarboxylase turns over rapidly in all of the cells.     

Growth inhibition of pathogenic yeast isolates by α-difluoromethylornithine: An inhibition of ornithine decarboxylase

A large body of evidence exists suggesting that polyamines can play essential roles in cellular growth and differentiation. We examined the ability of -difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, the major rate-limiting enzyme in polyamine biosynthesis, to inhibit the growth of Candida albicans, C. tropicalis, and C. parapsilosis. Substantial growth-inhibition was observed for all three species at DFMO concentrations ranging from 1 to 100 mM. C. tropicalis was significantly more susceptible to DFMO than C. albicans or C. parapsilosis. Depletion of cellular polyamine pools was seen in all 3 species following exposure to DFMO and polyamine depletion enhanced the susceptibility of the organisms to DFMO. The action of DFMO was specifically antagonized by exogenous polyamines. These data suggest that polyamines are important in the growth of Candida spp. and that inhibitors of polyamine biosynthesis may be useful as antifungal agents.     

Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled α-difluoromethylornithine

The binding of alpha-difluoromethylornithine, an irreversible inhibitor, to ornithine decarboxylase was used to investigate the amount of enzyme present in rat liver under various conditions and in mouse kidney after treatment with androgens. Maximal binding of the drug occurred on incubation of the tissue extract for 60min with 3mum-difluoromethyl[5-(14)C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, having M(r) about 100000 and subunit M(r) about 55000. Treatment of rats with thioacetamide or carbon tetrachloride or by partial hepatectomy produced substantial increases in ornithine decarboxylase activity and parallel increases in the amount of enzyme protein as determined by the extent of binding of difluoromethyl[5-(14)C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-(14)C]ornithine bound to protein. In all cases, the ratio of drug bound to activity was 26fmol/unit, where 1 unit corresponds to 1nmol of substrate decarboxylated in 30min. These results indicate that even after maximal induction of the enzyme in rat liver there is only about 1ng of enzyme present per mg of protein. When mice were treated with androgens there was a substantial increase in renal ornithine decarboxylase activity, the magnitude of which depended on the strain. There was an excellent correspondence between the amount of activity present and the capacity to bind labelled alpha-difluoromethylornithine in the mouse kidney extracts, but in this case the ratio of drug bound to activity was 14fmol/unit, suggesting that the mouse enzyme has a higher catalytic-centre activity. After androgen induction, the mouse kidney extracts contain about 170ng of enzyme/mg of protein. These results indicate that titration with alpha-difluoromethylornithine provides a valuable method by which to quantify the amount of active ornithine decarboxylase present in mammalian tissues, and that the androgen-treated mouse kidney is a much better source for purification of the enzyme than is rat liver.     

Inhibition of EMT6 tumor growth by interference with polyamine biosynthesis ; Effects of α-difluoromethyl- ornithine,an irreversible inhibitor or ornithine decarboxylase

α-Difluoromethylornithine (α-DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), retarded the growth rate of EMT6, a murine mammary sarcoma, in tissue culture. When female BALB/_C mice were inoculated subcutaneously with EMT6 cells, administration of α-DFMO as a 3% solution in the drinking water beginning 5 days after tumor inoculation resulted in an 80% inhibition of tumor weight gain by day 27 compared to controls. This treatment regimen, equivalent to 4.4 g α-DFMO/kg/day, decreased tumor ODC activity, stimulated S-adenosyl-L-methionine decarboxylase (SAM-DC) activity and markedly decreased tumoral putrescine and spermidine, but not spermine, concentrations. The tumor growth inhibitory effects of α-DFMO were similar to those obtained with 4 weekly doses of cyclophosphamide (100 mg/kg i.p. beginning on day 6 post-inoculation). The combination of cyclophosphamide plus α-DFMO caused the same or greater inhibition of tumor growth than either treatment alone. When the SAM-DC and diamine oxidase inhibitor, 1,1'-((methylethanediylidene)-dinitrilo) bis (3-amino-guanidine), was added to α-DFMO treatment, tumor SAM-DC activity, putrescine and spermidine concentrations, but not ODC activity, returned to control values and the anti-proliferative effects of α-DFMO were reversed. These results suggest that α-DFMO treatment is an effective non-toxic method of inhibiting tumor growth by a mechanism involving polyamine depletion.     

Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases’ cascade

Antizymes delicately regulate ornithine decarboxylase (ODC) enzyme activity and polyamine transportation. One member of the family, antizyme-1, plays vital roles in molecular and cellular functions, including developmental regulation, cell cycle, proliferation, cell death, differentiation and tumorigenesis. However, the question of how does it participate in the cell apoptotic mechanism is still unsolved. To elucidate the contribution of human antizyme-1 in haematopoietic cell death, we examine whether inducible overexpression of antizyme enhances apoptotic cell death. Antizyme reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells, acute T leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G₁ appearance, loss of mitochondrial membrane potential (Δψ _m ), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following conditional antizyme overexpression, all protein levels of cyclin-dependent kinases (Cdks) and cyclins are not significantly reduced, except cyclin D, before their entrance into apoptotic cell death. However, introduced cyclin D1 into Jurkat T tetracycline (Tet)-On cell system still couldn’t rescue cells from apoptosis. Antizyme doesn’t influence the expression of tumor suppressor p53 and its downstream p21, but it interferes in the expressions of Bcl-2 family. Inducible antizyme largely enters mitochondria resulting in cytochrome c release from mitochondria to cytosol following Bcl-xL decrease and Bax increase. According to these data, we suggest that antizyme induces apoptosis mainly through mitochondria-mediated and cell cycle-independent pathway. Furthermore, antizyme induces apoptosis not only by Bax accumulation reducing the function of the Bcl-2 family, destroying the Δψ _m , and releasing cytochrome c to cytoplasm but also by the activation of apoptosomal caspase cascade.     

Trophinin-mediated cell adhesion induces apoptosis of human endometrial epithelial cells through PKC-δ

Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-δ protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-δ associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-δ became tyrosine phosphorylated, dissociated from trophinin and entered the nucleus. In PKC-δ knockdown endometrial cells, GWRQ did not induce apoptosis. These results suggest that trophinin-mediated cell adhesion functions as a molecular switch to induce apoptosis through the PKC-δ pathway in endometrial epithelial cells. Thus, trophinin-mediated induction of apoptosis of endometrial epithelial cells, which function as a barrier to embryo invasion, allows trophoblast invasion of maternal tissue and embryo implantation in humans.Key words: blastocyst, embryo implantation, apoptosis, cell adhesion, signal transduction     

Cell kinetic status of mouse lens epithelial cells lacking αA- and αB-crystallin

alphaA- and alphaB-crystallins are small heat shock proteins and molecular chaperones that are known to prevent non-specific aggregation of denaturing proteins. Recent work indicates that alphaA-/- lens epithelial cells grow at a slower rate than wild-type cells, and cultured alphaB-/- cells demonstrate increased hyperproliferation and genomic instability, suggesting that these proteins may exert a direct effect on the cell cycle kinetics, and influence cell proliferation. However, the cell cycle parameters of alphaA/alphaBKO (double knockout) cells have not been analyzed. Here we investigate the cell cycle kinetics of synchronized mouse lens epithelial cultures derived from wild-type and alphaA/alphaB double knockout (alphaA/alphaBKO) mice using BrdU labeling of proliferating cells, and flow cytometric analysis. We also provide data on the changing pattern of expression of HSP25, a small heat shock protein in alphaA/alphaBKO and wild-type cells during the cell cycle. Using serum starvation to synchronize cells in the quiescent G0 phase, and restimulation with serum followed by BrdU labeling and flow cytometry, the data indicated that as compared to wild-type cells, a <50% smaller fraction of the alphaA/alphaBKO cells entered the DNA synthetic S phase of the cell cycle. Furthermore, there was a delay in cell cycle transit through S phase in alphaA/alphaBKO cells, suggesting that although capable of entering S phase, the alphaA/alphaBKO cells are blocked in G1 phase, and are delayed in their cell cycle progression. Immunoblot analysis with antibodies to the small heat shock protein HSP25 indicated that although HSP25 increased in G1 phase of wild-type cells, and remained elevated on further progression through the cell cycle, HSP25 accumulation was delayed to S phase in alphaA/alphaBKO cells. These data can be interpreted to indicate that mouse lens epithelial cell progression through the cell cycle is significantly affected by expression of alphaA and alphaB-crystallin.     

Effects of α-particles on survial and chromosomal aberrations in human mammary epithelial cells

We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of -particles emitted from radon daughters, we concentrated our studies on the efficiency of -particles. Confluent cultures of M/10 cells were exposed to accelerated -particles [beam energy incident at the cell monolayer=3.85 MeV, incident linear energy transfer (LET) in cell= 109 keV/µm] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for -particles (D₀ = 0.73± 0.04 Gy), while a shoulder was observed for x-rays (/ = 2.9 Gy; D₀ = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET -particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for cc-particles and linear-quadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges. The RBE for the induction of total chromosome aberrations (2.3 at 37% cell survival) was lower than that for cell survival, suggesting that chromosome damage at the first postirradiation mitosis is not sufficient to account for the increased efficiency of -particles in the induction of lethal effects. However, measured cell survival after -particle irradiation can be predicted from chromosome damage when cells at different population doubling numbers after irradiation are considered. In fact, a high percentage of -irradiated cells carried unstable chromosomal aberrations up to population doubling number about 5. On the other hand, x-ray-induced damage disappeared rapidly. These results suggest that -particle-induced reproductive death of human mammary epithelial cells is caused by chromosome damage in the first 5 generations following exposure, whereas the in-activation produced by low-LET radiation is mostly related to the aberrations at the first post-irradiation mitosis.     

Effects of α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on testosterone-induced regeneration of prostate and seminal vesicle in castrated rats

1. Castration of adult rats markedly decreases the amounts of polyamines (putrescine, spermidine and spermine) and of RNA and DNA in the ventral prostate and the seminal vesicle. 2. Daily injections of testosterone propionate to rats castrated 7 days previously increase polyamine and nucleic acid contents more rapidly in the seminal vesicle than in the ventral prostate. 3. After 7 days of androgen treatment, polyamine and nucleic acid contents of the seminal vesicle are significantly higher than those of intact animals. Nucleic acid, but not polyamine, contents return to normal values during the next 4 days of continued treatment. In the prostate, androgen treatment increases polyamine and nucleic acid contents to, but not above, normal values. 4. Repeated doses of alpha-difluoromethylornithine, a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase, totally blocked the testosterone-induced increase of putrescine and spermidine in the ventral prostate and of putrescine in the seminal vesicle. They slowed significantly the accumulation of spermine in the ventral prostate and of spermidine in the seminal vesicle. alpha-Difluoromethylornithine also retarded the testosterone-induced accumulation of RNA in the ventral prostate. However, no clear correlation was apparent between accumulation of polyamines and of nucleic acids in the two organs. 5. alpha-Difluoromethylornithine markedly slows the testosterone-induced weight gain of the prostate, but not of the seminal vesicle. Cytological studies suggest that this effect on the prostate is due to inhibition of the androgen-induced restoration of the secretion content of prostatic acini.     

Colocalization of α-lactalbumin and a major casein in secretory vesicles of rat mammary epithelial cells

Summary Whether both casein and noncasein (serum or whey) proteins of milk are contained within the same secretory vesicles of milk secreting mammary epithelial cells was explored. Antibodies to a major casein and to -lactalbumin of rat milk were localized in thin sections with colloidal gold-conjugated second antibodies. Antibodies to the casein component bound to an antigen present within lumina of Golgi apparatus cisternae and within secretory vesicles. This antigen was also recognized in structures within secretory vesicles and within alveolar lumina which were ultrastructurally identified as casein micelles. Antigens recognized by antibodies to -lactalbumin also were present in Golgi apparatus cisternae and within secretory vesicles. Both anti-casein and anti--lactalbumin antibodies recognized antigens within the same secretory vesicles. These observations show that one major noncasein protein of rat's milk is present in casein-containing secretory vesicles.     

Inhibition of Aurora-A kinase induces cell cycle arrest in epithelial ovarian cancer stem cells by affecting NFĸB pathway

Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer.     

Inhibition of Aurora-A kinase induces cell cycle arrest in epithelial ovarian cancer stem cells by affecting NFκB pathway

Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer.Key words: ovarian cancer stem cells, aurora-A kinase, cell cycle arrest, nuclear factor kappaB     

Activation of p53 in gastric cancer cells by tripchlorolide specifically induces apoptosis

There are two possible outcomes when DNA damage occurs in normal mammalian cells: either induction of cell-cycle checkpoint which inhibits the progress of the cell cycles as well as activates DNA repair pathways, or activation of apoptosis to eliminate damaged cells. The p53 tumour-suppressor gene plays a key role in selecting these pathways. In our present works, the human gastric cancer cell line AGS was treated with tripchlorolide, a potent antitumor compound purified from a Chinese herb Tripterygium Wilfordii Hook. Single cell gel electrophoresis (Comet assay) showed that the treatment of tripchlorolide resulted in DNA damage in AGS cells. The damaged AGS cells went through apoptosis, which was time- and dose- dependent.     

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGF-β is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-β signalling. To understand the role of TIF1γ in TGF-β signalling and its requirement for EMT, we analysed the TGF-β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-β-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-β signalling and EMT.     

Effects of α-difluoromethylornithine on pancreatic growth induced by caerulein

The role of ornithine decarboxylase and of polyamines was investigated on caerulein-induced pancreatic growth by the use of α-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase. By itself, DFMO did not affect the pancreatic gland at all but when combined with caerulein, it reduced the increases in DNA synthesis and DNA content initiated by the cholecystokinin analog. The general hypertrophic action of caerulein was not affected by DFMO but specific increases in amylase and chymotrypsin concentrations were observed after 2 days of caerulein. The effect on amylase concentration was further increased after 4 days but that on chymotrypsin was reversed, showing a significant decrease. These data suggest that the polyamines might be involved in pancreatic growth that is stimulated by caerulein and that their action could be mainly oriented towards cellularity. The specific decreases obtained in DNA synthesis and content brought about by DFMO support this observation.     

β3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells

Active RhoA localizes to plasma membrane, where it stimulates formation of focal adhesions and stress fibers. RhoA activity is inhibited by p190RhoGAP following integrin-mediated cell attachment to allow sampling of new adhesive environments. p190RhoGAP is itself activated by Src-dependent tyrosine phosphorylation, which facilitates complex formation with p120RasGAP. This complex then translocates to the cell surface, where p190RhoGAP down-regulates RhoA. Here we demonstrate that the epidermal growth factor receptor (EGFR) cooperates with β3 integrin to regulate p190RhoGAP activity in mouse mammary gland epithelial cells. Adhesion to fibronectin stimulates tyrosine phosphorylation of the EGFR in the absence of receptor ligands. Use of a dominant inhibitory EGFR mutant demonstrates that fibronectin-activated EGFR recruits p120RasGAP to the cell periphery. Expression of an inactive β3 integrin subunit abolishes p190RhoGAP tyrosine phosphorylation, demonstrating a mechanistic link between β3 integrin-activated Src and EGFR regulation of the RhoA inhibitor. The β3 integrin/EGFR pathway also has a positive role in formation of filopodia. Together our data suggest that EGFR constitutes an important intrinsic migratory cue since fibronectin is a key component of the microenvironment in normal mammary gland development and breast cancer. Our data also suggest that EGFR expressed at high levels has a role in eliciting cell shape changes associated with epithelial-to-mesenchymal transition.