Approval of DFMO for high-risk neuroblastoma patients demonstrates a step of success to target MYC pathway.

The "undruggable" MYC oncoproteins are deregulated in 70% human cancers. The approval of DFMO, an irreversible inhibitor of ornithine oxidase (ODC1) that is a direct transcriptional target of MYC, demonstrates that patients can benefit from targeting MYC activity via an indirect approach. However, the mechanism of action of DFMO needs further studies to understand how it works in post-immunotherapy neuroblastomas. Efforts to develop a more potent and safer drug to block MYC function will continue despite challenges.

Polyamines drive myeloid cell survival by buffering intracellular pH to promote immunosuppression in glioblastoma.

Glioblastoma is characterized by the robust infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs). It is not fully understood how TAMCs survive in the acidic tumor microenvironment to cause immunosuppression in glioblastoma. Metabolic and RNA-seq analysis of TAMCs revealed that the arginine-ornithine-polyamine axis is up-regulated in glioblastoma TAMCs but not in tumor-infiltrating CD8+ T cells. Active de novo synthesis of highly basic polyamines within TAMCs efficiently buffered low intracellular pH to support the survival of these immunosuppressive cells in the harsh acidic environment of solid tumors. Administration of difluoromethylornithine (DFMO), a clinically approved inhibitor of polyamine generation, enhanced animal survival in immunocompetent mice by causing a tumor-specific reduction of polyamines and decreased intracellular pH in TAMCs. DFMO combination with immunotherapy or radiotherapy further enhanced animal survival. These findings indicate that polyamines are used by glioblastoma TAMCs to maintain normal intracellular pH and cell survival and thus promote immunosuppression during tumor evolution.

Improving Eflornithine Oral Bioavailability and Brain Uptake by Modulating Intercellular Junctions With an E-cadherin Peptide.

Eflornithine has been used to treat second-stage human African trypanosomiasis. However, it has inadequate oral bioavailability and low blood-brain barrier permeation, thus requiring a lengthy and complicated intravenous infusion schedule. Here, we investigated the feasibility of using an intercellular junction-modulating E-cadherin peptide HAV6 to enhance the oral bioavailability and blood-brain barrier permeation of eflornithine. Eflornithine was not metabolized in liver microsomes, nor was it a substrate for the human efflux transporter P-glycoprotein. Furthermore, HAV6 and HAV6scr (sequence scrambled HAV6) were stable in simulated gastric fluid with pepsin and rat intestinal mucosal scrapings. Both peptides were stable in human plasma, albeit less stable in rat and mouse plasma. HAV6 increased eflornithine permeability across Madin-Darby canine kidney and Caco-2 cell monolayers (5- and up to 8.5-fold), whereas HAV6scr had little effect. Using an in situ rat brain perfusion model, HAV6, but not HAV6scr, significantly increased eflornithine concentrations in different brain regions up to 4.9-fold. In rats, coadministration of HAV6 increased eflornithine oral bioavailability from 38% to 54%, brain concentrations by up to 83%, and cerebrospinal fluid concentrations by 40%. In conclusion, coadministration of HAV6, either during intravenous infusion or as an oral formulation, has the potential to improve eflornithine-based treatment for second-stage human African trypanosomiasis.

Extracellular Spermine Activates DNA Methyltransferase 3A and 3B.

We first demonstrated that long-term increased polyamine (spermine, spermidine, putrescine) intake elevated blood spermine levels in mice and humans, and lifelong consumption of polyamine-rich chow inhibited aging-associated increase in aberrant DNA methylation, inhibited aging-associated pathological changes, and extend lifespan of mouse. Because gene methylation status is closely associated with aging-associated conditions and polyamine metabolism is closely associated with regulation of gene methylation, we investigated the effects of extracellular spermine supplementation on substrate concentrations and enzyme activities involved in gene methylation. Jurkat cells and human mammary epithelial cells were cultured with spermine and/or D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase. Spermine supplementation inhibited enzymatic activities of adenosylmethionine decarboxylase in both cells. The ratio of decarboxylated S-adenosylmethionine to S-adenosyl-L-methionine increased by DFMO and decreased by spermine. In Jurkat cells cultured with DFMO, the protein levels of DNA methyltransferases (DNMTs) 1, 3A and 3B were not changed, however the activity of the three enzymes markedly decreased. The protein levels of these enzymes were not changed by addition of spermine, DNMT 3A and especially 3B were activated. We show that changes in polyamine metabolism dramatically affect substrate concentrations and activities of enzymes involved in gene methylation.

A Label-Free Continuous Fluorescence-Based Assay for Monitoring Ornithine Decarboxylase Activity with a Synthetic Putrescine Receptor.

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6]uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>107 M-1) than to the substrate L-ornithine (340 M-1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters ( kcat = 0.12 s-1 and KM = 24 µM) and inhibition constants of the two ODC inhibitors α-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z' factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.

Triethylenetetramine modulates polyamine and energy metabolism and inhibits cancer cell proliferation.

Polyamine metabolism is an attractive anticancer drug target, since polyamines are absolutely required for cellular proliferation, and increased levels of polyamines and their biosynthetic enzyme ornithine decarboxylase (ODC) are associated with cancer. Triethylenetetramine (TETA) is a charge-deficient isosteric analogue of the polyamine spermidine (Spd) and a Cu(II)-chelating compound used for the treatment of Wilson's disease, and it has been implicated as a potential anticancer therapeutic drug. In the present study, we studied the effects of TETA in comparison with two other Cu(II)-chelators, D-penicillamine (PA) and tetrathiomolybdate (TTM), on polyamine metabolism in DU145 prostate carcinoma, MCF-7 breast carcinoma and JEG-3 choriocarcinoma cells. TETA induced antizyme, down-regulated ODC and inhibited [(14)C] Spd uptake. Moreover, it completely prevented α-difluoromethylornithine (DFMO)-induced increase in [(14)C] Spd uptake, and inhibited [(14)C] putrescine (Put) uptake and ODC activity in vivo Seven-day treatment of DU145 cells with TETA caused growth cessation by reducing intracellular polyamine levels and suppressing the formation of hypusinated eukaryotic translation initiation factor 5A (eIF5A). TETA or its N-acetylated metabolites also inhibited spermine (Spm), diamine and semicarbazide-sensitive amine oxidases and decreased the level of intracellular reactive oxygen species. Moreover, TETA inhibited the utilization of Put as energy source via the tricarboxylic acid (TCA) cycle, as indicated by decreased production of (14)CO2 from [(14)C] Put. These results indicate that TETA attacks multiple proven anticancer drug targets not attributed to copper chelation, which warrants further studies to reveal its potential in cancer chemoprevention and cure.

Hessian fly larval feeding triggers enhanced polyamine levels in susceptible but not resistant wheat.

BACKGROUND: Hessian fly (Mayetiola destructor), a member of the gall midge family, is one of the most destructive pests of wheat (Triticum aestivum) worldwide. Probing of wheat plants by the larvae results in either an incompatible (avirulent larvae, resistant plant) or a compatible (virulent larvae, susceptible plant) interaction. Virulent larvae induce the formation of a nutritive tissue, resembling the inside surface of a gall, in susceptible wheat. These nutritive cells are a rich source of proteins and sugars that sustain the developing virulent Hessian fly larvae. In addition, on susceptible wheat, larvae trigger a significant increase in levels of amino acids including proline and glutamic acid, which are precursors for the biosynthesis of ornithine and arginine that in turn enter the pathway for polyamine biosynthesis. RESULTS: Following Hessian fly larval attack, transcript abundance in susceptible wheat increased for several genes involved in polyamine biosynthesis, leading to higher levels of the free polyamines, putrescine, spermidine and spermine. A concurrent increase in polyamine levels occurred in the virulent larvae despite a decrease in abundance of Mdes-odc (ornithine decarboxylase) transcript encoding a key enzyme in insect putrescine biosynthesis. In contrast, resistant wheat and avirulent Hessian fly larvae did not exhibit significant changes in transcript abundance of genes involved in polyamine biosynthesis or in free polyamine levels. CONCLUSIONS: The major findings from this study are: (i) although polyamines contribute to defense in some plant-pathogen interactions, their production is induced in susceptible wheat during interactions with Hessian fly larvae without contributing to defense, and (ii) due to low abundance of transcripts encoding the rate-limiting ornithine decarboxylase enzyme in the larval polyamine pathway the source of polyamines found in virulent larvae is plausibly wheat-derived. The activation of the host polyamine biosynthesis pathway during compatible wheat-Hessian fly interactions is consistent with a model wherein the virulent larvae usurp the polyamine biosynthesis machinery of the susceptible plant to acquire nutrients required for their own growth and development.

Studies on cell migration model in intestinal epithelial restitution for pharmacological research.

OBJECTIVE: To set up a suitable IEC-6 migration model for pharmacological research and observe the effect of complex polysaccharide from Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao to IEC-6 cell migration. METHODS: The main conditions related to the establishment of the model, including the planting density of the cell, the observation time after scratching, the concentration of the auxiliary material Matrigel, the treatment of the serum-starvation, the concentration of alpha-difluoromethylornithine (DFMO), an inhibitor of the cell migration, were investigated respectively; and the effects of the tested medicines on the model were observed. RESULTS: 4 x 10(5) cell/mL was the suitable planting density of the cell in the 6-well plate; at the 24th hour after scratching was the appropriate time to count the migrating cells; and the proper concentration of Matrigel was 5%; the serum-starvation could evidently reduce the migrating cells, so the culture medium should contain the serum; 2.5 - 5 mmol/L DFMO was proper for inhibition of the cell migration. Complex polysaccharide from Astragalus membranaceus and spermidine both can promote cell migration. CONCLUSION: The established model of IEC-6 cell migration was suitable for intestinal epithelial restitution such as the researches on pathophysiological mechanisms is the effects of the medicines on the cell migration.

The change of antizyme inhibitor expression and its possible role during mammalian cell cycle.

Antizyme inhibitor (AIn), a homolog of ODC, binds to antizyme and inactivates it. We report here that AIn increased at the G1 phase of the cell cycle, preceding the peak of ODC activity in HTC cells in culture. During interphase AIn was present mainly in the cytoplasm and turned over rapidly with the half-life of 10 to 20 min, while antizyme was localized in the nucleus. The level of AIn increased again at the G2/M phase along with ODC, and the rate of turn-over of AIn in mitotic cells decreased with the half-life of approximately 40 min. AIn was colocalized with antizyme at centrosomes during the period from prophase through late anaphase and at the midzone/midbody during telophase. Thereafter, AIn and antizyme were separated and present at different regions on the midbody at late telophase. AIn disappeared at late cytokinesis, whereas antizyme remained at the cytokinesis remnant. Reduction of AIn by RNA interference caused the increase in the number of binucleated cells in HTC cells in culture. These findings suggested that AIn contributed to a rapid increase in ODC at the G1 phase and also played a role in facilitating cells to complete mitosis during the cell cycle.

Effect of difluoromethylornithine on thyroid function in rats.

BACKGROUND: A variety of stimuli cause a rapid increase in polyamine synthesis by increasing an enzyme ornithine decarboxylase required for the biosynthetic pathway of protein synthesis. Difluoromethyl ornithine is a selective inhibitor of this enzyme and hence arrests cell replication strikingly. Its effects on thyroid gland are studied with respect to change in animal's weight and levels of Triiodothyronine, Thyroxine and Thyroid stimulating hormone. The study was conducted to evaluate the inhibitory effects of Di-fluoromethyl ornithine (DFMO) administration on polyamine metabolism of thyroid gland in rats. METHODS: The study was conducted on rats weighing 248 to 320 grams, divided into control and DFMO treated group. A dose of 50 mg/rat was administered subcutaneously to the treated group for 5 consecutive days and placebo (normal saline) injections to control group. On sixth day, blood was collected by cardiac puncture and serum was separated. Serum T3, T4 and TSH were analyzed with the help of radioimmunoassay in both groups. RESULTS: In treated group there was a fall in T3, T4 concentration with significant rise in TSH concentration as compared to control group. CONCLUSION: DFMO (Difluoro methyl ornithine) decreases cellular proliferation of thyroid gland as is assessed by decrease in thyroid hormone levels. The hypothalamo pituitary thyroid axis however remains intact as is shown by a feedback rise in TSH concentration. DFMO can thus be employed for anti-neoplastic clinical trials on account of interference with activity of ODC (Ornithine Decarboxylase) fundamental for polyamine biosynthesis.

Effect of the polyamine analogue N1,N11-diethylnorspermine on cell survival and susceptibility to apoptosis of human chondrocytes.

Chondrocyte survival is closely linked to cartilage integrity, and forms of chondrocyte apoptotic death can contribute to cartilage degeneration in articular diseases. Since growing evidence also implicates polyamines in the control of cell death, we have been investigating the role of polyamine metabolism in chondrocyte survival and apoptosis. Treatment of human C-28/I2 chondrocytes with N(1),N(11)-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, inhibited polyamine biosynthesis and induced polyamine catabolism, thus rapidly depleting all main polyamines. DENSPM did not increase significantly caspase activity, but provoked a late cell death associated to DNA fragmentation. A short treatment with DENSPM did not reduce cell viability when given alone, but enhanced caspase-3 and -9 activation in chondrocytes exposed to tumor necrosis factor-alpha (TNF) and cycloheximide (CHX). A longer treatment with DENSPM however reduced caspase response to TNF plus CHX. Depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not cause cell death and contrasted apoptosis by decreasing caspase activities. In conclusion, following DENSPM treatment, C-28/I2 chondrocytes are initially sensitized to caspase 9-dependent apoptosis in the presence of TNF and CHX and may eventually undergo a late and mainly caspase-independent cell death in the absence of other stimuli. Moreover, these results indicate that a reduction of polyamine levels not only leads to inhibition of cell proliferation, but also of caspase-mediated pathways of chondrocyte apoptosis.

Effects of polyamine depletion by alpha-difluoromethylornithine on in vitro and in vivo biological properties of 4T1 murine mammary cancer cells.

Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anticancer therapy. Polyamine depletion by alpha-difluoromethylornithine (DFMO) has been shown to decrease pulmonary and bone metastasis from human breast cancer cell xenografts. Following these observations, this study was designed to test the effects of DFMO on in vitro and in vivo features of the highly invasive and metastatic 4T1 murine mammary cancer cells. DFMO inhibited proliferation, caused G1-S arrest, and suppressed in vitro invasiveness of 4T1 cells. In contrast to our previous findings with MDA-MB-435 cells, DFMO did not affect the activation of signal transducers and activator of transcription 3, c-Jun N-terminal kinase, and extracellular signal-regulated kinase, but decreased phosphorylation of p38. DFMO did not alter the expression of Twist. DFMO delayed the orthotopic growth of 4T1 xenografts in association with suppressed putrescine and spermidine levels but increased levels of spermine. DFMO did not affect pulmonary metastasis when primary tumors of control and DFMO-treated mice were matched for size. Interestingly, DFMO reduced Ki-67 expression only in the primary tumors but did not affect its expression in the metastatic tumors in the lung. Cleaved caspase-3 expression was not affected by DFMO in either the primary tumors or the pulmonary metastasis. In summary, DFMO treatment markedly inhibited in vitro proliferation and invasiveness of 4T1 cells and retarded the growth of orthotopic xenografts in mice. The failure of DFMO to inhibit pulmonary metastasis in this system appears to be due, at least in part, to its lack of antiproliferative effect at the metastatic sites.

Pathways involved in alanyl-glutamine-induced changes in neutrophil amino- and alpha-keto acid homeostasis or immunocompetence.

We examined the effects of DON [glutamine-analogue and inhibitor of glutamine-requiring enzymes], alanyl-glutamine (regarding its role in neutrophil immunonutrition) and alanyl-glutamine combined with L-NAME, SNAP, DON, beta-alanine and DFMO on neutrophil amino and alpha-keto acid concentrations or important neutrophil immune functions in order to establish whether an inhibitor of *NO-synthase [L-NAME], an *NO donor [SNAP], an analogue of taurine and a taurine transport antagonist [beta-alanine], an inhibitor of ornithine-decarboxylase [DFMO] as well as DON could influence any of the alanyl-glutamine-induced effects. In summary, irrespective of which pharmacological, metabolism-inhibiting or receptor-mediated mechanisms were involved, our results showed that impairment of granulocytic glutamine uptake, modulation of intracellular glutamine metabolisation and/or de novo synthesis as well as a blockade of important glutamine-dependent metabolic processes may led to significant modifications of physiological and immunological functions of the affected cells.

Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells.

Polyamine depletion prevents apoptosis by increasing serine/threonine phosphorylation leading to either inactivation or activation of pro- and anti-apoptotic proteins, respectively. Despite evidence that protein kinases are regulators of apoptosis, a specific role for protein phosphatases in regulating cell survival has not been established. In this study, we show that polyamine depletion inhibits serine/threonine phosphatase 2A (PP2A). Inhibition of PP2A in cells depleted of polyamines correlated well with increased phosphorylation of Bad at Ser112. Bad Ser112 phosphorylation in response to tumor necrosis factor (TNF)-alpha treatment decreased with time in cells grown in control as well as those grown in the presence of alpha-difluoromethylornithine plus putrescine. However, a sustained increase in the levels of Bad Ser112 phosphorylation was maintained in response to TNF-alpha treatment in cells grown in the presence of alpha-difluoromethylornithine. Inhibition of PP2A by okadaic acid and fostriecin or PP2A small interfering RNA transfection significantly decreased TNF-alpha-induced apoptosis in control and polyamine-depleted cells. Inhibition of PP2A by okadaic acid: 1) increased Bad and Bcl-2 phosphorylation at Ser112 and Ser70, respectively; 2) increased ERK activity; 3) prevented JNK activation; 4) prevented cytochrome c release, and activation of caspases-9 and -3 in response to TNF-alpha. Inhibition of MEK1 by U0126 prevented phosphorylation of Bad at Ser112. These results indicate that polyamines regulate PP2A activity, and inhibition of PP2A in response to polyamine depletion increases steady state levels of Bad and Bcl-2 proteins and their phosphorylation and thereby prevents cytochrome c release, caspase-9, and caspase-3 activation.

Inhibitors of polyamine metabolism: review article.

The identification of increased polyamine concentrations in a variety of diseases from cancer and psoriasis to parasitic infections has led to the hypothesis that manipulation of polyamine metabolism is a realistic target for therapeutic or preventative intervention in the treatment of certain diseases. The early development of polyamine biosynthetic single enzyme inhibitors such as alpha-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) showed some interesting early promise as anticancer drugs, but ultimately failed in vivo. Despite this, DFMO is currently in use as an effective anti-parasitic agent and has recently also been shown to have further potential as a chemopreventative agent in colorectal cancer. The initial promise in vitro led to the development and testing of other potential inhibitors of the pathway namely the polyamine analogues. The analogues have met with greater success than the single enzyme inhibitors possibly due to their multiple targets. These include down regulation of polyamine biosynthesis through inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase and decreased polyamine uptake. This coupled with increased activity of the catabolic enzymes, polyamine oxidase and spermidine/spermine N1-acetyltransferase, and increased polyamine export has made the analogues more effective in depleting polyamine pools. Recently, the identification of a new oxidase (PAO-h1/SMO) in polyamine catabolism and evidence of induction of both PAO and PAO-h1/SMO in response to polyamine analogue treatment, suggests the analogues may become an important part of future chemotherapeutic and/or chemopreventative regimens.

Polyamine synthesis and transport inhibition in a human anaplastic thyroid carcinoma cell line in vitro and as xenograft tumors.

Polyamines are essential cellular components for neoplastic transformation and cell proliferation. Antineoplastic efforts that inhibit polyamine synthesis are insufficient to induce cytotoxicity, due to compensatory induction of polyamine transport. Treatment of an anaplastic human thyroid carcinoma cell line (DRO90-1) with a novel polymeric spermine conjugate (polyspermine; PSpm) caused in vitro cytotoxicity and inhibited the growth of xenograft tumors at low concentrations. Similar in vitro antineoplastic effects were noted with two other human anaplastic thyroid carcinoma cell lines. This coincided with inhibition of polyamine uptake and synthetic enzyme activities, with reduced ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAM-DC) but increased spermidine/spermine N1-acetyltransferase (SSAT) activities, as measured in DRO90-1 cells. In subsequent studies using these cells, PSpm was effective in reducing the intracellular levels of all polyamines in vitro, resulting in cytotoxicity that was not reversed by administration of extracellular polyamines. Low-dose PSpm inhibited tumor growth in vivo, but high doses of PSpm potentiated xenograft tumor growth. PSpm degradation products produced with in vivo treatment may be produced that function as substrates for polyamine biosynthesis. These studies suggest that polyamine metabolism inhibition is a viable target for antineoplastic therapy of anaplastic thyroid carcinoma, although the in vivo response to PSpm suggests that this agent will have limited clinical utility.

Rapid induction of apoptosis by deregulated uptake of polyamine analogues.

Treatment of Chinese hamster ovary cells with alpha-difluoromethylornithine for 3 days, followed by exposure to cycloheximide, led to an unregulated, rapid and massive accumulation of polyamine analogues. This accumulation led to cell death by apoptosis within a few hours. Clear evidence of DNA fragmentation was seen in response to both N-terminally ethylated polyamines and to polyamines containing methyl groups on the terminal carbon atoms. Programmed cell death was induced within 2-4 h of exposure to 1 microM or higher concentrations of N1,N11-bis(ethyl)norspermine. The presence of cycloheximide increased the uptake of the polyamine analogues and therefore led to cell death at lower analogue concentrations, but it was not essential for the induction of apoptosis, since similar effects were seen when the protein synthesis inhibitor was omitted and the concentration of N1, N11-bis(ethyl)norspermine was increased to 5 microM or more. The induction of apoptosis was blocked both by the addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, or by the addition of the polyamine oxidase inhibitor N1-methyl-N2-(2,3-butadienyl)butane-1,4-diamine (MDL 72,527). These experiments provide evidence to support the concepts that: (1) polyamines or their oxidation products may be initiators of programmed cell death; (2) regulation of polyamine biosynthesis and uptake prevents the accumulation of toxic levels of polyamines; and (3) the anti-neoplastic effects of bis(ethyl) polyamine analogues may be due to the induction of apoptosis in sensitive tumour cells.

Reduced tissue ornithine increases the cytotoxicity of difluoromethylornithine.

BACKGROUND: Polyamines are low molecular weight cations that are essential for the growth of all cells. The polyamine inhibitor difluoromethylornithine (DFMO) will decrease tumor growth when administered parenterally; thrombocytopenia is the major dose-limiting toxicity. Since an essential amino acid-based total parenteral nutrition (TPN) regimen was shown to reduce the ornithine and polyamine content of a transplantable sarcoma in preliminary studies, the effect of the amino acid content of TPN on the antitumor activity of DFMO was evaluated. MATERIALS AND METHODS: Fischer 344 male rats were inoculated subcutaneously with a transplantable sarcoma and fed a restricted intake of Purina rodent chow-5001 (8 g/d, RI) for 12 days to induce malnutrition. Rats were then randomized to continue receiving the RI regimen or to receive one of two TPN regimens for an additional 6 days. Isocaloric TPN was formulated with essential amino acids (E) as the sole nitrogen source or with E and nonessential amino acids including arginine (ENA). DFMO (1000 mg/kg/d) was added to the infusate of one group of rats receiving each of the respective TPN regimens. RESULTS: The growth rate of the sarcoma was significantly decreased (P < 0.05) when DFMO was administered with E for 6 days but not when given to rats receiving ENA. DFMO-related thrombocytopenia was greater when administered with E as compared with rats given ENA. The plasma and tissue levels of DFMO were not affected by the TPN amino acid content nor did DFMO have any effect on plasma or tissue ornithine levels. The plasma and tissue levels of ornithine, however, were significantly lower for rats given E as compared with rats receiving ENA or those continued on RI. CONCLUSIONS: The results show that the cytotoxicity of DFMO was enhanced by an essential amino acid-based TPN. This increase was directly associated with a decrease in the plasma and tissue content of ornithine and polyamines.

Localization of ornithine decarboxylase in mouse teeth. An in vitro and in vivo study.

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, and thus in tissue growth and development, has been localized in mouse dental tissues, in vivo as well as in vitro by light and electron microscopic autoradiography with radiolabeled alpha-difluoromethylornithine ([3H]DFMO). Mandibular first molar germs from day-18 fetuses were incubated in vitro in the presence of [3H]DFMO and processed for autoradiography. For ODC localization in vivo, 3-day old puppies received [3H]DFMO by injection. As controls, puppies were injected either with unlabeled DFMO, or with cycloheximide before administration of isotope. Kidneys and mandibles were excised and processed for autoradiography. In vitro, labeling was found in all cell types of the tooth germ, but with a more intense labeling in ameloblasts and odontoblasts. In both these, radioactivity decreased from the tip of the cusps to the cervical loop. In vivo the binding of [3H]DFMO in cells of the ameloblast and odontoblast lineages, respectively, showed a gradual increase form the posterior end of the incisor to its anterior end. The distribution of radioactivity in the kidney was in accordance with findings by others. Both the kidney and tooth cell labeling decreased strongly after cycloheximide treatment. The results show that ODC is expressed in tooth-forming cells, and that ODC is not only present in differentiating cells but occurs at higher amounts in mature, secreting cells. The findings suggest that polyamines have a central role in tooth development.