DL-alpha-difluoromethylornithine Research Articles

Abstract 6725: Targeting urea cycle dysfunction to prevent and treat osteosarcoma

Abstract Introduction: Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents and young adults. Intensive multimodal treatment cures almost 75% of patients who present with localized disease; however, only 25% of patients who present with metastases become long term survivors, and those who suffer a metastatic relapse are almost never cured. Polyamine metabolism and signaling play important roles in multiple cancers but have not previously been studied in osteosarcoma. D, L-alpha-difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC), the initial, rate-limiting enzyme within the polyamine biosynthetic pathway, and has been studied in a number of different cancers as either a therapeutic or a chemopreventive agent. We investigated the role of polyamines in OS proliferation and metastasis and whether blocking the polyamine synthetic pathway with DFMO had therapeutic potential in the treatment of OS. Methods: We evaluated proliferation, apoptosis, clonogenic growth and the ability to grow under nonadherent conditions in vitro using established OS lines. To investigate the effect of DFMO on tumor growth in vivo, we implanted fragments of patient derived xenograft into the tibias of NOD/SCID/IL-2Rγ null mice. Upon confirmation of tumor growth, mice were randomized to either receive drinking water or drinking water supplemented with 2% DFMO. A third cohort of mice received water supplemented with 2% DFMO only after hindlimb amputation. Results: DFMO has a profound effect on the proliferation of OS cell lines in vitro. Utilizing caspase assays, we determined that DFMO does not induce apoptosis, but is cytostatic and inhibits proliferation. We found that DFMO prevents the clonogenic growth of 3 different OS cell lines in soft agar and prevents spheroid formation under non-adherent conditions. Interestingly, once established, spheroidss were not disrupted by the addition of DFMO. In vivo, administration of 2% DFMO after hindlimb amputation decreased local recurrence and limited metastasis (p=0.02). Conclusion: DFMO inhibits proliferation and clonogenic growth in vitro, and decreases distant metastasis in vivo. Since pediatric dosing and safety data are already established, our findings are readily translatable to clinical trials. Citation Format: Rachel Offenbacher, Paul Ciero, David Loeb. Targeting urea cycle dysfunction to prevent and treat osteosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6725.

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.

Polyamines control the growth of the fish-killing dinoflagellate Karenia mikimotoi in culture

Effects of intracellular polyamines on the growth of the harmful dinoflagellate Karenia mikimotoi were investigated in culture experiments using an axenic culture. Polyamines were analyzed with HPLC. Free norspermine was the most abundant polyamine during growth of K. mikimotoi. Cellular norspermidine contents increased significantly during the exponential growth phase with increasing growth rate. The maximum growth yield of K. mikimotoi was reduced by the polyamine biosynthetic inhibitor, d,l-alpha-difluoromethylornithine (DFMO) which inhibits ornithine decarboxylase. These results suggest that polyamines, especially free norspermine, play significant roles in the growth of K. mikimotoi.

Suppression and Regression of Choroidal Neovascularization by Polyamine Analogues

Polyamine analogues inhibit tumor growth in vitro and in vivo, and oligoamines with a chain length of 10, 12, or 14 are particularly potent. This study was conducted to investigate the effect of the decamines CGC-11144 and CGC-11150 in a mouse model of choroidal neovascularization (CNV). Mice with laser-induced rupture of Bruch's membrane were given intraperitoneal, intravitreous, or periocular injection of CGC-11144, CGC-11150, or vehicle, and after 14 days, they were perfused with fluorescein-labeled dextran, and the area of CNV was measured on choroidal flatmounts by image analysis. In some groups of mice, treatments were started 7 days after rupture of Bruch's membrane to determine the effect of the agent on established CNV. Electroretinograms (ERGs) were performed to assess the effects on retinal function, and histopathology was used to evaluate retinal structure. Intraperitoneal injection of 10 or 20 mg/kg CGC-11144 or CGC-11150 resulted in small but significant reductions in the area of CNV. Intravitreous injection of 20 microg CGC-11144 or CGC-11150 on days 0 and 7 after rupture of Bruch's membrane resulted in a approximately 40% reduction in the area of CNV, with a similar reduction after periocular injections of 0.2 mg CGC-11144 three times a week for 2 weeks. Both intravitreous and periocular delivery of CGC-11144 also caused significant regression of established CNV. Within 2 days of periocular injection of CGC-11144, there was apoptosis in CNV lesions, but not in normal blood vessels or other retinal cells. Periocular injections of d,l-alpha-difluoromethyl-ornithine (DFMO), which decreases polyamine levels by a different mechanism, also inhibited CNV. There was no decline in ERG amplitudes or abnormal retinal morphology after daily injections of 0.2 mg CGC-11144 for 2 weeks, but a single intravitreous injection compromised retinal structure and function. Periocular delivery of the polyamine analogues may be a useful approach for the treatment of CNV.

Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi.

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.

Melarsoprol refractory T. b. gambiense from Omugo, north-western Uganda.

Culture adapted T. b. gambiense isolated from Northwest Uganda were exposed to 0.001-0.14 microg/ml melarsoprol or 1.56-100 microg/ml DL-alpha-difluoromethylornithine (DFMO). Minimum inhibitory concentrations (MICs) of each drug were scored for each isolate after a period of 10 days drug exposure. The results indicate that T. b. gambiense isolates from Northwest Uganda had elevated MIC values for melarsoprol ranging from 0.009 to 0.072 microg/ml as compared with T. b. gambiense isolates from Cote d'Ivoire with MIC values ranging from 0.001 to 0.018 microg/ml or with T. b. rhodesiense from Southeast Uganda with MIC values from 0.001 to 0.009 microg/ml. All MIC values obtained fell below expected peak melarsoprol concentrations in serum of treated patients. However, it may not be possible to maintain constant drug concentrations in serum of patients as was the case in our in vitro experiments. Importantly, the MIC of 0.072 microg/ml exhibited by one of the isolates from Northwest Uganda was above levels attainable in CSF indicating that this isolate would probably not be eliminated from CSF of treated patients. PCR amplification of the gene encoding the P2-like adenosine transporter followed by restriction digestion with Sfa NI enzyme revealed presence of fragments previously observed in a trypanosome clone with laboratory-induced arsenic resistance. From our findings it appears that reduced drug susceptibility may be one factor for the frequent relapses of sleeping sickness after melarsoprol treatment occurring in Northwest Uganda.

P53 independent G(1) arrest induced by DL-alpha-difluoromethylornithine.

Ornithine decarboxylase (ODC), which catalyzes polyamine biosynthesis, plays an essential role in cell growth. DL-alpha-Difluoromethylornithine (DFMO), a synthetic inhibitor of ODC, inhibits cell growth. However, the exact mechanism by which polyamine depletion by DFMO results in growth inhibition remains to be elucidated. We clarified the mechanisms by which DFMO inhibits human gastric cancer cell (MKN45) growth. DFMO induced MKN45 cell G(1) phase arrest after 48 h, and the percentage of G(1) arrest cells continued to increase until 72 h. Expression of p21 and phosphorylation of Stat1 were significantly induced by DFMO at 24 h. Luciferase assay and gel shift assay showed specific binding of Stat1 to the p21 promoter, and promoter activity was activated at 24 h. In dominant negative p53 expressing cells, DFMO significantly induced p21 expression, arrested cells at G(1) phase, and suppressed cell growth effectively. These results suggest that DFMO induced MKN45 cell arrest at G(1) phase in a p53 independent manner, and Stat1 is, at least in part, involved in G(1) arrest.

P44/42 mitogen-activated protein kinase is involved in the expression of ornithine decarboxylase in leukaemia L1210 cells

The involvement of p44/42 mitogen-activated protein kinase (MAPK) in the induction of ornithine decarboxylase (ODC) was investigated by using PD98059, a specific MAPK-kinase (MEK1/2) inhibitor, and other signal-transduction inhibitors. In d,l-alpha-difluoromethylornithine (DFMO)-resistant L1210 cells stimulated to grow from quiescence, treatment with PD98059 inhibited p44/42 MAPK phosphorylation and the induction of ODC activity and protein. A marked reduction of the accumulation of mature ODC mRNA and its intron-containing precursor was observed, whereas ODC turnover was hardly affected. PD98059 also reduced the content of antizyme, but not that of antizyme mRNA. U0126, a novel and more potent inhibitor of MEK1/2, provoked a dose-dependent inhibition of ODC induction at lower concentrations with respect to PD98059. Other effective inhibitors of ODC induction proved to be genistein, manumycin A, herbimycin A, LY294002, wortmannin and KT5823, suggesting the involvement of other key proteins of signal-transduction pathways, i.e. Ras, Src, phosphatidylinositol 3-kinase and cGMP-dependent protein kinase, which may have a positive impact on MAPK. Cells kept in a DFMO-free medium, and thus containing high levels of putrescine and spermidine, showed enhanced MAPK phosphorylation and lower sensitivity to PD98059, compared with cells maintained in the presence of DFMO. In conclusion, these results indicate that the activation of p44/42 MAPK may favour the expression of ODC, and that polyamines, in turn, may affect the phosphorylation state of MAPK.

Pneumocystis carinii Polyamine Catabolism

DL-alpha-Difluoromethylornithine (DFMO) causes polyamines of the AIDS-associated opportunistic pathogen Pneumocystis carinii to diminish 15 times more rapidly than mammalian host cells. The proposed mechanism was that, unlike mammalian cells, P. carinii is unable to regulate polyamine catabolism when synthesis is blocked. To test this, the responses of the polyamine catabolic enzymes spermidine/spermine acetyltransferase (SSAT) and polyamine oxidase (PAO) were determined using a new high-performance liquid chromatography assay to measure the products of these enzymes. The specific activities in untreated Pneumocystis carinii were 1.78 +/- 0.5 pmol min(-1) mg protein(-1) for SSAT, similar to mammalian cells, and 6.42 +/- 0.8 pmol min(-1) mg protein(-1) for PAO, 19% of that of mammalian cells. DFMO treatment for 12 h caused reductions of only 11 and 4% in SSAT and PAO, respectively, despite polyamine reductions of 94, 96, and 90% for putrescine, spermidine, and spermine, respectively. The P. carinii SSAT K(m) value of 25 microM spermidine is 20% of that of mammalian cells, and the PAO K(m) value of 14 nM N(1)-acetylspermidine is 0.01% of that of mammalian cells. Acetylated polyamines continue to be lost from P. carinii even when exposed to DFMO. Collectively, these results support the hypothesis that P. carinii is unable to regulate polyamine catabolism.

Polyamine depletion arrests cell cycle and induces inhibitors p21(Waf1/Cip1), p27(Kip1), and p53 in IEC-6 cells.

The polyamines spermidine and spermine and their precursor putrescine are intimately involved in and are required for cell growth and proliferation. This study examines the mechanism by which polyamines modulate cell growth, cell cycle progression, and signal transduction cascades. IEC-6 cells were grown in the presence or absence of DL-alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the first rate-limiting enzyme for polyamine synthesis. Depletion of polyamines inhibited growth and arrested cells in the G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the level of p53 protein and other cell cycle inhibitors, including p21(Waf1/Cip1) and p27(Kip1). Induction of cell cycle inhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike many other cell lines. Although polyamine depletion decreased the expression of extracellular signal-regulated kinase (ERK)-2 protein, a sustained increase in ERK-2 isoform activity was observed. The ERK-1 protein level did not change, but ERK-1 activity was increased in polyamine-depleted cells. In addition, polyamine depletion induced the stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) type of mitogen-activated protein kinase (MAPK). Activation of JNK-1 was the earliest event; within 5 h after DFMO treatment, JNK activity was increased by 150%. The above results indicate that polyamine depletion causes cell cycle arrest and upregulates cell cycle inhibitors and suggest that MAPK and JNK may be involved in the regulation of the activity of these molecules.

Inhibition of breast cancer in nude mouse model by anti-angiogenesis.

The mechanism by which DL-alpha-difluoromethylornithine (DFMO) inhibits angiogenesis is generally thought to involve the inhibition of the rate-limiting enzyme, ornithine decarboxylase (ODC), leading to polyamine depletion in cells and the ultimate cytostatic effect on proliferating endothelial cells. Another mechanism for inhibiting tumor growth involves pentosan polysulfate (PPS) which binds heparin-binding growth factors, known to be crucial for tumor angiogenesis. To quantitate the combined anti-angiogenic effect of DFMO and PPS on tumors, blood vessels were stained using monoclonal antibodies against the platelet endothelial cell adhesion molecule (PECAM). When compared to untreated mice, DFMO/PPS-treated mice exhibited significantly lower (6-fold) blood vessel counts. Furthermore, mice receiving the combination drug treatment had prolonged life compared to untreated tumor-bearing mice, but less than normal tumor-free mice. The prolonged life span of drug treated tumor-bearing mice also correlated with reduced tumor burden in these mice. The use of single drug treatment results in rapid tumor growth and eventual death of tumor-bearing mice. We have demonstrated that there was significant difference in survival time which correlated to less tumor burden in treated groups of mice as compared to the controls. Inhibition of tumor angiogenesis by the drug combinations suggest that these compounds are anti-angiogenic agents for potential use in clinical trial.

Effects of DFMO on glioma cell proliferation, migration and invasion in vitro.

The polyamine inhibitor DL-alpha-difluoromethylornithine (DFMO) is a specific irreversible inhibitor of ornithine decarboxylase which is a rate-limiting enzyme in the polyamine bio-synthesis pathway. The present study describes the effects of DFMO on glioma cell proliferation, migration and invasion using multicellular spheroids from three glioma cell lines (GaMg, U-251 Mg and U-87 Mg). 10 mM DFMO reduced cell migration in the three cell lines by about 30-50%. 1 mM putrescine, added together with DFMO inhibited the DFMO effect. A stronger effect was observed in the growth assay where 10 mM DFMO reduced the spheroid growth, for all cell lines, by 90%. This effect was also reversed by adding 1 mM of putrescine. In vitro tumor cell invasion experiments indicated after 3 days of confrontation, an extensive invasion also after 10 mM DFMO treatment. The brain aggregate volumes were reduced to about the same extent as in the absence of drug, suggesting essentially no effects of DFMO on the invasive process. It is concluded that the tumor spheroids retained their ability to invade normal brain tissue even after DFMO exposure. However, DFMO inhibited spheroid growth and cell migration which supports the notion that cell growth, migration and invasion are biological properties that are not necessarily related to each other.

Alterations in ornithine decarboxylase characteristics account for tolerance of Trypanosoma brucei rhodesiense to D,L-alpha-difluoromethylornithine.

Ornithine decarboxylase (ODC), the target enzyme of D,L-alpha-difluoromethylornithine (DFMO), was investigated in four DFMO-tolerant Trypanosoma brucei rhodesiense isolates from East Africa and two DFMO-susceptible T. b. gambiense isolates from West Africa. Neither drug uptake nor inhibition of ODC activity by DFMO in cellular extracts differed in the two trypanosome subspecies. However, the specific ODC activity of the cellular extracts was three times as high in T. b. rhodesiense isolates as in T. b. gambiense isolates. Furthermore, a significant difference in the turnover rate of ODC was observed. The time required to induce a 50% reduction of T. b. rhodesiense ODC activity under cycloheximide pressure (tentative half-life) was about 4.3 h, whereas that required for T. b. gambiense ODC was longer than 18 h. We concluded that the higher specific ODC activity and faster enzyme turnover contributed to a substantial degree to the DFMO tolerance observed in the East African T. b. rhodesiense isolates.

Modulation of alterations in p53 tumor suppressor gene and its association with activation of ras proto-oncogenes during chemoprevention of colon cancer

Previously, we reported (Carcinogenesis 15: 1317-1323, 1994) a high rate of activating point mutations in I ns proto-oncogenes in azoxymethane (AOM)-induced colon tumors, and a significant suppression of these mutations by dietary administration of chemopreventive agents, D,L-alpha-difluoromethylornithine (DFMO) and piroxicam. To understand the role of p53 tumor suppressor gene in chemoprevention of colon cancer and to study the association of p53 gene alterations with activation of ras genes, we determined point mutations in conserved regions (exons 5-9) of p53 gene and analyzed the occurrence of double event of ms activation acid p53 mutation. Groups of male F344 rats were fed the modified AIN-76A diet containing 0, 4000 ppm DFMO, or 150 ppm piroxicam and administered s.c. AOM at a dose rate of 15 mg/kg body wt, once weekly, for 4 weeks. Vehicle controls received s.c. equal volume of normal saline. Animals were sacrificed 32 weeks after the last AOM or saline injection and their grossly visible colon tumors were analyzed to determine p53 mutations by PCR amplification based single strand conformation polymorphism (SSCP) and direct DNA sequencing. Our results demonstrate that about 57% tumors from animals fed the control diet contained predominantly missense but also nonsense mutations, whereas only 30% tumors from animals on piroxicam diet, and none (0%) from animals fed the DFMO diet had similar mutations. Analysis of data revealed that about half of the tumors from animals on control diet possessed both ms and p53 mutations together, only 27% of colon tumors from animals on piroxicam diet and none of the tumors from animals on DFMO diet exhibited both ms and p53 mutations. These results indicate that the administration of piroxicam, a non-steroidal anti-inflammatory drug, and DFMO, a irreversible inhibitor of ornithine decarboxylase, may inhibit selective proliferation of initiated cells containing activated las and/or mutant p53. Dietary DFMO exerted more pronounced inhibition of selective amplification of initiated cells containing mutated ras and/or p53.

Chemopreventive effect of difluoromethylornithine (DMFO) on mouse skin squamous cell carcinomas induced by Benzo(a)pyrene

The effect of the chemopreventive agent D,L-alpha-difluoromethylornithine (DFMO) on the incidence of skin squamous cell carcinoma was studied in SENCAR mice treated weekly with topical applications of benzo(a)pyrene (B(a)P) (0.15 mmol, 2 x /week) on the dorsal skin. Animals were randomized to receive either chow or chow supplemented with DFMO (1 g/1 kg) and studied at 10, 15, 20, 25, and 30 weeks of B(a)P treatment. Morphometric analyses at each timepoint evaluated the epidermal thickness (ET) and the number of epidermal nucleated layers (NL). The ET increased from 12-17 microns as early as 10 weeks after B(a)P treatment, reaching 22 microns at 20 weeks, and 27 microns at 25 weeks (130% increase). The NL also increased markedly. A relatively modest increase in ET was observed in animals treated with B(a)P and DFMO (16% at 15 weeks, 53% at 20 weeks, and 85% at 25 weeks) as compared to controls. The relative increase in NL showed a similar pattern. Although extensive epidermal hyperplasia was seen early, clear-cut focal premalignant lesions were not identifiable before week 20 of B(a)P treatment. At 20 weeks, the most frequently noted focal premalignant lesions in carcinogen-treated animals (without DFMO) were moderate dysplasias. At 25 and 30 weeks, a large increase was seen in the incidence of more advanced dysplastic lesions and invasive carcinomas. In the group treated with B(a)P and DFMO, a marked reduction in the number of carcinomas was observed at 25 and 30 weeks. At 25 weeks, DFMO reduced tumor yield from 5.8 to 3.2 carcinomas per mouse. At 30 weeks, the reduction was from 13.1 to 5.7 carcinomas per mouse (57% reduction). Collectively, these data emphasize the strong chemopreventive effect of DFMO against tumors in the mouse skin complete carcinogenesis model, as indicated by the reduction of overall skin tumor incidence and the decreased epidermal hyperplasia in DFMO-treated animals. Morphometrically defined increases in ET and NL can be used as early biomarkers of DFMO chemoprevention in mouse skin tumorigenesis.

Localization of S‐adenosylmethionine decarboxylase in murine tissues by immunohistochemistry

S-adenosylmethionine decarboxylase (AdoMetDC), one of three key enzymes in polyamine biosynthesis, is present in minute concentrations in adult tissues, whereas increased AdoMetDC activity occurs in conjunction with differentiation and growth. The occurrence of AdoMetDC in tissues has earlier been studied by biochemical technique only. In this work, an antiserum against recombinant human AdoMetDC was used to investigate the localization of the enzyme in different mouse tissues. In all tissues studied, immunolabeling was seen in cytoplasm, while cell nuclei were negative. In the kidney, AdoMetDC immunoreactivity was confined to the inner part of the cortex; the outer part of the cortex and the medulla displayed only a weak AdoMetDC immunoreaction. In the cortex, renal tubules were strongly reactive, while renal corpuscules were weakly immunolabeled. In developing teeth, AdoMetDC immunoreactivity displayed a gradient of distribution, the immunolabeling intensity being increased from the cervical region to the tip of the cusps. In the incisor, post-secretory ameloblasts were strongly labeled. In the oral mucosa, epithelial cells demonstrated stronger immunolabeling than did cells of the subjacent connective tissue. As for muscle cells, immunoreactive material was confined to the periphery of the fibers. In vitro, treatment with DL-alpha-difluoromethylornithine (DFMO) or methylglyoxal bis-(guanylhydrazone) (MGBG) led to an increase in AdoMetDC. It can be concluded that antibodies to recombinant human AdoMetDC provide a tool for the immunohistochemical localization of AdoMetDC, and that the distribution of the enzyme in the tissues studied gives further support to the importance of polyamines in the development and functions of these organs.

EFFECT OF CHEMOPREVENTIVE AGENTS ON POSTTRANSLATIONAL PLASMAMEMBRANE ASSOCIATION OF RAS-P21 DURING CHEMOPREVENTION OF AZOXYMETHANE-INDUCED COLON CARCINOGENESIS

Accumulating data suggest that activation of ms proto-oncogenes and inactivation of tumor suppressor genes induce malignant phenotype in colonic cells. However, the transforming ability of ras oncogenes critically depends on correct localization of ras-p21 in plasma membrane. In our previous studies, we demonstrated a strong correlation between the modulation of ras activation (both in terms of mutational activation and over-expression of ras genes) by chemopreventive agents and colon tumor outcome during different stages of azoxymethane (AOM)-induced colon carcinogenesis. In the present study, which is a part of our ongoing investigations on the role of ras in chemoprevention of colon cancer, we studied the effect of D,L-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, and piroxicam, a non-steroidal antiinflammatory drug (NSAID), on the post-translational membrane association of ras-p21 during AOM-induced colon carcinogenesis. Groups of male F344 rats were fed the modified AIN-76A diets containing 0, 150 ppm piroxicam or 4000 ppm DFMO, and administered s.c. AOM dissolved in normal saline at a dose rate of 15 mg/kg body weight/week for 4 weeks. Vehicle control groups received equal volume of normal saline. Groups of animals were then sacrificed at 4, 16, 24, and 32 weeks after last AOM or saline injection and their colonic mucosa and tumors were analyzed for cytoplasmic as well as membrane bound ras-p21 levels. AOM-treatment resulted in increasingly higher levels of membrane-bound ras-p21 with advancing stages of colon tumorigenesis without any significant changes in cytoplasmic ras-p21. Dietary DFMO significantly suppressed AOM-induced membrane-bound ras-p21 in a time-dependent manner. Administration of piroxicam though resulted in significant inhibition of membrane-bound ras-p21, but concomitantly increased the cytosolic levels of ras-p21. Inhibition of membrane-bound ras-p21 levels by DFMO and piroxicam strongly correlated with the suppression of AOM-induced colon tumorigenesis by these agents. Data from the present and earlier studies suggest that DFMO may afford chemoprevention by suppressing DNA and protein biosynthesis by depleting intracellular polyamines, whereas piroxicam may exert its antitumor activity by interfering with post-translational membrane localization of ras-p21, in addition to modulating arachidonic acid metabolism.

Inhibition of ornithine decarboxylase does not prevent intestinal smooth muscle hyperplasia in the rat

The objective of this research is to examine the relationship between intestinal smooth muscle cell proliferation and polyamine metabolism. Proliferation of muscle is induced by serosal application of benzyldimethyltetradecylammonium chloride (BAC) to a segment of rat jejunum. This treatment destroys all of the longitudinal muscle, one-half of the circular muscle, and the myenteric and extrinsic nerves. The remaining muscle cells undergo mitosis and, by 15 days, the number of muscle cells is increased in both the longitudinal and circular muscle layers. Within 12 h after BAC treatment, there is an increase in the activity of ornithine decarboxylase (ODC) that returns to preinjury levels by 4 days. The smooth muscle content of the three polyamines putrescine, spermidine, and spermine is increased 6 h after injury, returning to preinjury levels within 1 day. DL-alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, prevents the transient increases in both ODC activity and the three polyamines in the smooth muscle. In addition to preventing the transient increases in the three polyamines, DMFO has different effects on smooth muscle content of putrescine, spermidine, and spermine. Putrescine content is permanently depleted by DFMO. Spermidine and spermine content is initially decreased within 24 h by DFMO administration. In contrast to the persistent depletion of putrescine by DFMO, spermidine and spermine return to preinjury levels by 2 days. The maximal peak in DNA synthesis occurs 2 days after injury. In DFMO-administered animals, the maximal DNA synthesis occurs 5 days after injury. The increases in smooth muscle wet weight and DNA content 15 days after injury are not affected by DFMO.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of azoxymethane-induced mutational activation of ras protooncogenes by chemopreventive agents in colon carcinogenesis.

In our previous study, we demonstrated that azoxymethane (AOM) treatment significantly enhanced the expression of ras p21, the protein product of ras genes, and that the dietary administration of chemopreventive agents such as D,L-alpha-difluoromethylornithine (DFMO), a irreversible inhibitor of ornithine decarboxylase, and piroxicam, a non-steroidal anti-inflammatory drug (NSAID), exerted a significant inhibitory effect on AOM-induced ras p21 expression. In the present study, which is an extension of our earlier investigation, we have determined the effect of DFMO and piroxicam on mutational activation of ras protooncogenes during AOM-induced colon carcinogenesis. Groups of male F344 rats were fed the modified AIN-76A diet containing 0 or 150 p.p.m. piroxicam, or 4000 p.p.m. DFMO and administered s.c. AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt, once weekly, for 4 weeks. Vehicle control groups received s.c. equal volumes of normal saline. Groups of animals were then killed at 0, 4, 16, 24 or 32 weeks after last AOM or saline injection. AOM-induced colon tumors and colonic mucosa from AOM treated as well as saline treated animals were analyzed for point mutations in K- and H-ras protooncogenes by a combination of polymerase chain reaction mediated restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. Our results demonstrate that of the total 65 AOM-induced colon tumors analyzed, 45/50 (90%) obtained from AOM-treated animals fed the control diet, 4/11 (36%) from AOM-treated animals fed piroxicam diet, and 1/4 (25%) from AOM-treated animals fed the DFMO diet, contained single-point mutations occurring specifically at the second nucleotide of codon 12 which were identified exclusively as G to A transitions in case of K-ras, and G to A transitions and also G to T transversions in H-ras. Similar point mutations were identified in colonic mucosa of 21/30 (70%) of AOM-treated animals fed the control diet, 10/30 (33%) of AOM-treated animals fed piroxicam diet, and none of 30 (0%) of AOM-treated animals fed DFMO diet. These results indicate that the administration of piroxicam and DFMO may inhibit the selective amplification of AOM-induced initiated cells carrying mutated ras genes. Dietary DFMO exerted more pronounced inhibition of selective amplification of initiated cells containing AOM-induced mutant ras. Data suggest that determination of ras activation may be a useful marker for chemoprevention of colon cancer.

Effect of DL-Alpha-Difluoromethylornithine Pretreatments in Maize Callus Differentiation

The effect of pretreatments with DL-alpha-difluoromethylornithine (DFMO), an irreversible suicide inhibitor of the ornithine decarboxylase (ODC) activity, in plant differentiation, polyamine (PA) and amino acid contents of maize callus cultures was investigated. This study indicates that DFMO pretreatments can be used to improve regenerative response from maize callus cultures. These findings may also be useful in other recalcitrant cultures.