Inhibition of ornithine decarboxylase with 2-difluoromethylornithine: reduced incidence of dimethylhydrazine-induced colon tumors in mice

2-Difluoromethylornithine (DFMO) was administered to 1,2-dimethylhydrazine (DMH)-treated mice to reduce colonic polyamine levels and mucosal hyperplasia. Mice received 1% DFMO in drinking water throughout the experiment and were given injections of DMH (20 mg/kg) weekly for 28 weeks. DFMO inactivated 93% of colonic ornithine decarboxylase activity. Although DMH treatment did not induce colonic ornithine decarboxylase activity by Week 28, the putrescine content was increased 31% in DMH-treated mice (p less than 0.01). Concurrent treatment with DFMO depressed putrescine content (42 to 63%) and spermidine content (27 to 38%), but it increased spermine content (18 to 22%). At Week 28 of treatment with DMH alone, RNA content was increased 8.6% (p less than 0.01), DNA content 10% (p less than 0.01), DNA specific activity 24% (p less than 0.01), and crypt depth 20% (p less than 0.01), but not in mice receiving DMH and DFMO. At 28 weeks, 13 of 17 mice (76%) treated with DMH alone had histologically confirmed colon cancers; of mice treated with DMH and DFMO, two of 18 (11%) had colonic tumors. Throughout the experiment, 50 colon cancers developed in 16 DMH-treated mice (mean, 3.12 tumors/mouse); three mice treated with DMH and DFMO developed three colon cancers total (p less than 0.001). Reduction of colonic polyamine levels after DFMO treatment prevents proliferative changes induced by DMH and reduces the incidence of tumors.     

Role of polyamines in the growth of hormone-responsive experimental breast cancerin vivo

Summary We have provided evidence for a critical role of polyamines in the growth of the hormone-responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumorin vitro. The present experiments were designed to test whether polyamines are involved in the growth of this experimental tumorin vivo. To test this hypothesis, groups of rats bearing NMU-induced mammary cancers were randomly allocated to receive no treatment or escalating doses of the polyamine biosynthesis inhibitor -difluoromethyl-ornithine (DFMO) (0.5%, 1%, 2%, 3% in drinking water). DFMO inhibited tumor growth in a dose-dependent fashion and consistently reduced tumor putrescine level. To evaluate the time dependency of this effect, additional groups of rats received either no treatment or 2% DFMO for 3, 7, 14, and 21 days. At all times DFMO suppressed tumor putrescine level as well as spermidine to spermine ratio. Finally, exogenous administration of putrescine (200 mg/kg/i.p./day × 21 days) given concomitantly with DFMO restored tumor growth, partially repleted tumor putrescine level, and raised the spermidine to spermine ratio to control levels. Putrescine, given alone, had no significant effect on either tumor polyamine levels or tumor growth. Except for modest weight loss, no major toxicity was encountered. These results indicate that polyamines play an important role in the growth of the NMU rat mammary tumorin vivo. The interaction between polyamines and hormones in supporting NMU mammary tumor growthin vivo remains to be elucidated.     

Alterations in polyamine metabolism during continuous intravenous infusion of alpha-difluoromethylornithine showing correlation of thrombocytopenia with alpha-difluoromethylornithine plasma levels

Polyamine biosynthesis is important for cell proliferation and growth. The purpose of this study was to determine the biochemical and pharmacological parameters associated with host toxicity from a continuous infusion of alpha-difluoromethylornithine (DFMO). Twenty-five patients with metastatic carcinoma of the colon or rectum received continuous infusion of DFMO at a median dose of 8 g/m2/day (range, 6-14) for 28 days. DFMO plasma levels, RBC, plasma putrescine, spermidine, and spermine levels, and patient toxicities were evaluated. There was a significant decrease in RBC and plasma levels of putrescine, spermidine, and spermine following DFMO administration compared with the baseline RBC and plasma levels. Pearson correlation coefficient comparing nadir platelet count and steady-state DFMO level was statistically significant (n = 37; P less than 0.01; r = -0.53). Sustained suppression of circulating polyamine levels was also achieved with continuous DFMO infusion. The correlation between steady-state plasma DFMO levels and lowering of platelet count warrants prospective evaluation to determine its clinical usefulness.     

Effects of alpha-difluoromethylornithine and 5-fluorouracil on the proliferation of a human colon adenocarcinoma cell line

Because alpha-difluoromethylornithine (DFMO) reduces the incidence of experimental colon cancers, inhibits the growth of human lung cancer cells and human leukemia cells in culture, and in combination with methylglyoxal (bis)guanylhydrazone induces remission in children with leukemia, its effectiveness against a human colon adenocarcinoma cell line (Colo 205) was tested alone and in combination with 5-fluorouracil (5-FU). Both DFMO (2 X 10(-4) M) and 5-FU (10(-6) M) inhibited Colo 205 cell proliferation. Above 5 X 10(-4) M DFMO (p less than 0.001) and at 10(-4) M 5-FU (p less than 0.001), Colo 205 growth was completely inhibited. Although DFMO did not sensitize Colo 205 cells to a noninhibitory concentration of 5-FU, the effectiveness of inhibitory concentrations of 5-FU and DFMO in reducing Colo 205 cell growth was additive. DFMO (2 X 10(-4) M) caused 89 to 93% inhibition of ornithine decarboxylase activity (p less than 0.001) and reduced levels of putrescine (93%; p less than 0.01) and spermidine (57%; p less than 0.02). Growth rate and the intracellular putrescine and spermidine contents were restored by 10(-6) M putrescine. DFMO could be an effective chemotherapeutic agent against human colonic cancer because of its effects at such unusually low concentrations in vitro.     

Effects of alpha-difluoromethylornithine on the growth of experimental Wilms' tumor and renal adenocarcinoma

Because polyamines are essential for cellular growth and differentiation, and because human renal carcinomas have spermidine levels that are higher than those in normal renal tissue, effects of 2-difluoromethylornithine (DFMO) on the growth of experimental renal tumors were investigated. DFMO is a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme controlling polyamine biosynthesis. DFMO (2%) in drinking water was administered to BALB/c mice with intrarenal transplants of a renal adenocarcinoma cell suspension and to Wistar/Furth rats with s.c. transplants of a Wilms' tumor. At 28 days, renal carcinomas in DFMO-fed mice weighed 72% less than those in control animals (p less than 0.001). Wilms' tumor weight was not affected by DFMO feeding. DFMO caused 72 to 75% inactivation of ornithine decarboxylase activity and reduced putrescine levels in renal carcinoma and Wilms' tumor, reduced spermidine levels in Wilms' tumor, and apparently raised spermine levels in the latter as a consequence. DNA content was not affected by DFMO feeding. The mean number of lung metastases in DFMO-fed, renal carcinoma-bearing mice was 0.1 and in controls was 1.4 (p less than 0.001). DFMO feeding increased survival of mice bearing renal carcinomas by 3.0 +/- 0.8 (S.E.) days (p less than 0.05), i.e., from 30.5 +/- 0.8 days to 33.5 +/- 1.2 days. DFMO did not affect the growth of Wilms' tumor; however, in renal adenocarcinoma, it reduced growth, prevented lung metastases, and increased survival.     

Effect of intravenous alpha-difluoromethylornithine on the polyamine levels of normal tissue and a transplantable fibrosarcoma

The effect of a continuous i.v. infusion of alpha-difluoromethylornithine (DFMO) on the polyamine metabolism of tumor and normal host tissue was determined. Non-tumor-bearing Fischer 344 rats or rats bearing a transplantable fibrosarcoma received continuous infusions of DFMO through a central venous catheter at three dose levels. Treatment with DFMO resulted in a time- and dose-dependent, cytostatic effect on the growth of the tumor. In fibrosarcoma-bearing rats the tumor putrescine levels were reduced after 6 and 12 days of DFMO treatment. Tumor spermidine levels were consistently reduced after 6 and 12 days of treatment with the reduction being dose dependent. The decrease in tumor ornithine decarboxylase activity was dose dependent. Erythrocyte putrescine levels were decreased in tumor- and non-tumor-bearing rats, suggesting that DFMO reduces the tumor contribution to the erythrocyte pool. Erythrocyte spermidine levels of fibrosarcoma- and non-tumor-bearing rats were elevated at the lower DFMO doses administered for 12 days but returned to normal as the dose was increased. Erythrocyte spermine levels were elevated in both groups of rats at all DFMO doses. Although normal host tissue weights were not affected by treatment with DFMO, the putrescine and spermidine levels of liver, spleen, and kidney and ornithine decarboxylase activity of the liver and kidney were decreased. These data demonstrate that i.v. DFMO has a cytostatic effect toward a rapidly growing fibrosarcoma associated with the depletion of both tumor putrescine and spermidine levels.     

in vivo effects of 4-amidinoindan-l-one 2′-amidinohydrazone (CGP 48664A) and α-difluoromethylornithine (DFMO) on L1210 growth,cell-cycle phase distribution and polyamine contents

We studied the in vivo effects of 4-amidinoindan-l-one 2′-amidinohydrazone (CGP 48664A), α-difluoromethylornithine (DFMO) and a combination of CGP 48664A-DFMO on tumor growth, cell-cycle phase distribution and polyamine contents. DBA-2 mice were inoculated i.p. with 10⁵ L1210 cells on day 0, treated i.p. on days 1–4 and killed on day 5. As compared to controls, CGP 48664A, DFMO and the CGP 48664A-DFMO combination reduced L1210 cell numbers by 33, 43 and 85%, respectively. CGP 48664A did not affect cell-cycle phase distribution. DFMO and the CGP 48664A-DFMO combination caused a moderate and a heavy accumulation in G₀/G₁- and G₂/M-phases, respectively. Compared with controls, the CGP 48664A-DFMO combination reduced putrescine, spermidine and total polyamines, but did not affect spermine. Compared with CGP 48664A, the CGP 48664A-DFMO combination caused lower putrescine and total polyamines, higher spermine, but no change in spermidine. Compared with DFMO, the CGP 48664A-DFMO combination caused higher putrescine and spermidine, lower spermine, but no change in total polyamine levels. We conclude that CGP 48664A potentiates the cystostatic effect of DFMO in vivo. The resulting growth inhibition is accompanied by an accumulation in G₀/G₁- and G₂/M-phases and a reduction of putrescine and spermidine. The data suggest that perturbed polyamine composition rather than reduced spermidine or total polyamine pool size causes a profound growth inhibition. © 1995 Wiley-Liss, Inc.     

Antitumor effects of two polyamine antimetabolites combined with mitomycin C on human stomach cancer cells xenotransplanted into nude mice

The antitumor effects of alpha-difluoromethylornithine (DFMO), methylglyoxal-bis-guanylhydrazone (MGBG) and mitomycin C (MMC), administered separately or in various combinations, on human stomach cancer cells xenotransplanted into BALB/c nude mice were studied using the protocol of Battelle's Columbus Laboratories (Ovejera et al., 1978). DFMO (1,000 mg/kg in 2 divided doses) and MGBG (50 mg/kg) were given intraperitoneally (i.p.) for 7 consecutive days from the time when the tumor weighed about 100 mg. MMC (2 mg/kg) was given i.p. every other day from the same time. Animals treated with either DFMO or MGBG alone displayed tumor growth comparable to that seen in untreated controls. In mice treated with DFMO plus MGBG with or without MMC, or in mice treated only with MMC, tumor growth was significantly lower than in untreated mice. In the group which received only combined DFMO/MGBG there was a rapid regrowth of the tumor after termination of therapy. Tumor putrescine levels decreased within 4 days following the administration of DFMO; however, spermidine levels did not decline with either DFMO or MGBG treatment even after 7 days. When combined DFMO/MGBG was given, there was a significant decline in spermidine levels 7 days after the initiation of treatment. In contrast, when MMC alone was administered, putrescine and spermidine levels in the tumor did not differ from those in control mice. Spermine decreased markedly in tumor with the combined administration of DFMO/MGBG as well as with combined DFMO/MGBG/MMC, but decreased only slightly when MMC alone or MMC plus either DFMO or MGBG was administered. By the 7th treatment day, DNA biosynthesis in the tumor had dropped markedly in all groups except those receiving DFMO or MGBG alone.     

Chemoprevention of mouse colon tumors with difluoromethylornithine during and after carcinogen treatment

alpha-Difluoromethylornithine (DFMO) treatment has been shown to modify carcinogenesis in many experimental tumor models, including skin, breast, and colon. This study was designed to determine whether DFMO treatment can inhibit experimental mouse colon tumors after carcinogen treatment and whether an associated effect of DFMO on cell proliferation in colon mucosa occurs. Male CD1 mice (40 per group) received dimethylhydrazine (30 mg/kg/week x 6 weeks, s.c.) and various schedules of DFMO, 1% in drinking water: Group A, none; Group B, following dimethylhydrazine treatment; Group C, during dimethylhydrazine treatment; and Group D, continuously throughout the study. Measurements of RBC polyamine levels showed that DFMO treatment ablated putrescine levels and confirmed that a systemic biological effect was achieved. Analysis of tumor data showed a significant inhibitory effect of DFMO treatment on colon tumor (adenomas and adenocarcinomas) incidence in Groups B (24%) and D (20%) compared to control Group A (52%, P less than 0.05 A versus B, P less than 0.02 A versus D) and on squamous cell carcinomas of the anus in all groups (P less than 0.001 A versus B, P less than 0.05 A versus C, A versus D). No consistent effect of DFMO treatment on cell proliferation in colon mucosa was identified. This study supports the hypothesis that DFMO treatment alters events in the postinitiation phases of mouse colon tumorigenesis.     

Effect of concentration of D,L-2-difluoromethylornithine on murine mammary carcinogenesis

The appearance of chemically induced mammary gland carcinomas in virgin female Sprague-Dawley rats was blocked by the administration of D,L-2-difluoromethylornithine (DFMO) in drinking water during the stage of tumor promotion. Rats were given injections s.c. at 50 days of age with either 35 mg of 1-methyl-1-nitrosourea (MNU) per kg of body weight or the 0.9% NaCl solution in which the carcinogen was dissolved. At 57 days of age, the rats were each randomly allocated to one of 14 treatment groups. Ten groups (five solvent treated and five MNU treated) were assigned to treatments consisting of 0.00, 0.0625, 0.125, 0.25, or 0.50% (w/v) solution of DFMO in their drinking water; two MNU-treated groups were placed on or removed from DFMO treatment (0.5%; w/v) at 90 days post-carcinogen exposure; and two carcinogen-treated groups received either putrescine (0.5-g/kg diet) or putrescine and DFMO (0.5%; w/v) throughout the experiment. The study was terminated 183 days after carcinogen treatment. All doses of DFMO exerted a protective effect against the induction of mammary cancer; however, only the feeding of the 0.125% and the 0.5% solutions of DFMO resulted in a significant reduction in cancer incidence. The average number of cancers per rat was reduced, and cancer-free time was extended at all concentrations of DFMO. The protective effect of DFMO was sustained following withdrawal of treatment at 90 days post-MNU injection. Feeding putrescine in conjunction with DFMO treatment partially blocked the inhibitory activity of DFMO. DFMO treatment did not affect food or water intake; body weight gain; the weight of ovaries, uterus, adrenal glands, liver, kidney, or spleen; or the periodicity of the estrous cycle. These data provide evidence of an inhibitory effect of DFMO against mammary cancer induced by MNU which cannot be attributed to a systemic toxic effect of this compound.     

Reduced cytocidal efficacy for adriamycin in cultured human carcinoma cells depleted of polyamines by difluoromethylornithine treatment

We have studied the effect of pretreatment with difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, on the cytocidal responses of four human adenocarcinoma cell lines to Adriamycin (ADR). The cell lines utilized included HuTu-80 (duodenum), HT-29 (colon), ME-180 (cervix), and A-427 (lung). A 48-h DFMO pretreatment reduced putrescine and spermidine content to less than 10 and less than 1% of control levels and decreased spermine to between 70 and 30% of controls. Plating efficiency assays were used to generate ADR dose-response survival curves for DFMO-treated and control cultures. The DFMO pretreatment significantly protected human adenocarcinoma cells from the lethal effects of ADR. Addition of exogenous putrescine to the DFMO-treated cultures 24 h before treatment with ADR restored their cytocidal response to ADR to near control levels. Putrescine had no effect on cell survival in cultures that were not pretreated with DFMO. These observations suggest that DFMO-induced protection from ADR may be a specific consequence of DFMO-induced inhibition of polyamine biosynthesis. Alternatively, since ADR efficacy varies directly with cellular growth rates and DFMO inhibits proliferation, the protection may have resulted from DFMO-induced growth inhibition. Comparison of ADR uptake in DFMO-pretreated and control cells showed that the protection did not result from decreased intracellular accumulation of ADR.     

Influence of K-ras activation on the survival responses of Caco-2 cells to the chemopreventive agents sulindac and difluoromethylornithine.

The nonsteroidal anti-inflammatory drug sulindac and the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) are both potent inhibitors of colon carcinogenesis in experimental models of this disease. The combination of these two agents is undergoing evaluation as a strategy for colon cancer chemoprevention in humans with resected colon polyps. We evaluated the effects of the major sulfide and sulfone metabolites of sulindac and DFMO alone, or in combinations, on the growth and survival of Caco-2 colon cancer-derived cells and in clones of these cells transfected with an activated K-ras oncogene. Both the sulfide and sulfone metabolites of sulindac reduced cell viability, measured by colony-forming assays, primarily by inducing apoptosis. Expression of an activated K-ras oncogene caused cells treated with either sulindac sulfide or sulfone to undergo apoptosis earlier than nontransfected controls. However, clonogenic survival, measured 2 weeks after drug treatment, was the same in both Caco-2 and ras-transfected Caco-2 cells treated with sulindac metabolites. A 24-h treatment with DFMO caused a dose-dependent decrease in the colony-forming ability of cells expressing an activated K-ras but had no effect on the viability of the parental Caco-2 cells. The DFMO-dependent decrease in colony formation in K-ras-activated cells occurred in the absence of apoptosis. Assessment of cell survival by colony-forming assays indicated that these two agents acted in an additive manner when combined. These data indicate that K-ras can influence the kinetics of apoptosis induction by sulindac metabolites and cell survival in response to DFMO. However, cytotoxicity induced by these agents occurs via unique mechanisms. These studies suggest that the combination of DFMO and sulindac may be useful in human cancer prevention strategies.     

INHIBITORY EFFECT OF DEMO ON THE GROWTH OF TRANSPLANTED HUMAN COLON CANCER IN NUDE MICE

A human colon cancer cell line Hce- 8693 was heterotransplanted in nude mice. Polyamine blosythesis Inhibitor a- dlfiuoromethylomithine (DFMO ) show a marked reproducible inhibition in this model. The size and weight of transplanted tumor In DFMO group were smaller than those of the control group and the average inhibition rate was 72.8% (P < 0.001) . DFMO showed higher tumor inhibitory rate than 5-Fu (35. 4%) (P<0. 001) . Furthermore. DFMO demonstrated less severe bone marrow inhibition in the nude mice than 5-Fu (20. 0% Vs 53. 2%. P<0. 001) .There was no synergistic action in these two drugs at the experimental dose. The concentration of putrescine and spermidine in the plasma and tumor tissue in the DFMO group were 70% lower than those of the control group (P<0. 001) . These results indicate that the anti-tumor effect of DFMO might be explained by the inhibition of polyamine biosynthesis and this study provides an experimental basis for future clinical application of DFMO.     

Inhibition of mouse skin tumor promotion and of promoter-stimulated epidermal polyamine biosynthesis by alpha-difluoromethylornithine

Application of the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin leads to a manifold induction of ornithine decarboxylase (ODC) activity within 5 hr and an increased accumulation of putrescine. The relevance of these TPA-induced changes to the mechanism of tumor promotion was investigated using alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO applied to mouse skin (0.3 mg in 0.2 ml of solvent) or administered in the drinking water (1%) in conjunction with skin tumor promotion by TPA inhibited the formation of mouse skin papillomas by 50 and 90%, respectively. TPA-induced ODC activity and the accumulation of putrescine were almost completely inhibited. DFMO given in the drinking water decreased spermidine levels, but DFMO treatment by any route did not alter the spermine levels of mouse epidermis. DFMO decreased TPA-induced hyperplasia by 25 to 40%, and the TPA-caused increases in DNA synthesis and mitotic index were inhibited by 60 and 50%, respectively. Therefore, in mouse epidermis, enhanced cell proliferation can be dissociated from ODC induction and the accumulation of putrescine. At the tested dose levels and routes of administration, DFMO did not inhibit the inflammatory response to TPA in several tissues. These results provide evidence for an essential role of ODC induction and the accumulation of putrescine in tumor promotion by TPA and add strength to the proposal that DFMO may be a promising drug for the prevention and treatment of cancer in human beings.     

多胺生物合成抑制剂DFMO抗裸鼠移植人大肠癌的实验研究

A human colon cancer cell line Hce-8693 was heterotransplanted in nude mice. polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) showed a marked reproducible inhibition in this model. The size and weight of transplanted tumor in DFMO group were smaller than those of the control group and the average inhibition rate was 72.8% (P less than 0.001). DFMO showed higher tumor inhibitory rate than 5-Fu (35.4%) (P less than 0.001). Furthermore, DFMO demonstrated less severe bone marrow inhibition in the nude mice than 5-Fu (20.0% vs 53.2%, P less than 0.001). There was no synergistic action in these two drugs at the experimental doses. The concentration of putrescine and spermidine in the serum and tumor tissue in the DFMO group were 70% lower than those of the control group (P less than 0.001). These results indicate that the anti-tumor effect of DFMO might be explained by the inhibition of polyamine biosynthesis and this study provides an experimental basis for future clinical application of DFMO.     

Effects of DL-alpha-difluoromethylornithine on the growth and metastasis of B16 melanoma in vivo

The effects of DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on the growth of experimental mouse B16-F10 melanoma cells were investigated. DFMO (3%) in drinking water was administered to B16-F10 melanoma-bearing mice. At 24 days, B16-F10 melanomas in DFMO-fed mice weighed 75% less than those in control mice (p less than 0.001). DFMO reduced putrescine and spermidine levels in B16-F10 melanoma by 98% and 84%, respectively, and prolonged the mean survival time from 25.9 +/- 1.2 to 35.7 +/- 2.2 days (p less than 0.001). The effects of DFMO on experimental metastasis were also investigated. DFMO treatment resulted in a significant decrease in pulmonary metastasis induced by i.v. injection of B16-F10 melanoma cells.     

Urinary polyamine levels in cancer patients treated with D,L-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis

The polyamine biosynthesis inhibitor D,L-alpha-Difluoromethylornithine hydrochloride monohydrate (DFMO) has cytostatic and cytotoxic effects against various human tumor cell lines in vitro. We measured levels of the polyamines putrescine and spermidine in the urine of cancer patients undergoing "conventional" chemotherapy in a two-arm randomized phase I-II study with and without additional DFMO administered orally at a dose of 1.7 g/sq.m. t.i.d. The study group included 38 patients with carcinoma of the breast, stomach, prostate, or female genital organs or metastatic carcinoma of unknown origin. A control group of 32 patients with similar malignancies received "conventional" chemotherapy without DFMO. Polyamine levels were determined periodically in the urine of all patients. In DFMO-treated patients, a significant decrease in putrescine and spermidine levels was observed after 3 weeks of DFMO therapy (the first time point evaluated) that usually persisted throughout the course of treatment. Significant differences in polyamine levels between DFMO-treated and control patients were observed for patients in remission. Less significant differences were noted, however, for patients with static or progressive disease between DFMO-treated and control groups. DFMO activity appears to be reflected by a long-term decrease in urinary polyamine levels.     

Putrescine-dependent invasive capacity of rat ascites hepatoma cells.

The effects of inhibitors of polyamine synthesis on the invasive capacity of rat ascites hepatoma (LC-AH) cells were examined by in vitro assay of penetration of the LC-AH cells through a monolayer of calf pulmonary arterial endothelial (CPAE) cells. Pretreatment of LC-AH cells with alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, before seeding them onto a CPAE cell monolayer and culturing them for 24 h in the absence of DFMO decreased the number of penetrating tumor cells time and dose dependently (about 35% of the maximal inhibition) without affecting their viability or proliferative activity. DFMO treatment caused a marked decrease in the intracellular level of putrescine but not of spermidine or spermine. The DFMO-induced decreases in invasive capacity and putrescine level were almost completely reversed by the addition of putrescine to the medium during pretreatment with DFMO or invasion assay but were not affected by exogenous spermidine or spermine. No change in the invasive capacity was observed when the CPAE cells were treated with DFMO and the LC-AH cells with methylglyoxal-bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, which depressed the spermidine and spermine levels but increased the putrescine level in the LC-AH cells. These results suggest that intracellular putrescine modulates the in vitro invasive capacity of LC-AH cells.     

The effects of chronic ethanol administration on polyamine content during dimethylhydrazine-induced colorectal carcinogenesis in the rat

Chronic ethanol (EtOH) consumption has been implicated as aco-carcinogen, selectively promoting rectal tumor formation.We studied the effects of EtOH consumption on tumor formationand polyamine content (putrescine, spermidine and spermine)in proximal and distal colon and rectum of Sprague-Dawley ratstreated with the procarcinogen 1,2-dimethylhydrazine (DMH).Sixty-four adult male rats were pair fed nutritionally completeliquid diets with 36% of calories supplied as EtOH or isocaloriccarbohydrates. Both groups received 4 weeks of the liquid dietfollowed by 4 weeks of standard laboratory chow during which50% of the rats in each group received DMH (30 mg/kg) or vehicles.c. weekly. This cycle was repeated four times (32 weeks).Animals were sacrificed at the end of each 8 week cycle andnormal appearing and available tumor bearing tissue from proximaland distal colon and rectum was obtained for polyamine contentand histology. Five animals, unexposed to DMH or EtOH servedas baseline controls. There were no consistent regional differencesin putrescine, spermidine or spermine of baseline controls.A progressive decrease in tissue putrescine was seen in allthree regions of the control group and was significant at 24and 32 weeks versus baseline controls. In all three regions,chronic EtOH consumption prevented the decrease in tissue putrescine.Spermidine content was also significantly increased in the distalcolon of EtOH-treated animals compared to baseline values. Consistentchanges in spermine content were seen in any treatment groupor region. A significant increase in putrescine content of normalappearing and tumor-bearing tissue of DMH treated animals at32 weeks was noted. Chronic EtOH administration did not augmentrectal or colonic polyamine content in DMH-treated animals.Likewise, chronic EtOH consumption did not alter the number,size or distribution of large bowel tumors of DMH treated animals.     

Altered expression of c-myc,p16 and p27 in rat colon tumors and its reversal by short-term treatment with chemopreventive agents

Modulation of gene expression in tumors has the potential of being a surrogate end-point biomarker for chemoprevention. Thus, we determined the modulation by chemopreventive agents of the protein and mRNA expression of genes in rat colon tumors. Male F344 rats were administered three weekly injections of 15 mg/kg azoxymethane. Forty-seven weeks later, they received aspirin (600), calcium chloride (50 000), 2-(carboxyphenyl) retinamide (2-CPR, 315), alpha-difluoromethylornithine (DFMO, 3000), piroxicam (200), quercetin (33 600), 9-cis retinoic acid (9-cis RA, 30), rutin (3000), or sulindac (280) in their diet at the indicated mg/kg concentration for 7 days and were then killed. In colon tumors relative to the mucosa, the protein and mRNA levels of c-myc were increased, while the levels of p16 and p27 were decreased. Calcium chloride, DFMO, piroxicam and sulindac administered for 7 days decreased the mitotic index and reduced the protein and mRNA levels of c-myc in colon tumors. Calcium chloride, DFMO and piroxicam increased the protein and mRNA levels of p16 and along with sulindac increased the protein level of p27, but not its mRNA. The other agents failed to modulate both the mitotic index and the expression of the genes. The ability of the chemopreventive agents to prevent colon tumors was determined. Male F344 rats were administered three weekly injections of 15 mg/kg azoxymethane and 8 weeks later they were administered aspirin, 2-CPR, DFMO, piroxicam, 9-cis RA and rutin in their diet. The rats were killed 26 weeks after they started to receive the chemopreventive agents. The multiplicity of colon tumors was reduced by DFMO and piroxicam, increased by rutin and not affected by the other agents. Hence, agents that prevented colon cancer decreased the mitotic index and altered the expression of c-myc, p16 and p27 suggesting that modulation in the expression of these genes are potential biomarkers for chemopreventive activity.