Polyamines as mediators of the effect of prolactin and growth hormone on the growth of N-nitroso-N-methylurea-induced rat mammary tumor cultured in vitro in soft agar

This study evaluated the role of polyamines in mediating the effect of ovine prolactin (oPRL) and ovine growth hormone (oGH) on the growth of the N-nitroso-N-methylurea [(NMU) CAS:684-93-5]-induced rat (Sprague-Dawley) mammary cancer cultured in soft agar. oPRL and oGH had a dose-related growth-promoting effect when added to NMU-induced mammary tumors cultured in soft agar in the absence of serum. The effect of oPRL and oGH was blocked by alpha-difluoromethylornithine (DFMO), a suicide inhibitor of ornithine decarboxylase, the rate-limiting enzyme of polyamine biosynthesis. Exogenous administration of polyamines reversed the inhibitory effect of DFMO and completely restored the action of oPRL and oGH. Our results indicated that polyamines are essential in mediating the growth of the NMU-induced mammary tumor under these experimental conditions.     

Polyamines and estrogen control of growth of the NMU-induced rat mammary tumor

Recent in vitro evidence suggests that polyamines play an important role in the growth of the N-nitrosomethyl-urea (NMU)-induced rat mammary tumor, and that they may be involved in mediating the effect of estrogens on tumor growth. In support of this hypothesis, we here show that inhibition of polyamine biosynthesis with alpha-difluoromethyl-ornithine (DFMO) blocks the mitogenic effect of estradiol-17 beta (E2) added to NMU-mammary tumors grown in soft agar in the presence of the antiestrogen tamoxifen (Tam). Exogenous polyamine administration reversed the inhibitory effect of DFMO and restored E2 action. Administration of polyamine inhibitors to NMU-tumor-bearing rats induced significant inhibition of tumor growth, although tumor ornithine decarboxylase (ODC) was not consistently suppressed. Under our experimental conditions, such treatment did not potentiate the antitumor effect of Tam. Tam alone was found to suppress tumor ODC, suggesting a possible involvement of the polyamine pathway in its antitumor action. These data suggest that the polyamines may play an important role in the hormonal control of the growth of this experimental breast cancer.     

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.     

Role of polyamines in the synthesis of prolactin-regulated growth factors by experimental breast cancer in culture

Summary The present experiments were designed to test whether polyamines play an essential role in the synthesis of growth factors induced by ovine prolactin (oPRL), using the N-nitrosomethylurea (NMU)-induced rat mammary tumor cultured in the soft agar clonogenic assay. Conditioned media (CM) obtained from tumors treated with oPRL and the polyamine biosynthesis inhibitor -difluoromethylornithine (DFMO) (1 mM) no longer exerted the colony-stimulating effect which was observed with oPRL-CM. Such growth-promoting activity was restored with conditioned media obtained from tumors treated with oPRL, DFMO, and increasing concentrations of spermidine from 1 to 500µM. The colony-stimulating effect of the CM employed could not be accounted for by the contaminating presence in the media of oPRL, DFMO, and polyamines. These results indicate that in our system polyamines play an important role in the synthesis of oPRL-regulated growth factors.     

Role of polyamines in estradiol-induced growth of human breast cancer cells

The present study explored the possible involvement of polyamines in estradiol-stimulated proliferation of human breast cancer cells using the estrogen-responsive subline of T-47D cells (clone 11). 17 beta-Estradiol (10(-10) M) stimulated cell growth 2- to 4-fold. This estradiol-induced proliferation was associated with a peak (at 12-24 h) of activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme of polyamine biosynthesis. Estradiol-induced cell growth and ODC activity were observed only in the presence of 10% charcoal-treated fetal bovine serum, suggesting that serum factors are required for estrogen action. alpha-Difluoromethylornithine (DFMO, 0.1 mM), a specific inhibitor of ODC, blocked the estradiol-induced cell proliferation and ODC activity. Putrescine (0.1 mM) rescued the inhibitory effect of DFMO on growth of steroid-treated cells. Putrescine alone was not stimulatory to cells, and in combinations with estradiol, it did not augment the effect of estradiol. In addition, DFMO abolished the estradiol-induced growth of several other hormone-responsive cell lines but did not affect the proliferation of unresponsive cells. Hormone-responsive cells exhibited differential sensitivity to DFMO; resistant cell lines (e.g., MCF-7) were found to possess higher endogeneous levels of ODC than sensitive cell lines (e.g., T-47D and ZR-75-1). Our findings indicate that polyamines are essential, although not sufficient, in estrogen stimulation of human breast cancer cells.     

Individual and combined effects of alpha-difluoromethylornithine and ovariectomy on the growth and polyamine milieu of experimental breast cancer in rats

Despite considerable evidence suggesting a critical role of polyamines in the hormonal control of breast cancer growth in vitro, their role in in vivo tumor growth is not established. In these experiments, we evaluated the individual and combined effects of the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) and ovariectomy on the growth and cellular levels of ornithine decarboxylase (ODC) and polyamines of N-nitrosomethylurea-induced rat mammary tumors. Despite a similar suppressive effect on ODC activity, the two treatments had a different effect on polyamine levels. As expected, DFMO selectively suppressed putrescine, whereas spermidine and spermine levels were minimally or not affected at all. Since quantitatively putrescine contributes the least to overall polyamine pools, the DFMO effect on this latter parameter was modest. In contrast, ovariectomy, by suppressing the more abundant spermidine and spermine, produced a more profound suppression of total polyamine pools. This finding is in agreement with the notion that hormones not only control ODC activity, but also other enzymes involved in the synthesis of the distal polyamines. Ovariectomy was also more potent than DFMO administration in inhibiting N-nitrosomethylurea-induced mammary tumor growth. No major additive/synergistic effects were observed between DFMO and ovariectomy on tumor growth and cellular levels of ODC activity and polyamines. DFMO administration lowered the tumor level of progesterone receptors and appeared to potentiate the suppressive effect of ovariectomy. In contrast, neither treatment, alone or in combination, altered tumor levels of estrogen receptors. DFMO administration did not affect circulating levels of estradiol and prolactin or uterine and ovarian weights, thus suggesting that its effects were not indirectly mediated through alterations of the endocrine milieu of the host.     

Selectivity of polyamine involvement in hormone action on normal and neoplastic target tissues of the rat

The present experiments were designed to evaluate the polyamine involvement in hormonal actions on proliferation and receptor content of neoplastic tissue (hormone-responsive breast cancer) as well as on growth of normal endocrine target tissue (uterus) in the same animals. Administration of estradiol and perphenazine (to stimulate endogenous prolactin release) stimulated N-nitrosomethyl-urea (NMU)-induced rat mammary tumor growth following ovariectomy-induced tumor regression. Such hormonal activation of breast cancer growth was completely abolished by treatment with -difluoromethyl-ornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, which lowered tumor content of polyamines. The growth inhibitory effect of DFMO was partially reversible by exogenous putrescine administration. In contrast, the rise in cytosolic content of progesterone receptors induced by hormonal treatment was not affected by suppression of tumor polyamine levels by DFMO. Similarly, DFMO administration failed to influence the hormone-induced increase in uterine weight in the same animals. Thus, our data suggest selectivity of polyamine involvement in hormone actions, which, in our experimental system, seems to be restricted to the endocrine control of neoplastic cell proliferation.     

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 tamoxifen and alpha-difluoromethylornithine on clones of nitrosomethylurea-induced rat mammary tumor cells grown in soft agar culture

N-Nitrosomethylurea-induced mammary tumors were grown in a bilayer soft-agar culture system. The antiestrogen tamoxifen prevented formation of 50% of colonies formed in this in vitro system. Possible mediation of these antimitotic effects through the polyamine pathway was suggested by a similar inhibition of colony formation by difluoromethylornithine (DFMO), a suicide inhibitor of ornithine decarboxylase, and the lack of additivity of DFMO and tamoxifen. The cytostatic effect of DFMO was found to be dose dependent. The specificity of the DFMO effect through the polyamine pathway was indicated by the dose-dependent rescue of colony growth with exogenous administration of putrescine, the polyamine distal to the site of inhibition. A lack of alteration of colony size in proliferating clones was uniformly observed. These data indicate that the soft-agar culture technique can be successfully used to investigate the endocrine mechanisms affecting the growth of individual experimental mammary cancers. The data also suggest an important role of polyamines in mediating the growth of this mammary tumor model.     

Role of polyamines in the growth of hormone-responsive and -resistant human breast cancer cells in nude mice.

Recent in vitro data suggest that at least some hormone-independent breast cancer cells exhibit increased polyamine biosynthesis and resistance to antipolyamine therapy. To address this issue under conditions of in vivo growth, we tested the antiproliferative effect of the polyamine synthetic inhibitor alpha-difluoromethyl-ornithine (DFMO) on hormone-dependent (MCF-7) and -independent (MDA-MB-231, BT-20) breast cancer cell lines growing in nude mice. We observed that DFMO significantly inhibited the growth of established tumors to a similar extent in all cell lines, even though tumor regression was only observed with MCF-7 cells. DFMO, while inhibiting E2-supported MCF-7 breast cancer growth, did not inhibit E2-stimulated progesterone receptor synthesis. Cellular levels of polyamines were highest in MCF-7 cells and lowest in the BT-20 cell line. Tumor content of spermidine was similarly suppressed by DFMO treatment in the 3 cell lines, while the spermine level was unaffected. Cellular putrescine levels were suppressed in MCF-7 and BT-20 cells. Administration of DFMO prior to implantation of fragments of MCF-7 or MDA-MB-231 tumors in nude mice significantly inhibited tumor development to a similar extent. The action of DFMO seemed to be predominantly tumoristatic since new tumors develop in some mice upon discontinuation of the drug. We conclude that the hormone-independent breast cancer cell lines tested do not exhibit increased polyamine biosynthesis or resistance to antipolyamine therapy when grown in vivo in nude mice.     

Polyamines and the synthesis of estradiol-regulated growth factors in rat mammary cancer in culture

We have recently provided evidence to suggest that the polyamine pathway plays an essential role in the expression of the growth-promoting effect of estradiol (E₂) regulated growth factors in the N-nitrosomethylurea (NMU) induced rat mammary tumor culturedin vitro in the soft agar clonogenic assay. To further explore the interaction between the polyamine pathway and autocrine control of tumor growth by E₂, we tested whether, in our system, polyamines play a role in the synthesis of E₂-regulated growth factors. Conditioned medium (CM) obtained from tumors treated with E₂ and the polyamine biosynthesis inhibitor -difluoromethyl-ornithine (DFMO) (1 mM) no longer exhibited the colony-stimulating effect which was consistently observed with E₂-CM. Such growth promoting activity was restored in a dose-dependent fashion with CM obtained from tumors treated with E₂, DFMO, and increasing concentrations of spermidine (from 1 to 100µM). Conditioned medium obtained from tumors treated with DFMO with and without spermidine in the absence of E₂ had no discernible effects on colony formation. The colony stimulating effect of the CM employed could not be accounted for by the contaminating presence in the media of E₂, DFMO, or polyamines. These results indicate that, in our system, the polyamine pathway plays an important role in the synthesis of E₂-regulated growth factors.     

Effect of inhibition of polyamine biosynthesis by DL-alpha-difluoromethylornithine on the growth and melanogenesis of B16 melanoma in vitro and in vivo

The objective of the present study was to investigate the effect of polyamine depletion by alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on the growth and differentiation of B16 melanoma cells grown in culture and also as solid tumors in mice. Polyamine depletion by DFMO (2.5 mM) resulted in a complete inhibition of cell growth in culture and a 90% inhibition of viability of melanoma cells as determined by clonogenic assay at the end of 7 days after DFMO treatment. These results indicate that polyamine depletion induced by DFMO is cytotoxic to B16 melanoma cells in culture. Furthermore a 2- to 5-fold increase in tyrosinase activity and 10-fold accumulation of melanine were observed in polyamine depleted cells compared to control cultures. These effects of DFMO could easily be reversed by the addition of putrescine simultaneously with DFMO. Administration of different doses of DFMO in drinking water to B16 melanoma tumor bearing mice also resulted in an increase in tyrosinase activity and a dose dependent inhibition (86-90%) of tumor growth. Although one cannot rule out the possibility of induction of differentiated phenotype as a result of antiproliferative activity of DFMO, the data presented indicate that the unique sensitivity of melanoma to DFMO may be due to a combination of cell growth inhibition and concomitant induction of differentiation upon polyamine depletion. The results of the present study indicate that polyamines play an important role in growth and differentiation of melanoma and also provide an example of inhibition of tumor cell growth by induction of cellular differentiation.     

Differential effects of polyamine homologues on the prevention of DL-alpha-difluoromethylornithine-mediated inhibition of malignant cell growth and normal immune response.

Natural polyamines (putrescine, spermidine, and spermine) are ubiquitous cellular cations that play an important role in cell proliferation and differentiation. Ornithine decarboxylase is the first and a rate-limiting enzyme in the biosynthesis of polyamines. Polyamine depletion using DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, has been shown to suppress cell growth in a variety of settings, including those of tumor and lymphocyte proliferation. The objective of the present investigation was to examine the inhibitory effects of DFMO on a variety of murine in vitro immune responses, including lymphocyte proliferation in response to T-cell mitogen (concanavalin A), B-cell mitogen (lipopolysaccharide), and alloantigen as well as cytotoxicity. DFMO-mediated inhibition of cell proliferation in these cases correlated with depletion of intracellular polyamines. The inhibitory effects of DFMO were reversed by polyamine repletion with putrescine. Putrescine also reversed the growth-inhibitory effects of DFMO on 4 tumor cell lines that we tested: 28-13-3S, YAC-1, P-815, and K562. However, putrescine homologues exhibited a differential effect in preventing DFMO-mediated inhibition of cell growth in normal lymphocytes and cancer cell lines. Only putrescine homologues containing a shorter methylene chain were effective in preventing the growth-inhibitory action of DFMO on normal immune response. In contrast, only the longer chain homologue 1,5-diaminopentane overcame the effect of DFMO on tumor cell growth. These findings suggest that supplementation with selected polyamine homologues may sustain normal immune response in DFMO-treated individuals while effectively suppressing malignant cell growth. The potential clinical relevance of these observations is discussed.     

Antimetastatic activity of DL-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis, in mice

Our earlier studies indicated a role for polyamines (namely, putrescine, spermidine, and spermine) not only in tumor growth but also in tumor metastases. We have observed that administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, resulted in significant inhibition of visually detectable pulmonary metastases in mice implanted with Lewis lung carcinoma. The objective of the present study is to investigate the effect of DFMO on other spontaneous and experimental metastatic models and also to determine which step(s) in the tumor metastatic cascade is sensitive to DFMO. The results presented in this study with malignant mouse B16 amelanotic melanoma (B16a) showed a dose-dependent effect of DFMO on the inhibition of both tumor growth and grossly detectable pulmonary metastases. DFMO, when administered as 0.5, 1, and 2% solution in drinking water, resulted in 0, 24.5, and 60% inhibition of tumor growth, respectively, whereas at the same doses an inhibition of 55, 83, and 96% of visible metastases was observed. At treatment levels of 1 and 2% DFMO, 30 and 65% of the animals were free of metastases. DFMO, at 0.5%, did not show any effect on tumor growth, while a significant 55% inhibition of visible pulmonary metastasis was observed, suggesting a specific role for polyamines in tumor metastasis. DFMO treatment also resulted in a significant reduction of putrescine and spermidine levels with a slight increase in spermine concentration in the tumor tissue. DFMO administration did not inhibit the experimental metastases induced as a result of i.v. injection of B16 melanoma (line F10) tumor and Lewis lung carcinoma cells into the tail vein. These results provide preliminary evidence to indicate that tumor cell polyamine depletion by DFMO might affect the first step in the metastatic cascade, intravasation (i.e., prevent the invasion of metastatic tumor cells into lymphatics or blood vessels), although the effect of DFMO on other steps in the metastatic cascade cannot be ruled out.     

Polyamine involvement in the growth of hormone-responsive and -resistant human breast cancer cells in culture

Recent evidence indicates that hormone-responsive but not -resistant human breast cancer cells in culture are sensitive to the antiproliferative effect of the polyamine (PA) biosynthetic inhibitor alpha-difluoromethylornithine (DFMO). The present experiments were designed to investigate the potential differential involvement of the PA pathway in the growth of these different biological subtypes of human breast cancer. Thus, we evaluated the effect of DFMO on proliferation, ornithine decarboxylase (ODC) activity, and PA levels of the hormone-dependent MCF-7 and -independent MDA-MB-231 breast cancer cell lines. When tested at comparable cell density, the two cell lines had similar levels of ODC activity and PA. Administration of DFMO (0.01, 0.1, 1, 4 mM) for 6 days caused a similar dose-dependent inhibition of proliferation (up to approximately 15% of control) associated with suppression of ODC activity to undetectable levels at the highest dose. In both cell lines, putrescine and spermidine levels were maximally suppressed by doses of DFMO greater than 0.1 mM. Higher doses of DFMO (1 and 4 mM) also suppressed spermine levels to approximately 60% of control. In detailed time-course studies, DFMO administration (0.1 mM) similarly suppressed by 80% the rise in ODC observed in both cell lines following a medium change. At all time points, putrescine and spermidine levels were likewise suppressed to a similar extent. Addition of putrescine (0.1-2.5 mM) to DFMO-treated cells repleted cellular PA levels and restored growth to approximately 80% of control in both cell lines. We conclude that, under these experimental conditions, PA are similarly involved in the growth of the hormone-responsive MCF-7 and -resistant MDA-MB-231 human breast cancer cell lines.     

Effect of the ornithine decarboxylase inhibitor alpha, alpha-difluoromethylornithine on phorbol diester-induced inhibition of murine B lymphocyte differentiation

The tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits the differentiation of murine B lymphocytes to antibody-producing plasma cells, in unfractionated spleen cell cultures or enriched B lymphocyte cultures. To determine the role of polyamines in TPA-induced inhibition, unfractionated splenic lymphocytes, in culture with antigen, were incubated with alpha, alpha-difluoromethylornithine (DFMO, 0.10 mM), an irreversible inhibitor of ornithine decarboxylase (ODC). DFMO prevented the TPA-induced inhibition of antibody forming cell number in a 5-day in vitro immunization procedure as measured by a hemolytic plaque assay. In enriched B lymphocyte cultures, however, DFMO had no comparable effect. DFMO did not prevent TPA-induced inhibition of antibody production in unfractionated spleen cell cultures but itself inhibited the amount of antibody produced. Putrescine (0.1 mM), added on day 4 of immunization, reversed DFMO inhibition of antibody production but did not enable DFMO to prevent the TPA-induced inhibition. These findings suggest that TPA-induced inhibition of plasma cell number can be mediated indirectly through effects on T lymphocytes and/or macrophages or directly through effect on B lymphocytes.     

Increased survival of L1210 leukemic mice by prevention of the utilization of extracellular polyamines. Studies using a polyamine-uptake mutant, antibiotics and a polyamine-deficient diet.

When L1210 leukemia cells are inhibited in their polyamine synthesis by treatment with alpha-difluoromethylornithine (DFMO), their growth in culture is strongly suppressed. In striking contrast, the survival of L1210 leukemic mice is only marginally prolonged by DFMO treatment. This inconsistency is due to the fact, that in the mouse the tumor cells can utilize extracellular polyamines to compensate for the decrease in putrescine and spermidine synthesis caused by DFMO treatment. In the present study, we demonstrate that a reduction in the transport of polyamines into the tumor cells is a more effective means of increasing the therapeutic effect of DFMO than is a reduction in the supply of extracellular polyamines. DFMO treatment cured 30-75% of leukemic mice bearing mutant L1210-MGBGr cells deficient in polyamine uptake, but only slightly increased the survival time of leukemic mice bearing the parental L1210 cells despite the fact that the supply of extracellular polyamines was reduced (by feeding the mice a polyamine-deficient diet containing antibiotics). The effectiveness by which DFMO cured leukemic mice bearing L1210-MGBGr cells appeared to be sex dependent. Thus, 58% of the female mice, as compared to 30% of the male mice, were cured by DFMO treatment.     

Normal and malignant human urothelium: in vitro response to blockade of polyamine synthesis and interconversion

To assess the effect of polyamine blockade on urinary epithelium, growth of normal human urothelial cell cultures and human transitional cell carcinoma (TCC) cell lines in the presence of various inhibitors of polyamine synthesis was evaluated. All four human TCC cell lines tested were quite sensitive to the specific inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO), requiring less than or equal to 1.0 mM DFMO to double generation time. Alternatively, all three human urothelial cultures tested required 5-20-fold higher DFMO concentrations to achieve similar growth inhibition. The inhibitory effect of DFMO on TCC was found to act synergistically with that of the inhibitor of S-adenosylmethionine decarboxylase, methylglyoxal bis(guanylhydrazone), and additively with that of recombinant beta-serine interferon. Addition of individual polyamines entirely prevented the inhibitory effects of DFMO and/or methylglyoxal bis(guanylhydrazone) but not that of beta-serine interferon. It appears that inhibition of polyamine synthesis and/or interconversion holds promise in the management of TCC and that the in vitro model described will be of value in investigating such clinical applications.     

Effects of alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) on the growth of experimental renal adenocarcinoma in mice

alpha-Difluoromethylornithine (DFMO) and methylglyoxal bis-(guanylhydrazone) (MGBG) were tested against a murine renal adenocarcinoma, because polyamines are necessary for neoplastic cell growth and because human renal adenocarcinomas contain higher levels of spermidine than do normal renal cells; MGBG inhibits spermidine synthesis and has some activity against human renal tumors; DFMO irreversibly inhibits ornithine decarboxylase, the first rate-limiting enzyme controlling polyamine biosynthesis; and DFMO promotes intracellular accumulation of MGBG in experimental tumor models and human leukemia. DFMO (2%) in drinking water, MGBG (15 mg/kg i.p.), or a combination of DFMO and MGBG was administered daily to BALB/c mice (n = 80) with intrarenal transplants of renal adenocarcinoma cells. At 28 days, renal carcinomas weighed 64 and 73% less, respectively, in DFMO- and DFMO-MGBG-treated mice than in control animals (p less than 0.01). MGBG alone had no antigrowth effect. DFMO-MGBG reduced the total metastatic index (total number of metastases/total number of animals) to 1.2 versus 3.6 in control animals (p less than 0.01) and increased survival by 12.3 +/- 1.5 (S.E.) days, from 30.8 to 42.5 days (p less than 0.05). Compared with control, DFMO-, or MGBG-treated animals, DFMO-MGBG exposure reduced tumor growth and the number of metastases, prevented metastases in some animals (47%), and increased survival of mice bearing renal adenocarcinomas. DFMO also appeared to selectively increase the uptake of [14C]MGBG by tumor tissue, which may help to explain the enhanced synergistic antigrowth effect of DFMO and MGBG against this murine renal adenocarcinoma.     

alpha-Difluoromethylornithine alters calcium signaling in platelet-derived growth factor-stimulated A172 brain tumor cells in culture.

alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, inhibits the growth of brain tumor cell lines and is undergoing clinical trials as a treatment for brain tumors. Platelet-derived growth factor (PDGF) is thought to regulate the growth and development of precursors of both normal and neoplastic astrocytic cells; calcium signaling is thought to play a role in the transduction of PDGF signals. Using laser fluorescence image cytometry, flow cytometry, and spectrofluorometry, we studied the effect of DFMO on the calcium signals induced by PDGF in A172 human glioblastoma cells. Four days of treatment with 5 mM DFMO substantially shortened PDGF-induced calcium signals. The effect was reversed more than 10 h but less than 24 h after putrescine treatment, even though polyamines were repleted 4 h after putrescine and spermidine were added. DFMO did not substantially affect intracellular calcium release or the timing of the opening and closing of plasma membrane calcium channels. These findings support the notion that calcium signaling may be a target for inhibitors of polyamine metabolism.