Altered cell cycle phase distributions in cultured human carcinoma cells partially depleted of polyamines by treatment with difluoromethylornithine

We have studied the effects of partial polyamine depletion, induced by treatment with alpha-difluoromethylornithine (DFMO) on cell cycle phase distributions in five cultured human carcinoma cell lines. We used flow cytometry of cells stained with chromomycin-A3 and computer analysis to measure phase distributions of treated and control cultures. All five lines respond to 1-5 mM DFMO treatment with a total absence of measurable putrescine, a loss of greater than 90% of spermidine, and a 30-40% decline in spermine by 48 h after DFMO addition. The proliferation of all five lines is inhibited as well. Nonetheless, only four of the cell lines (HuTu-80, HT-29, MCF-7, and A-427) show a marked increase in the G1-phase fraction and decrease in the S-phase fraction as a consequence of DFMO treatment. Small, but significant, decreases in the G2-M populations of these cell lines also occurred after DFMO treatment. Exogenous putrescine (5-50 microM) reversed both the polyamine depletion and the perturbed phase distributions of DFMO-treated cultures but was without effect on phase distributions of cultures not treated with DFMO. The fifth cell line (ME-180) showed no effect of polyamine depletion on cell cycle phase distributions in DFMO-treated cultures and also no effect of exogenous putrescine on phase fractions of either control or DFMO-treated cells. These observations indicate that some human tumor cell lines are dependent upon adequate intracellular polyamine content for maintenance of cell cycle traverse. They also imply that human tumor cell lines are heterogeneous with regard to their cell cycle response to DFMO-induced polyamine deficiency.     

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

The polyamine inhibitor DL--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.     

Effect of alpha-difluoromethylornithine on 1,3-bis(2-chloroethyl)-1-nitrosourea and cis-diamminedichloroplatinum(II) cytotoxicity, DNA interstrand cross-linking, and growth in human brain tumor cell lines in vitro

The polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) has been shown to potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in 9L rat brain tumor cells and in non-central nervous system human cancer cells in vitro, but the effects on a human brain tumor cell line have not been reported. Because BCNU is one of the main chemotherapeutic agents used clinically for the treatment of brain tumors, the effect of DFMO treatment on cell growth and potentiation of cytotoxicity was studied in vitro in U-251 MG and SF-126 cells, human tumor cell lines derived from malignant glioma tissue. Pretreatment of U-251 MG with 1 mM DFMO depleted cells of putrescine and spermidine within 48 h but did not sensitize cells to BCNU treatment even after a pretreatment of 72 h. DFMO treatment had no effect on the number of interstrand cross-links formed in BCNU-treated cells. Even treatment with 5 mM DFMO for 72 h caused only the suggestion of potentiation of BCNU cell kill. In contrast, a 72-h pretreatment with 1 mM DFMO decreased the cytotoxic effect of cis-diammine-dichloroplatinum(II) and caused a 38% decrease in the number of DNA interstrand cross-links formed. The glutathione content and cell cycle distribution of U-251 MG cells were not affected by DFMO pretreatment. Because Phase II clinical trials with DFMO and BCNU have shown promise for the treatment of anaplastic astrocytomas in humans, a second brain tumor cell line, SF-126, was studied. In this cell line a consistent potentiation of BCNU cytotoxicity (dose enhancement of 1.2 at the 10% survival level) was observed in cells pretreated with 1 mM DFMO for 72 h.     

Decreased cytotoxicity of 1-beta-D-arabinofuranosylcytosine in 9L rat brain tumor cells pretreated with alpha-difluoromethylornithine in vitro

The effect of pretreating 9L rat brain tumor cells in vitro with 10 mM alpha-difluoromethylornithine (DFMO), an enzyme-activated, irreversible inhibitor of ornithine decarboxylase, on the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) was investigated using a colony-forming assay. DFMO-mediated polyamine depletion significantly decreased the cytotoxicity of ara-C. The addition of putrescine to DFMO-pretreated cells replenishes intracellular polyamine levels and prevents the decrease in ara-C cytotoxicity. Flow cytometric analysis showed that cells treated with ara-C alone entered S phase semisynchronously within 8 hr after the end of treatment. This phenomenon was virtually eliminated by DFMO pretreatment. Addition of putrescine to DFMO-pretreated cells before treatment with ara-C prevented this DFMO-induced cell cycle kinetic effect.     

Cytotoxic activity of a polyamine analogue, monoaziridinylputrescine, against the PC-3 human prostatic carcinoma cell line

We have previously demonstrated that prostate and prostate-derived rodent tumors can be manipulated into increasing their accumulation of radiolabeled putrescine by alpha-difluoromethylornithine (DFMO)-induced depletion of intracellular putrescine and spermidine. As methods which increase intracellular accumulation of cytotoxic agents often increase the chemotherapeutic effectiveness of the agent, we examined whether an alkylating derivative of putrescine would be cytotoxic to tumor cells. We present here our findings on the cytotoxicity of the aziridinyl derivative of putrescine (AZP) against prostatic cancer cells. The apparent Km for putrescine was 2.5 microM with or without DFMO pretreatment and the apparent Ki for AZP was 1 microM with or without DFMO pretreatment. Intracellular polyamine depletion by DFMO pretreatment resulted in a 3.7-fold greater accumulation of AZP compared to non-DFMO-treated cells. The growth inhibitory activity of AZP was increased with prior polyamine depletion by DFMO with the 50% effective dose decreasing from 18 microM to 2.1 microM. Putrescine was able to block the cytotoxic effect of AZP. Putrescine was also able to rescue the AZP-treated PC-3 cells for up to 6 h following a 1-h exposure to AZP. It appears that aziridinylputrescine behaves like putrescine in that it competes with putrescine for uptake into the cell and, like putrescine, has its uptake into the cell increased by prior polyamine depletion. It differs from putrescine in that it expresses cytotoxic activity and inhibits the growth of the human prostate-derived PC-3 cell line. This cytotoxic activity is also increased by prior polyamine depletion. The cytotoxic behavior of AZP is dependent both on the concentration and duration of exposure. Putrescine can rescue the cells from the effect of AZP. AZP is a potentially useful cytotoxic analogue that utilizes the polyamine transport system for its uptake into the cell.     

Time dependence of the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity caused by alpha-difluoromethylornithine-induced polyamine depletion in 9L rat brain tumor cells

Treatment of 9L rat brain tumor cells with 1.0 mM alpha-difluoromethylornithine (DFMO) produced a time-dependent depletion of cellular putrescine and spermidine. An increase in the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity, measured with a colony-forming efficiency assay, followed the time course of polyamine depletion, reaching its maximum at 48 hr, the time at which maximum polyamine depletion was achieved. Treatment with DFMO at concentrations as low as 0.05 mM for 48 hr effectively depleted putrescine and spermidine and potentiated BCNU cytotoxicity. Treatment for 96 hr with 0.01 mM DFMO produced a partial decrease in putrescine and spermidine levels and a moderate potentiation of BCNU cytotoxicity. The amount of polyamine depletion in 9L cells treated with 0.05, 0.1, and 0.5 mM DFMO was identical at both 48 and 96 hr, but potentiation of BCNU cytotoxicity was greater at 96 hr than at 48 hr. When 9L cells were treated for 48 hr with 1 mM DFMO and DFMO was then removed from the cultures, polyamine levels did not reach control levels by 96 hr after change of medium. The potentiation of BCNU cytotoxicity during this 96-hr period correlated with the extent of polyamine depletion. When 100 microM putrescine was added to the culture medium after DFMO pretreatment (1 mM), polyamine levels approached those of control cells by 24 hr, and the amount of potentiation of DFMO cytotoxicity decreased. These results show that potentiation of BCNU cytotoxicity correlates closely with the amount of DFMO-induced polyamine depletion in 9L cells.     

Polyamines in brain tumor therapy

Summary In the search for ways to augment current brain tumor therapies many have sought to exploit the fact that adult brain tissue is virtually lacking in cell division. This endorses a special appeal to therapeutic approaches which target the dependence on cell division for brain tumor growth. Polyamines play an essential role in the proliferation of mammalian cells and depletion results in inhibition of growth. As a result, there are investigations into the feasibility of controlling tumor growth by targeting the enzymes in polyamine metabolism with specific enzyme inhibitors. DFMO, an inhibitor of putrescine synthesis, is a cytostatic agent which in combination with tritiated radioemitters or cytotoxic agents such as, MGBG or BCNU is an effective antitumor agent, but the effectiveness of DFMOin vivo is reduced by tumor cell uptake of polyamines released into the circulation by normal cells and from gut flora or dietary sources. However, DFMO therapy combined with elimination of exogenous polyamines inhibits tumor growth but also results in body weight loss, reduced protein synthesis and evidence of toxicity. Furthermore, tumor growth recurs upon termination of treatment. In contrast, competitive polyamine analogs function in the homeostatic regulation of polyamine synthesis but fail to fulfill the requirements for growth and they continue to inhibit tumor growth for several weeks after cessation of treatment. Analogs are now in clinical trials. However, their action may be highly specific and differ from one cell type to another. We suggest that the effectiveness of polyamine based therapy would be enhanced by two approaches: local delivery by intracerebral microdialysis and tumor cell killing by internal radioemitters such as tritiated putrescine or tritriated thymidine which are taken up in increased amounts by polyamine depleted tumor cells. The growth inhibition by polyamine depletion prevents the dilution of the radioactive putrescine and thymidine. The overload of radioactivity kills the growth inhibited cells so that growth cannot recur when treatment terminates.     

Estradiol control of ornithine decarboxylase mRNA,enzyme activity,and polyamine levels in MCF-7 breast cancer cells: therapeutic implications

Previous studies have shown that natural polyamines - putrescine, spermidine, and spermine - play a key role in the mechanism of action of estrogens in breast cancer. Ornithine decarboxylase (ODC) is the first enzyme of the polyamine biosynthetic pathway. To examine estrogenic regulation of polyamine biosynthesis in breast cancer, we measured ODC mRNA, ODC activity, and polyamine levels in G₁ synchronized MCF-7 cells. ODC mRNA and activity increased four-fold over that of cells in G₁ phase between 8 to 16 h after the addition of estradiol. Polyamine levels showed a sharp increase by 8 h after the addition of estradiol and decreased by 12 h. We further examined whether synthetic homologs of putrescine or spermidine could replace natural polyamines in supporting MCF-7 cell growth. Treatment of MCF-7 cells with 1 mM difluoromethylornithine (DFMO), an inhibitor of ODC, suppressed putrescine, spermidine, and spermine levels by 74, 78, and 10%, respectively, within 48 h. Cells treated with DFMO for 48 h were supplemented with either putrescine or its homologs or spermidine or its homologs. Diaminopropane, diaminobutane (putrescine), and diaminopentane were capable of fully or partially reversing the growth inhibitory effects of DFMO, whereas diaminoethane had no significant effect. Among a series of triamines, H₂N(CH₂)_nNH(CH₂)₃NH₂ (where n = 2 to 8; abbreviated as APn n = 4 for spermidine, or AP4), spermidine was most effective in reversing the effects of DFMO, whereas compounds with shorter or longer methylene bridging regions were less effective. AP8 was ineffective in reversing the growth inhibitory effects of DFMO. At 10 µM concentration, AP8 also inhibited DNA synthesis by 66%, as measured by [³H]-thymidine incorporation. These data show that MCF-7 cells have a strong requirement for polyamines for their growth and that estradiol stimulates the polyamine cascade by inducing the ODC mRNA level. Our results also suggest that polyamine homologs such as AP8 might be potentially useful in breast cancer therapy.     

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.     

Tumor angiogenesis and polyamines: alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, inhibits B16 melanoma-induced angiogenesis in ovo and the proliferation of vascular endothelial cells in vitro.

alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, inhibited B16 melanoma-induced angiogenesis in chick embryo chorioallantoic membrane and subsequently the growth of the tumor on the chorioallantoic membrane. These inhibitions were reversed by exogenous putrescine and spermidine. DFMO also inhibited rapid neovascularization in yolk sac membrane of 4-day-old chick embryos and the inhibition was reversed by exogenous putrescine and spermidine. DFMO strongly inhibited DNA synthesis and proliferation of bovine pulmonary artery endothelial (BPAE) cells in culture and decreased their ornithine decarboxylase activity and intracellular polyamine concentrations. Addition of putrescine to the culture medium of DFMO-treated BPAE cells restored their intracellular putrescine and spermidine concentrations and their DNA synthesis and proliferation. Addition of spermidine to cultures of DFMO-treated BPAE cells restored their intracellular spermidine concentration and their DNA synthesis and proliferation. DFMO inhibited the proliferation of B16 melanoma cells in culture but the inhibitory effect was much less than that on BPAE cells. When one-half the monolayer of confluent cultures of BPAE cells had been peeled off, addition of DFMO to the cultures inhibited the proliferation and extension of the BPAE cells into the vacant area but had no effect on stationary cells in the remaining half of the monolayer, suggesting that it inhibited induction of proliferation of endothelial cells. These findings suggest that the antitumor activity of DFMO against solid tumors is probably due more to its inhibition of tumor-induced angiogenesis by inhibition of proliferation of endothelial cells induced by polyamine depletion than to a direct effect on tumor cell proliferation.     

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.     

Treatment of hamster pancreatic cancer with alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis

Polyamines are essential for cell division and growth. Inhibition of polyamine biosynthesis by alpha-difluoromethylornithine (DFMO) on the growth of hamster H2T pancreatic cancer was investigated both in vitro and in vivo. Cell-doubling time (TD) and survival fraction were determined after a single treatment with DFMO (5 mM). We examined the ability of putrescine to reverse the growth-inhibitory effect of DFMO. The TD for cells treated with DFMO in vitro was 49.6 +/- 5.7 versus 25.4 +/- 2.6 hours for control. The addition of putrescine to DFMO-treated H2T cells showed a reversal of the growth-inhibitory effect of DFMO. Cytotoxicity in vitro increased with prolonged treatment; the survival fraction after 24 hours of treatment was 32%; after 48 hours, 19%; after 72 hours, 13%; and after 92 hours, 8%. We performed two separate animal experiments. In experiment I, H2T cells were injected into the cheek pouch of male Syrian golden hamsters; controls did not receive DFMO. Continuous treatment with 3% DFMO in the drinking water was begun 7 days before, on the day of, or 7 days after tumor cell injection. In experiment II, 4 groups were treated identically to those in experiment I. An additional group of 10 hamsters received 3% DFMO and no tumor, and another additional group of 10 hamsters were housed individually with 3% DFMO begun 7 days after tumor cell injection. Tumor size, body weight, water, and food intake were measured. DFMO treatment in vivo significantly inhibited tumor size and inhibited growth of pancreatic cancer by as much as 50% of control. Our results demonstrate a significant antiproliferative effect of DFMO on the growth of pancreatic adenocarcinoma both in vitro and in vivo.     

Sensitization of Chinese hamster ovary cells to heat shock by alpha-difluoromethylornithine

When exposed to alpha-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, Chinese hamster ovary cells become increasingly sensitive to the cytotoxic effects of elevated temperatures (D.J.M. Fuller and E.W. Gerner, Cancer Res., 42:5046-5049, 1982). This sensitization becomes marked at times greater than 24 h after drug removal, and by 48 h, polyamine-depleted cells that have been exposed to 43 degrees C for 90 min have clonogenic survival values more than two orders of magnitude lower than control populations. Dose response studies demonstrate that, when measured 36 h after removal of the drug, hyperthermic cytotoxicity is maximally potentiated by exposure to DFMO for times as short as 2 to 4 h. A drug concentration of 1 mM for 8 h also elicits maximal response. An additional 8-h drug treatment 24 h after the first fails to further reduce survival in response to heat shock, suggesting the effects of the first exposure are persistent. Intracellular putrescine pools are depleted by the drug within 8 h, and spermidine levels continue to decline for up to 50 h. Consistent with these observations, ornithine decarboxylase (EC 4.1.1.17) activity is found to be reduced for up to 48 h after drug removal. The concomitant depression of spermidine is reflected in the elevation of S-adenosylmethionine decarboxylase (EC 4.1.1.50), which is substrate limited. Putrescine and spermidine show no sign of reaccumulation until approximately 4 days after DFMO exposure. Exposure to exogenous putrescine reversed the sensitization to heat shock induced by DFMO. This effect is quite specific for putrescine (1.4-diaminobutane) and is not replicated by other diamine homologues ranging from 1.3-diaminopropane to 1.8-diaminooctane. Polyamine-depleted cells express thermotolerance with kinetics similar to control cells although overall survival levels are lower. These results suggest that the mechanism of induction and expression of thermotolerance is independent of the role of acid-soluble polyamine pools in cellular responses to heat shock.     

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.     

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.     

Effects of difluoromethylornithine on proliferation,polyamine content and plating efficiency of cultured human carcinoma cells

Summary We investigated the effects of an irreversible inhibitor of ornithine decarboxylase, difluoromethylornithine (DFMO), on the proliferation, polyamine content, and plating efficiency of five human adenocarcinoma cell lines in vitro. DFMO inhibited the growth of all five lines when added at concentrations between 0.1 and 5.0 mM. The cell lines varied in their sensitivity to DFMO-induced cytostasis, HuTu-80 being most sensitive and HT-29 being most resistant. These differences appeared to be related to the ability of DFMO to prevent continued production of putrescine in the treated cells. Exogenous putrescine (5–50 M) reversed the growth inhibition for all five cell lines when added 48 h after DFMO treatment. The lowest concentration of exogenous putrescine (5 M) only restored intracellular polyamine content of DFMO-treated cells to control levels for the first 24 h after its addition. After that time, spermidine content declined once again to that observed for cells treated with DFMo alone. The higher concentrations of exogenous putrescine restored the content of all three polyamines to control levels for as much as 3 days after its addition, but did not cause a greater increase in cell growth rates that did 5 M putrescine. These data suggest that human adenocarcinoma cell proliferation is dependent on continued polyamine biosynthesis, but that the basal content of intracellular polyamines is greatly in excess of the minimum level required to support cell growth. In the 1.0–5.0 mM concentration range, DFMO treatment for 48 h caused a slight, but statistically significant, reduction in plating efficiency for three of our cell lines, and had no significant effect on the two others.Abbreviations ANOVA analysis of variance - DAH diaminoheptane - DFMO -difluoromethylornithine - DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - ODC ornithine decarboxylase - Pu putrescine - SD standard deviation - Sd spermidine - sp spermine This investigation was supported by PHS grant no CA-32758 awarded by the National Cancer Institute, DHHS, and by a grant from the American Cancer Society, Illinois Division, Inc. (82-6). Additional support was obtained from a Biomedical Research Support grant to Northwestern University Medical School (RR-05370) from the USPHS, NIH and from the Earle M. Bane Biomedical Research Fund     

Microbial flora in the gastrointestinal tract abolishes cytostatic effects of alpha-difluoromethylornithine in vivo

Although treatment with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) leads to depletion of intracellular polyamines and to related growth inhibition in vitro, its cytostatic effects in vivo are disappointing. This may be due to abolition of DFMO-induced growth inhibition by polyamines released during normal body cell turnover, to dietary polyamines, or to putrescine synthesized by the microbial flora in the GI tract. We studied selectively (aerobic) and totally (aerobic + anaerobic) GI tract-decontaminated LI210-bearing mice fed with 3 types of diet differing in their polyamine and carbohydrate residue contents and treated with combinations of intraperitoneal DFMO and oral deuterium-labelled putrescine. Our data show that, irrespective of diet type, total decontamination markedly potentiates the moderate tumor growth inhibition that is caused by DFMO alone. During total decontamination, growth-inhibited L1210 cells accumulate in the G0/G1 phase of the cell cycle. Although orally administered deuterium-labelled putrescine gave rise to deuterium labelling of L1210 putrescine, spermidine and spermine, the polyamine levels in our diets played only a minor role.     

Differential effect of alpha-difluoromethylornithine on the in vivo uptake of 14C-labeled polyamines and methylglyoxal bis(guanylhydrazone) by a rat prostate-derived tumor

The uptake of exogenously administered radiolabeled polyamines by a rat prostate-derived tumor line, the Dunning R3327 MAT-Lu, and various normal tissues was studied. Pretreatment of tumor cells in vitro with alpha-difluoromethylornithine (DFMO), a polyamine synthesis inhibitor, resulted in a markedly enhanced uptake of both [14C]putrescine and [14 C]spermidine. The in vitro uptake of [14C]putrescine by these cells was effectively inhibited by unlabeled spermine, spermidine, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1,4-diaminopentane, and 1,4-diaminobutane, but less effectively by 1,4-diamino-2,3-butene and 1,4-diamino-2,3-butyne. The diamines, 1,3-diaminopropane and 1,2-diaminoethane, were ineffective in inhibiting [14C]putrescine uptake in vitro into the R3327 MAT-Lu cell line. When tumor-bearing animals were pretreated with DFMO or with DFMO and 5-alpha-dihydrotestosterone propionate, the tumor and prostate uptake of [14C]putrescine and [14C]-cadaverine was enhanced but not substantially increased in other tissues. In contrast to the in vitro results, spermidine and spermine were not enhanced substantially by DFMO pretreatment into any tissue, and their uptake into the tumor actually decreased. Ethylenediamine, which does not utilize the polyamine transport system, did not have its uptake increased into any tissue following DFMO pretreatment. The chemotherapeutic agent, methylglyoxal bis(guanylhydrazone), which utilizes the polyamine transport system for uptake into cells, exhibited uptake behavior different from that of the polyamines. Thus, methylglyoxal bis(guanylhydrazone) uptake into the tumor was not significantly increased or decreased by DFMO or by DFMO + 5-alpha-dihydrotestosterone propionate pretreatment, and only the ventral, but not the dorsal-lateral, lobe of the prostate showed increased uptake of methylglyoxal bis(guanylhydrazone) following DFMO + 5-alpha-dihydrotestosterone propionate pretreatment.     

Possible factors in the potentiation of 1-(2-chloroethy)-3-trans-4-methylcyclohexyl-1-nitrosourea cytotoxicity by α-difluoromethylornithine in 9L rat brain tumor cells

Depletion of intracellular levels of polyamines in 9L rat brain tumor cells by α -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, significantly enhanced the cytotoxicity of 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl-1-nitrosourea (MeCCNU) in vitro as measured by a colony-forming efficiency assay. Administered as a single agent, DFMO was not cytotoxic to 9L cells. Treatment for 48 hr with 10, 1, 0.5 or 0.1 mM DFMO produced similar levels of polyamine depletion and similar potentiation of MeCCNU cytotoxicity. Restoration of intracellular polyamine levels by the addition of exogenous putrescine (1 mM) to treated cells prevented the potentiation of MeCCNU, which indicates that this phenomenon might be the result of polyamine depletion. DNA adduct formation in polyamine-depleted and control cell was studied with [14C]-MeCCNU; no difference in monoadduct formation was found between polyamine-depleted and control cells. Experiments to determine whether polyamine depletion has an effect on enzymes involved in the repair of alkylated bases showed that the activity of O⁶-methylguanine-DNA demethylase, 7-methylguanine-DNA glycosylase and 3-methyladenine-DNA glycosylace were unaffected by 48 hr of treatment with 10 mM DFMO. DFMO treatment causes a substantial increase in the intracellular content of decarboxylated S-adenosyl-l-methionine, which was reversed by addition of putrescine. The possibility that the elevation of decarboxylated S-adenosyl-l-methionine rather than the depletion of polyamines is responsible for the effects of DFMO is discussed.     

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.