Antitumor effect of a new multienzyme inhibitor of polyamine synthetic pathway, methylglyoxal-bis(cyclopentylamidinohydrazone), against human and mouse leukemia cells

Methylglyoxal-bis(cyclopentylamidinohydrazone) (MGBCP) has been synthesized as a multienzyme inhibitor for the polyamine-synthesizing pathway. This drug inhibited S-adenosylmethionine decarboxylase (EC 4.1.1.50), spermine synthase and spermidine synthase activities, competitively with S-adenosylmethionine, spermidine, and putrescine, respectively. MGBCP inhibited the growth of human leukemia Molt 4B and K 562 cells at 10 to 100 microM concentrations. Spermidine and spermine levels were markedly depressed in these MGBCP-treated leukemic cells, and the synthesis of protein, but not of DNA or RNA, was significantly diminished. In in vivo experiments, MGBCP depleted spermidine and spermine in the P388 leukemic ascites cells, and prolonged the survival time of mice bearing P388 leukemia. The S-adenosylmethionine decarboxylase-stabilizing effect of MGBCP in mouse liver, Molt 4B and K 562 cells was much less than that of the parent inhibitor methylglyoxal-bis(guanylhydrazone). Induction of ornithine decarboxylase activity by MGBCP in the cultured leukemic cells was also much less than that by methylglyoxal-bis(guanylhydrazone).     

Difluoro-phenyl-ethyl(4-aminopropylamidinohydrazone): an irreversible inhibitor for both ornithine and S-adenosylmethionine decarboxylases and its effect on human lymphoid leukemia Molt 4B cells

Difluoro-phenyl-ethyl(4-aminopropylamidinohydrazone) (DFPA) was synthesized as an irreversible inhibitor of ornithine and S-adenosylmethionine decarboxylases and analyzed for antiproliferative effects on leukemia cells. The compound irreversibly inhibited ornithine and S-adenosylmethione decarboxylases. This inhibition could not be reversed by dialysis or chromatography on Sephadex G-50. When human lymphoid leukemia Molt 4B cells were cultured in the presence of the inhibitor, the intracellular levels of polyamines (putrescine, spermidine and spermine) and the rate of cell proliferation were markedly depressed. In these polyamine-depleted and growth-retarded cells, the synthesis of protein, but not of DNA or RNA, was observed to be significantly inhibited.     

Effects of difluoromethylornithine and dicyclohexylammonium sulfate on the transformed state of AKR-MCA cells

The effect of two inhibitors of polyamine biosynthesis, difluoromethylornithine and dicyclohexylammonium sulfate, on the transformed fibroblastic cell line AKR-MCA and its parental counterpart AKR-2B was investigated. Treatment of monolayer AKR-MCA cells with either agent results in morphological changes akin to AKR-2B; the cells appear to be flattened with a polygonal shape. The ability of the inhibitors to alter the phenotype is lost when the cells are cocultured with polyamines. More specifically, putrescine and spermidine abrogate the effects of difluoromethylornithine while only spermidine is effective in reversing the dicyclohexylammonium sulfate induced phenomenon. Further evidence that these enzyme inhibitors are reversing the transformed state of AKR-MCA is obtained from soft agarose experiments. AKR-MCA cells will generate colonies only in the absence of either difluoromethylornithine or dicyclohexylammonium sulfate. Polyamine levels were determined in parental AKR-2B and AKR-MCA cells. The levels of putrescine and spermine were similar in both cell types. In contrast, significantly more (P less than or equal to 0.01) spermidine was expressed by the malignant line [7.3 +/- 0.8 (SD) nmol/10(6) cells] when compared with the untransformed AKR-2B (5.4 +/- 0.8 nmol/10(6)) cells. Intracellular putrescine and spermidine were sensitive to difluoromethylornithine, dicyclohexylammonium sulfate, and dimethylformamide, a planar, polar solvent which has been reported to "normalize" the transformed phenotype. AKR-MCA treated with difluoromethylornithine or dimethylformamide manifested time dependent reductions in both polyamines which preceded morphological changes. Dicyclohexylammonium sulfate similarly caused a 70% reduction in spermidine, but in contrast to the other agents there was a marked accumulation of putrescine. These data concur with the established molecular actions of the two enzyme inhibitors as blockers of ornithine decarboxylase (difluoromethylornithine) and spermidine synthase (dicyclohexylammonium sulfate). The normalizing capacity of dimethylformamide was not compromised by cotreatment with putrescine or spermidine. Both difluoromethylornithine and dicyclohexylammonium sulfate inhibited the growth of monolayer AKR-2B and AKR-MCA. In view of the well documented cytostatic effects of polyamine inhibitors, it is suggested that a decrease in growth by these agents triggers a more normal phenotype in AKR-MCA cells.     

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.     

Properties of L1210 cells resistant to alpha-difluoromethylornithine

L1210 cells were selected for resistance to the ornithine decarboxylase (ODC) inhibitor, alpha-difluoromethylornithine. When grown in the absence of the inhibitor, these cells possessed very high ornithine decarboxylase levels. These represented about 1 part in 300 of the soluble protein, which is several hundred times greater than the maximal value found in the original L1210 cells. The resistant cells contained at least 100-fold higher levels of ODC mRNA but the half-life of ODC (about 45 min) was not altered significantly. The resistant cells had much higher putrescine and cadaverine levels than control cells, but there was no significant difference in cellular spermidine or spermine content or in production of 5'-methylthioadenosine, which is a measure of polyamine synthesis. Addition of putrescine to the control or resistant cells had no effect on their content of spermidine and spermine but addition of decarboxylated S-adenosylmethionine increased the content of spermidine and spermine. These results indicate that ornithine decarboxylase is not the rate-limiting step in polyamine synthesis in these L1210 cells. The growth of the alpha-difluoromethylornithine-resistant L1210 cells was inhibited when their ability to synthesize spermidine and spermine was blocked by the addition of the S-adenosylmethionine decarboxylase inhibitor, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine. Treatment with this compound produced a reduction of more than 85% in the production of 5'-methylthioadenosine and led to a large increase in the content of putrescine and a substantial decline in the content of spermidine and spermine. These results indicate the potential value of S-adenosylmethionine decarboxylase inhibitors as therapeutic agents in conditions where ODC inhibitors are ineffective.     

Pharmacological properties of the ornithine decarboxylase inhibitor 3-aminooxy-1-propanamine and several structural analogues

Analogues of 3-aminooxy-1-propanamine proved to be highly potent and selective inhibitors of ornithine decarboxylase (ODC). The compounds competed with ornithine for the substrate binding site of ODC, but resulted in progressive and apparently irreversible inactivation of the enzyme. Diamine oxidase was inhibited by these compounds to a lesser extent than ODC; the compounds were not metabolized by this enzyme. Several derivatives were growth-inhibitory for human T24 cells and for other mammalian cells, the most active compound being 3-aminooxy-2-fluoro-1-propanamine (AFPA). Growth-arrested cells were largely depleted of putrescine and spermidine. Cellular growth arrest could be antagonized by supplementation with spermidine. Selection for resistance against AFPA led to cells with amplified ODC genes and overexpression of the message. Some of the derivatives were tumoristatic at well-tolerated doses in mice bearing solid T24 tumours. The antiproliferative activity of these compounds appears to be mediated by polyamine depletion.     

15-Deoxyspergualin, an antiproliferative agent for human and mouse leukemia cells shows inhibitory effects on the synthetic pathway of polyamines

The mechanism of the antitumor action of 15-deoxyspergualin (DSG) was investigated. DSG inhibited spermidine synthase noncompetitively with putrescine, spermine synthase competitively with spermidine and polyamine oxidase in vitro. Induction of ornithine decarboxylase (ODC) activity observed after subculture of human leukemia cells was blocked by the addition of DSG to the culture medium. In DSG-treated leukemia cells, putrescine, spermidine and spermine levels were markedly depressed. The synthesis of protein was also greatly diminished in these polyamine-depleted leukemic cells, whereas the depressions of DNA and RNA syntheses were minimum. In in vivo experiments, DSG depressed polyamine levels in P388 leukemic ascites cells, and prolonged the survival times of mice bearing the leukemia cells. These results suggest that inhibition of polyamine and protein biosyntheses by DSG is substantially responsible for its antitumor action on the tumor cells.     

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.     

Putrescine and spermidine uptake is regulated by proliferation and dexamethasone treatment in AR4-2J cells

Polyamines are essential for cell growth and differentiation. Their specific uptake contributes to the regulation of intracellular polyamine levels. In this study, we describe the modulation of this transport mechanism in a rat tumoral pancreatic acinar cell line (AR4-2J) and analyze the transport system characteristics of the normal rat pancreatic acini. Normal acini had a common carrier for spermidine and spermine, like AR4-2J cells, but not a specific putrescine carrier. Intracellular polyamine deprivation enhanced putrescine and spermidine uptake of AR4-2J cells with no modification of polyamine carrier affinity. Uptake was modulated during growth and decreased for both polymaines at confluence. AR4-2J cell differentiation with dexamethasone prevented cell proliferation and diminished uptake of both putrescine and spermidine without affecting their respective carrier affinities. Our data show, first, that the polyamine transport system could be modulated by polyamine metabolism with no change in its affinity characteristics. Second, in rat pancreatic acinar cells, neoplastic transformation was partly characterized by induction of a high-affinity putrescine carrier. This phenotype was not reversed by dexamethasone-induced cell differentiation.     

Polyamines and hormonal regulation of N-nitrosomethylurea-induced rat mammary tumor growth in vivo

We have observed that polyamines play an essential role in the expression of hormonal action on the growth of the N-nitrosomethylurea-induced mammary tumor cultured in soft agar. Since polyamine levels cannot be measured in this system, we could not determine whether tumor polyamine pools are under endocrine control. To test this hypothesis, the following experiments were conducted in N-nitrosomethylurea tumor-bearing rats in vivo. Both tamoxifen administration (200 micrograms/day) and ovariectomy produced dramatic reductions in tumor growth but neither treatment significantly altered tumor polyamine levels after either 7 or 21 days of treatment. Some decrease in tumor level of putrescine, spermidine, and spermine was observed 21 days after ovariectomy but it was not statistically significant. Exogenous administration of putrescine (300 mg/kg/day) reversed the antitumor effect of tamoxifen but did not prevent tumor regression induced by ovariectomy. This effect of putrescine was, however, variable in magnitude from experiment to experiment. To test whether the reversal of tamoxifen effect by putrescine might simply be due to interference with tamoxifen uptake by the tumor cells, we measured estrogen and progesterone receptors in the tumors of rats chronically treated with tamoxifen and tamoxifen plus putrescine. In both groups estrogen receptors are virtually undetectable, thus suggesting that putrescine had not inhibited tamoxifen entry into the cells and binding to estrogen receptors. Progesterone receptor levels were similarly high in both groups and not significantly different from control. These results indicate that at least under these experimental conditions N-nitrosomethylurea mammary tumor polyamine pools are not under ovarian hormone control. The mechanism by which putrescine reverses tamoxifen's effect remains unclear.     

Cyanidin 3-O-beta-D-glucoside isolated from skin of black Glycine max and other anthocyanins isolated from skin of red grape induce apoptosis in human lymphoid leukemia Molt 4B cells

Cyanidin 3-O-beta-D-glucoside (CG) was purified from black bean seed coat and other anthocyanins were prepared from red grape skin. These anthocyanins were identified by Mass, and 1H- and 13C-NMR. The effects of four anthocyanins on cell viability in human leukemia Molt 4B cells were investigated. The anthocyanins displayed strong growth inhibitory effects against human leukemia Molt 4B cells. Morphological changes showing apoptotic bodies were observed in the Molt 4B cells treated with these anthocyanins. The fragmentations by these anthocyanins of DNA to oligonucleosomal-sized fragments, that is a characteristic of apoptosis, were observed to be concentration-dependent. N-acetyl-L-cysteine, an antioxidant, suppressed the fragmentation of DNA by these anthocyanins. These findings suggest that growth inhibition of Molt 4B cells by these anthocyanins result from the induction of apoptosis by these compounds and that active oxygen is involved in the induction of apoptosis in the Molt 4B cells.     

Effects of inhibitors of polyamine biosynthesis on the growth and melanogenesis of murine melanoma cells

Both 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), and methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), strikingly stimulated melanotic expression of murine Cloudman S91 melanoma cells. The stimulation of tyrosinase (EC 1.10.3.1) activity and melanin formation by DFMO was closely associated with intracellular depletion of putrescine and spermidine developed in response to the drug. However, little or no evidence was obtained indicating that enhanced melanogenesis in response to MGBG was mediated through an inhibition of polyamine biosynthesis. Indirect inhibitors of ornithine decarboxylase, such as 1,3-diaminopropane and 1,3-diaminopropan-2-ol, but not putrescine, likewise inhibited the growth of the melanoma cells and stimulated their melanin production. The stimulation of melanogenesis by polyamine antimetabolites was not mediated by cyclic adenosine 3':5'-monophosphate, in contrast to the effect elicited by alpha-melanotropin. It is also unlikely that MGBG or the diamines acted as lysosomotropic agents capable of stimulating tyrosinase activity in situ, since the enzyme activity was stimulated by the drugs irrespective of whether assayed in cultured cells or using cell-free homogenates. None of the agents stimulated tyrosinase activity in vitro. The effect of DFMO and MGBG on melanoma cell proliferation was reversible, but the restoration of normal growth and melanin formation, especially in cells exposed to DFMO, was remarkably slow. The present results represent a further experimental model, in which the inhibition of polyamine accumulation is accompanied by signs of terminal differentiation.     

Specific induction of apoptosis by 1,8-cineole in two human leukemia cell lines,but not a in human stomach cancer cell line

We have investigated the effects of 1,8-cineole [the main component of essential oil prepared from bay-leaves Laurus nobilis L.)] on DNA of human leukemia cell lines, Molt 4B, HL-60 and stomach cancer KATO III cells. Specific induction of apoptosis by 1,8-cineole was observed in human leukemia Molt 4B and HL-60 cells, but not in human stomach cancer KATO III cells. Morphological changes showing apoptotic bodies were observed in the human leukemia HL-60 cells treated with 1,8-cineole. The fragmentations of DNA by cineole to oligonucleosomal-sized fragments that is a characteristic of apoptosis were concentration- and time-dependent in Molt 4B and HL-60 cells, but not in KATO III cells. The present study shows that the suppression growth by 1,8-cineole in the leukemia cell lines results from the induction of apoptosis by this compound.     

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.     

Altered polyamine profiles in prostatic hyperplasia and in kidney tumors.

Polyamine concentrations were evaluated in normal human prostatic tissue as well as hyperplastic prostate. Normal tissues had high concentrations of putrescine and spermine with intermediate spermidine concentrations, whereas there was a dramatic increase in the spermine concentration in patients with hypertrophy of the prostate. Although not highly significant, spermidine concentrations were elevated slightly in benign hyperplasia, whereas the putrescine content was decreased compared to normal tissue. Polyamine concentrations were measured also in human kidney tumors and corresponding healthy kidney tissue. The concentration of spermidine in renal carcinomas was significantly elevated when compared to histologically normal areas of the same kidney. The spermine concentration of the tumor was generally lower but not highly significant (p less than 0.01). These data suggest that polyamines are accumulated above normal levels in pathological conditions such as benign hyperplasia of the prostate and renal carcinoma. In both cases, spermidine turnover rate may be influenced by carcinogenesis.     

Physiological effects in bovine lymphocytes of inhibiting polyamine synthesis with ethylglyoxal bis(guanylhydrazone)

Previous results have suggested that ethylglyoxal bis(guanylhydrazone) is a more specific inhibitor of polyamine biosynthesis than the widely used methylglyoxal bis(guanylhydrazone). The physiological effects on mitogenically activated lymphocytes of polyamine depletion with ethylglyoxal bis(guanylhydrazone) were examined. In the presence of ethylglyoxal bis(guanylhydrazone) and the ornithine decarboxylase inhibitor alpha-difluoromethylornithine, the cellular contents of putrescine, spermidine, and spermine were decreased by 75 to 90, 65 to 80, and 40 to 60%, respectively, compared with control cultures. Inhibition of DNA synthesis in these polyamine-deficient cells was always greater than that of protein synthesis. Upon addition of spermidine to the deficient cells, the cellular spermidine content was restored within 4 hr, but the complete recovery of macromolecular synthesis took 10 to 20 hr. Thymidine kinase and DNA polymerase alpha activities in polyamine-deficient cells were lower than those in normal cells, whereas RNA polymerase II and leucyl transfer RNA synthase activities were nearly equal to those in normal cells. These results and studies with 2-dimensional gel electrophoresis raise the possibility that polyamines may regulate the synthesis of specific proteins. Decreased synthesis of replication proteins in polyamine-deficient cells may be one reason for the reduced synthesis of DNA.     

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.     

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     

Pre-treatment of a human T-lymphoblastoid cell line with L-asparaginase reduces etoposide-induced DNA strand breakage and cytotoxicity.

The effect of L-asparaginase (L-asp) pre-treatment on etoposide-induced DNA strand breakage and cytotoxicity was investigated. In a T-lymphoblastoid cell line, Molt 4, etoposide-induced DNA strand breaks, DNA-protein cross-links and cytotoxicity were reduced by pre-treatment with L-asp for 15 hr, but it did not cause these changes in a promyelocytic-leukemia cell line, HL-60, which is less sensitive than Molt 4 to L-asp. However, pre-treatment of Molt 4 cells with L-asp did not significantly alter the accumulation of [3H]-etoposide. Cell-cycle analyses showed an increase in G1-phase cells, a significant decrease in both S-phase cells and G2/M-phase cells pre-treated with L-asp in Molt 4 cells, but L-asp exposure did not result in any significant changes in HL-60 cells. On the other hand, L-asp pre-treatment did not affect topoisomerase-I (Topo-I) inhibitor, camptothecin (CPT)-induced DNA strand breaks or toxicity in Molt 4 cells. Our data imply that a decrease in S- and G2/M-phase cells following L-asp treatment may explain the reduction of etoposide-induced DNA lesions and cytotoxicity in Molt 4 cells, since topoisomerase-II (Topo-II) content or activity is a function of cellular proliferation status.     

Effects of a bis(benzyl)spermine analog on MCF-7 breast cancer cells in culture and nude mice xenografts

We studied the effects of a polyamine analog, N,N'-bis{3-[(phenylmethyl)amino]propyl}-1,7-diaminoheptane (MDL 27695) on MCF-7 cells, as part of an attempt to develop new drugs for breast cancer treatment. Using [H-3]-thymidine incorporation assay and long-term growth curves, we found that MDL 27695 inhibited the growth of MCF-7 cells in a dose-dependent manner in the low mu M range. G1 synchronized cells progressing in cell cycle showed delayed and inefficient entry into S phase in the presence of 4 mu M MDL 27695. Consistent with a G1 arrest, MDL 27695 significantly reduced estradiol-mediated increase in the expression of cyclin D1. HPLC analysis showed that treatment of MCF-7 cells with MDL 27695 reduced the accumulation of natural polyamines, putrescine, spermidine, and spermine, by 43, 38, and 45%, respectively, at 8 h after the initiation of cell cycle. This decrease in polyamine levels was not associated with a decrease in the activity of polyamine biosynthetic (ornithine decarboxylase, ODC; s-adenosylmethionine decarboxylase, SAMDC) or catabolizing (spermidine/spermine acetyltransferase, SSAT) enzymes. However, there was a 40% decrease in the uptake of putrescine and spermidine, in cells treated with MDL 27695. Our studies also showed that MDL 27695, at a dose of 20 mg/kg, caused a significant inhibition of tumor growth in nude mice harboring MCF-7 cell derived tumors, without overt symptoms of toxicity. These data indicate that the polyamine analog MDL 27695 is an efficient inhibitor of MCF-7 breast cancer cell growth in vitro and in vivo. Our results suggest that polyamines are critical factors in cell cycle regulation of breast cancer cells and potential targets for therapy.