Maternal behavior as a regulator of polyamine biosynthesis in brain and heart of the developing rat pup.

Rat pups removed from the mother and placed in a warm incubator for 1 hour or more show a 50 percent reduction in ornithine decarboxylase activity in the brain and heart. The decline is not caused by lack of nutrition. Instead, these studies suggest that active maternal behavior is necessary to maintain normal polyamine metabolism in brain and heart of the pup during development.

Ornithine decarboxylase: essential in proliferation but not differentiation of human promyelocytic leukemia cells.

The ornithine decarboxylase inhibitor DL-alpha-difluoromethyl ornithine inhibited a proliferation-associated increase in ornithine decarboxylase activity in cultured human promyelocytic leukemia cells, resulting in a marked suppression of cell proliferation and subsequent cell loss. It also inhibited increases in ornithine decarboxylase activity associated with the phorbol ester-induced conversion of promyelocytic HL-60 cells to monocyte-like cells and the retinoic acid-induced conversion to granulocyte-like cells. However, the inhibition of ornithine decarboxylase activity did not prevent cellular differentiation. These results suggest that polyamine biosynthesis has a specific role in cell proliferation rather than in inducing differentiation that is not accompanied by proliferation. The data also demonstrate that cessation of proliferation in HL-60 cells is not necessarily associated with differentiation.

An inhibitor of polyamine synthesis arrests cells at an earlier stage of G1 than does calcium deprivation.

Methylglyoxal bis(guanylhydrazone) completely inhibits the induction of thymidine kinase after serum stimulation of quiescent fibroblasts only if added within 3 h after serum, whereas calcium deprivation blocks this induction up to 12 h after serum stimulation. Experiments in which one of these blocks was imposed as the other was released confirmed that cells blocked by methylglyoxal bis(guanylhydrazone) are arrested at an earlier stage in G1 than cells blocked by calcium deprivation.

Persistently decreased hepatic levels of 5'-deoxy-5'-methylthioadenosine during regeneration of and chemical carcinogenesis in rat liver.

The hepatic levels of 5'-deoxy-5'-methylthioadenosine (MTA) were measured in the livers of adult male Sprague-Dawley rats a) killed at various times during the liver regeneration process, b) killed at times after partial hepatectomy when the liver mass had already been completely restored (hereafter called post-regeneration livers), or c) continuously fed 3'-methyl-4-dimethyl-aminoazobenzene (CAS: 55-80-1) up to the full development of hepatoma and killed at regular intervals during hepatocarcinogenesis. Hepatic MTA levels were always significantly decreased, although to different degrees in both in vivo models of hepatic growth and at all times during the investigation. Astonishingly, the MTA levels were also significantly decreased in the post-regeneration livers, in which there was also a significant increase in the activity of adenosylmethionine decarboxylase (S-adenosyl-L-methionine decarboxylase; EC 4.1.1.50) with normal levels of activity of ornithine decarboxylase (EC 4.1.1.17). These results demonstrate that a) the MTA content is always decreased in rat liver whenever this organ is involved in a proliferative process (whether controlled or uncontrolled); b) the decrease in hepatic MTA content is a biochemical feature necessary for, but by no means by itself sufficient for, hepatocyte proliferation to occur, since this decrease remains long after complete restoration of the liver mass; and c) the return of the hepatocytes to the normal biochemical program after restoration of the liver mass is not complete, even though these cells become quiescent, because there are still some biochemical abnormalities in the post-regeneration livers.

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.

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.

A selective effect of methylglyoxal-bis(guanylhydrazone) on the synthesis of mitochondrial DNA of cultured L1210 leukemia cells.

Methylglyoxal-bis(guanylhydrazone) (MGBG) is a polycationic drug which is useful in the chemotherapy of lymphoid and myeloid proliferative disorders. The drug has recently been shown to produce selective ultrastructural damage to the mitochondria of proliferating cell populations. It is important to understand the molecular basis for this action, since it may be related to the known ability of MGBG to block polyamine biosynthesis. Accordingly, the effect of MGBG treatment on the incorporation of [3H]thymidine into both mitochondrial and nuclear DNA has been examined. Exponentially growing L1210 leukemia cells were prelabeled with [14C]thymidine, treated with MGBG for 1.5 to 16 hr, and then pulse labeled with [3H]-thymidine. Incorporation of [3H]thymidine into mitochondrial DNA was selectively inhibited at 5 hr with concentrations of 1 to 10 microM MGBG. Nuclear DNA, however, was not similarly affected until 8 to 11 hr of drug treatment. Dye-CsCl gradients of mitochondrial DNA indicated that the inhibition of synthesis occurred in replicative forms of circular DNA. Uptake studies excluded the possibility of drug interference with cellular uptake of thymidine. Ultrastructural studies revealed a very close correlation between the dose-response curve for mitochondrial damage and that for MGBG inhibition of mitochondrial DNA synthesis. This correlation suggests a direct cause-and-effect relationship between inhibition of mitochondrial DNA synthesis and ultrastructural damage, but the possibility of both phenomena being related to another action by the drug, such as inhibition of polyamine biosynthesis, or a drug effect on mitochondrial function, must also be considered.

Biological properties of N4- and N1,N8-spermidine derivatives in cultured L1210 leukemia cells.

Eleven novel spermidine (SPD) derivatives were synthesized as potential anticancer agents and evaluated for their ability to compete with [3H]SPD for cellular uptake, to inhibit cell growth, to affect polyamine biosynthesis, to suppress enzyme activity, and to substitute for SPD in supporting growth of cultured L1210 leukemia cells. The compounds included a series of N4-SPD derivatives (N4-methyl-SPD, N4-ethyl-SPD, N4-acetyl-SPD, N4-hexyl-SPD, N4-hexanoyl-SPD, N4-benzyl-SPD, and N4-benzoyl-SPD) and a series of N1,N8-SPD derivatives [N1,N8-bis(ethyl)-SPD, N1,N8-bis(acetyl)-SPD, N1,N8-bis(propyl)-SPD, and N1,N8-bis(propionyl)-SPD]. Uptake studies revealed N4-alkyl derivatives to be the most effective competitive inhibitors of [3H]SPD uptake (Ki, 26 to 43 microM) followed by N1,N8-alkyl derivatives (Ki, 71 to 115 microM), then N4-acyl derivatives (Ki, 115 to greater than 500 microM), and N1,N8-acyl derivatives (Ki, greater than 500 microM). The data indicate the relative importance of the terminal amines and of charge as determinants of cellular uptake. Of the 11 derivatives, only N4-hexyl-SPD, N1,N8-bis(ethyl)-SPD, and N1,N8-bis(propyl)-SPD demonstrated antiproliferative activity at 0.1 mM with 50%-inhibitory concentration values at 48 h of 30, 40, and 50 microM, respectively. In the case of the N1,N8-SPD derivatives, recovery from growth inhibition was enhanced considerably by exogenous SPD, suggesting involvement of polyamine depletion. At 10 to 30 microM, both N1,N8-bis(ethyl)-SPD and N1,N8-bis(propyl)-SPD (but not N4-hexyl-SPD) inhibited polyamine biosynthesis as indicated by significant reductions in polyamine pools and in biosynthetic enzyme activities. The more effective of the two, N1,N8-bis(ethyl)-SPD, depleted intracellular putrescine and SPD and reduced spermine by approximately 50% at 96 h and decreased ornithine and S-adenosylmethionine decarboxylase activities by 98 and 62%, respectively. Since neither derivative (at 5 mM) directly inhibited these enzymes from untreated cell extracts by significantly more than SPD itself, it is suspected that they act by regulating enzyme levels. As a measure of regulatory potential of the derivatives, ornithine decarboxylase was assayed in cells treated for 24 h and compared to the effects of 10 microM SPD which reduced the enzyme activity by 80%. None of the N4-SPD derivatives affected ornithine decarboxylase activity, while N1,N8-bis(ethyl)- and (propyl)-SPD were nearly as effective as SPD. Apparently, the central amine of the molecule is critical for regulatory function.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Kinetics of cell proliferation and polyamine synthesis during Ehrlich ascites tumor growth.

The kinetics of cell proliferation and polyamine synthesis during Ehrlich ascites tumor growth were studied. The steady deceleration of the specific growth rate with increasing tumor mass that was observed was attributable to a prolongation of the cell cycle, particularly of the S and G2 phases. The cell cycle time (Tc) was 43.3 hr (TG1 equals 10.8, TS equals 26.8, and TG2 equals 5.7 hr) on the seventh day of growth and 76.0 hr (TG1 equals 14.0, TS equals 52.0, and TG2 equals 10.0 hr) on the tenth day of growth. The growth fraction showed a decrease from 0.77 to 0.60 during the 7- to 10-day tumor growth interval. The cell death rate remained low and essentially unchanged during this period. A high correlation was found between polyamine synthesis (ornithine decarboxylase activity) and the specific growth rate; the correlation coefficient was 0.985. There was also a high positive correlation between the cellular polyamine (spermidine and spermine) and nucleic acid content (spermidine: DNA equals 0.916, spermine: DNA equals 0.947, spermidine:RNA equals 0.907, and spermine: RNA equals 0.881). These observations suggest that there may be a functional coupling between polyamines and nucleic acids, and they support the hypothesis that polyamines play an important role in DNA replication and cell division.

Polyamines and biosynthetic enzymes in the rat intestinal mucosa and the influence of methylglyoxal-bis(guanylhydrazone).

The use of methylglyoxal-bis(guanylhydrazone) (MGBG) in the clinical treatment of myeloid and lymphoid disorders has been limited by severe host toxicity to renewing tissues, particularly the intestinal mucosa. Since the drug is a potent inhibitor of spermidine biosynthesis, the distributions of ornithine and S-adenosylmethionine decarboxylases and polyamine pools have been characterized in the rat intestinal mucosa in an attempt to discern the basis for MGBG toxicity. A method of epithelial cell isolation in which fractions of cells are sequentially collected in a villus tip-to-crypt gradient was used. Ornithine decarboxylase activity was highest in the villus tip region and unexpectedly lowest in the crypts, while S-adenosylmethionine decarboxylase activity showed the opposite pattern. Intracellular polyamine pools were uniform along the gradient corresponding to the villus length and increased appreciably in the crypt region. The relative concentrations of the individual polyamines were highest in the crypts, with spermidine and spermine being nearly equivalent in all regions. Twenty-four hr after a single i.p. injection of MGBG (50 mg/kg), S-adenosylmethionine decarboxylase activity increased markedly, especially in the crypt region (approximately 50-fold), while ornithine decarboxylase activity also increased but to a lesser extent. Putrescine pools were most affected by MGBG and were elevated 5- to 6-fold, especially in the crypt region. The results are consistent with an alteration of polyamine biosynthesis by MGBG being involved in the antiproliferative toxicity of the drug.

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.

Independent regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase in methylthioadenosine phosphorylase-deficient malignant murine lymphoblasts.

The control of polyamine synthesis in neoplastic cells is complex and incompletely understood. Using murine lymphoma cells deficient in methylthioadenosine (MTA) phosphorylase, we have analyzed the role of MTA in the regulation of ornithine decarboxylase and S-adenosylmethionine (SAM) decarboxylase, the two rate-limiting enzymes in the polyamine-biosynthetic pathway. The addition of MTA to the enzyme-deficient lymphoblasts induced within 1 to 3 h an increase in the activities of both decarboxylases and an accompanying rise in putrescine and decarboxylated SAM levels. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine blocked the MTA-triggered accumulation of putrescine but not decarboxylated SAM. In a reciprocal manner, the SAM decarboxylase inhibitor methylglyoxal bis(guanylhydrazone) prevented the accretion of decarboxylated SAM but not putrescine. The MTA-induced rise in SAM decarboxylase and ornithine decarboxylase activities preceded by several hours changes in spermidine or spermine pools. However, MTA decreased the flux through the polyamine-synthetic pathway, as estimated by the incorporation of radioactive ornithine into spermine. Similar changes in polyamine metabolism were observed in a secondary mutant deficient in MTA phosphorylase, but resistant to MTA toxicity. These results suggest that the velocity of polyamine synthesis, or the concentration of MTA itself, may regulate ornithine decarboxylase and SAM decarboxylase activities through separate, growth-independent mechanisms.

Effect of difluoromethylornithine, an inhibitor of polyamine biosynthesis, on the topoisomerase II-mediated DNA scission produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide in L1210 murine leukemia cells.

Treatment of mouse leukemia L1210 cells with the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) increased the magnitude of the DNA scission produced by the DNA intercalator 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). This enhanced DNA scission was protein concealed and protein associated, as was the m-AMSA-induced scission in cells unexposed to DFMO. The effect of DFMO required more than 6 hr to develop and was greater at 48 hr than at 24 hr of exposure to DFMO. Exogenously added putrescine partially reversed the effects of DFMO, while exerting no effect on m-AMSA-induced DNA scission in cells unexposed to DFMO. The cellular uptake of [14C]-m-AMSA was the same in DFMO-treated or untreated cells. The DNA scission and DNA-protein cross-linking produced by m-AMSA appear to represent the stabilization of an intermediate in the normal cycle of topoisomerase II function (Nelson, E.M., Tewey, K.M., and Liu, L.F., Proc. Natl. Acad. Sci. USA, 81: 1361-1365, 1984). Since polyamine depletion appears to affect the magnitude of this effect in cells, and since polyamines can alter topoisomerase II function in vitro, polyamines may be involved in topoisomerase function in vivo either directly or through secondary effects, such as alterations of the conformation of chromatin, the intracellular site at which topoisomerase acts.

Relative abilities of bis(ethyl) derivatives of putrescine, spermidine, and spermine to regulate polyamine biosynthesis and inhibit L1210 leukemia cell growth.

It has been shown previously (Porter et al., Cancer Res., 45: 2050-2057, 1985) that the N1,N8-bis(ethyl) derivative of spermidine has significant antiproliferative activity which appears to derive from its regulatory effects on the polyamine biosynthetic pathway, particularly on ornithine decarboxylase activity. In the present study, N1,N4-bis(ethyl)putrescine (BEP) and N1,N12-bis(ethyl)spermine (BESm) were compared with N1,N8-bis(ethyl)spermidine (BES) in their ability to inhibit cell growth and regulate polyamine biosynthesis. With cultured L1210 murine leukemia cells, the IC50 values at 48 h were approximately 2 mM for BEP, 30 microM for BES, and 1 microM for BESm making the latter the most effective polyamine inhibitor or analogue thus far identified. At concentrations which approximated IC50 values and yielded similar intracellular concentrations at 48 h (1500-2000 pmol/10(6) cells), the effects of the analogues on polyamine biosynthesis generally correlated with their antiproliferative activity. BEP, at 1 mM, exerted relatively minor effects on polyamine biosynthesis. By contrast, 100 microM BES totally eliminated ornithine decarboxylase activity, depleted putrescine and spermidine pools, and decreased spermine pools by 40%. AdoMet decarboxylase activity was lowered slightly. The most impressive effects were obtained with 10 microM BESm which decreased ornithine and AdoMet decarboxylase activities by 99 and 84%, respectively; depleted putrescine and spermidine pools; and decreased spermine pools by 73%. None of the analogues, at 1 or 3 mM, had significant direct inhibitory effects on the decarboxylase activities from untreated cells with the exception of BESm which inhibited ornithine but not AdoMet decarboxylase activity. Thus, the effects of the analogues on these enzymes in treated cells are presumed to be mainly mediated by regulatory mechanisms. In this regard, BESm was superior to BES since both ornithine and AdoMet decarboxylase activities were suppressed. Given its unique activities, BESm would seem to have potential as both an antiproliferative agent and also as an experimental probe for studying regulation of the polyamine pathway, particularly AdoMet decarboxylase.

Polyamine biosynthesis enzymes in the induction and inhibition of differentiation in Friend erythroleukemia cells.

O-Ornithine decarboxylase (ODC) activity in cultures of Friend erythroleukemia cells induced to differentiate with various compounds was examined. Based on ability to stimulate ODC activity, the inducers tested could be divided into two categories. Inducers of the first class, among which were dimethyl sulfoxide and hexamethylene bisacetamide, stimulated ODC activity and were inhibited by dexamethasone and the phorbol diester, 12-O-tetradecanoylphorbol-13-acetate. Specific inhibitors of polyamine biosynthesis, such as methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, and the ornithine analogs, alpha-methylornithine and alpha-hydrazinoornithine, also inhibited the induction of Friend erythroleukemia cell differentiation. The inhibition of induced differentiation by this class of compounds could be abrogated by spermine, spermidine, or putrescine. Inducers of the second class, among which were sodium butyrate, actinomycin D, and aminonucleoside of puromycin, had little or no stimulatory effect on ODC and were inhibited only by bromodeoxyuridine. The effect of bromodeoxyuridine, which inhibits inducers of both classes, was not abrogated by polyamines.

Anchorage dependency effects on difluoromethylornithine cytotoxicity in human lung carcinoma cells.

Difluoromethylornithine (DFMO), a specific, irreversible, enzyme-activated inhibitor of ornithine decarboxylase activity, the first and rate-limiting step in polyamine biosynthesis, has been shown to inhibit neoplastic cell proliferation in culture. In most cases, such inhibition is not accompanied by cell loss, with the exception of multiple cell lines of human small cell lung carcinoma (SCC), a human leukemia cell line (HL-60), and possibly the B16 melanoma cell line. The first two cell types grow as anchorage-independent suspension cultures, the HL-60 as single cells and the SCC as multicellular spheroid aggregates. Moreover, in the spectrum of human lung carcinoma cells in culture, the SCC cells respond in a cytotoxic manner to DFMO, whereas the non-small cell lung carcinoma (non-SCC) cells, which are anchorage dependent, show only growth inhibition, without actual cell loss. In the present study, we have investigated relationships between anchorage-dependent and -independent growth patterns of cells in culture and their response to DFMO treatment. Two non-SCC lung cancer cell lines, which normally grow as anchorage-dependent monolayers, show growth inhibition but no cell loss with the addition of DFMO. When these anchorage-dependent cells were forced to grow as multicellular aggregates, by coating the culture flask with Teflon, the cells developed an increased sensitivity to DFMO. They showed not only inhibition of cell proliferation but also cell death. Two SCC cell lines, which normally grow as anchorage-independent spheroids, developed adherence to the culture dishes coated with fibronectin. These cells, which show a cytotoxic response to DFMO during normal anchorage-independent growth, developed a decreased sensitivity to DFMO, showing only cell growth inhibition, but no cell death when treated during anchorage-dependent growth. Our data thus suggest that the state of anchorage dependence of lung cancer cells in culture is a critical factor in determining their response to polyamine depletion during treatment with DFMO.

Comparison of the biological effects of four irreversible inhibitors of ornithine decarboxylase in two murine lymphocytic leukemia cell lines.

The effects of the enzyme-activated irreversible inhibitors of ornithine decarboxylase, alpha-difluoromethylornithine, alpha-(fluoromethyl)dehydroornithine, alpha-(fluoromethyl)dehydroornithine methyl ester, and (2R,5R)-6-heptyne-2,5-diamine (RR-MAP), on cell growth and parameters related to polyamine biosynthesis were compared in L5178Y and L1210 cells under identical culture conditions. The two lines are murine lymphocytic leukemia cells which differ in their ability to metabolize 5'-methylthioadenosine, the by-product of polyamine biosynthesis: L5178Y cells contain a specific 5'-methylthioadenosine phosphorylase; L1210 cells do not. In L1210 cells, the 50% inhibitory concentrations (lC50S) of the various analogues were 3.0 mM for alpha-difluoromethylornithine, 0.2 mM for alpha-(fluoromethyl)dehydroornithine, 0.1 mM for alpha-(fluoromethyl)dehydroornithine methyl ester, and 0.01 mM for RR-MAP. L5178Y cells were somewhat more sensitive to the inhibitors with lC50 values of 0.5 mM for alpha-difluoromethylornithine, 0.06 mM for alpha-(fluoromethyl)dehydroornithine, 0.03 mM for alpha-(fluoromethyl)dehydroornithine methyl ester, and 0.002 mM for RR-MAP. In all cases, growth inhibition was fully prevented by exogenous putrescine. The effects of the inhibitors on parameters related to polyamine metabolism were compared at drug concentrations approximating the average of lC50 values for the two cell lines. Under these treatment conditions, polyamine pools were similarly affected by the various inhibitors. Typically, putrescine and spermidine were depleted, but effects on spermine pools differed according to the cell line, increasing slightly in L1210 cells and decreasing by about 50% in L5178Y cells. Spermine pools in L1210 cells could be reduced by RR-MAP at concentrations higher than the lC50 (i.e., 0.1 mM). Clonogenicity in soft agar was decreased about 50% by putrescine and spermidine depletion and was not further affected by spermine depletion. The inhibitors elevated S-adenosylmethionine decarboxylase activity in both cell lines with a 2-fold greater increase in L5178Y cells than in L1210 cells. Finally, the inhibitors decreased S-adenosylmethionine pools in L1210 cells by about 50% but had little effect on these pools in L5178Y cells with the exception of RR-MAP, which decreased S-adenosylmethionine pools by about 40%. Whether the different polyamine responses of the two cell lines are related to their ability to metabolize 5'-methylthioadenosine is uncertain. It is apparent, however, that the presence or absence of methylthioadenosine phosphorylase does not substantially modulate the antiproliferative activity of ornithine decarboxylase inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)

Possible interactions between the urea cycle and synthesis of pyrimidines and polyamines in regenerating liver.

Ornithine levels rise progressively in the liver of partially hepatectomized rats, probably as a consequence of the increased flow of metabolites through the urea cycle. Ammonia and urea concentrations in the blood and liber of partially hepatectomized animals are not significantly different from those of sham-operated rats. However, in regenerating livers, the ability to remove ammonia from the blood is close to its maximal limit. Ammonia overload leads to the production of large amounts of orotic acid and causes a marked elevation of hepatic ornithine decarboxylase activity. Among the pyrimidine precursors dihydroorotic acid injections increase the activity of the enzyme while orotic acid is without effect. A peak of labeled material that corresponds to dihydroorotic acid was identified by partition chromatography of acid-soluble extracts of livers of partially hepatectomized rats previously given injections of [14-C2 bicarbonate. The labeling of dihydroorotic acid from [14-C] bicarbibate is increased in the liver of rats given injections of ornithine. Despite the difficulties involved in studies of ornithine decarbozylase activity in vivo, our results suggest that mutual interactions between urea, pyrimidine, and polyamine synthesis take place during liver regeneration.

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

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