Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: Crosstalk between ODC and BCL-2

Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-α)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCδ) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKδ pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.     

Intrinsic and extrinsic factors in estrogen action in human breast cancer: role of polyamines and pituitary factors

Although polyamines are important in regulating proliferation of mammalian cells, their role in hormone induction of cell growth has not been delineated. In the estradiol-responsive human breast cancer cell line, T-47D clone 11, estradiol (10(-10) M) was able to stimulate cell proliferation and the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the biosynthesis of polyamines. alpha-Difluoromethylornithine (DFMO), a specific inhibitor of ODC, blocked the estradiol-induced cell proliferation and ODC activity. Exogenous addition of putrescine, the natural product of ODC, rescued the inhibitory effect of DFMO. In addition, DFMO abolished the estradiol-induced growth of several other estrogen-responsive human breast cancer cell lines but did not affect the growth of hormone-independent cell lines. Further, a serum factor was found to be required for estradiol to exert its effect. To gain insight into the nature of this and possibly other extrinsic factors involved, the effect of estradiol on the proliferation of T-47D cells transplanted into athymic nude mouse was evaluated. In this in vivo system, estradiol alone produced only moderate growth of the human breast tumor. The simultaneous transplantation of a prolactin (PRL)- and growth hormone (GH)-secreting rat pituitary tumor or normal rat pituitary glands at a different site dramatically potentiated the effect of estradiol on the growth of the breast tumor xenograft. Purified PRL or GH were without effect, indicating that the active pituitary factor is neither PRL nor GH. Further, conditioned medium from rat pituitary tumor cells potentiated the mitogenic effect of estradiol on T-47D and several other estrogen receptor-positive human breast cancer cell lines in vitro under serum-free condition. In conclusion, we have identified both intrinsic (polyamines) and extrinsic (pituitary/serum) factors that are importance for estrogen to exert its mitogenic action. The next goal will be to elucidate the mechanisms of action of these molecules in the modulation of estrogen action.     

Regulation of ornithine decarboxylase in B16 mouse melanoma cells: synergistic activation of melanogenesis by alphaMSH and ornithine decarboxylase inhibition

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by alpha-melanocyte-stimulating hormone (alphaMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, alphaMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or alphaMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor alpha-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with alphaMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to alphaMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by alphaMSH.     

Superinduction of ornithine decarboxylase by halogenated ribofuranosylbenzimidazoles.

1. The effect of dichlororibofuranosylbenzimidazole (DiCl-RB), an inhibitor of hnRNA synthesis and casein kinase-2 activity, on ornithine decarboxylase (ODC) was investigated in a difluoromethylornithine (DFMO) resistant, ODC overproducing cell line. 2. In cells growing in the absence of DFMO, DiCl-RB provoked a marked, but transient increase in ODC activity and immunoreactive ODC content. 3. The ODC response to DiCl-RB was prevented by cycloheximide and was not due to stabilization of the enzyme. 4. The dibromo derivative analogue (DiBr-RB) exerted similar effects on ODC, but was effective at lower concentrations. 5. The halogenated ribofuranosylbenzimidazoles were ineffective in cells growing in the presence of DFMO and containing higher levels of ODC protein.     

A new microbioassay for the measurement of lactogenic hormones in human serum

The standard Nb2 assay for biologically active prolactin has been modified to allow a rapid convenient microbioassay without loss of specificity or accuracy. Lactogenic hormones specifically stimulate the replication of Nb2 node rat lymphoma cells in suspension culture and form the basis of a currently available bioassay to measure prolactin and growth hormone in human serum. A new microbioassay was developed using microtest plates enabling a large number of samples to be assayed simultaneously whilst maintaining the overall sensitivity of the bioassay for lactogenic hormones. Growth of the Nb2 node lymphoma cells, measured by a light scattering technique using optical density on a spectrophotometer, was shown to be closely correlated with the cell number determined on a Coulter counter. Addition of excess anti-human prolactin and anti-human growth hormone completely inhibited the growth stimulatory effects of both human prolactin and human growth hormone. This new microbioassay (BA) and conventional radioimmunoassay (RIA) were used to measure lactogenic hormones in 48 normal subjects. There was a close correlation between the results of both assays for each hormone studied in the control sera. The mean basal BA/RIA ratio was 1.5 (range 0.8-2.0) for prolactin, 0.7 (range 0-4.5) for growth hormone and 1.3 (range 0.5-1.9) for total lactogenic activity.     

Epidermal growth factor potentiates the induction of ornithine decarboxylase activity by prostaglandins in embryonic palate mesenchymal cells: effects on cell proliferation and glycosaminoglycan synthesis

Epidermal growth factor (EGF) and prostaglandins (PGs) have been implicated in the regulation of a number of developmental processes in the mammalian embryonic palate. Normal palatal ontogenesis is dependent on the presence and quite possibly on the interaction of various hormones and growth factors. The interaction between EGF and PGs in regulation of murine embryonic palate mesenchymal (MEPM) cell growth and differentiation was therefore investigated by monitoring the activity of ornithine decarboxylase (ODC), the principle and rate limiting enzyme of polyamine biosynthesis. ODC activity is tightly coupled to the proliferative and differentiative state of eukaryotic cells and therefore serves as a reliable indicator of such cellular functions. Treatment of confluent cultures of MEPM cells with EGF (1-50 ng/ml) resulted in a dose-related increase in ODC activity, while similar treatment with either PGE2 or PGF2 alpha (at concentrations up to 1 microM) did not elicit a dose-dependent increase in enzyme activity. Concurrent treatment of MEPM cells with EGF (20 ng/ml) and either PGE2 or PGF2 alpha (0.1-10000 nM) resulted in a marked prostaglandin dose-dependent induction of ODC activity, suggesting a strong cooperative interaction between these factors. ODC activity was maximal by 4 to 8 hr and could be completely inhibited by preincubation of the cells with actinomycin D or cycloheximide, indicating that de novo synthesis of RNA and protein is necessary for enzyme induction. Stimulation of ODC activity by EGF and PGE2 in these cells was not positively correlated with the level of cellular DNA synthesis but did result in a ninefold increase in the synthesis of extracellular glycosaminoglycans (GAGs), a key macromolecular family implicated in palatal morphogenesis. Stimulation of GAG synthesis was significantly inhibited by the administration of 5 mM DFMO (an irreversible inhibitor of ODC), indicating that the marked increase in GAG production was dependent, in part, on the induction of ODC activity by EGF and PGE2. Qualitative analysis of the palatal GAGs indicated that synthesis of several major classes of GAGs was stimulated. Collectively these data demonstrate a cooperative interaction between EGF and PGs in the induction of ODC activity. Such activity may serve to regulate the synthesis of GAGs, which are instrumental in mammalian palatal ontogenesis.     

An evaluation of the involvement of polyamines in modulating MCF-7 human breast cancer cell proliferation and progesterone receptor levels by estrogen and antiestrogen

The present studies were undertaken to determine the importance of the polyamine biosynthetic pathway in cellular proliferation and hormone-regulated progesterone receptor synthesis in estrogen receptor-containing breast cancer cells. Treatment of MCF-7 cells with difluoromethylornithine (DFMO), the irreversible inhibitor of the enzyme ornithine decarboxylase (ODC), prevented estradiol-induced cell proliferation in a dose-dependent fashion. DFMO inhibition of estradiol-induced cell proliferation was completely recoverable by the addition of exogenous putrescine while putrescine alone did not stimulate proliferation of control cells. ODC activity was 4-fold greater in estrogen-treated cells and DFMO (5 mM) fully inhibited ODC activity. DFMO was able to suppress only slightly further the proliferation of antiestrogen (tamoxifen) treated cells and putrescine was able to recover this DFMO inhibition. In contrast to the suppressive effect of DFMO on cell proliferation, DFMO had no effect on the ability of estrogen to stimulate increased (4-fold elevated) levels of progesterone receptor. Hence, while ODC activity appears important for estrogen-induced cell proliferation, inhibition of the activity of this enzyme has no effect on the ability of estradiol to increase cellular progesterone receptor content.     

Therapy of murine squamous cell carcinomas with 2-difluoromethylornithine

Targeted overexpression of an ornithine decarboxylase (ODC) transgene to mouse skin (the K6/ODC mouse) significantly enhances susceptibility to carcinogenesis. While in most strain backgrounds the predominant tumor type resulting from initiation-promotion protocols is benign squamous papilloma, K6/ODC mice on a FVB/N background develop malignant squamous cell carcinomas (SCCs) rapidly and in high multiplicity after carcinogen treatment. We have investigated the utility of polyamine-based therapy against SCCs in this model using the ODC inhibitor 2-difluoromethylornithine delivered orally. At a 2% concentration in drinking water, DFMO caused rapid tumor regression, but in most cases, tumors eventually regrew rapidly even in the presence of DFMO. The tumors that regrew were spindle cell carcinomas, an aggressive undifferentiated variant of SCC. At 1% DFMO in the drinking water, tumors also responded rapidly, but tumor regrowth did not occur. The majority of DFMO-treated SCCs were classified as complete responses, and in some cases, apparent tumor cures were achieved. The enzymatic activity of ODC, the target of DFMO, was substantially reduced after treatment with 1% DFMO and the high SCC polyamine levels, especially putrescine, were also significantly lowered. Based on the results of BrdUrd labeling and TUNEL assays, the effect of DFMO on SCC growth was accompanied by a significant reduction in tumor proliferation with no increase in the apoptotic index. These results demonstrate that SCCs, at least in the mouse, are particularly sensitive to polyamine-based therapy.     

Inhibition of ornithine decarboxylase by retinoic acid and difluoromethylornithine in relation to their effects on differentiation and proliferation

Murine embryonal carcinoma F9 cells can be induced to differentiate by 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC). The differentiated phenotype is similar to that of retinoic acid (RA)-treated F9 cells. In contrast to F9 cells the differentiated cells secrete plasminogen activator and express keratin intermediate filaments. Both DFMO and RA reduce ornithine decarboxylase activity, polyamine levels and inhibit cell proliferation of F9 cells. These compounds also reduce ODC, polyamine levels and proliferation of mouse BALB/c 3T6 fibroblasts. RA inhibits the induction of ODC by insulin, serum and to a lesser extent that of epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of DFMO and RA can be distinguished by their response to putrescine. The induction of differentiation and the inhibition of cell proliferation by DFMO can be totally abolished upon the addition of putrescine, whereas the actions of RA are not affected at all. These results suggest that the inhibition of ODC and reduction of polyamines are not causal in the induction of differentiation and the inhibition of proliferation by RA.     

Stable ornithine decarboxylase in a rat hepatoma cell line selected for resistance to alpha-difluoromethylornithine

Ornithine decarboxylase (ODC) is extremely unstable in mammalian cells. This unusual characteristic facilitates rapid fluctuations in the activity of this enzyme in response to variations in its biosynthesis. Unfortunately, very little is known about the mechanism or regulation of this ODC-specific proteolytic pathway. This study describes the production and characterization of a variant of the rat hepatoma HTC cell line that is strikingly deficient in this pathway. This cell variant was induced by selection for growth in stepwise increasing concentrations (up to 10 mM) of the irreversible ODC inhibitor, alpha-difluoromethylornithine (DFMO). Resistance to this inhibitor appears to result from a combination of elevated (10X) ODC biosynthesis and inhibited degradation, producing greater than a 2000-fold increase in the level of ODC protein. In these variant cells (DH23b) inhibition of protein synthesis by cycloheximide did not result in rapid loss of enzyme activity or ODC protein determined by radioimmunoassay. Pulse-chase studies with [35S]methionine confirmed that this enzyme was not preferentially degraded, even when spermidine was added to the media. ODC purified from the variant cells was found to be identical to the control cell enzyme in size, isoelectric point, substrate binding kinetics, and sensitivity to the inhibitor DFMO. Also, as in the control cells, a major fraction of the ODC molecules extracted from DH23b cells was shown to be phosphorylated on a serine residue. The inability to detect physical or kinetic differences between the parent and the variant cell ODC suggests that the unusual stability of ODC in this cell is associated with a defect in a cellular mechanism for ODC-specific degradation.     

Interaction of Zn2+ and Eu3+ with bovine liver glutamate dehydrogenase.

Stepwise increments of the concentration of 2-difluoromethylornithine (DFMO), a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (ODC), resulted in a selection of cultured human IgG-myeloma cells (Sultan cell line) capable of growing in the presence of up to 3 mM-DFMO. This capacity was associated with 10-fold increase in ODC activity in the dialysed extracts of drug-resistant myeloma cells, markedly enhanced synthesis rate for ODC enzyme molecules, as revealed by a 20 min [35S]methionine labelling of cellular proteins, followed by specific immunoprecipitation and SDS/polyacrylamide-gel electrophoresis, dose-dependently increased expression of ODC mRNA in resistant cells (effective dose causing 50% inhibition), dose-dependent amplification of ODC gene sequences in a 9-kilobase-pairs EcoRI genomic DNA fragment, and (v) a 10-fold increase in the ED50 (effective dose causing 50% inhibition) for the anti-proliferative action of DFMO in these myeloma cells. These results represent one of the few gene amplifications described in cultured human cells.     

Differential regulation of pituitary hormone secretion and gene expression by thyrotropin-releasing hormone. A role for mitogen-activated protein kinase signaling cascade in rat pituitary GH3 cells

We examined the possible involvement of mitogen-activated protein (MAP) kinase activation in the secretory process and gene expression of prolactin and growth hormone. Thyrotropin-releasing hormone (TRH) rapidly stimulated the secretion of both prolactin and growth hormone from GH3 cells. Secretion induced by TRH was not inhibited by 50 microM PD098059, but was completely inhibited by 1 microM wortmannin and 10 microM KN93, suggesting that MAP kinase does not mediate the secretory process. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin, whereas expression of growth hormone mRNA was largely attenuated. The increase in prolactin mRNA stimulated by TRH was inhibited by addition of PD098059, and the decrease in growth hormone mRNA was also inhibited by PD098059. Transfection of the cells with a pFC-MEKK vector (a constitutively active MAP kinase kinase kinase), significantly increased the synthesis of prolactin and decreased the synthesis of growth hormone. These data taken together indicate that MAP kinase mediates TRH-induced regulation of prolactin and growth hormone gene expression. Reporter gene assays showed that prolactin promoter activity was increased by TRH and was completely inhibited by addition of PD098059, but that the promoter activity of growth hormone was unchanged by TRH. These results suggest that TRH stimulates both prolactin and growth hormone secretion, but that the gene expressions of prolactin and growth hormone are differentially regulated by TRH and are mediated by different mechanisms.     

p53 independent G(1) arrest induced by DL-alpha-difluoromethylornithine

Ornithine decarboxylase (ODC), which catalyzes polyamine biosynthesis, plays an essential role in cell growth. DL-alpha-Difluoromethylornithine (DFMO), a synthetic inhibitor of ODC, inhibits cell growth. However, the exact mechanism by which polyamine depletion by DFMO results in growth inhibition remains to be elucidated. We clarified the mechanisms by which DFMO inhibits human gastric cancer cell (MKN45) growth. DFMO induced MKN45 cell G(1) phase arrest after 48 h, and the percentage of G(1) arrest cells continued to increase until 72 h. Expression of p21 and phosphorylation of Stat1 were significantly induced by DFMO at 24 h. Luciferase assay and gel shift assay showed specific binding of Stat1 to the p21 promoter, and promoter activity was activated at 24 h. In dominant negative p53 expressing cells, DFMO significantly induced p21 expression, arrested cells at G(1) phase, and suppressed cell growth effectively. These results suggest that DFMO induced MKN45 cell arrest at G(1) phase in a p53 independent manner, and Stat1 is, at least in part, involved in G(1) arrest.     

Ornithine decarboxylase activity in human endometrium and endometrial cancer cells

Summary Ornithine decarboxylase (ODC) activities were significantly higher in proliferative endometrium during the estrogen-dominated follicular phase of the menstrual cycle than in secretory endometrium after the formation of the progesterone-secreting corpus luteum. The enzymatic activity was increased about fivefold by renewal of the medium during incubations of endometrial fragments or isolated endometrial glands. Endometrial adenocarcinoma cells (HEC-1, HEG-50), both in monolayers and suspension, also responded to medium renewal by increasing ODC activity about 10-fold after 4 h, with subsequent reduction to control levels after 7 h. These effects were blocked by actinomycin D and cycloheximide. Endometrial stromal cells exhibited highly variable ODC activities at different passages. Difluoromethylornithine (DFMO) and sodium molybdate had marked antiproliferative effects in HEC-50 cultures, reducing cell numbers to 10 to 20% of control values 11 d after plating and inhibiting ODC activity by approximately 80% on Day 7. The antiproliferative effect of DFMO, but not that of molybdate, was reversed by 10 μM putrescine, the product of ODC activity. In contrast to DFMO, molybdate had no effect on ODC activity of cell homogenates. Molybdate did not elicit antizyme formation in HEC-50 cells under conditions in which putrescine did. These results indicate that ODC activity, present in both epithelial and stromal cells, as shown analytically and also by autoradiography after labeling with [³H]DFMO, may be related to cell proliferation in vivo and that proliferation of human endometrial cancer cells in culture can be arrested by DFMO and by molybdate. This investigation was supported by PHS grant HD 07197, awarded by the National Institute of Child Health and Human Development and PHS grant CA 15648, awarded by the National Cancer Institute.     

B23 is a downstream target of polyamine- modulated CK2

Our previous studies have shown that the overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, increases the enzymatic activity of the polyamine-responsive enzyme casein kinase 2 (CK2). Because CK2 is known to preferentially associate with the nuclear matrix in response to other trophic stimuli, we investigated the effects of ODC overexpression on CK2 localisation and on the CK2-mediated phosphorylation of a known CK2 substrate, the nucleolar phosphoprotein B23. Immunofluorescence analysis of CK2 and B23 in primary keratinocytes revealed that ODC overexpression resulted in the colocalisation of CK2 with B23 at the nucleolar borders. ODC overexpression also increased CK2 kinase activity 2-fold at the nuclear matrix, a response which could be abrogated by treatment of K6/ODC transgenic keratinocytes with the ODC inhibitor α-difluoromethylornithine (DFMO). Levels of B23 protein were also elevated in ODC-overexpressing cells compared to normal cells or transgenic cells treated with DFMO. This increase in protein level was neither due to an increase in steady-state mRNA levels, nor was it due to increased stability of B23 protein. Phosphorylation of B23 was also increased in ODC-overexpressing cells, and this increased phosphorylation could be blocked by treatment of the cells with the CK2 kinase inhibitors apigenin or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). These data suggest that B23 may be a downstream effector of polyamines via phosphorylation by the protein kinase CK2.     

Consequences of aberrant ornithine decarboxylase regulation in rat hepatoma cells

DH23A cells, an α-difluoromethylornithine (DFMO)–resistant variant of rat hepatoma tissue culture cells (HTC), contain high levels of very stable ornithine decarboxylase (ODC). In the absence of DFMO, the high ODC activity results in a large accumulation of endogenous putrescine. Concomitant with the putrescine increase is a period of cytostasis and a subsequent loss of viable cells. In contrast, HTC cells with a moderate polyamine content can be maintained in exponential growth. This suggests that a moderate polyamine concentration is necessary for both optimal cell growth and survival. The cytoxicity observed in the DH23A cells is apparently not due to byproducts of polyamine oxidation or alterations in steady state intracellular pH or free [Ca²⁺]. It is possible to mimic the effects of high levels of stable ODC by treatment of cells with exogenous putrescine in the presence of DFMO. This suggests that overaccumulation of putrescine is the causative agent in the observed cytotoxicity, although the mechanism is unclear. These data support the hypothesis that downregulation of ODC may be necessary to prevent accumulation of cytotoxic concentrations of the polyamines. © 1994 Wiley-Liss, Inc.     

Increased ornithine decarboxylase activity and polyamine biosynthesis are required for optimal cytolytic T lymphocyte induction

The objective of the present investigation was to evaluate the requirement for increased ornithine decarboxylase (ODC) activity and polyamine biosynthesis in the induction of cytolytic T lymphocytes (CTL). In this regard, we have utilized alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO treatment completely abrogated Con A-induced NW T-cell ODC activity. Similarly, DFMO treatment reduced putrescine and spermidine biosynthesis 100 and 87% respectively by the end of a 48-hr incubation period. Polyamine depletion reduced the Con A-mediated polyclonal induction of CTL by 52 and 81% at 24 and 48 hr of culture, respectively. The effect of DFMO on CTL induction could be reversed by the addition of exogenous putrescine. These data indicate that the observed effects of DFMO on CTL induction were mediated through inhibition of polyamine biosynthesis. Therefore, increased ODC activity and polyamine biosynthesis are required for optimal CTL induction. Furthermore, polyamine depletion did not impair IL-2 production; however, IL-2-dependent proliferation was reduced. These data are the first to discriminate between the requirement for polyamines with regard to IL-2 responsiveness, rather than IL-2 production, during a primary T-cell mitogenic response.     

Ornithine decarboxylase induction and polyamine biosynthesis are required for the growth of interleukin-2- and interleukin-3-dependent cell lines

The objective of this study was to evaluate induction of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, and subsequent polyamine accumulation in interleukin-2 (IL-2)- and interleukin-3 (IL-3)-dependent growth. The CTLL-20 and FDC-P1 cell lines, which have been shown to be absolutely dependent on IL-2 and IL-3, respectively, were used in these studies. The CTLL-20 and FDC-P1 cells each had different temporal patterns of ODC induction following lymphokine stimulation. ODC levels increased rapidly in the FDC-P1 cells, peaking 4 hr after stimulation with IL-3. In contrast, peak ODC activity in the CTLL-20 cells occurred 18 hr following stimulation with IL-2 and reached eightfold higher levels than those observed in the FDC-P1 cells. Treatment with D,L-alpha-difluoromethylornithine X HCl X H2O (DFMO), a specific irreversible inhibitor of ODC activity, completely abrogated lymphokine-dependent ODC induction in both the CTLL-20 and FDC-P1 cell lines. Similarly, intracellular levels of the polyamines putrescine and spermidine were reduced in both cell lines following DFMO treatment. DFMO treatment reduced both IL-2- and IL-3-dependent proliferation in a dose-dependent manner. However, this inhibition could be reversed by the addition of exogenous putrescine. DFMO treatment had no effect on cell viability. Polyamine-depleted CTLL-20 and FDC-P1 cells showed decreased absorption of IL-2 and IL-3 activity, respectively. However, the addition of exogenous putrescine restored the ability of the cells to absorb the appropriate lymphokine. These data are the first to demonstrate that ODC induction and polyamine biosynthesis are required in lymphokine dependent growth.