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PubMed1993

Alterations in repair of alkylating agent-induced DNA damage in polyamine-depleted human cells.

Treatment of HeLa cells with the polyamine biosynthesis inhibitors difluoromethylornithine (DFMO) and/or methylglyoxal bis(guanylhydrazone) (MGBG) results in marked depression in levels of the cellular polyamines putrescine, spermidine and spermine. Cells in this polyamine-depleted state exhibited increased sensitivity to monofunctional alkylating agents, manifested as decreased cloning ability and retardation of the DNA excision repair process. DFMO treatment did not alter the initial level of interaction of radiolabeled alkylating agent with cellular DNA, but combined treatment with DFMO and MGBG reduced covalent binding, probably through effects on cell cycling. Polyamine supplementation had no effects on initial yield of DNA single-strand breaks in drug-treated cells. The repair defect appeared similar to that observed previously in polyamine-depleted cells following X-irradiation and UV irradiation, namely retarded sealing of DNA strand breaks. It was not possible to reverse the effects of these inhibitors by short periods of polyamine loading, despite the fact that all three polyamines could be restored to near-normal levels. These findings provide the first demonstration of altered response of polyamine-depleted cells to monofunctional alkylating agents and contribute to our understanding of altered responses of polyamine-depleted cancer cells to a variety of DNA-reactive chemotherapeutic drugs.

Alkylating Agents↗
PubMed1981

Comparative role of polyamines in division and plastid differentiation of Euglena gracilis.

Regulation of polyamine biosynthesis during growth and differentiation of Euglena gracilis was investigated. Increased activity of L-ornithine decarboxylase (EC 4.1.1.17), the enzyme which catalyzes the initial step in polyamine synthesis in Euglena, and accumulation of polyamines were observed prior to DNA replication in synchronous cultures of heterotrophically or photoautotrophically grown cells. In photoautotrophic cells three maxima of polyamine synthesis were observed during the light period of the cell cycle. The transition form quiescence to active growth was accompanied in heterotrophic Euglena by a very large stimulation of ornithine decarboxylase activity and polyamine synthesis; the decrease in growth potential of these cells was correlated with a decrease in polyamine levels. In contrast, differentiation of Euglena i.e. a shift from heterotrophic to photoautotrophic mode of living in the absence of division, led only to a minor stimulation of polyamine biosynthesis. Alpha-Methylornithine, an inhibitor of ornithine decarboxylase, blocked the growth of heterotrophic Euglena, and depletion of intracellular polyamines decreased the differentiation rate. Both events could be reversed by addition of putrescine to the growth medium. This study suggests that Euglena requires a minimal intracellular level of polyamines to grow and differentiate under optimal conditions. This requirement seems to be more stringent for cell division.

Carboxy-Lyases↗
PubMed1989

Polyamines induce precocious development in rats. Possible interaction with growth factors.

The study reports the effects of daily subcutaneous injections of the biogenic polyamines putrescine, spermidine and spermine (10 mg/kg each) given for a short postnatal period, on growth and development of rats. Polyamine treatment, while only slightly enhancing normal body weight gain, prevented the weight loss caused by surgical injury of 5-day-old animals. The treatment resulted in earlier eyelid and ear opening and in earlier maturation of righting and gripping responses. Increased number of neurons in the superior cervical ganglion that is caused by polyamine treatment, could not be prevented by castration of newborn rats, thus excluding the testes as a site through which polyamines may exert their action. An apparent increase in immunohistochemically detectable nerve growth factor was evident in iris and submaxillary salivary gland of polyamine-treated animals, but no change in epidermal growth factor immunohistochemistry was detected in the salivary gland. We conclude: (1) treatment of newborn rats with polyamines can accelerate somatic and neurobehavioral development; (2) further studies are required in order to verify and quantitate the effects of polyamines on growth factors, and (3) the results imply that exogenous polyamines may exert their growth-promoting effects on a number of cell types when these cells experience periods of polyamine dependence.

Animals↗
PubMed1990

Polyamine involvement in basal and estradiol-stimulated insulin-like growth factor I secretion and action in breast cancer cells in culture.

Recent evidence indicates that the polyamine pathway may play a significant role in the autocrine/paracrine control of breast cancer cell proliferation by hormones. To directly test this hypothesis, in the present experiments, we evaluated the polyamine involvement in immunoactive insulin-like growth factor I (IGF-I) secretion and IGF-I action using MCF-7 breast cancer cells cultured in serum-free medium in the presence and absence of estradiol (E2). Administration of the polyamine biosynthetic inhibitor, alpha-difluoromethylornithine (DFMO) induced a marked suppression of cellular ornithine decarboxylase (ODC) activity and polyamine levels which was associated with significant, although partial, inhibition of E2-stimulated growth. Exogenous putrescine administration repleted cellular polyamine pools and completely reversed the growth-inhibitory effect of DFMO. Despite these parallel changes in polyamine levels and proliferative activity, basal as well as E2-stimulated levels of immunoactive IGF-I measured in the conditioned media were unaffected by DFMO with and without exogenous putrescine administration. On the other hand, induction of polyamine depletion and repletion by the same treatments significantly (although partially) affected the proliferative action of exogenously added IGF-I. These findings indicate that polyamines, while not involved in immunoactive IGF-I production, play an important role, at least in part, in IGF-I action in this experimental system. Furthermore, we observed that the administration of a monoclonal antibody directed against IGF-I was able to partially block basal as well as of a monoclonal antibody directed against IGF-I was able to partially block basal as well as E2-stimulated MCF-7 cell proliferation. We conclude that immunoactive IGF-I is an important but not sole mediator of MCF-7 breast cancer growth under our experimental conditions. The polyamine pathway plays an important role in the expression of its proliferative action.

Antibodies, Monoclonal↗
PubMed1992

Sodium butyrate stimulates polyamine biosynthesis in colon cancer cells.

Differentiation inducers act through polyamine-dependent and independent pathways. Sodium butyrate (NaB) inhibits proliferation and induces terminal differentiation in human and murine cancer cell lines. An effect of this agent on polyamine biosynthesis has not been demonstrated previously. In the present study, we examined the effects of NaB on polyamine biosynthesis in mouse colon cancer (MC-26) cells. All studies were performed on exponentially growing cells, and ODC and polyamine transport measurements were performed as described previously. NaB inhibited the growth of MC-26 cells in a dose-dependent manner. Cell shape was significantly altered by treatment with NaB (development of dendritic-like processes and flattening and spreading out of cells on culture dishes). NaB stimulated ODC activity in a dose-dependent manner. The activity was elevated by 8 h after treatment, and at 48 h there was a ten-fold increase in activity (compared with control activity). The increase in ODC activity led to an increase in polyamine biosynthesis; putrescine, spermidine, and spermine levels in MC-26 cells were significantly elevated by 24 h after treatment with NaB. Polyamine uptake was similar in control cells and cells treated with NaB alone. Our finding of significant stimulation of polyamine uptake by NaB after inhibition of endogenous synthesis (by an ODC-dependent pathway) in DFMO-treated cells suggests that cellular requirements are increased for polyamines in NaB-treated cells. We conclude that polyamine-dependent processes are important in the mechanism of action of NaB in colon cancer cells.

Animals↗
PubMed2003

Abnormal growth of polyamine-deficient Escherichia coli mutant is partially caused by oxidative stress-induced damage.

Polyamines participate in numerous cellular processes and are required for normal cell growth in Escherichia coli. In this study, we constructed a new polyamine-deficient E. coli mutant and investigated the physiological function of polyamines during normal aerobic growth conditions. We showed that the requirement for sulfur-containing, branched chain, and aromatic amino acids, which was exhibited in the sodA sodB double mutant faced with severe oxidative stress, was also true of the polyamine-deficient mutant during normal aerobic cell growth. Sorbitol, sucrose, mannose, 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an antioxidant that functions as an oxygen radical scavenger including z.rad;O(2)(-), and thiamine partially relieved the cell growth defect caused by polyamine depletion in a dose-dependent manner. As was the case for the cells treated with paraquat, the mutant had an elongated shape compared with the polyamine-proficient wild type. Decreased aeration also relieved the cell growth defect of the polyamine-deficient mutant. Finally, we confirmed that chloromethyl-2('),7(')-dichlorofluorescin diacetate (DCFH-DA), which is oxidized in a fluorescent product in the presence of various oxidants, also fluoresce in the polyamine-deficient cells. These results showed that abnormal growth of the polyamine-deficient E. coli mutant results partially from oxidative stress-induced damage and the mutant thus exhibits the requirement for antioxidant or specific nutritional amino acid during normal aerobic growth.

Amino Acids↗
PubMed2006

Involvement of polyamines in apoptosis of cardiac myoblasts in a model of simulated ischemia.

Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy, and heart failure. The polyamines putrescine, spermidine, and spermine are polycations absolutely required for cell growth and division. However, increasing evidence indicates that polyamines, cell growth, and cell death can be tightly connected. In this paper, we have studied the involvement of polyamines in apoptosis of H9c2 cardiomyoblasts in a model of simulated ischemia. H9c2 cells were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation, that induces apoptosis. The activity of ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis that synthesizes putrescine, is rapidly and transiently induced in ischemic cells, reaching a maximum after 3 h, and leading to increased polyamine levels. Pharmacological inhibition of ornithine decarboxylase by alpha-difluoromethylornithine (DFMO) depletes H9c2 cardiomyoblasts of polyamines and protects the cells against ischemia-induced apoptosis. DFMO inhibits several of the molecular events of apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, caspase activation, downregulation of Bcl-xL, and DNA fragmentation. The protective effect of DFMO is lost when exogenous putrescine is provided to the cells, indicating a specific role of polyamine synthesis in the development of apoptosis in this model of simulated ischemia. In cardiomyocytes obtained from transgenic mice overexpressing ornithine decarboxylase in the heart, caspase activation is dramatically increased following induction of apoptosis, with respect to cardiomyocytes from control mice, confirming a proapoptotic effect of polyamines. It is presented for the first time evidence of the involvement of polyamines in apoptosis of ischemic cardiac cells and the beneficial effect of DFMO treatment. In conclusion, this finding may suggest novel pharmacological approaches for the protection of cardiomyocytes injury caused by ischemia.

Animals↗
PubMed2003

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines.

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.

Animals↗
PubMed1996

Motor depressant effects of systemically administered polyamines in mice: involvement of central NMDA receptors.

The systemic administration of polyamines (s.c.) produced a dose-dependent motor depression. With high doses the depressant effect was long-lasting and the animals showed signs of toxicity. ED50 values for spermine, spermidine and putrescine were 38, 90 and 251 mg/kg respectively. The motor depression induced by the systemic administration of N-methyl-D-aspartate (NMDA; 25 mg/kg i.p.) was used as a model for studying the interactions between polyamines and the NMDA receptor. Results indicate that (1) the motor effects elicited by NMDA are very similar to those induced by polyamines at ED50 doses; (2) polyamines, even at non-active doses, potentiate the motor depressant effect induced by NMDA; (3) the NMDA receptor antagonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine (MK-801; 0.5 mg/kg i.p.), abolishes the depressant effect elicited by NMDA and by polyamines, even at toxic doses; (4) amphetamine (1.5 mg/kg i.p.) does not counteract the motor depressant effects of NMDA or polyamines. On the other hand, the adenosine receptor antagonist, theophylline (30 mg/kg i.p.), counteracts NMDA- but not polyamine-induced motor depression. The concentration of polyamines in the brain is modified after their systemic administration at high doses and at the ED50 dose of putrescine. In conclusion, the data suggest that the NMDA receptor could be a target mediating the motor effect elicited by polyamines. They also show that the quantitative analysis of the motor effects elicited by non-convulsant doses of NMDA might be a powerful tool for studying in vivo the interaction between neurotransmission systems involved in the regulation of motor activity.

Amphetamine↗
PubMed1999

Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway.

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5' leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiations. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.

Adenosylmethionine Decarboxylase↗
PubMed2003

Polyamines reduce paraquat-induced soxS and its regulon expression in Escherichia coli.

Polyamines, ubiquitous polycationic compounds, are involved in many cellular responses and relieve paraquat-induced cytotoxicity in Escherichia coli. We constructed a new E. coli mutant strain, JIL528, which is deficient in the biosynthesis of both putrescine and spermidine, to examine the physiological role of polyamines under oxidative stress caused by paraquat. Putrescine and spermidine downregulate the expression of soxS induced by paraquat in a concentration-dependent manner. The product of SoxS is a key regulator governing cellular responses against oxidative stress in E. coli. The downregulation of soxS expression by polyamines was not shown in the soxR mutant background. Glucose-6-phosphate dehydrogenase (G6PDH; encoded by zwf) and manganese-containing superoxide dismutase (Mn-SOD; encoded by sodA) activities induced by paraquat were decreased by exogenous polyamines. The induction of the zwf expression by paraquat was also decreased by exogenous polyamines. The polyamine-deficient mutant strain JIL528 showed a higher soxS expression than its parent polyamine-proficient wild type BW1157, on exogenous supplementation of paraquat concentrations below 1 micromol/L. While the growth rate of the mutant was decreased, soxS expression was increased in a concentration-dependent manner above 0.01 micromol/L of paraquat. In contrast, growth inhibition of the mutant by paraquat was relieved, and soxS was no longer induced by exogenous putrescine (1 mmol/L). In conclusion, polyamines protect against paraquat-induced toxicity but downregulate soxS expression, suggesting that the protective role of polyamines against oxidative damage induced by paraquat results in soxS downregulation.

Biogenic Polyamines↗
PubMed2004

Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme.

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme (AZ). The synthesis of mammalian AZ was reported to involve a polyamine-induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. We identified a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian AZ. We show that synthesis of yeast AZ (Oaz1) involves polyamine-regulated frameshifting as well. Degradation of yeast ODC by the proteasome depends on Oaz1. Using this novel model system for polyamine regulation, we discovered another level of its control. Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome. Degradation of Oaz1, however, is inhibited by polyamines. We propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation.

Amino Acid Sequence↗
PubMed2001

Effect of polyamine depletion on caspase activation: a study with spermine synthase-deficient cells.

Activation of the caspase proteases represents a central point in apoptosis. The requirement for spermine for the processes leading to caspase activation has been studied in transformed embryonic fibroblasts obtained from gyro (Gy) mutant male mice. These cells lack spermine synthase activity and thus provide a valuable model to study the role of spermine in cell processes. Gy fibroblasts do not contain spermine and have a higher spermidine content. However, when compared with fibroblasts obtained from normal male littermates (N cells), Gy fibroblasts were observed to grow normally. The lack of spermine did not affect the expression of Bcl-2, and caspases 3 and 9 were activated by etoposide in both N and Gy cells, indicating that spermine is dispensable for caspase activation. Spermine deficiency did not significantly influence caspase activity in cells treated with etoposide, cycloheximide or staurosporine, but sensitized the cells to UV irradiation, which triggered significantly higher caspase activity in Gy cells compared with N cells. alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis that is able to deplete cells of putrescine and spermidine, but usually does not influence spermine content, was able to produce a more complete polyamine depletion in Gy cells. This depletion, which included spermine deficiency, dramatically increased caspase activation and cell death in Gy fibroblasts exposed to UV irradiation. On the other hand, in either N or Gy cells, DFMO treatment did not influence caspase activity triggered by staurosporine, but inhibited it when the inducers were cycloheximide or etoposide. In Gy cells depleted of polyamines by DFMO, polyamine replenishment with either spermidine or spermine was sufficient to restore caspase activity induced by etoposide, indicating that, in this model, polyamines have an interchangeable role in supporting caspase activation. Therefore, spermine is not required for such activation, and the effect and specificity of polyamine depletion on caspase activity may be very different, depending on the role of polyamines in the specific death pathways engaged by different stimuli. Some inducers of apoptosis, for example etoposide, absolutely require polyamines for caspase activation, yet the lack of polyamines, particularly spermine, strongly increases caspase activation when induced by UV irradiation.

Animals↗
PubMed2002

Antizyme induction by polyamine analogues as a factor of cell growth inhibition.

The polyamines spermidine and spermine and their diamine precursor putrescine are essential for mammalian cell growth and viability, and strategies are sought for reducing polyamine levels in order to inhibit cancer growth. Several structural analogues of the polyamines have been found to decrease natural polyamine levels and inhibit cell growth, probably by stimulating normal feedback mechanisms. In the present study, a large selection of spermine analogues has been tested for their effectiveness in inducing the production of antizyme, a key protein in feedback inhibition of putrescine synthesis and cellular polyamine uptake. Bisethylnorspermine, bisethylhomospermine, 1,19-bis-(ethylamino)-5,10,15-triazanonadecane, longer oligoamine constructs and many conformationally constrained analogues of these compounds were found to stimulate antizyme synthesis to different levels in rat liver HTC cells, with some producing far more antizyme than the natural polyamine spermine. Uptake of the tested compounds was found to be dependent on, and limited by, the polyamine transport system, for which all these have approximately equal affinity. These analogues differed in their ability to inhibit HTC cell growth during 3 days of exposure, and this ability correlated with their antizyme-inducing potential. This is the first direct evidence that antizyme is induced by several polyamine analogues. Selection of analogues with this potential may be an effective strategy for maximizing polyamine deprivation and growth inhibition.

Animals↗
PubMed1984

The role of polyamine depletion and accumulation of decarboxylated S-adenosylmethionine in the inhibition of growth of SV-3T3 cells treated with alpha-difluoromethylornithine.

The effects of alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, on cell growth rate, polyamine content and the content of decarboxylated S-adenosylmethionine in SV-3T3 transformed mouse fibroblasts were studied. DL-alpha-Difluoromethylornithine at 1 mM or higher concentrations decreased the growth rate by over 90% after 2 or more days of exposure, but the cells remained viable, although quiescent for at least 9 days. Addition of 10 microM-spermidine or -spermine or 50 microM-putrescine at any time throughout this period completely reversed the inhibition of growth. Treatment with alpha-difluoromethylornithine decreased putrescine and spermidine contents by more than 98% and that of spermine by 60%, but cells exposed to exogenous polyamines did not require complete replenishment of the polyamine pools to resume growth. In fact, a virtually normal growth rate was obtained in cells lacking putrescine, having 2% of normal spermidine content and 156% of normal spermine. These results suggest that the well-known increase in putrescine and spermidine in cells stimulated for growth is not essential for this to occur and that mammalian cells can utilize spermine as their only polyamine. A substantial reversal of the growth-inhibitory effect of alpha-difluoromethylornithine was produced by a number of polyamines not normally found in mammalian cells, including the spermidine analogues aminopropylcadaverine and sym-homospermidine, which were partially converted into their respective spermine analogues by addition of an aminopropyl group within the cell. The spermine analogue sym-norspermine was also effective, but the maximal growth rate produced by these unphysiological polyamines was only 60-70% of that produced by the normal polyamines. These results indicate that spermidine and spermine have the optimal length for activation of the cellular processes critically dependent on polyamines and should help in identifying these processes. Exposure to alpha-difluoromethylornithine leads to an enormous rise in the concentration of decarboxylated S-adenosylmethionine, which reached a peak at 530-fold after 3 days of exposure and steadily declined to 140-fold after 11 days. This increase was abolished by addition of exogenous polyamines, which rapidly decreased the activity of S-adenosylmethionine decarboxylase. The increase in decarboxylated S-adenosylmethionine is unlikely to be solely responsible for the decrease to the same extent by spermine, sym-norspermidine and sym-homospermidine, which produce 97%, 16% and 60% of the control growth rate, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosylmethionine Decarboxylase↗
PubMed1986

Polyamines and HeLa-cell DNA replication.

HeLa cells were synchronized for S-phase DNA synthesis by the double thymidine-block procedure. A comparison was made of the polyamine content and S-phase DNA synthesis in cells from control cultures and cultures to which an inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine, was added to the synchronization medium. Control cells showed a peak of synchronous DNA synthesis at 3 h and a maximum concentration of polyamines at 6-9 h after release of the second thymidine block. Cells from cultures containing the inhibitor were severely inhibited in the synthesis of DNA and contained no putrescine and only traces of spermidine while the spermine content was lowered by as much as 80%. Supplementation of cultures containing alpha-difluoromethylornithine with a polyamine, at the time of release of the second thymidine block, replenished the intracellular pool of the administered polyamine and partially restored S-phase DNA synthesis, with a lag of 3-6 h. Almost complete restoration of DNA synthesis in cells depleted of polyamines was achieved by the addition of a polyamine to cultures at least 10 h before release of the second thymidine block. The lag in initiation of synchronous S-phase DNA synthesis was eliminated in these cells. It is concluded that reversal by polyamines of the deficiency in S-phase DNA synthesis, in polyamine-depleted HeLa cells, is a time-dependent process indicative of the necessity for the replenishment of replication factors or their organization into an active replication complex.

DNA Replication↗
PubMed1997

Rapid induction of apoptosis by deregulated uptake of polyamine analogues.

Treatment of Chinese hamster ovary cells with alpha-difluoromethylornithine for 3 days, followed by exposure to cycloheximide, led to an unregulated, rapid and massive accumulation of polyamine analogues. This accumulation led to cell death by apoptosis within a few hours. Clear evidence of DNA fragmentation was seen in response to both N-terminally ethylated polyamines and to polyamines containing methyl groups on the terminal carbon atoms. Programmed cell death was induced within 2-4 h of exposure to 1 microM or higher concentrations of N1,N11-bis(ethyl)norspermine. The presence of cycloheximide increased the uptake of the polyamine analogues and therefore led to cell death at lower analogue concentrations, but it was not essential for the induction of apoptosis, since similar effects were seen when the protein synthesis inhibitor was omitted and the concentration of N1, N11-bis(ethyl)norspermine was increased to 5 microM or more. The induction of apoptosis was blocked both by the addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, or by the addition of the polyamine oxidase inhibitor N1-methyl-N2-(2,3-butadienyl)butane-1,4-diamine (MDL 72,527). These experiments provide evidence to support the concepts that: (1) polyamines or their oxidation products may be initiators of programmed cell death; (2) regulation of polyamine biosynthesis and uptake prevents the accumulation of toxic levels of polyamines; and (3) the anti-neoplastic effects of bis(ethyl) polyamine analogues may be due to the induction of apoptosis in sensitive tumour cells.

Alkylation↗
PubMed1998

Developmental effect of polyamine depletion in Caenorhabditis elegans.

Ornithine decarboxylase (ODC) catalyses the conversion of ornithine to putrescine, an obligate precursor to the polyamines spermidine and spermine. We reported previously that homozygous odc-1 (pc13) worms have no detectable ODC activity. Despite their inability to make polyamines, these mutant worms appear normal, but with a slight reduction in total brood size, when grown in complex medium that presumably contains polyamines. We now show that when ODC-deficient worms are transferred to polyamine-free medium, they show a strong phenotype. odc-1 worms have two different fates, depending upon the developmental stage at which polyamines are removed. If the polyamines are removed at the L1 larval stage, the mutant animals develop into adult hermaphrodites that produce very few or no eggs. In contrast, if mutant larvae at the later L4 stage of development are transferred to polyamine-deficient medium, they develop and lay eggs normally. However, approx. 90% of the eggs yield embryos that, although well differentiated, arrest at early stage 3. Either maternal or zygotic expression of ODC provides partial rescue of embryonic lethality. Supplementing deficient medium with the polyamine spermidine allows ODC-deficient worms to develop as on complex medium. Together, these findings suggest that ODC activity is most critically required during oogenesis and embryogenesis and, furthermore, that exogenous polyamines can override the requirement for ODC activity.

Animals↗