Characterization of a COS cell line deficient in polyamine transport.
ABSTRACT In the present study, we describe the isolation and characterization of a COS cell line deficient in polyamine uptake that may provide an important tool for the molecular cloning of polyamine transporter(s). The cells were selected by isolation for resistance against the cytotoxic agent, methylglyoxal bis(guanylhydrazone) (MGBG), which is entering the cells using the same transport system as the polyamines. The isolated cell line was capable of growing in the presence of 100 microM MGBG, which totally inhibited the growth of the wild-type cells. The transport of putrescine and spermidine was markedly decreased in the COS-MGBGr cells. The decrease in putrescine transport was mainly a result of a 14-fold decrease in Vmax, whereas the reduced spermidine uptake was due to a 3-4-fold decrease in Vmax as well as 12-fold increase in Km, indicating the existence of at least two separate transport systems. No major difference in polyamine content was seen between the parental and the COS-MGBGr cells when grown without MGBG. In the presence of MGBG, both cell lines exhibited an increase in putrescine content. Treatment with MGBG also resulted in a decrease in spermidine and spermine contents in the wild-type cells. In the COS-MGBGr cells, on the other hand, there were no statistically significant effects on the spermidine and spermine contents by MGBG treatment. In the wild-type cells, depletion of polyamines, e.g., by treatment with the ornithine decarboxylase inhibitor 2-difluoromethylornithine (DFMO), stimulated the uptake of polyamines (3-7-fold), whereas in the COS-MGBGr cells the effect of DFMO treatment on polyamine transport was only minor. In contrast to the growth-medium of the wild-type cells, large amounts of polyamines accumulated in the medium of the COS-MGBGr cells, presumably indicating that COS cells normally excrete polyamines and then salvage them using the polyamine transport system.
SourceAvailable from: Mervi T Hyvönen
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ABSTRACT: To determine whether intracellular uptake of spermine is necessary to induce haemoglobin synthesis in murine erythroleukaemia (MEL) DS 19 cells, we used single-step selection for resistance to N1,N12-bis(ethyl)spermine (BESM), a cytotoxic spermine analogue, to isolate clones deficient in polyamine transport. The cells were approximately 500-fold more resistant to BESM than parental cells and were unable to accumulate BESM, putrescine, spermidine or spermine. Addition of spermine to the polyamine-transport-deficient cells failed to induce haemoglobin synthesis. Hexamethylene-1,6-bisacetamide, a well-known differentiating agent, induced haemoglobin synthesis in both parental and resistant cells. Polyamine-transport-deficient cells transfected with DNA purified from the parental cell line were further selected for their ability to grow in the presence of alpha-difluoromethylornithine and putrescine. The transfectants had an active transport system for polyamines, and spermine added to their culture medium accumulated inside the cells and induced haemoglobin production. These findings indicate that intracellular spermine uptake is required to induce haemoglobin production in MEL cells.Biochemical Journal 01/1996; 312 ( Pt 3):933-8. · 4.78 Impact Factor
Article: Polyamines in living organisms[Show abstract] [Hide abstract]
ABSTRACT: Natural polyamines, putrescine, spermidine and spermine are ubiquitous cell components essential for normal cellular functions and growth. Chemically these compounds are very simple organic aliphatic cations and fully protonated under physiological conditions. There is a strong correlation between proliferation rate of the cells and their polyamine contents. Adjustments of intracellular concentrations of polyamines to physiological requirements are orchestrated by de novo synthesis, polyamine uptake and catabolic reactions. De novo synthesis can in principle be substituted by polyamine uptake from extracellular environment. Over accumulation of polyamines is controlled by release and by a feedback regulation system that involves synthesis of a protein, antizyme that leads to degradation of ornithine decarboxylase and repression of polyamine uptake. The development of specific polyamine biosynthesis inhibitors and structural analogues of polyamines have revealed that maintaining polyamine levels are a prerequisite for animal cell proliferation to occur. The interruption of polyamine biosynthesis or minimizing the uptake of exogenous polyamines via the polyamine transport system offers meaningful targets for treatment of certain hyperproliferative diseases, most notably cancer. The polyamines influence confusingly large number biological processes, yet despite several decades of intensive research work, their exact functions in living organisms remains obscure. In this review, the current state of scientific knowledge regarding polyamines, their functions and their metabolism in mammalian cells is presented. Doal poliaminler putresin, spermidin ve spermin hücrenin normal ifllev ve büyümesi için esas olan yayg›n hücresel bilefliklerdir. Bu bileflikler kimyasal yönden çok basit organik alifatik katyonlard›r ve fizyolojik koflullarda tamamen protonlanm›fl durumdad›rlar. Hücrenin çoalma h›z› ile poliamin içerii aras›nda çok yak›n bir iliflki vard›r. Poliamin konsantrasyonlar›n›n fizyolojik ihtiyaca göre ayarlanmas› yeni sentez, poliamin al›nmas› ve katabolik reaksiyonlar aras›ndaki uyumlulua bal›d›r. Yeni sentez prensip olarak hücre d›fl› ortam›ndan poliamin al›nmas› ile salan›r. Poliaminlerin fazla birikimi, sal›nmas› ve protein sentezi, ornitin dekarboksilaz y›k›m›na neden olan antizim ve poliamin al›m›n›n bask›lanmas›n› içeren feedback regulasyon sistemi taraf›ndan kontrol edilir. Spesifik poliamin biyosentez inhibitörlerinin ve poliaminlerin yap›sal analoglar›n›n geliflimi, hayvan hücre çoalmas›n›n meydana gelmesinin poliamin düzeyine bal› olduunu ortaya koymufltur. Poliamin biyosentezinin kesintiye urat›lmas› veya poliamin tafl›nma sistemi vas›tas› ile d›fl ortamdan poliaminlerin al›nmas›n›n azalt›lmas›, çok h›zl› hücre çoalmas›n›n söz konusu olduu belirli hastal›klar›n, en önemlisi kanserin, tedavisinde anlaml› sonuçlar vermifltir. Poliaminler çok fazla say›daki biyolojik olaylar› etkiler, onlarca y›ld›r yap›lan youn araflt›rmalara ramen, canl› organizmalardaki tam ifllevleri hala aç›k deildir. Bu derlemede poliaminlerle ilgili son bilimsel veriler, ifllevleri ve memeli hücrelerindeki metabolizmalar› sunulmufltur. Anahtar sözcükler: Poliamin, ODC, AdoMetDC, poliamin analoglar›, kanser