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Polyamines (PAs), such as spermidine (SPD) and spermine (SPM), are essential to promote cell growth, survival, proliferation, and longevity. In the adult central nervous system (CNS), SPD and SPM are accumulated predominantly in healthy adult glial cells where PA synthesis is not present. To date, the accumulation and biosynthesis of PAs in developing astrocytes are not well understood. The purpose of the present study was to determine the contribution of uptake and/or synthesis of PAs using proliferation of neonatal astrocytes as an endpoint. We inhibited synthesis of PAs using α-difluoromethylornithine (DFMO; an inhibitor of the PA biosynthetic enzyme ornithine decarboxylase (ODC)) and inhibited uptake of PAs using trimer44NMe (PTI; a novel polyamine transport inhibitor). DFMO, but not PTI alone, blocked proliferation, suggesting that PA biosynthesis was present. Furthermore, exogenous administration of SPD rescued cell proliferation when PA synthesis was blocked by DFMO. When both synthesis and uptake of PAs were inhibited (DFMO + PTI), exogenous SPD no longer supported proliferation. These data indicate that neonatal astrocytes synthesize sufficient quantities of PAs de novo to support cell proliferation, but are also able to import exogenous PAs. This suggests that the PA uptake mechanism is present in both neonates as well as in adults and can support cell proliferation in neonatal astrocytes when ODC is blocked. [ABSTRACT FROM AUTHOR]

Since antizyme (AZ) is known to inhibit cell proliferation and to increase apoptosis, the question arises as to whether these effects occur independently of polyamines. Intestinal epithelial cells (IEC-6) were grown in control medium and medium containing 5 mM difluoromethylornithine (DFMO) to inhibit ODC, DFMO + 5 µM spermidine (SPD), DFMO + 5 µM spermine (SPM), or DFMO + 10 µM putrescine (PUT) for 4 days and various parameters of growth were measured along with AZ levels. Cell counts were significantly decreased and mean doubling times were significantly increased by DFMO. Putrescine restored growth in the presence of DFMO. However, both SPD and SPM when added with DFMO caused a much greater inhibition of growth than did DFMO alone, and both of these polyamines caused a dramatic increase in AZ. The addition of SPD or SPM to media containing DFMO + PUT significantly inhibited growth and caused a significant increase in AZ. IEC-6 cells transfected with AZ-siRNA grew more than twice as rapidly as either control cells or those incubated with DFMO, indicating that removal of AZ increases growth in cells in which polyamine synthesis is inhibited as well as in control cells. In a separate experiment, the addition of SPD increased AZ levels and inhibited growth of cells incubated with DFMO by 50 %. The addition of 10 mM asparagine (ASN) prevented the increase in AZ and restored growth to control levels. These results show that cell growth in the presence or absence of ODC activity and in the presence or absence of polyamines depends only on the levels of AZ. Therefore, the effects of AZ on cell growth are independent of polyamines. [ABSTRACT FROM AUTHOR]

Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines—such as putrescine, spermidine and spermine—are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion—by means of α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase—abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells. [ABSTRACT FROM AUTHOR]