Neocortex patterning by the secreted signaling molecule FGF8.

A classic model proposes that the mammalian neocortex is divided into areas early in neurogenesis, but the molecular mechanisms that generate the area map have been elusive. Here we provide evidence that FGF8 regulates development of the map from a source in the anterior telencephalon. Using electroporation-mediated gene transfer in mouse embryos, we show that augmenting the endogenous anterior FGF8 signal shifts area boundaries posteriorly, reducing the signal shifts them anteriorly, and introducing a posterior source of FGF8 elicits partial area duplications, revealed by ectopic somatosensory barrel fields. These findings support a role for FGF signaling in specifying positional identity in the neocortex.

Increase in cap- and IRES-dependent protein synthesis by overproduction of translation initiation factor eIF4G.

The role of eIF4G during the initiation of protein synthesis was studied using mouse mammary carcinoma FM3A cells and FM4G cells that overproduce an N-terminally truncated form of eIF4G, which lacks the binding site of poly(A)-binding protein. An increase in eIF4G was correlated with an increase in protein synthesis and RNA helicase activity. Translation of mRNAshaving both short and long 5'-untranslated regions (5'-UTR) increased significantly in FM4G cells compared to that in FM3A cells. Both full-length and N-terminally truncated eIF4G transfectants of NIH3T3 cells formed colonies in soft agar and increased the saturation density of cell growth, indicating that both eIF4Gs function similarly. We also found that an internal ribosome entry site (IRES) exists in the 5'-UTR of ornithinedecarboxylase mRNA and that IRES-dependent protein synthesis increased in FM4G cells. Our results indicate that an increase in eIF4G contributes to the formation of active eIF4F similarly to that caused by an increase in eIF4E, as well as to a stimulation of IRES-dependent protein synthesis.

Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation.

Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation Cell proliferation and transformation induced by growth factor stimulation or by carcinogens, viruses, or oncogenes are characterized by an associated increase in polyamine levels, which is mediated by increased polyamine biosynthesis and enhanced uptake of polyamines. Polyamine biosynthesis is catalyzed particularly, in the level of ornithine decarboxylase (ODC). The elevation of cellular polyamine levels on the other hand accelerates the induction of ornithine decarboxylase antizyme (antizyme), which is involved not only in ODC-degradation, but in the negative regulation of polyamine transport. Taking advantage of these characteristics of antizyme, the potential of antizyme as a factor having anti-cell growth and anti-tumor activity was investigated. We show that antizyme can induce cell death associated with a rapid decline of intracellular polyamine contents. The possible anti-tumor activities of ectopically expressed antizyme were tested in p21H-ras (Val 12)-transformed NIH3T3 cells and several human malignant cell lines including a line with loss of p53 expression, and they were shown to be as sensitive as nontransformed NIH3T3 cells in vitro. The in vivo anti-tumor activity was also tested using nude mice inoculated with H-ras transformed NIH3T3 cells that had been transfected with inducible antizyme expression vector and the results showed that antizyme expression in vivo blocks tumor formation in these mice. These results suggest that ectopic antizyme expression is of possible therapeutic benefit in the treatment of cancer, which is mediated by ODC inactivation and intracellular polyamine depletion.

Transcriptional inhibition of the operon for the spermidine uptake system by the substrate-binding protein PotD.

Inhibition of spermidine uptake in Escherichia coli, which occurs in the presence of accumulated polyamines, has been studied using the spermidine uptake operon consisting of the potA, -B, -C, and -D genes. Transcription of the potABCD operon was inhibited by PotD, a spermidine-binding protein usually found in the periplasm, and the inhibitory effect of PotD was increased by spermidine. Transcription was not affected by bovine serum albumin, PotA, or PotF, suggesting that the effects of PotD are specific to the PotD protein. In the presence of 8 mM spermidine, a 50% inhibition of transcription was observed with a molar ratio of approximately 1:500 of template DNA:PotD. It was found that PotD bound to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream of the ATG initiation codon of the potA gene. Binding of PotD to the downstream site was stimulated by spermidine. Overexpression of PotD in Escherichia coli DH5alpha inhibited the uptake of spermidine, the synthesis of PotABCD mRNA, and expression of a lacZ reporter gene fused downstream of a potA gene containing the PotD binding sites. In cells overexpressing PotD, a large amount of PotD existed as PotD precursor in spheroplasts. Our results indicate that PotD precursor can also inhibit spermidine transport. The amino acid residues in PotD that are involved in its interaction with the potABCD operon were determined using mutated PotD proteins. Thr-35 and Ser-85 of PotD were found to be important for this interaction. These results suggest that transcription of the spermidine transport (potABCD) operon is inhibited in vivo by PotD precursor rather than PotD through its binding to two regions close to the transcriptional initiation site of the operon.

Formation of a complex containing ATP, Mg2+, and spermine. Structural evidence and biological significance.

The conformation of ATP in the presence of Mg2+ and/or spermine was studied by 31P and 1H NMR, to clarify how polyamines interact with ATP. Spermine predominantly interacted with the beta- and gamma-phosphates of ATP in the presence of Mg2+. A conformational change of the beta- and gamma-phosphate of ATP with spermine could not be observed in the absence of Mg2+ by 31P NMR. It was found by 1H NMR that the conformation of adenosine moiety of ATP was not influenced significantly by spermine. The binding of Mg2+ to ATP was slightly inhibited by spermine and vice versa. The results indicate that the binding sites of Mg2+ and spermine on ATP only partially overlap. The PotA protein, an ATP-dependent enzyme, was used as a model system to study the biological role of the ATP-Mg2+-spermine complex. The ATPase activity of PotA was greatly enhanced by spermine. Double reciprocal plots at several concentrations of spermine as an activator indicate that spermine interacts with ATP, but not with PotA. The activity of protein kinase A was also stimulated about 2-fold by spermine. The results suggest that a ternary complex of ATP-Mg2+-spermine may play an important role in some ATP-dependent reactions in vivo and in the physiological effects of endogenous polyamines.

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in early Xenopus embryos induces cell dissociation and inhibits transition from the blastula to gastrula stage.

Xenopus early embryos contain relatively low levels of S-adenosyl-methionine decarboxylase (SAMDC) and its mRNA. When SAMDC mRNA was injected into Xenopus embryos, it was preserved until the blastula stage and induced a large increase in SAMDC activity. The SAMDC-overexpressed embryos developed normally until the blastula stage but at the early gastrula stage cells which received the mRNA, dissociated autonomously and stopped synthesizing protein. In a hypotonic medium, the dissociated cells, and hence whole embryos, autolyzed. However, in isotonic media dissociated cells did not autolyze, although they did not divide and their DNA and RNA synthesis activity was greatly inhibited. The effects of SAMDC overexpression were abolished by coinjection of ethylglyoxal-bis(guanylhydrazone) (EGBG), a specific inhibitor of SAMDC. In SAMDC-overexpressed embryos the level of putrescine decreased and that of spermidine increased, though to limited extents, resulting in a considerable decrease in the putrescine/spermidine ratio. However, direct injection of spermidine did not mimic the effect of SAMDC overexpression, and putrescine coinjected with SAMDC mRNA to maintain the normal putrescine/spermidine ratio did not rescue the embryos. Conversely, the level of S-adenosylmethionine (SAM) greatly decreased and coinjection of SAM, which restored the level of SAM, rescued the embryos. We concluded that in SAMDC-overexpressed embryos a SAM-deficient state was induced and this caused cell dissociation and inhibition of transition from the blastula to gastrula stage. We suggest that the SAM-deficient embryos obtained in the present study provide a unique system for studying the cellular control mechanism underlying the blastula-gastrula transition.

Malignant transformation by overproduction of translation initiation factor eIF4G.

N4G3, a cell line that overexpresses translation initiation factor eIF4G, one of the components of eIF4F, was made by stable transfection of the human eIF4G cDNA into NIH3T3 cells. The cells expressed 80-100 times greater levels of eIF4G mRNA than did NIH3T3 cells. N4G3 cells formed transformed foci on a monolayer of cells, showed anchorage-independent growth, and formed tumors in nude mice. These results indicate that overexpression of eIF4G caused malignant transformation of NIH3T3 cells. It is also known that overexpression of eIF4E, another component of eIF4F, causes transformation of NIH3T3 cells. However, there was no difference in the amount of eIF4E protein between N4G3 and NIH3T3 cells, indicating that cell transformation does not involve a change in eIF4E levels. The results may be due to an effect of eIF4G on translational control of protein synthesis directed by mRNAs having long 5'-untranslated region.

Identification of regulatory region of antizyme necessary for the negative regulation of polyamine transport.

Antizyme is a negative regulator of ornithine decarboxylase (ODC) and of polyamine transport. Regions of antizyme necessary for the negative regulation of polyamine transport were determined by transfecting ODC-overproducing EXOD-1 cells with mutant antizyme genes containing different size deletions in the NH2- and COOH-terminal of antizyme (AZ69-227). When peptide 119-144 or peptide 211-216, which are responsible for the binding of ODC, were deleted from antizyme, the mutant antizyme could not reverse the inhibition of growth of EXOD-1 cells produced by spermine. In parallel with the decrease in antizyme effect on cell growth, spermine transport activity and the accumulation of spermine in EXOD-1 cells were not significantly altered by the mutant antizyme, whereas wild-type antizyme decreased spermine transport and accumulation. When the peptide 69-118, which is responsible for the degradation of ODC, was deleted from antizyme, the mutant antizyme showed a smaller effect compared with the normal antizyme in terms of the inhibition of spermine transport and the recovery from the spermine inhibition of cell growth. The results indicate that regions 119-144 and 211-216 in antizyme are necessary for the negative regulation of polyamine transport and that these regions overlap with ODC binding sites.

Spermidine regulation of protein synthesis at the level of initiation complex formation of Met-tRNAi, mRNA and ribosomes.

Spermidine regulation of protein synthesis (stimulation at low concentrations and inhibition at high concentrations) was studied using a mRNA with a GC-rich 5'-untranslated region. It was found that the initiation complex formation of mRNA, Met-tRNAi, and 40 S ribosomal subunits was regulated by spermidine. The inhibition of initiation complex formation at high spermidine concentrations was greater with 80 S ribosomes than with 40S ribosomal subunits. This was partially explained by the spermidine inhibition of initiation factor-dependent RNA helicase activity.

Enhancement of helicase activity and increase of eIF-4E phosphorylation in ornithine decarboxylase-overproducing cells.

In mouse FM3A ornithine decarboxylase (ODC) overproducing cells (EXOD-1), the amount of ODC protein was approximately 100-fold that of normal cells. Since it is well known that the translational efficiency of ODC mRNA is very low and that eIF-4E is a limiting factor for the mRNA recognition and the scanning of 40 S ribosomal subunits, we measured the amount and phosphorylation of eIF-4E in EXOD-1 cells. An increase in the phosphorylation of eIF-4E, its association with p220 protein, and an enhancement of RNA helicase activity were observed in the cells. These results support the hypothesis that phosphorylation of eIF-4E enhances RNA helicase activity through eIF-4F (4A, 4E, and p220) complex formation.

Inhibition of the growth of various human and mouse tumor cells by 1,15-bis(ethylamino)-4,8,12-triazapentadecane.

Effects of 1,15-bis(ethylamino)-4,8,12-triazapentadecane (BE3333), the least toxic bis(ethyl)pentaamine, on the growth of tumor cells were studied in in vitro systems and with tumor xenografts in mice. BE3333 suppressed ornithine decarboxylase and S-adenosylmethionine decarboxylase, induced spermidine/spermine N1-acetyltransferase, and thus decreased the amount of polyamines. BE3333 accumulated in cells at a concentration 3-5-fold that of spermine in control cells through the polyamine transport system. The accumulated BE3333 inhibited protein synthesis, especially mitochondrial protein synthesis, and decreased the amount of ATP. The inhibition of protein synthesis was correlated with the subsequent inhibition of cell growth. BE3333 showed inhibitory effects in in vitro systems against the growth of mouse FM3A mammary carcinoma cells, human SW480 and SW620 colon tumor cells, Lu-65A and A549 lung tumor cells, MCF-7 breast tumor cells, and MALME-3M and A375 melanoma cells at a range of 0.5-10 microM. Intravenous (30 mg/kg) or i.p. (50 mg/kg) daily injections of BE3333 for 5 or 7 days greatly suppressed the growth of human colon tumor SW620 xenotransplanted into nude mice. Similar antitumor activity was obtained with continuous infusion of BE3333 into the peritoneal cavity (80 mg/kg), but not with p.o. administration (200 mg/kg). BE3333 also showed inhibitory effects against the growth of lung tumors (Lu-65, Lx-1, Lc-1, and Lu-61), stomach tumors (Sc-6 and St-15), and melanoma (SEKI) xenotransplanted into nude mice. The results indicate that BE3333 is effective against both rapid- and slow-growing tumors, with reasonable short-term host toxicity.

Inhibition of cell growth by combination of alpha-difluoromethylornithine and an inhibitor of spermine synthase.

The inhibitory effect on cell growth of a combination of alpha-difluoromethylornithine (DFMO) and an inhibitor of aminopropyl transferase was examined. N-(3-aminopropyl)cyclohexylamine (APCHA) and trans-4-methylcyclohexylamine (4MCHA) were used as inhibitors of spermine and spermidine synthases, respectively. Combination of DFMO and APCHA showed strong inhibitory effects on the growth of FM3A cultured cells and P388 leukemia cells in mice, compared with DFMO alone. The prolongation of survival time of P388 leukemia-bearing mice by DFMO (1,500 mg/kg) was 1.12-fold, while that by DFMO (1,500 mg/kg) plus APCHA (25 mg/kg) was 1.30-fold. The prolongation of survival time nearly paralleled the decrease of P388 leukemia cells in mice. However, the antiproliferative effect of DFMO was not strengthened by 4MCHA in the above two experimental systems. In the FM3A cell culture system, both putrescine and spermidine contents were decreased by DFMO, but spermine content did not decrease significantly. When APCHA was added to the medium with DFMO, spermine content was decreased greatly but a compensatory increase in spermidine was observed. Spermidine content in P388 leukemia cells was also decreased by DFMO, the increase in spermine was suppressed but a compensatory increase in spermidine was observed. Nevertheless, the spermidine content remained significantly low compared with the value in non-treated P388 leukemia cells. Thus, the results indicate that the antiproliferative effect of DFMO was strengthened by APCHA due to the decrease in spermine content, and that the decrease in total amount of spermidine and spermine, especially the decrease in spermine, is necessary for inhibition of cell growth.