Molecular characterization of a third member of the guanylyl cyclase-activating protein subfamily.

The mammalian retina contains at least two guanylyl cyclases (GC1 and GC2) and two guanylyl cyclase-activating proteins (GCAP1 and GCAP2). Here we present evidence of the presence of a new photoreceptor-specific GCAP, termed GCAP3, which is closely related to GCAP1. The sequence similarity of GCAP3 with GCAP1 and GCAP2 is 57 and 49%, respectively. Recombinant GCAP3 and GCAP2 stimulate GC1 and GC2 in low [Ca2+]free and inhibit GCs when [Ca2+]free is elevated, unlike GCAP1, which only stimulates GC1. GCAP3 is encoded by a distinct gene present in other mammalian species but could not be detected by genomic Southern blotting in rodents, amphibians, and lower vertebrates. The intron/exon arrangement of the GCAP3 gene is identical to that of the other GCAP genes. While the GCAP1 and GCAP2 genes are arranged in a tail-to-tail array on chromosome 6p in human, the GCAP3 gene is located on 3q13.1, suggesting an ancestral gene duplication/translocation event. The identification of multiple Ca2+-binding proteins that interact with GC is suggestive of complex regulatory mechanisms for photoreceptor GC.

Cooperativity between the polyamine pathway and HER-2neu in transformation of human mammary epithelial cells in culture: role of the MAPK pathway.

Our experiments were designed to test the cooperativity between the polyamine pathway and HER-2neu in inducing transformation of human mammary epithelial cells in culture. Using the MCF-10A breast epithelial cell line, we observed that induction of overexpression of ornithine decarboxylase (ODC) (the first rate-limiting enzyme in polyamine biosynthesis) markedly potentiated the anchorage-independent growth stimulating effect of the beta2 isoform of neu differentiating factor (NDF) known to activate HER-2neu in MCF-10A cells. ODC overexpression, on the other hand, did not enhance growth in liquid culture, thus pointing to a specific effect on transformation rather than proliferation. ODC-overexpressing MCF-10A cells exhibited increased MAPK phosphorylation in response to administration of NDF and/or epidermal growth factor (EGF). In contrast, the phosphorylation of the members of the stress-activated protein kinase cascade p38 and SEK were not affected by ODC overexpression. Of note, in the absence of EGF and NDF, ODC overexpression failed to induce both clonogenicity and MAPK activation. These results suggest that increased polyamine biosynthetic activity critically interacts with HER-2neu in promoting human mammary cell transformation in culture and that the MAPK cascade is an important mediator of this interaction. If confirmed in future in vivo studies, our results may identify important new targets for the chemoprevention of human breast cancer.

Ornithine decarboxylase over-expression stimulates mitogen-activated protein kinase and anchorage-independent growth of human breast epithelial cells.

In these experiments we tested the hypothesis that constitutive activation of polyamine(PA) biosynthesis may contribute to mammary carcinogenesis. Spontaneously immortalized normal human MCF-10A breast epithelial cells were infected with the retroviral vector pLOSN containing a cDNA which codes for a truncated and more stable ornithine decarboxylase (ODC), the rate-limiting enzyme in PA synthesis. Upon chronic selective pressure with alpha-difluoromethyl-ornithine (DFMO) (an irreversible inhibitor of ODC), infected MCF-10A cells exhibited an approximately 250-fold increase in ODC activity, which persisted despite discontinuation of DFMO. ODC-over-expressing MCF-10A cells showed a modest decrease in S-adenosylmethionine decarboxylase and an increase in spermidine/spermineN1-acetyltransferase. Analysis of cellular PA profile revealed a selective accumulation of putrescine without alterations in spermidine and spermine contents. Lesser degrees of increased ODC activity were obtained reproducibly by re-exposing the cells to incremental small doses of DFMO. We observed a bell-shaped dose-related positive effect of ODC activity on clonogenicity in soft agar of MCF-10A cells. Since anchorage-dependent growth was actually reduced, such positive influence on this feature of transformation was not a non-specific consequence of a growth advantage provided by ODC over-expression. In addition, we observed a close parallelism between the dose-dependent effects of ODC expression on clonogenicity and activity of the ERK-2 kinase, a central element of the MAPK cascade. Our data demonstrate an interaction between PA and the MAPK signalling pathway and suggest that the latter may be involved in ODC-induced transformation of mammary epithelial cells.

Biochemical and growth-modulatory effects of the new S-adenosylmethionine decarboxylase inhibitor CGP 48664 in malignant and immortalized normal human breast epithelial cells in culture.

CGP 48664 [4-aminoindanon-1-(2'-amidino)hydrazone dihydrochloride monohydrate] is a newly introduced inhibitor of S-adenosylmethionine decarboxylase (SAMDC) with increased selectivity of action and reduced toxicity. We analyzed the biochemical and antiproliferative effects of this compound in a panel of hormone-dependent (3 clones of MCF-7, T47D) and -independent (MDA-MB-231, BT-20) human breast cancer cell lines in culture. For comparison, we also tested its effects in the spontaneously immortalized human breast epithelial cell line MCF-10A. All cell lines were highly sensitive to the growth-inhibitor effect of CGP 48664 with an IC50 between 0.1 and 0.5 microM. A dose-dependent bell-shaped increase in SAMDC was observed in normal and malignant breast cells resulting from enzyme stabilization by the inhibitor as supported by Western blot analysis. While ornithine decarboxylase (ODC) activity consistently increased, the effect of CGP 48664 on spermidine/spermine N'acetyltransferase (SSAT) was variable in the breast cancer cell lines. In contrast, the inhibitor consistently reduced SSAT activity level in the MCF-10A cell line and its derivative partially transformed by a mutated ras oncogene. As expected cellular putrescine levels were markedly increased by CGP 48664 administration, whereas spermidine and spermine contents were reduced. However, the degree of reduction was usually only moderate. Furthermore, exogenous polyamine administration was relatively ineffective in rescuing the antiproliferative effect of CGP 48664 in MCF-7 cells, while exerting a more complete rescue in the MDA-MB-231 cell line. We conclude that CGP 48664 exerts a potent growth-inhibitory effect on mammary cells in culture. However, its action may not always be entirely mediated through the polyamine pathway.

Polyamine profiles and growth properties of ornithine decarboxylase overexpressing MCF-7 breast cancer cells in culture.

To determine the direct influence of the polyamine (PA) pathway on breast cancer phenotype, we employed a transfection approach to induce overexpression of the PA biosynthetic enzyme ornithine decarboxylase (ODC) in the hormone-responsive MCF-7 breast cancer cell line. Using a modified calcium phosphate method and an ODC cDNA coding for a truncated and more stable enzyme, we were able to achieve a moderate to marked degree of ODC overexpression (up to 150-fold) in a transient transfection system. ODC-overexpressing MCF-7 cells exhibited a selective increase in cellular putrescine content, while the levels of spermidine and spermine remained unaffected. Under defined culture conditions, overexpression of ODC resulted in a consistent but modest increase in [3H]thymidine incorporation into DNA which was similar in the presence and absence of 17-beta-estradiol, TGF-alpha, and IGF-I. In the presence of serum, the effect of ODC overexpression on basal [3H]-thymidine incorporation into DNA was inconsistent, possibly as a result of subtle differences in culture conditions. Overall, our results support the hypothesis that activation of the PA biosynthetic pathway may confer a growth advantage to breast cancer cells.

Phenotypic features of breast cancer cells overexpressing ornithine-decarboxylase.

Polyamines (PA) have been shown to be critical mediators of estradiol-induced breast cancer cell proliferation. This finding suggests that constitutive activation of the PA pathway may promote tumor progression, possibly leading to hormone independence. To test this hypothesis, we transfected hormone-responsive MCF-7 breast cancer cells with a complementary DNA coding for ornithine-decarboxylase (ODC), the first rate-limiting enzyme in PA biosynthesis. Marked ODC overexpression observed in stably transfected clones was associated with a selective increase in cellular putrescine content, while spermidine and spermine levels were not altered. ODC-overexpressing MCF-7 cells were resistant to the antiproliferative effects of low but not high concentrations of the enzyme inhibitor, alpha-difluoromethylornithine. In agreement with our hypothesis, sensitivity to the growth-promoting action of estradiol was reduced by approximately one third (P < 0.001) in ODC-overexpressing MCF-7 cells compared with vector-only transfected clones. Basal growth under anchorage-dependent conditions was only marginally increased by ODC overexpression (P = 0.048), while clonogenicity in soft agar was actually reduced. These data suggest that activation of PA biosynthesis may contribute in part to the acquisition of estrogen independence by breast cancer cells. Since only putrescine content was increased as a result of ODC overexpression, these data may underestimate the overall influence of the PA pathway on breast cancer phenotype.

Effects of the S-adenosylmethionine decarboxylase inhibitor, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine, on cell growth and polyamine metabolism and transport in Chinese hamster ovary cell cultures.

The regulation of polyamine transport and the roles of polyamine transport and synthesis in cell growth were investigated using cultured Chinese hamster ovary (CHO) cells and CHOMG cells which are mutants lacking polyamine-transport activity. Metabolically stable methylated polyamine analogues were used to measure polyamine accumulation, and the irreversible S-adenosyl-L-methionine decarboxylase inhibitor, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (AbeAdo), was used to inhibit synthesis. Exposure to AbeAdo lead to a dose-dependent decrease in growth for both cell lines, although CHOMG cells were more sensitive. Intracellular putrescine levels were greatly increased in AbeAdo-treated CHO cells and to a lesser extent in CHOMG cells, whereas intracellular spermidine and spermine levels were substantially reduced in both. Treatment with AbeAdo increased putrescine content in the culture medium to a much greater extent in CHOMG cultures indicating that a portion of the excess putrescine synthesized in response to AbeAdo treatment is excreted, but that CHO cells salvage this putrescine whereas it is lost to CHOMG cells which cannot take up polyamines. AbeAdo treatment increased polyamine transport into CHO cells despite high intracellular putrescine, suggesting that spermidine and/or spermine, and not putrescine, are the major factors regulating transport activity. The accumulation of either 1-methylspermidine or 1,12-dimethylspermine was significantly increased by AbeAdo treatment. Accumulation was increased even further when protein synthesis was blocked by cycloheximide, indicating that a short-lived protein is involved in the regulation of polyamine uptake. In the presence of cycloheximide and AbeAdo or alpha-difluoromethylornithine, methylated polyamine derivatives accumulated to very high levels leading to cell death. These results show that the polyamine-transport system plays an important role in retaining intracellular polyamines and that down-regulation of the transport system in response to increased intracellular polyamine content is necessary to prevent accumulation of toxic levels of polyamines.

Effects of chronic 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxy- adenosine (AbeAdo) treatment on polyamine and eIF-5A metabolism in AbeAdo-sensitive and -resistant L1210 murine leukaemia cells.

We have previously reported that prolonged chronic exposure to the S-adenosyl-L-methionine decarboxylase (AdoMetDC) inhibitor, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxy-adenosine (MDL 73811, AbeAdo), leads to cytostasis of L1210 cells [Byers, Ganem and Pegg (1992) Biochem. J. 287, 717-724]. Further studies to investigate the mechanism by which these effects are brought about were carried out by comparing an L1210-derived cell line (R20) that is resistant to AbeAdo with the parent cells. The R20 cells were derived by two rounds of AbeAdo-induced cytostasis followed by rescue with exogenous polyamines. Cytostasis was induced in L1210 cells treated for 12 days with 10 microM AbeAdo; however, exposure to up to 40 microM AbeAdo did not induce cytostasis in R20 cells. Putrescine levels were elevated and spermine levels were depleted in both treated L1210 and treated R20 cells. Spermidine was depleted in treated L1210 cells but was only partly reduced in treated R20 cells. AdoMetDC activity was below the limit of detection in treated L1210 cells but, although greatly reduced, could be measured in the treated R20 cells. The resistance of the R20 cells to the effects of AbeAdo on cell growth and spermidine depletion correlated with reduced AbeAdo accumulation by R20 cells. In the absence of spermidine synthesis, unhypusinated eukaryotic translation initiation factor 5A (eIF-5A) accumulated in AbeAdo-treated L1210 cells. There was no detectable accumulation of unhypusinated eIF-5A in R20 cells. Unhypusinated eIF-5A accumulated during AbeAdo treatment was depleted in L1210 cells rescued by exogenous spermidine. These findings are consistent with the hypothesis that AbeAdo-induced cytostasis is due to the loss of hypusinated eIF-5A. However, spermine was able to rescue AbeAdo-treated L1210 cells without significantly reducing the unhypusinated eIF-5A accumulated during AbeAdo treatment, suggesting that only a small amount of the unmodified protein must be hypusinated to restore cell growth.

An early enlargement of the putrescine pool is required for growth in L1210 mouse leukemia cells under hypoosmotic stress.

Hypoosmotic stress is a potent inducer of ornithine decarboxylase (ODC) activity in a variety of mammalian cells, but the physiological relevance of this response has not been determined. To test whether an increased putrescine content confers a growth advantage at lower osmolarities, we compared the ability of L1210 mouse leukemia cells and of ODC-overproducing variants obtained from this cell line (D-R cells) to proliferate after a hypotonic shock (325----130 mosmol/kg). The growth rate of D-R cells at 130 mosmol/kg was greater than or equal to 5-fold higher than in L1210 cells; and unlike the ODC-overproducing strain, L1210 cells underwent up to a 90% loss of viability over time as seen after restoration of normosmotic growth conditions and by trypan blue exclusion tests. The addition of putrescine or L-ornithine stimulated the proliferation of both cell sublines up to 5-fold in a concentration-dependent manner, with a maximal effect observed at about 10 and 100 microM, respectively. Putrescine restored virtually normal growth rates in both sublines at osmolarities as low as 190 mosmol/kg. No other alpha,omega-diamine was active in that respect whereas spermidine was markedly inhibitory. Furthermore, D-R cells incubated at 130 mosmol/kg showed a marked growth inhibition by 1-aminooxy-3-aminopropane (potent ODC inhibitor to which they are resistant in isotonic media) as a result of putrescine but not spermidine depletion. Whereas ODC was strongly and rapidly induced by hypotonic shock there was a precipitous decline in S-adenosylmethionine decarboxylase activity. Putrescine synthesis and accumulation were nevertheless reduced in D-R cells incubated at 130 mosmol/kg because of a decreased availability of L-ornithine. When either putrescine or L-ornithine was added to hypotonic media, D-R cells accumulated putrescine massively for extended periods together with a reduction in spermidine and spermine contents. Putrescine transport patterns were altered by hypotonic shock, net excretion of the diamine being reduced by about 80%, with a concurrent enlargement of the intracellular pool. Finally, parental L1210 cells incubated with an irreversible inhibitor of S-adenosylmethionine decarboxylase for 24 h until hypotonic shock and supplemented with putrescine in the presence of the drug thereafter exhibited a greatly exaggerated growth stimulation by the diamine. These results demonstrate an essential role for an early increase in putrescine content in the growth adaptation of a mammalian cell line to a lower osmolarity.

Expression of a human gene for polyamine transport in Chinese-hamster ovary cells.

A molecular-genetic approach towards isolating mammalian polyamine-transport genes and their encoded proteins was devised involving the production of Chinese-hamster ovary (CHO) cells expressing a human polyamine-transport protein. CHO cells and a polyamine-transport-deficient CHO mutant cell line (CHOMG) were equally sensitive to the antiproliferative effects of alpha-difluoromethylornithine (DFMO), which blocked endogenous polyamine synthesis. Exposure to exogenous polyamines increased intracellular polyamine levels and reversed this DFMO-induced cytostasis in the CHO cells, but not in the CHOMG cells. CHOMG cells were therefore transfected with human DNA (isolated from HT-29 colon carcinoma cells) and cells expressing the human polyamine-transport system were identified by the ability of these cells to grow in a medium containing DFMO and polyamines. A number of different positive clones were identified and shown to have the capacity for polyamine uptake and an increased sensitivity to the toxic effects of the polyamine analogue methylglyoxal bis(guanylhydrazone). Differences in these properties between the clones are consistent with a multiplicity of polyamine-transport systems. Some clones also showed a change in growth characteristics, which may indicate a relationship between genes involved in the polyamine-transport system and in cell proliferation.

Effect of S-adenosyl-1,12-diamino-3-thio-9-azadodecane, a multisubstrate adduct inhibitor of spermine synthase, on polyamine metabolism in mammalian cells.

The effects of the potent spermine synthase inhibitor S-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDatad) on polyamine biosynthesis have been studied in transformed mouse fibroblasts (SV 3T3 cells) and in mouse leukemia cells (L1210). A dose-dependent decrease in intracellular spermine concentration was observed in both cell lines when grown in the presence of the inhibitor. A major difference in the effects seen in these two cell lines was the cytotoxicity observed in L1210 cells exposed to the inhibitor, which contrasted with little or no effects on growth of SV 3T3 cells treated similarly. Oxidative metabolism of the drug in L1210 cells was suggested by the fact that addition of aminoguanidine, an amine oxidase inhibitor, to the cell cultures ablated the cytotoxic effects of the inhibitor. Complete analysis of intracellular polyamines was carried out, together with analysis of S-adenosylmethionine, decarboxylated S-adenosylmethionine, and the inhibitor. These analyses revealed that, although the inhibitor had a dramatic effect on spermine biosynthesis in the cells studied, a compensatory increase in spermidine biosynthesis was observed. This resulted in no change in total polyamine concentrations in cells treated with inhibitors of either spermine synthase or spermidine synthase (Pegg et al., 1982) alone or in combination. In all cases, the concentration of the aminopropyl donor decarboxylated S-adenosylmethionine increased dramatically, thus allowing for the observed maintenance of total polyamine levels even in the presence of either one or both potent inhibitors of the aminopropyltransferases. Oxidative metabolism of the inhibitor complicates the interpretation of experiments carried out in the absence of amine oxidase inhibitors such as aminoguanidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of N1,N12-bis(ethyl)spermine and related compounds on growth and polyamine acetylation, content, and excretion in human colon tumor cells.

Exposure of human colon tumor (HT 29 cells) to N1,N12-bis(ethyl)spermine and analogs produced a rapid loss of intracellular polyamines. This loss was brought about predominantly by an increased excretion of spermidine. N1,N11-Bis(ethyl)norspermine and N1,N12-Bis(ethyl)spermine were potent inducers of spermidine/spermine N1-acetyltransferase, and this induction facilitated the efflux of polyamines by enhancing the conversion of spermine into spermidine. N1,N14-Bis(ethyl)homospermine, which did not induce spermidine/spermine N1-acetyltransferase, also caused the loss of spermidine from the cell but was less effective in bringing about the decline in intracellular spermine. These results indicate that cellular polyamine levels can be regulated by excretion of spermidine and that the bis(ethyl)spermine derivatives deplete intracellular polyamine content by interference with this process.

Increase in S-adenosylmethionine decarboxylase in SV-3T3 cells treated with S-methyl-5'-methylthioadenosine.

Treatment of SV-3T3 cells with the spermine synthase inhibitor S-methyl-5'-methylthioadenosine [AdoS+(CH3)2] led to a large increase in the activity of S-adenosylmethionine decarboxylase (AdoMetDC) without affecting ornithine decarboxylase. The elevation of AdoMetDC activity was due to an increased amount of enzyme protein, as demonstrated by radioimmunoassay and by immunoblotting. The increase in AdoMetDC protein was caused by at least three factors: an increase in the amount of translatable mRNA, an increased translation efficiency of the mRNA and an increase in the half-life of the protein. The depletion of spermine brought about by AdoS+(CH3)2 appeared to be responsible for the increased synthesis of AdoMetDC and for part of the decrease in its rate of degradation. An additional stabilization of the enzyme protein was probably due to the binding of AdoS+(CH3)2, which is also a weak inhibitor of AdoMetDC. These results demonstrate the importance of cellular spermine concentrations in regulating the activity of AdoMetDC, which is a key enzyme controlling polyamine synthesis.

Acetylation of decarboxylated S-adenosylmethionine by mammalian cells.

Decarboxylated S-adenosylmethionine was found to be a substrate for the nuclear acetyltransferases that act on polyamines and on histones. The rate of acetylation of decarboxylated S-adenosylmethionine was more than twice that of spermidine at saturating substrate concentrations, and decarboxylated S-adenosylmethionine was an active inhibitor of the acetylation of histones by nuclear extracts from rat liver. The acetylation of decarboxylated S-adenosylmethionine occurred in vivo in SV-3T3 cells exposed to the ornithine decarboxylase inhibitor 2-(difluoromethyl)ornithine. The decline in putrescine and spermidine brought about by exposure to 2-(difluoromethyl)ornithine was found to be accompanied by a large rise in the content of both decarboxylated S-adenosylmethionine and acetylated decarboxylated S-adenosylmethionine. These results indicate that decarboxylated S-adenosylmethionine is metabolized not only in the well-known reactions in which it serves as an aminopropyl donor for polyamine biosynthesis but also by acetylation in reaction with acetyl coenzyme A. Furthermore, the inhibition of histone acetylation by decarboxylated S-adenosylmethionine could contribute to the biological effects brought about by inhibitors of ornithine decarboxylase.

Vimentin synthesis by ocular lens cells.

A study of the synthesis of the intermediate filament protein, vimentin, is reported here. The following systems were examined: the epithelial cells of the organ-cultured rabbit lens, the epithelial and cortical fiber cells of the organ-cultured adult chicken lens and the epithelial cells of the rabbit lens grown in tissue culture. Vimentin is actively synthesized by all of the above mentioned cells.