Ornithine Decarboxylase mRNA Levels Research Articles

PC12 Variants with Altered Ornithine Decarboxylase Expression Patterns Exhibit Altered Signal Transduction Capabilities

Nerve growth factor (NGF) induced differentiation of primary and permanent neuronal cell cultures is accompanied by a rapid and transient induction of ornithine decarboxylase (ODC) mRNA and enzymatic activity; a similar ODC induction accompanies mitogenic effectors in many additional cell types. In an effort to assess the role of ODC activity in neuronal cell biology, we have used the ODC suicide substrate inhibitor difluoromethylornithine (DFMO) to select PC12 cell variants with altered ODC expression patterns and characterized the resulting phenotypes. The variants fall into three distinct classes based upon their patterns of ODC mRNA and ODC activity levels; however, all are severely compromised in their ability to respond properly to NGF treatment. Following NGF treatment, none of the variants exhibits any morphological differentiation. In addition, none of the variants is capable of properly inducing either c-fos mRNA (an "immediate early" response) or GAP-43 mRNA (a "late" response) following NGF treatment. Our data suggest that altered ODC metabolism can lead to inactivation of element(s) active very early in the normal NGF signal transduction cascade.

Ornithine Decarboxylase and S-Adenosylmethionine Decarboxylase Expression during the Cell Cycle of Chinese Hamster Ovary Cells

Cells in mitosis were harvested from exponentially growing Chinese hamster ovary cells by the mitotic detachment technique. Immediately after harvesting, the mitotic cells were seeded in tissue culture flasks and incubated at 37°C in a CO2 incubator. Care was taken not to perturb the progression of cells through the cell cycle. At every hour after seeding for 14 h, cells were collected for analysis of cell cycle distribution, cellular polyamine content, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities, and relative mRNA contents. The progression through the cell cycle was monitored by DNA flow cytometry. The putrescine, spermidine, and spermine levels were approximately doubled during the cell cycle: putrescine mainly during late S and G2, spermidine continuously during the entire cell cycle, and spermine mainly during G1 and S. The ODC activity was low in seeded mitotic cells and the enzyme was activated in late G1 and reached a plateau in S phase. A second burst in activity was observed during late S phase and maximal ODC activity was found at the S/G2 transition. The relative ODC mRNA level approximately doubled during the cell cycle and the increase in the relative level mainly took part during mid and late S phase. AdoMetDC activity increased in late G1 and a first maximum was observed during the G1/S transition. A second burst in activity was found in mid S phase. Maximal AdoMetDC activity was found in G2. The relative AdoMetDC mRNA approximately doubled during the cell cycle and the increase in the relative level mainly took place during late G1 and early S phase. Our results indicate that polyamine synthesis was regulated at transcriptional and translational/post-translational levels during the cell cycle of Chinese hamster ovary cells.

Ornithine decarboxylase gene expression is aberrantly regulated via the cAMP signal transduction pathway in malignant H-ras transformed cell lines.

We have tested the hypothesis that H-ras transformed cells contain alterations in signal pathways important in controlling the expression of ornithine decarboxylase (ODC), the highly regulated rate-limiting activity in the biosynthesis of polyamines. Mouse 10T1/2 fibroblasts and a series of 10T1/2 H-ras transformed cell lines were treated with stimulators of cAMP synthesis (forskolin and cholera toxin), a biologically stable analogue of cAMP (8-bromo-cAMP), and an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine). Elevations in ODC gene expression were noted in H-ras transformed cells that were not observed in parental 10T1/2 fibroblasts. The forskolin-mediated effects were not detected with 1,9-dideoxyforskolin, a compound structurally related to forskolin, which does not activate adenyl cyclase. The effects observed with cholera toxin were not detected when cells were treated with the purified subunits of this compound, indicating that the toxin-induced effects were cAMP-specific. Actinomycin D treatment prior to forskolin exposure reduced the elevation observed in ODC gene expression indicating the involvement of the transcriptional process. Furthermore, we observed that cycloheximide treatment of malignant but not benign H-ras transformed cells significantly elevated ODC message level. Treatment of malignant cells with both cycloheximide and forskolin together resulted in a further additive elevation in ODC message, but a similar treatment of benign tumor cells reduced the forskolin-mediated increase in ODC message. In addition, treatment of H-ras transformed cells with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) led to an elevation in ODC mRNA levels not observed in parental 10T1/2 fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade of ornithine decarboxylase enzyme protects against ischemic brain damage.

Polyamines are derived from ornithine by the actions of ornithine decarboxylase (ODC), which is the rate-limiting step in this pathway. Polyamines play a role in cell growth, neoplasia, differentiation, and response to injury. We have shown that transient cerebral ischemia gives rise to increased ODC mRNA and enzyme activity in the gerbil brain. ODC and polyamines are thought to be important in the generation of edema and the neuronal cell loss associated with cerebral ischemia. To test this theory, we examined the ODC activity, putrescine levels, and neuronal density in the CA1 region of the hippocampus following ischemia and reperfusion injury in the absence and presence of an inhibitor of ODC activity, alpha-difluoromethylornithine (DFMO). Pretreatment of animals with DFMO resulted in attenuation of the ODC activity following 5 min of ischemia and 4 h of reperfusion. In addition, DFMO prevented the increase in polyamine levels, as determined by measurement of putrescine in the ischemic brain. These alterations were not due to changes in ODC mRNA level. Further analysis revealed that DFMO treatment blocked the delayed neuronal cell death in the CA1 region of the hippocampus that accompanies ischemia and reperfusion injury. Administration of DFMO resulted in a dose-dependent beneficial effect upon neuronal cell survival. These results suggest that ODC enzyme activity and the production of polyamines play a significant role in the response of the brain to ischemic injury.

Expression patterns of ornithine decarboxylase and c-met in growing Yoshida AH-130 hepatoma.

The expression of the two proto-oncogenes ornithine decarboxylase and c-met was examined during various phases of growth of Yoshida AH-130 ascites hepatoma. Ornithine decarboxylase (ODC) and c-met mRNA levels declined progressively from day 5 (exponential growth-phase) until day 14 (quasi-stationary growth-phase). Transcription rate for both the genes remained constant between days 5 and 10, while decreasing at day 14. ODC activity was consistent with ODC mRNA level during hepatoma growth. In host liver, ODC mRNA accumulated 5 and 14 days after tumor transplantation, while c-met mRNA level was elevated until day 10 and diminished at day 14. ODC activity triplicated at day 14 in host liver. The progressive decline in the expression of ODC and c-met observed in hepatoma might be one of the mechanisms important for the control of tumor growth.

Post-Transcriptional Suppression of Human Ornithine Decarboxylase Gene Expression by Phorbol Esters in Human Keratinocytes

The induction of ornithine decarboxylase levels by the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in mouse skin has been shown to be integral to tumor promotion by TPA, and changes in ornithine decarboxylase activity indicate the proliferative state of many different cell types. However, in cultured human epidermal cells, TPA has been reported to be antiproliferative. Therefore, to elucidate pathways that TPA activates in cultured human skin cells, we have examined the levels at which TPA regulates ornithine decarboxylase gene expression in two immortalized human epidermal keratinocyte cell lines, and in normal neonatal keratinocytes. We have found that in cultured human keratinocytes, TPA cases a marked decrease in ornithine decarboxylase enzyme activity (50-90%), with no detectable effect on ornithine decarboxylase mRNA levels. TPA decreased steady-state levels of ornithine decarboxylase immunoreactive protein (approximately 50-67%), accounting for the 50-90% suppression of ornithine decarboxylase activity levels, as well as decreasing new synthesis of ornithine decarboxylase protein (48-50%). However, measurement of ornithine decarboxylase protein half-life showed no significant effect of TPA. Also, prolonged treatment of keratinocytes with phorbol esters abolished the suppression of ornithine decarboxylase activity by TPA. Our data, therefore, suggest that phorbol esters suppress ornithine decarboxylase gene expression predominantly by decreasing ornithine decarboxylase mRNA translatability.

Relationship of Ornithine Decarboxylase Activity and Histological Findings in Human Hepatocellular Carcinoma

To evaluate the correlation between the polyamine metabolism and the degree of malignancy in hepatocellular carcinoma, we measured ornithine decarboxylase activity and polyamine concentrations in neoplastic tissue and adjacent noncancerous tissue from resected specimens of liver from 30 patients. Ornithine decarboxylase activity, polyamines (putrescine, spermidine and spermine) and ornithine decarboxylase mRNA levels were significantly higher in hepatoma tissue than in noncancerous tissue. The activity of this enzyme in the tumor tissue had a negative correlation with the histological degree of differentiation judged according to a modification of the Edmondson and Steiner classification. Resected hepatoma tissue was stained immunohistochemically with antibodies for proliferating cell nuclear antigen (also called cyclin), a marker of cell proliferation. We noted correlation between ornithine decarboxylase activity and the number of cells stained for this antigen (r = 0.882, p < 0.001). These results indicate that ornithine decarboxylase activity is high in human hepatocellular carcinoma, leading to increased intracellular concentrations of polyamines. Ornithine decarboxylase activity also reflected the rate of tumor proliferation and was correlated with the histological findings.

Asparagine markedly induces the expression of ornithine decarboxylase gene in transformed mammalian cells but not in their untrasformed counterparts

We have previously shown that asparagine alone induces a 10–15-fold increase in ornithine decarboxylase (ODC) mRNA level in DF-40 mouse neuroblastoma cells. The induction is due to an accumulation of ODC mRNA through a post-transcriptional stabilization mechanism (Chen, Z.P. and Chen, K.Y. (1992) J. Biol. Chem., 267, 6946–6951). In the present study we showed that asparagine induced ODC gene expression in v-Ha- ras-transformed 3T3 ( ras-3T3) cells but not in 3T3 cells. Other growth related genes including c- src, c- ras, and c- fos were not affected by asparagine in ras-3T3 cells. Southern blot analysis indicated that the pronounced asparagine effect was not due to ODC gene amplification in ras-3T3 cells. The effect of asparagine on the induction of ODC mRNA could account for the significant increases in the ODC activity in ras-3T3 cells. We also examined the effect of asparagine on ODC gene expression in human KD cells and their transformed counterparts. Our findings strongly suggest that the induction of ODC mRNA by asparagine may represent a component of an altered growth regulatory program associated most prominently with cell transformation.

Post-transcriptional inhibition of ornithine decarboxylase induction by zinc in a difluoromethylornithine resistant cell line

Addition of Zn 2+_ to cell medium inhibited the induction of ornithine decarboxylase (ODC) activity in ODC overproducing L1210-DFMO r cells. A significant effect was observed at a concentration as low as 0.01 mM, however a more marked inhibition was caused by the addition of 0.1 mM Zn 2+. The inhibition of the induction of ODC activity was accompanied by a proportional decrease in the content of immunoreactive ODC protein, whereas the level of ODC mRNA, detemined by a solution hybridization RNase protection assay, was not affected signigicantly. Instead, some acceleration of ODC turnover was observed. the addition of 0.1 mM Co 2+ or Mn 2+, but not of other divalent metal ions, also inhibited ODC induction; differently from Zn 2+ however, these metals affected cell viability and/or cell growth. Removal of endogenous Zn 2+ by a chelator also provoked a strong decrease of ODC induction, which was reversed by Zn 2+. However, addition of Zn 2+ in excess of the chelator proved to be markedly inhibitory. These results indicate that both a restricted Zn 2+ availability and an enhanced presence of the metal can inhibit the induction of ODC in L1210-DFMO r cells.

Secretin inhibits induction of ornithine decarboxylase activity by gastrin in duodenal mucosa and IEC-6 cells

Ornithine decarboxylase (ODC) catalyzes the first rate-limiting step in polyamine biosynthesis, and increased ODC activity is one of the earliest biochemical events associated with the induction of cellular proliferation. The current study examines the regulation of ODC activity in rat duodenal mucosa and IEC-6 cells (a line of normal rat intestinal crypt cells) in response to the trophic hormone, gastrin, and its inhibitor, secretin. Rats were fasted 22 h before the various treatments, and ODC activity was measured in scraped duodenal mucosa. Gastrin significantly increased ODC activity within 3 h to 4.3 times control levels. The effect of gastrin was totally inhibited by 5 micrograms/kg secretin. In doses of 5 or 10 micrograms/kg, secretin had no effect on basal ODC. Epidermal growth factor (EGF) and refeeding fasted rats also significantly increased ODC activity in duodenal mucosa, but the effects of EGF and refeeding were not prevented by secretin. In cultured IEC-6 cells, ODC activity was significantly increased after exposure to gastrin, 5% dialyzed fetal bovine serum (FBS), EGF, and asparagine. Secretin in doses ranging from 10(-10) to 10(-6) M caused a linear and significant inhibition of the stimulation of ODC activity by gastrin. No dose of secretin affected basal ODC activity or enzyme activity stimulated by 5% dialyzed FBS, EGF, or asparagine in IEC-6 cells. The ODC mRNA levels in IEC-6 cells were also increased after exposure to gastrin. Administration of secretin significantly prevented the stimulated expression of the ODC gene in cells treated with gastrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth signaling and JAK2 association mediated by membrane-proximal cytoplasmic regions of prolactin receptors

Prolactin (PRL) has recently been demonstrated to induce tyrosine phosphorylation and activation of the cytoplasmic tyrosine kinase JAK2 in PRL-dependent Nb2 lymphoma cells. The present study represents an initial effort to identify cytoplasmic regions of the PRL receptor (PRLR) that are critical to growth signal generation and JAK2 activation. Variably truncated rat PRLRs were stably expressed in the murine 32D cell line. PRL-induced proliferation was mediated by the full-length receptor and the mutant G328, which contains the membrane-proximal Homology Boxes 1 and 2 characteristic of hematopoietin receptors. In contrast, mutant receptors lacking either Box 2 or both Boxes 1 and 2 were not capable of transmitting a growth signal. The mitogenic capacity of the PRLR variants correlated with JAK2 association and activation, as well as with induction of mRNA levels for the growth-related gene ornithine decarboxylase. We conclude that mitogenic competence of rat PRLRs resides within the first 94 amino acids of the cytoplasmic domain. The data suggest that Homology Box 1/Box 2 region is critical to JAK2 phosphorylation and association with the PRLR. These findings will serve as a basis for more detailed molecular characterization of PRLR domains essential for JAK2 interaction and growth signal generation.

Modulatory effect of dexamethasone on ornithine decarboxylase activity and gene expression: A possible post‐transcriptional regulation by a neutral metalloprotease

The intracellular effect of dexamethasone (DXME) on the activity and gene expression of ornithine decarboxylase (ODC) was studied in Syrian hamster embryo cells (SHE). The ODC activity (expressed as nmoles decarboxylated ornithine mg-1 protein h-1) was 4.61 +/- 0.14 in untreated cells, whereas it increased to 14.38 +/- 0.26 after 5 h treatment with 1.6 x 10(-7) M TPA. In contrast, DXME (2.5 x 10(-5) M) reduced the ODC activity by 50 per cent to 2.35 +/- 0.22. In cells co-treated for 5 h with TPA and DXME, ODC activity decreased to the level of the untreated cells. However, when DXME was added 3 h after TPA treatment for 2 h, in the continuous presence of TPA, the ODC activity unexpectedly increased further to 16.44 +/- 1.05. The modulation of ODC activity correlated partly with the level of ODC mRNA. Thus when cells were treated with TPA, the ODC mRNA increased threefold, whereas it decreased by 30 per cent when the cells were exposed to DXME. In TPA-DXME co-treated cells, as in TPA pretreated cells followed by DXME for 2 h, a decrease (31.25 per cent and 12.5 per cent respectively) was observed in ODC mRNA. In turnover studies, DXME was found to increase the stability of ODC; the discrepancy between ODC activity and ODC mRNA levels could result from an inhibitory effect of the corticoid on proteolysis of ODC. Studies of lysosomal protease showed that the activities of cathepsins L, B and H decreased following TPA treatment. DXME also inhibited cathepsin L and B activities, but stimulated cathepsin H.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of immediate early genes, interleukin-1β and ornithine decarboxylase messenger RNA in spinal cord by seizures

A number of studies have shown that seizures produced by a variety of stimuli such as metrazol, kindling or excitotoxin cause the induction of immediate early genes (IEG) and ornithine decarboxylase (ODC). The process is largely mediated through glutamate receptors since N-methyl-D-aspartate antagonists prevent this induction [ I ,2] and the response is localised to the site of stimulation. In this study we have investigated whether changes in gene expression occur during seizure activity in spinal cord through indirect activation of motor pathways. Seizures were induced by stereotaxic injection of kainate (1 l g ) into the nucleus basalis [3]. Cervical spinal cord and brain stem were removed at 1, 2, 4, 6, and 24 h after kainate injection, mRNA was prepared as previously described 141 and characterised by Northern blot analysis. Quantitation of mRNA was carried out by slot blot analysis and levels of c-fos, ODC and IL-1 0 mRNAs were related to 0-tubulin mRNA. Seizures were associated with a marked induction of c-fos mRNA from 1 h after injection, rising to a maximum at 6 h, where a 10-fold increase was detected. This increase was transient and decreased to control levels by 24 h. A parallel time course of induction of IL-10 mRNA and ODC mRNA was also seen in spinal cord reaching a maximum at 6 h. ODC mRNA increased 20fold at 6 h whereas IL-10 increased 10-fold. Levels of ODC mRNA in brain stem were increased 2-3-fold by kainate injection whilst levels of c-fos and IL-10 mRNA were largely unaffected. These results indicate that seizure produces an extensive induction of an IEG (c-fos), ODC and a cytokine (IL-1 0) affecting not only cortical areas responsible for initiating the seizure but also the spinal cord and brain stem through secondary activation.

Expression of ornithine decarboxylase gene in elderly human monocytes

The proliferative capacity of the immune system is impaired in elderly subjects and the expression of various genes involved in cell cycle progression is reduced in PHA stimulated lymphocytes during the aging process. Macrophages play a fundamental role in the immune system response. It has recently been demonstrated that the process of macrophage activation is accompanied by a rapid, transient rise of ornithine decarboxylase (ODC) mRNA levels. In fact, the ODC gene seems to be involved in macrophage activation and differentiation. The authors demonstrated that the steady-state levels of ODC mRNA and the correlated superoxide anion production are lower in the monocytes of elderly subjects with respect to those in young subjects used as control. These results confirmed the impaired immune function of the elderly.

Estradiol control of ornithine decarboxylase mRNA, enzyme activity, and polyamine levels in MCF-7 breast cancer cells: therapeutic implications.

Previous studies have shown that natural polyamines-putrescine, spermidine, and spermine-play a key role in the mechanism of action of estrogens in breast cancer. Ornithine decarboxylase (ODC) is the first enzyme of the polyamine biosynthetic pathway. To examine estrogenic regulation of polyamine biosynthesis in breast cancer, we measured ODC mRNA, ODC activity, and polyamine levels in G1 synchronized MCF-7 cells. ODC mRNA and activity increased four-fold over that of cells in G1 phase between 8 to 16 h after the addition of estradiol. Polyamine levels showed a sharp increase by 8 h after the addition of estradiol and decreased by 12 h. We further examined whether synthetic homologs of putrescine or spermidine could replace natural polyamines in supporting MCF-7 cell growth. Treatment of MCF-7 cells with 1 mM difluoromethylornithine (DFMO), an inhibitor of ODC, suppressed putrescine, spermidine, and spermine levels by 74, 78, and 10%, respectively, within 48 h. Cells treated with DFMO for 48 h were supplemented with either putrescine or its homologs or spermidine or its homologs. Diaminopropane, diaminobutane (putrescine), and diaminopentane were capable of fully or partially reversing the growth inhibitory effects of DFMO, whereas diaminoethane had no significant effect. Among a series of triamines, H2N(CH2)nNH(CH2)3NH2 (where n = 2 to 8; abbreviated as APn n = 4 for spermidine, or AP4), spermidine was most effective in reversing the effects of DFMO, whereas compounds with shorter or longer methylene bridging regions were less effective. AP8 was ineffective in reversing the growth inhibitory effects of DFMO. At 10 microM concentration, AP8 also inhibited DNA synthesis by 66%, as measured by [3H]-thymidine incorporation. These data show that MCF-7 cells have a strong requirement for polyamines for their growth and that estradiol stimulates the polyamine cascade by inducing the ODC mRNA level. Our results also suggest that polyamine homologs such as AP8 might be potentially useful in breast cancer therapy.

Inhibitory effect of silymarin, an anti-hepatotoxic flavonoid, on 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity and mRNA in SENCAR mice.

In recent years, considerable emphasis has been placed on identifying new cancer chemopreventive agents which could be useful for human populations. Silymarin, an anti-oxidant flavonoid isolated from artichoke, has been shown to possess significant activity against hepatotoxicity and other pharmacological and physiological disorders. Since many antioxidants inhibit tumor promotion, and because ornithine decarboxylase (ODC) is a well known biochemical marker of tumor promotion, we assessed the effect of skin application of silymarin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal ODC activity and ODC mRNA levels in SENCAR mice. Application of silymarin at doses of 0.5-18 mg (1-37 mumol)/mouse prior to that of TPA (2.5 micrograms) treatment resulted in significant inhibition of TPA-induced epidermal ODC activity in a dose- and time-dependent manner. Northern blot analysis revealed that topical application of silymarin at the dose of 2 mg/mouse resulted in almost complete inhibition of TPA-induced epidermal ODC mRNA. In other studies, silymarin also showed significant inhibition of epidermal ODC activity induced by several other tumor promoters, including free radical-generating compounds. Our data suggest that silymarin could be a useful anti-tumor promoting agent capable of ameliorating the tumor promoting effects of a wide range of tumor promoters.

Regulation of Ornithine Decarboxylase in the Kidney of Autoimmune Mice with the lpr Gene

The lymphoproliferative lpr gene confers a lupus-like disease with lymphadenopathy, antinuclear antibody production, and glomerulonephritis in MRL-lpr/lpr mice. Upregulation of ornithine decarboxylase (ODC) activity and polyamine levels have been observed in the kidney and lymphoid organs of this strain. Inhibition of ODC with 0.5-1.5% (w/v) difluoromethylornithine (DFMO) in drinking water prolonged life-span and ameliorated renal disease. Glomerulonephritis is a major cause of morbidity and mortality in human and murine lupus. In order to elucidate the mechanism(s) of ODC regulation in lupus nephritis, we characterized ODC at the protein and mRNA levels in 3 strains of autoimmune mice with the lpr genetic background (MRL-lpr/lpr, C3H-lpr/lpr and C57BL/6J-lpr/lpr) using Western blotting, enzyme kinetics, turnover rate measurements, Northern blot hybridization, and reverse transcription-polymerase chain reaction (RT-PCR). Normal BALB/c mice were used as a control. We found that ODC activity in the kidney of lpr strains was 4- to 6-fold higher than that of BALB/c mice. The intensity of the major ODC protein band at 54 kD in Western blot was 4-fold higher in MRL-lpr/lpr and C3H-lpr/lpr kidney compared to that of BALB/c kidney. Putrescine levels were 2- to 4-fold higher in kidney of lpr strains than that of BALB/c and DFMO-treated MRL-lpr/lpr mice. DFMO treatment significantly reduced ODC activity and polyamine levels. The half-life of ODC enzyme in MRL-lpr/lpr, C3H-lpr/lpr, B6-lpr/lpr and BALB/c mouse kidneys was 15, 5, 8 and 23 min, respectively. There was no significant difference in the Km values of different strains, whereas Vmax values differed significantly. There was no difference in the level of SAMDC, another enzyme involved in the polyamine biosynthetic pathway, in various strain. Steady-state levels of ODC mRNA were lower in lpr strains compared to that of BALB/c mouse. Our results suggest that the basis for up-regulation of ODC is not at the transcriptional level, but may involve post-transcriptional modification(s) in lpr strains. The link between aberrant regulation of ODC and the immunopathogenesis of murine lupus nephritis indicates novel targets for lupus therapy.

UVB radiation induces phosphorylation of the epidermal growth factor receptor, decreases EGF binding and blocks EGF induction of ornithine decarboxylase gene expression in SV40‐transformed human keratinocytes

We previously demonstrated that epidermal growth factor (EGF) induces a several-fold increase in ornithine decarboxylase (ODC) activity and the steady-state level of ODC mRNA in cultured SV40-transformed human keratinocytes (1). Pretreatment of cell cultures with ultraviolet B (UVB) radiation resulted in a reduction of EGF-induced ODC activity. To determine whether UVB inhibits the accumulation of ODC mRNA by EGF, cells were pretreated with 20 mJ/cm2 UVB or sham-irradiated and then incubated with 100 ng/ml EGF. Northern blot analysis revealed that UVB irradiation entirely blocked the EGF induction of ODC mRNA. Since the binding of EGF to its plasma membrane receptor is the first step in initiating a biological response, the effect of UVB on EGF binding was evaluated. UVB treatment of cultured keratinocytes resulted in an immediate and dose-dependent reduction of EGF binding. Scatchard analysis revealed that the reduction of EGF binding was due to a 52% decrease in the number of available receptors, from 6.2 x 10(4)/cell to 3.0 x 10(4)/cell. However, UVB decreased the EGF-binding affinity very little (Kd = 0.60 nM in control and Kd = 0.75 nM in UVB-treated Z114 cells). In addition, UVB did not alter the rate of EGF internalization. These data suggest that UVB blocks the signal transduction pathway of EGF that is involved in regulation of ODC gene expression. Immunoblot analysis of extracts from irradiated cells showed that UVB induced tyrosine phosphorylation of EGFR and that the quantity of EGFR protein was unaffected by UVB treatment. Phosphorylation of EGFR may be responsible for decreased binding of EGF to its receptor.

Retinoic acid regulates ornithine decarboxylase gene expression at the transcriptional level

Retinoic acid (RA) is important for normal mammalian development and growth. Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the biosynthesis of the polyamines, and we have previously shown that ODC mRNA levels are suppressed by RA in human skin cells. Using HeLa cells, we now show that treatment with 0.5 microM RA for 24 h suppresses endogenous ODC mRNA levels and the expression of a transfected ODC/chloramphenicol acetyltransferase plasmid (Kpn-ODCCAT), containing sequences from -1450 to +810 of the human ODC gene. Co-transfection with either the alpha-RA receptor (alpha-RAR) or a chimeric alpha-RA/oestrogen receptor (alpha-RAER) followed by treatment with the cognate hormone suppresses expression of Kpn-ODCCAT and Not-ODCCAT, which contains sequences from -250 to +514. Liganded alpha-RAR suppresses the activity of Kpn-ODCCAT more markedly than does liganded alpha-RAER (98% and 80% suppression, respectively), whereas both receptors have very similar effects on Not-ODCCAT expression (73% and 67% suppression, respectively). The unliganded alpha-RAR suppresses Kpn-ODCCAT by 76%, whereas unliganded alpha-RAER has no significant effect. These data show that RA regulates ODC-gene expression at the transcriptional level, and that alpha-RAR, but not alpha-RAER, can confer full hormonal responsiveness. This suggests that the activating function present in the alpha-RAR ligand-binding domain is required for full transcriptional regulation.

Cytosolic and nuclear spermidine acetyltransferases in growing NIH 3T3 fibroblasts stimulated with serum or polyamines: relationship to polyamine-biosynthetic decarboxylases and histone acetyltransferase

The expression (mRNA level of enzymic activity) of cytosolic and nuclear spermidine acetyltransferases was studied in NIH 3T3 fibroblasts, either (1) serum-starved and stimulated to grow by serum refeeding, or (2) treated with inhibitors of ornithine decarboxylase (ODC) (MDL 72.175) and S-adenosylmethionine decarboxylase (AdoMetDC) (MDL 73.811) and stimulated to grow by spermidine. Expression of the known growth-regulated genes for ODC, AdoMetDC and histone acetyltransferase was also examined. The mRNA for spermidine/spermine N1-acetyltransferase (SAT) accumulated after serum refeeding (between 6 and 16 h) and even more after spermidine addition (16 h). Histone acetyltransferase activity increased after both growth stimuli, whereas spermidine N8-acetyltransferase activity remained unchanged. After serum stimulation, the ODC mRNA level and activity rose between 6 and 16 h, whereas AdoMetDC mRNA accumulation occurred later (16 h) than the increase in enzyme activity (6 h). Stimulation of ODC and AdoMetDC activities was suppressed by the inhibitors added alone or in combination with spermidine, whereas mRNA accumulation was down-regulated by spermidine. These results indicate that the expression of SAT was growth-controlled and that SAT mRNA level was regulated by polyamines.