Ornithine Decarboxylase mRNA Expression Research Articles

Cytoskeletal active drugs modulate signal transduction in the protein kinase C pathway.

The cytoskeletal network of cells is postulated to play a role in the signal transduction pathways of growth promoting stimuli. We show here that cytoskeletal active drugs modulate the mitogenic signal transduction pathway of the tumor promoter TPA in 3T3-L1 cells. Compounds which act on microtubules (vinblastine sulfate) and microfilaments (cytochalasin B) have opposite effects on DNA synthesis. Vinblastine sulfate leads to stimulation, whereas cytochalasin B causes potent inhibition of DNA synthesis in response to TPA. These drugs are cell cycle specific and apparently exert their regulatory action distal to activation of protein kinase C by TPA. The expression of four genes necessary for DNA synthesis in response to tumor promoters was examined: two nuclear proto-oncogenes (c-myc and c-fos), a transcription factor (c-jun/AP-1) and a key enzyme involved in polyamine synthesis (ornithine decarboxylase). c-jun mRNA levels are not modulated during the action of cytoskeletal disrupting drugs on TPA-mediated mitogenesis, whereas c-myc and c-fos mRNA levels are similarly enhanced. Expression of ornithine decarboxylase mRNA and protein is increased by vinblastine sulfate but decreased by cytochalasin B in TPA treated cells. These data indicate that changes in cytoskeletal organization may play a role in regulating the levels of an enzyme critical for DNA synthesis.

Effect of basic fibroblast growth factor on ornithine decarboxylase activity and mRNA expression in a pancreatic tumoral cell line (AR4-2J).

Basic fibroblast growth factor (bFGF) is a potent mitogen for various cell types. Induction of ornithine decarboxylase (ODC) activity is one of the early events triggered in proliferating cells. Our aim was to study the effect of bFGF on ODC activity and ODC mRNA expression in a pancreatic tumoral cell line, AR4-2J. Following kinetic and dose-response studies, we found that maximal stimulation (150% over control) of ODC activity occurred after 3 h of bFGF treatment (10(-9) M), the EC50 being 20 pM. To elucidate the mechanism by which bFGF stimulates ODC activity, we measured the ODC mRNA levels by Northern blot hybridization using a 32P-labeled rat cDNA probe. In AR4-2J cells treated with bFGF at 10(-9) M over 120 min, ODC mRNA expression was transiently increased by 71.6% at 60 min. Furthermore, bFGF was also able to stimulate ODC mRNA synthesis in the presence of cycloheximide. In conclusion, in AR4-2J cells of pancreatic origin, bFGF stimulates ODC gene transcription. This effect contributes to the stimulation of ODC enzymatic activity and to the proliferative effect of bFGF on this cell line.

Effect of hypotonic stress on ornithine decarboxylase mRNA expression in cultured cells.

This report examines the effect of hypotonic stress on ornithine decarboxylase (ODC) activity and ODC mRNA concentrations in LLC-PK1 cells. Earle's balanced salts solution minus glucose (EBSS-G) with decreasing concentrations of NaCl was utilized as the ODC induction medium. Hypotonic EBSS-G increased both the concentration of ODC mRNA and the specific activity of ODC in LLC-PK1 cells. Actinomycin D and cycloheximide prevented the increase in enzyme activity resulting from hypotonic stress. Actinomycin D was also a potent inhibitor of ODC mRNA expression resulting from hypotonic stress. Cycloheximide had very little effect on the induction of ODC mRNA in cells incubated in hypotonic EBSS-G. The magnitude of the increase in both ODC mRNA concentrations and enzyme activity was dependent on the incubation time in hypotonic media. The increase in ODC mRNA concentrations preceded the elevation in enzyme activity. ODC mRNA concentrations and the specific activity of ODC increased as a function of decreasing media osmolarity. The addition of putrescine, spermidine, and spermine to EBSS-G containing reduced NaCl suppressed the increase in LLC-PK1 ODC activity related to hypotonic stress. In contrast, these polyamines did not prevent the increase in ODC mRNA resulting from hypotonic shock. Furthermore, it was demonstrated that hypotonic stress increases ODC mRNA levels and enzyme activity in four additional cell lines from two different species. Based on these results it is suggested that one or more signal transducers associated with cell volume expansion enhance expression of the ODC gene.

Prolactin induces growth-related gene expression in rat aortic smooth muscle in vivo

We examined the effects of in vivo administration of prolactin on growth-related gene expression in aorta. Optimal mRNA expression for both the proto-oncogene, c-myc and ornithine decarboxylase occurred at 22 mg/kg prolactin. For c-myc, this was seen as early as 15 min. Prolactin-induced ornithine decarboxylase mRNA expression began at 1 h. The results confirm work showing induction of these genes by prolactin in tissues where it is mitogenic and supports a role for prolactin in the trophic response of vascular smooth muscle.

Polyamine levels, ornithine decarboxylase (ODC) activity, and ODC-mRNA expression in normal and cancerous human colonocytes

Ornithine decarboxylase (ODC) and polyamines (putrescine, spermidine, and spermine) are crucial for cell proliferation. Recently, elevated ODC activity and polyamine levels have been suggested as biological markers for human colon cancer. In this study, we measured ODC activity and the levels of polyamines (putrescine, spermidine, spermine, and cadaverine) and acetyl-putrescine in human colonocytes isolated from cancerous areas compared to the adjacent normal colon tissue. In addition, ODC mRNA expression was compared between both groups. We found that colonocytes isolated from cancerous areas had significantly higher mean value of ODC activity, putrescine, spermidine, spermine, and cadaverine levels up to 1480%, 470%, 260%, 380%, and 510% respectively compared to colonocytes isolated from the adjacent normal colonic mucosa. No difference was found in acetyl-putrescine levels between cancerous and normal colonocytes. Steady-state levels of ODC mRNA were slightly elevated in cancerous colonocytes relative to normal colonocytes in two of three paired samples. However, the increase in ODC mRNA levels is not sufficient to account for the increase in ODC activity suggesting that colonocyte ODC activity is regulated post-transcriptionally.

THE ANTIPROLIFERATIVE EFFECT OF CYCLOSPORINE ON HEMATOPOIETIC AND LYMPHOBLASTOID CELL LINES-COMMON MECHANISTIC ELEMENTS WITH INTERFERON-ALPHA

Cyclosporine, but not its nonimmunosuppressive analog cyclosporine H (CsH), caused in a variety of hematopoietic cell types a growth arrest in the G0/G1 phase of the cell cycle. This arrest was associated with a significant reduction in the c-myc mRNA levels, which could be observed already 1 hr following CsA treatment. Similarity between the antiproliferative effects of CsA and IFN-alpha was observed. Thus, the IFN-alpha sensitive human B-lymphoblastoid cell line Daudi was also sensitive to CsA while an IFN-alpha resistant variant of Daudi cells was found to be resistant to CsA as well. Inhibition of protein synthesis with cycloheximide during IFN-alpha or CsA treatment blocked their ability to reduce the expression of c-myc. Depletion of protein kinase C (PKC) activity from cells by pretreatment of Daudi cells with phorbol.12-myristate 13-acetate (PMA) abolished the G0/G1 arrest induced by both CsA and IFN-alpha. Combinations of low concentrations of CsA and IFN-alpha had synergistic effects on cell-cycle distribution and on c-myc mRNA level, suggesting that CsA and IFN-alpha differ in some features of their antiproliferative action. This conclusion was supported by the observation that a CsA-resistant variant of Daudi cells was found to retain its sensitivity to IFN-alpha. In addition, reduction of ornithine decarboxylase mRNA expression was obtained with IFN-alpha but not with CsA. Taken together, our results suggest that CsA and IFN-alpha share some common element(s) in the pathways of their antiproliferative activity. The possible mechanisms of their antigrowth effects and the clinical significance of our findings are discussed.

Structural and functional studies on the human interleukin-6 receptor. Binding, cross-linking, internalization, and degradation of interleukin-6 by fibroblasts transfected with human interleukin-6-receptor cDNA

A cDNA coding for the human interleukin-6 receptor (IL-6-R) has been expressed stably in murine NIH/3T3 fibroblasts. Transfected cells exhibited a single class of binding sites for 125I-labeled recombinant human interleukin-6 (125I-rhIL-6) (Kd = 440 pM, 20,000 receptors per cell). Affinity cross-linking of 125I-rhIL-6 to the IL-6-R-expressing NIH/3T3 cells led to the detection of three 125I-rhIL-6-containing protein complexes with molecular masses of 100, 120, and 200 kDa suggesting a complex organization of the IL-6-R in the plasma membrane. IL-6 added to the transfected NIH/3T3 cells exerted growth inhibition. This anti-growth effect was observed by the measurement of cell numbers and ornithine decarboxylase mRNA expression. IL-6-R overexpressing fibroblasts internalized 125I-rhIL-6. Intracellular limited proteolysis of IL-6 could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A possible implication of skin fibroblasts in the catabolism of IL-6 is discussed.

Effect of ethanol on polyamine synthesis during liver regeneration in rats.

Ethanol consumption retards the hepatic regenerative response to injury. This may contribute to the pathogenesis of liver injury in alcoholic individuals. The mechanisms responsible for ethanol-associated inhibition of liver regeneration are poorly understood. To determine if the antiregenerative effects of ethanol involve modulation of polyamine metabolism, parameters of polyamine synthesis were compared before and during surgically induced liver regeneration in ethanol-fed rats and isocalorically maintained controls. After partial hepatectomy, induction of the activity of ornithine decarboxylase (ODC), the rate limiting enzyme for polyamine synthesis, was delayed in rats that had been fed ethanol. This was correlated with reduced levels of putrescine, ODC's immediate product. Increases in hepatic spermidine and spermine were also inhibited. Differences in ODC activity between ethanol-fed and control rats could not be explained by differences in the expression of ODC mRNA or by differences in ODC apoenzyme concentrations, suggesting that chronic ethanol intake inactivates ODC posttranslationally. Supplemental putrescine, administered at partial hepatectomy and 4 and 8 h thereafter, increased hepatic putrescine concentrations and markedly improved DNA synthesis and liver regeneration in ethanol-fed rats. These data suggest that altered polyamine metabolism may contribute to the inhibition of liver regeneration that occurs after chronic exposure to ethanol.

Rapid expression of ornithine decarboxylase mRNA in a macrophage-like cell line: cAMP repression of the requirement for prior protein synthesis.

Ornithine decarboxylase (ODC) activity was rapidly induced in the RAW264 macrophage-like cell line after treatment with bacterial lipopolysaccharide (LPS). ODC mRNA levels were determined by isolating cellular RNA, followed by Northern blot and dot blot analysis using a 32P-labeled cDNA probe. ODC mRNA levels increased within 1 hour of stimulation of RAW264 cells with 1.0 microgram/ml LPS. Transcription rate analysis on isolated nuclei indicated that an increase in transcription rate contributed to this increase in ODC mRNA. ODC mRNA levels continued to rise for 4 hours, peaking at eight times the basal level. ODC mRNA appeared as a single 2.2-kb band prior to stimulation. After stimulation, the 2.2-kb band intensified, and a second (2.7 kb) band was seen by Northern gel analysis. Similar induction was demonstrated when 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was used as the stimulus. The induction of ODC mRNA by either LPS or TPA was blocked by the addition of cycloheximide (25 micrograms/ml) or anisomycin (0.1 mM) to the cellular incubation mixture. This indicated that protein synthesis was required as a prerequisite to LPS or TPA induction of ODC mRNA. Experiments in which cycloheximide addition was delayed after LPS treatment indicated that some of the required protein synthesis occurred within the first 30 minutes and that complete expression of ODC mRNA was possible if protein synthesis continued for at least 2 hours before cycloheximide was added. Stimulation with 8-bromo-cAMP in addition to LPS has been shown to enhance the induction of ODC over that induced by LPS or TPA alone. It was not possible to block ODC mRNA induction with cycloheximide or anisomycin after treatment with the combined stimulus of LPS and cAMP or TPA and cAMP, indicating that protein synthesis was not required when cAMP was used as a coinducer. Thus, we have shown that in the same cell, ODC mRNA can be induced by two different pathways, one requiring protein synthesis and one not requiring protein synthesis.

Human myeloma cells acquire resistance to difluoromethylornithine by amplification of ornithine decarboxylase gene.

Stepwise increments of the concentration of 2-difluoromethylornithine (DFMO), a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (ODC), resulted in a selection of cultured human IgG-myeloma cells (Sultan cell line) capable of growing in the presence of up to 3 mM-DFMO. This capacity was associated with 10-fold increase in ODC activity in the dialysed extracts of drug-resistant myeloma cells, markedly enhanced synthesis rate for ODC enzyme molecules, as revealed by a 20 min [35S]methionine labelling of cellular proteins, followed by specific immunoprecipitation and SDS/polyacrylamide-gel electrophoresis, dose-dependently increased expression of ODC mRNA in resistant cells (effective dose causing 50% inhibition), dose-dependent amplification of ODC gene sequences in a 9-kilobase-pairs EcoRI genomic DNA fragment, and (v) a 10-fold increase in the ED50 (effective dose causing 50% inhibition) for the anti-proliferative action of DFMO in these myeloma cells. These results represent one of the few gene amplifications described in cultured human cells.