Ornithine decarboxylase protein diversity and activity modulation in HTC cells

1982 ◽  
Vol 105 (3) ◽  
pp. 1189-1197 ◽  
Author(s):  
John L.A. Mitchell ◽  
Gail K. Mitchell
Keyword(s):  
Ornithine Decarboxylase ◽  
Protein Diversity ◽  
Htc Cells

scholarly journals Osmotic stress induces variation in cellular levels of ornithine decarboxylase-antizyme

1998 ◽  
Vol 329 (3) ◽  
pp. 453-459 ◽  
Author(s):  
L. A. John MITCHELL ◽  
G. Gary JUDD ◽  
Aviva LEYSER ◽  
Chung-youl CHOE
Keyword(s):  
Steady State ◽  
Ornithine Decarboxylase ◽  
Osmotic Shock ◽  
Regulatory Protein ◽  
Feedback Regulation ◽  
Integral Role ◽  
Htc Cells ◽  
Steady State Levels ◽  
The Common ◽  
Translational Start

The polyamines, and especially putrescine, play an integral role in the physiological response of cells to varying extracellular osmotic conditions. Ornithine decarboxylase (ODC) synthesis and stability, as well as the activity of the polyamine transporter, had all been reported to be very sensitive to media osmolarity in different cells and tissues, yet the mechanism of this complex, co-ordinated response was not known. In this study we have determined that all these aspects of osmotic-shock response may be mediated by the common regulatory protein, ODC-antizyme. HTC cells were induced for antizyme and then exposed to media of reduced osmotic strength. Both antizyme activity and protein decreased rapidly, under these conditions, to new steady-state levels that depended upon the degree of reduction in media tonicity. This antizyme reduction was found to be due to a rapid increase in antizyme degradation, with a half-life decrease from 75 min down to 45 min occurring immediately upon exchanging media. In complementary experiments, increased media tonicity induced elevated antizyme levels and stability. The sensitivity of antizyme turnover to osmotic conditions was also observed in DH23b cells, which contain elevated levels of more stable antizyme. Interestingly, the two main antizyme proteins, AZ-1 and AZ-2 (presumably products from the first and second translational start sites), differed in their responses to these changing osmotic conditions. Just as feedback regulation of antizyme synthesis provides an effective mechanism for maintaining stable polyamine levels, these studies suggest that alteration in the rate of antizyme degradation may be the mechanism whereby cells adjust steady-state polyamine levels in response to stimulation or stress.

scholarly journals Ornithine decarboxylase antizyme in kidneys of male and female mice

1988 ◽  
Vol 254 (2) ◽  
pp. 367-372 ◽  
Author(s):  
Y Murakami ◽  
M Marumo ◽  
S I Hayashi
Keyword(s):  
Ornithine Decarboxylase ◽  
Half Life ◽  
Mouse Kidney ◽  
Repeated Injection ◽  
Male Mice ◽  
Protein Inhibitor ◽  
Male And Female ◽  
Female Mice ◽  
Htc Cells

Antizyme, a protein inhibitor of ornithine decarboxylase (ODC), was shown to be induced in mouse kidney by repeated injection of putrescine. Antizyme was also present as a complex with ODC in the kidney of untreated mouse. The amount of the renal ODC-antizyme complex was 3-fold higher in male mice than in female mice. On the contrary, the proportion of ODC present as a complex with antizyme was 24-fold higher in females than in males, and the decay of renal ODC activity after cycloheximide treatment was about 5-fold more rapid in females than in males. Administration of testosterone to female mice, a procedure known to prolong the half-life of renal ODC, increased both ODC activity and the content of ODC-antizyme complex, but decreased the antizyme/ODC ratio in the kidney. These results are consistent with the previous observation in HTC cells that the decay rate of ODC activity in the presence of cycloheximide correlated well with the proportion of ODC present as a complex with antizyme, suggesting the ubiquitous role of antizyme in ODC degradation.

Effect of GABA and isogabaculine on ornithine decarboxylase and putrescine metabolism

1982 ◽  
Vol 243 (1) ◽  
pp. C35-C38 ◽  
Author(s):  
K. A. Diekema ◽  
P. P. McCann ◽  
B. J. Lippert
Keyword(s):  
Ornithine Decarboxylase ◽  
Neuroblastoma Cells ◽  
Appreciable Amount ◽  
Gamma Aminobutyric Acid ◽  
Irreversible Inhibitor ◽  
Gaba Transaminase ◽  
Rat Glioma ◽  
Glioma C6 ◽  
Metabolic Route ◽  
Htc Cells

Isogabaculine (3-amino-1,3-cyclohexadienyl carboxylic acid; RMI 71,932), an irreversible inhibitor of GABA transaminase, when added to mouse neuroblastoma cells in spinner culture at the time of induction of cell proliferation, increased ornithine decarboxylase (ODC) activity threefold above that of normal control cells and twofold above that of GABA (gamma-aminobutyric acid)-treated cells. Isogabaculine did not affect ODC activity of rat glioma (C6) or rat hepatoma (HTC) cells. As determined by half-life measurements of ODC and intracellular GABA concentrations, isogabaculine apparently has a direct stabilizing effect on ODC in neuroblastoma cells that is unrelated to the accumulation of GABA due to GABA transaminase inhibition. Putrescine metabolism to GABA or spermidine was determined in C6, HTC, and neuroblastoma cells in the presence or absence of isogabaculine and/or GABA. Neither GABA nor isogabaculine treatment dramatically altered the metabolism of putrescine to GABA or spermidine in neuroblastoma, C6 glioma, or HTC cells. However, the appreciable amount of labeled GABA formed from putrescine indicated that this metabolic route may be more important than was previously thought.

scholarly journals Accumulation of ornithine decarboxylase-antizyme complex in HMOA cells

1985 ◽  
Vol 225 (3) ◽  
pp. 689-697 ◽  
Author(s):  
Y Murakami ◽  
K Fujita ◽  
T Kameji ◽  
S Hayashi
Keyword(s):  
Tissue Culture ◽  
Complex Formation ◽  
Ornithine Decarboxylase ◽  
New Method ◽  
Htc Cells ◽  
Hepatoma Tissue

A new method was developed for the assay of ornithine decarboxylase (ODC)-antizyme complex, in which alpha-difluoromethylornithine (DFMO)-inactivated ODC was used to release active ODC competitively from the complex. ODC-antizyme complex was present in the extracts of hepatoma tissue-culture (HTC) cells and of ODC-stabilized variant HMOA cells, in much larger amounts in the latter. Cellular amounts of the complex fluctuated after a change of medium in a similar manner in HTC and HMOA cells, increasing during the period of ODC decay. After treatment with cycloheximide, the decay of ODC-antizyme complex in HMOA cells was more rapid than the decay of free ODC, but it was much slower than the decay of free ODC or complexed ODC in HTC cells. Administration of putrescine caused a rapid increase in the amount of ODC-antizyme complex in both HTC and HMOA cells, but nevertheless the decay of total ODC (free ODC plus ODC-antizyme complex) was more rapid with putrescine than with cycloheximide. These results suggested the possibility that ODC is degraded through complex-formation with antizyme. In contrast with complexed antizyme, free antizyme was not stabilized in HMOA cells.

scholarly journals Proteasome pathway operates for the degradation of ornithine decarboxylase in intact cells

1996 ◽  
Vol 317 (1) ◽  
pp. 77-80 ◽  
Author(s):  
Yasuko MURAKAMI ◽  
Nobuyuki TANAHASHI ◽  
Keiji TANAKA ◽  
Satoshi ŌMURA ◽  
Shin-ichi HAYASHI
Keyword(s):  
Ornithine Decarboxylase ◽  
Proteasome Inhibitor ◽  
Mammalian Cells ◽  
Degradation Pathway ◽  
Dependent Manner ◽  
Intact Cells ◽  
Htc Cells ◽  
Proteasome Pathway ◽  
Hypertonic Shock

Ornithine decarboxylase (ODC) is degraded in an ATP-dependent manner in vitro by the 26 S proteasome in the presence of antizyme, an ODC destabilizing protein induced by polyamines. In the present study we examined whether the proteasome catalyses ODC degradation in living mammalian cells. Lactacystin, the most selective proteasome inhibitor, strongly inhibited the degradation of ODC that had been induced in hepatoma tissue-culture (HTC) cells by refeeding with fresh medium. Furthermore the inhibitor inhibited the rapid degradation of ODC that had been induced by hypotonic shock. Interestingly, hypertonic shock was found to increase the proportion of ODC present as a complex with antizyme (the ratio of ODC–antizyme complex to total ODC). Cycloheximide, which partly inhibits rapid ODC degradation caused by hypertonic shock, also partly inhibited the increase in the ratio of ODC–antizyme complex to total ODC. These results suggest that a common ODC degradation pathway, namely the antizyme-dependent and 26 S proteasome-catalysed ODC degradation pathway, is also operating in intact cells for osmoregulated ODC degradation.

The role of calcium in the induction of ornithine decarboxylase in rat HTC cells

Life Sciences ◽  
1981 ◽  
Vol 29 (7) ◽  
pp. 707-710 ◽  
Author(s):  
Zoe Nakos Canellakis ◽  
Theoharis C. Theoharides ◽  
Philip K. Bondy ◽  
Efthalia T. Triarhos
Keyword(s):  
Ornithine Decarboxylase ◽  
Htc Cells

scholarly journals Ornithine decarboxylase modification and polyamine-stimulated enzyme inactivation in HTC cells

1985 ◽  
Vol 228 (2) ◽  
pp. 297-308 ◽  
Author(s):  
J L A Mitchell ◽  
P Qasba ◽  
R E Stofko ◽  
M A Franzen
Keyword(s):  
Ornithine Decarboxylase ◽  
Gradient Elution ◽  
Culture Conditions ◽  
Enzyme Synthesis ◽  
Enzyme Inactivation ◽  
Rapid Loss ◽  
Enzyme Form ◽  
Htc Cells ◽  
Normal Enzyme ◽  
Marked Accumulation

Ornithine decarboxylase isolated from HTC cells was separated into two distinct charged states by salt-gradient elution from DEAE-Sepharose columns. This charge difference between the enzyme forms was maintained in partially purified preparations, but enzyme form II was observed to change to form I in a time-dependent polyamine-stimulated fashion in crude cell homogenates. The enzyme modification that produces this charge diversity between the alternative enzyme states was further investigated for its role in enzyme activity induction, protein stability and rapid turnover. Inhibition of new protein synthesis by cycloheximide resulted in a much more rapid loss of form I enzyme than of form II, suggesting that during normal enzyme turnover the latter enzyme state may be derived from the former. Culture conditions that favour the stabilization of this usually labile enzyme generally induced an increased proportion of the enzyme in the form II charge state. In particular, inhibitors of synthesis of spermidine and spermine induced the stabilization of cellular ornithine decarboxylase and promoted a marked accumulation in form II. Conversely, polyamines added to the cells in culture induced a very rapid loss in both forms of the enzyme, an effect that could not be attributed merely to an inhibition of new enzyme synthesis. It appears that the polyamines, but not putrescine, may be an essential part of the rapid ornithine decarboxylase inactivation process and that they may function in part by stimulating the conversion of the more stable enzyme form II into the less stable enzyme state, form I.

scholarly journals Hypusine modification in eukaryotic initiation factor 5A in rodent cells selected for resistance to growth inhibition by ornithine decarboxylase-inhibiting drugs

1996 ◽  
Vol 320 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Margaret E TOME ◽  
Eugene W GERNER
Keyword(s):  
Cell Lines ◽  
Ornithine Decarboxylase ◽  
Critical Factor ◽  
Initiation Factor ◽  
Synthetase Activity ◽  
Eukaryotic Initiation Factor ◽  
Variant Cell ◽  
Htc Cells ◽  
Main Effects ◽  
Selection Of

Selection of HTC cells in drugs that inhibit ornithine decarboxylase (ODC) has produced two cell lines, HMOA and DH23A/b, that contain increased amounts of more stable ODC. In addition to alterations in ODC, these cells appear to produce modified eukaryotic initiation factor 5A (eIF-5A) at different rates, a reaction that both requires spermidine and is essential for proliferation. Alterations to the modification of eIF-5A by spermidine cannot be accounted for by changes in eIF-5A protein or modified eIF-5A turnover. Deoxyhypusine synthetase activity is similar in the parental and variant cell lines and is unaltered by growth into plateau phase or by spermidine depletion. The increased rate of eIF-5A modification in DH23A/b cells is due to an increased accumulation of the unmodified eIF-5A precursor. Increased precursor accumulation is not due to increased eIF-5A transcription, but rather it can be attributed to a metabolic accumulation caused by growth under conditions of chronically limiting spermidine. Selection using drugs that inhibit ODC apparently does not cause alterations in the eIF-5A modification pathway. These data support the hypothesis that one of the main effects of spermidine depletion is depletion of the modified eIF-5A pool, and that this is a critical factor in the cytostasis often observed after depletion of cellular polyamines.

scholarly journals Mammalian cell polyamine homeostasis is altered by the radioprotector WR1065

1998 ◽  
Vol 335 (2) ◽  
pp. 329-334 ◽  
Author(s):  
John L. A. MITCHELL ◽  
Jennifer RUPERT ◽  
Aviva LEYSER ◽  
Gary G. JUDD
Keyword(s):  
Ornithine Decarboxylase ◽  
Mammalian Cells ◽  
Regulatory Protein ◽  
Polyamine Metabolism ◽  
Intact Cells ◽  
Polyamine Synthesis ◽  
Radiation Induced ◽  
Htc Cells ◽  
Low Levels ◽  
The Stability

Mammalian cells become more susceptible to radiation-induced death and mutagenesis when restricted in their production of the natural polyamines putrescine, spermidine and spermine. The effects of polyamine deprivation are reversed by N-(2-mercaptoethyl)-1,3-diaminopropane (WR1065), a simple aminothiol that has been extensively studied for its radioprotectant properties. Because this compound and its oxidized derivative WR33278 bear some resemblance to the polyamines, it was hypothesized that radioprotection by WR1065 or its metabolites is derived, at least in part, from their ability to supplement the natural polyamines. To evaluate the ability of these aminothiol compounds to emulate polyamine function in intact cells, rat liver hepatoma (HTC) cells were treated with radioprotective doses of WR1065; the ability of this compound to affect various aspects of normal polyamine metabolism was monitored. Although cellular WR1065 was maintained at levels exceeding those of the polyamines, this aminothiol did not have any polyamine-like effect on the initial polyamine biosynthetic enzyme, ornithine decarboxylase, or on polyamine degradative reactions. On the contrary, treatment with relatively low levels of WR1065 resulted in an unexpected increase in putrescine and spermidine synthesis. WR1065 treatment enhanced the stability, and consequently the activity, of ornithine decarboxylase. This stabilization seems to result from a WR1065-induced delay in the synthesis of antizyme, a critical regulatory protein required in the feedback modulation of polyamine synthesis and transport. The increase in cellular spermidine induced by WR1065 might explain its antimutagenic properties, but is probably not a factor in protection against cell killing by radiation. This is the first evidence that compounds can be designed to control polyamine levels by targeting the activity of the regulatory protein antizyme.

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