Rat uterine polyamine biosynthetic decarboxylase activities following multiple injections of estradiol-17 beta and/or estriol

A single injection of 0.5 micrograms estradiol-17 beta (E2) plus 0.5 micrograms estriol (E3) stimulated a different pattern in 22-24 day-old rat uterine ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMDC) activities than was induced by either a single injection of 0.5 micrograms E2 or multiple injections of 0.5 micrograms E3. Differences included alterations in enzyme activity peak timing as well as activity duration. Every 3 hour injections of 0.05 micrograms E2 induced maximum uterine ODC activity at 4, 24, 32, and 40 hours, intermediate activity at 48, 64, and 72 hours as well as a small peak by 56 hours. When 0.05 micrograms E2 plus 0.05 micrograms E3 were injected simultaneously every 3 hours, the ODC activity pattern was similar except that activity fell to intermediate levels by 40 hours. It is suggested that E3 alterations of E2 induced uterine enzyme activities (when monitored at frequent intervals) could be physiological alterations in uterine growth responses due to E2-E3 hormone interactions. However, there appeared to be no differences between E2 or E2 plus E3 induction of DNA synthesis and luminal epithelial cell height and cross-sectional area or ODC and SAMDC activities when measured at 24, 48, or 72 hours.     

Induction of ornithine decarboxylase, RNA, and protein synthesis in macrophage cell lines stimulated by immunoadjuvants

Early biochemical changes associated with adjuvant stimulation of macrophage protein synthesis were studied using two murine macrophage cell lines, PU5-1.8 and J774.1. An induction of ornithine decarboxylase (ODC) was detected 2 hours after exposure of PU5-1.8 and J774.1 cells to two crude immunoadjuvants, BCG cell walls (BCGcw) and lipopolysaccharides from Escherichia coli (LPS). The chemically defined immunoadjuvant glycopeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDPL) also promoted an increase in ODC activity at 2 hours that was maximal after 4 hours, while little or no effect was observed with the D-alanyl analog (MDPD) that is devoid of adjuvant activity. The increase in ODC activity promoted by BCGcw in PU5-1.8 and J774.1 cells returned toward control levels by 6 to 8 hours. BCGcw also stimulated RNA and protein synthesis which remained elevated for at least 24 hours and was associated with a decrease in DNA synthesis and cell proliferation. ODC induction by BCGcw and MDPL was enhanced by the addition of PGE2 in both cell lines. Indomethacin slightly depressed the magnitude of ODC stimulation by BCGcw in J774.1 cells but failed to alter the response of PU5-1.8 cells. Additional observations indicated that the induction of ODC by BCGcw in both cell lines was preceded by an activation of cyclic AMP-dependent protein kinase. These observations suggest that a cyclic AMP-mediated induction of ODC may be an early biochemical marker of adjuvant stimulation in macrophages.     

Polyamine biosynthetic decarboxylase activities following estradiol-17β or estriol stimulation of the immature rat uterus

Following a single intraperitoneal injection of 0.5 μg estradiol-17β (E2) into immature female rats uterine ornithine decarboxylase (ODC) activity increased to a peak at 4 hours postinjection. It decreased to intermediate levels by 6 hours and remained elevated until returning to control levels by 18 hours. When either 0.5 μg estriol (E3) or 0.05 μg E2 was injected, activity increased to a 4 hour ODC peak then decreased to control levels by 10 hours. The decrease to intermediate levels of ODC activity after dosing with 0.5 μg E2 occurred at the same time activity decreased to control levels following treatment with either 0.05 μg E2 or 0.5 μg E3.S-Adenosyl methionine decarboxylase (SAMDC) activity had increased by 4 hours following an injection of 0.5 μg E2 and remained elevated until 16 hours then decreased to control levels. An injection of 0.05 μg E2 or 0.5 μg E3 stimulated only a 4 hour peak after which time SAMDC decreased to control levels by 14 hours. After an Injection of 5.0 μg E2 SAMDC activity had increased by 4 hours and remained elevated for the remainder of the experiment (16 hours).Decreases in ODC activity following 4 and 10 hours may reflect a decrease in nuclear estrogen receptor levels. The ODC activity seen here following 0.5 μg E2 injection is similar in timing to that seen in other proliferating systems and may be due to a common mechanism.     

Polyamine- and insulin-like growth factor-I-mediated proliferation of porcine uterine endometrial cells: a potential role for spermidine/spermine N(1)-acetyltransferase during peri-implantation

Insulin-like growth factor-I (IGF-I) and the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) are progesterone-regulated genes with maximal expression at peri-implantation in the porcine uterine endometrium. However, while IGF-I stimulates cell proliferation, SSAT, by acetylating the naturally occurring polyamines (PA) spermine (SPM) and spermidine (SPD), typically functions as a cell growth inhibitor. The present study examined the functional relationships of IGF-I, SSAT, and PA in the control of endometrial cell proliferation. Northern blot analysis indicated that SSAT mRNA levels change with distinct pregnancy stages, in contrast to those for the PA biosynthetic enzyme ornithine decarboxylase (ODC). Primary cultures of luminal and glandular epithelial (LE, GE) and stromal (ST) cells isolated from Day 12 pregnant pig endometrium had IGF-I mRNA levels for ST > LE > GE cells. The mRNA levels for SSAT and ODC were transiently diminished by IGF-I treatment, but only in GE cells. By contrast, SPM and SPD increased SSAT mRNA levels in GE and ST cells, but increased ODC mRNA levels only in GE cells. IGF-I, putrescine (PUT), and SPM individually increased cellular DNA synthesis as measured by tritiated thymidine incorporation in GE and ST cells, while SPD had an effect only in ST cells. IGF-I enhanced the proliferative effect of each PA in GE cells, but only of SPD in ST cells. The mitogen-activated protein kinase inhibitor, PD98059, inhibited the induction by SPM of GE cell DNA synthesis but not that of IGF-I. Wortmannin, a phosphatidylinositol-3-kinase inhibitor had no effect on either IGF-I or SPM induction of GE cell DNA synthesis. The relative concentrations of SPM, SPD, and PUT in uterine luminal fluids differed, with the levels for each PA higher at pregnancy Day 12 than at 11.5. These results suggest that IGF-I and PA act through distinct signaling pathways to mediate cell-type-specific growth of early pregnancy pig uterine endometrium. Further, SSAT, through its control of intracellular PA levels, likely plays a modulatory role in the establishment of an optimal uterine environment for successful embryo attachment.     

Effect of diaminobutene and diaminopropane on diet-stimulated polyamine synthesis and cell proliferation in rat liver.

The effect of two putrescine analogs were studied on hepatic polyamine synthesis and cell proliferation, both of which were stimulated by food intake. Trans-1, 4-diamino-2-butene (diaminobutene), which is a potent competitive inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), repressed the induction of ODC and effectively inhibited the accumulation of putrescine in rat liver which was induced by the feeding of dietary protein. Unexpectedly, diaminobutene did not suppress DNA synthesis and mitotic activity in rat liver, suggesting that it can mimic the role of putrescine in cell proliferation. 1,3-Diaminopropane effectively repressed the induction of ODC caused by food intake and also suppressed DNA synthesis and mitotic activity without affecting the accumulation of RNA or protein. The suppression of mitotic activity by 1,3-diaminopropane was reversed by a single injection of putrescine, spermidine, spermine, or diaminobutene. It was concluded that rapid accumulation of polyamines, especially putrescine, was a prerequisite for the later enhancement of DNA synthesis and cell proliferation in rat liver caused by food intake.     

Effects of dexamethasone and indomethacin on estrogen-induced uterine growth

Certain aspects of estrogen-induced uterine growth are reminiscent of an inflammatory response. Dexamethasone (DEX) and indomethacin (IND), two anti-inflammatory agents that interfere with arachidonic acid metabolism, were examined with respect to their effects on several growth-associated responses of the uterus to estrogen. Ovariectomized rats were given a s.c. injection of either DEX (2 mg) or IND (8 mg) immediately prior to receiving a s.c. injection of estradiol (10 ωg). At 4 hr, DEX inhibited estrogen-stimulated uterine wet weight and ornithine decarboxylase (ODC) activity by 100% and 48%, respectively. At 24 hr, ³H-leucine incorporation into protein was inhibited 44% and ³H-thymidine incorporation into DNA was depressed 83%. Estrogen-stimulated increases in uterine protein/DNA ratio and epithelial microvilli density at 24 hr were not inhibited by DEX. IND inhibited estrogen-stimulated wet weight by 64% and ³H-thymidine incorporation into DNA by 42%, yet did not inhibit the increases in ODC activity, ³H-leucine incorporation into protein or protein/DNA ratio. These results suggest that the inflammation-like component of estrogen-induced uterine growth is mediated, at least in part, by arachidonic acid metabolites and is directed primarily toward stimulating cell division, and not cell growth.     

The development of competence in resting B cells. The induction of cyclic AMP and ornithine decarboxylase activity after direct contact between B and T helper cells.

In the present communication, an experimental approach is utilized that facilitates the study of biochemical processes induced in B cells after their interaction with Th cells. In this approach, Th cell clones are stimulated for 18 h upon anti-CD3-coated plates, fixed with paraformaldehyde, and added at a 2 to 3:1 ratio to small, resting B cells (isolated from Percoll gradients). Th cells not stimulated on anti-CD3-coated plates, but fixed with paraformaldehyde, serve as controls for these experiments. The activated, fixed Th cells induce a transient, sixfold increase in B cell levels of cAMP, as well as an increase in B cell expression of ornithine decarboxylase (ODC) activity. This enzyme initiates the synthesis of polyamines and has been shown to be increased as cells enter the growth phase. In addition, previous studies have shown that the cellular levels of ODC activity are controlled by a multi-tiered regulatory cascade. To examine this aspect, polyclonally stimulated B cells were studied. Such cells demonstrated a gradual increase in ODC mRNA levels that peaked between 6 and 15 h and can be partially explained by a three- to fourfold increase in mRNA stability but not by changes in the enzyme affinity for substrate. The increase in ODC mRNA occurs in the absence of protein synthesis, suggesting that the ODC gene is a member of the immediate/early gene family. Finally, the early increase in ODC mRNA was enhanced in cells in which cAMP levels were artificially elevated, suggesting the possibility that the cAMP-dependent signaling pathway participates during the regulation of this gene expression. The significance of these experimental results concerning the process of B cell activation is discussed.     

Polyamine biosynthesis in the developing rabbit palate

Levels of putrescine, spermidine, and spermine and their biosynthetic enzymes, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) were measured in the developing rabbit palate between day 14 and day 18 of gestation. DNA, RNA, and protein synthesis were also measured during this time period to determine if a correlation exists between polyamine biogenesis and macromolecular synthesis. ODC activity was found to be twice as high on day 14 as on the succeeding days of gestation, while SAMDC activity did not change significantly. Levels of putrescine and spermine were higher on day 14 by 22% and 30%, respectively, than levels on day 18. Spermidine concentration did not change. DNA synthesis remained relatively constant between days 14 and 18 of gestation, suggesting that there is no peak in cell proliferation during this period. RNA synthesis was elevated significantly on day 14 and protein synthesis was significantly higher on both days 14 and 16. This data indicates that there is no correlation between polyamine synthesis and cell proliferation during this period of palatal development, but polyamines could play a regulatory role in RNA and/or protein synthesis.     

Alpha-fetoprotein serum levels and the development of estrogen-sensitive cell multiplication in the hamster uterus

Subcutaneous injections of 5 or 25 micrograms estradiol-17 beta (E2)/kg in ovariectomized adult hamsters produced substantial increases in uterine wet weight, protein content and the mitotic indices of the glandular and luminal epithelia. However, no significant increase was seen in total uterine DNA. Intact hamsters from 2 to 25 days of age received a daily subcutaneous injection of 5 micrograms E2/kg for 2 consecutive days. Significant increases in uterine wet weight and protein content first occurred at 8 and 17 days, respectively. No significant increase was observed in uterine DNA. In a separate experiment, hamsters between 2 and 20 days of age received one subcutaneous injection of 5 micrograms E2/kg. Mitotic indices in the stroma were increased at 6 and 10 days of age. Mitotic indices in the luminal epithelium were significantly increased only at 6 days of age. Rocket immunoelectrophoresis revealed a sharp decline in serum alpha-fetoprotein (AFP) concentrations after 2 days of age. Estradiol concentrations in the sera of immature hamsters gradually decreased from 55 pg/ml at 0 days of age to 17 pg/ml at 20 days of age. These results provide a quantitative analysis of the effects of E2 upon cell proliferation in the hamster uterus. The correlation of declining AFP levels and the incipience of the mitotic response to estrogen suggests that AFP may directly inhibit estrogen-sensitive cell multiplication in the neonate. Other possible causes for the lack of a mitotic response in the uterus of the newborn hamster to the administration of E2 are also discussed.     

Stimulation of uterine ornithine decarboxylase in organ culture by decreasing osmolality: Possible relation to in vivo mechanisms

Ornithine decarboxylase (ODC) activity increases during many growth responses. Some cells and tissues in culture also exhibit elevated enzyme activity with decreasing osmolality of the culture medium. We have found that this also occurs with uterine tissue from ovariectomized rats. Organ culture incubation under hypotonic conditions caused maximal stimulation of uterine ODC activity at 4 hr. This stimulation was observed when either NaCl or sucrose was used to adjust the osmolality. Incubation under isotonic conditions also increased ODC activity relative to hypertonic conditions. This increase was similar in magnitude to that seen with unincubated uterine tissue from animals receiving systemic estradiol or intrauterine cholera toxin. Both estradiol and cholera toxin increase vascular permeability, and the resultant edema changes the extracellular microenvironment of the uterine cells. We suggest that this change somehow is mimicked by organ culture under hypotonic or isotonic conditions and is responsible for the stimulation of uterine ODC activity.     

Control of DNA synthesis and ornithine decarboxylase activity by hormones and amino acids in primary cultures of adult rat hepatocytes

The conditions for stimulation of ornithine decarboxylase (ODC) and DNA synthesis in primary monolayer cultures of non-growing, highly differentiated hepatocytes from adult rats were compared. The syntheses of ODC and DNA were not stimulated by hormones on the 1st day of culture, but they were induced markedly by insulin (10⁻⁸ M) and epidermal growth factor (EGF, 0.1 μg/ml) in cells cultured for 40 h. The effects of insulin and EGF were synergistic, and the ODC activity as well as the DNA synthesis in the presence of these hormones was comparable to that of cultured hepatocytes from partially hepatectomized liver. Other factors had different effects on the two processes. Dexamethasone induced ODC slightly, but it inhibited DNA synthesis strongly. Putrescine inhibited ODC activity, but it had no effect on DNA synthesis. Asparagine and glutamine induced ODC activity, but they inhibited DNA synthesis; their inhibitory effects on DNA synthesis were specific to primary cultured liver cells and were not seen in an established rat liver cell line or in mouse L cells. These results show that although there is some correlation between ODC induction and DNA synthesis, the former is not essential for cell growth. There was no indication of cell division under conditions where maximal ODC induction and DNA synthesis were observed. Cytofluorometry of cells treated with insulin and EGF showed that the DNA content increased from 2 N to 4 N, and to 8 N in some cells. Therefore, under the present culture conditions, mature liver cells could enter G2 phase through S phase, but could not enter M phase.     

Expression of ornithine decarboxylase in pancreatic development

We examined the relationship between ornithine decarboxylase (ODC) and growth and differentiation in the developing rat exocrine pancreas. The ODC activity profile showed 2 distinct stages of increases with the first occurred at 14–16 days of age, and a second at 21–23 days of age. Growth parameters evaluated as gains in tissue mass, protein and DNA content in the pancreas indicated a low growth rate soon after birth with a transition to a much more rapid growth rate around the age of 20–21 days, a time corresponded to the second rise in ODC activity. Differentiation parameters evaluated as the accumulation of trypsinogen, amylase and lipase showed different temporal changes. While the rate of accumulation of all three enzymes was relatively low following birth, a rapid rate of accumulation of trypsinogen and amylase started around 15–16 days, a time corresponding to the first rise in ODC activity. Lipase, however, did not show an increase in its accumulation until around age 20 days. These results indicate that a rise in ODC activity is closely associated with growth and differentiation in the developing rat pancreas. To further examine this issue, the steady state levels of ODC mRNA in developing rats were evaluated by Northern blots probed with an ODC cDNA. The developmental profile of ODC mRNA showed a broad peak with a pronounced shoulder occurring at 10 days of age. A higher peak was reached around 20 days of age, then dropped precipitously to a very low level at the age of 24 days. This temporal changes in the level of ODC mRNA show good relationship to the changes in ODC activity suggesting that the control of ODC expression occurs at least in part at the pre-translational level.     

EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells

The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5-10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4-day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF-treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF-treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF-induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.     

Mechanism of hepatic ornithine decarboxylase induction by the ornithine decarboxylase-inducing factor isolated from tumor ascites fluid: determination of target cells for the factor in the liver

The effect of the ODC-factor, which was partially purified from ascites fluid of mice bearing Ehrlich ascites hepatoma, on DNA synthesis in the normal mouse liver and spleen was studied, and target cells for the factor in the liver were examined. DNA synthesis in the liver increased about 4-fold over the basal level 39-42 h after the increase of ODC activity induced by injection of the factor into normal mice. This increase of DNA synthesis was inhibited by repeated injection of DAPol. The inhibition was completely reversed by the administration of an appropriate amount of putrescine at about the same time. TK activity also increased in parallel with DNA synthesis. Normal mice with and without treatment with the factor were used to examine which cell population in the liver is the real target for the factor. The livers were dispersed and three cell populations (heavy, medium, and light) were separated by centrifugation. The heavy and light cell populations were characterized as mature hepatocytes and a cell population consisting mainly of immature hepatocytes and non-hepatocytes by analysis of marker enzymes, pyruvate kinase isozymes, L and M2, respectively. The factor stimulated ODC induction, with concomitant increases in TK and DNA poly activities and DNA synthesis, most effectively in the light cell fraction followed in order by the medium and heavy fractions. A nutritional factor (a high protein diet), which is a potent inducer of liver ODC, appeared to act on liver in a different way from the ODC-factor, judging from the results of studies of both whole liver and the fractionated cell system described above. Autoradiography of [3H]thymidine incorporation into liver cells showed that DNA synthesis in mature hepatocytes as well as nonhepatocytes was enhanced by injection of the factor. Stimulation of non-hepatocytes seems to be suggestive evidence that an immunologic response of mice might be developed by the factor. In fact, ODC activity, DNA synthesis, and DNA poly activity (but not TK) in the spleen significantly increased in response to the factor and their increments were suppressed by DAPol, though less sensitively than those in the light cell fraction of the liver.     

Effect of maternal deprivation on polyamine metabolism in preweanling rat brain and heart

Ornithine Decarboxylase (ODC) the first and probably rate-limiting step in polyamine biosynthesis, is usually elevated in tissues with high rates of growth or protein synthesis and in preweanling rat brain is altered by hormones thought to play a role in stress such as thyroxine and cortisol. Maternal deprivation stress was examined for its effect on preweanling rat brain ODC activity. Ten day old rat pups were removed from the maternal cage, then alternately returned to the mother or placed in a warm environment after the method of Hall. Pups taken from the mother for as little as 1 hour show a significant decline in their whole brian ODC activity. The effect peaked at 2–4 hours of deprivation, at which time the brain ODC activity was 60% below that of the equally handled littermate controls. Two hours of deprivation produced a similar effect throughout preweanling development. The effect occurred in all brain regions and also in heart. Return to mother reversed rapidly the deprivation-induced ODC decline, with ODC activity overshooting to 300% of control 2 hours after return, then declining to baseline by 4 hours. Putrescine, the immediate product of the ODC reaction, declined 15% one hour after deprivation, and 50% in brain and heart after 15 hours. These data demonstrate that maternal deprivation alters polyamine metabolism in preweanling rat brain and heart.     

Induction of ornithine decarboxylase in macrophages by bacterial lipopolysaccharides (LPS) and mycobacterial cell wall material

Lipopolysaccharide from $\text{E. Coli}$ (LPS) and BCG cell walls (BCGcw) are recognized immunoadjuvants that directly stimulate some macrophage functions. The macrophage cell line J774.1 and peritoneal exudate cells (PEC) from mice can be stimulated by LPS or other adjuvants $\text{in vitro}$ to synthesize and release protein factor(s) that activate thymus-derived lymphocytes. We have utilized J774.1 cells and PEC to demonstrate that an increase in ornithine decarboxylase (ODC) activity is a marker of early biochemical changes in adjuvant-stimulated macrophages. BCGcw and LPS increased ODC within 2 hours in J774.1 cells as well as murine peritoneal exudate macrophages. Maximal increases in ODC were detected 4 hours after the addition of adjuvants to J774.1 cells. The marked increases (12–23 fold) in ODC observed with BCGcw (20 μg/ml) did not appear to involve an effect on cell proliferation which was suppressed by this adjuvant. Cycloheximide inhibited the induction of ODC by LPS and BCGcw in the macrophage cell line. Evidence that the induction of ODC may be promoted by an increase in cyclic AMP was provided by experiments demonstrating that prostaglandin E₁ (PGE₁) and 8-bromo-adenosine-3′:5′-monophosphate (8Br-cyclic AMP) can mimic the effects of LPS and BCGcw in J774.1 cells. These observations indicate that one of the early biochemical changes in macrophages promoted by adjuvants is an induction of ODC.     

Transplacental estrogen responses in the fetal rat: increased uterine weight and ornithine decarboxylase activity

The synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE2), are more potent than 17 beta-estradiol (E2) in inducing uterine weight gain in the neonatal rat, due to the binding of E2 to serum alpha-fetoprotein (AFP). However, all three hormones are equipotent in inducing neonatal uterine ornithine decarboxylase (ODC) activity. The present study assessed estrogen potency in fetal rats. Pregnant CD rats were injected sc daily on gestation days (GD) 16-20 with DES, EE2, or E2 in sesame oil. Both DES and EE2, but not E2, significantly increased uterine weight at birth, to more than twice that of controls. In addition, implants which continuously release E2 only slightly increased uterine weight at birth. Alternatively, dams were given a single estrogen injection on GD 20 and were sacrificed at various times after injection. Peak fetal uterine ODC activity occurred at 6-8 hours after maternal injection for all three estrogens. E2 had a relative potency about tenfold less than either DES or EE2 in stimulating fetal ODC activity, in contrast to equal potencies of the three estrogens in the postnatal rat uterus. Similar patterns were found following direct fetal injection with E2 or DES. In summary, these data demonstrate a transplacental induction of fetal uterine ODC activity and uterine weight gain by both DES and EE2. In addition, the lack of correlation between these endpoints in response to E2 suggests that they may be useful as selective indicators of potential toxicity of both natural and synthetic estrogens.