Prognostic value of ornithine decarboxylase and polyamines in human breast cancer: correlation with clinicopathologic parameters.

The polyamines putrescine, spermidine, and spermine and ornithine decarboxylase (ODC), the rate-limiting enzyme in their biosynthetic pathway, play an important role in cell proliferation, differentiation, and transformation. In the present study, we have analyzed polyamine concentrations and ODC activity in samples from benign breast diseases (n = 36), benign breast tissue adjacent to the primary carcinoma (n = 19), and breast carcinoma (n = 104). ODC activity in primary carcinoma was significantly higher (2.42 +/- 0.22 nmol CO2/h g; P < 0.001) than that found in benign breast (0.62 +/- 0.15 nmol CO2/h g) or in breast tissue adjacent to the primary carcinoma (0.52 +/- 0.16 nmol CO2/h g). The total polyamine content of breast cancer tissues was higher than in benign breast diseases (704.3 +/- 38.3 nmol/g wet weight versus 295.8 +/- 27.4 nmol/g wet weight) and correlated well with ODC activity (Pearson, r = 0.42; P < 0.001). ODC activity correlated with histological grade, peritumoral lymphatic or blood vessel invasion, S-phase fraction, and cathepsin D. Total polyamine concentration increased with S-phase fraction, cathepsin D, and aneuploidy. No significant correlation was found between ODC or polyamines and tumor size, lymph node involvement, or steroid receptor status. A major finding in our study was that ODC activity was an independent prognostic factor for recurrence and death. The results indicate that the estimation of ODC activity and polyamines in human breast carcinoma might be useful to determine tumor aggressiveness and suggest that ODC may have a potential value as both a prognostic factor and a chemoprevention target in human breast cancer.     

Colonic polyamine content and ornithine decarboxylase activity as markers for adenomas

Polyamine content (putrescine, spermidine, and spermine) or ornithine decarboxylase (ODC) activity was measured in normal-appearing colonic mucosa from patients undergoing colonoscopy. Comparisons were made between those with and those without adenomatous polyps. Colonic mucosal polyamine content was measured in 44 persons. Mean putrescine content was 1.25 +/- 0.26 (SE) nmol/mg protein in 22 patients with adenomatous polyps compared with 0.53 +/- 0.12 nmol/mg protein in patients without polyps (P less than 0.02). Tissue content of spermidine and spermine did not differ between these two groups. Ornithine decarboxylase activity was measured in tissue from 45 patients. Mean ODC activity was 2.84 +/- 0.73 pmol/hr/mg protein in 23 persons with adenomatous polyps compared with 1.15 +/- 0.18 pmol/hr/mg protein in persons without polyps (P less than 0.05). Mucosal putrescine and ODC activity are elevated in patients with adenomatous polyps compared with patients without polyps. These biochemical markers may prove helpful in improving surveillance methods for colorectal cancer and premalignant adenomatous polyps.     

Relationship between ornithine decarboxylase levels in anaplastic gliomas and progression-free survival in patients treated with DFMO-PCV chemotherapy

The purpose of this study was to assess the relationship between progression-free survival (PFS) in patients treated with DFMO + PCV (procarbazine, CCNU, vincristine) chemotherapy for malignant gliomas with tumor cell ornithine decarboxylase (ODC) activity. Formalin-fixed slides were obtained for study patients with anaplastic gliomas (AGs) and glioblastoma treated on protocol DM92-035. ODC levels were measured using an antibody to ODC coupled to Alexa 647 dye (Ab-ODC-Alexa 647). Ab-ODC-Alexa 647 intensity in transgenic murine hearts of differing ODC activity was used to calculate ODC activity in tumor cell nucleoplasm. In total, tumor specimens for 31 of 114 (27%) patients treated on the AG strata and 10 patients from the GBM strata were obtained. We found that tumor ODC level heterogeneity increased with increasing tumor malignancy. In a Cox proportional hazards model, PFS was found to be inversely related to median tumor ODC activity, with an unadjusted hazard ratio for median ODC group (>3.3 vs. 3.3 nmol/30 min/mug protein. Of AG tumors in which ODC activity was evaluated, 26% had ODC levels > 3.3 nmol/30 min/mug protein. This study shows that Ab-ODC-Alexa 647 fluorescence intensity can be used as a surrogate marker of ODC biochemical activity in AGs and can predict PFS to DFMO-based chemotherapy.     

Structure-activity relations of s-adenosylmethionine decarboxylase inhibitors on the growth of MCF-7 breast cancer cells

Summary SAMDC is a key enzyme in the biosynthesis of spermidine and spermine, 2 polyamines that are essential for cell proliferation. Inhibition of polyamine biosynthesis is often targeted as a therapeutic strategy to suppress cancer cell growth as these cells contain elevated levels of polyamines. We examined the effect of a new group of SAMDC inhibitors, CGP33829, CGP35753, CGP36958, CGP39937, and CGP48664, (obtained from CibaGeigy, Basel, Switzerland), and their parent compound, MGBG, on the proliferation of MCF-7 breast cancer cells. MGBG had minimal effects on the proliferation of MCF-7 cells up to 6 µM concentration. In contrast, CGP48664 and CGP39937, containing 2 aromatic rings that delocalize the electron system of the backbone of MGBG, were potent inhibitors with 50% growth inhibition at 0.5 µM concentration. Other CGP compounds were less effective in inhibiting cell growth. The ability of CGP48664 to inhibit MCF-7 cell proliferation was related to its ability to inhibit SAMDC and to consequently deplete spermidine and spermine levels in the cell. Exogenous spermidine and spermine could reverse the growth inhibitory effects of this compound. CGP compounds also increased the activity of ODC, another enzyme involved in polyamine biosynthesis. Northern blot analysis of mRNA from MCF-7 cells progressing in cell cycle after G1 synchronization did not show an increase in ODC mRNA level by CGP48664. These data demonstrate structure-activity relationships of a series of MGBG derivatives on cell growth, enzyme activities, and polyamine biosynthesis in a hormoneresponsive breast cancer cell line and suggest potential application of SAMDC inhibitors as therapeutic agents.     

Relation between induction of rat hepatic ornithine decarboxylase activity by tumor promoters 12-O-tetradecanoylphorbol-13-acetate and phenobarbital and levels of the polyamines putrescine, spermidine and spermine, in vivo; differential effects of retinyl-acetate

A single i.p. injection of 12-O-tetradecanoylphorbol-13-acetate(TPA) induced a transient increase in the levels of rat liverputrescine, spermidine and spermine. These polyamine concentrationscontinuously increased until 6 h, immediately following administrationof the tumor promoter. Phenobarbital (PB) induced an increaseof the putrescine and spermidine concentrations during the 8h post administration studied, while spermine reached a plateauafter 4 h. When retinyl-acetate (RA) was injected one hour priorto TPA, the increases of the polyamines were considerably inhibited.This treatment with RA partially inhibited further increaseof putrescine and spermine by PB. These findings are discussedin respect to the concomitant ornithine decarboxylase (ODC)activities, we have previously reported. I.p. injection of RAalone caused an elevation of ODC activity as well as putrescineand spermidine concentration within 2 h of exposure.     

Epidermal polyamine profiles after multiple exposures to ultraviolet radiation

In experimental studies of u.v.-skin-carcinogenesis u.v.-radiationis usually given in discrete amounts over a protracted periodof time. Epidermal polyamine profiles were investigated in hairlessmice after single and multiple exposures to ultraviolet-B (u.v.B).Hairless mice were irradiated with u.v.B from FS40 sunlampsand sacrificed after 1, 5, 10 or 20 days of daily irradiationwith 0.9 kJ/sq m u.v.B at 6, 24 or 48 h after the final irradiation.Epidermis was analyzed for ornithine decarboxylase (ODC) activity,and for its putrescine, spermidine and spermine content: Skinbiopsies were examined for histological changes. As previouslyreported epidermal ODC activity was induced 6 h after one irradiationwith u.v.B and reached a maximum activity at {small tilde}24h. In contrast after 5, 10 or 20 daily irradiations with u.v.Bthe epidermal ODC activity was maximal at {small tilde}6 h afterthe final irradiation and by 24 h had returned towards controllevels. The magnitude of the ODC activity measured 6 h afterirradiation increased with the number of irradiations. A similarpattern was seen with epidermal putrescine levels where a markedshift from a peak at {small tilde}24 h after one irradiationwith u.v.B to a peak at {small tilde}6 h after 20 days of irradiationwith u.v.B occurred. Spermidine levels increased as the numberof u.v.B exposures was increased and spermine levels tendedto decrease. The spermidine/spermine ratio increased most rapidlyduring the first 5 exposures, and remained elevated throughto 20 days of daily irradiation. Chronic irradiation with u.v.Bresults in rapid induction of ODC activity and putrescine accumulationin the epidermis, events also elevated by chemical or viraltransformation.     

Regulation of ornithine decarboxylase gene expression in MCF-7 breast cancer cells by antiestrogens

Ornithine decarboxylase (ODC) is an enzyme intimately related to cell growth regulation. The metabolic products of ODC, the polyamines, are known to play a vital role in the structure and function of biological macromolecules including nucleic acids and proteins. The activity of ODC is stimulated by estrogens in their target cells. In order to gain insight into the molecular mechanism of action of antiestrogens in human breast cancer, we studied the effect of tamoxifen and 4-hydroxytamoxifen on the concentration of ODC mRNA, ODC activity, and the polyamine levels in a hormone-responsive breast cancer cell line, MCF-7. ODC mRNA concentration was reduced to 40% of the controls after 6 h of treatment of the cells with 100 nM 4-hydroxytamoxifen, but tamoxifen had no significant effect on ODC mRNA after treating with even 1 microM concentration for 36 h. ODC activity was, however, reduced to 40 and 75% of the controls after 24 h of treatment with 4-hydroxytamoxifen and tamoxifen, respectively. There was a significant reduction in the concentration of putrescine to 63% of control in tamoxifen-treated cells, but spermidine and spermine levels were not affected. With 4-hydroxytamoxifen, putrescine, spermidine, and spermine levels were reduced to 41, 62, and 79% of the control, respectively. In addition, exogenous putrescine was able to reverse the growth inhibitory effects of 4-hydroxytamoxifen. Overall, these results indicate that ODC and polyamine levels in MCF-7 cells are controlled by antiestrogens, and that suppression of polyamine biosynthesis plays a critical role in the growth inhibitory effects of antiestrogens.     

Changes in epidermal polyamine biosynthesis and specific activity of DNA following a single application of 12-O-tetra-decanoyl-phorbol-13-acetate to hairless mouse skin

A single application of 12-O-tetradecanoyl-phorbol-13-acetate(TPA) to hairless mouse skin induces increased activity of epidermalL-ornithine carboxy-lyase (E.C. 4.1.1.17 [EC] ) (ODC) with a peakat 5 h, and S-adenosyl-L-methionine carboxy-lyase (E.C.4.1.1.50)(SAM-D) with a broad peak at 20 –36 h. The temporal sequenceof the accumulation of polyamines; i.e. putrescine, spermidineand spermine, and the rate of DNA synthesis was investigated.All four parameters were measured in the same tissue-samplesand multiple peaks of DNA synthesis and of individual polyamineswere demonstrated. In the period from 0 – 12 h, therewas an initial decrease in the rate of DNA synthesis. In thisperiod changes in the molar ratio of spermidine/spermine werenegatively correlated to the rate of DNA synthesis. From 12– 48 h, however, changes in the molar ratio of spermidine/spermine had an almost identical time course with rates of changeof DNA synthesis. Based on corresponding cell kinetic resultsit is suggested that the spermidine/ spermine ratio reachesa maximum peak during the S-phase of the cell cycle. The relationbetween the rate of DNA-synthesis and the spermidine/spermineratio as well as the ordered time sequence for the accumulationof putrescine and the induction of ODC and SAM-D activities,suggest a strong interdependence and a strict regulation ofthese events in hairless mouse epidermis induced to proliferateby TPA.     

Growth-inhibitory effects of the chemopreventive agent indole-3-carbinol are increased in combination with the polyamine putrescine in the SW480 colon tumour cell line

Background

Many tumours undergo disregulation of polyamine homeostasis and upregulation of ornithine decarboxylase (ODC) activity, which can promote carcinogenesis. In animal models of colon carcinogenesis, inhibition of ODC activity by difluoromethylornithine (DFMO) has been shown to reduce the number and size of colon adenomas and carcinomas. Indole-3-carbinol (I3C) has shown promising chemopreventive activity against a range of human tumour cell types, but little is known about the effect of this agent on colon cell lines. Here, we investigated whether inhibition of ODC by I3C could contribute to a chemopreventive effect in colon cell lines.

Methods

Cell cycle progression and induction of apoptosis were assessed by flow cytometry. Ornithine decarboxylase activity was determined by liberation of CO₂ from¹⁴C-labelled substrate, and polyamine levels were measured by HPLC.

Results

I3C inhibited proliferation of the human colon tumour cell lines HT29 and SW480, and of the normal tissue-derived HCEC line, and at higher concentrations induced apoptosis in SW480 cells. The agent also caused a decrease in ODC activity in a dose-dependent manner. While administration of exogenous putrescine reversed the growth-inhibitory effect of DFMO, it did not reverse the growth-inhibition following an I3C treatment, and in the case of the SW480 cell line, the effect was actually enhanced. In this cell line, combination treatment caused a slight increase in the proportion of cells in the G₂/M phase of the cell cycle, and increased the proportion of cells undergoing necrosis, but did not predispose cells to apoptosis. Indole-3-carbinol also caused an increase in intracellular spermine levels, which was not modulated by putrescine co-administration.

Conclusion

While indole-3-carbinol decreased ornithine decarboxylase activity in the colon cell lines, it appears unlikely that this constitutes a major mechanism by which the agent exerts its antiproliferative effect, although accumulation of spermine may cause cytotoxicity and contribute to cell death. The precise mechanism by which putrescine enhances the growth inhibitory effect of the agent remains to be elucidated, but does result in cells undergoing necrosis, possibly following accumulation in the G₂/M phase of the cell cycle.     

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 µM 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.     

Effect of retinoic acid pretreatment on 12-O-tetradecanoylphorbol-13-acetate-induced cell population kinetics and polyamine biosynthesis in hairless mouse epidermis

A single topical application of 17 nmol 12-O-tetradecanoyl-phorbol-13-acetate(TPA) to the skin of hairless mice induces characteristic transientalterations in the epidermal cell turnover and maturation (0–96h), associated in time with characteristic changes in the activitiesof L-ornithine carboxylyase (E.C. 4.1.1.17 [EC] ) (ODC) and S-adenosyl-L-methioninecarboxy-lyase (E.C. 4.1.1.50 [EC] ) (SAM-D) and in the accumulationof polyamines. The effects on these responses of local pretreatmentof the skin with retinoic acid 1 h prior to TPA were investigatedat selected time points. Retinoic acid inhibited the TPA-inducedODC activity and the ensuing accumulation of putrescine, butdid not alter the TPA-induced SAM-D activity or the molar ratioof spermidine/spermine. This pretreatment also decreased thenumber of dividing basal cells in the first TPA-induced synchronizedwave of proliferating cells. However, during the subsequentperiod of proliferation, the number of dividing cells in theretinoic acid pretreated group was comparatively increased.Hence, at four dose levels of retinoic acid (0.17, 1.70, 17.0and 170 nmol), which all inhibited the TPA-induced ODC effectively,there was no change in the total number of basal cells thatdivided during 16–48 h after TPA-application. The theoryis put forward that retinoic acid might exert its antitumorigeniceffect during tumor promotion with TPA by interfering with therate and/or quality of epidermal cell maturation, rather thanby inhibiting cell proliferation.     

Clinical application of18F-FUdR in glioma patients — PET study of nucleic acid metabolism

Summary Positron emission tomography was used to investigate the metabolism of nucleic acids by¹⁸F-fluoro-2-deoxyuridine (¹⁸F-FUdR) in 22 patients with gliomas. Sixteen cases of high grade glioma clearly demonstrated a region of high activity with a differential absorption rate (DAR) of 0.64 ± 0.34. Six cases of low grade glioma failed to reveal a positive image of the tumor and the DAR in tumor was 0.21 ± 0.042 (p < 0.01). This PET-¹⁸F-FUdR study succeeded in differentiating high and low grade gliomas from the view point of nucleic acid metabolism.     

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

Summary 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--estradiol, TGF-, 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.     

Properties of L1210 cells resistant to alpha-difluoromethylornithine

L1210 cells were selected for resistance to the ornithine decarboxylase (ODC) inhibitor, alpha-difluoromethylornithine. When grown in the absence of the inhibitor, these cells possessed very high ornithine decarboxylase levels. These represented about 1 part in 300 of the soluble protein, which is several hundred times greater than the maximal value found in the original L1210 cells. The resistant cells contained at least 100-fold higher levels of ODC mRNA but the half-life of ODC (about 45 min) was not altered significantly. The resistant cells had much higher putrescine and cadaverine levels than control cells, but there was no significant difference in cellular spermidine or spermine content or in production of 5'-methylthioadenosine, which is a measure of polyamine synthesis. Addition of putrescine to the control or resistant cells had no effect on their content of spermidine and spermine but addition of decarboxylated S-adenosylmethionine increased the content of spermidine and spermine. These results indicate that ornithine decarboxylase is not the rate-limiting step in polyamine synthesis in these L1210 cells. The growth of the alpha-difluoromethylornithine-resistant L1210 cells was inhibited when their ability to synthesize spermidine and spermine was blocked by the addition of the S-adenosylmethionine decarboxylase inhibitor, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine. Treatment with this compound produced a reduction of more than 85% in the production of 5'-methylthioadenosine and led to a large increase in the content of putrescine and a substantial decline in the content of spermidine and spermine. These results indicate the potential value of S-adenosylmethionine decarboxylase inhibitors as therapeutic agents in conditions where ODC inhibitors are ineffective.     

Spermine synthase overexpression in vivo does not increase susceptibility to DMBA/TPA skin carcinogenesis or Min-Apc intestinal tumorigenesis

Numerous studies have demonstrated a link between elevated polyamine biosynthesis and neoplastic growth, but the specific contribution of spermine synthase to epithelial tumor development has never been explored in vivo. Mice with widespread overexpression of spermine synthase (CAG-SpmS) exhibit decreased spermidine levels, increased spermine and a significant rise in tissue spermine:spermidine ratio. We characterized the response of CAG-SpmS mice to two-stage skin chemical carcinogenesis as well as spontaneous intestinal carcinogenesis induced by loss of the Apc tumor suppressor in Apc (Min) (/+) (Min) mice. CAG-SpmS mice maintained the canonical increases in ornithine decarboxylase (ODC) activity, polyamine content and epidermal thickness in response to tumor promoter treatment of the skin. The induction of S-adenosylmethionine decarboxylase (AdoMetDC) activity and its product decarboxylated AdoMet were impaired in CAG-SpmS mice, and the spermine:spermidine ratio was increased 3-fold in both untreated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated skin. The susceptibility to 7,12-dimethylbenz[a]anthracene (DMBA)/TPA skin carcinogenesis was not altered in CAG-SpmS mice, and SpmS overexpression did not modify the previously described tumor resistance of mice with targeted antizyme expression or the enhanced tumor response in mice with targeted spermidine/spermine-N ( 1) -acetyltransferase expression. CAG-SpmS/Min mice also exhibited elevated spermine:spermidine ratios in the small intestine and colon, yet their tumor multiplicity and size was similar to Min mice. Therefore, studies in two of the most widely used tumorigenesis models demonstrate that increased spermine synthase activity and the resulting elevation of the spermine:spermidine ratio does not alter susceptibility to tumor development initiated by c-Ha-Ras mutation or Apc loss.     

Nestin and CD133: valuable stem cell-specific markers for determining clinical outcome of glioma patients

Aim

Gliomas represent the most frequent neoplasm of the central nervous system. Unfortunately, surgical cure of it is practically impossible and their clinical course is primarily determined by the biological behaviors of the tumor cells. The aim of this study was to investigate the correlation of the stem cell markers Nestin and CD133 expression with the grading of gliomas, and to evaluate their prognostic value.

Methods

The tissue samples consisted of 56 low- (WHO grade II), 69 high- (WHO grade III, IV) grade gliomas, and 10 normal brain tissues. The expression levels of Nestin and CD133 proteins were detected using SABC immunohistochemical analysis. Then, the correlation of the two markers' expression with gliomas' grading of patients and their prognostic value were determined.

Results

Immunohistochemical analysis with anti-Nestin and anti-CD133 antibodies revealed dense and spotty staining in the tumor cells and their expression levels became significantly higher as the glioma grade advanced (p < 0.05). There was a positive correlation between the two markers' expression in different gliomas tissues (rs = 0.89). The low expression of the two markers significantly correlated with long survival of the glioma patients (p < 0.05). The survival rate of the patients with Nestin+/CD133+ expression was the lowest (p < 0.01), and the multivariate analysis confirmed that conjoined expression of Nestin+/CD133+ and Nestin-/CD133- were independent prognostic indicators of gliomas (both p < 0.01, Cox proportional hazard regression model).

Conclusion

These results collectively suggest that Nestin and CD133 expression may be an important feature of human gliomas. A combined detection of Nestin/CD133 co-expression may benefit us in the prediction of aggressive nature of this tumor.

     

Elevated levels of ornithine decarboxylase and polyamines in the kidneys of estradiol-treated male hamsters

The effects of 17 ß-estradiol (E₂) on the levels ofornithlne decarboxytase (ODC), S-adenosylmethionine decarboxylase(SAMDC), and of the polyamines putrescine, spermidine and sperminein the kidneys of castrated male hamsters were determined. Thei.p. injection of E₂ into male hamsters led to renal ODC levelsthree times above the control levels 6–12 h after treatment.Similarly, the renal ODC levels in hamsters treated with chronicdoses of E₂ for 60–180 days were 13–1.9 times thecorresponding enzyme levels in control, sham-operated animals.With a series of estrogen analogues, there was a direct correlationbetween the rise in renal ODC in vitro and the binding to renalestradiol receptor sites in vitro. The hamster kidney levelsof the polyamines putrescine, spermidine and spermine all declinedduring the 180-day experimental period. A single i.p. injectionof E led to a 70% increase in renal SAMDC activity 12 h aftertreatment. However, the administration of E for 180–270days was without effect on the normal age-dependent declinein SAMDC levels noted in kidneys. These results indicate that,like other carcinogens and promoters, E₂ increases the levelsof ODC and of polyamines in its target tissue and that the risein ODC is mediated by a specific estradiol-binding protein.     

Constitutively elevated levels of ornithine and polyamines in mouse epidermal papillomas

Epidermal papillomas were induced in CD-1 mice by a single topical application of 7,12-dimethylbenzanthracene (DMBA) followed by twice weekly applications of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in acetone. Control groups consisted of mice treated singly or chronically with acetone or TPA. TPA induced a rapid, yet transient 500- to 1000-fold increase in ornithine decarboxylase (ODC) activity which resulted in a 2- to 8.4-fold elevation of putrescine in both singly or chronically TPA-treated mouse epidermis 4-6 h after its application. After 24 h, levels of spermidine, but not spermine, were also elevated. The ODC and arginase activities in the 11 individual papillomas studied averaged 400- and 26-fold higher respectively than basal levels in epidermis. The activity of ODC in most papillomas, unlike ODC in epidermis, could be stimulated by guanosine 5'-triphosphate (GTP). Putrescine and spermidine levels in papillomas, especially those exhibiting highly GTP-stimulated ODC, were substantially higher compared to either normal or TPA-treated epidermis. Although epidermis contains a relatively high ornithine content, its level is even further elevated in papillomas, in some cases as much as 70-fold. The consequences of the constitutively elevated polyamine levels in papillomas caused by the loss of control over the normally tightly regulated polyamine biosynthetic pathway are not known, but could be important in regulating the balance between proliferation and differentiation in this self-renewing epithelial tissue.     

Individual and combined effects of alpha-difluoromethylornithine and ovariectomy on the growth and polyamine milieu of experimental breast cancer in rats

Despite considerable evidence suggesting a critical role of polyamines in the hormonal control of breast cancer growth in vitro, their role in in vivo tumor growth is not established. In these experiments, we evaluated the individual and combined effects of the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) and ovariectomy on the growth and cellular levels of ornithine decarboxylase (ODC) and polyamines of N-nitrosomethylurea-induced rat mammary tumors. Despite a similar suppressive effect on ODC activity, the two treatments had a different effect on polyamine levels. As expected, DFMO selectively suppressed putrescine, whereas spermidine and spermine levels were minimally or not affected at all. Since quantitatively putrescine contributes the least to overall polyamine pools, the DFMO effect on this latter parameter was modest. In contrast, ovariectomy, by suppressing the more abundant spermidine and spermine, produced a more profound suppression of total polyamine pools. This finding is in agreement with the notion that hormones not only control ODC activity, but also other enzymes involved in the synthesis of the distal polyamines. Ovariectomy was also more potent than DFMO administration in inhibiting N-nitrosomethylurea-induced mammary tumor growth. No major additive/synergistic effects were observed between DFMO and ovariectomy on tumor growth and cellular levels of ODC activity and polyamines. DFMO administration lowered the tumor level of progesterone receptors and appeared to potentiate the suppressive effect of ovariectomy. In contrast, neither treatment, alone or in combination, altered tumor levels of estrogen receptors. DFMO administration did not affect circulating levels of estradiol and prolactin or uterine and ovarian weights, thus suggesting that its effects were not indirectly mediated through alterations of the endocrine milieu of the host.     

Prolonged ornithine decarboxylase induction in regenerating carcinogen-treated liver

A cascade of events leading to hypertrophy has been proposed and implicated in growth regulation in a variety of normal and neoplastic cells and tissues. There is a tightly coupled temporal sequence: (a) cyclic adenosine 3':5'-monophosphate-dependent protein kinase (cAPK) activation; (b) ornithine decarboxylase (ODC) induction; and (c) the accumulation of the organic cation, spermidine, resulting in an increased spermidine/spermine ratio characteristic of both normal and neoplastic growth. The specific activation of type I cAPK has been implicated to ODC induction, and the amounts of type I and type II cAPK alter as a function of growth and transformation. Therefore, we wished to study the alterations in these biochemical parameters as well as that of a putative marker of preneoplastic hepatocytes, gamma-glutamyltranspeptidase, in a rapid multistep hepatocarcinogenesis system. We found a marked and prolonged increase in the cAPK ratio followed by a similar pattern of ODC induction after a single carcinogenic dose of diethylnitrosamine and again in response to partial hepatectomy. Liver foci were detectable within four days of partial hepatectomy in animals that received the entire carcinogen regimen, and the foci contained significant and increasing amounts of gamma-glutamyltranspeptidase activity. The increase in ODC activity was followed closely by an increased spermidine/spermine ratio. Total type I activity in the cytosol decreased most dramatically at the time of foci formation, suggestive of selective activation and turnover. These data suggest that the prolonged activation of cAPK and elevation of ODC may be necessary for hepatocarcinogenesis.