Tumor progression is accompanied by significant changes in the levels of expression of polyamine metabolism regulatory genes and clusterin (sulfated glycoprotein 2) in human prostate cancer specimens.

Using Northern blotting, the expression levels of the genes for polyamine metabolism regulatory proteins and clusterin have been measured in a series of 23 human prostate cancers (CaPs) dissected from radical prostatectomy specimens. Patient matched, nontumor tissue was dissected from benign areas of the gland. The results indicate that transcripts encoding ornithine decarboxylase (ODC), ODC antizyme, adenosylmethionine decarboxylase, and spermidine/spermine N1-acetyltransferase (SSAT) were significantly higher, whereas clusterin (sulfated glycoprotein 2) mRNA was significantly lower in tumors compared with the benign tissue. All mRNA levels were compared with those of histone H3 and growth arrest-specific gene 1, markers of cell proliferation and cell quiescence, respectively, and glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene. In poorly differentiated and locally invasive CaPs and in tumors with unfavorable prognosis or total prostate-specific antigen (PSA) levels > 10.0 ng/ml at diagnosis, an overall increase in the levels of H3 mRNA and a decrease in growth arrest-specific gene 1 mRNA was detected, indicative of higher proliferation activity, whereas the differences in expression levels for the polyamine metabolism and clusterin genes were higher. ODC and SSAT changes were positively correlated in normal tissue but not in high-grade cancer, whereas ODC antizyme and SSAT changes were positively correlated in more malignant CaPs but not in normal tissue. Tumor classification based on the changes in expression levels of all of the genes studied could be correlated to differentiation grade and local invasiveness classification systems in 72.2 and 83.3% of the cases, respectively. In a 1-year follow-up period, three patients whose CaPs ranked as less aggressive according to clinical staging, but classified as advanced cancers with the proposed molecular classification, showed increases in total PSA levels, indicative of tumor relapse. Thus, molecular classification, based on gene expression, may enhance the available prognostic tools for prostate tumors.

Gene relaxation and aging: changes in the abundance of rat ventral prostate SGP-2 (clusterin) and ornithine decarboxylase mRNAs.

Sulfated glycoprotein 2 (SGP-2) mRNA progressively increased in the ventral prostate of the aging rat, reaching, at 24 months, 4-fold higher than at 3 months. Ornithine decarboxylase (ODC) mRNA peaked at 6 months (4-fold increase), and at 12 and 24 months was maintained at higher levels than at 3 months. ODC enzymatic activity was enhanced at 6 months to a much smaller extent than its own mRNA, the values at 12 and 24 months dropping to below those at 3 months. Putrescine (Put), spermidine (Spd) and spermine (Sp) concentrations also peaked at 6 months (100% increase for Put, 50% for Sp and Spd). At 24 months, Put and Spd were diminished, and Sp was unchanged with respect to the 3-month values. Under the same conditions, glyceraldehyde-3-phosphate dehydrogenase mRNA did not undergo significant alterations.

Clusterin (SGP-2) gene expression is cell cycle dependent in normal human dermal fibroblasts.

In confluent human dermal fibroblasts brought to quiescence (G0) by serum starvation, the S phase peaked at 24 h after serum re-addiction and G2/M phase peaked at 36 h. This was confirmed by titration of h-gas1 mRNA (a marker of G0 phase) and histone H3 (a marker of S phase). Clusterin mRNA accumulation progressively increased in cells proceeding to confluence after seeding and to quiescence upon serum starvation, and peaked at around G0, in parallel with h-gas1 mRNA. At 6 h (roughly G1 phase) clusterin transcript formed a second peak, followed by a gradual decrease until 36 h. Correspondence of clusterin protein accumulation to its mRNA occurred solely with regard to the G0 peak but not to the second one. The possible meaning of the cell cycle related clusterin gene expression is discussed.

Coordinate changes of polyamine metabolism regulatory proteins during the cell cycle of normal human dermal fibroblasts.

In human dermal fibroblasts, brought to quiescence (G0) by serum starvation, the S phase peaked 24 h and G2/M phases 36 h after serum re-addition. Under the same conditions, ornithine decarboxylase mRNA peaked at 12 h, decreased markedly in S phase and remained low until 48 h. Conversely, ornithine decarboxylase antizyme transcript dropped to its lowest level at 12 h, while reaching its highest values between 24 and 48 h. Ornithine decarboxylase activity followed essentially the pattern of its mRNA, but relative changes were much greater. S-Adenosylmethionine decarboxylase transcript and enzyme activity also peaked at around 12 h, decreasing thereafter. Spermidine/spermine N1-acetyltransferase mRNA and activity reached the highest values at 36-48 h. Putrescine concentration increased up to 18 h and fell dramatically in the S phase, remaining low thereafter. Both spermidine and spermine reached peaks at 18 h and decreased in the S phase, but not nearly as much as putrescine. We discuss how this comprehensive study may help to understand the involvement of polyamines in the control of cell proliferation.

Differential induction of spermidine/spermine N1-acetyltransferase activity in cisplatin-sensitive and -resistant ovarian cancer cells in response to N1,N12-bis(ethyl)spermine involves transcriptional and post-transcriptional regulation.

The growth inhibition that occurs in cisplatin-sensitive 2008 human ovarian cancer cells in response to the spermine analogue, N1,N12-bis(ethyl)spermine (BESpm), is associated with a potent induction of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in polyamine catabolism. Conversely, in cisplatin-resistant C13* cells, which are less responsive to BESpm, enzyme induction does not occur at comparable levels after exposure to the bis(ethyl)-derivative. In this study, we investigated the molecular mechanisms underlying the differential induction of SSAT activity in cisplatin-sensitive and -resistant cells. Northern blot analysis revealed a difference in the level of SSAT mRNA expression in the two cell lines; in particular, 2008 cells treated with 10 microM BESpm for progressively increasing periods of time accumulated more heteronuclear (3.5 kb) and mature (1.3/1.5 kb) SSAT mRNAs than its resistant variant. SSAT mRNA accumulation paralleled enzyme activity and both were almost completely prevented in the two lines by co-treatment with 5 microg/ml actinomycin-D (Act-D), suggesting that transcription plays a major role in the analogue-mediated induction of SSAT. Moreover, when Act-D was added 48 h after BESpm exposure, SSAT mRNA and enzyme activity were stabilised in both cell lines. Therefore, the marked difference in the induction of SSAT activity seems to be related to increased enzyme synthesis, particularly in sensitive cells, whose SSAT protein turnover was also greatly reduced (half-life >12 h in 2008 cells versus 5 h in C13* cells) in the presence of BESpm. These findings suggest that cisplatin-resistance modulates the SSAT response to BESpm at transcriptional and post-transcriptional levels.

Increased levels of clusterin (SGP-2) mRNA and protein accompany rat ventral prostate involution following finasteride treatment.

Finasteride is a well-known inhibitor of the prostatic enzyme 5 alpha-reductase type 2 which prevents conversion of testosterone into 5 alpha-dihydrotestosterone, the active intraprostatic androgen, which causes prostate involution through a combination of cell atrophy and cell death. The drug is widely used to improve symptoms of benign prostatic hyperplasia in man. Clusterin, a glycoprotein which is generally up-regulated under conditions inducing cell atrophy or organ involution, is produced at a high level in the regressing rat ventral prostate following androgen ablation. According to several authors, clusterin does not respond to finasteride treatment, suggesting a different action of testosterone and 5 alpha-dihydrotestosterone. We show here that, under our conditions, finasteride was capable of inducing production of both clusterin mRNA and protein in the rat ventral prostate. In fact, by using different and converging techniques, such as Northern hybridization, in situ hybridization histochemistry and immunohistochemistry, we were able to show a strong induction of the clusterin gene in the epithelial cell population of the gland. The response to finasteride, which was similar to that seen with castration, occurred with a delay of a few days. In situ and immunohistochemistry experiments indicated that both orchidectomy and finasteride administration resulted in increased transition of the epithelial cells from the columnar to the cuboidal (atrophic) shape, and this was accompanied by an increased intensity of the signal for clusterin. Thus, it appears that induction of clusterin is part of the molecular process leading to prostate involution caused by either orchidectomy or finasteride administration.

Cadmium induction of renal and hepatic ornithine decarboxylase activity in the rat. Effects of sex hormones and involvement of the renin-angiotensin system.

We investigated the effect of sex hormones on the sex-dependent response of rat kidney ornithine decarboxylase (ODC) activity to cadmium (Cd) administration and the involvement of the renin-angiotensin system in mediating stimulation of the liver enzyme by the metal. The response of renal ODC to Cd, which occurs in intact adult males but not in females, is also detectable in prepubertal and castrated males. Upon treatment with 17 beta-estradiol, the basal levels of enzyme activity in intact or castrated adult males were enhanced and Cd administration failed to increase them further. In adult females the kidney enzyme became responsive after ovariectomy. Also, in prepubertal females renal ODC was induced by Cd, and this was prevented by treatment with 17 beta-estradiol. Under the same conditions, changes in the levels of Cd accumulation within the kidney, that might account for variations in the response of ODC activity, did not occur. Cd caused an increase in renin activity starting minutes after its injection. Captopril, which specifically inhibits the conversion of angiotensin I to angiotensin II, prevented completely the induction of liver ODC by this metal; stimulation of the enzyme by Co was not affected by the drug. A similar inhibitory effect was exerted by propranolol. Adrenalectomy had no influence on the response of hepatic ODC to Cd; the decarboxylase was unaffected by aldosterone administration. It is suggested that Cd may induce liver ODC through the increase in angiotensin II following stimulation of renin by the metal.

Affinity chromatography of yeast nicotinamide-adenine dinucleotide-specific isocitrate dehydrogenase on immobilized nicotinamide-adenine dinucleotide. Effects of ligands.

The method of affinity chromatography has been used for studying the effects of some ligands of yeast NAD-specific isocitrate dehydrogenase on the affinity of the enzyme for NAD+ immobilized on Sepharose 4B. In absence of ligands, the enzyme is eluted from NAD+-Sepharose columns by 0.1 M phosphate buffer, pH 7.6, in a highly purified form. The elution of enzyme is accelerated by NAD+ and, more effectively, by AMP; and retarded by isocitrate and citrate. The elution patterns show a rather irregular shape, probably due to the occurrence of aggregation processes of the enzyme protein.

Polymeric forms of yeast nicotinamide-adenine dinucleotide-specific isocitrate dehydrogenase in the presence of various ligands.

The molecular weight of NAD-specific isocitrate dehydrogenase, purified from baker's yeast, has been studied by molecular sieve chromatography. By elution of the enzyme from columns of Sepharose 6B with 0.05 M phosphate buffer, pH 7.6, a mol. wt of 151,000 was measured. Higher values of the mol. wt were measured in presence of the following ligands (mol. wt in parentheses): the substrate, isocitrate (224,000); the activators, citrate (203,000) and AMP (275,000); the inhibitor, NaCl (360,000). A mol. wt of 337,000 was measured when AMP, which antagonizes the inhibition by chloride, was present together with NaCl. The results indicate the absence of a correlation between the aggregation form of the enzyme in presence of the ligands and the effects of these ligands on the enzyme activity.

Genetic inactivation of ApoJ/clusterin: effects on prostate tumourigenesis and metastatic spread.

ApoJ/Clusterin (CLU) is a heterodimeric protein localized in the nucleus, cytoplasm or secretory organelles and involved in cell survival and neoplastic transformation. Its function in human cancer is still highly controversial. In this study, we examined the prostate of mice in which CLU has been genetically inactivated. Surprisingly, we observed transformation of the prostate epithelium in the majority of CLU knockout mice. Either PIN (prostate intraepithelial neoplasia) or differentiated carcinoma was observed in 100 and 87% of mice with homozygous or heterozygous deletion of CLU, respectively. Crossing CLU knockout with TRAMP (prostate cancer prone) mice results in a strong enhancement of metastatic spread. Finally, CLU depletion causes tumourigenesis in female TRAMP mice, which are normally cancer free. Mechanistically, deletion of CLU induces activation of nuclear factor-kB, a potentially oncogenic transcription factor important for the proliferation and survival of prostate cells.

Blockade of alpha-and beta -adrenergic receptors can prevent stimulation of liver ornithine decarboxylase activity by glucocorticoid or laparatomy.

Rat liver ornithine decarboxylase induction by dexamethasone or laparatomy, which is dramatically impaired by catecholamine depletion, is not affected by alpha-and beta -adrenergic blockers administered simultaneously 1 h prior to steroid injection or operation. However, if blockade is maintained for 24 h, an effect comparable to that of catecholamine depletion is obtained. Reciprocally, the response of the decarboxylase to catecholamines is severely compromised in adrenalectomized rats. Under the same conditions, induction of tyrosine aminotransferase by dexamethasone is not significantly affected by catecholamine availability, which altogether demonstrates that rat liver ornithine decarboxylase activity is specifically governed by the interaction between glucocorticoids and catecholamines.

Response of hepatic ornithine decarboxylase and polyamine concentration to surgical stress in the rat: evidence for a permissive effect of catecholamines on glucocorticoid action.

Laparatomy of the rat dramatically induced hepatic ornithine decarboxylase that reached a peak 4 h after the operation. A similar pattern was shown by putrescine concentration. Spermidine was also enhanced, while spermine maintained unchanged. Administration to the animals of either isoproterenol or glucocorticoids (hydrocortisone or dexamethasone) also caused dramatic elevation of liver ornithine decarboxylase. The effect of isoproterenol, but not that of glucocorticoids, was prevented by previous treatment with propranolol. The beta-blockade was unable to prevent the effect of laparatomy on the liver enzyme. This was obtained instead, by depleting the endogenous catecholamines with either alpha-methyl-p-tyrosine or reserpine. Under these conditions, administration of glucocorticoids had no effect on the hepatic enzyme.

Polyamine distribution and activity of their biosynthetic enzymes in the European sea bass (Dicentrarchus labrax L.) compared to the rat.

1. In the liver, heart and brain of the European sea bass, putrescine concentrations are much higher than in the equivalent rat tissues; spermidine and spermine levels are smaller. 2. Ornithine decarboxylase in the bass liver is more active, but less stable than that in the rat; stability is acquired upon partial purification. Bass liver adenosylmethionine decarboxylase activity is less than that found in the rat. Both are activated and stabilized by putrescine. 4. The activating effect of putrescine decreases as the assay temperature is decreased. This may explain the high level of putrescine but low levels of spermidine and spermine in the bass liver.

Manipulation of the expression of regulatory genes of polyamine metabolism results in specific alterations of the cell-cycle progression.

We have previously reported that cyclical phases of accumulation and depletion of polyamines occur during cell-cycle progression. Regulatory ornithine decarboxylase (ODC) catalyses the first step of polyamine biosynthesis. Ornithine decarboxylase antizyme (OAZ), induced by high polyamine levels, inhibits ODC activity and prevents extracellular polyamine uptake. Spermidine/spermine N1-acetyltransferase (SSAT) regulates the polyamine degradation/excretion pathway. Here we show that 24 h transient transfection of immortalized human prostatic epithelial cells (PNT1A and PNT2) with antisense ODC RNA or OAZ cDNA, or both, while effectively causing marked decreases of ODC activity and polyamine (especially putrescine) concentrations, resulted in accumulation of cells in the S phase of the cell cycle. Transfection with SSAT cDNA led to more pronounced decreases in spermidine and spermine levels and resulted in accumulation of cells in the G2/M phases. Transfection with all three constructs together produced maximal depletion of all polyamines, accompanied by accumulation of PNT1A cells in the S phase and PNT2 cells in the G0/G1 and G2/M phases. Accumulation of PNT1A cells in the S phase progressively increased at 15, 18 and 24 h of transfection with antisense ODC and/or OAZ cDNA. At 24 h, the DNA content was always reduced, as a possible outcome of altered chromosome condensation. A direct link between polyamine metabolism, cell proliferation and chromatin structure is thus proposed.

Ornithine decarboxylase in perfused rat heart.

In the isolated perfused rat hearts, the activity of tissue ornithine decarboxylase gradually decreases over 90 min. In contrast, the activity of S-adenosylmethionine decarboxylase, lactate dehydrogenase, and glutamate-oxalacetate transaminase stays unchanged after a small decrease during the first 10 min. Ornithine decarboxylase is released from the perfused heart under conditions in which neither the lower molecular weight S-adenosylmethionine decarboxylase nor polyamines leak out. Ten minutes of ischaemia did not change the rate of release of ornithine decarboxylase. Ischaemia followed by reperfusion (20 min) increased release of ornithine decarboxylase.