Isolation of tumour stem-like cells from benign tumours.

BACKGROUND: Cancerous stem-like cells (CSCs) have been implicated as cancer-initiating cells in a range of malignant tumours. Diverse genetic programs regulate CSC behaviours, and CSCs from glioblastoma patients are qualitatively distinct from each other. The intrinsic connection between the presence of CSCs and malignancy is unclear. We set out to test whether tumour stem-like cells can be identified from benign tumours. METHODS: Tumour sphere cultures were derived from hormone-positive and -negative pituitary adenomas. Characterisation of tumour stem-like cells in vitro was performed using self-renewal assays, stem cell-associated marker expression analysis, differentiation, and stimulated hormone production assays. The tumour-initiating capability of these tumour stem-like cells was tested in serial brain tumour transplantation experiments using SCID mice. RESULTS: In this study, we isolated sphere-forming, self-renewable, and multipotent stem-like cells from pituitary adenomas, which are benign tumours. We found that pituitary adenoma stem-like cells (PASCs), compared with their differentiated daughter cells, expressed increased levels of stem cell-associated gene products, antiapoptotic proteins, and pituitary progenitor cell markers. Similar to CSCs isolated from glioblastomas, PASCs are more resistant to chemotherapeutics than their differentiated daughter cells. Furthermore, differentiated PASCs responded to stimulation with hypothalamic hormones and produced corresponding pituitary hormones that are reflective of the phenotypes of the primary pituitary tumours. Finally, we demonstrated that PASCs are pituitary tumour-initiating cells in serial transplantation animal experiments. CONCLUSION: This study for the first time indicates that stem-like cells are present in benign tumours. The conclusions from this study may have applications to understanding pituitary tumour biology and therapies, as well as implications for the notion of tumour-initiating cells in general.

Dexamethasone inhibits the anti-tumor effect of interleukin 4 on rat experimental gliomas.

Retroviral-mediated gene transfer of the IL-4 gene into experimental gliomas can cause tumor rejection, supporting the clinical use of this form of gene therapy for glioblastomas (GBM). In a clinical setting, the administration of dexamethasone (dex) is a standard procedure for GBM patients. This led us to examine the effects of dex on IL-4 gene therapy. We injected intracranially Fischer 344 rats with phosphate-buffered saline, 9L gliosarcoma cells mixed with E86.L4SN(200) cells (retroviral producer cells, RPC, transducing IL-4 cDNA) and 9L cells mixed with PA317.STK.SBA cells (control RPC expressing the HSV-tk gene). The rats from each group were treated with 0, 50, 100 or 250 microg dex/kg/day released by osmotic pumps implanted subcutaneously. While 80-100% of rats receiving 9L cells mixed with IL-4 RPC and not treated by dex survived for at least 2 months following tumor injection, only 50% and 17% of rats receiving 50 or 100 microg/kg/day of dex, respectively, reached this time point. These results indicate that dex significantly diminished the anti-tumor effect of IL-4. Thus, in a clinical setting, IL-4 gene transfer should be performed when low levels of dex are administered or in the absence of dex.

Mxi1 inhibits the proliferation of U87 glioma cells through down-regulation of cyclin B1 gene expression.

Mxi1 is a Mad family member that plays a role in cell proliferation and differentiation. To test the role of Mxi1 on tumorigenesis of glioma cells we transfected a CMV-driven MXI1 cDNA in U87 human glioblastoma cells. Two clones were isolated expressing MXI1 levels 18- and 3.5-fold higher than wild-type U87 cells (clone U87.Mxi1.14 and U87.Mxi1.22, respectively). In vivo, U87.Mxi1.14 cells were not tumorigenic in nude mice and delayed development of tumours was observed with U87.Mxi1.22 cells. In vitro, the proliferation rate was partially and strongly inhibited in U87.Mxi1.22 and U87.Mxi1.14 cells respectively. The cell cycle analysis revealed a relevant accumulation of U87.Mxi1.14 cells in the G(2)/M phase. Interestingly, the expression of cyclin B1 was inhibited to about 60% in U87.Mxi1.14 cells. This inhibition occurs at the transcriptional level and depends, at least in part, on the E-box present on the cyclin B1 promoter. Consistent with this, the endogenous Mxi1 binds this E-box in vitro. Thus, our findings indicate that Mxi1 can act as a tumour suppressor in human glioblastomas through a molecular mechanism involving the transcriptional down-regulation of cyclin B1 gene expression.

Polyamine oxidase and tissue transglutaminase activation in rat small intestine by polyamines.

Polyamine degradation was studied in the small intestine from rats fed on a polyamine-supplemented diet. Lactalbumin diet was given to Hooded-Lister rats, with or without 5 mg rat(-1) day(-1) of putrescine or spermidine for 5 days. Polyamine oxidase activity increased with putrescine and spermidine in the diet, whereas spermidine/spermine N(1)-acetyltransferase and diamine oxidase activities were unchanged. We also studied the calcium-dependent and -independent tissue transglutaminase activities, since they can modulate intestinal polyamine levels. Both types of enzymes increased in the cytosolic fraction after putrescine (about 65%) or spermidine (80-100%). Our results indicate that exogenous polyamines stimulate intestinal polyamine oxidase and tissue transglutaminase activities, probably to prevent polyamine accumulation, when other pathways of polyamine catabolism (acetylation and terminal catabolism) are not activated.

Distribution and activity of transglutaminase in rat brain carcinogenesis and in gliomas.

Tissue transglutaminase is a calcium-dependent enzyme which may influence cell morphology, cytoskeletal processes and membrane functions. During rat brain carcinogenesis induced by transplacental administration of N-ethyl-N-nitrosourea to BD IX rats, cytosolic tissue transglutaminase activity was increased by about 140% at 30 days of extrauterine life and returned towards the control values at 3-5 months. In the particulate fraction, enzyme activity progressively increased, reaching values similar to those present in the developed gliomas. Tissue transglutaminase activity in gliomas had a behavior inverse to that observed in controls, with a decrease (about 50%) in the cytosol and a marked increase (380%) in the particulate fraction, indicating a redistribution of enzyme activity.

In vivo modulation of 73 kDa heat shock cognate and 78 kDa glucose-regulating protein gene expression in rat liver and brain by ethanol.

BACKGROUND: In cultured cells of various origin, ethanol induces the synthesis of 70 kDa family heat shock proteins (hsp70 family), which play a role in the protection of protein traffic and secretion, as well as in cytoskeleton organization. To assess whether ethanol also can induce such genes in vivo, we studied the behavior of hsp70, hsc73, and grp78 messenger ribonucleic acids (mRNAs) and related proteins in the liver and brain of rats acutely treated with ethanol. METHODS: Overnight fasted Sprague-Dawley rats (220-250 g) were acutely treated with a low (2 g/kg body weight) or a high (5 g/kg body weight) dose of ethanol as a 30% solution in saline or an equal volume of saline (controls) by gastric intubation. Animals were killed at various times after treatments (3-72 hr). Messenger RNA levels for different members of hsp70 family (hsp70; 73 kDa heat shock cognate, or hsc73; and 78 kDa glucose-regulating protein, or grp78) were determined by Northern blot analysis and hybridization with specific complementary deoxyribonucleic acid (cDNA) probes. The amounts of related proteins were assayed by Western blot analysis with specific antibodies. Autoradiograms and fluorograms were subjected to densitometric scanning. RESULTS: Ethanol (2 g/kg) caused a slight increase in hsc73 and grp78 mRNA levels only in the liver, without enhancing the amount of proteins. Ethanol (5 g/kg) increased the level of hsc73 and grp78 mRNAs and related proteins in the liver. In the brain, the amount of hsc73 mRNA was enhanced, but this did not change hsc73 protein. In addition, we observed an increase in cerebral grp78 transcript and related protein. Hsp70 gene was not induced in the examined tissues by either dose of ethanol. CONCLUSIONS: Hepatic and cerebral hsc73 and grp78 genes are responsive to ethanol in vivo, and their activation may signal the cell's effort to counteract the harmful action of ethanol.

Changes in hepatic polyamine catabolism in elderly rats.

AIMS/BACKGROUND: Given the important role of polyamines (putrescine, spermidine, spermine) in the modulation of macromolecular syntheses, gene expression and proteolysis, alterations in their metabolic pathways could be relevant during senescence. Since the few existing data address mainly polyamine biosynthesis, we studied the oxidative catabolism of polyamines in the liver of rats 3-36 months of age. METHODS: Polyamine oxidase activity was fluorimetrically measured using N1-acetylspermine as substrate. Spermidine/spermine N1-acetyltransferase and diamine oxidase were measured by radiochemical methods using labeled acetyl-coenzyme A and putrescine, respectively, as substrate. Polyamines were separated by HPLC and fluorimetrically quantified after post-column derivatization with o-phthaldialdehyde. RESULTS: Spermidine/spermine N1-acetyltransferase activity increased in 36-month-old rats and polyamine oxidase activity in 24- and 36-month-old rats. A decline in spermine and increases in spermidine and putrescine in elderly rats suggested an activation of the interconversion pathway of higher into lower polyamines. The activity of diamine oxidase, which degrades putrescine, was enhanced starting from 12 months of age. CONCLUSION: In the liver of aged rats, an increase in the catabolic enzymes leads to a reconversion of the higher polyamines to putrescine. This increased catabolism may represent an important age-related change and may contribute to impairment of the expression of growth-related genes in senescence.

Oxidative degradation of polyamines in rat pancreatic hypertrophy.

In the hypertrophic pancreas, we studied the oxidative degradation of polyamines, which are endogenous polycations important for cell division, growth and differentiation. To induce pancreatic hypertrophy, rats were fed on a semi-synthetic diet containing a daily dose of 42 mg phytohaemagglutinin per rat for 5 or 10 days. In the model, the activities of polyamine oxidase (the enzyme that degrades spermidine, spermine and mainly their acetyl derivatives) and diamine oxidase (the key enzyme of terminal catabolism of polyamines in vivo) increased by 100-180% and 90-100%, respectively, parallel to an elevation in polyamine content (40-100%). The results suggest that in pancreas hypertrophy, which does not exhibit stimulation of spermidine/spermine N1-acetyltransferase activity, increases in the activity of polyamine and diamine oxidases are related events that lead to putrescine formation and removal of excess polyamines.

Transglutaminase activity in rat brain after ethanol exposure.

The effect of acute and chronic ethanol treatment on the activity of tissue transglutaminase (a calcium-dependent enzyme that catalyzes the covalent association between proteins, as well as proteins and polyamines) was studied in homogenate and in the cytosolic fraction of rat brain (telencephalon and diencephalon). A single dose of ethanol (5 g/kg of body weight, by gastric intubation) caused a 2-fold increase in enzyme activity at 6 hr after the ethanol dose, with a return toward the basal values at 24 hr. In vitro experiments with ethanol or acetaldehyde showed that the increase in transglutaminase activity was due to ethanol per se and not to its metabolism. The enzyme stimulation was correlated with a decrease in the levels of the polyamine spermine, a physiological substrate for the enzyme. Similar results were also found in the brain from rats fed on an ethanol diet for 4 months. The enhancement in tissue transglutaminase activity may thus lead to a decline in spermine, a polyamine known to have important protective functions in the cell.

Response of intestinal transglutaminase activity to dietary phytohaemagglutinin.

The behaviour of the activity of tissue transglutaminase, a calcium-dependent enzyme, and the levels of polyamines which are physiological substrates for the enzyme, were studied in rat small intestine induced to grow by lectin phytohaemagglutinin. Transglutaminase activity greatly increased in the homogenates and the cytosolic fractions of the intestinal mucosa of lectin-treated rats compared to that of untreated animals. The measurement of enzyme activity in the presence of monodansylcadaverine, a competitive inhibitor of transglutaminase, testified that the assayed enzyme activity was authentic transglutaminase. As regards polyamines, the level of spermine did not change, whereas putrescine and spermidine contents were enhanced. The activation of transglutaminase, which was probably due to Ca2+ accumulation in enterocytes, could have a role in maintaining enterocyte adhesion and intestinal cell homeostasis, and/or repairing lectin-induced damages of microvilli of the gut epithelium.

Aging and polyamine acetylation in rat kidney.

The acetylation of polyamines was investigated in rat kidney as a function of age. The activity of cytosolic spermidine/spermine N1-acetyltransferase, the rate-limiting enzyme in polyamine interconversion, increased from 3 to 36 months of age. The activity of cytosolic spermidine N8-acetyltransferase, an enzyme probably related to polyamine excretion, also increased. The activity of polyamine oxidase, which catalyzes the oxidative cleavage of polyamine N1-acetyl derivatives into putrescine, decreased until 24 months, when an accumulation of N1-acetylspermidine occurred. Subsequently, at 36 months, polyamine oxidase activity returned toward high values, in concomitance with the disappearance of N1-acetylspermidine, an increase in spermidine and putrescine, and a decline in spermine was observed. Our results show that in rat kidney during aging there is an activation of the acetylation and interconversion of higher polyamines into putrescine, which is considered an alternative pathway of spermidine and putrescine formation.

Transglutaminase activity in rat liver after acute ethanol administration.

The acute effect of ethanol on hepatic transglutaminase (EC 2.3.2.13) activity and polyamine levels were investigated in the rat. A high dose of ethanol (5 g/kg body weight, given by gastric intubation) caused in homogenate and cytosolic fraction an inhibition of 50-70% from 3 to 24 h which thereafter was reversible. Such a decrease may be in part responsible for the observed enhancement in putrescine and spermidine contents observed at the same times. Pyrazole, an inhibitor of alcohol dehydrogenase, prevented the ethanol-induced reduction in transglutaminase activity. Disulfiram, an inhibitor of aldehyde dehydrogenase, allowed detection of an inhibitory effect on enzyme activity even at a low dose of ethanol (2 g/kg), which per se did not modify transglutaminase activity. The hepatic cytosolic fraction, incubated in the presence of various concentrations of acetaldehyde, showed a dose-dependent inhibition of transglutaminase activity. All of these results suggest that acetaldehyde, the first and toxic metabolite of ethanol, inhibits hepatic transglutaminase activity, probably by its binding to the active thiol site of the enzyme. The reduction in transglutaminase may lead to an alteration of cytoskeleton, since the enzyme is known to be involved in tubuline polymerization and microfilament assembly.

Polyamine acetylation in rat brain during N-ethyl-N-nitrosourea-induced cerebral carcinogenesis.

The behavior of cerebral polyamine acetylation was examined in rat brain during N-ethyl-N-nitrosourea-induced carcinogenesis. Before tumor development, treated brains exhibited an enhancement in cytosolic spermidine/spermine N1-acetyl-transferase activity with concomitant accumulation of N1-acetylspermidine and increases in putrescine and spermidine. This indicates a stimulation of the interconversion pathway of polyamines into putrescine, an important molecule for cell growth. Our data also show the presence of a cytosolic spermidine N8-acetyltransferase activity in fetal rat brain, with values similar to those previously observed in gliomas. The detection of cytosolic spermidine N8-acetyltransferase activity in tumors may thus represent the expression of a fetal gene that does not seem to have a particular function during carcinogenesis.

Diamine and polyamine oxidase activities in phytohaemagglutinin-induced growth of rat small intestine.

The activities of diamine and polyamine oxidases, two enzymes of polyamine catabolism, were studied in hyperplastic growth of rat small intestine induced by phytohaemagglutinin. This growth, evaluated by the elongation of Lieberkühn's crypts, was more extensive in the proximal than in the distal parts of the gut. The activity of diamine oxidase was significantly reduced in the proximal (70%), medial (45%) and the distal (25%) parts. The activity of polyamine oxidase was doubled. The concentrations of putrescine, cadaverine and spermidine were significantly elevated in the three intestinal parts studied, whereas those of histamine and spermine were unchanged. It appears that changes in the activities of diamine and polyamine oxidases may contribute to the increased putrescine content, which is necessary to maintain active polyamine turnover for sustaining growth of the gut.