Giuseppe Scalabrino

Polyamines in mammalian tumors. Part II

Publisher Summary This chapter investigates the links between polyamines and tumors. It is particularly interesting to modify the culture conditions to clarify the influence of the environmental milieu on the activities of the polyamine biosynthetic enzymes by enhancing or decreasing the levels of these enzymes to see the role of polyamines in the cell growth process. To this purpose, several studies have been carried out with rat hepatoma cells growing in culture. Studies of the effects of growth conditions on ornithine decarboxylase (ODC) activity presents evidence that dilution of high-density hepatoma cell cultures with fresh medium results in a very large and transient increase in enzyme activity. Furthermore, among the factors affecting the growth conditions of the cultured cells, the addition of fresh medium or serum to the culture has been demonstrated to be one of the most important for a variety of other cell lines. In spite of the decades of research, most of the role of polyamines in eliciting the peculiar neoplastic behavior and in permitting invasive neoplastic growth remains unclear.

The multi-faceted basis of vitamin B12 (cobalamin) neurotrophism in adult central nervous system: Lessons learned from its deficiency.

Glial cells, myelin and the interstitium are the structures of the mammalian central nervous system (CNS) mainly affected by vitamin B(12) (cobalamin, Cbl) deficiency. Most of the response to the damage caused by Cbl deficiency seems to come from astrocytes and microglia, and is manifested as an increase in the number of cells positive for glial fibrillary acidic protein, the presence of ultrastructural signs of activation, and changes in cytokine and growth factor production and secretion. Myelin damage particularly affects the lamellae, which are disorganized by edema, as is the interstitium. Surprisingly, rat Schwann cells (myelin-forming cells of the peripheral nervous system) are fully activated but the few oligodendrocytes (myelin-forming cells of the CNS) are scarcely activated. The presence of intramyelin and interstitial edema raises questions about the integrity of the blood-brain barrier and blood-cerebrospinal fluid (CSF) barrier. The results obtained in the CNS of Cbl-deficient rats indicate that cytokine and growth factor imbalance is a key point in the pathogenesis of Cbl-deficient neuropathy. In the rat, Cbl deficiency increases the spinal cord (SC) synthesis and CSF levels of myelinotoxic cytokines (tumor necrosis factor (TNF)-alpha and soluble (s) CD40:sCD40 ligand dyad) and a myelinotoxic growth factor (nerve growth factor), but decreases SC synthesis and CSF levels of a myelinotrophic cytokine (interleukin-6) and a myelinotrophic growth factor (epidermal growth factor, EGF). The in vivo administration of IL-6 or EGF, or agents antagonizing the excess myelinotoxic agent, is as effective as Cbl in repairing or preventing Cbl-deficiency-induced CNS lesions. An imbalance in TNF-alpha and EGF levels has also been found in the CSF and serum of patients with severe Cbl deficiency.

Cobalamin (vitamin B12) in subacute combined degeneration and beyond: traditional interpretations and novel theories

Subacute combined degeneration (SCD) is a neuropathy due to cobalamin (Cbl) (vitamin B(12)) deficiency acquired in adult age. Hitherto, the theories advanced to explain the pathogenesis of SCD have postulated a causal relationship between SCD lesions and the impairment of either or both of two Cbl-dependent reactions. We have identified a new experimental model, the totally gastrectomized rat, to reproduce the key morphological features of the disease [spongy vacuolation, intramyelinic and interstitial edema of the white matter of the central nervous system (CNS), and astrogliosis], and found new mechanisms responsible for the pathogenesis of SCD: the neuropathological lesions in TGX rats are not only due to mere vitamin withdrawal but also to the overproduction of the myelinolytic tumor necrosis factor (TNF)-alpha and the reduced synthesis of the two neurotrophic agents, epidermal growth factor (EGF) and interleukin-6. This deregulation of the balance between TNF-alpha and EGF synthesis induced by Cbl deficiency has been verified in the sera of patients with pernicious anemia (but not in those with iron-deficient anemia), and in the cerebrospinal fluid (CSF) of SCD patients. These new functions are not linked to the coenzyme functions of the vitamin, but it is still unknown whether they involve genetic or epigenetic mechanisms. Low Cbl levels have also been repeatedly observed in the sera and/or CSF of patients with Alzheimers disease or multiple sclerosis, but whether Cbl deficit plays a role in the pathogenesis of these diseases is still unclear.

In vitro inhibition of protein synthesis in rat liver as a consequence of ethanol metabolism

Abstract Ethanol depresses the incorporation of amino acids into cell proteins of rat liver slices. This inhibition is nearly concentration independent for alcohol concentrations between 3 and 150 mM, and disappears after removal of ethanol. The effect of ethanol on liver protein synthesis is lower in fasting than in fed rats. The inhibition of protein synthesis does not occur in guinea-pig kidney slices, a tissue virtually devoid of alcohol dehydrogenase, and is prevented in liver by pyrazol. Acetaldehyde brings about a concentration-dependent inhibition of protein synthesis both in rat liver and in guinea-pig kidney. Acetoin, up to 5 mM concentration, does not influence protein synthesis in liver. Sorbitol, which mimics ethanol as regards the shifting of the redox level in the hepatocyte, depresses protein synthesis in liver in relation to its concentration in the medium. Pyruvate partially removes the inhibition of cell protein synthesis induced by ethanol. The data show that the inhibition of cell protein synthesis in rat liver slices by ethanol is a consequence of alcohol metabolism. They also suggest that this inhibition is due to acetaldehyde as well as to the shifting of the redox level in the cell.

Polyamines in mammalian ageing: An oncological problem, too? A review

This review surveys the literature about changes in polyamine contents and levels of activity of the enzymes involved in the polyamine biosynthetic pathway in organs of ageing mammals. The literature about changes in the polyamine levels in physiological fluids in healthy ageing humans is also reviewed. Generally speaking, decreases in the levels of the main polyamines (noticeably putrescine and spermidine) are observed in different mammalian organs with ageing. The polyamine levels in serum and in urine of healthy human beings are also age-related, declining progressively with increasing age. Some major enzymes (i.e., ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) involved in the polyamine biosynthetic pathway show similar trends. Hormonal induction of ornithine decarboxylase activity is strongly reduced in organs of aged animals, as it is in neoplastic organs. There is also some evidence for an age-related decrease in the level of ornithine decarboxylase and its inducibility in mammalian cells cultured in vitro. Some in vitro effects of spermidine and spermine on aged structures or systems are briefly summarized. There is no evidence yet that this generally reduced capacity of mammalian aged organs for polyamine biosynthesis is one of the factors responsible for the well known high incidence of some neoplasias in elderly humans. In view of the typical stimulatory effects of the tumour promoters on polyamine biosynthesis in target tissues and the effects of senescence on the same metabolic pathway, it can be excluded that the ageing process has a tumour promoting effect by itself. However, although the exact mechanism responsible for the increased occurrence of some tumors during mammalian senescence is still obscure, there are enough experimental data (both in humans and in animals) to indicate that the reduced polyamine biosynthetic capacity of aged mammals can account for the slower course of some tumors in elderly patients.

Experimental and clinical evidence of the role of cytokines and growth factors in the pathogenesis of acquired cobalamin-deficient leukoneuropathy

Our experimental and clinical studies have highlighted the non-coenzyme functions of cobalamin (Cbl; vitamin B12). The neuropathy of the rat central nervous system (CNS) due to Cbl deficiency is associated with increases in CNS tissue and/or cerebrospinal fluid (CSF) levels of some neurotoxic molecules, and decreases in local and/or CSF levels of some neurotrophic molecules. The increased molecules are nerve growth factor (NGF), tumor necrosis factor (TNF)-alpha, and the soluble (s)CD40:sCD40 Ligand dyad; the decreased molecules are epidermal growth factor (EGF) and interleukin-6. The morphological lesions of the CNS white matter in Cbl-deficient (Cbl-Df) rats can be prevented to the same extent by treatments replacing Cbl or the deficient neurotrophic molecules, or treatment with agents that antagonize the excess neurotoxic molecules. Patients with neurological and/or hematological manifestations of severe Cbl deficiency also have high TNF-alpha levels and low EGF levels in CSF and serum. Cbl replacement treatment corrects cytokine and growth factor abnormalities in Cbl-Df patients and Cbl-Df rats, and so Cbl-Df CNS neuropathy is also due to an imbalance in local cytokine/growth factor networks. TNF-alpha and NGF levels are also increased in Cbl-Df rat liver, which is morphologically unaffected by Cbl deficiency. The increases in TNF-alpha and NGF levels increase nuclear factor-kappaB activity levels in both the CNS and liver, and this indirect regulation supports the idea that Cbl may modulate the expression of some cytokine/growth factor genes in rat CNS and other tissues. Finally, we have tried to harmonize our pathogenetic theory of cytokine and growth factor dysregulation with the biochemical interpretation.

Epidermal growth factor as a local mediator of the neurotrophic action of vitamin B12 (cobalamin) in the rat central nervous system

We have recently demonstrated that the myelinolytic lesions in the spinal cord (SC) of rats made deficient in vitamin B12 (cobalamin) (Cbl) through total gastrectomy (TG) are tumor necrosis factor‐α (TNF‐α)‐mediated. We investigate whether or not permanent Cbl deficiency, induced in the rat either through TG or by chronic feeding of a Cbl‐deficient diet, might modify the levels of three physiological neurotrophic factors—epidermal growth factor (EGF), vasoactive intestinal peptide (VIP), and somatostatin (SS)—in the cerebrospinal fluid (CSF) of these rats. We also investigated the ability of the central nervous system (CNS) in these Cbl‐deficient rats to synthesize EGF mRNA and of the SC to take up labeled Cbl in vivo. Cbl‐deficient rats, however the vitamin deficiency is induced, show a selective decrease in EGF CSF levels and an absence of EGF mRNA in neurons and glia in various CNS areas. In contrast, radiolabeled Cbl is almost exclusively taken up by the SC white matter, but to a much higher degree in totally gastrectomized (TGX) rats. Chronic administration of Cbl to TGX rats restores to normal both the EGF CSF level and EGF mRNA expression in the various CNS areas examined. This in vivo study presents the first evidence that the neurotrophic action of Cbl in the CNS of TGX rats is mediated by stimulation of the EGF synthesis in the CNS itself. It thus appears that Cbl inversely regulates the expression of EGF and TNF‐α genes in the CNS of TGX rats.—Scalabrino, G., Nicolini, G., Buccellato, F. R., Peracchi, M., Tredici, G., Manfridi, A., Pravettoni, G. Epidermal growth factor as a local mediator of the neurotrophic action of vitamin B12 (cobalamin) in the rat central nervous system. FASEB J. 13, 2083–2090 (1999)

Myelinolytic lesions in spinal cord of cobalamin-deficient rats are TNF-α-mediated

Repeated intracerebroventricular (i.c.v.) microinjection of tumor necrosis factor‐α (TNF‐α) into normal rats causes intramyelin and interstitial edema in the white matter of the spinal cord (SC). This response is identical to that observed in the SC white matter of rats made cobalamin (Cbl) deficient by total gastrectomy (TG). Immunoblot analysis showed that: 1) the level of the biologically active form of the TNF‐α protein (17 kDa) is higher in the SC of totally gastrectomized (TGX) rats 2 months after TG, i.e., at the postoperative time when edema is observed; 2) SC levels of TNF‐α protein (17 kDa) in 2‐mo‐TGX‐, Cbl‐treated rats are reduced to control. Repeated i.c.v. microinjections of anti‐TNF‐α antibodies, transforming growth factor‐β1 (TGF‐β1) or interleukin‐6 (IL‐6) into TGX rats, begun shortly after TG, substantially reduced both intramyelin and interstitial edema in the SC white matter. This study provides the first evidence that the hallmark myelin damage of Cbl‐deficient central neuropathy, which is a pure myelinolytic disease, is not caused directly by the withdrawal of the vitamin itself, but reflects enhanced production of the biologically active form of TNF‐α by SC cells. This study thus supports the view that TGF‐β1 and IL‐6 may act as neuroprotective agents in Cbl deficiency central neuropathy.—Buccellato, F. R., Miloso, M., Braga, M., Nicolini, G., Morabito, A., Pravettoni, G., Tredici, G., Scalabrino, G. Myelinolytic lesions in spinal cord of cobalamin‐deficient rats are TNF‐α‐mediated. FASEB J. 13, 297–304 (1999)

High tumor necrosis factor-α in levels in cerebrospinal fluid of cobalamin-deficient patients

We studied 14 patients with neurological manifestations of subacute combined degeneration (SCD) and 40 control patients not cobalamin (Cbl)‐deficient. The cerebrospinal fluid (CSF) markers of Cbl deficiency (Cbl and total homocysteine [tHCYS] levels) and the CSF levels of tumor necrosis factor (TNF)‐α and epidermal growth factor (EGF) were measured. Significantly higher levels of tHCYS and TNF‐α, and significantly lower levels of Cbl and EGF were found in the SCD patients. In human CSF, as in human serum and the rat central nervous system, decreased Cbl concentrations are concomitant with an increase in TNF‐α and a decrease in EGF‐levels. Ann Neurol 2004;56:886–890

Enhanced levels of biochemical markers for cobalamin deficiency in totally gastrectomized rats: uncoupling of the enhancement from the severity of spongy vacuolation in spinal cord.

The totally gastrectomized (TGX) rat is a new experimental model for studying the pathogenesis of cobalamin (Cbl)-deficient myelopathy, i.e., subacute combined degeneration, total gastrectomy (TG) serving as a surgical paradigm of human pernicious anemia. We determined the serum levels of some biochemical indicators of Cbl deficiency in TGX rats at 2 to 10 months after TG. Methylmalonic acid (MMA) rose within 2 months and progressively increased thereafter until the end of the investigation period. 2-Methylcitric acid (MCA) rose significantly by 6 months and showed a further increment 4 months later. Homocysteine was only clearly elevated much later than the serum MMA, i.e., 10 months after the operation. The concentrations of MMA, MCA, and cystathionine were increased in kidney, liver, and spinal cord (SC) of TGX rats at 10 months. Chronic treatment of TGX rats with Cbl greatly decreased the serum levels of all the metabolic indicators of Cbl deficiency. Chronic peroral administration of the antibiotic lincomycin to TGX rats in an attempt to suppress the enteric flora markedly decreased serum MMA levels. Only Cbl, however, given either for the first 2 months after TG or for the third and fourth postoperative months (i.e., after SC abnormalities had already appeared) significantly decreased the severity of spongy vacuolation in SC white matter, although not completely preventing or repairing the neuropathological damage. Therefore, neither the early impairment in TGX rats of the Cbl-dependent methylmalonyl-coenzyme A mutase reaction nor the more delayed impairment of the Cbl-dependent methionine synthase step, as reflected by changes in serum metabolite levels, seems to be causally related to the TG-induced spongy vacuolation in SC white matter.