Marzia Govoni

Nucleolar size indicates the rapidity of cell proliferation in cancer tissues.

In order to define the importance of the nucleolus in tumour pathology, the relationship between nucleolar size and function and tumour mass growth rate was studied in vivo. Ten established human cancer cell lines from colon carcinomas and neuroblastomas were inoculated subcutaneously in athymic mice and the doubling time (DT) of the xenograft tumour mass was calculated. The tumour DTs ranged from 3.2 to 15.7 days. Nucleolar size was evaluated in sections from formalin‐fixed and paraffin‐embedded tumour samples after silver staining for AgNOR proteins, using a specific image analysis system. The nucleolar area values were inversely related to the xenograft tumour mass DTs (r=−0.90; p<0.001). Nucleolar functional activity was also evaluated using rapid, intermediate, and slow growing tumours (one each). The values of RNA polymerase I activity measured in vitro were strongly related to the corresponding tumour DTs (r=−0.99; p=0.03). The labelling indices (LIs) of three proliferation markers, MIB1, PCNA, and bromodeoxyuridine (BrdU), were also evaluated. As revealed by the MIB1 and PCNA LIs, almost all the cells of the xenograft tumours were cycling (86.6±5.6 SD and 95.5±2.0 SD, respectively). Neither the MIB1, PCNA or BrdU LIs were related to the xenograft tumour mass DT, showing that the different growth rates of tumour xenografts were not due to different growth fractions, but were mainly related to different cell proliferation rates. The present data demonstrate that the size and function of the nucleolus are related to the cell proliferation rate of cancer tissue. Evaluation of nucleolar size after silver staining of AgNOR proteins represents a unique parameter for the histological assessment of rapidity of cell proliferation in tumour lesions. Copyright

Gene polymorphism affecting α1‐antichymotrypsin and interleukin‐1 plasma levels increases Alzheimer's disease risk

Plasma levels of α1‐antichymotrypsin (ACT) and inter‐leukin‐1β (IL‐1β) were increased in patients with probable Alzheimers disease (AD). A common polymorphism within ACT and IL‐1β genes affected plasma levels of ACT or IL‐1β, and AD patients with the ACT T,T or IL‐1β T,T genotype showed the highest levels of plasma ACT or IL‐1β, respectively. The concomitant presence of the ACT T,T and IL‐1β T,T genotypes increased the risk of AD (odds ratio: 5.606, confidence interval: 1.654–18.996) and decreased the age at onset of the disease. Ann Neurol 2000;48:388–391

Apolipoprotein E and α-1-antichymotrypsin allele polymorphism in sporadic and familial Alzheimer's disease

Abstract Alzheimer disease (AD) patients with both sporadic and familial forms of AD and non-demented controls were genotyped for common polymorphisms in the signal peptide for α-1-antichymotrypsin (ACT) gene and in two different regions of apolipoprotein E (APOE) gene. The ACT TT genotype was over-represented ( P=0.025 ) in patients with early onset of sporadic AD. In this patients group ACT TT genotype conferred a significant crude odds ratio for the disease (OR=2.09; 95% CI=1.09–4.00, P=0.025 ). After adjustment for the APOE ϵ4 and APOE −491 genotypes, logistic regression analysis confirmed that the ACT TT genotype resulted independently associated with early onset AD (adjusted OR=2.56; 85% C.I.=1.3–5.2, P=0.009 ). The frequency of APOE ϵ4 allele was increased in AD, as expected (OR=5.92, 95% CI=3.60–9.70, P=0.0001 ). On the contrary, the APOE −491 A/T genotypes were not associated with AD. No preferential association of the APOE ϵ4 allele or APOE −491 A/T genotypes with ACT A/T alleles was observed in AD. Present findings indicated that subjects with ACT TT genotype had an increased risk of developing AD and suggested that this genotype influenced the risk of an early onset of the disease by affecting the production of ACT molecules.

Different effects of ribosome biogenesis inhibition on cell proliferation in retinoblastoma protein- and p53-deficient and proficient human osteosarcoma cell lines.

Abstract.  Objectives: To evaluate the effects of rRNA synthesis inhibition on cell cycle progression and cell population growth according to the RB and p53 status. Material and methods: RB‐ and p53‐proficient U2OS cells and the RB‐ and p53‐deficient SAOS‐2 cells were used, rRNA transcription hindered by actinomycin D, and cell cycle analysed by flow cytometry. Results: One hour of actinomycin D treatment induced in U2OS cells a block at the cell cycle checkpoints G1‐S and G2‐M, which was removed only after rRNA synthesis was resumed. rRNA synthesis inhibition did not influence cell cycle progression in SAOS‐2 cells. No effect on cell cycle progression after actinomycin D‐induced rRNA inhibition was also found in U2OS cells silenced for RB and p53 expression. A mild perturbation of cell cycle progression was observed in U2OS cells silenced for the expression of either RB or p53 alone. We also treated U2OS and SAOS‐2 cells with actinomycin D for 1 h/day for 5 days. This treatment lightly reduced growth rate of the U2OS cell population, whereas cell population growth of SAOS‐2 cells was completely inhibited. A marked reduction of ribosome content occurred in SAOS‐2 cells after the long‐term actinomycin D treatment, whereas no modification was observed in U2OS cells. Conclusions: These results demonstrate that inhibition of ribosome biogenesis does not hinder cell cycle progression in RB‐ and p53‐deficient cells. A daily‐repeated transitory inhibition of ribosome biogenesis leads to a progressive reduction of ribosome content with the consequent extinction of cancer cell population lacking RB and p53.

Key role of the achievement of an appropriate ribosomal RNA complement for G1-S phase transition in H4-II-E-C3 rat hepatoma cells

Cell growth is closely related to cell proliferation and an adequate ribosome biogenesis appears to be necessary for cell duplication. In the present study, we have investigated the relationship between rRNA synthesis and cell cycle progression. For this purpose, in a first set of experiments, we evaluated the effect of rRNA synthesis variation on cycle duration in asynchronously growing H4‐II‐E‐C3 rat hepatoma cells. Cells were either treated with insulin or insulin plus actinomycin D (AMD). The hormone stimulated ribosome biogenesis, which was later followed by an increased synthesis of DNA and a shortening of cell doubling time (DT). Bivariate flow cytometry indicated that the reduced length of the cell cycle was mainly due to the shorter G1‐phase. AMD, at the concentration of 0.04 μg/ml, hindered ribosome biogenesis without affecting heterogeneous RNA production. A 12‐h reduction in ribosome biogenesis level by AMD caused a lowering of DNA synthesis and a lengthening of cell DT with a longer G1‐phase. In a second set of experiments, we analyzed the cell content variations of 28S and 18S rRNA transcripts during G1 phase in H4‐II‐E‐C3 cells, synchronized by serum deprivation, and then stimulated by serum, serum plus insulin, and serum plus insulin and AMD. In control cells, a progressive increase in rRNA content occurred until the highest value of rRNA content was reached 21 h after serum stimulation. In insulin‐treated cells, the highest rRNA value was reached at 12 h whereas in AMD‐treated cells, the rRNA quantity was constantly low until 18 h and then sharply increased at 21 h. In the three experimental conditions, the highest values of rRNA amount were reached at the end of G1 phase and were quite similar to one another. We also evaluated, by real‐time RT‐PCR, cyclin E mRNA expression, which appeared to sharply increase at those times in which the maximum increase in the rRNA content was observed. Our results indicated that the achievement of an appropriate amount of rRNA allows G1/S phase transition, probably by modulating the expression of cyclin E mRNA.

Polynucleotide:adenosine glycosidase activity of saporin-L1: effect on various forms of mammalian DNA.

Saporin-L1 from the leaves of Saponaria officinalis belongs to a group of plant polynucleotide:adenosine glycosidases, known as ribosome-inactivating proteins due to their property of depurinating the major rRNA. Previous experiments indicated that saporin-L1 and other ribosome-inactivating proteins depurinate also DNA [Barbieri et al. (1994) Nature 372, 324; and (1996) Biochem. J. 319, 507-513]. Here we describe the effects of highly purified nuclease-free saporin-L1 on mammalian nuclear and mitochondrial DNA. Saporin-L1 had less activity on mitochondrial DNA than on nuclear DNA. A low, although significant, depurination of both chromatin and whole nuclei was observed. Mitochondrial nucleic acids are heavily depurinated in intact mitochondria, although the contribute of mtDNA to the deadenylation events is not known. The kinetic constants for several substrates were determined.

Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

Many drugs currently used in chemotherapy work by hindering the process of ribosome biogenesis. In tumors with functional p53, the inhibition of ribosome biogenesis may contribute to the efficacy of this treatment by inducing p53 stabilization. As the level of stabilized p53 is critical for the induction of cytotoxic effects, it seems useful to highlight those cancer cell characteristics that can predict the degree of p53 stabilization following the treatment with inhibitors of ribosome biogenesis. In the present study we exposed a series of p53 wild-type human cancer cell lines to drugs such as actinomycin D (ActD), doxorubicin, 5-fluorouracil and CX-5461, which hinder ribosomal RNA (rRNA) synthesis. We found that the amount of stabilized p53 was directly related to the level of ribosome biogenesis in cells before the drug treatment. This was due to different levels of inactivation of the ribosomal proteins–MDM2 pathway of p53 digestion. Inhibition of rRNA synthesis always caused cell cycle arrest, independent of the ribosome biogenesis rate of the cells, whereas apoptosis occurred only in cells with a high rDNA transcription rate. The level of p53 stabilization induced by drugs acting in different ways from the inhibition of ribosome biogenesis, such as hydroxyurea (HU) and nutlin-3, was independent of the level of ribosome biogenesis in cells and always lower than that occurring after the inhibition of rRNA synthesis. Interestingly, in cells with a low ribosome biogenesis rate, the combined treatment with ActD and HU exerted an additive effect on p53 stabilization. These results indicated that (i) drugs inhibiting ribosome biogenesis may be highly effective in p53 wild-type cancers with a high ribosome biogenesis rate, as they induce apoptotic cell death, and (ii) the combination of drugs capable of stabilizing p53 through different mechanisms may be useful for treating cancers with a low ribosome biogenesis rate.

The hydroxy-methyl-glutaryl CoA reductase promoter polymorphism is associated with Alzheimer's risk and cognitive deterioration

A link between cholesterol and Alzheimers disease (AD) had been suggested. Hydroxy-methylglutaryl-coenzyme A reductase (HMGCR) is the rate limiting enzyme in the synthesis of cholesterol. A single nucleotide polymorphism (SNP) in the promoter of this gene, never described in Italian AD population, was investigated in case-control studies. Genotype distribution and allele frequency in two groups of AD patients and non demented controls were investigated. A cohort of AD patients were also followed up for 2 years, cognitive performances recorded and a possible influence of this SNP on the disease progression was tested. The CC genotype of the HMGCR gene was associated with a reduced risk of AD. Conversely the A allele of this polymorphism was over represented in AD patients. The presence of the A allele was also associated with an accelerated cognitive deterioration in AD patients followed up for 2 years. However, transfection experiments showed that this polymorphism did not directly influence functional activity in luciferase reporter gene assays. This polymorphism of the HMGCR gene appears to be linked to both AD risk and disease progression. Present findings reinforce the notion that abnormal regulation of cholesterol metabolism is a key factor in the pathogenesis of the disease.

Lactate dehydrogenase inhibitors can reverse inflammation induced changes in colon cancer cells.

&NA; The inflammatory microenvironment is an essential component of neoplastic lesions and can significantly impact on tumor progression. Besides facilitating invasive growth, inflammatory cytokines were also found to reprogram cancer cell metabolism and to induce aerobic glycolysis. Previous studies did not consider the possible contribution played in these changes by lactate dehydrogenase (LDH). The A isoform of LDH (LDH‐A) is the master regulator of aerobic glycolysis; it actively reduces pyruvate and causes enhanced lactate levels in tumor tissues. In cancer cells, lactate was recently found to directly increase migration ability; moreover, when released in the microenvironment, it can facilitate matrix remodeling. In this paper, we illustrate that treatment of human colon adenocarcinoma cells with TNF‐&agr; and IL‐17, two pro‐inflammatory cytokines, modifies LDH activity, causing a shift toward the A isoform which results in increased lactate production. At the same time, the two cytokines appeared to induce features of epithelial‐mesenchymal transition in the treated cells, such as reduction of E‐cadherin levels and increased secretion of metalloproteinases. Noteworthy, oxamate and galloflavin, two inhibitors of LDH activity which reduce lactate production in cells, were found to relieve the inflammation‐induced effects. These results suggest LDH‐A and/or lactate as common elements at the cross‐road between cancer cell metabolism, tumor progression and inflammation. At present, LDH inhibitors suitable for clinical use are actively searched as possible anti‐proliferative agents; our data lead to hypothesize for these compounds a wider potential in anticancer treatment. Graphical abstract Figure. No caption available.

Effects of camptothecin, an inhibitor of DNA topoisomerase I on ribosomal gene structure and function in TG cells

Summary— The effects of camptothecin treatment and topoisomerase I inhibition on ribosomal gene structure and function were investigated in TG cells, a human tumour cell line. 90‐ and 180‐min treatments with 25 μM camptothecin resulted in an increased DNA fragmentation and decreased activity of topoisomerase I in cell extracts. After 180‐min treatment, the incorporation of labelled uridine into total cell RNA was reduced to 39% and the ribosomal RNA synthesis to 10%, as compared to values of control cells. At the ultrastructural level, the nucleolar components appeared to be segregated; after selective DNA staining, with osmium‐amine complex, a part of the nucleolar chromatin of treated cells showed the presence of thin, extended DNA filaments, superimposable to those present in control cells.