The in vivo activity of different 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (vastatins) on neointimal formation induced by insertion of a flexible collar around one carotid artery of normocholesterolemic rabbits was investigated. The contralateral carotid artery served as a sham control. Pravastatin, lovastatin, simvastatin, and fluvastatin were given mixed with food at daily doses of 20 ing/kg body wt for 2 weeks starting on the day of collar placement. The treatment with vastatins did not modify rabbit plasma cholesterol concentrations. The neointimal formation was assessed by measuring the cross-sectional thickness of intimal and medial tissues of fixed arteries with light microscopy. Fourteen days after collar placement, intimal hyperplasia (mostly cellular) was pronounced in treated carotid arteries. The intimal/medial (I/M) tissue ratio was 12-fold higher in treated arteries than in arteries without the collar (0.36±0.04 versus 0.03±0.02). Animals treated with lovastatin (R=12), simvastatin (n=12), and fluvastatin (R=12) showed significantly less neointimal formation; I/M tissue ratios were 0.24±0.03, 0.20±0.03, and 0.17±0.03, respectively. The inhibition elicited by pravastatin (n=12, 032 ±0.03) did not reach statistical significance. or-Actin antibody immunofluorescence analysis of serial sections revealed that cells present in the hyperplastic intima were mostly myocytes. Rates of intimal myocyte proliferation were also measured by incorporation of 5-bromo-2'-deoxyuridine, a thymidine analogue, into replicating DNA. Immunofluorescence analysis showed that 5-bromo-2'-deoxyuridine was actively incorporated into intimal myocytes after insertion of the collar, with a labeling index (percent of labeled myocytes) of 2.15 after 14 days. Labeling indexes for pravastatin-, lovastatin-, simvastatin-, and fluvastatin-treated carotid arteries were 2.01, 1.32,1.23, and 1.20, respectively, suggesting a direct effect of vastatins on arterial myocyte proliferation. The different responsiveness shown by the vastatins tested may be attributed to the differences in their capacity to penetrate cell membranes and their potency in inhibiting the HMG CoA reductase enzyme. We conclude that the inhibition of carotid intimal myocyte proliferation by these vastatins is independent of their effect on plasma cholesterol concentrations. -11 suggesting that the hypolipidemic effect is

SummaryThe tumour suppressor p53 negatively controls cell cycle progression in response to perturbed ribosome biogenesis in mammalian cells, thus coordinating growth with proliferation. Unlike mammalian cells, p53 is not involved in the growth control of proliferation in yeasts and flies. We investigated whether a p53-independent mechanism of response to inadequate ribosome biogenesis rate is also present in mammalian cells. We studied the effect of specific inhibition of rRNA synthesis on cell cycle progression in human cancer cell lines using the small-interfering RNA procedure to silence the POLR1A gene, which encodes the catalytic subunit of RNA polymerase I. We found that interference of POLR1A inhibited the synthesis of rRNA and hindered cell cycle progression in cells with inactivated p53, as a consequence of downregulation of the transcription factor E2F-1. Downregulation of E2F-1 was due to release of the ribosomal protein L11, which inactivated the E2F-1-stabilising function of the E3 ubiquitin protein ligase MDM2. These results demonstrated the existence of a p53-independent mechanism that links cell growth to cell proliferation in mammalian cells, and suggested that selective targeting of the RNA polymerase I transcription machinery might be advisable to hinder proliferation of p53-deficient cancer cells.

The identification of biological markers of Alzheimer's disease (AD) can be extremely useful to improve diagnostic accuracy and/or to monitor the efficacy of putative therapies. In this regard, peripheral cells may be of great importance, because of their easy accessibility. After subjects were grouped according to diagnosis, the expression of conformationally mutant p53 in blood cells was compared by immunoprecipitation or by a cytofluorimetric assay. In total, 104 patients with AD, 92 age-matched controls, 15 patients with Parkinson's disease and 9 with other types of dementia were analyzed. Two independent methods to evaluate the differential expression of a conformational mutant p53 were developed. Mononuclear cells were analyzed by immunoprecipitation or by flow-cytometric analysis, following incubation with a conformation-specific p53 antibody, which discriminates unfolded p53 tertiary structure. Mononuclear cells from AD patients express a higher amount of mutant-like p53 compared to non-AD subjects, thus supporting the study of conformational mutant p53 as a new putative marker to discriminate AD from non-AD patients. We also observed a strong positive correlation between the expression of p53 and the age of patients. The expression of p53 was independent from the length of illness and from the Mini Mental State Examination value.

Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.

46BR.1G1 cells derive from a patient with a genetic syndrome characterized by drastically reduced replicative DNA ligase I (LigI) activity and delayed joining of Okazaki fragments. Here we show that the replication defect in 46BR.1G1 cells results in the accumulation of both single-stranded and double-stranded DNA breaks. This is accompanied by phosphorylation of the H2AX histone variant and the formation of ␥H2AX foci that mark damaged DNA. Single-cell analysis demonstrates that the number of ␥H2AX foci in LigI-defective cells fluctuates during the cell cycle: they form in S phase, persist in mitosis, and eventually diminish in G 1 phase. Notably, replicationdependent DNA damage in 46BR.1G1 cells only moderately delays cell cycle progression and does not activate the S-phase-specific ATR/Chk1 checkpoint pathway that also monitors the execution of mitosis. In contrast, the ATM/Chk2 pathway is activated. The phenotype of 46BR.1G1 cells is efficiently corrected by the wild-type LigI but is worsened by a LigI mutant that mimics the hyperphosphorylated enzyme in M phase. Notably, the expression of the phosphomimetic mutant drastically affects cell morphology and the organization of the cytoskeleton, unveiling an unexpected link between endogenous DNA damage and the structural organization of the cell.