Low-intensity pulsed ultrasound affects growth, differentiation, migration, and epithelial-to-mesenchymal transition of colorectal cancer cells.

Ultrasound (US) offers potentially important opportunities from a therapeutic point of view. Thus, the study of the biological effects of US on cancer cells is important to understand the consequences of these changes on the malignant phenotype. This study aimed to investigate the effects of low-intensity ultrasound (LIPUS) on the phenotype of colorectal cancer cell lines. Cell proliferation was evaluated by viability test and by evaluation of pERK expression, while cell motility using the scratch test. Cell differentiation was evaluated assessing alkaline phosphatase activity. Epithelial mesenchymal transition was assessed by analyzing the expression of Vimentin and E-Cadherin. Release and uptake of extracellular vesicles (EVs) were evaluated by flow cytometry. LIPUS effects on the organization of cytoskeleton were analyzed by confocal microscopy and by evaluation of Rho GTPase expression. No alterations in vitality and clonogenicity were observed when the intermediate (0.4 MPa) and the lowest (0.035 MPa) acoustic intensities were administered while the treatment with high intensity (1 MPa) induced a reduction of both cell viability and clonogenicity in both cell lines in a frequency-dependent manner. LIPUS promoted the differentiation of colon cancer cells, affected epithelial-to-mesenchymal transition, promoted the closure of a wound as well as increased the release of EVs compared with untreated cells. LIPUS-induced increase in cell motility was likely due to a Rho GTPase-dependent mechanism. Overall, the results obtained warrant further studies on the potential combined effect of LIPUS with differentiating agents and on their potential use in a clinical setting.

Aspirin inhibits cancer stem cells properties and growth of glioblastoma multiforme through Rb1 pathway modulation.

Several clinical studies indicated that the daily use of aspirin or acetylsalicylic acid reduces the cancer risk via cyclooxygenases (Cox-1 and Cox-2) inhibition. In addition, aspirin-induced Cox-dependent and -independent antitumor effects have also been described. Here we report, for the first time, that aspirin treatment of human glioblastoma cancer (GBM) stem cells, a small population responsible for tumor progression and recurrence, is associated with reduced cell proliferation and motility. Aspirin did not interfere with cell viability but induced cell-cycle arrest. Exogenous prostaglandin E2 significantly increased cell proliferation but did not abrogate the aspirin-mediated growth inhibition, suggesting a Cox-independent mechanism. These effects appear to be mediated by the increase of p21 waf1 and p27 Kip1 , associated with a reduction of Cyclin D1 and Rb1 protein phosphorylation, and involve the downregulation of key molecules responsible for tumor development, that is, Notch1, Sox2, Stat3, and Survivin. Our results support a possible role of aspirin as adjunctive therapy in the clinical management of GBM patients.

Controlled release of 18-ß-glycyrrhetic acid by nanodelivery systems increases cytotoxicity on oral carcinoma cell line.

The topical treatment for oral mucosal diseases is often based on products optimized for dermatologic applications; consequently, a lower therapeutic effect may be present. 18-ß-glycyrrhetic acid (GA) is extracted from Glycirrhiza glabra. The first aim of this study was to test the cytotoxicity of GA on PE/CA-PJ15 cells. The second aim was to propose and test two different delivery systems, i.e. nanoparticles and fibers, to guarantee a controlled release of GA in vitro. We used chitosan and poly(lactic-co-glycolic) acid based nanoparticles and polylactic acid fibers. We tested both delivery systems in vitro on PE/CA-PJ15 cells and on normal human gingival fibroblasts (HGFs). The morphology of GA-loaded nanoparticles (GA-NPs) and fibers (GA-FBs) was investigated by electron microscopy and dynamic light scattering; GA release kinetics was studied spectrophotometrically. MTT test was used to assess GA cytotoxicity on both cancer and normal cells. Cells were exposed to different concentrations of GA (20-500 µmol l-1) administered as free GA (GA-f), and to GA-NPs or GA-FBs. ROS production was evaluated using dichlorodihydrofluorescein as a fluorescent probe. Regarding the cytotoxic effect of GA on PE/CA-PJ15 cells, the lowest TC50 value was 200 µmol l-1 when GA was added as GA-NPs. No cytotoxic effects were observed when GA was administered to HGFs. N-acetyl Cysteine reduced mortality induced by GA-f in PE/CA-PJ15 cells. The specific effect of GA on PE/CA-PJ15 cells is mainly due to the different sensitivity of cancer cells to ROS over-production; GA-NPs and GA-FBs formulations increase, in vitro, this toxic effect on oral cancer cells.

Increased expression of CD133 is a strong predictor of poor outcome in stage I colorectal cancer patients.

OBJECTIVE: Stage I colorectal carcinomas display a highly variable behavior which is not accurately predicted by the available prognostic markers. CD133 is considered a useful marker to identify the so-called cancer stem cells in colorectal cancers (CRCs) and its expression has been shown to have prognostic significance in CRC patients. This study aimed to verify whether immunohistochemical evaluation of CD133 might correlate with the progression risk of stage I CRC patients. MATERIAL AND METHODS: Expression levels of the CD133 molecule were analyzed and compared in two series of stage I surgically resected CRC patients showing disease progression and death for the disease and patients with no evidence of disease progression after at least 6 years after surgery. RESULTS: A positive staining for CD133 was detected in 52% of the cases with poor prognosis and only in 9% of the group with good prognosis, and this difference was highly significant (p < 0.001). A significant correlation was detected between CD133 expression and histological parameters, such as tumor budding, vascular invasion, and presence of lymph node micrometastases but not tumor grading, gender, and age. Disease-free survival and cancer-specific survival of CD133 negative tumors were significantly longer compared to positive cases. In multivariate analyses, CD133 staining confirmed to be a predictor of shorter survival independent from vascular invasion but not from lymph nodes micrometastases. CONCLUSIONS: These findings demonstrate that CD133 immunostaining is a useful predictor of high risk progression in stage I CRC patients and might help to identify patients eligible for adjuvant chemotherapy.

Identification of Endothelin-1 and NR4A2 as CD133-regulated genes in colon cancer cells.

Several in vitro assays have been proposed to identify cancer stem cells (CSCs), including immunophenotyping, sphere assay and side population (SP) assay. CD133 antigen has been proposed as a CSC marker in colon cancer (CC). However, no functional data are available to date and conflicting results have been reported regarding its role as true CSC marker. Here we set out to identify a molecular signature associated with potential CSC. CD133(+) cells isolated from the CaCo-2 CC cell line were analysed by microarray molecular profiling compared to CD133(-) counterparts. Various differentially expressed genes were identified and the most relevant transcripts found to be over-expressed in CD133(+) cells were evaluated by quantitative RT-PCR in the CD133(+) fractions isolated from several CC cell lines. In the attempt to find a correlation between putative CSCs, isolated by means of CD133 immunophenotyping and the SP approach, we demonstrated a significant enrichment of CD133(+) cells within the SP fraction of CC cells, and comparison of the gene expression profiles revealed that Endothelin-1 (END-1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts are highly expressed in both CD133(+) and SP fractions of CC cells. Moreover, depletion of CD133 by siRNA induced a significant attenuation of END-1 and NR4A2 expression levels in CaCo-2 cells, while expression of all three molecules decreased during sodium butyrate-induced differentiation. In conclusion, we have identified a molecular signature associated with potential CSCs and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression in colon cancer cells.

Lycopene induces cell growth inhibition by altering mevalonate pathway and Ras signaling in cancer cell lines.

Several evidences suggest that cancer cells have abnormal cholesterol biosynthetic pathways and prenylation of small guanosine triphosphatase proteins. Tomato lycopene has been suggested to have beneficial effects against certain types of cancer, including that of prostate, although the exact molecular mechanism(s) is unknown. We tested the hypothesis that lycopene may exert its antitumor effects through changes in mevalonate pathway and in Ras activation. Incubation of the Ras-activated prostatic carcinoma LNCaP cells with a 24 h lycopene treatment (2.5-10 µM) dose dependently reduced intracellular total cholesterol by decreasing 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase expression and by inactivating Ras, as evidenced by its translocation from cell membranes to cytosol. Concomitantly, lycopene reduced the Ras-dependent activation of nuclear factor-kappaB (NF-κB). Such a reduction was parallel to an inhibition of reactive oxygen species production and to a decrease in the phosphorylation ofc-jun N-terminal kinase, extracellular signal-regulated kinase 1/2 and p38. These effects were also accompanied by an arrest of cell cycle progression and by apoptosis induction, as evidenced by a decrease in cyclin D1 and phospho-AKT levels and by an increase in p21, p27 and p53 levels and in Bax:Bcl-2 ratio. The addition of mevalonate prevented the growth-inhibitory effects of lycopene as well as its increase in Ras cytoplasmatic accumulation and the subsequent changes in NF-κB. The ability of lycopene in inhibiting HMG-CoA reductase expression and cell growth and in inactivating Ras was also found in prostate PC-3, colon HCT-116 and HT-29 and lung BEN cancer cells. These findings provide a novel mechanistic insight into the growth-inhibitory effects of lycopene in cancer.

A proteomic approach to characterizing ciglitazone-induced cancer cell differentiation in Hep-G2 cell line.

Drug induced cell differentiation represents a promising experimental model for proteomic analysis of cancer cells. In fact, by modulating and monitoring neoplastic cell differentiation it could be possible to identify cytodifferentiation related protein expression changes that can be subsequently utilized in vivo as potential cancer biomarkers. One main advantage of this approach is the significant reduction of biological variability normally observed in clinical biomarker research, with important implications also in prognosis and therapy. At this regard, a new class of differentiating agents is emerging, the so called PPAR-ligands, which however are characterized by a debated mechanism of action that has not been yet studied through a proteomic approach. To this aim, we investigated ciglitazone-induced differentiation of a human hepatocarcinoma HepG2 cell line, by monitoring biochemical and cellular parameters of cytodifferentiation and modifications of cellular protein profiles through 2-DE and MALDI-TOF analysis. Independent of the hypothesized mechanism of action of this intriguing PPARgamma agonist, results indicated that ciglitazone is a strong differentiating agent for the HepG2 cell line and that this process is associated with modifications of protein expression related to cell antioxidant systems, the cell cycle apparatus, signal transduction pathways, cellular stress and invasiveness. At last, considering these and other published data, a proteomic profile related to the cancer aggressiveness is beginning to emerge.

Loss of nuclear p27(kip1) and α-dystroglycan is a frequent event and is a strong predictor of poor outcome in renal cell carcinoma.

Expression levels of p27(kip1) , a negative regulator of the G1 phase of the cell cycle, and 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, were assessed by immunostaining in a series of renal cell carcinomas (RCCs) and their prognostic significance was evaluated. Expression of p27(kip1) as well as of the α-subunit of the dystroglycan (DG) complex, previously reported to be altered in RCC, was also evaluated by western blot analysis. Nuclear expression of p27(kip1) was reduced in a significant fraction of tumors and low p27(kip1) staining correlated with higher tumor grade (P < 0.01). Recurrence and death from clear cell RCCs were significantly more frequent in p27(kip1) -low expressing tumors and Kaplan-Meier curves showed a significant separation between high vs low expressor groups for both disease-free (P = 0.011) and overall (P = 0.002) survival. Low nuclear expression of p27(kip1) as well as loss of α-DG were confirmed to be independent prognostic parameters at a multivariate analysis and the simultaneous loss of both molecules defined a "high-risk" group of patients with increased risk of recurrence (RR = 28.7; P = 0.01) and death (RR = 12.9; P = 0.03). No significant correlation with clinical or pathological parameters was found for 8-OHdG staining. Western blot analyses suggested a post-translational mechanism for the loss of α-DG expression and demonstrated that cytoplasmic dislocation of the protein contributes to the loss of active nuclear p27(kip1) . Loss of nuclear p27(kip1) is a frequent event in human RCCs and is a powerful predictor of poor outcome which, in combination with low DG expression, could help to identify high-risk patients with clear cell RCC.

Evaluation of in vitro toxic effects of cement dusts: a preliminary study.

Cement is widely used for construction and several reports have suggested a potential toxicity of cement dusts although it has never been definitively assessed. To determine the cytotoxic and bioactive effects of cement dusts, cultures of normal rat fibroblasts were exposed to different types of cements and cell growth parameters, apoptosis and the occurrence of DNA damage (both in terms of DNA breaks and oxidative damage) were analyzed. Cells were exposed to cement extracts or cultured in direct contact with cement dusts and the results obtained were compared to cells cultured in fresh medium. A dose-dependent decrease in viable cells was observed with all tested cements. Different results were obtained in the cell-cement direct contact tests compared to the indirect contact tests performed using extracts. Inhibition of cell growth was associated in most cases with an accumulation of cells in the S-phase of the cell-cycle and the appearance of an apoptotic peak. DNA strand breaks, assessed by comet test, and increase in the levels of 8-OHdG, an important marker of DNA oxidative damage, always occurred by incubating cells in the presence of cement extracts or dusts. However, after removal of cement, a rapid damage repair was generally observed with an almost complete recovery within 12 hours. In conclusion, all cements analyzed in this study displayed a limited toxicity in vitro without significant differences amongst them. Overall, the results obtained indicate that cements should be treated as hazardous materials but they do not allow to make accurate predictions regarding the in vivo effects. Further studies are warranted to reach a better understanding of the potential toxic effects of cements, to identify the responsible mechanisms and to evaluate the possibility of modulating and/ or preventing them.

Magnesium deficiency affects mammary epithelial cell proliferation: involvement of oxidative stress.

Low Mg availability reversibly inhibited the growth of mammary epithelial HC11 cells by increasing the number of cells in the G0/G1 phase of the cell cycle. Because low Mg has been reported to promote oxidative reactions, we considered that low Mg-dependent growth arrest was mediated by oxidative stress. Surprisingly, both dichlorofluorescein-detectable reactive oxygen species and hydrogen peroxide-induced oxidative DNA damage were found to be lower in cells cultured in low Mg than in cells grown under control or high-Mg conditions. Gene expression profiling of low- and high-Mg cells showed the modulation of several genes, some regulating cell proliferation. In addition, low Mg cells also displayed overexpression of glutathione S-transferase (GST), leading to increased enzymatic activity. Of note, GST has been shown to modulate cell growth; therefore, we suggest that in low-Mg cells, GST upregulation might have a dual role in protecting against oxidative stress and in modulating cell proliferation.

Effect of beta-carotene-rich tomato lycopene beta-cyclase ( tlcy-b) on cell growth inhibition in HT-29 colon adenocarcinoma cells.

Lycopene beta-cyclase (tlcy-b) tomatoes, obtained by modulating carotenogenesis via genetic engineering, contain a large amount of beta-carotene, as clearly visible by their intense orange colour. In the present study we have subjected tlcy-b tomatoes to an in vitro simulated digestion and analysed the effects of digestate on cell proliferation. To this aim we used HT-29 human colon adenocarcinoma cells, grown in monolayers, as a model. Digested tomatoes were diluted (20 ml, 50 ml and 100 ml/l) in culture medium and added to the cells for different incubation times (24 h, 48 h and 72 h). Inhibition of cell growth by tomato digestate was dose-dependent and resulted from an arrest of cell cycle progression at the G0/G1 and G2/M phase and by apoptosis induction. A down-regulation of cyclin D1, Bcl-2 and Bcl-xl expression was observed. We also found that heat treatment of samples before digestion enhanced beta-carotene release and therefore cell growth inhibition. To induce with purified beta-carotene solubilised in tetrahydrofuran the same cell growth inhibition obtained with the tomato digestate, a higher amount of the carotenoid was necessary, suggesting that beta-carotene micellarised during digestion is utilised more efficiently by the cells, but also that other tomato molecules, reasonably made available during digestion, may be present and cooperate with beta-carotene in promoting cell growth arrest.

Docosahexaenoic acid induces apoptosis in lung cancer cells by increasing MKP-1 and down-regulating p-ERK1/2 and p-p38 expression.

Different agents able to modulate apoptosis have been shown to modify the expression of the MAP-kinase-phosphatase-1 (MKP-1). The expression of this phosphatase has been considered a potential positive prognostic factor in lung cancer, and smoke was shown to reduce the levels of MKP-1 in ferret lung. Our aim was to assess whether the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), known to inhibit the growth of several cancer cells mainly inducing apoptosis, may exert pro-apoptotic effect in lung cancer cells by modifying MKP-1 expression. We observed that DHA increased MKP-1 protein and mRNA expression and induced apoptosis in different lung cancer cell lines (mink Mv1Lu adenocarcinoma cells, human A549 adenocarcinoma and human BEN squamous carcinoma cells). We inhibited the pro-apoptotic effect of DHA by treating the cells with the phosphatase inhibitor Na(3)VO(4) or by silencing the MKP-1 gene with the specific siRNA. This finding demonstrated that the induction of apoptosis by DHA involved a phosphatase activity, specifically that of MKP-1. DHA reduced also the levels of the phosphorylated MAP-kinases, especially ERK1/2 and p38. Such an effect was not observed when the MKP-1 gene was silenced. Altogether, the data provide evidence that the DHA-induced overexpression of MKP-1 and the resulting decrease of MAP-kinase phosphorylation by DHA may underlie the pro-apoptotic effect of this fatty acid in lung cancer cells. Moreover, they support the hypothesis that DHA may exert chemopreventive action in lung cancer.

50-Hz extremely low frequency electromagnetic fields enhance cell proliferation and DNA damage: possible involvement of a redox mechanism.

HL-60 leukemia cells, Rat-1 fibroblasts and WI-38 diploid fibroblasts were exposed for 24-72 h to 0.5-1.0-mT 50-Hz extremely low frequency electromagnetic field (ELF-EMF). This treatment induced a dose-dependent increase in the proliferation rate of all cell types, namely about 30% increase of cell proliferation after 72-h exposure to 1.0 mT. This was accompanied by increased percentage of cells in the S-phase after 12- and 48-h exposure. The ability of ELF-EMF to induce DNA damage was also investigated by measuring DNA strand breaks. A dose-dependent increase in DNA damage was observed in all cell lines, with two peaks occurring at 24 and 72 h. A similar pattern of DNA damage was observed by measuring formation of 8-OHdG adducts. The effects of ELF-EMF on cell proliferation and DNA damage were prevented by pretreatment of cells with an antioxidant like alpha-tocopherol, suggesting that redox reactions were involved. Accordingly, Rat-1 fibroblasts that had been exposed to ELF-EMF for 3 or 24 h exhibited a significant increase in dichlorofluorescein-detectable reactive oxygen species, which was blunted by alpha-tocopherol pretreatment. Cells exposed to ELF-EMF and examined as early as 6 h after treatment initiation also exhibited modifications of NF kappa B-related proteins (p65-p50 and I kappa B alpha), which were suggestive of increased formation of p65-p50 or p65-p65 active forms, a process usually attributed to redox reactions. These results suggest that ELF-EMF influence proliferation and DNA damage in both normal and tumor cells through the action of free radical species. This information may be of value for appraising the pathophysiologic consequences of an exposure to ELF-EMF.

Direct effect of infliximab on intestinal mucosa sustains mucosal healing: exploring new mechanisms of action.

BACKGROUND: Infliximab is effective in inflammatory bowel disease through several mechanisms, possibly acting at the mucosal level. AIM: To assess the role of infliximab on intestinal mucosa and whether it contributes to mucosal healing. METHODS: Human colonic mucosal biopsies were incubated with or without infliximab. Cultured biopsies were evaluated for histological staining, CD68, CD3, E-cadherin and phospho-extracellular signal-regulated kinases (ERK) expression, and apoptosis. A scratch assay and MTT assay were performed with Caco2 cells in the presence of infliximab and/or tumour necrosis factor (TNF)-α or treated with supernatants obtained from human peripheral blood mononuclear cells or human intestinal fibroblasts treated with TNF-α and infliximab alone or in association. RESULTS: Infliximab-treated biopsies displayed a better histological appearance, reduced inflammation with an increase of E-cadherin, phospho-ERK and apoptosis. Supernatants showed lower TNF-α, IL-17, IL-6 and IL-8 concentration, with an increase in fibroblast-growth-factor. Motility at scratch assay and proliferation at MTT assay of Caco2 cells displayed differential modulation by TNF-α and infliximab, directly or through supernatants of human intestinal fibroblasts and human peripheral blood mononuclear cells exposed to them. CONCLUSION: Infliximab contributes to the mucosal healing process by acting directly at an intestinal mucosal level; infliximab indirectly affects epithelial cell migration and proliferation by acting on both fibroblasts and leukocytes.

Docosahexaenoic acid enhances the susceptibility of human colorectal cancer cells to 5-fluorouracil.

PURPOSE: Powerful growth-inhibitory action has been shown for n-3 polyunsaturated fatty acids against colon cancer cells. We have previously described their ability to inhibit proliferation of colon epithelial cells in patients at high risk of colon cancer. In the work reported here we investigated the ability of docosahexaenoic acid (DHA) to potentiate the antineoplastic activity of 5-fluorouracil (5-FU) in p53-wildtype (LS-174 and Colo 320) and p53-mutant (HT-29 and Colo 205) human colon cancer cells. METHODS: When in combination with DHA, 5-FU was used at concentrations ranging from 0.1 to 1.0 microM, much lower than those currently found in plasma patients after infusion of this drug. Similarly, the DHA concentrations (< or =10 microM) used in combination with 5-FU were lower than those widely used in vitro and known to cause peroxidative effects in vivo. RESULTS: Whereas the cells showed different sensitivity to the growth-inhibitory action of 5-FU, DHA reduced cell growth independently of p53 cellular status. DHA synergized with 5-FU in reducing colon cancer cell growth. The potentiating effect of DHA was attributable to the enhancement of the proapoptotic effect of 5-FU. DHA markedly increased the inhibitory effect of 5-FU on the expression of the antiapoptotic proteins BCL-2 and BCL-XL, and induced overexpression of c-MYC which has recently been shown to drive apoptosis and, when overexpressed, to sensitize cancer cells to the action of proapoptotic agents, including 5-FU. CONCLUSION: Our results indicate that DHA strongly increases the antineoplastic effects of low concentrations of 5-FU. Overall, the results suggest that combinations of low doses of the two compounds could represent a chemotherapeutic approach with low toxicity.

The activation of type 1 corticotropin releasing factor receptor (CRF-R1) inhibits proliferation and promotes differentiation of neuroblastoma cells in vitro via p27(Kip1) protein up-regulation and c-Myc mRNA down-regulation.

Our group has previously shown that corticotropin releasing factor (CRF) inhibits proliferation of human endocrine-related cancer cell lines via the activation of CRF type-1 receptors (CRF-R1). Tumors originating from the nervous system also express CRF receptors but their role on neoplastic cell proliferation was poorly investigated. Here we investigated the effect of CRF receptor stimulation on nervous system-derived cancer cells, using the SK-N-SH (N) human neuroblastoma cell line as an experimental model. We found that SK-N-SH (N) cells express functionally active CRF-R1, whose activation by CRF and the cognate peptide urocortin (UCN) is associated to reduced cell proliferation and motility, as well as neuronal-like differentiation. UCN did not interfere with cell viability and cell-cycle arrest. Those effects seem to be mediated by a mechanism involving the activation of cAMP/PKA/CREB pathway and the subsequent downstream increase in p27(Kip1) and underphosphorylated retinoblastoma protein levels, as well as reduced c-Myc mRNA accumulation.

Phenylbutyrate increases SMN expression in vitro: relevance for treatment of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease, characterized by degeneration of the anterior horn cells of the spinal cord. SMA presents with a highly variable phenotype ranging from very severe to mild (type I-III). No cure for SMA is available at present. All forms of SMA are caused by homozygous loss of the functional survival motor neuron (SMN1) gene. However, all patients have one or more copies of the SMN2 gene, nearly identical to SMN1. Both genes encode the SMN protein but the level produced by SMN2 is insufficient to protect from disease. Increasing SMN2 gene expression could be of considerable therapeutic importance. The aim of this study was to assess whether SMN2 gene expression can be increased by 4-phenylbutyrate (PBA). Fibroblast cell cultures from 16 SMA patients affected by different clinical severities were treated with PBA, and full-length SMN2 transcripts were measured by real-time PCR. In all cell cultures, except one, PBA treatment caused an increase in full-length SMN2 transcripts, ranging from 50 to 160% in type I and from 80 to 400% in type II and III cultures. PBA was found also effective in enhancing SMN protein levels and the number of SMN-containing nuclear structures (gems). These data show that SMN expression is considerably increased by PBA, and suggest that the compound, owing also to its favorable pharmacological properties, could be a good candidate for the treatment of SMA.

Regulation of magnesium content during proliferation of mammary epithelial cells (HC-11).

To study the role of Magnesium in the regulation of cell proliferation we characterized the proliferation behaviour of HC-11 mammary epithelial cells that were grown in media containing low to high Mg concentrations. Cells grown under control conditions (0.5 mM Mg in the medium) or in the presence of high (H) Mg (45 mM) displayed similar log-phases and reached confluence in 72h. In the presence of low (L) Mg (0.025 mM) the cells exhibited a reduced growth rate and did not reach confluence at 72h. Intra cellular total Mg increased from 12 to 36h of culture in all cells examined but returned to basal levels in those cells which reached confluence (i.e., control and H-Mg cells). Intra cellular Mg increased independent of mitosis-induced changes of volume and adenine nucleotides pools but correlated with an increased percentage of cells in the S phase and with total nucleic acid contents. These bell-shaped changes of intra cellular Mg were less evident in L-Mg cells, likely due to a combination of low Mg levels in the medium and decreased growth rate. Changes in membrane potential and pH were important factors that contributed to maintaining intra cellular Mg at physiologic levels in the face of increased or decreased availability of extra cellular Mg. H-Mg cells were depolarised and more acidic than control cells; conversely, L-Mg cells showed a pattern of hyperpolarization and alkalinization. These results lend support to the concept that Mg may be involved in regulating cell proliferation, and show that cells maintain adequate levels of intra cellular Mg, and hence their proliferation potential, even under conditions of extreme changes of extra cellular Mg.

Oxidative DNA damage as a marker of aging in WI-38 human fibroblasts.

Cause-effect relationships between oxidative stress, DNA damage and aging were investigated in WI-38 human diploid fibroblasts at 21, 41 or 58 population doublings (PDs), corresponding to young, middle age or old fibroblasts, respectively. Oxidative DNA damage was evaluated by immunohistochemical detection of 8-hydroxy-2'deoxyguanosine (8-OHdG) adducts or by single cell microgel electrophoresis (COMET assay). Aging was evaluated by growth rate, senescence-associated-beta-galactosidase (SA-beta galactosidase) activity, cell cycle distribution, and expression of p21. Our results demonstrate that (i) oxidative DNA damage is proportional to the age of cells (ii) DNA damage in old/58 PDs cells reflects both an increased susceptibility to oxidative stress, induced by acute exposure to sub-lethal concentrations of hydrogen peroxide (H(2)O(2)), and a reduced efficiency of repair mechanisms. We also show that mild chronic oxidative stress, induced by prolonged exposure to 5 microM H(2)O(2), accelerates aging in fibroblasts. In fact, this treatment increased 8-OHdG levels, SA-beta-galactosidase activity, and G0/G1 cell cycle arrest in middle age/41 PDs, making them similar to H(2)O(2)-untreated old/58 PDs cells. Although other mechanisms may concur in mediating the effects of H(2)O(2), these results lend support to the concept that oxidative stress may be a key determinant of aging. Measurements of oxidative DNA damage might therefore be exploited as reliable marker of cellular aging.