Comparison of ex vivo and in vitro human fibroblast ageing models.

Several studies have analyzed modulation of gene expression during physiological ageing with interesting, but often contradictory results, depending on the model used. In the present report we compare age-related metabolic and synthetic parameters in human dermal fibroblasts (HDF) isolated from young and old subjects (ex vivo ageing model) and cultured from early up to late cumulative population doublings (CPD) (in vitro ageing model) in order to distinguish changes induced in vivo by the aged environment and maintained in vitro, from those associated with cell senescence and progressive CPD. Results demonstrate that fibroblasts from aged donors, already at early CPD, exhibit an impaired redox balance, highlighting the importance of this parameter during ageing, even in the presence of standard environmental conditions, which are considered optimal for cell growth. By contrast, several proteins, as those related to heat shock response, or involved in endoplasmic reticulum and membrane trafficking, appeared differentially expressed only during in vitro ageing, suggesting that, at high CPD, the whole cell machinery becomes permanently altered. Finally, given the importance of the elastic component for a long-lasting connective tissue structural and functional compliance, this study focuses also on elastin and fibulin-5 synthesis and deposition, demonstrating a close relationship between fibulin-5 and ageing.

Biocompatibility of various root canal filling materials ex vivo.

AIM: To evaluate the biocompatibility of a resin-based endodontic filler (RealSeal) using the indirect cytotoxicity test. METHODOLOGY: Human gingival fibroblasts were cultured ex vivo. Pellets of the materials to be tested were incubated for 24, 48, and 72 h at 37 degrees C under sterile conditions to obtain their eluates. The fibroblasts were exposed to either diluted (50%) or undiluted eluates for 24 h. A culture medium with foetal calf serum was added to the control wells. Cell viability was estimated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method. The data concerning cell viability were statistically analyzed using one-way anova test and Bonferroni multiple comparisons test. RESULTS: Eluates obtained after 24 h of incubation with the resin filler did not reduce cellular viability. An increase in cellular viability, as compared with control cells, was observed in the gutta-percha group. The undiluted eluate from the polyether material was cytotoxic, causing an 82 +/- 4% decrease in cellular viability. Eluates obtained after 48 h of incubation with the resin filler increased cellular viability, whereas the polyether significantly reduced viability. Gutta-percha did not cause any detectable change. After 72 h of incubation the eluate of the resin filler caused an increase in cellular viability, as did gutta-percha, whereas polyether caused a significant decrease. CONCLUSIONS: RealSeal resin filler was nontoxic in this laboratory model. Further investigations are necessary to verify its usefulness in clinical applications.

Multidrug resistance protein-6 (MRP6) in human dermal fibroblasts. Comparison between cells from normal subjects and from Pseudoxanthoma elasticum patients.

Multidrug resistance protein-6 (MRP6) is a membrane transporter whose deficiency leads to the connective tissue disorder Pseudoxanthoma elasticum (PXE). In vitro dermal fibroblasts from normal and PXE subjects, homozygous for the R1141X mutation, were compared for their ability to accumulate and to release fluorescent calcein, in the absence and in the presence of inhibitors and competitors of the MDR-multidrug resistance protein (MRP) systems, such as 3-(3-(2-(7-choro-2 quinolinyl) ethenyl)phenyl ((3-dimethyl amino-3-oxo-propyl)thio) methyl) propanoic acid (MK571), verapamil (VPL), vinblastine (VBL), chlorambucil (CHB), benzbromarone (BNZ) and indomethacin (IDM). In the absence of chemicals, calcein accumulation was significantly higher and the release significantly slower in PXE cells compared to controls. VBL and CHB reduced calcein release in both cell strains, without affecting the differences between PXE and control fibroblasts. VPL, BNZ and IDM consistently delayed calcein release from both control and PXE cells; moreover, they abolished the differences between normal and MRP6-deficient fibroblasts observed in the absence of chemicals. These findings suggest that VPL, BNZ and IDM interfere with MRP6-dependent calcein extrusion in in vitro human normal fibroblasts. Interestingly, MK571 almost completely abolished calcein release from PXE cells, whereas it induced a strong but less complete inhibition in control fibroblasts, suggesting that MRP6 is not inhibited by MK571. Data show that MRP6 is active in human fibroblasts, and that its sensitivity to inhibitors and competitors of MDR-MRPs' membrane transporters is different from that of other translocators, namely, MRP1. It could be suggested that MRP1 and MRP6 transport different physiological substances and that MRP6 deficiency cannot be overcome by other membrane transporters, at least in fibroblasts. These data further support the hypothesis that MRP6 deficiency may be relevant for fibroblast metabolism and responsible for the metabolic alterations of these cells at the basis of connective tissue clinical manifestations of PXE.

Hyaluronan uptake by adult human skin fibroblasts in vitro.

Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism.

Hyaluronan affects protein and collagen synthesis by in vitro human skin fibroblasts.

Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1 microM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P < 0.05), and particularly in collagen (-40%; P < 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.

Pure-silica zeolites (Porosils) as model solids for the evaluation of the physicochemical features determining silica toxicity to macrophages.

The interaction between inhaled particles and alveolar macrophages plays a key role in silica-related diseases. It has been previously shown [Fubini, B., et al. (1999) Chem. Res. Toxicol. 12, 737-745] that a monocyte-macrophage cell line (J774) may be employed in the evaluation of the degree of cytotoxicity to alveolar macrophages of various silica dusts. In this paper, pure-silica zeolites (porosils) in microcrystalline form have been employed as "model solids" in an effort to show which physicochemical properties of the silica particle are playing a major role in the toxicity to macrophages. The samples employed covered four different porosil crystal structures (MFI, FAU, TON, and MTT) and also include a synthetic rodlike cristobalite (CRIS-rd). When compared at equal weight, the samples cover a wide range of cytotoxicity from inert to toxic as unheated mineral cristobalite [Fubini, B., et al. (1999) Chem. Res. Toxicol. 12, 737-745]. Mild grinding did not affect cytotoxicity. Calcined (open pores) and uncalcined (pore filled with template) TON exhibited the same cytotoxicity, indicating that only the outer surface is implied. The hydrophobic and/or hydrophilic character of TON, evaluated by adsorption calorimetry, is close to what has been previously found for silicalite and is consistent with a hydrophilic outer surface and hydrophobic pore walls. The potential for generating hydroxyl radicals from hydrogen peroxide varies among the various porosils that have been studied. A model is proposed for the correlation between inhibition of growth on proliferating cells and physicochemical properties varying from one to the other sample. The extent of external surface and the aspect ratio were related to the intensity of the cytotoxic effect, while the level of radical release was not. This suggests, on one hand, that comparison of toxicity among various dusts should be made at equal particle surface and, on the other, that in the model studied, free radical release does not play a crucial role in the primary event of toxicity to alveolar macrophages.

Abnormal phenotype of in vitro dermal fibroblasts from patients with Pseudoxanthoma elasticum (PXE).

Pseudoxanthoma elasticum (PXE) is a genetic connective tissue disease, whose gene and pathogenesis are still unknown. Dermal fibroblasts from patients affected by PXE have been compared in vitro with fibroblasts taken from sex and age-matched normal individuals. Cells were grown and investigated in monolayer, into three-dimensional collagen gels and in suspension. Compared with normal cells, PXE fibroblasts cultured in monolayer entered more rapidly within the S phase and exhibited an increased proliferation index; on the contrary, similarly to normal fibroblasts, PXE cells did not grow in suspension. Furthermore, compared with normal fibroblasts, PXE cells exhibited lower efficiency in retracting collagen type I lattices and lower adhesion properties to collagen type I and to plasma fibronectin. This behavior was associated with higher expression of integrin subunits alpha2, alpha5, alphav, whereas beta1 subunit as well as alpha2beta1 and alpha5beta1 integrin expression was lower than in controls. Compared to controls, PXE fibroblasts had higher CAM protein expression in accordance with their high tendency to form cellular aggregates, when kept in suspension. The demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities, is consistent with the hypothesis that the gene responsible for PXE might have a broad regulatory role on the cellular machinery.

Effect of Micromorphology and Surface Reactivity of Several Unusual forms of Crystalline Silica on the toxicity to a Monocyte-Macrophage Tumor Cell Line.

The fibrogenic or carcinogenic response to the inhalation of crystalline silica dusts is strictly related to the physicochemical properties of the particles, which, in turn, are mostly determined by the "origin" and the history of the dust. Several physicochemical properties have been reported to modulate silica pathogenicity. None of them simply correlate with the reported toxicity in all the systems used to study silica pathogenicity. This confirms, on the one hand, that several properties are implicated at the same time, and on the other that pathogenicity is the result of a multistage process. There is a general consensus on the key role played by alveolar macrophages in silica-related diseases. For this article the cytotoxicity of a large variety of silicas, including rather unusual forms, with controlled micromorphology and surface properties, has been studied on a mouse monocyte-machrophage tumor cell line successfully employed in previous studies on cristobalite (Fubini et al., 1999). When compared on a per unit surface basis, crystalline silicas were more cytotoxic than amorphous ones, with the notable exception of stishovite, the nonpathogenic crystalline polymorph, with octahedrally coordinated silicon atoms. Among the amorphous ones, a diatomaceous earth and a powdered silica glass exhibited an intermediate toxicity, higher than what was elicited by a pyrogenic silica. In this study a new class of crystalline silicas have been considered, pure-silica zeolites, which constitute a new morphological entity with which cells may be confronted. The cytotoxicity of these samples varies from inert to highly cytotoxic, covering all the range of toxicity covered by the traditional silica dusts. We discuss the influence of morphological properties and surface reactivity on the cytotoxicity of several pure-silica zeolites. The extent of exposed surface and the shape of the particles correlate with cell toxicity. The lower cytotoxicity of one "non-pathogenic quartz" and of an aluminum-coated Min-U-Sil quartz, compared with the original pathogenic Min-U-Sil quartz, suggest a depressive effect of the aluminum ions present at the surface of both quartzes. The extreme variability in the biological response to crystalline silicas is confirmed and a new class of materials is brought to the study of the mechanisms of silica pathogenicity.

Relationship between surface properties and cellular responses to crystalline silica: studies with heat-treated cristobalite.

A fibrogenic sample of cristobalite dust, CRIS (crystalline silica of mineral origin), was heated to 1300 degrees C (CRIS-1300) to relate induced physicochemical modifications to cytotoxicity. Heating did not affect dust micromorphology and crystallinity, except for limited sintering and decreased surface area of CRIS-1300. Thermal treatments deeply affected surface properties. Electron paramagnetic resonance showed surface radicals progressively annealed by heating, mostly disappearing at >/=800 degrees C. Surface hydrophilicity or hydrophobicity, evaluated with water vapor adsorption, still showed some hydrophilic patches in CRIS-800, but CRIS-1300 was fully hydrophobic. Heating modified the biological activity of cristobalite. Cytotoxicity, tested on proliferating cells of the mouse monocyte macrophage cell line J774, showed that CRIS was cytotoxic and CRIS-800 was still cytotoxic, but CRIS-1300 was substantially inert. Cytotoxicity of CRIS to the rat lung alveolar epithelial cell line, AE6, as measured by colony forming efficiency, was greatly reduced for CRIS-800 and eliminated for CRIS-1300. The rate of lactate dehydrogenase release by rat alveolar macrophages was lowered for CRIS-800, and release was completely inactivated for CRIS-1300. The absence of surface radicals and the onset of hydrophobicity may both account for the loss of cytotoxicity upon heating. Differences observed between CRIS-800 and CRIS-1300, both fully deprived of surface radicals, indicate that hydrophobicity is at least one of the surface properties determining the cytotoxic potential of a dust.

Construction and in vitro functional evaluation of a low-density lipoprotein receptor/transferrin fusion protein as a therapeutic tool for familial hypercholesterolemia.

A cDNA sequence encoding a soluble form of the human low-density lipoprotein receptor (LDL-R) was produced by RT-PCR amplification. This form of the receptor contains the N-terminal cysteine-rich domain, the EGF homology domain, and the serine/threonine-rich domain, but lacks the membrane anchor as well as the cytoplasmic domain. By the same technical approach a cDNA sequence encoding rabbit transferrin was generated. In-frame fusion of the two cDNAs produced a sequence encoding a chimeric protein potentially capable of binding LDL on the N-terminal side and the transferrin receptor on the C-terminal side. It was expected that LDL bound to the chimeric protein could be internalized, targeted to an acidic compartment, and processed through the pathway of the transferrin receptor. Cells transfected with the LDL-R/transferrin cDNA translate, glycosylate, and secrete the corresponding protein in the culture medium. The secreted protein binds LDL in a ligand-blotting experiment. Finally, the chimeric protein mediates the binding and internalization of LDL in mutant cells lacking the LDL receptor. In fact, Watanabe rabbit fibroblasts, incubated with the chimeric protein show a fourfold increase in LDL binding, a fivefold increase in LDL internalization, and a sixfold increase in LDL degradation, with respect to unincubated fibroblasts.

Coordinate changes of polyamine metabolism regulatory proteins during the cell cycle of normal human dermal fibroblasts.

In human dermal fibroblasts, brought to quiescence (G0) by serum starvation, the S phase peaked 24 h and G2/M phases 36 h after serum re-addition. Under the same conditions, ornithine decarboxylase mRNA peaked at 12 h, decreased markedly in S phase and remained low until 48 h. Conversely, ornithine decarboxylase antizyme transcript dropped to its lowest level at 12 h, while reaching its highest values between 24 and 48 h. Ornithine decarboxylase activity followed essentially the pattern of its mRNA, but relative changes were much greater. S-Adenosylmethionine decarboxylase transcript and enzyme activity also peaked at around 12 h, decreasing thereafter. Spermidine/spermine N1-acetyltransferase mRNA and activity reached the highest values at 36-48 h. Putrescine concentration increased up to 18 h and fell dramatically in the S phase, remaining low thereafter. Both spermidine and spermine reached peaks at 18 h and decreased in the S phase, but not nearly as much as putrescine. We discuss how this comprehensive study may help to understand the involvement of polyamines in the control of cell proliferation.

Clusterin (SGP-2) gene expression is cell cycle dependent in normal human dermal fibroblasts.

In confluent human dermal fibroblasts brought to quiescence (G0) by serum starvation, the S phase peaked at 24 h after serum re-addiction and G2/M phase peaked at 36 h. This was confirmed by titration of h-gas1 mRNA (a marker of G0 phase) and histone H3 (a marker of S phase). Clusterin mRNA accumulation progressively increased in cells proceeding to confluence after seeding and to quiescence upon serum starvation, and peaked at around G0, in parallel with h-gas1 mRNA. At 6 h (roughly G1 phase) clusterin transcript formed a second peak, followed by a gradual decrease until 36 h. Correspondence of clusterin protein accumulation to its mRNA occurred solely with regard to the G0 peak but not to the second one. The possible meaning of the cell cycle related clusterin gene expression is discussed.

Medium sized lactones with hypolipidaemic and antioxidant activity: synthesis and biological evaluation of promising dual-action anti-atherosclerosis drugs.

Macrocyclic lactones 1a-b have been synthesized and their potential therapeutic value evaluated. The key structural feature of these active 'chimera' compounds is the 12-membered lactone ring that brings together the well-known polysubstituted hydroquinone moiety of antioxidants and the alpha,alpha-dimethyl substituted acyl residue of gemfibrozil. Lactones 1a-b showed better activity than probucol, a classical phenolic antioxidant, in preventing the Cu++-induced oxidative modification of human LDL. The hypolipidaemic activity of the new lactones, evaluated as the inhibition of lipids biosynthesis in Hep-G2 cells, was comparable to that of gemfibrozil. These features, added to the lack of cytotoxicity, make this new class of medium sized lactones promising dual-action drugs useful as anti-atherosclerosis agents.

Growth properties and growth factor responsiveness in skin fibroblasts from centenarians.

Human fibroblast cultures, which have a finite replicative lifespan in vitro, are the most widely used model for the study of senescence at the cellular level. An inverse relationship between replicative capability and donor age has been reported in human fibroblast strains. We studied the growth capacity of fibroblast primary cultures derived from people whose lifespan was as closer as possible to the expected maximum human lifespan, i.e. people over one hundred. Our data suggest that outgrowth of fibroblasts from biopsies, growth kinetics at different population doubling levels, capability to respond to a classical mitogenic stimulus (such as 20% serum) and a variety of growth factors, were remarkably similar in fibroblasts from centenarians and young controls. On the whole, our data challenge the tenet of a simple and strict relationship between in vivo aging and in vitro proliferative capability of human fibroblasts, at least at the individual level.

Four novel mutations of sterol 27-hydroxylase gene in Italian patients with cerebrotendinous xanthomatosis.

We report the characterization of eight mutations of sterol 27-hydroxylase gene (CYP27) in five Italian patients with cerebrotendinous xanthomatosis, who were found to be compound heterozygotes. Four mutations (C --> T at nt 45 of exon 4, G(+1) --> A in intron 6, G(+5) --> T in intron 7, and G(-1) --> A in intron 7) are novel. The C --> T at nt 45 of exon 4 converts the arginine codon into a stop codon thus generating a truncated protein of 198 amino acids. The three splice site mutations reduced the content of CYP27 mRNA in skin fibroblasts to very low or undetectable levels and generated minute amounts of abnormal mRNAs. The G(+1) --> A transition in intron 6 produced three abnormal mRNAs. In the first, the 5' half of exon 6 joins to exon 7, skipping 89 bp of exon 6, and in the second, exon 5 joins directly to exon 7. The predicted translation products of these mRNAs are truncated proteins. In the third abnormal mRNA, exon 5 joins to exon 8 with an in-frame deletion of 246 bp. The G(+5) --> T transversion in intron 7 generates a single abnormal mRNA in which exon 6 joins directly to exon 8, with a frameshift and a premature stop codon. In the G(-1) --> A transition in intron 7, two mRNAs are generated. In the first, the retention of the whole intron 7 causes a frameshift and a premature stop codon; in the second, the joining of exon 7 to exon 8 is associated with an in-frame deletion of the first 6 nucleotides. All these novel mutations are predicted to produce structurally abnormal enzymatic proteins with no measurable biological activity.

Cell behavior and cell-matrix interactions of human palmar aponeurotic cells in vitro.

The present investigation has been performed to better characterize, in vitro, normal aponeurotic cells in comparison with dermal fibroblasts and with cells derived from Dupuytren's affected aponeuroses. Cells were cultured in monolayer and/or into three-dimensional collagen gels. Cell structure, adhesion, and spreading capability on different substrates, as well as integrin expression were investigated by light and electron microscopy and by flow cytometry. Cell-matrix interactions were also analyzed by gel retraction experiments in the presence, or absence, of RGD peptides and anti-integrin antibodies. Normal aponeurotic cells, compared with dermal fibroblasts, exhibited in vitro peculiar structural features, which were substantially maintained in Dupuytren's aponeurotic cells, irrespective of the substrate they were grown on. By contrast, the aponeurotic cell behavior was different in normal and diseased cells, these latter approaching that of dermal fibroblasts. Normal aponeurotic cells, in fact, were characterized by low efficiency in retracting the collagen gel, low alpha 2, alpha 1, and alpha 5 integrin subunit expression and low adhesion properties onto collagen and fibronectin, whereas cells isolated from the aponeuroses of Dupuytren's patients exhibited higher capability of retracting the collagen gel, increased adhesion properties toward collagen and fibronectin, and higher levels of integrin expression. No differences were observed between dermal fibroblasts from Dupuytren's patients or from normal subjects. These in vitro results are consistent with those previously obtained in situ, suggesting that palmar aponeurotic cells have a peculiar phenotype and that changes in cell-matrix interactions occur in Dupuytren's contracture. Moreover, by comparing data obtained from the retracted fibrotic cords and the still clinically unaffected aponeuroses of the same patients, it may be noted that Dupuytren's disease is not only confined to the clinically involved branches, but includes the whole aponeurosis of the affected hand.

Effect of heparin on cell proliferation: lack of correlation with heparin binding sites on cell membrane.

In this study an attempt was made to correlate the in-vitro anti-proliferative effect of heparin with the heparin binding on the cell surface. Cells with different sensitivities to the anti-proliferative effect of heparin (BHK-21, FAO, SMC, BAEC, A-431, V-79, and skin fibroblasts) were incubated with [3H]heparin either in the presence or in the absence of unlabelled heparin. A saturable binding was found only in BHK-21, FAO, SMC, BAEC and V-79. Scatchard analysis revealed the presence of a single class of binding sites. The binding of [3H] heparin was efficiently displaced by unlabelled heparin, pentosan polysulfate and low-molecular-weight heparin, but not by dermatan sulfate. Although the sensitivity to the anti-proliferative effect of heparin varied considerably among the cell types (BHK-21 > SMC, FAO > BAEC > V-79), there was no correlation between the reduction of proliferation of these cells and either their heparin binding capacity or the number of binding sites per cell.

c-fos/c-jun expression and AP-1 activation in skin fibroblasts from centenarians.

In vitro replicative senescence is characterized by an irreversible growth arrest due to the inability of the cell to induce some key regulators of cell cycle progression, such as c-fos and AP-1, in response to mitogenic stimuli. In vitro replicative senescence and in vivo aging have been assumed to be two related phenomena, likely controlled by overlapping or interacting genes. As a corollary, fibroblasts from centenarians, which have undergone a long process of senescence in vivo should have very limited proliferative capability. On the contrary, in a previous work we found that fibroblasts from centenarians exhibited the same capacity to respond to different mitogenic stimuli as fibroblasts from young donors. Here we provide evidences that the well preserved proliferative response is likely due to the fact that some pivotal regulators- c-fos, c-jun and AP-1-are still fully inducible, despite a long process of in vivo senescence. Our data therefore suggest that in vivo and in vitro aging are separate phenomena whose possible relationships, if any, have to be ascertained very carefully.

Antiproliferative activity of 3-aminobenzamide in A431 carcinoma cells is associated with a target effect on cytoskeleton.

3-Aminobenzamide (3-ABA) is an inhibitor of poly(ADP-ribose)polymerase (PARP), an enzyme involved in several cellular processes, and exerts its effects by acting at the cytoskeleton level. Here we show that 3-ABA has an antiproliferative effect on the human carcinoma cell line A431, as measured by different assays. 3-ABA was capable of inhibiting cell growth as well as colony formation, this inhibitory effect is reversible. Morphological analyses showed a series of cellular alterations, such as a remarkable increase of dendritic-like protrusions, quite unusual in epithelial cells, and suggestive of a differentiative triggering. Immunocytochemical studies suggested that a major target of 3-ABA was indeed the cytoskeleton. These data, together with those of the literature, indicate that 3-ABA, depending on cell histotype and drug concentration, is a versatile drug capable of exerting antiproliferative and cytostatic effects as well as cytotoxic and antiapoptotic effects, processes sharing an important involvement of cytoskeleton. These unique characteristics of 3-ABA may be of interest for cancer research.

Cerebrotendinous xanthomatosis caused by two new mutations of the sterol-27-hydroxylase gene that disrupt mRNA splicing.

Cerebrotendinous xanthomatosis (CTX) is an inherited sterol storage disease associated with the accumulation of cholestanol and cholesterol in various tissues. CTX is caused by a deficiency of sterol-27-hydroxylase, a mitochondrial enzyme that oxidizes the side chain of cholesterol in the pathway leading to the formation of bile acids. In the present study we report two mutations of sterol-27-hydroxylase gene (CYP27 gene) found in Italian CTX patients. Proband T.C. is homozygous for a G-->A transition at the first nucleotide of intron 7. This mutation causes the formation of minute amounts of an abnormal mRNA, in which exon 6 joins directly to exon 8 with the skipping of exon 7. The exon 6-exon 8 junction results in a frame shift, downstream from the codon for Arg362, which generates a string of 28 novel amino acids preceding a premature termination codon. Proband C.U. is homozygous for a G-->C transversion at the last nucleotide of exon 3. This mutation, which changes the consensus sequence of the 5' donor splice site, is associated with barely detectable levels of sterol-27-hydroxylase mRNA, of normal size, in proband fibroblasts. As both mutations change the sites for two restriction enzymes, rapid methods were devised for the identification of the healthy carriers among the probands' family members and for the screening of these mutations in other CTX patients.