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In the present study, we were interested to investigate how autistic traits (including systemizing and empathy) and academic degree influence individuals’ visuospatial abilities. To this end, 352 university students completed the Autism Spectrum Quotient (AQ), the Empathy Quotient, the Systemizing Quotient (SQ) and visuospatial tests measuring figure disembedding and mental rotation of two-dimensional figures. Engineering-design students (architecture and engineering) were the most accurate in disembedding and mentally rotating figures, followed by students of physical sciences (computer science, chemistry, physics, etc.) and fact-based humanities (languages, classics, law); biological (psychology and neuroscience, etc.) and systems-based social scientists (economics and commerce) were the least accurate. Engineering-design students also showed higher SQ scores with respect to the other four academic degree subjects, with students of biological sciences showing lower SQ scores. Importantly, results from a path analysis revealed that SQ (but not AQ) exerted an indirect effect on figure disembedding and mental rotations through the influence of the academic degree. Thus, the present findings reveal shadydifferences in systemizing degree and visuospatial performance withinsystemizing-based degree subjects.Implications for education are discussed.

Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; in vitro iodide is cytotoxic, inhibits cell growth, and induces morphological changes in thyroid cells of some species. In this study, we investigated the effect of iodide excess in an immortalized thyroid cell line (TAD-2) in primary cultures of human thyroid cells and in cells of nonthyroid origin. Iodide displayed a dose-dependent cytotoxicity in both TAD-2 and primary thyroid cells, although at different concentrations, whereas it had no effect on cells of nonthyroid origin. Thyroid cells treated with iodide excess underwent apoptosis, as evidenced by morphological changes, plasma membrane phosphatidylserine exposure, and DNA fragmentation. Apoptosis was unaffected by protein synthesis inhibition, whereas inhibition of peroxidase enzymatic activity by propylthiouracil completely blocked iodide cytotoxicity. During KI treatment, reactive oxygen species were produced, and lipid peroxide levels increased markedly. Inhibition of endogenous p53 activity did not affect the sensitivity of TAD-2 cells to iodide, and Western blot analysis demonstrated that p53, Bcl-2, Bcl-XL, and Bax protein expression did not change when cells were treated with iodide. These data indicate that excess molecular iodide, generated by oxidation of ionic iodine by endogenous peroxidases, induces apoptosis in thyroid cells through a mechanism involving generation of free radicals. This type of apoptosis is p53 independent, does not require protein synthesis, and is not induced by modulation of Bcl-2, Bcl-XL, or Bax protein expression.

HMG-CoA reductase inhibitors, such as Lovastatin and Simvastatin, cause cell cycle arrest by interfering with the mitogenic activity of mitogens present in culture media. Cells are induced to pause in G1 and can readily resume growth upon removal of the enzymatic block. Estrogens, acting via their nuclear receptor, are mitogens for different normal and transformed cell types, where they foster cell cycle progression and cell division. In estrogen-responsive MCF-7 human breast cancer cells, but not in non responsive cells, 17 beta-estradiol (E2) induces cells arrested with Lovastatin or Simvastatin to proliferate in the presence of inhibitor, without restoring HMG-CoA reductase activity or affecting the protein prenylation pattern. Mitogenic stimulation of G1-arrested MCF-7 cells with E2 includes primary transcriptional activation of c-fos, accompanied by transient binding in vivo of the estrogen receptor and/or other factors to the ERE and the estrogen-responsive DNA region of this proto-oncogene, as detected by dimethylsulphate genomic footprinting analysis. Mitogenic stimulation of growth-arrested MCF-7 cells by E2 occurs, under these conditions, without evident activation of ERK-1 and -2 kinases, and thus independently from the mitogen-responsive signal transduction pathways that converge on these enzymes.

The expression of the low-affinity receptor for IgE Fc epsilon RII) in the human monocyte-like U-937 cell line can be upregulated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and by IgE. TPA induces terminal differentiation of U-937 cells and causes a four- to fivefold increase in the number of Fc epsilon RII. TPA also modulates the expression of several other membrane markers of U-937 cells. IgE alone has a modest effect on the expression of Fc epsilon RII (about a 10% increase), while simultaneous treatment of U-937 cells with TPA and IgE has a cooperative effect, causing an eightfold increase in the number of Fc epsilon RII. Cycloheximide strongly suppresses the expression of Fc epsilon RII, both in TPA-stimulated and unstimulated cells; this effect can be partly reversed by culturing the cells in the presence of IgE. These results suggest that TPA induces the expression of newly synthesized receptors, while IgE causes an accumulation of preformed receptors.

Monocyte interaction with proteins of the extracellular matrix (ECM) is regulated by expression of specific cell-surface receptors. 12-O-tetradecanoyl phorbol-13-acetate (TPA) has been shown to induce the promonocytic cell line U937 to a more differentiated monocyte-like state. In this study we have analysed the attachment of U937 cells to ECM proteins and the effects of treatment with TPA on this process. Non-induced U937 cells attach to fibronectin- and Matrigel-coated surfaces without TPA stimulation, but TPA further increases adherence to these substrates as measured by an enhanced binding and by the lower concentration of proteins needed in the substrate to achieve 50% of maximal cell adhesion. Attachment to type I collagen was seen only with activated U937 cells, whereas no measurable attachment to bovine serum albumin, vitronectin, and type IV collagen was detected. TPA-activated U937 cells showed a two-fold increase in the expression of the RGD-dependent integrin receptors alpha 3 and alpha 5, and a reduction in the expression of alpha 4, another fibronectin-specific receptor, whereas the common beta 1 chain was unchanged. Attachment of U937 cells to fibronectin was primarily mediated by the alpha 3 and alpha 5 integrins, as revealed by the ability of GRGDS peptides to inhibit attachment, whereas the CS-1 peptide, containing the alpha 4 binding site, was largely ineffective in blocking attachment.

Cyclin D1 is a target for positive regulation by estrogens in growth-responsive cells, in which it mediates their mitogenic effects. Amplification and overexpression of the cyclin D1 gene (CCND1) might thus represent a genetic lesion inducing hormone-independent growth of transformed cells. Indeed, cyclin D1 overexpression has been found in up to 50% of primary breast cancers, and in about one-third of these cases, this is linked to amplification of the 11q13 chromosomal region, which also includes the CCND1 gene. These tumors are predominantly estrogen receptor-positive, and for this reason, these patients are often selected for adjuvant antiestrogen therapy. No information is available, however, as to whether cyclin D1 overexpression due to gene amplification might interfere with and reduce antiestrogen efficacy. This was investigated here by taking advantage of an experimental model that reproduces cyclin D1 overexpression resulting from increased CCND1 gene dosage in hormone-responsive human breast cancer cells. For this, MCF-7 cells stably transfected with a tet-inducible cyclin D1 expression vector were tested for their in vitro response to steroidal (ICI 182,780) and nonsteroidal (trans-4-hydroxytamoxifen) antiestrogens under condition of low (endogenous only) or high (exogenous) cyclin D1 levels. Results show that although cyclin D1 overexpression seems to interfere with the early cell cycle effects of antiestrogens, it does not prevent their cytostatic actions, so that growth of cyclin-overexpressing MCF-7 cells is still efficiently inhibited in vitro by these drugs.

The effects of the stable expression of E1A and/or middle T oncogenes on the proliferative activity of PC Cl3 normal thyroid cells are reported. The proliferation of PC Cl3 cells is mainly regulated by insulin and TSH in a stimulatory way and by somatostatin in an inhibitory fashion. The transformed cell lines, named PC Py and PC E1A Py, show an autonomous pattern of proliferation. The blockade of phosphotyrosine phosphatase activity with vanadate increased the proliferation rate of PC Cl3 under basal and stimulated conditions and completely prevented the inhibitory activity of somatostatin, suggesting that in PC Cl3 cells, a tonic tyrosine phosphatase activity regulates basal and stimulated proliferation, and that a somatostatin-dependent increase in this activity may represent a cytostatic signal. Conversely, in both PC Py and PC E1A Py, vanadate did not modify basal and stimulated proliferation. We analyzed tyrosine phosphatase activity in the different cell lines basally and under conditions leading to the arrest of cell proliferation: confluence (contact inhibition), growth factor deprivation (starvation), and somatostatin treatment. Under basal conditions, tyrosine phosphatase activity was significantly lower in PC Py and PC E1APy cell lines than that in the normal cells. The inhibition of the proliferation induced by contact inhibition or somatostatin treatment was accompanied by an increase in tyrosine phosphatase activity only in PC Cl3 cells. The reduction in tyrosine phosphatase activity in PC E1APy cells correlated with a significant reduction in the expression of R-PTPη, a tyrosine phosphatase cloned from PC Cl3 cells. Conversely, the expression of another receptor-like PTP, PTPμ, was unchanged. Thus, PTPη may be a candidate to mediate inhibitory signals (i.e. activation of somatostatin receptors or cell to cell contact) on the proliferative activity of PC Cl3 cells, and the reduction of its expression in the transformed cell lines may lead to an alteration in the control of cell proliferation.

We describe a two-dimensional model of activation of the behavioural systems according to which four prototypical activation styles can be identified within a system by positioning an individual on two main dimensions. A functional style, characterized by primary strategies of activation, a hyperactivated style, by hyperactivation strategies, an inhibited style, by deactivating strategies, and a fourth style, the problematic style, characterized by coexistence of hyperactivating and deactivating strategies. Here, we focus on the problematic style representing a dysfunctional expression of the behavioral systems leading to chaotic and unpredictable behaviors, and also relating to higher rates of psychopathology and reduced ability to participate to therapy. The present model could help the clinician to identify problematic behavioural activations undermining the therapeutic process.

Thyroid hormone (TH) is an important regulator of growth, development, and metabolism. Most of the active TH T3 is generated by peripheral TH metabolism mediated by the iodothyronine deiodinases. Type 3 deiodinase (D3) inactivates T3 via specific deiodination reactions. It is an oncofetal protein frequently expressed in neoplastic tissues and is a direct target of the sonic hedgehog (Shh) pathway in basal cell carcinomas (BCCs). However, the molecular mechanisms triggered by T3 in BCC are still mostly unrevealed. Here, we demonstrate that D3 action is critical in the proliferation and survival of BCC cells. D3 depletion or T3 treatment induce apoptosis of BCC cells and attenuate Shh signaling. This is achieved through a direct impairment of Gli2 protein stability by T3. T3 induces protein kinase A, which in turn destabilizes Gli2 protein via its C-terminal degron. Finally, in a mouse model of BCC, T3-topical treatment significantly reduces tumor growth. These results demonstrate the existence of a previously unrecognized cross talk between TH and Gli2 oncogene, providing functional and mechanistic evidence of the involvement of TH metabolism in Shh-induced cancer. TH-mediated Gli2 inactivation would be beneficial for therapeutically purposes, because the inhibition of Shh-Gli2 signaling is an attractive target for several anticancer drugs, currently in clinical trials.

A striking special ability in arranging picture stories was reported in an Asperger child (C.M.) showing an exceptional performance on Wechsler picture arrangement subtest. Neuropsychological examination did not disclose visuoperceptual and spatial defects, or working memory, attention and executive disorders, but revealed an attentional bias towards local details of complex structures. A specific assessment of C.M.'s understanding of picture stories demonstrated that, with respect to normal controls, he showed an enhanced ability to detect causal links among elements of a story. These findings provide support to the hypothesis that savantism can be related to strong systemizing in autism.

The melanocortin-4 receptor (MC4R) is involved in regulating energy homeostasis; mutations in this gene have been associated with 1-5% of early-onset human obesity. The aim of this study was to functionally characterize MC4R mutations identified in morbidly obese subjects living in Southern Italy. We studied their ligand binding, signaling pathway and subcellular localization. As expected, mutants Q43X and S19fsX51, which produce truncated forms of receptor, were devoid of activity. The activity of mutants W174C and A175T were very different even though the mutations are adjacent and are in the same transmembrane helix (TMH). In fact, the production and expression of mutant A175T on the plasma-membrane (PM) was similar to that of the wild-type (wt) receptor and the mutant retained 70% of wt receptor activity; on the contrary, the production of W174C mutant in the cytoplasm was similar to that of the wt receptor and mutant A175T but was only barely detectable on the PM and was devoid of activity. Confocal microscopy showed that W174C remained entrapped in the endoplasmic reticulum (ER) of the cells. Structural analysis showed that substitution of Trp174, located in the middle of TMH4 and 100% conserved in all known MC4Rs, with Cys could impair the relative orientation of TMH2 and TMH4 thereby affecting the overall protein architecture. Furthermore, co-expression studies showed that mutant A175T but not W174C had a dominant negative effect on the wt receptor activity. © 2010 Elsevier B.V.

The expression of the beta1 family of integrins was determined in thyroid follicular cells from patients with Graves' disease (GD). Integrin expression was quantitated by flow fluorocytometry of single cell suspensions with antibodies against the common beta1 chain and the alpha1-alpha6 subunits. Results indicated that also in thyroid glands of GD, as previously observed in nodular goiters, two follicular cell populations with different patterns of beta1 integrin expression coexist (VLAalpha3beta1 and VLAalpha1,3,5,6beta1). The VLAalpha1,3,5,6beta1 thyrocyte population in GD was more abundant than in nodular goiters, ranging from 40 to 70% of the total follicular cells and the overall expression of the beta1 integrins was a two-fold higher. In thyrocytes from patients with GD cultured in vitro, alpha3 and alpha2 expression was regulated by cell-to-cell contact as previously described in normal thyroid cells, while the expression of alpha1, alpha5 and alpha6 was quickly lost during the culture. Our data suggest that the integrin profile of the VLAalpha1,3,5,6beta1 thyrocyte population in GD is induced by micro-environmental conditions rather than being the expression of a constitutive phenotype.

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway that is responsible for the generation of NADPH, which is required in many detoxifying reactions. We have recently demonstrated that G6PD expression is induced by a variety of chemical agents acting at different steps in the biochemical pathway controlling the intracellular redox status. Although we obtained evidence that the oxidative stress-mediated enhancement of G6PD expression is a general phenomenon, the functional significance of such G6PD induction after oxidant insult is still poorly understood. In this report, we used a GSH-depleting drug that determines a marked decrease in the intracellular pool of reduced glutathione and a gradual but notable increase in G6PD expression. Both effects are seen soon after drug addition. Once G6PD activity has reached the maximum, the GSH pool is restored. We suggest and also provide the first direct evidence that G6PD induction serves to maintain and regenerate the intracellular GSH pool. We used HeLa cell clones stably transfected with the human G6PD gene that display higher G6PD activity than the parent HeLa cells. Although the activities of glutathione peroxidase, glutathione reductase, and catalase were comparable in all strains, the concentrations of GSH were significantly higher in G6PD-overexpressing clones. A direct consequence of GSH increase in these cells is a decreased reactive oxygen species production, which makes these cells less sensitive to the oxidative burst produced by external stimuli. Indeed, all clones that constitutively overexpress G6PD exhibited strong protection against oxidants-mediated cell killing. We also observe that NF-kappa B activation, in response to tumor necrosis factor-alpha treatment, is strongly reduced in human HeLa cells overexpressing G6PD.

The receptor (CXCR4) for the stromal-derived factor-1 (SDF1) and the urokinase-receptor (uPAR) are up-regulated in various tumors. We show that CXCR4-transfected cells migrate toward SDF1 on collagen (CG) and do not on vitronectin (VN). Co-expression of cell-surface uPAR, which is a VN receptor, impairs SDF1-induced migration on CG and allows migration on VN. Blocking fMLP receptors (fMLP-R), alpha-v integrins or the uPAR region capable to interact with fMLP-Rs, impairs migration of uPAR/CXCR4-transfected cells on VN and restores their migration on CG. uPAR co-expression also reduces the adherence of CXCR4-expressing cells to various components of the extracellular matrix (ECM) and influences the partitioning of beta1 and alpha-v integrins to membrane lipid-rafts, affecting ECM-dependent signaling. uPAR interference in CXCR4 activity has been confirmed in cells from prostate carcinoma. Our results demonstrate that uPAR expression regulates the adhesive and migratory ability of CXCR4-expressing cells through a mechanism involving fMLP receptors and alpha-v integrins.

Systemic sclerosis (SSc) is characterized by excessive fibrosis throughout the body. There are two major subsets of SSc, diffuse cutaneous Systemic sclerosis (dSSc) and limited cutaneous Systemic sclerosis (ISSc). Fibroblasts play a key role in SSc. The expression and function of the urokinase (uPA)-mediated plasminogen activation (PA) system, a well-characterized system of serine-proteases involved in several pathological processes, has been investigated in SSc fibroblasts. The expression of the components of the PA system, including uPA, its type-1 and type-2 inhibitors (PAI-1 and PAI-2) and its receptor (uPAR), was examined by Western blot in fibroblasts from patients affected by limited and diffuse forms of SSc. uPA and PAI-1 secretion increased only in fibroblasts from ISSc lesions compared to normal fibroblasts. PAI-2 levels were decreased in fibroblasts from both SSc forms. Interestingly, fibroblasts from areas not adjacent to the lesions (not-affected) of the diffuse form showed reduced levels of PAI-1 and increased uPAR expression. Adhesion experiments showed reduced adherence to VN of fibroblasts from ISSc lesions and from non-affected areas of the diffuse form, as compared to normal controls. These results suggest a role for uPA and PAI-1 in the ISSc form, likely related to the activation of latent forms of cytokines and to the accumulation of ECM components, whereas a role for uPAR can be hypothesized in the evolvement of the diffuse form, based on its up-regulation in the non-affected areas.

We describe a patient who suddenly developed prosopometamorphopsia after a childbirth; she claimed that the left half of well-known and unfamiliar faces looked distorted. Brain MR was normal, whereas SPECT showed hypoperfusion of the left infero-lateral occipital cortex. No visual recognition defects for objects or faces were present. In three matching tasks with half-faces (Experiment 1), chimeric faces (Experiment 2), or chimeric objects (Experiment 3), the patient was impaired only when she matched pairs of chimeric faces differing in their left half; the same results were obtained after 1 year. This is the first behavioural demonstration of selective chronic metamorphopsia for the left side of faces, and provides new insights for models of face processing.

The closing-in phenomenon (CIP) is often observed in patients with severe drawing disorders, but its cognitive bases are not well understood. We describe an experimental investigation aimed to clarify the nature of closing-in and its relationships with drawing disorders in a patient with corticobasal degeneration. In copying simple or complex stimuli (Experiment 1), the patient showed adherent and near types of closing-in, not affected by stimulus complexity, and produced distorted and often unrecognisable drawings. On the contrary, in drawing to dictation (without any available model), patients' performances significantly improved with respect to copying (Experiment 2). These data were consistent with the hypothesis that in some patients closing-in may develop from frontal-related release of approach behaviour even in the absence of relevant visuoperceptual impairments. By asking the patient to reproduce given spatial locations within circular frames (Experiment 3), we could further demonstrate the sparing of visuospatial processing and the frontal genesis of closing-in. These findings allowed us to speculate on the heterogeneous nature of closing-in.

We describe a follow-up study of a patient with a selective, progressive impairment of topographical orientation. The patient's topographical difficulties were evident only in unfamiliar surroundings at the beginning of the observation period but later on they were observed even at home. Serial neuropsychological tests demonstrated a progressive impairment of visuospatial abilities with sparing of the other cognitive domains; only at the last assessment, about six years after early disturbances and three years after the first evaluation, the patient developed the typical cognitive impairments of Alzheimer's disease (AD). This case represents a focal variant of AD not previously described and suggests that the neuronal pathways underlying spatial orientation may be selectively damaged by the degenerative process.

Thyroid transcription factor gene 1 (TTF-1) is a homeobox-containing gene involved in thyroid organogenesis. During early thyroid development, the homeobox gene Nkx-2.5 is expressed in thyroid precursor cells coincident with the appearance of TTF-1. The aim of this study was to investigate the molecular mechanisms underlying thyroid-specific gene expression. We show that the Nkx-2.5 C terminus interacts with the TTF-1 homeodomain and, moreover, that the expression of a dominant-negative Nkx-2.5 isoform (N188K) in thyroid cells reduces TTF-1-driven transcription by titrating TTF-1 away from its target DNA. This process reduced the expression of several thyroid-specific genes, including pendrin and thyroglobulin. Similarly, down-regulation of TTF-1 by RNA interference reduced the expression of both genes, whose promoters are sensitive to and directly associate with TTF-1 in the chromatin context. In conclusion, we demonstrate that pendrin and thyroglobulin are downstream targets in vivo of TTF-1, whose action is a prime factor in controlling thyroid differentiation in vivo.

The Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a hematopoietic growth factor that regulates the in vitro and in vivo proliferation and differentiation of hematopoietic cells through the interaction with a specific heterodimeric receptor complex (GM-CSFR), consisting of an alpha and a beta chain with molecular weights of 80 and 120 KDa, respectively. We have studied the expression of the GM-CSFR (alpha chain) on the surface of the human osteosarcoma cell line SaOS-2 and the in vitro effects of different concentrations (10, 100, and 200 ng/ml) of GM-CSF on GM-CSFR expression and the biological activity of SaOS-2 cells. Our data show that SaOS-2 cells express GM-CSFR and that GM-CSF can down-regulate the expression of its own receptor on these cells. Furthermore, to evaluate the biological effects of GM-CSF on SaOS-2 cells, we have investigated cell proliferation and differentiation of these cells treated with different doses of the growth factor through: (1) a morphological analysis of typical osteoblast differentiation markers such as osteopontin and BSP-II; (2) measurement of alkaline phosphatase (ALP) activity; (3) production of bone ECM components (collagen I, fibronectin, tenascin, and laminin); (4) production of interleukin-6 (IL-6) and osteocalcin in the culture medium. The results show that the in vitro treatment of SaOS-2 cells with recombinant human GM-CSF causes a decreased cell proliferation and an increased production of osteopontin, BSP-II, ALP, IL-6, and most but not all ECM components. These findings suggest that GM-CSF can regulate proliferation and differentiation of osteoblast-like SaOS-2 cells and could also play an unexpected role in the maturation of bone tissue.

The urokinase-type plasminogen activator (uPA) is able to cleave its cell surface receptor (uPAR) anchored to the cell membrane through a glycophosphatidylinositol tail. The cleavage leads to the formation of cell surface truncated forms, devoid of the N-terminal domain 1 (D1) and unmasks or disrupts, depending on the cleavage site, a sequence in the D1-D2 linker region (residues 88-92), which in the soluble form is a potent chemoattractant for monocyte-like cells. To investigate the possible role(s) of the cleaved forms of cell surface glycophosphatidylinositol-anchored uPAR, uPAR-negative human embrional kidney 293 cells were transfected with the cDNA of intact uPAR (uPAR-293) or with cDNAs corresponding to the truncated forms of uPAR exposing (D2D3-293) or lacking (D2D3wc-293) the peptide 88-92 (P88-92). Cell adhesion assays and co-immunoprecipitation experiments indicated that the removal of D1, independently of the presence of P88-92, abolished the lateral interaction of uPAR with integrins and its capability to regulate integrin adhesive functions. The expression of intact uPAR induced also a moderate increase in 293 cell proliferation, which was accompanied by the activation of ERK. Also this effect was abolished by D1 removal, independently of the presence of P88-92. The expression of intact and truncated uPARs regulated cell directional migration toward uPA, the specific uPAR ligand, and toward fMLP, a bacterial chemotactic peptide. In fact, the uPA-dependent cell migration required the expression of intact uPAR, including D1, whereas the fMLP-dependent cell migration required the expression of a P88-92 containing uPAR and was independent of the presence of D1. Together these observations indicate that uPA-mediated uPAR cleavage and D1 removal, occurring on the cell surface of several cell types, can play a fundamental role in the regulation of multiple uPAR functions.

Overexpression of the cyclin D1 (CCND1) gene, encoding a downstream effector of mitogenic signals that plays a central role in G1 phase progression, is often found in cancerous cells. In sporadic breast cancer (BC), this is one of the most frequent and early genetic lesions identified so far, found in more than 50% of the tumors. Inhibitors of the mevalonate/protein prenylation pathway belong to a new family of cancer therapeutic agents that act by blocking intracellular mitogenic signal transduction pathways, thereby preventing expansion of pre-cancerous foci and inhibiting growth of transformed cells. It is not known at present whether constitutively high intracellular levels of cyclin D1 might interfere with the cytostatic actions of mevalonate/protein prenylation inhibitors. This possibility was investigated here by assessing the cell cycle effects of Simvastatin, a non-toxic upstream inhibitor of the mevalonate pathway, on human BC MCF-7 cells expressing either normal or enhanced levels of cyclin D1 from of a stably transfected, tet-inducible expression vector. Results show that constitutive overexpression of this protein, such as that found in sporadic BCs, does not influence the growth inhibitory effects of Simvastatin in vitro. In addition, D1-overexpressing embryo fibroblasts were also found to be responsive to the cell cycle effects of mevalonate/protein prenylation pathway blockade, further suggesting that high intracellular levels of cyclin D1 do not prevent the cytostatic actions of compounds targeting this metabolic pathway.

Antiestrogens are widely used for breast cancer treatment, where they act primarily by inhibiting the mitogenic action of estrogens on tumor cells. The effects of the pure antiestrogen ICI 182,780 on estrogen-regulated cell cycle phase-specific events were investigated here in synchronously cycling human breast cancer (HBC) cells. In early G 1 -arrested MCF-7 or ZR-75.1 cells, 17β-estradiol (E2) induces rapid activation of the cyclin/Cdk/pRb pathway, as demonstrated by D-type G 1 cyclins accumulation during the first few hours of hormonal stimulation, followed by sequential accumulation of E, A and B1 cyclins and progressive pRb phosphorylation, as cells progress through the cell cycle. When added to quiescent cells together with E2, ICI 182,780 prevents all of the above hormonal effects. Interestingly, in mid-G 1 cells (2–8 h into estrogen stimulation) the antiestrogen causes rapid reversal of hormone-induced D-type cyclins accumulation and pRb phosphorylation, and still fully inhibits G 1 –S transition rate, while in late-G 1 cells it does not prevent S phase entry but still inhibits significantly DNA synthesis rate, S-phase cyclins accumulation and pRb hyperphosphorylation. These results indicate that pure antiestrogens prevent multiple estrogen-induced cell cycle-regulatory events, each timed to allow efficient G 1 completion, G 1 –S transition, DNA synthesis and cell cycle completion.

The urokinase-type plasminogen activator receptor (uPA-R) focuses the proteolytic activity of its ligand, the urokinase-type plasminogen activator (uPA), on the cell surface, and can also act as an adhesion receptor for vitronectin (VTN). uPA increases uPA-R affinity for VTN and is also able to cleave its receptor. We have previously shown that uPA-R is involved in the adhesion of normal thyroid cells to VTN. In the present report, we have investigated the effect of uPA on normal thyroid cell adhesion to some extracellular matrix (ECM) components. We show that a short-term treatment with uPA does not change normal thyroid cell adhesion to fibronectin (FNT), collagen (CGN), laminin (LMN) and VTN. The prolongation of uPA treatment increases cell adhesion to VTN, and, less efficiently, to other ECM components. Since the short term uPA treatment causes a partial cleavage of uPA-R, that does not increase with time, the observed increase in cell adhesivity cannot be related to the cleavage of uPA-R. We show that the adhesion improvement after the long term uPA treatment is instead due to a strong increase of the cell-surface expression of the integrin beta3 and a moderate increase of the integrin alpha(v). Both alpha(v) beta3 and alpha(v) beta1 are integrinic receptors for VTN.

Background & Aims: We have previously shown that autocrine insulin-like growth factor (IGF)-II synthesis through IGF-I receptor stimulates proliferation and inhibits differentiation of Caco-2 cells. To demonstrate whether differentiation of Caco-2 cells is dependent on cell growth status, we analyzed the effect of cell cycle arrest on differentiation of wild-type and IGF-II–overexpressing cells. Methods: Cells were treated with drugs that inhibit the progression either to S phase (l-b-D-arabinofuranosylcytosine or M phase (nocodazole). Cell differentiation was analyzed by assessing apolipoprotein A-1 and sucrase-isomaltase expression. Cell proliferation and DNA content were assessed by thymidine incorporation and fluorescence-activated cell sorter analysis, respectively. Cell cycle regulatory molecules were analyzed by assessing p21 and retinoplasma protein (pRb) expression and pRb phosphorylation. Results: Cell cycle block at G1-S phase was associated with increased expression of differentiation markers in both parental and IGF-II–transfected cells. On the contrary, cell cycle arrest at G2-M phase correlated with the expression of differentiation markers in parental but not in IGF-II–transfected cells. Constitutive IGF-II–expressing cells actively incorporated thymidine and showed an increase in the proportion of cells with >4N DNA ploidy in the presence of nocodazole. Nocodazole treatment of constitutive IGF-II–expressing cells stimulated p21 expression in the presence of hyperphosphorylated pRb. Conclusions: The data show that cell cycle arrest increases differentiation of Caco-2 cells. IGF-II–mediated proliferation may prevent cell differentiation through effects on control cell checkpoint proteins. GASTROENTEROLOGY 1999;116:1358-1366

In the present study the effects of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) on fibroblast growth and activity have been studied. In this regard the AA have evaluated in primary cultures of human gengival normal fibroblasts (PG1 cells): a)-the expression of GM-CSF receptor (GM-CSFR) (alfa unit) on the cell surface; b)-the in vitro effects of different doses of GM-CSF on the GM-CSFR expression and on the proliferation and activity of fibroblasts. PG1 cells have been stimulated in vitro with different concentrations of GM-CSF (10, 50, 80, 100 and 150 ng/ml) using promonocytic cell line U937 as positive control for GM-CSFR expression. GM-CSFR was investigated by flow cytometry, with mouse monoclonal antibody (mAb) against the alfa chain of the human GM-CSFR and fluorescein-conjugated goat antimouse immunoglobulin G (IgG). At high GM-CSF concentration (80 ng/ml) the AA observed: 1)-A marked increase of GM-CSFR expression evaluated as fluorescence intensity (about three fold in respect to the controls); 2)-Maximal increase of PG1 cells proliferation. Moreover immunofluorescence on fibroblasts obtained from culture plates showed increased actin stress fibers and fibronectin production with low stimulation by GM-CSF, while higher concentration of this cytokine determined increased proliferation of cells, but a decreased formation of actine fibers and vinculin plaques. These results demonstrate: 1)-The presence of GM-CSFR on the surface of fibroblasts; 2)-The proliferation and the synthesis activity of these cells (in vitro) are modulated by different concentration of GM-CSF. We hypothesize that GM-CSF until 80 ng/ml can upregulate the expression of the receptor. Therefore, on the basis of previous findings of high serum levels of GM-CSF in course of scleroderma, a disease characterized by fibroblast hyperactivity, a possible role of this cytokine in the pathogenic process of this disease can be hypothesized.

Inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, such as Simvastatin and Lovastatin, reduce the rate of DNA synthesis and proliferation of a wide variety of cell types in vitro, by inducing a cell cycle arrest in G1. In estrogen-free medium, DNA synthesis is reduced by more that 90% following exposure of normal and transformed human breast epithelia] cells to 20 microM Simvastatin or Lovastatin for 24 to 42 hrs. We show here that stimulation of estrogen responsive MCF-7 cells with nanomolar concentrations of 17beta-estradiol (E2) prevents inhibition of DNA synthesis by these compounds. The effect of the hormone is antagonized by both steroidal and non steroidal antiestrogens, and it is not detectable in estrogen receptor-negative MCF-10a cells. Cell cycle analysis demonstrates that HMG-CoA reductase inhibitors are unable to induce G1 arrest of MCF-7 cells in the presence of E2.

The C-myc gene encodes a nuclear protein whose precise function is still not folly understood. Introduction of a c-myc gene into a number of cell lines leads to an increase in their susceptibility to NK-cell lysis. It was reported earlier that c-myc can induce a decrease in the membrane expression of the MHC class-1 molecules and this may be one of the factors that render target cells relatively more susceptible to NK lysis. In this contribution, we show, in a human LCL line transfected with a constitutively active c-myc gene, an increased sensitivity to NK lysis, which correlates with an augmented effector-target binding ability of c-myc-transfected LCLs and with a high 1CAM-1 expression rather than with down-regulation of MHC class-l W6/32 epitope expression. © 1994 Wiley-Liss, Inc.