Modelling cancer cell budding in-vitro as a self-organised, non-equilibrium growth process.

Tissue self-organization into defined and well-controlled three-dimensional structures is essential during development for the generation of organs. A similar, but highly deranged process might also occur during the aberrant growth of cancers, which frequently display a loss of the orderly structures of the tissue of origin, but retain a multicellular organization in the form of spheroids, strands, and buds. The latter structures are often seen when tumors masses switch to an invasive behavior into surrounding tissues. However, the general physical principles governing the self-organized architectures of tumor cell populations remain by and large unclear. In this work, we perform in-vitro experiments to characterize the growth properties of glioblastoma budding emerging from monolayers. We further propose a theoretical model and its finite element implementation to characterize such a topological transition, that is modelled as a self-organised, non-equilibrium phenomenon driven by the trade-off of mechanical forces and physical interactions exerted at cell-cell and cell-substrate adhesions. Notably, the unstable disorder states of uncontrolled cellular proliferation macroscopically emerge as complex spatio-temporal patterns that evolve statistically correlated by a universal law.

Understanding biological dynamics: following cells and molecules to track functions and mechanisms.

The dissection of the molecular circuitries at the base of cell life and the identification of their abnormal transformation during carcinogenesis rely on the characterization of biological phenotypes generated by targeted overexpression or deletion of gene products through genetic manipulation. Fluorescence microscopy provides a wide variety of tools to monitor cell life with minimal perturbations. The observation of living cells requires the selection of a correct balance between temporal, spatial and "statistical" resolution according to the process to be analyzed. In the following paper ad hoc developed optical tools for dynamical tracking from cellular to molecular resolution will be presented. Particular emphasis will be devoted to discuss how to exploit light-matter interaction to selectively target specific molecular species, understanding the relationships between their intracellular compartmentalization and function.

An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.

Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.

Phosphorylation of SOS1 on tyrosine 1196 promotes its RAC GEF activity and contributes to BCR-ABL leukemogenesis.

Son of Sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the small GTPases RAC and RAS. Although the molecular mechanisms of RAS GEF catalysis have been unveiled, how SOS1 acquires RAC GEF activity and what is the physio-pathological relevance of this activity is much less understood. Here we show that SOS1 is tyrosine phosphorylated on Y1196 by ABL. Phosphorylation of Y1196 controls SOS1 inter-molecular interaction, is required to promote the exchange of nucleotides on RAC in vitro and for platelet-derived growth factor (PDGF) activation of RAC- and RAC-dependent actin remodeling and cell migration. SOS1 is also phosphorylated on Y1196 by BCR-ABL in chronic myelogenous leukemic cells. Importantly, in these cells, SOS1 is required for BCR-ABL-mediated activation of RAC, cell proliferation and transformation in vitro and in a xenograft mouse model. Finally, genetic removal of Sos1 in the bone marrow-derived cells (BMDCs) from Sos1fl/fl mice and infected with BCR-ABL causes a significant delay in the onset of leukemogenesis once BMDCs are injected into recipient, lethally irradiated mice. Thus, SOS1 is required for full transformation and critically contribute to the leukemogenic potential of BCR-ABL.

Expression of a dominant-negative retinoic acid receptor construct reduces retinoic acid metabolism and retinoic acid-induced inhibition of NIH-3T3 cell growth.

We have previously reported an unexpected relationship between retinoic acid-induced inhibition of cell growth and the ability of various cell lines to metabolize the retinoid. Here, we report that stable expression of the truncated retinoic acid receptor RAR alpha403, transduced in NIH-3T3 cells by a retroviral vector, rendered the cells resistant to retinoic acid for growth inhibition and reduced their ability to metabolize the retinoid at the same time as it blunted the induction of the target gene transglutaminase II. The data suggest that retinoic acid receptors mediate the growth-inhibitory action of retinoic acid as well as its metabolism and the induction of transglutaminase II.

Loss of retinoic acid receptors in mouse skin and skin tumors is associated with activation of the ras(Ha) oncogene and high risk for premalignant progression.

Retinoic acid receptor transcripts (RARalpha and RARgamma) are decreased in benign mouse epidermal tumors relative to normal skin and are almost absent in carcinomas. In this report, the expression of RARalpha and RARgamma proteins was analyzed by immunoblotting in benign skin tumors induced by two different promotion protocols designed to yield tumors at low or high risk for malignant conversion. RARalpha was slightly reduced in papillomas promoted with 12-O-tetradecanoylphorbol-13-acetate (low risk) and markedly decreased or absent in papillomas promoted by mezerein (high risk). However, mezerein also caused substantial reduction of RARalpha in nontumorous skin. RARgamma was not detected in tumors from either protocol and was greatly reduced in skin treated by either promoter. Both RARalpha and RARgamma proteins were decreased in keratinocytes overexpressing an oncogenic v-ras(Ha) gene, and RARalpha was underexpressed in a benign keratinocyte cell line carrying a mutated c-ras(Ha) gene. Introduction of a recombinant RARalpha expression vector into benign keratinocyte tumor cells reduced the S-phase population and inhibited [3H]thymidine incorporation in response to retinoic acid. Furthermore, transactivation of B-RARE-tk-LUC by retinoic acid was markedly decreased in keratinocytes transduced with the v-ras(Ha) oncogene (v-ras(Ha)-keratinocytes). Blocking protein kinase C function in v-ras(Ha)-keratinocytes with bryostatin restored RARalpha protein to near normal levels, reflecting the involvement of protein kinase C in RARalpha regulation. Both RARalpha and RARgamma are down-regulated in cultured keratinocytes by 12-O-tetradecanoylphorbol-13-acetate, further implicating PKC in the regulation of retinoid receptors. Our data suggest that modulation of RARs could contribute to the neoplastic phenotype in mouse skin carcinogenesis and may be involved in the differential promoting activity of mezerein and 12-O-tetradecanoylphorbol-13-acetate, particularly for selecting tumors at high risk for malignant conversion.

The effect of sphingosine on the release of fibronectin from human lung fibroblasts.

Previous work from our laboratory (Scheidl et al. (1992) Biochim. Biophys. Acta 1135, 295-300) has shown that sphingosine (Sph), a known inhibitor of protein kinase C (PKC) inhibited the release of fibronectin (FN) into the media of fibroblast cells in culture and that this effect was independent of PKC. We now report that the action of Sph was time dependent (maximum inhibition in 2 h), concentration dependent (0.5-10 microM, at which concentration Sph was not toxic), and rapidly reversible after removal of Sph. Incubating with [35S]methionine, we found Sph reduced FN release either by inhibition of FN synthesis or FN secretion. To distinguish between these two possibilities, cells were treated with monensin, an inhibitor of FN secretion, which together with Sph showed no additive effect on FN release. Cycloheximide also inhibited FN release, but this inhibition was additive to that with monensin. We concluded that Sph inhibited secretion of FN, not synthesis. Furthermore, intracellular FN declined less in Sph-treated than in untreated control cells. By cell-surface iodination with Na125I and lactoperoxidase, followed by immunoprecipitation with antibodies to FN and alpha 5 beta 1-integrin, we showed that labeled multimeric and dimeric cell-surface FN declined, whereas integrins remained unchanged upon Sph treatment. This result suggested that though the number of cell-surface receptors for FN was not affected by Sph, their affinity may be reduced. In addition, we found that either pre- or co-treatment for 30 min with Sph caused a robust inhibition of cell adhesion to FN-coated plastic. We conclude that Sph rapidly inhibits FN secretion, lowers cell-surface FN, and greatly reduces cell adhesion to a FN substratum.

The effect of sphingosine and phorbol ester on the signal transduction enzymes and fibronectin release in cell culture.

In testing the hypothesis that the stimulation of the release of fibronectin (FN) by 12-O-tetradecanoylphorbol 13-acetate (TPA) from human lung fibroblasts in culture is the result of activation of protein kinase C (PKC), we found that the PKC inhibitor sphingosine strongly inhibited FN release in presence and even in absence of TPA. However, a different PKC inhibitor, calphostin C, despite almost complete inhibition of PKC, had no effect on FN release. We concluded that sphingosine is a potent inhibitor of FN release from the cell surface, independent of its inhibition of PKC; and that TPA stimulates release of FN by a pathway other than activation of PKC. We found that the activation of PKC by TPA was accompanied by inhibition of the cAMP-dependent protein kinase (PKA). When PKA was inhibited by an antagonist (H8, a cAMP analogue) at a concentration specific for PKA inhibition, the release of FN was stimulated similar to the stimulation with TPA. Activation of PKA with forskolin resulted in decreased FN release. In conclusion, we have shown that: (1) sphingosine had a robust effect inhibiting the release of FN from fibroblasts, independent of its action on PKC; (2) TPA treatment of these cells resulted in inhibition of PKA; (3) inhibition of PKA stimulated FN release whereas its activation decreased this release. It is possible that PKA, by phosphorylating a protein, may function, directly or indirectly, in keeping FN attached to the cell surface of fibroblasts.

Somatostatin inhibits PDGF-stimulated Ras activation in human neuroblastoma cells.

The main physiological role of somatostatin (SST) is the control of hormone secretion. Recently, SST has been shown to exert antiproliferative effects on some human tumors via both direct and indirect mechanisms. We have previously found that in the human neuroblastoma cell line SY5Y the SST analogue lanreotide (BIM 23014) inhibited serum-stimulated cell proliferation and MAP kinase activity. Here, we examine the effect of SST on PDGF-induced Ras activation. We found that SST suppressed PDGF-induced Ras activation in a pertussis toxin (PTx)-independent and peroxovanadate-dependent manner. Ras-specific GTPase activating protein (GAP) activities were not altered by SST treatment. On the contrary, PDGF-induced PDGF receptor phosphorylation was decreased by SST in a PTx-independent, peroxovanadate-dependent manner, likely accounting for the SST-mediated inhibition of PDGF-induced Ras activation.

Ha-ras oncogene transformation abolishes retinoic acid-induced reduction of intracellular fibronectin.

All-trans-retinoic acid (RA) is a master regulator of cell differentiation and in this process it greatly influences cell adhesion and the elaboration of the extracellular matrix. Therefore, we were interested in the effect of RA on the biosynthesis of fibronectin (FN). RA reduced the level of intracellular FN in a time- and concentration-dependent fashion in NIH-3T3 cells, but not in NIH-3T3 cells transformed by an activated Ha-ras oncogene. Since the steady-state level of FN transcripts did not change after treatment of the cells with RA for various times or concentrations, RA probably acts at the translational level. In NIH-3T3 cells, RA had distinct effects on different receptors, from decreasing retinoic acid receptor (RAR)alpha to increasing RAR beta expression to no effect on RAR gamma. Transformation of NIH-3T3 cells with an activated Ha-ras oncogene downmodulated RAR expression and also abolished responsiveness to RA. A variety of approaches permitted the following conclusions: 1) RA-dependent FN downmodulation is mediated by RARs, 2) retinoid X receptors (RXRs) mediate the observed reduction of RAR alpha by RA, and 3) the blockade of RA responsiveness by Ha-ras-transfected cells cannot be overcome by overexpression of RAR alpha. These studies have identified fibronectin and RAR alpha as RA targets in fibroblast cells and have shown that oncogenic transformation renders the cells resistant to RA action.

Osteolytic bone metastasis is hampered by impinging on the interplay among autophagy, anoikis and ossification.

Here we show that the fate of osteolytic bone metastasis depends on the balance among autophagy, anoikis resistance and ossification, and that the hepatocyte growth factor (HGF) signaling pathway seems to have an important role in orchestrating bone colonization. These findings are consistent with the pathophysiology of bone metastasis that is influenced by the cross-talk of supportive and neoplastic cells through molecular signaling networks. We adopted the strategy to target metastasis and stroma with the use of adenovirally expressed NK4 (AdNK4) and Dasatinib to block HGF/Met axis and Src activity. In human bone metastatic 1833 cells, HGF conferred anoikis resistance via Akt and Src activities and HIF-1α induction, leading to Bim isoforms degradation. When Src and Met activities were inhibited with Dasatinib, the Bim isoforms accumulated conferring anoikis sensitivity. The proviability effect of HGF, under low-nutrient stress condition, was related to a faster autophagy deactivation with respect to HGF plus Dasatinib. In the 1833 xenograft model, AdNK4 switched metastasis vasculature to blood lacunae, increasing HIF-1α in metastasis. The combination of AdNK4 plus Dasatinib gave the most relevant results for mice survival, and the following molecular and cellular changes were found to be responsible. In bone metastasis, we observed a hypoxic condition - marked by HIF-1α - and an autophagy failure - marked by p62 without Beclin-1. Then, osteolytic bone metastases were largely prevented, because of autophagy failure in metastasis and ossification in bone marrow, with osteocalcin deposition. The abnormal repair process was triggered by the dysfunctional autophagy/anoikis interplay. In conclusion, the concomitant blockade of HGF/Met axis and Src activity seemed to induce HIF-1α in metastasis, whereas the bone marrow hypoxic response was reduced. As a consequence, anoikis resistance might be hampered favoring, instead, autophagy failure and neoformation of woven bone trabeculae. Mice survival was, therefore, prolonged by overcoming an escape strategy adopted by metastatic cells by disruption of tumor-stroma coevolution, showing the importance of autophagy inhibition for the therapy of bone metastasis.

Strength recovery after proximal humeral fractures treated with locking plate.

PURPOSE: The aim of this study is to understand whether the isokinetic strength could be a valid objective data of functional recovery evaluating a group of patients with proximal humeral fractures treated with open reduction and internal fixation with locking plate comparing the clinical and functional recovery (isokinetic strength) with the not involved side. METHODS: Seventy patients underwent surgery with locking plate placement for proximal humeral fractures. The strength of each patient's shoulders, both involved and not involved, was evaluated using isokinetic tests and Constant-Murley score. Finally, the study included 48 patients and the mean follow-up of 33 months. RESULTS: The functional outcome showed no significant differences between operated and not operated shoulder. CONCLUSIONS: This study shows that the assessment of the force can be supported by use of tools such as the evaluation with isokinetic machines with the advantage of having, in this way, an objective data on the functional recovery. LEVEL OF EVIDENCE: III.

Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement.

Dynamic assembly of actin filaments generates the forces supporting cell motility. Several recent biochemical and genetic studies have revealed a plethora of different actin binding proteins whose coordinated activity regulates the turnover of actin filaments, thus controlling a variety of actin-based processes, including cell migration. Additionally, emerging evidence is highlighting a scenario whereby the same basic set of actin regulatory proteins is also the convergent node of different signaling pathways emanating from extracellular stimuli, like those from receptor tyrosine kinases. Here, we will focus on the molecular mechanisms of how the machinery of actin polymerization functions and is regulated, in a signaling-dependent mode, to generate site-directed actin assembly leading to cell motility.

Retinoic acid and beta-carotene inhibit fibronectin synthesis and release by fibroblasts; antagonism to phorbol ester.

Previous work from our and other laboratories has shown that tumor promoters stimulated the loss of fibronectin (FN) from the cell surface of fibroblasts in culture; retinoic acid (RA) appeared to counteract this loss. We have now studied the action of RA and carotenoids on FN synthesis and release. Using mouse fibroblasts (C3H/10T1/2 cells), we found that RA inhibited release of FN into the medium in a time- and concentration-dependent manner (e.g. 90% inhibition in 48 h with 1 x 10(-6) M RA). RA caused inhibition of synthesis, as well as a time- and concentration-dependent decrease in FN mRNA. A second phenomenon we observed was the greatly increased binding of FN to the surface of the cells, both in dimeric and multimeric forms, caused by RA treatment. RA produced a striking inhibition of the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-stimulated FN release from the cell surface usually associated with tumor promotion. We postulate that the combined action of RA in causing decreased FN synthesis and increased FN binding to the cell surface is the reason for the apparent antagonism of RA to the TPA-stimulated release of FN. Surprisingly, beta-carotene (BC) and canthaxanthin (a non-provitamin A carotenoid) also inhibited the release of FN from these cells. The action of BC was specific, in that an antioxidant carotenoid (trans-methyl-bixin) and lycopene were inactive. BC also inhibited FN synthesis and thus inhibited the TPA-stimulated release of FN, similar to RA, but to a lesser extent. BC had no effect on the binding of FN to the cell surface.

Retinoic acid downregulates growth, fibronectin and RAR alpha in 3T3 cells: Ha-ras blocks this response and RA metabolism.

Retinoic acid (RA) reduced growth, fibronectin, and retinoic acid receptor (RAR alpha) in NIH 3T3 cells but not in cells transformed by the Ha-ras oncogene. RA lowered RAR alpha transcript and protein, increased RAR beta transcripts, and had no effect on RAR gamma. H-ras transformation downregulated RAR expression and abolished responsiveness to RA. Ha-ras-transformed cells were as active as normal NIH-3T3 cells in RA uptake but were unable to degrade it to medium oxidation product, so that, paradoxically, the resistant cells accumulated 20-30-fold as much RA as the sensitive cells. RA sensitivity/insensitivity correlated with RA metabolism/lack thereof in 15 cell lines in serum-free medium. These data suggest a relationship between RA inhibition of cell growth and intracellular RA metabolism.

Retinol bound to cellular retinol-binding protein is a substrate for cytosolic retinoic acid synthesis.

Retinol bound to cellular retinol-binding protein (CRBP) was found to be oxidized to retinoic acid by a soluble activity from calf liver. Cytosolic retinoic acid synthesis from retinol-CRBP was strictly dependent on the exogenous supply of either NAD or NADP. NAD-supported reactions carried out in the presence or in the absence of dimethyl sulfoxide yielded apparent Km and Vmax values for the retinol-CRBP complex of 3.5 +/- 0.6 microM, 611 +/- 49 pmol h-1 (mg of protein)-1, and 0.84 +/- 0.12 microM, 601 +/- 38 pmol h-1 (mg of protein)-1, respectively. The corresponding values for the oxidation of free retinol, dissolved in dimethyl sulfoxide, were 7.1 +/- 0.3 microM and 948 +/- 47 pmol h-1 (mg of protein)-1. Since the dissociation constant of the bovine retinol-CRBP complex is less than 10(-8) M, whereas the Km for retinol-CRBP is of the same order as the Km for free retinol, synthesis of retinoic acid from retinol-CRBP does not rely on prior dissociation of retinol. ApoCRBP proved to be a specific inhibitor of retinoic acid synthesis from CRBP-bound retinol. Its inhibitory effect was indistinguishable from the dilution of the radioactive retinol-CRBP substrate that was obtained by the addition of unlabeled holoCRBP. In contrast, the oxidation of CRBP-bound retinol was not inhibited by the addition of other retinoid binding proteins nor by the addition of either free retinol or retinol complexed with proteins distinct from CRBP. These results indicate that the protein moiety of holoCRBP is specifically recognized by the cytosolic enzyme system that catalyzes retinoic acid synthesis from CRBP-bound retinol.