AUTOCOUNTER, an ImageJ JavaScript to analyze LC3B-GFP expression dynamics in autophagy-induced astrocytoma cells.

An ImageJ JavaScript, AUTOCOUNTER, was specifically developed to monitor and measure LC3B-GFP expression in living human astrocytoma cells, namely T98G and U373-MG. Discrete intracellular GFP fluorescent spots derived from transduction of a Baculovirus replication-defective vector (BacMam LC3B-GFP), followed by microscope examinations at different times. After viral transgene expression, autophagy was induced by Rapamycin administration and assayed in ph-p70S6K/p70S6K and LC3B immunoblotting expression as well as by electron microscopy examinations. A mutated transgene, defective in LC3B lipidation, was employed as a negative control to further exclude fluorescent dots derived from protein intracellular aggregation. The ImageJ JavaScript was then employed to evaluate and score the dynamics changes of the number and area of LC3B-GFP puncta per cell in time course assays and in complex microscope examinations. In conclusion, AUTOCOUNTER enabled to quantify LC3B-GFP expression and to monitor dynamics changes in number and shapes of autophagosomal-like vesicles: it might therefore represent a suitable algorithmic tool for in vitro autophagy modulation studies.

Post-translational regulation of interleukin 1 beta secretion.

In view of the key role played by interleukin 1 (IL-1) beta in inflammation, its production is likely to be precisely regulated. Previous studies have shown that IL-1 beta biosynthesis is controlled at the transcriptional and translational levels. We have investigated whether post-translational events also play a role in regulating the production of bioactive IL-1 beta. IL-1 beta, which lacks a signal sequence, is released by activated monocytes through a novel pathway of secretion, alternative to the classical endoplasmic reticulum-Golgi route. Secretion of mature 17 kDA IL-1 beta is increased when pulse-labelled activated monocytes are chased in the presence of heat-aggregated immunoglobulins or of various drugs. Febrile temperatures inhibit secretion of mature IL-1 beta, but only reduce its synthesis: treatment with cycloheximide restores secretion. Processing of the 33 kDa precursor to the 17 kDa mature molecule is inhibited when the external pH is 8 or higher: under these conditions, release of unprocessed, biologically inactive 33 kDa IL-1 beta is observed. Thus, secretion of IL-1 beta is regulated by post-translational mechanisms which operate at the level of both proteolytic processing and extracellular export.

Secretion of thioredoxin by normal and neoplastic cells through a leaderless secretory pathway.

Thioredoxin, despite its function as an intracellular disulfide reducing enzyme and its lack of a signal sequence, has been found to play some roles extracellularly. Here we show that thioredoxin is actively secreted by a variety of normal and transformed cells, including fibroblasts, airway epithelial cells, and activated B and T lymphocytes. Neither brefeldin A nor dinitrophenol, two drugs that block transport through the exocytic pathway, inhibit secretion of thioredoxin, indicating that the latter does not follow the classical ER-Golgi route. The secretory mechanism for thioredoxin shares several features with the alternative pathway described for interleukin-1 beta, such as the potentiating effect on secretion of several unrelated drugs and the sensitivity to methylamine. However, unlike interleukin-1 beta, thioredoxin is not detected in membrane-bound compartments of secreting cells. In addition, when COS7 are transfected with plasmids encoding pro-interleukin-1 beta or thioredoxin, only the latter is detectable extracellularly.

Invasive activity, spreading on and chemotactic response to laminin are properties of high but not low metastatic mouse osteosarcoma cells.

We have examined the interactions of low (Os43 and OS48) and high (Os50/K8 and Os50/K12) metastatic cell lines derived from osteosarcomas (Os) of the Balb/c mouse with fibronectin (FN) and laminin (LN). All of these cell lines formed osteogenic tumors when transplanted subcutaneously into syngeneic mice. Os43 and Os48 cells gave rise to few metastases while the Os50/K8 and Os50/K12 cells were highly metastatic. In an in vitro chemoinvasion assay only the highly metastatic cells were able to invade a reconstituted basement membrane. Although the interactions of all cell lines with FN were quite similar, their response to LN differed considerably. Within each of the cell lines, chemotactic response to and cell spreading on LN were closely correlated. Highly metastatic Os cells migrated to and spread on LN substrates to a much greater extent than low metastatic cells. Os43 and particularly Os48 showed very much low migration to LN, similar to that of Balb/c 3T3 fibroblasts. They also spread poorly on LN, resembling the behavior of normal human bone cells which were used as a control. Thus, with these assays it is possible to distinguish the LN interactions associated with the metastatic phenotype of Os cells. The acquisition of LN recognition in tumor cells of bone origin may be related to their ability to invade and metastasize. This system may be valuable for the study of LN recognition molecules, their appearance, or changes with the metastatic phenotype.

Effect of vitamin A on chemotactic and chemoinvasive behaviour of an osteosarcoma cell line.

Vitamin A is known to be able to modulate cell growth and differentiation and to act as an inhibitor of the process of carcinogenesis in some experimental models. Here we have studied the effect of different concentrations of vitamin A on chemotactic and chemoinvasive behaviour of a metastatic osteosarcoma cell line. The cell proliferation was partially inhibited in the presence of 10(-5) M retinol after 4 days of incubation. Retinol effect on chemotactic and chemoinvasive activity of osteosarcoma cells seemed to be dose-dependent. The highest retinol concentration used (10(-5) M) had an inhibitory effect on migratory and invasive cell response. Lower retinol concentrations seemed to be able to enhance (10(-8) M) both chemotactic and chemoinvasive activity of osteosarcoma cells. Chemotaxis and chemoinvasion assays provide rapid and quantitative tools to study the "in vitro" behaviour of metastatic cells. Furthermore, they represent a mean to screen for drugs, hormones and other substances able to alter the metastatic phenotype.

Invasiveness and chemotactic activity of oncogene transformed NIH/3T3 cells.

The role of oncogenes in the acquisition of invasive and metastatic capabilities is controversial. Interactions with basement membranes are critical in the process of tumor invasion and metastasis. We compared the ability of 3T3 cells transformed by oncogenes involved in various stages of signal transduction to invade a reconstituted basement membrane in vitro and to grow in a three dimensional basement membrane gel (matrigel). Cell lines transformed by various oncogenes and oncoviruses: v-sis (a growth factor), v-erb-B (a truncated EGF receptor), Moloney sarcoma virus (v-mos: a protein kinase homologue), mutated c-ras oncogenes (G protein homologues), FBJ virus (v-fos: a nuclear protein) were investigated. All transformed cell lines were able to invade in the chemoinvasion assay, where a layer of matrigel is coated onto chemotaxis filters. FBJ/3T3 were the least invasive and SSV/3T3 the most invasive. Control 3T3 cells could not cross the matrigel barrier. All transformed cells grew on matrigel forming invasive, branching colonies, whereas control 3T3 were unable to grow in matrigel. Cells transfected with the v-erb-B gene grew as multilayers inside matrigel. Invasiveness and growth on matrigel were accompanied by a high chemotactic response to laminin (LN) in all transformed lines. These results suggest that invasion and growth on matrigel, together with migration to LN, are induced by a large spectrum of oncogenes. When 3T3 cells were transfected with v-sis oncogene under the transcriptional control of the metallothionein (MMT) promoter and exposed to Zn++, their in vitro invasiveness was specifically increased by around 3 fold. These findings provide further evidence supporting a direct role of the v-sis oncogene in the invasive phenotype.

High chemotactic motility and growth in hard agar of a variant of RSV-transformed fibroblasts are lost in late passages.

Cloning efficiency in hard agar (0.6%) and high chemotactic migration toward fibroblast conditioned medium have been shown to characterize metastatic tumor cells. We studied growth in 0.6% agar and chemotaxis of two lines of Rous Sarcoma virus-transformed Balb/c3T3 cells, B77/3T3 (low metastatic) and AA12 (high metastatic), and compared them to their non-transformed counterpart, in order to verify whether these properties were maintained during several subcultivations. Cells were cryopreserved at early passages and thawed for experiments. Both assays were performed on freshly thawed cells (4-6 weeks in culture) and on cells which had been cultured 15-20 weeks after thawing. B77/3T3, which are tumorigenic but low metastatic and which have a very low cloning efficiency in hard agar (0.1-1%), showed a chemotactic response to Balb/c3T3 conditioned medium about two-fold higher than control Balb/c3T3. This response did not change with time in culture. AA12 cells, a genetic unstable variant of B77/3T3 selected for its growth in hard agar (0.6%), had a high cloning efficiency in hard agar and showed a high chemotactic motility (three-fold the controls). High growth in 0.6% agar and high chemotaxis of AA12 were lost in late passages, where cells behaved as the controls. It seems that besides the already reported variation in anchorage-independent growth, genetically unstable tumor cells can also have important variations in chemotactic motility during subcultivations.

Decline of fibroblast chemotaxis with age of donor and cell passage number.

Human dermal fibroblasts have a limited life span in culture, which is manifested by a progressive decline of their proliferative activity. Here we show by the Boyden Chamber assay that the chemotactic response of human fibroblasts to fibroblast-conditioned medium and fibronectin declines during cellular aging in vitro and in vivo. The chemotactic response of human embryonic fibroblasts (HEF) declined progressively after the 25th passage. Virtually no chemotactic activity could be observed after the 40th passage in culture. Fibroblasts cultures from donors aged between 70-90 years had lost chemotactic activity by the 15th passage. Cells from patients suffering from progeroid syndromes of premature aging showed, even in early passages, a very low chemotactic response (20% of the HEF) and lost their chemotactic activity after a few subcultures. The response to the chemoattractant fibronectin also decreased with aging. Immunofluorescence studies indicated that the decline in chemotactic activity was accompanied by the formation of a thicker fibronectin network in the extracellular matrix of senescent human fibroblasts and progeroid cells than that observed in early passage embryonic cultures. Since fibroblast chemotaxis and synthesis of connective tissue components probably play an important role in tissue repair, our results could contribute to an understanding of age-related differences in the healing of skin wounds.

Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor.

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.

Interferons inhibit chemotaxis of transformed cells and their invasion of a reconstituted basement membrane.

Tumor cell migration and invasion are critical steps in the complex process of metastasis formation. It has been demonstrated that interferons (IFNs) inhibit the motility of human fibroblasts. In the present investigation we tested the effect of human leukocyte IFN and murine fibroblast IFN on the chemotactic migration of transformed and tumor-derived cells towards fibroblast conditioned medium. We were able to show that IFNs preferentially inhibit the chemotaxis of transformed and tumor-derived cell lines when compared to control fibroblasts. Inhibition was dose-dependent and most tumor cell strains were sensitive to concentrations of IFNs 10- to 100-fold lower than fibroblast cultures. Furthermore, leukocyte interferon was able to inhibit the invasion of transformed 3T3 fibroblasts through a gel of reconstituted basement membrane (Matrigel). These effects could be related to the antineoplastic activity of interferon.

SV40 transformed fibroblasts recognize the same 140 kD fibronectin chemotactic fragment as non-transformed cells.

SV40-virus-transformed human embryonal fibroblasts show an enhanced chemotactic response to the glycoprotein fibronectin. However, they recognize the same chemotactic active region as non-transformed fibroblasts. The result suggests that an enhancement of chemotaxis by fibroblasts which have been transformed with Simian Virus 40 is due not to the utilization of further chemotactic domains in the molecule, but to an increased sensitivity of the cells to the chemoattractant.

Comparison of the chemotactic response to conditioned medium of BALB/c3T3 fibroblasts and their SV 40 transformants.

The chemotactic migration of transformed cells out of their tissue of origin might represent an essential component of tumor invasion and metastasis. In the Boyden chamber assay SV 40 virus-transformed BALB-c3T3 fibroblast (SV/3T3) showed an increased chemotactic response to fibroblasts-conditioned medium in comparison to non-transformed BALB/c3T3 cells. This was verified for a large range of incubation times and cell densities. Fibroblast-conditioned medium contains components of the extracellular matrix of connective tissue. Since organs rich in connective tissue are often the site of metastasis, the enhanced chemotaxis of virus-transformed cells to conditioned medium could contribute to explain the malignancy of these cells.

Chemotactic response of SV40 virus transformed fibroblasts to conditioned medium: dependence on time and cell number.

The ability of tumor cells to move chemotactically in response to specific stimuli may be one of the many factors which distinguish malignant from non-malignant cells. In this work we demonstrated that a SV 40 virus transformed human fibroblast cell line (SV40/WI26) has an enhanced chemotactic response to fibroblast-conditioned medium (FCM) as compared to control human fibroblasts, and this was true for different migration times and different initial cell numbers. A similar behaviour has been described for SV40 transformed mouse fibroblasts (SV3T3). Furthermore, another SV40 transformed human fibroblast cell line (SV40/WI38) has been reported to have an enhanced chemotactic response to FCM compared to the normal counterpart. We conclude that the increased chemotactic response to FCM could be well correlated with the phenotype induced by fibroblast transformation with SV 40.