C Dragonetti

Effect of temperature on invasion of MO4 mouse fibrosarcoma cells in organ culture

Invasion by MO4 mouse fibrosarcoma cells into fragments of embryonic chick heart or lung in organ culture was studied histologically and ultrastructurally at various temperatures between 12 and 40°C. Invasion was absent for at least 7 days at or below temperatures of 29°C. Invasion was invariably observed at or above 30·5°C. Differences in invasion between 29 and 30·5°C could not be ascribed to differences in growth, migration, or microtubule assembly/disassembly of MO4 cells. Neither could they be explained through differences in the attachment of MO4 cells to the heart fragments. Possible explanations for the absence of invasion at lower temperature are: altered resistance of the extracellular matrix in heart or lung fragments, and deficient expression of fucosylated glycoproteins at the surface of MO4 cells. A population of MO4 cells plated from the parent line and adapted to grow at 28°C (MO4 28 cell line) did not differ in invasiveness from the parent MO4 cells.We conclude that the temperature dependence of invasion in organ culture might indicate as yet unexplored aspects of the mechanisms of tumour invasion.

Effect of inhibitors of glycosylation and carbohydrate processing on invasion of malignant mouse MO4 cells in organ culture.

Inhibitors of glycosylation and carbohydrate processing were used to investigate the role of carbohydrates exposed at the cell surface in invasion. Malignant mouse MO4 cells were confronted with embryonic chick heart in organ culture, an assay shown to be relevant for a number of aspects of invasionin vivo. Tunicamycin (1·0μg/ml), 2-deoxy-d-glucose (100mm),β-OH-norvaline (1·0mm), and Monensin (0·1μg/ml) reversibly inhibited the invasion of MO4 cells. At these concentrations the drugs also inhibited the growth of MO4 cells. 1-Deoxynojirimycin (10mm), swainsonine (0·4μg/ml), and Marcellomycin (0·1 μg/ml) permitted invasion. Marcellomycin also reversibly inhibited the growth of MO4 cells. These results show that drugs known to interfere with the glycosylation or processing of carbohydrate chains of glycoproteins in different ways have different effects on the invasion of MO4 cellsin vitro.

In vitro invasiveness of human bladder cancer from cell lines and biopsy specimens

Aggregates prepared from cell lines established from a human transitional cell carcinoma of the urothelium (Hu 456) or from apparently normal urothelium before (Hu 609) and after phenotypic transformation (Hu 609T) were confronted with fragments of embryonic chick cardiac muscle in organ culture. In this assay a correlation was found between in vitro invasiveness of animal cell lines and their capacity to produce invasive tumours in syngeneic animals [1, 10, 11]. The invasiveness of cells from established human urothelial lines was compared to the invasiveness of cells from fresh biopsy specimens of a normal urothelium, a non-invasive papilloma, and a metastasizing transitional cell carcinoma. Cells from all established lines (Hu 609, Hu 609T and HU 456) and from the biopsy specimens of the transitional cell carcinoma occupied and eventually replaced the cardiac muscle by contrast with cells from the normal urothelium or from the non-invasive papilloma. We concluded that the organ culture assay for invasiveness might be used to define malignancy of human bladder cell lines and to follow the various steps during the acquisition of invasiveness in vitro.

Invasiveness of malignant ST/A mouse lung cells in vitro

SummaryST/A mouse lung cells underwent apparently spontaneous malignant alteration in tissue culture. We have compared the capacity of these cells to form malignant tumours in syngeneic animals with their behaviour in vitro. ST-L1, ST-L22, and ST-L104 cells were malignant, whilst ST-L108 and ST-L109 cells were not. ST-L1 and ST-L22 cells showed anchoragedependence of growth, whilst ST-L104, ST-L108 and ST-L109 cells did not. ST-L22, ST-L104, ST-L108 and ST-L109 performed directional migration from a spheroid explanted on glass. This capacity was lost in ST-L1 cells, which produced so-called round-cell transformants. All but ST-L108 cells produced type C viral particles. The tumorigenic cell lines ST-L1, ST-L22 and ST-L104 invaded fragments of embryonic chick heart in three-dimensional culture, whereas the non-tumorigenic ST-L108 and ST-L109 cells did not. Furthermore, the histology of ST-L cells invading in three-dimensional culture resembled that of the invasive sarcomas which they produced in vivo. The present observations with ST-L cells confirm that invasiveness in three-dimensional culture is a reliable criterion for malignancy of tissue culture lines.

Tumorigenicity, invasiveness and metastatic capability of FR3T3 rat cells before and after transfection with bovine papilloma virus type 1 DNA.

Fischer rat FR3T3 cells were tested for tumorigenicity, invasive and metastatic capabilities before and after transfection, either with the entire bovine papilloma virus type 1 (BPV-1) genome or with a plasmid (pV69) containing a 69 per cent Bam H1-Hind III fragment of the BPV-1 genome as well as bacterial sequences. Cell lines were grouped as parental, pV69-transfectants, BPV-1 transfectants,in vitro derivatives, andin vivo derivatives. The tumorigenic, invasive and metastatic capabilities of these cell lines were examinedin vivo through s.c., and i.p. injections of cell suspensions and through s.c. implantations of cellular aggregates into syngeneic rats. Invasiveness was testedin vitro through confrontations with embryonic chick heart fragments in organ culture. All cell lines including parental lines, were found to be invasivein vitro and tumorigenicin vivo; all tumors were invasive. It is, therefore, not possible to draw conclusions about the role of BPV-1 gene sequences in the acquisition of the invasive phenotype. Transfection with BPV-1 genes conveyed the metastatic phenotype upon parental FR3T3 cells, which were themselves found to be non-metastatic. With regards to this, no differences were found between BPV-1 transfectants compared with pV69 transfectants. Untransfected cells became metastatic also through passagein vivo as an s.c. tumor. The expression of the metastatic phenotype was not noticeably correlated with alterations of growth characteristics of the cell lines. We concluded that the implication of BPV-1 gene sequences in conveying the metastatic phenotype upon FR3T3, if any, was indirect, presumably through alterations of the host cell genome. Our experiments illustrate the need for long-term observations with parental cell lines before drawing conclusions about the role of oncogenes in the acquisition of the malignant phenotype.

Heparan sulfate at the surface of HeLa cells.

SummaryHeLa cells, labeled with Na235SO4, release into the culture medium35SO4 bound to plasma membrane vesicles next to35SO4-glycoproteins and free35SO4. Plasma membrane vesicles, experimentally produced by treatment with formaldehyde, contain35SO4 and their surface can be stained with high iron diamine. Scanning of chromatograms of the trypsinate from labeled cells demonstrates radioactivity on the spot of heparan sulfate. It is concluded that HeLa cells synthesize heparan sulfate, which is incorporated at the plasma membrane and released by shedding of small vesicles.

Methods for Morphological and Biochemical Analysis of Invasion in vitro

ABSTRACT Invasiveness of malignant cells is demonstrated in vitro using three-dimensional shaker cultures of tissue fragments with aggregates of malignant cells. Both tissues are allowed to adhere to each other on a non-adhesive substrate. This method is used to examine the invasiveness of various cell lines with the aim of defining their malignancy. Cellular activities, which are presumed to be involved in invasion are studied : 1) Adhesion of malignant cells to the host tissue; 2) Destruction of the host tissue by invading cells; 3) Phagocytosis of material from the host by the malignant cells.

Colloidal iron binding to the surface of HeLa cells, spreading in monolayer culture

SummaryWe have used the colloidal iron (CI) binding technique, adapted for transmission electron microscopy, for semiquantitative evaluation of the negative charge density at the surface of HeLa cells in monolayer culture. The surface area increases when HeLa cells spread on the substrate. This increase brings about a decrease in the thickness of the CI rim, indicating a decrease in negative surface charge density. This phenomenon implicates lowering of the electrostatic repulsion, and explains the formation of intercellular contacts at the level of spread parts of the cell. Because of lack of penetration, CI particles are absent in regions of close apposition between cells and between cells and substrates. Absence of CI binding in broader intercellular or cell-substrate spaces was explained through masking of the anionic groups.

Histochemical evidence for sulfomucins at the surface of HeLa cells.

SummaryThe nature of the negatively charged groups present at the surface of HeLa cells was further investigated. Therefore we applied a series of light microscopic staining techniques, widely used for the demonstration of epithelial mucosubstances on tissue sections, to HeLa cells from suspension cultures. Our histochemical findings confirmed the presence of carboxylated substances at the surface of these cells. Furthermore we obtained conclusive evidence for the presence of sulfated molecules. Both substances seem to be closely related to epithelial sialomucins and sulfomucins.