Effects of human and salmon calcitonin on human articular chondrocytes cultivated in clusters.

The effects of different pharmacological concentrations (0, 5, 10, 100, and 1000 ng/mL) of synthetic human calcitonin (hCT) and salmon calcitonin (sCT) on the incorporation of [3H]thymidine and production of proteoglycans (PG) and type II collagen (coll II) by human articular chondrocytes during a 20-day period were studied in a tridimensional chondrocyte culture model. [3H]Thymidine uptake was measured in chondrocyte clusters, and specific PG and coll II RIAs were performed every 4 days on the culture medium and cell aggregates; total PG and coll II production were also assessed at different culture durations by adding the amounts found in culture media and their corresponding clusters. Incubation with hCT or sCT did not affect [3H]thymidine uptake regardless of the dose. For each culture period, PG and coll II release into culture medium, cluster content, and total production increased significantly in a dose-dependent manner. Cumulative curves for these parameters showed a progressive significant increase with culture duration at hCT and sCT doses of 0, 5, and 10 ng/mL. Cumulative curves obtained with 10, 100, and 1000 ng/mL were seldom significantly different from one another. No differences emerged between the use of hCT or sCT. Thus, CT exerted no proliferative effect on human articular chondrocytes in tridimensional culture, but displayed a dose-dependent and prolonged stimulatory effect on PG and coll II production. CT may possess chondroprotective properties in addition to its other known effects.

Proteoglycans synthesized by human chondrocytes cultivated in clusters.

Proteoglycan metabolism was studied by a specific human cartilage proteoglycan radioimmunoassay in human chondrocytes cultivated in clusters. In this culture system, after a few days, previously dissociated chondrocytes were aggregated. They then synthesized a new cartilage matrix and were morphologically differentiated; they had a round shape and were situated inside small individual cavities (lacunae). The amounts of proteoglycan released into culture medium and present in chondrocyte clusters were maximal on the third to fifth day of culture; production decreased and stabilized from the 10th day to the end of culture. During the first days of culture, monomeric proteoglycans were present in large proportion; they gradually decreased between the sixth and 11th day of culture. These results suggest a modified synthesis of proteoglycan and hyaluronic acid during cultivation.

Effects of peptidic glycosaminoglycans complex on human chondrocytes cultivated in three dimensions.

Human chondrocytes from the pelvic joint were cultivated in suspension; under these conditions, after a few days, cells aggregated. These chondrocytes were morphologically differentiated (round shape, situated inside cavities and surrounded by a matrix synthesized during cultivation) and biosynthetically differentiated (synthesis of type II collagen and cartilage proteoglycans (PG) (Bassleer et al. In vitro 22, 115-120, 1986). In this work, we present the metabolic and cellular effects of a peptidic-glycosaminoglycan (P-GAG) complex isolated from calf cartilage and bone marrow. We analyzed the effects of P-GAG on DNA synthesis (appreciated by 3H-thymidine incorporation into DNA), on type II collagen and on PG synthesis analyzed by specific radioimmunoassays. According to its final concentration in culture medium, P-GAG was able to stimulate proliferation or to favor the production of specific components of cartilage matrix, type II collagen and PG.

Metalloproteinases and serine proteases activities in mixed spheroids of mouse b16 melanoma-cells and fibroblasts.

A murine B16 melanoma parental line (B16) and two clones either pigmented (B16P) or non-pigmented (B16NP) were cocultured as aggregates with mouse 3T3 fibroblasts. In vivo, these mixed aggregates developed more aggressive tumors in the mouse than corresponding pure aggregates. In vitro, the three pure cell lines secreted TIMP-2 and tissue-type plasminogen activator (t-PA) activities. Coculture conditioned media contained gelatinases (A and B), their inhibitors (TIMP-1 and TIMP-2), both plasminogen activators (u-PA and t-PA) and a plasminogen activator inhibitor (PAI) which formed high MW t-PA/PAI complexes. In all cases, cell-associated extracts contained essentially u-PA activities. These proteolytic activities are involved in extracellular matrix degradation and could contribute to the greater tumorigenic and invasive capacities of these B16 lines when previously cultured with fibroblasts in 3 dimensions.

Analysis of tridimensional mixed cultures of mouse B16 melanoma cells and 3T3 fibroblasts.

The interactions between invasive malignant cells and normal fibroblastic cells were studied in a cellular spheroid model in vitro. Murine B16 melanoma cells (previously cultured in monolayer for a short period) and 3T3 mouse fibroblasts (greater than or equal to 130 passages in monolayers) were cultured under tridimensional conditions (pure or mixed spheroids). As compared to pure 3T3 or mixed spheroids, B16 spheroids were smaller and characterized by a higher proliferation rate, a lower degree of necrosis, and a less abundant extracellular matrix. Disintegration was observed in some pure 3T3 or mixed spheroids. Melanogenesis progressively increased inside B16 or mixed spheroids. By immunohistochemical methods and electron microscopy, laminin, fibronectin and collagen I, III and IV in extracellular matrix were studied in the three types of spheroids.

Effects of FeSO4 on B16 melanoma cells differentiation and proliferation.

The effects exerted by FeSO4, in the presence or absence of vitamin C, on melanogenesis and proliferation in mouse B16 melanoma cells in culture were analysed either in serum-free (MEM-N2) or in serum-supplemented media. These cellular parameters can be either stimulated or on the contrary inhibited, depending on the metal concentration, the presence or the absence of vitamin C and serum, and on the type of culture (subconfluent or clonal). Vitamin C toxicity for B16 cells was decreased in the presence of FeSO4.

Control of B16 melanoma cells differentiation and proliferation by CuSO4 and vitamin C.

The effects exerted by CuSO4, in the presence or absence of vitamin C, on melanogenesis and proliferation in mouse B16 melanoma cells in culture were analysed either in serum-free (MEM-N2) or in serum-supplemented media. The stimulation or the inhibition of these cellular parameters can be induced, depending on the metal concentration, the presence or absence of vitamin C, the composition of the culture medium and on the type of culture (subconfluent or clonal). Vitamin C toxicity for B16 cells was generally increased in serum-free medium, in clonal cultures, or in the presence of CuSO4.

Cytotoxic and mitogenic activities in culture media conditioned by mouse B16 melanoma cells and 3T3 fibroblasts.

Cytotoxic and mitogenic soluble factors are released into media conditioned by pure or mixed populations of mouse 3T3 fibroblasts and B16 melanoma cells cultivated in vitro. These activities are demonstrated by the use of MTT cell survival test and 3HTDR incorporation. Mitogenic (M.W. greater than 10,000) and cytotoxic factors (M.W. less than 1,000) are present and are generally more active on B16 cells than on fibroblasts. Their release into conditioned media is related to the rate of pigmentation in B16 cells and to the mode of cultivation (monolayers or cell aggregates).

Cytological effects of culture media conditioned by B16 melanoma cells and 3T3 fibroblasts.

Culture media conditioned (CM) by mixed populations of mouse B16 melanoma cells and 3T3 fibroblasts cultivated as monolayers exert cytological effects on B16 or 3T3 cells when treated separately in culture. By ultrafiltration of these CM, we show that a stimulatory activity on B16 melanoma cells proliferation is present in fractions with M.W. greater than 10,000 daltons. A strong cytotoxic activity for B16 melanoma cells and, to a lower degree, for 3T3 fibroblasts is detected in fractions with M.W. less than 1,000 daltons. The ultrastructural analysis of cells (B16 or 3T3) treated with cytotoxic fractions reveals in them mitochondrial swelling, blebs, broken membranes and dead cells.

In vitro cytotoxic activity of two potential anticancer drugs isolated from Strychnos: strychnopentamine and usambarensine.

The cytotoxicity and the selective antiprotozoal activity of some Strychnos alkaloids, namely strychnopentamine (SP) and usambarensine (US) (7) led us to analyze and compare their effects with emetine (EM) by using mouse B16 melanoma cells cultivated in vitro. We observed by cytological analysis and proliferation rate studies that these substances induce analogous cytotoxic effects in B16 cells, but at different concentrations i.e. formation of lamellar bodies in the cytoplasm, the which contain pre-melanosomes in the case of SP and US, vacuoles and blebs. At concentrations near their respective IC50, SP and US, but not EM, decreased colony formation. We showed by incorporation of labelled precursors that SP and US first inhibit RNA synthesis while EM initially acts on protein synthesis. These alkaloids increased melanin synthesis. Furthermore, only EM and SP caused hemolysis of sheep red blood corpuscles. This could explain why the rate of antiplasmodial activity is higher for SP and EM.

Evaluation of matrix metalloproteinases and serine proteases activities in three B16 melanoma cell lines with distinct tumorigenic potential.

Mouse B16 melanoma cells (B16, parental line) and two derived clones either pigmented (B16P) or non pigmented (B16NP) were cultured as monolayers (2D) or on agar, as aggregates (3D). The productions of gelatinases A and B (72 kDa and 92 kDa type IV collagenases) and their inhibitors (TIMP1 and TIMP2), plasminogen activators (PAs) and plasminogen activator inhibitors (PAI) were investigated. The B16 cell lines did not secrete any gelatinase, but they secreted TIMP2, tissue-type (t-PA), urokinase-type (u-PA) plasminogen activators and PAI-1 like activities. High levels of PAI activity were determined in conditioned media and cellular extracts of B16NP, which could account for the lower tumorigenic potential of these cells. In 3D cultures, the cellular extracts of the three cell lines contained essentially u-PA activity. This activity could contribute to the greater tumorigenic and invasive capacities of B16, B16P and B16NP when cultured in 3D.

Laminin and 67 kD laminin binding protein in mouse B16 melanoma cells and 3T3 fibroblast spheroids.

Multicellular spheroids which promote cell-cell and cell-matrix interactions were prepared in culture with mouse B16 melanoma cells (pigmented or non pigmented) alone or mixed with mouse 3T3 fibroblasts. Their volume and proliferation or necrosis rate were evaluated. As measured by dot blot immunoassay, laminin was mainly produced by fibroblasts rather than by melanoma cells. High levels of laminin B1 chain mRNA were detected only in spheroids composed of 3T3 fibroblasts. The levels of 67 kD laminin binding protein mRNA were high in all cell populations studied here.

Characterization of non pigmented B16 melanoma cell-derived cytotoxic factors.

We analyzed and tried to characterize substance(s) responsible for cytotoxic activities detected in culture media conditioned by non pigmented B16 melanoma cells (NPB16). The different cytological tests used showed that ultrafiltrated conditioned media (CM U1 fraction) contained several cytotoxic factors with a Mw lower than 1000 Da. These factors seemed to act either directly or indirectly on cell membranes, mitochondria, on the cell cycle and on protein and DNA synthesis. A cytotoxic activity could be found even after high dilution of CM U1. These cytotoxic factors were rapidly released by B16 cells in culture, independently of cell confluence. Their activities in the treated cells were also very fast and the cytotoxic effects were irreversible after only a few hours of treatment. These factors were not intermediate products during melanogenesis, neither polyamines, nor proteases. At least one of them seemed to be a small acidic and basic stable peptide without disulfide bounds but not heat stable. The synthesis of at least one of these cytotoxic factors was inhibited by cycloheximide and the cytotoxic activity was partially destroyed by pronase and trypsin, but not by pepsin. The cytotoxicity was not modified by copper complexants or free radical inhibitors (bovine serum albumin (BSA), tyrosine, superoxyde dismutase (SOD), catalase, vitamin E). Furthermore the levels of glutathione peroxydase activity and reduced glutathione did not change after treatment by CM U1 as compared to controls.

Effects of strychnopentamine on cells cultured in vitro.

This paper describes the powerful cytotoxic action exerted by strychnopentamine (SP), a dimeric indole alkaloid extracted from Strychnos usambarensis Gilg, on B16 melanoma cells and on non-cancer human fibroblasts cultured in vitro. SP strongly inhibits cell proliferation and induces cell death at a relatively low concentration (less than 1 microgram/ml) after 72 h of treatment in the two lines. Incorporation of [3H]thymidine and [3H]leucine by B16 cells significantly decreases after only 1 h of treatment at 0.5 microgram/ml. SP induces the formation of dense lamellar bodies and vacuolization in the cytoplasm, intense blebbing at the cell surface and various cytological alterations leading to cell death.

Fibronectin promotes lung colony formation in the mouse by B16 melanoma cells spheroids.

By microscopical observation and using an original method of automatic image analysis, we studied on histological sections the rate of lung colony formation after intravenous injection into the mouse of B16 melanoma cells previously cultivated in vitro as pure or mixed spheroids (B16 + 3T3 fibroblasts). The preincubation in vitro of pure spheroids with fibronectin significantly increased the percentages of lung section area occupied by tumors and the relative number of internal lung colonies. This effect of fibronectin was even more obvious when mixed spheroids were injected.

Characterization and tumorigenicity of spheroids composed of pigmented or non pigmented B16 melanoma cells.

A parental line of mouse B16 melanoma cells (B16) and two derived cloned lines, either pigmented (B16P) or non pigmented (B16NP), were cultured in vitro as spheroids. After 48 hrs, the pigmented cells (B16, B16P) formed smaller and looser aggregates, with higher rates of cell proliferation and lower amounts of extracellular matrix as compared to B16NP spheroids. The three lines were more tumorigenic when inoculated subcutaneously as spheroids than as isolated cells. Furthermore, B16P or B16 spheroids developed richly vascularized subcutaneous tumors and metastases more rapidly than B16NP aggregates. After intravenous injection of spheroids, the measurement with an image analyzer of the area of sections in lung colonies indicated that B16P colonies were larger and more numerous than those induced by B16NP cells.

Influence of laminin or fibroblasts upon colony formation in the mouse by B16 melanoma cell spheroids: a morphometric analysis.

By microscopical observation and using an original morphometric method, we analyzed on histological sections the rate of lung colony formation after the intravenous injection into the mouse of B16 melanoma cells previously cultivated in vitro as aggregates. After the injection of B16 pure spheroids, superficial lung colonies were more numerous than internal lung colonies. After the injection of mixed spheroids (B16 + 3T3 fibroblasts), the size of colony sections was increased. Addition of laminin to pure or mixed spheroids decreased the size of colony sections but increased the number of internal lung colonies.

Antimitotic activity of strychnopentamine, a bisindolic alkaloid.

Strychnopentamine has been tested for its cytotoxic and antitumor activities and compared with two other bisindolic alkaloids that possess an usambarane skeleton. The presence of a N-methylpyrrolidine group increases the antimitotic activity of this type of alkaloids.

Screening of cytotoxic activities of Strychnos alkaloids (methods and results).

The potential cytotoxic activities of 46 alkaloids isolated from different Strychnos species were tested on different cancer or normal cells cultured in vitro. The authors used a relatively simple microtest which gives good reproducibility. Most of the active compounds belong to the usambarane skeleton but other structure-activity relationships are being discussed.

Effects of trace metals on mouse B16 melanoma cells in culture.

The effects of fourteen metal ions (As3+, As5+, Cd2+, Co2+, Cr3+, Cr6+, Hg2+, Li+, Mg2+, Mn2+, Ni2+, Se4+, V5+, VO2+) on the proliferation and differentiation in mouse B16 melanoma cells cultivated in vitro were analyzed. Cell number assays, melanin, and protein measurements, a 3(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reduction test (MTT survival test), and a clonal growth assay were performed. At 10(-4)M, metal ions such as As3+, As5+, Cd2+, Cr6+, Se4+, V5+, VO2+, and, to a minor extent, Li+, Hg2+, and Co2+ significantly reduced the number of the B16 melanoma cells. For the same molar concentration, the order of the levels of cell toxicity of the metal compounds to B16 cells as measured by the MTT test was as follows: Hg2+ > Cr6+ = Cd2+ > As3+, As5+, > V5+, VO2+ > Se4+ = Ni2+ = Co2+ = Li+. An increased synthesis of melanin in B16 cells was noted after incubation with Co2+, Ni2+, Cd2+, and Li+, whereas Se4+ had, on the contrary, an inhibiting effect on melanogenesis.