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.

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.

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.

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.

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.

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 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.

Effects of cis-diamminedichloroplatinum (II) loaded liposomes on mouse Ehrlich tumor cells.

Cis-diamminedichloroplatinum II (cisplatin) heavily or lightly loaded (fluid, solid, negatively charged or neutral) liposomes were prepared. Cisplatin release from liposomes was observed only after long dialysis times or after liver lysosomal enzymatic disintegration in solution. Mouse Ehrlich tumor cells (ELT) cultured in vitro were treated with cisplatin, liposomes or cisplatin loaded liposomes, and the effects on the mitotic activity, the DNA content and the ultrastructure were compared. Cisplatin (1-10 micrograms/ml) had an antimitotic activity and modified the DNA content in ELT cells. Ribosome aggregation, perichromatin or interchromatin granule accumulation, and chromatin condensation or some degree of dispersion could be observed. Negatively charged fluid liposomes had an antimitotic activity and modified the DNA content in ELT cells at lower concentrations (0.3 mumoles/ml) than in the case of neutral fluid liposomes (1.5 mumoles/ml). Negatively charged solid liposomes were not toxic at these concentrations. Ultrastructural analysis of ELT cells treated in vitro with negatively charged fluid liposomes revealed their extracellular adsorption and their disintegration in phagolysosomes. A fusion between liposomes and the plasma membrane was not definitely demonstrated. Cisplatin loaded liposomes also had an antimitotic activity and modified the DNA content in ELT cells. These effects were similar to or more pronounced than those induced by free cisplatin. Ultrastructural analysis revealed some kind of electron dense material in phagolysosomes which was never observed after the treatment with free cisplatin or liposomes alone. Effects on nucleic acids were rarely observed.

Quantitative cytochemical analysis by microdensitometry of spontaneous or alpha-MSH-stimulated melanogenesis in B16 melanoma cells cultivated in vitro.

A spontaneous cyclic phenomenon characterized by successive waves of either high proliferative rate or intense melanogenesis is described in non-confluent B16 melanoma cells subcultivated during 2 months (or more). Dopaoxidase activity is quantified in individual cells after L-dopa reaction, by an original method of visible light absorption cytophotometry. A 24-hr treatment with alpha-MSH increases dopa-oxidase activity. This increase is also noted during the following 14 hr, in a fresh medium devoid of alpha-MSH, in which cell proliferation resumes after 24 hr. Phenylthiourea, cycloheximide or actinomycin D inhibit dopaoxidase activity, but also cell proliferation in alpha-MSH pre-stimulated cells. The effects of the two latter agents suggest that de novo synthesis of the enzyme takes place following alpha-MSH treatment.

Reevaluation by image analysis of the effects of fibroblasts, fibronectin or laminin upon colony formation in mouse lungs by B16 melanoma cells.

A recently described personal method based on image analysis of histological sections was used in order to quantify lung colony formation by B16 melanoma cells injected intravenously into the mouse. These tumor cells were preincubated in vitro either with fibronectin (FN), laminin (LN) or fibroblasts (FB), which are implicated in the process of invasion and metastasis. Thanks to this method, a more accurate analysis of lung colonies (section area and number) formed by tumor cells was realized. By image analysis, we show that when FB were mixed with B16 cells, a drastic increase of tumor sections number and area was induced. LN increased the tumor sections area, but not their number. No effect of FN on B16 cells was observed. LN and FN promoted tumor anchorage in the depth of the lungs while FB reduced the latter. These facts could explain the contradictory results obtained by simply counting macroscopically superficial lung colonies. When cultured in vitro, these B16 melanoma cells did not produce any type of IV collagenase, either alone or in the presence of LN or FN, but in cocultures (B16 with 3T3) and in fibroblasts cultures, this enzyme was present. This could explain, among other factors, why the rate of invasiveness exerted by B16 cells is higher when the latter are coinjected with FB.