The growth characteristics and the antigenic profile of two human melanoma established cell lines in uitro are reported. The growth curves of the two cell lines show some peculiar features which are also detected in their DNA distribution content. In the late stages of growth, in fact, the cell DNA content resumes the initial distribution, suggesting a partial recovery of the cell proliferating potential. When the two cell lines were tested by indirect immunofluorescence with monoclonal antibodies to human melanoma associated antigens, a rather constant expression of these epithopes was observed. Key terms: Melanoma lines, growth curves, distribution analysis of DNA content and monoclonal antibodiesMalignant transformation is associated with both functional and structural changes of the cell membrane and, in particular, the expression of tumor-associated antigens seems to be a general aspect of human and experimental tumors (4).The present scarcity of human malignant cell lines with a well-defined growth pattern and antigenic profile has thus far prevented understanding the relationship of membrane changes to cell growth, differentiation and metastatic potential.The aim of this paper was to study the growth characteristics and the kinetic parameters of two human melanoma cell lines (M10 and M14) against which monoclonal antibodies to three tumor-associated antigens have been developed. This preliminary study is a part of a wider research project aimed at the parallel evaluation of cell DNA content and antigenic profile, using biparametric cytofluorometry as a means to estimate the possible correlations between these two parameters in different stages of the cell cycle. Materials and MethodsCell Culture Technique and Flow Cytometry: The two lines M10 and M14, derived from human metastatic nodules were estab- For subcultures the monolayers were rinsed once with Earle's solution and incubated with 0.5 mM EDTA at 37°C. The cells were dispersed in RPMI 1640 + 10% fetal calf serum and counted in a model ZB1 Coulter counter; the subcultures were inoculated with 5 to 8 X lo4 cells in 5 ml. The plating efficiency was evaluated by seeding -800 cells in a flask culture plate (60 mm) incubated at 37°C in 5% C02:95% air 8-12 days. The colonies were stained with 3% methylene Blue and counted (9). Five replicate plates were used for each determination. Analyses of DNA distribution were carried out on cells at different stages of growth, either in the log or in the apparent plateau phase.The cells were collected using EDTA 0.5 mM and after centrifugation the pellet was resuspended in PBS-Dulbecco's Ca++ and Mg++ free (Grand Island Biological Co., Grand Island, NY). The ce11 suspension was transferred into a staining solution containing: 10 p g / d mithramycin, 10 gg/ml propidium iodide (Calbiochem-Behring, San Diego, CA), 0.1% Nonidet, 15 mM Magnesium Chloride in Tris-HC1 pH 7.5.The nuclear fluorescence was measured with a microfluorimeter ICP22 (Phywe) using a mercury 100 W lamp, excitation filter BG12 3mm coupled with a K590 ...

Flow cytometry is a widely recognized method of rapidly assessing the ploidy and proliferation status of experimental and solid tumors. In the present work, a variety of human cancers from various sites (lung, head and neck, etc.), of traditional interest in our laboratory, have been analyzed. In agreement with a number of recent reports, a general heterogeneity of human solid tumors can be evidenced. In particular: (a) solid tumors are characterized by a variable degree of aneuploidy; (b) the internal structure of solid tumors is highly heterogeneous especially with respect to the fraction of aneuploid malignant cells and their distribution through the cycle phases; and (c) some solid tumors are also characterized by the presence, to a variable extent, in the tumor of mass of multiple cell clones. Static fluorimetry of Feulgen-stained (mitotic) single cells offers a way to confirm this kind of observations.

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The proliferation patterns of bone marrow cells from 16 cases of acute nonlymphatic leukemia (ANLL) and 13 of dysmyelopoietic syndromes were analyzed with DNA-flow cytometry. In order to reduce the contamination by nonproliferating peripheral blood cells, all aspirates were submitted to a density centrifugation step. DNA content measurements were performed with a PHYWEICP 22 flow system apparatus. The distribution of cells in te cell cycle phases was calculated by means of an automated fitting procedure. No significant difference between the cell cycle distribution in ANLL and dysmyelopoietic syndromes was detectable. No relationship was observed between the size of the S phase compartment and the percentage of blasts in the original samples. Aspirates from ANLL were also cultured in vitro in liquid phase on dialysis membranes. A clear relationship was observed between the size of the phase compartment and in vitro cell growth since, with the exception of one case, all samples with less than 11% cells in S phase failed to proliferate in vitro. The proliferation profile of ANLL cells after 10 days in culture was similar to that of the original samples.