Synthesis of collagen and hyaluronic acid by fibroblast hybrids.

Very little is known about the mechanisms of control of differentiated functions in mammalian cells. The present work, concerned with the synthesis of collagen and hyaluronic acid, was undertaken with the aim of obtaining information on the control of these two differentiated functions in the fibroblast. Established cell lines of fibroblastic origin differ greatly in their ability to synthesize these substances. " 2 The use of the technique of somatic cell hybridization (review in ref. 3) enabled us to combine in a single cell the genomes of two fibroblasts differing in the rates of production of collagen and hyaluronic acid, and to study the dominance relationships between the genes involved in these functions. Materials and Methods. -The hybrid clones studied were produced in two crosses between a single line of fibroblasts synthesizing considerable amounts of collagen and hyaluronic acid, and two different fibroblast lines producing very little collagen and no detectable amounts of hyaluronic acid. The "parental" line common to the two crosses is 3T6 clone 7, isolated from 3T6, an established mouse cell line.4 Clone 7 synthesizes slightly smaller amounts of collagen and hyaluronic acid than the parent population 3T6 (cf. refs. 1 and 2). The two established cell lines producing very little collagen and no hyaluronic acid are NCTC 2555 and NCTC 24725 clone 6,6 previously shown to undergo hybridization.7 The karyological features of the parental lines used for the identification of hybrids of the two crosses (3T6-7 X 2555 and 3T6-7 X 2472-6) are as follows. The cells of line 3T6-7 contain among their 70-79 telocentric chromosomes (mode 73), 2-3 distinct "markers" recognizable in hybrids of both crosses: one medium-sized telocentric with a pronounced proximal secondary constriction, and one or two small telocentrics with a very strong median constriction appearing as a feebly staining "heterochromatic" gap. Cells of line 2555 (54-58 chromosomes, mode 57) are characterized by the presence of 17-20 biarmed chromosomes and the absence of "rabbit-ear" chromosomes, of which several are observed in normal mouse cells and in 3T6. Thus, hybrids of the cross 3T6-7 X 2555 are identified by the simultaneous presence of the two or three "markers" and the "rabbit-ear" chromosomes of line 3T6-7, and of the biarmed chromosomes contributed by line 2555. Cells of line 2472-6 contain 48-53 (mode 50) chromosomes, among them one extra-long telocentric and one biarmed chromosome. The hybrids of the cross 3T6-7 X 2472-6 were therefore easily identified by the presence of the 3T6-7 "markers" and of the extra-long and the biarmed chromosome contributed by 2472-6. Cultures of all cell lines were grown in the Dulbecco-Vogt modification of Eagle's medium, containing 10% calf serum. Ascorbic acid (50 ,&g/ml) was added to the medium at least several days before measurements of collagen synthesis.8 For hybridization, mixed cultures of the two cell lines to be crossed were grown at 290C.6 Samples of these cultures were periodically examined karyologically for the presence of hybrids. When the proportions of hybrid cells reached 20% in cross 3T6-7 X 2555 and 90% in cross 3T6-7 X 2472-6, the mixed cultures were cloned at 360C. Among the clones isolated, three from each cross were kept for further investigation.9 An uncloned hybrid population, resulting from the overgrowth of the mixed culture 3T6-7 X 2555 by the hybrid cells, was also available. The purity