Cells from embryonic chicken muscle were cultivated in serum-free medium. After two days, the suspended cells (almost all of which were nondividing myoblasts) were subcultured in serum-containing medium, either in gelatincoated tissue culture dishes (to promote reattachment) or in bacteriological dishes (to prevent reattachment). The extent of fusion was high in both suspended and reattached cultures.Newly synthesized proteins from day-5 cultures were resolved by two-dimensional electrophoresis and detected by autoradiography. Not Most normal cell types exhibit anchorage-dependent proliferation; by contrast, many transformed cell lines proliferate well in suspension, a characteristic that appears to be consistently correlated with tumorigenicity (1, 2). For 3T6 cells, which show strict anchorage dependence of proliferation, forced suspension has been shown to result in a rapid decrease in both mRNA synthesis and mRNA degradation and in a slower decrease in total protein synthesis (3). Some proteins may be synthesized in near-normal amounts in suspension (4) and some mRNA species, and the proteins they code for, are synthesized in greater-than-normal amounts during recovery after reattachment (4). For these cells, then, there is reason to believe that their pattern of gene expression is regulated by cell-substrate interactions.Myogenic precursor cells from avian or mammalian embryos proliferate readily in monolayer culture. With time, they give rise to nondividing, terminally differentiating "myoblasts" that synthesize and accumulate muscle-specific molecules and form syncytial "myotubes." These cells elongate, elaborate the specialized subcellular structures characteristic of functional muscle, and contract (for review, see refs. 5-7).The influence of the culture substrate on muscle differentiation has received much attention (5). In clonal cultures myotube formation is favored by collagen-coated substrates. At high density this requirement is overcome, probably by cellderived collagen (5). Holtzer et al. (7) state that myogenic cells "remain alive when cultured on a surface to which they are unable to stick but will not differentiate unless they become attached." On the other hand, it has been reported that, under conditions that prevent normal cell-substrate interaction, myogenic cells did not divide and migrate, but were able to fuse and to synthesize and assemble myofibrillar proteins (8). Recently, several authors have observed that chicken myogenic cells can fuse in suspension, forming multinucleated "myoballs" (9-12). These myoballs possess acetylcholine receptors, generate action potentials, contract, and, when allowed to attach to a substrate, elongate into myotubes (10,11).In this paper, we reexamine the question of the anchorage dependence of skeletal muscle differentiation. Samples of the same cell suspension are cultivated in the same medium either in suspension or attached to culture dishes and then compared directly with respect to: (i) extent of fusion; (ii) pattern of synthesized prote...
The results of the extension of a collaborative study for the detection of chemical-induced DNA damage in rat hepatocytes in vitro are presented in this report. Three coded compounds, i.e. 1,4-butanediol dimethanesulphonate, hydrazine sulphate and sodium dichromate, were tested for DNA repair synthesis by seven different laboratories, either using autoradiographic procedures or the liquid scintillation counting technique. Inter-laboratory standardization was intentionally not requested in order to investigate the validity of each study design under routine conditions. 1,4-Butanediol dimethanesulphonate was clearly positive in most laboratories; sodium dichromate was generally positive, while the results on hydrazine sulphate were contradictory.
A compound's mutagenicity in different Salmonella tester strains can suggest its mechanism of reaction with DNA. Clear confirmation of such a mechanism, however, requires a direct test of the compound's reaction with DNA, often relying on specific in vitro studies. We report the use of a rapid in vitro test designed to measure DNA unwinding, a characteristic of DNA intercalators and many frameshift mutagens. CGS 20928A, an adenosine antagonist, produced a significant (> 2-fold) increase in revertants only for Salmonella tester strain TA1537, and only without metabolic activation. These data indicated that the compound was a direct acting frameshift mutagen and possibly intercalated into DNA. Our DNA unwinding assay indicated that at concentrations of > 0.1 mM CGS 20928A behaved like known intercalating compounds in that it unwound DNA. These concentrations of compound are comparable to those found mutagenic to TA1537. By comparison, the frameshift mutagen and known intercalating compound 9-aminoacridine unwound DNA in this assay in a concentration dependent fashion between 6-12 microM. ICR-191, another acridine frameshift mutagen, also unwound DNA. A compound structurally related to CGS 20928A, which was not mutagenic in Salmonella tester strains, did not produce any DNA unwinding even at 10 mM. Because the assay uses microgram quantities of material, it should be ideal for screening small amounts of congeneric series suspected of frameshift mutagenicity.
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