Effects of 5-bromo-2'-deoxyuridine on beating heart cell cultures from neonatal hamsters

Abstract

1. Primary heart cell cultures from neonatal hamsters yielded a heterogeneous cell population, containing muscle cells undergoing progressive differentiation, as well as non-muscle cells. 2. Addition of 5-bromo-2'-deoxyuridine, at an early stage, to such cultures enhanced the formation of beating sheets of differentiated muscle cells. Accumulation of myosin heavy chains and creatine kinase also occurred in the presence of the analogue. 3. To obtain these effects, the analogue had to be added during the initial rapid growth phase of the cells. Division of the treated cells then ceased when the cell numbers had approximately doubled. 4. Similar results were obtained with other inhibitors of DNA synthesis. Thus improved muscle cell cultures can be obtained by preventing non-muscle cells from overgrowing the cultures. 5. One effect caused only by 5-bromo-2'-deoxyuridine was a large increase in the Ca2+-stimulated ATPase (adenosine triphosphatase) activity which sedimented at low ionic strength. This increase was not due to a greater content of myofibrillar myosin, or to myosin isoenzyme changes, because purified myosin prepared from treated and untreated cultures did not exhibit the increased Ca2+-stimulated ATPase activity.

Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Cross-striations, ultrastructure, and chronotropic response to isoproterenol.

“…Ara C produced stable NMC numbers, but for only 3 days (Coetzee and Gevers, 1978). Among the few other studies using cell counts, several report only total cells; these declined in rat heart cultures maintained without serum (Desmond and Harary, 1972) and increased in hamster cultures with BrdU, 0.1 mM (Coetzee et al, 1977). MCs from the 5-day chick heart continued to divide in the presence of BrdU, 0.07 mM (Chacko and Joseph, 1974).…”

“…Various culture modifications have been tried to inhibit NMC proliferation, including drugs (Coetzee et al, 1977;Coetzee and Gevers, 1978), radiation (Desmond and Harary, 1972), glutamine deprivation (Clark, 1976), and manipulation of the quantity or character of the serum or plasma supplementation (Clark et al, 1977;Jones et al, 1978;Van der Laarse et al, 1979). In no case has arrest of proliferation over several days and absence of MC toxicity been documented by counts of MCs and NMCs.…”

5-bromo-2'-deoxyuridine-stimulated calcium ion- or magnesium ion-dependent ecto-(adenosine triphosphatase) activity of cultured hamster cardiac cells

“…BrdUrd* exerts diverse and sometimes apparently contradictory effects on many mammalian cells grown in culture. We have reported a further effect of BrdUrd on a Ca2+-dependent ATPase activity in primary cultures of hamster heart cells (Coetzee et al, 1977). This Ca2+-dependent ATPase activity was not associated with myofibrillar myosin.…”

“…The materials used were as described in the preceding paper (Coetzee et al, 1977). Cell types other than hamster heart cells were provided by dUrd was purchased from The Radiochemical Centre, Amersham, Bucks., U.K., ouabain (Strophanthin-G) from BDH Chemicals, Poole, Dorset, U.K., verapamil hydrochloride (Isoptin) from Knoll A.G. Chemical Works, Ludwigshafen, West Germany, and p-nitrophenyl phosphate from Sigma Chemical Co., St. Louis, MO, U.S.A.…”

“…Primary heart cell cultures were established, cells harvested, counted and homogenized as described previously (Coetzee et al, 1977). Secondary, tertiary and quaternary cultures were obtained from primary heart cell cultures by trypsin treatment of monolayers and replating them in series at cell concentrations of 0.5 x 106 cells per 60mm-diameter dish.…”