The synthetic diacylglycerol l-oleoyl-2-acetylglycerol competes directly with [ H]phorbol 12,13-dibutyrate for common binding sites in monolayer cultures of Swiss 3T3 cells and rapidly stimulates the phosphorylation of a Mr 80,000 cellular protein that has recently been shown to reflect the activation of protein kinase C in intact cells. Thus, this diacylglycerol provided a useful tool to determine whether exogenously added diacyIglycerols can mimic the potent tumor promoter phorbol ester in eliciting DNA synthesis and cell division in quiescent cells. We found that OAG acts synergistically with insulin and other growth factors to stimulate reinitiation of cell proliferation, and several lines of evidence indicate that OAG shares with phorbol esters a common pathway of mitogenic action via stimulation of protein kinase C activity in intact 3T3 cells. The findings support the hypothesis that diacylglycerols represent endogenous analogs of phorbol esters and raise the possibility that diacylglycerols generated in the plasma membrane could act as a mitogenic signal for quiescent cells.Ca2+-sensitive phospholipid-dependent protein kinase (protein kinase C) is a widely distributed cyclic nucleotide-and calmodulin-independent phosphotransferase (1-3) that is activated by unsaturated diacylglycerols in the presence of phospholipids and physiological concentrations of Ca2+ (4)(5)(6). Recent studies with cell-free preparations (7-10) as well as with intact fibroblasts (11) have shown that the potent tumor promoters of the phorbol ester family (12) (13,14) and that 1,2-diacylglycerols compete with this radioactive ligand for common binding sites (15). It has been hypothesized that the high-affinity specific receptor for phorbol esters that was detected in a wide variety of cells and tissues (16-21) is a complex formed between protein kinase C, Ca2 , and membrane phospholipids (13,22) and that unsaturated diacylglycerols generated by phospholipid breakdown (22, 23) may represent the long-sought endogenous analogs of the phorbol esters and structurally related plant diterpenes (15,(23)(24)(25). A critical test of this hypothesis required experiments with intact cells.Phorbol esters are not only potent tumor promoters (12, 26) but also they induce a complex array of early biological responses in cultured cells (24,26), as well as stimulate DNA synthesis and cell division in quiescent cells (21,25,27). A crucial step for establishing that some or all these events are mediated through activation of protein kinase C and that this phosphotransferase system may play a central role in the regulation of cellular growth is to show that exogenously added diacylglycerols can mimic the action of the phorbol esters in stimulating reinitiation of DNA synthesis and cell division in quiescent cells. To test whether diacylglycerol and phorbol esters act as functional analogs, we chose untransformed Swiss 3T3 cells as a model system because of the considerable body of information available on their mitogenic response to phorbol ester...

Since the pioneering work of the Millers it has become clear that most chemical carcinogens require metabolism to reactive electrophiles and then exhibit their carcinogenic potential by reacting chemically with, and modifying, cellular macromolecules. At first modification of proteins was considered most likely to be of importance in carcinogenesis. Later, Brookes and Lawley demonstrated that the extent of binding of several polycyclic hydrocarbons to DNA, but not to RNA or protein isolated from the skin of mice treated topically with these compounds, correlated with their known carcinogenic potency to this tissue. Mammalian cells, particularly mouse embryo cells, treated with chemical carcinogens have often been used, and DNA has been involved almost exclusively from whole cells. However, mitochondria possess unique DNA which accounts for 0.1-1% of the total DNA present in mammalian cells, and three studies have shown that carcinogenic alkylating agents modify the michondrial DNA by a factor about five times greater than the nuclear DNA from the same cells. We demonstrate here that with six polycyclic aromatic compounds, all of which require metabolic activation and bind to DNA to a much smaller extent than direct than direct-acting alkylating agents, the binding to mitochondrial relative to DNA is dramatically increased by a factor of nearly 50 to over 500.

€544. A Convenient Solid for Calibration of the Gouy MagneticSusceptibility Apparatus. By B. N. FIGGIS and R. S. NYHOLM. THE measurement of magnetic susceptibilities by the Gouy method is a relative one, the apparatus being calibrated in terms of a substance of known susceptibility, for which water, nickel chloride solution, and powdered cupric sulphate pentahydrate or ferrous ammonium sulphate hexahydrate have been used. The low suceptibility of water is often inconvenient if small tubes are being calibrated. Nickel chloride solution requires accurate analysis before use and ferrous ammonium sulphate is often of questionable purity. This substance and copper sulphate do not pack well and several different values for the susceptibilities of both solids have been reported.The required properties for a calibrant are: (1) Readily available pure;(2) an accurately known and moderate susceptibility (xg = ;(3) stability in moist air;(4) xg must vary in a known and simple way, at least at room temperature; (5) easily and reproducibly packable into the Gouy tube. The complex mercury tetrathiocyanatocobaltate HgCo(CNS), offers some advantages and its susceptibility has therefore been accurately determined. at 20' 1 being used as reference, the gram susceptibility of the complex is 16.44 (-&O.OS) x at 20".As reported elsewhere,l it obeys the Curie-Weiss law, xg cc (T + lO)-l where T is expressed

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An analysis has been made of the C-H...O interactions in cyclopenta[a]phenanthrenes, for which structural data on fifteen 15,16-dihydrocyclopenta[a]-phenanthren-17-ones are available. These compounds mostly contain only one O atom, a carbonyl group at the 17-position, and therefore the only groups available for interactions are C-H groups. In addition, the crystal structure of a second polymorph of the 11-ethyl derivative is described. M(r) = 260.33, Pbca, a = 17.012 (2), b = 21.042 (2), c = 7.6465 (6) A, V = 2737.2 (4) A, Z = 8, Dx = 1.264 Mg m-3, Cu K alpha, lambda = 1.5418 A, mu = 0.56 mm-1, F(000) = 1104, T = 295 K, final R = 0.090 for 1669 reflections above 2 sigma (F). The conformation of the ethyl group is gauche [C(12)-C(11)-C(18)-C(19) = 75.8 (7) degrees], differing from the cis value of -1.3 (5) degrees for the Pnaa form. The molecular distortion in the Pbca polymorph is also larger than that in the Pnaa polymorph; this distortion is evidenced by torsion angles (13-20 degrees) in the bay region and by an out-of-plane displacement (0.8 A) of the C atom of the methylene portion of the ethyl group [the C atom attached to C(11)]. Packing diagrams and intermolecular distances were analyzed for all the dihydrocyclopenta[a]phenanthrenes for which structural data are available. There appear to be three types of packing. The first type consists of a dimer herringbone formed by the interactions of two molecules by way of the ketone group and the C-H of C(12) of the adjacent ring. The second type of packing also involves a dimer but involves C-H and O-C groups at either ends of the molecule. The third type is a layer structure and involves compounds that crystallize with a unit-cell length of 7.5-7.6 A (or, in a very planar structure, 13.8 A). The translational stacking (approximately 4 A apart) found in polycyclic aromatic hydrocarbons is not observed in the crystal structures of these dihydrocyclopenta[a]-phenanthrenes because of the bulk of methyl or methylene groups and the dipole moment of the carbonyl group.