A. Sivak

Phenotypic Expression of Transformation: Induction in Cell Culture by a Phorbol Ester

A biologically potent, tumor-promoting phorbol ester from Croton tiglium L. induces the appearance of transformed clones in a population of contact-inhibited fibroblasts of the mouse cell line 3T3. Mixed populations of cells with 10,000 3T3 cells and 100 virus-transformed cells were exposed to the phorbol ester and, in comparison with untreated cells, showed a marked increase in the numbers of transformed clones that grew. Exposure of 3T3 cultures to the carcinogen 7,12-dimethylbenz(a)anthracene alone or prior to exposure to the phorbol ester did not cause any increase in the number of transformed clones.

Activation of cell membrane enzymes in the stimulation of cell division.

Summary The tumor promoter, phorbol myristate acetate, increases the specific activities of the cell membrane enzymes Na + -K + -ATPase and 5′-nucleotidase in microsomal preparations from stationary cultures of BALB/c-3T3 mouse embryo fibroblasts. Under these conditions of in vitro exposure, the microsomal enzymes NADH diaphorase and glucose-6-phosphatase were not affected.

Cellular interactions of phorbol myristate acetate in tumor promotion.

Abstract The radioactivity from tritium-labeled phorbol myristate acetate is bound to mouse skin with a half-life of approx. 24 h. Autoradiographs revealed that most of the label was associated with the kerato-sebaceous layer over the squamous epithelial layer. The major portion of the radioactivity was recovered in the lipid-containing fractions of a Schmidt-Thannhauser extraction. In cell cultures of 3T3 mouse embryo fibroblasts, maximal binding of [3H]PMA was achieved in 18 h and was stable for up to 4 days in the presence of the labeled tumor-promoting agent. Autoradiographs of formalin-fixed cells showed extensive cytoplasmic labeling with no substantial nuclear activity. Methanol fixation resulted in complete removal of the tritium. Sedimentation analysis in Dextran gradients of subcellular fractions of 3T3 cells exposed to [3H]PMA demonstrated that the bulk of bound label was associated with a Na+-K+ ATPase-rich protein peak which has the properties of cell membranes.

Studies with carcinogens and tumor-promoting agents in cell culture.

Abstract Transformation into permanent cell lines of hamster embryo fibroblasts was induced by benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene. Cells of the transformed lines had altered karyotypes, and the benzo(a)pyrene-induced line was tumorigenic in hamsters. Several lines of mouse fibroblasts of altered morphology were obtained following pyrene and benzo(a)pyrene exposure, but these cells were not tumorigenic when injected in cortisonized mice. 7,12-Dimethylbenz(a)anthracene inhibited the cellular proliferation of an untransformed line (3T3) of mouse fibroblasts to a lesser extent than in a polyoma virus-transformed variant of the same line. At doses that were not toxic to growth of mass cultures, exposure to 7,12-dimethylbenz(a)anthracene caused a reduction in the subsequent cloning efficiency of the 3T3 line. Similar inhibitory effects were obtained with tobacco smoke condensate, a phorbol ester fraction from Croton tiglium L. and benzo(a)pyrene. The toxic effect of bcnzo(a)pyrene was demonstrable 20 generations after exposure. No transformation was observed in the 3T3 cell line with any chemical treatment. The toxic and transforming effects of carcinogens in cell culture systems is discussed.

Induction of cell division in BALB/c-3T3 cells by phorbol myristate acetate or bovine serum: effects of inhibitors of cyclic AMP phosphodiesterase and Na+-K+-ATPase.

SummaryCell division is induced in stationary cultures of BALB/c-3T3 mouse embryo cells without renewal of medium by addition of the tumor promoter, phorbol myristate acetate (PMA), or bovine serum. The addition of dbcAMP (10−3m) or other inhibitors of cAMP phosphodiesterase, papaverine (6.7×10−6m), Persantin (5×10−5m) or RO-20-1724 (10−4m), prevents cell replication induced by PMA or serum. In contrast, ouabain (10−4m) and N,N′-dicyclohexylcarbodiimide (10−5m), inhibitors of Na+−K+-ATPase activity, block the PMA-stimulated effect but do not inhibit serum-stimulated cell division. Several stages in the cell cycle are sensitive to dbcAMP addition. One is early in the G1 phase at the time of reinitiation of the cell cycle from a stationary (G0) phase, a second is associated with the G1-S transition, and a third with passage of cells from a post-S phase to mitosis. Based on observations of early morphological changes, responses of plasma membrane ezymes and effects of enzyme inhibitors, the stimulation of cell division in BALB/c-3T3 cells by PMA or serum appears to involve several membrane functions which may act in a cooperative manner.

Tumorigenicity of acridine orange.

ACRIDINE orange, 3,6-bisdimethylamino-acridine (A.O.), exhibits a variety of effects in biological systems. Drosophila melanogaster (Clark, 1953); it also inhibits tumor induction on mouse skin in two-stage carcinogenesis (Van Duuren et al., 1969), and causes photo-dynamic inactivation of tobacco mosaic virus (Sastry and Gordon, 1966) and other viruses. Moreover, acridine orange has been shown to inhibit protein and nucleic acid biosynthesis in cell culture systems (Zelenin and Liapunova, 1964; Scholtissek and Becht, 1966). Because of these varied properties, the mode of interaction of the dye with nucleic acids, particularly DNA, has been widely studied by a variety of physical methods (Van Duuren, 1969) and several modes of binding of dye to nucleic acid have been proposed (Drummond et al., 1965). The relationship between the mode of action of carcinogens, mutagens and tumor-initiating agents is a problem of continuing interest (Trainin et al., 1964; Van Duuren and Sivak, 1968). Since little is known about the tumor-initiating and carcinogenic activity of acridine orange, it was of interest to examine these properties in mice and rats. The present report gives the results of these experiments. MATERIALS AND METHODS Animals. Mice were ICR/Ha Swiss obtained from Millerton Research Farms (Millerton, N.Y.). Females were used in all experiments. The mice were vaccinated against ectromelia at age 6 weeks and started on test at age 8 weeks. All mice were housed on sterilized wood chips in metal cages, 10 to a cage. Rats were female eastern Sprague-Dawley obtained from Blue Spruce Farms (Altamont, N.Y.). The rats were 6 weeks old and weighed 120-125 g. when testing began. They were housed in suspended wire mesh cages, 2 to a cage. Both mice and rats were fed Purina Laboratory Chow and water ad libitum. The animal rooms were temperature controlled at 22-24° C. Biological testing methods. Animals were weighed and observed monthly for the duration of the experiment. Tumors were recorded and counted at each observation. Any animal judged clinically to be in poor condition was sacrificed before the end of the experiment. All animals were examined carefully post-mortem and tumors and other lesions were excised for histological examination. Tissue sections were fixed in 10% formalin, blocked in paraffin and stained with hematoxylin and eosin. Routine sections of liver were also taken in the mouse skin treatment groups which received acridine orange repeatedly. The duration

Chromosomal proteins in fixed metaphase cells.

SummaryThe usual Carnoy fixative (acetic acid: methanol, 1:3) for metaphase preparations removes most histone as well as other nuclear proteins from rat and mouse embryo fibroblasts. Acrylamide gel electrophoresis patterns of fixatives from treated cells and the residual cell pellets show that significant amounts of histones are extracted into the Carnoy fixative. In contrast, formalin treatment fixes histones in the cells and renders them unextractable by the usual procedures. Autoradiographic studies with H3-lysine-labeled cells and electrophoretic analysis of cell extracts and fixative confirm these findings. The demonstration of typical quinacrine and trypsin-G-banding patterns with cells from both fixation procedures suggeststhat chromosomal banding patterns are independent of either the presence or absence of basic histone proteins.

Classification of cell types: Agglutination and chromosomal properties

SummaryThe properties of agglutination by plant lectins, along with chromosome patterns, were examined in a variety of mammalian cell types. Untransformed adult and embryonic cells in culture, direct mouse spleen cell preparations, SV40-transformed 3T3 cells, and trypsinized 3T3 cells were all highly agglutinable with concanavalin A and with wheat germ agglutinin. In contrast, untreated cells of the contact-inhibited 3T3 line were alone among the cells tested in their low agglutinability. Chromosome analysis of the cultured cells showed that karyotypic variation from the diploid to an aneuploid state in mouse and rat embryo cultures was not accompanied by a change in agglutinability. Adult rat lung, adult monkey kidney, and embryonic human lung cells, which were all highly agglutinable, showed the normal diploid pattern. Thus, agglutination of cells by plant lectins appears to be a cellular property often associated with non-neo-plastic cells.

The tumor-promoting activity of tobacco leaf extract and whole cigarette tar.

IN recent work in this laboratory it was shown that an extract of flue-cured cigarette tobacco leaf (WTE) as well as cigarette smoke condensate (WCT) exhibit tumor-promoting activity (Van Duuren et al., 1966). In these experiments a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) was applied on mouse skin as initiator followed by repeated skin application of the promoting agents. While this procedure proved satisfactory for bioassay of a number of fractions and subfractions derived from tobacco leaf and tars, it was desirable also to explore systemic treatment with several initiating agents followed by skin application of the promoting agents. The present report describes the bioassay of several promoting agents with DMBA, benzo[a]pyrene (BP) and urethane as initiators. The initiators were given by subcutaneous and intraperitoneal injection in mice; the promoting agents were applied on skin. The results of these experiments are compared with our earlier results, in which both the initiator and promoter were applied on mouse skin (Van Duuren and Orris, 1965; Van Duuren et al., 1966).

Comparison of the biological activity of the tumor promotor phorbol myristate acetate and a metabolite, phorbolol myristate acetate, in the cell culture*

Phorbolol myristate acetate, a metabolite of the tumor promotor phorbol muristate acetate in mouse skin, has one-fiftieth the potency of the parent molecule for the induction of cell division in stationary cultures of BALB/c-3T3 mouse embryo cells. Similarly, in a mixed cell culture assay devised for detection of tumor-promoting agents, phorbolol myristate acetate exhibited only a small fraction of the activity of unmetabolized phorbol ester. The results indicate that the biological activity of phorbol esters either does not require metabolic conversion or is converted by the cells used in this system and that phorbolol myristate acetate would be a tumor promotor of low potency for mouse skin compared to phorbol myristate acetate.