Histone synthesis in vitro by cytoplasmic microsomes from HeLa cells.

HeLa cell microsomes incorporate labeled amino acids in vitro into acid-soluble proteins which have the same electrophoretic mobility as histones isolated from tile purified HeLa cell nuclei. The capacity to Svnthesize histones in vitro is dependent on deoxyribonucleic acid synthesis in the cells from which the microsonal fraction is prepared.

Antagonism of concanavalin A capping in phorbol ester-activated lymphocytes by calmodulin inhibitors and certain amino acid esters.

12-O-Tetradecanoylphorbol-13-acetate (TPA), an effective tumor promoter in mouse skin and comitogen in bovine lymphocytes, rapidly stimulates concanavalin A-mediated cap formation in the latter cells. The ability of different phorbol derivatives to facilitate the capping reaction correlates well with their potencies as lymphocyte comitogens and as tumor-promoting agents. This effect of TPA on capping in bovine lymphocytes, which is apparent within min, is neither mimicked nor altered by dibutyryl cyclic adenosine 3':5'-monophosphate or cyclic guanosine 3':5'-monophosphate. Cytochalasin D, a microfilament-disrupting agent, inhibits cap formation, thereby suggesting the participation of microfilaments in the response. Benzoyl tyrosine ethyl ester selectively inhibits the TPA-stimulated cap formation, whereas benzoyl tyrosinamide is inactive. A comparison of related amino acid derivatives reveals that their activities are dependent on the nature of both the amino acid side chain and the carboxyl end blocking group. Trifluoperazine and N-(6-aminohexyl)-5-chloronaphthalenesulfonamide, known inhibitors of the calmodulin-dependent processes, also selectively block the TPA-stimulated cap formation, whereas trifluoperazine sulfoxide, a less effective calmodulin antagonist, is relatively inactive. These data suggest that the stimulation of capping by TPA involves the activation of a calmodulin-dependent process which may also be regulated by the function of an esterase.

Retinoic acid inhibition of the comitogenic action of mezerein and phorbol esters in bovine lymphocytes.

12-O-Tetradecanoylphorbol-13-acetate (TPA) is an effective comitogen in phytohemagglutinin-treated bovine lymphocytes. Concurrent addition of 10(-8) M TPA gives a greater than 6-fold increase in DNA synthesis over cultures treated with the lectin alone. The delayed addition of phorbol ester, relative to the start of the lectin treatment, eliminates this synergistic action. Structure-function studies show that the comitogenic activity of different phorbol diesters runs parallel to their tumor-promoting activity. A nontoxic level (50 micronM) of retinoic acid selectively antagonizes this synergistic effect of phorbol ester. This inhibitory action requires the near-concurrent addition of retinoic acid with TPA. In contrast, the TPA-mediated induction of RNA and protein synthesis is unaffected by retinolic acid. A number of natural and synthetic retinoids were evaluated; none were as inhibitory as was retinoic acid. Lymphocyte cultures appear to provide a useful system for exploring the mechanisms of action of both TPA and retinoic acid.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-accelerated phospholipid metabolism by 5,8,11,14-eicosatetraynoic acid.

The tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), accelerates choline phospholipid synthesis in bovine lymphocytes by an oxygen-dependent mechanism. This action is prevented by high concentrations of the cyclooxygenase inhibitor, indomethacin (1 to 3 mM), suggesting a possible involvement of lipid oxidation in the response. The acetylenic analog of arachidonic acid, 5,8,11,14-eicosatetraynoic acid (ETYA), at concentrations in the 10 to 50 microM range also prevents the acceleration of the incorporation of [methyl-3H]choline hydrochloride into choline phospholipids, and it appears more selective in its action. This antagonistic effect of ETYA, an agent which inhibits both the cyclooxygenase and lipoxygenase routes of arachidonic acid oxidation, can be precluded by arachidonic acid but not by other unsaturated or saturated fatty acids. If ETYA is added to lymphocyte cultures after 1 hr of TPA treatment, the established rate of [methyl-3H]choline hydrochloride incorporation is unaffected, but further acceleration is blocked. The inhibition by ETYA cannot be counteracted by any of the prostaglandins, Types A, B, D, E, or F alpha, or by the prostacyclin compounds, PGI1 and 6,9-thia-PGI2. The thromboxane pathway also appears not to be involved since 9,11-azoprosta-5,13-dienoic acid (Azo I), a selective inhibitor of thromboxane synthetase, does not affect the TPA response. These results suggest that TPS may activate the lipoxygenase rather than the cyclooxygenase pathway for lipid oxidation and that an arachidonic acid hydroperoxide or a subsequent metabolite plays a key role in the stimulation of choline phospholipid synthesis by the tumor-promoting phorbol ester, TPA.

Role of nonhemoglobin heme accumulation in the terminal differentiation of Friend erythroleukemia cells.

The differentiation of murine erythroleukemia cells (T3C12 Friend cells) on treatment with dimethyl sulfoxide (DMSO) has been correlated with an early and extended overproduction of heme. The cessation of cell replication and the development of nuclear condensation, changes which are associated with the terminal differentiation of these cells, occur when the level of heme rises 0.1 nmol/10(6) cells over that amount which can be complexed by globin protein. A significant fraction of the excess heme is localized in the nuclei of the DMSO-treated cells. The addition of exogenous hemin facilitates the onset of terminal differentiation in the DMSO-treated T3C12 cells, whereas only the induction of globin gene expression is observed in the absence of DMSO. To study the role of heme in erythroid differentiation, a Friend cell variant (R10) has been isolated whose synthesis and accumulation of heme in response to DMSO is deficient. This variant grows logarithmically in the presence of DMSO and fails to terminally differentiate. When exogenous hemin is added, the DMSO-treated R10 cells stop replicating. In the absence of DMSO, however, hemin-treated R10 cells continue to grow and express their globin genes without exhibiting other signs of terminal differentiation. The combined results support the conclusion that both the DMSO treatment and the accumulation of excess heme are required to bring about the terminal differentiation of Friend erythroleukemia cells. Preliminary evidence is presented which suggests that in addition to the induction of heme and globin synthesis, DMSO also induces a heme activation process which is required for differentiation. The possible relationship of this process to the terminal differentiation of erythroid cells is discussed.

Effects of retinoic acid and juvenile hormone on the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate.

The tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), a highly active comitogen in phytohemagglutinin-treated bovine lymphocytes, induces an 11-fold increase in ornithine decarboxylase activity over cultures treated with the lectin alone. This synergistic action of TPA could be antagonized by the simultaneous addition of the acyclic sesquiterpene, insect juvenile hormone III. Retinoic acid (vitamin A acid), an inhibitor of the tumor-promoting action of TPA in mice, was also an effective antagonist but required administration to lectin-activated lymphocytes 1 hr prior to TPA. These data suggest that metabolic activation of retinoic acid is required in order to exert its antagonistic action. Comparison of the responses in the lymphocytes and mouse skin suggests that the lymphocytes provide an excellent system for studying the molecular processes through which phorbol esters and retinoids influence the growth and differentiation of both normal and premalignant cells.

Proposed coupling of chromatin replication, hormone action, and cell differentiation.

DNA replication in isolated nuclei is highly dependent on the availability of soluble proteins from the cell cytoplasm. Activity is distributed nonrandomly among the different proteins, and the range of proteins that are required for optimal DNA replication varies with the fractions of DNA being replicated. Support of DNA replication has been correlated with the uptake of these proteins by nuclei and their integration into an immature form of the newly replicated chromatin; the latter has been shown by density analysis to be richer in protein content than the bulk of nonreplicating chromatin. Pulse labeling of DNA in living cells has revealed that a similar protein-rich chromatin is formed as an intermediate in chromatin replication in vivo; however, this form rapidly matures by the exclusion of proteins. The dependency of DNA replication on the presence of soluble cytoplasmic proteins and the physical association of these entities with newly replicated chromatin prompt the proposal that availability of specific proteins may play an important role in determining the ultimate genetic expressability of the matured chromatin and thus the cell phenotype. The finding that dexamethasone, a steroid that regulates the expression of several genes and directs the differentiation of certain cells, can modify the uptake of proteins in isolated nuclei is in accord with this hypothesis.

Inhibition of skin tumor promotion by retinoic acid and its metabolite 5,6-epoxyretinoic acid.

The ability of 5,6-epoxyretinoic acid, a biologically active metabolites of retinoic acid, to inhibit both the induction of ornithine decarboxylase (ODC) activity and skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) was evaluated. Application of 5,6-epoxyretinoic acid either concurrently with or 1 hr after each application of TPA to the initiated mouse skin inhibited the formation of skin tumors as effectively as did retinoic acid. 5,6-Dihydroretinoic acid, which is a poor substrate for epoxidation, also inhibited skin tumor promotion. 5,6-Epoxyretinoic acid, 5,6-dihydroretinoic acid, and retinoic acid were equally effective in inhibiting the induction of ODC activity by TPA. Insect juvenile hormones inhibited neither the induction of ODC activity nor skin tumor promotion by TPA. These results indicate that (a) epoxidation of retinoic acid at the 5,6-position is not a rate-limiting modification for the anti-promoting activity of retinoic acid and that (b) inhibition of the induction by TPA of mouse epidermal ODC activity may be a simple test for screening the potential prophylactic activities of new retinoids.

Phorbol esters activate the pathway for phosphatidylethanol synthesis in differentiating HL-60 cells.

12-O-Tetradecanoylphorbol 13-acetate (TPA) has been shown to induce the formation of an unusual acidic phospholipid, phosphatidylethanol, in HL-60 cells. The synthesis of this lipid is dependent upon the presence of ethanol in the culture medium of TPA-treated cells; however, other exogenous alcohols can substitute as headgroup precursors with the formation of the corresponding phosphatidyl alcohol. The activation of the pathway for phosphatidyl alcohol synthesis appears to be mediated through protein kinase C. Studies of the time-course for the synthesis and accumulation of phosphatidylethanol suggest a possible involvement of the pathway for phosphatidyl alcohol synthesis in the TPA-induced differentiation of HL-60 cells.

Effects of hexachlorocyclohexane isomers on concanavalin A 'capping' in bovine lymphocytes.

The effects of alpha, beta, gamma, and delta isomers of hexachlorocyclohexane on concanavalin A 'capping' in bovine lymphocytes were evaluated, gamma and delta hexochlorocyclohexane inhibited 'capping' whereas the alpha and beta isomers were without effect. In addition, gamma-hexachlorocyclohexane has been shown to antagonize the maintenance of preformed 'caps' and cause the rapid dispersal of the concanavalin A-receptor complexes over the surface of cells by a temperature-dependent mechanism. The possible role of a gamma-hexachlorocyclohexane-sensitive process in the organization of microflow patterns in the lectin-activated lymphocyte membrane is discussed.

Inhibition of the mixed lymphocyte proliferative response by phorbol esters.

The phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate, a potent cocarcinogen in mice, blocks the induction of DNA synthesis in lymphocytes undergoing the mixed lymphocyte response. At 10(-7) M diester, the induced DNA synthesis is inhibited almost completely (99%). This action of the diester affects some early step in the response which is necessary for the triggering of cell replication; on-going DNA replication is not significantly affected. Phorbol 12,13-diacetate, a less potent analogue in tumor promotion in vivo, is also a less potent inhibitor of the mixed lymphocyte response (75% inhibition at 10(-6) M). Phorbol, the parent alcohol, is not effective in either system. The use of phorbol diesters in the molecular dissection of mixed lymphocyte responses is discussed.

Dissociation of the DNA replicase system of bovine lymphocyte nuclei.

Exposure of S-phase nuclei or subnuclear preparations from phytohemagglutinin-stimulated bovine lymphocytes to 0.02 M ATP caused an immediate and almost total loss of their ability to replicate DNA in vitro. Other ribonucleoside and deoxyribonucleoside triphosphates caused a similar inhibition of DNA replication. Levels of ATP which inhibit replication cause the release of DNA polymerases alpha and beta and small pieces of DNA from these nuclei. This release occurs both at 4 and 37 degrees C. The data support the conclusion that high levels of ATP or other nucleoside triphosphates inhibit DNA replication in nuclei by dissolution of the DNA replication complex. The limited success in reconstitution of the DNA replicase complexes is discussed.

DNA replication in nuclei isolated from bovine lymphocytes.

DNA replication was studied in nuclei isolated from phytohemagglutin-stimulated bovine lymphocytes. The mitogen treatment induced more than 60% of these cells to engage in DNA synthesis with a peak of synthetic activity occurring about 48 h after the addition of phytohemagglutinin. Throughout this response the ability of isolated nuclei and nuclear sonicates to synthesize DNA in vitro was proportional to the synthetic ability of the intact cells of origin. The subcellular systems appear to continue synthesis at replicative sites which were active in vivo. The rate of in vitro synthesis by both nuclei and nuclear sonicates was about two-thirds that of intact cells. The one cytoplasmic and two nuclear DNA polymerase activities separated from these cells were found to have properties similar to those of other eucaryotic cells.

A requirement for RNA, protein and DNA synthesis in the establishment of DNA replicase activity in synchronized HeLa cells.

DNA replicase activity of synchronized cultures of HeLa cells was assayed by a permeable cell technique during normal S-phase and under conditions of restricted RNA, protein, or DNA synthesis. Inhibition studies with puromycin, cycloheximide, actinomycin D, and 2-mercapto-1-(beta-4-pyridethyl)benzimidazole revealed that the establishment as well as the maintenance of DNA replicase activity in S-phase cells was dependent on the continued synthesis of both RNA and protein. Measurements during limitation of DNA replication by hydroxyurea, cytosine arabinoside, or restricted availability of thymidine indicate that a low level of DNA synthesis is required to activate or assemble some subunits of DNA replicase. Evidence for the existence of short-lived RNA and protein factors essential for DNA replicase activity is discussed.

Dissociation and reconstitution of the DNA replicase system of HeLa cell nuclei.

The DNA replication system of S-phase HeLa nuclei has been dissociated by cautious extraction at 0 degrees C with 0.25 M NaCl. Replicase activity has been reestablished by recombination of the fractions and reduction of the salt concentration. The reconstituted system, like the starting nuclei, depended on ATP, 4dNTP, MgCl2, the proper ionic strength and the soluble cytoplasmic protein fraction. The activity of the nuclear extract showed a cell cycle dependency and was elevated in the nuclei of cells at the G1 leads to S boundary. In the presence of Mg2+ the major activity of the nuclear extract precipitated during dialysis to reduce the salt concentration; this precipitate exhibited DNA polymerase alpha activity. Chromatography of the active extracts over phosphocellulose separated the replicase supporting factors into three fractions. The major activity eluted in the fraction containing the DNA polymerase alpha activity; the other two active fractions were devoid of polymerase activity. The fraction containing DNA polymerase alpha from the nuclear extracts supported DNA replicase activity in salt-extracted nuclei whereas an equivalent level of DNA polymerase alpha from the cytoplasm was not effective. The data suggest that the DNA polymerase alpha of the salt extracts of S-phase nuclei is either different than the cytoplasmic enzyme or is associated with some essential replicase-supporting factor.

Role of cytosol proteins in DNA chain growth and chromatin replication in Friend erythroleukemia cell nuclei.

The influence of cytosol proteins on the replication of DNA and chromatin in isolated nuclei from Friend erythroleukemia cells has been investigated. The overall process has been clearly shown to proceed stepwise. In the absence of cytosol proteins DNA chain growth tends to stop after the addition of approximately 200 nucleotides to the ends of growing chains. In the presence of cytosol proteins these sections grow to approximately 250 nucleotides, and participate in the stepwise extension of the replication process through adjacent nucleosomal sections of the template. Immediately following pulse labeling, the newly replicated DNA resides in a chromatin form which appears to be relatively resistant to digestion by micrococcal nuclease. During a chase interval, the association of the pulse-labeled DNA with nuclear proteins matures to a form which yields lengths of DNA upon digestion with micrococcal nuclease that correspond to mono-, di-, tri- and polynucleosomal units of chromatin. In the absence of cytosol proteins the nuclease resistant state of the labeled DNA tends to predominate and persist. The data support the view that DNA replication in a chromosomal setting proceeds stepwise over successive nucleosomal sections of template made accessible by the interaction of the cytosol proteins at or near the replication fork.

Localization of globin gene replication in Friend Leukemia cells to a specific interval of the S phase.

Murine erythroblastic leukemia cells, infected with Friend leukemia virus, were grown and synchronized in suspension culture by a double-block procedure involving medium depletion and treatment with excess thymidine. Replicating cultures were then caused to synthesize DNA during the early, middle, or late third of the S period with bromodeoxyuridine as a precursor. The bromodeoxyuridine density-labeled DNA and normal DNA were isolated by sedimentation in a cesium chloride density gradient and analyzed for the level of globin-specific DNA by hybridization with radioactive cDNA of the globin messenger RNA. Globin genes were found to be replicated near the end of the middle third of the S phase. The incorporation of bromodeoxyuridine into DNA also resulted in a lowered induction of hemoglobin synthesis in dimethyl sulfoxide-treated cells. In this case, the sensitivity was correlated with the introduction of bromodeoxyuridine into DNA replicated in early S phase. The possibility that this fraction of DNA, which is low in globin gene content, carries genes regulating the erythroid differentiation or the expression of globin genes is discussed.

A permeable cell system for studying DNA replication in synchronized HeLa cells.

Treatment of HeLa cells with a hypotonic buffer solution makes them permeable to nucleotides. Cells which are in S-phase at the time of treatment continue to synthesize DNA when supplied with the four deoxyriboside triphosphates, ATP, Mg2+, and the proper ionic environment. DNA replication extends from sites which were active in the cells prior to treatment. The product is confined to the nucleus and is sensitive to deoxyribonuclease. Under optimum conditions, up to 5% of the HeLa genome can be replicated from exogenous nucleotides. In synchronized cultures the level of DNA replicase activity, as measured in permeable cells at different points in the cell cycle, correlates with the rate of [14C] thymidine incorporation measured in the living, untreated cells.

Role of fatty acid structure in the reversible activation of phosphatidylcholine synthesis in lymphocytes.

Fatty acids rapidly accelerate (1.5-7.0-fold) the incorporation of [methyl-3H]choline chloride into the phosphatidylcholine fraction of bovine lymphocyte lipids. This ability of fatty acids to activate choline phospholipid synthesis has been correlated with certain structural features of fatty acids. Mono- and polyenoic unsaturated fatty acids of 18 and 20 carbons in length are highly active, whereas their saturated analogues are nearly inactive. Among the unsaturated fatty acids, the cis-isomers are active, while the trans-isomers are relatively ineffective. The delayed addition of bovine serum albumin (5 mg/ml) and other lipid-binding proteins to activated cells rapidly counteracts the lipid effects. The activated state of the cell membrane thus appears to be a dynamic one, requiring the continued interaction of the fatty acid with a lipid-sensitive target molecule of the cell surface that in turn appears to coordinate the enzymatic components of this pathway.

Analysis of the effects of fatty acids and related compounds on the synthesis of phosphatidylcholine in lymphocytes.

Phosphatidylcholine synthesis in cultured bovine lymphocytes is stimulated by cis-unsaturated fatty acids. This stimulation is correlated with an activation of the enzyme cytidyltransferase (EC 2.7.7.15) and its apparent translocation from the cytosol to the membrane/particulate of cells. In addition, these agents increase the levels of cytidine diphosphocholine - a product of the cytidyltransferase reaction and a precursor to phosphatidylcholine. Retinoic acid and 5,8,11,14-eicosatetraynoic acid both activate cytidyltransferase activity and raise cytidine diphosphocholine levels, yet they are ineffective as stimulators of overall phosphatidylcholine synthesis. The effects of all of these lipids are reversed by the delayed addition of bovine serum albumin. The data point to the view that cytidyltransferase activation is required but is not sufficient for stimulation of phosphatidylcholine synthesis: regulation at another step is suggested.