Gerald C. Mueller

Apoptotic death in adenocarcinoma cell lines induced by butyrate and other histone deacetylase inhibitors

n-Butyrate inhibits the growth of colon cancer cell lines. In the HCT 116 cell line, butyrate-induced growth inhibition is almost fully reversible, whereas in the VACO 5 cell line, a subpopulation undergoes apoptosis within 30 hr of treatment with butyrate. Concurrent treatment of VACO 5 cells with butyrate and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) accelerates and increases the incidence of cell death to nearly 100% of the population, whereas HCT 116 cells largely remain alive during treatment with this combination. The action of butyrate as an inhibitor of histone deacetylase was assessed in these cell lines by examining extracted core histones for their electrophoretic mobility in Triton/acid/urea gels. The concentrations of butyrate that were effective for inducing apoptosis were similar to the concentrations that caused hyperacetylation of core histones in the VACO 5 cell line. Furthermore, an examination of other carboxylic acids for induction of apoptosis revealed a rank order that corresponded to the order of potency in causing hyperacetylation of core histones. Specifically, the active acids were 3-5 carbons in length and lacked substitution at the 2-position. Isovaleric and propionic acids, in particular, proved to be effective inducers of both hyperacetylation and apoptosis at 5 mM concentrations, a finding of potential relevance to the unusual pancytopenia occurring after acidotic episodes in isovaleric and propionic acidemias. The duration of butyrate treatment required for chromatin fragmentation (10-20 hr) corresponded to the time required for histone H4 to become predominantly tetraacetylated. Furthermore, trichostatin A, a structurally dissimilar inhibitor of histone deacetylase, mimicked butyrate-induced apoptosis of VACO 5 cells and growth inhibition of HCT 116 cells. The dramatic enhancement of VACO 5 cell death by TPA, and the high level resistance of HCT 116 cells to butyrate were not evident from histone acetylation determinations. Thus, applications of butyrate for cytoreduction therapy will benefit from pharmacodynamic assessment of histone acetylation, but will require additional work to predict susceptibility to butyrate-induced death.

A nuclear system for DNA replication from synchronized HeLa cells

Abstract A nuclear system of DNA synthesis is described which appears to continue the replication process in vivo . In addition to intact nuclei, the system requires all 4 deoxynucleoside triphosphates, Mg 2+ , ATP, an inorganic salt and a heat-labile, SH-sensitive factor from the cytoplasmic fraction. There is also evidence for a second SH-sensitive process confined to the nucleus. The activity of the system correlated closely with the DNA synthetic activity of intact cells in synchronized cultures. The product was sensitive to deoxyribonuclease and, characteristic of replicating DNA, most of the product moved to the interphase during phenol extraction. That fraction of the product which was phenol extractable behaved like cellular DNA in CsCl and sucrose density gradients. A comparison of the nuclear system and the DNA polymerase found in the cytoplasm revealed different requirements for substrate concentration, pH, salt and ATP. The cytoplasmic DNA polymerase activity was not correlated with the cell cycle. Both systems were inhibited by PHMB and high levels of MgCl 2 . An analysis of the substrates utilized by the system revealed that all of the phosphate entering DNA was derived from the 5′α-position of the deoxynucleoside triphosphates without prior cyclization to the 3′-position.

Synergistic action of phorbol esters in mitogen-activated bovine lymphocytes☆

Abstract Phorbol esters act synergistically with phytohemagglutinin (PHA) and Concanavalin A to promote DNA synthesis in bovine lymphocytes. Studies of this response indicate that phorbol esters are useful tools for elucidating the cellular processes that are related to the action of mitogens.

Studies on the mechanism by which phytohemagglutinin rapidly stimulates phospholipid metabolism of human lymphocytes

The growth initiator phytohemagglutinin rapidly accelerates 32Pi incorporation into lymphocyte phosphatidylinositol. This study shows that phytohemagglutinin rapidly accelerates myo-[2-3H]inositol incorporation (1800%) but only slightly increases [2-3H]glycerol and inhibits [1-14C]oleic acid incorporation into phosphatidylinositol. A convenient method for isolating the lymphocyte plasma membrane and nuclear fractions is described. 3 min exposure of lymphocytes to phytohemagglutinin produces a 430% stimulation of 32Pi incorporation into phosphatidic acid of the plasma membrane fraction while in the nuclear fraction the stimulation is only 80%. Under these conditions, 32Pi incorporation into phosphatidylinositol is stimulated to nearly the same extent in all fractions. Direct addition of phytohemagglutinin to a lymphocyte plasma membrane fraction stimulates phosphatidic acid synthesis by diglyceride kinase. It is suggested that phytohemagglutinin stimulates the cyclic interconversion of phosphatidic acid and phosphatidylinositol.

Early- and late-replicating deoxyribonucleic acid complexes in HeLa nuclei.

Abstract The DNA which replicated early in the period of DNA synthesis in synchronized cultures of HeLa cells was labeled with [3H]thymidine. After several generations of random growth the cells were resynchronized and exposed to bromodeoxyuridine for various portions of the DNA-synthesis period. The 3H label was carried in the bromodeoxyuridine-containing, heavy hybrid of the DNA that replicated early in the second synchronized cell cycle.

Control of histone synthesis in HeLa cells

Abstract The coupling between DNA replication and histone synthesis during the S-phase of HeLa cells has been studied using metabolic inhibitors in living cells and a protein synthesizing polysome system from treated cells. Blocking DNA synthesis in vivo with hydroxyurea leads to a transient accumulation of newly synthesized histones in the cytoplasm and the concomitant suppression of histone synthesis. Both the histone synthesizing activity of the isolated polysomes and the [3H]uridine-labeled histone mRNA of this fraction are lost in a parallel manner. This loss can be prevented by blocking protein synthesis in vivo with cycloheximide prior to blocking DNA synthesis with hydroxyurea. In this state the [3H]uridine-labeled histone mRNA continues to accumulate in the polysome fraction. The data support the conclusion that DNA replication opens template sites for transcription into histone mRNAs which are subsequently translated by cytoplasmic polysomes. It is proposed that following the blockade of DNA synthesis the accumulating histones act both cytoplasmically to limit translation and in the nucleus to repress the further synthesis of histone mRNA.

Identification of transferrin as a lymphocyte growth promoter in human serum

Abstract Human transferrin was shown to greatly enhance the growth of lymphocytes in response to PHA and antigens in vitro. The growth promoting action of transferrin could not be supplanted by FeCl 3 . Transferrins from other species varied in their ability to support growth of human lymphocytes stimulated by PHA.

Molecular events in the reproduction of animal cells: III. Fractional synthesis of deoxyribonucleic acid with 5-bromodeoxyuridine and its effect on cloning efficiency

Abstract Synchronized cultures of HeLa cells were caused during a single cycle of DNA synthesis to replicate given fractions of their DNA with 5-bromodeoxyuridine (BdeU) instead of thymidine. It was observed that the introduction of 5-bromodeoxyuridine into the early replicating DNA complexes resulted in a striking decline in the cloning efficiency whereas the introduction of 5-bromodeoxyuridine into the late replicating DNA was without effect. The data suggest that the early replicating DNA of the HeLa nucleus plays an important role in survival.

Effect of puromycin in vivo on the synthesis of protein, RNA and phospholipids in rat tissues.

Abstract The intraperitoneal injection of 15 mg. puromycin every hour for 4 hr. into 250–300-g. rats reduced protein synthesis in vivo in uteri, liver, heart, kidney, and thymus to 10, 15, 36, 40, and 49% of controls, respectively. Under these same conditions ribonucleic acid (RNA) synthesis was not inhibited in any tissue studied except the thymus. The synthesis of phospholipid in the liver and uteri was not inhibited, but a stimulatory response to puromycin was observed. These results are discussed with regard to the use of puromycin in studying the role of protein synthesis in certain physiological processes.

Studies on the nature of replicating DNA of HeLa cells

Abstract Replicating DNA from synchronized HeLa cells pulse labeled with radioactive thymidine differed from non-replicating DNA by its partition to the interphase fraction during extraction with phenol or chloroform. Pulse labeling followed by a chase with non-isotopic thymidine demonstrated that DNA of the interphase fraction was converted to a form which was extractable in the aqueous phase. Blocking DNA synthesis with hydroxyurea prevented this conversion. Sonication of the interphase DNA demonstrated that the property causing the extraction of the DNA into the interphase was localized at or near the site of active replication. Centrifugation of the replicating DNA isolated from the phenol-water interphase revealed that the labeled material separated into two fractions: one which sedimented rapidly to the bottom of a sucrose gradient, but floated in CsCl density gradient; and a fraction which sedimented slightly slower than non-replicating DNA in sucrose gradient but exhibited the same buoyant density in CsCl. The rapidly sedimenting material was converted to a slowly sedimenting material with the same buoyant density as non-replicating DNA by heat, alkali, or sonication, but was unaffected by pronase. Evidence for attachment of a cellular component to replicating DNA is discussed.