Activity of MGCD290, a Hos2 histone deacetylase inhibitor, in combination with azole antifungals against opportunistic fungal pathogens.

We report on the in vitro activity of the Hos2 fungal histone deacetylase (HDAC) inhibitor MGCD290 (MethylGene, Inc.) in combination with azoles against azole-resistant yeasts and molds. Susceptibility testing was performed by the CLSI M27-A3 and M38-A2 broth microdilution methods. Testing of the combinations (MGCD290 in combination with fluconazole, posaconazole, or voriconazole) was performed by the checkerboard method. The fractional inhibitory concentrations were determined and were defined as <0.5 for synergy, >or=0.5 but <4 for indifference, and >or=4 for antagonism. Ninety-one isolates were tested, as follows: 30 Candida isolates, 10 Aspergillus isolates, 15 isolates of the Zygomycetes order, 10 Cryptococcus neoformans isolates, 8 Rhodotorula isolates, 8 Fusarium isolates, 5 Trichosporon isolates, and 5 Scedosporium isolates. MGCD290 showed modest activity when it was used alone (MICs, 1 to 8 microg/ml) and was mostly active against azole-resistant yeasts, but the MICs against molds were high (16 to >32 microg/ml). MGCD290 was synergistic with fluconazole against 55 (60%) of the 91 isolates, with posaconazole against 46 (51%) of the 91 isolates, and with voriconazole against 48 (53%) of the 91 isolates. Synergy between fluconazole and MGCD290 was observed against 26/30 (87%) Candida isolates. All 23 of the 91 Candida isolates that were not fluconazole susceptible demonstrated a reduced fluconazole MIC that crossed an interpretive breakpoint (e.g., resistant [MIC, >or=64 microg/ml] to susceptible [MIC,

Phorbol esters rapidly stimulate amiloride-sensitive Na+/H+ exchange in a human leukemic cell line.

The human, leukemic cell line, HL-60, undergoes differentiation in response to tumor-promoting phorbol esters. Recent studies have implicated stimulation of a Na+/H+ antiporter as an initial event in cellular differentiation and/or proliferation. The effects of phorbol esters on Na+-dependent H+ efflux from HL-60 cells were studied by pH-stat titration. Tumor-promoting phorbol diesters, but not the inactive parent alcohol, stimulated Na+-dependent H+ efflux in a rapid (within 1 min at 37 degrees C) and reversible manner. Stimulation was dependent on the concentration of extracellular sodium; lithium could substitute for sodium, but choline could not. Stimulation was dependent on the activity of extracellular protons and was inhibited completely by amiloride. The concentrations of phorbol diesters at which we observed half-maximal stimulation of Na+-dependent H+ efflux are very similar to the Kd reported in the literature for binding of these phorbol diesters to the phorbol ester receptor and the Km for phorbol diester activation of protein kinase C. Overall characterization of basal and phorbol ester-stimulated H+ efflux indicate that stimulation of a Na+/H+ antiporter constitutes a primary event in phorbol ester interaction with HL-60 cells.

Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation.

The uptake and fate of pinocytosed fluid were investigated in monolayers of pulmonary alveolar macrophages and fetal lung fibroblasts using the fluid-phase marker, [14C]sucrose. Initial experiments revealed that cellular accumulation of chromatographically repurified [14C]sucrose was not linear with incubation time. Deviation from linearity was shown to be due to constant exocytosis of accumulating marker. Chromatographic analysis revealed that the cells were unable to metabolize sucrose and were releasing it intact by a process that was temperature-sensitive but not dependent on extracellular calcium and magnesium. A detailed analysis of the kinetics of exocytosis was undertaken by preloading cells with [14C]sucrose for various lengths of time and then monitoring the appearance of radioactivity into isotope-free medium. Results indicated that modeling the process of fluid-phase pinocytosis and subsequent exocytosis required at least two intracellular compartments in series, one compartment being of small size and turning over very rapidly (t1/2 = 5 min in macrophages, 6--8 min in fibroblasts) and the other compartment being apparently larger in size and turning over very slowly (t1/2 = 180 min in macrophages, 430--620 min in fibroblasts). Computer-simulation based on this model confirmed that the kinetics of efflux faithfully reflected the kinetics of influx and that the rate of efflux completely accounted for the deviation from linearity of accumulation kinetics. Moreover, the sizes of the compartments and magnitude of the intercompartment fluxes were such that the majority of fluid internalized in pinocytic vesicles was rapidly returned to the extracellular space via exocytosis. This result provides direct experimental evidence for a process previously thought necessary based solely on morphological and theoretical considerations. Furthermore, the turnover of pinocytosed fluid was so dynamic that accumulation deviated from linearity even within the first few minutes of incubation. We were able to show that the kinetics of exocytosis allowed calculation of the actual pinocytic rate, a rate that was nearly 50% greater than the apparent initial rate obtained from the slope of the uptake curve over the first 10 min.

Pinocytosis and intracellular degradation of exogenous protein: modulation by amino acids.

Intracellular degradation of exogenous (serum) proteins provides a source of amino acids for cellular protein synthesis. Pinocytosis serves as the mechanism for delivering exogenous protein to the lysosomes, the major site of intracellular degradation of exogenous protein. To determine whether the availability of extracellular free amino acids altered pinocytic function, we incubated monolayers of pulmonary alveolar macrophages with the fluid-phase marker, [14C]sucrose, and we dissected the pinocytic process by kinetic analysis. Additionally, intracellular degradation of endogenous and exogenous protein was monitored by measuring phenylalanine released from the cell monolayers in the presence of cycloheximide. Results revealed that in response to a subphysiological level of essential amino acids or to amino acid deprivation, (a) the rate of fluid-phase pinocytosis increased in such a manner as to preferentially increase both delivery to and size of an intracellular compartment believed to be the lysosomes, (b) the degradation of exogenously supplied albumin increased, and (c) the fraction of phenylalanine derived from degradation of exogenous albumin and reutilized for de novo protein synthesis increased. Thus, modulation of the pinosome-lysosome pathway may represent a homeostatic mechanism sensitive to the availability of extracellular free amino acids.

Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination.

Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating spermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replication. In contrast, elevated levels of DNA ligase III mRNA were observed in primary spermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells.

Elevation of topoisomerase I messenger RNA, protein, and catalytic activity in human tumors: demonstration of tumor-type specificity and implications for cancer chemotherapy.

Topoisomerase I has been identified as an intracellular target of camptothecin, a plant alkaloid with anticancer activity. Various lines of evidence suggest that the sensitivity of cells to this drug is directly related to the topoisomerase I content. In humans, the levels of topoisomerase I have been shown to be elevated in colorectal tumors, compared to normal colon mucosa. The aim of our study was to determine whether (a) topoisomerase I levels are elevated in other solid tumors, (b) the elevated enzyme is catalytically active in these tumors, and (c) the increase in topoisomerase I levels in colorectal tumors is a result of increased transcription or translation. Topoisomerase I levels were quantitated in crude extracts from colorectal, prostate, and kidney tumors and their matched normal counterparts by Western blotting and by direct determination of catalytic activity, and mRNA levels were determined by Northern blotting. By Western blotting, colorectal tumors showed 5-35-fold increases in topoisomerase I levels, compared to their normal colon mucosa. In the case of prostate tumors, the increase was 2-10-fold, compared with benign hyperplastic prostate tissue from the same patients. However, no difference was observed in topoisomerase I levels in kidney tumors, compared to their normal counterparts. The catalytic activity of topoisomerase I was determined by a quantitative 32P-transfer assay in crude homogenates, without isolating nuclei. Colorectal and prostate tumors exhibited 11-40- and 4-26-fold increases, respectively, in catalytic activity. However, kidney tumors did not show any alteration in catalytic activity, compared to their normal matched samples. Thus, for all three tumor types there was a good correlation between enzyme levels and catalytic activity. Finally, colorectal tumors were analyzed for steady state mRNA levels. A 2-33-fold increase in mRNA levels was found in colorectal tumors, compared to normal colon mucosa. These results suggest that alterations in topoisomerase I expression in humans are tumor type specific and that the increase in topoisomerase I levels results from either increased transcription of the topoisomerase I gene or increased mRNA stability.

In vivo antitumor activity of two new seven-substituted water-soluble camptothecin analogues.

The development of camptothecin-like compounds as inhibitors of topoisomerase I for the treatment of resistant tumors has generated clinical excitement in this new class of drugs. We have developed two novel water-soluble camptothecin analogues which are specific inhibitors of topoisomerase I and are potent cytotoxins with significant antitumor activity. We added water-solubilizing groups off position 7 in the B ring of either 10,11-ethylenedioxy- or 10,11-methylenedioxy-20(S)-camptothecin. These water-soluble camptothecin analogues were demonstrated to be nanamolar inhibitors of the topoisomerase I enzyme in the cleavable complex assay. The compounds, GI147211 [7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camp tot hecin], and GI149893 [7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-cam pto thecin], were compared to topotecan, a known water-soluble inhibitor of topoisomerase I. Both GI compounds were found to be slightly more potent than topotecan as inhibitors of topoisomerase I in the cleavable complex assay and were 1.5-2 times more soluble. Tumor cell cytotoxicity assays using 5 separate cell lines demonstrated that both GI compounds were 5-10 times more potent than topotecan, although by comparison all three topoisomerase I inhibitors were unaffected by the multidrug resistance P-glycoprotein. The antitumor activity of all three topoisomerase I inhibitors was compared concomitantly in two human colon xenograft models. In both models, GI147211 and GI149893 were able to induce regression of established HT-29 and SW-48 colon tumors by as much as 60%. The antitumor activity of both compounds were also demonstrated in the MX-1 and PC-3 xenografts. Microscopic examination of selected tissues indicated that drug-induced toxicity was primarily limited to the gastrointestinal tract and was comparable among the three compounds. Further clinical development of this class of compounds is ongoing.

Rigid analogs of camptothecin as DNA topoisomerase I inhibitors.

Substituted 8-ethyl-2-(2-oxo-1,2-dihydroindol-3-ylidene)-8-hydroxy-2,3,5,8- tetrahydro-6-oxa-3a-azacyclopenta[b]naphthalene-1,4,7-triones were synthesized and evaluated as topoisomerase I inhibitors in an in vitro cleavable complex assay. The activity of these compounds may be attributed to their rigid, planar geometry, and an attempt was made to correlate the SAR in this series to known attributes of camptothecin.

Plant antitumor agents. 30. Synthesis and structure activity of novel camptothecin analogs.

A large number of camptothecin (CPT) analogs have been prepared in the 20S, 20RS, and 20R configurations with a number of ring A substituents. Topoisomerase I (T-I) inhibition data (IC50) have been obtained by standard procedures. In general, substitution at the 9 or 10 positions with amino, halogeno, or hydroxyl groups in compounds with 20S configuration results in compounds with enhanced T-I inhibition. Compounds in the 20RS configuration were less active in vitro and in vivo and those in the 20R configuration were inactive. Compounds with 10,11-methylenedioxy substitution on ring A displayed a marked increase in potency in the T-I inhibition assay. The activities of some of the analogs as determined in a variety of in vivo assays including the L-1210 mouse leukemia assay were, in general, in accord with T-I inhibition. A number of water-soluble analogs such as 20-glycinate esters, 9-glycinamides, or hydrolyzed lactone salts were prepared and tested in in vitro and in vivo assays. In general, these compounds were less active than CPT both in terms of T-I inhibition and life prolongation in the L-1210 assay. However, certain 20-glycinate esters showed good in vivo activity after iv administration.

Synthesis and antitumor activity of novel water soluble derivatives of camptothecin as specific inhibitors of topoisomerase I.

The synthesis and antitumor activities of the novel water soluble camptothecin derivatives 7-[(4-methylpiperazino)methyl]-10,11-(methylenedioxy)-(20S)-campto thecin trifluoroacetate (6) and 7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-camptot hecin trifluoroacetate (7) are described. The solubilities of compounds 6 and 7 were measured to be 4.5 and 5.8 mg/mL, respectively, in pH 5 acetate buffer in contrast to < 0.003 mg/mL for camptothecin in the same buffer. In the purified topoisomerase I cleavable complex enzyme assay, compounds 6 and 7 demonstrated potent inhibition of topoisomerase I with IC50's of 300 and 416 nM, respectively, in comparison to 679 nM for camptothecin and 1028 nM for topotecan. In human tumor cell cytotoxicity assays, compounds 6 and 7 demonstrated potent antitumor activity against ovarian (SKOV3), ovarian with upregulated MDRp-glycoprotein (SKVLB), melanoma (LOX), breast (T47D), and colon (HT29) with IC50's ranging from 0.5 to 102 nM. Compounds 6 and 7 induced tumor regressions in the HT29 human colon tumor xenograft model and demonstrated similar rank order of potency compared to in vitro assay results.

Water soluble inhibitors of topoisomerase I: quaternary salt derivatives of camptothecin.

Eleven water soluble 7-substituted quaternary ammonium salt derivatives of 10,11-(methylenedioxy)- and 10,11-(ethylenedioxy)-(20S)-camptothecin were synthesized via the Friedlander reaction followed by nucleophilic displacement with an aromatic amine. All of these compounds were more potent than camptothecin in the in vitro cleavable complex assay. These inherently charged camptothecin derivatives were cytotoxic against three different human tumor cell lines (SKOV3, an ovarian adenocarcinoma; SKVLB a multidrug resistant ovarian adenocarcinoma; and HT-29, a colon carcinoma). A selected group of five compounds was evaluated in the nude mouse HT-29 xenograft model. Two of these quaternary salts (17 and 18) were more efficacious than Topotecan in delaying tumor growth. In an extended in vivo model, 18 demonstrated tumor regression.

Topoisomerase I inhibition by the camptothecin analog Gl147211C. From the laboratory to the clinic.

The development of camptothecin-like compounds as inhibitors of DNA topoisomerase I for the treatment of solid tumors has generated clinical excitement in this new class of drugs. We have discovered, developed, and entered into clinical trial a novel, potent, and water-soluble camptothecin analog with significant antitumor activity. This compound, Gl147211C [7-(4-methylpiperaziinomethylene)-10, 11-ethylenedioxy-20(S)-camptothecin hydrochloride] is a specific inhibitor of DNA topoisomerase I. Compared to topotecan, Gl147211C is approximately three times as potent in the cleavable complex assay and approximately twice as soluble in aqueous medium. Human tumor cell line cytotoxicity assays indicated that Gl147211C was approximately 3- to 5-fold more potent than topotecan, while both compounds were relatively insensitive to the multidrug resistance P-glycoprotein. The in vivo preclinical antitumor activity of Gl147211C was compared to topotecan in an array of human tumor xenograft models in nude mice. In general, Gl147211C was able to induce regression of established tumors whereas topotecan was not. Microscopic evaluation of necropsied tissues indicated that drug-induced toxicity was mild, primarily limited to the gastrointestinal tract, and was comparable for both Gl147211C and topotecan. Based on these observations, Gl147211C moved through preclinical development and subsequently into Phase I clinical trial. A summary of Phase I trial results to date is provided.

Cell-cycle specific cytotoxicity mediated by rearranged ent-kaurene diterpenoids isolated from Parinari curatellifolia.

Two structurally novel cytotoxic ent-kaurene diterpenoids, 13-methoxy-15-oxozoapatlin and 13-hydroxy-15-oxozoapatlin, were isolated from the root bark of Parinari curatellifolia, together with the known compound, 15-oxozoapatlin, on the basis of bioactivity-guided chromatographic fractionation and found to demonstrate broad-spectrum cytotoxic activity against a panel of cultured human cancer cell lines. The structures of these compounds were determined by analysis of their spectroscopic data. The presence of an alpha, beta-unsaturated carbonyl group in 13-methoxy-15-oxozoapatlin suggested that the cytotoxic potential of this compound could be mediated through reaction with cellular nucleophiles by means of a Michael-type addition. The compound 13-methoxy-15-oxozoapatlin reacted with the nucleophiles L-cysteine and beta-mercaptoethanol. The adduct with beta-mercaptoethanol was isolated, structurally characterized and found to be approximately 5-fold less cytotoxic than 13-methoxy-15-oxozoapatlin itself. The compound 13-methoxy-15-oxozoapatlin did not interact with DNA nor guanosine, and it was not mutagenic for Salmonella typhimurium strain TM677. The effects of 13-methoxy-15-oxozoapatlin on the growth of human cancer cells were analyzed utilizing cultured ZR-75-1 breast cancer cells. Biosynthesis of DNA, RNA and protein was reduced in treated cells, and accumulation at the G2/M phase of the cell cycle was observed. The compound 13-methoxy-15-oxozoapatlin did not mediate antimitotic activity with dibutyryl cAMP-treated cultured astrocytoma cells, suggesting that the cell cycle effect is G2 specific. No antitumor activity was observed when athymic mice carrying KB cells were treated with 13-methoxy-15-oxozoapatlin. These data indicate that the cytotoxic activity of 13-methoxy-15-oxozoapatlin is mediated in part by covalent reaction with a cellular component (such as sulfhydryl-containing protein) by means of a Michael-type addition, and this results in the blockage of cell-cycle progression.

Association and metabolism of exogenously-derived lysophosphatidylcholine by cultured mammalian cells: kinetics and mechanisms.

The association and metabolism of exogenously-derived lysophosphatidylcholine (lysoPC) with cultured mammalian cells from a variety of sources was studied, and a mechanism was defined by computer modeling for Vero cells. Cell monolayers were incubated with radiolabeled lysoPC, and the kinetics of disappearance from the medium, association with the cells, and metabolism by the cells of lysoPC were monitored both in the absence and in the presence of fetal bovine serum. Exogenously-supplied lysoPC first associated with cell membranes, followed by an almost complete conversion to phosphatidylcholine (PC). The kinetics of partitioning and metabolism were identical regardless of whether the exogenously-supplied lysoPC was labeled with [methyl-3H]choline or with [1-14C]palmitate. A two-step mechanism, consisting of a reversible partitioning of exogenous lysoPC into the cell membrane followed by enzymatic reacylation of PC, was found to adequately describe the observed kinetics in the presence of 0 or 0.5% fetal bovine serum. The effect of temperature on the individual rate constants and on the overall process was examined. An Arrhenius plot indicated an acute temperature sensitivity between 15 and 23 degrees C, consistent with a dependence on the lipid phase of the membrane and a regional phase transition temperature characteristic of mammalian cells. The acute temperature sensitivity was almost entirely due to the temperature dependence of reacylation. A multistep mechanism was established by combining the kinetic constants determined under conditions of low exogenous protein with the binding constant between lysoPC and serum protein. This mechanism accurately predicts the rates of uptake of exogenously-derived lysoPC with cultured cells in the presence of serum concentrations between 0 and 10%. A survey of a variety of cultured cells indicated that the kinetics of association and metabolism of exogenously-derived lysoPC is cell-type specific.

Drug-induced alterations of tumor necrosis factor-mediated cytotoxicity: discrimination of early versus late stage action.

The killing of L-M cells by murine tumor necrosis factor (mTNF) was investigated by a combination of drug, antiserum neutralization, and kinetic studies. Kinetic studies with 125I-mTNF showed that the bulk of association of ligand with L-M cells peaked within 2 hr and the ligand was not degraded. Cell surface receptors were depleted (down regulated) by 6 hr when death commenced. The off-rates of acid-dissociable (surface bound) and acid-indissociable (internalized) compartments were found to be 9 min and 35 hr, respectively. Nevertheless, complete cell killing required the persistent presence of mTNF for up to 20 hr. This requirement was ablated by the concomitant addition of cycloheximide. Antiserum completely inhibited cytotoxicity when it was applied up to 4 hr after mTNF, but antiserum added 1 hr after mTNF was not neutralizing in the presence of cycloheximide. Thus, the inclusion of cycloheximide temporally dissociated early events (internalization and signal transduction) from lysis. Other drugs with and without cycloheximide were found to preferentially affect either early or later aspects of cell death. Phorbol myristate acetate and the ionophore A23187 were potent inhibitors of cytotoxicity, and staurosporine was a potent enhancer. These agents were more effective when added 1 hr before mTNF and cycloheximide than when added 1 hr after and likely affected early events in the cytolytic program. In contrast, chloroquine and cAMP likely affect more terminal aspects of cytotoxicity. Dibutyrylcyclic AMP, cholera, and pertussis toxins enhanced cytotoxicity. They were equipotent when added either before or after mTNF regardless of the presence of cycloheximide. Likewise, chloroquine was an equipotent inhibitor when added either before or after mTNF regardless of the presence of cycloheximide. Agents that primarily affect association events may be more likely to impinge on other TNF-mediated activities than agents that primarily affect lysis.

Cytosolic rat brain synapsin I is a diacylglycerol kinase.

The phosphorylation of diacylglycerol (DG), a reaction catalyzed by DG kinase, may be critical in the termination of effector-induced signals mediated by protein kinase C. Synapsin I is a principal target of intracellular protein kinases and is thought to be involved in the release of neurotransmitter from axon terminals. We present several lines of evidence which indicate that rat brain synapsin, in addition to this role, may function as a DG kinase. Purified rat brain DG kinase was digested with trypsin, which produced three major fragments whose sequence was identical to three regions in synapsin I. Using a rabbit anti-synapsin polyclonal antiserum, the elution profile of synapsin immunoreactivity coincided exactly with that of DG kinase activity in column fractions from the final step in the DG kinase purification procedure. As is the case with synapsin, the purified enzyme was a strongly basic protein with an isoelectric point greater than 10.0. Finally, incubating the DG kinase with highly purified bacterial collagenase, an enzyme that partially degrades the proline- and glycine-rich synapsin, resulted in the simultaneous loss of DG kinase activity and synapsin immunoreactivity. We conclude that cytosolic rat brain synapsin is capable of functioning as a DG kinase.

Macrophage phagocytosis: analysis of particle binding and internalization.

An improved radioassay for analysis of phagocytosis has been used to quantitate and characterize the binding and internalization of zymosan by monolayers of rabbit pulmonary alveolar macrophages. This method distinguishes zymosan particles reversibly bound to the cell surface from those internalized. The zymosan was radiolabeled with technetium 99m(99mTc), a gamma-emitter with a 6-h half-life. Use of 99mTc as the radiolabel also permitted simultaneous determinations of pinocytosis and cellular protein content using [14C]sucrose and [3H]amino acids, respectively. All endocytic data were normalized per adherent cell based on this latter measurement. A significant fraction of the cell-associated particles was bound to the cell surface but not internalized. Failure to correct for this compartment would have resulted in overestimation of phagocytic rate and total cellular capacity. Both binding and ingestion of functionally unopsonized zymosan were found to be saturable, temperature sensitive, dependent on glycolytic energy, dependent on a trypsin-sensitive membrane component, described by a maximal rate, and limited by a finite capacity. The time courses of both processes were found to be similar. These results led us to conclude that, in our system, both binding and internalization were active processes and that the limited capacity to ingest zymosan was not explained by a concomitant reduction in binding of the particle to the cell membrane. Furthermore, it was found that phagocytosis did not change the rate of fluid-phase pinocytosis, consistent with the concept that the cell membrane is a functional mosaic as has been previously found by others for phagocytic and transport sites in this cell type.

DNA triple-helix formation at physiologic pH and temperature.

Oligonucleotides that form a triple helix with duplex DNA offer a novel way to site specifically regulate gene expression in vivo. Triple helices formed by homopyrimidine oligomers containing both cytosine and thymine are stabilized by acid pH and low temperature, and there is little information about triplex formation with these oligomers at both pH 7.5 and 37 degrees C. Therefore, we examined the effect of changing various conditions on triplex formation at pH 7.5. A 30-mer oligonucleotide (composed of T and 5-methyl C) at submicromolar concentrations formed a triplex with its target duplex at pH 7.5 and 37 degrees C. Association of the 30-mer oligomer with the duplex was slow, with complete association requiring about 1 h. At 37 degrees C, a 21-mer oligomer bound weakly to the target duplex but both a 25-mer and the 30-mer readily formed a triplex. This relationship of triplex formation with length was temperature dependent, as at 25 degrees C the 21-mer behaved similarly to the longer oligomers. Increasing spermine concentrations (from 0.2 to 1 mM) increased the amount of triplex formed. Spermine may be important only for the association of the oligomer to the duplex, since decreasing the spermine concentration after the triplex formed did not reduce the amount of triplex detected. At 1 mM spermine, formation of the triple-helical complex was very dependent on the concentration of KCl; increasing the KCl from 50 to 100 mM prevented triplex formation. However, the inhibitory effect of KCl could be abrogated by raising the spermine concentration to 2 mM. Our observations indicate that a triple helix can form under physiologic conditions but its formation is affected by several competing interactions.

Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger.

The classic pathway for agonist-induced generation of diacylglycerol is via activation of a phospholipase C-mediated hydrolysis of the "phosphoinositides." We now report findings from a variety of cell types, which indicate that tumor-promoting phorbol diesters, serum, and platelet-derived growth factor activate within seconds the hydrolysis of phosphatidylcholine, as detected by the formation of diacylglycerol and phosphocholine. It is known that phorbol diesters do not stimulate hydrolysis of the phosphoinositides. Yet, in cells prelabeled with either [14C]oleate or [32P]orthophosphate, addition of the tumor promoter phorbol dibutyrate (PBt2) resulted in the rapid generation of both diacylglycerol and phosphatidate in a time- and dose-dependent manner. The fatty acid composition of the phosphatidate most resembled the fatty acid profile of phosphatidylcholine from the same cell type. Taken together, these findings suggested a role for protein kinase C in the generation of diacylglycerol (and phosphatidate) from phosphatidylcholine. To define further the pathways involved, the metabolism of cellular phosphatidylcholine was studied. In cells prelabeled with [3H]choline, addition of PBt2, but not 4 alpha-phorbol, stimulated the formation of intracellular phosphocholine within 45 sec. Furthermore, addition of platelet-derived growth factor (PDGF) or serum to "serum-starved" cells prelabeled with [3H]choline resulted in increased levels of intracellular phosphocholine within 15-30 sec. Thus, the data suggest that agonists that stimulate protein kinase C either directly (e.g., PBt2) or indirectly via activation of phosphoinositide hydrolysis (e.g., PDGF and serum) may stimulate degradation of phosphatidylcholine by phospholipase C in intact cells. However, prior down-regulation of protein kinase C by prolonged pretreatment of cells with PBt2 almost totally abolished subsequent stimulation of phosphatidylcholine degradation by PBt2 but only partially attenuated subsequent stimulation by PDGF and serum. These observations suggest that PDGF and serum act, at least partially, through a protein kinase C-independent mechanism. Lastly, the size of the cellular choline and CDP-choline pools were shown to be small and relatively insensitive to agonist addition, as compared to the size and behavior of the phosphocholine pool. Thus, the rapidly increased levels of phosphocholine (and diacylglycerol) arising in response to agonist addition appear to be derived directly from phosphatidylcholine by a phospholipase C-mediated mechanism.