In vitro inhibition of nitrogen mustard efficacy by postincubation of treated cells in serum protein.

Lettre-Ehrlich cells were loaded with sufficient HN2 to produce about a 98% cell kill. Postincubation of the HN2-loaded cells in PBS resulted in the loss of about 40% of their HN2 without changing the cytolytic effect, supporting the proposal that only bound drug was effective. Postincubation of the HN2-loaded cells in PBS which contained 2% bovine serum albumin or in cell-free mouse ascitic fluid (1.8% protein) resulted in the same relative cellular HN2 loss as well as a 79% decrease in the cell kill. The cytolytic effect of HN2 is believed to be dependent on the degree to which the drug crosslinks DNA in 2 sequential reactions. It seems likely that such crosslinking occurred in nearly all of the PBS-postincubated cells, as they were nearly all killed. By analogy, albumin postincubation apparently blocked the competition of such crosslinking.

Choline uptake is increased by Ca++ and liposomes+ Ca++ in ascites tumor cells.

Steady-state uptake of choline by Lettre-Ehrlich tumor cells in vitro, resulting in cell-to-medium ratios of 10 or more, is significantly increased by 0.2-1.0 mM Ca++ as well as by dipalmitoyl phosphatidyl choline multilamellar liposomes + Ca++. The increases occur in spite of a decrease in carrier affinity, as indicated by the Km, and therefore result either from increased carrier velocity or utilization of new carriers. About half of the labelled choline which is taken up is firmly bound to cells. That label which freely leaves cells is phosphocholine, thus, these cells utilize choline mainly in phospholipid synthesis. Choline and nitrogen mustard (HN2) share a plasma membrane carrier but the intracellular distribution of HN2 into DNA, RNA and protein, contrasts with that of choline, into phospholipid.

Modulation of the cellular toxicity of nitrogen mustard in murine cells.

A linear increase in cell uptake of nitrogen mustard, methyl-bis(beta-chloroethyl)amine (HN2), between 1 and 5 min, was observed after in vitro incubation of Ehrlich ascites tumor cells at 37 degrees C in phosphate-buffered saline containing HN2 followed by washing in 0 degrees C phosphate-buffered saline. After a second incubation in 37 degrees C phosphate-buffered saline without HN2, the cells lost about one-half of the drug which had been taken up, that which had not been covalently bound to macromolecules. The basal cytotoxic effect of HN2 on the cells was determined using a standard in vivo test for cell viability. Host survival was measured after 10(5) HN2-treated cells were injected i.p. into recipient mice, compared with injection of 10(5) untreated cells into paired control mice. Five min incubation of cells in vitro with multilamellar liposome vesicles (MLV) composed of L(alpha)dipalmitoyl-phosphatidyl choline in the presence of HN2, significantly increased tumor cell kill and mouse survival over HN2 alone. In contrast, added Ca2+ plus HN2 decreased cytotoxicity and survival. Significant increases in host survival following MLV treatment occurred without significant increase in total HN2 uptake and could be highly correlated with increased amounts of HN2 bound to DNA. Addition of vincristine (an inhibitor of microtubule polymerization) in the presence of HN2 also decreased the cytotoxic effect of HN2. The vincristine inhibition occurred, without altering total cell HN2 uptake, whether L(alpha)dipalmitoyl-phosphatidyl choline MLV were present or not. It is proposed that both Ca2+ and MLV act at membrane sites so as to alter the subcellular distribution and localization of HN2 and its accessibility to critical targets. This has been confirmed for MLV by demonstrating increased alkylation of DNA.

Relative muscarinic synaptic activation by anionic, neutral or cationic liposomes.

Liposomes made of dipalmitoyl phosphatidyl choline (DPC) and safflower oil fatty acids produce a contraction of the guinea pig ileum longitudinal muscle myenteric plexus preparation (LMMP). Stimulating the LMMP while liposomes are present yielded greater than control contractions, an effect which remained for many minutes after liposomes had been washed out. Neutral (DPC) liposomes produced a direct transitory decrease in basal tension and phasic activity. Stimulation in the presence of neutral liposomes resulted in slower attainment of maximal contraction. Cationic liposomes(DPC + oleylamine) had little direct effect, but slightly decreased stimulus evoked maximal contraction.

Calcium antagonism of anionic liposome actions at a muscarinic synapse.

Addition of 0.5 mM Ca++ to guinea pig ileum longitudinal muscle-myenteric plexus preparation, which is contracted under the influence of anionic liposomes, is followed by a rapid, temporary, relaxation. Such Ca++ addition produces no change in the field-stimulated preparation, eliminating direct Ca++ effects or neuronal acetylcholine release nor in the preparation stimulated by added acetylcholine, eliminating direct Ca++ effects on muscle. These eliminations leave the Ca++ interaction with liposomes, probably causing self-fusion, and loss from interaction with cholinergic neurons, as the mechanism for Ca++-induced relaxation.

Alterations of muscarinic synaptic activity by anionic liposomes.

Liposomes made of phosphatidyl serine and dioleylphosphatidic acid have two effects on contraction of the guinea pig ileum longitudinal muscle-myenteric plexus preparation. First, the liposomes directly stimulate contraction, apparently through acetylcholine release. Second, after the liposomes are removed from the Tyrode solution bathing the preparation, electrical field stimulation or acetylcholine addition produce contractions which are significantly less than control. The acetylcholine release effect is presynaptic, on the nerve terminal, while the decreased responsiveness effect is postsynaptic, at the level of the muscarinic receptor.

Differential enhancement of antitumor effectiveness by phospholipid vesicles (liposomes).

The simultaneous administration of dipalmitoylphosphatidylcholine liposomes and methyl-bis(beta-chloroethyl)amine (HN2) to Ehrlich ascites tumor-bearing mice results in prolongation of survival, reduction of toxicity, and increase in chemotherapeutic index when compared to HN2 alone. Delay for as little as 10 min in the administration of HN2 following the liposomes eliminates this enhancement of activity and, in fact, abolishes much of the chemotherapeutic activity of the alkylating agent itself. The enhancement phase of liposomal action correlates with a significant increase in HN2 uptake by tumor cells which cannot be due to entrapment of drug in the liposomes, while the reduced toxicity could reflect subsequent HN2 transport. Persistent membrane alteration is also seen in the contrasting case of the lipid-soluble alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea where advanced administration of the liposome preparation significantly increases the chemotherapeutic activity. The observed effects are also shown to be influenced by liposome composition. The hypothesis is advanced that, under the given experimental conditions, liposomes can cause persistent reorganization of cell membranes which follow a characteristic course and have specific features.

Relative enhancement by various liposomes of BCNU effectiveness against L-1210 leukemia in vivo.

When maximal effective dose (MED) of BCNU is injected together with liposomes to mice with L-1210 leukemia, the effectiveness of the drug is decreased. But when half the MED is injected with liposomes into such mice, survival is increased compared to mice injected with BCNU alone. Study of liposome composition on BCNU anti-leukemic effect revealed that negatively charged liposomes made of dioleylphosphadyl choline plus phosphatidyl serine given with half the MED of BCNU increased both long-term survival and mean survival time. This enhancement was not produced with negatively charged liposomes made of dioleylphosphatidic acid alone or in liposomes in which phosphatidyl serine plus dioleylphosphatidic acid was diluted with the neutral phospholipid, dioleylphosphatidyl choline.

Messenger ribonucleic acid metabolism in mammalian mitochondria. Isolation and characterization of polyribosomes from Ehrlich ascites mitochondria.

The Mg2+ precipitation procedure of R. D. Palmiter ((1974) Biochemistry 13, 3606) has been used for preparative scale isolation of polysomes from Ehrlich ascites mitochondria. Digitonin-washed metochondria used for isolating the polysomes contain no detectable reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and over 200-fold reduced hexokinase activity. The mitochondrial polysomes exhibit a heterogeneous sedimentation and appear to contain highly aggregated particlses ranging over hexamers. These polysomes are sensitive to RNase, (ethylenedinitrilo)tetraacetic acid and puromycin. Mitochondrial polysomes are active in portein synthesis when supplied with supernatant enzymes from the homologous mitochondrial source or from Escherichia coli. Cytoplasmic enzymes, however, appear to be completely inactive. Protein synthesis by mitochrondrial polysomes is sensitive to chloramphenicol and resistant to cycloheximide and emetine. The procedure yields particles containing intact rRNAs. The extent of cytoplasmic RNA contaminating the total mitochondrial RNA or mitochondrial polysomal RNA has been estimated to be negligible.

Messenger ribonucleic acid metabolism in mammalian mitochondria. Discrete poly(adenylic acid) lacking messenger ribonucleic acid species associated with mitochondrial polysomes.

The mRNA species released from mitochondrial polysomes prepared by the Mg2+ precipitation technique have been further characterized using various analytical techniques. Mitochondrial polysomes were dissociated by treatment with puromycin and chemically labeled with (3H) dimethyl sulfate. About 51% of steady-state mitochondrial mRNA bind to oligo(dT)-cellulose indicating the presence of poly(adenylic acid)(poly(A)) in this fraction. The poly(A)-containing mRNAs resolve into discrete bands of 9-16 Se, while the RNA fraction unable to bind to oligo(dT)-cellulose representing poly(A)-lacking mRNA contains 8-12 Se species. About 90% of poly(A) lacking RNA hybridizes with mitochondrial DNA and less than 7% hybridizes with nuclear DNA. The extent of hybridization of poly(A)-lacking RNA with mitochondrial DNA was not significantly affected by the presence of excess mitochondrial rRNA, cytoplasmic rRNA, or a tenfold concentration of poly(A)-containing RNA isolated from total mitochondrial RNA. Possible differences in sequence properties between poly(A)-containing and -lacking mitochondrial mRNAs were further verified using a solid phase-bound cDNA procedure. Poly(A)-containing mRNA released from mitochondrial polysomes shows over 85% sequance homology with oligo(dT)-cellulose-bound cDNA prepared against total mitochondrial poly(A)-lacking mitochondrial mRNA hybridizes with the cDNA providing direct evidence for the distinct sequence properties of the two mRNA species.

Comparison of the effects of nitrogen mustard on the functional properties of mitochondria from sensitive and resistant strains of Ehrlich ascites tumors.

In vivo treatment of sensitive tumor cells with nitrogen mustard (HN2) results in marked inhibitions of protein and nucleic acid synthesis by mitochondria subsequently isolated from these cells. This inhibition occurs at doses of drug which produce no apparent inhibition of total RNA synthesis. The inhibition gradually reverses itself in sensitive cells and is less severe and more rapidly reversed in resistant cells. The in vivo sensitivity of the mitochondria is in striking contrast to their in vitro behavior; isolated mitochondria resist 50 times the in vivo ID50 level of the drug. The sensitivity of protein and RNA synthesis in mitochondria is presumed to be related to the differences in organization between the nucleocytoplasmic processes and the mitochondrial. But the data also suggest that the in vivo effects on mitochondria are indirect, either acting via the cytoplasm or nucleus or via a carrier mechanism having mitochondrial affinity.

Distinctive effects of mechlorethamine (NSC-762), cyclophosphamide (NSC-26271), and BCNU (NSC-409962) on transcription in Ehrlich cells.

Comparisons have been made between mechlorethamine, cyclophosphamide (CP), and BCNU as to the effects of chemotherapeutic drug levels on the transcription of RNA in vivo in Ehrlich cells. Mechlorethamine causes a dose-dependent depression in the synthesis of large Hn RNA transcripts in the nucleus and in the amount of large polysomal mRNA in the cytoplasm without apparent disturbance of polyadenylation and transport. Because of compensating increases in smaller Hn RNA and mRNA chains there is no inhibition of total RNA synthesis. CP seems to accelerate total Hn RNA and mRNA formation but inhibits polyadenylation. CP also produces an excess of short chains with only a minor depression of the synthesis of longer transcripts and messages. BCNU inhibits both RNA synthesis and polyadenylation but without disturbance of the size distribution of the polynucleotide chains. The results with mechlorethamine, in particular, are not easily explained on the basis of premature chain termination at the sites of DNA alkylation.

Phosphorylation of DNA polymerase.

Both E. coli and calf thymus DNA polymerase can be phosphorylated by cAMP-dependent protein kinase and phosphorylation appears to stimulate the DNA polymerase reaction. Conversely, dephosphorylation of the polymerase molecule, by a protein phosphatase, inhibits the polymerase reaction.