Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells.

Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([3H]thymidine incorporation), and protein synthesis ([1-14C]leucine incorporation) in C2C12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.

Site-specific conjugation on serine right-arrow cysteine variant monoclonal antibodies.

We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third constant domain (C(H)3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chemical conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following reduction, reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence analysis. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment containing the engineered cysteine.

Development of class-switched, affinity-matured monoclonal antibodies following a 7-day immunization schedule.

Previously we reported making high-affinity monoclonal antibodies (MAbs) 13 days after the onset of Repetitive Immunizations Multiple Sites (RIMMS) strategy. The Ig subclass variety and affinity of these antibodies suggested that maturational processes had already begun within draining lymph nodes. We now demonstrate that this diversity can in fact be captured as early as Day 7. In the work reported here, somatic fusion of immune lymphocytes isolated from peripheral lymph nodes resulted in the isolation of affinity-matured MAbs reactive with cytosine deaminase. This model further demonstrates and substantiates at a cellular level the rapid development and maturation of T-cell-dependent B-cell responses occurring within draining lymph nodes following antigen challenge.

Samarium-153 and lutetium-177 chelation properties of selected macrocyclic and acyclic ligands.

We describe a simple in vitro characterization of chelation that is useful when choosing an appropriate ligand-metal combination for clinical applications. These properties include the effect of concentration on chelation efficiency, time to maximum chelation, and stability in acidic and serum environments. The macrocyclic ligands nitro-DOTA and nitro-PADOTA, the acyclic ligands nitro-CHX-A-DTPA, nitro-MX-DTPA, DTPA, and a novel terpyridine ligand, TMT-amine, were evaluated as chelate complexes of both intermediate energy beta-emitting lanthanides lutetium-177 and samarium-153. The data were compared to results obtained in a previously published study with yttrium-90. Acid lability, time to achieve maximum chelation, and stability in human serum are properties unique to each ligand-metal combination and should be evaluated prior to choosing an appropriate combination for therapeutic applications. Concentration dependence and duration of chelation are general properties of lanthanide and yttrium chelation that can be applied to an appropriate ligand-metal combination to achieve optimum chelation efficiencies.

Improved biodistribution, tumor targeting, and reduced immunogenicity in mice with a gamma 4 variant of Campath-1H.

Radiolabeled antibodies have shown promise for the treatment of lymphoma and for solid tumor targeting. Campath-1H is a humanized monoclonal antibody that reacts with the CD52 antigen present on human lymphoid and myeloid cells. Campath-1H is a gamma1 (G1) isotype that induces lymphopenia via an Fc-mediated mechanism(s). Isotype switches were engineered, and the resulting antibodies were expressed in NS0 mouse myeloma cells and biosynthetically radiolabeled with [35S]methionine. The forms included G1, G4, and a G4 variant that contained alanine substitutions at (EU numbering) Leu-235, Gly-237, and Glu-318. All isotypes bound antigen equivalently as assessed by target cell binding in vitro. The G4 variant had a greatly reduced capacity to interact with Fc receptor by virtue of reduced binding to THP-1 human myeloid cells and by a 1000-fold increase in EC50 to intermediate antibody-dependent cellular cytotoxicity. The pharmacokinetics of the isotypes were compared in CD-1 (nu/nu) mice bearing an experimental antigen-expressing tumor. The plasma half-life and tumor uptake were increased for the G4 variant. The G4 variant showed significantly less spleen, liver, and bone uptake but similar uptake in the lung, kidney, and stomach and lower tissue-to-blood ratios. Immunogenicity was assessed after repeated monthly administrations of unlabeled antibody in BALB/c mice. A 50% reduction in the incidence of anti-globulin response was observed for the G4 variant. These properties suggest that antibodies with reduced Fc receptor interaction merit additional study as potential targeting vehicles relative to other isotypes for radioimmunotherapy or situations where diminished normal tissue binding contributes to efficacy.

Generation of murine monoclonal antihuman milk fat globule membrane antibodies using immunoprecipitation and BIAcore analyses.

A selection of monoclonal antibodies was developed against deoxycholine-solubilized human milk fat globule membranes (HMFG). The antibodies were selected for their ability to immunoprecipitate 125I-labeled HMFG and then further analyzed by surface plasmon resonance on the BIAcore for their reactivity with HMFG and with a fusion protein containing a 4-mer of the muc-1 tandem repeat. Both the HMFG and the fusion protein proved to be robust surfaces for the analysis of crude supernatants. The BIAcore evaluation was useful in identifying true positives. BIAcore analyses of purified antibody preparations were used to determine binding characteristics such as affinity and intensity. The latter proved useful in selecting a panel for evaluation by immunohistochemistry for breast tissue reactivity. Four of 6 antibodies appeared to react more intensely with tumor compared with normal breast tissues. One of those antibodies reacted with the fusion protein 4-mer of the muc-1 tandem repeat.

Yttrium-90 chelation properties of tetraazatetraacetic acid macrocycles, diethylenetriaminepentaacetic acid analogues, and a novel terpyridine acyclic chelator.

Realization of the potential of yttrium-90 for the radioimmunotherapy of cancer depends on rapid and kinetically stable chelation. Conditions were evaluated that influenced the chelation efficiency of these select chelators for yttrium-90: the macrocyclic chelators 2-(rho-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tet raacetic acid (nitro-DOTA); alpha-(2-(rho-nitrophenyl)ethyl)-1,4,7,10,- tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic) acid (nitro-PADOTA); 2-(rho-nitrobenzyl)-1,4,7,10-tetraazacyclotridecane- N,N',N",N"'-tetraacetic acid (nitro-TRITA); the acyclic chelator diethylenetriaminepentaacetic acid (DTPA); its analogues N-[2-amino-3-(rho-nitrophenyl)propyl]-trans- cyclohexane-1,2-diamine-N,N',N"-pentaacetic acid (nitro-CHX-A-DTPA) and 2-methyl-6-(rho-nitrobenzyl)-1,4,7- triazaheptane-N,N,N',N",N"-pentaacetic acid (nitro-1B4M-DTPA or nitro-MX-DTPA); and a novel acyclic terpyridine chelator, 6,6"-bis[[N,N,N",N"- tetra(carboxymethyl)amino]methyl]-4'-(3-amino-4-methoxyphenyl)-2,2':6',2 "- terpyridine (TMT-amine). The chelators fell into two distinct classes. The acyclic chelators, DTPA, nitro-CHX-A-DTPA, nitro-MX-DTPA, and TMT-amine, chelated instantaneously in a concentration-independent manner. Chelation efficiency was affected minimally when the concentrations of trace metal contaminants were increased. In contrast, the macrocyclic chelators, nitro-DOTA, nitro-TRITA, and nitro-PADOTA, chelated yttrium-90 more slowly in a concentration-dependent manner where efficiency was maximal only when the chelator:metal ratio was greater than 3. Their chelation efficiency diminished in a concentration-dependent fashion as the concentrations of trace metal contaminants were increased. Optimum labeling efficiencies were obtained through application of these principles.(ABSTRACT TRUNCATED AT 250 WORDS)

Potent gastrin-releasing peptide (GRP) antagonists derived from GRP (19-27) with a C-terminal DPro psi [CH2NH]Phe-NH2 and N-terminal aromatic residues.

We have previously reported that octapeptides with a -DPro psi[CH2NH]Phe- NH2 C-terminus are potent GRP antagonists and have greatly enhanced in vivo stability. Now we report the detailed syntheses of such peptides and additional attempts to further increase metabolic stability. Replacement of the -DPro psi[CH2NH]Phe-NH2 with a "-DPro-statine"-Phe-NH2 led to less potent antagonistic activity. The introduction of ThiAla and BzthAla, to replace His and Trp, respectively, did not increase activity. A series of analogs having different aromatic residues at the N-terminal, other than 3-phenylpropionic acid, are equally potent. These residues show increased activity when hydrophilic substitutions are added to the aromatic ring. Replacement of the C-terminal Phe by DPhe and D2Nal is tolerated. Even though none of these peptides have higher activity than the original lead peptide, they are potentially more metabolically stable.

Development of potent gastrin-releasing peptide antagonists having a D-Pro-psi(CH2NH)-Phe-NH2 C terminus.

Gastrin-releasing peptide (GRP) is a 27-amino acid neuroendocrine hormone that may play a role in the pathophysiology of small cell lung carcinoma. GRP and bombesin, a structurally related peptide, stimulate the growth of some cultured cell types. C-terminal GRP peptide analogs were developed that inhibited 6 nM bombesin-induced [3H]thymidine incorporation into quiescent murine Swiss 3T3 cells, which routinely produced a 6-fold stimulation over the basal extent of incorporation. The peptides were also analyzed for their capacity to inhibit the binding of 50 pM 125I-labeled GRP to Swiss 3T3 cells. The combination of two chemical modifications, each antagonistic in itself, led to the creation of antagonists with orders of magnitude greater potency than either modification alone. (i) Antagonist analogs of the form -Leu26-psi(CH2NH)-Xaa27-NH2 [where Xaa is Leu, norleucine (Nle), or Phe; residues numbered after GRP], similar to those introduced by Coy and coworkers [for review, see Jensen, R. T. & Coy, D. H. (1991) Trends Pharmacol. Sci. 12, 13-19], were found to have nanomolar potencies. (ii) We found that an octapeptide C-terminal GRP analog having D-Pro adjacent to the C-terminal amino acid amide was antagonistic, with a potency of 40 nM. By combining both modifications, specific analogs were found with potencies > 1000-fold greater than our lead structure--[(4'-hydroxy)-3-phenylpropanoyl]-Pro-Arg-Gly-Asn-His-Tr p-Ala-Val - Gly-His-Leu-psi(CH2NH)-Nle-NH2--and greater than any antagonist previously reported. The analogs [(4'-hydroxy)-3-phenylpropanoyl]-His-Trp-Ala-Val-D-Ala-His-D-Pro- psi(CH2NH)-Phe-NH2 and 1-naphthoyl-His-Trp-Ala-Val-D-Ala-His-D-Pro-psi(CH2NH)-Phe-NH2 antagonized [3H]thymidine incorporation with IC50 values of approximately 0.3 nM and inhibited the binding of 125I-labeled GRP with IC50 values of approximately 1 pM. These peptides may be of use in the study of the physiology of GRP.

Conveyance of partial agonism/antagonism to bombesin/gastrin-releasing peptide analogues on Swiss 3T3 cells by a carboxyl-terminal leucine insertion.

Gastrin-releasing peptide (GRP) is a neuroendocrine hormone that may be involved in the pathophysiology of small cell lung carcinoma. We describe carboxylterminal peptide analogues of GRP and bombesin, a 14-residue amphibian homologue, that were modeled after the antagonist [Leu13-psi(CH2NH)-Leu14]bombesin and retained the psi bond. Three novel peptides contained a Leu insertion amino to the psi bond, i.e. ... Leu13Leu14 psi X (residues numbered after bombesin) where X = LeuNH2 or norleucine-NH2). The Leu-insertion analogues behaved as pure partial agonists/antagonists when examined for the ability to stimulate [3H]thymidine incorporation into quiescent Swiss 3T3 cells (agonist activity) and to diminish the agonist response of GRP (antagonist activity). A time course of [3H]thymidine incorporation into quiescent cells indicated maximal incorporation at 20-h post-peptide addition for bombesin and GRP and a Leu-insertion peptide, but the extent of the incorporation for the Leu-insertion peptide was half that of GRP and bombesin. The agonist dose responses of the Leu-insertion peptides (EC50 values of 1-10 nM) paralleled GRP and bombesin, but the maximal response of the Leu-insertion peptides, even at concentrations as high as 10(-4) M, was half the maximal value of GRP or bombesin. High concentrations of the Leu-insertion peptides antagonized 10 nM GRP (a concentration that produced a near-maximal GRP response) yielding a response that was half the maximal value of GRP and equivalent to the maximal response of the Leu-insertion peptides alone. Analogues of the form ... Leu13 psi X behaved as complete antagonists. The KD values of the Leu-insertion peptides for competitive binding versus 125I-GRP (2-50 nM) were as potent as parent ... Leu14 agonists. Stability studies indicated that peptide potencies for both agonist and antagonist activities diminished upon peptide incubation in medium or on cells. The results suggested that, for the Leu-insertion peptides, degradation into distinct products with different activities was not responsible for their partial agonist/antagonist behavior. Computer-generated molecular modeling studies indicated that the novel structures could adopt energy minimized conformations for either an agonist or an antagonist as proposed earlier (Coy, D.H., Heinz-Erian, P., Jiang, N.-Y., Sasaki, Y., Taylor, J., Moreau, J.-P., Wolfrey, W.T., Gardner, J.D., and Jensen, R. T. (1988) J. Biol. Chem. 263, 5056-5060).

A novel bombesin receptor antagonist (2258U89), potently inhibits bombesin evoked release of gastrointestinal hormones from rats and dogs, in vitro and in vivo.

Several bombesin-receptor antagonists are available that inhibit secretory and growth effects of bombesin, in vitro. In the present study, we examined the effects of a new class of bombesin receptor antagonists (modified GRP(15-27) peptides, with D-Pro26 and D-Ala24 moieties), on bombesin mediated effects, in vivo and in vitro. Of the 10 different compounds tested, BW-10 or 2258U89 ([de-NH2)Phe19,D-Ala24,D-Pro26 psi(CH2NH)Phe27]-GRP(19-27)) was most potent towards inhibiting bombesin binding to rat pancreatic acinar cancer cells with an ID50 of 0.5 nM. BW-10 (1 and 10 nM) significantly inhibited the gastrin response to 1 nM bombesin, from isolated rat stomach, in vitro, in a dose-dependent fashion. BW-10 (10-100 nmol/kg) was equally effective at significantly inhibiting bombesin evoked gastrin release in anesthetized rats, in vivo. [D-Phe6]Bombesin(6-13)-propylamide (BIM), a member of another class of antagonists, reported previously to be the most potent antagonist, in vitro, on the other hand, enhanced bombesin provoked gastrin release in rats. The antagonistic effects of BIM, in vivo, may thus be more selective. Intravenous infusion of BW-10 (10 nmol/kg/h) partially depressed gastrin and pancreatic polypeptide and completely abolished insulin released in response to bombesin, in conscious dogs. These results suggest that BW-10 functions as one of the most potent bombesin receptor antagonists, in vitro and in vivo, which could potentially be used as a therapeutic compound in treatment of some human diseases.

Tumoricidal effector mechanisms of murine BCG-activated macrophages: role of TNF in conjugation-dependent and conjugation-independent pathways.

The roles of secreted and membrane-associated TNFs were investigated in activated macrophage cytolysis of L929, EMT-6, and P815 targets. While all three targets were susceptible to cytolysis in coculture, an anti-TNF antiserum blocked lysis of L929 and EMT-6 but not of the P815 targets. Of the three targets, recombinant human or mouse TNF could only lyse the L929 target; despite the fact that a role for TNF was invoked in lysis of EMT-6 targets in coculture, the latter was strongly resistant to soluble rTNF, even at concentrations 30-40-fold higher than the Ka for its TNF-receptor. Cytolysis of the L929 target occurred when it was cocultured with BCG-activated macrophages even when these effector cells did not secrete TNF, either due to prior chemical crosslinking or to lack of exposure to a triggering level of lipopolysaccharide. Furthermore, by introduction of the anti-TNF antiserum over a dose-range, it was shown that macrophage cytolysis both of L929 and EMT-6 targets occurred in the absence of bioavailable, fluid-phase TNF. Thus, even for targets susceptible to fluid-phase TNF, TNF-dependent, direct macrophage-mediated cytolysis appears to be a function independent of secreted TNF and one that utilizes effector-target contact to express the action of a membrane form of the molecule.

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.

The effect of nicotinamide on microvascular density and thermal injury in rats.

The effects of nicotinamide on the microvasculature and wound healing were examined in rats subjected to thermal injury. Rats (250 g) were treated with 50 mg nicotinamide intraperitoneally twice daily for 21 days and then heart and brain biopsies were taken. Skin biopsies were removed from sites in and adjacent to the injury throughout the course of healing. Tissues were stained for alkaline phosphatase and capillary length density was determined by morphometric analysis. Significant increases were observed in the heart, brain, and dermal tissue of treated animals compared to controls. Capillary density in the injured skin was significantly greater when compared to the injured skin of saline-treated controls. The injuries of the rats that were treated systemically with nicotinamide healed significantly faster than saline-treated as determined by planimetric evaluation of the granulation bed and eschar.

Blockade of the type I somatomedin receptor inhibits growth of human breast cancer cells in athymic mice.

Insulin and insulin-like growth factors (IGIs) stimulate the growth of human breast cancer cells in vitro. The type I somatomedin receptor (SR) expressed in these cells may mediate the growth effects of these peptides. We have examined the role of this receptor on human breast cancer growth with a monoclonal antibody (alpha-IR-3) that blocks the receptor binding domain and inhibits IGF-I-induced growth. alpha-IR-3 inhibited clonal growth in vitro and blocked the mitogenic effect of exogenous IGF-I in both MCF-7 and MDA-231 breast cancer cell lines. Antibody-induced blockade of the type I SR also inhibited the estrogen-independent MDA-231 cells growing in vivo in nude mice, but growth of the estrogen-dependent MCF-7 cells was unaffected. IGIs are important growth regulators of MDA-231 breast cancer cells. Blockade of this growth stimulatory pathway may provide a new treatment strategy.

Reduction in tumor necrosis factor receptor affinity and cytotoxicity by glucocorticoids.

Micromolar concentrations of glucocorticoids rendered L-M cells (a murine tumorigenic fibroblast line) less sensitive to the cytotoxic activity of murine TNF. The potency of different steroids paralleled their known anti-inflammatory potency, and pretreatment was more effective than post treatment. Sex steroids and mineralocorticoids were ineffective. Dexamethasone also decreased the sensitivity of MCF-7 (a human mammary carcinoma line) to the cytotoxic activity of human recombinant TNF. Pretreatment of both cell lines reduced the affinity of specific cell surface receptors for the binding of their species 125I-TNF about 3-fold while retaining the same number of binding sites. The decrease in sensitivity was not due solely to the inhibition of early TNF-induced events (such as binding, internalization or signal transduction). Dexamethasone modestly enhanced inhibition beyond that of neutralizing antiserum alone when both were added midway in the L-M killing reaction (after receptor down regulation but before the onset of complete cell death).

The TNF receptor in TNF-mediated cytotoxicity.

The in vitro cytotoxic capacity (if not every pleiotropic property) of tumor necrosis factor (TNF) begins by interaction with specific high affinity cell surface receptors. The characterization of receptors and ligand kinetics is reviewed in relationship to cytotoxicity. Decreased receptor number and affinity correlate with sensitivity within a given cell line. In L-M cells (a sensitive tumorigenic fibroblast), TNF induces a biphasic downregulation of receptors. Internalized ligand and receptors are largely cleared before the onset of cell death. Drugs affecting cytotoxicity may act primarily on an early 'association' stage (ligand receptor interaction, internalization or perhaps signal transduction) or on a later 'lytic' stage. Phorbol myristate acetate is an example of the former, while chloroquine, cholera toxin and dibutyryl cyclic AMP are examples of the latter.

An antibody to the receptor for insulin-like growth factor I inhibits the growth of MCF-7 cells in tissue culture.

Alpha IR-3, a monoclonal antibody to the insulin-like growth factor I receptor which blocks insulin-like growth factor I binding and inhibits its activity, inhibits the binding of 125I-insulin-like growth factor I to MCF-7 cells (an estrogen dependent human breast carcinoma cell line) with an IC-50 of approximately 100 ng/ml. It also inhibits the growth of MCF-7 cells cultured in 5% calf serum with approximately the same IC-50. Inhibition of growth occurs both when cells are cultured in the presence and absence of estrogen and is more pronounced when cells are grown at a low density. These findings demonstrate a requirement for insulin-like growth factor I for optimal growth of MCF-7 cells and suggest that it is an autocrine growth factor in these cells.

Chemical identification of a tumor-derived angiogenic factor.

Neoplasms produce substances that induce blood vessel formation (angiogenesis). Fractions from ethanol extracts of the Walker 256 carcinoma were isolated by silica column chromatography and C18 reversed-phase high-performance liquid chromatography. Two of the isolated fractions induced neovascularization when tested in the rabbit corneal micropocket assay. One of the fractions was identified as nicotinamide by desorption-electron impact mass spectrometry, nuclear magnetic resonance spectroscopy, and gas chromatography-mass spectrometry. The second active fraction contained nicotinamide as part of a more complex, as yet unidentified, molecular arrangement. Microgram quantities of commercial nicotinamide induced neovascularization in the corneal micropocket assay and in the chick chorioallantoic membrane assay.

Tumoricidal effector mechanisms of murine Bacillus Calmette-Guérin-activated macrophages: mediation of cytolysis, mitochondrial respiration inhibition, and release of intracellular iron by distinct mechanisms.

Murine Bacillus Calmette-Guérin-activated macrophages mediate discrete cytotoxic effects in cocultured tumor target cells in vitro. These effects include: the loss of intracellular iron, in part associated with reversible inhibition of the Kreb's cycle enzyme, aconitase; cytostasis, associated with reversible lesions inflicted in the electron transport chain (ETC) of the mitochondria resulting in reversible loss of proliferative capacity; and cytolysis, manifested by eventual gross perturbation of the integrity of the plasma membrane. We demonstrate that these manifestations of cytotoxicity are the result of three independent mechanisms employing apparently distinct macromolecules for their commission. Analysis of target cells that are highly susceptible (L-929), highly resistant (L-1210), or have incomplete resistance (EMT-6) to the cytolytic effects of cocultured activated macrophages indicates that there is no consistent relationship between the release of intracellular 59Fe and 51Cr. Thus, perturbation of intracellular iron pools did not appear to be an obligatory step on the pathway to cytolysis. Further evidence for this dissociation was obtained by employing a specific heteroantiserum reactive with cytolytic molecule(s). This antiserum could block the cytolytic response (51Cr release of cocultured L-929 and EMT-6 targets) but had no effect on the extent of iron release from viable EMT-6 or L-1210 targets. Furthermore, the cytolytic factor itself was incapable of mediating effects on the ETC or in causing release of intracellular iron. Two lines of evidence suggested that effects on the ETC are not linked with loss of intracellular iron. First, the monokine respiration inhibitory factor was incapable of causing release of intracellular iron from target cells in which the mitochondria were strongly suppressed. Second, the kinetics of release of respiration inhibitory factor from endotoxin-triggered Bacillus Calmette-Guérin-activated macrophages indicate a retarded appearance compared to the time at which a factor mediating release of intracellular iron was detectable. Our results strongly suggest that these three distinct cytotoxic reactions are under differential control by the effector cell.