A randomized phase II study of two schedules of bryostatin-1 (NSC339555) in patients with advanced malignant melanoma: A National Cancer Institute of Canada Clinical Trials Group study.

PURPOSE: This study addressed the efficacy and toxicity of the novel compound Bryostatin-1 (NSC 339555), a novel agent with antineoplastic, hematopoietic and immunomodulatory activity in a variety of in vitro and in vivo systems. PATIENTS AND METHODS: This phase II study randomly assigned chemotherapy-naïve patients with untreated metastatic melanoma and measurable disease to two schedules of treatment: Arm A, 25 microg/m2 bryostatin-1 given as a 24 hour continuous infusion weekly or Arm B, 120 microg/m2 bryostatin-1 given as a 72 hour continuous infusion every 2 weeks. Although objective response was assessed using standard NCIC CTG criteria, antitumour activity was assessed using a multivariate endpoint incorporating both response (CR and PR) and early progression (PD at < or = 8 weeks). Seventeen patients were randomized to each arm. RESULTS: Arm A was better tolerated with 86.7% of 15 evaluable patients receiving > or = 90% of planned dose intensity versus 76.5% of 17 evaluable patients in Arm B. On Arm B, three patients experienced serious adverse events and three patients had to be removed from protocol therapy due to toxicity. The most common side effect was myalgia (33% grade 1-2 on Arm A versus 65% on Arm B with 5 patients experiencing grade 3 and one patient grade 4). Lethargy was more common on Arm A but more severe on Arm B. Other side effects such as nausea, diarrhea and headache were generally mild to moderate in nature and occurred with a similar frequency on both arms. Hematologic and biochemical toxicity were minimal. This trial was closed early because the protocol-stopping rule was met based on lack of required responses and on the number of early progressions on both arms. No partial or complete responses were seen; 3 patients randomized to Arm A had stable disease (duration 9-24 weeks) as did 4 patients (duration 10-38 weeks) randomized to Arm B. CONCLUSION: Arm A was better tolerated than Arm B. We conclude that bryostatin-1 has little efficacy in the treatment of metastatic melanoma with either of the schedules studied.

Immunolocalization of matrix metalloproteinases and their inhibitors in clinical specimens of bone metastasis from breast carcinoma.

Matrix metalloproteinases (MMPs) are essential in several stages of the metastatic process, and in normal bone development and remodeling. We explored whether the interaction between tumor cells and bone leads to changes in MMP and tissue inhibitor of MMP (TIMP) expression thus affecting osteolysis in metastatic bone disease. Using immunohistochemistry we have investigated the MMP/TIMP expression in tumor cells, fibroblasts, osteoblasts and osteoclasts. Thirty one specimens of bone metastasis from breast carcinoma were stained for MMP-1, -2, -9, MT1-MMP and TIMP-1, and -2 and compared with staining in normal breast tissue, primary breast carcinoma and normal bone. Specimens came from patients in three clinical scenarios: from open biopsies without or with pathological fracture, or bone marrow biopsies containing tumor from patients with pancytopenia but without clinical evidence of osteolysis. By bone histomorphometry the latter group showed a heavy tumor load not different from the open biopsy groups but displayed little active bone resorption and low numbers of osteoclasts. Cell type-specific MMP/TIMP expression was observed and the staining patterns were comparable between the three groups of patients. Though no major differences in the MMP/TIMP staining of tumor cells and fibroblasts were observed between bone metastasis and primary tumor, we showed that tumor cells do express MMPs capable of degrading bone matrix collagen. The number and activity of osteoclasts and osteoblasts was increased dramatically in bone metastases, their MMP/TIMP profiles, however, were not different from normal bone, suggesting that the mechanism of bone degradation by osteoclasts is not different from normal bone remodelling.

The activity of the Ets transcription factor PEA3 is regulated by two distinct MAPK cascades.

PEA3, a member of the Ets family of transcriptional regulatory proteins, binds to the PEA3 promoter element and stimulates transcription through this site. The activity of the PEA3 element is regulated by mitogens, activated receptor tyrosine kinases, and oncogenic members of the Ras signal transduction pathway. However, it is not clear whether PEA3 mediates transcriptional regulation by these agents because a number of different Ets proteins can functionally interact with the PEA3 element. To specifically learn whether the activity of PEA3 is regulated, we investigated the ability of constitutively-activated Ras (Ha-RasV12) and signaling proteins downstream of Ras to alter PEA3-dependent reporter gene expression in COS cells. Ha-RasV12 and activated proteins in both the extra-cellular regulated kinase (ERK) and the stress-activated protein kinase (SAPK) or Jun N-terminal kinase (JNK) cascades independently stimulated PEA3-mediated gene expression. Ha-RasV12 stimulation of PEA3 activity was reduced by dominant-negative mutants in each of these protein kinase cascades, suggesting that Ras activates PEA3 through both pathways. Furthermore, the ability of unique activators of each kinase cascade to stimulate PEA3-dependent gene expression was selectively reduced by dominant-negative mutants within the homologous but not the heterologous pathway. Hence two distinct mitogen-activated protein kinase (MAPK) cascades regulate PEA3 activity. PEA3 was phosphorylated in vivo at serine residues consistent with the possibility that it may be a direct target of MAPKs.

Activation of Escherichia coli F1-ATPase by lauryldimethylamine oxide and ethylene glycol: relationship of ATPase activity to the interaction of the epsilon and beta subunits.

The stimulation of the ATPase activity of Escherichia coli F1-ATPase by the detergent lauryldimethylamine oxide (LDAO) and the relationship of this activation to removal of the inhibitory epsilon subunit were studied. The detergent caused a dramatic decrease in the affinity of epsilon-depleted enzyme for epsilon subunit, suggesting that release of epsilon is involved in LDAO activation. However, even in the absence of any epsilon subunit, the detergent caused a 140% increase in activity, indicating activation by effects independent of epsilon. In contrast, the addition of 30% ethylene glycol to the reaction buffer caused a modest inhibition of the ATPase activity of epsilon-depleted F1-ATPase but rendered the enzyme insensitive to inhibition by epsilon subunit. This solvent prevented the cross-linking of epsilon to beta by a water-soluble carbodiimide, although epsilon remained linkable to both beta and gamma by dithiobis(succinimidyl propionate). Thus, epsilon was not dissociated from F1-ATPase, but its intimate interaction with the beta subunit was altered. These results suggest that the inhibitory action of epsilon is expressed through its interaction with beta. Kinetic analysis revealed that LDAO activated hydrolysis at both the high- and low-affinity promotional sites, with little change in Km values. Ethylene glycol caused a substantial increase in Km at the low-affinity promotional site and made the enzyme resistant to inhibition by aurovertin D.

The epsilon subunit and inhibitory monoclonal antibodies interact with the carboxyl-terminal region of the beta subunit of Escherichia coli F1-ATPase.

The epitopes of two classes of monoclonal antibody and the binding site for the epsilon subunit have been mapped to the carboxyl-terminal region of the beta subunit of Escherichia coli F1-ATPase using partial CNBr cleavage, weak acid hydrolysis, and Western blots. One class of antibody, B-I, inhibits ATPase activity; the other class, B-II, recognizes an epitope not exposed on the surface of intact F1. Data from two-dimensional gels and blots of beta cleaved with CNBr/weak acid showed that the B-I epitope lies between Asp-381 and the carboxyl-terminal Leu-459, and the B-II epitope lies between Asp-345 and Met-380. Weak acid hydrolysis of the beta-epsilon product obtained by cross-linking F1 with a water-soluble carbodiimide yielded a fragment containing epsilon and a 13-kDa carboxyl-terminal fragment of beta indicating that epsilon interacts with this portion of beta as well. Fab fragments from the B-I antibody beta-6 could be cross-linked to the epsilon subunit in native F1 by various cross-linking agents demonstrating that the antibody and the epsilon subunit occupy adjacent, nonoverlapping sites on the beta subunit. Implications of these results for the roles of the epsilon subunit and of the carboxyl-terminal region of the beta subunit in F1 are discussed.

Epsilon subunit of Escherichia coli F1-ATPase: effects on affinity for aurovertin and inhibition of product release in unisite ATP hydrolysis.

The epsilon subunit of Escherichia coli F1-ATPase is a tightly bound but dissociable partial inhibitor of ATPase activity. The effects of epsilon on the enzyme were investigated by comparing the ATPase activity and aurovertin binding properties of the epsilon-depleted F1-ATPase and the epsilon-replete complex. Kinetic data of multisite ATP hydrolysis were analyzed to give the best fit for one, two, or three kinetic components. Each form of F1-ATPase contained a high-affinity component, with a Km near 20 microM and a velocity of approximately 1 unit/mg. Each also exhibited a component with a Km in the range of 0.2 mM. The velocity of this component was 25 units/mg for epsilon-depleted ATPase but only 4 units/mg for epsilon-replete enzyme. The epsilon-depleted enzyme also contained a very low affinity component not present in the epsilon-replete enzyme. In unisite hydrolysis studies, epsilon had no effect on the equilibrium between substrate ATP and product ADP.P1 at the active site but reduced the rate of product release 15-fold. These results suggest that epsilon subunit slows a conformational change that is required to reduce the affinity at the active site, allowing dissociation of product. It is suggested that inhibition of multisite hydrolysis by epsilon is also due to a reduced rate of product release. epsilon-depleted F1-ATPase showed little of no modulation of aurovertin fluorescence by added ADP and ATP. Aurovertin fluorescence titrations in buffer containing ethylenediaminetetraacetic acid (EDTA) revealed that epsilon-depleted enzyme had high affinity for aurovertin (Kd less than 0.1 microM) regardless of the presence of nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation and inhibition of the Escherichia coli F1-ATPase by monoclonal antibodies which recognize the epsilon subunit.

The properties of two monoclonal antibodies which recognize the epsilon subunit of Escherichia coli F1-ATPase were studied in detail. The epsilon subunit is a tightly bound but dissociable inhibitor of the ATPase activity of soluble F1-ATPase. Antibody epsilon-1 binds free epsilon with a dissociation constant of 2.4 nM but cannot bind epsilon when it is associated with F1-ATPase. Likewise epsilon cannot associate with F1-ATPase in the presence of high concentrations of epsilon-1. Thus epsilon-1 activates F1-ATPase which contains the epsilon subunit, and prevents added epsilon from inhibiting the enzyme. Epsilon-1 cannot bind to membrane-bound F1-ATPase. The epsilon-4 antibody binds free epsilon with a dissociation constant of 26 nM. Epsilon-4 can bind to the F1-ATPase complex, but, like epsilon-1, it reverses the inhibition of F1-ATPase by the epsilon subunit. The epsilon subunit remains crosslinkable to both the beta and gamma subunits in the presence of epsilon-4, indicating that it is not grossly displaced from its normal position by the antibody. Presumably the activation arises from more subtle conformational effects. Antibodies epsilon-4 and delta-2, which recognizes the delta subunit, both bind to F1F0 in E. coli membrane vesicles, indicating that these subunits are substantially exposed in the membrane-bound complex. Epsilon-4 inhibits the ATPase activity of the membrane-bound enzyme by about 50%, and Fab prepared from epsilon-4 inhibits by about 40%. This inhibition is not associated with any substantial change in the major apparent Km for ATP. These results suggest that inhibition of membrane-bound F1-ATPase arises from steric effects of the antibody.

Column centrifugation generates an intersubunit disulfide bridge in Escherichia coli F1-ATPase.

Passage of F1-ATPase through a centrifuge column [Penefsky, H. S. (1979) Methods Enzymol. 56, 527-530] caused formation of a product with a relative molecular mass of 72,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The product was identified as cross-linked alpha and delta subunits by using Western blots and subunit-specific monoclonal antibodies. The cross-link was reversed by 50 mM dithiothreitol implying that it was a disulfide bridge. Formation of the cross-link was inhibited by 2 mM EDTA and was stimulated in some buffers by the addition of 10 microM CuCl2. Time course experiments indicated that the majority of the cross-link formed while the enzyme was passing through the column. Thus the cross-link induced by column centrifugation arose from the rapid, heavy-metal-ion-catalysed oxidation of two sulfhydryl groups, one on the alpha subunit and one on the delta subunit, to a disulfide. These results demonstrate that care must be exercised when running proteins through centrifuge columns as potentially deleterious disulfide formation can result. An anti-beta monoclonal antibody was capable of immunoprecipitating the entire enzyme including the cross-linked subunits, implying that the cross-linked alpha and delta subunits were still a part of F1. The formation of the cross-link affected neither the hydrolytic activity of the enzyme nor its susceptibility to inhibition by epsilon subunit. The cross-linked enzyme was unable to bind to F1-depleted membranes in experiments in which soluble F1 and membranes were separated by centrifugation. Column centrifugation did not generate the cross-link on membrane-bound enzyme. These results indicate that the alpha-delta cross-link results in a loss of binding affinity between F1 and F0.

Monoclonal antibodies to Escherichia coli F1-ATPase. Correlation of binding site location with interspecies cross-reactivity and effects on enzyme activity.

Twenty-one hybridoma cell lines which secret antibodies to the subunits of the Escherichia coli F1-ATPase were produced. Included within the set are four antibodies which are specific for alpha, six for beta, three for gamma, four for delta and four for epsilon. The antibodies were divided into binding competition subgroups. Two such competition subgroups are represented for the alpha, beta, and epsilon subunits, one for delta and three for gamma. The ability to bind intact F1-ATPase was demonstrated for some of the antibodies to alpha and beta, and for all of those to delta, while the antibodies to gamma and epsilon gave unclear results. All of the antibodies to alpha and beta which bound ATPase were found to have effects on the ATPase activity of purified E. coli F1-ATPase. One of those to alpha inhibited activity by about 30%. Another anti-alpha was mildly stimulatory. The four antibodies to beta which bound ATPase inhibited activity by 90%. In contrast, membrane-bound ATPase was hardly affected by the antibodies to alpha, but was inhibited by 40-60% by the antibodies to beta. The other antibodies to alpha and beta bound only free subunits, or partially dissociated ATPase, suggesting that their epitopes are buried between subunits in ATPase. These antibodies had no effects on activity. The ability of the antibodies to recognize ATPase subunits present in crude extracts from mitochondria, chloroplasts, and a variety of bacteria was tested using nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. One anti-beta specifically recognized proteins in the range of 50,000-60,000 daltons in each of the extracts, although the reaction with mitochondrial beta was weak. Some of the other antibodies had limited cross-reaction, but most were specific for the E. coli protein. In some species, those proteins which were recognized by the anti-beta ran with a higher apparent molecular weight than proteins which were recognized by an anti-alpha. All antibodies which exhibited cross-reactivity were found to recognize sites which were not exposed in intact ATPase, implying that the surfaces which lie between subunits are most highly conserved.