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Biomedical subjects

J S Lazo

Publications and source records attributed to J S Lazo.

At least 145 records · Page 8Linked to original sources
PubMed1987

Separation of the protective enzyme bleomycin hydrolase from rabbit pulmonary aminopeptidases.

Bleomycin (BLM) hydrolase inactivates the BLM class of antitumor antibiotics and protects against BLM-induced pulmonary fibrosis. This enzyme is poorly characterized but believed to be an aminopeptidase B. In the present report, both BLM hydrolase and aminopeptidase B from rabbit pulmonary cytosol were retained by arginyl-Sepharose and BLM-Sepharose affinity columns, further suggesting that these two enzymes are similar. When, however, BLM hydrolase was purified over 1800-fold by using our newly developed high-speed liquid chromatography assay for BLM hydrolase coupled with fast protein liquid chromatography, we found that this partially purified BLM hydrolase preparation lacked aminopeptidase B activity. Furthermore, BLM hydrolase was completely separated, by using anion-exchange Mono Q chromatography, from all the aminopeptidases identified in rabbit pulmonary cytosol: one aminopeptidase B, two aminopeptidases N, and one aminopeptidase with both aminopeptidase B and aminopeptidase N activities. Pulmonary BLM hydrolase also had a higher molecular weight than pulmonary aminopeptidase B. In contrast to aminopeptidase B, BLM hydrolase was not activated by NaCl and was much less stable at 4 degrees C. In addition, bestatin was a potent inhibitor of aminopeptidase B but had little effect on BLM hydrolase, while leupeptin was a potent inhibitor of BLM hydrolase but was less effective against aminopeptidase B. Thus, pulmonary BLM hydrolase and aminopeptidase B have affinity for each other's substrate, but they are clearly distinct enzymes on the basis of charge characteristics, molecular weight, stability, and sensitivity to inhibitors and activators.

Aminopeptidases↗
PubMed1987

High dose continuous infusion of bleomycin in mice: a new model for drug-induced pulmonary fibrosis.

Bleomycin (BLM) produces pulmonary fibrosis in mice when given as a single intratracheal injection, a single i.v. injection or multiple s.c. injections. All of these models are associated with significant disadvantages including a variable distribution of lesions, high mortality or a requirement for multiple procedures. We have developed a convenient method of BLM treatment that avoids these difficulties and yields extensive, reproducible pulmonary fibrosis in mice. Osmotic minipumps containing BLM (100 mg/kg) were implanted s.c. in C57Bl/6 mice and the drug was delivered as a continuous s.c. infusion over 1 week. No mortality occurred over the first 5 weeks after pump placement whereas i.v. BLM (80 mg/kg) produced 50% mortality within 2 weeks. BLM given by pump infusion produced a greater increase (P less than .05) in lung hydroxyproline after 6 weeks (70%) than a similar total dose given as multiple s.c. injections (40%). Lungs from pump-treated mice showed confluent subpleural fibrosis involving almost 50% of the pleural surface and evidence of subpleural alveolar collapse. Mice receiving i.v. or s.c. injections showed involvement of only 10 to 15% of the pleural surface. BALB/c mice were resistant to pulmonary fibrosis after pump implantation, indicating a murine strain difference in pulmonary responsiveness to BLM administered by constant infusion. This superior model for drug-induced pulmonary fibrosis uses a single procedure and provides an extensive, reproducible lung lesion. Additionally, our studies suggest that dysfunction of the pulmonary epithelium may play an important role in progressive pulmonary disease after BLM treatment.

Animals↗
PubMed1986

Endothelial injury caused by antineoplastic agents.

The vascular lining of the blood vessels to normal organs and malignant tissues would be expected to reflect the functional demands placed upon it. These functional requirements may be accomplished by specific biochemical macromolecules, some of which are localized on the plasma membrane of the endothelium. Considerable evidence exists that toxicity to at least some normal organs caused by antineoplastic agents is heralded by endothelial damage. This endothelial damage may reflect a specific drug-endothelium interaction, the mechanism and basis of which are not yet understood. The possibility also exists that destruction of solid tumors by currently employed antitumor agents is mediated in part by local loss of essential vasculature. Selective destruction of the tumor endothelium could be a rational and novel target to which drugs could be designed.

Animals↗
PubMed1986

Protein phosphorylation in cultured endothelial cells.

We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP-dependent protein kinase, three calcium/calmodulin-dependent protein kinases, protein kinase C, and at least one tyrosine kinase. No cyclic GMP-dependent protein kinase activity was found. The cells also contained numerous substrates for cyclic AMP-dependent protein kinase and protein kinase C. Fewer substrates were found for the calcium/calmodulin-dependent protein kinases. There was little difference between either protein kinase activities or substrates when pulmonary artery endothelium was compared to aortic endothelium grown under similar culture conditions. It is likely that these various protein kinases and their respective substrate proteins are involved in mediating several of the actions of the hormones and drugs which affect the vascular endothelium.

Animals↗
PubMed1986

Effect of Fluosol-DA/O2 on the antitumor activity and pulmonary toxicity of bleomycin.

The effect of an oxygen-carrying perfluorochemical emulsion on bleomycin antitumor activity and pulmonary toxicity was examined. Fluosol-DA (0.3 ml/mouse, i.v.), combined with bleomycin (10 or 15 mg/kg, i.p.) and a 2 h exposure to 95% oxygen (BFO) increased by five- to six-fold the tumor growth delay of FSaIIC fibrosarcoma compared to bleomycin alone (B). Only a slight increase in tumor growth delay was noted with the incomplete combinations of bleomycin and O2 (BO) and bleomycin and Fluosol-DA (BF). When bleomycin (10 mg/kg) was co-administered with 0.3 ml Fluosol-DA and 95% O2, cell survival was reduced ten-fold compared to that seen with bleomycin alone. In contrast, the surviving fraction of cells obtained from FSaIIC tumors treated in vivo indicated that 0.3 ml Fluosol-DA per mouse or a 2 h exposure to 95% O2 did not markedly alter the effects of bleomycin alone. The pulmonary effects of the BFO combination were assessed during the course of the therapy by bronchoalveolar lavage (BAL) analysis and pulmonary hydroxyproline (OH-Pro) content. Mice treated with this combination had a 20-fold increase in total numbers of cells obtained in the BAL compared to control animals. An increased cellularity in the lungs was also seen morphologically. The composition of the cells in the lavage fluid was altered after BFO but not BO treatment and reflected a neutrophilic influx. Furthermore, total protein recovered in the BAL fluid was increased 5-fold in the BFO treatment group compared to that in the control mice. Pulmonary OH-Pro, an index of collagen and fibrosis, was unaffected acutely after three treatments of either BFO or BO compared to control mice. Thus, Fluosol-DA and O2 can enhance the antitumor activity of bleomycin. The increased pulmonary cellularity suggests that this combination may also have adverse effects on lung tissue.

Animals↗
PubMed1986

Chemical modulation of bleomycin induced toxicity.

Both lidocaine (LIDO) and the calmodulin (CaM) antagonists, pimozide (PIM) and trifluoperazine (TFP), enhanced bleomycin (BLM) induced cytotoxicity and DNA damage. The toxicity with the combination of BLM and CaM antagonists represented true pharmacological synergism and was observed with the addition of the CaM antagonist either during or after BLM exposure. Additionally, the DNA damage of BLM and the BLM-like drugs, talisomycin S10b (TAL) or peplomycin (PEPLO), was also enhanced by CaM antagonists. LIDO, which similarly increased the lethal effects and DNA damage of BLM in L1210 cells, was also effective only during or after BLM exposure. The data presented here indicate that the modulation of toxicity seen with these drug combinations is reflected by changes in DNA integrity. Furthermore, these data suggest that the inhibition of DNA repair processes may be at least partially responsible for the enhanced toxicity and DNA damage when CaM antagonists or LIDO are added to BLM.

Animals↗
PubMed1986

Bleomycin inhibition of angiotensin-converting enzyme activity from serum, lungs, and cultured pulmonary artery endothelial cells.

The interaction between bleomycin (BLM) and angiotensin-converting enzyme (ACE) from bovine lungs, serum, and cultured pulmonary artery endothelial cells was examined. ACE activity from all sources was inhibited by metal-free BLM but not by metal-complexed BLM. The metal-free deamide metabolite of BLM, which is nontoxic, also inhibited ACE activity from bovine endothelial cell homogenates and serum. Incubation of intact cultured pulmonary artery endothelial cells for 1 h with metal-free BLM produced a concentration-dependent inhibition of cellular ACE activity, which was persistent, lasting at least 24 h after drug removal. In addition, the enzyme activity released into the culture medium was decreased. The inhibition of cellular ACE activity occurred in the absence of (1) cell death as measured by dye exclusion, (2) inhibition of total protein synthesis as measured by [3H]leucine incorporation, and (3) decreased cellular enzyme content as assayed using a monoclonal antibody against the enzyme. These results indicate that a brief exposure of intact cultured pulmonary endothelial cells to BLM can produce direct and prolonged inhibition of ACE activity. The inhibition of ACE activity does not relate directly to cell death but rather appears to result from metal chelation by BLM.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1986

Calmodulin: a potential target for cancer chemotherapeutic agents.

Calmodulin is a ubiquitous, calcium-binding protein that is responsible for many of the intracellular actions of calcium. Recent evidence suggests that calmodulin may regulate cellular proliferation and that its function may be altered in malignancy. The discovery that drugs such as phenothiazines antagonize the action of calmodulin led to the study of these antagonists against tumor cells. It is now appreciated that calmodulin antagonists are cytotoxic, can restore the sensitivity of resistant cells to drugs such as doxorubicin and vincristine, and can augment the cytotoxicity of bleomycin. This report addresses the possibility of developing new forms of chemotherapeutic agents that work by disrupting the function of this intriguing molecule.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1986

Increased lethality of calmodulin antagonists and bleomycin to human bone marrow and bleomycin-resistant malignant cells.

The effect of bleomycin and calmodulin antagonists on human cells was studied using a clonogenic assay. A 1-h exposure to nontoxic concentrations of the calmodulin antagonists melittin (0.5 microM), pimozide (5 microM), and chlorpromazine (20 microM) increased the lethality of bleomycin to human ovarian carcinoma cells (SK-OV). No increase was seen with chlorpromazine sulfoxide, which lacks calmodulin antagonistic activity. Maximum enhancement of bleomycin lethality by calmodulin antagonists was seen when the antagonist was present simultaneously with bleomycin rather than before or after bleomycin. The cytotoxicity of bleomycin to A-253 head and neck squamous carcinoma cells, which were 10-fold more sensitive to bleomycin alone compared to SK-OV cells, was not markedly altered by the presence of 20 microM chlorpromazine. Chlorpromazine, melittin, or pimozide also increased the toxicity of bleomycin to human granulocyte/macrophage and erythroid stem cell colonies. These results demonstrate that calmodulin antagonists can significantly increase the lethality of bleomycin to some but not all human tumor cells and that nonmalignant hematological human cells may also be affected by this combination.

Bleomycin↗
PubMed1985

Rapid distribution of adriamycin in the ascitic and pleural fluid of women with ovarian carcinomas.

Adriamycin has been detected by high-pressure liquid chromatography in ascites and pleural fluid within 30 min after intravenous injection of Adriamycin (50 mg/m2). The concentration of Adriamycin was approximately one-half of that in plasma. Significant concentrations of Adriamycin metabolites were seen in the ascites fluid of one patient 4 hr after intravenous drug injection. Individual variability in the achievable ascites and pleural concentrations of cytotoxic drugs may be important in determining the responsiveness of patients to chemotherapy and the described analytical technique should be useful in assessing the concentrations of Adriamycin and its metabolites in malignant ascites and pleural effusions.

Ascitic Fluid↗
PubMed1985

Enhanced bleomycin-induced DNA damage and cytotoxicity with calmodulin antagonists.

A wide variety of structurally different calmodulin antagonists enhanced the cytotoxicity of bleomycin A2 to leukemic L1210 cells. This potentiation occurred with nontoxic concentrations of calmodulin antagonists. The most potent blockers of L1210 calmodulin activity, melittin and mastoparan, were the most potent potentiators of bleomycin A2 cytotoxicity. Less potent agents such as pimozide, a diphenylbutylpiperidine, trifluoperazine and chlorpromazine, phenothiazines, and W-7, a naphthalene sulfonamide, required higher concentrations for potentiation of bleomycin A2-induced cytotoxicity, while homologs that lack anticalmodulin activity failed to increase the cytotoxicity seen with bleomycin A2. The potentiation of bleomycin A2 cytotoxicity was not due to an elevated cellular content of bleomycin A2 or to inhibition of bleomycin A2 inactivation. Using alkaline elution techniques, we found that pimozide increased bleomycin A2-induced DNA damage in intact L1210 cells. Pimozide did not, however, directly increase the formation of reactive species by bleomycin as measured by single or double strand breakage of covalently closed circular DNA. Thus, the potentiation of bleomycin cytotoxicity by these agents appears to be mediated by an increased damage to cellular DNA; this may be due to inhibition of DNA repair. The hypothesized calmodulin-dependent mechanism was not shared by all agents that caused breaks in DNA because no potentiation in cytotoxicity was observed when calmodulin antagonists were combined with either etoposide or X-irradiation.

Animals↗
PubMed1985

Lidocaine potentiation of bleomycin A2 cytotoxicity and DNA strand breakage in L1210 and human A-253 cells.

The survival of cultured L1210 cells exposed to bleomycin A2 (BLM A2) was markedly decreased by coincubation with the local anesthetic lidocaine. The potentiation occurred with concentrations of lidocaine that were nontoxic and was dependent upon both the concentration of lidocaine and BLM A2. A 1000-fold decrease in survival was seen with a 1-h exposure to 8 mM lidocaine and 10 microM BLM A2 compared to incubation with 10 microM BLM A2 alone. Prior exposure to lidocaine did not markedly alter BLM A2 cytotoxicity, while treatment with lidocaine immediately after BLM A2 exposure did, suggesting that increased cellular content of BLM A2 was not the mechanism of enhancement. Furthermore, lidocaine reduced the total amount of cell-associated radioactivity seen after incubation with [3H]BLM A2. The enhancement in L1210 cell cytotoxicity with lidocaine was not specific for the C- and N-terminal moieties of the BLM molecule. Other DNA-interacting antitumor agents, such as etoposide and mitomycin C, did not exhibit biologically significant alterations in their cytotoxicity when coincubated with lidocaine, although cis-diamminedichloroplatinum was significantly more toxic in the presence of lidocaine. The potentiation of BLM A2 cytotoxicity was not unique to murine tumor cells, since it was also seen with cultured human head and neck carcinoma (A-253) cells. Lidocaine did not increase directly BLM A2-induced breakage of DNA in vitro as measured by loss of form I pAT 153 DNA, but it did increase BLM A2-induced DNA strand breaks in intact L1210 cells coincubated with lidocaine and BLM A2. Exposure of L1210 cells to lidocaine after BLM A2 treatment also greatly increased DNA breakage consistent with possible inhibition of DNA repair. In addition, a modest reduction in the in vitro inactivation of BLM A2 by BLM hydrolase was found with lidocaine. We propose that inhibition of BLM metabolism and repair of BLM-induced DNA damage by lidocaine may have a role in the enhanced cytotoxicity.

Animals↗
PubMed1984

Pulmonary fate of [3H]bleomycin A2 in mice.

The pulmonary fate of radiolabeled bleomycin [S-methyl-3H]bleomycin A2 ( [3H]BLM A2) was studied in mice after intratracheal (i.t.) or s.c. injection. The loss of radioactivity from the lungs followed apparent first-order kinetics during the first 3 hr after i.t. drug instillation with a half-time of removal of 32 min. In addition, the initial pulmonary removal was linear with instilled doses ranging from 0.02 to 2.2 mg/kg. Radioactivity was detected in liver, kidneys, spleen and serum 1 hr after i.t. administration. Approximately 1% of the i.t. administered dose was detected in the lungs after 24 hr, indicating that the rate of removal of radioactivity slowed between 3 and 24 hr after i.t. drug instillation. Eighty percent of the radioactivity found in the lungs 1 hr after i.t. instillation was unmetabolized [3H]BLM A2, but by 24 hr less than 25% was unmetabolized drug and almost 40% was the nonfibrogenic metabolite, desamidobleomycin A2. After s.c. administration of 10 mg/kg of [3H]BLM A2, peak pulmonary levels were observed between 45 and 60 min and were less than 1% of the injected dose. Pulmonary levels comparable to those seen with a single fibrogenic i.t. dose (2.2 mg/kg) could not be obtained after a s.c. injection even with a toxic dose of the drug (100 mg/kg). In addition, twice weekly s.c. injections of unlabeled BLM A2 (10 mg/kg) for 5 weeks did not alter the amount of radioactivity seen in the lungs after a s.c. injection of [3H]BLM A2. Thus, the pulmonary fibrosis seen with i.t. BLM administration may reflect the high initial content of unmetabolized drug achieved in the lungs.

Animals↗
PubMed1984

Bleomycin hydrolase activity in pulmonary cells.

The metabolism of bleomycin (BLM) A2 by BLM hydrolase in the 105,000 X g supernatant fraction of homogenates obtained from freshly isolated and cultured pulmonary cells was assayed by high-pressure liquid chromatography. BLM A2 was converted solely to the less toxic desamido metabolite by the cytosol from isolated rabbit and bovine alveolar and interstitial macrophages, cultured rabbit and bovine pulmonary fibroblasts and cultured rabbit pulmonary artery endothelial cells. The BLM hydrolase activity in the cytosol from cultured rabbit fibroblasts had an apparent Km of 700 microM and Vmax of 33 nmol/hr/mg protein. The rate of BLM dA2 formation found with the cytosol of cultured rabbit pulmonary artery endothelial cells and pulmonary fibroblasts was 3 to 5 times greater per cell than that from the cytosol of rabbit alveolar and interstitial macrophages. Freshly isolated rabbit type II pneumocytes and bovine pulmonary artery endothelial cells grown in culture had undetectable levels of this inactivating enzyme activity. The expression of BLM hydrolase activity in rabbit pulmonary fibroblasts was stable for at least five passages in culture and was not significantly different over wide cell densities in culture. These data suggest that heterogeneity in the cellular distribution of BLM hydrolase activity exists in lungs. High levels of BLM hydrolase activity in the pulmonary endothelium or fibroblasts of some species may have an important role in determining the toxicity of BLM to the lungs.

Animals↗
PubMed1984

Antiproliferative actions of tamoxifen to human ovarian carcinomas in vitro.

The cytosolic estrogen receptor (ERc) and progestin receptor (PRc) levels were measured in 56 human epithelial ovarian tumors. The maximum ERc content in a tumor sample was 163 fmol/mg cytosolic protein. Forty-six of the tumor samples were evaluable for clonogenic growth in soft agar, and 19 samples produced 15 or more colonies per 10(5) cells plated. Four of the samples that grew had ERc levels of greater than 30 fmol/mg cytosolic protein. No correlation, however, between growth in soft agar and ERc or PRc content was observed. The antiproliferative properties of the antiestrogen, tamoxifen, were studied. Although no decrease in colony formation was observed after a 1-hr exposure to 0.2 or 2 microM tamoxifen, continuous exposure of cells to 2 microM tamoxifen reduced clonogenicity in 8 of 18 solid ovarian carcinomas examined. The maximum diminution in colony formation was approximately 50% of that of the control and was seen in 2 tumor samples. Both tumors that displayed the maximum response to continuous tamoxifen treatment had ERc and PRc levels greater than 30 fmol/mg cytosolic protein. None of the 14 tumors with ERc levels less than or equal to 30 fmol/mg cytosolic protein exhibited a decrease in colony formation of more than 50%. Exposure of cells for 1 hr to the combination of doxorubicin and tamoxifen produced a significant antagonism of the individual doxorubicin or tamoxifen antiproliferative effects in 7 of 9 samples examined. These data suggest that in a subset of human ovarian epithelial carcinomas tamoxifen alone can have some direct antiproliferative action on the clonogenic cells. The maximum antiproliferative effect of tamoxifen observed was related to ERc content in ovarian tumors.

Adult↗
PubMed1983

Prolonged reduction in serum angiotensin converting enzyme activity after treatment of rabbits with bleomycin.

Previous work from this laboratory (J. S. Lazo, J. D. Catravas, and C. N. Gillis, Biochem. Pharmacol. 30, 2577-2584, 1981; J. D. Catraveras, J. S. Lazo, and C. N. Gillis, J. Pharmacol. Exp. Ther. 217, 524-529, 1981) demonstrated that subacute bleomycin (BLM) administration (5 mg/kg) to rabbits three times weekly for 4 weeks produced a marked decrease in serum angiotensin converting enzyme (ACE) activity in addition to producing both morphological and biochemical evidence of pulmonary endothelial damage. In this work, the reversibility of the decreased serum ACE activity and the biochemical and morphological status of the pulmonary endothelium after a substantial drug-free period in rabbits was examined. Rabbits were injected sc three times weekly for 4 weeks with vehicle or BLM (5 mg/kg) and then maintained drug free for an additional 6 or 7 weeks. Serum ACE activity decreased over 60% during the BLM treatment but did not return to control levels at any time during the drug-free period. The pulmonary endothelium, however, appeared undamaged. No decrease in pulmonary ACE activity was detected either in vitro or in vivo nor was the single-pass pulmonary removal in vivo of [3H]norepinephrine and 5-[14C]hydroxytryptamine different between BLM-treated and control rabbits after the drug-free period. In addition, no evidence of pulmonary endothelial damage was observed by light and electron microscopy. Thus, reduction in serum ACE activity appears to be a durable reflection of BLM toxicity in rabbits that persists even in the absence of any detectable biochemical or morphologic endothelial injury.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1983

Lack of metabolism as the biochemical basis of bleomycin-induced pulmonary toxicity.

The biochemical basis for bleomycin-induced pulmonary toxicity was studied in vitro and in vivo with an improved HPLC system. The in vitro metabolism of bleomycin A2 to desamido-bleomycin A2 was measured in tissue homogenates from a species sensitive (mice) and relatively resistant (rabbits) to the pulmonary fibrogenic properties of bleomycin. Lung tissue from mice lacked detectable bleomycin hydrolase activity, whereas rabbit lung tissue homogenates had high levels of the enzyme activity, equaling that seen in rabbit kidneys and spleen. Injection of radiolabeled bleomycin A2 into mice demonstrated that only a small percentage of the total dose was taken up by any organ and that extensive metabolism of this drug occurred within 1 hr in liver, kidneys, and spleen but not in lungs in vivo. In addition, metabolites other than desamido-bleomycin A2 were prominent, and their relative amounts increased with time. Mice injected subcutaneously with bleomycin A2 developed pulmonary fibrosis, while animals treated with equivalent doses of desamido-bleomycin A2 did not, indicating that this metabolite is not as toxic to the lungs as is the parent compound. These results provide direct evidence that metabolism plays a major role in determining the toxic potential of bleomycin to the lungs.

Animals↗