Altered expression of extracellular superoxide dismutase in mouse lung after bleomycin treatment.

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung tissue and is believed to protect the lung from oxidative damage that results in diseases such as pulmonary fibrosis. This study tests the hypothesis that proteolytic removal of the heparin-binding domain of EC-SOD results in clearance of the enzyme from the extracellular matrix of pulmonary tissues and leads to a loss of antioxidant protection. Using a polyclonal antibody to mouse EC-SOD, the immunodistribution of EC-SOD in normal and bleomycin-injured lungs was examined. EC-SOD labeling was strong in the matrix of vessels, airways, and alveolar surfaces and septa in control lungs. At 2 d post-treatment, a slight increase in EC-SOD staining was evident. In contrast, lungs examined 4 or 7 d post-treatment, showed an apparent loss of EC-SOD from the matrix and surface of alveolar septa. Notably, at 7 d post-treatment, the truncated form of EC-SOD was found in the bronchoalveolar lavage fluid of bleomycin-treated mice, suggesting that EC-SOD is being removed from the extracellular matrix through proteolysis. However, loss of EC-SOD through proteolysis did not correlate with a decrease in overall pulmonary EC-SOD activity. The negligible effect on EC-SOD activity may reflect the large influx of intensely staining inflammatory cells at day 7. These results indicate that injuries leading to pulmonary fibrosis have a significant effect on EC-SOD distribution due to proteolytic removal of the heparin-binding domain and may be important in enhancing pulmonary injuries by altering the oxidant/antioxidant balance in alveolar interstitial spaces.

Sequential two-step cleavage of the retinoblastoma protein by caspase-3/-7 during etoposide-induced apoptosis.

During cellular apoptosis, retinoblastoma protein (RB) is subjected to cleavage near the carboxyl terminus by a caspase-3-like protease. In addition, an heretofore unidentified protease cleaves RB internally, generating fragments of 68 and 48 kDa. Internal cleavage abrogates the ability of RB to associate with E2F. To investigate the mechanism of RB internal cleavage, we developed and employed an in vitro cleavage assay. Incubation of in vitro translated (35)S-RB with apoptotic cell extracts led to RB cleavage at the C-terminus, followed by internal cleavage. The caspase peptide inhibitors z-VAD-FMK or z-DEVD-FMK blocked both cleavage events. Rapid C-terminal and internal cleavage were also observed when recombinant caspase-3 was added to (35)S-RB. Moreover, when caspase-3 was added to nonapoptotic cell extract, efficient internal cleavage of cellular RB was observed. Caspase-mediated internal cleavage occurred following RB residue aspartate(349) in the sequence DSID(349). This sequence is consistent with a DXXD recognition motif for caspase-3-like enzymes. Interestingly, we also observed RB internal cleavage in caspase-3-deficient MCF-7 cells, indicating that other caspases are capable of cleaving RB internally. Indeed, caspase-7, a member of the caspase-3 subfamily, was found to cleave (35)S-RB at both the carboxyl terminus, and following aspartate(349). By contrast, caspases that are not members of the caspase-3 subfamily failed to cleave RB. Taken together, our findings demonstrate that during apoptosis, a caspase-3-like protease is responsible for degradation and functional inactivation of RB by cleaving the protein internally following aspartate(349).

Purification and characterization of extracellular superoxide dismutase in mouse lung.

Extracellular superoxide dismutase (EC-SOD) is the major isozyme of SOD in arteries, but is also abundant in lungs. In particular, mouse lungs contain large amounts of EC-SOD compared to lungs in other mammals. This suggests that EC-SOD may have an amplified function in the mouse lung. This study describes the purification and characterization of mouse EC-SOD as well as its localization in mouse lung. Mouse EC-SOD exists primarily as a homotetramer composed of a pair of dimers linked through disulfide bonds present in the heparin-binding domains of each subunit. In addition, mouse EC-SOD can exist in active multimeric forms. We developed and utilized a polyclonal antibody to mouse EC-SOD to immunolocalize EC-SOD in mouse lung. EC-SOD labeling is strongest in the matrix of vessels, airways, and alveolar septa. This localization suggests that EC-SOD may have important functions in pulmonary biology, perhaps in the modulation of nitric oxide-dependent responses.

p53-independent dephosphorylation and cleavage of retinoblastoma protein during tamoxifen-induced apoptosis in human breast carcinoma cells.

We have investigated several molecular events that occur during the process of tamoxifen-induced apoptosis in human breast carcinoma cells. We show that the treatment of either MCF-7 (containing wild-type p53) or MDA-MB-231 cells (containing mutant p53) with tamoxifen resulted in apoptotic nuclear changes and an increase in the pre-G1 apoptotic population. This was accompanied by activation of the caspase enzymes, as evidenced by specific cleavage of poly(ADP-ribose) polymerase and retinoblastoma (RB) protein. The RB protein was cleaved at both an interior and carboxyl terminus cleavage site. In addition, dephosphorylation of RB was found at an early stage of tamoxifen-induced apoptosis in both cell lines. However, neither induction of p53 in MCF-7 cells nor induction of p21 in either cell line was detected, suggesting that tamoxifen-induced RB dephosphorylation and apoptosis are independent of the p53/p21 pathway. We also observed an increase in levels of the pro-apoptotic Bax protein, the inhibitory cytokine TGF-beta1 and the transcription factor c-Myc in tamoxifen-treated MDA-MB-231 cells, suggesting the possible involvement of these proteins during apoptosis in this system.

The one-ring open hydrolysis intermediates of the cardioprotective agent dexrazoxane (ICRF-187) do not inhibit the growth of Chinese hamster ovary cells or the catalytic activity of DNA topoisomerase II.

Dexrazoxane (ICRF-187), which is clinically used to reduce doxorubicin-induced cardiotoxicity, has growth inhibitory properties through its ability to inhibit the catalytic activity of DNA topoisomerase II. Because the bisdioxopiperazine dexrazoxane undergoes significant ring-opening hydrolysis under physiological conditions to form two one-ring open hydrolysis intermediates, a study was undertaken to determine if these two intermediates had either any growth inhibitory or topoisomerase II inhibitory effects. Neither of the one-ring open intermediates exhibited growth inhibitory effects towards Chinese hamster ovary cells nor were they able to inhibit topoisomerase II. Thus, it was concluded that only intact dexrazoxane is able to inhibit the catalytic activity of topoisomerase II.

Characterization of interior cleavage of retinoblastoma protein in apoptosis.

Previously we reported that at the onset of apoptotic execution, retinoblastoma protein (RB) was cleaved in its interior region, resulting in production of two major fragments, p48 and p68, and that the RB interior cleavage was mediated by a caspase-like activity. Here, we further characterized the RB interior cleavage process in human leukemia cells treated with the anticancer agent etoposide. We found that the RB interior cleavage activity was much more sensitive to two specific tetrapeptide caspase inhibitors, YVAD-CMK and DEVD-FMK, than the poly(ADP-ribose) polymerase cleavage activity, suggesting that two distinct caspases are involved in these processes. Several Asp residues are located in amino acids 341-421 of RB protein, and cleavage of any one of these sites by a caspase would generate a p48, which contains the amino terminus, and a p68 fragment, which contains the A/B pocket and the carboxyl terminus. This hypothesis was supported by the fact that the p48 and p68 fragments had selective binding affinity to different RB antibodies and that the p48 was found only in the low-salt-extracted cytoplasmic fraction, while the p68 was only in the nuclear fraction, of the apoptotic cells. However, the nuclear binding partner of the p68 RB fragment is not the transcription factor E2F-1 since a specific E2F-1 antibody coimmunoprecipitated only the unphosphorylated form of RB, but not the p68 fragment. Lastly, we confirmed that RB also underwent dephosphorylation and carboxyl terminal cleavage during apoptosis, as we and others reported previously.

Characterization of a Chinese hamster ovary cell line with acquired resistance to the bisdioxopiperazine dexrazoxane (ICRF-187) catalytic inhibitor of topoisomerase II.

A Chinese hamster ovary (CHO) cell line highly resistant to the non-cleavable complex-forming topoisomerase II inhibitor dexrazoxane (ICRF-187, Zinecard) was selected. The resistant cell line (DZR) was 1500-fold resistant (IC50 = 2800 vs 1.8 microM) to continuous dexrazoxane exposure. DZR cells were also cross-resistant (8- to 500-fold) to other bisdioxopiperazines (ICRF-193, ICRF-154, and ICRF-186), and somewhat cross-resistant (4- to 14-fold) to anthracyclines (daunorubicin, doxorubicin, epirubicin, and idarubicin) and etoposide (8.5-fold), but not to the other non-cleavable complex-forming topoisomerase II inhibitors suramin and merbarone. The cytotoxicity of dexrazoxane to both cell lines was unchanged in the presence of the membrane-active agent verapamil. DZR cells were 9-fold resistant to dexrazoxane-mediated inhibition of topoisomerase II DNA decatenation activity compared with CHO cells (IC50 = 400 vs 45 microM), but were only 1.4-fold (IC50 = 110 vs 83 microM) resistant to etoposide. DZR cells contained one-half the level of topoisomerase II protein compared with parental CHO cells. However, the specific activity for decatenation using nuclear extract topoisomerase II was unchanged. Etoposide (100 microM)-induced topoisomerase II-DNA complexes in DZR cells and isolated nuclei were similarly one-half the level found in CHO cells and in isolated nuclei. However, the ability of 500 microM dexrazoxane to inhibit etoposide (100 microM)-induced topoisomerase II-DNA covalent complexes was reduced 4- to 6-fold in both DZR cells and nuclei compared with CHO cells and nuclei. In contrast, there was no differential ability of aclarubicin or merbarone to inhibit etoposide-induced topoisomerase II-DNA complexes in CHO compared with DZR cells and isolated nuclei. It was concluded that the DZR cell line acquired its resistance to dexrazoxane mainly through an alteration in the topoisomerase II target.

Collateral sensitivity to the bisdioxopiperazine dexrazoxane (ICRF-187) in etoposide (VP-16)-resistant human leukemia K562 cells.

Etoposide (VP-16)-resistant K562 cells (K/VP.5) were 26-fold resistant to VP-16, due in part to a reduction in DNA topoisomerase II (topoisomerase II) protein levels. Compared with parental K562 cells, VP-16-resistant K/VP.5 cells were found to be 3.4-fold more sensitive to the effects of dexrazoxane (ICRF-187), a topoisomerase II inhibitor that does not stabilize topoisomerase II-DNA covalent complexes. In contrast, K/VP.5 cells were 4.0-fold cross-resistant to merbarone and showed no cross-resistance to fostriecin, two other topoisomerase II inhibitors that do not stabilize topoisomerase II-DNA covalent complexes. Preincubation with ICRF-187 resulted in greater inhibition of subsequent VP-16-induced topoisomerase II-DNA covalent complexes in K/VP.5 cells than in K562 cells. Conversely, preincubation with merbarone resulted in less inhibition of VP-16-induced topoisomerase II-DNA covalent complexes in K/VP.5 cells than in parental K562 cells. Preincubation with forstriecin had little effect on VP-16-induced topoisomerase II-DNA covalent complex formation in either cell line. The onset rates for ICRF-187 inhibition of VP-16-induced topoisomerase II-DNA complex formation were similar in sensitive and resistant cells. In addition, ICRF-187 had a comparable concentration-dependent inhibitory effect on the topoisomerase II catalytic activities of K562 and K/VP.5 cells. Together, our results indicate that collateral sensitivity to ICRF-187 in K/VP.5 cells is due to decreased topoisomerase II protein levels rather than to an alteration in topoisomerase II activity. Furthermore, results suggest that ICRF-187, merbarone, and fostriecin have different mechanisms of action that can be studied effectively in K/VP.5 and K562 cells.

Cyclothiazide modulates AMPA receptor-mediated increases in intracellular free Ca2+ and Mg2+ in cultured neurons from rat brain.

We investigated the modulation of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca2+ ([Ca2+]i) and intracellular free Mg2+ ([Mg2+]i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca2+]i were increased by 0.3-100 microM cyclothiazide, with an EC50 value of 2.40 microM and a maximum potentiation of 428% of control values. [Ca2+]i responses to glutamate in the presence of N-methyl-D-aspartate (NMDA) receptor antagonists were also potentiated by 10 microM cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca2+]i to both kainate and AMPA in the absence of extracellular Na+, and these Na(+)-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC50 value of 12.0 microM. Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg2+]i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.

Reduced phosphorylation of topoisomerase II in etoposide-resistant human leukemia K562 cells.

In this report we examine biochemical and genetic alterations in DNA topoisomerase II (topoisomerase II) in K562 cells selected for resistance in the presence of etoposide (VP-16). Previously, we have demonstrated that the 30-fold VP-16-resistant K/VP.5 cell line exhibits decreased stability of drug-induced topoisomerase II/DNA covalent complexes, requires greater ATP concentrations to stimulate VP-16-induced topoisomerase II/DNA complex formation, and contains reduced mRNA and protein levels of the M(r) 170,000 isoform of topoisomerase II, compared with parental K562 cells. K/VP.5 cells grown in the absence of VP-16 for 2 years maintained resistance to VP-16, decreased levels of topoisomerase II, and attenuated ATP stimulation of VP-16-induced topoisomerase II/DNA binding, compared with K562 cells. Sequencing of cDNA coding for two consensus ATP binding sites and the active site tyrosine in the K/VP.5 topoisomerase II gene indicated that no mutations were present in these domains. In addition, single-strand conformational polymorphism analysis of restriction fragments encompassing the entire topoisomerase II cDNA revealed no evidence of mutations in the gene for this enzyme in K/VP.5 cells. Nuclear extracts from K562 (but not K/VP.5) cells contained a heat-labile factor that potentiated VP-16-induced topoisomerase II/DNA covalent complex formation in isolated nuclei from K/VP.5 cells. Immunoprecipitated topoisomerase II from K/VP.5 cells was 2.5-fold less phosphorylated, compared with enzyme from K562 cells. Collectively, our data suggest that acquired VP-16 resistance is mediated, at least in part, by altered levels or activity of a kinase that regulates topoisomerase II phosphorylation and hence drug-induced topoisomerase II/DNA covalent complex formation and stability.