Biomedical subjects

J S Lazo

Publications and source records attributed to J S Lazo.

At least 91 records · Page 5Linked to original sources

PubMed1994

Human metallothionein isoform gene expression in cisplatin-sensitive and resistant cells.

Overexpression of metallothioneins (MTs) has been observed in some cis-diamminedichloroplatinum (CDDP)-resistant cells. We have developed oligonucleotide probes for each of the six non-neuronal human MT (hMT) isoforms and used them to assay hMT isoform expression in three pairs of CDDP-resistant and -sensitive human carcinoma cell lines, i.e., SCC25/CP versus SCC25 cells, H69/CP versus H69 cells, and SW2/CP versus SW2 cells. We found a 9-fold increase in basal hMT-IIa mRNA levels and a 5-fold increase in hMT-le mRNA levels in SCC25/CP cells, compared with SCC25 cells. Nuclear run-on studies also revealed a 3-fold increase in hMT-IIa transcription rate. Basal hMT-IIa steady state mRNA levels were 2-3.6-fold greater in H69/CP and SW2/CP cells, compared with their parental cells. No significant basal expression of hMT-Ia, -Ib, -If, or Ig was detected in any cells, suggesting that overexpression of these isoforms was not commonly associated with the CDDP-resistant phenotype. Levels of constitutively expressed hMT isoforms, as well as hMT-If, could be elevated by treatment of all cells with 100 microM zinc. The universal overexpression of hMT-IIa suggests a role of this particular isoform in CDDP resistance. Using our isoform-specific hMT-IIa probe and the demethylating agent 5'-azacytidine (AZC), we found that AZC pretreatment increased basal hMT-IIa mRNA levels in SCC25 but not SCC25/CP cells, suggesting that DNA hypomethylation was responsible for higher basal hMT-IIa mRNA levels in SCC25/CP cells. AZC had little or no effect on hMT-If or -Ig expression. Limited restriction analysis by methylation-sensitive enzymes, however, revealed no obvious differences in the methylation status of the hMT-IIa promoter in either SCC25 or SCC25/CP cells.

Azacitidine↗

PubMed1994

Bleomycin.

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Animals↗

PubMed1994

Interleukin-1 beta-mediated metallothionein induction and cytoprotection against cadmium and cis-diamminedichloroplatinum.

Metallothioneins (MTs) are major intracellular protein thiols that have been associated with resistance to the cytotoxicity of heavy metals and some cytotoxic anticancer drugs. The accumulation and subcellular location of MT and its functionality were examined in a human hormone-independent prostatic tumor cell line (DU-145) exposed to cytokines that participate in autocrine growth and the acute phase response. Incubation of DU-145 cells with interleukin-1 beta (IL-1 beta) caused a concentration-dependent increase in MT protein reaching a 2- to 3-fold maximum increase 24 to 48 hr after treatment with 10 U of IL-1 beta/ml; smaller increases were seen with human recombinant IL-1 alpha and tumor necrosis factor alpha. DU-145 cells constitutively expressed only MT-IIa and MT-Ie mRNA and the IL-1 beta-mediated increase in MT protein was preceded by a marked elevation solely in MT-IIa mRNA. Basal MT was immunolocalized by confocal microscopy primarily to the nucleus of DU-145 cells with some granular deposits near the cell surface. IL-1 beta treatment increased nuclear MT. DU-145 cells pretreated with IL-1 beta were 2- to 3-fold resistant to the toxicity of CdCl2 and cis-diamminedichloroplatinum(II) (cisplatin). Both MT induction and cytoprotection against CdCl2 and cisplatin were blocked by concomitant incubation with a recombinant human IL-1 receptor antagonist protein. These results suggest the responsiveness of human prostatic tumor cells to cytotoxic electrophilic anticancer drugs can be diminished by direct exposure to the cytokine IL-1 beta, possibly by a mechanism involving MT.

Cadmium↗

PubMed1994

Differential induction of etoposide-mediated apoptosis in human leukemia HL-60 and K562 cells.

Etoposide (VP-16) is one of several DNA-damaging agents that induce subcellular structural changes associated with apoptosis. VP-16 exerts its DNA-damaging and cytotoxic effects subsequent to interference with DNA topoisomerase II activity. VP-16 also stimulates c-jun and c-fos mRNA expression in some cell lines, including human leukemia K562 and HL-60 cells. To compare the temporal relationship between drug-induced c-jun expression and apoptosis, we examined cell morphology, cell viability, DNA integrity, and c-jun induction during VP-16 treatment of K562 and HL-60 cells. VP-16 (10 microM)-induced internucleosomal DNA damage and nuclear fragmentation were readily apparent within 6 hr in HL-60 cells but were absent in K562 cells treated for up to 24 hr. Some internucleosomal DNA damage was observed in K562 cells but only after treatment with 100 microM VP-16 for 24 hr. In contrast, VP-16-induced DNA single-strand breaks, VP-16-induced topoisomerase II/DNA covalent complex formation, and VP-16-mediated growth inhibition were similar in K562 and HL-60 cells. Also, the time course of VP-16-induced c-jun mRNA expression was comparable for both K562 and HL-60 cell lines. Western blot analysis of whole-cell lysates showed that Bcl-2 protein levels were 13-fold greater in HL-60 cells than in K562 cells. Thus, the resistance of VP-16-treated K562 cells to apoptosis was not attributable to protection by Bcl-2. Furthermore, the relatively high levels of Bcl-2 in HL-60 cells were not sufficient to protect these cells against apoptosis. Together, our results indicate that the temporal coupling of VP-16-induced DNA damage, c-jun expression, and apoptosis is cell type specific and suggest that different signaling pathways for apoptosis are operating in these two human leukemia cell lines.

Apoptosis↗

PubMed1993

NAD depletion after in vitro exposure of murine lung slices to bleomycin.

Bleomycin (BLM), a DNA-cleaving, antitumor antibiotic, causes pulmonary fibrosis. It also causes cell injury and activates the nuclear enzyme poly(ADP-ribose) polymerase (PAP; EC 2.4.2.30) in lung slices exposed to the drug in vitro. 3-Aminobenzamide (3-AB), a PAP inhibitor, prevents enzyme activation and cell injury. We have examined the potential role of ATP and NAD depletion in injury of BLM-sensitive C57B1/6N and -resistant BALB/cN murine lung slices treated with BLM or deprived of glucose, the major metabolic substrate of lung. Lung slices either were treated for 45 min with injurious concentrations of BLM (10-500 micrograms/mL) or were incubated without glucose, in the presence or absence of 2.5 mM 3-AB. Only the highest concentration of BLM, 500 micrograms/mL, caused any ATP depletion, and this 35% decrease was transient, occurring at 220 min in C57B1/6N slices. In contrast, glucose deprivation caused 50-70% ATP depletion in slices from both strains. BLM alone at 100 and 500 micrograms/mL caused a sustained 30-70% NAD depletion from 75 min through 400 min in C57B1/6N mouse lung slices. In the resistant BALB/cN lung slices, NAD depletion by BLM was only seen at 400 min. 3-AB almost completely antagonized NAD depletion in slices from both strains. In contrast to BLM, glucose deprivation did not decrease NAD levels unless 3-AB was present in C57B1/6N slices. Thus, ATP depletion may play a role in the injurious effects of glucose deprivation, but does not appear to be a major factor in pneumocyte injury caused by BLM. NAD depletion or other effects of PAP activation appear to account for the strain-selective, injurious effect of BLM on lung tissue.

Adenosine Triphosphate↗

PubMed1993

Structural basis for the sequence selectivity of DNA cleavage by bleomycins.

DNA strand scission by the bleomycin analogs talisomycin S10b and a novel fluorescent mimic, fluoromycin, has been characterized and compared with that of bleomycin A2. Both analogs were found to have essentially the same sequence specificity for DNA strand scission as bleomycin A2. As observed with bleomycin A2, the preferred sites of DNA cleavage by talisomycin S10b and fluoromycin were 5'-GpT-3' and 5'-GpC-3'. These results suggest that the DNA sequence selectivity of bleomycins remains unaltered upon modification of the C-terminal domain.

Base Sequence↗

PubMed1993

Evidence of differential cisplatin-DNA adduct formation, removal and tolerance of DNA damage in three human lung carcinoma cell lines.

The expression of intrinsic resistance to cisplatin in two lung cancer cell lines, one derived from a small cell carcinoma (SW1271) and the other from an adenocarcinoma (A549), relative to a drug-sensitive small cell line SW900, was characterized by: (i) expression of cross-resistance to mitomycin C and cadmium chloride, but increased sensitivity to adriamycin and etoposide; (ii) significantly decreased cisplatin uptake; (iii) elevated levels of glutathione which could be reduced by buthionine L-sulfoximine resulting in significant sensitization of the cells to cisplatin; (iv) a lack of consistent modification of metallothionein content and expression of levels of glutathione S-transferase, glutathione reductase and glutathione peroxidase or of activities of DT-diaphorase or catalase; (v) significantly reduced total DNA-platination levels immediately following a 1 h cisplatin treatment with 10 micrograms/ml (33.3 microM); (vi) increased removal of Pt-GG and Pt-AG adducts by the A549 cells, consistent with increased repair capacity, but a lack of removal of these major adducts by the SW1271 cells indicative of tolerance of this drug-induced DNA damage. These data therefore provide evidence of differential formation, repair and tolerance of DNA damage following exposure of three human lung carcinoma cell lines to cisplatin.

Adenocarcinoma↗

PubMed1993

Nuclear localization of bacterial Streptoalloteichus hindustanus bleomycin resistance protein in mammalian cells.

Prokaryotes produce a variety of toxins that affect genomic function of both eukaryotes and prokaryotes. The 375-base pair bacterial gene Streptoalloteichus hindustanus (Sh) ble encodes a small protein, Streptoalloteichus hindustanus bleomycin resistance protein (BRP), that inhibits in vitro DNA cleavage by the prokaryotic glycopeptide bleomycin, which is a clinically used anticancer drug. NIH/3T3 cells infected with a retroviral vector containing Sh ble (SH-9 cells) were highly resistant to the cytotoxicity of bleomycin-like drugs but not to the cytotoxicity of other, structurally unrelated, DNA-cleaving agents. Expression of BRP did not markedly alter total cellular content or distribution of bleomycin-like compounds. Fluorescently labeled bleomycin was primarily localized in cytoplasmic vesicles in NIH/3T3 and SH-9 cells, whereas BRP, which has no established nuclear localization sequence, was segregated to the nucleus and more specifically to euchromatin. This karyophilic BRP may intercept bleomycin in the nucleus.

3T3 Cells↗

PubMed1993

Bleomycins.

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Animals↗

PubMed1992

Sensitization of human cervical carcinoma cells to cis-diamminedichloroplatinum(II) by bryostatin 1.

Bryostatins are an important class of protein kinase C (PKC) activators. We have investigated the effect of bryostatin 1 on the antiproliferative activity of cis-diamminedichloroplatinum(II) (CP). A 24-h pretreatment of HeLa cells with 1 nM bryostatin 1 increased cellular sensitivity to CP by 4-fold. The effect of bryostatin 1 on the IC50 of CP (concentration of drug required to inhibit cell proliferation by 50%) was concentration-dependent and biphasic; the maximum effect of bryostatin 1 was seen with 1 nM, but higher concentrations of bryostatin 1 (greater than or equal to 10 nM) produced less CP sensitization. Although bryostatin 1 and phorbol esters caused an equivalent stimulation of HeLa cell PKC in cell-free systems, bryostatin 1 was less effective than phorbol esters in sensitizing cells to CP. Additionally, higher concentrations of bryostatin 1 (greater than or equal to 10 nM) antagonized CP sensitization by phorbol esters. Bryostatin 1 was even more potent than 12-O-tetradecanoylphorbol-13-acetate in inducing PKC down-regulation, and the maximum down-regulation was achieved with 10 nM bryostatin 1. Bryostatin 1 also increased cellular sensitivity to a CP analogue, cis-dichloro(ethylenediamine)platinum(II). A 24-h pretreatment with 1 nM bryostatin 1 increased cellular cis-[3H]DEP by 60%. The concentration- and time-dependent enhancement in CP sensitivity by bryostatin 1 was related to the increase in cis-[3H]DEP level. Thus, cellular accumulation of CP may be regulated by a PKC-dependent phosphorylation event.

Antineoplastic Agents↗

PubMed1992

In vivo circumvention of human colon carcinoma resistance to bleomycin.

Metabolic inactivation of bleomycin (BLM) by cysteine proteinase-like enzymes is thought to be a major mechanism of BLM tumor resistance. We now report that the human colon carcinoma COLO-205 is highly resistant to BLM and that E-64, a cysteine proteinase inhibitor, sensitizes COLO-205 to BLM. Treatment of COLO-205-bearing nude mice with either E-64 (40 mg/kg) or BLM (10 mg/kg) alone did not inhibit COLO-205 growth. However, pretreatment with E-64 prior to BLM prevented these xenografts from growing. Analysis by high performance liquid chromatography of in vivo BLM metabolism following [3H]BLM A2 treatment of COLO-205-bearing nude mice showed a different metabolic profile among the various organs and the tumor. Whereas [3H]BLM A2 was the only major radioactive peak detected in sera and tumors, several metabolites, including deamido-BLM A2, were found in kidney, liver, and lung as early as 15 min. Pretreatment of mice with E-64 inhibited tumor, kidney, and lung BLM A2 metabolism. Furthermore, pretreatment with E-64 increased BLM A2 accumulation in tumors (6.1-fold), kidney (4.0-fold), lung (2.8-fold), liver (1.8-fold), and serum (1.7-fold). E-64 pretreatment did not enhance the major toxicity of BLM, pulmonary fibrosis, as determined by both lung hydroxyproline levels and histopathology. Thus, the cysteine proteinase inhibitor E-64 affects the metabolic fate and the levels of accumulation of BLM in vivo. These results demonstrate that resistance of human COLO-205 tumors to BLM can be circumvented by E-64 without enhancement of the major side effect of BLM, suggesting a possible clinical use of this combination therapy.

Animals↗

PubMed1992

Structural requirements of lyngbyatoxin A for activation and downregulation of protein kinase C.

Structure-activity studies of novel synthetic analogues of lyngbyatoxin A reveal that the lactam ring but not the 7-linalyl moiety of lyngbyatoxin A is essential for the in vitro stimulation of protein kinase C (PKC). (-)-Indolactam V (ILV), which contains no hydrophobic substituent at C-7, or analogues containing either a linalyl or n-hexyl group at C-7 were equally efficacious in stimulating HeLa cell PKC in vitro and in competing with phorbol 12,13-dibutyrate for binding to PKC in intact cells. The hydrophobicity of alkyl groups at C-7, however, influenced the potency of these compounds to bind to and activate PKC. In addition, these compounds exhibited differences in their ability to translocate PKC. Lyngbyatoxin A (0.1 microM) like TPA induced a rapid translocation of PKC from the cytosol to the membrane and subsequently led to a sustained decrease in both cytosolic and membrane PKC activity. In contrast, (-)-n-hexylILV (0.1 microM) and (-)-ILV (1 microM) produced a transient and attenuated decrease in cytosolic PKC activity. At concentrations that produced half-maximal PKC stimulation, (-)-ILV did not cause any downregulation of PKC whereas lyngbyatoxin A and (-)-n-hexylILV led to 60% and 40% PKC downregulation, respectively. Western blot analyses with monoclonal antibodies to PKC isoforms indicated that reduction in PKC activity by chronic exposure to TPA or lyngbyatoxin A analogues could be explained by downregulation of PKC alpha. Constitutive expression of PKC beta and PKC gamma isoforms was low in HeLa cells and was not affected significantly by TPA or lyngbyatoxin A analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Synthesis and evaluation of fluoromycin: a novel fluorescence-labeled derivative of talisomycin S10b.

We have synthesized fluoromycin (FLM), a novel fluorescein-labeled derivative of talisomycin S10b (TLM S10b), and used it to evaluate cellular drug accumulation and distribution in bleomycin (BLM)-sensitive and -resistant cell lines. The fluorescence intensity of FLM was 300- to 400-fold greater than that of BLM A2, TLM S10b, or the lipophilic BLM analogue, liblomycin. FLM possessed an antiproliferative potency similar to liblomycin in BLM-sensitive human A-253 squamous carcinoma cells but was less potent than BLM A2 or TLM S10b. C-10E cells, a clone of A-253 cells with 40- to 50-fold resistance to BLM A2 and TLM S10b, were 50-fold resistant to FLM. A partially revertant cell population (C-10E ND) regained sensitivity to BLM A2, TLM S10b, and FLM. FLM like BLM cleaved pGEM-3Z plasmid DNA in vitro in a concentration-dependent manner. Flow cytometric analysis of FLM content in C-10E and C-10E ND cell lines showed 4-fold and 2-fold lower fluorescence intensity, respectively, compared with A-253 cells. Similar results were seen by fluorescence spectrophotometry with cell extracts. Fluorescence microscopy indicated heterogeneous distribution among A-253 cells with at least 50% of the cells exhibiting marked nuclear fluorescence localization. In contrast, C-10E cells displayed lower cellular fluorescence and predominantly cytoplasmic localization. C-10E ND cells exhibited a mixed population of nuclear and cytoplasmic vesicular localization with fluorescence levels that were intermediate between A-253 and C-10E cells. Thus, BLM-resistant cells have reduced levels of FLM and appear to have a lower nuclear:cytoplasmic ratio of FLM. FLM may be useful in studying the intracellular fate of BLM-like drugs as well as providing a tool to detect and isolate BLM-resistant cells.

Antineoplastic Agents↗

PubMed1992

Murine strain differences in acute lung injury and activation of poly(ADP-ribose) polymerase by in vitro exposure of lung slices to bleomycin.

The DNA-cleaving, antitumor antibiotic bleomycin (BLM) causes pulmonary fibrosis, but the essential early events initiating the fibrotic state have not been well characterized. Thus, we have directly examined BLM-mediated pulmonary cell injury by monitoring lactate dehydrogenase (LDH) release and nuclear poly(ADP-ribose) polymerase (PAP) activity, which is stimulated by DNA breakage, using lung slices isolated from BLM-sensitive (C57B1/6) and BLM-resistant (BALB/c) mice. Lung slices were incubated continuously with or without the PAP inhibitor, 3-aminobenzamide (3-AB), and exposed to BLM for 45 min. LDH release from C57B1/6 lung slices increased 2-fold by 8.5 h after treatment with BLM. In contrast, BLM failed to enhance cumulative LDH release by BALB/c mouse lung slices. Co-incubation of C57B1/6 lung slices with 3-AB prevented BLM-induced LDH release. Nuclear PAP was activated 3- to 4-fold 1.25 h after exposure of C57B1/6 lung slices to BLM but returned to control levels by 3.75 h. Nuclear PAP was only marginally affected at these times in BALB/c lung slices. Co-incubation of C57B1/6 slices with 3-AB prevented the early increases in PAP activity. These results demonstrate that murine strain sensitivity to acute cell injury and early PAP activation by BLM in lung slices parallels the in vivo sensitivity of lungs. In addition, 3-AB suppresses PAP activation and acute cell injury in lung slices. Differential activation of PAP appears to govern murine strain variation in response to BLM and is consistent with the hypothesis that activation of PAP participates in acute pneumocyte injury, initiating the process of BLM-induced fibrosis.

Animals↗

PubMed1992

Expression of pulmonary metallothionein genes in late gestational lambs.

Metallothioneins (MT) are low molecular weight proteins that are important in providing protection against heavy metals such as cadmium. Other precise physiologic roles for this family of proteins are less clear, but fetal hepatic cell proliferation and differentiation may be regulated through changes in MT levels and attendant MT-mediated regulation of zinc levels. The role of MT in other developing tissue, most notably lung, is far less clear. Although expression of MT has been reported to be extremely low in early postnatal and mature lung, we hypothesized that MT has a more ubiquitous role in organ development and that pulmonary MT levels may be elevated during periods of rapid lung growth. Thus, we studied expression of MT in late-gestation fetal lambs. Sheep are particularly useful because alveolarization of lung parenchyma occurs before birth (by d 120 of a normal 147-d gestation). Immunoreactive MT was localized to bronchial epithelium of fetal, newborn, and mature sheep. The intensity of staining was greatest in the 130-d gestational age (saccular) lung, where positive reaction product was noted in the cytoplasm and nucleus of alveolar epithelial and interstitial cells. We next evaluated MT expression in developing lung tissue using Northern blot analysis and 32P-cDNA probes against the 3'-untranslated regions of mRNA encoding each of four known functional sheep MT (sMT) isoforms. Expression of sMT-II, sMT-Ia, and sMT-Ib was restricted to the saccular stage (120-132 d gestational age), and sMT-Ic mRNA was not detected in pulmonary samples from any stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

In vivo sensitization of human lung carcinoma to bleomycin by the cysteine proteinase inhibitor E-64.

We recently demonstrated that the cysteine proteinase inhibitor, E-64, sensitizes human Burkitt's lymphoma (Daudi) to the antitumor action of bleomycin (BLM) by blocking its metabolism. We now report that E-64 sensitizes the BLM resistant human lung carcinoma A-549 by a mechanism unrelated to the inhibition of BLM metabolism. Treatment of A-549 tumor-bearing nude mice with either BLM (10 mg/kg) or E-64 (40 mg/kg) every other day for 10 days did not inhibit tumor growth. However, a 30 min pretreatment with E-64 prior to BLM caused complete and sustained inhibition of tumor growth. In contrast to our results with Burkitt's lymphoma, E-64 did not inhibit BLM metabolism but rather enhanced the tumor accumulation of BLM; within 10 min of BLM administration, tumors from E-64 pretreated mice showed a 6-fold higher accumulation of BLM A2 compared to non-pretreated xenografts. Furthermore, the level of tumor-associated BLM A2 remained 2-fold higher in E-64 pretreated mice 20 and 30 min after BLM administration. In E-64 pretreated mice, the plasma level of BLM was increased by 2-fold. These results demonstrate that the cysteine proteinase inhibitor, E-64, sensitized human lung carcinoma A-549 to BLM and, contrary to the expected mechanism, this effect of E-64 was not related to the inhibition of BLM metabolism.

Animals↗

PubMed1992

Neutralization of bleomycin hydrolase by an epitope-specific antibody.

Bleomycin hydrolase (BH) is a cysteine proteinase that terminates the pharmacological action of bleomycin (BLM). Amino acid sequence data obtained from a tryptic digest fragment of purified rabbit lung BH were used to synthesize a 14-amino acid peptide (LAVLEQEPIVLPAK; BHP14), which was conjugated to horseshoe crab hemocyanin and used to produce rabbit antiserum that was immunoreactive to both BHP14 and rabbit BH. Anti-BHP14 binding to BHP14 could be competitively blocked by the presence of either BHP14 or BH. Anti-BHP14 recognized both purified rabbit liver BH and postmicrosomal fraction from rabbit liver on Western blot, as a single band of M(r) approximately 48,000. Anti-BHP14 inhibited, in a concentration-dependent manner, BH activity in rabbit liver cytosolic fractions, as measured by deamido-BLM A2 formation. Thus, we have generated an epitope-specific neutralizing antibody to rabbit BH, which can block the metabolism of BLM by homogenates from rabbit tissue. These results suggest that the LAVLEQEPIVLPA epitope of rabbit BH is involved in the metabolism of BLM or is topologically near the active site. Furthermore, a BLM-resistant squamous carcinoma (C-10E) exhibited slightly more immunoreactivity, by enzyme-linked immunosorbent assay, to anti-BHP14 than did the parental A-253 cells, and a partially revertant (C-10E ND) cell line had intermediate anti-BHP14 binding. BH activity in these cell lines was in the same rank order as antibody binding, but differences in immunoreactivity were less than differences in enzymatic activity. Our epitope-specific neutralizing antibody should be useful in the further characterization of BH.

Amino Acid Sequence↗

PubMed1992

Bleomycins.

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Animals↗