Bleomycin (BLM) hydrolase catalyzes the inactivation of the antitumor drug BLM and is believed to protect normal and malignant cells from BLM toxicity. The normal physiological function of BLM hydrolase is not known. We now provide evidence for its membership in the cysteine proteinase family. BLM hydrolase was purified to homogeneity from rabbit lungs, and a partial amino acid sequence was determined from a tryptic digest peptide. On the basis of this sequence a 36-mer oligonucleotide was synthesized. The 36-mer oligonucleotide probe hybridized to a single mRNA species of 2.5 kb from several species and was used to isolate an 832-bp cDNA insert from a lambda gt11 rabbit liver cDNA library. This insert encoded the tryptic digest peptide previously identified in rabbit lung BLM hydrolase by amino acid sequencing. Analysis of the predicted amino acid sequence coded by the 832-bp BLM hydrolase cDNA fragment indicated no significant homology with any currently known proteins except for a 15 amino acid portion, which displayed remarkable homology with the active site of cysteine proteinases. Within this active-site region, 10 of the amino acid residues of papain and 9 of aleurain, cathepsin H, and cathepsin L were identical with those of rabbit liver BLM hydrolase. The catalytic cysteine of thiol proteinases was also conserved in BLM hydrolase, and cysteine proteinase specific inhibitors, such as E-64, were found to be potent inhibitors of BLM hydrolase activity. Furthermore, bleomycin hydrolase exhibited cathepsin H like enzymatic activity. Bleomycin hydrolase had, however, no significant cathepsin B or L activities.(ABSTRACT TRUNCATED AT 250 WORDS)
A patient is described with acute myelocytic leukemia refractory to conventional therapy, who also became highly resistant to methotrexate (MTX) after repeated courses of this drug. Leukemia cells from this patient were found to contain an elevated level of dihydrofolate reductase (DHFR) activity, with no change in the affinity of the enzyme for MTX. A sensitive "dot blot" assay revealed a fourfold increase in the gene copy number of DHFR. Southern blot analysis with a human DHFR cDNA probe confirmed this increase in the gene copy number, and demonstrated a similar restriction pattern with Eco R1, Hind III, and Pst 1 as seen with a highly amplified human leukemia cell line, K562. Additional DHFR fragments were detected, not seen in the K562 blot, suggesting the presence of pseudogenes, or a result of gene rearrangements occurring as part of the amplification process. Resistance to MTX in this patient was therefore ascribed to gene amplification and overproduction of DHFR.
IntroductionThe blast crisis of chronic myelogenous leukemia (CML) is refractory to most forms of cancer chemotherapy, but may be amenable to drugs that differentiate rather than kill leukemic cells. One mechanism implicated in resistance to cytodestructive drugs is overexpression of P-glycoprotein, the MDR] gene product. While several classes of drugs sensitize multidrug-resistant (MDR) cells by interfering with the function of P-glycoprotein in vitro, few sensitizers have been effective in vivo. We have developed a preclinical model of MDR/ CML uncomplicated by other mechanisms of drug resistance to evaluate the effects of MDR] overexpression on cytodestructive and differentiation therapy and the ability of sensitizers to restore chemosensitivity in this disease. The CML-derived cell line K562 was transfected with a human MDR] cDNA from the pHaMDR1 /A expression vector and selected with vinblastine. Resistant K562 clones were 20-30-fold resistant to vinblastine, were cross-resistant to doxorubicin and etoposide, and remained sensitive to cytosine arabinoside, 6-thioguanine, hydroxyurea, and mechlorethamine. Resistance was associated with decreased cellular accumulation of cytotoxic drug and was reversed by cyclosporin A and trans-flupenthixol. The MDR phenotype did not adversely affect the ability of K562 cells to produce fetal hemoglobin in response to hemin, and was associated with increased responsiveness of cells to differentiate with cytosine arabinoside. Upon differentiation, the resistant clones increased MDR] mRNA and P-glycoprotein. These studies suggest that the overexpression of the MDR] gene in CML may not adversely affect the ability to undergo erythroid differentiation and that these resistant K562 cell lines are good models for studying drug resistance mediated by P-glycoprotein in CML. (J. Clin. Invest. 1993. 91:2207-2215
A subline of human leukemia cells (K-562), highly resistant to methotrexate, was developed by stepwise selection in the presence of increasing concentrations of this drug. The ED50 of these resistant cells was 1 mM compared to 10 nM for the parental line. Comparison of certain folate-requiring enzymes from crude extracts of the parent and resistant cells showed a 240-fold elevation of dihydrofolate reductase activity in the resistant cells with no significant increase in the levels of the other enzymes. Purified dihydrofolate reductase from the resistant cells had the same physical and kinetic properties as the enzyme from the sensitive cells. Southern blot analysis showed a marked increase in the number of dihydrofolate reductase genes in the resistant line. The genomic organization of the human dihydrofolate reductase gene was determined by hybridization with specific cDNA sequences from a human cDNA to DNA fragments from K-562 cells generated by restriction endonucleases. The human dihydrofolate reductase gene contained at least four intervening sequences and was approximately 30 kb in size. Northern blot studies demonstrated an increase of dihydrofolate reductase mRNA species; the predominant message was 3.8 kb. Karyotype analysis revealed three elongated marker chromosomes, derived from chromosomes 5, 6, and 19 which contained homogeneous staining regions, which were not present in the parent cell line.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.