Protection of mammalian cells from the toxicity of bleomycin by expression of a bleomycin-binding protein gene from Streptomyces verticillus.
A gene, blmA, encodes a bleomycin (Bm)-binding protein, designated BLMA, from Bm-producing Streptomyces verticillus and confers resistance to Bm in Streptomyces and Escherichia coli cells. In the present study, by transfection of the gene into COS-1 cells with a plasmid designated pEF-BOS/blmA, which contains a strong promoter from the human polypeptide chain elongation factor 1alpha, we transiently overproduced BLMA at a high level of approximately 4% of the whole cell protein. Although NIH/3T3 cells transfected with pEF-BOS/blmA, designated NIH/3T3-BR cells, stably expressed BLMA, its expression level was about 0.1% of the total protein. Using an anti-BLMA monoclonal antibody reported previously [Sugiyama et al. (1995) FEBS Lett. 362, 80-84], we revealed that BLMA is localized in the nucleus of pEF-BOS/blmA-transfected COS-1 and NIH/3T3-BR cells. Semi-permeabilized nuclear transport experiments showed that BLMA penetrates the nuclear envelope by energy- and transporter-independent passive diffusion, suggesting that the karyophilic nature of BLMA may be due to the acidic nature of the protein. NIH/3T3-BR cells were 130-fold more resistant to Bm than the host cells. NIH/3T3 cells exhibited a swollen nuclear envelope and a malformed spindle body and overexpressed at least 4 kinds of stress proteins including calreticulin and mitochondrial matrix protein P1 when exposed to 25 microg/ml of Bm, whereas NIH/3T3-BR cells grew without morphological alteration and expressed no stress proteins under the same conditions. Furthermore, reverse transcription-polymerase chain reaction and Northern blot analysis showed that the expression of interleukin-6, an inflammatory cytokine, is activated by addition of Bm in NIH/3T3 cells, but not in the NIH/3T3-BR cells. These results suggest that BLMA contributes to protection of mammalian cells from the inflammatory effect of Bm.
Kumagai
T
T
Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, Minami-ku, Hiroshima, 734-8551, Japan.
Sugiyama
M
M
D016475:3T3 Cells; D000123:Acetyltransferases; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001761:Bleomycin / Q000633:toxicity*; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics; D008322:Mammals; D051379:Mice; D009685:Nuclear Envelope / Q000378:metabolism; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D015850:Interleukin-6; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D001761:Bleomycin; C087954:bleomycin N-acetyltransferase; D000123:Acetyltransferases