Purification, cDNA cloning, and expression of human sorcin in vincristine-resistant HOB1 lymphoma cell lines.
Human sorcin, M(r) 22-kDa/pI 5.3, was identified in HOB1 lymphoma cells resistant to 1.0 microM vincristine and polyclonal antibody against it was produced. Using antibody to precipitate sorcin from 32P-labeled cell extract, the SDS-PAGE-resolved human sorcin was not phosphorylated in contrast to the report of A. M. van der Bliek et al. ((1986) EMBO J. 5, 3201-3208) in which the authors emphasized hamster sorcin might be phosphorylated through a cAMP-dependent protein kinase. Although sorcin had not been found among proteins of parental HOB1 cells labeled by [35S]-methionine and resolved by 2-D -PAGE, it could be immunoprecipitated from the cytosolic extract of the same cells. In vitro phosphorylation study did not reveal an enhanced phosphoprotein located in the area of 22 kDa, either. A 521-bp human sorcin cDNA fragment synthesized by PCR was used to screen cDNA library constructed from mRNA of HOB1/VCR1.0 cells. Human sorcin cDNA encoded 198 amino acids, 10 among which have been replaced in hamster sorcin. Of the 10 amino acid changes, 6 involve serine's in human sorcin compared to hamster's 1. These data suggest a difference in phosphorylation state between the two species. The results of Southern blot indicate the sorcin gene was amplified in HOB1/VCR1.0 cells under the pressure of high concentration of vincristine but not secondary to the mdr gene amplification. 2-step PCR, exhibiting the gene could be latently expressed in parental HOB1 cells, confirms the above result of immunoprecipitation. Interestingly, HOB1/VCR0.5 cells (resistant to 0.5 microM vincristine) do not show amplification of the gene but do have increased expression of the protein. When this cell line was cultured in 0.1 microM vincristine for a period of time, the protein's expression amount reverted to the latent level. These results suggest that sorcin should have a place in mediating resistance of HOB1 cells to vincristine.
Lee
W P
WP
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, R.O.C.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D018432:Drug Resistance, Multiple / Q000235:genetics; D005784:Gene Amplification; D015870:Gene Expression; D006801:Humans; D008223:Lymphoma / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D010766:Phosphorylation; D013045:Species Specificity; D014407:Tumor Cells, Cultured; D014750:Vincristine / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D009363:Neoplasm Proteins; C503113:SRI protein, human; D014750:Vincristine