Purification, cDNA Cloning, and Expression of Human Sorcin in Vincristine-Resistant HOB1 Lymphoma Cell Lines

Abstract

Human sorcin,Mr22-kDa/pI5.3, was identified in HOB1 lymphoma cells resistant to 1.0 μMvincristine (designated HOB1/VCR1.0). The protein was purified and polyclonal antibody against it was produced. Using antibody to precipitate sorcin from32P-labeled cell extract, the SDS–PAGE-resolved human sorcin was not phosphorylated in contrast to the report of A. M. van der Blieket 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 vitrophosphorylation 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 themdrgene 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 μMvincristine) do not show amplification of the gene but do have increased expression of the protein. When this cell line was cultured in 0.1 μMvincristine 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.