Characterization of a membrane-associated protein kinase of multidrug-resistant HL60 cells which phosphorylates P-glycoprotein

Cells containing increased levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug-resistant phenotype. In the present study we have analyzed protein kinases capable of phosphorylating P-glycoprotein in membranes of HL60 cells isolated for resistance to vincristine. Analysis of this system demonstrates that in isolated membranes the protein kinase inhibitor staurosporine greatly reduces P-glycoprotein phosphorylation. In contrast, the kinase inhibitor H-7 does not affect this reaction. Fractionation of solubilized membrane proteins from sensitive and resistant cells on DEAE-cellulose reveals a major protein kinase (PK-1) which exhibits optimal activity in the presence of Mn2+ and histone H1. This enzyme fraction does not contain detectable levels of protein kinase C or cAMP-dependent protein kinase. PK-1 phosphorylation of two endogenous proteins is, however, greatly enhanced in the presence of phosphatidylserine or phosphatidyl-inositol. In reaction mixtures containing Mg2+ or Mn2+ in the absence of phospholipid, PK-1 from resistant cells phosphorylates an endogenous protein of 180 kilodaltons (P180), which exhibits an electrophoretic mobility identical to P-glycoprotein. In parallel experiments with PK-1 from sensitive cells there is no detectable phosphorylation of a P180 protein. P180 phosphorylated by PK-1 from resistant cells is immunoprecipitated by antibody against P-glycoprotein. Additional studies demonstrate that PK-1 is capable of phosphorylating specific synthetic peptides which correspond to the sequence of P-glycoprotein. Peptide phosphorylation occurs at both serine and threonine residues. These studies thus identify a novel membrane-associated protein kinase in HL60 cells which is capable of phosphorylating P-glycoprotein. This enzyme may have an important role in regulating levels of multidrug resistance.     

Adriamycin resistance in HL60 cells in the absence of detectable P-glycoprotein

Previous studies have shown that the development of multi-drug resistance in cell lines treated with chemotherapeutic agents is closely associated with the overexpression of a 170-180 kilodalton surface membrane glycoprotein (P-glycoprotein). In the present study a monoclonal antibody against the P-glycoprotein was used to determine if this protein is overexpressed in multi-drug resistant HL60 cells. Using either indirect immunofluorescent staining or immunoblot analysis P-glycoprotein could not be detected in HL60 cells isolated for resistance to adriamycin. In contrast HL60 cells isolated for resistance to vincristine contain the P-glycoprotein and the amount of this material increases with increasing levels of resistance. These studies thus demonstrate adriamycin resistance in P-glycoprotein negative HL60 cells. Furthermore adriamycin and vincristine are found to have distinct effects in inducing overexpression of P-glycoprotein in the HL60 cell line. This information could be useful in the development of therapeutic strategies for the treatment of certain forms of cancer.     

Calcium-activated potassium channels expressed from cloned complementary DNAs.

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.     

Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa

The effects of controlled stress, i.e. cooling, upon the distribution of actin in ram spermatozoa were examined to investigate the hypothesis that cytoskeletal proteins are involved in the maintenance of sperm plasma membrane integrity. The normal distribution of actin on the spermatozoon was initially determined. A monoclonal antibody (IgM) interacted exclusively with the post-acrosomal region and the principal piece of the flagellum. By the use of a polyclonal antibody, actin was detected on the acrosome (excluding the equatorial segment), the post-acrosomal region and the whole of the flagellum. The actin was present in its non-filamentous form. Spermatozoa fixed at 39 degrees C and then treated for the immunofluorescent detection of actin with the monoclonal antibody were mostly unstained (proportion stained = 4.4% (+/- 1.6; s.e.m.); n = 8 ejaculates). Provided spermatozoa were permeabilized by greater than 0.025% Triton X-100 before immunofluorescence, actin was localized in the postacrosomal region of all sperm heads, and to a minor extent on the principal piece of the flagellum. Use of the polyclonal antibody confirmed that the post-acrosomal antigen was unmasked by detergent treatment. Slow cooling, over 2-h periods to various temperatures between 5 and 15 degrees C, also induced an increase in the proportion of cells showing post-acrosomal actin immunoreactivity. Cooling through the temperature range 15 to 10 degrees C markedly increased the proportion of immunoreactive cells (mean +/- s.e.m.; 12 +/- 4.5% at 15 degrees C; 27 +/- 4.5% at 10 degrees C; n = 4 ejaculates). Further cooling to 5 degrees C failed to elicit increased staining. Ultrastructural examination of cooled spermatozoa confirmed that a subpopulation of spermatozoa exhibited post-acrosomal actin immunoreactivity after cooling. These results are compatible with the suggestion that actin fulfills a stabilizing function in spermatozoa.     

人α1/158Ⅴ型(α1c型)干扰素基因在大肠杆菌中的表达及其产物的初步纯化

采用DNA聚合酶链反应(PCR)技术,将本实验室从中国人胎肝细胞染色体DNA中发现和分离的IFN-α1/158V基因的原始克隆,改造成适于进行非融合蛋白原核表达的结构形式,并在大肠杆菌中获得高效表达。测得重组IFN-α1/158V的抗病毒活性为1.9×10~7单位/升菌液。随后又采用以单克隆抗体亲和层析为主的纯化流程对表达产物进行初步纯化,获得了在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带的纯化产物。