ABSTRACT
Leukoregulin is an immunologic hormone whose anticancer action includethe inhibition of cellular proliferation and cytolysis of tumor cells eitherdirectly or indirectly by enhancing the tumor cell's susceptibility to dest-ruction mediated by natural killer cells.The optimal condition for the ind-uction of leukoregulin Were studied by using orthogonal experimental design.The optimal concentration is 1x10~7/ml for human spleen cells;3.1?g/mlfor phytohemagglutinin(PHA);12.5ng/ml for 12-0-tetradecanoyl-phorbol-13-acetate;15% for boyin serum.Leukoregulin activity prepared by 70 hincubation is maximum,the experiment also confirmed that the inducingleukoregulin effect of PHA-PⅢis significantly high.
ABSTRACT
The cellular sources of leukoregulin (LR) and lymphotoxin (LT) from human peripheral blood leukocytes or spleen cells were examined The ratios of LT activity to LR activity in lymphokine preparations from different individuals were not constant but varied over 640-fold, suggesting that LR and LT activities were mediated by distinctly different entities.Purified LR was used to examine the relative susceptibilities of human or mice cells to LR.Most human tumor cells were very sensitive to LR while human normal cells and mice normal or tumor cells were less sensitive or resistant.Crude preparations of human interferon (Hu IFN)-? or Hu IFN-? were more cytotoxic to human tumor cells than partially purified natural Hu IFN-? or highly purified rHu IFN-?D, and crude or purified LR were more cytotoxic to human tumor cells than Hu IFN-? Hu IFN-?.Hu IFN-? activity but not cytotoxic activity could be removed from preparatons containing Hu IFN-?,LR and LT activities with the aid of monoclonal antibody to Hu IFN-?. Natural killer cytotoxic factor (NKCF) supernatants from the co-culture of human spleen cells and NK susceptible cell line K562 or Molt-4 were strongly cytotoxic to various tumor cell lines.The NKCF activity was completely adsorbed after incubation with Molt-4 cells and completely inactivated by incubating at pH2 for 24 h.The putative receptor for LR or LT was studied using adsorption assay.The present study reveals that human LR activity can be adsorbed from crude LR supernatants by incubation with native or formalin-treated K562 or SMMC-7721 cells at 37℃ or 4℃; LT activity can be adsorbed with L929 cells.Therefore, LR appears to be a cytotoxic/cytostatic factor that is distinct from LT, 1FN and NKCF.
ABSTRACT
In order to provide highly purified preparation of human ieukoregulin (LR) for the exploration of its anticancer mschatiism, crude preparations of LR and lymphotoxin(LT) derived from human spleen cell cultures induced with phytohem-agglutinin and diterp;ne ester l2-O-tetradecanoylphorbol-i3-acetate were purified by using four purification nmhois : (a) Con A-Sepharose 48 affinity chromatography, by which crude human LR or LT was purified to maximum specific activity of 85 333 U/mg protein or 13333 U/mg protein with a recovery of 28.1% or 31.9%, respectively 5 (b) DEAE-Sjphadex A-50 ion-exchange chromatography, by which partially purified LR or LT obtained after Con A-Sepharose 4B affinity chromatography was further purified 2.44-fold or 3.8o-fold with a recovery of 96% or 92%, respectively ; (c) DEAE-22-celluiose ion-exchange chromatography, by which crude human LR or LT was purified to maximum specific activity of 11 294 U/mg protein or 13 176 U/mg protein with a recovery of 87.5% or 89.6%.respectively ; (d) Blue-Sepharose CL-6B affinity chromatography, by which crude human LR or LT was purified to maximum specific activity of 51.89 U/mg protein or 2594 U/mg protein with a recovery of 53.75% or 5t3.G7%, respectively.Our data indicate that Con A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50 ion-exchange chromatography.DEAE-22-cellulose ion-exchange chromatography and Blue-Sepharose CL-6B affinity chromatography may be useful in the purification of LR or LT.