thymosin and Leukemia--Erythroblastic--Acute

Non-porous reversed phase (NPRP) high-performance liquid chromatography (HPLC) has been used as a rapid method to separate proteins from whole cell lysates of human erythroleukemia (HEL) cells. Using phosphate-buffered saline (PBS) as a lysis buffer to extract proteins from HEL cells, more than 100 proteins of molecular weight up to 30 kDa were separated by the NPRP HPLC method, using a programmed acetonitrile:H2O gradient. The separated proteins were collected as liquid fractions as they eluted, and were further separated on the NPRP column with a different gradient to separate coeluting peaks. The isolated protein fractions were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to determine the molecular weight of the protein. The proteins were cleaved by chemical or enzymatic digestion to produce peptide maps, which were analyzed by pulsed delayed extraction MALDI-MS. The peptide maps were matched against a database search to determine the protein identity. In some cases, several enzymes were used in order to find exactly one match against the database. This methodology is demonstrated for several proteins isolated from HEL cells and identified via database matching.

Topics: Calmodulin; Chromatography, High Pressure Liquid; Databases, Factual; DNA-Binding Proteins; Humans; Leukemia, Erythroblastic, Acute; Neoplasm Proteins; Peptide Mapping; Protein Isoforms; Ribonucleoproteins; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thymosin; Tumor Cells, Cultured

We investigated the effects of the systemic administration of thymosin alpha 1 plus relatively low doses of human recombinant interleukin-2 or very low doses of interferon alpha,beta in untreated and cyclophosphamide (CY)-treated DBA/2 mice challenged either subcutaneously or intravenously (i.v.) with Friend erythroleukemia cells (FLC). Both treatments resulted in the complete regression of subcutaneous tumor and cured a significative percentage of mice. They also increased the survival time of mice i.v. injected with large numbers of FLC. Neither immunotherapy alone nor CY, alone or in combination with single cytokines, produced similar effects. The antitumor action of these combined chemoimmunotherapy protocols seems to involve activation of the immune response since (a) a synergistic increase of the cytotoxicity of spleen cells was demonstrated in treated mice; (b) selective in vivo depletion of asialo-GM1, CD4, or CD8-positive cells abrogated this antitumor activity; and (c) a high lymphoid cell infiltration was found at the tumor site and in the livers of treated mice.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; CD4 Antigens; CD8 Antigens; Cyclophosphamide; Cytotoxicity, Immunologic; Disease Models, Animal; Friend murine leukemia virus; G(M1) Ganglioside; Interferon-alpha; Interferon-beta; Interleukin-2; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Male; Mice; Mice, Inbred DBA; Thymalfasin; Thymosin; Tumor Virus Infections