A myelomonocytic leukaemia cell line, WEHI-3, releases into its growth medium factors which stimulate the development of pluripotential cells, granulocyte/macrophage progenitor cells, megakaryocytic and erythroid progenitor cells. Also present is a factor which is essential for the continued proliferation in vitro of a variety of haemopoietic precursor cell lines of a granulocytic nature (FDC-P cells). Characterization of this growth factor has demonstrated that it is a glycoprotein of apparent Mr 25 800, in which the carbohydrate component appears to be important for activity. After several purification steps, there is an increase in specific activity of approx. 4000-fold over the starting material. At each stage of purification, the factor necessary for the proliferation of FDC-P cells 'co-purifies' with activity which stimulates the proliferation and development of normal multipotential haemopoietic cells as well as megakaryocytic, erythroid and granulocytic committed progenitor cells. This 'co-purification' occurs to the extent that the multilineage stimulating factor and the FDC-P growth factor can be eluted from the same region of sodium dodecyl sulphate/polyacrylamide gels. Thus, evidence so far, using different starting methods and purification regimes, suggests that one molecule may have multiple activities on diverse cell types.
The aconitase of Escherichia cofi was purified to homogeneity, albeit in low yield (0.6%). It was shown to be a monomeric protein of Mr 95000 or 97500 by gel filtration and SDS-PAGE analysis, respectively. The N-terminal amino acid sequence resembled that of the Baciflus subtifis enzyme (cifB product), but the similarity at the DNA level was insufficient to allow detection of the E. cofi acn gene using a 456 bp cifB probe. Phages containing the acn gene were isolated from a A-E. cofi gene bank by immunoscreening with an antiserum raised against purified bacterial enzyme. The acn gene was located at 28 min (1350 kb) in the physical map of the E cofi chromosome by probing Southern blots with a fragment of the gene. Attempts to locate the gene using the same procedure with oligonucleotide probes encoding segments of the N-terminal amino acid sequence were complicated by the lack of probe specificity and an inaccuracy in the physical map of Kohara ef d (Cef150, [495][496][497][498][499][500][501][502][503][504][505][506][507][508] 1987). Aconitase specific activity was amplified some 20-200-fold in cultures transformed with pGS447, a derivative of pUC119 containing the acn gene, and an apparent four-fold activation-deactivation of the phagemid-encoded enzyme was observed in late exponential phase. The aconitase antiserum cross-reacted with both the porcine and Salmonella typhimurium (Mr 120000) enzymes.
Summary Four established human breast tumour cell lines with different biologic properties were selected for study and requirements for their clonogenic growth in semisolid cultures were identified. The conventional conditions were modified by factors that enhanced colony formation of 3 or more of these cell lines. The modified culture conditions were then applied to the growth in agar of primary breast tumours. A 5-fold improvement in plating efficiency was observed when cultures of 105 primary tumours grown under these modified conditions were compared to those of 52 tumours grown earlier under conventional conditions, and a 4-fold improvement resulted from the addition of hormones and conditioned medidum in 26 tumours cultured simultaneously under both conditions. The biologic relevance of these clonogens recovered in vitro was substantiated by a 70% concordance of in vitro and in vivo tumour sensitivity to anticancer drugs.
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