A simple one-step isolation technique significantly enriched mouse fetal liver cells that respond to interleulkin 3 (IL-3), a multilineage hematopoietic growth factor.The fetal liver cell subpopulation isolated with monoclonal antibody AA4 contained 50-to 100-fold higher frequencies of multipotential (CFU-mix) or restricted (CFU-G/M, BFU-E) erythroid/myeloid precursors as well as precursors that differentiate to become mature B lymphocytes [CFU-mix = erythroid and myeloid colony-forming unit(s); CFU-G/M = CFU-granulocyte/macrophage; BFU-E = burst-forming uniterythroid]. The B-lymphocyte precursors could be cloned in single-cell cultures when IL-3-containing supernatants were present. Growth of these clones was supported by purified IL-3 but not by purified IL-2. Stable growth has been maintained for >6 mo in the presence of IL-3. Such clones express on their cell surface low amounts of class I major histocompatibility complex antigens and high amounts of AA4, GF1, and leukocyte common glycoprotein 200 under an atmosphere of 5% CO2 in air. WEHI-3 supernatant, which contains IL-3 and additional factors (1, 3-10), was stored at -70'C following centrifugation at 500 x g for 15 minand passage through a 0.22-gm filter. This supernatant, added at 10%6 (vol/vol) to IMDM containing 5% fetal calf serum, will be referred to as WEHI-3-conditioned medium (CM). Purified IL-3 and IL-2 (12, 13) were provided by J. Watson (University of Auckland). Monoclonal Antibodies (mAbs) and Quantitation of Cell Surface Determinants. Expression of cell surface determinants recognized by various mAbs was quantitated in a fluorescent antibody binding assay using rat anti-mouse mAbs as described (14,15). mAbs M1/89.18 (16) and D468HLK (17), which recognize leukocyte common glycoprotein (LGP) 200 and rat major histocompatibility complex (MHC) antigens, respectively, were also used.Cell-Separation Techniques. Subpopulations of fetal liver cells were obtained by "panning" on mAb-coated polystyrene dishes (14). Those cells removed after incubation and in the subsequent two rinses were pooled to produce the nonadherent (depleted) population. The adherent (selected) population was composed of cells that remained bound after 8-10 rinses and were recovered by repeatedly pipetting 5-10 ml of medium to gently resuspend cells. 7414The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Seven mesothelioma cell lines, established from patients with pleural mesothelioma, exhibited substantial heterogeneity regarding in vitro morphology and growth characteristics. Media conditioned by these cell lines and by MeT5A normal mesothelial cells were examined for (i) colony formation on human bone-marrow cells, (ii) hematopoietic growth-factor content and (iii) mitogenic activity on mesothelioma cells. Colony-stimulating activity was produced only by the ZL34 cell line. Analysis of conditioned media by ELISA revealed that all mesothelioma cell lines constitutively produced IL-6, while the MeT5A normal mesothelial cells did not; in addition, GM-CSF and G-CSF were detected in the supernatant of the ZL34 cell line. Using a 3H-thymidine incorporation assay, we showed that all mesothelioma cell lines produced mitogenic activity in the culture supernatant, in contrast to the MeT5A normal mesothelial cells. The mitogenic effect of the hematopoietic growth factors detected in mesothelioma culture supernatants was tested on mesothelioma cells and on MeT5A normal mesothelial cells: IL-6, GM-CSF and G-CSF did not stimulate any DNA synthesis. Our results suggest that these hematopoietic growth factors do not act as autocrine growth factors. A common feature of this panel of mesothelioma cell lines is the production of IL-6; although the biological significance of the aberrant production of cytokines by mesotheliomas remains unclear, IL-6 might be involved in paraneoplastic syndromes such as thrombocytosis.
It has been suggested that oestriol protects against breast cancer, because in some experiments on uterine growth it is only weakly active, and partially inhibits the effects of oestradiol-17beta. When its effects are measured 24 h after a single injection, oestriol behaves as a typical impeded oestrogen with low potency and a flat dose-response line. This does not result from failure to stimulate certain critical stages of growth but from failure to sustain the products of growth. We found that oestriol induced all phases of uterine growth including DNA synthesis and cell division. It was as effective as oestradiol in stimulating early increases in protein synthesis and uterine weight, and half as effective in stimulating epithelial cells to replicate DNA and divide. However, epithelial cell numbers did not increase after a single injection of oestriol because cell death rate increased at the same time as mitotic rate, apparently as a result of the more rapid loss of oestriol from the uterus. Repeated injections of oestriol prevented premature cell death and produced as much uterine hypertrophy and hyperplasia as oestradiol-17beta. These results support the thesis that the oestrogenic potency of a substance is largely determined by the duration of its occupation of receptors. Thus in situations of continuous production, (e.g. pregnancy) oestriol would be as active as oestradiol and unlikely to exert any significant 'buffering' or protective action. The findings are also discussed in relation to a new model for the regulation of cell proliferation.
Myeloproliferative virus, derived from Moloney sarcoma virus, causes erythroleukemia and myeloid leukemia in adult mice. This virus is also capable of fibroblast transfornation in vitro. The virus consists of two separable biological entities which have been cloned. The helper virus component caused no visible changes in adult mice, whereas the defective virus induced both spleen focus formation and a large increase in erythroid precursor cells but retained the sarcoma virus property of transforming fibroblasts in vitro. Thus, myeloproliferative virus is the first murine sarcoma virus which induces erythroleukemia in adult animals.
A structure‐activity relationship study of human interleukin‐3 (huIL‐3) was performed by functional analysis of huIL‐3 deletion and substitution variants combined with epitope mapping of huIL‐3 specific neutralizing monoclonal antibodies (mAb). Analysis of the huIL‐3 variants was accomplished by defining their capacity to compete with wild‐type huIL‐3 for binding to the huIL‐3 receptor and to induce the proliferation of the huIL‐3 dependent cell line M‐O7. HuIL‐3 variants with either 14 amino acids (aa) deleted from the N‐terminus or eight aa from the C‐terminus retained full biological activity in vitro. An huIL‐3 variant, with 18 N‐terminal aa deleted, exhibited a greater than 7‐fold reduced receptor binding capacity and proliferative activity. No biological activity could be detected with a variant where 22 C‐terminal aa have been deleted. Neutralizing mAb recognizing presumed discontinuous epitopes failed to interact with the latter deletion variant indicating a possible location of their epitopes within the C‐terminal region. Computer‐aided structure prediction and sequence homology analysis of this region indicated the presence of an amphiphilic alpha‐helix with highly conserved residues like Lys110 and Leu111. Substitution of Lys110 with either Glu or Ala resulted in variants with a 10‐fold reduced activity in the receptor binding assay and the proliferation assay. Further variants, where Leu111 was substituted by Pro or Met, were totally inactive in these assays. Analysis of the binding of the two neutralizing mAb to these substitution variants showed that they did not bind to either of the Leu111 variants suggesting that Leu111 is part of an active site. Based on our results, a possible model for the structure of the huIL‐3 molecule can be constructed with two active sites in close proximity.
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