The cytokine receptor repertoire specifies autocrine growth factor production in factor-dependent cells.

Mechanisms of helper virus-induced growth factor-independence were examined in FDC-P1 cells and FDC-P1 cells expressing the erythropoietin receptor (FDER cells). Retroviral mutagenesis of FDC-P1 cells led to factor-independent (FI) colonies from which cell lines could readily be established; whereas control cells exhibited at least 20 to 40-fold lower rates of factor-independence. From 44 independent experiments using either FDC-P1 or FDER cells, 205 autonomous cell lines were obtained. Sixteen colonies displayed a novel ("satellite-inducing") appearance in agar and produced up to 4.1 x 10(5) U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (some with altered GM-CSF transcript sizes) and/or interleukin-3 (IL-3). Retroviral mutagenesis of FDER cells increased the repertoire of autocrine growth factors now responsible for stimulating autocrine proliferation: 3% of FI cell lines produced erythropoietin (Epo) (0.5 U/mL). Unexpectedly, in every autonomous FDC-P1 cell line, reverse transcriptase-PCR demonstrated expression of a growth factor normally required for proliferation. Thus, a profound selection for cells able to produce growth factors as the mechanism for achieving autonomous proliferation was documented. The ectopic expression of a receptor lacking a cognate ligand ("orphan") followed by retroviral mutagenesis and selection for autocrine mutants may offer an effective method for identifying new ligands.

Localization of genes for two members of the JAK family of protein tyrosine kinases to murine chromosomes 4 and 19.

Genes encoding two members of the JAK family of protein tyrosine kinases, Jak-1 and Jak-2, have been mapped to mouse Chromosomes (Chrs) 4 and 19 respectively. These placements are consistent with the previously described location of human JAK1 and JAK2, which lie in regions of synteny on human Chrs 1p31-3 (JAK1) and 9p24 (JAK2). The location of Jak-2 in the mouse genome extends the region of homology between mouse Chr 19 and human Chr 9.

Localization of two mouse genes encoding the protein tyrosine kinase receptor-related protein RYK.

We have mapped the gene encoding the murine RYK growth factor receptor protein tyrosine kinase by genetic linkage analysis with recombinant inbred strains of mouse. Two distinct Ryk loci (Ryk-1 and Ryk-2) were identified. Ryk-1 mapped to Chromosome (Chr) 9, whereas Ryk-2 mapped to Chr 12. A similar arrangement of RYK-related loci was previously determined in the human. Synteny has already been established between murine Chr 9 in the region of Ryk-1, and human chromosome 3q11-12, the location of the human RYK-1 gene. However, the Ryk-2/RYK-2 loci on murine Chr 12 and human Chr 17p13.3 define a new region of synteny.

Cloning of a murine IL-11 receptor alpha-chain; requirement for gp130 for high affinity binding and signal transduction.

An adult mouse liver cDNA library was screened with oligonucleotides corresponding to the conserved WSXWS motif of the haemopoietin receptor family. Using this method, cDNA clones encoding a novel receptor were isolated. The new receptor, named NR1, was most similar in sequence and predicted structure to the alpha-chain of the IL-6 receptor and mRNA was expressed in the 3T3-L1 pre-adipocytic cell line and in a range of primary tissues. Expression of NR1 in the factor-dependent haemopoietic cell line Ba/F3 resulted in the generation of low affinity receptors for IL-11 (Kd approximately 10 nM). The capacity to bind IL-11 with high affinity (Kd = 300-800 pM) appeared to require coexpression of both NR1 and gp130, the common subunit of the IL-6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) receptors. The expression of both NR1 and gp130 was also necessary for Ba/F3 cells to proliferate and M1 cells to undergo macrophage differentiation in response to IL-11.

No evidence for GM-CSF receptor alpha chain gene mutation in AML-M2 leukemias which have lost a sex chromosome.

Sixty-five percent of acute myeloid leukemias subtype M2 (AML-M2), displaying a translocation between chromosomes 8 and 21 (t8;21) have also lost one or other copy of the sex chromosomes (XO). This finding has led to the hypothesis that a recessive oncogene may be present in the DNA common to both sex chromosomes, that is the pseudoautosomal region. The alpha chain of the receptor for the hemopoietic growth factor GM-CSF has recently been mapped to the human pseudoautosomal region and, given the role of this molecule in the control of normal hemopoiesis, it is thought to be a good candidate gene. This paper examines the structure, expression, and sequence of the GM-CSF receptor alpha chain gene (GMR alpha) in three primary AML-M2,XO samples and one AML-M2,XO cell line. Results of this work show no gross rearrangement or loss of the remaining allele of GMR alpha, detectable expression of its transcript and protein, and no changes at the nucleotide sequence level. Thus, we find no evidence to support a role for the GM-CSF receptor alpha chain molecule in the development of AML-M2,XO leukemias.

Complex binding of leukemia inhibitory factor to its membrane-expressed and soluble receptors.

The complex interaction of leukemia inhibitory factor (LIF) with its specific receptor present on the cell surface, in isolated membranes and in solution, has been examined in detail. Several aspects of this complexity have been highlighted, including the presence of high- and low-affinity murine LIF receptors, biphasic dissociation of human LIF from apparently homogeneous high- or low-affinity human LIF receptors, and unusual species cross-reactivity. The unusual species cross-reactivity observed between murine and human LIF has also been exploited to map an important receptor binding epitope on human LIF.

Leukaemia inhibitory factor and interleukin 6 are expressed at very low levels in the normal adult mouse and are induced by inflammation.

We have assessed the limitations of the polymerase chain reaction (PCR) as a semiquantitative technique for assessing very low level gene expression. Using PCR, the in vivo expression patterns of the cytokines Leukaemia Inhibitory Factor (LIF) and Interleukin 6 (IL-6) in the normal adult mouse, have been examined. We show that both LIF and IL-6 mRNA are constitutively expressed, albeit at extremely low levels, in most tissues. While it is unclear whether this low level of expression is of biological significance, it is possible that it reflects a local mode of action of these potent polyfunctional molecules. Lipopolysaccharide, the bacterial cell wall product responsible for endotoxic shock, when administered in vivo, was capable of inducing the expression of both LIF and IL-6 in all of the tissues examined. In addition, LIF and IL-6 expression was induced in lung tissue by in vitro culturing in serum-free media. This induction of LIF and IL-6, by LPS and culturing, may reflect the role of these molecules as mediators of the acute phase response to tissue damage.

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins.

Delineation of positive and negative control elements within the promoter region of the murine leukemia inhibitory factor (LIF) gene.

Leukaemia inhibitory factor (LIF) is a multifunctional growth and differentiation factor with activities in both the adult and the embryo. The expression of LIF appears to be tightly regulated, as the levels of constitutive expression in most tissues and cell lines is extremely low. In this report we have identified three sequence elements within the 5'-flanking region of the murine LIF gene which control the constitutive action of the LIF promoter. A nested set of DNA fragments from the LIF gene 5'-flanking region was placed upstream of the chloramphenicol acetyltransferase (CAT) gene and assayed for their ability to direct chloramphenicol acetyltransferase (CAT) expression in STO-fibroblasts. The essential promoter of the LIF-gene, giving rise to low levels of CAT expression, was found to require the major start-site of transcription (+1), a TATA-box (-31) and up to 72 additional 5' nucleotides (-32 to -103). A negative regulatory element which abolished CAT-activity was identified between positions -360 and -249. The SV40 enhancer element was able to override this apparent negative element. In addition, an apparent positive control element in the LIF 5'-flanking region, between positions -860 and -661 was identified which was also able to override this negative effect.

A human pseudoautosomal gene encodes the ANT3 ADP/ATP translocase and escapes X-inactivation.

We report that the human ANT3 ADP/ATP translocase gene is a pseudoautosomal gene located proximal to the GM-CSF receptor alpha chain gene (CSF2RA). An ANT3-homologous locus, likely corresponding to a pseudogene, maps to chromosome 9. The ANT3 gene is transcribed from the centromere to the telomere and contains in its first intron a CpG island mapped 1300 kb from the telomere. This gene is transcribed from the Y chromosome and from the active and inactive X chromosomes. This gene thus escapes X-inactivation as predicted for genes belonging to the pseudoautosomal region.

Structure and expression of the GM-CSF receptor alpha and beta chain genes in human leukemia.

In this paper we report on the structure and expression of the genes encoding the alpha and beta chains of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in human leukemia. The alpha chain gene is highly polymorphic in normal individuals and no evidence for rearrangement within this locus was found in 47 hemopoietic, nine non-hemopoietic malignancies and five human cell lines. Using the polymerase chain reaction the gene for the alpha chain was shown to be expressed in 18/18 primary myeloid as well as 8/8 primary lymphoid leukemias analysed. To investigate the integrity of the mRNA, polymerase chain reactions (PCR) using a combination of oligonucleotides spanning the entire coding region of the alpha chain were performed. Normal sized fragments were generated with all combinations of oligonucleotides from all but one leukemia. One chronic lymphoid leukemia displayed an apparent alteration at the 3' end of the 3' untranslated region of the alpha chain mRNA. No polymorphisms were detected in the beta chain gene which was also not rearranged in any of the samples analysed. The beta chain mRNA was expressed in 17/18 primary myeloid and 7/8 primary lymphoid leukemias and in those leukemias there was no evidence for any lesions in the mRNA, as judged by PCR fragment size. Thus gross structural lesions in the genes encoding the GM-CSF receptor alpha and beta chains appear to be infrequent in hemopoietic neoplasms.

Arrangement and localization of the human GM-CSF receptor alpha chain gene CSF2RA within the X-Y pseudoautosomal region.

The gene encoding one subunit of the receptor for the hemopoietic growth factor, GM-CSF, has been previously localized to the short arm of the human sex chromosomes. By pulsed-field gel electrophoresis, the precise localization of this gene, CSF2RA, within the pseudoautosomal region has been determined. The gene is located 1180 to 1300 kb from the telomere, in close proximity to the CpG island B5. The CSF2RA gene spans at least 45 kb, and a representation of most of the gene on three overlapping cosmid clones has been obtained. The exon(s) encoding the first 35 bp of cDNA sequence lies outside these cosmids. The CSF2RA gene is characterized by abundant hypervariable sequences, and a number of informative restriction fragment length polymorphisms have been defined.

Presence of single-stranded DNA in PCR products of slow electrophoretic mobility.

PCR products were characterized by electrophoresis, blotting and hybridization. In addition to the bands of expected size, bands of slower electrophoretic mobility were often detected. The slower bands completely disappeared when the PCR products were subjected to slow cooling, treated with S1 nuclease or run on an alkaline gel, whereas the bands of expected size were unaffected. The slower bands are therefore likely to contain single-stranded DNA.

Synergistic suppression: anomalous inhibition of the proliferation of factor-dependent hemopoietic cells by combination of two colony-stimulating factors.

Cells of the continuous murine hemopoietic cell line FDC-P1 expressing macrophage-colony-stimulating factor (M-CSF) receptors following retroviral insertion of murine c-fms cDNA proliferated clonally when stimulated by granulocyte/macrophage (GM)-CSF, multipotential CSF, or M-CSF. However, M-CSF combined with either GM-CSF or multi-CSF, even at low CSF concentrations, strongly inhibited colony formation, with loss of clonogenicity in affected cells accompanied by increased macrophage differentiation. Stimulation by these CSF combinations did not induce short-term changes in CSF receptor expression or internalization. FDC-P1 cells expressing another inserted tyrosine kinase receptor, basic fibroblast growth factor receptor, did not exhibit suppression when GM-CSF was combined with fibroblast growth factor. This phenomenon of synergistic suppression may have relevance for the future clinical use of combinations of CSFs, because a potentially similar suppression is also observable with some normal macrophage progenitor cells.

Cross-species comparison of the sequence of the leukaemia inhibitory factor gene and its protein.

Leukaemia inhibitory factor (LIF) is a pleiotropic growth factor active in diverse cell systems in both the adult and the embryo. The LIF gene from a number of mammalian species is highly conserved. The ovine and porcine LIF genes were cloned, sequenced and compared to the previously published murine and human LIF gene sequences. While the coding regions of the LIF gene are highly conserved, the non-coding regions are largely non-conserved. In a region of approximately 340 bp, the 5' end of the translational initiation codon is highly conserved (84%). This region includes four conserved TATA boxes, two transcriptional start-sites identified in the murine gene and the minimal region required to function as the LIF promoter. A sequence in the murine gene adjacent to this highly conserved region which appears to contain a negative control element is, however, poorly conserved between the four species compared, except for a sequence of 16 conserved nucleotides. Within the largely non-conserved first intron, there is a block of approximately 150 nucleotides which is highly conserved between all four species (approximately 72%). However, a sequence in intron 1 of the murine LIF gene which corresponds to an alternative exon of a putative variant LIF transcript is very poorly conserved between species, with only relics of this exon evident in the other three species. A comparison of the five LIF protein sequences available (murine, rat, human, ovine and porcine) revealed that the protein displays a high degree of similarity, ranging from 74% between mouse and sheep to 92% between rat and mouse. Several large blocks of absolutely conserved amino acid sequence were identified. The ovine LIF gene was modified to allow production of recombinant ovine LIF in yeast cells, which was shown to be biologically active on murine cells.

Localization of the human GM-CSF receptor beta chain gene (CSF2RB) to chromosome 22q12.2-->q13.1.

The gene for the beta-chain of the human GM-CSF receptor (CSF2RB) has been mapped to chromosome 22 by PCR analysis of a series of human x rodent somatic cell hybrids. In situ hybridization to normal human chromosomes and two translocations involving chromosome 22 and the chromosome expressing the rare fragile site FRA22A place the gene in the region 22q12.2-->q13.1, proximal to the fragile site.

Molecular biology of the leukaemia inhibitory factor gene.

Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine that has been identified and characterized in several laboratories by virtue of a number of different biological activities. LIF is encoded by a unique gene located at 11A1 in the mouse and at 22q12 in man. However, loci related to sequences in the 3' untranslated region of the mRNA have been detected and located elsewhere in the genome. The LIF gene from four mammalian species has been cloned and sequenced; the sequences are highly conserved within the coding regions and largely non-conserved within the non-coding regions. However, a number of non-coding segments displaying high interspecies similarity are evident; these are candidate control regions. Intriguingly, an exon corresponding to the 5' end of a variant LIF transcript in the mouse that encodes a potentially matrix-associated form of LIF is not conserved in the human, ovine and porcine genes. The promoter region of the LIF gene contains four well-conserved TATA elements, and two start sites of transcription have been identified. Three regions within the 5' flanking region have been identified as important for the function of the LIF promoter, including a candidate repressor sequence. The LIF gene is transcribed at only very low levels in normal tissues, but its expression can be increased by various stimuli.