FMS receptor for M-CSF (CSF-1) is sensitive to the kinase inhibitor imatinib and mutation of Asp-802 to Val confers resistance.

The kinase inhibitor imatinib is used in the treatment of chronic myeloid leukaemia, where it targets the intracellular Bcr-Abl tyrosine kinase, and gastrointestinal stromal tumours, where it targets either the KIT or PDGF tyrosine kinase receptors. Here, we report that imatinib is also an effective inhibitor of the closely related FMS receptor for macrophage colony stimulating factor and that mutation of Asp 802 of FMS to Val confers imatinib resistance. Imatinib readily reverted the transformed phenotype of haemopoietic and fibroblast cell lines that express the oncogene v-fms and also inhibited the growth of the Bacl.2F5 macrophage cell line. The cellular IC50 value of imatinib for FMS was similar to those for Bcr-Abl and KIT. Consequently, imatinib may also prove effective for the treatment of diseases whose progression is dependent upon macrophage-colony stimulating factor, this includes certain aspects of cancer and inflammation.

Mae mediates MAP kinase phosphorylation of Ets transcription factors in Drosophila.

The evolutionarily conserved Ras/mitogen-activated protein kinase (MAPK) cascade is an integral part of the processes of cell division, differentiation, movement and death. Signals received at the cell surface are relayed into the nucleus, where MAPK phosphorylates and thereby modulates the activities of a subset of transcription factors. Here we report the cloning and characterization of a new component of this signal transduction pathway called Mae (for modulator of the activity of Ets). Mae is a signalling intermediate that directly links the MAPK signalling pathway to its downstream transcription factor targets. Phosphorylation by MAPK of the critical serine residue (Ser 127) of the Drosophila transcription factor Yan depends on Mae, and is mediated by the binding of Yan to Mae through their Pointed domains. This phosphorylation is both necessary and sufficient to abrogate transcriptional repression by Yan. Mae also regulates the activity of the transcriptional activator Pointed-P2 by a similar mechanism. Mae is essential for the normal development and viability of Drosophila, and is required in vivo for normal signalling of the epidermal growth factor receptor. Our study indicates that MAPK signalling specificity may depend on proteins that couple specific substrates to the kinase.

Quantitative measurement of transcript levels throughout human preimplantation development: analysis of hypoxanthine phosphoribosyl transferase.

We have developed a competitive reverse transcription-polymerase chain reaction (RT-PCR) sensitive enough to detect and quantify as little as 2-fold differences in gene expression in individual oocytes and embryos throughout human preimplantation development. This RT-PCR assay can be tailored for the examination of any specific gene and so will give a unique insight into human preimplantation development. This technique was used to quantify the level of hypoxanthine phosphoribosyl transferase (HPRT) expression during preimplantation development and to correlate this with embryo sex. The amount of HPRT transcripts present in the unfertilized oocyte was equivalent to 7.7 fg of competitor cDNA. At the 4-cell stage there is a significant drop (P: = 0.0006) to approximately 1.2 fg. There was no detectable difference in the HPRT levels between female and male embryos following 2 days of in-vitro culture. In contrast HPRT gene expression was higher in day 3 female embryos than in males. This is the first study to quantify gene transcripts throughout each stage of human preimplantation development and it indicates that the accumulated HPRT transcripts present in the unfertilized human oocyte undergo extensive destruction following fertilization. This work also suggests that X-inactivation occurs beyond the 8-cell stage of human preimplantation development.

Evidence that downregulation of the M-CSF receptor is not dependent upon receptor kinase activity.

The downregulation of tyrosine kinase receptors attenuates signalling and is thought to be dependent upon intrinsic receptor kinase activity, largely because down-regulation is inhibited by a kinase-inactivating mutation of an invariant lysine residue of the receptors for EGF, insulin, M-CSF and PDGF. We confirmed that this mutation inhibited the degradation of the M-CSF receptor. However, two different kinase inactivating mutations of the invariant amino acids Gly 591 and Glu 633 did not prevent M-CSF-induced receptor degradation, so demonstrating that receptor kinase activity is not essential for this process. Three other kinase-inactivating mutations were found to cause constitutive receptor degradation in the absence of M-CSF, most probably by disrupting the structure of the activating loop of the kinase domain. It is known that extensive movement of the A-loop is necessary for kinase activation and is normally induced by ligand-binding. It is therefore suggested that some aspect or consequence of the change in structure of the A-loop caused by ligand binding also activates receptor downregulation, so ensuring that downregulation is coupled to but is not necessarily dependent upon receptor kinase activity.

Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation.

Expression of a receptor for human macrophage-colony stimulating factor (M-CSF or CSF-1), containing a point mutation which changes an aspartate to a valine at position 802 of the activating loop of the kinase domain, potently transforms the haemopoietic cell line FDC-P1 yet prevents Rat-2 fibroblast transformation. In order to understand this apparent paradox, aspartate 802 was changed by cassette mutagenesis to each of the other 19 amino acids. All hydrophobic amino acid substitutions were transforming when tested in FDC-P1 cells yet inactivating when tested in Rat-2 fibroblasts. These same amino acid substitutions also activated receptor degradation, strongly suggesting a causal relationship between receptor degradation and inactivation in fibroblasts. Point mutations or small deletions of Y708 within the kinase insert region of the mutant D802V receptor partly inhibited receptor degradation. The more stable D802V receptor derivatives were able to transform both FDC-P1 cells and Rat-2 fibroblasts, so establishing that the cell specific effect of the c-fmsD802V activating loop mutation is attributable to receptor degradation which accompanies kinase activation and prevents the transformation of Rat-2 but not of FDC-P1 cells.

Biological activity of the receptor for macrophage colony-stimulating factor in the human endometrial cancer cell line, Ishikawa.

Previously we found that the Ishikawa endometrial cancer cell line expresses macrophage colony-stimulating factor (M-CSF) and c-fms transcripts and that its proliferation is enhanced by the addition of recombinant M-CSF. This suggested that Ishikawa cells are constitutively stimulated by M-CSF. In support of this we now show that Ishikawa cells secrete M-CSF and that known stimulators of M-CSF production increase the amount detected in Ishikawa cell conditioned medium. Using retroviral infections to introduce and express exogenous c-fms genes in Ishikawa cells we also demonstrate proliferation to be partially inhibited by a dominant negative, mutant c-fms gene, yet enhanced approximately 3-fold by a normal c-fms gene, under conditions in which the only source of M-CSF was that produced by the cells. The data provide evidence for the existence of an active M-CSF/receptor loop in these endometrial cancer cells and suggests the possibility of such activity in tumours of the endometrium and ovary that aberrantly express M-CSF and fms genes.

Selection of activating mutations of c-fms in FDC-P1 cells.

FDC-P1 haemopoietic cells were used to select mutations of c-fms that constitutively activate the receptor for macrophage-colony stimulating factor (M-CSF or CSF-1). One mutation changed Ser 929 to Gly within a Ser/Gly rich region of the C-terminal tail and a second changed a nearby, highly conserved Leu 926 for Pro. A third mutation (D802V) changed Asp 802 to Val within the alpha L12/beta 9 region of the tyrosine kinase domain, so supporting the crystallographic evidence that this region triggers kinase activation. A c-kit mutation exactly equivalent to D802V was previously identified in a leukamic cell line and was demonstrated here to be transforming. Surprisingly, although D802V potently transformed FDC-P1 cells, it could not induce Rat-2 fibroblast foci, even in the presence of M-CSF. It is suggested that the accelerated receptor degradation induced by D802V may account for its cell specific effect.

Evidence for cell-specific differences in transformation by N-, H- and K-ras.

Although Ras plays a fundamental role in cellular proliferation, differentiation and transformation, clear functional differences between the three major Ras proteins (N-, H- and K-Ras) have not as yet been demonstrated. In this study, chimeric constructs were used to compare directly transformation by N-, H- and K-ras oncogenes. In Rat-2 and NIH3T3 fibroblasts, transformation assays (anchorage independence, focus-formation and growth in 1% FCS) showed that H12-Ras was more transforming than N12-Ras or K12-Ras. By contrast, in the human multipotent haemopoietic cell line, TF-1, N12-Ras exhibited greater biological activity. Northern blotting and protein analyses indicated that these findings were not the result of differences in expression or stability of p21Ras. Using further H-ras/N-ras chimeric constructs, we found that the greater transforming activity of H12-Ras in fibroblasts was not due to the hypervariable-CAAX region, but rather to unique sequences between amino acids 84 and 143. These data demonstrate cell specific differences in the intrinsic transforming potential of N-ras, H-ras and K-ras oncogenes.

Evidence that ras and myc mediate the synergy between SCF or M-CSF and other haemopoietic growth factors.

We previously reported that M-CSF could mimic the synergistic effect of SCF upon myeloid FDC-P1 cells that were first infected with a c-fms retrovirus, which encodes the human M-CSFr. We now report that an M-CSFr with a mutation of its autophosphorylation site at position 809 was, in response to M-CSF, unable both to synergize with IL-3 or GM-CSF and to induce c-myc; whereas a mutant receptor with a deletion of its kinase insert was unaffected for these processes. The expression of an exogenous c-myc proto-oncogene or a 12H-ras oncogene lowered the requirement of FDC-P1 cells for IL-3 or GM-CSF, in a similar manner to M-CSF or SCF addition. Furthermore, the expression of either of these genes complemented the defective M-CSFr F809. These results strongly support a role for ras and myc in the synergistic action of M-CSF and, by implication, of SCF, which implies that these signalling intermediates are rate-limiting for the action of IL-3 and GM-CSF and possibly other haemopoietic growth factors.

Synergy between SCF or M-CSF with IL-3 or GM-CSF in FDC-P1 cells: a sensitive assay of transforming mutations of c-fms.

Stem cell factor (SCF) was found to stimulate the growth of the haemopoietic cell line FDC-P1 in synergy with either interleukin 3 (IL-3) or granulocyte-macrophage-colony stimulating factor (GM-CSF). Similarly, macrophage colony-stimulating factor (M-CSF) was shown to synergize with IL-3 or GM-CSF, following the infection of FDC-P1 cells with a recombinant retrovirus which encoded the receptor for M-CSF (M-CSFr). These results raise the possibility that signal transduction pathways which are controlled by SCF in FDC-P1 cells, can be activated by M-CSF if its receptor is illicitly expressed. FDC-P1 cells that expressed the M-CSFr were responsive to as little as 100 U/ml of M-CSF when added in combination with IL-3 or GM-CSF. This sensitive assay was used to demonstrate that transforming deletions of the C-terminal tail of the M-CSFr and two-point mutations within the same region that converted tyrosine 969 to either phenylalanine or to cysteine, allowed the mutant M-CSF receptors to synergize with IL-3 or GM-CSF in the absence of M-CSF. These mutations were found to be more evidently transforming in FDC-P1 cells than in Rat-2 fibroblasts. The possible relevance of these results to leukaemia and to gynaecological malignancies is discussed.

Proto-splice site model of intron origin.

It is proposed that nuclear pre-mRNA introns (classical introns) were first generated as by-products during the evolution of alternative splicing. They were formed whenever two splice sites within the coding sequence of ancestral genes were used at a frequency that removed the coding constraint from the intervening sequence. Once introns had evolved, it is suggested that they were spread by the splicing machinery which inserted them into proto or cryptic-splice sites of other genes by reverse splicing, so giving rise to genes that have introns yet are not alternatively spliced. It is argued that 5' and 3' splice sites evolved from common ancestral splice sites, referred to as proto-splice sites, that were bidirectional and had a core consensus sequence of C or A, A, G, R, which remains today as the immediate flanking sequence of most introns. The ancestral splicing machinery, although inefficient, would have been capable of generating vast mRNA diversity by splicing between proto-splice sites. Natural selection would be expected to have preserved mutations that increased the amounts of advantageously spliced mRNA. It is argued that this process drove the evolution of present 5' and 3' splice sites from a subset of proto-splice sites and also drove the evolution of a more efficient splicing machinery. The positions of most introns that evolved directly from the coding sequence would be expected to correlate with protein structure.

Expression of v-fms and c-fms in the hemopoietic cell line FDC-P1.

A hemopoietic cell line FDC-P1 that requires either IL-3 or GM-CSF to survive and proliferate was infected with retroviruses that expressed either c-fms, which encodes the receptor for M-CSF, or v-fms, which is an oncogenic derivative of c-fms. The expression of c-fms allowed FDC-P1 to grow in the absence of IL-3 or GM-CSF provided that M-CSF was present. The M-CSF did not, however, induce macrophage differentiation. The expression of v-fms allowed FDC-P1 to grow in the absence of any added hemopoietic growth factors, including M-CSF, although the addition of M-CSF enhanced v-fms activity. V-fms cell lines grew to a higher cell density in suspension and were tumorigenic.

Evidence that introns arose at proto-splice sites.

The unexpected discovery of introns raised many questions about gene evolution. We provide evidence that actin and tubulin introns were gained between the G and R of the conserved coding sequence C/AAGR that is known to flank introns in general and which we call a proto-splice site. We conclude that the tubulin and actin introns are less ancient than the coding sequence and so could not have been involved in the primary evolution of the tubulin and actin genes.

Sequence analysis of the complete Caenorhabditis elegans myosin heavy chain gene family.

The sequences of three myosin heavy chain (MHC) genes from Caenorhabditis elegans, myo-1, 2 and 3, are presented. These genes, together with unc-54, comprise the entire nematode sacromeric MHC family. Comparison of nematode MHC sequences and sarcomeric, smooth and non-muscle MHCs from other organisms highlights conserved sequence features of the MHC rod believed to be important for thick filament assembly. These include: conservation of sequence differences between individual 28 amino acid repeats; invariant placements of large aromatic residues, such as tryptophan, in the rod sequences; conservation of "weak spots" in the hydrophobic seam; and conservation of non-uniform charge distributions along the length of the rod. The rod sequences of the body wall isoforms A and B are more closely related to each other than to the pharyngeal isoforms C and D, suggesting that structural constraints have been imposed by their location within the same thick filament. We have also identified the major transcriptional start site for gene unc-54. Surprisingly, there are no TATA or other known transcription factor elements immediately upstream from the unc-54 start site, or in the upstream regions of the other genes of the C. elegans MHC gene family.

lep operon proximal gene is not required for growth or secretion by Escherichia coli.

Leader peptidase is an essential enzyme of Escherichia coli and is required for protein export. The structural gene for leader peptidase (lep) is separated from its promoter by an upstream gene of unknown function (lepA). The gene lepA was shown by the use of minicell analysis and overproduction to encode a protein of 74,000 daltons. To determine whether this 74,000-dalton protein functions in protein export, a mutant of E. coli H560 was constructed which has a 1.5-kilobase-pair deletion in the lepA gene. The lepA deletion mutant had no apparent defect for growth or protein export, indicating that lepA is nonessential and that the two cotranscribed genes lepA and lep probably have unrelated functions.