Constitutive c-myc oncogene expression blocks mouse erythroleukaemia cell differentiation but not commitment

Mouse erythroleukaemia cells (also called Friend cells) can be isolated from the spleen of certain strains of mice that have been infected with the Friend virus complex. The cells resemble proerythroblasts and, when exposed to dimethyl sulphoxide (DMSO) or a variety of other chemicals, can be induced to undergo a programme of differentiation which closely resembles the final stages of normal erythropoiesis. This includes the cessation of proliferation and large increases in the production of messenger RNA for both alpha- and beta-globin. In addition, DMSO induces a rapid (less than 2 h) decrease in c-myc mRNA levels. The c-myc oncogene is expressed in the majority of proliferating normal cells and altered expression of the gene has been implicated in the genesis of a wide variety of tumours. To study the influence of oncogene activation on differentiation, we have transfected viral-promoter-driven c-myc genes into mouse erythroleukaemia cells. Constitutive c-myc expression was found to block DMSO-induced differentiation.     

Expression of a transfected human c-myc oncogene inhibits differentiation of a mouse erythroleukaemia cell line

The Friend-virus-derived mouse erythroleukaemia (MEL) cell lines represent transformed early erythroid precursors that can be induced to differentiate into more mature erythroid cells by a variety of agents including dimethyl sulphoxide (DMSO). There is a latent period of 12 hours after inducer is added, when 80-90% of the cells become irreversibly committed to the differentiation programme, undergoing several rounds of cell division before permanently ceasing to replicate. After DMSO induction, a biphasic decline in steady-state levels of c-myc and c-myb messenger RNAs occurs. Following the initial decrease in c-myc mRNA expression, the subsequent increase occurs in, and is restricted to, the G1 phase of the cell cycle. We sought to determine whether the down-regulation is a necessary step in chemically induced differentiation. Experiments reported here indicate that expression in MEL cells of a transfected human c-myc gene inhibits the terminal differentiation process.     

Expression of human fibroblast interferon gene in Escherichia coli

The human fibroblast interferon gene was inserted in a thermoinducible expression plasmid under control of the phage lambda PL promoter. The primary translation products predicted on the basis of the plasmid constructions were hybrid proteins starting with beta-lactamase or phage MS2 polymerase information followed by the total preinterferon. On induction, antiviral activity, whose physico-chemical, immunological and biological characteristics closely corresponded to those of authentic human fibroblast interferon, was synthesized. Processing to a size compatible with mature but unglycosylated authentic product was observed.     

Isolation and structure of a human fibroblast interferon gene

Chimaeric plasmids containing double-stranded cDNA copies of mRNA induced in human fibroblasts by poly I . C were screened by an RNA selection method. A series of clones to which human fibroblast interferon mRNA selectively hybridized was identified. From the nucleotide sequence of the gene, the complete amino acid sequence of human fibroblast interferon was deduced. The protein is 166 amino acids long and is preceded by a 21-amino acid signal sequence.     

Heterogeneity of poly(I) x poly(C)-induced human fibroblast interferon mRNA species

Three classes of human interferons (IFNs) have been defined on the basis of their immunological properties: the 'Le' or 'alpha' IFN, mainly derived from leukocyte or lymphoblastoid cells; the 'F' or 'beta' IFN, mainly derived from fibroblast cultures; and the 'T', 'immune' or 'gamma' IFN, mainly derived from mitogen- or antigen-stimulated lymphoid cells. Whereas several individual species of Le IFN have been purified to homogeneity, it is generally considered that F IFN represents a single protein. Thus current efforts to clone human fibroblast IFN mRNA sequences are based on the observation that F IFN mRNA sediments in sucrose gradients as a single RNA species of size corresponding to 12-14 S (refs 7-10). We show here, using gel electrohporesis of mRNA, that two populations of translationally active human fibroblast IFN mRNA molecules exist--an abundant '14 S' species and a scarce '11 S' species. Microinjection of either species of mRNA into Xenopus oocytes leads to the synthesis of biologically active F-type human IFN. These data agree with and complement recent RNA hybridization studies of Weissenbach et al.     

Loss of mouse fibroblast cell response to phorbol esters restored by microinjected protein kinase C

The phorbol esters in addition to being among the most potent mouse skin tumour promoters profoundly affect many different biological systems. It is postulated that they act through activation of protein kinase C, but substantial heterogeneity in their pharmacological and binding behaviour in some systems has caused concern about whether this is their only target. Evidence linking protein kinase C activation with biological responses to the phorbol esters includes similarity in structure-activity relations for binding and response; in vitro phosphorylation of specific proteins by protein kinase C at the same sites at which phorbol ester treatment induces phosphorylation in intact cells; and correlation in certain cell types between down regulation of protein kinase C on chronic phorbol ester treatment and loss of cellular responsiveness to the phorbol ester. Here we report that microinjection of purified protein kinase C into Swiss 3T3 fibroblasts pretreated with the phorbol ester phorbol 12,13-dibutyrate (PDBu) restores the mitogenic response of the cells to PDBu, directly establishing the involvement of protein kinase C in this response.     

A point mutation in the A gamma-globin gene promoter in Greek hereditary persistence of fetal haemoglobin

Hereditary persistance of fetal haemoglobin (HPFH) is a benign condition characterized by the production in adulthood of more than 1% fetal haemoglobin (HbF, alpha 2 gamma 2) in the absence of erythropoietic stress. Several genetic types have been discerned based on the level of HbF produced, the relative contributions of the duplicated fetal (G gamma and A gamma) globin genes, and the presence or absence of deletions involving the beta and delta genes in cis to the mutation. Greek HPFH is a non-deletion variety in which heterozygotes produce 10-20% HbF, predominantly due to overproduction of the A gamma chain. We have cloned a 40-kilobase (kb) region of the beta-globin cluster from a Greek HPFH allele and report here that a point mutation (G----A) occurs 117 base pairs (bp) 5' to the cap site of the A gamma-globin gene, just upstream of the distal CCAAT sequence. The corresponding region of the G gamma-globin gene is normal. We discuss the implications of this finding for the developmental regulation of globin gene expression.     

Isolation and partial sequence of recombinant plasmids containing human alpha-, beta- and gamma-globin cDNA fragments.

Human globin cDNA-derived recombinants with plasmid pCR1 have been prepared for use as specific hybridisation probes and for the partial sequencing of alpha-, beta- and gamma-globin genes.     

A gene expression map of human chromosome 21 orthologues in the mouse

The DNA sequence of human chromosome 21 (HSA21) has opened the route for a systematic molecular characterization of all of its genes. Trisomy 21 is associated with Down's syndrome, the most common genetic cause of mental retardation in humans. The phenotype includes various organ dysmorphies, stereotypic craniofacial anomalies and brain malformations. Molecular analysis of congenital aneuploidies poses a particular challenge because the aneuploid region contains many protein-coding genes whose function is unknown. One essential step towards understanding their function is to analyse mRNA expression patterns at key stages of organism development. Seminal works in flies, frogs and mice showed that genes whose expression is restricted spatially and/or temporally are often linked with specific ontogenic processes. Here we describe expression profiles of mouse orthologues to HSA21 genes by a combination of large-scale mRNA in situ hybridization at critical stages of embryonic and brain development and in silico (computed) mining of expressed sequence tags. This chromosome-scale expression annotation associates many of the genes tested with a potential biological role and suggests candidates for the pathogenesis of Down's syndrome.     

Zfy gene expression patterns are not compatible with a primary role in mouse sex determination

The Y chromosome determines maleness in mammals. A Y chromosome-linked gene diverts the indifferent embryonic gonad from the default ovarian pathway in favour of testis differentiation, initiating male development. Study of this basic developmental switch requires the isolation of the testis-determining gene, termed TDF in humans and Tdy in mice. ZFY, a candidate gene for TDF, potentially encodes a zinc-finger protein, and has two Y-linked homologues, Zfy-1 and Zfy-2, in mice. Although ZFY, Zfy-1 and Zfy-2 seem to map to the sex-determining regions of the human and mouse Y chromosomes, there is no direct evidence that these genes are involved in testis determination. We report here that Zfy-1 but not Zfy-2 is expressed in differentiating embryonic mouse testes. Neither gene, however, is expressed in We/We mutant embryonic testes which lack germ cells. These observations exclude both Zfy-1 and Zfy-2 as candidates for the mouse testis-determining gene.     

Human gamma-globin genes silenced independently of other genes in the beta-globin locus.

Erythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5' to the epsilon gene. The location of the LCR and its role in directing high-level expression of the globin genes has led to the suggestion that competition from the beta gene for interaction with the LCR has a major role in silencing the fetal gamma genes during adult life. We have now constructed lines of transgenic mice containing the human A gamma globin gene linked to the LCR. We observe high-level expression of the transgene in the embryonic stages but silencing of the gene in adult animals. We conclude that the gamma gene is not deregulated by the presence of the LCR and that competition from the beta gene is not required for silencing of the gamma genes in adult life. The silencing is therefore likely to be mediated by stage-specific factors binding to sequences immediately flanking the genes.     

Parental imprinting of the mouse H19 gene.

THE mouse H19 gene encodes one of the most abundant RNAs in the developing mouse embryo. It is expressed at the blastocyst stage of development, and accumulates to high levels in tissues of endodermal and mesodermal origin (H. Kim, unpublished result). After birth the gene is expressed in all tissues except skeletal muscle. It lacks a common open reading frame in the 2.5-kilobase RNA, but has considerable nucleotide sequence similarity between the genes of rodents and humans. Expression of the gene in transgenic mice results in late prenatal lethality, suggesting that the dosage of its gene product is strictly controlled. The H19 gene maps to the distal segment of mouse chromosome 7, in a region that is parentally imprinted, a process by which genes are differentially expressed on the maternal and paternal chromosomes. We have now used an RNase protection assay that can distinguish between H19 alleles in four subspecies of Mus, to demonstrate that the H19 gene is parentally imprinted, with the active copy derived from the mother. This assay will be of general use in assaying allele-specific gene expression.     

Selective activation of p53-mediated tumour suppression in high-grade tumours

Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related death worldwide, with an overall 5-year survival rate of only 10-15%. Deregulation of the Ras pathway is a frequent hallmark of NSCLC, often through mutations that directly activate Kras. p53 is also frequently inactivated in NSCLC and, because oncogenic Ras can be a potent trigger of p53 (ref. 3), it seems likely that oncogenic Ras signalling has a major and persistent role in driving the selection against p53. Hence, pharmacological restoration of p53 is an appealing therapeutic strategy for treating this disease. Here we model the probable therapeutic impact of p53 restoration in a spontaneously evolving mouse model of NSCLC initiated by sporadic oncogenic activation of endogenous Kras. Surprisingly, p53 restoration failed to induce significant regression of established tumours, although it did result in a significant decrease in the relative proportion of high-grade tumours. This is due to selective activation of p53 only in the more aggressive tumour cells within each tumour. Such selective activation of p53 correlates with marked upregulation in Ras signal intensity and induction of the oncogenic signalling sensor p19(ARF)( )(ref. 6). Our data indicate that p53-mediated tumour suppression is triggered only when oncogenic Ras signal flux exceeds a critical threshold. Importantly, the failure of low-level oncogenic Kras to engage p53 reveals inherent limits in the capacity of p53 to restrain early tumour evolution and in the efficacy of therapeutic p53 restoration to eradicate cancers.     

G to A substitution in the distal CCAAT box of the A gamma-globin gene in Greek hereditary persistence of fetal haemoglobin

The sequence 5'TTGGPyCAAT 3' (the 'CCAAT box') is a constituent of the promoter region of many eukaryotic and prokaryotic genes and is believed to play a part in promoter function. A characteristic of the two fetal human globin genes (A gamma and G gamma) is a duplication of a 12-base pair (bp) sequence containing the CCAAT box. Here we report a G----A substitution in the TTG sequence of the distal CCAAT box of the A gamma-globin gene in an individual with the A gamma (Greek) type of hereditary persistence of fetal haemoglobin (HPFH). This represents the first report of a natural mutation of the CCAAT box in a eukaryotic gene. The fact that this transition is associated with inappropriate expression of the A gamma gene in adult life suggests that the CCAAT box (or its surrounding sequences) may have a role in the developmental control of gamma-globin genes.     

Oxygen activation and the conservation of energy in cell respiration.

Many of the membrane-associated oxidases that catalyse respiratory reduction of O2 to water simultaneously couple this exergonic reaction to the translocation of protons across the inner mitochondrial membrane, or the cell membrane in prokaryotes, a process by which metabolic energy is conserved for subsequent synthesis of ATP. The molecular mechanism of O2 reduction and its linkage to H+ translocation are now emerging. The bimetallic haem iron-copper reaction centre in this family of enzymes is the critical structure for catalysis of both these processes.