Oncogene-blocking therapies: new insights from conditional mouse tumor models.

Identification of oncogene dependent signaling pathways controlling aggressive tumor growth has led to the emergence of a new era of oncogene-blocking therapies, including Herceptin and Gleevec. In the recent years conditional mouse tumor models have been established that allow switching-off the expression of specific oncogenes controlling tumor growth. The results may have two important implications for oncogene-blocking therapies: (i) downregulation of oncogenes, for instance HER2, MYC, RAS, RAF, BCR-ABL or WNT1, usually leads to a rapid tumor remission. However, it was observed that the initial remission was followed by recurrent tumor growth in most studies. Interestingly, different oncogenes controlled tumor growth in the recurrent than in the primary tumors. This could explain the astonishing clinical observation that inhibitors of a broader spectrum of protein kinases (so-called: "dirty inhibitors") may be superior over highly specific substances. Due to their additional "unspecific" inhibition of a broader spectrum of kinases, they may hamper the escape mechanisms by antagonizing also the pathways controlling recurrent tumor growth. (ii) Experiments with cell systems that allow switching-on oncogene expression point to a so far possibly underestimated cancer drug target: the dormant tumor cell. Oncogene expression (for instance: NeuT or RAS) led to a phenomenon named oncogene-induced senescence or dormancy. Dormant cells are unresponsive to mitogenic stimuli. Importantly, such cells are not at all ready to die, but can remain viable for extended periods of time. Recently, dormant tumor cells have been shown to be more resistant to stresses such as hypoxia or exposure to cytostatic drugs. It still is a matter of debate if and under which conditions dormant tumor cells can be "kissed to life". If these cells contribute to carcinogenesis, it will be important to identify substances specifically killing senescent cells. This review will focus on the possible relevance of senescence both as a pre-oncogenic condition and also for therapy.

4-Epidoxycycline: an alternative to doxycycline to control gene expression in conditional mouse models.

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.

Selective rescue of early haematopoietic progenitors in Scl(-/-) mice by expressing Scl under the control of a stem cell enhancer.

The stem cell leukaemia gene (Scl) encodes a basic helix-loop-helix transcription factor with a pivotal role in both haematopoiesis and endothelial development. During mouse development, Scl is first expressed in extra-embryonic mesoderm, and is required for the generation of all haematopoietic lineages and normal yolk sac angiogenesis. Ectopic expression of Scl during zebrafish development specifies haemangioblast formation from early mesoderm. These results suggest that SCL is essential for establishing the transcriptional programme responsible for the formation of haematopoietic stem cells and have focused attention on the transcriptional regulation of Scl itself. Previous studies have identified a panel of Scl enhancers each of which directed expression to a subdomain of the normal Scl expression pattern. Among them, a 3' enhancer directed expression during development to vascular endothelium and haematopoietic progenitors but not to Ter119(+) erythroid cells. The expression in haematopoietic stem cells, however, remained undetermined. We demonstrate that this 3' enhancer directs lacZ expression in transgenic mice to most foetal and adult long-term repopulating haematopoietic stem cells, and therefore functions as a stem cell enhancer. Consistent with these results, expression in Scl(-/-) embryos of exogenous Scl driven by the stem cell enhancer rescued the formation of early haematopoietic progenitors and also resulted in normal yolk sac angiogenesis. By contrast, erythropoiesis remained markedly deficient in rescued embryos. This observation is consistent with the inactivity of the stem cell enhancer in erythroid cells and reveals an essential role for SCL during erythroid differentiation in vivo.

Transcriptional regulation of the stem cell leukemia gene by PU.1 and Elf-1.

The SCL gene, also known as tal-1, encodes a basic helix-loop-helix transcription factor that is pivotal for the normal development of all hematopoietic lineages. SCL is expressed in committed erythroid, mast, and megakaryocytic cells as well as in hematopoietic stem cells. Nothing is known about the regulation of SCL transcription in mast cells, and in other lineages GATA-1 is the only tissue-specific transcription factor recognized to regulate the SCL gene. We have therefore analyzed the molecular mechanisms underlying SCL expression in mast cells. In this paper, we demonstrate that SCL promoter 1a was regulated by GATA-1 together with Sp1 and Sp3 in a manner similar to the situation in erythroid cells. However, SCL promoter 1b was strongly active in mast cells, in marked contrast to the situation in erythroid cells. Full activity of promoter 1b was dependent on ETS and Sp1/3 motifs. Transcription factors PU.1, Elf-1, Sp1, and Sp3 were all present in mast cell extracts, bound to promoter 1b and transactivated promoter 1b reporter constructs. These data provide the first evidence that the SCL gene is a direct target for PU.1, Elf-1, and Sp3.

Transcription of the SCL gene in erythroid and CD34 positive primitive myeloid cells is controlled by a complex network of lineage-restricted chromatin-dependent and chromatin-independent regulatory elements.

The SCL gene (also known as TAL-1) encodes a basic helix-loop-helix transcription factor that is essential for the development of all haematopoietic lineages, and ectopic expression of which results in T cell leukaemia. SCL is expressed in normal pluripotent haematopoietic stem cells and its expression is maintained during differentiation along erythroid, mast and megakaryocytic lineages, but is extinguished following commitment to other cell types. The mechanisms responsible for this pattern of expression are poorly understood, but are likely to illuminate the molecular basis for stem cell development and lineage commitment. We have identified multiple lineage-restricted DNase I hypersensitive sites in a 45 kb region spanning the murine SCL locus. Committed erythroid cells and CD34 positive primitive myeloid cells exhibited both shared and unique DNase I hypersensitive sites whereas none were found in T cells. The function of each hypersensitive site was studied using both transient and stable reporter assays in erythroid, primitive myeloid and T cells. Multiple positive and negative regulatory elements were characterised and found to display lineage-specificity, promoter-specificity and/or chromatin-dependence. These results represent the first description of key components of a complex network of regulatory elements controlling SCL expression during haematopoiesis.

Distinct mechanisms direct SCL/tal-1 expression in erythroid cells and CD34 positive primitive myeloid cells.

The SCL/tal-1 gene (hereafter designated SCL) encodes a basic helix-loop-helix transcription factor which is pivotal for the normal development of all hematopoietic lineages and which is expressed in committed erythroid, mast, and megakaryocytic cells as well as in hematopoietic stem cells. The molecular basis for expression of SCL in stem cells and its subsequent modulation during lineage commitment is of fundamental importance for understanding how early "decisions" are made during hematopoiesis. We now compare the activity of SCL promoters 1a and 1b in erythroid cells and in CD34 positive primitive myeloid cells. SCL mRNA expression in CD34 positive myeloid cells did not require GATA-1. Promoter 1a activity was weak or absent in CD34 positive myeloid cells and appeared to correlate with the presence or absence of low levels of GATA-1. However, promoter 1b, which was silent in committed erythroid cells, was strongly active in transient assays using CD34 positive myeloid cells, and functioned in a GATA-independent manner. Interestingly, RNase protection assays demonstrated that endogenous promoter 1b was active in both erythroid and CD34 positive myeloid cells. These results demonstrate that fundamentally different mechanisms regulate the SCL promoter region in committed erythroid cells and in CD34 positive myeloid cells. Moreover these observations suggest that in erythroid, but not in CD34 positive myeloid cells, promoter 1b required integration in chromatin and/or additional sequences for its activity. Stable transfection experiments showed that both core promoters were silent following integration in erythroid or CD34 positive myeloid cells. Our data therefore indicate that additional regulatory elements were necessary for both SCL promoters to overcome chromatin-mediated repression.

Lineage-restricted regulation of the murine SCL/TAL-1 promoter.

The SCL/TAL-1 gene encodes a basic helix-loop-helix transcription factor that is expressed in multipotent hematopoietic progenitors before lineage commitment. Its expression is maintained during differentiation along erythroid, mast, and megakaryocytic lineages, but is repressed after commitment to nonexpressing lineages. To begin to address the molecular mechanisms underlying this complex pattern of expression, we have studied the regulation of the murine SCL promoter in erythroid and T-cell lines. Analysis of the methylation and chromatin structure of the SCL promoter region showed that SCL mRNA expression correlated with DNase hypersensitive sites and methylation status of the promoter. Transient reporter assays showed that promoter 1a was active in erythroid cells but not in T cells. Sequences between -187 and +26 were sufficient for lineage-restricted activity of promoter 1a. A joint promoter construct containing both promoter 1a and promoter 1b also exhibited lineage-restricted activity. Conserved GATA (-37), MAZ (+242), and ETS (+264) motifs were all shown to contribute to SCL promoter activity in erythroid cells, but several other motifs were not required for full promoter activity. The pattern of complexes binding to the +242 MAZ and +264 ETS sites were the same in erythroid and T cells. However, GATA-1 bound the -37 GATA site in erythroid cells, whereas in T cells GATA-3 was only able to bind weakly, if at all. Moreover, GATA-1 but not GATA-2 or GATA-3 was able to transactivate SCL promoter 1a in a T-cell environment. These results suggest that inactivity of SCL promoter 1a in T cells reflected the absence of GATA-1 rather than the presence of trans-dominant negative regulators.

Discordant regulation of SCL/TAL-1 mRNA and protein during erythroid differentiation.

The SCL/TAL1 gene was originally identified by virtue of its rearrangement and transcriptional activation in patients with T cell acute lymphoblastic leukaemia. It encodes a helix-loop-helix transcription factor, is not normally expressed in T cells, but is expressed in erythroid, mast, megakaryocytic and progenitor cells. Over-expression of sense and antisense constructs have implicated SCL as a positive regulator of erythroid differentiation. In addition we have previously shown that SCL mRNA levels undergo biphasic modulation during induced erythroid differentiation of murine erythroleukaemia (MEL) cells with a transient early fall followed by a late rise. In this paper we have studied expression of the SCL protein during erythroid differentiation and also the molecular basis for the raised SCL mRNA levels that accompany erythroid differentiation. We have generated an anti-SCL antiserum and used it to demonstrate that an early transient fall in SCL protein does not occur during induced differentiation of MEL cells. Furthermore SCL protein levels underwent a late fall in three different models of erythroid differentiation and in two models of myeloid differentiation. The fall in SCL protein levels during induced erythroid differentiation contrasted with the concomitant marked rise in SCL mRNA levels. These observations have significant implications for the mechanism by which SCL may regulate erythropoiesis. In addition we have demonstrated that the stability of SCL mRNA was only marginally enhanced during erythroid differentiation of MEL cells, whereas the activity of a luciferase reporter construct driven by the SCL promoter was increased 11- to 17-fold. Up-regulation of transcription therefore accounted for most of the increase in SCL mRNA levels during erythroid differentiation.

Expression of lineage restricted transcription factors precedes lineage specific differentiation in a multipotent haemopoietic progenitor cell line.

Lineage commitment and differentiation are likely to be coordinated by the combined effects of multiple transcription factors acting on numerous different target genes. The mechanisms by which lineage-restricted patterns of transcription factor expression are established are therefore of particular relevance to our understanding of the role of transcription factors both in normal development and in oncogenesis. Here, we report that the genes for the lineage-restricted transcription factors SCL, GATA-1 and GATA-2 are expressed in all multipotent, IL-3-dependent, haemopoietic progenitor cell lines tested. Moreover, a liquid differentiation assay has been used to demonstrate down regulation of SCL, GATA-1, GATA-2 and PU-1 during differentiation into non-expressing lineages. These data support the concept that multiple lineage-restricted transcription factors are expressed prior to lineage commitment.

Transcription factors and the regulation of haemopoiesis: lessons from GATA and SCL proteins.

One of the central issues of developmental biology concerns the molecular mechanisms whereby a multipotent cell gives rise to distinct differentiated progeny. Differences between specialised cell types reflect variations in their patterns of gene expression. The regulation of transcription initiation is an important control point for gene expression and it is, therefore, not surprising that transcription factors play a pivotal role in mammalian development and differentiation. Haemopoiesis offers a uniquely tractable system for the study of lineage commitment and differentiation. The importance of transcription factors in the normal regulation of haemopoiesis is underlined by the frequency with which transcription factors are targeted by leukaemogenic mutations. Studies of the function and regulation of haemopoietic transcription factors, especially those expressed in lineage-restricted patterns, should greatly increase our understanding of the molecular control of haemopoiesis. In this review we have focused on insights provided by recent studies of the GATA and SCL proteins.

Structure of the gene encoding the murine SCL protein.

We have determined the molecular structure of the gene encoding the murine SCL protein (helix-loop-helix transcription factor). The gene consists of seven exons spanning approx. 20 kb. The intron/exon structure, coding region sequences and sequences present at the splice junctions were highly conserved between mouse and human. The 5' flanking sequence contains CCAAT and TATA consensus motifs with several putative binding sites for SP-1, AP-1 and GATA-1. Multiple mRNA transcripts were generated by alternate exon usage. The transcripts differed primarily in the 5' untranslated region (UTR), but potentially also encode a smaller SCL protein. Despite the high degree of conservation between species, the heptamer/nonamer signal sequences in the 5' region of the human SCL gene (the frequent site of SCL disruption in human leukemia) were poorly represented in the murine sequence. In keeping with this, structural abnormalities of murine SCL were uncommon in murine leukemias that express the SCL transcript.

Isolation and molecular characterization of the swinepox virus thymidine kinase gene.

Swinepox virus (SPV), the only member of the Suipoxvirus genus, shows little antigenic relatedness or DNA homology to members of the other poxvirus genera. A SPV thymidine kinase (TK) gene was detected and mapped to the left end of the HindIII G fragment using degenerate oligonucleotide probes. Cloning and sequencing of a 1.8-kb HindIII-BamHI fragment containing the SPV TK gene revealed an open reading frame (ORF) of 181 amino acids yielding a predicted polypeptide of Mr 20.6 kDa with significant homology to both poxvirus and vertebrate thymidine kinases. Comparison with other TK protein sequences showed that the SPV thymidine kinase was closely related to the TK genes of avipoxviruses (52.0%) and vertebrates (57.1-59.7%). The TK gene from African swine fever virus (ASF) showed little homology (30.5%) to the SPV TK gene suggesting that these two viruses are not closely related though they share many biochemical features and infect a single, common mammalian host (swine). The SPV TK gene, like that of other poxviruses, is transcribed early, and when cloned into a TK- strain of vaccinia converted the virus to a TK+ phenotype. BUdRR mutants of SPV contained frameshift, deletion, and missense mutations in the TK ORF.

Escherichia coli thymidine kinase: nucleotide sequence of the gene and relationships to other thymidine kinases.

The thymidine kinase (TK)-encoding gene (tdk) of Escherichia coli is located at min 27 of the E. coli genetic map. Sequence analysis of this region revealed an open reading frame of 205 codons. Identification of this region as the E. coli tdk gene was confirmed by its similarity to other TK-encoding genes. The E. coli amino acid (aa) sequence showed significant similarity to the corresponding TK polypeptides of vertebrates and large DNA viruses, but showed no similarity to known herpes virus TK enzymes. Mapping of highly conserved positions among all sequences indicates the importance of these residues for catalytic activity and may facilitate further functional studies. Using a distance matrix method, the evolutionary relationships among the TK aa sequence of poxviruses, eukaryotes and prokaryotes were analyzed and a potential phylogenetic tree was established.

Characterization and gene cloning of neurotactin, a Drosophila transmembrane protein related to cholinesterases.

Monoclonal antibodies have served to characterize neurotactin, a novel Drosophila protein for which a role in cell adhesion is postulated. Neurotactin is a transmembrane protein, as indicated by epitope mapping and amino acid sequence. Similarly to other cell adhesion molecules, neurotactin accumulates in parts of the membrane where neurotactin-expressing cells contact each other. The protein is only detected during cell proliferation and differentiation, and it is found mainly in neural tissue and also in mesoderm and imaginal discs. Neurotactin has a large cytoplasmic domain rich in charged residues and an extracellular domain similar to cholinesterase that lacks the active site serine required for esterase activity. The extracellular domain also contains three copies of the tripeptide leucine-arginine-glutamate, a motif that forms the primary sequence of the adhesive site of vertebrate s-laminin.

Sequence and evolutionary relationships of African swine fever virus thymidine kinase.

The thymidine kinase gene of African swine fever virus was mapped in a 1.4-kb EcoRI-PstI fragment located in the left half of the Eco RI K fragment of African swine fever virus DNA by using degenerate oligonucleotide probes derived from regions of the thymidine kinase sequence conserved in several poxviruses, man, mouse, and chicken. The nucleotide sequence of this region revealed an open reading frame of 196 codons, whose translated amino acid sequence showed significant similarity to the thymidine kinases of vaccinia virus, variola virus, monkeypox virus, shope fibroma virus, fowlpox virus, capripox virus, man, mouse, and chicken. The similarity scores obtained after comparison of known thymidine kinase sequences indicated that the African swine fever virus thymidine kinase is more distantly related than the poxvirus thymidine kinases to their cellular homologs. The evolutionary implications of these findings are discussed.