A highly selective telomerase inhibitor limiting human cancer cell proliferation.

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.

Vitamin D(3) enhances the expression of I-mfa, an inhibitor of the MyoD family, in osteoblasts.

I-mfa (inhibitor of the MyoD family) is a transcription modulator that binds to and suppresses the transcriptional activity of MyoD family members. I-mfa transcripts are expressed in sclerotome, suggesting a role of I-mfa in skeletogenesis. The aim of this study was to examine the expression and regulation of I-mfa in osteoblasts. We found that I-mfa is expressed at a low level in an osteoblast-like cell line, MC3T3E1, and a pluripotent differentiation modulator, 1,25-dihydroxyvitamin D(3), specifically enhanced I-mfa mRNA expression. This effect was completely blocked by the presence of an RNA polymerase inhibitor, but not by a protein synthesis inhibitor, suggesting that 1,25-dihydroxyvitamin D(3) upregulates transcription of the I-mfa gene without requirement for new protein synthesis. Western blot analysis indicated that 1,25-dihydroxyvitamin D(3) increased the I-mfa protein levels severalfold in MC3T3E1 cells. I-mfa expression was also observed in primary mouse calvaria cells and ROS17/2.8 cells and 1,25-dihydroxyvitamin D(3) enhanced I-mfa expression in these cells. These data indicate that I-mfa is a novel transcriptional regulator gene expressed in osteoblasts and that its level is under the control of 1,25-dihydroxyvitamin D(3).

Human RERE is localized to nuclear promyelocytic leukemia oncogenic domains and enhances apoptosis.

RE repeats encoded (RERE) was identified recently as a protein with high homology to the atrophin-1 protein, which appears to be causal in the hereditary neurodegenerative disorder termed dentatorubral-pallidoluysian atrophy (DRPLA) caused by an abnormal glutamine expansion. We have independently identified RERE in a search for genes localized to the translocation breakpoint region at chromosome 1p36.2 in the neuroblastoma cell line NGP. Here we show that neuroblastoma tumor cell lines display reduced abundance of RERE transcripts. Furthermore, we detected RERE protein mainly in the nucleus, where it colocalizes with the promyelocytic leukemia protein in promyelocytic leukemia oncogenic domains (PODs). Overexpression of RERE recruits a fraction of the proapoptotic protein BAX to PODS: This observation correlates with RERE-induced apoptosis, which occurs in a caspase-dependent manner. These results identify RERE as a novel component of PODs and suggest an important role of RERE in the control of cell survival.

Cell-type-dependent induction of eotaxin and CCR3 by ionizing radiation.

Eotaxin is an eosinophil-specific C-C chemokine that is implicated in the pathogenesis of eosinophilic inflammatory diseases, such as asthma and atopic dermatitis, by acting specifically on its receptor CCR3. Using RT-PCR analysis, we show that the expression of eotaxin is upregulated upon treatment with ionizing radiation (IR) in human dermal fibroblasts, but not in the bronchial epithelial cell line A549. In contrast, the gene encoding CCR3 is markedly induced in both cell types. None of the genes coding for other CCR3 ligands are significantly induced by IR in these cell types. cDNA array analysis of irradiated versus nonirradiated A549 cells and human dermal fibroblasts confirm and extend these results, and support the observation that regulation of eotaxin/CCR3-induction by IR occurs in a selective and cell-type-dependent manner. They further suggest that the induction of signaling via eotaxin and CCR3 may be an important step leading to eosinophilia in patients with radiation exposure.

Requirement of the mouse I-mfa gene for placental development and skeletal patterning.

The bHLH-repressor protein I-mfa binds to MyoD family members, inhibits their activity, and blocks their nuclear import and binding to DNA. In situ hybridization analysis demonstrated that mouse I-mfa was highly expressed in extraembryonic lineages, in the sclerotome, and subsequently within mesenchymal precursors of the axial and appendicular skeleton, before chondrogenesis occurs. Targeted deletion of I-mfa in a C57Bl/6 background resulted in embryonic lethality around E10.5, associated with a placental defect and a markedly reduced number of trophoblast giant cells. Overexpression of I-mfa in rat trophoblast (Rcho-1) stem cells induced differentiation into trophoblast giant cells. I-mfa interacted with the bHLH protein Mash2, a negative regulator of trophoblast giant cell formation, and inhibited its transcriptional activity in cell culture. In contrast, I-mfa did not interfere with the activity of the bHLH protein Hand1, a positive regulator of giant cell differentiation. Interestingly, I-mfa-null embryos on a 129/Sv background had no placental defect, generally survived to adulthood, and exhibited delayed caudal neural tube closure and skeletal patterning defects that included fusions of ribs, vertebral bodies and abnormal formation of spinous processes. Our results indicate that I-mfa plays an important role in trophoblast and chondrogenic differentiation by negatively regulating a subset of lineage-restricted bHLH proteins.

I-mf, a novel myogenic repressor, interacts with members of the MyoD family.

During embryogenesis, cells from the ventral and dorsal parts of the somites give rise to sclerotome and dermomyotome, respectively. Dermomyotome contains skeletal muscle precursors that are determined by the MyoD family of myogenic factors. We have isolated a novel myogenic repressor, I-mf (Inhibitor of MyoD family), which is highly expressed in the sclerotome. In contrast, MyoD family members are concentrated in the dermomyotome. We demonstrate that I-mf inhibits the transactivation activity of the MyoD family and represses myogenesis. I-mf associates with MyoD family members and retains them in the cytoplasm by masking their nuclear localization signals. I-mf can also interfere with the DNA binding activity of MyoD family members. We postulate that I-mf plays an important role in the patterning of the somite early in development.

Rem-1, a putative direct target gene of the Myb-Ets fusion oncoprotein in haematopoietic progenitors, is a member of the recoverin family.

The Myb-Ets oncoprotein encoded by the E26 avian leukaemia virus represents a fusion of two transcription factors which cooperate in transforming multipotent haematopoietic progenitors (MEPs) in vitro and in vivo. Previous studies with a temperature sensitive mutant in ets (ts1.1 E26) have suggested that the Ets part of the Myb-Ets fusion protein blocks multilineage differentiation of transformed MEPs, by regulating specific target genes. Using this system in a differential screening approach we have now identified a new gene, called rem-1, as a target for the E26 virus. Following shift of ts1.1 mutant transformed cells to the nonpermissive temperature a decreased expression of rem-1 was observed which increased upon downshift. The finding that this reexpression did not require new protein synthesis suggests that the Ets component of the fusion protein directly regulates rem-1 transcription. Rem-1 is related to a family of EF-hand-containing calcium-binding proteins that are predominantly expressed in the brain and in retinal cells. This family includes recoverin and visinin, proteins that have been implicated in regulating photoreception. Rem-1 is likewise expressed in these tissues but in addition in haematopoietic cells and in the gut. Enforced expression of rem-1 in ts1.1-transformed MEP cells, using a retroviral vector, showed that this gene is not sufficient to block their differentiation, but that it may provide them with a growth advantage.

A functional Ets DNA-binding domain is required to maintain multipotency of hematopoietic progenitors transformed by Myb-Ets.

Earlier work demonstrated that the Myb-Ets fusion protein of E26 avian leukemia virus induces the proliferation of multipotent hematopoietic progenitors (MEPs). These progenitors differentiate spontaneously at low frequencies along the erythroid lineage, and following the introduction of kinase/ras-type oncogenes or treatment with TPA, they are induced to differentiate along the myelomonocytic and eosinophilic lineages. Here, we show that the ts1.1 mutant of E26 encodes an Ets DNA-binding domain that is both defective and thermolabile for binding of specific DNA sequences. Correlating with this, ts1.1 MEP colonies transformed at the permissive temperature exhibit elevated levels of erythroid cells and eosinophils, whereas at the nonpermissive temperature they are induced to differentiate along the erythroid and myelomonocytic lineages and, to a lesser extent, along the eosinophil lineage. Induction of the former two lineages cannot be separated by pulse shift experiments and is essentially completed 2.5 days after temperature shift. Our results indicate that the Ets portion of the Myb-Ets fusion protein inhibits the lineage commitment of multipotent hematopoietic progenitors, probably via binding to regulatory DNA sequences of specific target genes.

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements.

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

DNA binding by c-Ets-1, but not v-Ets, is repressed by an intramolecular mechanism.

The E26 avian retrovirus causes an acute leukemia in chickens and transforms both myeloid and erythroid cells. The virus encodes a 135 kDa fusion protein which contains amino acid sequences derived from the viral Gag protein and the two cellular transcription factors c-Myb and c-Ets-1p68. Previously we have shown that like v-myb, v-ets on its own is also active in transformation, but only within the erythroid lineage. To understand better the mechanisms involved in the oncogenic activation of c-Ets-1p68, we used the polyoma PEA3 element, a known Ets binding site, to compare the sequence-specific DNA binding and transactivating properties of v-Ets and c-Ets-1p68. Using Ets protein synthesized in rabbit reticulocyte lysate in gel retardation assays, we detected little binding of c-Ets-1p68 to an oligonucleotide containing the PEA3 motif whereas v-Ets bound strongly. However, in transient cotransfection assays in chicken embryo fibroblasts both c-Ets-1p68 and v-Ets transactivated transcription from a heterologous promoter linked to PEA3 elements. Interestingly, fragments of c-Ets-1p68 with strong DNA binding activity could be produced by limited proteolysis, indicating that the DNA binding domain is repressed within the full-length molecule. By deletion mapping the DNA binding domain was localized to the most highly conserved region of the Ets-related proteins known as the ETS domain. The C-terminus as well as a region in the middle of the polypeptide chain are involved in repression of DNA binding in c-Ets-1p68. Significantly, v-Ets contains a 16 amino acid substitution at the C-terminus. Our results suggest that intramolecular repression of DNA binding is a regulatory mechanism in c-Ets-1p68 which is lost in v-Ets.