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.

Human telomerase contains two cooperating telomerase RNA molecules.

Telomerase uses a short stretch of its intrinsic RNA molecule as template for telomere repeat synthesis. Reverse transcription of the RNA template is catalyzed by the telomerase reverse transcriptase (TERT) protein subunit. We demonstrate that human telomerase reconstituted from recombinant TERT and telomerase RNA runs as a dimer on a gel filtration column and that it contains two telomerase RNA molecules. Significantly, a telomerase heterodimer reconstituted from wild-type and mutant telomerase RNA is barely active when compared with the wild-type homodimer. We conclude that the telomerase RNA templates in the active enzyme are interdependent and functionally cooperate with each other. We discuss models that may explain the biological and enzymatic roles of telomerase dimerization.

Mouse fibroblast-activation protein--conserved Fap gene organization and biochemical function as a serine protease.

The human fibroblast-activation protein (FAP), a member of the serine protease family, was discovered as an inducible type-II cell-surface glycoprotein selectively expressed by reactive stromal fibroblasts of epithelial cancers and healing wounds. Antibodies directed against human FAP have a clinical use for antibody-based tumor imaging. As part of an effort to generate animal models of FAP expression in epithelial tumorigenesis and wound healing, we previously cloned the cDNA encoding the mouse FAP homolog. In this study, we used PCR/restriction-fragment length polymorphism, identified in interspecific back-crosses between Mus musculus and Mus spretus, to map the Fap gene locus to a region of mouse chromosome 2, known to be syntenic to the previously identified FAP gene locus on human chromosome 2q23. The Fap gene spans approximately 60 kb and contains 26 exons ranging in size from 46 bp to 195 bp. This genomic organization is very similar to that of the human FAP locus. Similar to the gene encoding dipeptidyl peptidase IV (DPP IV), the nucleotides encoding the serine protease consensus motif, WGWSYGG, are split between two exons, a feature distinct from classical serine proteases. Consistent with the similarity to DPP IV, a chimeric FAP fusion protein expressed in a baculovirus system has dipeptidyl peptidase activity.

The disulfide bridges of the immunoglobulin-like domains of Fc gamma RIIIB are essential for efficient expression and biological activity.

The immunoglobulin G receptor Fc gamma RIIIB belongs to the immunoglobulin superfamily as two extracellular domains show homology to the immunoglobulin domains. Since some residues in these domains, such as the two cysteines, are supposed to form an intrachain disulfide bridge are so commonly conserved, they may be of importance for correct folding. Site-directed mutagenesis and expression in BHK21 confirmed this supposition for the Fc gamma RIIIB. Replacing both cysteines in the first and/or second domain by serines reduced the surface expression level by 50%, whereas the ligand binding capability was 20-30% of that seen in cells expressing the wild-type receptor. Replacing one of the four cysteines resulted in the loss of surface expression. Exchanging the conserved tryptophan in the first domain by phenylalanine only slightly affected the ligand binding (25%), whereas the surface expression remained unchanged.

The Rhizobium meliloti exoZl exoB fragment of megaplasmid 2: ExoB functions as a UDP-glucose 4-epimerase and ExoZ shows homology to NodX of Rhizobium leguminosarum biovar viciae strain TOM.

A 2.6 kb ClaI-BamHI DNA fragment of megaplasmid 2 of Rhizobium meliloti 2011 was found to carry genes involved in exopolysaccharide synthesis and infection of alfalfa nodules. The analysis of the nucleotide sequence of this DNA fragment revealed the existence of two open reading frames (ORFs) running in opposite directions. Plasmid integration mutagenesis showed that these ORFs are organized as two monocistronic transcription units. One of the ORFs represents a new exo gene designated exoZ, which is involved in, but not essential for, the production of acidic exopolysaccharide. However, exoZ is not necessary for nodule formation with alfalfa. The ExoZ protein was found to show homology (23.3%) to the NodX protein of the R. leguminosarum biovar viciae strain TOM, known to be essential for nodulating the primitive Afghanistan pea. The second identified ORF corresponds to the exoB locus. The deduced amino acid sequence of the ExoB protein is homologous (39.6%) to that of the Escherichia coli GalE protein. In R. meliloti, exoB codes for a UDP-glucose 4-epimerase. A deficiency in the activity of this enzyme fully accounts for all the multiple carbohydrate defects that have been observed in exoB mutants.

Molecular basis of IgG Fc receptor III defect in a patient with systemic lupus erythematosus.

By flow cytometry analysis we could show a decreased expression of Fc gamma receptor type III (Fc gamma RIII) on granulocytes of a patient with systemic lupus erythematosus (SLE). Therefore, we constructed a leukocyte cDNA library from this patient with the aim of investigating this defect on the molecular level. Using an Fc gamma RIII cDNA probe we isolated 15 Fc gamma RIII cDNA clones, which were all characterized by sequencing. Our sequence data revealed that the patient was heterozygous for Fc gamma RIII (NA-1/NA-2). Only clone 5 (NA-2) was a full-length cDNA clone. In contrast to the wild-type Fc gamma RIII the signal sequence is mutated, lacking the hydrophobic region essential for co-translational transport across the endoplasmic reticulum membrane. The predicted transport defect leading to the lack of membrane expression could be confirmed by immunofluorescence staining after expression of this cDNA clone in BHK cells. The cDNA clones 6 and 8 (NA-1) lack the first 45 bp of the signal sequence, but considering the flow cytometry data the signal sequence must be functional allowing the membrane expression of this receptor allele. The part of the cDNA sequence of all isolated clones coding the mature Fc gamma RIII is identical to the wild-type sequence. Therefore, we conclude that the decreased expression of Fc gamma RIII on granulocytes of this SLE patient is due to the transport defect of one of the receptor alleles.