Novel mRNA isoforms and mutations of uridine monophosphate synthetase and 5-fluorouracil resistance in colorectal cancer.

The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer. The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells. Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment. We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors. We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived. A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells. The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line. We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors. The observed mutations, aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer.

Cancer-associated somatic DICER1 hotspot mutations cause defective miRNA processing and reverse-strand expression bias to predominantly mature 3p strands through loss of 5p strand cleavage.

Our group recently described recurrent somatic mutations of the miRNA processing gene DICER1 in non-epithelial ovarian cancer. Mutations appeared to be clustered around each of four critical metal-binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3' end of the 5p miRNA strand of a pre-mRNA hairpin. To investigate the effects of these cancer-associated 'hotspot' mutations, we engineered mouse DICER1-deficient ES cells to express wild-type and an allelic series of the mutant DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal-binding site mutations were compared to each other and to wild-type DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation-carrying cells were distinct from both wild-type and DICER1-deficient cells. Further, miRNA profiles showed that mutant DICER1 results in a dramatic loss in processing of mature 5p miRNA strands but were still able to create 3p strand miRNAs. Messenger RNA (mRNA) profile changes were consistent with the loss of 5p strand miRNAs and showed enriched expression for predicted targets of the lost 5p-derived miRNAs. We therefore conclude that cancer-associated somatic hotspot mutations of DICER1, affecting any one of four metal-binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p strand miRNA. We further propose that this resulting 3p strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations.

A tripartite complex composed of ETV6-NTRK3, IRS1 and IGF1R is required for ETV6-NTRK3-mediated membrane localization and transformation.

ETV6-NTRK3 (EN), a chimeric tyrosine kinase generated by t(12;15) translocations, is a dominantly acting oncoprotein in diverse tumor types. We previously showed that insulin-like growth factor 1 receptor (IGF1R) is essential for EN-mediated oncogenesis and that insulin receptor substrate 1 (IRS1) is constitutively tyrosine phosphorylated and bound by EN in transformed cells. Given that IRS1 is also an adapter for IGF1R, we hypothesized that IRS1 might localize EN to IGF1R at the membrane to activate phosphatidylinositol 3-kinase (PI3K)-Akt, which is critical for EN oncogenesis. In this study, we examined EN/IRS1/IGF1R complexes in detail. We find that both IRS1 and kinase active IGF1R are required for EN transformation, that tyrosine phosphorylated IRS1 is present in high molecular weight complexes with EN and IGF1R, and that EN colocalizes with IGF1R at the plasma membrane. Both IGF1R kinase activity and an intact cytoplasmic Y950 residue, the IRS1-docking site of IGF1R, are required, confirming the importance of the IGF1R/IRS1 interaction for EN oncogenesis. The dual specificity IGF1R and insulin receptor (INSR) inhibitor, BMS-536924, blocks EN transformation activity, cell survival and its interaction with IRS proteins, and induces a striking shift of EN proteins to smaller sized molecular complexes. We conclude that a tripartite complex of EN, IRS1 and IGF1R localizes EN to the membrane and that this is essential for EN-mediated transformation. These findings provide an explanation for the observed IGF1R dependency of EN transformation. Blocking IGF1R kinase activity may, therefore, provide a tractable therapeutic strategy for the many tumor types driven by the EN oncoprotein.

TANK2, a new TRF1-associated poly(ADP-ribose) polymerase, causes rapid induction of cell death upon overexpression.

Tankyrase (TANK1) is a human telomere-associated poly(ADP-ribose) polymerase (PARP) that binds the telomere-binding protein TRF1 and increases telomere length when overexpressed. Here we report characterization of a second human tankyrase, tankyrase 2 (TANK2), which can also interact with TRF1 but has properties distinct from those of TANK1. TANK2 is encoded by a 66-kilobase pair gene (TNKS2) containing 28 exons, which express a 6.7-kilobase pair mRNA and a 1166-amino acid protein. The protein shares 85% amino acid identity with TANK1 in the ankyrin repeat, sterile alpha-motif, and PARP catalytic domains but has a unique N-terminal domain, which is conserved in the murine TNKS2 gene. TANK2 interacted with TRF1 in yeast and in vitro and localized predominantly to a perinuclear region, similar to the properties of TANK1. In contrast to TANK1, however, TANK2 caused rapid cell death when highly overexpressed. TANK2-induced death featured loss of mitochondrial membrane potential, but not PARP1 cleavage, suggesting that TANK2 kills cells by necrosis. The cell death was prevented by the PARP inhibitor 3-aminobenzamide. In vivo, TANK2 may differ from TANK1 in its intrinsic or regulated PARP activity or its substrate specificity.

Functional requirement of p23 and Hsp90 in telomerase complexes.

Most normal human diploid cells have no detectable telomerase; however, expression of the catalytic subunit of telomerase is sufficient to induce telomerase activity and, in many cases, will bypass normal senescence. We and others have previously demonstrated in vitro assembly of active telomerase by combining the purified RNA component with the reverse transcriptase catalytic component synthesized in rabbit reticulocyte extract. Here we show that assembly of active telomerase from in vitro-synthesized components requires the contribution of proteins present in reticulocyte extracts. We have identified the molecular chaperones p23 and Hsp90 as proteins that bind to the catalytic subunit of telomerase. Blockade of this interaction inhibits assembly of active telomerase in vitro. Also, a significant fraction of active telomerase from cell extracts is associated with p23 and Hsp90. Consistent with in vitro results, inhibition of Hsp90 function in cells blocks assembly of active telomerase. To our knowledge, p23 and Hsp90 are the first telomerase-associated proteins demonstrated to contribute to telomerase activity.

Telomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation.

The telomerase reverse transcriptase component (TERT) is not expressed in most primary somatic human cells and tissues, but is upregulated in the majority of immortalized cell lines and tumors. Here, we identify the c-Myc transcription factor as a direct mediator of telomerase activation in primary human fibroblasts through its ability to specifically induce TERT gene expression. Through the use of a hormone inducible form of c-Myc (c-Myc-ER), we demonstrate that Myc-induced activation of the hTERT promoter requires an evolutionarily conserved E-box and that c-Myc-ER-induced accumulation of hTERT mRNA takes place in the absence of de novo protein synthesis. These findings demonstrate that the TERT gene is a direct transcriptional target of c-Myc. Since telomerase activation frequently correlates with immortalization and telomerase functions to stabilize telomers in cycling cells, we tested whether Myc-induced activation of TERT gene expression represents an important mechanism through which c-Myc acts to immortalize cells. Employing the rat embryo fibroblast cooperation assay, we show that TERT is unable to substitute for c-Myc in the transformation of primary rodent fibroblasts, suggesting that the transforming activities of Myc extend beyond its ability to activate TERT gene expression and hence telomerase activity.

Expression of mouse telomerase reverse transcriptase during development, differentiation and proliferation.

We have identified the mouse telomerase reverse transcriptase component (mTERT) and demonstrate both substantial sequence homology to the human ortholog (hTERT), and the presence of reverse transcriptase and telomerase specific motifs. Furthermore, we show functional interchangeability with hTERT in in vitro telomerase reconstitution experiments, as mTERT produces strong telomerase activity in combination with the human telomerase RNA component hTR. The mouse TERT is widely expressed at low levels in adult tissues, with greatest abundance during embryogenesis and in adult thymus and intestine. The mTERT component mRNA levels were regulated during both differentiation and proliferation, while mTR levels remained constant throughout both processes. Comparison of mTERT and mTR levels to telomerase activity indicates that mTERT expression is more tightly linked to the regulation of telomerase activity during these processes than is mTR. In contrast to the situation in human cell cultures, mTERT transcript levels are present at readily detectable levels in primary cultured cells and are not upregulated following crisis. The widespread expression of mTERT in primary cells and mouse tissues could explain the increased frequency of spontaneous immortalization of mouse cells in culture and tumorigenesis in vivo.

Extension of life-span by introduction of telomerase into normal human cells.

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT.

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation. In addition to the human telomerase RNA component (hTR; ref. 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Telomerase catalytic subunit homologs from fission yeast and human.

Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

Telomere control of replicative lifespan.

The replicative capacity of cells may limit the lifespan of key systems in the body. It has long been known that normal human cells have a finite lifespan when placed in cell culture, and their lifespan is dependent on the age of the individual donor. The mechanism of the genetic program that times this process has been elusive. The telomere hypothesis of cell aging proposes that the length of the telomeric repeat array at chromosomal termini can time replication number and signal cell cycle arrest when critical telomere lengths are obtained. The erosion of telomeric DNA in normal tissues appears to be due to the lack of expression of components of the telomere maintenance system. Telomerase, the key enzyme involved in telomere replication, is not expressed in somatic tissues, but is expressed in germ cells, where telomere length is stably maintained, so that viable chromosomes can be transmitted to the next generation. Evidence is reviewed that correlates telomere length, telomerase activity, and the manipulation of telomere length with cell replicative capacity and cellular immortalization. Strong circumstantial evidence exists that indicates a role for telomere biology in the control of replicative capacity and in tumorigenesis.

The implications of telomerase biochemistry for human disease.

The replication of linear chromosome termini (telomeres) cannot be completely replicated by conventional DNA polymerases. Telomerase is a special DNA polymerase used by most eukaryotes to solve the telomere and replication problem. Telomerase is necessary for indefinite cell division in most immortal cells, but apparently unnecessary for the normal function of most somatic tissues. Telomerase may play a critical role in some genetic diseases, in regulating the lifespan of normal cells, and in tumorigenesis. This article reviews the structure and reaction mechanism of mammalian telomerase and how it may be exploited to control some human diseases.

Recognition of a chromosome truncation site associated with alpha-thalassaemia by human telomerase.

Telomeres define the ends of chromosomes; they consist of short tandemly repeated DNA sequences loosely conserved in eukaryotes (G1-8(T/A)1-4). Telomerase is a ribonucleoprotein which, in vitro, recognizes a single-stranded G-rich telomere primer and adds multiple telomeric repeats to its 3' end by using a template in the RNA moiety. In conjunction with other components, telomerase may balance the loss of telomeric repeats due to DNA replication. Another role of telomerase may be the de novo formation of telomeres. In eukaryotes like Tetrahymena, this process is an integral part of the formation of macronuclear chromosomes. In other eukaryotes this process stabilizes broken chromosomes. A case of human alpha-thalassaemia is caused by a truncation of chromosome 16 that has been healed by the addition of telomeric repeats (TTAGGG)n. Using an in vitro assay, I show here that human telomerase correctly recognizes the chromosome 16 breakpoint sequence and adds (TTAGGG)n repeats. The DNA sequence requirements are minimal and seem to define two modes of DNA recognition by telomerase.

The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats.

I have identified an activity in crude HeLa cell extracts that satisfies the requirements for a human telomere terminal transferase or telomerase. It catalyzes the addition of a 6 nucleotide repeating pattern to oligonucleotide primers containing human or nonhuman telomeric repeat sequences. Direct sequence analyses of reaction products reveal the added sequence to be TTAGGG in all cases. Under optimal conditions 65-70 repeats can be synthesized. The enzyme has the properties of a ribonucleoprotein. Telomerase has previously been observed only in ciliated protozoans, which possess 10(4) - 10(7) macronuclear telomeres. The identification of telomerase in HeLa cells with only approximately 100 telomeres indicates that telomerase-mediated telomere maintenance is conserved throughout eukaryotes.

A Tetrahymena intron nucleotide connected to the GTP/arginine site.

We have substituted all nucleotides at intron nucleotide 260 (N260) in transcripts related to the self-splicing Tetrahymena rRNA. Substitution slightly affects the binding and reaction of GTP with this group I catalytic center; kcat/Km varies over a three-fold range. The base of N260 therefore communicates with the rG site, but is unlikely to bond directly to GTP. Different nucleotides at this position also alter the binding of L-arginine to the intron, measured by inhibition of the reaction with GTP. Effects of similar small magnitude on interaction of RNA with both GTP and L-arginine support the previous argument from kinetic and structural comparison (Yarus, M. (1988) Science 240, 1751) that placed the two ligands of the RNA in the same binding site. G260 RNA shows the greatest affinity for both D- and L-arginine, but uniquely lacks stereoselectivity for the amino acid. Therefore G260 alters spatial relations within the G site, otherwise conserved in C260, U260, and A260 RNA's. Guanyl urea was used as a probe for the G/guanidino H-bonding part of the rG/arginine site. G260 RNA's dissociation constant for guanyl urea is similar to that of the other RNA's, suggesting that G260 RNA is unaltered at the G/guanidino end of the rG/arginine binding site. To account for all observations, we suggest that the G260 substitution alters the relative location of the RNA backbone near the 5' exon-intron junction, making this location more flexible and closer to the alpha-NH3+'s of L- and D-arginine.

Telomeric repeats of Tetrahymena malaccensis mitochondrial DNA: a multimodal distribution that fluctuates erratically during growth.

The linear mitochondrial DNA (mtDNA) of Tetrahymena malaccensis has tandem 52-base-pair repeats at its telomeres. The mtDNA has a multimodal distribution of telomeres. Different groups in the distribution have different numbers of telomeric repeats. The standard deviation of the size of each end group is independent of the mean size of the end group. The two sides of the mtDNA have different multimodal distributions of repeats. Cloned cell lines have multimodal distributions of mtDNA telomeres distinct from that of the original cell line. The number of telomere end groups and the average size of the end groups change in an erratic fashion as the cells are passaged and do not reach a stable equilibrium distribution in 185 generations. We propose that the mean size of a telomere end group and the size distribution of an end group are independently regulated. The system controlling the average size of end groups may be defective in T. malaccensis, since a closely related species (T. thermophila) does not have a multimodal distribution of mtDNA telomeres. T. hyperangularis, which has different telomeric repeats on each side of its mtDNA, has a multimodal distribution of mtDNA telomeres on only one side, suggesting that the mechanism controlling the average number of repeats in an end group can be sequence specific. These mitochondrial telomeres provide a new example of the more general phenomenon of expansion and contraction of arrays of repeated sequences seen, for example, with simple-sequence "satellite" DNAs; however, the mitochondrial telomeres change on a very short time scale.

Mitochondrial telomeres: surprising diversity of repeated telomeric DNA sequences among six species of Tetrahymena.

The DNA sequences at the ends of the linear mtDNA of 6 species of Tetrahymena encompassing 13 strains were determined. All the strains have variable numbers of a tandemly repeated DNA sequence, 31 bp to 53 bp in size, at their mtDNA termini. Based upon the size and nucleotide sequence of the terminal repeats, the telomeres can be separated into four classes. T. pigmentosa, hyperangularis, and hegewischi have different telomeric repeats on the two ends of their mtDNAs. The only conserved feature of the mtDNA termini is the presence of tandem repeats. The function of the repeats might be to promote unequal crossing over during recombination, thereby overcoming the problem of telomere replication for these linear DNAs.

Phylogenetic relationships and altered genome structures among Tetrahymena mitochondrial DNAs.

Tetrahymena thermophila mitochondrial DNA is a linear molecule with two tRNAs, large subunit beta (LSU beta) rRNA (21S rRNA) and LSU alpha rRNA (5.8S-like RNA) encoded near each terminus. The DNA sequence of approximately 550 bp of this region was determined in six species of Tetrahymena. In three species the LSU beta rRNA and tRNA(leu) genes were not present on one end of the DNA, demonstrating a mitochondrial genome organization different from that of T. thermophila. The DNA sequence of the LSU alpha rRNA was used to construct a mitochondrial phylogenetic tree, which was found to be topologically equivalent to a phylogenetic tree based on nuclear small subunit rRNA sequences (Sogin et al. (1986) EMBO J. 5, 3625-3630). The mitochondrial rRNA gene was found to accumulate base-pair substitutions considerably faster than the nuclear rRNA gene, the rate difference being similar to that observed for mammals.