WO1998037181A2 - Gene de sous-unite catalytique de telomerase et proteine codee - Google Patents

Gene de sous-unite catalytique de telomerase et proteine codee Download PDF

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WO1998037181A2
WO1998037181A2 PCT/US1998/003404 US9803404W WO9837181A2 WO 1998037181 A2 WO1998037181 A2 WO 1998037181A2 US 9803404 W US9803404 W US 9803404W WO 9837181 A2 WO9837181 A2 WO 9837181A2
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hest2
telomerase
cells
dna
protein
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Christopher M. Counter
Matthew Meyerson
Robert A. Weinberg
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Whitehead Institute For Biomedical Research
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)

Definitions

  • telomere ends of linear chromosomes are replicated by the ribonucleoprotein enzyme telomerase (Blackburn, E.H. Annu . Rev. Biochem. 53 , 163-94 (1984); Zakian, V.A. , Science 270 , 1601-7 (1995); Greider,
  • RNA subunit of this enzyme has now been described in multiple species (Blasco, M.A. et al . , Science 269, 1267-70 (1995);
  • Candidate protein subunits have been isolated in the ciliate Tetrahymena (Collins, K.et al . , Cell 81 , 677-86 (1995) but not, however, in any genetically tractable organism.
  • Telomerase is repressed in normal human somatic cells but is re-activated during tumor progression. This re- activation is not reflected by changes in the levels of previously cloned genes encoding telomerase subunits.
  • telomere length regulation in eukaryotes and proteins which are physically associated with the respective active telomerase enzyme and, thus, are each a component of the respective telomerase holoenzyme.
  • the gene, RNA transcript and encoded protein are a yeast gene and its encoded RNA transcript and protein.
  • the gene, RNA transcript and encoded protein are human. Also described herein is a human cDNA which encodes the human telomerase catalytic protein subunit .
  • the yeast telomerase gene is present on chromosome XII and its disruption alters telomere maintenance, as demonstrated by the decrease in telomere length in transposon- insertion mutants.
  • the sequence of the gene referred to herein as EST2 (SEQ ID NO. : 1) , the deduced amino acid sequence of the encoded Est2 protein, also referred to as Est2p, (SEQ ID NO.: 2) and the hEST2 RNA transcript (SEQ ID NO. : 36) are provided.
  • the DNA has been shown to be essential for telomerase activity in yeast and the encoded protein has been shown, by the methods described herein, to be physically associated with telomerase and a constituent of active telomerase complex in yeast.
  • the yeast telomerase DNA EST2 is a subject of the present invention; this includes isolated DNA comprising DNA selected from the following: DNA of SEQ ID NO.: 1; DNA which is the complement of SEQ ID NO. : 1, DNA which hybridizes to DNA of SEQ ID NO. : 1 or a complement thereof; DNA which localizes to yeast chromosome XII; and DNA which encodes the amino acid sequence of SEQ ID NO. : 2.
  • Isolated yeast Est2p is also the subject of this invention. This includes isolated Est2p of SEQ ID NO. : 2 and Est 2p encoded by EST2 DNA as defined herein.
  • the gene described herein is useful to identify genes encoding telomerase proteins in other eukaryotes, particularly in vertebrates, including mammals and especially humans. All or a portion of the gene described can be used. The encoded portion can be used to produce antibodies (monoclonal or polyclonal) which bind the Est2 protein and can, in turn, be used to identify corresponding proteins (proteins physically associated with telomerase enzymatic activity) in other eukaryotes.
  • hEST2 Also described herein is a human cDNA, which was originally named hEST2 and has been renamed hTERT , that encodes the human telomerase catalytic protein subunit, the encoded hEST2 RNA and the encoded hEST2 protein.
  • the human cDNA which was originally named hEST2 and has been renamed hTERT , that encodes the human telomerase catalytic protein subunit, the encoded hEST2 RNA and the encoded hEST2 protein.
  • the human cDNA which was originally named hEST2 and has been renamed hTERT , that encodes the human telomerase catalytic protein subunit, the encoded hEST2 RNA and the encoded hEST2 protein.
  • DNA described herein is a human homologue of yeast and ciliate genes which encode telomerase catalytic subunits; it shares significant sequence similarity with the telomerase catalytic subunit genes, yeast EST2 and Euplotes pl23. hEST2 RNA expression reflects telomerase activity.
  • RNA transcript is expressed in primary human tumors, cancer cell lines and telomerase-positive tissues, but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues.
  • hEST2 message is absent in pre-crisis, telomerase-negative transformed cells, but is readily detectable in post-crisis, telomerase-positive immortalized cells. Taken together, these observations are evidence that the induction of hEST2 mRNA expression is required for the telomerase activation that occurs during cellular immortalization and tumor progression .
  • the encoded human protein comprises six of the seven conserved sequence motifs which define the polymerase domains of members of the reverse transcriptase family and also includes the invariant aspartic acid residues required for telomerase enzymatic activity.
  • hEST2 protein comprises such motifs which define polymerase domains, beyond these it shows no sequence similarity with reverse transcriptases. It is more closely related to the telomerase catalytic subunits of yeast and ciliates than to other reverse transcriptases. In its domains that lie N- terminal to the polymerase domain, hEST2 shows clear relatedness to both pl23 and Est2p. Many of the sequence identities in the N-termini of the three proteins are in a region just before motif 1.
  • telomerase motif a unique motif, referred to as the telomerase motif; in hEST2 this motif extends from amino acid residue 556 to amino acid residue 565 of SEQ ID NO.: 3, with absolute invariant sequence extending from amino acid residue 560 to amino acid residue 565 of SEQ ID NO.: 3.
  • hEST2 is more closely related to telomerase reverse transcriptases than to non-telomerase reverse transcriptases.
  • the three telomerase catalytic subunits form a subgroup within the reverse transcriptase family.
  • the present invention relates to isolated DNA which encodes an RNA transcript which is expressed in primary human tumors, cancer cell line and telomerase-positive tissue.
  • the isolated DNA comprises hEST2 DNA such as DNA comprising SEQ ID NO. : 35 (DNA comprising the nucleotide sequence of SEQ ID NO. : 35) .
  • Isolated DNA of the present invention encodes an RNA transcript which is not detectable in telomerase-negative cell lines or in differentiated telomerase-negative tissues.
  • isolated DNA of the present invention encodes an RNA transcript which is not detectable in pre- crisis, telomerase-negative transformed cells and is detectable in post-crisis, telomerase-positive immortalized cells.
  • the DNA comprises hEST2 cDNA, such as SEQ ID NO. : 35 or DNA which hybridizes thereto or to a complement of hEST2 cDNA (such as a complement of SEQ ID NO. : 35) under conditions of high stringency.
  • a further subject of the present invention is an isolated RNA transcript encoded by isolated DNA of the present invention.
  • telomere shortening A method of altering telomerase function and, thus, of altering telomere shortening is also described.
  • expression and/or function of the telomerase- associated protein is altered (enhanced or reduced) , resulting in altered (enhanced or reduced) cell lifespan.
  • Expression of the telomerase-associated protein is enhanced, for example, by introducing DNA encoding the protein into cells.
  • telomerase-associated protein is introduced into cells .
  • Expression of the telomerase-associated protein is reduced, for example, by introducing into cells an agent which inhibits production or function of the protein, an agent which destroys the expressed protein or a dominant negative form of the protein.
  • the present invention also provides a method of increasing the lifespan of a cell or, alternatively, of reducing the lifespan of a cell, such as that of cancer cells or transformed cells.
  • telomerase activity is maintained or increased within the cells, such that telomeres are maintained and as the cells age, telomere shortening does not occur or occurs to a lesser extent than would otherwise be the case (if telomerase activity were not maintained or increased) .
  • Reduction of lifespan of cells, such as tumor cells is accomplished by introducing into the cells an inhibitor of the telomerase protein subunit described herein.
  • a method of assessing cells or aiding in the assessment of cells for malignancy or an increased likelihood of progression to malignancy is also the subject of this invention.
  • cells to be assessed are obtained from an individual (e.g., a human) in need of such an assessment.
  • the cells are processed in such a manner that DNA, RNA or both in the cells are rendered available for annealing or hybridization with complementary polynucleotides or oligonucleotides (DNA or RNA) , such as probes or primers, thereby producing processed cells.
  • DNA or RNA complementary polynucleotides or oligonucleotides
  • the processed cells are combined with DNA or RNA required for telomerase enzymatic activity (DNA or RNA encoding a protein required for telomerase enzymatic activity) , a complement of the required DNA or RNA or a characteristic portion or fragment of the DNA or RNA, such as the telomerase motif or all or a portion of the 5 ' end of hEST2 which is not shared with the yeast or Euplotes gene. If hybridization occurs, it is indicative of the presence of telomerase protein-encoding DNA or RNA in the cells and of activation of telomerase, which is also indicative of malignancy or an increased likelihood of progression to malignancy.
  • the hEST2 DNA encodes an
  • RNA transcript which is expressed in primary tumors, cancer cell lines and telomerase - positive tissues and is readily detectable in post-crisis, telomerase-positive immortalized cells, but not detectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. Detection of hEST2 DNA and/or of the RNA transcript, thus, makes it possible to detect DNA which encodes a transcript required for telomerase activation that occurs during cellular immortalization and tumor progression and to detect the RNA transcript itself.
  • the invention is a method of diagnosing or aiding in the diagnosis of development of malignancy (cancer, tumor formation) in an individual, in which the occurrence (presence or absence) and/or quantity of a telomerase protein (e.g., hEST2 protein) in cells is assessed.
  • a telomerase protein e.g., hEST2 protein
  • cells are obtained from an individual in need of diagnosis of malignancy and processed, if necessary, in such a manner that proteins in the cell are available for detection, such as by binding with antibodies or antibody fragments.
  • the processed cells are combined or contacted with antibodies that recognize (bind) the telomerase protein (e.g., antibodies that bind Est2p or hEST2 protein) under conditions appropriate for antibody binding to occur.
  • Whether binding occurs is determined; optionally, the extent to which binding occurs can also be determined. If binding of anti-telomerase protein antibodies (e.g., anti- hEST2 protein antibodies) to a component of processed cells occurs, it is indicative of the presence of hEST2 protein in the cells and of malignancy or an increased likelihood of development or malignancy in the individual.
  • anti-telomerase protein antibodies e.g., anti- hEST2 protein antibodies
  • the present invention is a method of reducing expression of hEST2 RNA and hEST2 protein in cells of an individual (e.g., a human or other mammal).
  • the method comprises administering to the individual a drug selected from the group consisting of drugs which inhibit (directly or indirectly) or bind hEST2 RNA and prevent or reduce production of hEST2 protein and drugs which inhibit hEST2 protein function or activity.
  • the drug is administered in a therapeutically effective amount (an amount sufficient to have the desired effect of reducing expression of hEST2 RNA and hEST2 protein) and under conditions appropriate for entry into cells, in which they have the desired effect.
  • the present invention is a method of treating cancer in an individual, in need of such treatment.
  • a drug which inhibits or binds hEST2 RNA (or DNA) and prevents or reduces production of hEST2 protein or a drug which inhibits hEST2 protein function or activity is administered to the individual under conditions appropriate for entry into cells and in a therapeutically effective amount (an amount sufficient to have the desired effect of inhibiting hEST2 RNA and/or preventing or reducing production of hEST2 protein or inhibiting hEST2 protein function or activity) , with the result that cancer in the individual is treated (reduced, reversed or prevented from advancing) .
  • a further embodiment of the invention is a method of altering (increasing or reducing) lifespan of cells in culture or in an individual . In the method in which lifespan is increased, hEST2 DNA is introduced into cells
  • hEST2 RNA transcript and encoded protein are produced in sufficient quantity to increase the lifespan of cells in the cultured cells or the individual .
  • lifespan is decreased
  • hEST2 protein function, activity or production is reduced (partially or totally) . This is done, for example, by introducing into cells in culture or administering to an individual in whom cell lifespan is to be decreased a drug which inhibits or binds hEST2 RNA and prevents or reduces production of hEST2 protein; a drug which inhibits hEST2 protein function, activity or production; or a drug which inhibits or binds hEST2 DNA and prevents or reduces production of hEST2 RNA transcripts .
  • the drug is administered to an individual under conditions appropriate for entry into cells in sufficient quantity to have the desired effect (in sufficient quantity to decrease lifespan of the cells by reducing hEST2 protein function, activity or production) .
  • the drug can be, for example, a small organic molecule, an enzyme which degrades hEST2 protein, an enzyme inhibitor (e.g., a telomerase or hEST2 enzyme inhibitor) , an hEST2 transcriptional regulator; an antisense molecule or a dominant negative form of hEST2 protein.
  • DN mutant yeast strain
  • the RAD 52 coding sequence of a haploid strain of genotype MATa leu 2-3 , 112 lys2 -201 trpl -1 ura3 -
  • DNR in which the absence of RAD52 was complemented by introducing the plasmid pYPCR+ 35 , which encodes the RAD52 gene under it own promoter and the selectable marker URA3 , is also described. Also described are mutant yeast strain DNRtlc ⁇ , in which the TLCl gene in DNR cells was replaced with the LEU2 selectable marker gene and Y0035, in which • li ⁇
  • the Est2 gene has been mutated with the addition of a TN3 LEU2 transposon in the DNR genetic background.
  • the mutant yeast strain described herein requires telomerase activity for viability. It is useful to carry out a rapid and automatable biological assay for telomerase inhibitors and, thus, for inhibitors of telomere biosynthesis. The assay is also the subject of the present invention.
  • the mutant yeast strain is also useful to identify agents which are recombination inhibitors.
  • the mutant yeast strain referred to as DN, the RAD52 recombination gene has been replaced by a HTS gene.
  • disruption of the TLCl telomerase RNA subunit gene or the MIT1 putative telomerase protein subunit gene in the rad52 mutants leads to a delayed cell death.
  • Yeast lacking both recombination activity and telomerase activity die after about 20 to 60 cell generations. Telomerase activity can be disrupted by enzyme inhibitors, as well as by genetic inactivation.
  • test compounds are added, at different concentrations, to the DNA mutant strain (the test strain) and to a wild-type (control) strain. Periodically, such as each day, cultures of the two strains are diluted (e.g., 1:1000). Compounds that kill the yeast instantly or that kill the DNA and wild-type yeast with identical delays, are eliminated. Compounds that kill the test yeast strain significantly before the control yeast strain are telomerase inhibitors . This biological assay has several advantages over available methods.
  • Figure 2 shows alignment of the predicted amino acid sequence of hEST2 , also referred to as hTERT, (SEQ ID NO.: 3) with the yeast Est2p (SEQ ID NO. : 2) and Euplotes pl23 (SEQ ID NO.: 4) homologues. Amino residues within shaded blocks are identical between at least two proteins . Identical amino acids within the reverse transcriptase (RT) motifs (Poch et al . , EMBO J. 8:3867-3874 (1989); Xiong and Eickbush, EMBO J. 9:3353-3362 (1990)) are in black boxes, an example of a telomerase-specific motif in an outlined gray box, and all other identical amino acids in gray boxes. RT motifs are extended in some cases to include other adjacent invariant or conserved amino acids. The sequence of the expressed sequence tag AA281296 is underlined.
  • Figures 3A-3F show the alignment of RT motifs 1 to 6 of telomerase subunits hEST2 (SEQ ID NO. : 5-10) , pl23 (SEQ ID NO.: 11-16) and Est2p (SEQ ID NO.: 17-22) with S. cerevisiae group II intron-encoded RTs a2-Sc (SEQ ID NO. : 23-28) and al-Sc (SEQ ID NO.: 29-34).
  • the consensus sequence of each RT motif is shown (h: hydrophobic residues, p: small polar residues, c: charged residues) .
  • telomeres and RT consensus Amino acids that are invariant among telomerases and the RT consensus are boxed in black, while those that are invariant among telomerases and similar to the RT consensus are boxed in gray. Open boxes identify highly conserved residues unique to either telomerases or to non-telomerase RTs . Asterisks denote amino acids essential for polymerase catalytic function.
  • Figure 4 is an ideogram of human chromosome 5p showing linkage of hEST2 to sequence-tagged site (STS) markers
  • the calculated distance between hEST2 and these STS markers is in centiRays (cR) with LOD scores in parentheses.
  • Figures 5A-5B show the nucleotide sequence of hEST2 cDNA in which the start codon ATG is underlined (SEQ ID NO. : 35) .
  • Figure 6 is the amino acid sequence of hEST2 (SEQ ID NO . : 3 ) .
  • Figures 7A, 7B and 7C are the nucleotide sequence of partially or alternatively spliced hEST2 message RNA (SEQ ID NO. : 36) .
  • Figures 8A-8F show the expression of hEST2 in normal human tissues and cancer cell lines. Duplicate RNA blots were probed with an hEST2 probe (top panels) or with a ⁇ - actin probe as internal control (bottom panels) . The 4.4 kb, 6 kb, and 9.5 kb transcripts are hEST2-specific .
  • Figures 9A-9D shows hEST2 expression in primary human tumors. Rnase protection assays are shown for hEST2 and ⁇ - actin controls.
  • linear chromosomes of eukaryotic cells offer the biological advantages of rapid recombination, assortment, and genetic diversification.
  • linear DNA is inherently more unstable than circular forms .
  • the eukaryotic chromosome has evolved to include a DNA-protein structure, the telomere, which caps chromosome ends and protects them from degradation and end-to-end fusion (Blackburn, Ann . Rev. Biochem. 53:163-194 (1984); Zakian, Science 270:1601-1607 (1995)).
  • telomeres The DNA component of telomeres consists of tandem repeats of guanine-rich sequences that are essential for telomere function (Blackburn, Ann. .Rev. Biochem . 53:163-194
  • telomerase enzyme is essential for complete replication of telomeric DNA because the cellular DNA-dependent DNA polymerases are unable to replicate the ultimate ends of the telomeres due to their requirement for a 5' RNA primer and their unidirectional mode of synthesis. Removal of the most terminal RNA primer following priming of DNA synthesis leaves a gap that cannot be replicated by these polymerases (Oiovnikov, Dokl . Akad. Nauk SSSR
  • Telomerase surmounts this problem by de novo addition of single-stranded telomeric DNA to the ends of chromosomes (Greider and Blackburn, Cell 43:405-413 (1985), Greider and Blackburn, Nature 337:331-337 (1989) ; Yu et al . , Nature 344 : 126 -132 (1990); Greider, In Telomeres, eds.
  • telomerase enzymes that have been characterized to date are RNA-dependent DNA polymerases which synthesize the telomeric DNA repeats using an RNA template that exists as a subunit of the telomerase holoenzyme (Greider, In Telomeres, eds. Blackburn and Greider, Cold Spring Harbor Laboratory Press, pp. 35-68 (1995)).
  • the genes specifying the RNA subunits of telomerases have been cloned from a wide variety of species, including humans (Feng et al . ,
  • telomere shortening leads to progressive telomere shortening as cells pass through successive cycles of replication (Yu et al . , Nature 344:126-132 (1990); Singer and Gottschling, Science
  • telomerase activity is readily detectable in germline cells and in certain stem cell compartments.
  • enzyme activity is not detectable in most somatic cell lineages (Harley et al . , Cold Spring Harb . Symp .
  • telomeres of most types of human somatic cells shorten with increasing organismic age and with repeated passaging in culture, similar to the situation seen in protozoan and yeast cells that have been deprived experimentally of a functional telomerase enzyme (Harley et al . , Nature 345:454-460
  • telomere shortening continues until a subsequent point is reached that is termed crisis, where telomeres have become extremely short (Counter et al . , EMBO J. 11:1921-1929 (1992) and J. Virol .
  • the crisis phenotype is reminiscent of that observed in yeast and Tetrahymena cells in which telomerase function has been experimentally perturbed.
  • telomere-negative human cells like that of their yeast and ciliate counterparts, is ultimately limited by the . length of telomeres.
  • the immortal cells that escape crisis are characterized by readily detectable levels of telomerase activity and by stable telomeres (Counter et al . , EMBO J. 11 1921-1929 (1992) and J. Virol .
  • telomere activity is readily detected in the great majority of human tumor samples analyzed to date (Counter et al . , Proc . Natl . Acad. Sci . , USA 92:2900-2904
  • telomere shortening serves as an important anti-neoplastic mechanism used by the body to block the expansion of pre-cancerous cell clones.
  • tumor cells transcend the crisis barrier and emerge as immortalized cell populations by activating previously unexpressed telomerase, enabling them to restore and maintain the integrity of their telomeres (Counter et al . , EMBO J. 12:1921-1929 (1992) and Proc . Natl . Acad . Sci . , USA
  • telomere-associated protein TPl/TLPl does not reflect the level of telomerase activity (Harrington et al . , Science 275:973-977 (1997);
  • telomere levels increase 100- to 2000-fold during the immortalization of human cells, the level of hTR message increases at most two-fold
  • telomere activation has remained elusive . Described herein is work which has shown that mutations in a gene of eukaryotic origin (in an open reading frame required for telomere maintenance) results in loss (absence) of telomerase enzymatic activity in cells and that the encoded protein is physically associated with the active telomerase enzyme, thus showing that the DNA encodes a component of a eukaryotic telomerase holoenzyme.
  • DNA of the present invention includes cDNA, genomic DNA, recombinantly produced DNA and chemically synthesized DNA. It can be obtained from a source in which it occurs in nature or can be produced using known chemical or recombinant DNA methods. The following is a discussion of work carried out relating to yeast and human telomerase protein.
  • EST2 encodes a component of the yeast telomerase holoenzyme.
  • telomeres and telomerase Interest in telomeres and telomerase has heightened in recent years with the discovery that telomerase is present at low, almost undetectable, levels in most human somatic tissues and is readily detectable in germline cells and in the vast majority of tumor cell samples (Counter, CM. et al . , Proc . Natl . Acad. Sci . USA 91 , 2900-4 (1994); Kim, N.W. et al . , Science 266, 2011-5 (1994). Somatic cell lineages which lack telomerase lose telomere segments progressively as they pass through successive replication cycles, limiting their lifespan (Harley, C.B. et al.,
  • telomerase enzymes of ciliates the formation of thousands of mini-chromosomes present in their macro-nuclei seems to require correspondingly high amounts of telomerase, dwarfing the amounts available for study in metazoan cells.
  • the telomerase enzyme in Tetrahymena has been reported to be composed of two proteinaceous and one R ⁇ A subunit, the latter responsible for templating the telomeric DNA segments (Greider, C.W. & Blackburn, E.H., Nature 377, 331-7 (1989); Collins, K. et al . , Cell 81 ,
  • telomere The protein subunits of telomerase have not been described in any other organism, although the genes specifying the RNA subunits of telomerase have been cloned in a wide range of other species (Blasco, M.A. et al . , Science 269, 1967-70
  • EST2 encodes a protein of unknown function (Lendvay, T.S. et al., Genetics 144 , 1399- 1412 (1996)); EST1 (Lundblad, V. & Szostak, J.W. , Cell 57, 633-43 (1989)) and CDC13 (Garvik, B. et al . , Mol . Cell Biol .
  • KEM1 appears to encode a nuclease that acts on telomeres (Liu, Z. & Gilbert, W.,
  • telomerase protein subunits A search for genes encoding telomerase protein subunits was initiated because the protein subunits of the yeast telomerase have not yet been identified, and because there are no obvious homologs of the Tetrahymena telomerase protein subunits encoded in the yeast genome (Collins, K. et al . , Cell 81 , 677-86 (1995);
  • telomere loss in yeast cells lacking the telomerase RNA component was compensated by the actions of a second telomere-maintenance system that utilizes the DNA recombination machinery.
  • An essential component of this machinery is the product of the RAD52 gene.
  • telomere mutant yeast could be identified by their requirement for RAD52 function.
  • a yeast strain carrying an inactive chromosomal rad52 ⁇ allele that was complemented by a plasmid-borne wild-type RAD52 gene linked to a URA3 gene was generated, in order to determine if mutants in telomerase function could be revealed by a screen for mutants that require RAD52 function.
  • RAD52 function was inactivated through eviction of the RAD52-encoding plasmid (by counter- selecting against the URA3 marker with 5-fluoro-orotic acid (FOA) , cell viability was unaffected for the succeeding 80 cell generations (Boeke, J.D. et al., Mol . Gen . Genet . 197, 345-6 (1984) .
  • telomerase- deficient cells perish upon the loss of JAD52 function and that this phenotype is therefore useful to screen for mutants carrying lesions in a variety of genes affecting telomerase function.
  • RAD52 plasmid were mutagenized by homologous recombination with a library of yeast genomic fragments, each of which carried one or more copies of an inserted mini- Tn3::lac__::LEC72 transposon (Burns, N. , et al., Genes Dev. 8 ,
  • yeast clones were generated. These mutagenized yeast cells were cultured for 40 generations to allow the telomeres of any cells lacking telomerase function to shorten substantially. A portion of the cell colonies was replica-plated and the RAD52 plasmid was evicted from one of each replica pair to identify cell clones that required RAD52 for continued viability.
  • the 245 remaining RAD52-dependent cell clones were tested for changes in telomere structure by Southern hybridization analysis of their telomeres. Of these 245 haploids, 16 cell clones carried short telomeres identical to those seen, in yeast lacking a functional TLCl gene.
  • Tetrad analysis of the diploid derivatives of these clones was performed to determine whether the rad52 synthetic lethal phenotype was caused by the transposon insertion or by an adventitious unlinked mutation elsewhere in the yeast genome. In each case, the lethality co-segregated with the transposon insertion, demonstrating that this phenotype is derived directly from the genetic disruption effected by a single inserted transposon.
  • Saccharomyces Genome Database http//genome-www. Stanford. edu/Saccharomyces) ) .
  • the predicted amino acid sequence encoded by the gene identified on chromosome XII is presented in Figures IA and IB (SEQ ID NO. : 2) ; the predicted protein includes 884 amino acids with an estimated mass of 102 kilodaltons.
  • the predicted gene product does not share significant sequence similarity with any sequences in the databases available through the National Center for Biotechnology Information BLAST server.
  • EST2 a novel gene, involved in telomere length regulation.
  • the open reading frame identified on chromosome XII is identical to EST2 ; therefore, this gene is referred to herein as EST2.
  • est2 transposon-insertion mutants show that they have the same delayed cell death phenotype, dependence on RAD52 , and telomere shortening as was displayed by cells carrying mutations in their TLCl gene. Diploids heterozygous for disrupted alleles of RAD52 and EST2 were sporulated and the resulting tetrads were analyzed for growth in culture and for telomere length. In 33 tetrads analyzed, the vast majority of the est2::TN3 rad52 ⁇ double mutant spore products became inviable by approximately 20 generations in culture, and the remainder died by approximately 40 generations.
  • est2::Tn3 mutants with wild-type RAD52 began to decline by approximately 60 generations, and this phenotype was more pronounced after approximately 80 generations.
  • the EST2 wild-type spore products remained completely viable at all generations, regardless of their RAD52 genotype.
  • telomere shortening was comparable to that observed in tlcJ::TN3 mutants.
  • TLCl has been the only yeast gene known to be a subunit of telomerase and to be required for telomere activity in vi tro .
  • the est2::TN3 gene mutants are phenotypically indistinguishable from tlc2::TN3 mutants.
  • telomerase assay was used to show that extracts from wild-type yeast catalyze the elongation of a single- stranded telomeric primer by four to sixteen nucleotides in an RNase-sensitive fashion, indicating telomerase activity.
  • extracts derived from yeast bearing transposons inserted into EST2 lacked detectable telomerase activity, like tlcl ⁇ mutants, which are known to be telomerase- negative (Co n, M. & Blackburn, E.H., Science 269, 396-400 (1995) ) .
  • Est2 is either a protein subunit of telomerase or an upstream regulator required for telomerase activity. It was possible to distinguish between these possibilities by replacing the endogenous EST2 gene with a variant encoding a protein with three influenza hemagglutinin (HA) epitope stages at its carboxy terminus
  • ⁇ EST2 -HA This modified EST2 allele and, thus, its product, is fully functional.
  • the EST2-HA yeast show no overt phenotype, their telomeres are wild-type length, and extracts from these yeast exhibit normal levels of telomerase activity. Extracts from EST2-HA and control yeast were incubated with excess anti-HA antibody, after which the immunoprecipitates and resultant supernatants were separated and assayed for telomerase activity.
  • the anti-HA antibody immunoprecipitated telomerase activity from extracts of yeast expressing the tagged Est2-HA protein, but not from extracts expressing normal Est2 protein.
  • EST2 shares no homology with either of the two genes reported to encode the Tetrahymena telomerase protein subunits (Collins, K. et al . , Cell 81 , 677-86
  • Est2 protein of Tetrahymena may not yet have been identified.
  • telomere length is maintained by two mechanisms.
  • telomeric DNA repeats are synthesized by the ribonucleoprotein enzyme telomerase.
  • This RNA-dependent DNA polymerase (or "reverse transcriptase") copies the DNA repeats from an RNA template that is a component of the enzyme .
  • telomere length can be maintained by an alternative mechanism in yeast and possibly also in humans: the homologous recombination pathway used for double strand break repair.
  • mutants of the telomerase subunits in conjunction with mutants of the recombination pathway have been shown to become inviable after growth for 40 to 80 generations.
  • This synthetic lethal phenotype was used as a screen for telomerase mutants in a background of recombination mutants and, as a result, a novel telomerase gene was identified.
  • Drug screens which work in the same fashion as demonstrated herein for the genetic screen are useful to identify telomerase inhibitors in a recombination mutant background, and recombination inhibitors in a telomerase mutant background.
  • Telomere maintenance inhibitors which can be a telomerase inhibitor alone, a recombination inhibitor alone, or a combination, can be identified by the method described herein, using appropriate mutant eukaryotic cells, such as the mutant yeast strain described herein. Such telomerase inhibitors can be further assessed, using known methods, to confirm their effectiveness as anti- fungal drugs or as anti-cancer drugs.
  • Normal human somatic cells lack a pathway to maintain telomere lengths, and their telomeres shorten continuously.
  • Immortal human cells whether in tissue culture or the vast majority of malignant tumors in the human body, have developed a mechanism to maintain telomeres. Telomere maintenance occurs as a result of activation of the telomerase enzyme, activation of a recombination pathway, or possibly both.
  • mutant strain of the yeast Saccharomyces cerevisiae that requires telomerase activity for viability has been generated.
  • This strain can be used in a rapid and automatable biological assay for telomerase inhibitors .
  • the mutant yeast strain, named DN the RAD52 recombination gene has been replaced by a HIS3 gene.
  • MIT1 EST2 telomerase protein subunit gene in the rad52 mutants has been shown to lead to a delayed cell death.
  • Yeast lacking both recombination activity and telomerase activity die after about 20 to 60 cell generations. Telomerase activity can be disrupted by enzyme inhibitors as well as by genetic inactivation, and, therefore, the mutant strains described can be used to identify telomerase inhibitors.
  • test compounds are added at different concentrations to the DN mutant strain and to a wild-type control strain. Each day, the cultures are be diluted (e.g., 1:1000). Compounds that kill the yeast instantly, or that kill the DN and wild-type yeast with identical delays, are eliminated.
  • telomerase inhibitors Compounds that kill the DN yeast significantly before the wild-type yeast are telomerase inhibitors .
  • the method described is a biological assay and has several advantages over available methods. It is fast, automatable, and can be performed easily in large quantities. As a biological assay, it reflects the physiological function of the drugs. Furthermore, it allows testing of biochemical and uptake parameters in a single step. Finally, this assay will also exclude inhibitors of other metabolic enzymes, such as RNA and DNA polymerases and is highly specific for telomerase inhibitors .
  • High-throughput screening can be accomplished by growing the yeast in microtiter dishes. Aliquoting of media, dilution of test drug compounds, and dilution of yeast cultures can be performed robotically. Growth can be measured by the optical density of the yeast culture at 595-600 nm; growth measurements and preliminary analysis of the data can also be automated.
  • the telomerase inhibitor screen has several applications, such as to identify inhibitors of fungal telomerase enzymes, mammalian telomerase enzymes and reverse transcriptase .
  • telomerase inhibitors effective in Saccharomyces will likely function in Candida, as well as in other pathogenic yeast.
  • anti-fungal drugs There are only a few effective anti-fungal drugs currently available; many are inadequate because of their serious side effects. Thus, a novel approach to anti-fungal drugs could be clinically valuable for the treatment of systemic fungal infections.
  • telomere function is likely to be conserved between diverse species.
  • inhibitors of yeast telomerase could also inhibit mammalian telomerase, and can be tested for their abiltiy to act as anti-cancer agents.
  • These compounds can also serve as lead compounds for the identification of such inhibitors and for modification to produce more effective inhibitors (e.g., inhibitors which are longer-lived, exhibit great inhibitory effects, resist degradation by cellular enzymes) .
  • telomerase inhibitors 3
  • Telomerase inhibitors identified by the present screen can be assessed, using known methods, for their ability to act as retroviral reverse transcriptase.
  • inhibitors of the telomere recombination pathway are identified by their ability to kill telomerase mutant yeast but not wild-type yeast.
  • the screen is similar to the telomerase inhibitor screen described above .
  • test compounds are added at different concentrations to a telomerase mutant strain (e.g., tlcl or mi l/est2) , referred to as a test strain, and to a wild-type control strain.
  • a test strain e.g., tlcl or mi l/est2
  • a wild-type control strain e.g., tlcl or mi l/est2
  • the cultures are diluted 1:1000.
  • Compounds that kill the yeast instantly, or that kill the telomerase mutant and wild-type yeast with identical delays, are eliminated.
  • Compounds that kill the telomerase mutant yeast significantly before the wild-type yeast are recombination inhibitors.
  • the ability of the compounds to inhibit recombination can be confirmed using known methods.
  • the recombination inhibitors have several uses.
  • combination therapy of a recombination inhibitor with a telomerase for treating fungal infections and/or malignant neoplasms.
  • recombination inhibitors can be used to potentiate the toxic effects of radiation treatment and of chemotherapeutic agents that induce DNA double- strand breaks.
  • Yeast strains were generated by replacing the RAD52 coding sequence of the haploid strain L3853 (gift of G. Fink; genotype MATa leu2 -3 , 112 lys2 -201 trpl -1 ura3 - 52 his3 -200) with the HIS3 gene using a PCR-based homologous gene disruption method (Baudin, A. et al . ,
  • RAD52 in this strain was complemented by introducing the plasmid pYPCR+ 35 , which encodes the RAD52 gene under its own promoter and the selectable marker URA3 , thereby generating strain DNR.
  • the TLCl gene was replaced with the
  • LEU2 selectable marker by the above PCR procedure in L3853 and DNR cells, yielding the strains tlcl ⁇ and DNRtlc ⁇ , respectively.
  • Yeast mutagenesis Approximately 230 ⁇ g of the appropriately prepared yeast::mini-Tn3::lacZ::LJ_T72 genomic library (Burns, N. , et al., Genes Dev. 8, 1087-105 (1994)) were introduced by homologous recombination into approximately 7xl0 6 cells of strain DNR. Approximately lxl0 6 Ura + Leu + clones representing successful transposon insertions were pooled and replated to a total number of -lxlO 6 clones to "age" the yeast for the rad52 ⁇ synthetic lethal screen. 1x10 s of such yeast were then reseeded for the rad52 ⁇ synthetic lethal screen, as described in the text.
  • Genomic DNA was isolated and digested with the restriction enzyme Xhol, which liberates the telomeric DNA (Guthrie, C, & Fink, G.R. , Guide to
  • Tetrad analysis Diploid derivatives of the transposon-mutagenized yeast lacking the pYCPR- plasmid were sporulated and tetrads were dissected according to standard methods (Guthrie, C, & Fink, G.R., Guide to yeast genetics and molecular biology (Academic Press, New York,
  • Yeast extracts 6 litres of yeast cultures were harvested at an optical density of 0.4-0.6 at 600 nm, resuspended in TMG buffer, disrupted with a bead beater and centrifuged for 90' at 100,000gr at 4°C as described (Cohn,
  • Telomerase assay Telomerase activity was assayed essentially as outlined by Cohn and Blackburn with the following two modifications: first, 4 ⁇ g of crude S100 yeast extract was incubated with the oligonucleotide
  • TMGN buffer TMGN
  • TMGN resuspended in 50 ⁇ l TMGN.
  • a duplicate extract was diluted and left untreated on ice for 2 hours. 4 ⁇ g of supernatant or untreated extracts and 10 ⁇ l of beads were assayed for telomerase activity.
  • hEST2 hTERT
  • hEST2 RNA expression levels as an important mechanism used in a variety of developmental contexts to determine the amount of telomerase activity present in specific cell lineages.
  • the levels of the catalytic subunit of telomerase may represent a rate-limiting determinant of enzyme activity in many types of cells.
  • up-regulation of hEST2 message may be an important mechanism through which telomerase becomes activated during both cellular immortalization and the progression of malignant tumors.
  • hEST2 also referred to as hTERT
  • the human telomerase subunit described here shares extensive sequence similarities with the catalytic subunits of the yeast and ciliate telomerase enzymes.
  • telomere subunit Lingner and co-workers. (Lingner et al . , Science 275:561- 567 (1997)).
  • experiments that will provide additional proof that the gene described herein encodes the human telomerase subunit include those that demonstrate that mice lacking the mouse mEST2 homologue also lack telomerase activity; that the hEST2 protein is physically associated with a ribonucleoprotein complex that exhibits telomerase activity; and that alteration of critical residues in the domain of hEST2 that is homologous to RTs inactivates its catalytic function.
  • telomerases described to date include motifs that indicate they are distant homologues of a variety of RTs. They also share several unique, telomerase-specific sequence motifs. Yeast, ciliates and mammals represent highly diverged branches of the phylogenetic tree, which suggests that the catalytic subunit of the telomerase was developed early in eukaryotic evolution. It is possible that it was present in the cell that became ancestral to all contemporary eukaryotes. Even earlier, it appears that this enzyme shares ancestry with the precursors of the RTs specified by a variety of transposons and viruses.
  • telomere activity might be modulated.
  • the expression of the hEST2 mRNA may be repressed in many post-embryonic cell lineages, depriving cells in these lineages of the telomerase catalytic subunit. This in turn may underlie the observed progressive telomeric shortening associated with aging in the cells in many of these lineages.
  • telomerase enzyme activity when transformed cells escape from crisis or when tumors progress toward malignancy may, in many cases, be explained mechanistically by the de-repression of hEST2 mRNA expression.
  • this de-repression occurs in both transformed embryonic kidney cells and lymphocytes when they emerge from crisis and begin to exhibit telomerase activity.
  • the enhanced hEST2 RNA expression is achieved at the transcriptional or post-transcriptional level.
  • telomere activity Described here is a correlation between hEST2 mRNA levels and assayable telomerase activity. These two manifestations of hEST2 gene expression are present in a constant, predictable ratio. This provides the basis for assessing cells for hEST2 mRNA as an indicator of telomerase activity or assessing cells for telomerase activity as an indicator of hEST2 mRNA occurrence or level. It is possible that other mechanisms besides the presently observed modulation of hEST2 mRNA levels may intervene to modulate telomerase activity.
  • telomere-negative cells As described in Example 8, over expression of hEST2 in previously (normally) telomerase-negative cells is sufficient to impart telomerase activity to these cells, providing clear proof that hEST2 is the human telomerase catalytic subunit .
  • the data demonstrate that the ectopic expression of hTERT in otherwise telomerase-negative human cells is necessary and sufficient for induction of telomerase activity.
  • the physical association of hTERT with telomerase activity confirms that hTERT is a telomerase subunit. Up-regulation of the hTERT gene is the sole barrier to activation of telomerase in the tested cells.
  • telomere catalytic subunit provides an entree into understanding one of the essential steps in human tumor pathogenesis -- that leading to cell immortalization. Enzymatic assays have demonstrated that telomerase is often activated at a relatively late step in tumor progression (Harley et al . , Nature 345:458-460 (1994)). This step may occur when evolving, pre-malignant cell clones have surmounted the senescence barrier and subsequently, having exhausted their telomeric ends, encounter the successive barrier of crisis.
  • telomerase may enable cells to breach these barriers to further clonal expansion, thereby conferring great selective advantage on the rare ' cell that has acquired the ability to resurrect the long-repressed telomerase activity.
  • activation of telomerase may represent an essential step in tumor progression.
  • Such dependence on telomerase activity means that this enzyme represents an attractive target of drugs designed to interfere with malignant cell proliferation.
  • hEST2 message is up-regulated in human tumors and in immortalized cells lends further credence to this idea.
  • the identification of hEST2 as the candidate telomerase catalytic subunit provides a biochemical reagent for identifying such drugs.
  • telomerase catalytic subunit gene a human gene, hEST2, which is a telomerase catalytic subunit gene.
  • the gene transcript (mRNA) is expressed in primary human tumors, cancer cell lines and telomerase-positive tissues, but is undetectable, using the assays described herein, in cell lines known to be telomerase negative and in differentiated telomerase- negative tissues.
  • the hEST2 gene shares significant sequence similarity with a yeast telomerase catalytic subunit gene (S. cerevisiae EST2) and a ciliate telomerase catalytic subunit gene (Euplotes pl23) , as shown in Figure 2.
  • the three telomerase enzymes are members of the reverse transcriptase family of enzymes .
  • RNA transcript or RNA message encoded by hEST2 DNA of SEQ ID NO. : 35 or by DNA which encodes hEST2 protein of SEQ ID NO. : 3 is also the subject of this invention.
  • hEST2 is a single-copy gene which is approximately 40 kb in size and was shown to localize (to be present on) chromosome 5, particularly subband 5pl5.33. (See Figure 4) Expression levels in normal human tissues and human cancer cell lines were assessed, as described in Example 3. This assessment revealed two major RNA species (4.4 kb and 9.5 kb) and one minor RNA species (approximately 6 kb) . hEST message was detectable in several normal tissues, including thymus, testis and intestine; the latter two tissues are known to be telomerase-positive and the telomerase status of thymus has not been reported. Using the assays described herein, hEST2 transcript was not detected in most other normal human tissues, including heart, brain, placenta, liver, skeletal muscle and prostate, all of which have been reported to be telomerase-negative.
  • the hEST2 transcript or message As described herein, the hEST2 transcript or message
  • telomere is expressed in primary human tumors and cancer cell lines, but is not detectable in telomerase-negative cell lines and differentiated (normal, nontumorigenic) telomerase-negative tissues.
  • hEST2 message is not detectable in pre-crisis, telomerase-negative transformed cells, but is readily detectable in post-crisis, telomerase-positive immortalized cells.
  • the work described herein provides a method of differentiating between telomerase-positive cells and telomerase-negative cells, as well as for monitoring a change in telomerase content of cells. As a result, as described below, the present work provides a method of identifying cells which are telomerase positive and, thus, of identifying cells which are tumor or cancer cells or are en route to becoming tumor cells (malignant) .
  • a method of identifying telomerase-positive cells is the subject of the present invention. Also the subject of the present invention is a method of identifying cells which are transformed, malignant, cancerous, tumor or post- crisis cells or are en route to or likely to become transformed, malignant, cancerous, tumor or post-crisis cells.
  • malignant or malignancy is used herein to refer to transformed, cancer, cancerous, tumor, tumorigenic and post-crisis cells.
  • the phrase "a method of identifying malignant cells in an individual” encompasses a method of identifying cancer cells, transformed cells, tumor cells and/or post-crisis cells in an individual.
  • the present invention provides a method of diagnosing, aiding in the diagnosis of or predicting an increased likelihood of the occurrence of cancer, tumor formation and/or the development of malignancy in an individual, particularly a mammal and, specifically, a human.
  • telomerase content of cells is assessed by detecting or measuring hEST2 DNA, hEST2 transcript ⁇ hEST2 message) or h__ST2-encoded protein
  • hEST2 protein (hEST2 protein) .
  • the quantity of hEST2 DNA, hEST2 transcript or hEST2 protein can be determined; alternatively, the occurrence (presence or absence) of hEST2 DNA, hEST2 transcript or hEST2 protein can be detected.
  • the presence of hEST2 DNA, hEST2 message and/or hEST2 protein in cells is indicative of the presence of malignant cells or malignancy in the individual.
  • hEST2 DNA, hEST2 RNA (or both) is detected or measured in tissue or cells obtained from an individual. This can be carried out using known methods, such as hybridization (e.g., in situ hybridization) or amplification methods. All or a portion of hEST2 DNA or
  • RNA can be used in such a method as a probe (e.g., to detect hEST2 DNA or RNA) or a primer (e.g., to amplify hEST2 DNA) .
  • a probe e.g., to detect hEST2 DNA or RNA
  • a primer e.g., to amplify hEST2 DNA
  • telomere up-regulation of hEST2 RNA is associated with activation of telomerase during cell immortalization and may be an important mechanism through which telomerase becomes activated during cellular immortalization, the progression of malignant tumors or both.
  • detection and/or measurement of hEST2 RNA provides a means of assessing cells (e.g., mammalian, such as human cells) for malignancy or the likelihood of progression to malignancy.
  • detection of hEST2 RNA, even without quantification of hEST2 RNA levels, in cells indicates that telomerase is activated and that the cells are malignant or have an increased likelihood of progression to malignancy (relative to cells in which hEST2
  • RNA is not detected) .
  • Quantification of hEST2 RNA can be carried out as well. Higher concentrations of hEST2 RNA cells are expected to indicate a more advanced stage in malignancy or a greater likelihood of progression to malignancy than is the case with lower hEST2 RNA levels in cells.
  • hEST2 RNA levels determined by assessing an individual's cells can be compared with pre-established hEST2 RNA levels. For example, hEST2 RNA levels determined for an individual ' s cells can be compared with hEST2 RNA levels in cells known to be malignant and/or at established stages of malignancy (e.g., a pre-established reference or standard) .
  • the levels of hEST2 RNA determined for an individual can be compared with hEST2 RNA levels determined for the same individual in prior assessments, in which case the individual serves as his or her own standard or reference.
  • assessment of hEST2 RNA can be carried out using known methods, such as in si tu hybridization, Rnase protection assay, reverse transcriptase (RT) PCR, Southern blot analysis or Northern blot analysis.
  • RT reverse transcriptase
  • the presence of malignancy or an increased likelihood of malignancy is assessed in a method in which a nucleic acid (e.g., DNA) anneals to nucleic acid (e.g., RNA) .
  • a nucleic acid e.g., DNA
  • RNA nucleic acid
  • the method can be Northern blot analysis, Rnase protection assay or RT PCR.
  • One embodiment is as follows: Cells to be assessed for hEST2 DNA or RNA are obtained from the individual and processed to render
  • hEST2 DNA and/or RNA in the cells available for annealing with or hybridization of complementary nucleic acid sequences DNA or RNA (e.g., with complementary poly- or oligonucleotides (DNA or RNA) ) . All or a portion of hEST2 DNA or hEST2 RNA can be used as the hybridization probe or sequence.
  • hEST2 protein shares sequence similarities and identities with yeast pl23 and Euplotes Est2p, particularly in a region just before motif 1 ( Figure 2, boxed region) . As described, this region appears to be unique to telomerases.
  • DNA or RNA comprising DNA or RNA encoding region (s) of similarity and/or identify with yeast pl23 and/or Euplotes Est2p such as region (s) unique to telomerase, can be used in the hybridization assay or other assay carried out to detect or measure hEST2 DNA or RNA in an individual's cells.
  • DNA or RNA comprising DNA or RNA which encodes a region or regions unique to hEST2 protein can be used as the probe.
  • the hEST2 can be used as the probe.
  • DNA or RNA is used as a primer in an amplification method.
  • the cells processed to render DNA and/or RNA available for annealing (hybridization) with complementary poly- or oligonucleotides are combined with all or a portion of hEST2 DNA or RNA under conditions appropriate for hybridization of complementary nucleic acids (DNA, RNA) to occur. Whether annealing (hybridization) occurs is then determined. If hybridization occurs (forming complexes of cellular DNA or RNA and the hEST2 poly- or oligonucleotides) , it is an indication that the cells contain hEST2 DNA or hEST2 RNA.
  • Poly- or oligonucleotides used in the method can be, for example, DNA which encodes the telomerase motif described herein (e.g., DNA which encodes amino acid residues 556 to 565 of SEQ ID NO. : 3 or amino acid residues 560 to 565 of SEQ ID NO. : 3) or amino acid residues 1 to 50 of hEST2 protein or a portion thereof.
  • hEST2 RNA is upregulated (expressed) , it is indicative of activation of telomerase and of malignancy or an increased likelihood of progression to malignancy.
  • the amount of hEST2 RNA can also be determined (by determining, for example, the extent to which hybridization with hEST2
  • DNA or RNA occurs) and used to assess the extent of telomerase activity and the stage of malignancy. This detection and/or measurement of hEST2 DNA or RNA can be carried out at various intervals over time to assess the status of the malignancy (e.g., progression, reversal).
  • hEST2 protein is analyzed (detected and/or quantified) .
  • This can be done using known methods, such as enzymatic assays or immunoassays, which indicate hEST2 protein is present in cells assessed and, optionally, quantify the protein.
  • hEST2 telomerase activity can be assessed by determining whether extension of a telomeric primer occurs when a sample (e.g., cells, cell fractions or cell component (s) ) from an individual is combined with the telomeric primer under conditions appropriate for hEST2 protein telomerase activity and telomeric extension.
  • antibodies which recognize (bind) hEST2 protein can be used to determine if the protein is present in cells or other sample obtained from an individual .
  • cells are obtained from an individual and processed or treated to render proteins in the cells available for binding with antibodies, thus producing processed cells.
  • the processed cells are combined or contacted with antibodies which bind hEST2 protein and whether binding occurs between antibodies which bind hEST2 protein (hEST2 protein-binding antibodies) and protein in the cells is determined. If hEST2 protein-binding antibody/protein binding occurs (to form complexes) , it is indicative of the presence of hEST2 protein in the cells and, thus, of malignancy or an increased likelihood of progression to malignancy in the individual (in cells of the individual) .
  • the antibodies can be monoclonal antibodies or polyclonal antibodies (e.g., polyclonal sera) and can be specific for (bind or recognize only) hEST2 protein or can be nonspecific for hEST2 protein (bind or recognize hEST2 protein and other protein (s) ) .
  • Humanized antibodies can also be used.
  • Antibodies which recognize a motif or epitope unique to hEST2 and/or to members of the class to which hEST2 belongs are particularly useful . For example, antibodies which specifically bind the telomerase motif, represented in Figure 2, which is common to hEST2 protein, yeast pl23 protein and/or Euplotes EST2 can be produced, using known methods, and used to assess cells for hEST2 protein.
  • antibodies which specifically bind a motif which is unique to hEST2 protein can be used.
  • the assessment can be detection (determination of presence or absence) or measurement (quantification) .
  • the presence of hEST2 protein is indicative of malignancy or at least of an increased likelihood of progression to malignancy.
  • Antibodies which bind or recognize (specifically or non-specifically) hEST2 protein are also a subject of the present invention.
  • Nucleic acid probes and primers (poly- or oligonucleotides) comprising hEST2 DNA or RNA are also the subject of this invention.
  • the poly- or oligonucleotides will vary in length and need to be of sufficient length to bind to and remain bound to hEST2 DNA or hEST2 RNA under the conditions used. They will generally be at least four to six bases in length and can comprise the entire hEST2 DNA or hEST2 RNA (alone or with additional non hEST2 DNA or RNA) .
  • the probes or primers will hybridize to at least a characteristic portion of hEST2 DNA or hEST2 RNA (a portion which is present in members of the class to which hEST2 belongs) , thus making it possible to identify hEST2 DNA or hEST2 RNA substantially to the exclusion of other proteins.
  • a probe or primer can comprise DNA or RNA which encodes a characteristic motif or region of hEST2 protein, such as the telomerase motif (e.g., amino acids 556 to 565 or 560 to 565) or the amino terminal amino acid sequence of hEST2 protein not present in Est2p or pl23. (See Figure 2) .
  • a probe or primer encoding amino acid residues 1 to 50 (inclusive) of hEST2 protein (SEQ ID NO. : 3) or a portion thereof can be used.
  • Methods of altering hEST2 DNA transcription and expression, methods of altering hEST2 protein function and methods of identifying agents which alter (enhance or reduce) transcription, expression or function are also the subject of this invention.
  • the subject of the present invention are a method of increasing or shortening the lifespan of cells in culture, ex vivo or in vivo; agents or drugs (DNA, RNA, drugs, small organic molecules, enzymes, for example) useful for lengthening or shortening cell lifespan and methods of identifying agents which enhance lifespan.
  • a method of inhibiting hEST2 protein comprises introducing into cells an agent which inhibits hEST2 protein, directly or indirectly; as a result, function of the enzyme is abolished or the enzyme is inactivated.
  • hEST2 protein is inhibited directly, for example, by introducing into cells an agent which binds to or otherwise "ties up" hEST2 protein such that it is less active or inactive (cellular telomerase activity is reduced or eliminated) .
  • a drug or agent which inactivates hEST2 or inhibits hEST2 catalytic function (and concomitantly reduces or eliminates telomerase activity) by degrading it or preventing it from being produced can be introduced into cells in which hEST2 protein is to be inhibited.
  • Such drugs or agents can be, for example, a small organic molecule or a dominant negative protein.
  • Agents which prevent production of hEST2 message or its further processing (e.g., to produce DNA) or a dominant negative form of the telomerase protein can also be introduced into cells to inhibit hEST2 protein. Methods and agents which inhibit hEST2 catalytic function are useful for treatment of cancer and, thus, are useful as anti-cancer therapies.
  • hEST2 DNA, hEST2 RNA or hEST2 protein is inhibited (partially or totally) in cells, lifespan of the cells will be shortened (shorter than it would be if the DNA, RNA or protein were not inhibited) .
  • an agent or drug which inhibits hEST2 transcription or expression or hEST2 protein function in the cell will also shorten the lifespan of the cell because telomerase activity (and, thus, chromosomal extension or maintenance) will be inhibited.
  • the agent is useful as a therapeutic for treating or preventing malignancy in individuals in whom malignant cells or cells with increased likelihood to progress to malignancy are present.
  • agents include, for example, enzyme (telomerase or hEST2 protein) inhibitors, enzymes which degrade hEST2 protein, hEST2 transcriptional regulators, antisense molecules (e.g., DNA, RNA, PNA) and dominant negative mutant forms of hEST2.
  • enzyme telomerase or hEST2 protein
  • hEST2 transcriptional regulators enzymes which degrade hEST2 protein
  • antisense molecules e.g., DNA, RNA, PNA
  • dominant negative mutant forms of hEST2 e.g., DNA, RNA, PNA
  • a drug or agent is introduced into the individual in such a manner that it enters cells of the individual in sufficient quantity to have the desired effect (increase or decrease in cell lifespan) .
  • a drug which prevents production of hEST2 transcript of hEST2 protein function is introduced into an individual, using known methods, in sufficient quantities to enter cells, such as a tumor, precancerous or cancerous cells, whose lifespan is to be decreased (e.g., made shorter than would be the case in the absence of the drug) .
  • a drug which enhances cell lifespan can be introduced into an individual in such a manner that it enters cells in sufficient quantity to enhance hEST2 protein expression or prolong its activity (e.g., by blocking its degradation).
  • the present invention is also a method of enhancing or increasing the lifespan of cells in culture, in which telomerase activity or function is enhanced, such as by introducing hEST2 DNA or hEST2 protein into the cells or by activating an endogenous hEST2 gene.
  • telomerase activity or function is enhanced, such as by introducing hEST2 DNA or hEST2 protein into the cells or by activating an endogenous hEST2 gene.
  • the lifespan of normal human or other mammalian cells can be extended (immortalized human or other mammalian cells can be produced) and the resulting cells used for therapeutic purposes (e.g., grafting of tissue (such as skin) or of organs) , screening or assay methods or production of proteins or other cellular products.
  • the lifespan of epithelial cells, keratinocytes or endothelial cells can be extended by introducing hEST2 DNA into the cells, in which the hEST2 DNA is expressed or introducing hEST2 protein into the cells.
  • the resulting cells with longer lifespan can be transplanted into or grafted onto an individual (e.g., as skin grafts, as systems for delivery of therapeutic proteins, such as hormones and enzymes) , to whom they provide therapeutic benefit.
  • cells whose lifespan is to be extended are cultured under conditions appropriate for their viability and hEST2 DNA or hEST2 protein is introduced into them.
  • the resulting cells are maintained under conditions appropriate for expression of hEST2 DNA and/or activity of hEST2 protein, with the result that the lifespan of the cells is enhanced.
  • enhanced lifespan cells such as keratinocytes
  • an individual e.g., as a skin graft
  • EXAMPLE 1 Cloning of hEST2 (hTERT) The expressed sequence tag database (dbEST) was searched for sequences related to—the yeast protein Est2p (Lendvay et al . , Genetics 144 : 1399 - 1412 (1996)) and the
  • the resulting 377 bp fragment was used to probe 1 ZAP phage cDNA libraries derived from the human Jurkat T-cell lymphoma (Stratagene, La Jolla, CA) and human Nalm-6 pre-B cell leukemia cell lines (Weissbach et al . , J " . Biol . Chem. 259:20517-20521 (1994)). A total of seven independent clones were isolated from these two libraries. Three of these cDNA clones together with plasmid 712562 were sequenced completely in both directions. The remaining clones were sequenced in specific regions. Reprobing of the Jurkat library with the 5 ' -most 500 bp region identified from the hEST2 cDNA clones yielded one new clone containing an insert that overlapped with already determined sequences .
  • Rapid amplification of cDNA ends was performed by PCR amplifying testis Marathon-ready cDNA (Clontech, Palo Alto, CA) with flanking primer AP-1 (Clontech) and hEST2 primer R0096 (5' -CAAGAAACCCACGGTCACTCGGTCCACGCG-
  • DNA sequencing reactions were performed with the AmpliTaq FS Prism ready reaction cycle sequencing kit (Perkin-Elmer/ABI) and electrophoresed on a 373 A Stretch ABI DNA sequencer. The resulting sequences were assembled into a contig using the program Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, MI) . The 1132 amino acid ORF identified in this contig was aligned with pl23 and Est2p using the Pattern- Induced Multi-sequence Alignment program version 1.4 (R. F. Smith, Baylor College of Medicine & T. F. Smith, Boston University) using the server http: //dot .imgen.bcm.tmc.edu : 933l/multi-align/multi-align. h tml, with minor modifications to the final alignment. BLASTP searches were performed against the non-redundant protein databases using the server http : //www. ncbi .nlm.nih. gov/BLAST/ .
  • the identified expressed sequence tag provided only a fragment of the putative human telomerase open reading frame. Therefore, a human Jurkat T-cell lymphoma and a human Nairn-6 pre-B cell leukemia cDNA library were screened -55 -
  • CCCGCGAI ⁇ SC (SEQ ID NO. : 46) , is similar to the consensus GCC (A/G) CCAUGG (SEQ ID NO.: 47) characteristic of translation initiation sites (Kozak, Cell 44:238-292
  • the predicted 127 kDa protein shares extensive sequence similarity with the entire seq ⁇ ences of the Euplotes and yeast telomerase subunits ( Figure 2) and extends beyond the amino- and carboxyl-termini of these proteins.
  • a BLAST search reveals that the probabilities of these similarities occurring by chance are 1.3 x 10 "18 and 3 x 10 ⁇ 13 , respectively.
  • the probability of similarity between the yeast and Euplotes telomerases in a protein BLAST search is 6.9 x 10 "6 .
  • the human gene was initially named hEST2 ⁇ human EST2 homologue) to reflect its clear relationship with the yeast gene, the first of these genes to be described.
  • the current name for the gene is hTERT, which stands for human TElomerase
  • EST2 Reverse Transcriptase .
  • EST2 was named because of the phenotype of gver shortening telomeres caused by its mutant alleles (Lendvay et al . , Genetics 144:1399-1412 (1996)) and was later demonstrated to encode the yeast telomerase catalytic subunit (Counter et al . , Proc. Natl . Acad. Sci . , USA 94 : 9202 -9201 (1997); Lingner et al . , Science 275:561-
  • hEST2 ⁇ hTERT is a member of the reverse transcriptase (RT) family of enzymes ( Figures 2 and 3A-3F) . Seven conserved sequence motifs, which define the polymerase domains of these enzymes, are shared among the otherwise highly divergent RT family (Poch et al . , EMBO J. 8:3867-3874 (1989); Xiong and
  • pl23 and Est2p share six of these motifs with, most prominently, the a2-Sc enzyme, an RT that is encoded within the second intron of the yeast C0X1 gene (Kennell et al . , Cell 73:133-146
  • telomerase catalytic subunit like its yeast and ciliate counterparts, belongs to the RT superfamily of enzymes.
  • hEST2 shares some sequence similarity with RTs, it is not a conventional RT. Rather, it is far more closely related to the telomerase catalytic subunits of yeast and ciliates than to other RTs. Whereas the BLAST probability of sequence similarity between hEST2 and the telomerase subunits of the single-cell eukaryotes arising by chance is 10 "13 to 10 -18 , the chance probability of sequence similarity with the next most closely related RT, a2-Sc, is 0.12. Beyond the motifs that define the polymerase domains of these various enzymes, hEST2 shows no sequence similarity with RTs.
  • BLAST searches identify clear relatedness between hEST2 and both pl23 and Est2p, the chance occurrence of these similarities being 1.6 x 10 "9 and 1.8 x 10 " " respectively.
  • Many of the sequence identities in the N-termini of hEST2, pl23 and Est2p reside in a region just before motif 1 ( Figure 2, boxed region) . This sequence is not found in RTs, nor is it apparent in other proteins, suggesting the presence of motifs that may be unique to telomerases. Identification of the catalytic subunits of yet other telomerases will be required to validate this possibility.
  • hEST2 is more closely related to the already described telomerases than it is to non-telomerase RTs .
  • sequence similarity of the region encompassing the RT motifs between hEST2 and the catalytic subunits of yeast and Euplotes has a probability of chance occurrence of 5.7 x 10 "6 and 1.9 x 10 "5 respectively compared to 0.0056 for a2-Sc, the next most closely related non-telomerase RT.
  • telomeres Within the RT motifs are several amino acids that are invariant among the telomerases but divergent between telomerases and non-telomerase RTs, or alternatively nearly invariant among non-telomerase RTs but divergent between these RTs and telomerases ( Figures 3A-3F ) .
  • hEST2 , Est2p, and pl23 form a clearly defined subgroup within the RT family.
  • hEST2 is a human homologue and very likely an orthologue of the microbial enzymes described to date. Sequencing of a number of cDNA clones has revealed two distinct forms of hEST2 transcripts. See Figures 5A-5B and
  • Figures 7A-7C Four independent cDNA clones, isolated from three independently generated libraries deriving from distinct cell types, lack an identical 182 bp segment within the open reading frame. The absence of this segment leads to a shift in reading frame that introduces a premature termination codon. Both forms of the hEST2 transcript were detected by RT-PCR in a variety of human cell types. Information on the intron-exon boundaries of hEST2 is not available. The simplest interpretation of these data is that the sequence difference between the two groups of cDNAs reflects the existence of two alternatively spliced mRNAs of the hEST2 gene. The physiological consequences of the expression of the potential non-functional hEST2 transcript are obscure at present.
  • hEST2 cDNA was used as a probe in Southern blot analyses of human genomic DNA. These reveal hEST2 to be a single-copy gene with an estimated size of 40 kb . All the genomic sequences reactive with the cDNA probe appear to be components of this -40 kb locus, suggesting that there are no other closely related genes in the human genome .
  • hEST2 was localized to a specific chromosomal region by analyzing two independent panels of hamster-human radiation hybrid (RH) cells with two markers spanning different regions of hEST2. Initial mapping using the
  • Genebridge 4 RH panel placed both hEST2 markers between sequence-tagged sites (STS) WI-9907 and D5S417 ( Figure 4) . Independent confirmation of this localization was then obtained by mapping carried out with a second panel , the Stanford G3 RH panel. This second mapping placed both hEST2 markers next to STS marker AFMA139YA9 (GDB locus
  • D5S678 which itself is localized between the above-mentioned markers WI-9907 and D5S417. These markers are present at the telomeric end of chromosome 5p (Hudson et al . , Science 270:1945-1954 (1995)).
  • FISH fluorescence in situ hybridization
  • Chromosome 5p is one of the most common targets for amplification in non-small-cell lung cancers; it is amplified in -70% of tumors (Balsara et al . , Cancer Res .
  • telomerase becomes activated during tumor progression, and, as discussed above, this activation has been associated with the immortalization of tumor cells. A variety of mechanisms might be invoked to explain such activation, among which is the induction of the expression of one or more telomerase subunits .
  • the telomerase holoenzyme is presumed to exist as a multi-subunit ribonucleoprotein complex and, therefore, the levels of any one of the subunits, including those of the catalytic
  • SUBSUME SHEET (RULE 26) subunit described here might be rate-limiting in determining enzyme activity.
  • the components of the telomerase holoenzyme might be expressed constitutively and subject to various types of post-translational modification that would govern their activity.
  • transcript levels of the telomerase RJSIA subunit and of the TP1/TLP1 gene do not necessarily correlate with telomerase activity (Feng et al . , Science 269 : 1236 -1241 (1995); Avilion et al . , Cancer Res . 55:645-650 (1996); Blasco et al . , Nat . Genet . 22:200-
  • RNA blots prepared from a panel of normal human tissues and human cancer cell lines were probed with cDNA fragments deriving from two independent, non-overlapping regions of the hEST2 gene. This probing revealed two major RNA species migrating near the 4.4 kb and the 9.5 kb markers, as well as a minor species of -6 kb ( Figures 8A-8F) .
  • RNA species were recognized by both probes, confirming that each represents an hEST2 mRNA.
  • hEST2 message was detectable in several normal tissues including thymus, testis, and intestine.
  • testis Kelm et al . , Science 255:2011-2015 (1994); Wright et al . , Dev. Genet . 18:173-179 (1996)
  • intestine Hiyama et al . , Int . J. Oncol . 9:453-458 (1996) are known to be telomerase-positive, while the telomerase status of the thymus has not been reported.
  • hEST2 mRNA was strongly expressed in a variety of cancer cell lines, most strikingly in the leukemic cell lines HL-60 and K-562 ( Figures 8C and 8F) . It is unclear why only very low levels of hEST2 message were observed in
  • HeLa cells on this particular Northern blot ( Figures 8C and 8F) ; reanalysis of independently prepared HeLa cell RNA both by Northern blot and by RNase protection demonstrated that hEST2 mRNA is present in HeLa cells at high levels comparable to those seen in K-562 cells.
  • the two major and one minor hEST2 transcript appear to be expressed in the same relative proportions in all cell types that yielded detectable hEST2 mRNA.
  • EXAMPLE 5 hEST2 Expression in Primary Human Cancers
  • the expression of hEST2 mRNA in cancer cell lines suggested that hEST2 transcript levels might be elevated in primary tumors as well.
  • RNA was extracted from a variety of tumor samples as well as from normal control tissues and analyzed this for the presence of hEST2 mRNA using an RNase protection assay.
  • 11 of 11 tumor samples examined showed detectable levels of hEST2 message ( Figures 9A-9D) .
  • hEST2 RNA was undetectable in normal breast and ovarian tissue but was expressed at significant levels in 2 of 2 breast tumors as well as 2 of 2 breast tumor-derived cell lines, and in 4 of 4 ovarian tumors .
  • the hEST2 transcript was detected at high levels by the RNase protection assay in testis and at moderate levels in colon.
  • four colon tumor samples and a testicular tumor sample were found to express detectable levels of hEST2 RNA ( Figures 9A-9D) ; two of the colon tumors showed significantly elevated levels as well.
  • telomerase activity and hEST2 RNA level were analyzed in a panel of non-immortalized and immortalized cell lines.
  • TRAP telomerase assay Two mortal fibroblast strains, WI-38 and IMR-90, were found to lack detectable telomerase activity.
  • telomerase activity was readily detectable in three immortal cell lines, HeLa, 293, and K-562.
  • telomerase-negative cells also lacked detectable hEST2 message as gauged by an RNase protection assay, while the immortal telomerase-positive cell lines expressed significant levels of hEST2 RNA. These results indicate that hEST2 message levels correlate closely with telomerase activity.
  • hEST2 RNA expression and telomerase activity are present in immortal transformed cells, " but absent in mortal normal cells.
  • These findings support the role of induction of hEST2 expression in the activation of telomerase that occurs during cellular immortalization. That this is the case was shown by analyzing hEST2 transcript levels by RNase protection, and comparing the levels of hTR and telomerase activity in pre-crisis cells prior to the up-regulation of telomerase, and in post-crisis telomerase-positive immortal cells from two different transformed human cell populations: Epstein-Barr virus-transformed B lymphocytes (B4 cells) and SV40-T antigen transformed embryonic kidney cells (HA1 cells) .
  • B4 cells Epstein-Barr virus-transformed B lymphocytes
  • HA1 cells SV40-T antigen transformed embryonic kidney cells
  • hEST2 RNA was undetectable in the pre-crisis cells but was clearly present in the post-crisis, telomerase-positive cells. Induction of hEST2 message is the rate-limiting step for the activation of telomerase during immortalization .
  • telomere up-regulation of hEST2 RNA is associated with the activation of telomerase during cell immortalization. It remained unclear whether conditions that repress telomerase activity in cultured cells might similarly operate by shutting down expression of the hEST2 mRNA in these cells.
  • telomere activity has been shown to decline over a period of two to five days (Sharma et al . , Proc . Natl . Acad . Sci . , USA 92:12343-12346 (1995); Bestilny et al . , Cancer Res . 55:3796-3802 (1996);
  • EXAMPLE 8 Ectopic Expression of hTERT mRNA in Telomerase- Negative Cells and Assessment of Expression on Levels of Telomerase Activity
  • the hTERT cDNA was introduced into a mammalian expression construct carrying the CMV promoter.
  • the C-terminus of the vector-encoded protein was marked with an influenza virus hemagglutinin (HA) epitope tag, yielding the plasmid pCI -neo- hTERT -HA.
  • HA hemagglutinin
  • Such a modification does not affect the catalytic activity of the Est2p protein of S. cerevisiae (Counter, CM., et al . , "The catalytic subunit of yeast telomerase", Proc. Natl . Acad. Sci . USA, 94 : 9202 - 9201 (1997) .
  • the immortal GM847 cells are telomerase-negative (Bryan, T.M. , et al . , "Telomere elongation in immortal human cells without detectable telomerase activity", EMBO
  • GM847 cells employ a telomerase-independent mechanism to maintain telomere length (Murnane, J.P., et al . , "Telomere dynamics in an immortal human cell line", EMBO J. , - 67-
  • telomerase-negative human somatic cells which lack replicative immortality
  • these GM847 cells could be propagated indefinitely following transfection, allowing us to study the properties of clonally isolated cell populations that have stably acquired the introduced hTERT gene.
  • a number of stably transfected GM847 cell clones were generated with either the control vector or the hTERT-HA expression vector, mRNA expression of hTERT was analyzed by
  • hTERT-HA transcript was detected only in GM847 sublines stably transfected with an hTERT-HA expression plasmid, but not in untransfected telomerase-positive control cell lines 293 and HL-60, in the parental GM847 line or in GM847 sublines transfected with the empty vector.
  • the cells expressing hTERT-HA did not express the endogenous hTERT transcript , despite the fact that this RNA is clearly detected in telomerase- positive cells.
  • hTR RNA subunit of telomerase does not correlate with enzyme activity, this RNA was detected in all cells tested, irrespective of whether the cells had telomerase activity.
  • An actin control probe demonstrates comparable loading of RNA from each cell line. Moreover, the specificity of the probes used was demonstrated by their failure to protect tRNA. Expression of hTERT-HA was also analysed at the protein level, by immunoblotting with an anti-HA antibody probe directed against the HA tag of the vector-encoded hTERT protein.
  • hTERT A ⁇ 130 kDa product corresponding to the predicted size of hTERT was detected in those lines derived from GM847 cells that were stably transfected with the hTERT-HA expression construct, but not in those cell clones that had been transfected with the empty vector.
  • the antibody likewise did not detect endogenous (untagged) hTERT known to be expressed in 293 cells (Meyerson, M., et al . "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during
  • telomerase activity was measured in these different cell lines by assaying the ability of a cellular extract to elongate a primer in a telomerase-specific manner. The products of this in vi tro reaction are subsequently detected by specific PCR amplification, yielding a ladder of products differing from one another by 6 bp (Kim, N.W. , et al . , "Specific association of human telomerase activity with immortal cells and cancer" , Science, 255:2011-2015 (1994)).
  • telomerase activity was detected in 293 cells, a cell line with one of the highest levels of telomerase activity known. This activity was sensitive to heat treatment of the extract, which inactivates telomerase (Kim, N.W., et al . , "Specific association of human telomerase activity with immortal cells and cancer", Science, 255:2011-2015 (1994)). In contrast, almost no telomerase products were detectable following assay of extracts from untransfected GM847 cells or from GM847 sublines stably transfected with the empty control vector.
  • telomere activity was not due to the presence of a PCR-inhibiting activity, as an internal control was specifically PCR-amplified, (Kim, N.W., and Wu, F. "Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP)", Nucleic Acids Res . ,
  • telomerase activity was readily detectable in those clones of GM847 cells that were stably transfected with the hTERT-HA expression vector and this activity was sensitive to heat inactivation.
  • the levels of telomerase activity observed in these transfectants approached those seen in extracts from 293 cells.
  • the restoration of telomerase activity in the cells transfected with hTERT-HA was not due to the up-regulation of the endogenous hTERT gene, as evidenced by failure to detect the corresponding mRNA in these cells.
  • ectopic expression of hTERT in previously telomerase-negative cells is sufficient to generate telomerase activity at levels comparable to those found in immortalized telomerase- positive cells.
  • telomerase activity detected in cells transfected with the hTERT-HA expression vector was physically associated with ectopically produced hTERT-HA, confirming that hTERT is, as predicted, a constituent of the telomerase holoenzyme.
  • Telomerase activity could be immunoprecipitated with an anti-HA monoclonal antibody from extracts of either 293 cells or GM847 cells that ectopically express hTERT-HA.
  • the telomerase activity was not immunoprecipitated with an antibody directed against an irrelevant antigen (anti-p53) , nor was it immunoprecipitated when either antibody was incubated with extracts prepared from control vector-transfected GM847 cells.
  • telomerase activity is specifically co-immunoprecipitated with hTERT-HA. Whether the observed induction of telomerase activity following ectopic hTERT expression was unique to GM847 cells was also determined. This was done by transiently transfecting WI-38 normal human fibroblasts, which lack detectable levels of telomerase activity and hTERT message but express the hTR gene (Meyerson, M. , et al., "hEST2 , the
  • Extracts derived from both populations of fluorescing cells were assayed for telomerase activity. Whereas fibroblasts transfected with pGreenLantern-1 and pCI-neo vector alone lacked enzymatic activity, those co-transfected with the hTERT-HA expression vector were clearly telomerase-positive. Transfection with pCI-neo -hTERT-HA of IMR-90 cells, another telomerase- negative normal human fibroblast cell strain that does not normally express hTERT (Kim, N.W. , et al . , "Specific association of human telomerase activity with immortal cells and cancer", Science, 266 : 2011-2015 (1994); (Meyerson, M. , et al . , "hEST2, the Putative Human Telomerase
  • telomere activity confirms that hTERT is a telomerase subunit.
  • forced expression of hTERT sufficed to impart telomerase activity indicates that levels of hTR mRNA and TP-1 or other still unidentified components of the telomerase holoenzyme are not rate-limiting determinants of telomerase activity in these cells.
  • Up-regulation of the hTERT gene is therefore the sole barrier to activation of telomerase in the tested cells. It still remains to be determined if telomerase activity can be restored in this fashion in all telomerase-negative cells.
  • results described here show that activity is conferred by ectopic expression of hTERT in cell types representative of two known classes of telomerase-negative cells: telomerase-negative immortal cell lines and normal mortal human cell strains.
  • telomerase-negative immortal cell lines and normal mortal human cell strains.
  • expression of hTERT mRNA is the rate-determining step for telomerase activation in other human cells lacking enzyme activity. This makes the regulation of transcription from the hTERT promoter a potential target for modulation during tumorigenesis and cell immortalization.
  • PCR was carried out for 35 cycles of 94°C for 0.5 min., 69°C for Ml or 65°C for M2 for 0.5 min. and 72°C for 1.5 min.
  • the results of the PCR screening were analyzed using the statistical program RHMAP provided through the following two e-mail servers on the World Wide Web: http://shgc-www.stanford.edu and http : //www-genome . wi .mit . edu/cgi-bin/contig/rhmapper .pi .
  • HL-60 differentiation assays cells were pelleted and resuspended in normal growth medium plus all- trans retinoic acid (Sigma) at a final concentration of 1 ⁇ M.
  • RNA samples were prepared in the RNA Stat-60 solution (Tel-Test "B", Friendswood, Texas) according to the manufacturer's protocol .
  • Duplicate filters containing poly A(+) -selected mRNAs from various human tissues and cell lines were incubated according to the manufacturer's instructions with two independent hEST2 probes.
  • One probe was derived by
  • RNAse Protection Analysis Radiolabelled RNA probes for RNAse protection analysis were synthesized using [ - 32 P]UTP, T7 RNA polymerase and the MAXIscript kit (Ambion, Austin, TX) . DNA templates for probe synthesis were created as follows. For the hEST2 probe, the insert from plasmid clone 712562 was subcloned into pUHD 10-3 (a gift from M. Gossen) , and template DNA was synthesized by PCR using a forward plasmid-specific primer and a reverse primer containing 18bp of hEST2 complementary sequence (5 ' -TCTCTGCGGAAGTTCTG) (SEQ ID NO.: 52) and the T7 promoter sequence.
  • ⁇ -actin probe template DNA was synthesized by PCR on a human ⁇ -actin cDNA insert (Clontech) .
  • the hEST2 and the ⁇ -actin control probes were hybridized in the same reaction tube.
  • the hTR probe was synthesized directly from the linearized pGRN83 plasmid (Feng et al . , Science 269:1236-1241 (1995)).
  • RNA and radiolabelled RNA probes were coprecipitated in ethanol, resuspended in hybridization buffer, hybridized at 68° C, then digested with RNases A and Tl . Samples were then re-precipitated and analyzed on a 6% denaturing polyacrylamide gel.
  • telomerase repeat amplification protocol (TRAP) assays were performed as described (Kim et al . , Science 256:2011-

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Abstract

Cette invention concerne de l'ADN isolé codant la sous-unité catalytique d'une holoenzyme de télomérase eucaryotique telle que la sous-unité catalytique d'une holoenzyme de télomérase humaine ou de levure; le transcript d'ARN qui est exprimé dans des tumeurs humaines primitives, des lignées cellulaires cancéreuses et des tissus télomérase-positifs; et la protéine de sous-unité catalytique codée. L'invention concerne également des procédés qui permettent d'évaluer le caractère malin des cellules ou la probabilité accrue que les cellules deviennent malignes et des méthodes de diagnostic ou facilitant le diagnostic du développement du caractère malin des cellules chez un individu.
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