Affiliations of authors: A. P. Cuthbert, J. Bond, D. A. Trott, A. Marriott, G. Khoudoli, R. F. Newbold, Human Cancer Genetics Unit, Department of Biology and Biochemistry, Brunel University, Uxbridge, U.K; S. Gill, J. Broni, C. S. Cooper, Molecular Carcinogenesis Section, The Haddow Laboratories, The Institute of Cancer Research, Royal Cancer Hospital, Surrey, U.K.; E. K. Parkinson, Beatson Institute for Cancer Research, CRC Beatson Laboratories, Glasgow, U.K.
Correspondence to: Robert F. Newbold, Ph.D., Department of Biology and Biochemistry, Brunel University, Uxbridge UB8 3PH, U.K. (e-mail: robert.newbold{at}brunel.ac.uk).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent comparisons of the activity of the enzyme telomerase in normal and immortal human cells have led to the formulation of an attractive molecular model to explain replicative cellular senescence and immortalization (9). Telomerase, an RNA-dependent DNA polymerase, was first identified in primitive eukaryotes (10). Telomerase compensates for telomere loss due to the "end replication problem" (11), exonuclease attack (12), and possibly oxidative damage (13). Most normal human cells, other than germ cells, do not have detectable telomerase activity and, as a result, telomeres shorten at rates ranging from 40 to 200 base pairs (bp) per cell division depending on the cell type (14). In contrast, the vast majority of human cancers possess active telomerase (9) and thus are able to maintain their telomeres. Based on these findings, it has been proposed (15) that the progressive loss of telomeres provides a "counting" mechanism for determining cellular replicative potential. In this model, the induction of senescence is ultimately triggered in cells with critically shortened telomeres via the activation of some, as yet unknown, cell cycle inhibitory pathway.
The mechanism by which telomerase is repressed in normal human cells appears to operate at the level of transcriptional regulation of the catalytic subunit (i.e., the protein component) of the telomerase ribonucleoprotein (16). In very recent studies (17,18), reconstitution of telomerase activity in normal human cells by ectopic expression of the cloned (16,19-21) gene encoding the catalytic subunit resulted in cells with a substantially increased proliferative capacity. Furthermore, in a previous investigation, Feng et al. (22) showed that expression of antisense sequences directed to the RNA component of human telomerase produced telomere shortening and growth crisis in a subclone of the immortal HeLa cell line. However, despite such strong evidence supporting a causal relationship between telomerase and immortalization in vitro, direct proof that the telomerase activity seen in human cancer cells is the result of a distinct genetic event in the process of carcinogenesis (i.e., resulting in re-expression of the telomerase catalytic subunit) is still lacking. It remains formally possible that the targets for malignant transformation in vivo are rare telomerase-positive cells [e.g., stem cells or progenitor cells; see (23)] and, therefore, that telomerase activity in cancer results from clonal selection rather than induction (24,25).
To resolve this and other important questions concerning the role of telomerase in cell senescence and cancer, we have adopted a somatic cell genetic approach based on microcell-mediated monochromosome transfer to identify genes in the normal human genome that induce replicative senescence and/or telomerase repression when introduced into human tumor cells. For this purpose, we have developed a unique panel of highly stable monochromosome human : mouse "donor" hybrid cell lines representing the complete normal human chromosome complement (26) and have shown that these can be used effectively in the functional identification and accurate subchromosomal localization of novel antiproliferative genes (27). Our recent screen of these chromosomes (28) using a human head and neck carcinoma cell line as a recipient for monochromosome transfer revealed strong telomerase repressive activity specifically associated with the introduction of a single, cytogenetically intact copy of human chromosome 3. In the present study, we used monochromosome transfer to investigate in detail the effects of human chromosome 3 on telomerase activity in an early-passage human breast cancer cell line derived from a primary intraductal carcinoma and attempted to relate the extent of telomerase repression to the timing of growth arrest (replicative senescence) induced in these cells by the new chromosome. In addition, we undertook a detailed analysis of the structural integrity of the introduced chromosome 3 in nonresponsive (i.e., immortal) hybrid segregants to identify regions on the short arm of chromosome 3 (frequently deleted in human cancers) where the candidate telomerase regulator gene(s) may reside.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A newly established breast cancer cell line, 21NT (provided by R. Sager and V. Band, Dana-Farber Cancer Institute, Boston, MA) (29), was used as the recipient for monochromosome transfer studies. This cell line was derived from a primary intraductal carcinoma of the breast and is one of a series of well-characterized lines from the primary tumor and metastatic deposits of a single 36-year-old patient. Early-passage cells were cultured in modified Eagle medium alpha supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 50 µg/mL gentamicin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 1.0 µg/mL insulin, 2.8 µM hydrocortisone, and 12.5 ng/mL epidermal growth factor as described by Band et al. (29). Mouse(A9) : human monochromosome hybrid donor cell lines A9-Hytk3, A9-Hytk8, and A9-Hytk20 carrying a selectable fusion gene marker, Hytk (Hy, bacterial hygromycin phosphotransferase; tk, herpes simplex virus thymidine kinase) tagged on normal human chromosome 3 (Hyg-3), 8 (Hyg-8), and 20 (Hyg-20), respectively, derived by us previously (26), were maintained in Dulbecco's modified Eagle medium containing 10% FBS and 400 U/mL hygromycin B (Calbiochem Corp., San Diego, CA). Monochromosome hybrid donors A9+3 (30) and A9+12 (31) carrying a neo (neo = bacterial neomycin-resistance gene)-tagged human chromosome 3 (Neo-3) and 12 (Neo-12), respectively, were obtained from Coriell Cell Repositories (Camden, NJ) and were maintained in 400 µg/mL geneticin [G418) (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD).
Microcell-Mediated Monochromosome Transfer and Cytogenetic Analysis of Hybrids
Microcell-mediated chromosome transfer of normal human chromosomes into 21NT cells was carried out as described by us (26,32) previously. Microcell hybrids were selected from postfusion cells in medium supplemented with either hygromycin B (400 U/mL) for hyg (hyg = hygromycin-resistance gene)-tagged chromosomes or G418 (800 µg/mL) for neo (neo = neomycin resistance gene)-tagged chromosomes. Preliminary cytogenetic characterization of the subset of 21NT hybrids that did not undergo growth arrest was performed by chromosome fluorescence in situ hybridization (FISH) by use of digoxigenin-labeled chromosome-specific probes (Oncor, Inc., Gaithersburg, MD). Fluorescein isothiocyanate fluorescence was observed using an Olympus epifluorescence microscope attached to an uncooled CCD camera system (Sony; Cologne, Germany).
Assay of Telomerase Activity in 21NT Cells and Monochromosome Hybrids
Telomerase activity was assayed using the standard telomere repeat
amplification protocol (TRAP) (33) and/or the TRAP-eze
telomerase detection kit (Oncor, Inc.). 21NT colonies and
monochromosome hybrid derivatives were lysed in situ in 200
µL of ice-cold lysis buffer. Protein concentrations were determined
with a Coomassie blue protein assay kit (Pierce Chemical Co., Rockford,
IL). Reaction mixtures for the standard TRAP included 200 ng of
protein, 5 attograms of an internal standardeither a 150-bp myogenin
complementary DNA (cDNA) (33) or, in the case of the TRAP-eze
assay, the shorter (36-bp) internal DNA standard included with the
kitto facilitate quantitative comparisons of telomerase activity and
to control for Taq polymerase inhibitors, 25 µM
deoxynucleotide triphosphates, and 4 µCi each of 10 mCi/mL
[-32P]deoxycytidine triphosphate and
[
-32P]deoxythymidine triphosphate. Reaction mixtures
were incubated for 1 hour at room temperature prior to polymerase chain
reaction (PCR) amplification of telomerase extension products. PCR
conditions were one cycle of 90 °C/90 seconds,
94 °C/35 seconds followed by 35 cycles of
94 °C/30 seconds, 50 °C/30 seconds, and
72 °C/45 seconds. The final extension step was
72 °C/60 seconds. PCR products were resolved in 10%
nondenaturing polyacrylamide gels and visualized by use of a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Signal intensities
of telomerase products were standardized against the internal control
and subjected to semiquantitative evaluation using ImageQuanTTM
software (Molecular Dynamics).
Subchromosomal Localization of Candidate Telomerase Repressor Gene(s)
A total of 44 polymorphic dinucleotide markers, mapped to the short arm of chromosome 3, were selected from the Genethon Human Genetic Linkage Map (34) and the Human Genome Database (Johns Hopkins University, Baltimore, MD). The relative order of these markers was established from data available in the Genetic Location Database (35). Cytogenetic information was also taken into consideration in cases where linkage data were weaker. Markers were assessed for informativeness by defining the allelotypes of both chromosome 3 donor hybrids (A9-Hytk3 and A9+3) and the 21NT (recipient) cell line. DNA was isolated from hybrid and parental cells (control) by proteinase K digestion and phenol/chloroform extraction (36). DNA samples were diluted to 6 ng/µL and 30 ng was added to 15-µL aliquots of PCR mix containing 200 nmol of each primer (Advanced Biotechnologies, Epsom, Surrey, U.K.). One of each primer pair was labeled with one of three fluorescent tags; HEX, TET, or 6-FAM. PCR was carried out using conditions that yielded a single product of the appropriate size. PCR amplification products were mixed and separated by electrophoresis on an ABI 377 DNA sequencer (Applied Biosystems Inc. [ABI], Foster City, CA). Data were collected using the Genescan Collection and Analysis Software (ABI, version 1.2.1). The resultant files were transferred to Genotyper (ABI version 1.1.1) for analysis and allele calling.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Normal human chromosomes 3 (Hyg-3 and Neo-3), 8 (Hyg-8), 12
(Neo-12), and 20 (Hyg-20) were transferred from A9 donor hybrids into
21NT cells by microcell fusion, and the proliferative potential of
resulting hybrid clones (in selection medium) determined by direct
(daily) observation under phase-contrast microscopy. Hybrid colonies
generated with donors carrying chromosomes 8, 12, and 20 (Table
1) proliferated continuously and could be picked and
established as continuous cultures (>100 population doublings), with
no sign of replicative senescence or diminished growth rate relative to
the parent control cultures. In marked contrast, the vast majority
(>90%) of hybrid clones produced by use of either of the
monochromosome 3 donors (Hyg-3 or Neo-3) exhibited delayed growth
arrest. Cells within individual colonies initially proliferated
normally with a marginally increased mean doubling time of
approximately 50 hours (compared with around 40 hours for the parental
21NT cells). This was followed by a pronounced reduction in growth rate
during which cells showed morphologic changes characteristic of
senescing epithelial cells (37), including enlargement,
multinucleation, vacuolation, and positive senescence-associated
(SA)-ß-galactosidase staining (38) (data not shown).
Complete growth arrest most commonly occurred after 10-12 population
doublings (colony size: 1000-5000 cells), although 57 of 257 hybrids
recovered from six experiments with the Hyg-3 donor chromosome (Table
1)
displayed longer division potentials of 15-18 population doublings
before entering growth crisis. Twenty-four of these clones eventually
generated immortal variants, apparently via a rare event, after 58-161
days of crisis. Only 26 of the 257 Hyg-3 hybrids proliferated
continuously from the outset, and these were cryopreserved 11-27 days
after picking colonies. Very similar results were obtained with the
Neo-3 donor chromosome (100 of 111 hybrids entered growth arrest).
|
Telomerase activity present in 21NT breast cancer cells and in
monochromosomal hybrid derivative clones (constructed using normal
human chromosomes 3, 8, or 20) was determined using the TRAP assay
(33). Hybrid colonies were lysed in situ to permit
analysis of telomerase in cells during their normal division phase
prior to reduction in growth rate and senescence. Strong repression of
telomerase activity was observed (Table 2) in 36 (78%) of 46
Hyg-3/21NT hybrids and in 20 (67%) of 30 Neo-3/21NT hybrids. In
contrast, TRAP assays performed on Hyg-8/21NT and Hyg-20/21NT hybrid
clones at an equivalent stage of development revealed low levels of
telomerase activity in only eight (11%) of 73 and two (8%) of
24 clones, respectively (Table 2);
these proportions
could be wholly accounted for by the interclonal variability in
telomerase activity in the parental 21NT cell line (13% of subclone
extracts possessed activity 10% or lower than that of a mass
culture extract). Telomerase activity was judged as being repressed if
TRAP ladder intensities were less than 10% of the signal generated
by an extract of a representative colony of 21NT parental cells
(quantified relative to the internal standard following
phosphorimaging). In practice, telomerase activity in the majority
of monochromosome 3 hybrids was less than 2% of the control. Fig.
1
shows typical results (TRAP gels and ImageQuanTTM
analysis of TRAP ladder signal intensities) obtained with five
Hyg-3/21NT hybrids (four of which were telomerase repressed), five
Neo-3/21NT hybrids (three of which were repressed) plus, as controls,
10 Hyg-8/21NT hybrids and the parent 21NT cell line. The 21NT extract
was derived from a colony of similar size to the hybrids and a 10-fold
dilution of the 21NT sample gave a corresponding reduction in the
signal intensity of the TRAP ladder. The range of telomerase activities
observed in the Hyg-8/21NT hybrids was similar to that detected in
randomly isolated 21NT colonies. Failure to generate TRAP ladders after
known positive extracts were treated with ribonuclease confirmed the
specificity of the assay for the ribonucleoprotein telomerase.
Reproducible standard internal control (SIC) signal intensities
eliminated the possibility that the weak TRAP ladders seen with a
number of extracts were caused by Taq polymerase inhibitors. In
addition, mixing weakly positive or negative samples with positive
samples produced no evidence for the presence of enzyme inhibitors in
the former extracts (data not shown).
|
|
PCR and FISH Analysis of Microcell Hybrids for the Presence of Transferred Chromosome 3 Material
Preliminary evidence of successful monochromosome 3 transfer into
21NT cells was obtained by PCR using primers directed at the selectable
marker (neo or Hytk). Amplification of the expected sequence occurred
in all of the DNA samples from 20 randomly selected clones tested (data
not shown). Insufficient division potential in hybrids destined to
undergo delayed growth arrest (i.e., the majority of hybrids) precluded
metaphase FISH analysis. In parental 21NT metaphase spreads (modal
chromosome number = 54), two large submetacentric chromosomes were
fully painted (Fig. 2). Chromosome 3 material was
also detected in single discrete regions of three other 21NT
chromosomes (two interstitial in location and one a translocation). Of
six immortal (i.e., revertant or segregant) Hyg-3/21NT hybrids tested,
four carried an additional submetacentric chromosome 3 (Fig. 2).
The
remaining two hybrids possessed novel subchromosomal fragments but were
without an additional intact chromosome 3 indicating that, in these
clones, the introduced copy of chromosome 3 had undergone breakage and
translocation of fragments to 21NT chromosomes.
|
Around one in 11 chromosome 3/21NT hybrids escaped the induced growth arrest after a prolonged growth crisis. A total of 36 of these revertant hybrids was established from 14 experiments. To investigate further the dependence of 21NT cell immortality on the presence of active telomerase, extracts from these hybrids were subjected to TRAP immediately following their emergence from crisis. Telomerase activity in all samples was comparable to that observed in the parental cell line. Similarly, all of 48 immortal Hyg-3/21NT hybrid clones that proliferated continuously in the absence of crisis proved strongly telomerase positive. Thus, in all cases, hybrid immortality was strongly linked to the presence of functional telomerase.
To obtain evidence of a causal link between telomerase repression and
delayed growth arrest in 21NT/monochromosome 3 hybrids, actively
proliferating Hyg-3/21NT and Neo-3/21NT hybrid colonies of unknown fate
were isolated by the use of cloning cylinders and tested simultaneously
for telomerase activity and division potential. Ten Hyg-20/21NT hybrid
colonies were included in the study as controls. One half of each
colony was lysed for TRAP analysis; the remaining cells were
successfully returned to culture with the exception of clone 16.4 that
failed to re-establish. The results are shown in Fig. 3.
Seven of 10 monochromosome 3 hybrids underwent
growth arrest and TRAP analysis of proliferating cell lysates revealed
telomerase to be repressed in all of these clones. Interestingly, one
initially repressed hybrid (clone 16.1) entered growth arrest but
reverted to immortality after crisis. This coincided precisely with
restoration of telomerase activity (data not shown). Functional
telomerase activity detected in two immortal clones (15.2 and 16.5)
that displayed no evidence of crisis was 35% and 120% of
parental cell levels, respectively. All 10 Hyg-20/21NT control hybrids
were immortal and nine of these had high levels of telomerase.
|
Based on our previous experience in mapping human cell growth arrest genes (27), immortal 21NT hybrids that arose during this investigation either (a) by continuous exponential proliferation of hybrid clones or (b) from a rare event during crisis (see above) were regarded as potential segregants that may have suffered loss of a critical telomerase repressor gene (or genes) on chromosome 3. Our recent analysis (28) of the structural integrity of a transferred normal chromosome 3 in rare, telomerase-positive, head and neck carcinoma (BICR31) hybrids revealed no evidence of deletions on 3q, the long arm of chromosome 3, but did show nonrandom deletions in the 3p21 region of the short arm. Therefore, in this study, deletion mapping of 21NT segregants was focused on the short arm of chromosome 3.
Allelotypes were obtained from 125 segregants recovered from 14
independent experiments, in which both the Hyg-3 and the Neo-3
chromosomes had been used to demonstrate telomerase repression and
delayed growth arrest in 21NT microcell hybrids. Thirty-eight
informative polymorphic markers were employed for mapping. Examples of
results for four such microsatellite markers (D3S3521, D3S1263,
D3S3623, and D3S3697) illustrating unambiguous allele discrimination by
Genescan analysis are shown in Fig. 4. For each
marker, scans (i) through (v) depict examples of (i) a hybrid with the
introduced allele retained, (ii) with the introduced allele deleted,
(iii) the allelotype of the 21NT parent (recipient) cell line, (iv)
that of DNA of different human origin (genomic DNA derived from a
chronic lymphocytic leukemia cell line), and (v) that of the donor
hybrid used (A9-Hytk3 or A9+3). The peaks representing the different
sequence-tagged site (STS) alleles are assigned a number (boxed in the
figure) by GeneScan.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have shown that telomerase can be strongly repressed in a well-characterized breast carcinoma cell line by the experimental introduction of a normal copy of human chromosome 3 and that repression is tightly associated with the induction of delayed growth arrest of the tumor cells. Additionally, by fine mapping the structural integrity of the introduced chromosome in the subset of monochromosome 3 hybrids that retained normal levels of telomerase activity, we have obtained strong evidence supporting the existence of a gene or genes that negatively regulate telomerase in normal cells. This analysis has defined two candidate regions on the short arm of chromosome 3 where telomerase repressor gene(s) may be located.
The definitive identification, and subsequent molecular cloning, of gene(s) that repress telomerase in human cells will have important consequences in relation to providing definitive answers to the two questions posed above. For example, the availability of cloned telomerase repressor sequences as probes should permit the mechanism of inactivation (i.e., mutational or epigenetic) of such genes in a variety of human cancers to be investigated directly and the timing of inactivation to be determined. In addition, the availability of vectors expressing telomerase repressor gene cDNA(s) will enable the efficacy of induced telomerase repression at arresting the growth of various human cancer cells to be properly evaluated and the rate at which immortal variants emerge in mass cultures of stably repressed transfectants to be accurately quantified.
The division potential of our monochromosome 3/21NT hybrids (10-18 population doublings) was consistent with the short mean TRF length we observed in the parent cells (around 3 kb). In a previous study, Ohmura et al. (42) also associated chromosome 3-induced growth arrest in RCC23 renal carcinoma cells with telomerase repression and telomere shortening, although the fate of only three clones was followed to terminal division. In their experiments, the greater division potential of these three RCC23 hybrids (23-43 population doublings) is not unexpected given the longer TRFs (around 6 kb) observed in the parent tumor cell line. Work to date, therefore, strongly points to the presence of one or more telomerase repressor genes, located on chromosome 3, which restore the program of replicative senescence in fully malignant human carcinoma cells.
Our experiments reported here have established a clear-cut relationship between telomerase repression and the induction of permanent growth arrest in a large number of independently derived monochromosome 3 hybrids. All hybrid clones with repressed telomerase either senesced or entered a pronounced growth crisis. No telomerase-negative clones were observed that retained immortality, and all clones that reverted to immortality via a rare event (after crisis) were telomerase positive. Alternative pathways of human telomere maintenance (possibly recombinational) have been implicated in a number of immortal cell lines, including a subset of simian virus 40 (SV40) large T-antigen-immortalized diploid fibroblasts (43) and a small proportion of human tumor cell lines (44). These cell lines generally have low or undetectable levels of telomerase activity and hypervariable telomere lengths. Clearly, from a therapeutic perspective, it is important to know, for a representative panel of tumor cell populations, whether and at what frequency such variants emerge under conditions of stringent telomerase repression. In this study, we observed no evidence of reversion to immortality via an alternative (nontelomerase-related) pathway in nearly 107 cumulative hybrid cell divisions. It should be stressed that our experimentally induced (and, in many cases, strong) repression of telomerase activity was achieved by only a single normal copy of chromosome 3. Therefore, in normal diploid somatic cells, it is reasonable to expect that both alleles of a telomerase repressor gene would need to be functionally deleted for telomerase to be activated. Further work with a telomerase repressor gene cDNA vector (with which multiple-copy transfection could be readily achieved) or with powerful chemical inhibitors of telomerase will facilitate quantification of events (e.g., leading to activation of an alternative pathway) occurring at rates below 10-7 per cell division.
The two nonrandom genetic losses (at 3p21.3-p22 and 3p12-21.1) identified in this investigation, by allelotype analysis of rare immortal revertants, involve regions that commonly suffer loss of heterozygosity (LOH) and homozygous deletion in human cancers. The large (proximal) deletion (SCDR-2) at 3p12-p21.1 defined in six hybrids spans breast cancer-specific allele losses and several nonrandom homozygous deletions identified in breast, cervix, colon, lung, and renal carcinomas (45). The putative tumor suppressor gene FHIT (fragile histidine triad) has been mapped to 3p14.2 (46) and therefore could in theory be considered as a candidate, although the role of FHIT as a tumor suppressor gene is unclear (47). Interestingly, a deletion involving 3p13-p14.2, associated with human papillomavirus E6- and E7-immortalization of human uroepithelial cells, also overlaps this region (48). Similarly, Fusenig and Boukamp (49) have associated this region of chromosome 3p with immortalization in the minimally transformed human skin keratinocyte cell line HaCaT (50).
The distal deletion (SCDR-1) at 3p21.3-p22 defined by six other hybrid segregants is also of possible relevance to human cancer. Two clones were exclusively deleted at D3S3623 and the informativeness of adjacent markers in one of these indicated a minimal region that overlaps discrete homozygous deletions identified in lung cancer cell lines and tumors (51-53). This region is distal to a cluster of overlapping lung cancer-specific deletions at 3p21.3 that has also been associated with tumor suppressive activity in mouse A9 cell hybrids carrying a fragment of human chromosome 3 (54). Homozygous deletions at 3p21.3-p22 have not been reported in breast cancers. However, allelotyping studies have identified nonrandom LOHs involving this region (55) that were associated with tumor aneuploidy and poor patient prognosis.
Allelotype analysis of the head and neck carcinoma cell line, BICR31, in which we demonstrated chromosome 3-mediated repression of telomerase (28), revealed that most 3p microsatellites had undergone LOH (56). Although the absence of matched normal DNA precluded definitive allelotyping of the 21NT parent cell line, the great majority of markers were found to be heterozygous, even though G banding (29) and FISH analysis indicated that extensive rearrangement of chromosome 3 had occurred. However, many tumor types, including breast cancer, have shown allelic losses in both regions of deletion identified in our chromosome 3/21NT hybrid segregants.
Our observation that two contiguous interstitial 3p deletions (rather than a single nonrandom genetic loss) were associated with immortality and functional telomerase was somewhat unexpected. The results could indicate the presence of two genes necessary for repressing telomerase, either of which may be deleted to cause derepression, and which encode elements of a single telomerase regulatory pathway. Alternatively, only one (possibly 3p21.3-p22) of the regions may represent the subchromosomal location of a single telomerase repressor, particularly since 3p14 has been well documented as a fragile site (47). This notwithstanding, we believe that the mapping information we have obtained to date provides sufficient grounds to justify the initiation of a positional cloning program to isolate the gene(s). To this end, we are currently screening (by spheroplast transfer) yeast artificial chromosomes (57) from contigs mapping to the two regions of interest for telomerase repressive function in 21NT cells.
![]() |
NOTES |
---|
Supported by UK Cancer Research Campaign Project Grant SP2133/0202 and Contract F14PCT950008 from the European Commission (Nuclear Fission Safety Programme).
We thank Vimla Band for providing 21NT cells, Paul Simpson for guidance with fluorescence in situ hybridization, and Andrew Silver for assistance with telomere length determinations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Newbold RF, Overell RW. Fibroblast immortality is a prerequisite for transformation by EJ c-Ha-ras oncogene. Nature 1983;304:648-51.[Medline]
2 Rheinwald JG, Beckett MA. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultures from human squamous cell carcinomas. Cancer Res 1981;41:1657-63.[Abstract]
3 Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res 1961;25:585-621.
4 Goldstein S. Replicative senescence: the human fibroblast comes of age. Science 1990;249:1129-33.[Medline]
5 Newbold RF, Overell RW, Connell JR. Induction of immortality is an early event in malignant transformation of mammalian cells by carcinogens. Nature 1982;299:633-5.[Medline]
6 Namba M, Nishitani K, Hyodoh F, Fukushima F, Kimoto T. Neoplastic transformation of human diploid fibroblasts (KMST-6) by treatment with 60Co gamma rays. Int J Cancer 1985;35:275-80.[Medline]
7 Newbold RF, Cuthbert AP, Themis M, Trott DA, Blair AL, Li W. Cell immortalization as a key, rate-limiting event in malignant transformation: approaches toward a molecular genetic analysis. Toxicol Lett 1993;67:211-30.[Medline]
8 Sager R. Genetic suppression of tumor formation. Adv Cancer Res 1985;44:43-68.[Medline]
9 Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011-5.[Medline]
10 Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985;43(2 Pt 1):405-13.[Medline]
11 Watson JD. Origin of concatemeric T7 DNA. Nat New Biol 1972;239:197-201.[Medline]
12 Markarov VL, Hirose Y, Langmore JP. Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening.Cell 1997;88:657-66.[Medline]
13 von Zglinicki T, Saretzki G, Docke W, Lotze C. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res 1995;220:186-93.[Medline]
14 Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, et al. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 1992;11:1921-9.[Abstract]
15 Harley CB. Telomere loss: mitotic clock or genetic time bomb? Mutat Res 1991;256:271-82.[Medline]
16 Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 1997;90:785-95.[Medline]
17
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin
GB, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 1998;279:349-52.
18 Holzman D. Geron extends human cell lifespan with telomerase. Genet Eng News 1998;18:1.
19
Nakamura TM, Morin GB, Chapman KB, Weinrich SL,
Andrews WH, Lingner J, et al. Telomerase catalytic subunit homologs from fission yeast and
human. Science 1997;277:955-9.
20
Harrington L, Zhou W, McPhail T, Oulton R, Yeung DS, Mar
V, et al. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes Dev 1997;11:3109-15.
21
Kilian A, Bowtell DD, Abud HE, Hime GR, Venter DJ, Keese
PK, et al. Isolation of a candidate human telomerase catalytic subunit gene, which reveals
complex splicing patterns in different cell types. Human Mol Genet 1997;6:2011-9.
22 Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu CP, et al. The RNA component of human telomerase. Science 1995;269:1236-41.[Medline]
23
Harle-Bachor C, Boukamp P. Telomerase activity in the
regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived
skin keratinocytes. Proc Natl Acad Sci U S A 1996;93:6476-81.
24 Greaves M. Is telomerase activity in cancer due to selection of stem cells and differentiation arrest? Trends Genet 1996;12:127-8.[Medline]
25 Shay JW, Wright WE. The reactivation of telomerase activity in cancer progression. Trends Genet 1996;12:129-31.[Medline]
26 Cuthbert AP, Trott DA, Ekong RM, Jezzard S, England NL, Themis M, et al. Construction and characterization of a highly stable human:rodent monochromosome hybrid panel for genetic complementation and genome mapping studies. Cytogenet Cell Genet 1995;71:68-76.[Medline]
27 England NL, Cuthbert AP, Trott DA, Jezzard S, Nobori T, Carson DA, et al. Identification of human tumour suppressor genes by monochromosome transfer: rapid growth-arrest response mapped to 9p21 is mediated solely by the cyclin-D-dependent kinase inhibitor gene, CDKN2A (p16INK4A). Carcinogenesis 1996;17:1567-75.[Abstract]
28 Newbold RF. Genetic control of telomerase and replicative senescence in human and rodent cells. Ciba Found Symp 1997;211:177-97.[Medline]
29 Band V, Zajchowski D, Swisshelm K, Trask D, Kulesa V, Cohen C, et al. Tumor progression in four mammary epithelial cell lines derived from the same patient. Cancer Res 1990;50:7351-7.[Abstract]
30 Ning Y, Lovell M, Taylor L, Pereira-Smith OM. Isolation of monochromosomal hybrids following fusion of human diploid fibroblast-derived microcells with mouse A9 cells. Cytogenet Cell Genet 1992;60:79-80.[Medline]
31 Ning Y, Lovell M, Cooley LD, Pereira-Smith OM. "PCR karyotype" of monochromosomal somatic cell hybrids. Genomics 1993;16:758-60.[Medline]
32 Newbold RF, Cuthbert AP. Mapping human senescence genes using interspecific monochromosome transfer. In: Freshney RI, Freshney MG, editors. Culture of immortalized cells. New York (NY): Wiley-Liss; 1996. p. 53-75.
33 Wright WE, Shay JW, Piatyszek MA. Modifications of a telomeric repeat amplification protocol (TRAP) result in increased reliability, linearity and sensitivity. Nucleic Acids Res 1995;23:3794-5.[Medline]
34 Dib C, Faure S, Fizames C, Samson D, Drouot N, Vignal A, et al. A comprehensive genetic map of the human genome based on 5264 microsatellites. Nature 1996;380:152-4.[Medline]
35
Collins A, Frezal J, Teague J, Morton NE. A metric map of
humans: 23,500 loci in 850 bands. Proc Natl Acad Sci U S A 1996;93:14771-5.
36 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York (NY): Cold Spring Harbor Laboratory Press; 1989.
37 Edington KG, Loughran OP, Berry IJ, Parkinson EK. Cellular immortality: a late event in the progression of human squamous cell carcinoma of the head and neck associated with p53 alteration and a high-frequency of allele loss. Mol Carcinog 1995;13:254-65.[Medline]
38 Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 1995;92:9363-7.[Abstract]
39 Starling JA, Maule J, Hastie ND, Allshire RC. Extensive telomere repeat arrays in mouse are hypervariable. Nucleic Acids Res 1990;18:6881-8.[Abstract]
40
de Lange T. Activation of telomerase in a human tumor. Proc Natl Acad Sci U S A 1994;91:2882-5.
41 Shay JW, Wright WE, Werbin H. Loss of telomeric DNA during aging may predispose cells to cancer. Int J Oncol 1993;3:559-63.
42 Ohmura H, Tahara H, Suzuki M, Ide T, Shimizu M, Yoshida MA, et al. Restoration of the cellular senescence program and repression of telomerase by human chromosome 3. Jpn J Cancer Res 1995;86:899-904.[Medline]
43 Bryan TM, Englezou A, Gupta J, Bacchetti S, Reddel RR. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J 1995;14:4240-8.[Abstract]
44 Bryan TM, Englezou A, Dalla-Pozza L, Dunham MA, Reddel RR. Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Nat Med 1997;3:1271-4.[Medline]
45 Kok K, Naylor SL, Buys CH. Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. Adv Cancer Res 1997;71:27-92.[Medline]
46 Ohta M, Inoue H, Cotticelli MG, Kastury K, Baffa R, Palazzo J, et al. The FHIT gene, spanning the chromosome 3p14.2 fragile site and acid renal carcinoma-associated t(3-8) breakpoint, is abnormal in digestive-tract cancers. Cell 1996 ;84:587-97.[Medline]
47
Mao L. Tumor suppressor genes: does FHIT fit?
[editorial]. J Natl Cancer Inst 1998;90:412-4.
48
Vieten L, Belair CD, Savelieva L, Julicher K, Brocker F,
Bardenheuer W, et al. Minimal deletion of 3p1314.2 associated with immortalization of
human uroepithelial cells. Genes Chromosomes Cancer 1998;21:39-48.[Medline]
49 Fusenig NE, Boukamp, P. Multiple stages and genetic alterations govern immortalization, malignant transformation, and tumor progression of human skin keratinocytes. Mol Carcinog. In press 1998.
50 Boukamp P, Stanbridge EJ, Foo DY, Cerutti PA, Fusenig NE. C-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy. Cancer Res 1990;50:2840-7.[Abstract]
51 Yamakawa K, Takahashi T, Horio Y, Murata Y, Takahashi E, Hibi K, et al. Frequent homozygous deletions in lung cancer cell lines detected by a DNA marker located at 3p21.3-p22. Oncogene 1993;8:327-30.[Medline]
52 Roche J, Boldog F, Robinson M, Robinson L, Varella-Garcia M, Swanton M, et al. Distinct 3p21.3 deletions in lung cancer and identification of a new human semaphorin. Oncogene 1996;12:1289-97.[Medline]
53 Murata Y, Tamari M, Takahashi T, Horio Y, Hibi K, Yokoyama S, et al. Characterization of an 800 kb region at 3p22-p21.3 that was homozygously deleted in a lung cancer cell line. Hum Mol Genet 1994;3:1341-44.[Abstract]
54 Todd MC, Xiang RH, Garcia DK, Kerbacher KE, Moore SL, Hensel CH, et al. An 80 kb P1 clone from chromosome 3p21.3 suppresses tumor growth in vivo. Oncogene 1996;13:2387-96.[Medline]
55 Eiriksdottir G, Bergthorsson JT, Sigurdsson H, Gudmundsson J, Skirnisdottir S, Egilsson V, et al. Mapping of chromosome 3 alterations in human breast cancer using microsatellite PCR markers: correlation with clinical variables. Int J Oncol 1995;6:369-75.
56 Loughran O, Clark LJ, Bond J, Baker A, Berry IJ, Edington KG, et al. Evidence for the inactivation of multiple replicative lifespan genes in immortal human squamous cell carcinoma keratinocytes. Oncogene 1997;14:1955-64.[Medline]
57 Huxley C. Transfer of YACs to mammalian cells and transgenic mice. In: Setlow JK, editor. Genetic engineering. New York (NY): Plenum Press; 1994. p. 65-91.
Manuscript received May 1, 1998; revised October 19, 1998; accepted October 30, 1998.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |