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Technical Advance |
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From the Graduate Program of Biochemistry, Cell and MolecularBiology,* the Brady UrologicalInstitute, the Departments ofPathology and Oncology,¶ The Johns Hopkins University School of Medicine, and The Johns HopkinsUniversity Bloomberg School of Public Health,|| Baltimore,Maryland; and The MathWorks Incorporated,Natick, Massachusetts
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Abstract |
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10 to 15). Thus, small tissue samples, including
clinical biopsies, can be easily accommodated. In
addition, the cells under study need not be actively cycling
and there is no requirement for tissue disaggregation or cell culture.
This method provides a more accurate assessment of telomere lengths
than Southern blotting because confounding contributions from undesired
cell types within tissue samples are avoided. Using this
technique, we were able to perform the first comparison of
relative telomere lengths in matched tumor versus normal
epithelial cells within archival human prostate
tissues.
Telomeres are essential structures composed of specialized terminal DNA sequence repeats complexed with telomere-binding proteins located at the ends of every human chromosome.6 Telomeric DNA tracts that, in humans are typically composed of 1000 to 2000 tandem repeats of the hexanucleotide sequence TTAGGG, are dynamic entities. Telomeres are subject to shortening during cell division because of their incomplete replication during DNA synthesis (the end replication problem).7-10 In addition, telomere shortening may also result from unrepaired single-strand breaks caused by oxidative damage.11-13 Conversely, telomeres may increase in length, either through action of the enzyme telomerase,14,15 or, at times, via amplification or genetic recombination events.16-20
Telomere maintenance is a critical factor in allowing continuous cell proliferation.21-24 This is underscored by the fact that at least 85% of human cancers, and a majority of established cell lines, possess active telomerase, whereas activity is rarely detected in normal differentiated human somatic cells.25-27 Also, forced expression of telomerase in cells destined to undergo senescence results in their immortalization.21-24
Recently, Lansdorp and colleagues28-30 developed a fluorescence in situ hybridization (FISH)-based method for quantitative telomere length measurement applicable to metaphase chromosome spreads. This technique has since been applied to interphase hematopoietic cells, cells in culture, and flow cytometry.31-36 This method has also been applied to mouse skin samples.37,38 However, the telomeres of inbred mouse strains are typically an order of magnitude longer than their human counterparts and no validation or detailed method has been presented for the quantification of telomeres in paraffin-embedded tissue samples.
Inspired by the Maude Abbott lecture delivered by the late Roger C. Haggitt, M.D., at the 2000 Annual Meeting of the United States and Canadian Academy of Pathology in New Orleans, we adapted the Lansdorp method28-30 and validated it for human formalin-fixed paraffin-embedded archival material. We also present the novel coupling of this technique with simultaneous localization of bound antibodies (immunofluorescence) allowing us to assess telomere lengths in specific cells of interest within tissue sections.
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Materials and Methods |
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The established human prostate cancer cell line LNCaP (clone FGC,
androgen-sensitive/nodal metastasis origin) was obtained from the
American Type Culture Collection (ATCC) (catalog no. CRL-1740; ATCC,
Rockville, MD). Androgen-independent sublines of the LNCaP.FGC human
prostate cell line, as well as the parental androgen-sensitive control
line from which they were derived, were generously provided by Dr. Joel
Nelson (Department of Urology, University of Pittsburgh School of
Medicine, Pittsburgh, PA). Cells were grown in
25-cm2
or 75-cm2
plastic
tissue-culture flasks (Falcon/Becton and Dickenson Co., Franklin Lakes,
NJ.) in a humidified incubator (37°C, 84% humidity, 5%
CO2/ambient air atmosphere) either in RPMI-1640
medium with phenol red supplemented with 10% fetal bovine serum (Life
Technologies, Inc., Rockville, MD), or in hormone-depleted medium
consisting of RPMI-1640 without phenol red supplemented with 10%
charcoal-stripped serum (HyClone Laboratories Inc., Logan, UT). Cells
were passaged weekly at 75% confluence using 0.25% trypsin/0.02%
ethylenediaminetetraacetic acid (Life Technologies, Inc.) for
detachment, followed by inactivation with serum-containing media
(normal or charcoal-stripped as appropriate) and seeding of new flasks
using a split ratio of 1:10. All lines were tested using a polymerase
chain reaction-based mycoplasma detection kit obtained from the ATCC
(catalog no. 90-1001K) and found to be free of mycoplasma
contamination. Additional cell lines used in this study were also
obtained from the ATCC. Media and passaging procedures followed the
accompanying ATCC recommendations.
Cell Block Preparation
Cell cultures were grown as described to 50 to 75% confluence, detached, and fixed in 10% (v/v) phosphate-buffered formalin (J.T. Baker, Phillipsburg, NJ) for 15 hours at 25°C. Fixed cells were pelleted by centrifugation at 500 x g for 10 minutes, washed once in 1x phosphate-buffered saline (PBS), and pelleted again. The cell pellets were resuspended in an equal volume of 0.8% agarose at 42°C. The agarose/cell mixture was then transferred to a 0.6-ml microfuge tube and allowed to solidify. Agarose/cell plugs were then embedded into the same paraffin block, thus producing a single block containing all of the LNCaP sublines possessing different telomere lengths.
Tissue Samples
Human tissues were obtained from the department of surgical pathology at The Johns Hopkins University School of Medicine. Two tissue microarrays were constructed for in situ hybridization/immunostaining. The first consisted of an assortment of normal human tissues obtained from excess tissues from surgical specimens from adults. The second consisted of matched samples of prostate carcinoma and normal prostate epithelium from radical prostatectomy specimens (n = 20 cases). Normal tissues were fixed in 10% neutral-buffered formalin and subjected to standard processing and paraffin embedding. Radical prostatectomy specimens were obtained fresh from the operating room. Specimens were immediately inked, processed by vigorous injection of formalin, and followed by microwave treatment.39 Prostates were then sectioned and tissues were further fixed for 1 to 4 hours and then subjected to standard tissue processing and paraffin embedding. For prostate specimens, patient ages varied between 50 and 70 years (median, 60.2 years). For tissue microarray construction, representative areas containing morphologically defined lesions or normal tissues were circled on the glass slides and used as a template. Arrays were constructed using a manual Tissue Puncher/Arrayer (Beecher Instruments, Silver Spring, MD) as previously described.40 For the normal tissue array, a total of 114 tissue cores (0.6-mm diameter) were arrayed in a single block using a spectrum of different human tissues. For the prostate tissue microarray, a total of 200 samples (0.6 mm in diameter cores) from 20 patients were arrayed into a single block using five samples each of tumor and normal tissue from each patient. The tissue microarray blocks were sectioned at 4 µm and stained by telomere-FISH (TEL-FISH) and/or immunofluorescence.
TEL-FISH and Telomere/Immunostaining-FISH (TELI-FISH)
Deparaffinized slides were hydrated through a graded ethanol series, placed in deionized water, followed by deionized water plus 0.1% Tween-20 detergent. Slides were then placed in citrate buffer (catalog no. H-3300; Vector Laboratories, Burlingame, CA), paired to form capillary gaps and steamed for 14 minutes (Black and Decker Handy Steamer Plus; Black and Decker Corp., Towson, MD), removed, and allowed to cool at room temperature for 5 minutes. Slides were then placed in PBS with Tween (PBST) (catalog no. P-3563; Sigma Chemical Co., St. Louis, MO) for 5 minutes. Slides were then placed in a protease solution consisting of 0.5 mg/ml of protease (Protease Type VIII, catalog no. P-5380; Sigma) in PBST for 1 minute at room temperature. Slides were thoroughly rinsed with deionized water, followed by 95% ethanol for 5 minutes, and then air-dried. Twenty-five µl of a Cy3-labeled telomere-specific peptide nucleic acid (PNA) [0.3-µg/ml PNA in 70% formamide, 10 mmol/L Tris, pH 7.5, 0.5% B/M Blocking reagent (catalog no. 1814-320; Boehringer-Mannheim, Indianapolis, IN)] was applied to the sample, which was then coverslipped, and denaturation was performed by incubation for 4 minutes at 83°C. Slides were then moved to a dark, closed container for hybridization at room temperature for 2 hours. Coverslips were then carefully removed and the slides were washed twice in PNA wash solution [70% formamide, 10 mmol/L Tris, pH 7.5, 0.1% albumin (from 30% albumin solution, catalog no. A-7284; Sigma)], followed by 3 x 5-minute washes in TBS. At this point slides were either counterstained with 4'-6-diamidino-2-phenylindole (DAPI) (250 ng/ml in deionized water, Sigma Chemical Co.) for 1 minute at room temperature, mounted with Prolong anti-fade mounting medium (catalog no. P-7481; Molecular Probes Inc., Eugene, OR), and imaged, or were processed for indirect immunofluorescence as follows. Slides were rinsed in PBST followed by application of primary antibody (anti-cytokeratin antibody 34ßE12, catalog no. 30904; Enzo Diagnostics, Farmingdale, NY) and incubated overnight at 4°C. Slides were then rinsed in PBST followed by application of fluorescent secondary antibody labeled with Alexa Fluor 488 (Molecular Probes) diluted 1:1000 in Dulbecco’s PBS, and incubated at room temperature for 30 minutes. Slides were then rinsed in PBST, and thoroughly rinsed in deionized water. Slides were drained and stained with DAPI. Slides were then rinsed well in deionized water, drained, mounted with Prolong anti-fade mounting medium (catalog no. P-7481, Molecular Probes Inc.), coverslipped, and imaged or stored at 4°C until used. Other primary antibodies used included: rabbit polyclonal anti-prostatic acid phosphatase (1:20,000; DAKO Corp., Carpinteria, CA), mouse monoclonal anti-p63 cocktail (cocktail of clones 4A4 and 63P02; Lab Vision, Fremont CA), and rabbit polyclonal anti-glutathione S-transferase pi (1:2000, DAKO). The PNA probe complementary to the mammalian telomere repeat sequence was obtained from Applied Biosystems (Framingham, MA), and has the sequence (N-terminus to C-terminus) CCCTAACCCTAACCCTAA with an N-terminal covalently linked Cy3 fluorescent dye. The analogous specificity control PNA, complementary to the Caenorhabditis elegans telomere sequence, was a generous gift from Dr. Carol Greider (Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD).
Telomere Length Measurements by Southern Blotting
Telomere length measurements by the telomere repeat fragment (TRF) Southern technique followed previously published methods.41 Proteinase K, phenol, and DNA molecular weight markers were obtained from Life Technologies, Inc. Restriction enzymes MspI (catalog no. 106S) and RsaI (catalog no. 167S) were obtained from New England Biolabs, Beverly, MA. Unless otherwise stated, chemicals routinely used in the preparation of buffers, and so forth were purchased from Sigma Chemical Co. In brief, high-molecular weight genomic DNA was prepared from cell pellets and 1 µg of each purified DNA stock was then subjected to agarose gel electrophoresis to check for evidence of degradation or shearing. In all samples >99% of ethidium bromide-staining material was 23 kbp or greater in length. One to two µg of this DNA was then digested to completion with 1 U each of RsaI and MspI as per the manufacturer’s instructions. DNA fragments and appropriate molecular weight standards were then separated by electrophoresis, blotted, and probed with a 24-mer oligonucleotide probe complementary to the telomeric repeat sequence (TTAGG)4 (Bio-Synthesis Inc., Lewisville, TX) using standard methods.
Phosphorimage exposures of telomere-probed Southern blots were analyzed with the program Image Quant (Molecular Dynamics, Sunnyvale, CA). The digitized signal data were then transferred to Microsoft Excel (Redmond, WA), and were the basis for calculating mean TRF length using the formula: L = (ODi)/(ODi/Li), where ODi = integrated signal intensity at position i, and Li = length of DNA fragment in position i. Mean fragment lengths were calculated throughout a range of 2 to 20 kbp. To correct for discrepancies in DNA loading and to confirm complete digestion, membranes were stripped and rehybridized with the microsatellite oligonucleotide probe (CAC)5.
Microscopy
Slides were imaged with a Zeiss Axioskop epifluorescence microscope equipped with short-arc mercury lamp illumination (Carl Zeiss Inc., Thornwood, NY) and a x100/1.4 NA oil immersion Neofluar lens. Fluorescence excitation/emission filters were as follows: Cy3 excitation, 546 nm/10 nm BP; emission, 578 nm LP (Carl Zeiss Inc.); DAPI excitation, 330 nm; emission, 400 nm via an XF02 fluorescence set (Omega Optical, Brattleboro, VT); Alexa Fluor 488 excitation, 475 nm; emission, 535 nm via a combination of 475RDF40 and 535RDF45 filters (Omega Optical). Fluorescent images were captured with a cooled charge-coupled device camera (Micro MAX digital camera; Princeton Instruments, Trenton, NJ). Integration times typically ranged from 500 ms to 800 ms for Cy3 signal capture, 50 ms to 100 ms for DAPI counterstain, and 100 ms to 200 ms for Alexa Fluor 488-conjugated antibodies. At the beginning of an imaging session, optimum exposure times were determined, and all exposure times were held constant thereafter, such that all cells within a comparison set experienced identical exposure times. In all cases, telomeric signals were within the linear response range of the charge-coupled device camera, which was confirmed by use of fluorescent microbead intensity standards (InSpeck microscope image intensity calibration fluorescent microspheres, catalog no. I-7223; Molecular Probes, Inc.). Assessment of decreases in signal intensity from multiple telomeric spots within the same field after sequential, timed exposures indicated that the extent of signal loss because of photobleaching of the hybridized Cy3 telomeric probe was linear, and <4% per 1000 ms.
Image Analysis
Quantitation of the digitized fluorescent telomere signals was accomplished by use of a semiautomated algorithm written with the image analysis software package IPLabs (Scanalytics, Inc., Fairfax, VA). For a given x1000 image, cells that contained detectable telomere signals that were in sharp focus were chosen for analysis. Image processing was performed as follows. For a given nucleus, the raw Cy3 telomere image was filtered with a Laplacian of Gaussian filter after background subtraction. This corrected image was then segmented on gray-value thresholding for contouring of telomeric spots that were then binarized; creating a mask that was applied to the original telomere fluorescence data. Telomeric signals identified by the segment mask, having sizes greater than the threshold for background noise, and that were contained within the area inscribed by the nuclear DAPI signal, were then measured, and the data for each telomeric spot within that particular nucleus was tabulated. The total DAPI fluorescence signal for each nucleus was also quantified. Tabulated data were then exported to Microsoft Excel (Microsoft Corp.) and STATA 6.0 for Microsoft Windows (Stata Corp., College Station, TX) or SAS (SAS Institute, SAS Institute, Cary, NC) for further data handling and statistical analysis, respectively.
For human prostate tissue samples, cancer cells were differentiated from normal cells by first identifying them on hematoxylin- and eosin-stained adjacent sections. Using the fluorescence microscope, cancer tissues were distinguished on the actual TELI-FISH slide by their epithelial appearance and lack of a basal epithelial layer, as assessed by staining with basal cell-specific anti-cytokeratin antibody 34ßE12 using a separate color for localization (Signet Laboratories, Dedham, MA). Normal appearing glands that contained an intact basal cell layer within the same tissue section served as internal positive controls for 34ßE12 staining.
Data Analysis
For each nucleus, the individual Cy3 telomere signals were summed and this total was divided by the total DAPI fluorescence signal for that nucleus, thus correcting for potentially confounding differences in nuclear cutting planes and ploidy. For the LNCaP panel, as well as additional validation sets, this procedure was performed using data from a minimum of 10 nuclei per cell line/subline. These ratios were multiplied by 10,000 and plotted against the mean TRF values determined separately by Southern analysis on these same cell populations. Pair wise comparisons (Wilcoxon rank sum) between the ratios for these cell lines were conducted and plotted using STATA 6.0.
For patient samples (Table 1)
,
comparisons of the mean ratio of telomeric signal to DAPI between tumor
and normal tissue were done using the t-test (SAS).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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To develop a set of positive controls for telomere length
assessment in tissue sections, we subjected a series of LNCaP prostate
cancer cell sublines to androgen withdrawal for varying lengths of
time, resulting in four sublines, each containing a different mean
telomere repeat length.42
To simulate standard pathology
slides, cell pellets from each cell subline were fixed with formalin
and processed into paraffin. A paraffin block containing all four
sublines was sectioned and stained by TEL-FISH. Representative images
of the hybridization reaction localizing the fluorescent telomeric PNA
probe in these cells are shown in Figure 1
. The four sublines were qualitatively
scored, at the microscope, for relative telomere length by four
separate observers in a blinded manner, and each observer correctly
determined the rank order [the probability of correctly ordering the
cell lines in this manner by chance is (1 in 4)4
=
0.000003]. Control hybridizations using a PNA probe complementary to
the C. elegans telomere sequence (TTAGGC) failed to produce
any discrete nuclear signals, thus, confirming the specificity of the
hybridization reactions using the PNA probe complementary to the
mammalian sequence (data not shown).
|
. Pairwise comparisons (Wilcoxon rank
sum) showed that all sublines differed significantly from one another
(all P values <0.01). Linear least squared fitting of the
data showed that the measurements of telomere lengths by TEL-FISH were
positively correlated (r = 0.997) with data
obtained by the standard Southern blot technique (TRF analysis)
performed on these same cell populations (Figure 2C
, large circles).
The linear trend line intersects the x axis at a TRF length
of 2 kbp. This value is in good agreement with published estimates
of the amount of subtelomeric DNA present in telomeric restriction
fragments.10,43-46
Because these subtelomeric sequences
are not composed of the canonical telomere repeat, the PNA probe does
not hybridize to them and, therefore, they do not interfere with
telomere length analysis by TEL-FISH. In a separate series using five
different cell lines, including two of nonprostatic origin, a positive
correlation was again obtained (r = 0.946) with
a nearly identical x intercept. The combined results of
these assays, representing nine different lines/sublines in total, are
plotted together in Figure 2C
.
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Telomeres were readily visualized in various human formalin-fixed
paraffin-embedded tissues (Figure 3
; a to
c). An optimized protocol for staining multiple human tissues in a
single tissue microarray was developed using a wide range of tissue
types, as well as tissue microarrays containing matched normal and
cancerous human prostate samples. We found that steam heating followed
by a brief protease digestion step gave bright telomere signals in both
the epithelium and stroma from the following tissues that were tested:
testis, urinary bladder, prostate, kidney, ovary, endometrium,
fallopian tube, uterine cervix, breast, lung, skin, tonsil, lymph node,
laryngeal epithelium, thymus, spleen, pancreas, gall bladder, thyroid,
peripheral nerve, colon, ileum, stomach, esophagus, liver, skeletal
muscle, and smooth muscle. Although cytoplasmic background
autofluorescence was generally minimal, at times the liver and skeletal
muscle showed significant levels of this background signal. However,
even in those tissues, this did not hamper the interpretation of
telomere signals because the nuclei showed very little
autofluorescence.
|
; compare telomere signals in
the host stroma and vasculature to those in the resident human prostate
cancer xenograft). Combined TEL-FISH and Immunofluorescence (TELI-FISH)
Using the TRF-Southern method, telomeres have previously been
shown to be shorter in human prostate cancer versus matched
adjacent normal tissues.41,50,51
However, these previous
studies used a complex mixture of cell types including stromal,
endothelial, and inflammatory cells whose telomeres were averaged using
the Southern blotting method. Thus, the telomere length estimates in
such complex mixtures are not necessarily reflective solely of
epithelial cells in either the tumor or the normal
sample.50
Because prostate cancer is derived from
epithelial cells, it is of interest to compare telomere lengths in
prostate cancer cells to non-neoplastic prostate epithelial cells
in situ. Although the fluorescent telomeric signals in
formalin-fixed sections are easily discernable, fluorescence microscopy
limits one’s ability to distinguish specific cell and tissue types. To
help facilitate the delineation of normal prostate epithelium from
adenocarcinoma, we wished to combine telomere length assessment
in situ with immunofluorescence. We therefore combined the
TEL-FISH assay with immunofluorescence (TELI-FISH). Figure 3, e and f
,
shows representative images of normal and cancerous human prostate
glands, respectively, from a tissue microarray stained for basal
cell-specific cytokeratins (green, antibody 34ßE12), DNA (blue,
DAPI), and telomeres (red, Cy3-labeled anti-telomeric PNA). As can be
seen, basal cells are easily identifiable in the normal gland, in the
green channel, while strong telomere signals are detected in the red
channel; in this specimen tumor cells show little or no telomeric
signal whereas the normal epithelial cells show bright telomere
signals. Also, bright telomere signals were identified in the prostatic
stroma. The performance of other antibodies, including those specific
for prostatic acid phosphatase, glutathione S-transferase
pi, and p63, was also assessed. To minimize loss of antigen, we omitted
the protease digestion step of the protocol. We saw no loss of
telomeric signals because of omission of this step, and all antibodies
performed as expected in combination with the PNA probe (data not
shown). An additional 16 prostate cancer cases were evaluated by the
TELI-FISH procedure using a tissue microarray consisting of matched
tumor and normal tissues from prostate cancer patients. Of the 17 total
cases examined, 10 had statistically significantly shorter telomeres in
the tumor than in the adjacent normal tissue (Table 1
,
P < 0.01). Tumor telomeres were also shorter in three
additional cases, however these differences were not statistically
significant (Table 1)
. In two other cases, the normal and tumor
telomere lengths were essentially identical, whereas in the remaining
two cases; the tumor telomeres were statistically significantly longer
than those in the corresponding normal epithelium (Table 1)
.
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Discussion |
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5 x
105
cells minimum), which is then digested with
one or more frequent cutting restriction endonucleases that do not
recognize the telomere repeat sequence. The resulting telomeric
restriction fragments (TRFs) are then sized by electrophoretic
separation, followed by Southern blotting and probing with a
telomere-specific probe. On visualization, one sees a smeared
distribution pattern of TRFs because, in part, of the dynamic nature of
telomeric DNA that produces both intercellular and intracellular
heterogeneity in telomere lengths. In addition, variation exists in the
amount of subtelomeric sequences (composed in part of telomere-related
sequences) that remain undigested, inflating the length estimate of
true telomeric repeats by 2 to 4 kbp.10,43-46
Other
drawbacks of this technique include the fact that information on the
distribution of telomere lengths at the level of single cells is not
obtainable, nor is one able to identify specific cell subtypes within
the tissue under study—a notable problem in most tissue samples, where
cellular heterogeneity is the rule, and different proportions of cell
subtypes having different telomere lengths may easily skew the
results.50
In addition, there is no universally agreed on
standard for the reporting of telomere length estimates from TRF
analysis, with the distribution mean, median, and peak intensity all
having been used.
Telomere quantitation by slot-blot represents a variation of the above
Southern technique that allow for the use of far fewer cells (1000
cells) as well as omission of the DNA isolation step.57,58
Unfortunately, information on the telomere size distribution is lost in
this approach because the measurement produces a single value
representing the total amount of telomeric DNA present in the sample.
FISH technology, using telomere-specific oligonucleotide probes, has also been used to quantitate telomeres, overcoming several of the disadvantages inherent in Southern-based techniques. Initially, this was performed semiquantitatively; by comparing the percent detectability of telomeric signals in interphase cells.54 A fully quantitative method (Q-FISH), using metaphase chromosome spreads, was later developed by Lansdorp and colleagues28-32,59,60 that was, in turn, applied by DePauw and colleagues,33 to interphase hematopoietic cells.28,29-31,32,33,59,60 Notable advantages of this approach include the following: 1) the use of a superior peptide nucleic acid (PNA) hybridization probe,61 2) the need for far fewer cells (<30), and 3) information is obtained on the telomere lengths of specific individual chromosomes. Unfortunately, one is restricted primarily to the use of cells proliferating in vitro, from which metaphase spreads can be made (eg, hematopoietic cells and cells growing in culture). Also, as with the Southern-based approaches, information regarding specific cell phenotypes is typically unobtainable. Similar methods using PRINS (primed in situ nucleic acid synthesis) in place of FISH have also been developed.62-65
One group has reported the application of the Q-FISH technique to mouse skin sections. However, no detailed protocol was presented and, as previously mentioned, inbred mice can have very long telomeres making them relatively easy to detect.37,38
FISH has also been combined with flow cytometry for the detection of repetitive DNA sequences66,67 and this methodology has been adapted to the quantitation of telomeres in interphase cells (flow-FISH).34-36 Advantages of this method include its high-throughput nature and ability to examine nondividing cells. Flow-FISH has become the method of choice for measuring telomeres in inbred mouse strains that possess extremely long telomeres.47,68-70 However, this method is primarily restricted to the use of sizeable numbers of hematopoietic cells, cultured cells, or nucleated blood cells. In addition, the FISH denaturation conditions tend to preclude the detection of secondary signals, such as cell surface antigens, although enrichment strategies using flow-sorting before FISH may be an option.71
Although all of the methods outlined above have particular strengths, we desired a new quantitative method that would be applicable to standard formalin-fixed human tissue specimens to exploit the vast array of archival human pathological material available. To this end, we developed a combined FISH/immunofluorescence method (TELI-FISH) applicable to standard formalin-fixed, paraffin-embedded human tissue specimens. We validated this technique using a series of human cancer-cell culture lines possessing known telomere lengths, and applied it to a human prostate tissue microarray containing matched normal/tumor pairs. For the first time, we were able to directly compare telomere lengths in prostate cancer cells to those in matched normal appearing epithelial cells. The data were in general agreement with previous studies using complex cell mixtures.41,50,51
Internal standards could potentially be incorporated into the experimental protocol, allowing for the measurement of absolute, as opposed to relative, telomere lengths. If tissue microarrays are being used, then a panel of cell lines having known telomere lengths could be included on the array, allowing the generation of a standard curve. This would also allow comparisons to be made between different experiments and different laboratories.
Our method of direct assessment of telomere lengths in fixed tissue samples should be useful for testing hypotheses implicating telomere shortening in age-related pathologies, tumorigenesis, cancer progression, and may eventually provide useful diagnostic or prognostic information. TEL-FISH could also be useful for predicting the potential efficacy of telomerase inhibition therapies in cancer using diagnostic biopsy specimens because tumors with very long telomeres, or having large intratumoral cell-cell telomere length heterogeneity, may be resistant to such therapies.
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Acknowledgements |
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Footnotes |
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Supported by the Public Health Service (National Institutes of Health/National Cancer Institute grants K08CA78588, CA84997, DK07552, National Cancer Institute SPORE grant no. P50CA58236) and the Charlotte Geyer Foundation (to A.M.D.).
Accepted for publication December 20, 2001.
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M. J. Morton, S. Zhang, A. Lopez-Beltran, G. T. MacLennan, J. N. Eble, R. Montironi, M.-T. Sung, P.-H. Tan, S. Zheng, H. Zhou, et al. Telomere Shortening and Chromosomal Abnormalities in Intestinal Metaplasia of the Urinary Bladder Clin. Cancer Res., October 15, 2007; 13(20): 6232 - 6236. [Abstract] [Full Text] [PDF]
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 T. Tsuji, K. Aoshiba, and A. Nagai Alveolar Cell Senescence in Patients with Pulmonary Emphysema Am. J. Respir. Crit. Care Med., October 15, 2006; 174(8): 886 - 893. [Abstract] [Full Text] [PDF]
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 C. Matthews, I. Gorenne, S. Scott, N. Figg, P. Kirkpatrick, A. Ritchie, M. Goddard, and M. Bennett Vascular Smooth Muscle Cells Undergo Telomere-Based Senescence in Human Atherosclerosis: Effects of Telomerase and Oxidative Stress Circ. Res., July 21, 2006; 99(2): 156 - 164. [Abstract] [Full Text] [PDF]
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 V. Russo, P. Berardinelli, A. Martelli, O. Di Giacinto, D. Nardinocchi, D. Fantasia, and B. Barboni Expression of Telomerase Reverse Transcriptase Subunit (TERT) and Telomere Sizing in Pig Ovarian Follicles J. Histochem. Cytochem., April 1, 2006; 54(4): 443 - 455. [Abstract] [Full Text] [PDF]
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M. Gomez, J. Wu, V. Schreiber, J. Dunlap, F. Dantzer, Y. Wang, and Y. Liu PARP1 Is a TRF2-associated Poly(ADP-Ribose)Polymerase and Protects Eroded Telomeres Mol. Biol. Cell, April 1, 2006; 17(4): 1686 - 1696. [Abstract] [Full Text] [PDF]
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S. Lantuejoul, J. C. Soria, L. Morat, P. Lorimier, D. Moro-Sibilot, L. Sabatier, C. Brambilla, and E. Brambilla Telomere Shortening and Telomerase Reverse Transcriptase Expression in Preinvasive Bronchial Lesions Clin. Cancer Res., March 1, 2005; 11(5): 2074 - 2082. [Abstract] [Full Text] [PDF]
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A. K. Meeker, J. L. Hicks, C. A. Iacobuzio-Donahue, E. A. Montgomery, W. H. Westra, T. Y. Chan, B. M. Ronnett, and A. M. De Marzo Telomere Length Abnormalities Occur Early in the Initiation of Epithelial Carcinogenesis Clin. Cancer Res., May 15, 2004; 10(10): 3317 - 3326. [Abstract] [Full Text] [PDF]
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E. Montgomery, P. Argani, J. L. Hicks, A. M. DeMarzo, and A. K. Meeker Telomere Lengths of Translocation-Associated and Nontranslocation-Associated Sarcomas Differ Dramatically Am. J. Pathol., May 1, 2004; 164(5): 1523 - 1529. [Abstract] [Full Text] [PDF]
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L.-Q. Sun, D. W. Lee, Q. Zhang, W. Xiao, E. H. Raabe, A. Meeker, D. Miao, D. L. Huso, and R. J. Arceci Growth retardation and premature aging phenotypes in mice with disruption of the SNF2-like gene, PASG Genes & Dev., May 1, 2004; 18(9): 1035 - 1046. [Abstract] [Full Text] [PDF]
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A. K. Meeker, J. L. Hicks, E. Gabrielson, W. M. Strauss, A. M. De Marzo, and P. Argani Telomere Shortening Occurs in Subsets of Normal Breast Epithelium as well as in Situ and Invasive Carcinoma Am. J. Pathol., March 1, 2004; 164(3): 925 - 935. [Abstract] [Full Text] [PDF]
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 W. C. Hahn Role of Telomeres and Telomerase in the Pathogenesis of Human Cancer J. Clin. Oncol., May 15, 2003; 21(10): 2034 - 2043. [Abstract] [Full Text] [PDF]
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A. K. Meeker, J. L. Hicks, E. A. Platz, G. E. March, C. J. Bennett, M. J. Delannoy, and A. M. De Marzo Telomere Shortening Is an Early Somatic DNA Alteration in Human Prostate Tumorigenesis Cancer Res., November 15, 2002; 62(22): 6405 - 6409. [Abstract] [Full Text] [PDF]
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N. T. van Heek, A. K. Meeker, S. E. Kern, C. J. Yeo, K. D. Lillemoe, J. L. Cameron, G. J. A. Offerhaus, J. L. Hicks, R. E. Wilentz, M. G. Goggins, et al. Telomere Shortening Is Nearly Universal in Pancreatic Intraepithelial Neoplasia Am. J. Pathol., November 1, 2002; 161(5): 1541 - 1547. [Abstract] [Full Text] [PDF]
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