- Email: [email protected]
8 Direct, in situ assessment of telomere length variation in human cancers and preneoplastic lesions
Direct, in situ Assessment of Telomere Length Variation in Human Cancers and Preneoplastic Lesions Alan Meeker, Wesley R, Gage, Angelo De Marzo, and Anirban Maitra
Introduction
alterations in key cancer-associated genes (Fearon and Vogelstein, 1990). Given the extremely low basal mutation rate of normal human somatic cells, it has been proposed that an underlying genetic instability must exist in cancer progenitor cells, resulting in the generation of a sufficient number of such clonal genetic changes (Loeb, 1991). Although it is well recognized that tumor-associated genetic instability operates at the level of the chromosomes (Lengauer et al., 1998; Loeb, 2001), the precise timing of chromosomal instability during tumorigenesis has not been well characterized. One path to chromosomal instability is via telomere dysfunction (Gisselsson et al., 2000). Telomeres are composed of specialized deoxyribonucleic acid (DNA) tandem repeats complexed with telomere-binding proteins, located at the ends of linear chromosomes (Blackburn, 1991). Telomeres stabilize chromosomes by preventing deleterious recombinations and fusions; they also keep cells from recognizing their chromosomal termini as DNA double-strand breaks. Telomeric DNA tracts are dynamic entities, subject to shortening during cell division as a result of their incomplete replication
Grossly abnormal karyotypes, displaying both numeric and structural changes, are a nearly universal finding in human epithelial malignancies, reflecting either a transient or ongoing state of chromosomal instability (Lengauer et al., 1998). This observation may be interpreted as a manifestation of a mutator phenotype acting at the chromosomal level and likely appears early in tumorigenesis (Shih et al., 2001). Several genes involved in the maintenance of chromosomal stability have been identified, and, as such, they represent candidate mutational targets for karyotype destabilization (Hartwell, 1992). However, defects in such genes have so far been implicated in only a small subset of human cancer cases, and these primarily affect chromosome number. Thus, the molecular mechanisms underlying chromosomal instability, particularly those involved in the generation of complex chromosomal rearrangements, in the majority of human cancers remain a mystery. The transition from normal to cancer is thought to require the accumulation of multiple somatic genomic Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 2: Molecular Pathology, Colorectal Carcinoma, and Prostate Carcinoma
83
Copyright 9 2005 by Elsevier (USA) All fights reserved.
84 (referred to as the "end replication problem") (Levy et al., 1992). In addition, telomeres may shorten as a
result of cell turnover in the presence of unrepaired DNA strand breaks caused by oxidative damage (von Zglinicki, 2000). Critically short telomeres become dysfunctional and, as demonstrated more than 50 years ago, loss of telomere function can be a major mechanisms for the generation of chromosomal abnormalities (McClintock, 1941). Chromosome end-to-end fusions ensue, producing dicentric, multicentric, and ring chromosomes that missegregate or break during mitosis, leading to a series of so-called breakage-fusion-bridge (BFB) cycles capable of generating aneusomies and the various types of structural abnormalities typically seen in human solid tumor karyotypes (Blasco et a/.,1997; O'Hagan et al., 2002). It has been postulated that dysfunctional telomeres could play a causal role in tumorigenesis by instigating chromosomal instability, thus promoting neoplastic transformation (Bacchetti, 1996; Hastie et al., 1990). Results from telomerase knockout mouse models, in which animals possessing critically short telomeres exhibit an increased cancer incidence, support this concept (Blasco et al., 1997; Rudolph et al., 2001). The combined observations of short telomeres, plus the frequent activation of telomerase in human cancers, suggest that the majority of tumors undergo critical telomere shortening at some point during their development. This could simply be a consequence of the end-replication problem combined with extensive cell turnover occurring during tumor expansion. However, if telomere shortening occurs early, it could be playing an important role during the initiation stage of tumorigenesis. Thus, the timing of the occurrence of telomere shortening during human cancer development is a critical question. The vast majority of epithelial malignancies appear to develop from morphologically defined precursor lesions, termed intraepithelial neoplasia (IEN). Examinations to date have revealed evidence of gross genetic instability in IEN lesions, supporting an early role for genetic changes in malignant transformation (Qian et al., 1999; Shih et al., 2001). If telomere dysfunction is a major cause of this genetic instability, then signs of this dysfunction should likewise be evident in these early premalignant lesions. To test this, we developed and validated an in situ method for telomere length assessment telomere length fluorescent in situ hybridization (TEL-FISH) in formalin-fixed and paraffin-embedded (FFPE) human tissues (Meeker et al., 2002a). Application of this method to preinvasive precursor lesions of several human epithelial cancers~including those of prostate, pancreas, breast, large intestine, bladder, uterine cervix, esophagus, and oral cavity---demonstrated its utility (Meeker et al., 2004). We found clear evidence of
I Molecular Pathology telomere length abnormalities, primarily telomere shortening, and telomere length heterogeneity in the majority of IEN lesions from these human epithelial tissues (Figure 14). In this review we describe the detailed method for analyzing telomere lengths in archival tissue sections using this fluorescence in situ hybridization (FISH) protocol.
MATERIALS 1. ChemMate slides (Cat. No. 12-548-6A, Fisher Scientific, Newark, DE). 2. Xylene. 3. Ethanol (absolute, 95%, 70%). 4. A source of deionized water. 5. 1% Tween-20 detergent. 6. Citrate buffer (Target unmasking solution; Cat. No. H-3300, Vector Laboratories, Inc., Burlingame, CA). 7. Capillary gap tray (automated processor slide holder, or equivalent, Dako Corp., Carpinteria, CA). 8. Black and Decker Handy Steamer Plus (Black and Decker, Towson, MD). 9. Phosphate buffer saline with Tween (PBST) (Cat. No. P-3563, Sigma, St. Louis, MO). 10. Protease Type VIII (Cat. No. P-5380, Sigma) (optional; see later in this chapter). 11. Prolong Anti-fade Mounting Media (Cat. No. P-7481, Molecular Probes, Eugene, OR). 12. 1 M Tris (pH 7.5) (Cat. No. 15567-027, Gibco/ BRL, Grand Island, NY). 13. B/M Blocking Reagent (10% in maleic acid; as per manufacturer's instructions; Cat. No. 1096-176, Boehringer-Mannheim, Indianapolis, IN). 14. 100% Formamide (Cat. No. 1814-320, Boehringer-Mannheim). 15. Bovine-albumin solution (Cat. No. A-7284, Sigma). 16. DAPI[4'-6-diamidino-2-phenylindole] (Cat. No. D-8417, Sigma). 17. Anti-mouse immunoglobulin G (IgG) fraction Alexa Fluor 488 (Molecular Probes, Cat. No. A-11029), or anti-rabbit IgG fraction Alexa Fluor 488 (Molecular Probes, Cat. No. A-11034) secondary antibody. 18. Peptide nucleic acid (PNA) telomere-specific hybridization probe, custom synthesized. Sequence = (N-terminus to C-terminus) CCCTAACCCTAACCCTAA with an N-terminal covalently linked fluorescent dye~here we use Cy3. (Applied Biosystems, Framingham, MA). 19. Fluorescence microscope equipped with appropriate fluorescence filter set. Here, we use Omega Optical, XF38 filter set (Omega Optical Inc., Brattleboro, VT) for Cy3 visualization. 20. Slide warmer.
8 Direct, In situ Assessment of Telomere Length Variation
85
Figure 14 Telomeric fluorescence in situ hybridization) (TEL-FISH) for telomere signal intensities demonstrate intense fluorescent signals in normal colonic epithelium and in stroreal fibroblasts, whereas striking reduction in telomere signal intensity is observed in adenomatous (precancerous) epithelium.
METHOD 1. Prepare unstained slides. The specimens used are tissues that have undergone routine neutral-buffered formalin fixation followed by paraffin embedding. Ethanol-fixed tissues are also suitable. Typically, 4 or 5 gM thick sections are cut from the paraffin blocks and applied to ChemMate slides. These slides are suitably treated and marked to allow for capillary gap formation during stream treatment. Heating at high temperature or for prolonged periods should be avoided because this can cause increased background auto fluorescence. 2. Preheat slides to 65~ for 10 min to melt paraffin. 3. Transfer slides to staining rack and place them in xylene 2 x for 5 min each to remove paraffin (at this time, turn on slide rack warmer and steamer to preheat). 4. Hydrate slides through a graded ethanol s e r i e s ~ absolute x 2, 95% x 2, and one change of 7 0 % ~ a n d dip until clear (DUC). 5. Place slides in one change of deionized water, then place them in one change of deionized water with 0.1% Tween, DUC. 6. Pair slides to form capillary gaps between tissue sides, and then place into a cap gap tray (automated processor slide holder, Dako or equivalent) containing citrate buffer (target unmasking solution); steam for 14 min (Black and Decker Handy Steamer Plus). 7. Remove slides from steamer and let cool to room temperature (~5 min).
8. Place slides into PBS with Tween (PBST) x 5 min. If you are not digesting the tissue with protease: Rinse in deionized water, 70% ethanol, 95% ethanol, and let air-dry. Then, proceed to Denaturation (Step 9). Otherwise, perform protease steps (a-d), and then proceed to Denaturation. Protease Treatment
Depending on the tissue, degree of fixation, or particular antibody being used, the incubation time, protease type, and concentration may require optimization. a. Place slides in protease solution (Protease Type VIII, 0.5 mg/ml in PBST) for 1 min at room temperature in a Coplin (50 ml) or PAP jar (holds 4 slides, 20 ml). b. Rinse slides thoroughly with deionized water 4-5 times. c. Place slides in 95% EtOH for 5 min. d. Air-dry slides. Sample Denaturation
Adjust temperature on slide warmer to prepare for denaturation. Do this by placing a small box (e.g., slide box) upside down over the heater surface with a surface-reading thermometer underneath the box. Cover the box with aluminum foil and allow to
86
I Molecular Pathology
equilibrate to temperature. Check thermometer, and adjust setting of slide warmer as necessary to obtain proper denaturation temperature under the enclosure. Remove tube of Prolong Anti-fade Mounting Media (Molecular Probes) from freezer to thaw. Note: For this and all subsequent steps, keep slide(s) in darkness. 9. Dilute PNA telomere hybridization probe in PNA diluent buffer to a final concentration of 300 ng/ml. Carefully, so as to avoid introducing bubbles, place 35 ~tl of diluted PNA probe onto the specimen. Apply coverslip, again without introducing air bubbles, and denature at 83~ • 4 min in the dark (inverted slide box on preheated slide warmer). PNA Diluent:
(recipe makes 1 ml; 35 ~1 required per slide) 0.29 ml distilled water 10 ~1 1 M Tris (pH 7.5) (Cat. No. 15567-027, Gibco/BRL) 5 ~tl B/M Blocking Reagent, prepared as per manufacturer 0.7 ml 100% Formamide
10. Move slides to a dark closed container and hybridize for 2 hrs at room temperature. To thawed tube of Prolong, add 1 ml glycerol (supplied) and mix well. Mix occasionally to dissolve solid. 11. Carefully remove coverslips from slides and wash in darkness with PNA Wash Solution: 2 x 15 rain each. PNA Wash Solution:
(50 ml) 35 ml formamide 15 ml distilled water 0.5 ml 1 M Tris (pH 7.5) 165 ~tl 30% bovine serum albumin
12. TBST wash 3 x 5 min each. Note: If NOT conducting a double label (FISH + fluorescent antibody), skip to Step 17 of this protocol.
Immunofluorescence Section (for Antibody/FISH Double Label) 13. Rinse slides 1 x in PBST. 14. Apply appropriately diluted primary antibody. Incubate 45 min at room temperature or overnight at 4~ 15. Rinse slides in PBST. 16. Apply appropriate fluorescent secondary antibody diluted 1:100 in Dulbecco's PBS. Incubate for 30 min at room temperature. 17. Rinse slides in PBST. 18. Drain slides and stain with DAPI for 1 rain (1:10,000 dilution in water of a 5 mg/ml stock solution).
19. Rinse with PBST. 20. Rinse slides well in distilled water. 21. Drain slides and mount while still wet with coverslip using 1-2 drops of Prolong. Anti-fade Mounting Media solution or equivalent anti-fade solution. Avoid bubbles. Viewing: Fluorescent telomere signals are best viewed with 40X or higher oil immersion objectives. For Cy3-1abeled PNA probes, we find we get good signals using an Omega Optical XF38 filter set (Omega Optical Inc.): Emission: OG 590 (Omega XF3016) Dichroic: DRLP 570 (Omega XF2015) Excitation: DF10 546 (Omega XF3016) Storage: Slides kept refrigerated (4~ in the dark will retain signals for at least several weeks.
RESULTS AND DISCUSSION The method described herein was developed to allow high-resolution telomere length assessment in human FFPE tissue sections, thus making the vast resources of archival tissues available for telomere length analysis. Validation of the method (Meeker et al., 2002a) showed that intensity of the fluorescent telomeric (TEL-FISH) signals is linearly related to telomere length as determined independently via Southern blot analysis of telomeric restriction fragments. Unlike Southern analysis, TEL-FISH can be performed on very small, fixed specimens. Furthermore, providing single cell resolution completely avoids the confounding effects of cell type heterogeneity typically present in clinical specimens. It is important to note that because TEL-FISH can be combined with standard immunofluorescence and tissue architecture is maintained, direct comparisons between different regions or specific cell types are easily accomplished. Further information regarding regions of interest can be obtained by performing standard hematoxylin and eosin (H&E) staining following examination of the TEL-FISH slides by fluorescence microscopy. Finally, because the telomeric signal intensity is directly related to telomere length, quantitation is possible via standard image analysis techniques (Meeker et al., 2002a). TEL-FISH has proved useful in assessing telomere length abnormalities in cancerous and precancerous lesions. The vast majority (-95%) of IEN lesions examined, the earliest identifiable cancer precursors, are composed largely of cells possessing telomere length abnormalities, with most lesions displaying abnormally
8 Direct, In situ Assessment of Telomere Length Variation short telomeres (Figure 14). It therefore appears that the telomere shortening frequently observed in malignant epithelial tumors has already occurred by the preinvasive stage (Meeker et al., 2002a,b; Meeker et al., 2004; van Heek et al., 2002). Indeed, when both were present, invasive cancers and accompanying IEN lesions exhibited similar degrees of telomeric shortening. Telomere length status may provide utility in predicting prognosis, as a novel endpoint for cancer chemoprevention studies, in the pathologic diagnosis of human cancer precursor lesions, and for prediction and monitoring patient response to anti-telomerase therapies. It is our hope that the method described here will prove to be a useful research tool for addressing these questions.
References Bacchetti, S. 1996. Telomere maintenance in tumor cells. Cancer Surv. 28:197-216. Blackburn, E.H. 1991. Structure and function of telomeres. Nature 350:569-572. B lasco, M.A., Lee, H.W., Hande, M.E, Samper, E., Lansdorp, EM., DePinho, R.A., and Greider, C.W. 1997. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91(1):25-34. Fearon, E.R. and Vogelstein, B. 1990. A genetic model for colorectal tumorigenesis. Cell 61(5):759-767. Gisselsson, D., Pettersson, L., Hoglund, M., Heidenblad, M., Gorunova, L., Wiegant, J., Mertens, E, Dal, C., in, E, Mitalman, E, and Mandanl, N. 2000 Chromosomal breakage-fusionbridge events cause genetic intratumor heterogeneity. Proc Natl. Acad Sci. USA 97(10):5357-5362. Hartwell, L. 1992. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 71(4):543-546. Hastie, N., Dempster, M. Dunlop, M., Thompson, A.M., Green D.K., and Allshire, R.C. 1990. Telomere reduction in human colorectal carcinoma and with ageing. Nature 346:866-868. Lengauer, C., Kinzler, K.W. 1998. Genetic instabilities in human cancers. Nature 396(6712):643-649. Levy, M.Z., Allsopp, R.C., Futcher, A.B., Greider, C.B., and
87
Hartey, C.B., 1992. Telomere end-replication problem and cell aging. J. Mol. Biol. 225(4):951-960. Loeb, L.A. 1991. Mutator phenotype may be required for multistage carcinogenesis. Cancer Res. 51(12):3075-3079. Loeb, L.A. 2001. A mutator phenotype in cancer. Cancer Res. 61(8):3230-3239. McClintock, B. 1941. The stability of broken ends of chromosomes in Zea mays. Genetics 26:234-282. Meeker, A.K., Gage, W.R. Hicks, J.L., Simon, I., Cottman, J.R., Platz, E.A., March, G.E., and De Marzo A.M. 2002. Telomere length assessment in human arichival tissues: Combined telomere fluorescence in situ hybridization and immunostaining. Am. J. Pathol. 160(4):1259-1268. Meeker, A.K., Hicks, J.L., Platz, E.A., March, G. E., Bennett, C. J., Delannay, M.J., De Marzo, A.M. 2002b. Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. Cancer Res. 62(22):6405-6409. Meeker, A.K., Hicks, J.L., Iacobuzio-Donahue, C.A., Montgomery, E.A., Westra, W.H., Chan, T.Y., Ronnett, B.M., and De Marzo, A.M., 2004. Telomere length abnormalities occur early in the initiation of epithelial carcinogenesis. Clin. Cancer Res. 10(10):3317-3326. O'Hagan, R.C., Chang, S., Maser, R.S., Mohan, R., Artandi, S.E., Chin, L., and DePinho, R.A. 2002. Telomere dysfunction provokes regional amplification and deletion in cancer genomes. Cancer Cell 2(2): 149-155. Qian, J., Jenkins, R.B., and Bostwick, D.G. 1999. Genotic and chromosomal alterations in prostatic intraepithelial neoplasia and carcinoma detected by fluorescence in situ hybridization. Eur. Urol. 35(5-6):479--483. Rudolph, K.L., Millard, M., Bosenberg, M.W., and DePinho, R.A. 2001. Telomere dysfunction and evolution of intestinal carcinoma in mice and humans. Nat. Genet. 28(2):155-159. Shih, I.M., Zhou, W., Goodman, S.N., Lengauer, C., Kinzler, K.W., and Vogelstein, B. 2001. Evidence that genetic instability occurs at an early stage of colorectal tumorigenesis. Cancer Res. 61(3):818-822. van Heek, N.T., Meeker, A.K., Kern, S.E., Yeo, C.J., Lillemoe, K.D., Cameron, J.L., Offerhaus, G.J., Hicks, J.L., Wilentz, R.E., Goggins, M.G., De Marzo, A.M., Hruban, R.H., and Maitra, A. 2002. Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia. Am. J. Pathol. 161(5): 1541-1547. von Zgylinicki, T. 2000. Role of oxidative stress in telomere length regulation and replicative sensescence. Ann. N. Y. Acad. Sci. 908:99-110.
Introduction
alterations in key cancer-associated genes (Fearon and Vogelstein, 1990). Given the extremely low basal mutation rate of normal human somatic cells, it has been proposed that an underlying genetic instability must exist in cancer progenitor cells, resulting in the generation of a sufficient number of such clonal genetic changes (Loeb, 1991). Although it is well recognized that tumor-associated genetic instability operates at the level of the chromosomes (Lengauer et al., 1998; Loeb, 2001), the precise timing of chromosomal instability during tumorigenesis has not been well characterized. One path to chromosomal instability is via telomere dysfunction (Gisselsson et al., 2000). Telomeres are composed of specialized deoxyribonucleic acid (DNA) tandem repeats complexed with telomere-binding proteins, located at the ends of linear chromosomes (Blackburn, 1991). Telomeres stabilize chromosomes by preventing deleterious recombinations and fusions; they also keep cells from recognizing their chromosomal termini as DNA double-strand breaks. Telomeric DNA tracts are dynamic entities, subject to shortening during cell division as a result of their incomplete replication
Grossly abnormal karyotypes, displaying both numeric and structural changes, are a nearly universal finding in human epithelial malignancies, reflecting either a transient or ongoing state of chromosomal instability (Lengauer et al., 1998). This observation may be interpreted as a manifestation of a mutator phenotype acting at the chromosomal level and likely appears early in tumorigenesis (Shih et al., 2001). Several genes involved in the maintenance of chromosomal stability have been identified, and, as such, they represent candidate mutational targets for karyotype destabilization (Hartwell, 1992). However, defects in such genes have so far been implicated in only a small subset of human cancer cases, and these primarily affect chromosome number. Thus, the molecular mechanisms underlying chromosomal instability, particularly those involved in the generation of complex chromosomal rearrangements, in the majority of human cancers remain a mystery. The transition from normal to cancer is thought to require the accumulation of multiple somatic genomic Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 2: Molecular Pathology, Colorectal Carcinoma, and Prostate Carcinoma
83
Copyright 9 2005 by Elsevier (USA) All fights reserved.
84 (referred to as the "end replication problem") (Levy et al., 1992). In addition, telomeres may shorten as a
result of cell turnover in the presence of unrepaired DNA strand breaks caused by oxidative damage (von Zglinicki, 2000). Critically short telomeres become dysfunctional and, as demonstrated more than 50 years ago, loss of telomere function can be a major mechanisms for the generation of chromosomal abnormalities (McClintock, 1941). Chromosome end-to-end fusions ensue, producing dicentric, multicentric, and ring chromosomes that missegregate or break during mitosis, leading to a series of so-called breakage-fusion-bridge (BFB) cycles capable of generating aneusomies and the various types of structural abnormalities typically seen in human solid tumor karyotypes (Blasco et a/.,1997; O'Hagan et al., 2002). It has been postulated that dysfunctional telomeres could play a causal role in tumorigenesis by instigating chromosomal instability, thus promoting neoplastic transformation (Bacchetti, 1996; Hastie et al., 1990). Results from telomerase knockout mouse models, in which animals possessing critically short telomeres exhibit an increased cancer incidence, support this concept (Blasco et al., 1997; Rudolph et al., 2001). The combined observations of short telomeres, plus the frequent activation of telomerase in human cancers, suggest that the majority of tumors undergo critical telomere shortening at some point during their development. This could simply be a consequence of the end-replication problem combined with extensive cell turnover occurring during tumor expansion. However, if telomere shortening occurs early, it could be playing an important role during the initiation stage of tumorigenesis. Thus, the timing of the occurrence of telomere shortening during human cancer development is a critical question. The vast majority of epithelial malignancies appear to develop from morphologically defined precursor lesions, termed intraepithelial neoplasia (IEN). Examinations to date have revealed evidence of gross genetic instability in IEN lesions, supporting an early role for genetic changes in malignant transformation (Qian et al., 1999; Shih et al., 2001). If telomere dysfunction is a major cause of this genetic instability, then signs of this dysfunction should likewise be evident in these early premalignant lesions. To test this, we developed and validated an in situ method for telomere length assessment telomere length fluorescent in situ hybridization (TEL-FISH) in formalin-fixed and paraffin-embedded (FFPE) human tissues (Meeker et al., 2002a). Application of this method to preinvasive precursor lesions of several human epithelial cancers~including those of prostate, pancreas, breast, large intestine, bladder, uterine cervix, esophagus, and oral cavity---demonstrated its utility (Meeker et al., 2004). We found clear evidence of
I Molecular Pathology telomere length abnormalities, primarily telomere shortening, and telomere length heterogeneity in the majority of IEN lesions from these human epithelial tissues (Figure 14). In this review we describe the detailed method for analyzing telomere lengths in archival tissue sections using this fluorescence in situ hybridization (FISH) protocol.
MATERIALS 1. ChemMate slides (Cat. No. 12-548-6A, Fisher Scientific, Newark, DE). 2. Xylene. 3. Ethanol (absolute, 95%, 70%). 4. A source of deionized water. 5. 1% Tween-20 detergent. 6. Citrate buffer (Target unmasking solution; Cat. No. H-3300, Vector Laboratories, Inc., Burlingame, CA). 7. Capillary gap tray (automated processor slide holder, or equivalent, Dako Corp., Carpinteria, CA). 8. Black and Decker Handy Steamer Plus (Black and Decker, Towson, MD). 9. Phosphate buffer saline with Tween (PBST) (Cat. No. P-3563, Sigma, St. Louis, MO). 10. Protease Type VIII (Cat. No. P-5380, Sigma) (optional; see later in this chapter). 11. Prolong Anti-fade Mounting Media (Cat. No. P-7481, Molecular Probes, Eugene, OR). 12. 1 M Tris (pH 7.5) (Cat. No. 15567-027, Gibco/ BRL, Grand Island, NY). 13. B/M Blocking Reagent (10% in maleic acid; as per manufacturer's instructions; Cat. No. 1096-176, Boehringer-Mannheim, Indianapolis, IN). 14. 100% Formamide (Cat. No. 1814-320, Boehringer-Mannheim). 15. Bovine-albumin solution (Cat. No. A-7284, Sigma). 16. DAPI[4'-6-diamidino-2-phenylindole] (Cat. No. D-8417, Sigma). 17. Anti-mouse immunoglobulin G (IgG) fraction Alexa Fluor 488 (Molecular Probes, Cat. No. A-11029), or anti-rabbit IgG fraction Alexa Fluor 488 (Molecular Probes, Cat. No. A-11034) secondary antibody. 18. Peptide nucleic acid (PNA) telomere-specific hybridization probe, custom synthesized. Sequence = (N-terminus to C-terminus) CCCTAACCCTAACCCTAA with an N-terminal covalently linked fluorescent dye~here we use Cy3. (Applied Biosystems, Framingham, MA). 19. Fluorescence microscope equipped with appropriate fluorescence filter set. Here, we use Omega Optical, XF38 filter set (Omega Optical Inc., Brattleboro, VT) for Cy3 visualization. 20. Slide warmer.
8 Direct, In situ Assessment of Telomere Length Variation
85
Figure 14 Telomeric fluorescence in situ hybridization) (TEL-FISH) for telomere signal intensities demonstrate intense fluorescent signals in normal colonic epithelium and in stroreal fibroblasts, whereas striking reduction in telomere signal intensity is observed in adenomatous (precancerous) epithelium.
METHOD 1. Prepare unstained slides. The specimens used are tissues that have undergone routine neutral-buffered formalin fixation followed by paraffin embedding. Ethanol-fixed tissues are also suitable. Typically, 4 or 5 gM thick sections are cut from the paraffin blocks and applied to ChemMate slides. These slides are suitably treated and marked to allow for capillary gap formation during stream treatment. Heating at high temperature or for prolonged periods should be avoided because this can cause increased background auto fluorescence. 2. Preheat slides to 65~ for 10 min to melt paraffin. 3. Transfer slides to staining rack and place them in xylene 2 x for 5 min each to remove paraffin (at this time, turn on slide rack warmer and steamer to preheat). 4. Hydrate slides through a graded ethanol s e r i e s ~ absolute x 2, 95% x 2, and one change of 7 0 % ~ a n d dip until clear (DUC). 5. Place slides in one change of deionized water, then place them in one change of deionized water with 0.1% Tween, DUC. 6. Pair slides to form capillary gaps between tissue sides, and then place into a cap gap tray (automated processor slide holder, Dako or equivalent) containing citrate buffer (target unmasking solution); steam for 14 min (Black and Decker Handy Steamer Plus). 7. Remove slides from steamer and let cool to room temperature (~5 min).
8. Place slides into PBS with Tween (PBST) x 5 min. If you are not digesting the tissue with protease: Rinse in deionized water, 70% ethanol, 95% ethanol, and let air-dry. Then, proceed to Denaturation (Step 9). Otherwise, perform protease steps (a-d), and then proceed to Denaturation. Protease Treatment
Depending on the tissue, degree of fixation, or particular antibody being used, the incubation time, protease type, and concentration may require optimization. a. Place slides in protease solution (Protease Type VIII, 0.5 mg/ml in PBST) for 1 min at room temperature in a Coplin (50 ml) or PAP jar (holds 4 slides, 20 ml). b. Rinse slides thoroughly with deionized water 4-5 times. c. Place slides in 95% EtOH for 5 min. d. Air-dry slides. Sample Denaturation
Adjust temperature on slide warmer to prepare for denaturation. Do this by placing a small box (e.g., slide box) upside down over the heater surface with a surface-reading thermometer underneath the box. Cover the box with aluminum foil and allow to
86
I Molecular Pathology
equilibrate to temperature. Check thermometer, and adjust setting of slide warmer as necessary to obtain proper denaturation temperature under the enclosure. Remove tube of Prolong Anti-fade Mounting Media (Molecular Probes) from freezer to thaw. Note: For this and all subsequent steps, keep slide(s) in darkness. 9. Dilute PNA telomere hybridization probe in PNA diluent buffer to a final concentration of 300 ng/ml. Carefully, so as to avoid introducing bubbles, place 35 ~tl of diluted PNA probe onto the specimen. Apply coverslip, again without introducing air bubbles, and denature at 83~ • 4 min in the dark (inverted slide box on preheated slide warmer). PNA Diluent:
(recipe makes 1 ml; 35 ~1 required per slide) 0.29 ml distilled water 10 ~1 1 M Tris (pH 7.5) (Cat. No. 15567-027, Gibco/BRL) 5 ~tl B/M Blocking Reagent, prepared as per manufacturer 0.7 ml 100% Formamide
10. Move slides to a dark closed container and hybridize for 2 hrs at room temperature. To thawed tube of Prolong, add 1 ml glycerol (supplied) and mix well. Mix occasionally to dissolve solid. 11. Carefully remove coverslips from slides and wash in darkness with PNA Wash Solution: 2 x 15 rain each. PNA Wash Solution:
(50 ml) 35 ml formamide 15 ml distilled water 0.5 ml 1 M Tris (pH 7.5) 165 ~tl 30% bovine serum albumin
12. TBST wash 3 x 5 min each. Note: If NOT conducting a double label (FISH + fluorescent antibody), skip to Step 17 of this protocol.
Immunofluorescence Section (for Antibody/FISH Double Label) 13. Rinse slides 1 x in PBST. 14. Apply appropriately diluted primary antibody. Incubate 45 min at room temperature or overnight at 4~ 15. Rinse slides in PBST. 16. Apply appropriate fluorescent secondary antibody diluted 1:100 in Dulbecco's PBS. Incubate for 30 min at room temperature. 17. Rinse slides in PBST. 18. Drain slides and stain with DAPI for 1 rain (1:10,000 dilution in water of a 5 mg/ml stock solution).
19. Rinse with PBST. 20. Rinse slides well in distilled water. 21. Drain slides and mount while still wet with coverslip using 1-2 drops of Prolong. Anti-fade Mounting Media solution or equivalent anti-fade solution. Avoid bubbles. Viewing: Fluorescent telomere signals are best viewed with 40X or higher oil immersion objectives. For Cy3-1abeled PNA probes, we find we get good signals using an Omega Optical XF38 filter set (Omega Optical Inc.): Emission: OG 590 (Omega XF3016) Dichroic: DRLP 570 (Omega XF2015) Excitation: DF10 546 (Omega XF3016) Storage: Slides kept refrigerated (4~ in the dark will retain signals for at least several weeks.
RESULTS AND DISCUSSION The method described herein was developed to allow high-resolution telomere length assessment in human FFPE tissue sections, thus making the vast resources of archival tissues available for telomere length analysis. Validation of the method (Meeker et al., 2002a) showed that intensity of the fluorescent telomeric (TEL-FISH) signals is linearly related to telomere length as determined independently via Southern blot analysis of telomeric restriction fragments. Unlike Southern analysis, TEL-FISH can be performed on very small, fixed specimens. Furthermore, providing single cell resolution completely avoids the confounding effects of cell type heterogeneity typically present in clinical specimens. It is important to note that because TEL-FISH can be combined with standard immunofluorescence and tissue architecture is maintained, direct comparisons between different regions or specific cell types are easily accomplished. Further information regarding regions of interest can be obtained by performing standard hematoxylin and eosin (H&E) staining following examination of the TEL-FISH slides by fluorescence microscopy. Finally, because the telomeric signal intensity is directly related to telomere length, quantitation is possible via standard image analysis techniques (Meeker et al., 2002a). TEL-FISH has proved useful in assessing telomere length abnormalities in cancerous and precancerous lesions. The vast majority (-95%) of IEN lesions examined, the earliest identifiable cancer precursors, are composed largely of cells possessing telomere length abnormalities, with most lesions displaying abnormally
8 Direct, In situ Assessment of Telomere Length Variation short telomeres (Figure 14). It therefore appears that the telomere shortening frequently observed in malignant epithelial tumors has already occurred by the preinvasive stage (Meeker et al., 2002a,b; Meeker et al., 2004; van Heek et al., 2002). Indeed, when both were present, invasive cancers and accompanying IEN lesions exhibited similar degrees of telomeric shortening. Telomere length status may provide utility in predicting prognosis, as a novel endpoint for cancer chemoprevention studies, in the pathologic diagnosis of human cancer precursor lesions, and for prediction and monitoring patient response to anti-telomerase therapies. It is our hope that the method described here will prove to be a useful research tool for addressing these questions.
References Bacchetti, S. 1996. Telomere maintenance in tumor cells. Cancer Surv. 28:197-216. Blackburn, E.H. 1991. Structure and function of telomeres. Nature 350:569-572. B lasco, M.A., Lee, H.W., Hande, M.E, Samper, E., Lansdorp, EM., DePinho, R.A., and Greider, C.W. 1997. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91(1):25-34. Fearon, E.R. and Vogelstein, B. 1990. A genetic model for colorectal tumorigenesis. Cell 61(5):759-767. Gisselsson, D., Pettersson, L., Hoglund, M., Heidenblad, M., Gorunova, L., Wiegant, J., Mertens, E, Dal, C., in, E, Mitalman, E, and Mandanl, N. 2000 Chromosomal breakage-fusionbridge events cause genetic intratumor heterogeneity. Proc Natl. Acad Sci. USA 97(10):5357-5362. Hartwell, L. 1992. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 71(4):543-546. Hastie, N., Dempster, M. Dunlop, M., Thompson, A.M., Green D.K., and Allshire, R.C. 1990. Telomere reduction in human colorectal carcinoma and with ageing. Nature 346:866-868. Lengauer, C., Kinzler, K.W. 1998. Genetic instabilities in human cancers. Nature 396(6712):643-649. Levy, M.Z., Allsopp, R.C., Futcher, A.B., Greider, C.B., and
87
Hartey, C.B., 1992. Telomere end-replication problem and cell aging. J. Mol. Biol. 225(4):951-960. Loeb, L.A. 1991. Mutator phenotype may be required for multistage carcinogenesis. Cancer Res. 51(12):3075-3079. Loeb, L.A. 2001. A mutator phenotype in cancer. Cancer Res. 61(8):3230-3239. McClintock, B. 1941. The stability of broken ends of chromosomes in Zea mays. Genetics 26:234-282. Meeker, A.K., Gage, W.R. Hicks, J.L., Simon, I., Cottman, J.R., Platz, E.A., March, G.E., and De Marzo A.M. 2002. Telomere length assessment in human arichival tissues: Combined telomere fluorescence in situ hybridization and immunostaining. Am. J. Pathol. 160(4):1259-1268. Meeker, A.K., Hicks, J.L., Platz, E.A., March, G. E., Bennett, C. J., Delannay, M.J., De Marzo, A.M. 2002b. Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. Cancer Res. 62(22):6405-6409. Meeker, A.K., Hicks, J.L., Iacobuzio-Donahue, C.A., Montgomery, E.A., Westra, W.H., Chan, T.Y., Ronnett, B.M., and De Marzo, A.M., 2004. Telomere length abnormalities occur early in the initiation of epithelial carcinogenesis. Clin. Cancer Res. 10(10):3317-3326. O'Hagan, R.C., Chang, S., Maser, R.S., Mohan, R., Artandi, S.E., Chin, L., and DePinho, R.A. 2002. Telomere dysfunction provokes regional amplification and deletion in cancer genomes. Cancer Cell 2(2): 149-155. Qian, J., Jenkins, R.B., and Bostwick, D.G. 1999. Genotic and chromosomal alterations in prostatic intraepithelial neoplasia and carcinoma detected by fluorescence in situ hybridization. Eur. Urol. 35(5-6):479--483. Rudolph, K.L., Millard, M., Bosenberg, M.W., and DePinho, R.A. 2001. Telomere dysfunction and evolution of intestinal carcinoma in mice and humans. Nat. Genet. 28(2):155-159. Shih, I.M., Zhou, W., Goodman, S.N., Lengauer, C., Kinzler, K.W., and Vogelstein, B. 2001. Evidence that genetic instability occurs at an early stage of colorectal tumorigenesis. Cancer Res. 61(3):818-822. van Heek, N.T., Meeker, A.K., Kern, S.E., Yeo, C.J., Lillemoe, K.D., Cameron, J.L., Offerhaus, G.J., Hicks, J.L., Wilentz, R.E., Goggins, M.G., De Marzo, A.M., Hruban, R.H., and Maitra, A. 2002. Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia. Am. J. Pathol. 161(5): 1541-1547. von Zgylinicki, T. 2000. Role of oxidative stress in telomere length regulation and replicative sensescence. Ann. N. Y. Acad. Sci. 908:99-110.