Localization of Telomerase hTERT Protein and hTR in Benign Mucosa ...

Anatomic Pathology / HTERT PROTEIN AND HTR IN CERVICAL MUCOSA

Localization of Telomerase hTERT Protein and hTR in Benign Mucosa, Dysplasia, and Squamous Cell Carcinoma of the Cervix Michael Frost, MBBS,1 Joanna B. Bobak, PhD,1 Roberto Gianani, MD,1 Nam Kim, PhD,2 Scott Weinrich, PhD,2 David C. Spalding, PhD,3 Laura G. Cass, PhD,3 L. Chesney Thompson, MD,4 Takayuki Enomoto, MD, PhD,5 Daniel Uribe-Lopez, BS,1 and Kenneth R. Shroyer, MD, PhD1 Key Words: Telomerase; Human telomerase reverse transcriptase; hTERT; Human telomerase RNA component; hTR; Cervix; Mib-1

Abstract Telomerase has been detected by telomerase repeat amplification protocol (TRAP) assay in cervical dysplasia and squamous cell carcinoma but not in most normal cervical tissues. In the present study, the cellular localization of the protein catalytic subunit of telomerase (hTERT) and the RNA component (hTR) were investigated by a sensitive immunohistochemical technique and by in situ hybridization, respectively. hTERT protein was detected in all diagnostic categories of cervical specimens. hTERT was localized predominantly to the lower suprabasal levels of normal squamous mucosa but was detected throughout virtually all levels of the lesional epithelium in lowgrade squamous intraepithelial lesions (LSILs), highgrade squamous intraepithelial lesions (HSILs), and squamous cell carcinoma (SCC). Telomerase expression correlated with hTERT detection in SCC and HSIL but was not detected by TRAP assay in most samples of normal mucosa or LSIL. The distribution of hTR correlated with the localization of hTERT in HSIL and SCC but was restricted to the basal and suprabasal cell layers in normal mucosa and LSIL.

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Cervical cancer is the most common fatal malignant neoplasm of women in many developing Third World countries but in the United States, it currently ranks about tenth, owing to the success of diagnostic cytopathology in the detection of potential precursor lesions.1-3 The great majority of cervical cancers are squamous cell carcinomas (SCCs), and epidemiologic and laboratory data have shown that they are preceded by intraepithelial precursor lesions that are composed of atypical or dysplastic squamous cells.4 Cervical preneoplastic lesions can be divided into low-grade squamous intraepithelial lesions (LSILs), which include condyloma and mild dysplasia, and high-grade squamous intraepithelial lesions (HSILs), comprising moderate dysplasia, severe dysplasia, and carcinoma in situ.5 Molecular markers of premalignant potential could one day have an important role in the detection and clinical management of patients with precursors of SCC. Telomerase is a ribonucleoprotein complex with terminal transferase activity that is expressed in the vast majority of human malignant tumors and malignant cell lines but not in most corresponding benign tissues.6 Telomerase is composed of an RNA component (hTR), a catalytic protein subunit (hTERT), and the telomerase-associated protein TEP1.7-9 Telomerase activity has been detected in a high proportion of cervical SCCs and HSILs but is expressed in a relatively low proportion of samples of normal cervical mucosa.10-12 Takakura and coworkers13 found concordance between telomerase activity and the detection of hTERT messenger RNA (mRNA) by reverse transcriptase–polymerase chain reaction (RT-PCR) in most cases of SCC, while telomerase activity and hTERT mRNA were not detected in normal cervical mucosa. By contrast, the levels of hTR and TEP1 mRNA were nonspecific, displaying wide expression in © American Society of Clinical Pathologists

Anatomic Pathology / ORIGINAL ARTICLE

glutathione S transferase (GST)-hTERT (aa 549-831) fusion protein. Western blot analysis of the products of a TNT in vitro transcription-translation system (Promega, Madison, WI), programmed with hTERT plasmid, demonstrated specificity of the 4B1 antibody for the hTERT protein ❚Image 1❚.

Materials and Methods Tissue Samples Benign cervical squamous mucosa, SILs, and cervical SCCs were obtained from cervical cone biopsy and hysterectomy specimens as previously described.12 Two pathologists (K.R.S. and M.F.) reviewed each case using standard diagnostic criteria 22,23 and selected a total of 55 histologic regions from 39 patients for the localization of hTERT protein as follows: nonneoplastic cervical squamous mucosa samples, 25; LSIL, 7; HSIL, 13; and SCC, 10. In situ hybridization for hTR was performed on a representative subset of the cases, including 7 sections of normal or benignreactive cervical mucosa, 3 LSILs, 8 HSILs, and 4 SCCs. Telomerase Assay Telomerase expression was evaluated in a previous study by the TRAP assay.12 Western Blot Analysis of 4B1 Antibody A mouse monoclonal IgG2A kappa antibody, designated 4B1, was produced by immunization with a © American Society of Clinical Pathologists

TTNT+hTERT NT+ hTERT plasmid

Immunohistochemical Localization of hTERT Four-micrometer sections of formalin-fixed paraffinembedded tissue blocks were collected on glass slides (Superfrost Plus, VWR Scientific, West Chester, PA) and baked overnight at 60°C. Following passage through xylene and graded alcohols, a 15-minute microwave antigen retrieval was performed in a pressure cooker containing citrate buffer. Avidin, biotin, hydrogen peroxide, and protein blocks were applied, followed by 4B1 at dilutions of 1:500 to 1:1,500 for 15 minutes at room temperature. The sections were then processed using the Catalyzed Signal Amplification (CSA) system (DAKO, Carpinteria, CA). This is a highly sensitive immunohistochemical staining procedure based on the catalyzed reporter deposition process first described by Bobrow and coworkers24 and Adams.25 Briefly, the sections were incubated with a biotinylated secondary antibody, streptavidin-biotin-horseradish peroxidase complex, biotinylated tyramide, and streptavidin peroxidase,

plasmid TTNT+No NT+ No plasmid

normal and neoplastic samples. Yashima et al14 found hTR by in situ hybridization in all cases of normal cervical mucosa, metaplastic squamous epithelium, and LSIL and showed that this expression was limited to the basal cell layer. By contrast, HSIL and SCC contained hTR throughout all cell layers of the lesional mucosa.14 Similarly, Nakano and coworkers15 demonstrated colocalization of hTR and hTERT mRNA in cervical SCCs and SILs, as well as in normal squamous mucosa, where these components were limited to the basal and suprabasal cell layers. However, telomerase activity, as detected by telomerase repeat amplification protocol (TRAP) assay, was limited essentially to carcinomas.15 Expression of hTERT is regulated by transcriptional repression and alternate splicing of hTERT mRNA, with only the full-length hTERT mRNA (457 base pairs) correlating with telomerase activity.16 The 5'-regulatory region of the hTERT gene contains numerous binding sites, including Sp1 sites and an E box, which is a potential Myc oncoprotein binding site.17-19 The molecular chaperones, p23 and heat shock protein 90 (Hsp90), recently have been shown to bind to hTERT and contribute to assembly of the functional telomerase molecule,20 and protein kinase Calpha seems to induce telomerase activity by catalyzing the phosphorylation of human TEP1 (hTEP1) and hTERT.21 In the present study, we evaluated the correlation of hTERT and hTR distribution in benign cervical mucosa, cervical dysplasia, and SCCs with the detection of telomerase activity and Ki-67, a marker of cell proliferation.

210 kd 132 kd

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❚Image 1❚ Western gel using 4B1 antibody. 4B1 antibody was used on sodium dodecyl sulfate gel of material derived from a TNT in vitro transcription-translation system. The TNT reaction that was programmed with human telomerase reverse transcriptase (hTERT) plasmid showed staining of an approximately 130-kd band, consistent with the size of the full length hTERT protein (127 kd). Bands at the bottom of the gel represent nonspecific reaction between TNT protein products and the reagents used in the Western blot.

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Frost et al / HTERT PROTEIN AND HTR IN CERVICAL MUCOSA

with intervening tris(hydroxymethyl)aminomethane– buffered saline with polysorbate (Tween; TBST) rinses. Diaminobenzidine was used for chromogenic visualization. Negative controls included the substitution of a subclassmatched monoclonal antibody that was generated against an irrelevant antigen. To demonstrate specificity of the antihTERT monoclonal antibody, a blocking study was performed whereby the primary antibody solution was immunoabsorbed with 375 ng of hTERT fusion protein. This resulted in a virtually complete block of staining. Blocking with 20 µg of a nonspecific protein (bovine serum albumin) had no effect on staining.

for 30 minutes at 55°C, and rinsed in TBST. One hundred to one hundred fifty microliters of anti-FITC horseradish peroxidase conjugate (1:400) (DAKO) was added and incubated at room temperature for 30 minutes in a humid chamber. The slides were rinsed in TBST, and fluorescyl tyramide reagent was added and incubated at room temperature for 15 minutes. The slides were then washed in TBST and water, cover glasses were applied, and the slides were viewed using a Leitz Dialux 20 fluorescence microscope.

Ki-67 Antigen Immunostaining Sections were reacted with Mib-1 monoclonal antibody to Ki-67 (Immunotech, Margency, France; PN IM 0505), diluted 1:40 in TBST, using an automated cell stainer (Ventana Medical Systems Inc, Tucson, AZ). Following incubation with primary antibody (32 minutes at 37°C), the sections were processed using an avidin-biotin system coupled with horseradish peroxidase and diaminobenzidine. The negative control for the immunohistochemical assay consisted of incubation for 37 minutes at 37°C of paired histologic sections with mouse serum (1 µg/mL in TBST) in substitution for the primary antibody. Any nuclear staining was interpreted as positive. In each case, the intramucosal distribution of staining and the percentage of positive cells were assessed.

Immunohistochemical localization of hTERT protein revealed a reproducible pattern of staining in cervical tissues. The negative control resulted in a faint diffuse nonspecific staining in several cases that was much less intense than the positive signal obtained with 4B1. A total of 55 histologic regions, from 39 patients, were analyzed for the immunohistochemical expression of hTERT protein with 4B1 antibody. The corresponding frozen tissues from each case were analyzed previously for telomerase expression by the TRAP assay. The immunohistochemical expression of Ki-67 antigen was evaluated in 53 samples with Mib-1 monoclonal antibody. There was insufficient tissue in the residual tissue blocks for Mib-1 localization in 2 cases. In situ detection of hTR was performed on 22 cases that encompassed a representative subset of all diagnostic categories. hTERT was detected in 20 (80%) of 25 sections of normal or benign-reactive cervical mucosa. hTERT staining was predominantly confined to the lower portions of the suprabasal squamous mucosa. In a few sections, hTERT staining also was detected in many of the basal cells. The subcellular localization of staining was nuclear in most cases ❚Image 2B❚. By contrast, 4 cases displayed a predominantly cytoplasmic pattern of staining that extended to the uppermost layers of the squamous mucosa. hTERT was detected in 6 (86%) of 7 sections of LSIL in a predominantly suprabasal pattern; however, the cellular localization was both cytoplasmic and nuclear in all 6 cases. In 2 cases, the staining was confined to the lower two thirds of the mucosa, while the remaining 4 cases showed almost full-thickness mucosal staining for hTERT. Among cases with a histologic diagnosis of LSIL, sections with marked human papillomavirus–associated nuclear atypia showed the strongest staining intensity ❚Image 2E❚. hTERT was detected in 11 (85%) of 13 sections of HSIL, with almost full-thickness distribution of expression in nearly all cases. The parakeratotic cell layer overlying HSIL was negative for hTERT. Only 1 case of HSIL showed an absence of staining in the basal cell layer that

hTR In Situ Hybridization Paraffin-embedded tissue sections were collected and deparaffinized as described above. Target retrieval was performed in 1× target retrieval solution (DAKO) for 10 minutes, using a decloaking chamber (Biocare Medical, Walnut Creek, CA). Following incubation in 0.001% pepsin, 0.2N hydrochloric acid for 20 minutes at room temperature, the slides were rinsed in ultrapure water and immersed in 0.3% hydrogen peroxide in methanol for 20 minutes. Fifteen microliters of antisense hTR fluorescein isothiocyanate (FITC)–conjugated probe (DAKO) was applied to the sections and allowed to hybridize in a humid chamber at 55°C for 2 to 16 hours. Negative control histologic sections were incubated with the sense hTR FITC-conjugated probe and processed in parallel with the antisense hybridized sections. The sense and antisense probes are 425 and 456 base single-stranded DNAs, respectively, generated by cycled primer extension of a plasmid (pGRN8983, Geron Corp, Menlo Park, CA) containing the mature hTR sequence. A section of testis was included with each set of slides and processed as a positive control with both sense and antisense hTR probes. Following hybridization, the slides were soaked in TBST, incubated in 1× stringent wash solution (DAKO) 728

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Results

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A

D

B

E

C

F

❚Image 2❚ Correlation between histologic diagnosis, human telomerase reverse transcriptase (hTERT) distribution, and Mib-1 staining. A (H&E), B (hTERT), and C (Mib-1), Normal squamous mucosa. hTERT nuclear staining extends from basal cell layers to mid-portion of mucosa, while Mib-1 is restricted to the parabasal cell layers (original magnification ·25). D (H&E), E (hTERT), and F (Mib-1), Low-grade squamous intraepithelial lesion, condyloma. hTERT and Mib-1 staining correlate with nuclear atypia and extend into the upper third of the squamous mucosa (original magnification ·25).


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G

J

H

K

I

L

G (H&E), H (hTERT), and I (Mib-1), High-grade squamous intraepithelial lesion, severe dysplasia. hTERT and Mib-1 staining are detected throughout all layers of the dysplastic squamous mucosa (original magnification ·25). J (H&E), K (hTERT), L (Mib-1), Squamous cell carcinoma. Nuclear and cytoplasmic staining for hTERT is detected in all tumor cells except those that show terminal squamous differentiation in squamous pearls (arrow). A randomly distributed subpopulation of the tumor cells is stained for Mib-1 (original magnification ·50).

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was similar to that seen in sections of normal cervical mucosa and LSIL. A heterogeneous pattern of hTERT subcellular localization was identified, with individual cases showing nuclear, cytoplasmic, and mixed patterns of staining ❚Image 2H❚. hTERT was detected in all SCCs (10/10 [100%]) with a predominantly cytoplasmic localization, although a nuclear component also was detected. Foci of squamous differentiation in squamous pearls failed to stain for hTERT ❚Image 2K❚. Telomerase staining showed variable concordance with the detection of telomerase activity by TRAP assay, depending on the histologic diagnosis ❚Table 1❚. The concordance of hTERT distribution with TRAP results was addressed only in samples that did not contain concurrent higher grade lesions. Histologic features from representative sections are shown ❚Image 2A❚ ❚Image 2D❚ ❚Image 2G❚ ❚Image 2J❚. Telomerase activity was detected in 5 (33%) of 15 hTERT-positive samples of normal or benign-reactive cervical mucosa and in 1 (25%) of 4 hTERT-positive LSIL cases. By contrast, telomerase activity was detected in 7 (78%) of 9 hTERT-positive HSIL samples and in 10 (100%) of 10 hTERT-positive SCCs. Telomerase activity was detected by TRAP assay in 1 sample of hTERT-negative normal cervical mucosa but was negative in the other 4 samples that did not stain for hTERT. Mib-1 staining was restricted to the immediate 1 to 3 suprabasal cell layers of normal or benign-reactive cervical mucosa ❚Image 2C❚ . The majority of sections of LSIL showed Mib-1–positive staining confined to the lower third of the mucosa; however, some LSILs displayed almost fullthickness staining ❚Image 2F❚. All HSILs ❚Image 2I❚ and SCCs ❚Image 2L❚ displayed full-thickness staining for Mib-1. In situ hybridization for hTR revealed a reproducible pattern of localization in cervical tissues. In situ hybridization with the antisense hTR probe showed a strong nuclear signal. No specific signal was generated by the sense probe. hTR was detected in 21 of 22 sections that were included in the study but was negative in a single section of HSIL. HSILs ❚Image 3D❚ and SCCs ❚Image 3F❚ displayed predominantly full-thickness epithelial localization of hTR, similar to

the distribution of hTERT protein and Ki-67. By contrast, normal and benign-reactive cervical mucosa and LSILs showed a basal and suprabasal cell distribution of hybridization within the lower portions of the mucosa ❚Image 3B❚. Histologic features from representative sections are shown ❚Image 3A❚ ❚Image 3C❚ ❚Image 3E❚.

Discussion hTERT protein was detected by a sensitive immunohistochemical method in the majority of histologic sections of normal or benign-reactive cervical mucosa, LSIL, and HSIL and was detected in all SCCs. The distribution of hTERT correlated with the histologic diagnosis. hTERT was confined to the lower portions of the squamous mucosa in most samples of normal cervical mucosa. By contrast, hTERT was detected throughout virtually all layers of the lesional epithelium in most LSILs, HSILs, and SCCs. The subcellular localization was predominantly nuclear in nonneoplastic and LSIL cases and nuclear and cytoplasmic in HSILs and SCCs. The histologic distribution hTERT correlated with Ki-67 expression in HSILs and SCCs, with full-thickness staining of the lesional epithelium. In normal mucosa and LSILs, hTERT staining extended closer to the mucosal surface than the Mib-1 staining. Although hTERT was detected in the majority of sections of normal or benign cervical mucosa, LSILs, HSILs, and SCCs, telomerase activity was detected by TRAP assay predominantly in highgrade dysplastic lesions and in carcinoma. hTR localization correlated with both hTERT and Mib-1 staining in HSILs and SCCs but showed a more restricted pattern of localization to the basal and suprabasal cell layers of benign cervical mucosa and LSILs. The immunohistochemical localization of hTERT protein in the cervix has not been evaluated previously. The results of the present study are comparable to previous results in a range of vulvar biopsy specimens stained with anti-hTERT (4B1) antibody; normal samples showed suprabasal staining, while the level of staining paralleled the

❚Table 1❚ Correlation of Detection of hTERT, hTR, Telomerase Activity, and Mib-1 Labeling With Histologic Diagnosis Histologic Diagnosis Nonneoplastic Low-grade squamous intraepithelial lesion High-grade squamous intraepithelial lesion Squamous cell carcinoma

hTERT Positive (%)

hTR Positive (%)

Telomerase Activity*/hTERTPositive Cases (%)

Average Mib-1– Positive Cells (%)

20/25 (80) 6/7 (86)

7/7 (100) 3/3 (100)

5/15 (33) 1/4 (25)

16 37

11/13 (85)

7/8 (88)

7/9 (78)

61

10/10 (100)

4/4 (100)

10/10 (100)

59

hTERT, human telomerase reverse transcriptase; hTR, human telomerase RNA component. * Telomerase activity detected by telomerase repeat amplification protocol assay.


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A

B

C

D

E

F

❚Image 3❚ Correlation between histologic diagnosis and human telomerase RNA component (hTR) in situ hybridization. A and B, Normal squamous mucosa. The hTR signal is restricted to the basal cell layer (original magnification ·50). C and D, High-grade squamous intraepithelial lesion, moderate dysplasia. hTR is detected in squamous cells throughout the lower two thirds of the dysplastic mucosa. Note the absence of hTR signal in the superficial parakeratotic layer (original magnification ·25). E and F, Squamous cell carcinoma. Nuclear signal for hTR is detected in virtually all malignant cells (original magnification ·50). A, C, and E, H&E-stained sections. B, D, and F, hTR in situ hybridization.

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degree of dysplasia in vulvar intraepithelial neoplasia cases.26 Tahara et al27 reported the detection of hTERT protein by an immunohistochemical method in colonic adenocarcinoma and nonneoplastic crypt epithelium, where it was confined to the lower portion of the crypts, while Yasui and coworkers 28 detected hTERT protein in the majority of gastric carcinomas but found weak or no staining in nonneoplastic mucosal cells. The immunohistochemical localization of hTERT has been problematic, possibly owing to a low copy number of this protein on a per-cell basis. In our hands, the localization of hTERT with the 4B1 antibody required the use of the CSA detection system. This system is highly sensitive but may be associated with correspondingly higher levels of background staining than may be achieved with conventional immunoperoxidase methodologic approaches. In the present study, we saw some evidence of nonspecific staining of normal endocervical columnar cells and stromal mesenchymal cells. In addition, an apparently specific staining of stromal lymphocytes was seen that was most prominent in germinal centers. Previous studies that used a radiolabeled in situ hybridization system found that hTR is restricted to the basal cell layer of normal cervical mucosa, metaplastic squamous epithelium, and LSIL.14 Similar to the findings of the present study, these authors found that hTR showed a full-thickness pattern of distribution in HSILs and SCCs, with decreased expression in areas of squamous maturation.14 Similarly, Nakano et al15 reported colocalization of hTR RNA and hTERT mRNA in the basal and parabasal cells of histologically normal cervical squamous epithelium, higher levels of expression in dysplastic epithelium, and full-thickness expression in SCCs. The immunohistochemical detection of hTERT protein in most HSILs and all SCCs is not unexpected, considering that the majority of such samples are TRAP positive. However, the detection of hTERT staining in normal mucosa and a high proportion of LSILs was not anticipated because previous studies had shown that only a minority of those cases are TRAP positive. Possible explanations for the lack of concordance between hTERT protein detection and telomerase activity in our study could include the generation of alternatively spliced forms of hTERT mRNA or the posttranslational modification of hTERT protein that could result in immunohistochemically detectable variants of hTERT protein that are functionally inactive. An alternative explanation could be that there is telomerase activity in at least some of these hTERT-positive cells but that the levels are below the threshold of detection of even the sensitive TRAP assay. Consistent with this concept, studies by Härle-Bachor et al29 and Yasumoto et al30 showed that telomerase activity could be detected in normal human © American Society of Clinical Pathologists

epidermal cells and uterine ectocervical keratinocytes, but only after fractionation to separate the telomerase-positive basal cells from the residual telomerase-negative squamous cells. The predominantly nuclear localization of hTERT in nonneoplastic and LSIL samples as found in the present study contrasts with the nuclear and cytoplasmic localization of hTERT that was found in HSILs and SCCs. The disruption of normal nuclear targeting mechanisms for translocation of hTERT to the nucleus, as has been described for estrogen receptors, may be responsible for these different staining patterns and the variable concordance with TRAP positivity.31,32 Our findings support the concept of a population of normal stem cells in the lower layers of the cervical squamous mucosa that contains detectable amounts of hTERT protein and hTR RNA. Although this stem cell population of the normal cervical mucosa seems to be largely undetected by the TRAP assay, it may be responsible for the lack of TRAP specificity seen in various studies. The increasing proportions of hTERT-positive cells in SILs and SCCs suggest that hTERT is an integral component of the dysplasia-carcinoma sequence. A possible application of hTERT protein immunostaining may be in the area of cervical cytology. As the majority of cells in Papanicolaou smears are derived from superficial squamous cells, the specificity of hTERT staining for the detection of HSILs may be greater than that seen in surgical biopsy specimens owing to decreased representation of nonneoplastic cells from the lower portion of the mucosa. Thus, in this specific application, the detection of hTERT in cytologically equivocal cells could be useful in the diagnosis of the cytologic precursors of SCC. We found a reproducible distribution of hTERT protein in benign, premalignant, and malignant cervical mucosa that correlated with histologic diagnosis, Mib-1 staining, and hTR detection. Although the detection of hTERT protein seemed to correlate with telomerase activity in HSILs and SCCs, hTERT also was detected in LSILs and normal or benign-reactive cervical mucosa. From the Departments of 1Pathology and 4Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, CO; 2Geron, Menlo Park, CA; 3DAKO, Carpinteria, CA; and the 5Department of Obstetrics and Gynecology, Osaka University Faculty of Medicine, Osaka, Japan. Dr Shroyer was supported by grants R01 CA78442-01A1 and R44 CA67564-02A1, and Drs Kim and Weinrich were supported by grant R43 CA67564, from the National Cancer Institute, Bethesda, MD. Address reprint requests to Dr Shroyer: Dept of Pathology, University of Colorado Health Sciences Center, 4200 E 9th Ave, B-216, Denver, CO 80262.

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18. Takakura M, Kyo S, Kanaya T, et al. Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells. Cancer Res. 1999;59:551-557. 19. Horikawa I, Cable PL, Afshari C, et al. Cloning and characterization of the promoter region of human telomerase reverse transcriptase gene. Cancer Res. 1999;59:826-830. 20. Holt SE, Aisner DL, Baur J, et al. Functional requirement of p23 and Hsp90 in telomerase complexes. Genes Dev. 1999;13:817-826. 21. Li H, Zhao L, Yang Z, et al. Telomerase is controlled by protein kinase Calpha in human breast cancer cells. J Biol Chem. 1998;273:33436-33442. 22. Wright TC, Kurman RJ, Ferenczy A. Carcinoma and other tumors of the cervix. In: Kurman RJ, ed. Blaustein’s Pathology of the Female Genital Tract. New York, NY: Springer-Verlag; 1994:244-254. 23. Wright TC, Kurman RJ, Ferenczy A. Carcinoma and other tumors of the cervix. In: Kurman RJ, ed. Blaustein’s Pathology of the Female Genital Tract. New York, NY: Springer-Verlag; 1994:287-289. 24. Bobrow MN, Litt GJ, Shaughnessy KJ, et al. The use of catalyzed reporter deposition as a means of signal amplification in a variety of formats. J Immunol Methods. 1992;150:145-149. 25. Adams JC. Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem. 1992;40:1457-1463. 26. Wada H, Enomoto T, Yoshino K, et al. Immunohistochemical localization of telomerase hTERT protein and analysis of clonality in multifocal vulvar intraepithelial neoplasia. Am J Clin Pathol. 2000;114:371-379. 27. Tahara H, Yasui W, Tahara E, et al. Immuno-histochemical detection of human telomerase catalytic component, hTERT, in human colorectal tumor and non-tumor tissue sections. Oncogene. 1999;18:1561-1567. 28. Yasui W, Tahara H, Tahara E, et al. Expression of telomerase catalytic component, telomerase reverse transcriptase, in human gastric carcinomas. Jpn J Cancer Res. 1998;89:10991103. 29. Härle-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;93:6476-6481. 30. Yasumoto S, Kunimura C, Kikuchi K, et al. Telomerase activity in normal human epithelial cells. Oncogene. 1996;13:433-439. 31. Devin-Leclerc J, Meng X, Delahaye F. Interaction and dissociation by ligands of estrogen receptor and Hsp90: the antiestrogen RU 58668 induces a protein synthesis-dependent clustering of the receptor in the cytoplasm. Mol Endocrinol. 1998;12:843-854. 32. Dauvois S, White R, Parker MG. The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling. J Cell Sci. 1993;106:1377-1388.
