© 2002 American Thoracic Society
Expression of Human Telomerase Reverse Transcriptase in LymphangioleiomyomatosisPathology Section and Pulmonary Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland Correspondence and requests for reprints should be addressed to Zu-Xi Yu, M.D., Ph.D., Pathology Section, National Heart, Lung, and Blood Institute, Building 10/2N240, National Institutes of Health, 10 Center Drive MSC-1518, Bethesda, MD 20892-1518. E-mail: yuz{at}helix.nih.gov
Telomerase synthesizes nucleotide hexameric repeats (telomeres) at the ends of chromosomes, replacing base sequences that are lost from these sites during each mitotic cycle and protecting these ends against the action of exonucleases and ligases. Therefore, telomerase is essential for maintaining cellular replication. To evaluate the role of telomerase in the proliferation of abnormal smooth muscle cells (lymphangioleiomyomatosis [LAM] cells) in LAM, we performed immunostaining and in situ hybridization studies to identify telomerase protein and messenger RNA (mRNA), respectively, in pulmonary (n = 18) and extrapulmonary (n = 4) lesions from 22 women with LAM (14 untreated and 8 treated with progesterone or tamoxifen). Immunoreactivity and hybridization signals for telomerase were observed in 5 to 20% of LAM cells, mostly of the spindle-shaped type, in 21 of the 22 patients, and were less intense in the treated group. Other types of cells were unreactive in both groups. Telomerase colocalized in the same cells with  -smooth muscle actin, but only rarely with HMB-45 antibody (a marker for epithelioid LAM cells); colocalization with proliferating cell nuclear antigen was incomplete. The telomerase-positive LAM cells may constitute the sources of renewal of LAM cells. Modulation of telomerase may be involved in the control of LAM cell proliferation.
Key Words: lymphangioleiomyomatosis • lung • telomerase • smooth muscle cells
Lymphangioleiomyomatosis (LAM) is a rare disorder that mainly affects young women and is characterized by (1) proliferation of abnormal smooth muscle cells (LAM cells) in pulmonary interstitum and along axial lymphatics of the thorax and abdomen, (2) multiple thin-walled pulmonary cysts, and (3) a high incidence of angiomyolipomas, which most often occur in the kidneys (1). The LAM cells tend to form nodules, in which two subtypes of cells are recognized: small, spindle-shaped cells that are centrally located; and larger, peripherally located cells that have an epithelioid morphology, contain receptors for estrogen and progesterone, and are reactive with HMB-45 antibody, a marker for developing melanosomes. These characteristics are acquired during the transition of spindle-shaped cells to epithelioid cells. LAM is not considered to be a neoplastic disorder. However, the nature of this proliferation is poorly understood, and LAM cells can demonstrate some degree of invasiveness: the infiltrates of LAM cells sometimes extend beyond the fibrous tissue capsules of lesions of extrapulmonary LAM. Immunostaining for proliferating cell nuclear antigen (PCNA), a marker of mitotic activity, has demonstrated that the spindle-shaped LAM cells proliferate much more rapidly than the epithelioid cells (reviewed in 1). However, there is no report of telomerase activity in LAM cells. Telomeres are present at the ends of chromosomes and consist of up to 10 to 20 kilobases of hexameric nucleotide repeats (TTAGGG) (2). They play a key role in stabilizing chromosomes upon replication by protecting their ends against the action of exonucleases and ligases. Loss of telomeric sequences is an important mechanism that limits the number of cell divisions and induces cellular senescence. In all normal somatic cells, each cycle of cell division and DNA replication results in a loss of 50–200 terminal nucleotides from each chromosome, as DNA polymerase cannot completely replicate the ends of linear DNA molecules (3). Telomerase is expressed in most human neoplastic cells and immortalized cell lines but is inactive in normal somatic cells except for germ cells and stem cells (4, 5). Human telomerase has two essential components: (1) a telomerase RNA component (hTERC), which serves as a template for the synthesis of telomeric repeats, and (2) telomerase reverse transcriptase (hTERT), which provides the catalytic activity and is associated with other proteins, such as telomerase protein 1 (hTEP1) (6). hTERC is expressed in all cells, but the expression of hTERT is limited to cells that show telomerase activity (7). Other experiments also strongly suggest that hTERT is the major determinant of human telomerase activity (8). Biochemical methods for the measurement of telomerase activity are well-established (4). The recent development of a rabbit polyclonal antibody against TERT has made it possible to investigate telomerase activity at the cellular level by means of immunohistochemical staining (9). To evaluate the role of telomerase activity in the proliferation of LAM cells, we have employed immunohistochemical and in situ hybridization methods for the localization of this enzyme, and we have determined the relationship between the expression of TERT and various characteristics of the LAM cells.
Tissue Samples The study group consisted of 18 women ranging from 17 to 51 years of age (mean age, 36 years) with well-documented LAM. The patients were divided into two groups according to whether their tissue samples had been taken before treatment, at the time of their initial evaluation (n = 10), or at pulmonary transplantation after prolonged therapy with progesterone or tamoxifen (n = 8). The durations of the illness, estimated from the onset of symptoms to the time when the diagnosis was made, ranged from 1–24 months in an untreated group and from 7 months to 22 years in a treated group. Medroxyprogesterone was the most commonly used regimen, and the frequent dose was 400 mg/month. We also examined tissues from four untreated patients with extrapulmonary LAM, including one mediastinal lymph node and three retroperitoneal masses, as well as one angiomyolipoma of the kidney. These patients ranged from 27 to 60 years of age (mean age, 43 years). The study was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute.
Preparation of Tissues
Single Antibody Labeling
Dual Antibody Labeling
In Situ Hybridization
Immunoreactivity for TERT was observed in tissues from all 10 untreated cases of pulmonary LAM as well as in four cases of extrapulmonary LAM and in one AML (Figures 1A–1D). A positive reaction also was recognized in seven of eight treated cases of pulmonary LAM. In untreated cases, TERT was localized in the nuclei as well as in the cytoplasm of 10% of spindle-shaped LAM cells (Figure 1A). This reactivity was rarely present in the epithelioid LAM cells. In treated cases, the intensity of the reaction for TERT was lower than that in untreated cases (Figure 1B). In the cases of extrapulmonary LAM and AML (Figures 1C and 1D), the reaction for TERT was positive in the smooth muscle–like LAM cells and appeared similar to that observed in untreated cases of pulmonary LAM. Semiquantitative analysis of hTERT was performed on 12 well-stained cases, including 6 untreated and 6 treated patients (Table 1). TERT-positive cells in untreated group were 1.9% to approximately 14.6% and 0.8% to approximately 10.6% in the treated group, respectively. Reactivity for TERT was not detected in normal cells or tissues, including bronchial and vascular smooth muscle cells, adipocytes in the angiomyolipoma, or the endothelial cells lining vascular channels in the extrapulmonary LAM lesions. Control procedures gave negative results (Figure 1E).
Dual-immunofluorescent staining and confocal microscopy showed that the reaction for TERT was positive mainly in cells that also showed reactivity for -smooth muscle actin (Figure 2B). This reactivity was recognized in a very few -smooth muscle actin–negative LAM cells. Furthermore, TERT colocalized with PCNA in the nuclei of some LAM cells in untreated cases (Figure 2C). However, this colocalization was rare in treated cases, in which reactivity for PCNA was observed in only very few LAM cells. Dual staining for TERT and HMB-45 antibody showed that these two reactions colocalized only very rarely, thus confirming that the vast majority of the TERT-positive cells were of the spindle-shaped type (Figure 2A).
In situ hybridization showed a signal for hTERT in the cytoplasm of approximately 5% of spindle-shaped LAM cells in all lesions of LAM and 10–20% of the LAM cells in the AML and lesions of extrapulmonary LAM (Figures 3A–3D). The signal was more intense in extrapulmonary LAM lesions and the AML than in pulmonary lesions. In the latter, it was less intense and frequent in the treated cases. Vascular and bronchial smooth muscle cells were negative. Reactivity with the sense probe was negative in all cases (Figure 3E).
This study demonstrates the presence of telomerase in 5–20% of pulmonary and extrapulmonary LAM cells, as indicated by the localization of the hybridization signal for hTERT messenger RNA (mRNA) and the immunohistochemical reactivity for TERT. The patterns of colocalization with other LAM cell markers indicate that the telomerase activity is limited to the spindle-shaped type of LAM cells. The observation of reactivity for telomerase in both the nucleus and the cytoplasm of LAM cells is consistent with synthesis of this enzyme in the endoplasmic reticulum and subsequent transport to the nucleus (9). The finding that telomerase-positive LAM cells are less frequent in tissues obtained after prolonged therapy with progesterone or tamoxifen correlates with the proposed decrease in LAM cell proliferation occurring under these circumstances. The present authors are not aware of data on the immunohistochemical reactivity of normal or neoplastic smooth muscle cells for telomerase. Two biochemical studies (11, 12) have demonstrated that telomerase is not functional in normal uterine smooth muscle cells or uterine leiomyomas. Ulaner and colleagues (12) showed that certain normal ovarian tissues and myometrial leiomyomas lacked telomerase activity despite expressing hTERT containing complete A and B transcriptase motifs, which are parts of the reverse transcriptase sequence of hTERT (7). They concluded that a functional telomerase complex is regulated at multiple levels, including hTERT transcription and alternative splicing of hTERT transcripts. c-Myc enhances telomerase activity (13). The TERT promoter contains c-Myc binding sites, and c-Myc expression results in increased promoter activity with increased hTERT mRNA transcription and telomerase activity (14). Our finding that c-Myc is immunohistochemically demonstrable in spindle-shaped LAM cells (15) correlates with the presence of telomerase in these cells. Because estrogen can increase c-Myc mRNA and protein levels in a hormone-responsive human breast cancer cell line (16), hormonal therapy may influence hTERT transcription through altered c-Myc accumulation, a potential mechanism for therapeutic action of these agents in LAM. Of relevance to LAM are recent studies (17, 18) showing that telomerase activity is increased by estrogen and decreased by progesterone and tamoxifen in tissues sensitive to the biologic effects of these agents, including breast, uterus, ovary, and normal and neoplastic cell lines derived from these organs. The activation of telomerase by estrogen is mediated by upregulation of hTERT mRNA. The estrogen-responsive element in the hTERT promoter binds to estrogen receptors and is responsible for the transcriptional activation by ligand-activated estrogen receptors. Telomerase activity has been shown to be inhibited by protein kinase C inhibitors (19) and by protein phosphatase 2A (20). The association of TRF1 and tankyrase with human telomere protein complexes also has been found to inhibit telomerase function (21, 22). It remains to be determined whether these or other factors participate in the regulation of telomerase activity in LAM cells. The small percentage of telomerase-positive cells in LAM may be related to the fact that cellular replication is much slower in this disorder than in malignant neoplasms. The decrease in the reactivity for telomerase in cells from patients with LAM treated with progesterone or tamoxifen could be due to downregulation of telomerase by these drugs. Histochemical studies of telomerase in tumors have been limited to pulmonary adenocarcinomas (9) and malignant mesotheliomas (23). In both, more than 80% of the tumor cells showed reactivity for telomerase and a much lower reactivity for PCNA. However, among LAM cells, the frequency of these reactivities was much lower, and their colocalization was much less consistent. Although these findings indicate that the telomerase-positive cells are capable of replicating, telomerase activity is not to be considered a measure of mitosis. In summary, this study demonstrates the presence of telomerase in 5–20% of LAM cells, mostly of the proliferative spindle-shaped type. Only a small percentage of LAM cells is telomerase positive, and the telomerase does not colocalize with PCNA. Possibly, the telomerase-positive cells can function as sources for replication of LAM cells. Thus, modulation of telomerase activity may be of importance in LAM cell proliferation; however, this remains to be established.
The authors thank Mrs. C. Jane Bell for her editorial assistance in the preparation of this manuscript and Dr. Martha Vaughan for helpful discussions and critical review of the manuscript. Received in original form May 10, 2001; accepted in final form March 6, 2002
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