Interferon regulatory factor-1 (IRF-1) is a mediator for interferon-γ induced attenuation of telomerase activity and human telomerase reverse transcriptase (hTERT) expression

Abstract

Constitutive activation of the telomerase is a key step in the development of human cancers. Interferon-γ (IFN-γ) signaling induces growth arrest in many tumors through multiple regulatory mechanisms. In this study, we show that IFN-γ signaling represses telomerase activity and human telomerase reverse transcriptase (hTERT) transcription, and suggest that this signaling is mediated by IRF-1. Ectopic expression of IRF-1 attenuated hTERT promoter activity. Murine embryonic fibroblasts (MEFs) genetically deficient in IRF-1 (IRF-1−/−) showed an elevated level (>15 times) of hTERT promoter activity as compared to the hTERT promoter activity of wild-type MEFs. The telomerase activity and hTERT expression in IRF-1−/− MEFs were downregulated by IRF-1 transfection. Interestingly, less extent of telomerase repression was observed in HPV E6 and E7 negative, p53 mutant HT-3 cells than in HPV 18 E6 and E7 positive HeLa cells (intact p53). These findings provide evidence that IRF-1 is a potential mediator of IFN-γ-induced attenuation of telomerase activity and hTERT expression.

Introduction

Telomeres are composed of tandem arrays of short DNA sequences, TTAGGG, several kilobases in length. In the absence of telomerase activity, the telomere gradually shortens during replication, due to the loss of 5′ ends in double-stranded DNA (Blackburn, 1991; Makarov et al., 1997). Recently, several hypotheses concerning telomere erosion as a primary cause of cellular senescence have been proposed (Allsopp and Harley, 1995; Holt and Shay, 1999). Blocking or avoiding cellular senescence is a required step for the infinite cell proliferation present in immortalized cancer cells. Stable maintenance of telomere length is required for the long-term proliferation of cancer (Shay, 1995; Shay and Wright, 1996; Holt and Shay, 1999).

Telomerase, an RNA-dependent DNA polymerase, synthesizes telomeric repeats using the 3′ telomeric end as a primer and an RNA template. This process maintains telomere length (Morin, 1989; Weinrich et al., 1997). A high level of telomerase activity has been reported in 85% of malignant human cells. In comparison, only 30% of benign tumor tissue cells and 0.5% of normal cells exhibit a high level of telomerase expression (Shay and Bacchetti, 1997). It is generally accepted that there is a link between cellular proliferation or senescence and telomerase activity. Activated telomerase is detected in highly proliferative normal cells, such as early embryonic stem cells and basal keratinocytes in the adult epidermis (Harle-Bachor and Boukamp, 1996; Wright et al., 1996). However, telomerase activity is not present in quiescent tissues. These findings indicate a pivotal role for telomerase in expanding the life span of immortalized cancer cells.

Cervical cancer is a major cancer type that affects women in developing countries. Over 70% of all cervical carcinoma is associated with high-risk human papillomavirus (HPV 16 and HPV 18) that encode two viral oncogene products E6 and E7 (Schiffman, 1992; Bosch et al., 2002). These two proteins are known to exert their oncogenic functions by interfering with two cellular tumor suppressive proteins p53 and Rb. The HPV 16 E6 protein binds to the p53 protein and promotes its degradation (Scheffner et al., 1990; Werness et al., 1990). HPV 16 E7 protein interferes with binding of Rb to E2F leading to activate transcription of genes essential for cell cycle progress (Dyson et al., 1989; Boyer et al., 1996). Recent studies have shown that expression of E6 and E7 proteins in primary keratinocytes led to activation of telomerase and immortalization, suggesting that E6 and E7 are implicated in regulation of telomerase (Veldman et al., 2001; Baege et al., 2002).

Interferon (IFNs) have been successfully used in the treatment of various cervical cancer patients, and the mechanism of their antitumor action is an active area of research. IFNs are a family of multifunctional cytokines that trigger immunomodulatory, antiviral, antiproliferative, and antitumor activities (Deiss et al., 1995; Maciejewski et al., 1995; De Maeyer and De Maeyer-Guignard, 1998). IFN-γ induces p53-independent apoptosis and cell cycle arrest (Ossina et al., 1997; Kano et al., 1999). More than 30 IFN-inducible genes have been identified (Farber, 1992; Lengyel, 1993a, b). A number of those genes are involved in tumor suppressive functions. Specifically, interferon regulatory factor-1 (IRF-1) is one of the candidates for tumor suppressive proteins induced by IFNs (Harada et al., 1993,1994). IRF-1 plays an important role in apoptosis, growth arrest, and cell differentiation (Harada et al., 1993; Tanaka et al., 1994; Tamura et al., 1995). Analysis of IRF-1-deficient mice and of ectopic expression of IRF-1 has shown that IRF-1 may prevent oncogene-induced malignant transformation and trigger programmed cell death (Tanaka et al., 1994; Nozawa et al., 1999). Furthermore, clinical studies have indicated that deletions or rearrangements of the IRF-1 gene are consistently observed in leukemia cells of patients with acute myelogenous leukemia (50%) and with preleukemic myelodysplastic syndrome (30%) (Willman et al., 1993; Lengyel, 1993a, b). Esophageal and gastric cancers are also characterized by the loss of an IRF-1 allele. Likewise, a functionally inactivating point mutation of the IRF-1 gene has been reported in human gastric cancers (Ogasawara et al., 1996; Nozawa et al., 1998). Taken together, the above studies suggest that IRF-1 plays a critical role as a tumor suppressor in certain types of cancers.

In the present study, we provide evidence that IRF-1 plays an important role in mediating IFN-γ-induced inhibition of human telomerase reverse transcriptase (hTERT) expression and telomerase activity. Telomerase repression by IFN-γ/IRF-1 signaling was stronger in HPV 18 E6 and E7 positive HeLa cells than in HPV E6/E7 negative HT-3 cells, indicating that this pathway may be involved in downregulating HPV 18 E6 and E7 proteins.

Results

Detection of telomerase activity in human cervical cancers

Telomerase activity is undetectable in most normal human somatic cells. In contrast, telomerase becomes highly activated in various types of human cancers and in immortalized cell lines. We examined telomerase activity in human cervical cancer tissues, most of which were human papillomavirus (HPV) positive, in three normal cervical tissues and in two human cervical cancer cell lines, HeLa and HT-3. The three normal cervical tissues showed undetectable telomerase activity. In contrast, both cervical cancer tissues and cervical cancer cell lines showed high telomerase activity (Figure 1).

Figure 1
figure1

Telomerase activity in cervical cancer tissues and cell lines. A, telomerase activity profile from normal and tumor tissues of cervical patients and cervical cancer cell lines. Telomerase activity was determined by TRAP assay. CHAPS extracts 1 μg pretreated with RNase A (200 μg/ml) was used to inactivate the RNA component of telomerase. 1–3, normal tissues; 4–7, Tumor tissues; 8–9, HeLa and HT-3 cell lines; 10, TSR8, quantitative control

IFN-γ and IRF-1 suppressed telomerase activity and hTERT transcription

Both HPV 18 E6 and E7 positive HeLa and E6 and E7 negative HT-3 cell lines have high levels of telomerase activity as well as of hTERT mRNA expression, as determined by TRAP and RT–PCR assays. IFN-γ treatment, with doses that ranged from 25 to 250 U/ml, led to downregulation of telomerase activity in both cell lines in a dose-dependent manner (data not shown). After exposure of HeLa cells to 250 U/ml IFN-γ, a dramatic decline in both telomerase activity and hTERT mRNA level was observed at 24 h when the level reached a plateau of about 55 and 70%, respectively, of the levels seen in untreated cells (Figure 2a–c). HT-3, a HPV E6 and E7 protein negative cell line also showed downregulated hTERT expression and telomerase activity by IFN-γ (Figure 2) but to a lesser extent than HeLa cells (Figure 2a–c).

Figure 2
figure2

Effects of IFN-γ on telomerase activity and hTERT levels on HeLa and HT-3 cells. Cells were cultured in the presence of IFN-γ (250 U/ml) for the indicated times. Telomerase activity and hTERT expression were determined using TRAP assay (a) and RT–PCR (b). Respectively the relative telomerase activity (•) and hTERT level (▪) were indicated (c), as described in the ‘Materials and methods’ section. Data shown are representative of three independent experiments

Next, we asked whether IRF-1, an IFN-γ inducible antioncogenic protein is a downstream mediator of IFN-γ signaling to hTERT transcription (Harada et al., 1993). HeLa and HT-3 cells were treated with IFN-γ at various time intervals. Total protein was extracted and subjected to Western blot analysis for IRF-1 expression. IRF-1 was induced within 6 h after IFN-γ treatment and its level was sustained for 48 h (Figure 7a). SKOV3 is HPV negative cell line. Growth arrest and reduction of tumorigenecity, as a consequence of telomerase inhibition, were observed in several cell lines with inactivated p53. So we used the SKOV3 cell line to show whether the downregulation of hTERT expression and telomerase activity by IFN-γ/IRF-1 is mediated by a p53-dependent or independent pathway. Transfection of IRF-1 into both cell lines also led to a significant suppression of telomerase activity and hTERT expression. This effect was noticeable after 36 h in HeLa and 48 h in HT-3 and SKOV3 cell lines (Figure 3). Consistent with IFN-γ treatment, the observed inhibitory action on telomerase activity and hTERT expression was stronger in HeLa cells than in HT-3 cells (Figure 3). These results indicate that IRF-1 mediates IFN-γ inducible repression of telomerase activity and hTERT expression.

Figure 7
figure7

Effect of IFN-γ or IRF-1 on the expression of IRF-1, p53, E6, E7, and cMyc (a,b), and effect of E6/E7 on hTERT promoter activity (c). Western blot analysis was performed on total cell lysates (20 μg) using antiserum against represented protein products after IFN-γ treatment (a), and IRF-1 effect of E7 expression were performed by Western (Ba) and RT–PCR analysis (Bb). Cells were transfected with 2 μg of human telomerase 3396 bp promoter reporter gene construct and 2 μg of expression vector, pcDNA3.1-E6 or -E7 (c). Luciferase activity was assessed 48 h after transfection and then normalized to β-gal activity. Values are means ±s.d. from triplicate samples and are representative of two independent experiments

Figure 3
figure3

Effects of IRF-1 on telomerase activity and hTERT levels on HeLa, HT-3 and SKOV3 cells. Cells were transfected with 2 μg of expression vector, pcDNA3.1-IRF-1, for the indicated time points. Telomerase activity (a) and hTERT expression (b) were determined using TRAP assay and RT–PCR, respectively. The relative telomerase activity (•) and hTERT level (▪) were represented in (c). Data shown are representative of three independent experiments

IFN-γ and IRF-1 repressed the hTERT promoter

To confirm whether IFN-γ affects hTERT transcriptional activation, we transfected a 3396 bp hTERT promoter region reporter gene construct (2 μg) into HeLa and HT-3 cells. IFN-γ treatment (250 U/ml) was performed 24 h after transfection, and luciferase activity was assessed 48 h after transfection. As shown in Figure 4a, IFN-γ-treated cells showed a significant decrease (65% for Hela cells, 40% for HT-3) in the hTERT promoter activity.

Figure 4
figure4

IFN-γ/IRF-1 downregulates hTERT promoter activity in U937, HeLa and HT-3 cells. Cells were transfected with 2 μg of human telomerase 3396 bp promoter reporter gene construct in the presence and absence of IFN-γ (a), or various amounts of pcDNA-IRF-1 (0, 0.5, 1, and 1.5 μg) (b). IFN-γ treatment (250 U/ml) was performed 24 h after transfection. Luciferase activity was assessed 48 h after transfection and then normalized to β-gal activity. Values are means ±s.d. from triplicate samples and are representative of two independent experiments. All IFN-γ or IRF-1-treated cells were significantly different from the control untreated cells (one-way ANOVA, P<0.05). And the difference between the HeLa and HT-3 cells were also significant (two-way ANOVA, P<0.05)

Next, we asked whether IRF-1 could decrease the activity of the hTERT promoter. To accomplish this, IFN-sensitive U937 monocytic cell lines, which constitutively express IRF-1 gene product, Hela and HT-3 cells were cotransfected with 2 μg of 3396 bp hTERT promoter region reporter construct and various amounts of pcDNA-IRF-1 (0, 0.5, 1, and 1.5 μg). Transiently transfected IRF-1 suppressed luciferase activity in a dose-dependent manner in U-937 promonocytic cells (Figure 4b). HeLa cells showed 60% suppression, while the suppressing effect in HT-3 cells was relatively weak (31%) (Figure 4b). And the two-way ANOVA test showed that the difference of hTERT promoter activity between HeLa and HT-3 cells were statistically significant (P<0.05).

Telomerase activity and hTERT transcription in IRF-1-deficient MEFs

To confirm the suppression of telomerase activity caused by IRF-1, wild-type and IRF-1−/− MEF cells were cultured in the presence of IFN-γ. Wild-type MEFs showed downregulation of telomerase activity, but IRF-1−/− MEFs did not (Figure 5a and b). As expected, the IRF-1 protein was dramatically induced in wild-type MEFs upon IFN-γ treatment but no change was detected in IRF-1−/− MEFs (Figure 5c). To determine the basal level of hTERT promoter activity, the 3396 bp hTERT promoter region reporter construct was transfected into primary MEFs from wild-type and IRF-1 deficient mice. IRF-1−/− MEFs showed a hTERT promoter activity that was 15 times higher than that present in wild-type MEFs. IRF-1−/− MEFs transfected with IRF-1 showed about 45% less hTERT promoter activity (Figure 6).

Figure 5
figure5

INF-γ effects in MEF cell lines. (a) Wild-type (WT) or IRF-1−/− primary embryonic fibroblasts cells was cultured in the presence of IFN-γ (250 U/ml) for the indicated times. Telomerase activity was determined using TRAP assay (a). The relative telomerase activity of IRF-1 deficient MEF cells (•) and WT MEF cells (▪) were represented in (b). Data shown are representative of three independent experiments. Western blot analysis was performed on total cell lysates (20 μg) using IRF-1 anti-serum after IFN-γ treatment (c)

Figure 6
figure6

Transcriptional activity of human telomerase promoter is upregulated in IRF-1−/− cells. (a) Human telomerase 3396 bp promoter reporter gene construct (2 μg) was transfected into wild-type (WT) or IRF-1−/− primary embryonic fibroblasts, then determined the basal level of hTERT promoter activity. (b) Cells were transfected with 2 μg of human telomerase 3396 bp promoter reporter gene construct and various amounts of pcDNA-IRF-1 (0, 0.1, 0.25, 1, 2, and 5 μg). Luciferase activity was assessed 48 h after transfection and then normalized to β-gal activity. Values are means ±s.d. from triplicate samples and are representative of two independent experiments

IFNγ/IRF-1 repressed telomerase through downregulation of HPV 18 E6 and E7 proteins but not c-Myc

Consistent with a previous report (Kim et al., 2000), IFN-γ treatment resulted in a decreased level of both E6 and E7 proteins. Interestingly, the level of p53 was unaffected in HT-3 cells, which do not have E6 and E7 proteins. An HPV 18 E6 and E7 protein-positive cell line, HeLa, showed a clear increase in p53 to a level similar to that found in HT-3 cells 12 h after IFN-γ treatment (Figure 7a), which can be explained by the roles of HPV E6 protein in promoting degradation of p53. Adenoviral introduction of IRF-1 downregulated both HPV 18 E7 (HeLa) and HPV 16 E7 (Caski) protein and also mRNA levels (Figure 7b). hTERT promoter activities were increased by transient transfection with E6 or E7 in HeLa, HT3, wild-type MEFs and IRF-1−/− MEFs (Figure 7c). These results suggest that HPV 18 E6 and E7 proteins are associated with increases in telomerase activity and hTERT expression, and that IFN-γ/IRF-1 signaling to hTERT expression may be mediated through HPV 18 E6 and E7 proteins in Hela cells.

c-Myc activates hTERT transcription by binding to the hTERT promoter region (Wang et al., 1998; Wu et al., 1999). IFNs suppress c-Myc expression post-transcriptionally in some cell systems (Jonak and Knight, 1984; Dron et al., 1986). To determine whether c-Myc is involved in IFN-γ-induced downregulation of telomerase activity in human cervical cancers, we examined c-Myc expression levels after IFN-γ treatment. Interestingly, the amount of c-Myc protein level was not changed by IFN-γ (Figure 7a). Similarly, IRF-1 expression did not affect c-Myc expression in the cervical cancer cell lines tested (data not shown).

Relation between telomerase activity and G1 arrest

IFN-γ causes cell growth arrest at the G1 phase of the cell cycle (Bromberg et al., 1996; Horvath et al., 1996). Because several previous reports have shown that the levels of telomerase activity may be related to proliferation and cell cycle regulation (Greider, 1998; Akiyama et al., 1999; Shiratsuchi et al., 1999), we investigated whether the effect of IFN-γ on telomerase activity depends upon cell cycle arrest. HeLa and HT-3 cells were treated with mimosine, which is known to arrest cells at the G1/S transition by inhibition of the eucaryotic initiation factor 5-A and upregulation of p21WAF1. Treatment of the two cell lines with mimosine for 24 h resulted in a nearly complete cell cycle arrest of over 95% in G1-phase, and with interferon-γ also showed over 85% cell cycle arrest in G1-phase as shown by the analysis of cellular DNA content by flow cytometry (data not shown). Although the majority of IFN-γ-treated cells showed downregulation of telomerase activity during the G1 phase of the cell cycle, cells whose cycle had been arrested by mimosine did not show any change in telomerase activity for up to 72 h (Figure 8). These results indicate that telomerase downregulation is not caused by cell cycle arrest, and also that G1 arrest by IFN-γ is not indispensable for modulating telomerase activity.

Figure 8
figure8

Effects of mimosine on telomerase activity and hTERT levels on HeLa and HT-3 cells. Cells were cultured in the presence of 0.5 mM mimosine for the indicated times. Telomerase activity and hTERT expression were determined using TRAP assay (a) and RT-PCT (b), respectively. The relative telomerase activity (•) and hTERT level (▪) were represented in (c). Data shown are representative of three independent experiments

Regulation of hTERT transcription by IRF-1 not by direct binding to hTERT promoter

To determine whether the IRF-1 binds to two putative IRF-1 binding sites on hTERT promoter and directly regulates the transcriptional activation, we constructed first and/or second putative IRF-1 binding site deleted or mutated hTERT promoters and used for transfection and reporter assays. The hTERT full promoter was downregulated by IRF-1 expression and all of the mutated promoters also showed repression of the hTERT promoter (data not shown), suggesting that the two putative binding sites are not the binding site of IRF-1. It is possible that an alternative binding site may exist or that IRF-1 indirectly regulates the transcriptional activation.

Discussion

Telomerase activation is one of the hallmarks of cancers. We herein provide evidence that IRF-1 downregulates telomerase activity and hTERT expression. Lack of IRF-1 gene via gene knockout resulted in significant activation of telomerase activity. Consistently, increase of IRF-1 by ectopic transfection or by IFN-γ treatment suppressed hTERT gene expression and telomerase activity.

The function of IRF-1 as an antioncogenic transcription factor is well known although the underlying mechanism is not well understood (Harada et al., 1993; Tanaka et al., 1994). Our studies using MEFs deficient in IRF-1 transfected with hTERT promoter reporter system indicate that IRF-1 is critically involved in downregulation of hTERT expression. Consistently, IRF-1−/− mice show an impairment of tumor suppression (Tanaka et al., 1994). Taken together these findings suggest a mechanism that IRF-1 exerts its antioncogenic action by negatively regulating hTERT mRNA expression and telomerase activity.

HPV 16 E6 and E7 proteins have been implicated in immortalization of primary keratinocytes possibly by activation of telomerase or maintenance of telomere length (Veldman et al., 2001; Baege et al., 2002). Consistent with recent studies, IFN-γ treatment or IRF-1 expression decreased HPV 18 E7 and E6 protein levels in HeLa cells, and this decrease may contribute to the repression of telomerase activity. Further, overexpression of E6 or E7 increased hTERT promoter activities in all cell lines we tested (Figure 7c). These results suggest that IFN-γ/IRF-1 signaling may repress hTERT expression through modulation of HPV 18 E6 or E7 protein. Interestingly, similar repression of telomerase activity and hTERT expression were observed even in the HPV E6 and E7 negative cell line, HT-3 after IFN-γ treatment or IRF-1 expression, but to a lesser extent than in the HPV 18 E6 and E7 positive HeLa cell line (Figures 2, 3 and 4). These findings indicate that HPV 18 E6 and E7-independent IFN-γ/IRF-1 signaling to hTERT transcription exists as well.

Growth arrest and reduction of tumorigenecity, as a consequence of telomerase inhibition, were also observed in several cell lines with inactivated p53 (Hahn et al., 1999; Zhang et al., 1999). Consistent with these findings our present results showed that IRF-1 repressed telomerase activity and hTERT expression in HT-3 (HPV E6 and E7 negative, mutated p53 and Rb) and SKOV-3 (p53 null) cell lines (Figures 3 and 4), which indicates IFN-γ/IRF-1 signaling may downregulate hTERT expression and telomerase activity by a p53-independent pathway. However, inhibition of hTERT promoter activity was less in HT-3 cells (mutated p53 and Rb) than in HeLa cells (intact p53) following IFN-γ treatment or IRF-1 expression (Figure 4). This result indicates that p53 signaling pathway may also affect the hTERT expression.

When the two putative IRF-1 binding sites on the hTERT promoter were deleted or mutated, a dramatic decrease in promoter activity by IRF-1 was still observed (data not shown). These results suggest that the putative sites are not IRF-1 binding sites and that IRF-1 may indirectly regulate the transcriptional activation of the hTERT promoter. It has been reported that IRF-1 does not bind to a consensus DNA element but may require a specific DNA sequence such as a composite element (Crowe and Nguyen, 2001). Alternatively, IRF-1 could be part of a multiprotein complex: an IRF association domain has been identified in IRF-1 that may be responsible for such an interaction with other transcription factors (Chapman et al., 2000). Although we did not find a functional IRF-1 binding site on the hTERT promoter, it is still possible that IRF-1 can bind to another yet-to-be identified site and directly regulates the hTERT promoter. Future studies will clarify these questions.

In previous studies, the overexpression of c-Myc (Cong et al., 1999; Wu et al., 1999) activated hTERT promoter activity and increased hTERT mRNA transcription and telomerase activity in normal and tumor cell lines. However, we could not observe downregulation of c-Myc in IFN-γ-treated or IRF-1-transfected cervical cancer cell lines. These results suggest that inhibition of hTERT mRNA transcription and telomerase activity may not be caused by downregulation of c-Myc.

There has been a controversy regarding the relation between cell cycle arrest and telomerase activity. In myeloma cells, cells arrested in G1 by IFN-α have lower telomerase activity compared to cultured myeloma cells without IFN-α, while in sorted S-phase cells the activity of telomerase is higher (Shiratsuchi et al., 1999). Similar results were reported in IFN-α treated Daudi cells (Akiyama et al., 1999). In contrast to these findings, several groups have demonstrated that telomerase activity was regulated by factors other than cell cycle machinery. For example, the telomerase activity in human pancreatic cancer cells is directly inhibited by p53, rather than through other cell cycle arrest and apoptosis pathways (Zhang et al., 1999). Xu et al (2000) recently reported that there is a rapid decline in hTERT and telomerase activity in human malignant and nonmalignant hematopoietic cells treated with IFN-α, and that cell cycle arrest in early S phase by aphidicholin does not inhibit telomerase activity in malignant hematopoietic cells (Kusumoto et al., 1999). In our study, the G1 arrest triggered by mimosine did not lead to a downregulation of telomerase activity or to a decrease in hTERT mRNA levels (Figure 8). This lack of effect on telomerase by mimosine implies that G1 arrest is not the critical factor in the downregulation of telomerase activity, at least in human cervical cancers.

In conclusion, we demonstrate that IRF1 is a key regulator of telomerase repression in both HPV-positive and -negative cervical cancer cells of different p53 status. In HPV-positive cells, IFN-γ/IRF-1 signaling represses HPV E6 and E7 expressions that are implicated in hTERT expression. Further, telomerase suppression of IFN-γ/IRF-1 signaling was less in the HPV-negative cell line. We propose a possibility that inhibition of telomerase activity and HPV E6 and E7 expressions are an important mechanism through which IRF-1 exerts its tumor-suppressing function.

Materials and methods

Tissues

Cervical cancer biopsies were obtained from patients at the Department of Obstetrics and Gynecology, Samsung Medical Center, Seoul, Korea. Clinical staging criteria were assigned by The International Federation of Gynecology and Obstetrics (IFGO). The stages of cancer were Ib to IIa. All samples were squamous cell carcinomas.

Cell culture and mice

HeLa and HT-3 are human cervical cancer cell lines. HeLa is an HPV 18 E6 and E7-positive cell line, and HT-3 is an HPV E6 and E7 negative cell line with mutated p53 and Rb (Scheffner et al., 1991). SKOV-3 is a p53-null human ovarian cancer cell line. U937 is an interferon-sensitive human myelomonocytic lymphoma cell line. These cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (Life Technologies Inc., Gaithersburg, MD, USA), Fungizon (Life Technologies Inc., Gaithersburg, MD, USA), anti-PPLO (Life Technologies Inc., Gaithersburg, MD, USA), streptomycin, and penicillin G at 37°C with humidified atmosphere of 5% CO2. All mice were housed in cages with filter tops in a laminar-flow hood, fed food and acid water ad libitum, and bred in a SPF facility. Light and dark ratio was 12 : 12 h. Mice with targeted mutations in the IRF-1 gene had been backcrossed to C57BL/6 seven to ten times. Primary mouse embryonic fibroblasts (MEFs) from IRF-1 heterozygote mating were prepared from embryos at the day 13.5 of development (E13.5), and cultured in DMEM with 10% FBS.

Plasmids and cell transfections

Human telomerase 3396 bp promoter subcloned into pGL3-luc (Promega, USA) was provided by TK Kim (Oh et al., 1999). MEFs were used at early passage (three to five passages) for transfection in six-well plates. MEF transfection experiments were carried out using Effectene (Qiagen) according to the manufacturer's instructions. An SV40-pRL (Promega) was cotransfected to normalize the transfection efficiency. The deletion mutant promoters were obtained by PCR from pGL3hTERT-luc as template using the following primers: 5′-IndexTermGATTGAGCCCCTTCCCTATC-3′ for the 2302 bp promoter mutant, and 5′-IndexTermCCATGCCCAGCTCAGAATTTA-3′ for the 1733 bp promoter mutant. The right primer for all of deletion mutants was 5′-IndexTermAGGAACCAGGGCGTATCTCTT-3′ on the vector. The products of amplification were then cloned in the SmaI/HindIII site of the pGL3-basic vector. For the site-directed mutagenesis of the putative IRF-1 binding sites of the hTERT promoter, we used the Quickchange™ Site-Directed Mutagenesis kit (Stratagene) and followed the manufacturer's protocol. The integrity of each construct was verified by sequencing. Transient transfection analysis was performed with 70–80% confluent cultures in six-well plates.

Construction of adenovirus expressing IRF-1

Adenovirus was generated as described by Kim et al (1999). Briefly, pΔACMV-IRF-1 was generated by the insertion of 1.8 kb EcoR1 fragment of human IRF-1 from pVL1932-IRF-1 (kind gift from Dr Takashi Fujita, The Tokyo Metropolitan Institute of Medical Science, Japan) into pΔACMVp(A). pΔACMV-IRF-1 and adenovirus backbone vector, pJM17 were cotransfected into a packaging cell line, 293 using Fugene 6 transfection reagent (Boehringer Mannheim). A replication competent virus (RCV) negative clone was propagated in 293 cells and purified through two rounds of CsCl density gradient centrifugation. The titer of the virus stock was determined by plaque assay on lawns of 293 cells. AdCMV-βgal was used as an internal control.

RT–PCR analysis of hTERT mRNA

Expression of hTERT mRNA was analysed by reverse transcription (RT)–PCR amplification. Total RNA was prepared from cell lines using TRIzol (Gibco-BRL) according to the manufacturer's protocol. One μg of Total RNA (1 μg) was reverse transcribed at 37°C for 45 min in the presence of random hexamers and Moloney murine leukemia virus reverse transcriptase (Gibco-BRL). Analysis of the expression of telomerase subunit was performed by RT–PCR amplification as described previously (Nakamura et al., 1997). A 145 bp hTERT fragment was amplified using the primer pair 5′-IndexTermCGGAAGAGTGTCTGGAGCAA-3′ and 5′-IndexTermGGATGAAG-CGGAGTCTGGA-3′.

Telomerase assay

Extracts from cell lines and cervical cancer tissues were prepared using CHAPS lysis buffer. Telomerase activity was detected by TRAPEZE Telomerase Detection Kit (Intergen Co.) according to the manufacturer's protocol. Telomerase activity was calculated by computing the ratio of the entire ladder to the signal of the amplified internal control. We used 0.5 μg protein extract for each sample and a 36-bp internal standard was used as an internal control.

Luciferase assay

Transient transfection of luciferase reporter plasmids was performed using Lipo-fectAMINE (Life Technologies, Inc.) according to the manufacturer's protocol. Luciferase assays were performed using the standard luciferase assay system (Promega). All experiments were performed in triplicate.

Western blot analysis

Cells were lysed and equal amounts of cell extracts (20 μg) were electrophoresed on 12% SDS–PAGE, electrotransferred onto a nitrocellulose membrane, and probed with antibodies. The antibodies for IRF-1, and HPV type 18 E7 were purchased from Santa Cruz Biotechnology, Inc., P53 was from Neomarkers (CA, USA) and HPV type18 E6 was from Chemicon (CA, USA). Detection was performed using the enhanced chemiluminescence system (Amersham).

Determination of cell cycle arrest by flow cytometry

Cells were prepared and labeled with propidium iodide using standard methods for cell cycle analysis. Flow cytometric analysis was carried out on a fluorescence-activated cell scanner (FAC-Scan; Becton Dickinson) using Lysis II software.

Statistical analysis

hTERT promoter activity data were analyzed by one-way ANOVA test and the difference of hTERT promoter activity between the Hela and HT-3 cells was analyzed by two-way ANOVA test.

References

  1. Akiyama M, Iwase S, Horiguchi-Yamada J, Saito S, Furukawa Y, Yamada O, Mizoguchi H, Ohno T and Yamada H . (1999). Cancer Lett. 142, 23–30.

  2. Allsopp RC and Harley CB . (1995). Exp. Cell. Res., 219, 130–136.

  3. Baege AC, Berger A, Schlegel R, Veldman T and Schlegel R . (2002). Am. J. Pathol., 160 1251–1257.

  4. Blackburn EH . (1991). Nature, 350, 569–573.

  5. Bosch FX, Lorincz A, Munoz N, Meijer CJ and Shah KV . (2002). J. Clin. Pathol., 55, 244–265.

  6. Boyer SN, Wazer DE and Band V . (1996). Cancer Res., 56, 4620–4624.

  7. Bromberg JF, Horvath CM, Wen Z, Schreiber RD and Darnell Jr JE . (1996). Proc. Natl. Acad. Sci. USA, 93, 7673–7678.

  8. Chapman RS, Duff EK, Lourenco PC, Tonner E, Flint DJ, Clarke AR and Watson CJ . (2000). Oncogene, 19, 6386–6391.

  9. Cong YS, Wen J and Bacchetti S . (1999). Hum. Mol. Genet., 8, 137–142.

  10. Crowe DL and Nguyen DC . (2001). Biochim. Biophys. Acta., 1518, 1–6.

  11. De Maeyer E and De Maeyer-Guignard J . (1998). Int. Rev. Immunol., 17, 53–73.

  12. Deiss LP, Feinstein E, Berissi H, Cohen O and Kimchi A . (1995). Genes Dev., 9, 15–30.

  13. Dron M, Modjtahedi N, Brison O and Tovey MG . (1986). Mol. Cell. Biol., 6, 1374–1378.

  14. Dyson N, Howley PM, Munger K and Harlow E . (1989). Science, 243, 934–940.

  15. Farber JM . (1992). Mol. Cell Biol., 12, 1535–1545.

  16. Greider CW . (1998). Proc. Natl. Acad. Sci. USA, 95, 90–92.

  17. Hahn WC, Stewart SA, Brooks MW, York SG, Eaton E, Kurachi A, Beijersbergen RL, Knoll JHM, Meyerson M and Weinberg RA . (1999). Nat. Med., 5, 1164–1170.

  18. Harada H, Kitagawa M, Tanaka N, Yamamoto H, Harada K, Ishihara M and Taniguchi T . (1993). Science, 259, 971–974.

  19. Harada H, Kondo T, Ogawa S, Tamura T, Kitagawa M, Tanaka N, Lamphier MS, Hirai H and Taniguchi T . (1994). Oncogene, 9, 3313–3320.

  20. Harle-Bachor C and Boukamp P . (1996). Proc. Natl. Acad. Sci. USA, 93, 6476–6481.

  21. Holt SE and Shay JW . (1999). J. Cell. Physiol., 180, 19–18.

  22. Horvath CM, Stark GR, Kerr IM and Darnell Jr JE . (1996). Mol. Cell Biol., 16, 6957–6964.

  23. Jonak GJ and Knight Jr E . (1984). Proc. Natl. Acad. Sci. USA, 81, 1747–1750.

  24. Kano A, Haruyama T, Akaike T and Watanabe Y . (1999). Biochem. Biophys. Res. Commun., 257, 672–677.

  25. Kim J, Hwang ES, Kim JS, You E, Lee SH and Lee J . (1999). Cancer Gene Ther, 6, 172–178.

  26. Kim KY, Blatt L and Taylor MW . (2000). J. Gen. Virol., 81, 695–700.

  27. Kusumoto M, Ogawa T, Mizumoto K, Ueno H, Niiyama H, Sato N, Nakamura M and Tanaka M . (1999). Clin. Cancer Res., 5, 2140–2147.

  28. Lengyel P . (1993). Proc Natl Acad Sci USA, 90(13), 5893–5895.

  29. Maciejewski JP, Selleri C, Sato T, Cho HJ, Keefee LK, Nathan CF and Young NS . (1995). J. Clin. Invest., 96, 1085–1092.

  30. Makarov VL, Hirose Y and Langmore JP . (1997). Cell, 88, 657–666.

  31. Morin GB . (1989). Cell, 59, 521–529.

  32. Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrews WH, Lingner J, Harley CB and Cech TR . (1997). Science, 277, 955–959.

  33. Nozawa H, Oda E, Nakao K, Ishihara M, Ueda S, Yokochi T, Ogasawara K, Nakatsuru Y, Shimizu S, Ohira Y, Hioki K, Aizawa S, Ishikawa T, Katsuki M, Muto T, Taniguichi T and Tanaka N . (1999). Genes Dev., 13, 1240–1245.

  34. Nozawa H, Oda E, Ueda S, Tamura G, Maesawa C, Muto T, Taniguichi T and Tanaka N . (1998). Int. J. Cancer, 77, 522–527.

  35. Ogasawara S, Tamura G, Maesawa C, Suzuki Y, Ishida K, Satoh N, Uesugi N, Saito K and Satodate R . (1996). Gastroenterology, 110, 52–57.

  36. Oh S, Song Y, Yim J, Kim TK . (1999). J. Biol. Chem., 274, 37473–37478.

  37. Ossina NK, Cannas A, Powers VC, Fitzpatrick PA, Knight JD, Gilbert JR, Shekhtman EM, Tomei LD, Umansky SR and Kiefer MC . (1997). J. Biol. Chem., 272, 16351–16357.

  38. Scheffner M, Munger K, Byrne JC and Howley PM . (1991). Proc. Natl. Acad. Sci. USA, 88, 5523–5527.

  39. Scheffner M, Werness BA, Huibregtse JM, Levine AJ and Howley PM . (1990). Cell, 63, 1129–1136.

  40. Schiffman MH . (1992). J. Nat. Cancer Inst., 84, 394–398.

  41. Shay JW . (1995). Mol. Med. Today, 1, 378–384.

  42. Shay JW, Bacchetti S . (1997). Eur. J. Cancer, 33, 787–791.

  43. Shay JW and Wright WE . (1996). Trends Genet., 12, 129–131.

  44. Shiratsuchi M, Muta K, Umemura T, Nishimura J, Nawata H and Kozuru M . (1999). Lymphoma, 34, 349–359.

  45. Tamura T, Ishihara M, Lamphier MS, Tanaka N, Oishi I, Aizawa S, Matsuyama T, Mak TW, Taki S and Taniguichi T . (1995). Nature, 376, 596–599.

  46. Tanaka N, Ishihara M, Kitagawa M, Harada H, Kimura T, Matsuyama T, Lamphier MS, Aizawa S, Mak TW and Taniguichi T . (1994). Cell, 77, 829–839.

  47. Veldman T, Horikawa I, Barrett JC and Schlegel R . (2001). J. Virol., 75, 4467–4472.

  48. Wang J, Xie LY, Allan S, Beach D and Hannon GJ . (1998). Genes Dev., 12, 1769–1774.

  49. Weinrich SL, Pruzan R, Ma L, Ouellette M, Tesmer VM, Holt SE, Bodnar AG, Lichtsteiner S, Kim NW, Trager JB, Taylor RD, Carlos R, Andrews WH, Wright WE, Shay JW, Harley CB and Morin GB . (1997). Nat. Genet., 17, 498–502.

  50. Werness BA, Levine AJ and Howley PM . (1990). Science, 248, 76–79.

  51. Willman CL et al. (1993). Science, 259, 968–971.

  52. Wright WE, Piatyszek MA, Rainey WE, Byrd W and Shay JW . (1996). Dev. Genet., 18, 173–179.

  53. Wu KJ, Brandori C, Amacker M, Simon-Vermont N, Polack A, Lingner J and Dalla-Favera R . (1999). Nat. Genet., 21, 220–224.

  54. Xu D, Erickson S, Szeps M, Gruber A, Sangfelt O, Einhorn S, Pisa P and Grander D . (2000). Blood, 96, 4313–4318.

  55. Yasui W, Tahara H, Tahara E, Fujimoto J, Nakayama J, Ishikawa F, Ide T and Tahara E . (1988). Jpn. J. Cancer Res., 89, 1099–1103.

  56. Zhang X, Mar V, Zhou W, Harrington L and Robinson MO . (1999). Genes Dev., 13, 2388–2399.

Download references

Acknowledgements

We thank Dr Takashi Fujita (Tokyo Metropolitan Institute of Medical Science, Japan) for the IRF-1 DNA and Dr Seon-Woo Kim (Biostatistics Unit in Samsung Biomedical Center, Korea) for statistical analysis. This work was supported by SRC and G7 funds from the Korea Science and Engineering Foundation.

Author information

Correspondence to Je-Ho Lee.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lee, S., Kim, J., Lee, H. et al. Interferon regulatory factor-1 (IRF-1) is a mediator for interferon-γ induced attenuation of telomerase activity and human telomerase reverse transcriptase (hTERT) expression. Oncogene 22, 381–391 (2003). https://doi.org/10.1038/sj.onc.1206133

Download citation

Keywords

  • telomerase
  • hTERT
  • interferon-γ (INF-γ)
  • IRF-1

Further reading