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(CANCERRESEARCH 57, 549-554,February1, 19971 Telomerase Activity Is Associated with Cell Cycle Deregulation in Human Breast Cancer1 GöranLandberg, Niels Hilmer Nielsen, Na Nilsson, Stefan 0. Emdin, Jenny Cajander, and GöranRoos2 Departments ofPa:hology (G. L, N. H. N., P. N., I. C., G. RI, Oncology (N. H. N.], and Surgety (S. 0. El, Umed University. 5-90187 Umed, Sweden ABSTRACT Deregulation of the cell cycle by abnormal expression of one or several cell cycle regulatory proteins is a common finding In malignant tumors and might be a prerequisite for cancer development. Telomerase activity Is an immortalization marker that is found in most cancers and for which an association with an active cell cycle has been Implicated. In the tissue of 106 human breast cardnemas, we analyzed the relationship between telomerase activity levels and defects In the cell cycle machinery with a focus en the retinoblastoma protein (pRB) pathway(s). The fraction ef telemerase-posltlve tumors was 85%, and large differences in telomerase activity were found. Overexpression of cydlin Dl and/er cydlin E, in combination with a normal pRB, was a typical feature oftumors with high telomerase activity levels. Down-regulation ofpl6@'4 was not related per se to telomerase activity, but tumors with low p16'@4 j@combination with cydlin Dl or E overexpresslon demonstrated high activity. Tumor cell proliferation, determined by Kl-67 expression, correlated significantly to telemerase activity levels. There was, however, not a strict association between proliferation rate and telemerase activity, because tumors with Inactivated pRB had the highest Kl-67 fractions but Intermediate telom erase activity. Also, cyclin Dl everexpresslon was associated with high telomerase levels without an increase in tumor cell proliferation. The present study indicates that telomerase activation occurs preferentially in breast cancers with certain cell cycle regulatory defects and that telom types (25, 26). There is also an association between telomerase erase activity levels may depend on the specific defect(s). INTRODUCTION After a certain number of cell divisions, normal cells activate a senescence program characterized by morphological changes, growth arrest in G1, and shortened telomeres. Telomeres, which consist of TTAGGG repeats with associated proteins, shorten 50—100bp after each cell cycle round due to incomplete replication of the 3'-ends. At a critically short telomere length, cells enter the senescence stage and eventually they will die. Thus, the life span of a normal cell popula tion is limited, and a strong association between the replicative ca pacity of normal cells and telomere length has been demonstrated, which forms the basis for the telomere hypothesis of senescence (1—4).The telomere loss may lead to DNA breaks with activation of p53-dependent or -independent DNA damage pathways, induction of CDK3 inhibitors (e.g., p21 and p2'l) and the final G@ block of senescence (5). In support of this hypothesis, a temporary rescue from the senescence program can be achieved by inactivation of the pRB or p53 tumor suppressor genes, leading to a prolonged life span but usually not to immortalization (6—9).During this period of prolonged life, a further decrease in telomere length takes place until a second stage of crisis characterized by genomic instability is reached. Rare cells will survive this state and become immortalized, a process most Received 8/12/96; accepted 12/3/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. @ I was supported by grants from the Swedish Cancer Foundation, Medical Faculty of UmeA, Sahlbergs Foundation, Lions Cancer Research Foundation (UmeA, Sweden), and a special grant from the VSsterbotten County Council. 2 To whom requests for reprints should be addressed. Fax: +46-90-7852829; E-mail: [email protected]. 3 The abbreviations used are: CDK, cyclin-dependent kinase; RB, likely due to loss of normal gene function, because hybrids between normal and immortalized cells usually have a limited life span (10). One typical feature of inunortalized cells in vitro is the presence of active telomerase, which can synthesize telomeric DNA repeats using an internal RNA template, compensating for the loss of telomeric DNA after each cell division cycle (11, 12). Thus, telomerase activity has been demonstrated in the vast majority ofpermanent cell lines, but immortal cell lines without detectable activity have been described (7, 13—16).Active telomerase was first shown in vivo in advanced ovar ian carcinoma and hematopoietic B-cell neoplasias (17, 18) with the conventional primer-extension telomerase assay (1 1). Using a more sensitive method, the TRAP assay, telomerase activity has been detected in high frequency in various tumors (19, 20—24). Thus, telomerase activity appears to be a frequently appearing marker in human cancer. The regulation of telomerase activity is poorly understood, but cell line data indicate that a telomere length-dependent mechanism is the major pathway. However, normal lymphocytes up-regulate a notable telomerase activity upon antigen and mitogen stimulation in vitro as well as in vivo, indicating that telomere length-independent mecha nisms for telomerase activation can be important for specific cell retinoblastoma; pRB, RB protein; TRAP, telomeric repeat amplification protocol; ITAS, internal TRAP assay standard. activity and cell proliferation, because terminal differentiation and cell cycle exit is followed by down-regulation of the telomerase activity (27, 28). The pRB has a central role in the control of the cell cycle by suppressing important transcription factors, such as E2F, which is released after inactivation of pRB by phosphorylation (29). CDKS and associated cyclins are responsible for the phosphorylation of pRB, and the coordinate expression and association of CDKS, cyclins, and CDK inhibitors drives cells through the cell cycle (30). In human tumors, deregulation of the cell cycle machinery is common, and abnormal expression of one or several cell cycle-regulatory proteins might be a prerequisite for all cancer development (31). In a fraction of breast cancers, the cyclin Dl gene is amplified and the protein overex pressed, potentially inducing inactivation of pRB by phosphorylation and S-phase progression (32, 33). RB inactivation, caused by muta tions and deletions, is found in breast cancer and represents the most severe cell cycle-regulatory defect with a complete lack of cell cycle control (30, 3 1). Cyclin E is also often overexpressed in various malignancies (34, 35), including 25% of breast cancers (36). The functional consequences of a deregulated cyclin E is not fully under stood, but apart from an abnormal phosphorylation of pRB and release of E2F, other yet undefined targets might be affected (37, 38). Only a few studies have been directed toward the relationship between telomerase activity in tumors and the status of different cell cycle regulators. In lung cancer, an association between telomere length alterations and loss of heterozygosity in p53 and RB was suggested, but no data on telomerase activity were presented (39). In cell lines from four immortalization complementation groups, no correlation between telomerase activity and abnormalities in RB, p53 and pl6'@4 could be demonstrated, but all cell lines were defective in at least one of these components (40). In the present study, we demonstrate an association between telomerase activity levels and the status of pRB, cyclin Dl, cyclin E, and p16IN@@in a material of breast 549 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research. TELOMERASE ACTIVITYANDCELLCYCLEREGULATION cancer, a tumor type with a high frequency of telomerase-positive cells (20, 41). Our data indicate that telomerase activation occurs preferentially in tumors with certain deregulations in the cell cycle machinery. (Amersham International). Membranes were blocked in PBS containing 5% dried milk and 0.1% Tween-20 and probed with monoclonal mouse antibodies against cyclin E (HE 12, Santa Cruz Biotechnology, Inc.), cyclin Dl (DCS-6), and pl6@4 (@55Ø; DCS antibodies were kind gifts from Dr. Jiri Bartek, Copenhagen, Denmark), and mouse anti-actin antibodies (Boehringer Mann heim) for 1 h. After a second incubation with peroxidase-conjugated MATERIALS AND METHODS Samples. Diagnostic tumor samples were collected from 106 patients with antimouse antibodies (Amersham Corp.), proteins were detected using an enhanced chemiluminescent detection system (Amersham Corp.). Relative protein concentrations were determined as described earlier (36). breast carcinoma. According to the classification from The International Union Briefly, different cell line standards were loaded on each gel with the tumor Against Cancer, 28 patients had stage 1, 60 patients stage II, two patients stage stage. samples, and after densitometric quantification (Molecular Dynamics, Inc.) of the enhanced chemiluminescence films, relative protein concentrations were before sampling. calculated by dividing optical densities from tumor specimens by that of the ifi, and eight patients stage IV cancer. Eight patients had an unknown No patient had been treated with radiation or chemotherapy All tumors were taken care of immediately after excision, and at least two pieces were snap frozen in liquid nitrogen and stored in — 80°Cuntil extracts for immunoblotting and telomerase activity analysis were prepared. Adjacent tissue pieces were formalin fixed, paraffin embedded, and used for routine morphological examination and immunohistochemical cell line standard. Immunohistochemistry. Breast cancer specimens were fixed in formalin and embedded in paraffin according to the clinical routine. Fixation time in formalin varied slightly between different samples but were in general between 15 and 24 h. Three-pm staining. The presence paraffin sections were prepared from 73 available of tumor cells in samples used for telomerase assay and immunoblouing studies was verified by Giemsa-stained imprints from each sample. Telomerase Assay. Preparationof tumorextracts,theTRAPassay,andthe quantifications of telomerase activity were performed as detailed previously (13, 42, 43). Briefly, frozen tumor tissue was, depending on the sample size, cases, and the slides were dried and thereafter deparaffinized in xylene and rehydrated in alcohol and water according to standard procedures. To obtain adequate pRB and Ki-67 staining, slides were boiled (96°C)in a microwave oven for a total of 15 mm (5 mm X 3) using a citrate buffer at pH 6.0. homogenized in 50—200 @l of ice-cold lysis buffer, which was composed of 10 mM Tris-HC1 (pH 7.5), 1 mM MgCl2, 1 mxi EGTA, 0.5% 3-[(3-cholamido istry antibodies (C15; Santa Cruz Biotechnology, Inc.), and antigen-antibody visu propyl)dimethylammonio]-l-propanesulfate (Pierce Chemical Co.), 10% glyc erol, 5 mM f3-mercaptoethanol (Sigma Chemical Co.), and 0.1 mi@iphenyl alization as well as H&E counterstaining were performed according to the Ventana program. Ki-67 stainings were processed manually using MIB-l methylsulfonyl antibodies (Immunotech) and avidin-biotin complex method kits (Vector Lab oratories, Inc.). fluoride (Boehringer Mannheim). After a 30-mm incubation on ice, the lysate was centrifuged for 20 mm at 13,000 X g, and the supernatant was snap frozen in liquid nitrogen and stored at —80°C. Protein concentration was measured using the bicinchoninic acid protein assay kit (Pierce Chemical lyophilized at the bottom and sequestered by a wax barrier (Ampliwax; Perkin-Elmer Corp.). Protein extracts were diluted appropriately, and an aliquot of 0.005—5@g protein was assayed in 50 @lof reaction mixture composed of 50 staining Microscopic Co.). PCR tubes for the TRAP assay contained 0.1 @agof the CX primer (5'-CCCTFACCC'VFA-CCCTFACCCTAA-3') Microwave-treated @M of each deoxynucleotide triphosphate, 0.1 @gof TS primer (5'-AATCCGTCGAGCAGAGTT-3'), 5 attogram of a l50-bp ITAS (43), 0.5 @M T4 gene 32 protein (Boebringer Mannheim), 2 pCi each of [a32P]dCTP and [a@2P]dUP, 3000 Ci/mmol slides were processed in an automatic immunohistochem machine (Ventana Evaluation. 320—202; Ventana, Stained sections were Inc.) using anti-pRB microscopically exam mcd, and the staining intensity of pRB in tumor cells was compared to that in adjacent normal cells. A tumor was considered to be pRB abnormal if there was no or very weak nuclear pRB staining in all tumor cells. The percentage of Ki-67-positive cells was determined by calculating at least 300 tumor cells crossing randomly distributed lines. Statistical Methods. Comparison between groups was performed using log-rank and x@ (Pearson) correlation analysis. All calculations were per formed using SPSS version 6.0 (SPSS, Inc., Chicago, IL). (Amersham Corp.), and 2 units of Taq polymerase (Boehringer Mannheim). RESULTS Following a 30-rain incubation at room temperature, the samples were sub jected to 31 PCR cycles of94°C for 30 s, 50°Cfor 30 s, and 72°Cfor 45 s. The PCR products were examined by electrophoresis on 10% nondenaturing poly acrylamide gels. The gels were fixed in 0.5 M NaC1, 40 mM sodium acetate (pH 4.2), and 50% ethanol for 25 mm and exposed without drying to phosphor screens. Evaluations and calculations were done with a Molecular Imager and the Molecular Analyst software (Bio-Rad Laboratories, Inc.). To control the telomerase activity origin of the six-nucleotide ladder, protein extracts were incubated with RNase (Boebringer Mannheim) for 20 mm at 37°Cprior to mixing with the reaction mixture. To make semiquantitative assessments of relative telomerase activity levels, only extract dilutions in which the intensity of the ITAS was approximately the same as the intensity of the ITAS in the lysis buffer alone were quantitated. The relative telomerase activity in a sample was derived from the ratio in intensity between the ITAS and the telomerase ladder in a lane. The activity value assigned for a sample was the mean of at least two experiments and was calculated for 0.5 @.tg protein. Western Blotting and Cyclin Dl, Cyclist E, and p16@4 PrOtein Deter mination. Tissue samples of approximately50 mg were homogenized and sonicated for 2 x 15 s in lysis buffer [0.5% NP4O,0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HC1 (pH 7.5), 150 mM NaCl, with the following protease and phosphatase inhibitors: 20 mg/mi leupeptin; 20 mg/mi aprotinin; 10 mg/mi pepstatin; 1 mM phenylmethylsulfonyl fluoride; 1 mM sodium fluoride; and 1 nmi EDTAJ with adequate cooling during the procedure. Samples were centrifuged at 14,000 X g for 30 mm at 4°C,and aliquots of the supernatant were stored at —80°Cuntil further analysis. Total protein concen tration was determined by a bicinchoninic acid protein assay (Pierce Chemical Co.). Electrophoresiswas performedon 11%SDS-polyacrylamidegels using 40-mg protein samples per lane (Bio-Rad Minigel System, Bio-Rad Labora tories, Inc.), and proteins were then transferred to mtrocellulose membranes Telomerase Activity. Taq polymerase-inhibitory activity was re vealed in a significant number of samples demonstrated as no PCR amplification product of the internal standard. In all cases with Taq polymerase inhibition, the internal standard was amplifiable after dilution of the cell extracts and estimation of activity levels could be achieved. Fig. 1 shows examples of tumors with differences in telom erase activity and with Taq inhibition. By the semiquantitative deter mination large differences in telomerase activity were demonstrated, with values ranging from 0 to 153 as illustrated in Fig. 2. The mean telomerase activity level for all tumors was 10.2 (median, 1.6). Below an activity of 0.2, no telomere repeat ladder was visible by the eye, and therefore, a cutoff at 0.2 was used to discriminate telomerase positive from -negative tumors, giving 90 (84.9%) positive and 16 (15.1%) negative tumors at 0.5 @g tumor extract. The highest telom erase activity levels in the tumors were comparable to the activity found in permanent cell lines (data not shown). Telomerase Activity in Relation to Cydlin Dl and Cyclin E Expression. Cylin Dl and cyclin E expression varied considerably between individual tumors, as described separately (36). In summary, both cycin Dl and cyclin E showed biphasic distributions, and the majority of the tumors expressed low protein levels, whereas a frac tion of the tumors expressed higher and more heterogeneous levels of one of the cycins or both in combination. The majority of tumors with high cycin E levels expressed low cydin Dl . Tumors defined to overexpress cyclin E or Dl were selected with cutoff points based on 550 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research. @ @ @ -@ In . C4 In V) 0 @q 0 0 IL, TELOMERASE ACflVITY AND CELL CYCLE REGULATION their protein distribution in the tumor samples. Examples of cyclin E, cydlin Dl, and actin immunoblots of five breast tumors and a control cell line are presented in Fig. 3A. Correlation analysis revealed a significant association between cycin Dl protein content and telom erase activity level (r = 0.3416; P = 0.001; n = 100; not shown in figures). No such association was found for cyclin E (r = 0.13 15; P = 0.196; n = 100). To evaluate telomerase activity in relation to the expression of cell @ @ @ ‘@ ,, ‘-4 — — e A — e@ _______ Cydin E @-—. Cydlln Dl Actin —@1 —@ — — — B @ ____________ p16Th11@@ @ 11 @ .p i@I Fig. 3. Western blots of selected breast cancer samples and control cell lines. A, cyclin E, cyclin Dl, and actin expression in five tumors (the same samples as shown in Fig. 1), demonstrating obvious differences in protein expression of the two cyclins. Cyclin E demonstrates several bands shown previously to represent biologically active isoforms (35), all of which were included in the densitometric quantification. B. pl6@4 seven tumors showing large differences in protein expression. Fig. 1. TRAP assay data on five breast cancer samples. U210, telomerase negative. U210 and U214 indicate Taq polymerase inhibition at a protein concentration of 5 .sg (but U214 is clearly positive) and show amplification of the internal standard after dilution of the extract.U208,U211,and U212demonstratevariabletelomeraseactivity. A L. .@ = z o@ —,@ 50 Relative @ 100 telomerase activity B I. E = z Relative telomerase activity and pRB Staining. DV0W, and p16LNK4 high demonstrated Fig. 2. Histograms showing relative telomerase activity in 106 breast cancer samples. A, disalbutin Activity Seven out of 73 tumors (9.6%) studied for pRB expression by immunohistochemistry lacked pRB, whereas nontumor cells stained positive (Fig. 5). pRB-negative tumors, which were regarded as had intermediate telom erase levels (Table 1), and the telomerase activity was considerably lower compared to tumors with cyclin D or E defects (Table 2). Combination of Various Cell Cycle Defects and Telomerase Activity. Table 2 shows relative telomerase activity in different tu mor groups subdivided according to the expression of cyclin Dl, cycin E, pl6'@4, and pRB. Tumors lacking detectable cell cycle defects, i.e., tumors with cyclin DV0@@, cyclin and @norma1 had the lowest telomerase activity levels (Table 2). The highest activity was demonstrated in p@normaltumors with high cyclin Dl or/and cyclin E expression. For tumors with high cyclin Dl or cycin E levels, a low expression of pl6tN@@seemed to increase the telomerase activity. p@abnoflTUL1 tumors, all of which were cyclin Elu5t@, cycin 4) @ cycle regulators, the tumors were subdivided into four groups, with cutoffs at relative telomerase activity levels of 0.2, 0.8, and 8. Sub dividing the tumors according to cyclin Dl and cyclin E levels demonstrated that overexpression of each cyclin was associated strongly with intermediate to high telomerase activity levels (Table 1). All tumors (n = 5) with high levels of both cyclin Dl and E had high telomerase activity. Telomerase Activity and p16'@4 Expression. pl6tN@@protein levels were estimated by densitometry of immunoblots (Fig. 3B), and the tumors were divided in three groups (low, intermediate, and high) according to protein levels. pl6'@―expression did not correlate with telomerase activity (r = —0.1256; P = 0.211; n = 101), and tumors with intermediate or high p161N@@ expression did not deviate sigmf icantly from low p16IN@@expressers regarding telomerase activity (Table 1 and 2). Interestingly, the highest telomerase activities were found in tumors with low expression of pl6tN@@(Fig. 4), indicating that down-regulation of pl6@?4@@ in combination with other cell cycle defects (Table 2) might contribute to maximum telomerase activity in tumors. Telomerase 150 levels in ofall samples; B, distribution ofsamples with a relative telomerase activity between 0 and 3.5. intermediate telomerase activ ity levels (Table 2). A summary of the data is given in Fig. 6, indicating various cell cycle defects in relation to telomerase when the tumors were subdivided using a cutoff at 0.8 of relative activity. 551 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research. TELOMERASE ACTIVITYANDCELLCYCLEREGULATION @Statistical Table I Telomerase activity in breast cancer extracts in relation to the expression of cyclin DI, cyclin E, pI6―@4, cross-table analysis (x@according to Pearson) comparing tumor distribution within the different subgroups defined by relative telomerase activity (@0.8 and >0.8) and expression showeda of the cell cycle regulators showed a significant difference for cyclin Dl (P = 0.021) and cyclin E (P < 0.001) but not for @16@N1@ (P = 0.698), whereas pRB borderline value (P = 0.059).Cydin Dla AlteredRelative Telomerase activity Low Cydin E― High Low p16INK4a High Low @p@b Intermediate and high Normal activitycO.2 telomerase 15133 181 >0.2—0.8 0>0.8— @ @ @8 1Tumors >8 100Totalnumber with telomerase activity >0.8 (%) a Determined by densitometry of Western ll@ 4J 15136 2lJ 112 lJ 14126 l2J 2112 101 10123 l3J 0 25 l8J 6 27 1 18J 11 1 12123 28 16J 10 1 24 l4J 23 2lJ 15 9J 24 58 82 55 92 63 67 65 78 28 80 26 70 36 66 o 7 blots. b Evaluated by immunohistochemistry. C T'@'@ assay; relative activity in relation to internal standard, given as number of tumors activityMean telomerase Median Eb0w, @[email protected] 37p16b0@v pl6)@i5I@ Dl@―, Dlhigh, @@@norma1 panflOITh@l, pl6bow D1―@―, panflonnal, pl6intcrmediatc E―@', p@nOflflaI @ pannormal, p16'°― EhI5I@, p@flOflfl@1, @16intcrmed1ate Dlhi5I@,E―@―, p@flOflfl@l @ @p@abnorn@aI (all 7All 106Dl, tumors10.2 and D1b0w)12.1 0.7 1.5 70 6.3 7.0 1.3 2.5 23 13 18.8 22.3 11.7 13.4 15 11 9.1 8.5 4 24.9 21.6 13 26.6 23.3 9 22.1 20.1 5.2 11.7 21.6 2.5 1.6 4 5 cyclin Dl; E, cyclin E. @ each Telomerase Activity and Tumor Cell Proliferation. Expression of the nuclear antigen Ki-67 is characteristic for cycling cells, and in the present material, the fraction of Ki-67-positive cells ranged from 4 to 84%, with a mean of 25. 1%. In the total tumor material tested for Ki-67, a significant and positive correlation to telomerase activity level was found (r = 0.39; P = 0.001; not shown in figures). However, regarding tumor subgroups with different cell cycle defects, another picture emerged as illustrated in Fig. 6, because cycin D1to@ tumors had higher telomerase activity than cydlin DV0―tumors with a comparable Ki-67 index. More strikingly, cases dem onstrated the highest proliferation rate but a telomerase activity lower than cyclin Dihigh expressers (Fig. 7). Thus, it was concluded that telomerase activity levels in breast cancer could be dissociated from the tumor cell proliferation rate and seemed to be connected to the expression of specific cell cycle regulators. @‘l4O -@e 120 @lO0 DISCUSSION @ category. cell extract dilutions, potential Taq polymerase-inhibitory activities can be revealed, and by using an internal standard, the importance of such inhibition could be verified as in the study of Hiyama et a!. (20). Our telomerase activity data were calculated for a protein content of 0.5 ,ag, which means that if an extract was diluted 10 times to achieve amplification of the internal standard, the activity value obtained was multiplied by 10. Further analysis supported the assumption that semiquantitative determinations can be useful and of biological sig nificance, because specific tumor subgroups with defined defects in the cell cycle machinery had clearly different telomerase activity levels. The M1/M2 model proposed by Shay et a!. (9) states that a cell must bypass the M1 (senescence stage) and M2 checkpoints before immor talization and that activation of telomerase can occur at M2. The senescence stage (M @) can be overcome by inactivation of the pRB or p53 tumor suppressor proteins, leading to a prolonged life span but usually not immortalization (6—8).This means that inactivation of the p53 or pRB pathways could provide a tumor with the capability for many extra cell divisions and a higher chance for activation of telomerase. Our data gave some support for this scenario concerning pRB inactivation. Regarding p53, it is well recognized that breast cancers frequently harbor p53 gene mutations and that these also might be of prognostic significance. Previous studies on human mammary epitheial cells have indicated that p53, but notably not RB, is involved in the M1 mechanism (45). In the present tumor material, the p53 gene and protein status were unknown, but studies have been initiated in our Table 2 Relative subgroup?Tumor telomerase activity levels in different breast cancer NumberDlb0@@@, groupRelative in I. 4) E In the present study of 106 breast cancers, semiquantitative deter mination of telomerase activity levels revealed a spectrum from negative to strongly positive tumors, and the activity levels were demonstrated to be associated with defective cell cycle regulation. The finding of 85% telomerase-positive tumors is in agreement with recent data on breast cancer showing 73—93%positive cases (20, 41). Regarding the fraction of telomerase-positive cases, breast cancer is similar to other epithelial malignancies analyzed with the TRAP assay (19, 20, 22—24). Semiquantitative determinations of telomerase activity, as per formed in the present study, have recently been published for malig nant and nonmalignant lymphoid cells and skin disorders (26, 44). By 80 060 4)40 .#; @20 ..: 0 .• ... 0.2 ... 0.4 0.6 0.8 1.0 p16@4protein Fig. 4. Dot plot showing 1.2 1.4 1.6 1.8 2.0 level @l6tN@@4 levels in relation to relative telomerase activity. Samples with the highest telomerase activity values were found in tumors with low expression of pl6@4. 552 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research. TELOMERASE ACI1VITY AND CELL CYCLE REGULATION suggests that down-regulation of pl6@4 alone, which represents a rather moderate cell cycle defect with preserved cycin Dl-CDK4/6 regulation, is insufficient to reach M2 crisis and activation of telom erase. There was nevertheless an additive effect of low @165N@@ and overexpression of cyclins E or Dl on telomerase activity, suggesting that the @16JNK4 level is important together with other cell cycle defects. This was highlighted by the fact that all four tumors with a relative telomerase activity above 60 expressed low pl6@@4 protein levels. We have shown recently that cyclin E is overexpressed in a fraction of breast cancers (36). Cyclin E could potentially have functions similar to cycin Dl and be involved in the transformation of cells (54), even though no experimental evidence for cyclin E as an oncogene has been published. The highest telomerase activity levels were found in tumors with cycin E overexpression, suggesting that cycin E deregulation represents a cell cycle defect of importance for telomerase activity. The present study is the first demonstrating an association between telomerase activity and defects in the cell cycle machinery in a human tumor type, and our data also indicate that such defects might influ ence the expression levels of telomerase. We found that this associ ation for breast cancer and the relevance of the findings for other tumor types remains to be established. Tumors of diverse origin have different patterns concerning their spectrum of cell cycle defects, and it is conceivable that differences will be found regarding the effects on telomerase activity. The data presented here are compatible with the M11M2 model, but there is a possibility that certain cell cycle proteins @ . Cyclin E@― Fig. 5. Immunohistochemical staining of pRB in breast cancer. A, tumor with pRB expression in tumor cells as well as in benign cells. B, pan-negative tumor, with positive . Cycli Cyclin Dl―1ti' reaction in surrounding benign cells. -#----— @- @ pRBnormaI laboratory to explore its possible impact on activation and regulation of telomerase. The fraction of p@thnoflfl@1tumors was somewhat lower than found previously in breast cancer (46, 47), and all tumors were telomerase positive but with intermediate activity, indicating that other factors are important for maintenance of high activity levels. To define the RB status, we used immunohistochemistry, which is considered to be a reliable method because of difficulties for mutated pRB to enter the nucleus as well as a complete lack of staining in tumors with ho mozygous RB deletions. In support of our immunohistochemical data, all tumors expressed high pl6@4 levels, indicating a stabilization of pl6@4 through association with CDK4/6 due to the inactivation of RB and down-regulation of cycin Dl (48, 49). We have also observed a low fraction of hyperphosphorylated pRB as well as frequent loss of heterozygosity at two intragenic RB polymor phisms in pRBa@@@@0rmM tumors supporting the immunohistochemical approach (50). In the pRB-regulatory pathway(s), both cydin Dl and pl6@4 are key factors, and a potential deregulated expression might cause func tional inactivation of pRB by phosphorylation and unordered entrance into S phase (31). There are several clinical and experimental data indicating that cyclin Dl is an oncogene (51—53),and the protein is overexpressed in a substantial part of breast tumors (33, 53). In the present study, the expression of cycin Dl was associated with telom erase activity. @16INK4 down-regulation was in our material the most common cell cycle defect (65%), giving an increased phosphoryla tion/inactivation of pRB with enhanced proliferation pressure (31), but we found no significant correlation to telomerase activity. This • Cyclin E@@W Cyclin D11w Cyclin DIM@@ —k. . Cyclin or Cyclin D1―@' + :II::::::::@.c:zz::::I p16b0@@ • . pRba@@@@ % = fraction of tumors with relative telomerase activity > 0.8 Fig. 6. A model for acquisition of telomerase activity in breast cancer in relation to various defects in the expression of the cell cycle regulators cyclin Dl, cyclin E, pl6'@4, and pRB. The percentages given represent the fraction of tumors with relative telomerase activity levels >0.8 (see also Table 1). 80 UKI-67(%)—0 activityi—L@@1I-Lr-@ Tdomerue 60 S @40 20 0 C,dMElsw CydINEIIIØI C@tIInEISW Cyd1EW@I pRBâsssm@ CyduiDl I.w C@ Dl low CydinDl hi@i CydkiDl hid. Fig. 7. Histogram exploring the relationship between tumor cell proliferation, defined as Ki-67 antigen-positive cells, and relative telomerase activity in subgroups of breast cancer with different cell cycle defects. 553 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research. TELOMERASE ACTIVITY AND CELL CYCLE REGULATION can have specific effects on telomerase regulation, an important issue for future experimental studies on telomerase regulation. C. A., Nochols, G., Khaled, Z., Telang, N. T, Narayanan, R. Differentiation of immortal cells inhibits telomerase activity. Proc. Natl. Acad. Sci. USA, 92: 12343— 12346, 1995. 28. Holt, S. E., Wright, W. 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Telomerase Activity Is Associated with Cell Cycle Deregulation in Human Breast Cancer Göran Landberg, Niels Hilmer Nielsen, Pia Nilsson, et al. Cancer Res 1997;57:549-554. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/57/3/549 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1997 American Association for Cancer Research.