Molecular changes accompanying senescence and immortalization of cultured human mammary epithelial cells

The International Journal of Biochemistry & Cell Biology 34 (2002) 1382–1394

Review

Molecular changes accompanying senescence and immortalization of cultured human mammary epithelial cells Paul Yaswen∗ , Martha R. Stampfer Department of Cell and Molecular Biology, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mailstop 70A-1118 Berkeley, CA 94720, USA Received 12 December 2001; received in revised form 10 March 2002; accepted 26 March 2002

Abstract Limits on the proliferative potential of cultured normal human cells may be consequences of pathways that exist to suppress tumorigenicity. Human mammary epithelial cells (HMEC) employ several mechanisms to prevent unlimited growth. One mechanism may be activated by stress, and is associated with upregulated expression of p16INK4a . In serum-free medium, some HMEC arise spontaneously which do not express p16. These “post-selection” HMEC are capable of long-term proliferation, but ultimately cease growth when their telomeres become very short. As they approach a growth plateau, termed agonescence, post-selection HMEC populations accumulate chromosome abnormalities. In contrast to the crisis exhibited by cells lacking functional p53, agonescent cells can be maintained as viable cultures. Although transduction of hTERT, the catalytic subunit of telomerase, into post-selection cells can, by itself, efficiently produce immortality and avoid agonescence, the errors that produce telomerase reactivation during carcinogenesis are not known. The block to endogenous telomerase reactivation in HMEC is extremely stringent. However, if one predisposing error is present, the probability greatly increases that additional error(s) required for immortalization may be generated by genomic instability encountered during agonescence. In p53(+) HMEC immortalized after chemical carcinogen exposure, the events involved in overcoming agonescence can be temporally separated from activation of telomerase. We have used the term “conversion” to describe the gradual process that leads to telomerase activation, telomere length stabilization, decreased p57KIP2 expression, and increased ability to grow uniformly well in the presence or absence of TGF␤. In the presence of active p53, conversion may represent a rate-limiting step in immortal transformation. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Immortality; Telomerase; TGF␤; p16INK4a ; p57KIP2

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. p16INK4a -associated senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Agonescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: HMEC, human mammary epithelial cells; PD, population doublings; SA-␤gal, senescence-associated ␤-galactosidase; EL, extended life; CKI, cyclin dependent kinase inhibitor ∗ Corresponding author. Tel.: +1-510-486-4192; fax: +1-540-486-4475. E-mail address: p [email protected] (P. Yaswen). 1357-2725/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 0 4 7 - X

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4. Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1387 4.1. Changes in p57KIP2 during conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1388 4.2. Acquisition of resistance to TGF␤-mediated growth arrest during conversion . . . . . . . . . . . . . . 1389 5. Immortalization requires multiple errors in distinct pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1389 5.1. c-myc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1389 5.2. p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1390 5.3. ZNF217 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1390 5.4. Conclusions and hypotheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1391 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393

1. Introduction Human epithelial cancers show a steep age-dependent increase in incidence [1]. This sharp increase is thought to be due to accumulated changes in gene expression and function that over time favor the development of growth autonomy and invasiveness in susceptible cells. Considerable resources have been devoted to identifying differences between normal and cancer cells in the belief that knowledge of the specific changes that “drive” cancer progression will facilitate the development of new targeted therapies. A recurrent problem with this strategy is that cancer cell genomes and phenotypes are usually very unstable, and without additional information, changes contributing to cancer pathophysiology can not be distinguished from neutral consequences of genetic drift. For this reason, various tractable experimental systems have been set up to try to determine the effects of individual changes. Cultured human cells have been used historically to study: (a) cellular responses to specific changes under conditions where other potential variables are controlled; (b) phenotypes that may be uniquely human and thus not amenable to study using animal models. A general observation, first noted by Hayflick nearly 40 years ago [2], is that cells cultured from normal human somatic tissues have limited capacities for cell division before undergoing a series of poorly characterized changes culminating in a viable, but non-proliferative state called replicative senescence. Senescence is marked by the appearance of large, flattened vacuolated cells, and occurs despite a steady supply of nutrients and growth factors. Circumstantial evidence has indicated that senescence of cultured human cells may model a key mechanism of tumor suppression: (a) cells from normal

human somatic tissues always senesce, whereas cells from tumor tissues may not; (b) tumor viruses often contain genes that extend proliferative life spans of infected cells; (c) overcoming senescence is required for tumorigenic transformation of cultured cells; (d) telomerase activity, which can facilitate immortal growth, is suppressed in somatic tissues and cells that senesce, but is associated with most tumors and immortal cells. Recent work in numerous laboratories has begun to elucidate the molecular mechanisms responsible for causing and maintaining replicative senescence, and for overcoming it. In this brief review, we summarize what is known about senescence and immortalization of one particular cell type, the human mammary epithelial cell (HMEC), from which most breast cancers are thought to originate. Much of what is summarized is drawn from our own work using a system developed over the past 25 years.

2. p16INK4a -associated senescence HMEC cultures are most often derived from reduction mammoplasty tissue, and are usually grown as adherent monolayers directly on plastic or glass substrates [3]. Cells migrate from epithelia-enriched organoids to cover the surrounding substrate. In the serum-containing MM medium originally developed in our lab, HMEC cultures normally proliferate for ∼15–30 population doublings (PD) before ceasing active growth, looking morphologically senescent, and exhibiting senescence-associated ␤-galactosidase (SA-␤gal) activity [4] (Figs. 1 and 2). The mean TRF length (a measure of telomere length) at this terminal growth arrest is ∼6–8 kbp. In an early effort to simulate the carcinogenic process in vitro in our laboratory, normal HMEC cultures in MM medium were

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Fig. 1. Morphological features of growing vs. senescent HMEC. Cells from specimen 184 are shown during: (A) pre-selection growth phase; (B) p16-associated senescence; (C) post-selection growth phase; (D) agonescence. The bar in panel (A) equals 100 ␮m. Note the greatly increased size and vacuolated appearance (arrows) of senescent/agonescent cells.

exposed to repeated sublethal doses of the common carcinogen, benzo(a)pyrene (BaP) [5]. This treatment yielded several extended life (EL) cultures with increased proliferative potential compared to untreated controls. Later, a serum-free medium (MCDB 170) was developed for normal HMEC growth [6]. In this medium, most cells stop growing after just 10–20 PD. However, a cell population that is capable of long-term growth (50–100 PD total) arises spontaneously in these cultures at frequencies which vary depending on specimen and culture conditions. This outgrowth of cells in growth-arrested cultures has been termed self-selection. In 1998, several groups reported that elevated protein expression of the cyclin dependent kinase inhibitor (CKI) p16INK4a is associated with the senescent HMEC appearing after 15–25 PD, and that p16 is absent in the post-selection HMEC with greater growth potential [7–9]. No changes in other CKIs were reported. Although no mutations were found in the p16 gene in post-selection cells, the p16 promoter was found to be heavily methylated. The inverse correlation between p16 expression and proliferative potential suggested that p16 is at least partially

responsible for the first block to continuous growth encountered by HMEC, and that absence of p16 permits the extended proliferative capacity of the post-selection HMEC. Subsequent testing indicated that EL cultures examined also did not express p16. In one case, an EL culture shared a nonsense mutation in the p16 protein coding sequence with the immortal line derived from it [10]. This data provided additional evidence supporting the role of p16 in mediating the first senescence barrier and implicating it as a possible target of carcinogen-induced changes. The causes of p16 upregulation in pre-selection HMEC and downregulation in post-selection HMEC remain to be identified. It is unlikely that telomere shortening is solely responsible for the upregulation of p16, since: (a) the introduction of exogenous hTERT (the catalytic subunit of telomerase) fails to abrogate p16 expression or the associated growth arrest [8,11]; (b) studies of post-selection HMEC indicate that mean TRF length can become shorter than the ∼6–8 kbp found in senescing pre-selection cells without impairing growth [12]. Studies in various cell types have suggested that p16 may accumulate

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Fig. 2. Schematic diagram of HMEC culture system and derivation of immortal cells. These diagrams show the derivation and characteristics of: (A) untreated cultures; (B) cultures exposed to the chemical carcinogen benzo(a)pyrene; (C) immortalized cultures. In (A), HMEC derived from reduction mammaplasty tissue were originally cultured in serum-containing MM medium. In this medium, HMEC cultures normally only proliferate for ∼15–25 population doublings (PD) before ceasing growth, exhibiting SA-␤gal activity and looking morphologically senescent. Mean TRF length at this terminal growth arrest is ∼6–8 kbp. Cells at this stage exhibit high levels of p16. Later on, a serum-free medium (MCDB170) was developed. In this medium, most cells stop growing after just 10–20 PD, again with high p16 levels. However, a cell population arises spontaneously in these cultures that is capable of long-term growth (50–100 PD total). The outgrowth of cells in growth-arrested cultures has been termed self-selection. The selected cells do not express p16. Agonescence of post-selection HMEC occurs after 50–100 PD with mean TRF of ∼5 kbp, SA-␤gal expression, and no p16 expression. Agonescing post-selection HMEC have been found to accumulate chromosome abnormalities and show increasing evidence of cell death as they approach this second growth arrest. In (B), repeated exposure of normal cultures in MM medium to sublethal doses of a known chemical carcinogen, benzo(a)pyrene (BaP), yielded several extended life cultures with increased growth potential. In virtually, in all cases these cultures underwent agonescence after several additional population doublings, just like post-selection cultures. The extended life cultures also did not express p16. In a few extremely rare instances (C), B(a)P-exposed extended life cultures yielded immortal cell lines without any additional treatments. The original immortal lines, designated 184A1 and 184B5 continued to display normal p53. Later on, two immortalized lines were derived from the same extended life population by insertional mutagenesis into the p53 gene. These lines have been useful for determining the effects of p53 loss in HMEC. The immortalized HMEC could maintain growth in the presence of TGF␤—which arrests the growth of normal HMEC, but not breast tumor cells.

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Fig. 3. The p16-associated growth arrest is influenced by external oxygen concentration. (A) The number of population doublings vs. days in culture is plotted for a pre-selection HMEC culture from specimen 184 that was divided in two, and grown in the presence of low (3%; 䊉) or high (ambient; 䊏) oxygen levels. The cells were passaged when confluent and replated at identical seeding densities. In each case, the last point plotted corresponds to the passage at which there was no net increase in cell number after four additional weeks in culture; (B) levels of total p16 protein in the cells at each passage were assayed by immunoblotting. Note that although incubation in the presence of low oxygen delayed the increase in p16 levels, it did not prevent the increase.

in response to particular forms of stress, including cumulative oxidative damage [13–15]. Cultured cells are routinely incubated in the presence of 20% oxygen found in the ambient atmosphere. When O2 levels are lowered to more physiologically relevant levels, the increase in p16 accumulation and senescence can be delayed in HMEC (Fig. 3). Cumulative oxidative damage may also result from intrinsic metabolic or pathologic processes. The mechanism by which the presence of this damage is relayed to the transcriptional machinery responsible for p16 upregulation is far from clear, but may involve members of the Ets family of transcriptional regulators [16]. The mechanism by which p16 is inactivated by epigenetic means in post-selection HMEC is even less well understood. It is not yet clear, e.g. whether cells with methylated p16 genes are present in normal tissue, and can be

selected for using appropriate culture conditions, or whether a methylase capable of de novo methylation is aberrantly expressed or targeted in some senescing cultures. Despite our evidence, and that of others [15], indicating that the timing and stringency of the growth arrest associated with high p16 expression can be manipulated by altering culture conditions (e.g. medium components, oxygen levels, feeder layers), the p16-associated senescence can not be dismissed as an artifact of cell culture. The fact that the p16/Rb pathway has been shown to be mutated or inactivated in many tumors in vivo indicates that it plays an important role in tumor suppression and that it is worth studying using cell culture models. Epigenetic silencing of p16 has been associated with many diverse human cancers [17]. De novo methylation of the CpG island encompassing the p16 promoter has been found in approximately one-third of primary breast tumors and breast cell lines [17]. In human lung samples, p16 inactivation is also found in mildly dysplastic lesions adjacent to squamous cell carcinomas [18] suggesting that, at least in this tissue, it is an early event that may be permissive for further oncogenic changes.

3. Agonescence When post-selection p16(−) HMEC cease growth, SA-␤gal is expressed, and the mean TRF length is ∼5 kbp. There is no induction of p16 expression, nor alterations in p53 or p21 expression. However, in contrast to the pre-selection HMEC, as post-selection HMEC begin to lose proliferative potential, they accumulate chromosome abnormalities, particularly telomeric associations, and show mitotic failures [19]. There is some cell death, although most cells remain viably arrested at all phases of the cell cycle. These latter criteria have necessitated the creation of a new term, “agonescence,” to distinguish this state from the senescence described in fibroblasts or pre-selection HMEC [20]. In the absence of carcinogenic or oncogenic insults, agonescence is extremely stringent. In our lab, untreated post-selection HMEC have never yielded spontaneous immortal clones in numerous experiments using large numbers of cells. Agonescence also differs from what has been described as crisis, or M2, in cultures treated with viral oncogenes: (a)

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in the long-term viability of most of the population, and (b) in the absence of immortal transformants. The presence of functional p53 at agonescence may account for these differences. The widespread karyotypic abnormalities seen at agonescence are most certainly due to the presence of critically shortened telomeres, resulting from the absence of telomerase activity. In contrast to preselection HMEC, post-selection HMEC are readily immortalized by transduction with hTERT [8,11], suggesting that short telomeres are the main impediment to unlimited growth of these cells. In agonescent cells, lack of sufficient telomeric repeats on individual chromosomes may impair formation of the t-loop structure [21], allowing the chromosome ends to be perceived as double-stranded DNA breaks by the DNA damage recognition machinery [22]. Attempts to repair these exposed ends may result in telomeric associations, and mitotic catastrophe as cells attempt to divide. The stringency of the proliferative block observed in agonescent p16(−) HMEC suggests that it cannot be overcome by altering the activity of a single gene, and thus is likely to be enforced by two or more independent mechanisms. The chances that all these mechanisms will be inactivated by rare genetic or epigenetic events in a given cell undergoing agonescence are probably infinitesimal. However, if certain predisposing errors are already present in an HMEC population prior to agonescence, the probability increases that immortal lines may appear at agonescence when additional genomic errors are generated. Carcinogen-treated EL cultures, like post-selection HMEC, cease growth when the mean TRF declines to ∼4–5 kbp. However, in a few extremely rare instances, EL cultures have yielded immortal cell lines without any additional treatments [5]. Two lines, 184A1 and 184AA4, were derived from an EL culture designated 184Aa and one line, 184B5, appeared in the EL culture 184Be. Karyotypic analysis indicated distinct clonal origins, and a very low level of genomic instability, in both 184A1 and 184B5 [23]. 184AA4 exhibited numerous abnormal karyotypes when first assayed; however later passages contained fewer abnormalities (Stampfer et al., submitted for publication). Like most breast tumor cells, none of these lines has a known defect in the expression or phosphorylation of Rb [24], or in the sequence of p53 [25]. Similar to their finite life span EL precursors,

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these lines lack expression of p16 and contain a stable form of the p53 protein.

4. Conversion A major finding of our work with carcinogen-treated HMEC lines is that the events involved in overcoming agonescence can be temporally separated from activation of telomerase. Surprisingly, early passages of the immortal 184A1 and 184B5 lines show little or no telomerase activity, and their telomeres continue to shorten during initial passages after their emergence [26]. When the mean TRF length decreases to ≤3 kbp, the populations begin to display slow heterogeneous growth, with very low colony forming efficiencies [26]. These mixtures of slow growing and non-proliferative cells are seen in both parental and repeatedly subcloned populations of 184A1 and 184B5. We have termed the early passages of these lines “conditionally immortal” because although mass cultures have repeatedly displayed indefinite growth, most individual cells are initially incapable of sustained growth, despite their clonal origins. With continued culture, the conditionally immortal cell populations very gradually display more uniform proliferative potential, accompanied by increasing expression of telomerase activity and eventual stabilization of telomere lengths. They also gradually acquire the ability to maintain growth in the presence of the pleiotropic cytokine TGF␤. We have used the term “conversion” to describe the gradual process that leads to: (a) activation of telomerase; (b) stabilization of telomere length; (c) ability to grow uniformly well in the presence or absence of TGF␤. Unlike agonescence or crisis, conversion is characterized by low initial labeling index and gradual recovery of good growth over multiple passages. The gradual change in phenotype in individual clones and subclones seen during conversion is inconsistent with clonal expansion of individual genetic mutants in an otherwise genomically stable population. This suggests that at least part of conversion is due to epigenetic mechanism(s). If overcoming agonescence does not require reactivation of telomerase activity, then what does it involve? Two clues are that: (a) the mean TRF length continues to decrease in the conditionally immortal HMEC that have overcome agonescence; (b)

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conditionally immortal HMEC may not express large numbers of gross genomic changes despite prolonged passage with extremely short telomeres. It is possible therefore, that overcoming agonescence involves the induction of a chromosome capping function that compensates for the lack of telomeric repeats and/or prevents telomeric associations. Though this function may only temporarily alleviate the problems associated with short telomeres, it may be sufficient to allow further changes that facilitate conversion. The slowly dividing cells with extremely short telomeres present during conversion may constitute a pool of cells accumulating random errors. If a corresponding stage occurs in tissue, cells undergoing conversion may be present as slow growing microscopic lesions that may be capable of giving rise to faster growing more malignant cells at any time. Thus the study of conversion and its consequences should be of paramount concern. 4.1. Changes in p57KI P 2 during conversion We have used the conditionally immortal HMEC to study the relationships among various phenotypes that change during conversion. The precipitous drop in colony forming efficiency and growth rate observed when mean TRF lengths decrease to less than 3 kbp suggested the imposition of a specific impediment to continuous growth in these cultures. Examination of the CKI p57KIP2 revealed a tight association between p57 expression and slow growth [27]. Increased expression of p57 first becomes detectable in carcinogen-exposed conditionally immortal HMEC that have overcome agonescence. Initially, p57 expression is confined to cells arrested in G0 by blockage of EGF receptor signal transduction. The conditionally immortal cells displaying good growth with mean TRF > 3 kbp are able to downregulate p57 at the mRNA level upon mitogenic stimulation and entry into G1. However, when mean TRF decreases to ≤3 kbp, p57 expression becomes apparent in growth medium containing a full complement of growth factors. This lack of p57 downregulation in fully supplemented cultures correlates precisely with the period of slow heterogeneous growth that ensues. p57 expression is subsequently gradually lost as the cultures complete conversion and resume good growth. Changes in p57 regulation must account for: (a) increased expression during G0 in conditionally

immortal cells; (b) stable expression in conditionally immortal cells with extremely short telomeres in complete growth medium; (c) absence of expression in fully immortal cells during G0 and in complete growth medium. Increased p57 expression may be an initial consequence of the undefined change(s) involved in enabling HMEC to overcome agonescence. The subsequent strong correlation among extremely short telomeres, upregulated p57 expression, and onset of slow, heterogeneous growth in conditionally immortal HMEC suggests a model in which the development of extremely short telomeres causes changes that ultimately result in increased steady state p57 levels and loss of proliferative capacity. Gene transduction experiments provide some direct evidence for this model and support the hypothesis that p57 is at least partially responsible for growth inhibition in conditionally immortal HMEC with critically short telomeres. Expression of exogenously introduced hTERT genes under the control of a retroviral LTR in early passage conditionally immortal 184A1 with mean TRF > 3 kbp causes telomeres to lengthen while preventing both the slow heterogeneous growth phase and accumulation of p57 [27]. Conversely, the same cells transduced with exogenous p57 genes exhibit premature slow heterogenous growth and show morphological changes similar to cultures with extremely short telomeres [27]. Although p57 is found primarily in the nucleus, there is no evidence that it binds telomeric structures or proteins. Therefore, the mechanism by which telomere length influences p57 expression is likely to be indirect. While it is possible that critically short telomeres trigger a DNA damage response that includes activation of factors necessary for p57 accumulation, there is no evidence that p57 accumulation is a general response to DNA damage. Thus, cells must become competent for p57 expression by some other mechanism before p57 expression can be increased by the presence of critically shortened telomeres. Loss of p57 expression in fully immortal HMEC can involve both genetic and epigenetic mechanisms. The p57 gene is imprinted with preferential expression of the maternal allele [28]. Thus loss of the maternal allele by itself can severely reduce p57 expression. The major (presumably maternal) p57 allele initially expressed is frequently lost in conditional immortal 184A1 cells that manage to proliferate during

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the initial slow heterogeneous growth phase of the conversion process [27]. It remains to be determined how the p57 gene is deleted so frequently in cells that do not exhibit general genomic instability. Possibly, the telomeric proximity of the p57 gene on chromosome 11p15.5 makes it especially vulnerable to deletion events in cells with critically short telomeres. Expression data indicate that deletion of the initially expressed p57 allele coincides with upregulation of the previously imprinted paternal p57 allele. Activation of the paternal allele may be a compensatory response to the loss of the maternal allele; the result of a feedback mechanism to prevent unlimited growth. Alternatively, a chromosomal region enforcing epigenetic silencing of the paternal gene may be lost or modified directly or indirectly as a result of the mutagenic process that results in the loss of the maternal allele. In contrast to the genetic inactivation observed for the maternal allele, loss of the remaining p57 allele in 184A1 has never been observed. In addition, loss of heterozygosity has not been observed for either p57 allele in 184B5 cells undergoing conversion to the fully immortal phenotype. In such instances, growth inhibition may not be as acute or abrupt and epigenetic mechanisms of p57 downregulation may be favored.

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HMEC suggests that the effect of hTERT is likely to be indirect, possibly involving cumulative changes in chromatin structure and/or soluble factors. hTERT expression might indirectly change the abundance, modification, and/or spatial arrangements of signaling molecules involved in TGF␤ growth inhibition through altering telomere association with nuclear matrix, or affecting the activities of telomere-associated proteins. Understanding how hTERT expression causes TGF␤ resistance will require more detailed examination of the components and interactions of the active telomerase complex and the TGF␤ signaling pathway.

5. Immortalization requires multiple errors in distinct pathways Although transduction of hTERT into post-selection HMEC demonstrates that agonescence can be avoided by activation of telomerase activity, the crucial question that remains is what errors produce telomerase reactivation during carcinogenesis in vivo. To address this question, we have begun to assess the roles in immortalization of pathologically relevant genes, i.e. genes known to undergo changes in expression or function in breast cancers.

4.2. Acquisition of resistance to TGFβ-mediated growth arrest during conversion

5.1. c-myc

Some features of conversion, such as the gradual acquisition of TGF␤ resistance, may be consequences of the reactivation of telomerase activity [11]. Expression of exogenous hTERT induces resistance to TGF␤-mediated growth arrest in both post-selection finite life span HMEC and conditionally immortal HMEC. However, while hTERT induces TGF␤ resistance quickly in finite life span HMEC, the induction is gradual in conditionally immortal cultures. The mechanism responsible for hTERT inducing either rapid or gradual TGF␤ resistance remains to be elucidated. Experiments using hTERT mutants indicate that in addition to being catalytically active, telomerase must be capable of telomere maintenance in vivo to confer TGF␤ resistance. Our data also indicate that there is no correlation between telomere length and TGF␤ resistance. The incremental acquisition of TGF␤ resistance in conditionally immortal

Amplification of c-myc has been reported in 10–30% of human breast cancers [29,30], and overexpression of murine c-myc has been reported to immortalize post-selection HMEC [31]. Studies of the hTERT promoter have revealed sequences that match the consensus for c-myc binding sites [32,33] and several studies suggest that c-myc can activate telomerase by inducing hTERT expression [31,34,35]. A consistent change associated with the conversion of HMEC to full immortality is deregulated expression of the c-myc oncogene [36]. Like finite life span HMEC, early passage conditionally immortal HMEC exhibit reduced levels of c-myc mRNA and protein during G0. However, in fully immortal HMEC, the overall level of c-myc expression is increased, and equivalent levels of c-myc are expressed in G0-arrested and cycling cell populations. We have observed very rare immortalization of post-selection

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HMEC transduced with human c-myc, however, unlike Wang et al. [31], we did not detect telomerase activity until several passages after transduction (unpublished results). This data suggests that deregulated c-myc may not be sufficient for immortalization or rapid activation of telomerase activity, but suggests that when combined with other rare unknown events, it may play a role in both processes. These other events could be generated during the period of chromosomal aberrations that characterize agonescence. 5.2. p53 Although a number of studies [37–41] have implicated p53 inactivation in immortalization of HMEC, our studies of HMEC immortalized after chemical carcinogen exposure indicate that p53 inactivation, per se, is not necessary for HMEC immortalization. However, p53 inactivation may hasten full immortalization. Both p57 expression and the pace of conversion appear to be dependent on p53 function. We have recently generated two additional immortal HMEC lines, designated 184AA2 and 184AA3, in which both copies of the p53 gene have been inactivated or lost (Stampfer et al., submitted for publication). In the p53 (−/−) lines, the mean TRF length did not decline below 3.5 kbp and p57 was not detected even at the earliest passages that could be assayed. Although these lines expressed some aspects of conversion, the process was greatly accelerated. Direct evidence that the absence of p53 was responsible for these differences was obtained using a retrovirus carrying a dominant negative p53 genetic suppressor element [42]. Conditionally immortal 184A1 cells transduced with this construct displayed upregulated telomerase activity, reduced p57 content and accelerated growth +/− TGF␤. These data suggest that p53 may suppress telomerase activity in cells that have overcome agonescence, until relieved by unknown events that occur during conversion. This suppression could be mediated through direct p53 protein interaction with the telomerase complex [43] or indirectly through transcriptional inhibition of hTERT [44]. Where examined, loss of p53 has been found to occur early in breast cancer, in ductal carcinomas in situ [45]. This finding is consistent with a model in which p53 loss precedes and promotes full immortalization.

5.3. ZNF217 The oncogene ZNF217 was originally identified based on its location on chromosome 20q13.2, an amplicon common in breast cancers. Extra copies of this chromosomal region occur in approximately 18% of breast tumors and 40% of breast cancer cell lines [46]. Amplification appears to be an early event in breast cancer [47] and is associated with poor prognosis [48]. ZNF217 encodes a conserved member of the C2H2 Kruppel family of transcription factors, with a DNA-binding domain of eight C2H2 zinc fingers and a separate proline-rich domain. Members of the Kruppel family have been implicated in both neoplastic and developmental disorders [49]. We investigated the functional consequences of ZNF217 overexpression by transducing the gene into finite life span 184 and EL 184Aa HMEC [50]. The ZNF217-transduced HMEC showed no initial growth advantage over the control cultures, but continued to grow beyond the point where the control population growth-arrested at the agonescence barrier. Numerous foci of small, mitotic, SA-␤gal negative cells appeared among the SA-␤gal positive agonescent cells. Growth was at first slow and heterogeneous, but became faster and more uniform with continued passage. After ∼5–15 passages, varying among experiments, most cells were SA-␤gal negative and grew well. In five independent experiments, ZNF217-transduced cultures gave rise to immortalized cells. Telomerase activity was not initially detectable in the ZNF217-transduced 184 and 184Aa cultures that maintained growth past agonescence, and the mean TRF length continued to decrease. Telomerase activity became detectable within 10 passages and then gradually increased, and mean TRF length stabilized at ∼4 kbp. When assayed for growth in TGF␤, ZNF217-transduced 184 and 184Aa were initially completely growth-inhibited prior to and just after overcoming agonescence. With increasing passage, there was a very gradual increase in the number of cells with progressively better growth capacity in TGF␤. Southern analysis of retroviral integration sites in ZNF217-transduced HMEC growing past agonescence suggested that these cultures were rapidly overgrown by distinct clonal populations. To determine whether distinct chromosomal alterations might be conferring growth advantages on clones immortalized

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with ZNF217, DNA from three different immortalized cultures was used for quantitative measurement of DNA copy number using comparative genomic hybridization [46]. Analysis showed low level regional DNA-sequence copy number variations on chromosomes 1q and 8q common to all three cell lines. The region amplified on 8q included the c-myc oncogene. In addition, each line showed unique regions of high and low level DNA-sequence copy number variations. These sites of regional copy number variation, some of which have also been frequently observed in breast cancer cell lines and primary tumors [46], could contain genes that cooperate with ZNF217 in facilitating immortalization. To determine whether loss of p53 function contributed to the immortalization of the ZNF217-transduced HMEC, p53 function was assayed by measuring p53 expression after exposure to the DNA damaging agent actinomycin D, and p53-dependent induction of GADD45 transcripts following UV irradiation. Induction of p53 similar to that in the finite life span cells was observed in all three ZNF217-transduced immortalized HMEC tested, and GADD45 mRNA levels were increased 4 h after UV exposure in both finite life span 184 and ZNF217 immortalized 184Aa. Rb was also present and underwent normal cycles of phosphorylation and dephosphorylation in these cells. Our work supports the hypothesis that ZNF217 gene amplification can contribute to tumor progression through involvement in overcoming agonescence. The low frequency of ZNF217-immortalized cells seen in our experiments, and the presence in these cells of additional chromosomal alterations suggest that ZNF217 overexpression/amplification probably disrupts only one of several pathways inhibiting immortality. The slow gradual changes in telomerase activity and growth in ZNF217-transduced cells after they have overcome agonescence resemble the changes seen during conversion of HMEC immortalized after chemical carcinogen exposure. Thus, the ZNF217- and carcinogen-immortalized HMEC show many similarities. 5.4. Conclusions and hypotheses (a) Current evidence indicates that HMEC employ several mechanisms to prevent unlimited growth. Some of these mechanisms are triggered by

(b)

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critically short telomeres, while others may be triggered independently of telomere length. In at least two cases (p16, p57), putative proteins immediately responsible for growth arrest may be downregulated through epigenetic changes. HMEC in which p16 is downregulated or the associated Rb pathway is inactivated, undergo a distinct set of degenerative changes collectively termed agonescence. During agonescence, growth arrest is associated with short telomeres and chromosomal abnormalities. In contrast to the crisis exhibited by cells lacking functional p53, agonescent cells can be maintained as viable cultures and do not yield immortal cells under normal conditions. Agonescence can be overcome by rare, as yet undefined, events that allow cells to successfully proliferate in the presence of extremely short telomeres. Inactivation of p53, per se, is not required for immortalization of HMEC. However, early passage p53(+) immortally transformed HMEC lines are only conditionally immortal, with no telomerase activity. Conditionally immortal HMEC exhibit low colony forming efficiency and slow growth when their mean TRF lengths decline below 3 kbp. The growth constraint is tightly associated with the failure to downregulate p57. Conversion of conditionally immortal HMEC to telomerase(+) cells displaying good uniform growth is gradual, even in subcloned populations, and may involve genetic as well as epigenetic changes in expression of p57 and other regulatory molecules.

Based on the conclusions above, we propose the following model (Fig. 4). Two major senescence pathways must be compromised for normal finite life span HMEC to achieve immortality. One pathway may be activated primarily by environmental and intrinsic stress and is associated with upregulated expression of p16. Loss of p16 expression, or other derangements in the Rb pathway, allow this senescence block to be averted. The second pathway is activated by critically short telomeres that result from suppression of telomerase activity, and is associated with the karyotypic abnormalities characteristic of agonescence. At least two alterations are required to overcome agonescence. In cultured HMEC under no selective pressures, the

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Fig. 4. Model for steps involved in immortal transformation of cultured HMEC. Senescence/selection occurs while telomeres are still comparatively long and correlates with high p16 expression. p16 inactivation by epigenetic or genetic means allows cells to proliferate for an extended number of population doublings, before agonescence occurs in cells with shorter telomeres. Agonescence is stringent, and is only rarely overcome by unknown events that allow the cells to continue proliferating with short telomeres. Cells overcoming agonescence are conditionally immortal, lacking telomerase activity, and exhibiting decreasing colony forming efficiency as telomeres become critically short. The subsequent slow, heterogenous growth phase correlates with sustained p57 expression. During conversion to full immortality, p57 inactivation by epigenetic and/or genetic means is accompanied by derepression of telomerase activity and stabilization of telomere lengths.

likelihood that all the necessary errors would occur in the same cell, even under the conditions at agonescence where widespread genomic errors are generated, is exceedingly small. Thus, the growth arrest observed in agonescent p16(−) HMEC is extremely stringent. However, if one predisposing error is already present, e.g. inactivated p53, deregulated c-myc, or overexpressed ZNF217, the probability greatly increases that the required additional error(s)

may be generated by the genomic instability produced by agonescence. In the absence of viral oncogenes or inactivated p53, overcoming the conditional immortal growth constraint (conversion) may also represent a rate-limiting step in immortal transformation. At present, there is no data clearly demonstrating that agonescence or conversion occurs during aging or carcinogenesis in vivo. However, the proposed model could at least partly explain the steep age-dependent

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increase in human epithelial cancers. Repression of telomerase in long-lived animals such as humans may prove to be a proverbial two-edged sword. In a majority of cells that have not accumulated any predisposing mutations, agonescence would provide a stringent limit to the number of replications a single lineage could undergo, and thus limit the opportunity for deleterious mutations to accumulate within that lineage. However, with advancing age, rare mutations that do arise despite the limit on replicative life span could be complemented by chromosomal aberrations arising during agonescence. The generation of widespread chromosomal aberrations during agonescence could account for the presence of abnormal karyotypes in most human carcinomas, even those that are p53(+). Where examined, e.g. in ductal carcinomas in situ, karyotypic abnormalities and telomerase are both seen during early stages of cancer progression [51,52]. This correlation suggests that the generation of chromosomal aberrations during agonescence, leading to reactivation of telomerase, may be the preferential route by which most epithelial tumors progress.

[9]

[10]

[11]

[12]

[13]

[14]

[15]

References [16] [1] R.A. DePinho, The age of cancer, Nature 408 (2000) 248– 254. [2] L. Hayflick, The limited in vitro lifetime of human diploid cell strains, Exp. Cell Res. 37 (1965) 614–636. [3] M. Stampfer, R.P. Yaswen, Culture models of human mammary epithelial cell transformation, J. Mam. Gland Bio. Neo. 5 (2000) 365–378. [4] G.P. Dimri, et al., A novel biomarker identifies senescent human cells in culture and in aging skin in vivo, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 9363–9367. [5] M.R. Stampfer, J.C. Bartley, Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo(a)pyrene., Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 2394–2398. [6] S.L. Hammond, R.G. Ham, M.R. Stampfer, Serum-free growth of human mammary epthelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract, Proc. Natl. Acad. Sci. U.S.A. 81 (1984) 5435–5439. [7] A.J. Brenner, M.R. Stampfer, C.M. Aldaz, Increased p16INK4a expression with onset of senescence of human mammary epithelial cells and extended growth capacity with inactivation, Oncogene 17 (1998) 199–205. [8] T. Kiyono, S.A. Foster, J.J. Koop, J.K. McDougall, D.A. Galloway, A.J. Klingelhutz, Both Rb/p16INK4a inactivation

[17]

[18]

[19]

[20]

[21]

[22]

1393

and telomerase activity are required to immortalize human epithelial cell, Nature 396 (1998) 84–88. L.I. Huschtscha, J.R. Noble, A.A. Neumann, E.L. Moy, P. Barry, J.R. Melki, S.J. Clark, R.R. Reddel, Loss of p16INK4 expression by methylation is associated with life span extension of human mammary epithelial cells, Cancer Res. 58 (1998) 3508–3512. A.J. Brenner, C.M. Aldaz, Chromosome 9p allelic loss and p16/CDKN2 in breast cancer and evidence of p16 inactivation in immortal breast epithelial cells, Cancer Res. 55 (1995) 2892–2895. M. Stampfer, J. Garbe, G. Levine, S. Lichsteiner, A. Vasserot, P. Yaswen, Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor ␤ growth inhibition in p16INK4 (−) human mammary epithelial cells, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 4498–4503. J. Garbe, M. Wong, D. Wigington, P. Yaswen, M.R. Stampfer, Viral oncogenes accelerate conversion to immortality of cultured human mammary epithelial cells, Oncogene 18 (1999) 2169–2180. Q.M. Chen, Replicative senescence and oxidant-induced premature senescence: beyond the control of cell cycle checkpoints, Ann. New York Acad. Sci. 908 (2000) 111–125. O. Toussaint, E.E. Medrano, T. von Zglinicki, Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes, Exp. Gerontol. 35 (2000) 927–945. R.D. Ramirez, C.P. Morales, B.S. Herbert, J.M. Rohde, C. Passons, J.W. Shay, W.E. Wright, Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions, Genes Dev. 15 (2001) 398–403. N. Ohtani, et al., Opposing effects of Ets and Id proteins on p16INK4a expression during cellular senescence, Nature 409 (2001) 1067–1070. J.G. Herman, A. Merlo, L. Mao, R.G. Lapidus, J.-P.J. Issa, N.E. Davidson, D. Sidransky, S.B. Baylin, Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylaton in all common human cancers, Cancer Res. 55 (1995) 4525–4530. S.A. Belinsky, K.J. Nikula, W.A. Palmisano, R. Michels, G. Saccomanno, E. Gabrielson, S.B. Baylin, J.G. Herman, Aberrant methylation of p16INK4a is an early event in lung cancer and a potential biomarker for early diagnosis, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 11891–11896. S. Romanov, K. Kozakiewicz, C. Holst, M.R. Stampfer, L.M. Haupt, T. Tlsty, Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes, Nature 409 (2001) 633–637. T.D. Tlsty, S.R. Romanov, B.K. Kozakiewicz, C.R. Holst, L.M. Haupt, Y.G. Crawford, Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence, J. Mam. Gland Biol. Neoplasia 6 (2001) 235–243. J.D. Griffith, L. Comeau, S. Rosenfield, R.M. Stansel, A. Bianchi, H. Moss, T. de Lange, Mammalian telomeres end in a large duplex loop, Cell 97 (1999) 503–514. H. Vaziri, M.D. West, R.C. Allsopp, T.S. Davison, Y.S. Wu, C.H. Arrowsmith, G.G. Poirier, S. Benchimol, ATM-

1394

[23]

[24]

[25] [26]

[27]

[28]

[29]

[30]

[31] [32]

[33]

[34]

[35]

[36]

[37]

P. Yaswen, M.R. Stampfer / The International Journal of Biochemistry & Cell Biology 34 (2002) 1382–1394 dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase, EMBO J. 16 (1997) 6018–6033. K. Walen, M.R. Stampfer, Chromosome analyses of human mammary epithelial cells at stages of chemically-induced transformation progression to immortality, Cancer Gen. Cyto. 37 (1989) 249–261. C. Sandhu, et al., TGF␤ stabilizes p15INK4b protein, increases p15INK4b /cdk4 complexes and inhibits cyclin D1-cdk4 association in human mammary epithelial cells, Mol. Cell Biol. 17 (1997) 2458–2467. T. Lehman, et al., p53 mutations in human immortalized epithelial cell lines, Carcinogenesis 14 (1993) 833–839. M.R. Stampfer, A. Bodnar, J. Garbe, M. Wong, A. Pan, B. Villeponteau, P. Yaswen, Gradual phenotypic conversion associated with immortalization of cultured human mammary epithelial cells, Mol. Biol. Cell 8 (1997) 2391–2405. T. Nijjar, D. Wigington, J.C. Garbe, A. Waha, M.R. Stampfer, P. Yaswen, p57/KIP2 loss of heterozygosity and expression during immortal conversion of human mammary epithelial cells, Cancer Res. 59 (1999) 5112–5118. S. Matsuoka, et al., Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor p57KIP2 , on chromosome 11p15, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 3026–3030. E.M. Berns, J.G. Klijn, K.L. van Stevern, H. Portengen, E. Noordegraaf, J.A. Foekens, Prevalence of amplificatio of the oncogenes c-myc, HER2/neu, and int-2 in 1000 human breast tumors: correlation with steroid receptors, Eur. J. Cancer 28 (1992) 697–700. Y. Harada, T. Katagiri, I. Ito, F. Akiyama, G. Sakamoto, F. Kasumi, Y. Nakamura, M. Emi, Genetic studies of 457 breast cancers. Clinicopathologic parameters compared with genetic alterations, Cancer 74 (1994) 2281–2286. J. Wang, L.Y. Xie, S. Allan, D. Beach, G.J. Hannon, Myc activates telomerase, Genes Dev. 12 (1998) 1769–1774. I. Horikawa, P.L. Cable, C.A. Afshari, J.C. Barrett, Cloning and characterization of the promoter region of human telomerase reverse-transcriptase gene, Cancer Res. 59 (1999) 826–830. Y.S. Cong, J. Wen, S. Bacchetti, The human telomerase catalytic subunit hTERT: organization of the gene and characterization of the promoter, Hum. Mol. Genet. 8 (1999) 137–142. K.-J. Wu, C. Grandori, M. Amacker, N. Simon-Vermot, A. Polack, J. Lingner, R. Dalla-Favera, Direct activation of TERT transcription by c-myc, Nature Genet. 21 (1999) 220–224. R.A. Greenberg, et al., Telomerase reverse-transcriptase gene is a direct target of c-myc but is not functionally equivalent in cellular transformation, Oncogene 18 (1999) 1219–1226. M.R. Stampfer, C.H. Pan, J. Hosoda, J. Bartholomew, J. Mendelsohn, P. Yaswen, Blockage of EGF receptor signal transduction causes reversible arrest of normal and transformed human mammary epithelial cells with synchronous reentry into the cell cycle, Exp. Cell Res. 208 (1993) 175–188. V. Band, J. DeCaprio, L. Delmolino, V. Kulesa, R. Sager, Loss of p53 protein in human papillomavirus type 16

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

E6-immortalized human mammary epithelial cells, J. Virol. 65 (1991) 6671–6676. D.E. Wazer, Q. Chu, X.-L. Liu, Q. Gao, H. Safaii, V. Band, Loss of p53 during radiation transformation of primary human mammary epithelial cells, Mol. Cell Biol. 14 (1994) 2468– 2478. J.W. Shay, G. Tomlinson, M.A. Piatyszek, L.S. Gollahon, Spontaneous in vitro immortalization of breast epithelial cells from a patient with Li-Fraumeni syndrome, Mol. Cell Biol. 15 (1995) 425–432. Q. Gao, S.H. Hauser, X.-L. Liu, D.E. Wazer, H. Madoc-Jones, V. Band, Mutant p53-induced immortalization of primary human mammary epithelial cells., Cancer Res. 56 (1996) 3129– 3133. L.S. Gollahon, J.W. Shay, Immortalization of human mammary epithelial cells transfected with mutant p53 (273his ), Oncogene 12 (1996) 715–725. V.S. Ossovskaya, et al., Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 10309–10314. H. Li, Y. Cao, M.C. Berndt, J.W. Funder, J.-P. Liu, Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro, Oncogene 18 (1999) 6785–6794. D. Xu, et al., Downregulation of telomerase reversetranscriptase mRNA expression by wild type p53 in human tumor cells, Oncogene 19 (2000) 5123–5133. S.J. Done, N.C.R. Arneson, H. Özçelik, M. Redston, I.L. Andrulis, p53 mutations in mammary ductal carcinoma in situ but not in epithelial hyperplasias, Cancer Res. 58 (1998) 785–789. A. Kallioniemi, et al., Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 2156– 2160. M. Werner, A. Mattis, M. Aubele, M. Cummings, H. Zitzelsberger, P. Hutzler, H. Hofler, 20q13.2 amplification in intraductal hyperplasia adjacent to in situ and invasive ductal carcinoma of the breast, Virchows Arch. 435 (1999) 469– 472. C. Collins, et al., Positional cloning of ZNF217 and NABC1: genes amplified at 20q13.2 and overexpressed in breast carcinoma, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 8703– 8708. T. Pieler, E. Bellefroid, Perspectives on zinc finger protein function and evolution-an update, Mol. Biol. Reports 20 (1994) 1–8. G. Nonet, M.R. Stampfer, K. Chin, J.W. Gray, C.C. Collins, P. Yaswen, The ZNF217 gene amplified in breast cancers promotes immortalization of human mammary epithelial cells, Cancer Res. 61 (2001) 1250–1254. J.W. Gray, C. Collins, Genomic changes and gene expression in human solid tumors, Carcinogenesis 21 (2000) 443– 452. A. Bednarek, A. Sahin, A.J. Brenner, D.A. Johnston, C.M. Aldaz, Analysis of telomerase activity in breast cancer: positive detection at the in situ breast carcinoma stage, Clin. Cancer Res. 3 (1997) 11–16.