A Brief History of HMEC Life, Aging, Death, and Immortality in our Culture Systems

An Overview of Growth, Aging, Senescence, and Immortality in our HMEC Culture System

Introduction

The goal of our studies since 1976 has been to understand the normal processes governing growth, aging, and senescence of HMEC, and how these normal processes are altered during immortal and malignant transformation. To address these goals, my lab has generated a large variety of HMEC types, ranging from primary organoid material to cultured cells at various stages of transformation (Chart 1). Examination of these cultures by my lab and many colleagues has elucidated significant differences between normal and abnormal HMEC, and produced a new, molecularly defined model of the senescence barriers encountered by cultured HMEC (Figure 1, Table 1). These senescence barriers function to suppress tumorigenesis, thus understanding how these barriers are overcome can provide insight into the mechanism of human malignant progression in vivo, and into possible therapeutic interventions. Our HMEC culture system has been shown to accurately model many aspects of early stage breast carcinogenesis in vivo, and can serve as an experimentally tractable model to examine factors that influence human cellular aging and carcinogenesis.

Model

We postulate that cultured finite lifespan HMEC encounter two mechanistically distinct barriers to indefinite proliferation, stasis (stress-associated senescence) and telomere dysfunction due to telomere attrition (replicative senescence). Finite HMEC are also vulnerable to oncogene-induced senescence (OIS). Errors are needed to bypass or overcome the stasis and replicative senescence barriers. Once cells become immortal (telomerase expressing) they are no longer vulnerable to OIS, and a gain-of-function oncogenic error can be sufficient to confer malignant properties.

Fig.1. Model of senescence barriers encountered by cultured HMEC

Table.1. Molecular properties of senescence barriers encountered by cultured HMEC

Stasis is a stress-associated barrier, mediated by the retinoblastoma (RB) pathway, that is independent of telomere length and extent of replication. The onset of stasis in cultured HMEC correlates with increased expression of p16,but not p21 [1-4] . The number of population doublings (PD) achieved prior to stasis is very variable and depends upon culture conditions; we have observed a range of ~10-60 PD [3-6] . Molecular correlates that can identify stasis, in addition to p16 expression, include arrest in G1, low labeling index (LI), non-critically short telomeres and normal karyotypes [2-4] . These parameters are consistent with an RB-mediated arrest and the absence of a significant DNA damage response (DDR). Cells at stasis express senescence-associated β-galactosidase (SA-β-Gal) activity and a senescent morphology. Stasis can be bypassed or overcome in cultured HMEC by multiple types of single alterations (genetic and/or epigenetic) in pathways governing RB, and does not require loss of p53 function [1, 6-9] . Overcoming stasis may correlate with hyperplasia/atypical hyperplasia in vivo, which commonly display clonal growth and errors in the RB pathway (e.g., loss of p16 expression, mutated RB, overexpressed cyclin D1) [1, 10-13] . Gross genomic aberrations are not common at this stage in vivo [14] , and are not associated with overcoming stasis in vitro [2, 4] .

We postulate that stasis can also be enforced by p53-dependent p21 in response to DNA damaging stresses such as oxidative damage or radiation. Although neither cultured HMEC or their isogenic mammary fibroblasts express p21 at stasis, other cell types may be more vulnerable to DNA damage inducing stresses in culture, express p21, and show greater evidence of a DDR at stasis. HMEC in vivo may also experience p53-inducing stresses. The p53-dependent type of stasis arrest does not require critically short telomeres nor genomic instability, and inactivation of p53 (or p21) function may facilitate overcoming this arrest [15, 16] . Reactivation of telomerase is neither necessary nor sufficient to overcome stasis.

Telomere dysfunction due to telomere attrition (replicative senescence) occurs in post-stasis HMEC (cells that have bypassed or overcome stasis) due to ongoing proliferation producing progressively shortened telomeres, in the absence of sufficient telomerase activity. When telomeres become critically short (mean TRF ≤ 5 kb), genomic instability and a DDR is elicited. Where wild-type p53 is present, most cells show a viable arrest; this barrier has been termed agonescence [2, 3, 17] . Karyotypic analysis of HMEC at agonescence has shown that virtually all metaphases exhibit gross chromosomal abnormalities, predominantly telomere associations [2] . This result is not consistent with a hypothesis that a p53-dependent senescence arrest due to telomere attrition occurs as soon as one uncapped telomere is present [18, 19] . When p53 is non-functional a viable arrest is not possible, and crisis-associated massive cell death occurs [3] . Agonescence can be distinguished from stasis in HMEC by the presence of critically short telomeres and genomic instability, higher LI (~15%), arrest at all phases of the cell cycle, and presence of a DDR (Table 1). HMEC at agonescence as well as at stasis display a senescent morphology and SA-β-Gal, so these properties do not readily distinguishable between these two molecularly distinct senescence barriers. Crisis can be distinguished from agonescence in HMEC by a higher LI (~40%) and the absence of a viable arrest. Since most human epithelial and fibroblast cells induced to transform to immortality in culture have had prior inactivation of p53 function (e.g., using viral oncogenes or inhibitors of p53 function) only crisis was observed in such cultures at replicative senescence.

The barrier due to telomere attrition can be overcome by the expression of sufficient telomerase to maintain stable telomere lengths. Overcoming telomere dysfunction may correlate with DCIS in vivo, which commonly display short telomeres, genomic instability, and telomerase reactivation [14, 20] . That most DCIS contain critically shortened telomeres indicates that the precursor cells did not express sufficient telomerase for telomere maintenance.

Cultured finite lifespan HMEC are vulnerable to oncogene-induced senescence (OIS) [21, 22] . HMEC that have attained immortality via reactivation of endogenous telomerase are no longer vulnerable to OIS, and show gain of malignancy-associated properties when exposed to oncogenes such as Raf-1, Ras or ErbB2 [21, 23, 24] . HMEC immortalized by exogenous hTERT transduction may appear initially sensitive to OIS, but can maintain growth [25] . The molecular correlates of OIS in HMEC differ from those seen in cells at stasis or telomere dysfunction (Table 1). OIS in HMEC does not require p16 or p53 function, and is independent of telomere length; it’s molecular properties are consistent with a DDR [21, 22] .

Finite Lifespan Cells

Pre-stasis HMEC: HMEC derived from reduction mammoplasties, milk, benign tumors, and non-tumor mastectomy tissues have been grown in either serum-containing (MM, M85, M87A) or serum-free (MCDB 170) media (Chart 1) [4-6, 26, 27] . Depending upon the media and culture conditions, active proliferation has ceased after ~10-60 PD. In media that support fewer PD, levels of p16 expression increase earlier; virtually all cells express p16 at stasis in all media used [1, 4] . The molecular profile of the HMEC at stasis is similar regardless of their PD potential or growth media (Table 1), with one noticeable difference. HMEC grown in serum-containing media have a typical senescent morphology of large flat vacuolated cells, whereas HMEC that had been grown in serum-free MCDB170 exhibit a more elongated morphology showing abundant stress fibers [4, 6, 27] . We believe this difference is due to the serum-free medium being more stressful for cultured HMEC, consistent with the early rise of p16 and the low PD potential of HMEC initiated in MCDB170 (commercial MEGM [Lonza} and M171 [Life Technologies] are based on our original MCDB170) [1, 6] . This difference in morphology may have led other investigators to consider this stasis arrest distinct, and refer to it as “M0” [28, 29] .

Our more recently developed M85/M87A media support long-term growth of the normal pre-stasis HMEC (Figures 2,3) [4] . The pre-stasis HMEC we now distribute were grown in these media. Populations contain a mixture of cells with markers of myoepithelial, luminal, and progenitor lineages; later passage cultures show fewer luminal cells [30] . While not yet carefully examined, cells with luminal markers may also cease proliferation as a consequence of terminal differentiation rather than p16(+) stasis. Senescent cells remain genomically stable. We have examined gene transcript profiles, global promoter methylation, and DDRs as a function of passage from several individual’s HMEC [4, 9] . As expected, gene expression changes significantly with passage, while no obvious differences were seen for promoter methylation. Some interindividual differences could be detected in gene expression and extent of DDRs. Previous studies have shown interindividual differences in carcinogen metabolism [31] , leading us to recommend that at least 2 individuals be examined to determine normal HMEC properties. We have also observed differences in lineage markers correlated with the age of the specimen donor [30] . With increasing age, HMEC from 4^th passage pre-stasis strains and from uncultured dissociated organoids showed a decline of myoepithelial cells, and an increase of luminal cells that exhibited molecular features usually ascribed to myoepithelial cells (increased expression of integrin α6 and keratin (K)14). The proportion of c-Kit expressing cells (putative progenitors) also increased. These data suggest that the observed age-associated increase in luminal breast cancer could be connected to changes that occur normally with aging in the human breast. Myoepithelial cells are thought to be tumor-suppressive and progenitors are putative etiological roots of some breast cancers. Thus during the aging process, the potential target cell population may increase while there is a simultaneous decrease in the cells thought to suppress tumorigenic activity.

Figure 2. Growth of pre-stasis HMEC in M85 or M87 ± oxytocin (X) or BSA (A). A. Primary cultures from three reduction mammoplasty specimens were started from organoids and grown in M85 ± X. The number of PD in primary culture cannot be accurately determined; growth is shown starting from passage 2. Growth of 184 HMEC in MM is shown for comparison. All media contained cholera toxin from passage 2. B. Frozen stocks of second pasasge184 HMEC were grown in media with (M85) or without (M87) conditioned media ± lipid rich BSA (A), in the presence of oxytocin. 250MK are cells derived from milk, grown in MM for primary culture, then switched to M85+X at second passage. Note the rapid initial growth in these new media formulations. These results indicate that it is possible to generate large batches of early passage pre-stasis HMEC from individual donors. [4]

Figure 3. Expression of markers associated with proliferation (LI) and senescence (p16, SA-b-Gal) in pre-stasis 184 HMEC with increasing passage. All cells except for 9p-X were grown in M85 with oxytocin; stasis in this population was at passage 15. The 9p-X culture was grown in M85 without oxytocin; stasis was at passage 10. Cultures examined are from the growth curve shown in Fig. 2. Note the reciprocal relationship between the small cells with a positive LI, and the larger, often vacuolated cells (senescent morphology) that are positive for p16 and SA-b-Gal, and negative for LI. Size marker = 200 microns. [4]

We have cultured pre-stasis HMEC from over 150 individuals in serum-containing media and have not observed even a single instance of a cell spontaneously overcoming the stasis barrier. However, early experiments that exposed primary cultures of specimen 184 HMEC grown in MM to the chemical carcinogen benzo(a)pyrene (BaP) resulted in the emergence of HMEC colonies that maintained growth after the bulk of the cultures ceased proliferation at stasis [7, 32] . These BaP post-stasis populations (originally called Extended Life), ceased growth after an additional 10-40 PD, with the exceptional of very rare cells that became immortal cell lines (see below). All three BaP post-stasis cultures that have been examined showed loss of p16 expression, associated either with mutation or promoter silencing [1, 13] . We have very limited quantities of BaP cultures available for distribution on a collaborative basis.

When the HMEC are cultured in the highly stressful serum-free MCDB170 medium, a small number of cells are able to overcome stasis in the absence of additional oncogenic exposures [6] . These post-stasis cells show methylation of the p16 promoter and absence of p16 expression, as well as nearly 200 other mostly cancer-associated changes in promoter methylation (in contrast to the BaP post-stasis cultures, which display only ~10 changes) [1, 9] . We originally called the emergence of these post-stasis cells “selection” and this class of post-stasis HMEC “post-selection”. We now recognize that selection (what other labs latter termed “M0”) is a stasis arrest. Although the pre-stasis populations may be heterogeneous with respect to a cell’s ease in silencing p16 to become post-selection [33] , we believe the post-selection cells are induced by growth in the stressful (oncogenic) serum-free MCDB170 medium, i.e., post-stasis cells are not present in the starting normal pre-stasis cultures. This is based on the total absence in over 30 years of our work of any post-stasis cell emerging from normal pre-stasis HMEC grown in any of our serum-containing media, as well as the absence or reduction of post-selection HMEC emerging from pre-stasis HMEC grown in MCDB170 when there are small changes in media composition (e.g., absence of a cAMP stimulator) or methodology (e.g., sub-culturing cells approaching stasis/selection rather than waiting 2-3 weeks at stasis without subculture for the post-selection cells to emerge; we presume the induction of the p16(-) cells is occurring during this time when no cell divisions are observed). It is likely that post-stasis cells pre-exist in some breast tissues; p16(-) HMEC have been seen in apparently normal breast tissues in vivo [33] . These rare cells have been called vHMEC; the nature of the error(s) leading to the silencing of p16 in vHMEC in vivo is not known; the term vHMEC has also been used by others to refer to p16(-) post-stasis cells in culture that are specifically post-selection. Of note, it has been suggested that the aberrant post-selection post-stasis HMEC (which are sold commercially as normal primary HMEC, e.g., Lonza CC-2551 and Life Technologies A10565) may be on a pathway to metaplastic cancer [34] .

Post-selection post-stasis p16(-) HMEC grow actively for an additional ~30-70 PD, depending on the individual. They express wild-type p53 that is present in a stable form [3, 35, 36] . As they near agonescence, they exhibit a senescent morphology, SA-β-Gal, a DDR, and genomic instability [2, 3] . If p53 function is inactivated (e.g., using the genetic suppressor element GSE22 [37] ) cells continue to proliferate for an additional ~2-4 passages, with increasing evidence of cell death and debris (Figure 4) [3] . The telomere dysfunction barrier is very stringent in post-selection HMEC. We have never seen any unperturbed cell at agonescence spontaneously immortalize. We have also never seen any immortalization at crisis in post-selection HMEC with p53 function inactivated by GSE22, but rare immortalization at crisis using DN-p53 constructs has been reported by others [38, 39] . This stringency is likely due, in part, to the molecular nature of this barrier; cells that fail to maintain a G1 or G2 arrest with critically short telomeres will eventually die or become non-proliferative as a consequence of the genomic instability and mitotic catastrophes. Unlike an arrest based upon blocking cell cycle progression (e.g., elevated levels of CKIs at stasis), the widespread chromosomal derangements present at telomere dysfunction are not reversible. Overcoming this barrier differs from overcoming stasis in that escaped cells will have acquired genomic abnormalities and may retain some degree of genomic instability [14] .

Figure 4. Growth and morphology of post-stasis post-selection 184 with and without functional p53. 184B HMEC were transduced with GSE22-containing or control (Babe) vectors at passage 5. (A) growth curves of 184B-Babe and 184B-GSE22. Note the additional PD in the cultures lacking functional p53. We believe growth rates are similar ± p53, but the absence of p53-mediated growth inhibition allows more cells to continue to proliferate to crisis, leading to apparent faster growth of the population as cells near telomere dysfunction. (B) 184B-Babe at agonescence, 2 months after plating at passage 15, contains mostly large, flat cells with some vacuolization; the cell population can retain this morphology and viability for over a year. (C) 184-GSE22, two weeks after plating at passage 15, shows areas of small proliferating cells and many very large flat cells (arrows). (D) 184B-GSE22, four months after plating at passage 15, shows mostly large multi-nucleated, vacuolated cells and abundant cell debris. All photographs are at the same magnification. [3]

More recently, we have generated another type of post-stasis HMEC using shRNA to p16 (p16sh) [40] . As expected, direct inhibition of p16 using p16sh led to widespread bypass of stasis in the exposed pre-stasis cultures. Post-stasis p16sh HMEC show few differences in their pattern of promoter methylation or gene transcripts compared to their precursor pre-stasis cultures ( [1, 9] unpublished). They grow for an additional ~20 PD until agonescence. Unlike post-selection post-stasis cells, p16sh post-stasis HMEC have generated rare clonal immortal lines during the period of genomic instability at agonescence [40] .

We have large supplies of post-selection HMEC available for distribution from women of various ages. It’s important to recognize that these cells are not normal, and acquire genomic instability as they are propagated in culture. In the past, due to the inability to attain long-term culture of normal pre-stasis HMEC, we provided post-selection HMEC for studies on finite HMEC. Since it is now possible to grow large quantities of normal pre-stasis HMEC, we recommend that studies aiming to understand normal HMEC behavior use normal HMEC and not the aberrant post-selection HMEC. For some experimental purposes, post-selection or other post-stasis HMEC may be preferable, e.g., examining the requirements for and mechanisms of overcoming the telomere dysfunction barrier, or assaying cells at different stages in progression. We can provide limited amounts of the less aberrant p16sh post-stasis HMEC for specific requests.

The telomere attrition barrier can be overcome or bypassed by the expression of sufficient telomerase to maintain stable telomere lengths. Based on our experience and the reports of others, reactivation of sufficient telomerase in finite lifespan HMEC is difficult to achieve using pathological relevant agents (i.e., not hTERT transduction or viral oncogenes), and may require multiple errors. This may reflect the fact that long-lived animals such as humans have evolved mechanisms for stringent repression of telomerase in normal adult non-stem cells, presumably for tumor suppression. In contrast, cells from small short-lived animals such as mice do not show such stringent telomerase repression, and, lacking the replicative senescence barrier, readily immortalize once they overcome stasis [41, 42] . We have postulated that immortalization and telomerase reactivation may be a rate-limiting step in human epithelial carcinogenesis, and so believe that great caution should be exercised in extrapolating mechanisms of rodent malignant progression to humans. One of the goals of our long-term program in developing an HMEC model system of transformation has been to make available experimentally tractable human cells for examination of this crucial step in human malignant progression, since this step cannot be accurately modeled in mice. Overcoming telomere dysfunction may correlate with DCIS in vivo, which commonly displays short telomeres and genomic instability, and may show telomerase reactivation. We have hypothesized that the genomic instability associated with agonescence and crisis can give rise to errors permissive for telomerase reactivation, and that the generation of breakage-fusion-bridge (BFB) cycles prior to immortalization may underlie some of the genomic instability and “passenger” errors seen in many carcinomas [3, 43] . Additionally, the extensive genomic instability prior to immortalization may introduce unknown errors that can contribute to the ultimate cancer cell phenotype, including level of aggressiveness. Our hypotheses are consistent with recent publications indicating that many properties of invasive tumors are already present in their pre-invasive DCIS lesions, such as tumor markers, gene expression profiles, gene methylation, PIK3CA mutations, and genomic errors [44-48] .

I want to add a few comments about nomenclature since this issue has frequently come up in discussions. It’s my general experience in science that functionally distinct molecules or molecular processes are given distinct names. Confusion could result if there were not distinct names for different, though closely related family members (e.g., growth factors and their receptors) or related mechanisms (e.g., apoptosis, anoikis, mitotic catastrophe). One of our overall goals is to try to model the many different in vivo pathways a normal cell can take to become malignant. Such information may assist individualized clinical interventions. Our data thus far indicate that molecular properties differ among different pathways, starting with early stage carcinogenesis. Pre-stasis HMEC differ from post-stasis HMEC, although both are finite, and as discussed above, there are significant differences among the various post-stasis types. Since these different post-stasis cultures are functionally different, we have given them distinct names. Similarly, since agonescence is molecularly and morphologically distinct from crisis, although both result from telomere attrition, we believe it important that there be distinct names. Confusion may also arise if similar mechanisms are given distinct names, e.g., we view what we are defining as stasis as having also been referred to as M0, M1, M^INT, M1.5, premature senescence, replicative senescence, and culture shock. What we are calling telomere dysfunction due to telomere attrition (agonescence/crisis) has been called replicative senescence, crisis, M2, and M1. Indeed, it was this situation that prompted our initial efforts to generate molecularly defined nomenclature for the senescence barriers [2] . I encourage everyone to employ the molecularly defined nomenclature we have presented here for our HMEC culture system.

Immortally Transformed Cell Lines

We have generated a variety of immortally transformed lines, mostly from specimen 184, more recently from specimens 240L, 122L and 805P, using various oncogenic agents (see: Chart 1 and Cell Types Generated) [3, 7-9, 21, 32, 40, 49-53] . Most of these lines were derived from post-stasis cultures, although in a few instances (involving hTERT or c-Myc transduction) lines emerged from perturbations of pre-stasis populations. As described in more detail below, these studies have led us to propose that attaining a fully immortal potential (i.e., synthesizing sufficient telomerase to maintain stable telomere lengths) involves several steps [3, 8, 21, 40, 49, 53] . Even after HMEC have acquired the errors allowing them to bypass/overcome both stasis and telomere dysfunction, and express hTERT, the resultant cells with indefinite proliferative potential still progress through further changes. We have called this process “conversion”. As described below, conversion is most prominent in cells that immortalize while retaining functional p53. Consequently, this process has not been widely studied, as most in vitro immortalized human epithelial and fibroblast cells had p53 inactivated prior to immortalization. While we have gained much information about the molecular properties associated with conversion, much about this process remains unknown.

Our first immortal lines were obtained from the BaP post-stasis cultures, 184Aa and 184Be [7, 8, 32, 49] . Extremely rare immortal lines have appeared at agonescence (184A1, 184AA4, 184AA8, 184B5, 184BE1). These cultures had been exposed to BaP, and likely harbor additional errors beyond the loss of p16 expression. Rare errors produced by the genomic instability at agonescence may complement pre-existing errors to allow telomerase reactivation. More frequent but still rare clonal lines appeared at agonescence following transduction of the breast cancer–associated oncogene ZNF217 into the 184Aa population (184AaZN1-3 [51] ). More frequent immortal clonal outgrowths at crisis were seen when p53 was inactivated in 184Aa using GSE22 (184AaGS1-2). Uniform immortalization was obtained following transduction of c-Myc into three different BaP post-stasis cultures (184AaMY1, 184BeMY1, 184CeMY1 [40] .

No post-selection HMEC has been observed to spontaneously immortalize. Rare immortal lines have appeared at agonescence following overexpression of either breast cancer associated oncogene, c-Myc or ZNF217 (184SMY1, 184ZN4-7) [40, 51] . We hypothesize that rare errors generated by the genomic instability at agonescence may complement ZNF217 or c-Myc to allow telomerase reactivation. Overexpression of both c-Myc and ZNF217 in post-selection HMEC was able to produce clonal immortal lines in repeat experiments (184ZNMY1-4, unpublished). Some of these lines immortalized early, prior to agonescence, and show no chromosomal copy-number changes by CGH.

More recently, we have targeted early passage pre-stasis cells grown in M85/M87A for transformation [40] . Our preliminary studies indicate that transduction of c-Myc can produce rare clonal escape from stasis leading to clonal immortalized lines. Transduction with p16sh (i.e., p16sh post-stasis cultures) produced rare clonal lines around the time of telomere dysfunction induced genomic instability. When post-stasis p16sh cultures were transduced with c-Myc, apparently uniform immortalization occurred. These new M85/M87A-derived lines are not yet well characterized and are available upon specific request.

Figure 5. Conversion of newly immortal p53(+) HMEC lines is associated with changes in many key properties. (A.B.) The p53(+) 184A1 line undergoing conversion exhibits changes in growth capacity (CFE) and expression of p57, expression of telomerase activity and mean TRF length, gains the ability to maintain growth in the presence of TGFβ, and becomes resistant to OIS [8, 21, 49, 51, 54] . When pre-conversion 184A1 is transduced with GSE22, there is a rapid increase in telomerase activity associated with stabilization of TRF length [8] .

Observations of our initial immortally transformed lines (with functional p53 and no transduced myc) led us to describe the process we have called conversion [8, 21, 43, 49-54] . Conversion has been most extensively studied in the immortal 184A1 line, which first appeared ~passage 8 in the 184Aa BaP post-stasis population, and had a mean TRF value of ~5 kb when first examined at passage 11 (Figure 5). We noted that cells that overcame agonescence gained the potential to express telomerase, but initially displayed little telomerase activity, and had ongoing telomere erosion with proliferation. When telomeres got extremely short (<3 kb), the conversion process ensued. Expression of the CKI p57^Kip2 initially abruptly increased and then slowly declined, associated with initial slow-heterogeneous growth and then gradual re-attaining of uniform good growth. Telomerase activity gradually increased, and the faint very short telomeres seen during conversion gradually became stabilized with a mean TRF of ~3-7 kb. As telomerase activity increased, the immortal lines gradually developed the ability to maintain growth in the presence of TGFb; this change is a direct consequence of the hTERT expression, as transduction of hTERT into post-selection HMEC confers the ability to maintain growth in TGFβ in addition to producing uniform immortalization [52] . A significant change that is associated with conversion and telomerase expression but not initial immortal potential is the loss of vulnerability to OIS [21] .

When we obtained immortal HMEC lines that lacked functional p53 (184AA2, 184AA3) we noted that they showed some initial telomerase activity, no p57 expression, and quickly attained good uniform growth ± TGFβ. Their mean TRF length stabilized at ~4-5 kb and never declined to the very low levels seen in the p53(+) lines. The role of p53 in repressing telomerase activity in newly immortal lines was then demonstrated by inactivating p53 (using GSE22) in pre-conversion 184A1 (Figure 5) [8] . Endogenous telomerase activity was quickly expressed and mean TRF lengths stabilized; existing p57 expression was rapidly reduced. GSE22 transduction into the finite lifespan precursors of the immortal lines did not induce significant telomerase activity indicating that abrogation of p53 function alone is not sufficient for telomerase reactivation in post-stasis HMEC. These results suggest that the newly immortal p53(+) lines have the potential to express telomerase, but expression is low due to a p53-mediated repression (unpublished data have also indicated that newly immortal p53(+) lines express low telomerase activity which can be inhibited). We hypothesize that fully immortal p53(-) lines are expressing an accelerated but molecularly similar conversion process as occurs in the p53(+) lines. The resulting cultures express similar properties, e.g., short stable telomeres, resistance to OIS, and hundreds of promoter methylation changes [9] , but the p53(-) lines never encountered the extremely short telomeres and p57 expression shown by the p53(+) lines. Newly immortal 184AA2 and 184AA3 did both initially briefly show slower growth and lower TRAP activity than at later passages.

Our current speculation is that conversion may reflect a need to alter chromosome conformation at the telomeres when cells transition from a finite state (no stable telomere length maintenance) to one where sufficient telomerase maintains the short stable telomeres. As well studied in yeast, immortal cells can have “counting” mechanisms to maintain telomeres within a limited size range [55] . Since most human carcinoma cells, as well as our immortal HMEC lines, maintain telomeres within a short range (mean TRF ~3-7 kb) [49, 56, 57] , some type of “counting” mechanism likely is involved. Short stable telomeres are not seen in normal telomerase expressing human cells such as stem cells and lymphocytes [58] , suggesting that active processes may be required for conversion to the distinct telomeric state seen in the immortalized and cancer-derived cells.. Functional p53 may present a partial barrier to the conversion process until very short telomeres provoke a structural change at the telomeric ends. Since the majority of breast cancers express wild-type p53, it is possible that the slower p53(+) version of the conversion process may be relevant to early-stage breast carcinogenesis in vivo. We have speculated that the low levels of telomerase expression coupled with extremely short telomeres could make newly immortal p53(+) breast cancers particularly vulnerable to therapeutic interventions targeting telomere dynamics.

In general, we have seen that different methods of producing immortal HMEC can yield cell lines with significantly different phenotypes. These methods may vary in the extent to which they model human malignant progression in vivo. Most of the immortalized lines generated thus far are from younger women and have phenotypes most similar to the basal subtype of human breast cancers. This subtype has a poor prognosis, but represents only a minority of breast cancer; it is more common in younger breast cancer patients. Possibly, the prior difficulty in growing normal human HMEC with luminal or progenitor properties has contributed to the limited phenotypes of the immortalized lines. We are currently developing new lines, using pathologically relevant agents and HMEC from older specimen donors, that are more reflective of the spectrum of human breast cancers seen in vivo.

Once the HMEC are immortally transformed and no longer vulnerable to OIS, the introduction of one or two oncogenes can further transform these cells towards malignancy (anchorage-independent growth, growth factor independence, and/or tumorigenicity in nude mice) [21, 23, 24] . Finite lifespan HMEC cannot be rendered malignant by the same oncogenes. Comparisons of non-malignant immortal lines with oncogene-exposed derivatives that had gained anchorage-independent growth did not show major differences in gene transcript profiling or global promoter methylation, in contrast to the major differences seen between all finite and all immortalized cultures [9, 59] . These data are consistent with the acquisition of immortality, rather than the acquisition of malignancy, as the step in human carcinogenesis most associated with molecular alterations.

Note: Immortal HMEC have been actively transformed to immortality. Normal human somatic cells are finite, and vulnerable to multiple tumor suppressor barriers (stasis, replicative senescence, OIS). Immortality (expression of sufficient telomerase to maintain stable telomere lengths) is the most common alteration from normal associated with human solid cancers. We believe that attaining immortality is likely the most rate-limiting step in human carcinogenesis – immortally transformed lines such as 184A1 and MCF10A have acquired the errors that allowed them to overcome all tumor suppressor barriers, so that the overexpression of one oncogene can confer malignancy. Our immortal lines cluster with tumor derived lines and not finite HMEC in properties such as gene expression, promoter methylation, and resistance to OIS and TGFβ growth inhibition. Immortal lines may be non-malignant, but they are NOT normal, “normal”, or untransformed. Please do not refer to immortal HMEC (or any immortalized human cells) as normal or non-transformed. I sometimes despair about how we will be able to understand and develop therapeutics for early stage human epithelial carcinogenesis when immortal cell lines such as 184A1 and MCF10A, or TERT-immortalized post-selection HMEC, are routinely referred to in the literature as normal or un-transformed, employed as “normal” controls, or used as a starting point to study “early stage” carcinogenesis. I view this as similar to calling telomerase(+) DCIS “normal”, and using it as a normal control for cancer or the starting point for studying early stage carcinogenesis.