Our recent studies on the process of immortalization of cultured human mammary epithelial cells (HMEC) have uncovered a previously undescribed, apparently epigenetic step, termed conversion. When first isolated, clonally derived HMEC lines of indefinite lifespan showed little or no telomerase activity or ability to maintain growth in the presence of TGFβ. Cell populations whose mean terminal restriction fragment length had declined to <3 kb also exhibited slow heterogeneous growth, and contained many non-proliferative cells. With continued passage, these conditionally immortal cell populations very gradually converted to a fully immortal phenotype of good growth±TGFβ, expression of high levels of telomerase activity, and stabilization of telomere length. We now show that exposure of the early passage conditionally immortal 184A1 HMEC line to the viral oncogenes human papillomavirus type 16 (HPV16)-E6, -E7, or SV40T, results in either immediate (E6) or rapid (E7; SV40T) conversion of these telomerase negative, TGFβ sensitive conditionally immortal cells to the fully immortal phenotype. Unlike conditional immortal 184A1, the HPV16-E7 and SV40T exposed cells were able to maintain growth in TGFβ prior to expression of high levels of telomerase activity. A mutated HPV16-E6 oncogene, unable to inactivate p53, was still capable of rapidly converting conditional immortal 184A1. Our studies provide further evidence that the transforming potential of these viral oncogenes may involve activities beyond their inactivation of p53 and pRB functions. These additional activities may greatly accelerate a step in HMEC immortal transformation, conversion, that would be rate-limiting in the absence of viral oncogene exposure.
Spontaneous transformation of cultured normal human cells to an indefinite lifespan occurs extremely rarely, if at all (Harris, 1987). Immortal transformation following exposure to physical carcinogens such as chemical carcinogens or irradiation has also been difficult to achieve in vitro. Consequently, most studies of human cell immortalization have utilized viral oncogenes to transform normal finite lifespan cells, since exposure to specific oncogenes can greatly increase the incidence of obtaining immortal cell lines. Models of human cellular immortalization have therefore been based largely on results obtained following viral oncogene exposure. These models have emphasized the capacity of viral oncogenes such as HPV-E6 and -E7, SV40T, and adenovirus E1A and E1B to inactivate the p53 tumor suppressor gene and/or the retinoblastoma protein (RB). An assumption underlying such models is that the processes responsible for immortal transformation are similar in cells immortalized with and without exposure to viral oncogenes.
In vivo, immortal transformation is thought to be a critical step in malignant progression (Bacchetti, 1996). Normal human somatic cells have a finite lifespan, whereas cells derived from tumor tissues can give rise to cell lines of indefinite lifespan. Tumor cells may need to overcome the proliferative constraints imposed by replicative senescence in order to accumulate the multiple derangements necessary for invasion and metastasis. Since viral oncogenes are not associated with most human cancers, and escape from replicative senescence may be critical for carcinogenesis, it is important to determine whether similar processes are involved in immortal transformation with and without use of viral oncogenes.
We have developed a model system of immortalization that uses cultured human mammary epithelial cells (HMEC) (Stampfer and Yaswen, 1994). Primary cultures of finite lifespan HMEC obtained from reduction mammoplasty tissue specimen 184 were exposed to the chemical carcinogen benzo(a)pyrene. Treated cells gave rise to several different extended life (EL) cultures. The EL cells subsequently lost proliferative capacity, however two cells, derived from two separate EL cultures (184Aa and 184Be), maintained proliferation. These two cells gave rise to the two immortally transformed cell lines 184A1 and 184B5 (Stampfer and Bartley, 1985, 1988). Karyotypic analysis indicates distinct clonal origins, and a very low level of genomic instability, in both cell lines (Walen and Stampfer, 1989). No defect has been found in either immortal line in the expression or phosphorylation of RB, or in the sequence of p53 (Lehman et al., 1993; Sandhu et al., 1997). Neither line displays sustained anchorage-independent growth, growth factor independence, or tumorigenicity. Similar to their finite lifespan EL precursors, both immortal lines lack expression of the cyclin dependent kinase inhibitor (CKI) p16 and display a stable form of p53 protein (Brenner et al., 1998; Lehman et al., 1993). Thus, 184A1 and 184B5 allow examination of the process of human cell immortal transformation without the possible confounding factors of viral oncogene exposure or loss of p53. They also indicate that it is possible to obtain an indefinite lifespan without loss of either p53 or RB.
Recent studies in our laboratory have uncovered a previously undescribed step, which we have termed conversion, involved in the immortal transformation of 184A1 and 184B5 (Stampfer et al., 1997). Although both lines showed an indefinite lifespan when grown in mass culture, early passages cultures displayed little or no telomerase activity, continued telomere shortening with passage, and an inability to maintain growth in the presence of TGFβ. Cell populations whose mean TRF (terminal restriction fragment) had declined to <3 kb also showed poor heterogeneous growth, with most individual cells losing proliferative capacity. When the telomeres became critically short, ∼2 – 2.5 kb mean TRF, these early passage conditionally immortal cells underwent a very gradual conversion characterized by increasing expression of telomerase activity, stabilization of telomere length, increasing number of cells displaying progressively better growth in TGFβ, and attainment of uniform good growth. The very gradual nature of this conversion process, and its reproducible manifestation in both mass cultures and clonal isolates of these immortal lines, have led us to suggest that conversion is an epigenetic process. Furthermore, we have proposed that there is an inherent epigenetic mechanism to reactivate telomerase when telomere length becomes critically short. This program is not normally encountered due to the multiple checkpoints which a stringent replicative senescence program imposes to prevent growth with shortened telomeres. The conditionally immortal cells have already overcome the replicative senescence block. Their ability to maintain proliferation with extremely short telomeres enables them to encounter the presumably epigenetic conversion process.
Since this gradual conversion process had not been previously reported, and the overall process of immortal transformation in these chemically transformed lines did not correlate with the reported descriptions of immortal transformation following viral oncogene exposure, we have examined the consequences of exposing early passage conditionally immortal 184A1 to specific viral oncogenes. Our results indicate the HPV16-E6, -E7 and SV40T oncogenes are capable of immediate or rapid conversion of conditionally immortal 184A1 populations to a fully immortal phenotype. Immortalization with these oncogenes may therefore obscure detection of conversion, a potentially rate-limiting step in immortal and malignant transformation. Additionally, a mutant HPV16-E6 oncogene lacking the ability to inactivate p53 was still able to rapidly convert conditionally immortal 184A1.
Infection of conditionally immortal 184A1 with viral oncogenes
The indefinite lifespan line 184A1 appeared in the EL 184Aa culture at passage (p) 9, distinguishable from 184Aa by faster growth, greater refractility, and smaller cell size. The EL 184Aa culture had emerged at p6 from 184 HMEC exposed to benzo(a)pyrene in primary culture (Stampfer and Bartley, 1985, 1998, see Materials and methods). Early passage 184A1 (p9 – 15) displays good uniform growth in the serum-free medium MCDB 170, but shows a steep decline in colony forming efficiency and labeling index (LI) per colony around passages 16 – 17, when the mean TRF has decliened to ∼3 kb (Stampfer et al., 1997). No telomerase activity or sustained growth in TGFβ has been detected in the good growing early passage 184A1 populations. 184A1 between passages 16 – 30 is characterized by poor heterogeneous growth, followed by a gradual increase in growth capacity±TGFβ leading to populations with good uniform growth by around passages 40 – 45.
We now sought to determine the effect of viral oncogenes, known to be capable of immortalizing HMEC, on the process of conversion in conditionally immortal 184A1. Early passage (p12) good growing 184A1 were infected with retroviral vectors containing the HPV16-E6, HPV16-E7, or SV40T genes, or control LXSN-based retrovirus. Infected cells were obtained following selection in G418 for 10 days. The infected (184A1-E6, 184A1-E7, 184A1-T) and control (184A1-LXSN) cultures were then maintained in MCDB 170 medium with periodic assays for telomerase activity, mean TRF length, and growth in the absence or presence of TGFβ, as described in Materials and methods. The oncogene-exposed cultures were assayed for the presence of the p53 protein within 4 – 5 passages following retroviral infection (Figure 1). As previously described (Lehman et al., 1993), finite lifespan post-selection 184 HMEC (184Ep p13) contain abundant wild type p53 in a stable form, while fibroblasts obtained from 184 breast tissue (184Fb p7) show a faint band characteristic of short-lived p53 protein. Both late passage fully immortal 184A1 (184A1 p56) and early passage conditionally immortal 184A1-LXSN contain abundant p53 levels similar to the finite lifespan 184 HMEC. The 184A1-E6 population showed very low levels of p53 at p15, presumably due to degradation of p53 by the ubiquitin-dependent proteolytic pathway (Scheffner et al., 1993). Conditional immortal 184A1 exposed to HPV16-E6 mutant oncogenes which do not inactivate p53 function (184A1-E6W132R and 184A1-E6JH26) showed levels of p53 protein expression similar to control 184A1 and post-selection 184 HMEC. The 184A1-E7 population showed a small increase in p53 protein levels while the 184A1-T population displayed high levels of p53, presumably due to stabilization of the p53 protein (Oren et al., 1981).
HPV16-E6 causes immediate conversion of conditionally immortal 184A1 to a fully immortal phenotype
As previously reported for early passage conditionally immortal 184A1, no telomerase activity was detected in the p12 184A1-LXSN culture, and the mean TRF length declined from 5 kb at p13 to a faint signal of 2.5 – 2 kb at passages 21 – 28 (Figures 2a and 3a). No 184A1-LXSN colonies showed any sustained growth in TGFβ through p28 (Figures 2c and 3c and Table 1). The 184A1-E6 culture was assayed for telomerase activity at p12 following G418 selection. High levels of activity were seen at p12, and at all passages examined thereafter (Figures 2b and 4a). Unlike the control cultures and consistent with its expression of high telomerase activity, the 184A1-E6 population maintained a mean TRF of ∼5 kb with continued passage to p19 (Figures 2a and 4b). 184A1-E6 also maintained uniform good growth through p25, with no evidence of the slow heterogeneous growth normally seen in conditionally immortal 184A1 when it reaches p16, and displayed by the 184A1-LXSN population (Figure 2c and Table 1). 184A1-E6 cultures initially showed both patchy growth similar to 184A1 and areas of more tightly packed smaller cells (Figure 5d), but by p21 184A1-E6 consisted mainly of tightly packed colonies (Figure 5e). Assays for growth in TGFβ demonstrated that 184A1-E6 was already capable of good growth in TGFβ at p13 (Figure 2c and Table 1). Thus, as soon as it could be assayed, early passage 184A1 transduced with the HPV16-E6 gene showed the phenotype of a converted, fully immortal HMEC line, i.e., high telomerase activity, stable telomere length, and good growth in the absence or presence of TGFβ. Some function(s) of the E6 gene are therefore capable of completely overcoming or circumventing the conversion process.
The several phenotypic differences shown by the fully immortal HMEC, compared to their conditionally immortal precursor populations, could be acquired through independent or obligately coordinated mechanisms. To test the possibility that the E6 gene might utilize more than one functional domain to confer all the phenotypic differences displayed by fully immortal HMEC, we examined the effect of mutated E6 genes on expression of telomerase activity and growth±TGFβ in early passage 184A1. We first introduced five HPV16 E6 substitution mutants, HPV16-E6 Cys-63-Gly, Cys-63-Arg, Cys-63-Ser, Cys-106-Arg, and Trp-132-Arg, whose protein products have been characterized as having low or no binding to the p53 protein, and no degradation of p53 (Dalal et al., 1996). 184A1 at p14 was transduced with these mutants along with wild type E6 and the vector alone control. When assayed at p15, cells transduced with the mutant E6 genes as well as the LXSN control showed no telomerase activity whereas the cells transduced with the wild type E6 had high telomerase levels (Figure 4a and data not shown). When mass cultures were assayed for growth in TGFβ at p16, the cells containing wild type E6 maintained growth whereas the cells with mutant E6 or LXSN controls showed no TGFβ resistant growth (data not shown). Both the mutant E6 and LXSN control populations exhibited slow heterogeneous growth by p17. 184A1 at p13 was then transduced with the amino terminal HPV16-E6 mutant E6JH26, which has also been reported not to bind or target p53 for degradation and not to affect p53 transactivation, but which does bind the E6-associated protein E6-AP and has been shown to activate low levels of telomerase in finite lifespan human keratinocytes and mammary cells (Klingelhutz et al., 1996; Mietz et al., 1992). With this mutant, moderate levels of telomerase activity were detected when first assayed at p14, and high levels were present by p17 (Figure 4a and data not shown). Assays for growth in TGFβ indicated no growth at p14, but by p17 100% of cells showed moderate to good growth (Table 1). The 184A1-E6JH26 population also maintained uniform good growth with no evidence of the growth constraint encountered by the control population at p17. Thus the HPV16-E6 mutants did not produce independent acquisition of the phenotypes present in fully immortal HMEC. However, the data with the HPV16-E6JH26 mutant indicate that the E6 oncogene does not need the ability to inactivate p53 to rapidly convert conditionally immortal 184A1 to full immortality, and suggest that some other E6 function is responsible for this behaviour.
HPV16-E7 and SV40-T oncogenes greatly accelerate conversion of conditionally immortal 184A1 to a fully immortal phenotype
When first assayed at p12, no telomerase activity was detected in the 184A1-E7 population (Figures 3b and 4a). Low levels of activity were detected at p18 and increased thereafter. By passages 25 – 29, 184A1-E7 showed high levels of telomerase activity, in contrast to the still undetectable activity at p28 in the 184A1-LXSN control population. The 184A1-T population exhibited an even more accelerated expression of telomerase activity. Very low levels could be detected at passages 12 – 15, and high levels were detected by p23 (Figure 3b). Consistent with the telomerase activity data, the mean TRF length of both 184A1-E7 and 184A1-T showed an initial decline from ∼5 Kb at p13 to faint signals of ∼3.5 kb at p21, followed by stabilization of telomere length (Figures 3a and 4b). This result differs from the lack of any telomere shortening in the immediately telomerase positive 184A1-E6 population, and from the continued telomere shortening to a faint mean TRF of ∼2 kb seen in the 184A1-LXSN control population with no telomerase activity at these passage levels. As described previously, telomerase activity (using 2 μg protein/assay) was first detectable in 184A1 at p30, and high activity was detected at >p40 (Stampfer et al., 1997).
Examination of the growth of 184A1-E7 and 184A1-T indicated that both cultures experienced some heterogeneous growth between passages 16 – 20. The 184A1-T cultures were remarkable for their rapid growth from the time the virus was introduced. Passage times for routine subculture at split ratios of 1 : 8 were about 5 – 7 days from passages 14 – 24, compared to approximately 7 days for the uniformly growing 184A1-E6 population. However, at passages 17 – 19, the 184A1-T populations showed the presence of large flat vacuolated cells similar to those observed in the 184A1 control populations during the period of slow heterogeneous growth (Figure 5b and g). When assayed for growth as colonies, many colonies with poor or non-sustained growth were visible at these passages, as seen by the decreased LI measured in colonies at p17 (Figure 3c and Table 1). The overall mass culture maintained rapid growth despite the presence of these non-proliferative cells, and by p21 the 184A1-T population showed uniform good growth. The morphology of the growing 184A1-T cells was not notably different from fully immortal 184A1. The 184A1-E7 population grew at a slower rate than either 184A1-T or 184A1-E6, with subculture about every 10 days from passages 14 – 24. From passages 17 – 19 they also displayed many non-proliferative cells and heterogeneous colonies (Figure 5f and Table 1). Unlike 184A1-T, p21 cells did not yet display uniform growth, but uniform good growth was observed by p29. The morphology of 184A1-E7 population showed consistently smaller cells and more patchy cell growth than 184A1-LXSN or 184A1-T (Figure 5f). In contrast to the viral oncogene exposed cultures, the control 184A1-LXSN populations continued to display slow heterogeneous growth through p28, with subculture approximately every 2 – 3 weeks.
While the above results with 184A1-E7 and 184A1-T are consistent with a model in which the oncogenes are able to accelerate a conversion process, the results measuring growth in the presence of TGFβ suggest that these oncogenes are also able to circumvent some aspects of the conditional immortal phenotype (Figure 3c and Table 1). Unlike the 184A1-LXSN colonies, which still showed no sustained growth in TGFβ at p28, the majority of 184A1-E7 and 184A1-T cells were able to maintain some growth in the presence of TGFβ at the earliest passages tested. Approximately 30 – 40% of 184A1-E7 colonies had a LI>25% from passages 14 – 25, indicating that this capacity was rapidly conferred by the expression of the viral oncogene, even in the absence of telomerase activity at p13. Similarly, approximately 80 – 90% of the 184A1-T colonies displayed >25% LI between passages 13 – 21, with an increasing percentage of colonies showing >50% LI. This result differs from our previously observed results with the uninfected 184A1 and 184B5 cell lines, where acquisition of the ability to maintain good growth in TGFβ during the conversion process was tightly associated with expression of high levels of telomerase activity (Stampfer et al., 1997), and suggests that these two phenotypes of fully immortal HMEC can be acquired independently in the presence of specific viral oncogenes.
HPV16-E6 induction of telomerase activity in finite lifespan HMEC
Our previous studies suggested that induction of telomerase activity in conditionally immortal cells is an inherent epigenetic consequence of continued growth with critically shortened telomeres. The above data with 184A1-E7 and 184A1-T is consistent with this hypothesis in that telomerase activity increased gradually as the mean TRF value decreased to ∼3.5 kb. However, HPV16-E6 was able to induce immediate high levels of telomerase activity in conditionally immortal 184A1 cells that had a mean TRF similar to senescent HMEC, i.e., ∼5 kb. Previous reports have also indicated that HPV16-E6 may induce low levels of telomerase activity in finite lifespan human epithelial cells (Klingelhutz et al., 1996). We therefore examined our finite lifespan HMEC cultures to determine whether HPV16 E6 could induce telomerase activity, and if so, whether it was dependent upon specific parameters of the HMEC population; i.e., growth conditions, age in culture, or telomere length.
Four different sets of finite lifespan 184 HMEC were examined following infection with HPV16-E6 or control vector and selection for 10 days in G418 (see Materials and methods): (1) pre-selection cells grown in the serum-free MCDB 170; this population was infected at p2 when still proliferative, and assayed at p3 when it contained mostly poorly growing, flat, striated cells as described (Hammond et al., 1984), and had a mean TRF of ∼8 – 7 kb (data not shown); (2) post-selection cells grown in the serum-free MCDB 170, infected and assayed at p9, p18, and p20. These populations had active cell division, with the mean TRF at passages 9 and 20 approximately 7 and 5.5 kb respectively (Stampfer et al., 1997 and data not shown); senescence occurred at ∼p22; (3) EL 184Aa, the precursor of the immortal 184A1 line, grown in MCDB 170, infected and assayed at p8 and p13. Both these populations had active cell division, mean TRF approximately 6 and 5.2 kb respectively (Stampfer et al., 1997 and data not shown); senescence occurred at ∼p15; (4) 184 g HMEC actively growin in the serum containing MM medium, infected at p3 and assayed at p4; this MM-grown population undergoes a senescence-like arrest around p6, and exhibited a mean TRF between 8 – 8.8 kb when assayed at passages 3, 4, 5 and 6 (data not shown). As previously reported (Stampfer et al., 1997), no telomerase activity was detected in the post-selection 184 or 184Aa without the E6 oncogene, nor was there detectable activity in the poorly growing p3 pre-selection populations grown in MCDB 170 (Figure 6 and data not shown). Introduction of the E6 gene resulted in low telomerase activity in the post-selection 184 p9 and in the 184Aa p8 and p13 cells. Repeated independent infections showed no telomerase activity in the near-senescent, but still proliferating, p18 and p20 post-selection 184 cells, nor in the poorly growing pre-selection p3 cells. These results show no apparent correlation between the level of telomerase expression induced by E6 and the age in culture or mean TRF of the cell population, and are in agreement with previous reports that the E6 oncogene can induce low levels of telomerase activity in telomerase negative finite lifespan human epithelial cells (Klingelhutz et al., 1996). In contrast to the cells grown in the serum-free medium, early passage 184 grown in MM showed a low level of telomerase activity, even in the absence of the E6 gene. This low telomerase activity was further increased to a medium level after the introduction of E6 (Figure 6). These data agree with previous reports which indicate that certain populations of cultured HMEC may have low levels of telomerase activity (Belair et al., 1998; Klingelhutz et al., 1996). Together, these data show that the HPV16-E6 oncogene is capable of rapid induction of telomerase activity in specific populations of young to near-senescent finite lifespan HMEC with mean TRFs ranging from ∼8 – 5 kb. They also indicate that the growth conditions of the cultured HMEC can influence whether or not low levels of telomerase activity are present in the uninfected as well as E6 infected populations.
The long term effects of HPV16-E6 exposure on finite lifespan 184 HMEC and EL 184Aa were also observed (data not shown). Exposure of post-selection p12 184 HMEC to HPV16-E6 recapitulated what has been described for immortalization by this viral oncogene (Band et al., 1991; Shay et al., 1993b; Wazer et al., 1995). We observed an extension of lifespan of approximately 2 – 4 passages, followed by a brief slowdown in growth and appearance of non-proliferative cells (crisis), followed by emergence of cell populations with good growth±TGFβ, telomerase activity, and an apparent indefinite lifespan. Exposure of p10 EL 184Aa resulted in a brief crisis period at the passage senescence would normally occur, followed by emergence of cell populations with good growth±TGFβ, telomerase activity, and an apparent indefinite lifespan.
We have recently described a novel step, termed conversion, which occurs as part of the immortalization process of chemical carcinogen exposed HMEC that have already overcome replicate senescence (Stampfer et al., 1997). The transition from a finite lifespan EL culture to a cell line with indefinite growth potential was not associated with immediate expression of telomerase activity or attainment of uniform good growth. Rather, both mass cultures and clonal derivatives of the three p53+/+ immortal HMEC lines that we have generated displayed a very gradual conversion from a phenotype of no telomerase activity as well as no ability to grow in TGFβ, to one with high telomerase activity and uniform ability to maintain active growth in the absence or presence of TGFβ. The period of slow heterogeneous growth required for the conversion of the early passage conditionally immortal cell lines to full immortality extended over 1 – 2 years of in vitro growth, and was tightly associated with expression of the CKI p57 (Yaswen et al., submitted). In contrast, the results described in this paper show that exposure of the early passage conditionally immortal cell line 184A1 to the viral oncogenes HPV16-E6, HPV16-E7, or SV40T resulted in a phenotype of full immortality, i.e., high telomerase activity and uniform good growth±TGFβ, within a much shorter time period. The HPV16-E6 oncogene rendered the 184A1 cells fully immortal within one passage of exposure, while the HPV16-E7 and SV40T large T oncogenes produced fully immortal 184A1 cells within 2 – 4 months.
The virtual lack of spontaneous immortal transformation of normal human cells in culture, coupled with the rarity of carcinogen induced in vitro transformation, has led to the use of viral oncogene exposure in order to achieve more reproducible and efficient immortalization. Exposure of HMEC to the SV40T oncogene yields inefficient but reproducible immortalization (Bartek et al., 1991; Shay et al., 1993a), while exposure to the high risk HPV-E6 and -E7 oncogenes provides reproducible and efficient immortalization (Band et al., 1990, 1991; Shay et al., 1993b; Band, 1998). Similarly, other human epithelial cell types have been readily transformed to immortality following exposure to HPV-E6 and -E7 (Choo et al., 1993; Klingelhutz et al., 1994; Viallet et al., 1994; Woodworth et al., 1996). The HPV16-E6 oncogene alone is also capable of efficient immortalization of the post-selection HMEC (Wazer et al., 1995) which do not express p16 (Brenner et al., 1998) and which may consequently not require functional inactivation of the RB protein by -E7 for immortalization.
Based largely on these viral oncogene mediated models of human cell transformation, previous investigators have suggested that immortal transformation involves overcoming at least two blocks, M1 and M2 (Shay et al., 1993c; Holt et al., 1997). This model postulates that at the first block, M1, shortened telomeres signal activation of p53 and RB controlled cell cycle checkpoints that cause a viable arrest in a G0 or G1-like state. Overcoming this block yields EL cultures, and has been ascribed to loss of normal RB and p53 function, consistent with the ability of these viral oncogenes to functionally inactivate RB and p53. The EL cells eventually reach the M2 block and undergo what has been described as a crisis, i.e., loss of proliferative capacity and death. Overcoming M2 has been considered to involve a rare mutation which occurs during crisis. Since EL cell telomeres continue to shorten with passage (Counter et al., 1992, 1994), while the immortally transformed cell lines show telomerase activity and stabilized telomere length, it has been postulated that this mutation involves reactivation of telomerase activity. A similar model has been used to describe the immortalization of post-selection HMEC following functional inactivation of the p53 gene (Gollahon and Shay, 1996). Our chemical carcinogen induced HMEM immortalization system shows a process of immortalization that differs from this M1/M2 model. Our data indicate that HMEC immortalization in vitro can occur without loss of either p53 or RB. This result is consistent with the in vivo data, in that the majority of human breast cancers, which do show telomerase activity, do not exhibit mutations in either p53 or RB (Thor et al., 1992; Hartmann et al., 1995; Sjogren et al., 1996; Lee et al., 1988; T'Ang et al., 1988). Our data also indicate that overcoming the block at replicative senescence and acquisition of an indefinite lifespan was not coincident with reactivation of telomerase. Telomerase reactivation appeared to be an epigenetic consequence of the unknown event that permitted continued proliferation beyond the shortened telomere length (∼5 kb mean TRF) that would normally signal replicative senescence. Telomerase activity increased gradually in our p53+/+ HMEC lines only after mean TRF lengths decreased to <2.5 kb (Stampfer et al., 1997).
Additionally, the relationship among what has been called EL and crisis in the viral oncogene models, and EL and conversion in our system is not clear. Our carcinogen induced EL cultures and our post-selection normal HMEC senesce at approximately the same mean TRF length, ∼5 kb, and show no obvious differences in the p53 or RB pathways. Both these populations lack expression of p16 and have a stabilized form of wild-type p53. However, some differences do exist between the EL and post-selection cells. We show here that the HPV16-E6 oncogene is able to reactivate telomerase in near senescent but proliferative EL 184Aa, but was consistently unable to do so in near senescent, proliferative post-selection 184 HMEC. Similarly, preliminary results from our laboratory indicate that late passage 184Aa, but not late passage 184 HMEC, can be made immortal by overexpression of human c-myc. We hypothesize that the EL cultures which gave rise to immortal cell lines harbor immortalization predisposing defects not present in post-selection HMEC. However, these defects are not the changes in p53 or RB associated with the M1 model block. We do not observe an obvious crisis-like event in the generation of our carcinogen exposed HMEC immortal cell lines. Virtually all cells in our EL cultures senesced and died. The extremely rare conditionally immortal cells that overcame replicative senescence then underwent an epigenetic conversion process which extended over a much longer time frame than has been described for crisis. Conversion is characterized by very gradual emergence of cells with progressively better growth capacity, rather than the more rapid emergence during crisis of cells which express good uniform growth.
These differences between viral oncogene and carcinogen mediated immortal transformation of HMEC suggest that caution should be exercised in generalizing among model systems. While the nature of the events occurring during immortal and malignant transformation of human breast epithelial cells in vivo are still not understood, development of primary breast cancers is a slow process estimated to take 8 years (Renan, 1993). HMEC obtained from primary tumors are generally characterized by poor growth in culture (Ethier, 1996), rather than the uniform rapid growth seen in HMEC immortalized in vitro by high risk HPV oncogenes. It is possible that the gradual conversion process seen in the carcinogen induced model system may more accurately model the process of in vivo malignant progression.
We have postulated that the reactivation of telomerase which occurs during HMEC conversion to full immortality is an inherent epigenetic response to the presence of critically short telomeres. In our three p53+/+ HMEC lines, 184A1, 184B5, and 184AA4 (Stampfer et al., 1997), reactivation occurs only after the cell populations' mean TRF has declined to ⩽2.5 kb and the populations exhibit slow heterogeneous growth accompanied by high levels of p57 expression. We have also generated two p53−/− lines from the EL 184Aa culture (Stampfer et al., in preparation). These populations undergo only a brief period of slow heterogeneous growth and show no expression of p57 (Stampfer et al., ibid.). Additionally, the mean TRF of these lines did not decline below 3.5 kb and strong telomerase activity was present by passage 23. The results reported here for the HPV16-E7 and SV40T infected conditionally immortal 184A1 are similar to these p53−/− lines in showing only a brief period of heterogeneous growth, mean TRF⩾3.5 kb, and strong telomerase activity by passages 22 – 24. While the SV40T oncogene is known to inactivate p53, we do not know what function of the E7 oncogene is responsible for these results. Unlike the increased level of p53 seen in 184A1-T and the decreased level in 184A1-E6, no major changes in p53 protein levels were seen in 184A1-E7. Nonetheless, all these lines showed a gradual increase in telomerase activity associated with initial telomere loss (from a mean TRF of ∼5 kb to 4 – 3.5 kb) and then telomere length stabilization by passage 20. Based on these data, we hypothesize that the epigenetic reactivation of telomerase and acquisition of good uniform growth can occur with much greater efficiency in these p53 negative and viral oncogene exposed cultures.
In the non-oncogene exposed conditional immortal HMEC lines, conversion was associated with a gradual and coordinate acquisition of the fully immortal properties of high telomerase activity and uniform good growth±TGFβ. In the oncogene exposed conditional immortal 184A1, we found that these properties did not appear to be obligately connected. While the wild-type and mutant HPV16-E6 we examined produced either all or none of this phenotype coordinately, the HPV16-E6 and SV40T exposed 184A1 showed an immediate ability to maintain growth in the presence of TGFβ, but not immediate high levels of telomerase activity. The HPV16-E7 and E1A oncogenes have been reported to have the capacity to bind and inactivate the CKI p27 (Mal et al., 1996; Zerfass-Thome et al., 1996), which has been associated with TGFβ growth inhibition in our HMEC system and other cell types (Polyak et al., 1994; Sandhu et al., 1997). This additional function of the E7 oncogene may account for its ability to rapidly confer TGFβ resistance to conditionally immortal 184A1. Although a similar activity has not been reported for the SV40T oncogene, our results suggest that it too may be capable of inactivating some aspect of the TGFβ growth inhibition pathway. Consistent with this hypothesis, we have seen that TGFβ exposure prevents the downregulation of p27 that occurs as TGFβ sensitive cells progress into G1, but has no effect on p27 downregulation in the TGFβ resistant fully immortal HMEC lines (Yaswen and Stampfer, unpublished results). In contrast, TGFβ has no effect on expression of the p57 during conversion.
Unlike HPV16-E7 and SV40T, the HPV16-E6 oncogene was able to confer immediate high levels of telomerase activity in conditionally immortal 184A1 that still had a mean TRF of 5 kb. This rapid induction of high telomerase activity by E6, as well as its ability to induce lower levels of telomerase activity in finite lifespan HMEC with mean TRF values of 8 – 5 kb, suggest that it acts through a mechanism other than an epigenetic turn-on of telomerase activity resulting from critically short telomeres. Telomerase activity is present in both tumor cells and cells with a high self-renewal capacity. In tumor cells and immortal cell lines, telomerase activity is associated with telomeres that are generally shorter than those found in finite lifespan cells (Bacchetti, 1996). However, telomerase positive cells with high self-renewal capacity do not necessarily exhibit shorter telomeres (Chiu and Harley, 1997; Vaziri and Benchimol, 1998). Possibly, the mechanism by which E6 reactivates telomerase is more closely related to the regulation of telomerase activity in finite lifespan telomerase positive cells than to the reactivation of telomerase which occurs during malignant progression. We do not know what function of E6 confers the capacity to induce telomerase activity, but since an E6 mutant unable to inactivate p53 retained this capacity, it is a function separate from the inactivation of p53.
There were differences in the ability of E6 to induce telomerase activity in our finite lifespan HMEC depending upon the culture conditions employed. Induction did not occur in the poorly growing pre-selection 184 nor in the near senescent, proliferative, post-selection 184. Additionally, different HMEC cultures varied in their basal expression of telomerase activity. The p16 expressing 184 HMEC grown in the serum-containing MM medium contained low levels of telomerase activity in the absence of any oncogene exposure. Exposure to E6 caused a further increase to medium levels of telomerase activity. These results indicate that the effects of the E6 oncogene on telomerase activity depend to some extent on the cell type in which it is introduced. However, there was no strict correlation with whether or not the cultures were proliferative. The detection of some telomerase activity in the early passage MM-grown 184, but not in the highly proliferative post-selection 184, may also indicate that culture conditions, or selection for growth of specific breast epithelial cell types in culture, can influence whether this activity is present. Previous reports have shown both the presence and absence of low levels of telomerase activity in HMEC, and other human epithelial cells in culture (Belair et al., 1998; Klingelhutz et al., 1996). We suggest that both situations exist, and it is the cells being examined that differ. However, we see no correlation between proliferative capacity and the expression of telomerase activity in the finite lifespan HMEC, as the HMEC population with the most long-term proliferative capacity, the post-selection cells, consistently show no telomerase activity.
In summary, we have shown that the viral oncogenes HPV16-E6, -E7, and SV40T are able to circument or greatly accelerate the very gradual conversion to full immortality normally seen in our p53+/+ HMEC cell lines. The ability of these oncogenes to simultaneously inactivate many cellular checkpoints is likely responsible for their capacity to achieve reproducible immortalization of HMEC and other human cells. These capacities extend beyond their known effects on inactivation of p53 and RB, particularly since a mutant E6 oncogene unable to inactivate p53 was still capable of causing rapid conversion of the conditionally immortal 184A1 line. While use of these viral oncogenes may help elucidate the mechanisms of immortal transformation of human cells, model systems of immortalization that employ viral oncogenes with such pleotrophic effects may not permit detection of what might otherwise be a rate limiting step in human epithelial cell immortalization and malignant progression, i.e., the very gradual conversion of conditionally immortal cells to full immortality.
Materials and methods
Finite lifespan 184 HMEC were obtained from reduction mammoplasty tissue of a 21-year-old individual and showed no epithelial cell pathology. When cultured in serum-free MCDB 170 medium (MEGM, Clonetics Corporation, San Diego, CA, USA), HMEC undergo a self-selection process after approximately 10 – 20 Pd, in which only a small number of cells maintain good growth (Hammond et al., 1984; Stampfer, 1995; Stampfer and Yaswen, 1994). The good growing post-selection HMEC cultures show an absence of p16 mRNA and protein expression, correlated with methylation of the p16 promoter (Brenner et al., 1998). Post-selection 184 HMEC senesce around passage 22, equivalent to approximately 80 population doublings. In the serum-containing medium, MM, the HMEC population ceases active growth after approximately 15 – 25 Pd, coincident with expression of high levels of p16 (Stampfer, 1982, 1985; Brenner et al., 1998). Extended lifespan 184Aa emerged from 184 HMEC following benzo(a)pyrene exposure of primary cultures growing in MM as described (Stampfer and Bartley, 1985, 1988). 184Aa first appeared as a single colony at passage 6, and showed complete loss of growth potential by passage 16 when grown in MCDB 170. Indefinite lifespan 184A1 appeared in a 184Aa culture at passage 9. Both 184Aa and 184A1 show no p16 protein expression due to mutations in the p16 gene (Brenner and Aldaz, 1995). Except where otherwise indiciated, cells were grown and subcultured in MCDB 170 as described (Stampfer, 1985).
Human recombinant TGFβ1 was purchased from R&D Systems (Minneapolis, MN, USA) and used at 5 ng/ml in the presence of 0.1% bovine serum albumin (Sigma). The ability to maintain growth in the absence or presence of TGFβ was assayed in the following manner. To detect colony forming efficiency, growth capacity and heterogeneity of single cell derived colonies, 200 – 2000 cells were seeded per 100-mm dish. Cultures were maintained for 14 – 20 days after seeding. [3H]thymidine (0.5 – 1.0 μCi/ml) was then added for 24 h 4 – 7 h following refeeding, and labeled cells were visualized by autoradiography as described (Stampfer et al., 1993). CFE was determined by counting the number of colonies of >50 cells, and growth capacity by counting the percentage of labeled nuclei in these colonies. Uniform good growth was defined as a labeling index (LI) of >50%. To determine growth capacity in TGFβ, TGFβ was added to some cultures for 10 – 14 days once the largest colonies contained 100 – 250 cells. Growth capacity per colony was determined as above. To detect very rare TGFβ resistant cells in mass cultures, 1×105 cells were seeded per 100-mm dish, and TGFβ was added 24 h later for 10 – 14 days; control cultures received bovine serum albumin alone. Cells were then labeled and prepared for autoradiography as above. Cultures±TGFβ were also visually monitored at least twice weekly for growth, mitotic activity, and morphology. These observations were recorded, and representative photographs taken.
Retroviruses carrying the wild-type HPV16-E6 gene, mutant HPV160-E6 genes (Cys-63-Gly, Cys-63-Arg, Cys-63-Ser, Cys-106-Arg, and Trp-132-Arg), and HPV16-E7 gene (provided by Vimla Band, Tufts University) in the LXSN vector were harvested from stable producer cells lines grown in DME/F12 supplemented with 10% fetal bovine serum. Retroviruses carrying the SV40-T antigen gene (provided by Judy Campisi, LBNL), or the HPV16-E6 mutant JH26 (provided by Denise Galloway, U Washington) in the LXSN vector were produced by transient transfection of producer cells grown in DME/F12 supplemented with 10% fetal bovine serum (Finer et al., 1994). In all cases, retroviral supernatants were collected in serum free MCDB 170 media containing 0.1% bovine serum albumin. Viral supernatants were filter sterilized and stored at −80°C. Viral infection of 184, 184Aa, and 184A1 cultures was in MCDB 170 media containing 0.1% bovine serum albumin and 2.0 μg/ml polybrene (Sigma). Transduced cells were grown for 24 h in standard media then shifted to selective media containing 300 μg/ml G418 (Gibco) for 10 days.
Telomerase assays and TRF analysis
Cells for telomerase assays were fed 24 h prior to harvesting. Cell extracts were prepared by a modification of the detergent lysis method (Kim et al., 1994) and protein concentrations were determined using the Coomassie protein assay reagent (Pierce, Rockford, IL, USA). Telomerase activity was measured using the TRAP-EZE telomerase detection kit (Oncor, Inc.) using 2 μg of protein per assay. The 32P-labeled telomerase products were detected using the PhosphorImager system (Molecular Dynamics, Sunnyvale, CA, USA) and semi-quantitation was performed by comparing PCR signals from the HMEC extracts to signals from cell extracts of 293 cells (an adenovirus-transformed human kidney cell line).
DNA isolation and mean telomere restriction fragment (TRF) analysis were performed as previously described (Bodnar et al., 1996) with one modification. Genomic DNA was isolated from cells and 3 μg was digested and resolved on 0.5% agarose gels. The separated DNA was transferred to a membrane and hybridized overnight to a 32P-labeled telomere specific probe (CCCTAA)3, and washed to remove nonspecific hybrids. Signal was detected using a Molecular Dynamics PhosphoImager system and quantitated using the Imagequant software program. Mean TRF length was calculated as described (Allsopp et al., 1992).
Protein lysates for Western blot analysis of p53 were collected from cells fed 24 h prior to harvest. Cells cultured on 100-mm plates were washed once with PBS and lysed in SDS buffer containing 4% SDS, 20% glycerol and 0.126M TRIS, pH 6.8 plus protease inhibitors (20 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A; Boehringer Mannheim, Indianapolis, IN, USA). Lysates were boiled for 10 min and passed several times through a 26G needle to shear the DNA. Protein concentrations were determined using the BCA protein assay reagent (Pierce). Protein samples of 50 μg were resolved on a 10% polyacrylamide gel and transferred to Immobilon-P transfer membrane (Millipore Corporation, Bedford, MA, USA). The membrane was blocked in TBST (25 mM TRIS, pH 7.4, 137 mM NaCl, 2 mM KCl, 0.05% Tween-20) containing 5% nonfat powdered milk and incubated with monoclonal Ab-6 against p53 (Oncogene Research Products, Cambridge, MA, USA). Binding of primary antibody was detected using HRP conjugated anti-mouse IgG (Transduction Laboratories, Lexington, KY, USA) and visualized by chemiluminescence.
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We thank Vimla Band (Tufts University), Judy Campisi (LBNL), and Denise Galloway (U Washington) for providing viral constructs. This work was supported by NIH grant CA-24844 (MRS, PY), and the Office of Energy Research, Office of Health and Biological Research, US Department of Energy under Contract No. DE-AC03-76SF00098 (MRS, PY).
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Garbe, J., Wong, M., Wigington, D. et al. Viral oncogenes accelerate conversion to immortality of cultured conditionally immortal human mammary epithelial cells. Oncogene 18, 2169–2180 (1999). https://doi.org/10.1038/sj.onc.1202523
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