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Carcinogenesis |
Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, East Lansing, Michigan 48824
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30 cumulative population doubling level (cpdl)] showed a low level of telomerase activity as measured by the telomeric repeat amplification protocol assay. For all 11 of the Type I EL clones and the single Type II EL clone that became immortal, telomerase activities were invariably activated at middle passages (60 cpdl) or late passages (100 cpdl). For the four Type II EL clones randomly selected from the nine Type II clones that did not become immortal, the telomerase activities were found to be further diminished at mid-passage, before the end of the life span. Thus, normal HBECs do have a low level of telomerase activity, and Type I HBECs with stem cell characteristics are more susceptible to telomerase activation and immortalization, a basis on which they may be major target cells for breast carcinogenesis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normal somatic cells have a finite life span. Telomere shortening as a consequence of the end-replication problem has been proposed as a mitotic clock for cellular senescence (8) . Telomerase, a ribonucleoprotein complex with reverse transcriptase activity that uses a RNA template to add TTAGGG hexonucleotide repeat onto the end of chromosomes, is capable of maintaining the telomere length and replicative activity of cells (9) . Whereas normal human cells or adult tissues, in general, lack telomerase activity, the great majority of human tumors from various tissues and immortal cell lines show telomerase activity (10 , 11) . A current predominant hypothesis proposes that the reexpression of telomerase occurs in most tumors and is probably a critical event responsible for continuous tumor cell growth (12) , recognizing the existence of alternative mechanism for immortalization (13, 14, 15) . Additional studies have revealed that telomerase activity is expressed in human germ-lines (testes and ovaries) (12) , human embryonic stem cells (16) , human lymphocytes and hematopoietic progenitor cells (17, 18, 19, 20) , candidate stem cells from the fetal liver (21) , human epidermal cells expressing a basal cell marker (22) , and from the basal layer (23) and in human endothelial (24) and uroepithelial cells (25) . The latter two cell types showed proliferation-dependent expression of telomerase (24 , 25) . The role of telomerase in malignant transformation has been questioned by a recent study that observed the presence of telomerase activity in both normal and tumorigenic human cells including breast epithelial cells (25) . However, this study did not quantitatively measure telomerase activity during the course of neoplastic transformation.
We have developed a cell culture method to grow two types of normal HBECs4 from reduction mammoplasty (26) . One type of cell (Type II HBECs), similar to those commercially available or used by most other laboratories, shows basal epithelial cell markers. The other cell type (Type I HBECs) expresses luminal epithelial phenotypes and estrogen receptors (26 , 27) . Importantly, Type I HBECs also show stem cell characteristics (i.e., the ability to differentiate into other cell types by cyclic AMP-inducing agents and to form budding/ductal structures in Matrigel). Because telomerase activity in human stem cells has not been well studied, our first objective is to determine whether telomerase is expressed in our two types of cells. Furthermore, it offers an opportunity to examine whether the HBECs with stem cell characteristics (Type I HBECs) are more susceptible to telomerase activation and immortalization.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Growth factor-reduced Matrigel (Becton Dickinson Labware, Bedford, MA) was used to study mammary organoid structure formation with Type I and Type II HBECs. Approximately, 1–1.5 x 106 cells were plated on 35-mm culture dishes or two-chamber Lab-Tek culture slides with a layer of Matrigel. Matrigel remains solid in the 37°C humidifed incubator where cells were allowed to aggregate for 1 day. After cells attached to Matrigel, the medium was changed or a second layer of Matrigel was placed on top of the first layer. Culture medium was changed once every 2 days.
Development of SV40 Large T-Antigen-transformed Type I and Type II HBECs.
The normal HBECs were transfected with a plasmid carrying an origin-defective SV40 genome expressing a wild-type large T-antigen [PRNS-1; a gift from Johng S. Rhim, Uniformed Services University of the Health Sciences, Bethesda, (M15SV1-11 and M15SV21-30 were derived from Type I and Type II HME 15, respectively)] by Lipofectin (Life Technologies, Inc., Gaithersburg, MD). The actively proliferating colonies were selected by their resistance to G418 (0.4 mg/ml for M15SV1-11 from Type I HBECs and 0.15 mg/ml for M15SV 21–30 from Type II HBECs). The proliferation potential of transformed clones was determined by their total cpdls using the formula cpdl = ln(Nf/Ni)/ln 2, where Ni and Nf are initial and final cell numbers, respectively, and ln is the natural log. The initial cell number was 2 x 105 for each propagation.
During the course of determining the potential cpdl for each SV40-transformed cell line, the populations of cells at different cpdls were preserved in liquid nitrogen. For the telomerase assay, the cells at early (22–30 cpdl), middle (50–60 cpdl), and late (100–110 cpdl) passages were grown and harvested to prepare cell lysates.
PCR-based Telomerase Assay.
Cells grown to about 50–70% confluence were harvested by trypsinization. After cell counts, the cells were centrifuged to remove trypsin solution. The cell pellet for each culture was washed with 10 ml of PBS and then centrifuged to remove PBS. Cells were then suspended at 1 x 106 cells/ml in PBS and aliquoted to Eppendorf tubes. After cells were centrifuged and PBS was carefully removed, the cell pellets were stored at -85°C. For the telomerase assay, the cell pellet was thawed and resuspended in 200 µl of 1x CHAPS lysis buffer/106 cells and left on ice for 30 min. The samples were spun in a microcentrifuge at 12,000 x g for 20 min at 4°C. The cell lysate for each sample was aliquoted to several new tubes and stored at -85°C. The original lysate represents a concentration of 5,000 cells/µl. Further dilution of the cell lysate was adjusted based on the level of telomerase activity for the individual cell line. Telomerase activity was examined by the TRAP assay (10)
using the TRAPez Telomerase Detection Kit (Oncor, Gaithersburg, MD). This protocol includes primers of a 36-bp internal control (I.C.) for quantitating the amplification efficiency, thus providing a positive control for accurate quantitation of telomerase activity within a linear range close to 2.5 logs. Each analysis included a negative control (CHAPS lysis buffer instead of cell lysate), a heat-inactivated control (the sample was incubated at 85°C for 10 min before the assay), and a positive cell line control (breast carcinoma cell line MCF-7). For RNase treatment, 10 µl of extract were incubated with 1 µg of RNase for 20 min at 37°C. The products of the TRAP assay were resolved by electrophoresis in a nondenaturing 12% PAGE in a buffer containing 54 mM Tris-HCI (pH 8.0), 54 mM boric acid, and 1.2 mM EDTA. The gel was stained with Syber Green (Molecular Probes, Inc., Eugene, OR) and visualized by a 302-nm or 254-nm UV transilluminator. Images were captured and analyzed by AlphaImager (Alpha Innotech Corp., San Leandro, CA) or NucleoVision760 (NucleoTech Corp., San Mateo, CA). Quantitation of the products generated from TRAP assay was calculated using the following formula: TPG (total product generated units) = ((x - xo)/c)/((r - ro)/cR) x 100, where x and xo represent signals corresponding to the TRAP product ladder bands of non-heat-treated and heat-treated sample lanes, respectively, r and ro represent signals from 1x CHAPS lysis buffer control (i.e., primer-dimer/PCR contamination control) and TSR8 (DNA quantitation control), respectively. c and cR are the signal from the internal standard (TSK1) in non-heat-treated samples and the signal from TSR8 quantitation control, respectively.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, whereas Type II cells with basal epithelial phenotypes formed a spherical organoid (Fig. 1D)
. When the two types of cells were plated together on Matrigel, they formed a ductal and terminal end bud-like structure (Fig. 1F)
. Because mammary stem cells are known to be present in the end bud for ductal morphogenesis and elongation (31
, 32)
, the ability of Type I HBECs to form these structures strongly indicates that the Type I HBEC population contains mammary epithelial stem cells that are capable of giving rise to luminal and basal epithelial cells.
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3 x 106 Type I HBECs, whereas 10 clones were derived from 4 x 106 Type II HBECs. All these Type I and Type II clones were able to bypass senescence and acquire an EL (i.e., more than 30 cpdl; normal HBECs never grew more than 20 cpdl). Therefore, the ability of SV40 large T-antigen-transformed Type I and Type II HBEC clones to acquire EL appears to be comparable. However, the frequency at which EL clones became immortal differs significantly between Type I and Type II HBEC-derived clones. All of the 11 (100%) EL clones derived from Type I cells have become immortal (i.e., actively proliferating after more than 100 cpdl). In comparison, only 1 of 10 (10%) EL clones derived from Type II cells has become immortal (Table 1)
. Excluding the immortal clone, the average life span of EL clones from Type II HBECs was 43 ± 5 cpdl. It is clear that Type I HBECs with stem cell characteristics were more susceptible to immortalization after SV40 large T-antigen transfection.
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, telomerase activity was present in both Type I (Lanes 2–4) and Type II (Lanes 6–8) HBECs. However, the level of activity was weak compared with that of the breast carcinoma cell line MCF-7 (Lane 14). Using one-tenth of the cell number in the assay (250 versus 2500 cells), the telomerase activity in MCF-7 was significantly higher than that in Type I and Type II HBECs. In contrast, telomerase activity was undetectable in human breast stromal fibroblasts at early passages (Lanes 10–12). The telomerase activities in normal and in immortal or tumorigenic HBECs are dependent on enzyme activity from the catalytic subunit and RNA template, as shown by the elimination of the activity by heat inactivation or RNase treatment (Fig. 2, A and B)
. To exclude the possibility that the low level of telomerase activity from primary HBECs may be due to the existence of potential telomerase inhibitors, assays with mixed cell lysates from both HBECs and MCF-7 were carried out (Fig. 3, A and B)
. Because HBECs did not affect the telomerase activity of MCF-7 cells in the lysate mixtures, no telomerase inhibitor was detected in either Type I or Type II HBECs.
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. The same level of telomerase activity was found in both early-passage (before 50 cpdl) SV40-transformed Type I and Type II clones as in normal HBECs. There was a dramatic increase of the activity around 50–60 cpdl for clones that became immortal, and the activity remained high thereafter. In contrast, Type II EL clones without telomerase activation always stopped proliferating at crisis, as shown later (Fig. 6)
. The fact that the activated levels of telomerase activity were found in all immortal cell lines and the fact that the majority of Type II EL clones did not become immortal indicate that the low level of telomerase activity detected in normal HBECs is insufficient for immortalization.
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30 cpdl), middle passage (around the potential crisis period, 50–60 cpdl), and late passage (immortalization; >100 cpdl), respectively, were assayed. All 11 Type I HBEC-derived clones showed a significant increase in telomerase activity at middle or late passage (Table 1
; only the results from seven clones are shown in Figs. 4
and 5
). However, among Type II HBEC-derived clones, the only one that became immortal has shown activated telomerase activity (Fig. 6)
. Four of the nine Type II clones that did not become immortal were randomly selected for the telomerase activity assay. None of these clones showed telomerase activation at mid-passage before the advent of crisis. As summarized in Table 1
, all Type I and Type II clones with activated telomerase activity eventually became immortal. These results suggest that Type I HBEC clones have a high potential for telomerase activation, which results in a high rate of immortalization.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have previously shown that Type I and Type II HBECs differ substantially in their response to an oncogenic (SV40) stimulus; i.e., Type I cells were AIG+, whereas SV40-transformed Type II cells totally lack the ability to grow in soft agar (AIG-; Ref. 26 ). We were able to confirm that the SV40-transformed Type I HBEC clones obtained in this study are capable of AIG (eight of eight clones5 ). Therefore, this study provides additional and stronger evidence that Type I HBECs are more susceptible to tumorigenic initiation by acquiring two major and common tumor cell phenotypes, i.e., AIG+ and immortality.
Whereas it is reasonable to expect Type I cells with stem cell characteristics to have telomerase activity, the presence of telomerase activity in Type II HBECs seems difficult to reconcile with previous reports that normal breast tissues (10)
and normal HBECs (33)
do not have telomerase activity. It is possible that our Type II cells are newly derived from Type I cells. Therefore, they could be considered as progenitor cells for basal epithelial cells. Indeed, type II cells can be derived from Type I cells after treatment with cyclic AMP-inducing agent (26)
. The newly derived cells are similar to early-passage Type II cells from breast tissue in cell phenotype and proliferation potential. The low telomerase activity in normal HBECs and early-passage SV40-transformed HBECs is not due to the presence of telomerase inhibitors, as demonstrated in Fig. 3
, or to the lower proportion of cycling cells because all cell cultures were harvested at near log phase of cell growth. However, this low level of telomerase activity in both Type I and Type II cells seems insufficient for unlimited growth for SV40-transfected cells, as shown by the requirement of telomerase activation in immortalized clones and the incapability of most Type II EL clones to become immortal.
The ability of Type I HBECs to form budding/ductal structures strongly suggests the stem cell characteristics of a proportion of these cells. The similarity of phenotypes between breast carcinoma cells and Type I cells (i.e., deficiency in gap junctional intercellular communication, expression of estrogen receptors, and luminal epithelial cell markers) further indicates that breast cancers could be derived from Type I cells as a result of blocked differentiation, consistent with the oncogeny as blocked or partially blocked ontogeny theory of carcinogenesis (2) .
The differential susceptibility of different HBEC types to immortalization by human papilloma virus (34) and by the catalytic component of telomerase, hTERT (33) , has been reported previously. None of these cells are similar to the Type I HBECs.
The high susceptibility of SV40-transfected Type I cells to immortalization may provide a basis for the idea that stem cells are major targets for neoplastic transformation. In turn, the high potential of susceptibility to telomerase activation seems to be a mechanism that Type I cells become immortal at higher rate than Type II HBECs. However, the mechanism that telomerase is more easily activated in Type I cells remains unknown. Our results show that it is not due to a higher level of telomerase activity in Type I cells than Type II cells. In human tumor cells, the activity of telomerase has been shown to be cell cycle dependent, with the highest level of activity detected in S phase, and the lowest level found in cells arrested at G2-M phase (35) . Overexpression of cyclin D1 and/or cyclin E was a typical feature of breast cancers with high telomerase activity (36) . It is likely that telomerase expression may be partially under the same control that regulates G1 to S-phase transition. In this respect, our previous observation that the expression of cyclin D1 was higher in Type I HBECs than in Type II cells (27) may be relevant.
A previous report concludes that telomerase activity may not be a biomarker for malignant transformation because it is present in both normal and tumor cells (25) . This conclusion may be misleading, because there is no quantitative comparison, and the comparison was made between tumor cells and unrelated normal cells. Using a well-characterized quantitative assay, we found that although both normal and immortal or tumorigenic cells have telomerase activity, the activities in immortal or tumorigenic cells are dramatically higher than that seen in normal cells. Indeed, the transition from low to high telomerase activity may be an indicator of when immortal cells are present in the population during the course of immortalization.
This study also has implications concerning the mechanism of carcinogenic initiation of breast epithelial cells. Because the function of SV40 large T-antigen is to inactivate p53 and pRb and to induce the CCAAT box binding factor that transactivates cell cycle-regulating genes such as cdc2 (37) , alteration in cell cycle regulation seems to be the major event to acquire an EL for normal HBECs. The subsequent conversion of a cell with EL to an immortal cell clearly involves the activation of telomerase, as shown in this study. This is consistent with a recent report that both Rb/P16INK4a inactivation and telomerase activity are required to immortalize HBECs (33) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the United States Army Medical Research and Materiel Command under DAMD17-96-1-6099 and the National Cancer Institute (CA21104). 
2 Present address: Laboratory of Public Health, College of Veterinary Medicine, Seoul National University, Kwonsun-ku, Suwon, 441-744, Korea.
3 To whom requests for reprints should be addressed, at Food Safety and Toxicology Building, Michigan State University, East Lansing, MI 48824. Phone: (517) 353-6347; Fax: (517) 432-6340; E-mail: changcc{at}pilot.msu.edu
4 The abbreviations used are: HBEC, human breast epithelial cell; EL, extended lifespan; cpdl, cumulative population doubling level; TRAP, telomeric repeat amplification protocol; AIG, anchorage-independent growth; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
Received 6/ 1/99. Accepted 10/19/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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H), respectively. The Type I HBECs, Type II HBECs, and fibroblasts used in this assay were all derived from the mammary tissue of one patient. As a positive control, the breast carcinoma cell line MCF-7 showed a high level of telomerase activity (Lane 14, 250 cells). Low levels of telomerase activity were detected in both normal Type I (Lanes 2–4) and Type II (Lanes 6–8) HBECs; the activity in fibroblasts was undetectable (Lanes 10–12). B, RNA dependency of the weak telomerase activity detected from primary Type I and Type II HBECs (lysate from 5000 cells), and the high activity from SV40-immortalized Type I HBECs (M13SV1) and two breast cancer cell lines (MCF-7 and MDA-MB-231). The extract equivalent to 100 cells of lysate was used in each assay. I.C., the internal control for quantitating the amplification efficiency in the TRAP assay.
