Article |
Address correspondence to Victoria L. Seewaldt, Box 2628, Duke University Medical Center, Durham, NC 27710. Tel.: (919) 668-2455. Fax: (919) 668-2458. E-mail: seewa001{at}mc.duke.edu
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Abstract |
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Key Words: extracellular matrix; mammary epithelial cells; apoptosis; p53; 3/ß1-integrin
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Introduction |
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The mechanism by which ECM-mediated signal transduction events might result in changes in gene expression is a subject of current investigation. It has been shown that rECM regulates both biomechanical and biochemical signaling events and conversely that alterations in cell morphology can alter the response of cells to rECM (Folkman and Moscona, 1978; Roskelley et al., 1994). It is hypothesized that because malignant cells have an altered response to rECM, ECM signaling pathways may utilize tumor suppressor checkpoints critical for cellular organization and polarity (Petersen et al., 1992; Howlett et al., 1994; Weaver et al., 1997).
Integrins are heterodimeric cell surface receptors that link ECM to structural and functional components within the cell (Hynes, 1992). There is increasing evidence that integrins are important regulators of normal mammary morphology, since mammary carcinoma cells frequently demonstrate atypical patterns of integrin expression including loss, downregulation, or improper localization (Koukoulis et al., 1991; Glukhova et al., 1995; Lichtner et al., 1998). In the normal mammary gland, the 3/ß1 integrin is expressed at the basal surface of luminal epithelial cells (Koukoulis et al., 1991; Glukhova et al., 1995). In contrast, invasive breast carcinomas demonstrate weak staining and redistribution of the
3/ß1 integrin. Recently, the
3/ß1 integrin has attracted considerable interest, since its function appears to be versatile. For example, the integrin
3/ß1 functions as a cell adhesion receptor for laminin-5 (epiligrin), a major ECM protein present in basement membrane (Xia et al., 1996). The
3/ß1-integrin is recruited to focal adhesion contacts in cultured cells and thereby plays an important role in linking ECM to components of the actin cytoskeleton (Carter et al., 1990b; Grenz et al., 1993; DiPersio et al., 1995). Integrin
3/ß1 is a critical mediator of intracellular adhesion (Kawano et al., 2001). Studies in keratinocytes suggest that
3/ß1 plays a critical role in cell spreading and migration and promotes gap junctional communication (Carter et al., 1990b; Xia et al., 1996; DiPersio et al., 1997). Recently,
3/ß1-integrin has been shown to be involved in the initiation apoptosis (Sato et al., 1999). Taken together, these studies illustrate the multifaceted role of
3/ß1-integrin in mediating interactions between ECM and epithelial cells and perhaps in initiating apoptosis.
Tissue homeostasis is maintained by a dynamic equilibrium between cellular proliferation and cell death (Evan and Littlewood, 1998). Apoptosis is considered to be the predominant mechanism of cell death and plays a central role in controlling cell number and eliminating cells sustaining DNA damage (Ashkenazi and Dixit, 1998). The role of the tumor suppressor p53 in ECM-induced growth arrest, polarity, and apoptosis is unknown. TP53 is a cell cycle "checkpoint" gene critical for cell cycle regulation, and it is functionally inactivated in human cancer at a high frequency (Hansen and Oren, 1997). Mutations of the TP53 gene are detected commonly in breast cancers and are associated with an increased risk of malignancy (Ashkenazi and Dixit, 1998; Levesque et al., 1998). Aberrant expression of p53 in mammary epithelial cells may be a biomarker predicting risk for subsequent breast carcinogenesis. Accumulation of p53 protein in mammary epithelial cells is detected frequently in women at high risk for the development of breast cancer (Fabian et al., 1996) and associated with an increased risk of progression to breast cancer in women with benign breast disease (Rohan et al., 1998).
Retrovirally mediated introduction of human papillomavirus type 16 (HPV-16) E6 protein into cells provides a model for the isolated loss of p53 function. The E6 protein of the cancer-associated HPV-16 binds to p53 and targets it for degradation through the ubiquitin pathway (Demers et al., 1996). We employed this approach and antisense (AS) oligondeoxyucleotides (ODNs) to acutely suppress p53 function in normal human mammary epithelial cells (HMECs) in order to model p53 loss in the context of ECM signaling.
Our results showed that control HMECs expressing p53 underwent rECM-mediated growth arrest and formed a polarized epithelium. In contrast, HMECs with HPV-16 E6 and ODN-suppressed p53 expression underwent rECM-induced growth arrest followed by apoptosis. p53- HMEC-E6 cells passaged in non-rECM culture rapidly acquired resistance to rECM-mediated growth arrest, polarity, and apoptosis after 8 10 passages in culture. Treatment of early passage p53- HMEC-E6 cells with either 3- or ß1-integrin function-altering antibodies (Abs) blocked rECM-mediated growth arrest and induction of apoptosis. Observations in our model system suggest that rECM may play an important role in the induction of apoptosis in early passage p53- HMECs via an
3/ß1 signaling pathway.
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Results |
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Northern blot analysis was performed on p53+ HMEC-P parental cells, p53- HMEC-E6 cells, and p53+ HMEC-LXSN controls (passage 11 and 18) to test for HPV-16 E6 mRNA expression. Expression of the LTR-initiated E6 mRNA transcript was observed in p53- HMEC-E6 cells at passage 11 and 18 but not in parental or vector controls (Fig. 1 a).
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Cytogenetic analysis of early and late passage p53+ and p53- HMECs
Cytogenetic analysis of p53+ HMEC-P parental cells, p53+ HMEC-LXSN controls, and early passage p53- HMEC-E6 cells was performed using both spectral karyotyping (SKY) and DAPI staining to (a) test whether parental and vector control cells exhibited a normal karyotype and (b) verify that early passage p53- HMEC-E6 cells did not exhibit specific karyotypic abnormalities.
38 early passage p53+ HMEC-P parental cells (passage 8) were karyotyped. 35 (92%) metaphase cells had a normal diploid karyotype. Three cells (8%) exhibited random chromosome loss.
Similarly, 22 early passage p53+ HMEC-LXSN control cells (passage 10) were karyotyped. 20 (91%) p53+ HMEC-LXSN metaphase cells had a normal diploid karyotype. Two cells (9%) exhibited random chromosome loss.
22 late passage p53+ HMEC-LXSN control cells (passage 17) were karyotyped. 18 (82%) p53+ HMEC-LXSN metaphase cells had a normal diploid karyotype. Three cells (13%) exhibited random chromosome loss. One cell (5%) was tetraploid (92 chromosomes). These results are consistent with karyotypes reported for late "phase a" (pregrowth plateau) HMECs (Romanov et al., 2001).
A total of 21 early passage p53- HMEC-E6 metaphase cells (passage 10) were karyotyped. Two cells were analyzed by SKY, and 19 cells were analyzed using inverted and contrast-enhanced DAPI staining. The majority of cells (12 cells, 57%) had a normal diploid chromosome content, three cells had random chromosome loss (14%), and the remaining cells were aneuploid. In three cells (14%), multiple losses of whole chromosomes occurred, resulting in the chromosomes number of 30, 32, and 36, respectively. The other three cells (14%) were either tetraploid (92 chromosomes) or hypotetraploid (90 and 80 chromosomes). Only two cells, both near diploid and both studied using DAPI staining, displayed structural chromosome changes: inv(20)(p11q13.1) in one cell, and del(X)(p21), dic(14;19)(q32;q13.4), and a marker chromosome in another.
In contrast to early passage cells, late passage p53- HMEC-E6 (passage 18) were markedly abnormal with numerical and structural chromosome aberrations. A total of 35 metaphase cells were analyzed: 27 using SKY and 8 using inverted and contrast-enhanced DAPI staining (Table I). These results have been published previously (Seewaldt et al., 2001). No cell had a normal diploid karyotype. The predominant types of structural changes were deletions, whole arm translocations, and dicentric chromosomes with breakpoints in the pericentromeric and/or telomeric regions. Although a majority of the 35 cells contained complex chromosomal rearrangements, each resistant cell analyzed was unique. This suggests that chromosome aberrations observed in the late passage p53- HMEC-E6 cell population resulted from a generalized event causing karyotypic instability that is inconsistent with the outgrowth of a mutant clone.
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As observed previously (Seewaldt et al., 1997b), day 7 p53+ HMEC-LXSN cells grown in rECM exhibited an acinus-like structure consistent with normal nonlactating mammary glandular epithelium (Fig. 4 a). There was no evidence of apoptosis. Day 14 p53+ HMEC-LXSN cells likewise did not exhibit evidence of apoptosis (Fig. 4 b).
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A second HMEC strain, AG11134, was tested to ensure that these observations were not HMEC strain specific. Similar to observations made in HMEC strain AG11132 above, (a) AG1134-LXSN controls underwent growth arrest and formed an acinus-like structure in contact with rECM at day 7 (unpublished data), and (b) early passage AG11134-E6 cells exhibited morphologic evidence of apoptosis at day 7 (unpublished data).
The terminal deoxynucleotidyl transferase (TdT) method was also used to detect the presence or absence of apoptotic strand breaks in day 7 rECM culture. The 3'-hydroxyl termini of apoptotic-induced strand breaks were labeled with biotin-dUTP by exogenous TdT and were detected in situ by HRP-conjugated streptavidin. Day 7 rECM growth-arrested early passage p53+ HMEC-LXSN controls (passage 10) did not demonstrate evidence of apoptosis (unpublished data). In contrast, early passage day 7 p53- HMEC-E6transduced cells (passage 10) grown in contact with rECM exhibited apoptotic strand breaks (unpublished data).
These observations indicate that whereas early passage p53+ HMEC-LXSN controls undergo rECM-induced growth arrest, early passage p53- HMEC-E6 cells undergo rECM-mediated growth arrest on day 6 followed by induction of apoptosis on day 7. Results from this in vitro model of rECM-induced apoptosis in HMECs suggest an association between HPV-16 E6induced suppression of p53 function and the induction of rECM-mediated apoptosis.
Acute suppression of p53 by an AS approach in HMECs promotes sensitivity to rECM-mediated apoptosis
Since HPV-16 E6 may have effects other than the suppression of p53, an AS approach was used to test whether the acute suppression of p53 function promotes sensitivity to rECM-mediated apoptosis. p53 protein expression was suppressed using a p53 AS ODN in HMECs. Western blot analysis demonstrated almost complete suppression of p53 protein expression in HMECs treated with the p53 AS ODN (p53- HMEC-AS) and no suppression of p53 protein expression in HMECs treated with a scrambled sequence of the p53 AS ODN (p53+ HMECscrambled AS [scrAS]) (Fig. 5).
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Discussion |
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Abnormal p53 expression in benign breast tissue is associated with the subsequent development of breast cancer and may represent a very early event in breast carcinogenesis (Fabian et al., 1996; Levesque et al., 1998; Rohan et al., 1998). Interactions between mammary epithelial cells and ECM play a critical role in maintaining normal tissue homeostasis and are likely to be disrupted during breast carcinogenesis. In this report, we describe an in vitro system for investigating interactions between HMECs with suppressed p53 protein expression and rECM as a potential model of early mammary carcinogenesis.
Retrovirally mediated expression of HPV-16 E6 and AS ODNs were used to acutely inhibit p53 protein expression in HMECs (Figs. 1 and 5). The combination of these approaches allowed us to utilize both viral and nonviral methods to suppress p53. We observed that early passage p53+ HMEC control cells underwent rECM-mediated growth arrest on day 67 and formed acinus-like structure (Figs. 3, 4, and 10). In contrast, early passage p53- HMEC-E6 cells and early passage p53- HMEC-AS cells proliferated until day 6 (Figs. 3, 4, and 6) and then underwent apoptosis on day 7 as evidenced by EM and by in situ TdT staining (Figs. 4 and 7; unpublished data). These observations suggest that the acute suppression of p53 function in HMECs by HPV-16 E6 and by AS ODNs may promote sensitivity to rECM-induced apoptosis.
ECM has been shown to provide essential signals for mammary epithelial cell survival and in their absence cells undergo apoptosis (Streuli et al., 1991; Strange et al., 1992; Pullan et al., 1996). The critical relationship between ECM signaling and p53 expression is highlighted by a recent report that ECM survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis (Ilic et al., 1998). However, there is significant evidence that growth arrest, differentiation, and survival signals may also promote apoptosis in genetically damaged cells (Seewaldt et al., 1995; Hong and Sporn, 1997; Mancini et al., 1997; Seewaldt et al., 1997b).
Our observation that the acute suppression of p53 in HMECs promotes sensitivity to apoptosis is consistent with several reports in human and mouse primary fibroblasts that the acute suppression of p53 results in apoptosis sensitivity (Hawkins et al., 1996; Wahl et al., 1996; Lanni et al., 1997). The mechanism by which the acute loss of p53 function might promote apoptosis is unknown. However, we observe that immediately after suppression of p53 by HPV-16 E6, HMECs exhibited a high percentage of tetraploid cells (14%) and an increased sensitivity to apoptosis. The tumor suppressor p53 is a critical component of the spindle checkpoint that ensures the maintenance of diploidy, and an increase in tetraploidy has been observed in cultured fibroblasts from p53-deficient mouse embryo fibroblasts (Cross et al., 1995). We speculate that it is possible that a loss in the spindle checkpoint might promote the induction of apoptosis when early passage p53- HMEC cells are exposed to either survival or growth arrest signals. Alternatively, p53 also plays an important role in differentiation, and therefore lack of p53 might result in cellular disregulation that promotes cell death. We observe that early passage p53- HMECs are sensitive to the induction of apoptosis by other agents that only promote growth arrest in p53+ HMECs at equimolar concentrations. For example, we observed that although tamoxifen promotes G1/S-phase arrest in early passage p53+ HMEC controls, tamoxifen promoted apoptosis in early passage p53- HMECs (Dietze et al., 2001; Seewaldt et al., 2001). Taken together, these observations provide evidence that the acute suppression of p53 might promote the induction of apoptosis when cells are exposed to growth arrest or survival signals.
Although early passage p53 HMEC-E6 cells underwent apoptosis when cultured in rECM, late passage cells were resistant to rECM-induced growth arrest, did not exhibit epithelial polarity, and failed to undergo apoptosis (Fig. 9). Loss of epithelial polarity is based on morphologic appearance by EM and lack of polarized expression of E-cadherin (Figs. 9 and 10). The development of apoptosis resistance correlated with the appearance of complex karyotypic abnormalities. Unlike early passage cells, late passage p53- HMEC-E6 cells continued to proliferate in rECM, formed multilayered aggregates of cells, and subsequently did not undergo apoptosis (Figs. 8 and 9). Based upon observations in our in vitro system, we propose that resistance to rECM-mediated growth arrest and polarity may promote resistance to apoptosis.
We investigated which component of rECM may be proapoptotic. Early passage p53- HMEC-E6 cells grown in collagen did not form acinar structures and did not undergo apoptosis (Fig. 11). Based on these observations, we speculate that the presence of collagen I/IV in a 1:1 ratio in our model system is not sufficient to induce apoptosis nor promote an acinar structure. When laminin was added to collagen I/IV gels, early passage p53- HMEC-E6 cells formed organized branched tubular structures that terminated in spherical cell clusters and underwent apoptosis (Fig. 11). Early passage p53- HMEC-E6 cells cultured in laminin suspension, in the absence of a plastic substratum for adhesion, formed similar three dimensional structures and likewise underwent apoptosis. This suggests that contact with laminin is critical for the induction of apoptosis in early passage p53- HMEC-E6 cells.
Integrin 3ß1 is a critical mediator of intracellular adhesion and an important receptor for laminin-5 (Xia et al., 1996; Kawano et al., 2001). Recently,
3/ß1-integrin has been shown to play a potential role in the initiation apoptosis in T cells (Sato et al., 1999). Resistance to rECM-mediated apoptosis in late passage p53- HMEC-E6 cells was associated with altered expression of
3-integrin (Figs. 12 and 13). Redistribution of
3-integrin has been seen previously by other investigators in association with loss of mammary epithelial cell polarity in rECM culture and is consistent with our findings (Weaver et al., 1997). Treatment of early passage p53- HMEC-E6 cells with
3- and ß1-integrin function-altering Abs blocked rECM-mediated growth arrest and inhibited the induction of apoptosis (Figs. 14 and 15). Taken together, these observations suggest an important role for
3/ß1 signaling in rECM-mediated growth regulation and apoptosis.
Previous investigators have tested the ability of 3- and ß1-integrin blocking Abs to mediate growth of breast cell lines in rECM and in collagen and fibrin gels (Howlett et al., 1995; Alford et al., 1998). HMT-3522, a nontumorigeneic breast cell line, demonstrated decreased proliferation in rECM culture when treated with either the inhibitory anti
3-integrin Ab P1B5 or antiß1-integrin Ab AIIB2 (Howlett et al., 1995). In contrast, we observed dysregulated proliferation when we treated HMECs with either the inhibitory anti
3-integrin Ab P1B5 or antiß1-integrin Ab JB1A. The induction of stimulatory or inhibitory functions by Abs directed to defined integrin subunits has been observed previously for both antiß1- and anti
3-integrin Abs (Lenter et al., 1993; Driessens et al., 1995; Lichtner et al., 1998) and is felt to be highly cell type specific. We hypothesize that utilization of nonimmortalized cells with low levels of
6ß4-integrin expression may account for differences between our results and those obtained by Howlett et al. (1995).
In conclusion, we have shown that whereas p53+ HMEC-LXSN cells undergo growth arrest and form polarized epithelium when grown in contact with rECM, p53- HMEC-E6 and p53- HMEC-AS cells that have acutely lost p53 function undergo apoptosis. Resistance to rECM-mediated growth arrest and polarity results in resistance to rECM-mediated apoptosis and is associated with altered expression of 3-integrin. Treatment of apoptosis-sensitive early passage p53- HMEC-E6 cells with either
3- or ß1-integrin function-altering Abs results in loss of rECM-mediated growth arrest and resistance to rECM-mediated apoptosis. We suggest that sensitivity and resistance to rECM-mediated apoptosis in p53- HMECs is dependent on the ability to form a polarized epithelium and may require
3/ß1-integrin signaling.
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Materials and methods |
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Retroviral transduction
The LXSN16E6 retroviral vector containing the HPV-16 E6 coding sequence (provided by D. Galloway, Fred Hutchinson Cancer Research Center, Seattle, WA) has been described previously (Halbert et al., 1991; Demers et al., 1994). HMECs (passage 9) were plated in four T-75 tissue culture flasks (Corning) in standard medium and grown to 50% confluency. Transducing virions from either the PA317-LXSN16E6 or the control PA317-LXSN (without insert) retroviral producer line were added at a multiplicity of infection of 1:1 in the presence of 4 µg/ml polybrene (Sigma-Aldrich) to log-phase cells grown in T-75 flasks. The two remaining T-75 flasks were not infected with virus. After 48 h, the two flasks containing transduced cells and one flask with untransduced cells were passaged 1:3 (passage 10) and selected with standard medium containing 300 µg/ml G418. Cells were grown in G418 containing standard medium for 1 wk until 100% of control untransduced cells were dead. The transduction efficiency was high during selection, cells were passaged 1:3 at the completion of selection (passage 11), and cells were maintained in the absence of selection before immediately proceeding to apoptosis experiments. The fourth flask of unselected untransduced parental control cells was passaged in parallel with the selected transduced experimental and vector control cells. Parental AG11132 cells were designated HMEC-P, transduced cells expressing the HPV-16E6 construct were designated p53- HMEC-E6, and vector control clones were designated p53+ HMEC-LXSN. All cells were maintained in standard medium after transfection in the absence of G418 selection to ensure that any chromosomal abnormalities or apoptosis resistance observed was not due to continued exposure to G418. All experiments were performed on mass cultures.
p53 oligonucleotides
The p53 AS oligonucleotide is an 18-mer targeting the region of the initiation codon (six base pairs immediately before the first and the first four coding codons): 5'-CGGCTCCTCCATGGCAGT-3'. This AS ODN has been used previously by several investigators to suppress p53 function (Bi et al., 1994; Capoulade et al., 2001). The p53 control ODN (5'-CGGCTCCTCATGGCAGT-3') was chosen to be a scrambled sequence of the AS ODN to ensure identical nucleotide content and minimize differences potentially attributable to nucleic acid content (Capoulade et al., 2001). In all ODNs, the first and the last three nucleotides were phosphorothiolate modified to increase stability in vitro.
Early passage p53+ HMEC-P parental cells were plated in T-75 plates in standard medium. After allowing 24 h for attachment, cell cultures were treated for 72 h with either 0.1 µM active or scrAS p53 ODNs. The culture medium was replaced by new standard medium containing fresh ODNs every 24 h. Western blot analysis was performed to confirm suppression of p53 expression as described below. The resulting film images were digitized and quantitated using Eastman Kodak, Co. 1D image analysis software.
rECM culture was as follows: cells were trypsinized, and 104 cells were resuspended in 100 µl rECM containing 0.1 µM of either active or p53 scrAS ODN on ice. rECM cultures were prepared as described below. rECM cultures were overlayed with standard medium containing 0.1 µM of either active or scrambled p53 ODNs. Overlay medium was changed every 24 h to ensure a fresh supply of ODNs. The diameter of the growing colonies was determined, and cell colonies were prepared for EM as described below. To measure p53 protein expression in cells grown in rECM culture, colonies were released from the matrix by 60-min incubation at 37°C with dispase (5,000 U/ml caseinolytic activity; Collaborative Research). Released cells were washed once using ice-cold PBS with 5 mM EDTA and twice with PBS alone. The resulting pellet was tested for p53 protein expression by Western blot analysis as described below.
Western blotting
Preparation of cellular lysates and immunoblotting were performed as previously described (Seewaldt et al., 1995, 1997b). p53 expression was detected using a 1:100 dilution of mouse antihuman p53 Ab-2 (Oncogene Research Products) and detected by ECL Western blotting detection reagents (Amersham Pharmacia Biotech) as described by the manufacturer.
Northern blot analysis
RNA was extracted with guanidium isothiocyanate and subjected to Northern blotting in formaldehyde denaturing gels as described previously (Seewaldt et al., 1995). 10 mg of RNA were loaded per lane. Molecular probes used in the Northern blot analysis were as follows: the human p53 probe was a 1.9-kb BamH1 fragment (Seewaldt et al., 1997b), and the 36B4 probe was a 700-bp PstI fragment that was used as a loading and transfer control probe (Seewaldt et al., 1995).
HMEC culture in reconstituted ECM
Mammary epithelial cells were grown in rECM by methods developed by Bissell and others (Folkman and Moscona, 1978; Howlett et al., 1994; Roskelley et al., 1994; Seewaldt et al., 1997b). 100 µl of rECM (MatrigelTM; Collaborative Research) or growth factordepleted rECM (growth factorreduced MatrigelTM; Collaborative Research) were added per well to a 48-well plate and allowed to gel at 37°C for 20 min. p53- HMEC-E6transduced cells and p53+ HMEC-LXSN vector controls were trypsinized, counted, and pelleted in a sterile microcentrifuge tube. Approximately 104 cells were resuspended in 100 µl rECM on ice, gently overlaid on the initial undercoating of ECM, and allowed to gel at 37°C for 20 min. Standard medium was then added, and wells were inspected to ensure an equal distribution of cells in each well. Cells were grown for 514 d in culture. Medium was changed daily.
For integrin-blocking experiments, 104 p53+ HMEC-LXSN vector control cells (passage 11) or p53- HMEC-E6 cells (passage 10) were pelleted and resuspended in 100 µl standard medium containing either Abs to
3- and ß1-integrins (Chemicon International) or control nonimmune mouse IgG for 15 min at room temperature (RT). Final concentration of
3-integrin blocking Ab (CDW496, clone P1B5) was 10 µg/ml, ß1-integrin blocking Ab (CD29, clone JB1A) was 20 µg/ml, and ß1-integrin stimulatory Ab (CD29, clone B3B11) was 10 µg/ml. 100 µl rECM was added to the cell suspension, gently mixed, and overlaid as described above. 1 ml standard medium containing the above respective concentration of blocking Ab was added to each well and changed every other day. Cells were grown for 59 d in rECM culture.
Collagen/laminin morphogenesis assays
Collagen and collagen/laminin gels were prepared by a modification of methods developed by Alford et al. (1998). Collagen type I (Sigma-Aldrich) and human placental collagen type IV (Sigma-Aldrich) were solubilized in 0.018 N acetic acid for a final concentration of 3 mg/ml each. Three parts collagen type I were mixed with one part collagen type IV. The collagen I/IV solution was neutralized by mixing 8 vol of collagen solution with 1 vol of sterile PBS and 1 vol of sterile 0.1 M NaOH for a final pH of 7.4. 100 µl of neutralized collagen I/IV solution were added per well to a 48-well plate and allowed to gel at 37°C for 20 min. Approximately 104 early passage p53- HMEC-E6 cells and p53+ HMEC-LXSN controls were resuspended in 100 µl neutralized collagen I/IV solution on ice, gently overlaid on the initial undercoating of collagen, and allowed to gel at 37°C for 20 min. Standard medium was then added, and wells were inspected to ensure an equal distribution of single cells suspended in each well. Cells were grown for 59 d in culture and then prepared for EM as described previously (Seewaldt et al., 1997b). For collagen/laminin gels, nine parts collagen I/IV were added to one part human placental laminin (Sigma-Aldrich), and gels were prepared as above. Laminin cultures were prepared as follows: 48-well plates were coated with 100 µl neutralized collagen I/IV solution and baked at 65°C for 24 h. This was repeated three times. 100 µl of human placental laminin were added per well and baked at 50°C until the laminin solution hardened. This was repeated three times. Approximately 104 early passage p53- HMEC-E6 cells were suspended in a 1:1 mixture of standard medium and human placental laminin. Cells were grown for 7 d and prepared for EM as previously described (Seewaldt et al., 1997b).
Transmission EM
p53- HMEC-E6 cells and p53+ HMEC-LXSN vector control cells were grown in contact with rECM as described above, and EM was preformed as described previously (Seewaldt et al., 1997b).
Cell growth determination in rECM culture
Cell growth was determined by the following criteria: the size of growing spherical cell colonies was measured with an eye piece equipped with a micrometer spindle. For both p53+ HMEC-LXSN vector controls and p53- HMEC-E6transduced cells, the 20 largest colonies were measured.
Detection of apoptosis by in situ TUNEL
p53- HMEC-E6 cells and p53+ HMEC-LXSN vector control cells were grown in contact with rECM as described above for 59 d. Cells were then fixed in PBS with 10% formalin and embedded in paraffin. Sections were deparaffinized and quenched in methanol containing 2.1% hydrogen peroxide. Antigen retrieval was achieved by placing slides in 10 mM citric acid at 95°C for 10 min. Nuclear proteins were stripped with 20 µg/ml proteinase K, and slides were washed in deionized water. Positive controls were immersed in DN buffer (30 mM Tris, pH 7.2, 140 mM sodium cacodylate, 4 mM magnesium chloride, 0.1 mM DTT) for 5 min at RT and then incubated with DNAseI (Roche) in DN buffer for 10 min at RT. Negative controls were treated with 5% FBS. All samples were immersed in TdT buffer (30 mM Tris, pH 7.2, 140 mM sodium cacodylate, 1.0 mM cobalt chloride) for 5 min at RT. Sections were covered with TdT/Bio-14-dATP solution (800 µl TdT buffer containing 120 U terminal transferase and 50 nM Bio-14-dATP [GIBCO BRL]), incubated for 1 h at RT, and then the reaction was terminated with PBS. Sections were blocked with 2% BSA for 10 min at RT and treated with ABC solution (Elite). Sections were allowed to complex on ice for 30 min, incubated for 30 min at 37°C, and were washed in PBS. Sections were stained with DAB (2% nickel chloride, 0.1% hydrogen peroxide) for 3 min at RT and counterstained.
E-cadherin immunostaining
Early and late passage p53- HMEC-E6 cells and p53+ HMEC-LXSN vector control cells were grown in rECM as described for 6 d and embedded in OCT (Miles). Cells were snap frozen, and 5-µm sections were obtained. Sections were fixed in for 30 min at RT with 3.7% formaldehyde in PBS and were blocked with 0.5% heat-denatured BSA (HD-BSA) in PBS for 1 h at RT. Cells were then incubated for 30 min with mouse antihuman E-cadherin Ab (BD Signal Transduction Laboratories), diluted in PBS with 0.5% HD-BSA for 30 min at RT, and then washed six times with PBS at RT. For Immunofluorescence, cells were incubated with FITC-conjugated goat antimouse Ab at a 1:200 Ab dilution (Santa Cruz Biotechnology, Inc.) in PBS with 0.5% HD-BSA for 30 min at RT and washed. Sections were mounted in 30% glycerol in PBS and visualized for immunofluorescence using a ZEISS LSM 410 fluorescence microscope.
Immunodetection of integrin expression
Early and late passage p53- HMEC-E6 cells and p53+ HMEC-LXSN vector control cells were grown on glass coverslips for 48 h in standard medium. Cells were fixed in for 20 min at RT with 2% formaldehyde in 0.1 M sodium cacodylate and 0.1 M sucrose at pH 7.2, permeabilized with 0.1% Triton X-100 for 10 min at RT, and blocked with 0.5% HD-BSA in PBS for 1 h at RT. Cells were then incubated with a primary Ab diluted in PBS with 0.5% HD-BSA for 1 h at RT and washed six times with PBS at RT. Abs against integrin subunits 2 (P1H5),
3 (P1F2, P1B5), and ß1 (P4C10) were a gift from William Carter (Fred Hutchinson Cancer Research Center) and have been described previously (Wayner and Carter, 1987; Wayner et al., 1988; Carter et al., 1990a,b). Abs against integrin
6 (GoH3) and ß4 (3E1) were obtained from Chemicon International. For Immunofluorescence, cells were incubated with either FITC-conjugated goat antimouse Ab at a 1:1,500 dilution or goat antirat Ab at a 1:4,000 dilution (Dako) in PBS with 0.5% HD-BSA for 1 h at RT and washed. Sections were mounted in a solution containing 25 mg/ml of 1,4-diazobicyclo-(2,2,2)octane in glycerol and visualized for immunofluorescence using a ZEISS LSM 410 fluorescence microscope.
For rECM culture, early and late passage p53- HMEC-E6 cells and p53+ HMEC-LXSN vector control cells were grown in contact with rECM for 6 d in standard medium. Cells were embedded in OCT, snap frozen, and sectioned as described above. Sections were fixed in for 20 min at RT with 2% formaldehyde in 0.1 M sodium cacodylate, 0.1 M sucrose, pH 7.2, and blocked with 0.5% HD-BSA in PBS for 1 h at RT. Sections were stained with Abs against integrin subunits 3 (P1F2) and ß1 (P4C10) and visualized as described above.
Cytogenetic analysis of early passage transduced and parental HMECs
Cultures of p53+ HMEC-P parental cells (passage 10), p53+ HMEC-LXSN vector controls (passage 10), p53- HMEC-E6 cells (passage 10), and p53- HMEC-E6 cells (passage 18) were checked for sufficient numbers of dividing cells and exposed to colcemid (GIBCO BRL) at a final concentration of 0.010.02 µg/ml for 23 h. Subsequently, the cells were released from flasks by trypsinization, exposed to hypotonic solution, and fixed as described previously (Mrózek et al., 1993). Chromosome preparations were made, and after appropriate aging slides were subjected to SKY, a method that enables simultaneous display of all human chromosomes in different colors (Schröck et al., 1996). Additional slides were also stained with DAPI (Vector Laboratories) alone. For SKY, the slides were hybridized with the SKY probe mixture containing combinatorially labeled painting probes for each of the autosomes and sex chromosomes (Applied Spectral Imaging) for 4245 h at 37°C. The hybridization and detection procedures were performed according to the manufacturer's protocol (Applied Spectral Imaging), and chromosomes were counterstained with DAPI in antifade solution. The multicolor hybridizations were visualized with the SpectraCube SD 200 system (Applied Spectral Imaging) mounted on the ZEISS Axioplan 2 epifluorescence microscope using a custom-designed optical filter (SKY-1; Chroma Technology). The DAPI images of all metaphase cells were acquired using a DAPI-specific optical filter. Spectral analysis and classification were performed using SkyView 1.2r visualization and analysis software (Applied Spectral Imaging). The assignment of breakpoints in structural abnormalities was made on comparison of images of SKY classified chromosomes with the images of the same chromosomes stained with DAPI that were inverted electronically and contrast enhanced by SkyView 1.2r software. Karyotypic abnormalities were classified according to the recommendations of the International System for Human Cytogenetic Nomenclature (Mitelman, 1995).
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Acknowledgments |
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This work is supported by National Institutes of Health/National Cancer Institute grants R01CA88799 (to V.L. Seewaldt), 2P30CA14236-26 (to V.L. Seewaldt), and 5P30CA16058 (to K. Mrózek), National Institutes of Health/National Institute of Diabetes and Digestive Kidney Diseases grant 2P30DK 35816-11 (to V.L. Seewaldt), American Cancer Society award CCE-99898 (to V.L. Seewaldt), a Charlotte Geyer award (to V.L. Seewaldt), a V-Foundation award (to V.L. Seewaldt), and a Susan G. Komen Breast Cancer award (to V.L. Seewaldt).
Submitted: 1 November 2000
Revised: 15 August 2001
Accepted: 14 September 2001
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