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Cell Growth & Differentiation Vol. 13, 205-213, May 2002
© 2002 American Association for Cancer Research

Insulin-like Growth Factor Binding Protein-related Protein 1 Inhibits Proliferation of MCF-7 Breast Cancer Cells via a Senescence-like Mechanism1

Heather-Marie P. Wilson, Roger S. Birnbaum, Martin Poot, LeBris S. Quinn and Karen Swisshelm2

Department of Pathology, University of Washington School of Medicine, Seattle, Washington 98195 [H-M. P. W., M. P., K. S.]; Research Service and Geriatric Research, Education, and Clinical Center, Veterans Administration Puget Sound Health Care System, Tacoma, Washington 98493 [R. S. B.]; and Division of Gerontology and Geriatric Medicine, Department of Medicine, University of Washington, Seattle, Washington 98195 [R. S. B., L. S. Q.]


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Elevated insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) mRNA in senescent human mammary epithelial cells suggested that the IGFBP-3 gene product may inhibit cell proliferation. To test this hypothesis, we used a retroviral vector to express IGFBP-rP1 cDNA in the IGFBP-rP1-deficient MCF-7 breast cancer cell line. Compared with control vector-transduced cells, cumulative cell numbers for IGFBP-rP1-transduced polyclonal or clonal cell cultures were reduced by 39 and 74%, respectively, after 1 week. Medium conditioned by IGFBP-rP1-producing cultures reduced cumulative cell numbers in parental MCF-7 cultures by 20% compared with medium from cultures of a control vector-transduced cell line. Nuclear fragmentation analysis and cell proliferation assays completed in the presence of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone excluded apoptosis as the responsible mechanism. The percentage of cells containing senescence-associated ß-galactosidase activity was doubled compared with control cell cultures. Flow cytometry analysis indicated that twice as many noncycling cells were present in the IGFBP-rP1-transduced MCF-7 cell cultures compared with controls. These findings indicate that IGFBP-rP1 is an inhibitor of MCF-7 breast cancer cell proliferation and may act via a cellular senescence-like mechanism.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
IGFBPs3 are potential positive and negative regulators of IGF signaling (1) . In addition to modulating IGF signaling, IGF-independent actions of several IGFBPs have been documented (2, 3, 4) . IGFBP-rPs are members of the IGFBP superfamily (1) and include mac25, connective tissue growth factor, nephroblastoma overexpressed, and CYR61 (referred to as IGFBP-rPs 1–4, respectively). IGFBP-rP1, previously cloned independently and named mac25, tumor-derived adhesion factor, prostacyclin-stimulating factor, or IGFBP-7, shares ~30% identity at the amino acid level with the IGFBPs (5, 6, 7) but exhibits low affinity for the known IGFBP ligands, IGF-I and IGF-II (8) .

Replicative senescence is a process that limits the capacity for cell division in nontransformed cells (9 , 10) . Changes in expression or activity of components of the IGF system have been associated with senescence (11, 12, 13, 14) . Increasing IGFBP-3 levels, associated with inhibition of cell proliferation (11) , are observed in conditioned medium of human diploid fibroblasts with increasing donor age (13) , in vitro senescence (14) , and increasing confluency (12) . Proteolytic degradation of growth enhancing IGFBPs, which may decrease the ability of these proteins to present IGFs to the IGF receptors on the cell surface, is increased in senescent human diploid fibroblast cells (12) .

IGFBP-rP1 mRNA expression is 3–8-fold higher in senescent versus proliferating normal HMECs, prompting us to speculate that IGFBP-rP1 may possess antiproliferative capabilities (5) . A potential tumor suppressor role for IGFBP-rP1 is supported by data revealing down-regulation of IGFBP-rP1 protein in primary prostate cancer versus normal prostate stroma and glandular epithelium (15) . Moreover, IGFBP-rP1 inhibits growth of immortalized or malignant human prostate epithelial cells in soft agar and tumor formation in nude mice by inducing apoptosis (16) . IGFBP-rP1 mRNA is induced in differentiating granulosa cells, which eventually enter replicative senescence (17) . Truncated murine mac25, lacking the IGF binding domain, attenuated clonal growth of human Saos II cells, which suggests that the physiological role of IGFBP-rP1 may be independent of IGF signaling (18) . Collectively, these previous findings led us to hypothesize that overexpression of IGFBP-rP1 may suppress the proliferative potential of ER-positive, IGFBP-rP1-negative breast cancer cells. To test this hypothesis, we stably expressed IGFBP-rP1 in the MCF-7 human breast carcinoma cell line and assessed cell proliferation parameters.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Expression of IGFBP-rP1 in MCF-7 Breast Cancer Cells.
MCF-7 breast cancer cells were retrovirally transduced with the LXSN control vector and with the IGFBP-rP1 expression vector (LIGFBP-rP1SN) and selected as described in "Materials and Methods." Transduced cells were assessed for IGFBP-rP1 mRNA expression by Northern blot analysis (Fig. 1A)Citation . We detected the endogenous IGFBP-rP1 transcript (1.1 kb) in Hs578T breast cancer cells (positive control) but, as reported previously (5 , 19) , not in the parental MCF-7 cells (negative control). Polyclonal and clonal LXSN-transduced MCF-7 cells likewise did not express IGFBP-rP1 mRNA. Both polyclonal and clonal IGFBP-rP1-transduced MCF-7 cell populations showed a 4.2-kb transcript corresponding to the predicted retroviral transcript size.



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  		Fig. 1. Analysis of IGFBP-rP1 expression in retrovirally transduced MCF-7 breast cancer cells. A, Northern blot analysis of total mRNA reveals that the endogenous IGFBP-rP1 transcript is detected at 1.5 kb in Hs578T breast cancer cells used as a positive control. The retrovirally generated IGFBP-rP1 transcript is detected at 4.2 kb in polyclonal (P1 and P2) and clonal (cl.) IGFBP-rP1-transduced MCF-7 cells. The lower panel is the same Northern blot probed for 36B4, a loading control. B, Western blot analysis of conditioned media. A Mr 31,000 protein was detected in 10 µl (L, low) and 100 µl (H, high) of conditioned medium from Hs578T breast cancer cells (positive control). Immunoreactive proteins of Mr ~33,000 and Mr 34,000 were detected in medium from two polyclonal and one clonal IGFBP-rP1-transduced MCF-7 cell cultures but not in control vector-transduced cells (negative controls). Human recombinant IGFBP-rP1 (hr-rP1) protein was loaded at 5, 2.5, and 1.25 ng/lane as a reference for IGFBP-rP1 protein expressed in IGFBP-rP1-transduced MCF-7 cells.

 
Western blot analysis detected endogenous IGFBP-rP1 as a Mr 31,000 protein in ER-negative Hs578T cells used as a positive control for immunodetection (Fig. 1B)Citation . IGFBP-rP1 protein was not detected in concentrated conditioned medium from either parental or control vector-transduced (LXSN) MCF-7 cells (Fig. 1B)Citation . Immunoreactive IGFBP-rP1 protein bands with relative mobilities of Mr 33,000 and Mr 34,000 were detected in conditioned medium from IGFBP-rP1-transduced MCF-7 cells (Fig. 1B)Citation . The detection of higher molecular weight immunoreactive IGFBP-rP1 is consistent with the presence of multiple phosphorylation sites predicted by the primary sequence. Fig. 1BCitation is representative of IGFBP-rP1 protein expression observed in all cell passages tested and used in the following experiments.

IGFBP-rP1 Suppresses Proliferation of MCF-7 Cells.
Cell counts were performed to determine whether transduction with vector alone would change the rate of proliferation of MCF-7 cells. After 7 days of culture, cumulative cell numbers present in parental and the two polyclonal control vector-transduced cell lines (Fig. 2A)Citation were not significantly different (P > 0.68), indicating that introduction of vector alone had no effect on cell growth in MCF-7 cells.



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  		Fig. 2. Reduction of cumulative cell numbers in IGFBP-rP1-transduced MCF-7 breast cancer cells over 7 days. A, parental, control-P1, and control-P2 cells have similar cumulative cell numbers after 7 days in culture. Bars, SE. B–E, cumulative cell numbers were determined on days 1, 2, 3, 5, and 7. The mean cell number was calculated; bars, SE. B, control-P1 and rP1-P1 cultures. C, control-P2 and rP1-P2 cultures. D, control-cl.1 and rP1-cl.1 cultures. E, control-cl.2 and rP1-cl.2 cultures. The observed differences between control and IGFBP-rP1-transduced MCF-7 cells were significant in polyclonal and clonal cultures. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

 
We assessed the effect of IGFBP-rP1 on cell proliferation by determining cumulative cell numbers of control vector- and IGFBP-rP1-transduced MCF-7 polyclonal and clonal cell lines on days 1, 2, 3, 5, and 7 (Fig. 2, B–E)Citation . In day 7 polyclonal cultures, IGFBP-rP1-transduced cells showed a 39% reduction (P = 0.007) in cell number compared with LXSN-transduced cells (Fig. 2, C and D)Citation . A more pronounced difference in cell number was detected in the IGFBP-rP1 clonal cell lines, which exhibited a 74% reduction (P < 0.001) in cumulative cell number on day 7 (Fig. 2, D and E)Citation .

To determine whether secreted IGFBP-rP1 protein could reduce cumulative cell numbers in nontransduced MCF-7 cells, parental MCF-7 cells were exposed to conditioned medium from control-cl.1 and rP1-cl.1 MCF-7 cells. Cumulative cell numbers from cultures of nontransduced (parental) MCF-7 were counted after a 6-day incubation in the presence of conditioned medium from IGFBP-rP1-secreting MCF-7 cells. The cultures receiving conditioned medium that contained IGFBP-rP1 had 20% fewer cells on day 7 than cultures incubated in conditioned medium from LXSN-transduced MCF-7 cells (Fig. 3)Citation . Although we cannot rule out the possibility that other factors stimulated by secreted IGFBP-rP1 are responsible for the observed growth inhibition, these data suggest a growth-inhibitory role for secreted IGFBP-rP1.



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  		Fig. 3. Response of parental MCF-7 breast cancer cells to secreted IGFBP-rP1 protein in conditioned medium from transduced clonal cell lines. Cumulative cell numbers were ascertained on day 7. The cell number in cultures exposed to medium from IGFBP-rP1 cultures are presented as a percentage of cells present in cultures exposed to LXSN-conditioned medium; bars, SE. The observed difference was significant; **, P < 0.01.

 
The lower cell numbers in IGFBP-rP1-transduced cultures could have been the result of a lower rate of proliferation, higher levels of cell death, or an increased fraction of cells undergoing senescence. Additional experiments were performed to distinguish among these possibilities.

Nuclear Fragmentation Is Not Altered in the Presence of IGFBP-P1.
To determine whether enhanced cell death contributed to the lower cell numbers observed in IGFBP-rP1-transduced cultures, we quantitated apoptotic cells using a nuclear fragmentation assay via staining with the nuclear dye Hoechst 33258 (Fig. 4)Citation . Apoptotic cells constituted <1% of any of the populations on day 7 (Fig. 4A)Citation . Moreover, we detected no significant differences in the numbers of apoptotic cells in IGFBP-rP1-transduced MCF-7 cells versus control vector-transduced on days 1, 3, or 7, indicating that IGFBP-rP1 inhibited proliferation by a mechanism other than apoptosis (Fig. 4B)Citation .



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  		Fig. 4. Nuclear fragmentation assay of apoptotic cells. A, polyclonal (P) and clonal (cl.) data are the mean percentage of cells determined on day 7; bars, SE. B, mean percentage of cells determined in clonal cultures on days 1, 3, and 7; bars, SE. No significant differences in the percentages of apoptotic cells were detected.

 
Caspase Inhibition Does Not Block IGFBP-rP1-mediated Inhibition of Proliferation.
Additional evidence that IGFBP-rP1 inhibits cell proliferation independent of caspase-mediated apoptosis was derived from cumulative cell number assays using the pan-caspase inhibitor, zVAD-fmk. Caspases are key mediators of programmed cell death (20 , 21) and can be activated by TNF-{alpha} (22) . Control-cl.1 and rP1-cl.1 cell cultures were first tested for the ability of zVAD-fmk to inhibit growth in the presence of TNF-{alpha} (Fig. 5, A and B)Citation . Cells remaining attached to the tissue culture plate were scored as viable cells and were counted after trypsinization. Comparisons of attached cells between untreated, TNF-{alpha}-treated, and TNF-{alpha}/zVAD-fmk-treated cells revealed that TNF-{alpha}-treated cultures contained fewer viable cells than untreated cultures. Cells cultured in the presence of zVAD-fmk and treated with TNF-{alpha} had more viable cells than cultures treated with TNF-{alpha} alone, providing evidence that caspase blockade by zVAD-fmk inhibited apoptosis in MCF-7 cells.



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  		Fig. 5. Cumulative cell numbers are unaltered in the presence of caspase inhibitor zVAD-fmk. A and B, control-cl.1 and rP1-cl.1 were assessed for the effect of zVAD-fmk on MCF-7 cells. Results are shown as the percentage of the control for each time point. A, control-cl.1 cultures. B, rP1-cl.1 cultures. C and D, cumulative cell numbers from control-cl.1 and rP1-cl.1 cultures were counted on days 1, 3, and 7, and the SEs were determined. C, untreated cells. D, cells treated with 25 µM zVAD-fmk on days 1 and 3. Significant differences were detected between control-cl.1 and rP1-cl.1 in untreated and zVAD-fmk-treated cell growth assays; ***, P < 0.001.

 
Significant differences in cell numbers were observed between control-cl.1 and rP1-cl.1 cultures after 7 days in culture (Fig. 5, C and D)Citation in the presence and absence of zVAD-fmk. A difference of 54% was observed in untreated cells (P = 0.003), whereas cells grown in the presence of zVAD-fmk showed a difference of 60% (P = 0.0004). These results indicate that cell proliferation was altered by IGFBP-rP1 via a mechanism independent of caspase-mediated apoptosis.

SA-ß-Galactosidase Activity Is Increased by IGFBP-rP1.
IGFBP-rP1-transduced cells displayed a larger, flattened surface area with an increased cytoplasmic:nuclear ratio compared with LXSN-transduced (control) and parental (nontransduced) MCF-7 cells (Fig. 6)Citation . These morphological changes were similar to those reported for senescent cells (23 , 24) . Therefore, we tested the transduced cells for SA-ß-galactosidase activity at pH 6, a marker of replicative senescence (25) . The proportion of cells exhibiting SA-ß-galactosidase activity was elevated 2-fold in IGFBP-rP1-transduced cell cultures compared with LXSN-transduced MCF-7 breast cancer cells (Fig. 7)Citation . In polyclonal cultures, the percentage of cells exhibiting SA-ß-galactosidase staining increased from 34.0% in control vector-transduced cells to 68.7% in IGFBP-rP1-transduced cell populations (P < 0.0001). In clonal populations, cells exhibiting SA-ß-galactosidase staining likewise increased from 41.8% in control vector-transduced to 82.9% in IGFBP-rP1-transduced populations (P < 0.0001). The increased presence of SA-ß-galactosidase activity and changes in morphology to a senescent phenotype suggested that the cellular changes that occurred in response to IGFBP-rP1 expression were associated with replicative senescence.



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  		Fig. 6. Morphology is altered in IGFBP-rP1-transduced MCF-7 breast cancer cells. A, LXSN-transduced cells (control-cl.1) exhibit morphology characteristic of the nontransduced parental line. Cells are tightly packed and have a cobblestone appearance. B and C, IGFBP-rP1-transduced cells (rP1-cl.1) are enlarged and flattened. All photomicrograph panels of viable cultures were x200.

 


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  		Fig. 7. Higher numbers of IGFBP-rP1-transduced MCF-7 cells contain pH 6 SA-ß-galactosidase activity. Polyclonal and clonal columns, mean percentage of cells; bars, SE. The observed differences were significant; **, P < 0.01; ***, P < 0.005.

 
IGFBP-rP1-transduced Cultures Contain a Higher Proportion of Noncycling Cells.
Although IGFBP-rP1-transduced cell lines grew more slowly (Fig. 2)Citation , we were unable to detect any differences in the length of the cell cycle using propidium iodine staining (not shown). This method yields information concerning cell cycle parameters of cycling cells; however, it does not resolve noncycling cells present in the population. Therefore, a BrdUrd-Hoechst proliferation assay was performed to determine the percentage of noncycling cells in each cell line (26) . BrdUrd quenches Hoechst staining dye, and as a result, Hoechst fluorescence decreases with progressive cycles of BrdUrd incorporation. With the addition of ethidium bromide for nuclear staining, bivariate flow cytograms were generated (not shown) that allowed us to trace the dividing cells through multiple cell cycles and permitted the distinction of G1 cells from G0 cells that have never entered S-phase (27) . There were a higher proportion of IGFBP-rP1-transduced cells remaining in the initial G0-G1 phase than among control cells (Fig. 8)Citation . We observed a 1.6–3.3-fold increase in the population of noncycling cells in both the clonal and polyclonal IGFBP-rP1-transduced MCF-7 cell lines relative to control vector-transduced cell lines. Such a scenario would be expected to contribute to reduced cell numbers and provides additional evidence that IGFBP-rP1-transduced cells are undergoing senescence.



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  		Fig. 8. Higher proportion of noncycling cells in IGFBP-rP1-transduced MCF-7 cell cultures determined with a Hoechst-BrdUrd proliferation assay. Noncycling cell numbers are shown as a percentage of the control (LXSN); bars, SE. Significant differences were detected between control-LXSN and rP1-transduced cell cultures; *, P < 0.05.

 
We cannot rule out the possibility that a small change in cell cycle transit occurred in addition to the observed increased population of cells arrested at G0-G1 in IGFBP-rP1-transduced MCF-7 cells. A difference of 1 h in a single cell cycle is difficult to detect but may contribute to observed differences in cumulative cell numbers after 7 days in culture.


   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
The objective of this study was to determine whether IGFBP-rP1, a gene up-regulated during senescence of normal HMECs, would inhibit the proliferation of human breast cancer cells. MCF-7 breast cancer cells, a cell line lacking endogenous IGFBP-rP1 expression, was transduced with IGFBP-rP1 cDNA, and the resulting expression of IGFBP-rP1 correlated with a marked reduction in cumulative cell numbers over a 7-day period, a change to a senescent morphology, and an increase in the presence of SA-ß-galactosidase activity. Furthermore, BrdUrd/Hoechst flow cytometry revealed an augmentation in the number of noncycling cells in IGFBP-rP1-transduced cultures. Morphological evidence of apoptosis was not observed, and the addition of the caspase inhibitor, zVAD-fmk, failed to restore IGFBP-rP1-transduced cell numbers to control levels, whereas the same agent did reverse TNF-{alpha}-induced apoptosis in these cells. We report that a senescence-like mechanism is responsible for the ability of IGFBP-rP1 to suppress proliferation in MCF-7 breast cancer cells.

We speculate that the negative effect of IGFBP-rP1 on cell proliferation is likely to be more dramatic what we have presented in this report, because for technical reasons we could analyze only proliferating clonal and polyclonal populations that arose after transduction to study the antiproliferative effects of IGFBP-rP1. During derivation of populations of transduced cells and clones, cells undergoing senescence would not be expanded for analysis. Consistent with this, we observed that over three orders of magnitude more LXSN-transduced clonal colonies (control) arose than in sister cultures transduced with the IGFBP-rP1 expression vector. Cells in polyclonal cultures with higher proliferation rates and possibly lower IGFBP-rP1 expression, or less sensitivity to the protein inhibitory function of IGFBP-rP1, were more likely to be selected with each passage, consistent with our observation of more dramatic results in the clonal versus polyclonal IGFBP-rP1-transduced cell lines.

We could not discern a clear correlation between apparent IGFBP-rP1 protein expression levels and the degree of growth inhibition observed. Because the samples were normalized per ml of conditioned medium and not per cell, the signal could result from a greater number of cells. However, immunoblots using samples normalized per cell number also did not reveal a correlation between IGFBP-rP1 expression levels and looked identical to the immunoblot presented in Fig. 1BCitation . Therefore, another possibility is that the effect of IGFBP-rP1 is maximal at lower concentration or, as stated above, there is heterogeneity in the response of individual clones to the growth-inhibiting effects of IGFBP-rP1.

Production of IGFBP proteins in mammary tissue is regulated by hormones and relates to ER status in tumors (28 , 29) . An inverse correlation exists between IGFBP-rP1 mRNA expression and ER status in human breast cancer cells (5 , 19) . It has been hypothesized that IGFBP-rP1 may elicit growth inhibition independent of IGF binding (18 , 30) ; however, the mechanism by which this may occur has not been elucidated. Studies of IGFBP-3 and its ability to induce apoptosis independent of IGF in the MCF-7 human breast cancer cell line suggested that this could have been a mechanism for IGFBP-rP1 (31) . Indeed, IGFBP-rP1 inhibited growth of the malignant human prostate epithelial cell subline M12 via apoptosis and induced an altered morphology (16) . However, our results show IGFBP-rP1 inhibited proliferation of MCF-7 breast cancer cells independent of caspase-initiated programmed cell death. Thus, IGFBP-rP1 is likely to participate in a distinct pathway of replicative stasis from IGFBP-3, which induces apoptosis in both ER-negative (Hs578T) and ER-positive (MCF-7) breast cancer cells (31 , 32) . A recent study demonstrating up-regulation of IGFBP-rP1 mRNA and protein in senescent human prostate epithelial cells (33) supports our previous studies of IGFBP-rP1 mRNA up-regulation in senescent HMECs (5) . The studies we report are the first to indicate that IGFBP-rP1 may inhibit cell proliferation via a senescence-like mechanism.

We observed that IGFBP-rP1 was secreted as a higher molecular weight protein from retrovirally transduced MCF-7 cells, suggestive of posttranslational modification. We also observed a higher molecular weight IGFBP-rP1 when we transduced the ER-positive BT474 breast cancer cell line with IGFBP-rP1 (data not shown). Analysis of the amino acid sequence predicted eight potential phosphorylation sites and one N-glycosylation site. The endogenous IGFBP-rP1 protein expressed by Hs478T breast cancer cells has been shown to be composed of a Mr 27,000 core protein and Mr 4,000 of N-linked sugars (34) , suggesting that either MCF-7 cells process sugars differently from Hs578T cells or that the protein is already N-glycosylated, and the additional Mr 33,000 and Mr 34,000 species are the result of protein phosphorylation, as reported for IGFBP-1 (35 , 36) , IGFBP-3 (36 , 37) , and IGFBP-5 (36) .

We have demonstrated that with IGFBP-rP1, a gene up-regulated in normal senescent HMECs (5) , identification and expression of molecules up-regulated during senescence may provide a potential therapeutic model for the treatment of breast cancer. Up-regulation of this pathway or its components may enhance tumor growth inhibition when combined with current breast cancer treatment protocols.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cell Culture.
PE501 and PA317 virus packaging cell lines were cultured in {alpha}MEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FBS. Hs578T and MCF-7 cells (American Type Culture Collection, Manassas, VA) were cultured in {alpha}MEM supplemented 5% FBS, HEPES, sodium pyruvate, nonessential amino acids, insulin, epidermal growth factor, and hydrocortisone ({alpha}MEM/5% FBS) as described previously (38) . For analysis of secreted IGFBP-rP1, serum-free medium consisted of {alpha}MEM supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 1x nonessential amino acids, insulin, epidermal growth factor, hydrocortisone, and 0.05% BSA (Sigma, St. Louis, MO).

Generation of IGFBP-rP1 Retroviral Vector Constructs and Transduced MCF-7 Cells.
IGFBP-rP1 cDNA was ligated into the BamHI site of the pLXSN plasmid (39 , 40) . The PE501 (ecotropic) and PA317 (amphotropic) retroviral packaging cell lines were used sequentially to generate amphotropic virus as described by Miller et al. (40) . Both empty vector and IGFBP-rP1 expression vectors contained a selectable marker for transduced cells, the neomycin phosphotransferase gene (NEO), driven by an internal SV40 promoter. MCF-7 cells were transduced by LXSN or LIGFBP-rP1SN virus in the presence of 4 µg/ml Polybrene (Sigma). After 72 h incubation with virus, the transduced cells were selected in medium containing 1 mg/ml G418 (Calbiochem, La Jolla, CA) and maintained in 0.75 mg/ml G418.

The following cell lines were generated from MCF-7 breast cancer cells and used in the experiments described in this report: parental (nontransduced control); two lines each of LXSN and LIGFBP-rP1SN polyclonal cell cultures (control-P1, control-P2, rP1-P1, and rP1-P2); one clonal cell line each of LXSN (control-cl.1) and LIGFBP-rP1SN (rP1-cl.1), collected from the second passage after transduction.

Polyclonal populations were generated by culturing MCF-7 cells in the presence of retrovirus for 3 days, followed by selection with G418 in {alpha}MEM/5% FBS. The surviving cells were used as polyclonal cell lines. Clonal populations were generated by plating transduced cells on 100-mm tissue culture dishes in the presence of G418. When clones had grown to ~50 cells, they were trypsinized and transferred to 24-well plates.

All experiments used cultures that were between 4 and 10 passages after transduction. We did not use cells prior to four passages because the IGFBP-rP1 transduced cells grew slowly and it took a minimum of four passages to acquire enough cells necessary to complete our studies. We did not use cell cultures beyond 10 passages after transduction because continuous passaging selected for subpopulations within the IGFBP-rP1-transduced cell lines, which had higher proliferation rates (data not shown).

Northern Analyses.
Transduced polyclonal and clonal populations of MCF-7 cells were grown to 90% confluence on 100-mm dishes, and RNA was isolated using the Ultraspec-II RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX). Total RNA (10 µg) was separated on formaldehyde-agarose gels and transferred to Zetaprobe membranes (Bio-Rad, Hercules, CA) using standard techniques (38) . The human acid ribosomal phosphoprotein P0 with estradiol-independent mRNA expression (36B4) was used as a control for loading and transfer (41) . Full-length IGFBP-rP1 cDNA and 36B4 probes were labeled with [32P]{alpha}-dCTP using the Random Primed DNA Labeling kit (Roche Molecular Biochemical, Indianapolis, IN). The final wash was done with 1x SSC/0.1% SDS at 65°C for both IGFBP-rP1 and 36B4.

Immunoblotting and Protein Analyses.
For Western blot analysis of secreted IGFBP-rP1, parental and transduced MCF-7 cell populations were plated to near confluency (5.5 x 104 cells/cm2 on 35-mm tissue culture dishes) in {alpha}MEM/5% FBS. On day 5, cultures were washed once with PBS, and serum-containing medium was replaced with 2 ml of serum-free medium. Twenty-four h later (day 6), 700 µl of conditioned medium from each of the seven MCF-7 cultures and 10 µl (L) and 100 µl (H) of conditioned medium from Hs578T breast cancer cells (positive control) were collected and immediately concentrated (42) onto 0.2 µm nitrocellulose membrane (Bio-Rad). Concentrated proteins were eluted by boiling in 12 µl of 1x sample buffer (0.5 M Tris, 10% glycerol, 8 M urea, and 2% SDS). Baculovirus-generated human recombinant IGFBP-rP1 protein (hr-rP1; R. Rosenfeld, Oregon Health Science University, Portland, OR) at 5, 2.5, and 1.25 ng/lane served as a control and for reference of protein expressed in IGFBP-rP1-transduced MCF-7 cells. The proteins were separated on a 15% SDS-PAGE precast gel with a 4% stacking gel (Bio-Rad). Proteins were transferred onto Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad) at 100 V for 1 h in transfer buffer (39 mM glycine, 48 mM Tris, 0.04% SDS, and 20% methanol). The membrane was incubated with 10% hydrogen peroxide for 10 min, blocked 1 h in 1% BSA/0.5% nonfat dry milk (Bio-Rad) in 0.05% Tween 20/PBS (TPBS), and incubated overnight at 4°C with rabbit anti-human recombinant IGFBP-rP1 (43) at 1:2500 in blocking solution. Blots were washed in TPBS and incubated for 3 h at room temperature with goat antirabbit IgG conjugated with horseradish peroxidase (Pierce, Rockford, IL) at 1:40,000 in blocking solution. The IGFBP-rP1 protein was detected with SuperSignal Chemiluminescent Substrate (Pierce) and CL-Xposure film (Pierce).

Cumulative Cell Number Assay.
Parental and retrovirally transduced MCF-7 cell lines (described above) were plated on 60-mm tissue culture plates at 2 x 103 cells/cm2. In each experiment, duplicate plates were trypsinized, and cells were counted on days 1, 2, 3, 5, and 7 using a hemocytometer. Two independent experiments per cell line were completed using polyclonal cultures, control-P1 and control-P2, rP1-P1 and rP1-P2. Three independent experiments were performed using clonal cultures, control-cl.1 and control-cl.2, rP1-cl.1 and rP1-cl.2.

Cell Number Assay of Nontransduced MCF-7 Grown in Conditioned Medium.
To generate conditioned medium with and without secreted IGFBP-rP1, control-cl.1- and rP1-cl.1-transduced MCF-7 cell lines were cultured on 100-mm tissue culture plates until confluent. Medium was replaced with 10 ml of fresh {alpha}MEM/5% FBS for 24 h and centrifuged before use to spin out cellular debris.

To assay the effects of secreted IGFBP-rP1 protein, parental (nontransduced) MCF-7 cells were plated on 60-mm tissue culture plates at 2 x 103 cells/cm2. Twenty-four h after plating and each subsequent day (days 1–6), medium was replaced with 4 ml of a 1:1 mixture of conditioned medium from the confluent cultures and fresh medium. On day 7, triplicate plates of recipient cells receiving either control-cl.1 or rP1-cl.1 were harvested after trypsinization and counted using a hemocytometer. Three independent experiments were performed for this assay.

Nuclear Fragmentation Assay for Apoptosis.
Cells were plated at 2 x 103 cells/cm2 on 60-mm tissue culture plates and cultured for 7 days, the end point of the cell number assay. On day 7, cells were harvested by trypsinization. To avoid loss of nonadherent cells, the conditioned medium, PBS wash, cells and trypsin, and medium used to rinse the plates were combined and pelleted by centrifugation. Cells were resuspended in methanol:acetic acid (3:1) and fixed at -20°C for a minimum of 24 h. Cells were applied to ethanol-cleaned glass slides and stained with 20 µM Hoechst 33258 (Sigma) in PBS for 30 min. Cells were analyzed for fragmented nuclei using a Zeiss epifluorescence microscope with a neo-fluor 40–0.75 objective lens. Approximately 500 cells/plate were analyzed using duplicate plates for each cell line in each experiment. The percentage of cells with fragmented nuclei was determined from two independent experiments.

Control-cl.1 and rP1-cl.1 cells were plated at 2 x 103 cells/cm2 on 6-well tissue culture plates. Trypsinized cells, conditioned medium, PBS wash, and medium used to rinse the wells were combined and pelleted by centrifugation on days 1 and 3. Cells were fixed and analyzed as described above. Triplicate samples were analyzed at each time point.

Cumulative Cell Number Assay in the Presence of Apoptosis-inducer TNF-{alpha} and Caspase-inhibitor zVAD-fmk.
To determine whether the pan-caspase inhibitor zVAD-fmk (Bachem, Torrance, CA) inhibits apoptosis in MCF-7 cells, cells were exposed to the apoptosis-inducer TNF-{alpha} (R&D Systems, Minneapolis, MN) and examined in a cell proliferation assay (below). Control-cl.1- and rP1-cl.1-transduced MCF-7 cells were each plated into 6-well tissue culture plates at 2 x 103 cells/cm2. On day 1, cells were treated at a final concentration of 25 µM zVAD-fmk. An additional 25 µM zVAD-fmk was added on day 3 to cultures designated for counts on day 7. TNF-{alpha} (30 ng/ml) was added with cycloheximide (10 µg/ml) to cultures with and without zVAD-fmk and incubated for 5 h before counting. The numbers of viable cells were determined and compared between untreated, TNF-{alpha}-treated, and TNF-{alpha}/zVAD-fmk-treated cultures.

Control-cl.1- and rP1-cl.1-transduced MCF-7 cells were each plated on 6-well tissue culture plates at 2 x 103 cells/cm2. On day 1, the caspase inhibitor zVAD-fmk was added to selected wells to give a final concentration of 25 µM. On day 3, an additional 25 µM fresh zVAD-fmk was added directly to cell cultures designated for cell counts on day 7. Cultures were treated with trypsin, and cells were counted using a hemocytometer for each time point on days 1, 3, and 7. Cultures were analyzed in triplicate/cell line at each time point.

SA-ß-Galactosidase Activity.
A histochemical assay of ß-galactosidase activity at pH 6 (25) was used to assess a potential senescent phenotype in cell cultures. Cell lines were plated at 2 x 103 cells/cm2 on 35-mm tissue culture dishes and cultured for 3 days. On day 3, cells were washed twice with PBS, fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS, rinsed twice with PBS, and stained with a solution containing 0.1% 5-bromo-4-chloro-3-indolyl-ß-D-galactoside, 40 mM citric acid, sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2 that was overlaid onto the cells and incubated at 37°C overnight. The cells were rinsed twice with sterile H2O the following day. Approximately 500 cells were counted from each plate, and the proportion of cells exhibiting a medium- to dark-blue stain, indicative of SA-ß-galactosidase activity, was scored. Cells were analyzed at day 3 while still sparse enough to prevent false-positive staining associated with increasing cell density (44) . Duplicate plates/cell line were used in each experiment. Two independent experiments were completed.

Flow Cytometric Assay of Noncycling Cells.
A modified BrdUrd-Hoechst quenching technique was used to track dividing cells. This method allows quantification of the fraction of noncycling cells present in cell cultures that have progressed through three cell cycles. Cells were plated at 1.1 x 104 cells/cm2 on a 6-well plate. BrdUrd (Sigma) at 100 µM in {alpha}MEM/5% FBS medium was added the following day. Cells were incubated in the presence of BrdUrd for 92 h, harvested by trypsinization, pelleted, resuspended in 0.5 ml {alpha}MEM/5% FBS containing 10% DMSO, and stored at -20°C until analyzed. Samples were thawed, pelleted, and resuspended in staining buffer containing 1.2 µg/ml Hoechst 33258 and analyzed by flow cytometry using procedures described previously (26 , 27) . Cells were analyzed for cell number versus Hoechst fluorescence using a Coulter Epics Elite flow cytometer (Beckman Coulter Corporation, Fullerton, CA) equipped with two argon lasers. The first laser was tuned to 488 nm (15 mW output), and the second was tuned to 360 nm UV (10 mW output). The mean percentage of the noncycling cells was determined. With the BrdUrd-Hoechst quenching assay, cells were quantitated at the end of replicative senescence when they were no longer cycling and dividing. The proportion of cells detected by this assay would be lower than the proportion of cells detected by the SA-ß-galactosidase activity assay, which stains cells approaching replicative senescence. Triplicate plates/cell line were used for each experiment. Three independent experiments were completed for this assay.

Statistical Analysis.
For all experimental data, mean and SE were calculated, and significance of difference was determined using a two-sided Student’s t test.


   Acknowledgments
 
We thank the following for sharing reagents: Dr. D. A. Miller for the LXSN retroviral vector and PE501 and PA317 cell lines; Dr. R. Rosenfeld and E. Wilson for human recombinant IGFBP-rP1 protein and anti-IGFBP-rP1 antibody. We acknowledge the critical readings and discussions by S. Hosier, M. Chaisson, C. Beckham, and Drs. L. Chen, J. Paik, K. Sommer, C. Franklin, N. E. Olson, H. J. Deeg, L. A. Loeb, and R. Palmiter.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by predoctoral fellowship DAMD 17-96-1-6247 (to K. S. and H-M. P. W.) from the United States Army Materiel and Command, pilot grant P20 CA/PS 66186 (to K. S.) from the Seattle Breast Cancer Foundation, and Nathan Shock Center of Excellence in the Basic Biology of Aging Grant 2 P30 AG 13280. Back

2 To whom requests for reprints should be addressed, at Department of Pathology, Box 357470, University of Washington, Seattle, WA 98195-7470. Phone: (206) 616-3182; Fax: (206) 543-3644; E-mail: kswiss{at}u.washington.edu Back

3 The abbreviations used are: IGFBP, insulin-like growth factor binding protein; IGFBP-rP1, IGFBP-related protein 1; HMEC, human mammary epithelial cell; ER, estrogen receptor; TNF, tumor necrosis factor; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone; SA-ß-galactosidase, senescence-associated ß-galactosidase; BrdUrd, 5bromodeoxyuridine; {alpha}MEM, {alpha}-minimal essential medium; FBS, fetal bovine serum. Back

Received for publication 7/ 9/01. Revision received 2/13/02. Accepted for publication 4/ 2/02.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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