Primary cells suppress oncogene-dependent apoptosis - Nature

brief communications

Primary cells suppress oncogene-dependent apoptosis Dominik M. Duelli* and Yuri A. Lazebnik*† *Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA †e-mail: [email protected]

Oncogenes that promote cell-cycle progression also sensitize cells to agents that induce apoptosis. Sensitization is thought to be caused by the induction of proapoptotic factors. Alternatively, sensitization may require the inactivation of inhibitors that ordinarily provide protection against cell death. Here we show that the adenoviral oncogene E1A sensitizes cells to an anti-cancer drug by at least two pathways. One establishes a link between the drug and pro-apoptotic factors, but is not sufficient for sensitization without the second pathway, which suppresses inhibitors of apoptosis.

poptosis1 is cell suicide that requires a specialized machinery, a central part of which is a family of proteases called caspases2. Understanding how oncogenes sensitize cells, and why cancer cells survive despite this sensitization, may provide a strategy with which to kill tumour cells selectively. The prevailing view is that oncogenes predispose cells to apoptosis by inducing expression of pro-apoptotic factors3–5. However, an alternative possibility is that normal cells are intrinsically protected from apoptosis by inhibitors that are inactivated in response to oncogene expression. We tested this hypothesis using a cellfusion-complementation assay. Although primary fibroblasts are resistant to anti-cancer drugs, fibroblasts that express E1A die by apoptosis6–8. This experimental system has been extensively used to study how oncogenes promote apoptosis. E1A is thought to sensitize cells by deregulating a set of transcriptional regulators4. To test whether E1A induces pro-apoptotic factors or represses inhibitors of apoptosis, we fused human fibroblasts expressing E1A (IMR90E1A) with parental primary cells (IMR90) and immediately treated the cells with etoposide, an anti-cancer drug that induces DNA damage. We found that the heterokaryons (the fusions between IMR90 and IMR90E1A cells) were resistant to apoptosis (Fig. 1a, b), whereas fusions between IMR90E1A cells remained sensitive (Fig. 1b). Heterokaryons not only failed to exhibit the morphological changes characteristic of apoptosis, but also excluded trypan blue, indicating that they remained viable (data not shown). Hence, fusion to primary cells suppressed E1A-dependent apoptosis. The effect of primary cells was not limited to cells expressing E1A. Myc also sensitizes fibroblasts to anti-cancer drugs9. Heterokaryons between IMR90 and cells expressing c-myc (IMR90 myc) also were resistant to apoptosis (Fig. 1b). Furthermore, fusion of IMR90 cells to the cancer cell lines, HeLa and Jurkat, which are sensitive to etoposide, also prevented apoptosis (Fig. 1b and data not shown). E1A-dependent apoptosis requires activation of caspase-9, which is achieved by its binding to cofactors APAF-1 and cytochrome c (cyt c)10,11. Cyt c must be released from mitochondria to participate in caspase-9 activation12,13. Consistent with their failure to undergo apoptosis, IMR90 cells do not release cyt c in response to etoposide10. Therefore we tested whether cyt c is released in heterokaryons using IMR90E1A cells expressing a dominant

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negative mutant of caspase-9 (IMR90E1Ac9; ref. 10). In these cells the morphological and biochemical changes that are triggered by caspase-9 in response to cyt c are prevented10, facilitating reliable monitoring of cyt c release. Indeed, the release of cyt c was blocked in heterokaryons (Fig. 1c; see also Supplementary Information, Fig. S1a). The degree of inhibition was directly proportional to the number of primary cells that were fused to each IMR90E1Ac9 cell (see Supplementary Information, Fig. S1b). Hence, fusion to primary cells blocked E1A-dependent apoptosis by preventing cyt c release. The failure of cyt c release in heterokaryons may be due to several factors — E1A could be inactivated, E1A-induced proapoptotic factors required for cyt c release could be diluted by fusion to primary cells, or primary cells may contain an inhibitor of apoptosis that blocks cyt c release. The first possibility is unlikely because E1A was present in the nuclei of heterokaryons, including 70% of nuclei derived from primary cells (see Supplementary Information, Fig. S2a). We consider the dilution hypothesis unlikely because culturing of heterokaryons for up to 17 h before treamtent of cells with etoposide did not sensitize heterokaryons to the drug (see Supplementary Information, Fig. S2b). We could not monitor heterokaryons for more than 17–18 h because they become hybrid cells (synkaryons) once they go through mitosis. However, because during a cell cycle the cell content doubles, and 17 h is 70% of the cycle, we reasoned that this time would be sufficient to replenish the apoptotic factors if they were diluted. By monitoring synkaryons we observed that they become sensitive to etoposide by 96 h after fusion (data not shown). Hence, a reasonable explanation is that primary cells express an inhibitor of cyt c release. The expression of this inhibitor must be repressed by E1A to induce drug sensitivity. The inhibitor provided by primary cells protects heterokaryons until it is degraded or inactivated, even if E1A-induced repression is immediate. We hereafter refer to this inhibitory activity provided by primary cells as IODA (inhibitor of oncogene-dependent apoptosis). To determine whether IODA is indeed constitutively expressed in primary cells, we tested whether enucleated primary cells can block cyt c release. We enucleated cells using a standard protocol that removes the nucleus by centrifugation, leaving the cytosol intact (Fig. 1d; see Methods). Enucleated IMR90 cells blocked cyt c release in heterokaryons nearly as efficiently as did intact cells (Fig. 1d, e), which is consistent with the idea that IODA, or its precursor, is constitutively expressed in primary cells. To determine whether IODA prevents E1A from activating pro-apoptotic factors that trigger cyt c release, we focused on the Bax protein. It has been reported that E1A-induced expression of Bax contributes to sensitization14, and that Bax can directly induce release of cyt c by translocating to mitochondria15–18. We found that, in our system, E1A did not affect the abundance of Bax, but it enabled Bax to translocate to mitochondria in response to etoposide treatment. In untreated cells, Bax was distributed diffusely (Fig. 2a, b). However, after etoposide treatment, Bax concentrated 859

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Figure 1 IODA is constitutively present in normal cells and prevents oncogene-dependent apoptosis. a, Fusion to primary cells prevents oncogenedependent apoptosis. Primary cells (IMR90) labelled with CellTracker Green CMFDA, and E1A-expressing cells (IMR90E1A) labelled with CellTracker Orange CMTMR, were fused and treated with etoposide (upper panels). DNA was stained with Hoechst 33342 (DNA, lower panels). Left panels, an IMR90 cell (contains green dye, arrowhead,) and an IMR90E1A cell (contains orange dye, star) that remained unfused; right panels, a heterokaryon (contains both orange and green dyes) derived from an IMR90 and an IMR90E1A cell. Cells with condensed chromatin (star, upper-left panel) were scored as apoptotic. Similar experiments were carried out with IMR90 cells expressing c-myc (IMR90myc) and HeLa cells. b, Quantification of the results in a. c, Cytochrome c (cyt c) is not released in heterokaryons. Cells were fused and treated

with etoposide as in a, except that IMR90E1Ac9 cells were labelled with CellTracker Blue CMAC (upper panels). Cytochrome c localization in primary cells (left panels), IMR90E1Ac9 (middle panels) and the heterokaryons (lower panels) treated with etoposide was detected by immunofluorescence (lower panels). d, e, The nucleus of a primary cell is not required to block cytochrome c release in heterokaryons. Primary cells were enucleated, stained with CellTracker Green CMFDA (upper-left panel; enucleated cell indicated as IMR90∆N), fused to IMR90E1Ac9 cells stained with CellTracker Blue CMAC and treated with etoposide. Cyt c release was detected by immunofluorescence (right panel, shows a fusion between an enucleated IMR90 and an IMR90E1Ac9 cell), and cells with released cyt c were scored. DNA was strained as in a. e, Quantification of the results in d.

in the mitochondria of E1A-expressing cells, but remained diffuse in primary cells (Fig. 2a, b). Hence, expression of E1A allows the formation of a link between DNA damage — the primary effect of etoposide — and Bax translocation. Bax also translocated in heterokaryons (Fig. 2a, b). Furthermore, Bax translocated indiscriminately to mitochondria from primary and E1A-expressing cells (Fig. 2c), indicating that IODA does not sever the link between DNA damage and Bax translocation, but prevents the subsequent steps that are required for cyt c release. These observations also indicate that cell fusion does not prevent E1A from activating at least some pro-apoptotic factors. This conflicts with the idea that cyt c in heterokaryons remained in mitochondria because pro-apoptotic factors were inactivated by dilution. The components of IODA remain to be identified. IODA may be a Bcl-2 like protein, because overexpression of Bcl-2 can also prevent cyt c release without interfering with Bax translocation19. However, we found that, in our system, E1A does not repress expression of Bcl-2 or of the Bcl-2-like proteins Bcl-XL, Mcl-1 and Bcl-w (data not shown). Bcl-2 and Bcl-X are phosphorylated in some cell lines, which is indicated by a change in mobility during electrophoresis20,21. We detected no such change in any of the Bcl2-like proteins tested in response to expression of E1A or etoposide treatment (data not shown). Hence, we conclude either that IODA

is not one of these proteins, or that these proteins are modified in a way that we did not detect. Alternatively, IODA may sequester factors that are required in addition to Bax for cyt c release22,23. Although IODA seems to act upstream of or at cyt c release, the subsequent steps of apoptosis in normal cells may also be inhibited. The inhibitors may be inhibitor-of-apoptosis proteins (IAPs), the activities of which could be negated in transformed cells by pro-apoptotic components such as Smac/DIABLO24,25, or through other pathways. Irrespective of the identity of IODA, our results indicate that E1A facilitates drug-induced release of cyt c by at least two pathways. One pathway enables the link to be formed between DNA damage and translocation of Bax to mitochondria, and another pathway must inactivate inhibitors that prevent cyt c release in normal cells. It is reasonable to speculate that transformed cells that retain IODA would be resistant to pro-apoptotic stimuli. Genetic studies have indicated that the ability of E1A and Myc to deregulate the cell cycle and promote apoptosis are tightly linked. E1A is known to promote cell-cycle progression by inactivating cell-cycle repressors26. Here we have shown that E1A and Myc promote apoptosis by inactivating repressors of apoptosis. Thus, it is tempting to speculate that repressors of cell-cycle progression and of apoptosis are controlled by the same regulators that the oncogenes inactivate. These regulators serve as a tripwire that ensures that derepression of the cell cycle leads to derepression of

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Figure 2 E1A allows etoposide-induced translocation of Bax to mitochondria. a, IODA does not interfere with E1A-dependent translocation of Bax. Primary cells (IMR90) labelled with CellTracker Green CMFDA, and E1A-expressing cells (IMR90E1Ac9) labelled with CellTracker Blue CMAC (cell tracking), were fused, and either were left untreated or were treated with etoposide. The localization of Bax in primary cells (upper panels), IMR90E1Ac9 cells (middle panels) and heterokaryons (lower panels) was detected by immunofluorescence (anti-Bax). b, Cells were scored for Bax translocation. c, Bax translocates indiscriminately to the mitochondria of primary and E1A-expressing cells. IMR90 cells labelled with MitoTracker

Green FM were fused to IMR90E1Ac9 cells labelled with CellTracker Blue CMAC (left panel), or conversely, IMR90 cells labelled with CellTracker Blue CMAC were fused to IMR90E1Ac9 cells labelled with MitoTracker Green FM (right panel). Cells were treated with etoposide and Bax localization was detected as described in Methods, using a secondary antibody conjugated to Alexa594 (red fluorescence). Images are overlays of CellTracker Blue CMAC, MitoTracker Green FM and Bax staining; areas of overlap between MitoTracker and Bax staining are yellow. In control experiments, Bax translocated to >50% of mitochrondria of IMR90E1Ac9 cells, but not to mitochondria of IMR90 cells (data not shown).

the apoptotic machinery. The apoptotic machinery can remain repressed in proliferating transformed cells if IODA is replaced by other anti-apoptotic molecules, such as products of adenoviral E1B or overexpressed endogenous Bcl-2.

2. Thornberry, N. A. & Lazebnik, Y. Caspases: enemies within. Science 281, 1312–1316 (1998). 3. Guo, M. & Hay, B. Cell proliferation and apoptosis [see comments]. Curr. Opin. Cell Biol. 11, 745–752 (1999). 4. Lowe, S. W. & Lin, A. W. Apoptosis in cancer. Carcinogenesis 21, 485–495 (2000). 5. Attardi, L. D., Lowe, S. W., Brugarolas, J. & Jacks, T. Transcriptional activation by p53, but not induction of the p21 gene, is essential for oncogene-mediated apoptosis. EMBO J. 15, 3693–3701 (1996). 6. Rao, L. et al. The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and bcl-2 proteins. Proc. Natl Acad. Sci. USA 89, 7742–7746 (1992). 7. Lowe, S. W. & Ruley, H. E. Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis. Genes Dev. 7, 535–545 (1993). 8. Samuelson, A. V. & Lowe, S. W. Selective induction of p53 and chemosensitivity in RB-deficient cells by E1A mutants unable to bind the RB-related proteins. Proc. Natl Acad. Sci. USA 94, 12094–12099 (1997). 9. Evan, G. et al. Induction of apoptosis in fibroblasts by c-myc protein. Cell 69, 119–128 (1992). 10. Fearnhead, H. O. et al. Oncogene-dependent apoptosis is mediated by caspase-9. Proc. Natl Acad. Sci. USA 95, 13664–13669 (1998). 11. Rodriguez, J. & Lazebnik, Y. Caspase-9 and APAF-1 form an active holoenzyme. Genes Dev. 13, 3179–3184 (1999). 12. Yang, J. et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129–1132 (1997). 13. Kluck, R. M., BossyWetzel, E., Green, D. R. & Newmeyer, D. D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132–1136 (1997). 14. McCurrach, M. E., Connor, T. M. F., Knudson, C. M., Korsmeyer, S. J. & Lowe, S. W. bax-deficiency promotes drug resistance and oncogenic transformation by attenuating p53-dependent apoptosis. Proc. Natl Acad. Sci. USA 94, 2345–2349 (1997). 15. Wolter, K. G. et al. Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139, 1281–1292 (1997). 16. Jurgensmeier, J. M. et al. Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl Acad. Sci. USA 95, 4997–5002 (1998). 17. Narita, M. et al. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc. Natl Acad. Sci. USA 95, 14681–14686 (1998). 18. Gross, A., McDonnell, J. M. & Korsmeyer, S. J. BCL-2 family members and the mitochondria in apoptosis [in process citation]. Genes Dev. 13, 1899–1911 (1999). 19. Putcha, G. V., Deshmukh, M. & Johnson, E. M. Jr BAX translocation is a critical event in neuronal apoptosis: regulation by neuroprotectants, BCL-2, and caspases. J. Neurosci. 19, 7476–7485 (1999). 20. Haldar, S., Jena, N. & Croce, C. M. Inactivation of Bcl-2 by phosphorylation. Proc. Natl Acad. Sci. USA 92, 4507–4511 (1995). 21. Guan, R. J. et al. 30 KDa phosphorylated form of Bcl-2 protein in human colon. Oncogene 12, 2605–2609 (1996).

Methods Cell tracking and immunofluorescence. Primary IMR90 embryonic human lung fibroblasts, and HeLa and Jurkat cells were from ATCC, Hanassas, Virginia. E1A, c-myc and caspase 9 dominant negative mutants were introduced into IMR90 cells using the retroviral MARX-IV system as described10. Cells were labelled with CellTracker vital dyes (Molecular Probes) according to the manufacturer’s instructions. Cyt c and Bax were detected with monoclonal antibodies 6H2.B4 and 6A7 (Pharmingen), respectively, and visualized with antimouse Alexa546 or Alexa594 (Molecular Probes). Images were obtained using a CCD (charge-coupled device) camera, and assembled using Oncore Software (Oncore, Gaithersburg, Maryland) and Adobe Photoshop (Adobe, San Jose, California). Data are means from 3 experiments in each of which at least 100 heterokaryons were scored.

Cell fusion and induction of apoptosis. Fusion partners were labelled with different tracking dyes, washed, detached, mixed, plated onto glass coverslips (Corning Costar, Corning, New York) and placed into 6-well plates (Falcon, Becton Dickinson, Franklin Lakes, New Jersey) at 6 × 105 cells/well. Cells were allowed to attach and were then washed and fused by adding 50% PEG 1000 (J.T. Baker Chemicals, Phillipsburg, New Jersey), pH 7.0, in serum-free DMEM for 1 min at room temperature. Cells were washed several times in DMEM and immediately transferred into DMEM lacking phenol red (Sigma) and containing 10% FBS and etoposide. For experiments with IMR90E1A cells or Jurkat cells, cells were treated with 50 µM etoposide for 24 h. IMR90myc and HeLa cells were treated with 100 µM etoposide for 48 h. Both attached and detached cells were stained with the DNA dye Hoechst 33342 (Sigma) to assess nuclear morphology. Cells with condensed chromatin were scored as apoptotic. Viability was measured by exclusion of trypan blue.

Enucleation. IMR90 cells were enucleated as described27. Briefly, cells were plated on TPX-slides (Corning Costar, Cambridge, Massachusetts) and enucleated by centrifugation at 11,000g at 37 °C for 30 min in the presence of 60 µg ml–1 cytochalasin B in 10% FBS in PBS. Cells were allowed to recover for 3 h before fusion. Sixty to eighty per cent of cells were enucleated using this procedure. Enucleated cells excluded trypan blue for at least 40 h after enucleation. RECEIVED 14 APRIL 2000; REVISED 5 JULY 2000; ACCEPTED 28 JULY 2000; PUBLISHED 19 OCTOBER 2000.

1. Evan, G. & Littlewood, T. A matter of life and cell death. Science 281, 1317–1322 (1998).


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brief communications 22. Nomura, M. et al. Apoptotic cytosol facilitates Bax translocation to mitochondria that involves cytosolic factor regulated by Bcl-2. Cancer Res. 59, 5542–5548 (1999). 23. Kluck, R. M. et al. The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol. J. Cell Biol. 147, 809–822 (1999). 24. Du, C., Fang, M., Li, Y., Li, L. & Wang, X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33–42 (2000). 25. Verhagen, A. M. et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102, 43–53 (2000). 26. Dyson, N. & Harlow, E. Adenovirus E1A targets key regulators of cell proliferation. Cancer Surv. 12, 161–195 (1992).

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27. Bols, N. C. & Kuhn, K. The enucleation of cells on plastic Leighton coverslips. Stain Technol. 56, 103–108 (1981).

ACKNOWLEDGEMENTS We thank J. Rodriguez for IMR90myc cells, and G. Hannon, M. Hastings, D. Helfman, A. Krainer and members of the Lazebnik laboratory for critical reading of the manuscript. This work was supported by National Institutes of Health grant no. CA13106-25, a Seraph Foundation grant to Y.L., and a Roche Research Foundation fellowship to D.M.D. Correspondence and requests for materials should be addressed to Y.L.


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Figure S1 Cytochrome c release is blocked in heterokaryons. a, IMR90 cells labelled with CellTracker Green CMFDA and E1A-expressing cells (IMR90E1Ac9) labelled with CellTracker Blue CMAC were fused, and treated with etoposide for 24 h. Cytochrome c (cyt c) was detected by immunofluorescence and cells were scored for cytochrome c release. b, The frequency of cells that retain cyt c is proportional to the number of IMR90 cells fused to an IMR90E1A cell. IMR90 and IMR90E1Ac9 cells were treated and scored as in a.

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Figure 2 E1A is present in nuclei derived from both IMR90 and IMR90E1Ac9 cells. a, Primary cells (IMR90) labelled with CellTracker Green CMFDA and E1Aexpressing cells (IMR90E1Ac9) labelled with CellTracker Blue CMAC were fused, and treated with etoposide for 24 h. E1A protein (arrows) was detected using monoclonal anitbody M58 (ref. 1), and anti-mouse immunoglobulin G Alexa 594 (red). Star indicates the nucleus derived from an IMR90 cell. b, Preincubation of heterokaryons for 17 h before etoposide treatment does not affect their sensitivity to etoposide. IMR90 and IMR90E1A cells were dyed with CellTrackers and fused as described in a. They were treated with etoposide either immediately or 17 h after fusion.

1. Harlow, E., Franza, B. R. Jr & Schley, C. Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products. J. Virol. 55, 533–546 (1985).


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