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[Cancer Research 60, 3957-3964, July 15, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

E1A Overcomes the Apoptosis Block in BCR-ABL+ Leukemia Cells and Renders Cells Susceptible to Induction of Apoptosis by Chemotherapeutic Agents1

Thorsten Stiewe2, Keiarasch Parssanedjad2, Helmut Esche, Bertram Opalka and Brigitte M. Pützer3

Institute of Molecular Biology (Cancer Research) [T. S., K. P., H. E., B. M. P.] and Department of Internal Medicine, West German Cancer Center [B. O.], University of Essen Medical School, Hufelandstrasse 55, D-45122 Essen, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A crucial function of the BCR-ABL chimeric gene in chronic myeloid leukemia is the prolongation of cell survival by inhibition of apoptosis. BCR-ABL expression confers cross-resistance to multiple genotoxic anticancer drugs by inhibition of the apoptotic response to DNA damage in association with cell cycle arrest at the G2-M restriction point. Previous reports indicated that BCR-ABL exerts its antiapoptotic effect against various apoptotic stimuli upstream to the cleavage and activity of caspase-3. Here we show that the adenovirus E1A protein induces substantial apoptosis in BCR-ABL expressing K562 and LAMA-84 leukemia cells. This apoptotic activity of E1A is accompanied by processing of caspase-3 and cleavage of poly(ADP-ribose) polymerase and can be significantly blocked by z-VAD-fmk Z-Val-Ala-Asp(OCH3)-CH2F and the caspase-3-specific inhibitor Z-DEVD-FMK Z-Asp(OCH3)-Glu-Val-Asp(OCH3)-CH2F. Moreover, E1A renders K562 cells, which are particularly resistant to cell death irrespective of the inducing agent, susceptible to induction of apoptosis by the chemotherapeutic agents etoposide and daunorubicin. Counteracting the DNA damage-induced inactivation of cdc2 kinase, E1A reverses the drug-induced G2-M arrest. These results indicate that solitary delivery of E1A significantly antagonizes BCR-ABL-induced antiapoptotic functions and circumvents the inherent resistance to DNA damage-induced apoptosis, supporting the use of E1A in combination with chemotherapeutic agents as a promising therapeutic strategy for successful treatment of Philadelphia chromosome-positive leukemia in vivo.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CML4 is a hematopoietic disorder that is characterized by the presence of excessive numbers of mature myeloid cells in the peripheral blood and bone marrow, considered to be a consequence of an expanded population of progenitor cells (1) . The cytogenetical hallmark of CML is the presence of the Ph chromosome, which results from a mostly reciprocal translocation of c-ABL from chromosome 9 to the BCR gene on chromosome 22, generating a chimeric BCR-ABL protein that has elevated levels of ABL tyrosine kinase activity (2) .

When the BCR-ABL gene is transduced into hematopoietic cell lines, they become growth factor independent and have increased proliferative capacity. Such studies have demonstrated that the mitogenic ability of BCR-ABL is mediated in part through activation of a Ras (3) and phosphatidylinositol 3'-kinase-dependent signaling pathways (4) , suggesting that the massive expansion of malignant cells in CML may be due to BCR-ABL-induced proliferation. However, there is much support that the clonal expansion evident in this malignant disorder is not a result of deregulated cellular proliferation (5, 6) but rather occurs via prolongation of cell survival by prevention of apoptotic cell death (7, 8) . Supporting the role of the BCR-ABL protein-tyrosine kinase as a negative regulator of apoptosis, deregulated kinase activity confers cross-resistance to multiple anticancer agents by inhibition of the apoptotic response to DNA damage (9, 10) . BCR-ABL can protect growth factor-dependent hematopoietic cell lines from apoptosis induced by factor withdrawal (11, 12) and Fas-mediated apoptosis (13) . In turn, recent observations indicated restoration of susceptibility to apoptosis and enhancement of survival through inhibition of BCR-ABL expression by antisense oligonucleotides (9, 14) , by the tyrosine kinase inhibitor CGP57148 (15) , and Fas-mediated down-modulation of BCR-ABL (16) , confirming the anti-apoptotic function of the chimeric protein.

The importance of apoptosis in maintaining hematopoietic homeostasis is evident from the consequences of its deregulation. Apoptosis occurs under physiological conditions, e.g., during T-cell maturation in the thymus, and is characterized by cell shrinkage, chromatin condensation, and DNA fragmentation (17) . A variety of apoptotic stimuli cause the preapoptotic mitochondrial release of cytochrome c into the cytosol, which mediates activation of caspase-3 from a precursor and cleavage of PARP (18) , resulting in execution of the whole program of apoptosis. However, recent findings indicated that BCR-ABL expression blocks apoptosis upstream of procaspase-3 activation (19) by preventing the cytosolic accumulation of cytochrome c and other preapoptotic mitochondrial perturbations in, for example, etoposide-treated K562 cells (20, 21) .

One of the hallmarks of apoptosis is that it is genetically regulated. A number of products of tumor suppressor genes, proto-oncogenes, and some viral genes are known to regulate this process (reviewed in Refs. 22, 23 ), making it open to genetic manipulation and thereby raising the possibility of therapeutic intervention. The adenovirus 5 E1A oncogene products interact with and perturb the function of key regulators of cell proliferation, such as the RB protein (24) . The result of these interactions is induction of cellular DNA synthesis but also loss of cell viability and induction of apoptosis, which impedes both the transformation of primary rodent cells and productive adenovirus infection of human cells (25, 26) . The ability of E1A to direct apoptosis is thought to be related to its ability to cause the release of the transcription factor E2F-1 from RB binding (27) . Enforced overexpression of E2F-1 has been shown to trigger apoptosis in quiescent fibroblasts and to suppress tumor growth in glioma cells (28, 29) .

E1A produces two major mRNAs, encoding proteins of 289 and 243 residues (289R and 243R), respectively, which differ only by the 46-amino acid conserved region 3 in the 13S protein known to activate expression of other early viral genes. The protein products of the E1A gene can induce apoptosis by both p53-dependent and -independent mechanisms. In the presence of wild-type p53, expression of the E1A 12S transcript leads to an increase in the levels of p53, resulting in the deregulation of Bax and Bcl-2 (30) , which correlates with the induction of apoptosis (31–34) . Within the viral context, however, the E1A 13S transcript can also induce apoptosis independently of p53, which appears to be dependent on conserved region 3 and the early region E4 (35, 36) . Our group recently showed that both E1A proteins are capable of inducing substantial apoptotic cell death in the absence of other adenoviral genes in cells lacking p53 (37) . Previous reports demonstrated that E1A expression enhances the sensitivity to apoptosis by ionizing radiation and various cytotoxic agents in murine embryonic fibroblasts, murine keratinocytes, and human ovarian cancer cells (31, 38, 39) .

In this study, we investigated the apoptotic activity of the E1A protein in the BCR-ABL-expressing leukemia cell lines K562 and LAMA-84, respectively. Our data indicate that E1A alone is capable of inducing substantial apoptosis by antagonizing the BCR-ABL-induced block within the apoptosis cascade of CML cells. E1A overcomes the inherent resistance to DNA damage induced apoptosis by bypassing the drug-induced BCR-ABL-mediated G2-M arrest and renders Ph-positive leukemia cells susceptible to induction of apoptosis by chemotherapeutic agents. Our results provide support for use of E1A in combination with chemotherapeutics as a promising approach for elimination of Ph-positive leukemias in patients.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Drug Treatment.
K562 and LAMA-84 Ph+ cell lines, derived from patients during the blast crisis phase of CML, were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). K562 and LAMA-84 cells both contain b3-a2 P210bcr-abl (40) . Cells were cultured in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 µg/ml penicillin, and 100 units/ml streptomycin in a 37°C incubator containing 5% CO2. Culture media and supplements were obtained from Life Technologies (Karlsruhe, Germany). Etoposide and daunorubicin were obtained from Sigma-Aldrich (Deisenhofen, Germany) and dissolved in acidified ethanol or sodium chloride solution, respectively. For treatment with chemotherapeutic agents, 7 x 106 cells were treated by continuous exposure to 0.2 µg/ml daunorubicin or etoposide at the indicated concentrations for 2 days. Drug treatment of transfected cultures was started 24 h after transfection.

Plasmids and DNA Transfection.
The adenovirus 5 E1A cDNAs were cloned into pRc/RSV (Invitrogen, Groningen, the Netherlands) for expression from the RSV promoter. A 498-bp fragment from the human cdc2 promoter was amplified by PCR using the following oligonucleotides as primers: 5'-TTAGGTCACTGAAATGTGCTCCTTG-3' (forward, bp -466 to -441) and 5'-CAATTTCCAAGAGCCAGCTTTGAAG-3' (reverse, bp +8 to +33). The fragment was subsequently cloned blunt-ended into the SmaI site of pGL3basic (Promega, Mannheim, Germany). Plasmid DNA was prepared by the alkaline lysis method and purified by CsCl-ethidium bromide density gradient centrifugation. Transfections were performed as described by using the electroporation method (41) .

Western Blotting.
Cell lysates were prepared after transfection, and protein levels were analyzed by Western blot essentially as described (27) . The antibodies used were directed against E1A (M73; Calbiochem, Bad Soden, Germany), CPP32 17-kDa subunit (E-8; Santa Cruz Biotechnology, Heidelberg, Germany), PARP 85 kDa (7D3–6; PharMingen, San Diego, CA), cdc2 (9112; New England Biolabs, Schwalbach, Germany), phospho-cdc2 (Tyr-15; 9111; New England Biolabs), and PKA{alpha} cat, the {alpha} catalytic subunit of protein kinase A (sc-903; Santa Cruz Biotechnology). CPP32 or PARP cleavage products, cdc2, phospho-cdc2, and PKA{alpha} cat were detected by using cell lysate from GFP-positive, transfected cells sorted out by flow cytometry analysis. Immune complexes were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Braunschweig, Germany).

Clonogenic Assay.
The ability to grow in soft agarose was determined as described previously (42) . Briefly, 10 µg of plasmid DNA expressing E1A or control vector were transfected together with the puromycin-N-acetyltransferase-expressing plasmid into 7 x 106 K562 cells by electroporation. Twenty-four hours after transfection, cells were washed and plated in a six-well plate in culture medium containing puromycin (1 µg/ml) and 0.35% agarose overlying a 0.7% agarose layer. The cells were incubated at 37°C for 3 weeks, after which puromycin-resistant colonies were counted under light microscopy.

Flow Cytometry Analysis.
Ten micrograms of plasmid DNA expressing E1A were transfected into 7 x 106 cells. Where required, the peptide caspase-inhibitors z-VAD-fmk or Z-DEVD-FMK (Calbiochem) were added simultaneously with the apoptotic-triggering signal at a final concentration of 50 µM. To measure the transfection efficiency, 2 µg of GFP reporter plasmid encoding the membrane-localized enhanced GFP were cotransfected to ensure optimal fluorescence intensity in combination with ethanol fixation (43) . To quantitate apoptosis by flow cytometry, floating and adherent cells were harvested 72 h after transfection, fixed in ethanol, and stained for DNA content with PI. Cells were measured for green fluorescence intensity (channel FL-1) and PI fluorescence (channel FL-3) in a fluorescence-activated cell sorter (FACSVantage; Becton Dickinson, Mountain View, CA) using CELLquest software (Becton Dickinson). The cells that did not express GFP were used to set the baseline to allow the gating of the GFP-positive cells. The percentage of apoptotic cells seen in the population by electroporation alone (typically 2–6%) was subtracted.

Luciferase Assay.
K562 cells were cotransfected by electroporation with 1 µg of the pGL3-basic (Promega) or pGL3-cdc2 firefly luciferase reporter plasmid and 2 µg of the E1A expression plasmid or the pRc/RSV control vector plasmid (Invitrogen), respectively. In all transfections 1 µg of pRL-TK (Promega) encoding for Renilla luciferase under the control of the herpes simplex virus TK promoter region was cotransfected to account for differences in transfection efficiency. Treatment with 5 µM etoposide was initiated 24 h after transfection. Cells were collected 48 h after transfection in passive lysis buffer (Promega). Firefly and Renilla luciferase activities were determined by a premanufactured dual luciferase reporter assay system (Promega). To account for differences in transfection efficiencies, firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent the SD within a representative experiment. Each experiment was repeated at least three times.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
E1A Induces Substantial Cytotoxicity and Apoptosis in Ph-positive Chronic Myeloid Leukemia Cells.
BCR-ABL has been shown to contribute to the protection of hematopoetic cells from the induction of apoptosis by cytokine withdrawal (14) , Fas ligation (13) , and treatment with cytotoxic drugs (9) . By contrast, E1A expression has been shown previously to induce apoptosis and enhance in vitro cytotoxicity to ionizing radiation and chemotherapeutic agents (38, 44) . The ability of E1A to mediate cytotoxicity in BCR-ABL-positive K562 erythroleukemia cells was analyzed by clonogenic survival in soft agarose. As shown in Fig. 1ACitation , the numbers of formed colonies were markedly decreased in the E1A 13S-transfected cells compared with mock-transfected cells. To investigate whether the observed loss of viability in Ph-positive leukemia cells on overexpression of E1A protein is due to apoptosis, we analyzed K562 (Fig. 1BCitation , I–III) and a second BCR-ABL positive cell line, LAMA-84 (Fig. 1Citation B, I, IV, and V), transiently transfected with the E1A 13S cDNA using FACS analysis of PI-stained cells. This flow cytometric assay measures the apoptotic rate at the time of harvesting rather than cumulative apoptosis (45) . Transfected cells were gated on the basis of the expression of GFP, which was cotransfected as a transfection marker (cotransfection rate of ~95%), and apoptosis was measured by the accumulation of cells with a sub-G1 DNA content 72 h after transfection. Quantification of sub-G1 cells revealed a significant, ~3–5-fold increase in apoptotic cells in both E1A-transfected BCR-ABL-positive leukemia cell lines (Fig. 1Citation B, I, III, and V) compared with cells transfected with the control vector (Fig. 1Citation B, I, II, and IV). E1A expression in these cells produced typical apoptotic features with striking changes in the nuclear morphology, characterized by intense staining of condensed chromatin and nuclear fragmentation as analyzed by fluorescence microscopy (data not shown). Because K562 cells are negative for p53 expression (46, 47) , the observed apoptotic response in the p53-negative cell system is consistent with our data, demonstrating apoptosis induction by solitary delivery of E1A in the absence of functional p53 as well as other adenoviral gene products (37) . As an indication of the transfection efficiency, E1A expression in K562 cells was monitored by Western blot analysis (Fig. 1CCitation ).



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  		Fig. 1. Ectopic expression of E1A triggers apoptosis in Ph-positive chronic myeloid leukemia cells. A, clonogenic assay of E1A 13S cDNA- and control plasmid-transfected (mock) K562 cells after selection for stable transfectants with puromycin for 3 weeks. The average number of resulting colonies is shown in the left panel; representative phase-contrast micrographs are shown on the right. B, FACS analysis of E1A 13S cDNA-transfected (I, III, and V) and control plasmid-transfected (mock; I, II, and IV) PI-stained K562 (I–III) and LAMA-84 (I, IV, and V) cells, respectively. The percentage of apoptotic cells by electroporation alone (EP) is as indicated (I). Transfected cells were gated on the basis of GFP expression. Apoptosis was measured by the accumulation of cells with a sub-G1 DNA content 72 h after transfection. The diagram represents the mean of three independent experiments. C, Western blot analysis of E1A expression in transfected K562. 293 cells are shown as a positive control.

 
Induction of Apoptosis by E1A Is Mediated by Caspase-3 Activation, Which Is Specifically Blocked in BCR-ABL-expressing Leukemia Cells.
Previous studies have indicated that caspase activation plays a critical role in the initiation of the active phase of apoptosis (48–50) . In addition, it has been suggested that the blockage of cytochrome c release and caspase-3 activation is a mechanism by which the deregulated BCR-ABL tyrosine kinase prevents apoptotic cell death (19, 21, 51) . These observations prompted us to investigate the effect of E1A treatment on processing of procaspase-3 (pro-CPP32) into the active 17- and 12-kDa subunits. To detect particularly short-lived proteins in a relatively small number of E1A-transfected cells in front of the untransfected background, the effect of E1A expression in K562 cells was determined by Western blot analysis using whole-cell extracts prepared from the GFP-positive, transfected population sorted out by FACS. In these cells, stimulation of apoptosis by E1A triggered processing of procaspase-3, as revealed by the appearance of an ~17-kDa product, which corresponds to the 17-kDa subunit of activated caspase-3 (Fig. 2ACitation ). As shown in Fig. 2ACitation , induction of apoptosis by E1A was also accompanied by cleavage of the 116-kDa intact form of PARP to the 85-kDa fragment intimately linked to the induction of apoptosis in other systems. Interestingly, the apoptotic activity of E1A in K562 leukemia cells, measured by flow cytometry analysis at 72 h after transfection, was significantly antagonized by the wide-spectrum caspase inhibitor z-VAD-fmk, resulting in an ~50% reduction of relative apoptosis (Fig. 2BCitation ). In addition, the same inhibitory effect on E1A-induced apoptosis was evident in E1A-transfected cells treated with the caspase-3-specific inhibitor Z-DEVD-fmk. These observations indicated that transiently expressed E1A is sufficient to abolish the antiapoptotic function of BCR-ABL by initiating the caspase cascade.



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  		Fig. 2. Apoptosis induction by E1A in BCR-ABL-positive K562 cells involves activation of caspase-3. A, activation of caspase-3 (CPP32) and PARP cleavage in cells transiently transfected with E1A or control plasmid (mock) was analyzed by Western blot. Equal amounts of whole-cell extracts from GFP-positive cells sorted out by FACS were separated by SDS-PAGE. Full-length caspase-3 and the cleaved 17-kDa subunit as well as PARP (116 kDa) and the 85-kDa proteolytic cleavage product are indicated by arrows. B, the inhibition of E1A-mediated apoptosis by z-VAD-fmk and the caspase-3-specific inhibitor Z-DEVD-fmk (50 µM) was quantitated by flow cytometry. Relative apoptosis 72 h after transfection is as indicated. Apoptosis (as determined by cells with a sub-G1 DNA content) was calculated by subtraction of the percentage of apoptotic cells seen in the population by electroporation alone. Apoptosis in mock-transfected cells (4.22 ± 1%) was set as 1. The graph represents the mean of two independent experiments.

 
E1A Converts the BCR-ABL-mediated G2 Arrest after Treatment with Chemotherapeutic Agents to Induction of Apoptosis.
Previous studies have shown that Ph-positive human chronic myeloid leukemia cells such as K562 are particularly resistant to cell death via apoptosis, irrespective of the inducing agent used (10, 51) . BCR-ABL expression prevents the apoptotic deletion of damaged cells by prolongation of cell cycle arrest in the G2-M phase (52) . The importance of G2 delay as a critical determinant of radio- or chemosensitivity was recently acknowledged by data indicating that abrogation of G2-M arrest in Ph-positive cells with caffeine neutralized the protective effect of BCR-ABL kinase (10) . Thus, to achieve maximal eradication of CML cells in vivo, the use of proteins able to reduce G2-M arrest after genotoxic damage may be beneficial. Based on our results described above, indicating that E1A is a potent inducer of apoptosis in K562 cells, we sought to investigate whether apoptosis induction by E1A is also correlated with increased susceptibility to chemotherapeutic drugs. If G2-M arrest is tightly linked to the apoptotic protection against DNA-damaging agents afforded by BCR-ABL kinase expression, this effect should be abolished by E1A. Mock- and E1A 13S cDNA-transfected K562 cells were treated by continuous exposure with 5 µM etoposide and 0.2 µg/ml daunorubicin, respectively, over 2 days, and flow cytometry analysis was performed 72 h after transfection. Treatment with either drug alone at the particular dose as well as higher doses (up to 68 µM etoposide or 1 µg/ml daunorubicin) did not result in the induction of significant amounts of apoptosis, which is consistent with previous reports (20, 53) . In contrast, compared with E1A-transfected cells in the absence of chemotherapeutic agents, the population of sub-G1 cells significantly increased after treatment of E1A-expressing cells with either of these agents (Fig. 3Citation , A–D). At 72 h after transfection ~70% of K562 cells treated with E1A plus etoposide showed a sub-G1 DNA content, compared with only 30% (Fig. 3, A and B)Citation of cells introduced with E1A alone. This sensitization to etoposide by E1A could also be observed in LAMA-84 leukemia cells (data not shown), indicating that E1A renders BCR-ABL-positive cells susceptible to apoptotic cell death by chemotherapeutic agents. A less pronounced but significant effect was apparent in E1A-transfected cells treated with daunorubicin (Fig. 3, C and D)Citation . As shown by Lock and Ross (54) , our results also demonstrated that the majority of BCR-ABL kinase-expressing cells arrest in G2-M after etoposide treatment of mock-transfected cells (Figs. 3Citation B, left bottom panel, and 4A). The same effect was evident after treatment with daunorubicin (Fig. 3Citation D, left bottom panel). In contrast, in drug-treated BCR-ABL-positive cells, which express E1A, induction of apoptosis was associated with a marked decrease of cells in G2-M (Fig. 3, BCitation and D, right bottom panel). A maximum in apoptosis induction by E1A with most of K562 cells in the sub-G1 fraction (76.6%) versus only a very small fraction of cells arresting in G2-M (13.2%) was already achieved at low etoposide doses between 5 and 20 µM (Fig. 4BCitation ). Together, these data suggest that temporary expression of E1A is sufficient to convert preexisting resistance to genotoxic damage by BCR-ABL-mediated cell cycle block to induction of apoptosis.



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  		Fig. 3. E1A sensitizes BCR-ABL-expressing K562 cells to chemotherapeutic agents. K562 cells were transiently transfected with mock or E1A plasmid DNA (A and B) in the absence or presence (over 2 days) of 5 µM etoposide or 0.2 µg/ml daunorubicin (C and D). For FACS analysis, transfected cells were gated on the basis of GFP expression. Apoptosis was measured by the accumulation of cells with a sub-G1 DNA content at 72 h after transfection. E, clonogenic assay of E1A 13S cDNA- and control plasmid-transfected (mock) K562 cells after selection in the absence or presence of 5 µM etoposide for stable transfectants with puromycin for 3 weeks. Shown are representative phase-contrast micrographs.

 


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  		Fig. 4. Effect of E1A expression on cell cycle distribution of BCR-ABL-positive hematopoietic cells after etoposide treatment. A, K562 cells transfected with control plasmid DNA. B, K562 cells transfected with E1A 13S. Cells were maintained in the presence of etoposide over 2 days at the indicated doses. The fraction of cells with a sub-G1 DNA content and cells in G2-M was quantified by flow cytometric analysis of GFP-positive PI-stained cells 72 h after transfection. The results of the cell cycle analysis are the mean of three experiments.

 
To assess the combined effect of E1A expression and treatment with DNA-damaging agents on the long-term proliferative capacity of BCR-ABL-positive cells, we analyzed clonogenic survival of E1A or mock-transfected cells in the absence or presence of 5 µM etoposide (Fig. 3ECitation ). Whereas mock transfection gave rise to numerous, highly proliferating colonies, the number of viable colonies was significantly reduced by E1A expression alone. We note that E1A expressing colonies are variable in size, most likely due to differences in E1A expression levels. The numerous colonies surviving etoposide treatment alone were barely visible and comprised only 10–20 cells on average. This antiproliferative effect of etoposide is consistent with the observed cell cycle arrest at the G2-M transition. Importantly, no viable, proliferating colonies could be observed on combination treatment with E1A and etoposide, indicating that only the combination of E1A expression and etoposide treatment is efficient to completely inhibit clonogenic survival.

E1A Interferes with Etoposide-induced cdc2 Inactivation.
To investigate the mechanism of E1A-induced reversal of BCR-ABL-mediated inhibition of DNA damage-induced apoptosis, we first sought to analyze the effect of E1A expression on the tyrosine kinase activity of BCR-ABL. We therefore transfected K562 cells in the absence or presence of etoposide with an E1A-expressing plasmid and monitored the pattern of phosphotyrosine proteins by Western blotting using a phosphotyrosine-specific antibody. There were certain changes in the phosphorylation pattern with an E1A-induced increase or decrease in the phosphorylation levels of different protein species (data not shown). However, the changes did not correlate with the differences observed when K562 cells were treated with a BCR-ABL-selective tyrosine kinase inhibitor (data not shown), suggesting that these changes are not specific for BCR-ABL tyrosine kinase activity.

Although E1A does not seem to directly inhibit BCR-ABL kinase activity, E1A might interfere with downstream functions responsible for the protection from DNA damage-induced apoptosis. G2-M arrest induced by various stimuli including irradiation and etoposide has been shown to be linked to the inhibition of p34cdc2 (cdc2) kinase (54) . In addition, BCR-ABL-mediated resistance to radiation-induced apoptosis correlates with increased tyrosine phosphorylation (i.e., inhibition) of cdc2 (10) . E1A has been shown to induce cdc2 expression and kinase activity by transactivation of the human cdc2 promoter (55, 56) . Thus E1A might specifically interfere with the BCR-ABL-mediated delay of G2-M transition after DNA damage by activating cdc2. We therefore analyzed the effect of E1A expression on cdc2 promoter activity in BCR/ABL-expressing leukemia cells. K562 cells were transiently transfected with a reporter plasmid containing a 498-bp fragment from the human cdc2 promoter linked to the reporter gene firefly luciferase either in the absence or presence of an E1A expression plasmid. To account for differences in transfection efficiency, the Renilla luciferase construct pRL-TK was cotransfected, and expression of firefly luciferase was normalized to Renilla luciferase activity. As shown in Fig. 5ACitation , transient expression of E1A efficiently induced transcription from the cdc2 promoter, consistent with published data obtained in different cells (56–58) . Etoposide treatment of K562 cells, previously shown to induce G2-M arrest, slightly elevated the basal activity of the cdc2 promoter but did not interfere with transactivation by E1A.



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  		Fig. 5. E1A interferes with etoposide-induced cdc2-inactivation. A, Cdc2 promoter activity was analyzed by luciferase assay. K562 cells were transiently cotransfected with pGL3-basic or pGL3-cdc2 luciferase constructs and E1A or control plasmid, respectively. Twenty-four hours after transfection cells were exposed to 5 µM etoposide as indicated. Luciferase activity was determined 48 h after transfection. Promoter activity of pGL3-basic was normalized to 1.0, and the activities of the remaining transfections reactions were expressed relative to this. Error bars represent the SD within a representative experiment. Each experiment was repeated at least three times. B, expression levels of cdc2, Tyr-15-phosphorylated cdc2 (p-cdc2), and E1A in K562 cells transiently transfected with E1A or control plasmid were determined by Western blot analysis. Cotransfection of GFP reporter plasmid served as a transfection marker. Twenty-four hours after transfection cells were exposed to 5 µM etoposide as indicated. Forty-eight hours after transfection GFP-positive cells were sorted out by FACS. Equal amounts of whole-cell extracts from GFP-positive cells were separated by SDS-PAGE. Equal loading of the gel was confirmed by reprobing with anti-PKA{alpha} cat antiserum.

 
To investigate whether this effect of E1A expression on cdc2 promoter activity correlates with changes in cdc2 protein expression, lysates of K562 cells transfected with E1A or the empty control vector in the absence or presence of etoposide were monitored for cdc2 expression by Western blotting. Indeed, consistent with the luciferase assay data, E1A-expressing K562 cells exhibited a pronounced increase in total cdc2 protein level 48 h after transfection (Fig. 5B, ICitation Citation ). Furthermore, an increase in cdc2 protein levels could also be seen in etoposide-treated cells regardless of the presence of E1A. Because cdc2 kinase activity is regulated by phosphorylation on Tyr-15 (59) , and increased tyrosine phosphorylation correlates with BCR-ABL-mediated protection from radiation-induced apoptosis in K562 cells (10) , we sought to investigate the effect of E1A expression on cdc2 Tyr-15 phosphorylation. As seen in Fig. 5Citation B, II, etoposide treatment significantly increased the levels of phosphorylated cdc2 in wild-type K562 cells. However, this increase in phosphorylated (i.e., inactive) cdc2 was completely abolished by expression of E1A.

Taken together, our data indicate that E1A increases cdc2 expression by activating the human cdc2 promoter and specifically interferes with inactivation of cdc2 kinase activity by tyrosine phosphorylation, which has been made responsible for the BCR-ABL-mediated protection from DNA damage-induced apoptosis.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A critical determinant of the efficacy of antineoplastic therapy is the response of malignant cells to DNA damage induced by anticancer agents. CML is characterized by a chronic phase consisting of an abnormal expansion of the myeloid compartment followed by an acute blast crisis (6) . At this invariably fatal stage, the inherent resistance of Ph-positive cells to cytotoxic therapy is a major impediment to the management of CML. As a cause of resistance, prolongation of the hematopoietic cell survival by inhibition of apoptosis has been proposed to be an integral component of BCR-ABL-induced leukemia (9, 14) . Thus, modulation of the apoptotic pathway represents a logical target for therapeutic intervention.

In the present study, we investigated the adenovirus E1A protein for its ability to convert particularly apoptosis-resistant BCR-ABL-positive leukemia cells (9) derived from patients in blast crisis into apoptosis-sensitive ones. This approach was suggested by observations indicating that E1A has tumor suppressor activities in a variety of solid human cancers both in vitro and in vivo (60–62) . Furthermore, E1A-mediated tumor suppression in these cells has been shown to be associated with enhanced cytotoxicity and the induction of apoptosis by both p53-dependent and -independent pathways (37, 39, 44) . Our present data show for the first time that E1A overexpression also results in a significant increase of apoptotic cell death in BCR-ABL-expressing hematopoietic K562 and LAMA-84 cells, respectively. The Ph-positive K562 cell line was shown to be resistant to various anticancer agents and to undergo etoposide-induced cell death only at a high dose of drug after prolonged exposure (20, 53) . We demonstrate that introduction of E1A in K562 leukemia cells clearly enhanced the induction of apoptosis by topoisomerase II inhibitors etoposide and also by daunorubicin at a low concentration and short exposure time. This substantial synergy between E1A and etoposide and the rather additive enhancement of apoptosis between E1A and daunorubicin is consistent with other studies that show enhanced apoptosis and increased chemosensitivity of E1A-transfected keratinocytes and cancer cell lines to DNA-damaging agents in the absence of p53 (63) .

Previous reports have indicated that a variety of antileukemic drugs including etoposide cause the preapoptotic mitochondrial release and cytosolic accumulation of cytochrome c (20) , which mediates the cleavage and activation of caspase-3 involved in the execution of apoptosis (20, 64) . Cells that overexpress BCR-ABL, however, receive all of the measurable damage induced by cytotoxic drugs but are unable to couple this damage to the apoptotic pathway (20, 65) . In Ph-positive cells, BCR-ABL expression results in the inhibition of the mitochondrial perturbations, thereby blocking the generation of caspase-3 activity and apoptosis (19, 21) . On the basis of previous findings, the induction of p53-independent apoptosis by E1A in the viral context requires other E4 gene products (35) but also involves a mechanism that includes activation of caspase-3 (66) . Because E4orf4 is the only E4 product capable of independent cell killing but does not require activation of known zVAD-fmk-inhibitable caspases (67) , it is likely that the processing of caspase-3 observed by Boulakia et al. (66) in p53-null cells is a direct consequence of the E1A function, which does not require cooperation with other viral proteins to cause cell death. Consistently, we have recently shown that the induction of apoptosis by the solitary delivery of E1A 13S into p53-null human tumor cells can be linked to caspase activity (37) . Our present data demonstrate that the apoptotic activity of E1A in hematopoietic K562 cells is accompanied by processing of caspase-3, and cleavage of PARP and can be significantly blocked by z-VAD-fmk and the caspase-3-specific inhibitor Z-DEVD-fmk. Thus, transient expression of the E1A protein is apparently sufficient to antagonize the BCR-ABL-induced block in the apoptosis signaling pathway triggered by DNA-damaging agents.

The delay in the apoptosis signaling cascade in BCR-ABL-expressing K562 cells treated with etoposide is known to be associated with the induction of cell cycle arrest in the G2-M phase, shown in the present paper and by Lock and Ross (54) , which leads to apoptosis protection. In contrast, etoposide treatment of E1A-expressing K562 cells correlates with the inhibition of G2-M arrest and substantially increased chemosensitivity, resulting in increased apoptosis of BCR-ABL-positive cells and significant reduction of long-term clonogenic survival. As our data indicate, E1A does not seem to interfere directly with the tyrosine kinase activity of BCR-ABL, because the changes observed in the pattern of phosphotyrosine proteins do not correlate with the changes induced by a BCR-ABL-selective tyrosine kinase inhibitor. However, E1A might interfere with downstream functions responsible for the protection from DNA damage-induced apoptosis. G2-M arrest induced by various stimuli including etoposide has been shown to be linked to the inhibition of p34cdc2 (cdc2) kinase (54) . In mammalian cells, G2-M transition is controlled by cdc2 kinase activity (68) , which is normally regulated by phosphorylation of cdc2 protein by the inhibitory kinase Wee1 and dephosphorylation by the activating phosphatase cdc25C. In some systems, increased phosphorylation of cdc2 in cells expressing Wee1 kinase occurred in association with protection from apoptosis (69) . On the other hand, induction of apoptosis is associated with premature activation (by dephosphorylation) of cdc2 kinase (70) . Previous studies have revealed that the human cdc2 promoter is transcriptionally activated by E1A proteins in cycling cells, which is mediated through two CCAAT box binding motifs (56–58) . With regard to this aspect, we detected increased cdc2 promoter activity and cdc2 protein levels in E1A-transfected K562 cells irrespective of the presence of etoposide. Thus E1A-induced up-regulation of cdc2 expression is apparently responsible for overriding the etoposide-associated inhibition of cdc2 kinase. In addition, etoposide-induced Tyr-15 phosphorylation (i.e., inactivation) of cdc2 was completely abolished in the presence of E1A. This may result at least in part from a rapid cleavage of Wee1 (71) , which has been shown to be a substrate of the caspase-3-like protease during Fas-induced apoptosis (71) . On the other hand, inhibition of etoposide induced cdc2 phosphorylation may also be due to activation of the phosphatase cdc25C. Indeed, CCAAT box motifs responsible for the E1A-mediated transactivation of the cdc2 promoter are also present in the cdc25C promoter, and nuclear extracts from human E1A-immortalized 293 cells bind the CCAAT elements of the cdc25C promoter to form specific DNA-protein complexes (57) . Together, these data strongly suggest that E1A overrides BCR-ABL-mediated DNA damage induced G2-M arrest by inhibition of cdc2 inactivation as a potential mechanism for E1A-induced cell death in CML.

The ability of E1A gene products to induce apoptosis in the presence of p53 is well established (32) . E1A interactions with RB and p300 have been linked to perturbation of cell cycle and apoptosis induction (34, 72) resulting in up-regulation of the ARF gene product (73) and stabilization of p53. In this context, ectopic expression of E2F1 as a critical downstream effector of RB, and E1A activity directly correlates with increased expression of ARF (74) . Our results in CML cells and those of other studies in cancer cells from solid tumors demonstrate that enhanced apoptosis and chemosensitivity by E1A can be unrelated to the amount of p53 present (63) . Investigation of E1A targets in the cell cycle regulatory pathway revealed that E1A-induced apoptosis was preceded not only by a rise in p53 but also by a precipitous drop in topoisomerase II{alpha}, suggesting that E1A can activate or induce components responsible for degrading topoisomerase II{alpha} (75) . Previous findings demonstrated that enforced E2F1 expression in myeloid progenitor cells confers preferential sensitivity to p53-independent apoptosis mediated by the topoisomerase II inhibitors etoposide and doxorubicin (76, 77) . On the basis of those and our data, we can speculate that E2F1 may be a potential target in E1A-expressing CML cells to sensitize for chemotherapeutic agents such as etoposide and daunorubicin to undergo p53-independent apoptosis.

Even when it is likely that multiple pathways are involved in the regulation of cell growth and apoptosis and also the response to cytotoxic agents in BCR-ABL-positive tumors, by targeting the final common pathway we may be able to create a potent therapeutic strategy to improve survival of CML patients. From our data, E1A-based therapy may represent a potent new approach by itself and when administered in conjunction with conventional cytotoxic drugs for the treatment of CML in the final stage of blast crisis.


   ACKNOWLEDGMENTS
 
We thank S. Zimmermann and B. Pollmeier for technical assistance and K. Lennarz for support in flow cytometry analysis.


   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 This work was supported by the Interne Forschungsförderung Essen program of the Medical Faculty of the University of Essen. Back

2 These authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Institute of Molecular Biology, University of Essen Medical School, Hufelandstrasse 55, D-45122, Germany. Phone: (49) 201-723-3158; Fax: (49) 201-723-5974; E-mail: brigitte.puetzer{at}uni-essen.de Back

4 The abbreviations used are: CML, chronic myelogenous leukemia; Ph, Philadelphia; RB, retinoblastoma; RSV, Rous sarcoma virus; PARP, poly(ADP-ribose) polymerase; GFP, green fluorescent protein; PI, propidium iodide; FACS, fluorescence-activated cell sorting; TK, thymidine kinase; CPP32, caspase-3. Back

Received 11/22/99. Accepted 5/16/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S. Madhusudan, A. Tamir, N. Bates, E. Flanagan, M. E. Gore, D. P. J. Barton, P. Harper, M. Seckl, H. Thomas, N. R. Lemoine, et al.
A Multicenter Phase I Gene Therapy Clinical Trial Involving Intraperitoneal Administration of E1A-Lipid Complex in Patients with Recurrent Epithelial Ovarian Cancer Overexpressing HER-2/neu Oncogene
Clin. Cancer Res., May 1, 2004; 10(9): 2986 - 2996.
[Abstract] [Full Text] [PDF]

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 Genes Dev.Home page
A. Cuconati, C. Mukherjee, D. Perez, and E. White
DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells
Genes & Dev., December 1, 2003; 17(23): 2922 - 2932.
[Abstract] [Full Text] [PDF]

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J. L. Cook, T. A. Miura, D. N. Ikle, A. M. Lewis Jr., and J. M. Routes
E1A Oncogene-induced Sensitization of Human Tumor Cells to Innate Immune Defenses and Chemotherapy-induced Apoptosis in Vitro and in Vivo
Cancer Res., June 15, 2003; 63(12): 3435 - 3443.
[Abstract] [Full Text] [PDF]

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 J. Virol.Home page
J. A. Mahr, J. M. Boss, and L. R. Gooding
The Adenovirus E3 Promoter Is Sensitive to Activation Signals in Human T Cells
J. Virol., December 20, 2002; 77(2): 1112 - 1119.
[Abstract] [Full Text] [PDF]

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