Protein tyrosine kinase Lyn mediates apoptosis induced by topoisomerase II inhibitors in DT40 cells

Akihiro Maruo, Isao Oishi, Kiyonao Sada, Masashi Nomi, Tomohiro Kurosaki1, Yasuhiro Minami and Hirohei Yamamura

Department of Biochemistry, Kobe University, School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
1 Department of Molecular Genetics, Institute for Hepatic Research, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi 570-0074, Japan

Correspondence to: H. Yamamura


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Several sets of non-receptor protein tyrosine kinases (PTK) play important roles in apoptosis induced by various extracellular stresses. Anti-cancer drugs induce cellular DNA damage and cytotoxic events, leading to apoptotic cell death. We utilized the established chicken B cell line, DT40 cells and their derived mutants, lacking the respective PTK [DT40/Syk(–), DT40/Lyn(–) and DT40/Btk(–)], to examine a role of these PTK in apoptotic processes induced by anti-cancer drugs. All anti-cancer drugs examined induced apoptosis of wild-type DT40 cells. Interestingly,DT40/Lyn(–), but not DT40/Syk(–) and DT40/Btk(–) cells, become resistant to apoptosis induced by adriamycin and etoposide, topoisomerase II (Topo II) inhibitory agents, compared to wild-type DT40 cells, as assessed by DNA fragmentation and TUNEL analyses. Ectopic expression of Fyn, another Src family member, in DT40/Lyn(–) cells restores largely the susceptibility of the cells against Topo II inhibitor-induced apoptosis. Furthermore, it was found that Topo II inhibitors activate c-Jun N-terminal kinase (JNK) slightly in both wild-type and DT40/Lyn(–) cells to similar extents. Collectively, these results suggest that Lyn is involved in Topo II inhibitor-induced apoptotic signaling in DT40 cells independent of JNK.

Keywords: anti-cancer drug, apoptosis, protein tyrosine kinase, topoisomerase


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Previous studies demonstrate that non-receptor protein tyrosine kinases (PTK) play crucial roles in a wide variety of cellular responses, including activation, proliferation, differentiation and apoptosis of cells (1,2). It has been well established that the Src family and Syk/ZAP-70 family of PTK, among non-receptor PTK, play important roles during activation of T and B cells (36). Furthermore, it has been reported that distinct sets of non-receptor PTK are activated by different stresses, eventually leading to apoptosis. In fact, it has been shown that oxidative stress, induced by addition of hydrogen peroxide, activates Syk, ZAP-70, Lck and Src (710), while hyperosmotic stress activates Syk, Lyn and Pyk2 (7,11,12). Radiation also induces the activation of non-receptor PTK; ionizing radiation activates Syk, Lyn, Abl and Btk (1317), and UVC radiation activates ZAP-70, Lyn, Src and Pyk2 respectively (8,11,12,17,18).

Genetic analyses have elucidated critical regulatory roles of non-receptor PTK in stress-induced signalings (19 20). Abl is required for ionizing radiation-induced cell cycle arrest at the G1 phase via tyrosine phosphorylation-dependent down-regulation of Cdk2 activity (14). Abl-deficient cells fail to undergo G1 arrest following ionizing radiation, yet ectopic expression of Abl restores the ability of these cells to down-regulate Cdk2 and undergo G1 arrest (15). It has also been shown that Abl is required for ionizing radiation-induced activation of c-Jun N-terminal kinase (JNK) (1315). Although ionizing radiation activates Syk, Lyn and Btk in chicken B cell line, DT40 cells, Btk-deficient DT40 cells, but not Syk- or Lyn-deficient DT40 cells, become resistant to ionizing radiation-induced apoptosis (17). Thus, it becomes evident that Btk acts as a positive regulator of ionizing radiation-induced apoptosis. Similar genetic approaches utilizing DT40 cells, lacking expression of the respective non-receptor PTK, have revealed that Syk is a negative regulator of osmotic stress-induced apoptosis, while Lyn is a positive regulator of UVC radiation-induced apoptosis (11).

Anti-cancer drugs, widely utilized in chemotherapy, can also be considered as cellular stresses. Although most anti-cancer drugs can induce apoptosis of cancer cells eventually, their modes of action are different from each other. Some of them exhibit their effects by directly interacting with cellular DNA. For example, 1-ß-D-arabinofuranosylcytosine (Ara-C) mis-incorporates into cellular DNA and inhibits replication by site-specific termination of DNA strands (21), while cis-diamminedichloroplatinum(II) (CDDP; cisplatin), an alkylating agent, is believed to interact covalently with chromosomal DNA, thereby inhibiting DNA synthesis (22). Several anti-cancer agents have been identified as inhibitors of DNA topoisomerases. Camptothecin (CPT) binds to the topoisomerase I (Topo I)–DNA complex, resulting in inhibition of the re-ligate step of the catalytic cycle (23). Etoposide (VP-16) acts as a Topo II inhibitor by interfering with the re-ligation step of the double-strand DNA–Topo II cleavable complex (2426). Adriamycin (ADR) stabilizes the cleavable complex between Topo II and DNA, resulting in the formation of single- and double-strand breaks, although its mode of action as an anti-cancer drug is rather complex (25,26).

In contrast to the mechanism of anti-cancer drug action, the mechanism of anti-cancer drug-induced signal transmission, eventually leading to apoptosis, remains largely unknown. It has been reported that structurally and functionally unrelated anti-cancer drugs [including Ara-C, CDDP, ADR, VP-16 and a microtubule destabilizing agent, vinblastine (VBL)] can activate JNK (13,14,2729). Furthermore, it has been shown that Abl is required for JNK activation induced by CDDP and Ara-C (13,29). However, it remains unclear whether non-receptor PTK, other than Abl, are involved in anti-cancer drug-induced stress signaling and apoptosis.

In this study, we examined the role(s) of non-receptor PTK, including Syk, Lyn and Btk, in anti-cancer drug-induced apoptotic signaling by utilizing a chicken B cell line, DT40 cells and their derived mutants lacking the expression of the respective PTK [DT40/Syk(–), DT40/Lyn (–) and DT40/Btk(–)]. Here we show that DT40/Lyn(–), but not DT40/Syk(–) and DT40/Btk(–) cells, become resistant to apoptosis induced by ADR and VP-16, selective inhibitors of Topo II, when compared with wild-type DT40. Ectopic expression of mouse Fyn, another member of the Src family PTK, in DT40/Lyn(–) cells significantly promotes apoptosis in response to the Topo II inhibitors. Taken together, these results indicate that Lyn acts as a positive regulator of the apoptotic response induced by the Topo II inhibitors and that Fyn compensates at least partly for this function of Lyn. We also discuss our findings in the light of their possible clinical application.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells and reagents
Chicken B cell line, DT40 cells, were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) chicken serum, 100 U/ml penicillin and 100 µg/ml kanamycin in a humidified 95% air, 5% CO2 atmosphere. The parent culture was maintained in continuous logarithmic growth between 5 and 10x105 cells/ml. DT40-derived mutants lacking the respective PTK, DT40/Lyn(–), DT40/Syk(–) and DT40/Btk(–), were established as described previously (30). The cDNA encoding mouse Fyn was cloned into pApuro expression vector and transfected into DT40/Lyn(–) cells by electroporation, and selected in the presence of 0.5 µg/ml puromycin (31). Anti-phosphotyrosine mAb (4G10) was from Upstate Biotechnology (Lake Placid, NY). Mouse anti-human JNK-1 mAb was purchased from PharMingen (San Diego, CA). Western blot chemiluminescence reagents (Renaissance) were from DuPont'/NEN (Boston, MA). Glutathione–Sepharose 4B was from Pharmacia (Uppsala, Sweden). GST expression vector containing the N-terminal fragment (amino acids 1–79) of c-Jun was a generous gift from Dr Hibi (Osaka University, Japan). The MEBSTAIN Apoptosis kit was purchased from MBL (Nagoya, Japan). Camptothecin (CPT) was kindly gifted from Daiichi pharmaceutical (Tokyo, Japan)/Yakult Honsha (Tokyo, Japan). ADR, VP-16 and CDDP were purchased from Nippon Kayaku (Tokyo, Japan). VBL was purchased from Kyorin (Tokyo, Japan). Ara-C was from Nippon Sinyaku (Kyoto, Japan).

Preparation of GST fusion protein pGEX3X
c-Jun (amino acids 1–79) encodes a GST fusion protein containing the JNK binding domain and the serine residues (at positions 63 and 73), the phosphorylation of which correlates well with the increased transcriptional activity of c-Jun. Escherichia coli XL1 Blue were transformed with this GST fusion protein expression vector. Proteins were purified following the protocol recommended by the manufacturer (Pharmacia). The amounts of purified proteins were estimated by SDS–PAGE and subsequent staining with Coomassie brilliant blue.

Assays for JNK activity
Cells (10x105/ml, 10 ml) in RPMI 1640 media were treated with the respective anti-cancer drugs under the conditions indicated and were harvested. Cells were solubilized with 0.5 ml of lysis buffer [50 mM Tris–HCl, pH 7.4, 2% (v/v) Triton X-100, 5 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, 2 mM PMSF, 10 µg/ml leupeptin and aprotinin] and insoluble materials were removed by centrifugation at 16,000 g for 15 min at 4°C. Cell lysates were immunoprecipitated with anti-JNK-1 antibody (1 µg/sample) and Protein A–Sepharose 4B for 2 h at 4°C. Half an aliquot of anti-JNK-1 immunoprecipitates was subjected to kinase assay (see below) and the remaining aliquot was subjected to immunoblot analysis with anti-JNK-1 antibody (see below). Anti-JNK-1 immunoprecipitates were washed 3 times with lysis buffer, once with washing buffer (50 mM HEPES and 10 mM MgCl2, pH 7.6) and once with kinase buffer (10 mM HEPES, pH 7.6, 10 mM MgCl2, 10 µM cold ATP and 10 µM sodium orthovanadate). Immune complex kinase assays were performed in 30 µl of kinase assay buffer containing 1 µCi of [{gamma}-32P]ATP (3000 Ci/mmol) and 5 µg of GST–c-Jun as a substrate. After a 20 min incubation at 30°C, the reaction was terminated by an addition of SDS sample buffer followed by boiling for 5 min. The samples were separated by SDS–PAGE. Autoradiography was carried out utilizing an image analyzer (Fuji BAS 2000).

Immunoblot analysis
Half an aliquot of the respective immunoprecipitates (see above) was washed 3 times with the lysis buffer, once with 10 mM HEPES, pH 8.0, containing 500 mM NaCl, and once with the same HEPES buffer without NaCl. The immunoprecipitated proteins were separated by 10% SDS–PAGE, transferred electrically to PVDF membranes and then blotted with anti-JNK-1 antibody. Immunoreactive proteins were visualized by using ECL.

DNA fragmentation analysis
Cells (5x105/ml, 10 ml) in RPMI 1640 media were treated with the indicated concentration of the respective anti-cancer drugs for the periods stated. Cells were harvested and then lysed in 0.5 ml of lysis buffer [10 mM Tris–HCl, pH 7.5, 10 mM EDTA, 150 mM NaCl, 1% (v/v) Triton X-100 and 0.1 mg/ml proteinase K] for 20 min at room temperature followed by a 30 min incubation with 0.1 mg/ml RNase A at 50°C. DNA fragmentation was analyzed on 2% agarose gels in the presence of 0.5 µg/ml ethidium bromide.

TUNEL assays
Cells (2x105/ml, 10 ml) in RPMI 1640 media were treated with the respective anti-cancer drugs under the conditions indicated. By using the MEBSTAIN kit (MBL), TUNEL assays were performed following the protocol recommended by the manufacturer. In brief, cells were washed and smeared by using the cytospin (10x105 cells per slide). Each slide was air-dried for 1 h using a fan, then fixed with 4% (w/v) paraformaldehyde at 4°C for 15 min. After fixation, cells were permeabilized with 0.5% (v/v) Tween 20 containing 0.5% (w/v) BSA for 15 min at room temperature. DNA fragmentation was nick end-labeled with biotinylated dUTP, mediated by TdT at 37°C for 1 h and subsequently stained with FITC-conjugated avidin. Samples were visualized by using a fluorescence microscopy (Axioplan; Zeiss, Oberkochen, Germany).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Anti-cancer drugs induce morphological changes characteristic of apoptosis
It has been reported that anti-cancer drugs induce apoptotic cell death of a wide variety of cell types. Apoptosis accompanies the unique morphological changes characterized by chromatin condensation, membrane blebbing and apoptotic bodies (32). Treatment of wild-type DT40 cells with the respective anti-cancer drugs, Ara-C (10 µM, 16 h) (Fig. 1Go, middle), VP-16 (5 µM, 2 h) (Fig. 1Go, right), ADR (500 ng/ml, 8 h), CDDP (10 µg/ml, 12 h), CPT (10 µg/ml, 12 h) and VBL (1 µg/ml, 2 h) (data not shown), results in the appearance of chromatin condensation, membrane blebbing and apoptotic bodies—characteristic features of apoptosis. To examine the role(s) of a series of non-receptor PTK in anti-cancer drug-induced apoptotic processes, DT40/Lyn(–), DT40/Btk(–) and DT40/Syk(–) cells, in addition to wild-type DT40 cells, were subjected to treatment with different classes of anti-cancer drugs described above. It was found that Ara-C (Fig. 1Go), CDDP, CPT and VBL (data not shown) induce apoptosis of all the cells examined to essentially identical extents as assessed by their morphological changes. Interestingly, DT40/Lyn(–), but not DT40/Btk(–) and DT40/Syk(–) cells, exhibited resistance to apoptosis induced by Topo II inhibitors, VP-16 (Fig. 1Go) and ADR (data not shown), when compared to wild-type DT40 cells. These microscopic analyses suggest that Lyn plays a role in Topo II inhibitor (VP-16 and ADR)-induced apoptosis of DT40 cells as a positive regulator.



View larger version (99K):
[in this window]
[in a new window]
 
Fig. 1. Morphological changes in DT40 and DT40-derived mutants following anti-cancer drug treatment. DT40 cells and the mutants (1x105/ml, 5 ml) were untreated (left), or treated with 10 µM Ara-C (middle) or 5 µM VP-16 (right) for the indicated periods and their morphological changes were examined by microscopic observation.

 
Inhibition of Topo II inhibitor-induced apoptosis by Lyn
To further examine the role(s) of non-receptor PTK in anti-cancer drug-induced apoptosis, we monitored DNA fragmentation of wild-type DT40 cells and their derived mutants, a typical biochemical marker of apoptosis, following treatment with the respective anti-cancer drugs. Consistent with our microscopic analyses, DNA fragmentation induced by Topo II inhibitors [VP-16 (5 µM, 4 h) and ADR (200 ng/ml, 10 h)] was significantly inhibited in DT40/Lyn(–), but not in DT40/Btk(–) and DT40/Syk(–) cells, when compared to wild-type DT40 cells (Fig. 2A and BGo). No apparent difference in the extents of DNA laddering was observed in these cells following treatments with other anti-cancer drugs, including Ara-C (Fig. 2CGo), CDDP, CPT and VBL (data not shown). To further confirm that DT40/Lyn(–) cells acquire resistance to the Topo II inhibitor-induced apoptosis, compared to DT40/Btk(–), DT40/Syk(–) as well as wild-type DT40 cells, we performed the TUNEL assays following treatment of the respective cells with Topo II inhibitors, VP-16 and ADR. Cytoplasmic spotty staining as well as prominent nuclear staining were observed in wild-type DT40 cells upon treatment with 5 µM VP-16, 4 h (Fig. 2DGo) or 200 ng/ml ADR, 8 h (data not shown). Essentially identical results were obtained in DT40/Btk(–) and DT40/Syk(–) cells (data not shown). On the other hand, most of the DT40/Lyn(–) cells were unaffected by VP-16 (Fig. 2DGo) and ADR (data not shown) under the same condition, and only a small fraction of DT40/Lyn(–) cells exhibited such spotty stainings. Taken together, these results indicate that Lyn exhibits the pro-apoptotic effect selective to the Topo II inhibitors among the anti-cancer drugs examined.




View larger version (82K):
[in this window]
[in a new window]
 
Fig. 2. Topo II inhibitor-induced DNA fragmentation was selectively inhibited in DT40/Lyn(–) cells. (A–C) DT40, DT40/Lyn(–), DT40/Btk(–) and DT40/Syk(–) cells (5x105/ml, 10 ml) were treated with 5 µM VP-16, 4 h (A) or 200 ng/ml ADR, 10 h (B) or 10 µM Ara-C, 12 h and collected by centrifugation. The {lambda} phage DNA digested with restriction endonucleases EcoT14I is indicated as a DNA molecular mass marker (the right lane). Cellular DNA was extracted and analyzed by a 2% agarose gel, containing ethidium bromide to detect DNA laddering. (D) Cells treated with the respective anti-cancer drugs were collected and applied onto the slide (1x105/ml, 50 µl per slide). Then TUNEL assay was performed as described in Methods.

 
To characterize in detail the resistance of DT40/Lyn(–) cells against apoptosis induced by the Topo II inhibitors, we examined whether these drugs induce DNA fragmentation in DT40 and DT40/Lyn(–) cells in time- and dose-dependent manners. To this end, DT40 and DT40/Lyn(–) cells were exposed to VP-16 (5 µM) for the indicated periods, and the extents of DNA fragmentation were analyzed. As shown in Fig. 3Go(A), in wild-type DT40 cells, DNA fragmentation was detectable within 2 h after VP-16 treatment and DNA laddering reached a maximal level at 4 h after drug treatment, while apparent DNA fragmentation was not observed in DT40/ Lyn(–) cells up to 8 h after treatment with VP-16. Subsequently, DT40 and DT40/Lyn(–) cells were exposed to VP-16 for 6 h with different concentrations, and were subjected to DNA fragmentation analysis. As shown in Fig. 3Go(B), in wild-type DT40 cells, apparent DNA fragmentation was first detectable with 2 µM VP-16 and the intensity of fragmented DNA was increased in a dose-dependent manner. On the other hand, in DT40/Lyn(–) cells, DNA fragmentation was only detectable at a high concentration of VP-16 (10 µM) (Fig. 3BGo).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3. Dose- and time-dependent DNA fragmentation induced by the Topo II inhibitor, VP-16. (A) DT40 (left) and DT40/Lyn(–) (right) cells (5x105/ml, 10 ml) were treated with 5 µM VP-16 and harvested at the indicated time points. The right lane is a DNA molecular mass marker. (B) DT40 (left) and DT40/Lyn(–) (right) cells (5x105/ml, 10 ml) were treated with VP-16 6 h at the indicated concentration. DNA fragmentation assay was performed as described in Methods. The right lane is a DNA molecular mass marker.

 
Lyn is not required for JNK activation induced by Topo II inhibitors
It has recently been reported that Pyk2 and c-Abl positively regulate the activation of JNK in response to osmotic stress and genotoxic stresses, including anti-cancer drug treatment (1214). Furthermore, JNK activation has been shown to correlate well with apoptosis induced by several extracellular stresses (34,35). Hence, we examined whether Lyn regulates Topo II inhibitor-induced apoptosis by modulating JNK activity. JNK activity was determined by a JNK assay using GST–c-Jun (1–79 amino acids) as an exogenous substrate as described in Methods. As shown in Fig. 4Go(A), treatment of DT40 cells with ADR (500 ng/ml, 2 h) and VP-16 (5 µM, 2 h) induced slight increases in JNK activity (1.12 ± 0.199- and 1.94 ± 0.744-fold respectively) over the control, while another anti-cancer drug, VBL (1 µg/ml, 2 h), or osmotic stress (0.2 M NaCl, 15 min) induced drastic increases in JNK activity (10.38 ± 6.31- and 16.8 ± 8.61-fold, respectively) (Fig. 4A, Gotop). Importantly, JNK activities in DT40 and DT40/Lyn(–) cells were comparable following treatment with ADR and VP-16 (Fig. 4A and BGo) as well as NaCl and VBL (data not shown). Immunoblot analysis with anti-JNK antibody revealed that the amounts of JNK in each sample for JNK assay were comparable (Fig. 4AGo, bottom). These results indicate that Lyn is not involved in Topo II inhibitor-induced JNK activation.




View larger version (50K):
[in this window]
[in a new window]
 
Fig. 4. Lyn is not required for Topo II inhibitor-induced activation of JNK. (A) DT40 and DT40/Lyn(–) cells (5x105/ml, 10 ml) were treated with ADR, VP-16, VBL or NaCl for the indicated conditions, and JNK was immunoprecipitated by anti-JNK-1 antibody. JNK activity was monitored by in vitro phosphorylation of GST–c-Jun (top). Protein levels of JNK immunoprecipitated from various cell lysates were estimated by anti-JNK immunoblotting analysis (bottom). (B) DT40 cells were treated with the drugs at indicated conditions and then the JNK assay was performed. The mean values from several separate experiments are shown with error bars indicating the SD.

 
Fyn can compensate largely the role of Lyn in Topo II inhibitor-induced apoptotic processes
Previous studies demonstrate that the function of Lyn in B cell receptor-mediated signaling can be compensated by the other Src family PTK, including Fyn and Lck (30,31). In fact, it has been shown that abnormality in Ca2+ mobilization and tyrosine phosphorylation observed in DT40/Lyn(–) cells following B cell receptor engagement can be restored largely by ectopic expression of Fyn in DT40/Lyn(–) cells (30). Therefore, we were interested in examining whether ectopic expression of Fyn in DT40/Lyn(–) cells can restore the susceptibility of the cells to Topo II inhibitor-induced apoptotic processes. For this purpose, wild-type DT40, DT40/Lyn(–) and DT40/Lyn(–)/Fyn cells were treated with the Topo II inhibitors, VP-16 and ADR, and their apoptotic features were analyzed. The microscopic analysis showed that DT40/Lyn(–)/Fyn, but not DT40/Lyn(–), cells exhibit morphological changes, characteristic of apoptosis, to similar extents as wild-type DT40 following treatment with 5 µM VP-16, 2 h (Fig. 1Go) or 200 ng/ml ADR, 10 h (data not shown).

The DNA fragmentation analysis also revealed that apparent DNA laddering was found in wild-type DT40 and DT40/Lyn(–)/Fyn, but not DT40/Lyn(–) cells, upon treatment with VP-16 and ADR (Fig. 5A and BGo), although the intensity of fragmented DNA was somewhat weaker in DT40/Lyn(–)/Fyn cells compared with wild-type DT40 cells. We next performed the TUNEL assays to further assess the ability of Fyn to restore the susceptibility of DT40/Lyn(–) cells to Topo II inhibitor-induced apoptosis. As shown in Fig. 5Go(C, top), DT40/Lyn(–)/Fyn cells exhibited cytoplasmic spotty staining as well as prominent nuclear staining to a similar, yet somewhat decreased, extent with wild-type DT40 cells following VP-16 treatment (5 µM, 4 h). To evaluate the compensatory function of Fyn, the percentage of TUNEL-positive apoptotic cells was examined in DT40, DT40/Lyn(–) and DT40/Lyn(–)/Fyn cells following VP-16 treatment. As shown in Fig. 5Go(C, bottom), the percentage of TUNEL-positive apoptotic cells in DT40/Lyn(–) /Fyn cells was significantly higher than that of DT40/Lyn(–) cells, yet still lower than that of wild-type DT40 cells. Collectively, these results indicate that Fyn can compensate at least partly for the function of Lyn in Topo II inhibitor-induced apoptotic processes.




View larger version (50K):
[in this window]
[in a new window]
 
Fig. 5. Fyn compensates partially for the function of Lyn. (A and B) DT40, DT40/Lyn(–), DT40/Lyn(–)/Fyn cells (5x105/ml, 10 ml) were treated with 2 µM VP-16, 4 h (A) or 200 ng/ml ADR, 10 h (B) and collected by centrifugation. DNA fragmentation assay was performed as described in Methods. The right lane is a DNA molecular mass marker. (C) DT40, DT40/Lyn(–) and DT40/Lyn(–)/Fyn cells (1x105/ml, 5 ml) were treated with VP-16 (5 µM ) for 4 h. TUNEL assay was performed as described in Methods. TUNEL-positive cells were counted from five independent fields (300 cells on total) and are represented as the percentage of apoptotic cells. The white (untreated) and black (5 µM VP-16, 4 h) bars represent mean values of percentage TUNEL-positive cells. The error bar represents the SD of five determinations.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Accumulating evidence demonstrates that several sets of non-receptor PTK, including Btk, Syk, ZAP-70, Lyn, Abl and Pyk2, play important roles in extracellular stress-induced signal transduction, eventually leading to apoptosis (68,11,1316), although it remains largely unknown how the respective non-receptor PTK regulate the stress-induced apoptotic processes. Recent studies utilizing genetic approaches have elucidated some if not all of the functional roles of Abl in stress signaling elicited by DNA-damaging agents (13,14). It was found that cells deficient in Abl fail to activate JNK and to arrest these cells at G1 phase following exposure to DNA-damaging agents, and that activation of JNK as well as G1 arrest can be restored by an ectopic expression of Abl in these cells (15). Thus, it has become evident that Abl is required for both the activation of JNK and G1 arrest induced by DNA-damaging agents. Our similar genetic analyses, utilizing a chicken B cell line, DT40 cells and the DT40-derived mutants lacking either Syk or Lyn, have revealed that Syk is a negative regulator of osmotic stress-induced apoptosis, while Lyn is a positive regulator of UVC radiation-induced apoptosis (11).

In this study, we have systematically examined the roles of non-receptor PTK, Btk, Syk and Lyn, in apoptotic processes induced by different types of anti-cancer drugs using wild-type DT40 cells and their derived mutants lacking the expression of Btk, Syk or Lyn. It was found that all the anti-cancer drugs examined induce apoptosis of wild-type DT40 cells as assessed by microscopic observation, DNA fragmentation and TUNEL analyses (Figs 1 and 2GoGo). Interestingly, apoptosis induced by Topo II inhibitors, ADR and VP-16, among the anti-cancer drugs examined, was inhibited in DT40/Lyn(–), but not in wild-type DT40, DT40/Btk(–), or DT40/Syk(–) cells (Figs 1 and 2GoGo), indicating that Lyn plays a critical role(s) in Topo II inhibitor-induced apoptotic processes. Furthermore, it was found that this function of Lyn can be compensated by another Src family member, Fyn (Fig. 5Go), suggesting possible functional redundancy among Src family PTK. However, it is important to note that an abnormal Ca2+ response as well as tyrosine phosphorylation observed in the same DT40/Lyn(–) cells upon B cell receptor stimulation can be restored largely by an ectopic expression of Fyn and Lck, but not of Src, indicating that some functional specificity among Src family PTK may exist (30,31). Thus, it will be of interest to test whether other Src family PTK, including Lck, can compensate for the function of Lyn in Topo II inhibitor-induced apoptosis.

Topo II is an essential nuclear enzyme involved in DNA replication and gene transcription by regulating the topological status of DNA in eukaryotic cells (35). Topo II acts by passing a double-stranded DNA helix through a transient double-strand break site and then religating the strand break. There are two Topo II isoforms, {alpha} and ß, encoded by the distinct genes, that mediate Topo II activity (36,37). It has recently been shown that Topo IIß is transiently distributed to the cytoplasm during the mitotic stage, while Topo II{alpha} is associated tightly with chromosomes constantly throughout the cell cycle (38,39), suggesting that each isoform plays specific roles in mediating DNA topology in the cell. Topo II is also a critical intracellular target of several clinically important anti-cancer agents. In fact, the anthracyclin ADR and the epipodophyllotoxin VP-16 interact with Topo II to inhibit the religation step of the enzyme (40,41), thereby stabilizing cleavable enzyme–DNA complexes that precede apoptosis. However, sensitivity of these two isoforms of Topo II against these anti-cancer drugs is not fully understood. Our results, showing that Lyn plays a role in the Topo II inhibitor-induced apoptosis (Figs 1–3 and 5GoGoGoGo), suggest that Lyn may interact functionally with Topo II. It is possible that Lyn may mediate the Topo II inhibitor-induced apoptotic signaling. Our results also indicate that Fyn, another Src family member, can compensate this function of Lyn (Fig. 5Go). It has been reported that mitomycin C, CDDP and Ara-C, anti-cancer drugs with DNA alkylating activities, can induce the activation of Lyn, and that Lyn may contribute to mitotic arrest by associating with and phosphorylating cdc2 (42,43). Although under our experimental conditions we failed to detect apparent activation of Lyn following treatment of DT40 cells with Topo II inhibitors when monitored by an in vitro kinase assay (data not shown), it is still possible that Lyn may phosphorylate a particular substrate in vivo upon treatment with Topo II inhibitors. Alternatively, biochemical activity other than tyrosine kinase activity of Lyn may be responsible for its function in Topo II inhibitor-induced apoptotic processes. Further study will be required to understand the role of Lyn in Topo II inhibitor-induced apoptotic signaling.

Recently, much attention has been paid on the roles of JNK in the apoptotic processes induced by various extracellular stresses (33,34). Blocking the JNK pathway abrogates apoptosis induced by extracellular stresses, including hydrogen peroxide, heat shock, UVC and {gamma} irradiation. The fact that a wide variety of anti-cancer drugs can also activate JNK (13,14,26,27,29) led us to examine the role of Lyn in Topo II inhibitor-induced JNK activation. We have observed reproducibly weak activation (1.12- to 1.94-fold) of JNK upon treatment of DT40 cells with ADR or VP-16, although another anti-cancer drug, VBL, induced activation of JNK at higher levels (10.38-fold) (Fig. 4A and BGo). Importantly, this weak, yet reproducible activation of JNK by the Topo II inhibitors, ADR and VP-16, was not affected by the presence or absence of Lyn (Fig. 4A and BGo).

Previous studies demonstrate that c-Abl is required for JNK activation and subsequent apoptosis induced by DNA damaging agents. In fact, ionizing radiation and alkylating agents, including Ara-C (15), CDDP and mitomycin C (13,14), induce the activation of c-Abl, leading to the subsequent activation of JNK. Thus, c-Abl appears to be involved in the stress signaling induced by DNA damaging agents, in particular, alkylating drugs. On the other hand, our results indicate that Lyn is involved in the Topo II inhibitor-induced apoptosis independent of JNK.

An important aspect of cancer therapy by anti-cancer drugs as well as {gamma} irradiation is how to discriminate cancer cells from normal unaffected cells. Our previous and present in vitro studies have revealed that non-receptor PTK, Btk and Lyn, determine the sensitivity of a particular cell (in this case DT40) to apoptosis induced by {gamma} irradiation and Topo II inhibitors respectively. Such in vitro studies may give us a hint to discriminate cancer cells from normal cells, provided that expression levels of a particular molecule (in this case non-receptor PTK) are different between these cells, although possible differences in the effects of anti-cancer treatments in vitro and in vivo have to be considered with caveats.


    Acknowledgments
 
This work was supported by grants provided by a grant-in-aid for general scientific research (09470043) and for scientific research on priority areas (09273104) from the Ministry of Education, Science, Sports and Culture, Japan; by Daiichi Pharmaceutical; by Nippon Boehringer Ingelheim, Kawanishi Pharma Research Institute; by the Kato Memorial Bioscience Foundation; by the Naito Foundation; by Kanae Foundation For Life and Socio-Medical Science; and Yamanouchi Foundation for Research on Metabolic Disorders.


    Abbreviations
 
Ara-C1-ß-D-Arabinofuranosylcytosine
ADRadriamycin
CDDPcis-diamminedichloroplatinum(II)
CPTcamptothecin
JNKc-Jun N-terminal kinase
PTKprotein-tyrosine kinase
Topo IItopoisomerase II
VBLvinblastine
VP-16etoposide

    Notes
 
Transmitting editor: K.-i. Arai

Received 30 September 1998, accepted 10 May 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Ullrich, A. and Schlessinger, J. 1990. Signal transduction by receptors with tyrosine kinase activity. Cell 61:203.[ISI][Medline]
  2. Schlessinger, J. and Ullrich, A. 1992. Growth factor signaling by receptor tyrosine kinases. Neuron 9:383.[ISI][Medline]
  3. Weiss, A. and Littman, D. R. 1994. Signal transduction by lymphocyte antigen receptors. Cell 76:263.[ISI][Medline]
  4. Kurosaki, T. 1997. Molecular mechanisms in B cell antigen receptor signaling. Curr. Opin. Immunol. 9:309.[ISI][Medline]
  5. Klausner, R. D. and Samelson, L. E. 1991. T cell antigen receptor activation pathways: the tyrosine kinase connection. Cell 64:875.[ISI][Medline]
  6. Yanagi, S., Kurosaki, T. and Yamamura, H. 1995. The structure and function of non-receptor tyrosine kinase p72Syk expressed in hematopoietic cells. Cell Signal. 7:185.[ISI][Medline]
  7. Qin, S., Minami, Y., Hibi, M., Kurosaki, T. and Yamamura, H. 1997. Syk-dependent and -independent signaling cascades in B cells elicited by osmotic and oxidative stress. J. Biol. Chem. 272:2098.[Abstract/Free Full Text]
  8. Schieven, G. L., Mittler, R. S., Nadler, S. G., Kirihara, J. M., Bolen, J. B., Kanner, S. B. and Ledbetter, J. A. 1994. ZAP 70 tyrosine kinase, CD45, and T cell receptor involvement in UV and H2O2 induced T cell signal transduction. J. Biol. Chem. 269:20718.[Abstract/Free Full Text]
  9. Nakamura, K., Hori, T., Sato, N., Sugie, K., Kawakami, T. and Yodoi, J. 1993. Redox regulation of a src family protein tyrosine kinase p56lck in T cells. Oncogene 8:3133.[ISI][Medline]
  10. Barchowsky, A., Munro, S. R., Morana, S. J., Vincenti, M. P. and Treadwell, M. 1995. Oxidant-sensitive and phosphorylation-dependent activation of NF-kappa B and AP-1 in endothelial cells. Am. J. Physiol. 269:L829.[Abstract/Free Full Text]
  11. Qin, S., Minami, Y., Kurosaki, T. and Yamamura, H. 1997. Distinctive functions of Syk and Lyn in mediating osmotic stress- and ultraviolet C irradiation-induced apoptosis in chicken B cells. J. Biol. Chem. 272:17994.[Abstract/Free Full Text]
  12. Tokiwa, G., Dikic, I., Lev, S. and Schlessinger, J. 1996. Activation of Pyk2 by stress signals and coupling with JNK signaling pathway. Science 273:792.[Abstract]
  13. Kharbanda, S., Ren, R., Pandey, P., Shafman, T. D., Feller, S. M., Weichselbaum, R. R. and Kufe, D. W. 1995. Activation of the c-Abl tyrosine kinase in the stress response to DNA-damaging agents. Nature 376:785.[ISI][Medline]
  14. Yuan, Z. M., Huang, Y., Whang, Y., Sawyers, C., Weichselbaum, R., Kharbanda, S. and Kufe, D. 1996. Role for c-Abl tyrosine kinase in growth arrest response to DNA damage. Nature 382:272.[ISI][Medline]
  15. Yuan, Z. M., Huang, Y., Ishiko, T., Kharbanda, S., Weichselbaum, R. and Kufe, D. 1997. Regulation of DNA damage-induced apoptosis by the c-Abl tyrosine kinase. Proc. Natl Acad. Sci. USA 94:1437.[Abstract/Free Full Text]
  16. Yang, C., Maruyama, S., Yanagi, S., Wang, X., Takata, M., Kurosaki, T. and Yamamura, H. 1995. Syk and Lyn are involved in radiation-induced signaling, but inactivation of Syk or Lyn alone is not sufficient to prevent radiation-induced apoptosis. J. Biochem. 118:33.[Abstract]
  17. Uckun, F. M., Waddick, K. G., Mahajan, S., Jun, X., Takata, M., Bolen, J. and Kurosaki, T. 1996. BTK as a mediator of radiation-induced apoptosis in DT-40 lymphoma B cells. Science 273:1096.[Abstract]
  18. Devary, Y., Gottlieb, R. A., Smeal, T. and Karin, M. 1992. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell 71:1081.[ISI][Medline]
  19. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz Friedman, A., Fuks, Z. and Kolesnick, R. N. 1996. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 380:75.[ISI][Medline]
  20. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J. and Greenberg, M. E. 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270:1326.[Abstract]
  21. Kufe, D. W., Munroe, D., Herrick, D., Egan, E. and Spriggs, D. 1984. Effects of 1-beta-D-arabinofuranosylcytosine incorporation on eukaryotic DNA template function. Mol. Pharmacol. 26:128.[Abstract]
  22. Eastman, A. 1990. Activation of programmed cell death by anticancer agents cisplatin as a model system. Cancer Cells2:275.[ISI][Medline]
  23. Chen, A. Y. and Liu, L. F. 1994. DNA topoisomerases: essential enzymes and lethal targets. Annu. Rev. Pharmacol. Toxicol. 34:191.[ISI][Medline]
  24. Chen, G. L., Yang, L., Rowe, T. C., Halligan, B. D., Tewey, K. M. and Liu, L. F. 1984. Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J. Biol. Chem. 259:13560.[Abstract/Free Full Text]
  25. Liu, L. F. 1989. DNA topoisomerase poisons as antitumor drugs. Annu. Rev. Biochem. 58:351.[ISI][Medline]
  26. Tewey, K. M., Rowe, T. C., Yang, L., Halligan, B. D. and Liu, L. F. 1984. Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science 226:466.[ISI][Medline]
  27. Osborn, M. T, and Chambers, T. C. 1996. Role of the stress-activated/c-Jun NH2-terminal protein kinase pathway in the cellular response to adriamycin and other chemotherapeutic drugs. J. Biol. Chem. 271:30950.[Abstract/Free Full Text]
  28. Nehme, A., Baskaran, R., Aebi, S., Fink, D., Nebel, S., Cenni, B., Wang, J. Y., Howell, S. B. and Christen, R. D. 1997. Differential induction of c-Jun NH2-terminal kinase and c-Abl kinase in DNA mismatch repair-proficient and -deficient cells exposed to cisplatin. Cancer Res. 57:3253.[Abstract]
  29. Kharbanda, S., Pandey, P., Ren, R., Mayer, B., Zon, L. and Kufe, D. 1995. c-Abl activation regulates induction of the SEK1/stress-activated protein kinase pathway in the cellular response to 1-beta-D-arabinofuranosylcytosine. J. Biol. Chem. 270:30278.[Abstract/Free Full Text]
  30. Takata, M., Sabe, H., Hata, A., Inazu, T., Homma, Y., Nukada, T., Yamamura, H. and Kurosaki, T. 1994. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO J. 13:1341.[Abstract]
  31. Takata, M. and Kurosaki, T. 1995. The catalytic activity of Src-family tyrosine kinase is required for B cell antigen receptor signaling. FEBS Lett .374:407.[ISI][Medline]
  32. Tomei, L. D. and Cope, F. O. 1991. Apoptosis: The Molecular Basis of Cell Death. Curr. Commun. 3.
  33. Zanke, B. W., Boudreau, K., Rubie, E., Winnett, E., Tibbles, L. A., Zon, L., Kyriakis, J., Liu, F. F. and Woodgett, J. R. 1996. The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat. Curr. Biol. 6:606.[ISI][Medline]
  34. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J. and Woodgett, J. R. 1994. The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369:156.[ISI][Medline]
  35. Hochhauser, D. and Harris, A. L. 1993. The role of topoisomerase II alpha and beta in drug resistance. Cancer Treat. Rev. 19:181.[ISI][Medline]
  36. Drake, F. H., Hofmann, G. A., Bartus, H. F., Mattern, M. R., Crooke, S. T. and Mirabelli, C. K. 1989. Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II. Biochemistry 28:8154.[ISI][Medline]
  37. Chung, T. D., Drake, F. H., Tan, K. B., Per, S. R., Crooke, S. T. and Mirabelli, C. K. 1989. Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes. Proc. Natl Acad. Sci. USA 86:9431.[Abstract]
  38. Heck, M. M., Hittelman, W. N. and Earnshaw, W. C. 1988. Differential expression of DNA topoisomerases I and II during the eukaryotic cell cycle. Proc. Natl Acad. Sci. USA 85:1086.[Abstract]
  39. Nakano, H., Yamazaki, T., Miyatake, S., Nozaki, N., Kikuchi, A. and Saito, T. 1996. Specific interaction of topoisomerase II beta and the CD3 epsilon chain of the T cell receptor complex. J. Biol. Chem. 271:6483.[Abstract/Free Full Text]
  40. Osheroff, N. 1989. Effect of antineoplastic agents on the DNA cleavage/religation reaction of eukaryotic topoisomerase II: inhibition of DNA religation by etoposide. Biochemistry 28:6157.[ISI][Medline]
  41. Robinson, M. J. and Osheroff, N. 1990. Stabilization of the topoisomerase II–DNA cleavage complex by antineoplastic drugs: inhibition of enzyme-mediated DNA religation by 4'-(9-acridinylamino)methanesulfon-m-anisidide. Biochemistry 29:2511.[ISI][Medline]
  42. Kharbanda, S., Yuan, Z. M., Taneja, N., Weichselbaum, R. and Kufe, D. 1994. p56/p53lyn tyrosine kinase activation in mammalian cells treated with mitomycin C. Oncogene 9:3005.[ISI][Medline]
  43. Yuan, Z. M., Kharbanda, S. and Kufe, D. 1995. 1-beta-D-arabinofuranosylcytosine activates tyrosine phosphorylation of p34cdc2 and its association with the Src-like p56/p53lyn kinase in human myeloid leukemia cells. Biochemistry 34:4908.[ISI][Medline]