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Previous Article | Next Article 
Molecular and Cellular Biology, June 2002, p. 4189-4201, Vol. 22, No. 12
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.12.4189-4201.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Agnieszka Bronisz,1,
Maciej Malecki,1,&
Janusz Blasiak,3 Richard Fishel,4 and Tomasz Skorski1*
Center for Biotechnology, College of Science and Technology, Temple University,1 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania,4 Department of Clinical Cytobiology, Medical Center for Postgraduate Education, Warsaw,2 Department of Molecular Genetics, University of Lodz, Lodz, Poland3
Received 1 August 2001/ Returned for modification 20 September 2001/ Accepted 18 March 2002
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FTKs and other oncogenic tyrosine kinases such as v-Src and HER-2/neu activate multiple signaling pathways responsible for protection from apoptosis, induction of growth factor-independent proliferation, transformation, and resistance to therapeutic drugs and to 
-radiation (25, 41, 43, 53, 54, 61, 85). Resistance to DNA-damaging agents is a cause for failure in the therapy of human cancer, including hematological malignancies. Several mechanisms of resistance to DNA damage have been suggested (18), including overexpression of the P-glycoprotein family of membrane transporters, which decrease the intracellular accumulation of the drugs (e.g., MDR1), changes in cellular proteins involved in detoxification (e.g., glutathione S-transferase) or activation of chemotherapeutic drugs (e.g., NADP), elevation of expression of antiapoptotic proteins regulating caspase-3 (e.g., Bcl-2 and Bcl-xL), transient cell cycle arrest, and modulation of DNA repair. Existing evidence indicates that the last three mechanisms may be engaged in drug resistance induced by BCR/ABL kinase (2, 4, 17, 46, 73).
BCR/ABL enhanced the expression and phosphorylation of the RAD51 protein (73). It is notable that RAD51 has been proposed to play a central role in homology-dependent recombinational repair (HRR) of DNA double-strand breaks (DSBs) (63, 75, 76). BCR/ABL-dependent stimulation of RAD51 facilitated HRR of DSBs and induced drug resistance in leukemic cells (73). DSBs are considered the principal lethal DNA damage resulting from treatment with radiomimetic and cross-linking drugs. DSBs can be generated when replication forks encounter the drug-induced DNA lesions (34). HRR mechanisms are known to resolve such replication fork-associated DSBs (3). It is of great importance that cells recognize DSBs and act upon them rapidly and efficiently, because cell death or impaired cell function can occur if these are left unrepaired or are repaired inaccurately.
In addition to DNA repair mechanisms, cell cycle checkpoint activation processes are initiated in response to DNA damage (14). More specifically, DNA damage signals to arrest cell cycle progression, giving the cell more time to repair what might otherwise be a fatal lesion. Numerous studies have implicated a G2/M checkpoint, because its disruption sensitized tumor cells to genotoxic agents (8, 9). Delay in the G2/M transition appears to be necessary for resistance to DNA damage in BCR/ABL-positive cells (4, 46, 77).
Mechanisms preventing mitochondrion-dependent activation of caspase-3 were also implicated in drug resistance of BCR/ABL cells (2, 17). BCR/ABL can inhibit apoptosis by modulation of Bcl-2 family members (1, 59, 60), which regulate activation of caspase-3 (26). Some FTKs can enhance expression of antiapoptotic members of the family, such as Bcl-xL (1, 33) and Bcl-2 (67), and inhibit the proapoptotic function of Bad (59). These events may prevent the release of cytochrome c from the mitochondria and activation of caspase-3 (2, 17, 31).
Taken together, previous reports implicated DNA repair, activation of a cell cycle checkpoint, and dysregulation of Bcl-2 family members in resistance to DNA damage (2, 4, 17, 46, 73). In this work we demonstrate that these mechanisms can work in concert to induce drug resistance in FTK-transformed cells.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-BCR/ABL and pSR-NPM/ALK retroviral constructs have been described (45, 72). The MigR1-BCR/ABL retroviral construct was obtained from Warren Pear (University of Pennsylvania, Philadelphia, Pa.). The cDNAs for TEL/ABL, TEL/JAK2, and TEL/TRKC(L) were obtained from D. Gary Gilliland (Brigham and Women's Hospital, Boston, Mass.) and subcloned into the pMSCV retroviral plasmid. The pSR-TEL/PDGFßR retroviral construct was received from Martin Carroll (University of Pennsylvania, Philadelphia, Pa.). Human RAD51 cDNA in the sense (S) and antisense (AS) orientations was cloned as RAD51-internal ribosome entry site (IRES)-green fluorescent protein (GFP) into the retroviral vector MigR1 (73). In addition, RAD51(S) and RAD51(AS) cDNAs were cloned into the pLXSP-puro and pMX-puro retroviral constructs. The pGL3-Basic vector containing the RAD51 promoter region and the pMX retroviral construct carrying the STAT5 dominant-negative mutant (STAT5-DNM, 
STAT5B) have been described (45, 73). Cells. The murine growth factor-dependent myeloid cell line 32Dcl3 and BCR/ABL-expressing clones have been described (45). The murine growth factor-dependent pro-B lymphoid cell line BaF3 and the BCR/ABL-transformed clone were obtained from Richard Van Etten (Harvard Medical School, Boston, Mass.). BaF3 cell lines transfected with NPM/ALK or with empty virus (BaF3-neo) were obtained from Steven Morris (St. Jude Children's Research Hospital, Memphis, Tenn.). BaF3 cells expressing TEL/JAK2, TEL/ABL, and TEL/TRKC(L) were obtained from D. Gary Gilliland, and BaF3 cells expressing TEL/PDGFßR were obtained from Martin Carroll. 32Dcl3 cells and BCR/ABL-positive cells expressing ectopic RAD51 were obtained after retroviral infection with the pMX-RAD51 retroviral construct and selection of the clones in puromycin. Overexpression of RAD51 protein was confirmed by Western analysis. 32Dcl3 cells overexpressing Bcl-xL were obtained from Daniel Link (Washington University School of Medicine, St. Louis, Mo.) and electroporated with the pMX-RAD51 plasmid. Clones overexpressing both Bcl-xL and RAD51 were obtained after selection in puromycin. Western analysis confirmed the elevated expression of Bcl-xL and RAD51.
Cell lines were maintained in Iscove’s Modified Dulbecco medium supplemented with 10% fetal bovine serum (FBS) and 15% WEHI-conditioned medium. Bone marrow mononuclear cells (BMCs) from C57BL/6 mice (The Jackson Laboratory, Bar Harbor, Maine) were obtained 6 days after treatment with 5-fluorouracil (67, 71). BMCs from CML blast crisis (CML-BC) patients were obtained after informed consent was given, and CD34+ cells were isolated (69). Primary hematopoietic cells were maintained in the presence of recombinant human interleukin-3 (IL-3) and stem cell factor as described (67, 69, 71). Draa-40 cells (52) were cultured in alpha minimal essential medium (-MEM) supplemented with 10% FBS. Tk-ts13 hamster fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS.
Drug resistance assays. Cisplatin (Platinol-AQ; Bristol-Myers Squibb Co., Princeton, N.J.) or mitomycin C (Sigma Chemical Co., St. Louis, Mo.) was added to cells growing in semisolid medium (103/ml) (MethoCult H4230; StemCell Technologies Inc., Vancouver, Canada) supplemented with IL-3. Colonies were scored after 7 days. Results are represented as the percentage of colony-forming cells after drug treatment in comparison to the untreated control group. Viable cells in suspension cultures were detected by trypan blue dye exclusion after 48 h of incubation with the drug. Results represent the percentage of drug-treated cells excluding trypan blue in comparison to the untreated cells.
Western analysis. Cells were solubilized in lysis buffer (10 mM HEPES [pH 7.5], 150 mM NaCl, 1% NP-40, 10% glycerol, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 1 mM Na3VO4, and 10 µg each of aprotinin and leupeptin/ml). Cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and examined by Western analysis with the following antibodies: anti-RAD51 (3C10; Upstate Biotechnology, Lake Placid, N.Y.), antiphosphotyrosine (PY20 from Oncogene Research Products, Cambridge, Mass., and 4G10 from Upstate Biotechnology, Lake Placid, N.Y.), antiactin (C11; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), anti-Bcl-2 (N-17; Santa Cruz), and anti-Bcl-x (Transduction Laboratories, Lexington, Ky.).
Luciferase assay. The RAD51 promoter transactivation assay was performed as described (73) with modifications. Tk-ts13 cells were transiently transfected by calcium phosphate (67, 68) with plasmids encoding FTKs or empty plasmid, STAT5-DNM or empty plasmid, as well as the RAD51 reporter plasmid containing the fused CpG-rich region of the human RAD51 promoter, the simian virus 40 (SV40) promoter, and the luciferase gene (RAD51-SV40-luc). The negative control plasmid encoded the SV40 promoter fused to the luciferase gene only (SV40-luc). A ß-galactosidase plasmid was also transfected into the Tk-ts13 cells as a transfection efficiency control. A total of 20 µg of cDNA/10-cm plate was used (FTK:DNM ratio, 1:4). At 48 h after transfection, serum-free medium containing 0.1% bovine serum albumin was added to the cells, and 12 h later luciferase was quantified by the luciferase assay system (Promega, Madison, Wis.). Transfection efficiency was normalized by measuring ß-galactosidase activity. Transactivation units were calculated as a ratio of the counts from RAD51-SV40-luc to the counts from SV40-luc in particular groups.
GFP-positive cells. Cells were infected with retrovirus-based particles encoding GFP as described (45), except that ecotropic Bosc23 (obtained from Warren Pear) and amphotropic Phoenix (American Type Culture Collection [ATCC], Manassas, Va.) packaging cells were used to infect murine and human cells, respectively. Transduced cells were collected after 48 to 72 h of cocultivation with the packaging cell line. GFP-positive cells were isolated by fluorescence-activated cell sorting (Becton Dickinson).
Inhibition of BCR/ABL kinase. ABL kinase inhibitor STI571 (16) was obtained from Novartis Pharma AG (Basel, Switzerland). Cells (106/ml) were incubated for 24 h with 1 µM STI571 in the presence of IL-3, then washed, and used for experiments.
HRR assay. Draa-40 cells (generously provided by M. Jasin, Sloan-Kettering Cancer Center, New York, N.Y.) have integrated one or two copies of the modified gene for GFP (SceGFP) as a recombination reporter and a fragment of the GFP gene as a donor for homologous repair (52). SceGFP has an inactivating insertion containing the restriction site for the rare-cutting I-SceI endonuclease. When I-SceI is expressed in vivo, a DSB results. An HRR event with a donor GFP gene fragment restores functional GFP expression, readily detected by flow cytometry. Cells were electroporated as described (52) with 100 µg of the I-SceI plasmid, 40 µg of the plasmid carrying FTK or not (control), and 40 µg of the RAD51 antisense plasmid or empty plasmid, and 48 h later 5 x 104 cells were analyzed by flow cytometry for the expression of GFP.
Modifications of G2/M cell cycle. G2/M delay was induced by nocodazole (46). Briefly, 15 ng of nocodazole (Sigma, St. Louis, Mo.) per ml was added to the cell culture 4 h before the drug. Twenty-four hours later, nocodazole and the drugs were removed by washing and replacing the culture medium. This protocol caused transient accumulation of cells in G2/M cell cycle detectable after 12 h and 24 h but not after 48 h of nocodazole treatment (data not shown). DNA damage-dependent G2/M delay was abolished by treatment with caffeine (84). Briefly, cisplatin or mitomycin C was added at 0 h, followed by addition of 2 mM caffeine at 12 h.
Cell cycle analysis. Cells (106) were fixed in 70% ethanol for 15 min at 4°C, washed, and incubated in 1 ml of phosphate-buffered saline (PBS) containing 0.1% NP-40 and 1 mg of DNase-free RNase (Boehringer Mannheim Co., Indianapolis, Ind.) per ml for 10 min at room temperature. DNA was stained by propidium iodide. Cells were analyzed with a FACSCalibur (Becton Dickinson) using the CellQuest program (70).
Immunofluorescence. RAD51 was detected by immunofluorescence as described (73). Briefly, cytospin slides were prepared from GFP+ CML-BC cells infected with the IRES-GFP or RAD51(AS)-IRES-GFP retrovirus. Cells were fixed with PBS containing 0.02% Triton X-100 and 4% formaldehyde. RAD51 was detected by mouse anti-RAD51 monoclonal antibody (Upstate Biotechnology), followed by tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse immunoglobulin secondary antibody (Molecular Probes Inc., Eugene, Oreg.). Negative controls were performed without primary antibodies. DNA was counterstained with the DNA fluorochrome 4',6'-diamidino-2-phenylindole (DAPI). Cells were visualized with a Nikon Eclipse E300 fluorescence microscope equipped with a digital camera. Images were prepared with Adobe Photoshop.
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RESULTS |
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RAD51 plays an essential role in FTK-induced drug resistance. Our previous findings implicated RAD51 in resistance to radiomimetic and cross-linking drugs in BCR/ABL cells (73). Western analysis showed that cells transformed by various FTKs, except TEL/TRKC(L), expressed five to eight times more RAD51 than parental cells growing in the presence of IL-3 (Fig. 2A, panel IL-3+). High levels of RAD51 were also detected in these cells growing in the absence of IL-3 (Fig. 2A, panel IL-3-). STAT5-mediated transactivation of the RAD51 promoter and inhibition of RAD51 cleavage by caspase-3 might contribute to elevated expression of RAD51 in BCR/ABL-transformed cells (73). Interestingly, cells expressing TEL/TRKC(L), which did not activate STAT5 (41), also did not elevate RAD51 protein (Fig. 2A).
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Because RAD51 overexpression appeared to be directly associated with drug resistance in BCR/ABL-transformed cells (73), we sought to determine if RAD51 plays a more general role in drug resistance of the cells transformed by FTKs able to activate STAT5. Genetic studies of mammalian RAD51 are complicated by the fact that it appears to be intimately involved in cellular proliferation (75) and the mouse gene knockout model displays both an embryo- and cell-lethal phenotype (40, 78). To circumvent these problems, we developed a derivative of retroviral construct MigR1 (50) to efficiently transduce isogenic cell lines and transiently affect RAD51 expression. The MigR1 derivative encodes a variant of the murine myeloproliferative sarcoma virus long terminal repeat (LTR) promoter upstream of the RAD51 antisense [RAD51(AS)] gene, an IRES, and the GFP gene.
Parental and FTK-transformed cells were infected with the MigR1 (control) or MigR1-RAD51(AS) plasmid, respectively, and GFP+ cells were isolated by fluorescence-activated cell sorter. RAD51 protein expression in FTK (BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFßR, and NPM/ALK)-positive GFP+ cells transfected with RAD51 antisense cDNA was downregulated to the levels detectable in GFP+ parental cells transfected with empty plasmid (Fig. 2C, lower panel). Expression of RAD51 in GFP+ TEL/TRKC(L) cells was similar to that in parental cells. GFP+ cells were incubated with cisplatin or mitomycin C, and viable cells were detected by using the trypan blue exclusion test. The clonogenic assay could not be applied at this time because downregulation of RAD51 may have an adverse effect on hematopoietic cell proliferation after 5 to 7 days of culture (data not shown).
Both tests, the clonogenic assay and trypan blue exclusion assay, demonstrated concordance for determining drug resistance and survival following drug treatment (73). Inhibition of RAD51 expression reduced the resistance to cisplatin and mitomycin C in FTK (BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFßR, and NPM/ALK)-transformed cells (Fig. 2C, upper panels). Diminished levels of RAD51 did not significantly alter the sensitivity of parental cells and TEL/TRKC(L)-positive cells to these drugs. Taken together, these observations and previous findings (73) clearly implicate RAD51 in the drug resistance of FTK-positive hematopoietic malignancies overexpressing RAD51.
Drug resistance induced by the BCR/ABL
RAD51 pathway was associated with enhanced HRR of DSBs (73). Thus, we examined if other FTKs are also able to stimulate HRR. The frequency of DSB repair in cells can be quantitated by using a stable cell line which contains heteroallelic nonfunctional fragments of the GFP protein (52). In this system, recombination is induced by transfection of the I-SceI restriction enzyme, which results in a DSB within one of the GFP parental alleles. HRR of this DSB can result in the expression of a functional GFP protein, which may be identified as GFP-positive cells (52). We observed a significant (P < 0.05) increase in the percentage of GFP+ cells after cotransfection with FTKs (BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFßR, NPM/ALK) and I-SceI compared to empty vector and I-SceI (Fig. 2D). Moreover, cotransfection of the RAD51(AS) with the I-SceI resulted in a significant (P < 0.05) reduction of GFP repair induced by these FTKs (Fig. 2D). Interestingly, TEL/TRKC(L), which did not elevate RAD51 expression, exhibited a reduced ability to stimulate GFP repair (Fig. 2D). Taken together, these results link RAD51-dependent recombination repair with the drug resistance of FTK-transfected cells overexpressing RAD51.
Elevated level of RAD51 is not the only factor contributing to drug resistance in FTK-transformed cells. To determine if RAD51 is solely responsible for the drug resistance of FTK-transformed cells, we took advantage of the BCR/ABL-selective inhibitor STI571 (16). Inhibition of BCR/ABL kinase activity by STI571 (Fig. 3A, lower panel, compare lanes 3 and 4) was associated with abrogation of drug resistance (Fig. 3B) and downregulation of RAD51 protein (Fig. 3A, upper panel, compare lanes 3 and 4). This observation, however, did not quantitate the contribution of RAD51 to the drug resistance phenotype because other genes and mechanisms are also dysregulated after inhibition of the BCR/ABL kinase (21, 48).
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Collaboration between RAD51, G2/M delay, and Bcl-xL in drug resistance.
Previous findings indicated that BCR/ABL stimulates RAD51, Bcl-xL (but not Bcl-2), and G2/M delay, which contributes to the resistance to genotoxic stress (1, 4, 73). We investigated if these processes are regulated by other FTKs. Western analysis and the DSB repair assay demonstrated that RAD51 is stimulated by the FTKSTAT5 pathway (Fig. 2A and 2B). The levels of Bcl-xL but not Bcl-2 protein were also upregulated by FTKs able to activate STAT5 (Fig. 4A). Parental BaF3 and FTK (BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFßR, and NPM/ALK)-positive cells growing in the presence of IL-3 accumulated in G2/M phase 12 h after drug treatment, but only the latter cells remained accumulated in G2/M after 16 h (percentages: parental, 12% ± 2%, versus BCR/ABL, 39% ± 5%; TEL/ABL, 33% ± 4%; TEL/PDGFßR, 37% ± 4%; TEL/JAK2, 44% ± 4%; and NPM/ALK, 40% ± 3%; P < 0.05) and 24 h (percentages: parental, 10% ± 3%, versus BCR/ABL, 29% ± 4%; TEL/ABL, 27% ± 4%; TEL/PDGFßR, 25% ± 5%; TEL/JAK2, 32% ± 4%; and NPM/ALK, 28% ± 3%; P < 0.05) (Fig. 4B). Similar G2/M delay was induced in BCR/ABL-transformed 32Dcl3 cells after treatment with cisplatin and mitomycin C (data not shown). TEL/TRKC(L)-transformed cells exhibited accumulation in S phase at 12 h after the treatment, but at 16 h and 24 h a significant proportion of these cells was also accumulated in G2/M (28% ± 4% and 31% ± 3%, respectively; P < 0.05 in comparison to parental cells) (Fig. 4B). At 48 h after treatment, FTK-positive cells did not display G2/M accumulation (data not shown). Thus, all FTK-positive cells examined displayed G2/M delay upon DNA damage.
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These processes (RAD51, Bcl-xL, and G2/M delay) are regulated independently, because stimulation of a single factor did not affect the others. For example, overexpression of RAD51 did not influence Bcl-xL or G2/M, overexpression of Bcl-xL did not affect RAD51 or G2/M, and G2/M delay had no effect on RAD51 or Bcl-xL (Fig. 5A and B). Individually, elevated expression of RAD51 (Fig. 5A, lane 3) or Bcl-xL (Fig. 5A, lane 4) and G2/M delay (Fig. 5B, panel 5) caused only moderate drug resistance in parental cells (Fig. 5C).
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Role of RAD51, Bcl-xL, and G2/M delay in drug resistance of BCR/ABL-positive primary leukemia cells. Since the role of RAD51, Bcl-xL, and the G2/M checkpoint in FTK-dependent drug resistance has been described in cell lines, the relevance of these mechanisms in primary cells needed to be addressed. RAD51 was found to be overexpressed in CML cells (73), which exhibited drug resistance (e.g., an average of 23.5% ± 3% and 18% ± 1.5% of CD34+ bone marrow cells obtained from three CML-BC patients survived treatment with 1.5 µg of cisplatin and 0.06125 µg of mitomycin C, respectively, in comparison to 7.5% ± 2% and 5% ± 2% of bone marrow cells from three normal donors, respectively [data not shown]; P < 0.05).
Downregulation of RAD51 protein in CML cells by expression of the RAD51 antisense construct (Fig. 6A, left panel) increased their sensitivity to cisplatin and mitomycin C (Fig. 6A, right panel). Expression of BCR/ABL also induced drug resistance in primary murine bone marrow cells (e.g., 50.5% ± 5% and 32% ± 5.5% of BCR/ABL-positive cells [Fig. 6C, right panel, bars 3 and 7, respectively] versus 16.5% ± 2.5% and 2% ± 1.5% of normal cells [data not shown] were able to form colonies after treatment with 0.375 µg of cisplatin per ml or 0.1 µg of mitomycin C per ml, respectively; P < 0.05).
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Prolonged G2/M accumulation of BCR/ABL-positive cells in comparison to control cells observed at 48 h after the treatment was accompanied by inhibition of the former cells, exhibiting the signature of apoptosis (subdiploid amount of DNA) (4.5% ± 2.5% in comparison to 30.5% ± 5%, respectively, P < 0.05). Disruption of G2/M delay by caffeine (for example see Fig. 6C, right panel box) significantly increased drug sensitivity of BCR/ABL cells (Fig. 6C, right panel). In conclusion, all three mechanisms, overexpression of RAD51 and Bcl-xL and G2/M delay, seem to work in the primary hematopoietic cells transformed by BCR/ABL and possibly also by other FTKs, such as TEL/ABL, TEL/JAK2, TEL/PDGFßR, and NPM/ALK.
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DISCUSSION |
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Here, we report that members of the family of BCR/ABL-related FTKs [BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFßR, NPM/ALK, and TEL/TRKC(L)] are able to induce drug resistance. FTK-transformed cells displayed similar or sometimes even more DNA strand breaks than normal cells after incubation with cisplatin or mitomycin C. These drugs do not react with DNA in a manner that would lead directly to strand breaks, and therefore the breaks must arise as indirect, secondary DNA lesions in the attempt to repair primary damage (82). When a replication fork encounters a drug-induced DNA lesion, it causes stalling of the replication process. To resolve this problem, single-strand breaks and/or DSBs occur; the latter are usually lethal if not repaired (34).
DSBs associated with replication forks are predominantly repaired by RAD51-dependent homologous recombination (3). This process is crucial for reinitiation of DNA replication on collapsed replication forks. We postulate that FTKs can stimulate HRR, resulting in drug resistance. In accordance with this hypothesis, we report that RAD51 was upregulated by various FTKs (BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFßR, and NPM/ALK) in a STAT5-dependent manner and played an essential role in facilitation of HRR (73; this work). This process might efficiently eliminate DSBs caused by cisplatin and mitomycin C and diminish signaling to apoptosis. In comparison, nontransformed cells express basal levels of RAD51, and thus DSBs are resolved with lower efficiency.
Interestingly, expression of RAD51 and drug resistance of TEL/TRKC(L)-transformed cells were significantly reduced in comparison to those in cells transformed by other FTKs (BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFßR, and NPM/ALK). Although TEL/TRKC(L) is a potent activator of the phospholipase C, phosphoinositol 3-kinase, SHC, and mitogen-activated protein kinase, in striking contrast to other FTKs, it does not activate STAT5 (41). The observation implicates the FTKSTAT5downstream effector (e.g., RAD51 and Bcl-xL) pathway in drug resistance (73, 74). This hypothesis is strengthened by the recent evidence that a STAT5 dominant-negative mutant was able to sensitize BCR/ABL-positive cells to DNA damage-induced apoptosis (29, 65).
In general, there appears to be a fundamental correlation between the expression of RAD51 and experimental resistance to therapeutic drugs (79, 81). Elevated levels of RAD51 have been linked to chlorambucil resistance in B-cell chronic lymphocytic leukemia (10) and with enhanced survival of pancreatic adenocarcinoma after treatment with the DSB-inducing therapeutic drug calicheamicin 1 (42). Conversely, downregulation of RAD51 increased the radiosensitivity of prostate cancer cells and malignant glioma cells (13, 49).
RAD51 has also been suggested to play an essential role in the repair of chromosome breaks in normal proliferating cells (75). Therefore, a basal level of RAD51 appears to be required for the processing of spontaneous DNA lesions. Oncogenes that alter the expression, phosphorylation, nuclear localization, and/or function of RAD51 may enhance global DNA repair efficiency and lead to drug resistance. In addition to its role in drug resistance, elevated expression or phosphorylation of RAD51 may be responsible for intrachromosomal or interchromosomal deletions and chromosomal translocations, usually mediated by homologous recombination between regions of shared homology (e.g., Alu sequences) (for a review, see reference 5). This hypothesis is supported by the recent finding that large submicroscopic deletions in regions with high overall density of Alu sequences repeats have been detected in BCR/ABL-positive leukemias (36). RAD51-dependent homologous recombination can also facilitate loss of heterozygosity by gene conversion mechanisms. Moreover, in certain conditions RAD51 can permit exchange between DNA strands containing short nonhomologous sequences (7). Thus, even modest changes in the cellular levels or the activity of RAD51 may not only cause drug resistance but also promote genomic instability.
Elevated expression of RAD51 induced drug resistance, but not of the same magnitude as that observed in FTK-positive cells. This observation suggested that additional mechanisms are stimulated by FTKs which contribute to drug resistance. Previous reports implicated upregulation of Bcl-xL (1) and G2/M delay (4, 46) in BCR/ABL-dependent drug resistance. Bcl-xL can prevent apoptosis by inhibition of the release of cytochrome c from mitochondria and subsequent activation of caspase-3 (31). G2/M delay seems to provide more time to repair potentially lethal DNA lesions (8). We report here that FTKs [BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFßR, and NPM/ALK but not TEL/TRKC(L)] enhanced Bcl-xL expression and that all FTK-positive cells tested displayed G2/M delay upon drug treatment. Thus, at least three mechanisms can eventually contribute to drug resistance induced by FTKs: RAD51-mediated HRR, elevation of Bcl-xL expression, and G2/M delay. Each mechanism was able to induce modest drug resistance in parental cells. Interestingly, simultaneous overexpression of RAD51 and Bcl-xL and induction of G2/M delay caused the strongest drug resistance. These observations indicate that various mechanisms may work in concert to protect leukemic cells from apoptosis induced by DNA damage.
Enhanced expression of RAD51 and Bcl-xL combined with G2/M delay induced drug resistance in parental cells of the magnitude similar to that observed in cells transformed by FTKs able to activate STAT5. It is tempting to speculate that combination of these factors is sufficient for the drug resistance, especially because there is genetic evidence suggesting that cells in G2 phase repair their DSBs by using primarily the HRR mechanism (20). This hypothesis, however, should be tempered by the fact that overexpression of RAD51 and Bcl-xL proteins in parental cells (even if comparable to that observed in FTK-transformed cells) has been driven by viral promoters. Moreover, in some circumstances ectopically overexpressed Bcl-xL could partially downmodulate HRR induced by elevated levels of RAD51 (58). Also, the mechanism of G2/M delay caused by nocodazole may be at least partially different from that induced by DNA damage.
G2/M phase is generally controlled by two checkpoints, at the G2M transition (G2 checkpoint) and at the metaphaseanaphase transition (M checkpoint) (14, 62). G2 checkpoint depends on the mechanisms regulating the Cdc2-cyclin B1 complex, such as ATMChk1serine phosphorylation of Cdc25C phosphatase, driving it outside the nucleus and preventing activation of Cdc2-cyclin B1, and p53p21 and 14-3-3
, resulting in inactivation and sequestration of the Cdc2-cyclin B1 complex in the cytoplasm (8). The M checkpoint is controlled by the mitotic spindle assembly mechanisms (62). Genetic and biochemical evidence showed that the G2 and M checkpoints could be activated by DNA lesions to prolong G2/M phase and reduce the cytotoxicity of DNA damage (9, 32, 39, 84).
The mechanisms of G2/M delay in FTK-positive cells have not been characterized, but regulation of Cdc2-cyclin B1 could be involved (46). Although there is no direct evidence that FTKs can affect the M checkpoint, CML cells contain elevated levels of MAD2 and BUB1 (T. Skorski, unpublished observation), which inhibit the anaphase-promoting complex and cause mitotic spindle arrest (62). Thus, it seems reasonable to speculate that both the G2 and M checkpoints may contribute to G2/M delay in FTK-positive cells treated with DNA-damaging agents. Nocodazole, a mitotic spindle inhibitor (83), does not affect the Cdc2-cyclin B1 complex, but requires BUB1 and MAD2 to exert cell cycle delay (46, 80). Therefore, the G2/M delay induced in parental BaF3 cells by nocodazole probably does not exactly mimic that observed in FTK-positive cells after DNA damage, but may demonstrate some similarities. Although nocodazole arrests cells after chromosome condensation, cells can decondense their chromosomes and return to G2 phase before reentering M phase after removal of nocodazole (56). A similar decondensation of chromosomes occurs when G2/M checkpoints are triggered by DNA damage (57), perhaps allowing better access of DNA repair machinery to the damaged DNA. In agreement with these observations, we (this work) and others (32) showed that nocodazole-induced G2/M delay reduced the genotoxic effect of drugs such as ethyl methanesulfonate, cisplatin, and mitomycin C when examined after 48 h (apoptosis) and 7 days (clonogenicity). In contrast, colcemid did not provide any protective effect shortly (6 h) after DNA damage induced by irradiation (12). This discrepancy may depend on differences in the experimental protocols and/or in the DNA lesions caused by cytostatic drugs and irradiation.
Therefore, results from our experimental model may lead to overestimation of the role of RAD51, Bcl-xL, and G2/M delay in the induction of resistance of FTK-positive leukemias and lymphomas to the cross-linking and radiomimetic drugs. We cannot rule out other mechanisms, such as G1 cell cycle arrest (14), elevated expression of Bcl-2 (55), and repair of DSBs by nonhomologous end joining (47). However, we (this work) and others (4, 46) did not detect a pronounced G1 arrest in FTK-transformed cells in response to DNA damage; conversely, the role of G2/M was emphasized. Bcl-xL but not Bcl-2 was reported to have a significant inhibitory effect on the activation of caspase-3 in BCR/ABL-transformed cells treated with cytostatics (1), but we cannot exclude that other antiapoptotic pathways may also contribute to drug resistance (59). The recent finding that BCR/ABL downregulates DNA-protein kinase catalytic subunits (15) diminishes the potential role (if any) of nonhomologous end joining in the drug resistance of FTK-transformed cells. Although RAD51-mediated HRR seems to represent the major pathway of repair of lethal DNA lesions in these cells (3), mechanisms such as single-strand annealing, nucleotide excision repair, and base excision repair may be also regulated by the FTKs and contribute to DNA repair.
The collaboration between RAD51, Bcl-xL, and G2/M delay to induce drug resistance was described for murine hematopoietic cell lines. Since these three components are also functional in primary leukemia cells expressing BCR/ABL, it seems reasonable to speculate that they may collaborate to induce drug resistance in these cells and possibly in other FTK-positive hematological malignancies.
In conclusion, we hypothesize that FTKs, depending on their ability to activate STAT5, may induce common mechanisms of drug resistance, consisting of the stimulation of RAD51 (facilitated DSB repair), Bcl-xL (inhibition of caspase-3 activation), and/or prolongation of G2/M cell cycle phase (more time for DNA repair). These mechanisms can work in concert to participate in therapeutic drug resistance in leukemia/lymphoma cells and to promote genomic instability leading to a more malignant phenotype of the disease (Fig. 7).
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant CA89052 from the National Cancer Institute, by American Cancer Society grant RPG-98-348-01-LBC, and by Medical Center for Postgraduate Education grant 501-2-1-03-97/07 (all to T.S.). T.S. is a Scholar of the Leukemia and Lymphoma Society. A.S. is a recipient of the fellowship from the Leukemia Research Foundation and is also supported by a grant from the Elsa U. Pardee Foundation. A.B. and M.M. were recipients of fellowships from the Batory Foundation.
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FOOTNOTES |
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Present address: Department of Molecular Genetics, University of Lodz, Lodz, Poland.
Present address: Department of Cell Biology, Cancer Center, Warsaw, Poland.
Present address: Laboratory of Molecular Neuropathology, Medical Research Center, Polish Academy of Sciences, Warsaw, Poland.
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). Downregulation of RAD51 in GFP-positive cells was determined by immunofluorescence analysis visualizing the levels of endogenous RAD51 (left panel). Drug sensitivity was assessed by the trypan blue exclusion test after 48 h of exposure to the indicated concentrations of cisplatin or mitomycin C. (B) BCR/ABL enhances expression of Bcl-xL in primary bone marrow cells. Murine bone marrow cells were infected with BCR/ABL-IRES-GFP virus (GFP+BCR/ABL) or IRES-GFP (GFP) empty virus. Bcl-xL expression was examined by Western analysis in GFP-positive cells. (C) BCR/ABL causes G2/M delay essential for drug resistance. GFP+BCR/ABL-positive cells and GFP-positive control cells were treated with 0.375 µg of cisplatin per ml in the presence of IL-3, and cell cycle analysis was performed after 0, 12, 24, and 48 h (left panel). GFP+BCR/ABL cells cultured in the presence of IL-3 were left untreated (