Chlorambucil Drug Resistance in Chronic Lymphocytic Leukemia: the Emerging Role of DNA Repair1

454 Vol. 7, 454–461, March 2001 Clinical Cancer Research

Minireview Chlorambucil Drug Resistance in Chronic Lymphocytic Leukemia: The Emerging Role of DNA Repair1

Lawrence Panasci,2 Jean-Pierre Paiement, indicates that this disease is initially often very sensitive (hy- Garyfallia Christodoulopoulos, persensitive) to these anticancer agents, to a greater extent than Alexandre Belenkov, Areti Malapetsa, and virtually all epithelial malignancies. A homogeneous monocel- lular population of malignant B lymphocytes is easily obtained Raquel Aloyz from CLL patients, thus providing a relatively unique opportu- Lady Davis Institute for Medical Research, Sir Mortimer B. Davis- nity to study clinically derived cells. We and others have pre- Jewish General Hospital, Montreal, Quebec, H3T 1E2 Canada viously demonstrated that there is a strong correlation between in vitro cytotoxicity of chlorambucil (measured by the MTT Abstract assay) and in vivo response in CLL patients (2–4). Therefore, CLL is an excellent malignancy for in vitro studies that should Various mechanisms have been implicated in nitrogen have direct clinical applicability. mustard drug resistance. The role of these mechanisms in the development of chlorambucil drug resistance in chronic lymphocytic leukemia (CLL) is discussed. We review these Resistance to the NMs mechanisms with emphasis on the emerging role of DNA The NMs are an important group of alkylating agents with repair, and specifically, recombinational repair. Inhibition activity against several human tumors (5–8). Many NM ana- of these repair processes may lead to new therapies, not only logues are transported by carrier-mediated systems into cells in CLL, but in other malignancies as well. and alkylate DNA, RNA, and proteins (9–11). Alkylation of DNA, and more specifically, the formation of DNA ICLs has CLL been considered to be responsible for the cytotoxicity of NMs A model of drug resistance with direct relevance to clinical (12–14). Resistance to the NMs in murine and human tumor practice is a malignancy with easy access to a homogeneous cells has been reported to be secondary to: (a) alterations in the population of malignant cells that represents the clinical status transport of these agents (15); (b) alterations in the kinetics of of the patients. CLL3 is characterized by the proliferation and the DNA cross-links formed by these agents (13, 14, 16); (c) accumulation of B lymphocytes that appear to be mature but are cytoplasmic metabolism of the chloroethyl alkylating moiety to biologically immature. In some patients, CLL has an indolent the inactive hydroxyethyl derivative (17) via GSH/GST (18– course and does not require treatment for many years. When 20); (d) overexpression of metallothionein, which confers re- treatment is necessary, single-agent chemotherapy with a NM, sistance to cis-platinum and cross-resistance to melphalan (21); usually chlorambucil, is the standard initial therapy, although (e) changes in apoptosis (22); and (f) altered DNA repair activity fludarabine, a new exciting agent, may be incorporated in front- (see Fig. 1; Ref. 23). There have been previous reports of line treatment. At least 60–80% of patients respond to NM alterations in the kinetics of DNA ICL formation and removal therapy, often for years, but eventually all patients become associated with resistance to the NMs (13, 14, 16), whereas resistant to these agents (1). Furthermore, many patients with others have found no differences in the ability of sensitive or CLL respond well to low-dose chlorambucil treatment, which resistant cells to remove NM-induced cross-links (24, 25). In this review of NM drug resistance in CLL, preference will be given to investigations that describe protein levels and/or activity rather than mRNA expression. Investigations that describe Received 10/5/00; revised 12/15/00; accepted 12/18/00. mRNA expression will be discussed when studies of activity The costs of publication of this article were defrayed in part by the and/or protein levels are not available. 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. NM Drug Resistance in CLL 1 Supported by a grant from the Leukemic and Lymphoma Society. L. P. is a recipient of the Gertrude and Stanley Vineberg Clinical Scientist Transport and Metabolism. The precise mechanisms Award. responsible for the development of resistance to the NMs in the 2 To whom requests for reprints should be addressed, at Jewish General clinical setting are not known. However, there may be multiple Hospital, 3755 Cote Ste. Catherine Road, Montreal, Quebec H3T 1E2, mechanisms that could differ as a function of the type of cancer. Canada.Phone:(514) 340-8248;Fax:(514) 340-8302;E-mail:LPanasci@ Using CLL as an interesting and easily available clinical hotmail.com. 3 The abbreviations used are: CLL, chronic lymphocytic leukemia; NM, model, we have investigated the transport, metabolism, and nitrogen mustard; GSH, glutathione; GST, GSH S-transferase; ICL, DNA interaction of melphalan in malignant B-lymphocytes interstrand cross-link; DSB, double-strand break; HR, homologous re- obtained from CLL patients (26). We have determined that combinational repair; NHEJ, nonhomologous DNA end-joining; DNA- lymphocytes from chlorambucil-resistant CLL patients are PK, DNA-dependent protein kinase; ATM, (gene mutated in) ataxia telangiectasia; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- cross-resistant to melphalan as compared with untreated CLL lium bromide; TUNEL, terminal deoxynucleotidyl transferase (Tdt)- lymphocytes, and that there is an excellent correlation between mediated nick end labeling. chlorambucil sensitivity and melphalan sensitivity in these pa- Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2001 American Association for Cancer Research. Clinical Cancer Research 455

Fig. 1 Mechanisms of drug -defective muta ,ء .resistance tions of this protein are known to hypersensitize to chloram- -p53 mu ,ءء ;bucil cytotoxicity tations result in chlorambucil resistance; X, DNA-PK increased activity correlates with chlorambucil resistance; ᭹, increased Rad51 foci formation correlates with chlorambucil resistance.

tients (2, 27). In general, chlorambucil-resistant CLL lympho- phalan transport or the intracellular levels of intact melphalan. cytes are 5- to 6-fold resistant in vitro as compared with sensi- There was no evidence of a difference in melphalan metabolism tive lymphocytes using the MTT assay (2). We found no in untreated patients’ lymphocytes versus resistant patients’ significant difference between untreated and treated resistant lymphocytes using TLC (26). In a separate study, chlorambucil patients’ lymphocytes in either the kinetic parameters of mel- uptake and metabolism was investigated in 17 CLL patients

including 3 patients who were clinically resistant to chloram- resistance to glucocorticoids and nucleoside analogues has been bucil. The pattern of drug uptake by CLL lymphocytes of a correlated with altered apoptosis. This initiation leads to various resistant patient was similar to that obtained from untreated biological effects that ultimately converge on the Bcl-2 family patients. Chlorambucil metabolism, as measured by a HPLC pathway. At this point, a cell can be protected from apoptosis or assay, suggested that drug decomposition occurred by a simple be committed to the process. Activation of the cysteine pro- chemical breakdown and that chlorambucil resistance was not teases known as caspases results in DNA degradation. Bcl-X explained by differences in intracellular metabolism (28). Thus, and the Bcl-2:bax ratio are important factors in the chemoresis- it is unlikely that alterations of drug uptake or metabolism are tance to apoptosis (38). responsible for NM resistance in CLL. Expression of apoptosis-regulating proteins in CLL was GSH and GST. Alterations in GSH and GST have been examined in a prospective fashion and correlated with in vitro correlated with NM drug resistance. NMs are a good substrate chemosensitivity using the TUNEL assay and clinical response for GST-␣ (29). However, transfection of GST (␣ or ␮ classes) (39). The CLL patients were previously untreated and enrolled in MCF-7 cells did not result in NM drug resistance (30, 31). on an Eastern Cooperative Oncology Group (ECOG) trial ac- Transfection of ␥-glutamylcysteine synthatase subunits into cording to which they were randomized to receive fludarabine, COS-7 cells resulted in a 2.6-fold increase in GSH levels and a chlorambucil, or both agents. The combined arm was discon-

2-fold increase in melphalan resistance at the LD99 concentra- tinued because of unacceptable toxicity. Also, chlorambucil in tion (32). Expression of a rat GST cDNA in rat mammary vitro sensitivity could not be determined using the TUNEL carcinoma cells resulted in a 6- to 30-fold increase in NM drug assay because of technical problems. However, analysis of DNA resistance (33). integrity has been correlated with chemotherapy response and GST activity was analyzed in a group of CLL patients who high risk/refractory disease in CLL (40, 41). In the ECOG trial, were either untreated or clinically resistant to chlorambucil. in vitro chemosensitivity of fludarabine did not correlate with There was a 2-fold increase in GST activity in the resistant the levels of any of the apoptosis-regulatory proteins (Bcl-2, patients’ lymphocytes, the predominant isozyme being GSTpi Bax, Mcl-1, Bak, Bag-1, and caspase-3). The in vitro chemo- (34). However, it has been suggested that GSTpi overexpression sensitivity data did not correlate with response. However, there is probably an accompaniment rather than a cause of resistance was a significant (P Ͻ 0.01) association between higher Mcl-1 (29). DiSimone et al. (35) found that GST activity was increased levels and failure to achieve complete response, as compared in treated patients and was probably not related to alkylating with partial response or no response, in a group of 37 patients. agent resistance. In contrast, others found no difference in GST Also, a weaker (P Ͻ 0.04) association between poor clinical activity or GSH levels between untreated and treated CLL response and higher Bag-1 protein was observed. These results patients (36, 37). We found no differences in GST activity and were not biased by the type of chemotherapy. Neither Bcl-2 GSH levels between sensitive CLL lymphocytes and chloram- levels nor the Bcl-2:Bax ratio correlated with clinical response bucil-resistant CLL lymphocytes as defined by both the MTT (39). assay and the clinical parameters (2). Thus, there is somewhat Pepper et al. (42, 43) reported a correlation between a conflicting data as concerns the role of GSH/GST in NM drug greater Bcl-2:Bax ratio and resistance to chlorambucil-induced resistance in CLL. However, the lack of evidence for altered apoptosis. However in 25 CLL patients, Johnston et al. (44) intracellular metabolism of melphalan or chlorambucil in resist- found no correlation between chlorambucil or fludarabine sen- ant CLL lymphocytes along with the conflicting evidence of sitivity, as measured by the MTT assay and the levels of the GSH/GST involvement suggest that this detoxification system is Bcl-2 protein or Bcl-2:Bax ratio. We also found no significant unlikely to be the predominant mechanism of NM drug resist- correlation between Bcl-2 protein levels or the Bcl-2:Bax ratio ance in CLL. and the in vitro (MTT assay) or the in vivo response to chloram- Another potential detoxification system that may play a bucil or melphalan in 28 patients (14 untreated and 14 treated role in NM resistance involves metallothionein. We found no resistant patients; Ref. 45). Morabito et al. (46) found no cor- differences in metallothionein mRNA expression between sen- relation between Bcl-2 protein levels in previously untreated sitive and resistant CLL lymphocytes (27). CLL lymphocytes and in vitro chlorambucil cytotoxicity deter- Apoptosis vis-a`-vis NM Drug Resistance in CLL. CLL mined by the MTT assay. is a malignancy of B-lymphocytes that are, at least at early The design of these studies was different. Kitada et al. (39) Ͼ stages, largely ( 95%) mature cells in the G0-G1 phase of the looked prospectively to identify innate drug resistance, whereas cycle. The malignant B-lymphocytes accumulate in the body others examined previously treated resistant CLL patients and because they survive an abnormally long time because of a thus, examined acquired drug resistance. Therefore, the role of dysregulation in the programmed cell death process. Pro- the various proteins involved in apoptosis, such as Bcl-2, Bax, grammed cell death results in apoptosis with its characteristic Bag, and Mcl-1, in chlorambucil drug resistance in CLL is not changes. The Bcl-2 family has a critical role in this process. clear, and additional investigations in acquired and innate drug There are three groups of Bcl-2 proteins. There is a dynamic resistance should be done. Also, the role of the MTT assay relationship between the group I, pro-apoptotic family (Bax and vis-a`-vis assays measuring apoptosis in predicting chlorambucil

Bak), the group II antiapoptotic proteins (Bcl-2, Bcl-XL, Mcl-1, response in CLL needs to be examined prospectively. A1, and Bcl-W), and the group III proapoptotic proteins (Bad, p53 Mutations vis-a`-vis Chlorambucil Drug Resistance.

Bcl-Xs, and Bik). The cell death pathway involves cell death There is a remarkable concordance in the literature as concerns signals from various agents, including chemotherapeutic drugs, p53 mutations in CLL. Only 10–15% of CLL patients have p53 that initiate apoptosis. Resistance to chlorambucil and cross- mutations but in the vast majority of cases, they are associated

with innate chlorambucil resistance (44–49). The overall fre- from DNA treated with chlorambucil (59). We measured quency of p53 immunohistochemical positivity in CLL was also 3-methyladenine-DNA-glycosylase activity in CLL extracts and 15% and correlated with a poorer response to therapy (50). p53 found a significantly (ϳ1.7-fold) higher activity in lymphocytes mutations are not a major factor in acquired drug resistance (45). from resistant CLL patients as compared with those from un- The mechanism by which a p53 mutation results in innate treated CLL patients. Because this activity may vary with cell chlorambucil resistance is not known. proliferation, it was corrected for differences in DNA synthesis DNA Cross-Links vis-a`-vis NM Drug Resistance in using [3H]thymidine incorporation (there were differences in CLL. Chlorambucil-induced alkylation of CLL lymphocyte DNA synthesis between the two groups although the vast ma- DNA results predominantly in the development of purine-drug jority of malignant B lymphocytes are nonproliferative), and this complexes (28). The NMs, including chlorambucil, may also resulted in no significant difference in enzyme activity between form intrastrand and/or ICLs at N-7 guanines (51). The ICLs are the two groups (60). Moreover, overexpression of the human considered to be important in the cytotoxicity of these drugs alkyl-N-purine DNA glycosylase in CHO cells did not result in (12–14). NM resistance, which suggested that alkyl-N-purine DNA gly- There are technical problems involved in quantitating NM- cosylase was not a rate limiting enzyme in NM drug resistance induced ICLs. NMs produce thermolabile glycosylic bonds (N- (61). Furthermore, mouse embryonic stem cells bearing null 7-guanine adducts), which yield apurinic sites and which, in mutations in this enzyme are not hypersensitive to the NMs (62). turn, can cause strand breaks and/or breaks of cross-links (re- Possible insights into mechanism(s) of ICL repair are viewed in Ref. 52). Strand breaks can interfere with molecular gained by examining NM hypersensitivity in DNA repair mu- size-based assays. The ethidium bromide fluorescence assay has tants. Significant NM and/or mitomycin C hypersensitivity the advantage that strand breaks are less likely to influence the (varying from moderate to severe) is found in several DNA quantification of cross-links (53). A widely used assay to deter- repair mutants including ERCC-1, ERCC-4 (XPF), XRCC-2, mine DNA cross-links is the alkaline elution assay (54–56). XRCC-3, Rad54, Ku70, Ku86, and DNA-PKcs (63–67). This technique involves molecular size differences. However, This analysis, along with information gained from studying the strand breaks induced by NMs may complicate interpreta- cross-link removal in both bacteria and Saccharomyces cerevi- tion of repair of ICLs when using this assay. siae, and the possibility that DNA DSBs are repaired in a similar Using the ethidium bromide fluorescence assay, we origi- fashion to ICLs, has resulted in the proposed model that nucle- nally reported (26) that DNA ICL formation at 4 h post- otide excision repair, via the ERCC-1/ERCC-4 endonuclease, melphalan incubation (a time point believed to be associated results in an incision 5Ј to the ICL and that recombinational with maximal cross-link formation) was decreased in malignant repair is involved in further processing of the lesion (reviewed B lymphocytes from resistant CLL patients. However, when we in Refs. 68, 69). As concerns repair of DSBs, nonhomologous examined cross-link formation and removal at 0, 4, and 24 h DNA end-joining uses no, or very limited, sequence homology after a 35-min melphalan incubation, there was evidence of a to rejoin ends directly, whereas homologous recombination re- greater amount of cross-links at time 0 in malignant B lympho- quires extensive regions of DNA homology. HR would be cytes from resistant CLL patients as compared with those from necessary for error-free repair of ICLs, whereas an illegitimate untreated CLL patients. Moreover, the untreated patients’ lym- or NHEJ mechanism of repair could result in deletional repair of phocytes developed a greater amount of cross-links at 4 h ICLs. It is also conceivable that all three types of repair (nucle- without evidence of removal at 24 h, whereas there was evi- otide excision, HR, and NHEJ) are implicated in the repair dence of progressive removal of DNA cross-links at 4 and 24 h simultaneously or are involved, depending on the phase of the in lymphocytes from resistant CLL patients. This suggests that cell cycle, in the processing of ICL. The various genes impli- enhanced DNA repair is implicated in this process (57). In cated in NHEJ include the components of DNA-PK, XRCC-4, another study, a patient with advanced CLL was treated with i.v. and ATM. NHEJ is a major mechanism of DSB repair in cyclophosphamide, and DNA ICLs in the lymphocytes were mammalian cells (reviewed in Refs. 70, 71). HR in human cells measured by the alkaline elution technique. Maximal DNA ICL implicates the HsRad51 family of proteins including HsRad51, formation occurred 12 h after injection. However, the level of HsRad52, Rad51B, Rad51C, Rad51D, HsRad54, XRCC-2, and cross-links was just above the sensitivity of the assay at 12 and XRCC-3. Rad51 binding to DNA requires the precedent binding 24 h after drug administration (58). Also, using the alkaline of Rad52. In addition, other Rad51 protein members may be elution technique, Johnston et al. (37) examined DNA cross-link involved in the assembly of Rad51 complex. Interactions of formation in CLL lymphocytes at 6 h after an in vitro incubation Rad51 with BRCA2, c-Abl kinase, and p53 have also been with chlorambucil. They found that the lymphocytes from two detected (reviewed in Refs. 69, 72). resistant CLL patients had as many DNA cross-links as the To gain insight into possible mechanisms of DNA cross- lymphocytes from patients sensitive to chlorambucil. link removal in NM-resistant CLL, Bramson et al. analyzed in DNA Repair of NM DNA Cross-Links in CLL. The vitro cross-resistance in CLL (2). Chlorambucil-resistant CLL mechanism of removal of DNA ICLs in mammalian cells is lymphocytes were completely cross-resistant to melphalan and poorly understood. There are several different DNA repair sys- mitomycin C; partially cross-resistant to cis-platinum and not tems that could be involved in the repair of NM-induced DNA cross-resistant to UV light nor methylmethane sulfonate. Be- ICLs, including base excision repair, nucleotide excision repair, cause UV radiation damage is repaired by nucleotide excision and recombinational repair. The mammalian base excision re- repair and methylmethane sulfonate is repaired by base excision pair enzyme, alkyl-N-purine DNA glycosylase (3-methylad- repair, it appears that these repair systems are not up-regulated enine-DNA-glycosylase), can excise damaged guanine bases in NM drug resistance in CLL (2). Also, ERCC-1 protein levels

were not increased in NM-drug-resistant CLL lymphocytes (27). kinase superfamily. Other members include ATM and the cell Nucleotide excision repair activity was very low in most CLL cycle checkpoint protein, ATR (71). Recently, loss of heterozy- lymphocytes, including the majority of those obtained from gosity or mutations of the ATM gene and a decrease in ATM previously treated CLL patients (73). protein levels have been found in ϳ30–40% of B-cell CLL NHEJ. Because ionizing radiation results in DSBs that patients. These factors appear to be associated with a shorter are largely repaired by NHEJ (70) and because DSBs are prob- survival, at least in younger patients (82–85). The association of ably repaired in a similar fashion to ICLs, cross-resistance ATM with chlorambucil drug resistance in CLL has not been studies between chlorambucil and ionizing radiation may be investigated to date. informative. Indeed, it appears that there is evidence of cross- HR. The involvement of nucleotide excision repair and resistance between ionizing radiation and chlorambucil in CLL HR in repair of ICLs is inferred from the fact that the mutant cell lymphocytes (74). lines with the greatest sensitivity (10- to 100-fold) to alkylating DNA-PK, a nuclear serine/threonine kinase, is a protein agents that produce ICLs are those that are deficient in, or complex that includes a catalytic subunit of Mr 460,000, DNA- lacking, XRCC2, XRCC3, ERCC1, and ERCC4/XPF (63, 67, PKcs, and a DNA binding subunit, the Ku autoantigen (a dimer 69). The nucleotide excision repair complex (ERCC-1/ERCC-4) of the Ku70 and Ku86 proteins). Ku binds to DSBs and other in mammalian cells makes dual incisions 22–28 bp apart on the discontinuities in the DNA and recruits DNA-PKcs to the dam- same strand, 5Ј to the ICL (86). This would then be followed aged site (70, 71). The active DNA-PK complex can then by HR. phosphorylate many DNA-bound proteins in the vicinity (75). Several human genes implicated in HR have been charac- Because mutations in DNA-PK result in X-ray and alkylating terized including HSRad52, HsRad51, Rad51B, Rad51C, agent sensitivity (64, 65), and because X-ray resistance develops Rad51D, HsRad54, XRCC2, and XRCC3 (reviewed in Refs. 69, in parallel with chlorambucil resistance in CLL (74), determi- 72). A recent model of interaction in yeast proposes that Rad52 nation of DNA-PK activity in CLL should be informative. In a interacts with RPA, followed by Rad52 association with Rad51. preliminary report with a small sample of CLL patients, an This leads to the assembly of Rad51 and associated proteins increase in DNA-PK activity was found in resistant samples onto ss-DNA which then initiate recombinational DSB repair (76). In collaboration with Catherine Muller and Bernard Salles (87). XRCC3 is necessary for the assembly of Rad51 foci, and (Institute of Pharmacology and Structural Biology, Toulouse, these proteins physically interact (67, 88). In fact, if all of the France), our laboratory examined DNA-PK activity in a group interactions described occur in one complex, then HsRad51, of 34 patients (18 patients resistant to chlorambucil both in vitro XRCC3, Rad51C, Rad51B, Rad51D, and XRCC2 are complexed and in vivo). There was an excellent linear correlation between together (reviewed in Ref. 69). Rad54 appears to be required DNA-PK activity and in vitro chlorambucil cytotoxicity (r, after the association of the above mentioned proteins and Rad54 0.875; P ϭ 0.0001). The increased DNA-PK activity was inde- may assist Rad51 in interacting with damaged DNA (89). pendent of other clinical and biological factors. The regulation The percentage of Rad51 foci-positive cells and the num- of DNA-PK activity was associated with increased DNA-bind- ber of foci per cell increased after treatment of human fibroblast ing activity of its regulatory subunit, Ku, and increased Ku and lymphoblast cells with methylmethane sulfonate, ␥ radia- protein levels. Interestingly, most untreated CLL patients have tion, and UV radiation (90). In view of the critical role of the very low levels of DNA-PK activity, which suggests that, ini- Rad51 protein in HR and its probable involvement in repair of tially, resistance in CLL may be simply a state in which tumor ICL, we investigated HsRad51 foci formation after in vitro cells lose an abnormal sensitivity to alkylating agents. In ϳ25% chlorambucil treatment of CLL lymphocytes. In vitro chloram- of the samples from untreated CLL patients, a variant (truncat- bucil treatment induced HsRad51 expression as measured by ed) form of the Ku86 protein was associated with very low increased immunopositive staining in all of the CLL samples. In DNA-PK activity and hypersensitivity to chlorambucil (77). the chlorambucil-resistant CLL lymphocytes, there was a linear Wortmannin, a nonspecific inhibitor of DNA-PK, which also correlation between induction of HsRad51 foci at 5.4 ␮M inhibits other phosphatidylinositol 3-kinases, sensitized CLL chlorambucil and the in vitro LD50 concentration of chloram- lymphocytes to the effects of chlorambucil. Moreover, there was bucil. Interestingly, there were no differences in HsRad51 pro- a significant correlation between the synergistic sensitization tein levels between sensitive and resistant CLL lymphocytes and fold decrease in DNA-PK activity, but because Wortmannin (91). also inhibits other phosphatidylinositol 3-kinases, these results must be interpreted with caution (78). The immunohistochemical expression of Ku autoantigen Summary of Results with Recombinational Genes

and DNA-PKcs was examined in various human tissues. There The regulation of DNA-PK activity appears to be tightly was a large variation in expression depending on the specific associated with the development of chlorambucil drug resist- tissue type (79). This supports our observations that there is a ance in CLL. In particular, low DNA-PK activity is associated variation in DNA-PK expression in human tissues. with hypersensitivity to chlorambucil. Furthermore, increased Although it appears reasonable that increased DNA-PK levels of DNA-PK activity are associated with chlorambucil activity is associated with increased repair of NM-induced ICLs resistance in CLL. Moreover, in resistant samples, stimulation and thus, increased drug resistance, it is possible that other of HsRad51 foci formation by chlorambucil correlates with mechanisms are involved, including a role for DNA-PK with chlorambucil drug resistance (i.e., the more resistant CLL lym- respect to apoptosis (80, 81). phocytes demonstrate a greater HsRad51 foci formation). Be-

DNA-PKcs is a member of the phosphatidylinositol 3- cause HsRad51 protein levels were not different between sen-

sitive and resistant CLL lymphocytes, the increased Rad51 foci 7. Fisher, B., Carbone, P., Economou, S. G., Frelick, R., Glass, A., formation after chlorambucil treatment in resistant CLL samples Lerner, H., Redmond, C., Zelen, M., Band, P., Katrych, D. L., Wolmark, suggests that these foci represent an active DNA repair process N., Fisher, E. R., and Participating NSABP Investigators. L-Phenylala- nine mustard (L-PAM) in the management of primary breast cancer. involving other Rad-51 related proteins. A plausible hypothesis N. Engl. J. Med., 292: 117–122, 1975. to explain these results is that low DNA-PK activity defines 8. Young, R. C., Chabner, B. A., Hubbard, S. P., Fisher, R. I., Bender, a hypersensitive state, whereas high DNA-PK activity, along R. A., Anderson, T., Simon, R. M., Canellos, G. P., and DeVita, V. T. with increased homologous recombination as determined by Advanced ovarian adenocarcinoma. N. Engl. J. Med., 299: 1261–1266, HsRad51 foci formation, contributes to the resistant state 1978. in CLL. 9. Goldenberg, G. J., Land, H. B., and Cormack, D. V. Mechanism of cyclophosphamide transport by L5178Y lymphoblasts in vitro. Cancer Res., 34: 3274–3282, 1974. Future Studies 10. Tew, K. D., and Taylor, D. M. Studies with cyclophosphamide The role of apoptosis in chlorambucil drug resistance labeled with phosphorus-32: nucleic acid alkylation and its effect on should be examined in more detail. In particular, the role of DNA synthesis in rat tumor and normal tissues. J. Natl. Cancer Inst. various proteins involved in apoptosis need to be further defined (Bethesda), 58: 1413–1419, 1977. in both prospective and retrospective studies of innate and 11. Hoes, I., Lemiere, F., Van Dongen, W., Vanhoutte, K., Esmans, acquired drug resistance. Also, a comparative prospective trial E. L., Van Bockstaele, D., Berneman, Z., Deforce, D., and Van der Ј of the MTT assay versus assays of DNA integrity in predicting Eeckhout, E. G. Analysis of melphalan adducts of 2 -deoxynucleotides in calf thymus DNA hydrolysates by capillary high-pressure liquid chlorambucil response in CLL would be informative. chromatography-electrospray tandem mass spectroscopy. J. Chro- The mechanism of low DNA-PK activity in sensitive CLL matogr. B. Biochem. Sci. Appl., 736: 43–59, 1999. lymphocytes involves decreased Ku protein levels and a variant 12. Ross, W. E., Ewig, R. A., and Kohn, K. W. Differences between form of Ku86, as compared with resistant samples. The regula- melphalan and nitrogen mustard in the formation and removal of DNA tion of this process needs to be further investigated at both the cross-links. Cancer Res., 38: 1502–1506, 1978. translational and transcriptional levels. Inhibition of DNA-PK 13. Zwelling, L., Michaels, S., Schwartz, H., Dobson, P. P., and Kohn, activity by small molecules and/or antisense technology in com- K. W. DNA crosslinking as an indicator of sensitivity and resistance of L1210 leukemia cells to cis-diaminedichloroplatinum (II) and L-pheny- bination with NM chemotherapy may improve the therapeutic lalanine mustard. Cancer Res., 41: 640–649, 1981. index of the latter compounds. As concerns HR, the implication 14. Parsons, P. G., Carter, F. B., Morrison, L., and Regius, Mary. of other proteins such as Rad54, XRCC-2, and XRCC-3 needs Mechanism of melphalan resistance developed in vitro in human mel- to be examined. Recently, a model of repair of DSB has been anoma cells. Cancer Res., 41: 1525–1534, 1981. proposed in which either Ku or HsRad52 binds DSBs, thus 15. Moscow, J. A., Swanson, C. A., and Cowan, K. H. Decreased directing entry into NHEJ or HR, respectively (92). 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