characterization of glutathione -s-transferase expression ...€¦ · study. purification and...

(CANCER RESEARCH 50. 3562-3568, June 15, 1990]

Characterization of Glutathione -S-Transferase Expression in Lymphocytes fromChronic Lymphocytic Leukemia Patients1

John C. Schisselbauer,2 Robert Silber, Esperanza Papadopoulos, Kevin Abrains, Frank P. LaCreta, and

Kenneth D. TewDepartment of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 fJ. C. S., F. P. L., K. D. T.], and Hematology Division, New York UniversityMedical Center, New York, New York 10016 [R. S., E. P., K. A.]

ABSTRACT

Chronic lymphocytic leukemia ((I.I.) is a disease state which frequently responds to alkylating agent chemotherapy but ultimately becomes refractory through acquired resistance mechanisms. In the presentstudy, we have examined the expression of glutathione S-transferases(GST) in both CLL and normal control lymphocytes, as these enzymeshave been implicated in mechanisms of natural and acquired resistance.Lymphocyte GST was purified from samples by high-pressure liquidaffinity chromatography, and subunits were identified by two-dimensionalgel electrophoresis and Â¡mmunoblottingby using polyclonal antibodiesspecific for individual subunits. Analysis of CLL lymphocyte GST activityusing the general substrate l-chloro-2,4-dinitrobenzene showed a statistically significant 2-fold increase in cells from chlorambucil-resistantpatients over those from untreated patients and normal individuals.Furthermore, chlorambucil therapy was seen to cause a 1.3- to 1.5-foldelevation of enzyme activity in three previously drug-naive patients.Analysis of GST isozyme subunits indicated that 95% of the CLL patientsexamined were positive for the ITisozyme, and this appeared quantitatively to be the major isozyme present. The a and n Â¡so/vineswere alsoexpressed in 63 and 53% of the patients, respectively. Examination ofcontrol lymphocytes, as well as separated B- and T-cell subpopulations,yielded similar results. The present study indicates that a high degree ofinterindividual variation occurs and that the pattern of CLL lymphocyteGST expression differs from that of other tumor tissues. While therewere no obvious correlations between the disease state or stage andisozymes expressed, the quantitative increase in GST activity in chlorambucil-resistant CLL patients may be of relevance to the overall resistant phenotype.

INTRODUCTION

Chronic lymphocytic leukemia is a disease state characterizedby the proliferation of abnormal, developmentally immature B-cells (1, 2) for which the chemotherapeutic regimen of choiceincludes chlorambucil (3). While most patients initially show agood response to this agent, the subsequent development ofresistance may hamper further treatment. Several possiblemechanisms for nitrogen mustard resistance have been investigated in either cultured cell systems or freshly isolated lymphocytes from CLL3 patients. Mechanisms found to be operative

in various cultured cell systems include elevated levels of cellularglutathione in L-phenylalanine mustard-resistant LI210 cells(4), elevated levels of metallothionein in chlorambucil-resistanthuman epithelial and mouse fibroblast cell lines (5), and meta-

Received 1/16/90; revised 3/16/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by NIH Grant CA43830-08 and a Bristol-Myers drug resistance

grant to K. D. T.; FCCC institutional Grants CA06927 (CORE grant), RR05539(BRSG), and an appropriation from the Commonwealth of Pennsylvania; andNIH Grant CAI 1655 to R.S.

2To whom requests for reprints should be addressed, at Department of

Pharmacology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia.PA 19111.

3The abbreviations used are: CLL, chronic lymphocytic leukemia; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; CDNB, 1-chloro-2,4-dinitrobenzene; TBS-T, tris-buffered saline (50 mM Tris, pH 7.5; 400mM NaCI) containing 0.05% (v/v) Tween 20; TBS-A, Tris-buffered saline containing 3% (w/v) bovine serum albumin.

bolic inactivation of drug in chlorambucil-resistant Yoshidaascites sarcoma cells (6). As chlorambucil uptake and effluxappear to occur by simple diffusion in cultured cells (7) andfreshly isolated lymphocytes (8), transport-based mechanismsof resistance do not appear to be operative for chlorambucil.

Several studies have demonstrated alterations in glutathioneS-transferases associated with the alkylator-resistant phenotypein cultured cells (for review see Ref. 9). These enzymes havebeen shown to be fundamentally important in processes ofmetabolic detoxification (10, 11) and are suggested to play aprotective role in mutagenesis and carcinogenesis (12). Additionally, alterations in transferase expression have been associated with transformation of tissues to the neoplastic state,and are suggested to be useful as metabolic markers for tumorsin a number of tissues (13). While few studies have examinedthe actual production of metabolites from antineoplastic agentscatalyzed by GST (14), altered levels of these enzymes havebeen implicated in mechanisms of resistance to several chemotherapeutic agents, including alkylators (15, 16; for review seeRef. 9), and it has been suggested that GST may be involved inthe metabolic inactivation of L-phenylalanine mustard by leu-kemic LI210 cells resistant to this agent (17). However, fewstudies of alkylating agent resistance have been performed infreshly isolated lymphocytes from CLL patients. An early studyby Hill and Harrap (18) provided evidence that resistant patients could degrade chlorambucil at the benzene ring whilesensitive individuals were unable to metabolize the drug. Amore recent paper by Bank et al. (8) reports the production oftwo metabolites from radiolabeled chlorambucil in lymphocytesfrom CLL patients detected by HPLC. A second paper byPanasci et al. ( 19) notes that lymphocytes from a patient treatedwith chlorambucil also metabolize the related compound mel-phalan, while there was no evidence found for melphalan metabolism in patients treated with the latter agent alone. In thepresent study, we have examined the glutathione 5-transferasecomposition of lymphocytes from both chlorambucil-treatedand untreated CLL patients, as well as control lymphocytes inorder to characterize transferase expression in CLL, as theseenzymes may be involved in alkylating agent resistance in thisdisease.

MATERIALS AND METHODS

Isolation and Purification of Lymphocytes. Purification and analysisof samples were carried out in a blinded fashion. Lymphocytes isolatedfrom 19 patients diagnosed with various stages of B-cell CLL wereanalyzed in detail for GST content. These and additional samples wereassayed for enzyme activity. Lymphocytes were isolated by centrifuga-tion of heparinized blood on a discontinuous Ficoll-Hypaque gradient(20). Resistance was defined as a failure to have a clinical or hemato-logical response following therapy with standard doses of chlorambucil.Isolated lymphocytes were washed twice with isotonic saline, pelletedby centrifugation at 500 x g, and stored frozen at -80Â°Cprior to use.

Lymphocytes were also obtained from healthy control donors by leu-kapheresis (in order to obtain sufficient yield) and purified as above.

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GLUTATHIONE S-TRANSFERASES IN CLL LYMPHOCYTES

Separation of B- and T-cells was carried out as described previously(21). As samples were stored frozen (-80Â°C)prior to use, aliquots from

one patient (patient 17) were examined before and after 3 months ofstorage at -80Â°C as an internal control to verify both reproducibility

and stability of the transferases under the conditions used in the presentstudy. Purification and analysis of the subunits present using eitherfresh or stored lymphocytes from patient 17 yielded identical GSTsubunit profiles.

HPLC Purification of Lymphocyte Glutathione S-Transferase. Frozenlymphocyte pellets containing 3 x IO8 to 1 x IO9 cells were rapidly

thawed, suspended, and homogenized for 50 s on ice in 10 ml of buffer(10 mM Tris-HCl, pH 7.8), using a Polytron homogenizer (Model PM7/20, Brinkmann Instrument Co., Westbury, NY). Supernatant fractions were obtained from the homogenate by centrifugation at 10,000x g for 20 min, followed by centrifugation of the latter supernatant for1 h at 100,000 x g to yield the crude cytosolic fraction. This latterfraction was filtered through a 0.2-nm acrodisc membrane (GelmanSciences, Ann Arbor, MI) and purified by using a HPLC glutathioneaffinity column as described previously (22). The purified transferasefraction was dialyzed and concentrated overnight against 10 HIMTris-HCl (pH 7.8), using a Micro-ProDicon dialysis/concentrator with a M,10,000 cutoff membrane (Bio-Molecular Dynamics, Beaverton, Oregon). This removed S-hexylglutathione, which was used to elute boundtransferase from the column. Aliquots of the above fractions were savedand assayed for protein (23) and transferase activity using CDNB assubstrate (24).

Two-Dimensional Gel Electrophoresis. Samples of the HPLC-puri-fied, dialyzed transferase fraction were analyzed by 2-dimensional gelelectrophoresis. Non-equilibrium isoelectric focusing was performed inthe first dimension by loading 5 to 10 ng of purified material onto theacidic end of a 3.8% polyacrylamide tube gel [12 cm long, containing2% pH 3-10 ampholytes (Pharmacia)] and focusing at 450 V for 4 h(25). Tube gels were then attached to 12.5% polyacrylamide slab gelsusing melted 1% agarose, and electrophoresis in the second dimensionwas carried out under standard conditions (26). Protein was visualizedby silver staining techniques (27). Where possible, duplicate gels wererun and one of the pair was transferred by electroblotting to a nitrocellulose membrane (28) for immunostaining.

Western Blot Analysis. Upon completion of blotting, membraneswere washed twice for 10 min with TBS-T, to remove blotting buffercomponents. This was followed by a 1-h incubation in TBS-A in orderto reduce nonspecific binding of antisera to membranes. Blots weresubsequently incubated with one of four different polyclonal primaryantibodies specific for individual GST subunits: rabbit anti-humanplacenta! GST (w), rabbit anti-rat liver GST Ya, rabbit anti-rat liverGST Yb, (BioPrep, Dublin, Ireland), or rabbit anti-rat Ya/Ybi (prepared as described previously; Ref. 29), the latter being cross-reactivewith both a and p (but not ir) class subunits. Primary antibody bindingwas carried out by incubating blots with the above antibodies in TBS-A for l h at the following dilutions: 1:5000 for ?r, a, or ÃŸ,and 1:500for a/fi class-detecting antibodies. Blots were next rinsed with TBS-Tthree times for 10 min each, and incubated for l h with the secondaryantibody, goat anti-rabbit IgG conjugated to horseradish peroxidase(Bio-Rad, Richmond, CA; diluted 1:500 with TBS-A). Finally, blotswere washed 3 times with TBS-T (10 min each) and bound antibodywas visualized by incubation with 4-chloro-l-napthol [Bio-Rad; prepared by dissolving 60 mg in 20 ml methanol, which was subsequentlyadded to 160 ml of Tris-buffered saline containing 0.033% (v/v) hydrogen peroxide].

RESULTS

Isozymes Detected in CLL Patients. Detailed analysis oflymphocyte GST isozymes was carried out on 19 CLL patientsdiagnosed in various stages of the disease and with differingdegrees of response to alkylating agent therapy (Table 1). Totalcumulative dose of chlorambucil received by treated patientsranged from 332 to 2672 mg of drug. All of these individualswere diagnosed as having the B-cell form of the disease, which

occurs in greater than 95% of all CLL cases (1). GST wereisolated from these lymphocytes and compared to control lymphocytes obtained from normal donors with respect to overalltransferase activity and isozymic composition. Purified fractions utilized for 2-dimensional analyses were enriched approximately 300-fold for transferase activity (CDNB as substrate)over the crude homogenate. A degree of variability was seenamong different individuals in terms of the pattern of transferase subunits expressed, as can be seen in Table 2, which containsrepresentative data from several individuals. Identification ofsubunits present was based primarily on immunoreactivity,secondarily on relative molecular weight, and finally on thebasis of relative pi, since non-equilibrium isoelectric focusingused here provides only approximate values for pi. A sample2-dimensional gel and companion Western blot are shown inFigs. 1 and 2 for patients 12 and 14, respectively. It can be seenfrom Fig. 1 that sometimes spots present in the transferaseregion of a gel do not stain strongly with transferase subunitantibodies (spots 3 and 4), possibly due to low levels for a givensubunit. However, in such cases, incubation of blots with eitherhigher concentrations of antibody (1:500 dilutions ofÂ« and /Â¿)or with an antibody cross-reactive to both Â«and n subunits (a//u), resulted in positive immunostaining which could not bedetected with lower titers of anti-tv or anti-/Â¿antibodies. Thus,both gels and blots were performed where possible to detectand classify which subunits were present in a given sample.

Several patients were seen to have multiple spots present fora given subunit (Table 2; Fig. 2). As many as five forms of aand ir, and three of Â¡iwere detected in some samples (e.g.,patient 4; Fig. 2). Such spots have the same molecular weight,which is characteristic for a given subunit, but different pivalues. This suggests a "microheterogeneity" among specific

subunits in a given individual. Additionally, low-molecular-weight spots which did not react with GST antibodies wereoccasionally seen below TTin some samples (Fig. 2). Most likelythese represented proteolytic products of ir, as a downwardstreak of protein can be seen linking them to TT.

The proportion of patients expressing a given major subunittype is given in Table 3, which indicates that 95% expressedthe GST 7Tgene family, with the a and ÃŸclasses being expressedat lesser, but significant frequencies (63 and 53%, respectively).Various subunit combinations were also expressed, but againat significantly lower frequencies than that for -Â¡ralone (Table

3). Thus, the single most characteristic feature of CLL lymphocytes is the presence of GST ?r, which was seen, in most cases,to be both qualitatively and quantitatively the major subunitexpressed.

Comparison to Control Lymphocytes. Differences in transferase composition between CLL and normal lymphocytes wereexamined by using mixed lymphocytes from normal individualsas a control. In some instances, analysis of GST from purifiedB- and T-cell fractions of normal donors was attempted, since

the ratio of B:T cells in CLL is high. Data for the controls arepresented in Table 4. The v subunit was expressed in all of thecontrols examined, a result similar to that obtained for CLLlymphocytes. Three of four mixed lymphocyte samples (T- andB-cells) expressed all three cytosolic forms (ir, a, and n). Comparison of purified B- with T-cells from the same individualyielded identical results in one individual (control 1, Table 4),but possibly a difference in a second individual (control 7) inthe presence of a second subunit (^) not seen in T-cells fromthe same subject. This may have been due to differences insample size, as studies of normal B- and T-cell GST werehampered both by the much lower yield of lymphocytes from

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Table 1 CLL patient characteristics

Patient12345678910111213141516171819SexMMMMFFMFFMFFMFFMMMFAge70607868567750587469865269716253775157"

Dose of drug given per course wasvariable*NT, not treated;Chlor, chlorambucil; Res,Rai

stage000000IIIIIIIIIIIIVIVdepending

onresistant;NR,Treatment

statusNT*NTNTNTNTNTNTNTNTNTChlorNTChlorChlorChlorNTChlorChlorChlorprotocol.not

resistant.Courses"6323191216Total

dose(mg)56033259636859026721860ResistancestatusResResNRNRNRResResTable

2 Analysis of trans) "erasesubunits present in lymphocytes from CLLpatientsPatient4811*1213Â»141618*19*SexMFFFMFMMFAge685886526971535157Spot1234567112345612345612121231234561234Approximatemolecularwt23,00025,00025,00025,00025,00025,00026,00023,00023,00023,00023,00023,00023.00025,00023,00023,00025,00026,00026,00026,00023,00026,00023,00025,00023,00025,00026,00023,00025,00025,00025,00025,00026,00023,00023,00025,00026,000Approximatepi5.86.16.46.67.27.67.76.55.55.96.15.86.26.76.16.77.15.97.17.16.58.06.17.06.87.17.26.06.96.97.37.27.54.55.96.76.3Immunoreactivity"TCa/tÃ-Â«/Ma/Ma/MÂ«/MÂ«/MrTTTvrNDTâ€¢*NDMMMira/nâ€¢xa_â€”-ra/Ma/Ma/MÂ«/Ma/M_â€”--Subunit

identityvaaaaaMTT1rTIfaTTaMMMTMâ€¢XaTaMâ€¢KaaaaMTTaM

Â°a/n, rabbit anti-rat GST antibody which cross-reacts with a and ÃŸsubunits in rat and human; ND, not detected; 3-10 fig protein analyzed in purified transferase

fractions; insufficient lymphocytes for Western blot analysis of patients 16 and 19.* Individuals who show disease states resistant to chlorambucil chemotherapy.

normal donors and loss of cells during purification of B- from Transferase Activity in CLL and Control Lymphocytes. Trans-T-cells. Two additional normal T-cell samples showed only the ferase-specific activity was assayed in the crude cytosolic ex-7Tform of the isozyme, but the low cell number and amount of tracts (SIOK; supernatant from 10,000 x g centrifugation) ofpurified GST analyzed may have resulted in insufficient sensi- lymphocytes obtained from the CLL patients analyzed in detailtivity to detect quantitatively minor subunits. (listed in Table 1) as well as from additional patient samples

3564




65

base acidFig. 1. Analysis of lymphocyte glutathione S-transferases from patient 12

(female, age 52, Rai Stage I, untreated). Two-dimensional polyacrylamide gelelectrophoresis of HPLC-purified transferase fraction (a) and sequential companion Western blots for n (b) and IT(c). Immunoreactivity with a was not detected.Transferase spots are labeled as follows: / and 2, ir; 3, a; and 4, 5. and 6, p.

65 4 Â¿2

65 4 32

base acidFig. 2. Analysis of lymphocyte glutathione .S-transferases from patient 4

(male, age 68. Rai Stage 0, untreated). Two-dimensional polyacrylamide gelelectrophoresis of H PLC-purified transferase fraction (a) and matching sequentialWestern blots immunostained with a/p cross-reactive antibody (A) and ir (c).Spots are labeled as follows: /, ir; 2-6, a; and 7, Â¡a.

for which enzyme activity alone was determined due to insufficient sample size. Enzyme activity of CLL lymphocytes wascompared to that of control lymphocytes (purified B-cells, T-cells, and mixed B- and T-cells) utilizing the general substrateCDNB. A great deal of interindividual variation was seen andspecific activities of the crude homogenate fraction were nearlyidentical to those measured in the SI OK fraction for a givenindividual.

When CLL patients were divided into three groups (untreated, chlorambucil responsive, and refractory), the resistantindividuals showed a statistically significant 2-fold increase in

crude cytosol transferase activity over both nontreated CLLpatients and normal individuals (Table 5). The very similarvalues for enzyme activity seen in lymphocytes from nontreatedpatients and normal individuals suggest that the increasedactivity of the resistant group may be a result of enzymeinduction in response to drug treatment. This possibility isfurther supported by the finding that three individuals monitored before and after treatment showed a 1.3- to 1.5-foldincrease in enzyme activity following treatment. (Pre- and post-treatment values obtained were 87.1 and 109.3; 71.5 and 97.4;and 82.1 and 121.0 nmol/mg/min, respectively).

Chlorambucil-resistant Individuals. Four of the patients stud-

Table 3 Summary of subunit distribution among CLL patients

Subunits

No. of patientsexpressing subunit

IndividualTaMCombinationsT,

a, andÃŸaandn7TandÂ«7Tand fj.18/19(95)12/19(63)10/19(53)7/19(37)05/19(26)2/19(11)

ied in detail showed clinical resistance to chemotherapy withchlorambucil. Two of these patients (patients 18 and 19) expressed all three of the known cytosolic transferase subunits (TT,a, and fi\ Table 2). One of these, patient 19, showed a GSTpattern that was similar to that of a stage II untreated individual(patient 16; Fig. 3), although the two differed in that theresistant individual expressed a more acidic form of ^ (spot 3).Of the two remaining individuals (patients 13 and 11), bothexpressed Ktogether with a second subunit. Two of the resistantindividuals showed multiple spots for particular subunits (patients 18 and 11; Table 2).

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6 (T-cells)

7 (T-cells)

7 (B-cells, cytosol)*


Table 4 Analysis of glutathione S-transferase composition in normal lymphocytes

ControlSpot1

(T-cells)1234561

(B-cells, cytosol)1232

(T + B-cells)1234563

(T + B-cells)124

(T + B-cells)12345675

(T + B-cells) 123456Approximate

molecularwt23,00023,00025,00026,00026,00026,00023,00023,00023,00025,00026,00026,00023,00026,00023,00023,00023,00025,00025,00025,00026,00023,00023,00023,00025,00026,00026,000ApproximatePi5.36.57.15.35.96.74.54.66.06.24.55.16.67.54.75.66.84.75.16.56.65.45.86.86.55.46.0Immunoreactivity"TÂ«/MÂ«InrVTMNDNDTÂ«/â€žrTNDNDNDa/MMTTNDa/MNDNDSubunitidentity7TCa*â€¢MMÂ»aMÂ«â€¢Â»Â»aMMTiÂ«TTÂ»aaaM**irorMM

23,000

23,000

5.6

5.2

T

a/it" alii, rabbit anti-rat GST antibody which cross-reacts with a and Â»subunits in rat and human; ND, not detected; 1-10 Mgprotein analyzed in purified transferase

fractions; 100 **gprotein analyzed in partially purified B-cell cytosolic fractions.* Some contamination with monocytes.

Table 5 Transferase assay data for crude cytosolic fractions

LymphocytesampleCLL,

nontreatedCLL, treated, nonresistantCLL, treated, resistantNormal B-cellsNormal T-cellsNormal B -t-T-cellsSpecific

activity(nmol/mg/min)"51.3

Â±8.5(16)67.4 Â±6.9 (8)

103.0Â± 13.6(12)Â»

37.8 (44.6, 30.9)54.1 Â±2.6(3)47.5 Â±8.7 (4)

" Data presented are for the 19 individuals studied in detail (listed in Table 1)

as well as 17 additional patients for whom enzyme activity alone was determined.Supernatant fromlO.OOO x g centrifugaron was assayed with l-chloro-2,4-dini-trobenzene as substrate; data given are mean Â±SEM (n), except for "normal B-cells" where n = 2; individual values are listed in parentheses.

* Significantly different from CLL, nontreated group and normal B- + T-cellgroup. P = 0.002 and 0.04, respectively, using Student's / test.

DISCUSSION

The most consistent feature of lymphocyte transferaseexpression, whether from normal or CLL cells, was the presenceof the T subunit. No evidence was found for elevated levels ofT expression in the tumor cells, which suggests that v is not areliable marker for the neoplastic state in CLL, in contrast tothe situation for rodent liver (13) and human tissues such ascolon (30), stomach (31), cervix (32), and kidney (33). Incontrast to *-, the a and n subunits were not always present in

CLL lymphocytes. Transferase M>which is known to follow apolymorphic distribution in human lymphocytes, has been sug

gested to play a protective role against cigarette smoke-inducedlung cancer (34). The value observed for Â¿iexpression in CLLof 53% is similar to that previously reported for normal livertissue (60%; Ref. 35) and lymphocytes from control smokerswithout cancer (59%; Ref. 34), but is significantly higher thanthe values of 35 and 36% reported for lymphocytes fromsmokers with lung cancer (34) and normal nonsmoking individuals (36), respectively. This indicates that the expression of aparticular transferase isoform may not only be tissue and tumorspecific, but may also reflect environmental factors to whichcells are exposed. Whether Â¿iis induced in smokers withoutcancer or instead is expressed at intrinsically higher frequenciesin healthy individuals remains to be answered.

Expression of a occurred at a frequency of 63%, slightlyhigher than that found for n- while various combinations ofsubunits were expressed at equal or lower frequencies in CLLlymphocytes. Comparison of these values to control lymphocytes (B- plus T-cells) suggested that the combination of TT,a,

and M might be expressed at a higher frequency in normal,rather than CLL lymphocytes, although the relatively low sample size for this control group warrants caution in drawing suchconclusions. Studies of B- and T-cell subpopulations from

normal individuals were seriously hampered due to the necessityfor leukapheresis of these individuals to obtain sufficient lymphocyte yields, the low percentage of B-cells present, and cell

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a

acidFig. 3. Two-dimensional polyacrylamide gel electrophoresis of lymphocyte

GST from (a) patient 16 (male, age 53, Rai Stage II, untreated) and (b) patient19, (female, age 57, Rai Stage IV, extremely resistant to chlorambucil). Notesimilarity of pattern expressed. Spots are labeled as follows: /, ir; 2, a; and 3, p.

losses during subsequent separation of B- and T-cells.Examination of 12 chlorambucil-resistant individuals showed

a statistically significant 2-fold increase in crude fraction trans-ferase activity over untreated CLL patients. This increased GSTactivity suggests that metabolic events in some way involvingGST may be responsible for resistance to this agent in CLL.While Bank et al. (8) were unable to detect any gross differencesin chlorambucil metabolism between lymphocytes of resistantand nonresistant patients, they did report the production of twoapparent metabolites of chlorambucil by CLL lymphocytes,detected by HPLC. Panasci et al. (19) found no evidence ofmetabolism of the related analogue melphalan in CLL lymphocytes incubated with the drug, except in one patient who wascurrently receiving chlorambucil therapy. While the latter groupfound a 2- to 5-fold decrease in DNA cross-links in resistantover untreated CLL lymphocytes incubated with melphalan,Bank et al. found a paradoxical increase in binding of chlorambucil to DNA of lymphocytes from resistant individuals. Noevidence of altered transport for either drug was found in theseformer studies. In the present work, there was no obviouscorrelation between resistance and transferase subunits presentwhen compared against either nonresistant CLL or controllymphocytes. This does not, however, preclude the possibilityof quantitative alterations in a given subunit, or alterations incompartmentalized forms.

We were able to detect strong signals on our 2-dimensionalgels and Western blots using 5-10 /Â¿gof highly purified transferase protein. In another study, Jones et al. (36) performedanalyses utilizing crude cytosolic fractions that were pooledfrom several individuals and partially purified by a differenttechnique, apparently requiring a minimum of 25 /Â¿gproteinfor reliable detection. In the present study, the use of leuka-pheresis allowed us to examine interindividual differences intransferase expression among normal as well as CLL lymphocytes which could not be detected in the former study. Jones et

al. failed to detect the presence of basic (a class) subunits inhuman lymphocytes using an anti-t antibody, while pointingout that this could be due to a low level of expression for thissubunit. This latter suggestion is consistent with our findings,as a in general appeared to be present at much lower levelsthan 7Tor n class subunits in our samples. Furthermore, wedetected w in 3 normal T-cell samples analyzed while Jones etal. did not, using a human lung A antibody. Thus differences inthe immunoreactivity of these antibodies, as well as the use ofa more highly purified transferase fraction here, could accountfor discrepancies between these two studies.

The presence of multiple pi forms for a given subunit in CLLlymphocytes is consistent with data reported for GST isolatedfrom other human tissues (37). Vander Jagt (38) reported thepresence of 13 distinct forms of apparent a in human liver andSteisslinger and Pfleiderer (39) reported 6 basic forms in humankidney and placenta, as well as 2 acidic forms in human kidney.In the present study, we have observed variable numbers ofspots for a given subunit with up to 5 different forms (withrespect to pi) being expressed for a given subunit in lymphocytes. While the multiple pi forms observed here could resultfrom carbam-oylation (40), this is unlikely here as samples must be heatedin the presence of urea for carbamoylation to occur, nor wouldthis explain the results obtained for other tissues (37-39). Insome instances, we have also observed protein spots having thesame pi as ITbut of a lower molecular weight on 2-dimensionalgels. Their overall appearance would be most consistent withproteolytic degradation products of ir, although these did notcross-react with TTor any of the other GST subunit antibodies.

In a number of instances, the predominant form of GSTexpressed in nonhepatic tissues appears to be ir. From our data,this would seem to be the case for CLL and normal lymphocytes, although we have observed high levels of expression ofbasic or neutral subunits in prostate (37), colon, and ovary.4

Both basic and neutral subunits were found in lymphocytesfrom normal, as well as all CLL groups examined here, although no obvious correlation was seen between lymphocytephenotype and transferase phenotype expressed or clinical patient status.

In summary, our investigation suggests that transferaseexpression in normal and CLL lymphocytes varies on an individual basis. This may explain the variable response observedwhen drug-resistant human neoplasms are treated in vitro withthe GST inhibitor ethacrynic acid in combination with nitrogenmustard or doxorubicin (41). No obvious correlation was seenbetween the pattern of GST expressed and disease stage ordegree of resistance. However, despite the lack of an obvioustransferase pattern associated with resistance, the elevated lymphocyte enzyme activity of resistant over untreated CLL patients and normal individuals is highly suggestive that metabolicevents, in some way involving GST, may contribute, at leastpartially, to the observed resistance.

ACKNOWLEDGMENTS

The authors would like to thank Eileen Walsh for technical assistanceand Donna Platz for assistance in the preparation of this manuscript.

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1990;50:3562-3568. Cancer Res John C. Schisselbauer, Robert Silber, Esperanza Papadopoulos, et al. Lymphocytes from Chronic Lymphocytic Leukemia Patients

-Transferase Expression inSCharacterization of Glutathione

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