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Failure to induce resistance to cytotoxic drugs in normal lymphocytes
Leukemin Research Vol. 20, No. 8, pp. 64-648, 1996. Copyright 0 1996 Ekvier Science Ltd. All rights reserved Printed in Great Britain 0145-2126196 $15.00 + 0.00
Pergamon PII: SO145-2126(96)00024-O
FAILURE
TO INDUCE RESISTANCE TO CYTOTOXIC NORMAL LYMPHOCYTES
DRUGS IN
Paul Bentley, Alun Thomas and JoseThomas Departments of HaematologyandThoracicMedicine,LlandoughHospital,Penarth,SouthGlamorgan,U.K. (Received 19 January 1996.Accepted 9 February
1996)
Abstract-Lymphocytes from patients who had received chemotherapy for lung cancer were examined for evidence of drug resistance using an in vitro assay for sensitivity to the effects of chlorambucil and etoposide. There was no evidence of induced resistance to the effects of chlorambucil or etoposide in the patients’ lymphocytes when compared to the sensitivity of the normal control subjects’ lymphocytes. In four patients, in whose lymphocytes sensitivity to chlorambucil was measured before and after a course of treatment, there was no significant change in the in vitro drug sensitivity. These findings are in contrast to those made previously using the lymphocytes of patients with chronic lymphocytic leukaemia and indicate that acquired drug resistance is a property of malignant cells and is not inducible in normal lymphocytes. Copyright 0 1996 Elsevier Science Ltd. Key words:
Normal
lymphocytes,
chemotherapy
Introduction
resistance.
become progressively resistant to the effects of chlorambucil in vitro after each course of cytotoxic chemotherapy. In the present study the effect of chemotherapy, administered for the treatment of lung cancer, on the sensitivity of normal lymphocytes was examined. Patients who had received etoposide were selected for study and the in vitro effects of etoposide and chlorambucil were examined in separategroups of patients and compared with the findings in the lymphocytes obtained from normal control subjects.
Resistance to chemotherapy is recognised to be the major obstacle to the successful treatment of many tumours. Many diverse cellular mechanismshave been proposed for this resistance and these include transmembranedrug export (via the product of the multidrug resistancegene m&-l), neutralization of cytotoxic drugs by cytoplasmic constituents, loss of topoisomeraseII activity, enhancementof DNA repair and inhibition of apoptosis. These mechanismsare available not only to tumour cells but also to normal tissues[14]. A notable feature of drug resistance in malignant cells is its enhancement following chemotherapy [5]; increased expression of mdr-1 and glutathione-s-transferase and topoisomeraseII suppressionhave each been shown to be induced in malignant cells by chemotherapy [6,7]. It hasbeen previously shown [8] that the sensitivity of lymphocytes to cytotoxic drugs can be measured by inhibition of DNA incorporation and the results bear a close relationship to clinical responsivenessin chronic lymphocytic leukaemia (CLL). The peripheral blood lymphocytes of the patients in that study were found to
Patients and Methods
Abbreviations: MBq, megabequerel;“H, tritiated; CLL, chronic lymphocytic leukaemia;mdr-1, multidrug resistance gene;P-gp, P-glycoprotein Correspondence to: D. P. Bentley, LlandoughHospitaland Community NHS Trust, Penlan Road, Penarth, South GlamorganCF64 2xX, U.K. 645
The study was approved by the South Glamorgan Health Authority Ethics Committee. The patients had received treatment according to local protocols for the managementof small-cell lung cancer. The total amount of etoposide received by each patient prior to the study is summarized in Table 1, together with the interval in days since the last dosewas administered.Most patients had received additional treatment including carboplatin or radiotherapy. In nine instancesthe patient’s lymphocytes were tested against chlorambucil and in eight against the effects of etoposide in vitro. Lymphocytes from five normal subjects were used for controls in the chlorambucil study and from eight normal subjects in the etoposide study. Lymphocytes were separatedfrom fresh venous blood samplesusing discontinuousdensity gradient centrifugation (Histopaque-1077, Sigma
646 Table
Patient number
P. Bentley et al. 1. Patient dosage data and chlorambucil Total dose etoposide received (mg)
Days since last dose
700 2100 1400 2100 2800 700 2100 2800 2100
35 29 9 11 18 1 5 37 12
and etoposide sensitivities lymphocytes
700 3500 5600 2100 2100 700 2800 2800
and the normal
Chlorambucil
Normals n=5 10 11 12 13 14 15 16 17
in the patients’
Mean S.D. Mean S.D.
Etoposide
IDso w/ml
Slope
2.4 4.6 2.2 2.9 3.5 3.1 2.7 1.8 3.3 2.9 f0.8 3.2
-0.33 - 0.21 -0.16 -0.30 -0.32 - 0.32 -0.29 -0.29 -0.40 -0.29 f 0.07 - 0.22 * 0.03
+0.38
ID50
7 3 3 150 6 109 240 120 Normals
n=8
Chemical Co., Poole, Dorset, U.K.) under sterile conditions. Cell cultures at a final concentration of 1 x 106/ml were performed in Eagles Minimum Essential Medium (Gibco Ltd, Paisley, U.K.) supplemented with 0.22% sodium bicarbonate, 5% fetal calf serum, 10,000 IU/lOO ml sodium benzyl penicillin and 5000 pg/lOO ml streptomycin. To each suspension 0.2 ml of autologous serum was added together with 50 ~1 of a solution of 0.4 @ml pokeweed mitogen. Chlorambucil solution (Sigma) was made up from pure powder by dissolving 24 mg in 1 ml of ethanol which was then diluted to 100 ml in phosphate-buffered saline (PBS). This stock solution was stored in aliquots at -20°C. Etoposide was obtained as a pharmaceutical preparation (Vepesid, Bristol-Myers Pharmaceuticals, Hounslow, Middlesex, U.K.). Chlorambucil solutions were addedto cultures to achieve final concentrations of between 1 and 10 @ml and etoposide solutions were addedto achieve final concentrations of between 0.1 and 1 l.@rnl. PBS was added to further cultures without inhibitor and sodium azide added to separatecultures as a blank. Cultures were performed in triplicate, except those without inhibitor which were performed in quintuplicate, at 37°C in 5% carbon dioxide in humidified air for 66 h. [3H]Thymidine (0.074 MBq; Amersham International plc, Amersham, Bucks, U.K.)
control subjects’
Mean S.D. Mean S.D.
ethl
0.25 0.32 0.32 0.18 0.45 0.21 0.19 0.18 0.26 f 0.09 0.22 * 0.04
Slope
-2.4 - 1.5 - 2.4 -2.6 -2.9 -2.2 -3.1 -2.1 -2.3 f0.5 -2.9 * 0.54
in 0.1 ml PBS was then addedto each culture which was then incubated for a further 24 h. Lymphocytes were harvested by filtration through 2.5 cm glass microfibre discs (Whatman Labsales,Maidstone, Kent, U.K.), the dried discs immersedin liquid scintillation fluid (Instagel, Canberra Packard Ltd., Cambourne, U.K.) and the samplescounted using a Wallac 1410 liquid scintillation counter. The percentage 3H uptake in each culture was calculated taking the uptake in the control cultures without inhibitor as 100%. Results The summarized results are shown in Table 1. In the normal control subjects, 50% inhibition of DNA synthesis (1~~~) was achieved at a mean (&S.D.) chlorambucil concentration of 3.2 f 0.38 @ml and in the patients the mean (+ S.D.) chlorambucil 1~~~ was 2.9 f 0.8 @ml. This difference is not significant (Mann-Whitney two sample tests U = 15.0, P = 0.35). The respective mean (+ S.D.) slope of the curves in the normal subjectswas -0.22 f 0.03 and for the patients was -0.29 f 0.07. The difference between these is not significant (Mann-Whitney U = 8.0, P = 0.6). Five sequential studies were undertaken prior to treatment and following a course of chemotherapy. In three of
Chemotherapy sensitivity of normal lymphocytes
Post-treatment
Pre-treatment
Fig. 1. Chlorambucil IDA,, for four patients measured before and after a course of etoposide. One patient was studied on two
occasions.
these there was a fall in the IDLE following treatment and in only one did the 1~~~ increase.These resultsare shown in Fig. 1. In the etoposide study the mean (+ S.D.) ~~~~ for the normal subjectswas 0.22 f 0.04 pg./ml whereas in the patients the mean (kS.D.) was 0.26 + 0.09 &ml. The difference between these groups is not significant (Mann-Whitney U = 25.0, P = 0.50). The slope of the dose-responsecurves against etoposidehad a mean (f S.D.) value in the normal subjects of -2.9 + 0.54 and in the patients -2.3 + 0.5. There is no significant difference between these two groups (Mann-Whitney U = 18.0, P = 0.16). Discussion The pleiotropic resistance of tumours to cytotoxic chemotherapy is characteristically inducible by exposure to chemotherapy. Solid tumours become progressively resistant despite initial sensitivity, acute leukaemia in relapse following chemotherapy-induced remission showspoorer responsesand the lymphocytes of patients with chronic lymphocytic leukaemia can be shown to become relatively more resistantto chlorambucil in vitro after the patient has received treatment [5,8]. Furthermore resistance can be induced in tumour cell lines cultured in the presenceof a cytotoxic drug [9-111. The recognisedmechanismsof drug resistancecan be
641
identified to explain many of these effects. Increased expression of the multidrug resistancegene mdr-1 and its product P-glycoprotein (P-gp) have been observed in leukaemias [12, 131, lymphomas [14], myeloma [15] and in solid tumours following treatment and related to clinical resistance [7]. Reduced expression of topoisomeraseII has been demonstrated in CLL [16] and theselymphocytes have increasedlevels of glutathiones-transferase [17]. Human tumour cell lines show increasedmdr-1 expression after drug exposure and this effect is rapidly induced but may remain stablefor many weeks [9]. Normal rodent cells can be induced to drug resistance in tissue culture [18, 191. Normal human peripheral blood lymphocytes are known to express mdr-1 [24,201, have low expressionof topoisomeraseII [16] and detectable glutathione levels [21] and glutathione-stransferase activity [22,23]. Thus, the potentially inducible mechanismsfor drug resistance are available to the normal cell. Furthermore mdr-1 hasbeen shown to be inducible in the lymphocytes of normal subjects in response to lipophilic solvents [24] and normal lymphocytes can be shown to have effective DNA repair mechanisms[25]. No evidence of induction of chemotherapy resistance was demonstrable in the normal lymphocytes obtained from the patients in this study despite lengthy and often multiple exposure to chemotherapy. There was no change in the characteristics of the dose-response curves following chemotherapy; selection of more resistant subclones by elimination of sensitive cells would have led to a decreasein the slope of the curves and any induction of drug resistance would have increased the 1~~~. Etoposide (an epipodophyllotoxin) resistance is mediated by a number of mechanisms, particularly by drug efflux via P-gp and by topoisomerase II suppression,whereas the mechanism of chlorambucil resistance is more obscure [26]. It has been suggested[20] that the effect of acquired drug resistance might be transient, explaining why low levels of mdr-I RNA is found in malignant cells and yet stable, chemotherapy-resistant tumour cell lines have been produced [9]. In the present study, patients were investigated between 1 and 240 days after receiving chemotherapy without any discernible difference in the results. The lack of induction of drug resistance in normal lymphocytes in patients receiving etoposide and other chemotherapeutic agents for solid tumour indicates an entirely different behaviour between normal lymphocytes and tumour cells. Acquired drug resistancetherefore appearsto be a feature of the neoplastic cell that is not found in its normal counterpart and probably results from the high mutation rate characteristic of tumours. Had such resistancebeen inducible in normal cells this
648
P. Bentley et al.
would have implications for the scheduling of chemotherapy but the lack of tolerance developing in normal cells emphasizes the need to develop strategies to prevent the emergence of drug resistance in tumours. References 1. Abbaszadegan M. R., Futscher B. W., Klimecki W. T., List A. & Dalton W. S. (1994) Analysis of multidrug resistance-associated protein (MRP) messenger RNA in normal and malignant hematopoietic cells. Cancer Res. 54, 4676. 2. Chaudhary P. M., Mechetner E. B. & Roninson I. B. (1992) Expression and activity of the multidrug resistance P-glycoprotein in human peripheral blood lymphocytes. Blood 80, 2735. 3. Drach D., Zhaos S., Drach J., Mahadevia R., Gattringer C., Huber H. & Andreeff M. (1992) Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype. Blood 80, 2729. 4. Klimecki W. T., Futscher B. W., Grogan T. M. & Dalton W. S. (1994) P-Glycoprotein expression and function in circulating blood cells from normal volunteers. Blood 83, 24.51. 5. Silber R., Degar B., Costin D., Newcomb E. W., Mani M., Rosenberg C. R., Morse L., Drygas J. C., Canellakis Z. N. & Potmesil M. (1994) Chemosensitivity of lymphocytes from patients with B cell chronic lymphocytic leukemia to chlorambucil, fludarabine, and camptothecin analogs. Blood 84, 3440. 6. Gekeler V., Frese G., Noller A., Handgretinger R., Wilisch A., Schmidt H., Muller C. P., Dopfer R., Klingebiel T., Diddens H., Probst H. & Niethammer D. (1992) Mdrllpglycoprotein, topoisomerase, and glutathione-S-transferase pi gene expression in primary and relapsed state adult and childhood leukaemias. Br. J. Cancer 66, 507. 7. Sanfilippo O., Ronchi E., De Marco C., Di Fronzo G. & Silvestrini R. (1991) Expression of P-glycoprotein in breast cancer tissue and in vitro resistance to doxorubicin and vincristine. Eur. J. Cancer 27, 155. 8. Bentley D. P. & Blackmore J. A. (1992) The inhibition of DNA synthesis in chronic lymphocytic leukaemia cells by chlorambucil in vitro. Br. J. Cancer 65, 171. 9. Chaudhary P. M. & Roninson I. B. (1993) Induction of multidrug resistance in human cells by transient exposure to different chemotherapeutic drugs. J. Nut1 Cancer Inst. 85, 632. 10. Evans C. D., Mirski S. E., Danks M. K. & Cole S. P. C. (1994) Reduced levels of topoisomerase II alpha and II beta in a multidrug-resistant lung-cancer cell line. Cancer Chemother. Pharmacol. 34, 242. 11. Hasegawa S., Abe T., Naito S., Kotoh S., Kurnazawa J., Hipfner D. R., Deeley R. G., Cole S. P. C. & Kuwano M. (1995) Expression of multidrug resistance-associated protein (MRP), MDRl and DNA topoisomerase II in human multidrug-resistant bladder cancer cell lines. Br. J. Cancer 71, 907. 12. Herweijer H., Sonneveld P., Bass F. & Nooter K. (1990) Expression of mdr 1 and mdr 3 multidrug-resistance genes
13.
14.
15.
16.
17.
18.
19.
20.
21. 22.
23. 24.
25.
26.
in human acute and chronic leukemias and association with stimulation of drug accumulation by cyclosporine. J. Natl Cancer Inst. 82, 1133. Holmes J., Jacobs A., Carter G., Janowska-Wieczorek A. & Padua R. A. (1989) Multidrug resistance in haemopoietic cell lines, myelodysplastic syndromes and acute myeloblastic leukaemia. Br. J. Huematol. 72, 40. Miller T. P., Grogan T. M., Dalton W. S., Spier C. M., Scheper R. J. & Salmon S. E. (1991) P-glycoprotein expression in malignant lymphoma and reversal of clinical drug resistance with chemotherapy plus high-dose verapamil. J. C&z. Oncol. 9, 17. Salmon S. E., Grogan T. M., Miller T., Scheper R. & Dalton W. S. (1989) Prediction of Doxorubicin resistance in vitro in myeloma, lymphoma, and breast cancer by Pglycoprotein staining. J. Nat1 Cancer Inst. 81, 696. McKenna S. L., Whittaker J. A., Padua R. A. & Holmes J. A. (1993) Topoisomerase II expression in normal haemopoietic cells and chronic lymphocytic leukaemia: drug sensitivity or resistance? Leukemia 7, 1199. Schisselbauer J. C., Silber R., Papadopoulos E., Abrams K., LaCreta F. P. & Tew K. D. (1990) Characterization of glutathione s-transferase expression in lymphocytes from chronic lymphocytic leukemia patients. Cancer Rex 50, 3562. Gupta R. S. (1983) Genetic, biochemical, and crossresistance studies with mutants of Chinese hamster ovary cells resistant to the anticancer drugs, VM-26 and VP-16213. Cancer Rex 43, 1568. Pommier Y., Kerrigan D., Schwartz R. E., Swack J. A. & McCurdy A. (1986) Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors. Cancer Res. 46. 3075. Holmes J. A., Jacobs A., Carter G., Whittaker J. A., Bentley D. P. & Padua R. A. (1990) Is the mdr 1 gene relevant in chronic lymphocytic leukemia? Leukemia 4, 216. Perry R. R., Mazetta J., Levin M. & Barranco S. C. (1993) Glutathione levels and variability in breast tumors and normal tissue. Cnncer 72, 783. Szarka C. E., Pfeiffer G. R., Hum S. T., Everley L. C., Balshem A. M., Moore D. F., Litwin S., Goosenberg E. B., Frucht H., Engstrom P. F. & Clapper M. L. (1995) Glutathione s-transferase activity and glutathione s-transferase u expression in subjects with risk for colorectal cancer. Cancer Res. 55, 2789. Tiirikainen M. I., Elonen E., Syrjall M. T., Jansson S.-E. & Krusius T. (1994) Flow cytometric analysis of glutathiones-transferase-rr in acute leukemia. Leukemia 8, 978. Hegewisch-Becker S., Szudra A. & Hossfeld D. K. (1993) Exposure to lipophilic industrial solvents leads to increased P-glycoprotein expression in peripheral blood cells. Br. J. Haematol. 85, 220. Thomale J., Seiler F., Miiller M. R., Seeber S. & Rajewsky M. F. (1994) Repair of 06-alkylguanines in the nuclear DNA of human lymphocytes and leukaemic cells: analysis at the single-cell level. Br. J. Cancer 69, 698. Kalken C. van, Pinedo H. M. & Giaccone G. (1991) Multidrug resistance from the clinical point of view. Eur. J. Cancer 27, 1481.
Pergamon PII: SO145-2126(96)00024-O
FAILURE
TO INDUCE RESISTANCE TO CYTOTOXIC NORMAL LYMPHOCYTES
DRUGS IN
Paul Bentley, Alun Thomas and JoseThomas Departments of HaematologyandThoracicMedicine,LlandoughHospital,Penarth,SouthGlamorgan,U.K. (Received 19 January 1996.Accepted 9 February
1996)
Abstract-Lymphocytes from patients who had received chemotherapy for lung cancer were examined for evidence of drug resistance using an in vitro assay for sensitivity to the effects of chlorambucil and etoposide. There was no evidence of induced resistance to the effects of chlorambucil or etoposide in the patients’ lymphocytes when compared to the sensitivity of the normal control subjects’ lymphocytes. In four patients, in whose lymphocytes sensitivity to chlorambucil was measured before and after a course of treatment, there was no significant change in the in vitro drug sensitivity. These findings are in contrast to those made previously using the lymphocytes of patients with chronic lymphocytic leukaemia and indicate that acquired drug resistance is a property of malignant cells and is not inducible in normal lymphocytes. Copyright 0 1996 Elsevier Science Ltd. Key words:
Normal
lymphocytes,
chemotherapy
Introduction
resistance.
become progressively resistant to the effects of chlorambucil in vitro after each course of cytotoxic chemotherapy. In the present study the effect of chemotherapy, administered for the treatment of lung cancer, on the sensitivity of normal lymphocytes was examined. Patients who had received etoposide were selected for study and the in vitro effects of etoposide and chlorambucil were examined in separategroups of patients and compared with the findings in the lymphocytes obtained from normal control subjects.
Resistance to chemotherapy is recognised to be the major obstacle to the successful treatment of many tumours. Many diverse cellular mechanismshave been proposed for this resistance and these include transmembranedrug export (via the product of the multidrug resistancegene m&-l), neutralization of cytotoxic drugs by cytoplasmic constituents, loss of topoisomeraseII activity, enhancementof DNA repair and inhibition of apoptosis. These mechanismsare available not only to tumour cells but also to normal tissues[14]. A notable feature of drug resistance in malignant cells is its enhancement following chemotherapy [5]; increased expression of mdr-1 and glutathione-s-transferase and topoisomeraseII suppressionhave each been shown to be induced in malignant cells by chemotherapy [6,7]. It hasbeen previously shown [8] that the sensitivity of lymphocytes to cytotoxic drugs can be measured by inhibition of DNA incorporation and the results bear a close relationship to clinical responsivenessin chronic lymphocytic leukaemia (CLL). The peripheral blood lymphocytes of the patients in that study were found to
Patients and Methods
Abbreviations: MBq, megabequerel;“H, tritiated; CLL, chronic lymphocytic leukaemia;mdr-1, multidrug resistance gene;P-gp, P-glycoprotein Correspondence to: D. P. Bentley, LlandoughHospitaland Community NHS Trust, Penlan Road, Penarth, South GlamorganCF64 2xX, U.K. 645
The study was approved by the South Glamorgan Health Authority Ethics Committee. The patients had received treatment according to local protocols for the managementof small-cell lung cancer. The total amount of etoposide received by each patient prior to the study is summarized in Table 1, together with the interval in days since the last dosewas administered.Most patients had received additional treatment including carboplatin or radiotherapy. In nine instancesthe patient’s lymphocytes were tested against chlorambucil and in eight against the effects of etoposide in vitro. Lymphocytes from five normal subjects were used for controls in the chlorambucil study and from eight normal subjects in the etoposide study. Lymphocytes were separatedfrom fresh venous blood samplesusing discontinuousdensity gradient centrifugation (Histopaque-1077, Sigma
646 Table
Patient number
P. Bentley et al. 1. Patient dosage data and chlorambucil Total dose etoposide received (mg)
Days since last dose
700 2100 1400 2100 2800 700 2100 2800 2100
35 29 9 11 18 1 5 37 12
and etoposide sensitivities lymphocytes
700 3500 5600 2100 2100 700 2800 2800
and the normal
Chlorambucil
Normals n=5 10 11 12 13 14 15 16 17
in the patients’
Mean S.D. Mean S.D.
Etoposide
IDso w/ml
Slope
2.4 4.6 2.2 2.9 3.5 3.1 2.7 1.8 3.3 2.9 f0.8 3.2
-0.33 - 0.21 -0.16 -0.30 -0.32 - 0.32 -0.29 -0.29 -0.40 -0.29 f 0.07 - 0.22 * 0.03
+0.38
ID50
7 3 3 150 6 109 240 120 Normals
n=8
Chemical Co., Poole, Dorset, U.K.) under sterile conditions. Cell cultures at a final concentration of 1 x 106/ml were performed in Eagles Minimum Essential Medium (Gibco Ltd, Paisley, U.K.) supplemented with 0.22% sodium bicarbonate, 5% fetal calf serum, 10,000 IU/lOO ml sodium benzyl penicillin and 5000 pg/lOO ml streptomycin. To each suspension 0.2 ml of autologous serum was added together with 50 ~1 of a solution of 0.4 @ml pokeweed mitogen. Chlorambucil solution (Sigma) was made up from pure powder by dissolving 24 mg in 1 ml of ethanol which was then diluted to 100 ml in phosphate-buffered saline (PBS). This stock solution was stored in aliquots at -20°C. Etoposide was obtained as a pharmaceutical preparation (Vepesid, Bristol-Myers Pharmaceuticals, Hounslow, Middlesex, U.K.). Chlorambucil solutions were addedto cultures to achieve final concentrations of between 1 and 10 @ml and etoposide solutions were addedto achieve final concentrations of between 0.1 and 1 l.@rnl. PBS was added to further cultures without inhibitor and sodium azide added to separatecultures as a blank. Cultures were performed in triplicate, except those without inhibitor which were performed in quintuplicate, at 37°C in 5% carbon dioxide in humidified air for 66 h. [3H]Thymidine (0.074 MBq; Amersham International plc, Amersham, Bucks, U.K.)
control subjects’
Mean S.D. Mean S.D.
ethl
0.25 0.32 0.32 0.18 0.45 0.21 0.19 0.18 0.26 f 0.09 0.22 * 0.04
Slope
-2.4 - 1.5 - 2.4 -2.6 -2.9 -2.2 -3.1 -2.1 -2.3 f0.5 -2.9 * 0.54
in 0.1 ml PBS was then addedto each culture which was then incubated for a further 24 h. Lymphocytes were harvested by filtration through 2.5 cm glass microfibre discs (Whatman Labsales,Maidstone, Kent, U.K.), the dried discs immersedin liquid scintillation fluid (Instagel, Canberra Packard Ltd., Cambourne, U.K.) and the samplescounted using a Wallac 1410 liquid scintillation counter. The percentage 3H uptake in each culture was calculated taking the uptake in the control cultures without inhibitor as 100%. Results The summarized results are shown in Table 1. In the normal control subjects, 50% inhibition of DNA synthesis (1~~~) was achieved at a mean (&S.D.) chlorambucil concentration of 3.2 f 0.38 @ml and in the patients the mean (+ S.D.) chlorambucil 1~~~ was 2.9 f 0.8 @ml. This difference is not significant (Mann-Whitney two sample tests U = 15.0, P = 0.35). The respective mean (+ S.D.) slope of the curves in the normal subjectswas -0.22 f 0.03 and for the patients was -0.29 f 0.07. The difference between these is not significant (Mann-Whitney U = 8.0, P = 0.6). Five sequential studies were undertaken prior to treatment and following a course of chemotherapy. In three of
Chemotherapy sensitivity of normal lymphocytes
Post-treatment
Pre-treatment
Fig. 1. Chlorambucil IDA,, for four patients measured before and after a course of etoposide. One patient was studied on two
occasions.
these there was a fall in the IDLE following treatment and in only one did the 1~~~ increase.These resultsare shown in Fig. 1. In the etoposide study the mean (+ S.D.) ~~~~ for the normal subjectswas 0.22 f 0.04 pg./ml whereas in the patients the mean (kS.D.) was 0.26 + 0.09 &ml. The difference between these groups is not significant (Mann-Whitney U = 25.0, P = 0.50). The slope of the dose-responsecurves against etoposidehad a mean (f S.D.) value in the normal subjects of -2.9 + 0.54 and in the patients -2.3 + 0.5. There is no significant difference between these two groups (Mann-Whitney U = 18.0, P = 0.16). Discussion The pleiotropic resistance of tumours to cytotoxic chemotherapy is characteristically inducible by exposure to chemotherapy. Solid tumours become progressively resistant despite initial sensitivity, acute leukaemia in relapse following chemotherapy-induced remission showspoorer responsesand the lymphocytes of patients with chronic lymphocytic leukaemia can be shown to become relatively more resistantto chlorambucil in vitro after the patient has received treatment [5,8]. Furthermore resistance can be induced in tumour cell lines cultured in the presenceof a cytotoxic drug [9-111. The recognisedmechanismsof drug resistancecan be
641
identified to explain many of these effects. Increased expression of the multidrug resistancegene mdr-1 and its product P-glycoprotein (P-gp) have been observed in leukaemias [12, 131, lymphomas [14], myeloma [15] and in solid tumours following treatment and related to clinical resistance [7]. Reduced expression of topoisomeraseII has been demonstrated in CLL [16] and theselymphocytes have increasedlevels of glutathiones-transferase [17]. Human tumour cell lines show increasedmdr-1 expression after drug exposure and this effect is rapidly induced but may remain stablefor many weeks [9]. Normal rodent cells can be induced to drug resistance in tissue culture [18, 191. Normal human peripheral blood lymphocytes are known to express mdr-1 [24,201, have low expressionof topoisomeraseII [16] and detectable glutathione levels [21] and glutathione-stransferase activity [22,23]. Thus, the potentially inducible mechanismsfor drug resistance are available to the normal cell. Furthermore mdr-1 hasbeen shown to be inducible in the lymphocytes of normal subjects in response to lipophilic solvents [24] and normal lymphocytes can be shown to have effective DNA repair mechanisms[25]. No evidence of induction of chemotherapy resistance was demonstrable in the normal lymphocytes obtained from the patients in this study despite lengthy and often multiple exposure to chemotherapy. There was no change in the characteristics of the dose-response curves following chemotherapy; selection of more resistant subclones by elimination of sensitive cells would have led to a decreasein the slope of the curves and any induction of drug resistance would have increased the 1~~~. Etoposide (an epipodophyllotoxin) resistance is mediated by a number of mechanisms, particularly by drug efflux via P-gp and by topoisomerase II suppression,whereas the mechanism of chlorambucil resistance is more obscure [26]. It has been suggested[20] that the effect of acquired drug resistance might be transient, explaining why low levels of mdr-I RNA is found in malignant cells and yet stable, chemotherapy-resistant tumour cell lines have been produced [9]. In the present study, patients were investigated between 1 and 240 days after receiving chemotherapy without any discernible difference in the results. The lack of induction of drug resistance in normal lymphocytes in patients receiving etoposide and other chemotherapeutic agents for solid tumour indicates an entirely different behaviour between normal lymphocytes and tumour cells. Acquired drug resistancetherefore appearsto be a feature of the neoplastic cell that is not found in its normal counterpart and probably results from the high mutation rate characteristic of tumours. Had such resistancebeen inducible in normal cells this
648
P. Bentley et al.
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