- Email: [email protected]
Chapter 21 Expression of monoclonal antibody-defined lung tumour antigens in drug resistant lung tumour cell lines
87
Lung Cancer, 4 (1988) 87-89 Elsevier CHAPTER21 EXPRESSION OF MONOCLONAL ANTIBODY-DEFINED LUNG TUMOUR ANTIGENS IN DRUG RESISTANT LUNG TUMOUR CELL LINES Julie G. Reeve, Jonathan J. Shaw, Peter R. Twentyman And Norman M. Bleehen MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, England, UK. Correspondence to J.G. Reeves.
ABSTRACT The expression of monoclonal antibody (Moab)defined lung turnout antigens has been investigated in in vitro-derived multidrug-resistant (MDR) variants of small cell (SCLC) and non-small cell (NSCLC) lung tumour cell lines. The majority of Moabs tested reacted equally well with MDR and drug-sensitive SCLC and NSCLC cells. However, in contrast to drug sensitive SCLC cells, MDR-SCLC cells failed to express the glycolipid antigen defined by Moab LCAl/LC38, showed increased expression of 2 glycoproteins, defined by Moabs LAM8 and SWA 4, and increased expression of the sugar epitope defined by Moab MOv15. Drug resistant large cell lung cancer cells also showed increased expression of the glycoprotein defined by Moab SWA 4 compared to the parent line which failed to react with this Moab. No significant differences were observed in the expression of lung tumour antigens by drug sensitive and resistant adenocarcinoma cells.
H69, which show the drug resistance phenotype in these cells to be a complex, multicomponent system involving hyperexpression of plasma membrane Pglycoprotein 10. In an effort to design novel strategies for the treatment and diagnosis of lung cancer, many workers have generated monoclonal antibodies (Moabs) to lung tumour cell surface antigens 11-13.
The prospective applications for the clinical use of such Moabs are, for the most part, dependent upon continued antigen expression before, during and after chemotherapy. In view of the plethora of cell surface effects exerted by ADM, the present study investigates lung tumour antigen expression in in vitro derived drug resistant SCLC and non-SCLC cells.
A
INTRODUCTION The anthracycline antibiotics are used in the treatment of a broad spectrum of human tumours. Although the biochemical mechanisms of action of these agents has been attributed to intercalation with DNA and consequent inhibition of DNA and RNA biosynthesis 1, there is a persuasive body of evidence to show that these drugs exert a vadety of effects on the cell sudace. This is particularly true for Adriamycin (ADM) which has been shown to affect plasma membrane physical and biochemical properties including membrane fluidity 2-5. More recently, ADM has been shown to cause upregulation of epidermal growth factor receptors in actively growing 3T3 and HeLa cells 6. Furthermore, in vitro derived multidrug-resistant (MDR) tumour cells are often characterised by a variety of diverse membrane alterations which may include increased or decreased expression of plasma membrane glycoproteins, altered plasma membrane lipid structural order and altered carbohydrate content 7. Overproduction of a high molecular weight family of glycoproteins, The P-glycoproteins, commonly observed in in vitro derived drug resistant cell lines, has also been observed ih viv0 in MDR human ovarian carcinomas 8 and in MDR acute nonlymphoblastic leukaemias 9, indicating that certain drug-related alterations in membrane composition observed in vitro are of clinical significance. Chemotherapy, which may include the anthracycline antibiotics is often used in the treatment of small cell lung cancer (SCLC) in man. Although objective remissions can be achieved in many cases treated with intense cytotoxic therapy, subsequent relapse is common and often attributable to drug resistant cells, either existing prior to or arising during treatment. That such cells may have altered plasma membrane composition is suggested by our recent studies of in vitro-derived MDR variants of SCLC cell line NCI-
;.]t,
]B
Figure l(a): Reactivity of NCI-H69 parent line with Moab LCAllLC38. Approximately 10% of the cells are stongly stained by the Moab in an indirect immunofluorescent assay.
Figure l(b): Reactivity of drug resistant variant H69/LX4 with Moab LCA1/LC38. Cells expressing the glycolipid defined by the Moab are not present in the drug resistant cells line.
0169-5002/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
88 MATERIALS AND METHODS
TABLE I Monoclonal antibody-defined lung tumour antigens showing altered expression in drug resistant cells.
Cells Details regarding the derivation of ADM-resistant variants of SCLC and NSCLC cell lines are given elsewhere TM. Briefly, the drug resistant variant, H69/LX4(LX4), of SCLC cell line NCI-H69 was adapted to grow continuously in 0.4 ug/ml ADM, the resistant variant (L23R) of large cell carcinoma line COR-L23 in 0.2ug/ml ADM, and the drug resistant variant (MOR-R) of adenocarcinoma cell line MOR in 0.2ug/ml ADM. All drug resistant variants are crossresistant to vincristine and colchicine. Cells were maintained in RPMI 1640 medium with 10% foetal calf serum, penicillin and streptomycin (All Gibco Europe Ltd). Indirect Immunofluorescence Assay
ANTIBODY
ANTIGEN DEFINED
% CELLS STAINED AND INTENSITY
H69 LCAI/ LC38
CELLS LX4 L23C L23R
GLYCOLIPID
10S
0
NT
NT
LAM8
90-153-KD GLYCOPROTEIN
0
10S
NT
NT
MOv15
SUGAR EPITOPE
10S
80S
40S
40S
SWA 4
90-110-KD GLYCOPROTEIN
30W 90S
0
5S
To investigate the expression of monoclonal antibody-defined lung tumour antigens in drug resistant and sensitive cell lines, unfixed cells were
screened against monoclonal antibodies using an indirect immunofluorescence assay. Cell lines growing as aggregates in suspension 9H69P and LX4) were disaggregated by gentle pipetting to yield a suspension of single cells and small clumps. Cells growing as adherent monolayers were treated with Versene (0.02%, Gibco Biocult) for 15 minutes at 37oc. Cells were washed thrice with phospate buffered saline (PBS) and incubated with FITCconjugated rabbit anti-mouse/rat lg antiserum (Dako, High Wycombe, UK) diluted 50-fold in PBS for 30 rain at 37oc. After copious washing cells were coverslipped in Hydramount (National Diagnostics New Jersey, USA) and viewed with an Olympus fluorescence microscope.
A
RESULTS, The majority of Moabs tested reacted equally well with drug sensitive and drug resistant SCLC and non-SCLC cells. However, it can be seen from Table I that compared to the parent cell line, the drug resistant variant (LX4) of SCLC cell line NC1-H69, fails to express the glycolipid defined by Moab LCA1/LC38(Fig la,b), and shows increased expression of the sugar epitope defined by Moab MOv15, the glycoprotein defined by LAM8 and the glycoprotein defined by Moab SWA 4 (Fig 2a,b).
]]
Moab SWA 4 which fails to react with non-SCLC cell lines (Dr. R.A, Stahel, personal communication), showed no reactivity with large cell carcinoma L23C cells (Table 1, Fig 3a). However, when ADMresistant L23R cells were screened against Moab SWA 4, antibody reactive cells were clearly identified in approximately 5% of the population (Fig. 3b). No significant, consistent alterations were observed in the expression of Moab-defined lung tumour antigens in the drug resistant variant of adenocarcinoma cell line MOR (data not shown).
t
DISCUSSION
Figure 2(a): Reactivity of NCI-H69 parent line with Moab SWA4. Approximately 20% of cells weakly express the antigen defined by this Moab.
Figure 2(b): Reactivity of H69/LX4 cells with Moab SWA4. The drug resistant vadant shows increased expression of the 90-110 kD glycoprotein defined by this Moab, wirh approximately 80% of the cells stained strongly by the antibody.
The present study demonstrates that alterations in lung turnout antigen expression can occur in in vitroderived drug resistant SCLC and non-SCLC cells. Therapy-related modulation of antigen expression may result in increased or decreased levels of normally expressed antigens, or in the "switching on" or unmasking of antigens not normally expressed in the untreated population. Such changes may be part of a general alteration in the composition of membranes in MDR cells as suggested by studies showing changes in membrane lipid composition upon development of drug resistance 15. However, it is also noteworthy that the increased expression of
89 (1978). Adriamycin: A proposal on the specificity of drug action. Biochem. Biophys. Res. Commun. 84, 802.
&
3.
Murphree, S.A., Tritton, T.R., Smith, P.L. and Sartorelli, A.C. (1981). Adriamycin-induced changes in the surface membrane of sarcoma 180 ascites cells. Biochem. Biophys. acta 649, 317.
4.
Crane, F.L., Mackellar, W.C., Morre, D.J. at al. (1980). Adriamycin affects plasma membrane redox functions. Biochem. Biophys. Res. Commun. 93, 746. Gosalvez, M , van Rossum, G.D.V. and Blance, M.F. (1979). Inhibition of sodium-potassiumactivated adenosine 5'-triphosphatase and ion transport by Addamyc!n. Cancer Res. 39, 257.
6.
]3
Zuckier, R. and Tritton, T.R. (1983). Adriamycin causes up-regulation of epidermal growth factor receptors in actively growing cells. Exp. Cell Ras. 148, 155. Riodan, J.R. and Ling, V. (1985). Genetic and biochemical characterisation of multidrug resistance. Pharmac. That. 28, 51. Bell, D.R., Gerlach, J.H., Kartner, N., Buick, R.N. et al. (1980). Detection of P-glycoprotein in ovarian cancer: A molecular marker associated with multidrug resistance. J. Clin. OncoL 3, 311.
9.
Ma. D.D.F.. Davey, R.A., Harman, D.H. et al. (1987). Detection of multidrug resistant phenotype in acute n o n - l y m p h o b l a s t i c leukaemia. Lancet i, 135.
10. Reeve, J.G., and Ling, V. (1985). Genetic and biochemical characterisation of multidrug resistance. Pharmacol. Ther. 28, 51.
Figure 3(a): Reactivity of large cell carcinoma cell line CDR-L23 with Moab SWA4. This cell line fails to express the antigen defined by the Moab.
Figure 3(b): Reactivity of drug resistant variant L23R with Moab SWA4. Approximately 5% of the population show reactivity with the antibody indicating the emergence of antigen-positive cells in the drug resistant cell line.
high molecular weight glycoproteins is commonly observed in MDR cell lines. By far the most striking membrane alteration in several MDR cells is the presence of P-glycoprotein 7, hyperexpression of which is thought to result in alterations in the energydependent efflux mechanism in MDR cells. Hence the alterations in cell surface expression in MDR cells described in the present study may be more directly implicated in the maintenance of the drug resistance phenotype by contributing to any of the variety of pleiotropic changes in membrane function which have been observed in MDR cells. interestingly, the 90-110KD glycoprotein defined by Moab SWA4 is hyperexpressed in drug resistant SCLC cells and apparently expressed de novo in drug resistant large cell carcinoma cells. We are currently evaluating the relevance of the expression of this antigen to the drug resistance phenotype by investigating the relationships between antigen
expression and reduced intracellular accumulation and in vitro drug sensitivity.
11. Cuttita, F., Rosen, S., Gazdar, A.F. and Minna, J.D. (11981). Monoclonal antibodies which demonstrate specificity for several types of human lung cancer. Proc. Natl. Acad. SOL (Wash.) 131,497. 12. Mulshine, J.L, Cuttita, F., Bibro, M. et al. (1983). Monoclonal antibodies that distinguish nonsmall cell from small cell lung cancer. J. Immunol. 131,497.
drug
13. Reeve, J.G., Wulfrank. D.A., Steward, J. et al. (1987). Proteins associated with multidrug resistance in human small cell lung cancer cell lines. Cancer Res. Submitted 1987.
The findings presented in this study may have implications for the application of monaclonal antibodies to the diagnosis, monitoring and treatment of lung cancer, and perhaps indicate that stability of antigen expression before and after chemotherapy is an important consideration in the selection of Moabs for such purposes.
14. Twentyman, P.R., Fox, N.E., Wright, K.A., and Bleehen, N.M. (1986). Derivation and preliminary characterisation of adriamycin resistant lines of human lung caner cells. Br. J. Cancer 53, 529.
REFERENCES
15. Rintoul, O.A. and Centre, M.S. (1984). Involvement of plasma membrane lipid structural order in adriamycin resistance in Chinese hamster cell lungs. Cancer Res. 44, 4978.
Di Marco, A. (1975). Adriamycin (NSC-123127): mode and mechanism of action. Cancer Chemother. Rep. Part III, 6, 91. 2.
Tritton, T.R., Murphree, S.A. and Sartorelli, A.C.
Lung Cancer, 4 (1988) 87-89 Elsevier CHAPTER21 EXPRESSION OF MONOCLONAL ANTIBODY-DEFINED LUNG TUMOUR ANTIGENS IN DRUG RESISTANT LUNG TUMOUR CELL LINES Julie G. Reeve, Jonathan J. Shaw, Peter R. Twentyman And Norman M. Bleehen MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, England, UK. Correspondence to J.G. Reeves.
ABSTRACT The expression of monoclonal antibody (Moab)defined lung turnout antigens has been investigated in in vitro-derived multidrug-resistant (MDR) variants of small cell (SCLC) and non-small cell (NSCLC) lung tumour cell lines. The majority of Moabs tested reacted equally well with MDR and drug-sensitive SCLC and NSCLC cells. However, in contrast to drug sensitive SCLC cells, MDR-SCLC cells failed to express the glycolipid antigen defined by Moab LCAl/LC38, showed increased expression of 2 glycoproteins, defined by Moabs LAM8 and SWA 4, and increased expression of the sugar epitope defined by Moab MOv15. Drug resistant large cell lung cancer cells also showed increased expression of the glycoprotein defined by Moab SWA 4 compared to the parent line which failed to react with this Moab. No significant differences were observed in the expression of lung tumour antigens by drug sensitive and resistant adenocarcinoma cells.
H69, which show the drug resistance phenotype in these cells to be a complex, multicomponent system involving hyperexpression of plasma membrane Pglycoprotein 10. In an effort to design novel strategies for the treatment and diagnosis of lung cancer, many workers have generated monoclonal antibodies (Moabs) to lung tumour cell surface antigens 11-13.
The prospective applications for the clinical use of such Moabs are, for the most part, dependent upon continued antigen expression before, during and after chemotherapy. In view of the plethora of cell surface effects exerted by ADM, the present study investigates lung tumour antigen expression in in vitro derived drug resistant SCLC and non-SCLC cells.
A
INTRODUCTION The anthracycline antibiotics are used in the treatment of a broad spectrum of human tumours. Although the biochemical mechanisms of action of these agents has been attributed to intercalation with DNA and consequent inhibition of DNA and RNA biosynthesis 1, there is a persuasive body of evidence to show that these drugs exert a vadety of effects on the cell sudace. This is particularly true for Adriamycin (ADM) which has been shown to affect plasma membrane physical and biochemical properties including membrane fluidity 2-5. More recently, ADM has been shown to cause upregulation of epidermal growth factor receptors in actively growing 3T3 and HeLa cells 6. Furthermore, in vitro derived multidrug-resistant (MDR) tumour cells are often characterised by a variety of diverse membrane alterations which may include increased or decreased expression of plasma membrane glycoproteins, altered plasma membrane lipid structural order and altered carbohydrate content 7. Overproduction of a high molecular weight family of glycoproteins, The P-glycoproteins, commonly observed in in vitro derived drug resistant cell lines, has also been observed ih viv0 in MDR human ovarian carcinomas 8 and in MDR acute nonlymphoblastic leukaemias 9, indicating that certain drug-related alterations in membrane composition observed in vitro are of clinical significance. Chemotherapy, which may include the anthracycline antibiotics is often used in the treatment of small cell lung cancer (SCLC) in man. Although objective remissions can be achieved in many cases treated with intense cytotoxic therapy, subsequent relapse is common and often attributable to drug resistant cells, either existing prior to or arising during treatment. That such cells may have altered plasma membrane composition is suggested by our recent studies of in vitro-derived MDR variants of SCLC cell line NCI-
;.]t,
]B
Figure l(a): Reactivity of NCI-H69 parent line with Moab LCAllLC38. Approximately 10% of the cells are stongly stained by the Moab in an indirect immunofluorescent assay.
Figure l(b): Reactivity of drug resistant variant H69/LX4 with Moab LCA1/LC38. Cells expressing the glycolipid defined by the Moab are not present in the drug resistant cells line.
0169-5002/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
88 MATERIALS AND METHODS
TABLE I Monoclonal antibody-defined lung tumour antigens showing altered expression in drug resistant cells.
Cells Details regarding the derivation of ADM-resistant variants of SCLC and NSCLC cell lines are given elsewhere TM. Briefly, the drug resistant variant, H69/LX4(LX4), of SCLC cell line NCI-H69 was adapted to grow continuously in 0.4 ug/ml ADM, the resistant variant (L23R) of large cell carcinoma line COR-L23 in 0.2ug/ml ADM, and the drug resistant variant (MOR-R) of adenocarcinoma cell line MOR in 0.2ug/ml ADM. All drug resistant variants are crossresistant to vincristine and colchicine. Cells were maintained in RPMI 1640 medium with 10% foetal calf serum, penicillin and streptomycin (All Gibco Europe Ltd). Indirect Immunofluorescence Assay
ANTIBODY
ANTIGEN DEFINED
% CELLS STAINED AND INTENSITY
H69 LCAI/ LC38
CELLS LX4 L23C L23R
GLYCOLIPID
10S
0
NT
NT
LAM8
90-153-KD GLYCOPROTEIN
0
10S
NT
NT
MOv15
SUGAR EPITOPE
10S
80S
40S
40S
SWA 4
90-110-KD GLYCOPROTEIN
30W 90S
0
5S
To investigate the expression of monoclonal antibody-defined lung tumour antigens in drug resistant and sensitive cell lines, unfixed cells were
screened against monoclonal antibodies using an indirect immunofluorescence assay. Cell lines growing as aggregates in suspension 9H69P and LX4) were disaggregated by gentle pipetting to yield a suspension of single cells and small clumps. Cells growing as adherent monolayers were treated with Versene (0.02%, Gibco Biocult) for 15 minutes at 37oc. Cells were washed thrice with phospate buffered saline (PBS) and incubated with FITCconjugated rabbit anti-mouse/rat lg antiserum (Dako, High Wycombe, UK) diluted 50-fold in PBS for 30 rain at 37oc. After copious washing cells were coverslipped in Hydramount (National Diagnostics New Jersey, USA) and viewed with an Olympus fluorescence microscope.
A
RESULTS, The majority of Moabs tested reacted equally well with drug sensitive and drug resistant SCLC and non-SCLC cells. However, it can be seen from Table I that compared to the parent cell line, the drug resistant variant (LX4) of SCLC cell line NC1-H69, fails to express the glycolipid defined by Moab LCA1/LC38(Fig la,b), and shows increased expression of the sugar epitope defined by Moab MOv15, the glycoprotein defined by LAM8 and the glycoprotein defined by Moab SWA 4 (Fig 2a,b).
]]
Moab SWA 4 which fails to react with non-SCLC cell lines (Dr. R.A, Stahel, personal communication), showed no reactivity with large cell carcinoma L23C cells (Table 1, Fig 3a). However, when ADMresistant L23R cells were screened against Moab SWA 4, antibody reactive cells were clearly identified in approximately 5% of the population (Fig. 3b). No significant, consistent alterations were observed in the expression of Moab-defined lung tumour antigens in the drug resistant variant of adenocarcinoma cell line MOR (data not shown).
t
DISCUSSION
Figure 2(a): Reactivity of NCI-H69 parent line with Moab SWA4. Approximately 20% of cells weakly express the antigen defined by this Moab.
Figure 2(b): Reactivity of H69/LX4 cells with Moab SWA4. The drug resistant vadant shows increased expression of the 90-110 kD glycoprotein defined by this Moab, wirh approximately 80% of the cells stained strongly by the antibody.
The present study demonstrates that alterations in lung turnout antigen expression can occur in in vitroderived drug resistant SCLC and non-SCLC cells. Therapy-related modulation of antigen expression may result in increased or decreased levels of normally expressed antigens, or in the "switching on" or unmasking of antigens not normally expressed in the untreated population. Such changes may be part of a general alteration in the composition of membranes in MDR cells as suggested by studies showing changes in membrane lipid composition upon development of drug resistance 15. However, it is also noteworthy that the increased expression of
89 (1978). Adriamycin: A proposal on the specificity of drug action. Biochem. Biophys. Res. Commun. 84, 802.
&
3.
Murphree, S.A., Tritton, T.R., Smith, P.L. and Sartorelli, A.C. (1981). Adriamycin-induced changes in the surface membrane of sarcoma 180 ascites cells. Biochem. Biophys. acta 649, 317.
4.
Crane, F.L., Mackellar, W.C., Morre, D.J. at al. (1980). Adriamycin affects plasma membrane redox functions. Biochem. Biophys. Res. Commun. 93, 746. Gosalvez, M , van Rossum, G.D.V. and Blance, M.F. (1979). Inhibition of sodium-potassiumactivated adenosine 5'-triphosphatase and ion transport by Addamyc!n. Cancer Res. 39, 257.
6.
]3
Zuckier, R. and Tritton, T.R. (1983). Adriamycin causes up-regulation of epidermal growth factor receptors in actively growing cells. Exp. Cell Ras. 148, 155. Riodan, J.R. and Ling, V. (1985). Genetic and biochemical characterisation of multidrug resistance. Pharmac. That. 28, 51. Bell, D.R., Gerlach, J.H., Kartner, N., Buick, R.N. et al. (1980). Detection of P-glycoprotein in ovarian cancer: A molecular marker associated with multidrug resistance. J. Clin. OncoL 3, 311.
9.
Ma. D.D.F.. Davey, R.A., Harman, D.H. et al. (1987). Detection of multidrug resistant phenotype in acute n o n - l y m p h o b l a s t i c leukaemia. Lancet i, 135.
10. Reeve, J.G., and Ling, V. (1985). Genetic and biochemical characterisation of multidrug resistance. Pharmacol. Ther. 28, 51.
Figure 3(a): Reactivity of large cell carcinoma cell line CDR-L23 with Moab SWA4. This cell line fails to express the antigen defined by the Moab.
Figure 3(b): Reactivity of drug resistant variant L23R with Moab SWA4. Approximately 5% of the population show reactivity with the antibody indicating the emergence of antigen-positive cells in the drug resistant cell line.
high molecular weight glycoproteins is commonly observed in MDR cell lines. By far the most striking membrane alteration in several MDR cells is the presence of P-glycoprotein 7, hyperexpression of which is thought to result in alterations in the energydependent efflux mechanism in MDR cells. Hence the alterations in cell surface expression in MDR cells described in the present study may be more directly implicated in the maintenance of the drug resistance phenotype by contributing to any of the variety of pleiotropic changes in membrane function which have been observed in MDR cells. interestingly, the 90-110KD glycoprotein defined by Moab SWA4 is hyperexpressed in drug resistant SCLC cells and apparently expressed de novo in drug resistant large cell carcinoma cells. We are currently evaluating the relevance of the expression of this antigen to the drug resistance phenotype by investigating the relationships between antigen
expression and reduced intracellular accumulation and in vitro drug sensitivity.
11. Cuttita, F., Rosen, S., Gazdar, A.F. and Minna, J.D. (11981). Monoclonal antibodies which demonstrate specificity for several types of human lung cancer. Proc. Natl. Acad. SOL (Wash.) 131,497. 12. Mulshine, J.L, Cuttita, F., Bibro, M. et al. (1983). Monoclonal antibodies that distinguish nonsmall cell from small cell lung cancer. J. Immunol. 131,497.
drug
13. Reeve, J.G., Wulfrank. D.A., Steward, J. et al. (1987). Proteins associated with multidrug resistance in human small cell lung cancer cell lines. Cancer Res. Submitted 1987.
The findings presented in this study may have implications for the application of monaclonal antibodies to the diagnosis, monitoring and treatment of lung cancer, and perhaps indicate that stability of antigen expression before and after chemotherapy is an important consideration in the selection of Moabs for such purposes.
14. Twentyman, P.R., Fox, N.E., Wright, K.A., and Bleehen, N.M. (1986). Derivation and preliminary characterisation of adriamycin resistant lines of human lung caner cells. Br. J. Cancer 53, 529.
REFERENCES
15. Rintoul, O.A. and Centre, M.S. (1984). Involvement of plasma membrane lipid structural order in adriamycin resistance in Chinese hamster cell lungs. Cancer Res. 44, 4978.
Di Marco, A. (1975). Adriamycin (NSC-123127): mode and mechanism of action. Cancer Chemother. Rep. Part III, 6, 91. 2.
Tritton, T.R., Murphree, S.A. and Sartorelli, A.C.