Br. J. Cancer Br. J. Cancer
(1991), XIV, Suppi. XIV, 63, Suppl. (1991), 63,
'."
43-45 43-45
©
Macmillan Press Ltd., 1991
Macmillan
Press
Ltd.,
1991
Establishment and characterisation of two new small cell lung cancer cell lines one from a patient with previous familial retinoblastoma -
F.G. Hay, L.W. Duncan & R.C.F. Leonard Imperial Cancer Research Fund Medical Oncology Unit and Department of Clinical Oncology, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK. Summary Two new small cell lung cancer (SCLC) cell lines have been established. The first, WX330, has been developed from a pleural effusion collected at presentation from a patient with extensive disease. The second, WX331, originated from a liver metastasis collected at post mortem from a patient who died of his SCLC disease. WX330 has been in culture for 4 years and WX331 for 11 months. Both cell lines have typical cytological features of SCLC, WX330 contains dense core granules on electron microscopic examination and both are tumourigenic in nude mice. Cell lines were analysed phenotypically using antibodies submitted to the Second International Workshop on SCLC Antigens by the immunoperoxidase method. WX330 is reactive with a wide range of SCLC and epithelial associated antibodies and WX331 with a more restricted group of antibodies of predominantly neuroendocrine and SCLC specificity. WX330 also reacts with an antibody against the multiple drug resistance-associated glycoprotein.
A continuing challenge in research in small cell lung cancer (SCLC) is to identify material for in vitro study which is representative of the clinical situation. The common experience of therapy has been the problem of tumour resistance to cytotoxic drugs in a disease that is initially, usually chemosensitive. Acquired resistance and, more infrequently, primary drug resistance are therefore major clinical problems and are the basic cause of incurability of SCLC. With the increasing availability of specific genetic probes, early observations on cytogenetic defects in SCLC have been confirmed in cell line studies. The major finding has been that virtually 100% of lung cancer cell lines show deletions of genetic material on the short arm of chromosome 3 (Naylor et al., 1987). However, a high proportion of cell lines have also been shown to exhibit RB (retinoblastoma) gene deletions (Harbour et al., 1988). This important cell proliferation control gene has been implicated in a variety of tumours. Despite the curability of retinoblastoma, there have been no well-studied cases of retinoblastoma being followed by SCLC. The purpose of this paper is to describe the establishment and characterisation of two cell lines, one derived from a patient who had clinically resistant disease and the second cell line from a metastasis in the liver in a patient who had had familial retinoblastoma but who died due to SCLC. Materials and methods Clinical background WX330 was derived from a pleural effusion collected at presentation from a patient with extensive SCLC. The patient was subsequently found to be unresponsive to chemotherapy (Etoposide/Vindesine) and died 6 months after presentation. WX331 was derived from a solid tumour metastasis of the liver obtained at post mortem. The 27 year old patient presented with limited stage SCLC, responded initially to chemotherapy (Adriamycin/cyclophosphamide/etoposide) before relapsing 8 months later with progressive disease. The patient had a previous history of familial retinoblastoma. Establishment of cell lines WX330 The cells from the pleural effusion were sedimented by centrifugation (1,000 g for O min), resuspended in RPMI
Correspondence: F.G. Hay.
1640 and red cells removed by Ficoll-Hypaque gradient separation. Interface cells were washed and checked for viability by vital dye exclusion. Aliquots of i05cellsml-' were cultured at 37°C, 90% humidity and 5% CO2 in RPMI + 10% Foetal calf serum (FCS) with added insulin (2.5 jig ml1 '), streptomycin (100 ,pg ml1 ), pyruvate (2 mM), penicillin (100 IU ml-') and glutamine (8 mM). Once the cell line was established and the tumour cells were proliferating, the medium was changed to HITES serum-free defined medium (Carney et al., 1980) to remove contaminating fibroblast cells. WX331 The tumour was cut into small pieces and the tumour cells were teased out. Thereafter, the resulting cell suspension was pelleted by centrifugation (1,000g for 10 min), washed in RPMI and passed over a Ficoll-Hypaque density gradient. The interface cells were washed and checked for viability by vital dye exclusion as before. Aliquots of 105 cells were cultured in HITES serum-free defined medium or HITES + 2+% FCS at 37°C, 90% humidity and 5% CO2. Measurement of clonogenicity The soft agar assay (Courtney et al., 1978) was used without the addition of irradiated feeder cells. Semi-liquid cultures were set up in agar (0.3% v/v). Fresh medium (1 ml) was added weekly. Colonies (> 50 cells) were scored after 28
days. Measurement of doubling times Cells were cultured in 35 mm wells. Duplicate cells were harvested daily and the cells counted. Population doubling times were calculated for cells in exponential phase of growth. Each experiment was repeated three times.
Tumourogenicity studies Cells in exponential growth in culture were harvested, centrifuged (1,000 g for O min), the pellet was washed and resuspended in phosphate buffered saline (PBS) at a density of I07 cells ml-'. 0.25 ml of cell suspension was injected subcutaneously into the flanks of nude mice. All animals were observed weekly until their tumours grew to a mean diameter of 1 cm or for at least 2 months in the absence of tumours. Biochemical assays The biochemical markers L-dopa decarboxylase (DDC) and creatinine kinase (CK) were measured by established methods (Gazdar et al., 1980; Gazdar et al., 1981).
44
F.G. HAY et al.
Immunohistochemistry The immunoperoxidase studies of the cell lines were performed using the Avidin-Biotin complex (ABC) system (Dako, UK). Briefly, cells were placed onto multispot slides (Hendley, (Essex) Ltd, UK) at approximately 2 x 104 cells per spot. Cells were fixed in methanol/acetone (1:1) for 10min then they were incubated at room temperature with 0.5% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity. After washing in TBS (Tris 0.5 M, pH 7.6, diluted in saline 1:10), they were incubated with rabbit serum: TBS (1:10) for 20min followed by the appropriate dilution of mouse monoclonal antibody (mab) for 30 min. Thereafter biotinylated rabbit anti-mouse preparation was applied (1:200) for 30min followed by the ABC complex also for 30 min. All steps were separated by three washes in TBS. The peroxidase was localised by treatment of the slides with freshly prepared 0.1% 3,3'diaminobenzidine and 0.01% hydrogen peroxide in Trisimidazole (pH 7.6) buffer for 10 min. After washing in water these samples were then counterstained with haematoxylin. For rat mabs an indirect immunoperoxidase system was used. The Workshop panel of mabs was used. In addition, the cell lines and the original material used in their derivation, were studied using HMFG2, CAM 5.2, AUA1, SM3 and UJ13A obtained from ICRF, London, EGFR1 and EGFRF4 (Gullick et al., 1986), 123C3 (Schol et al., 1988), and JSB1 (Scheper et al., 1988). Electron microscopy For transmission electron microscopy the tumour cells were pelleted and incubated in 2% glutaraldehyde in PBS. Post fixation was performed in 2% osmium tetroxide in PBS. The material was embedded in Epon and further preparations were according to routine procedures. Results Establishment and morphological characteristics of the cell lines WX330 grew very slowly at first but after the fifth passage became well established and has now reached passage 20. The cell line is routinely maintained in RPMI + 10% FCS, but also grows in HITES serum-free defined medium if
required. The cells grow as large aggregates of tightly packed cells which frequently undergo central necrosis with the formation of hollow sphericules. The doubling time of this cell line is now 57 h with a colony forming efficiency of 14.3% in agar. WX330 is resistant to storage in liquid nitrogen and is tumourogenic in nude mice, giving rise to tumours with an undifferentiated histology. The origin of both the cell line and the xenografted tumour from human SCLC cells has been confirmed by light and electron microscopy. The second cell line, WX331, became established very quickly after being put directly into culture in HITES serumfree defined medium. This cell line has now been in continuous culture for 11 months, grows as loose aggregates of cells, has a doubling time of 31 h, a colony forming efficiency of 33.7% in agar, recovers readily from liquid nitrogen storage and is tumourogenic in nude mice. This cell line has also been confirmed to be of the human SCLC type by light and electron microscopy. Values of L-dopa decarboxylase in WX330 were 2043 + 93.8 uIU mg' protein, creatinine kinase 2.26 ± 0.17 U mg-' protein. CKBB constituted 95% of total CK and CKMM 5%. WX331 was not tested. Both cell lines were immunophenotyped using the complete panel of mabs of the Second International Workshop. Results are shown in Table I. The pleural effusion which was used to derive WX330 stained with the anti-cytokeratin mab CAM 5.2, epithelial mabs AUA1 (cluster 2) HMFG2 and cluster 1 mabs 123C3 and UJ13A. The early passages of WX330 were also reactive with these mabs. At passages 12, 16, 17, 18 and 20, reactivity with CAM 5.2 had been lost and reactivity with EGFRF4 (internal portion of the epidermal growth factor) and with JSBl had emerged. A cell suspension prepared from the solid tumour sample used in the derivation of WX331 was reactive with CAM 5.2, HMFG2, SM3 and 123C3. Passages 1-4 of the WX331 cell line were strongly reactive with the cluster 1 mabs, 123C3 and UJ13A and reactive to a lesser extent with HMFG2, AUA1 and SM3. This cell line did not react with the JSBI or the EGFRF4 mabs. Discussion In this paper, we describe the establishment and characterisation of two new SCLC cell lines. WX330 grows in the manner of a classic SCLC cell line having a growth forma-
Table I Reactivity of WX330 and WX331 to Workshop antibodies
WX330 and WX331. Antibody name (and Workshop number)
WX330 alone. Antibody name (and Workshop number) S-L 11.14(34) SEN 6(60)
2
RNL 1(4), Moc 1(12), Moc 191(21), NCC-LU 246(3 1), NCC LU 243(48), SEN 36(61), NE 150(74), NE 25(77) Moc 31(11), Moc 58(14), PE35(75)
4
SWA1 1(59), SWA21(66), SWA22(67)
Cluster 1
Moc 151(61), Moc 181(20), S-L 4.20(85),
SL 2.21(90) 5
5A 6 7 8 Unclustered
SEN 31(64), SWA20(70) Mov 15(45), NCC LU 152(47)
NCC-CO-450(98) CAM 5.2(3, 56, 28, 71), Moc 171(18), MLuC3(43), NSE H124(38), NCC-RAS-044(57)
A-80(37) 43/9F(7), lA9-D4(8), 6A3-C8(9), Moc 172(9), BS 150(22), BS 153(24), SMI(27), Muc 54(29), Muc 18(30), MBRI(40), MLcC2(42), MLuC4(44), 443A6(46), NCC-LU-279(55), Fib 75(78), Mc 5(79), HMFG1(81), 2A6(83), S-L 3.5(35)
NEW SLCL CELL LINES
tion of tight sperical aggregates, a relatively long doubling time, high levels of the BB-isoenzymes of creatine kinase and dense core granules visible by electron microscopy. This cell line does, however, have a relatively high colony forming efficiency for a classic SCLC cell line. This cell line was reactive with a wide variety of the Second International Workshop panel of mabs and staining of the original tumour sample and later passages of the cell line indicated that the phenotype of this cell line is relatively stable. The reactivity of the cell line with the multiple drug resistance mab, JSBl, may reflect the non-responsiveness to chemotherapy of the patient from whom the cell line was derived. In vitro drug studies should define the relative resistance levels as against in vitro-induced resistance and also the pleiomorphism of the pattern. WX331 has biological characteristics which suggest that it
45
may be of the variant type of SCLC. It grows in loose clusters, has a short doubling time and a very high colonyforming efficiency in agar. Dense core granules were not apparent on electron microscopic examination. The CK and DDC values for this cell line are not yet available. WX331 should be a valuable resource for further elucidating the genetic lesions that link 13q (the retinoblastoma (RB) growth regulatory gene) to SCLC. By comparing changes seen in the human derived tissue with original germ-line DNA and with germ-line DNA from his affected siblings we hope to detect the functional changes at 13q or at other candidate loci which are specific to SCLC as opposed to RB. We thank Dr J. Plumb for performing biochemical assays, Dr M. McIntyre for advice in the interpretation of histological details and Ms J. Kerr for secretarial assistance.
References CARNEY, D.N., BUNN, P.A., GAZDAR, A.F., PAGAN, J.A. & MINNA,
HARBOUR, J.W., SHINN-LIANG LAI, WHANG-PENG, J., GAZDAR,
J.D. (1980). Selective growth of small cell carcinoma of the lung obtained from patient biopsies in serum free hormone supplemented medium. Proc. Natl Acad. Sci. USA, 78, 3185.
A.F., MINNA, J.D. & KAYE, F.J. (1988). Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science, 241, 353.
COURTNEY, V.D., SELBY, P.J., SMITH, I.E., MILLS, J. & PECKHAM,
NAYLOR, S.L., JOHNSON, B.E., MINNA, J.D. & SUKAGUCHI, A.Y.
M.J. (1978). Growth of human tumour cell colonies from biopsies using two soft agar techniques. Br. J. Cancer, 38, 77. GAZDAR, A.F., CARNEY, D.N., RUSSELL, E.K. & 5 others (1980). Establishment of continuous, clonable cultures of small-cell carcinoma of the lung which have amine precursor uptake and decarboxylation cell properties. Cancer Res., 40, 3502.
(1987). Loss of heterozygosity of chromosome 3p markers in small-cell lung cancer. Nature, 329, 451. SCHEPER, R.J., BULTE, J.W.M., BRAKKEE, J.G.P. & 8 others (1988). Monoclonal antibody JSBI detects a highly conserved epitope on the P-glycoprotein associated with multi-drug-resistance. Int. J. Cancer, 42, 389.
GAZDAR, A.F., ZWEIG, M.H., CARNEY, D.N., VAN STEIRTEGHEN,
SCHOL, D.J., MOOI, W.J., VAN DER GUGTEN, A.A., WAGENAAR,
A.C., BAYLIN, S.B. & MINNA, J.D. (1981). Levels of creatine kinase and its BB isoenzyme in lung cancer specimens and cultures. Cancer Res., 41, 2773.
S.J.Sc. & HILGERS, J; (1988). Monoclonal antibody 123C3, identifying small cell carcinoma phenotype in lung tumours recognises mainly, but not exclusively, endocrine and neurone-supporting normal tissues. Int. J. Cancer, 2, 34.
GULLICK, W.J., MARSDEN, J.J., WHITTLE, N., WARD, B., BOBROW, L. & WATERFIELD, M.D. (1986). Expression of epidermal growth
factor receptors on human cervical, ovarian and vulval carcinomas. Cancer Res., 46, 285.
(1991), XIV, Suppi. XIV, 63, Suppl. (1991), 63,
'."
43-45 43-45
©
Macmillan Press Ltd., 1991
Macmillan
Press
Ltd.,
1991
Establishment and characterisation of two new small cell lung cancer cell lines one from a patient with previous familial retinoblastoma -
F.G. Hay, L.W. Duncan & R.C.F. Leonard Imperial Cancer Research Fund Medical Oncology Unit and Department of Clinical Oncology, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK. Summary Two new small cell lung cancer (SCLC) cell lines have been established. The first, WX330, has been developed from a pleural effusion collected at presentation from a patient with extensive disease. The second, WX331, originated from a liver metastasis collected at post mortem from a patient who died of his SCLC disease. WX330 has been in culture for 4 years and WX331 for 11 months. Both cell lines have typical cytological features of SCLC, WX330 contains dense core granules on electron microscopic examination and both are tumourigenic in nude mice. Cell lines were analysed phenotypically using antibodies submitted to the Second International Workshop on SCLC Antigens by the immunoperoxidase method. WX330 is reactive with a wide range of SCLC and epithelial associated antibodies and WX331 with a more restricted group of antibodies of predominantly neuroendocrine and SCLC specificity. WX330 also reacts with an antibody against the multiple drug resistance-associated glycoprotein.
A continuing challenge in research in small cell lung cancer (SCLC) is to identify material for in vitro study which is representative of the clinical situation. The common experience of therapy has been the problem of tumour resistance to cytotoxic drugs in a disease that is initially, usually chemosensitive. Acquired resistance and, more infrequently, primary drug resistance are therefore major clinical problems and are the basic cause of incurability of SCLC. With the increasing availability of specific genetic probes, early observations on cytogenetic defects in SCLC have been confirmed in cell line studies. The major finding has been that virtually 100% of lung cancer cell lines show deletions of genetic material on the short arm of chromosome 3 (Naylor et al., 1987). However, a high proportion of cell lines have also been shown to exhibit RB (retinoblastoma) gene deletions (Harbour et al., 1988). This important cell proliferation control gene has been implicated in a variety of tumours. Despite the curability of retinoblastoma, there have been no well-studied cases of retinoblastoma being followed by SCLC. The purpose of this paper is to describe the establishment and characterisation of two cell lines, one derived from a patient who had clinically resistant disease and the second cell line from a metastasis in the liver in a patient who had had familial retinoblastoma but who died due to SCLC. Materials and methods Clinical background WX330 was derived from a pleural effusion collected at presentation from a patient with extensive SCLC. The patient was subsequently found to be unresponsive to chemotherapy (Etoposide/Vindesine) and died 6 months after presentation. WX331 was derived from a solid tumour metastasis of the liver obtained at post mortem. The 27 year old patient presented with limited stage SCLC, responded initially to chemotherapy (Adriamycin/cyclophosphamide/etoposide) before relapsing 8 months later with progressive disease. The patient had a previous history of familial retinoblastoma. Establishment of cell lines WX330 The cells from the pleural effusion were sedimented by centrifugation (1,000 g for O min), resuspended in RPMI
Correspondence: F.G. Hay.
1640 and red cells removed by Ficoll-Hypaque gradient separation. Interface cells were washed and checked for viability by vital dye exclusion. Aliquots of i05cellsml-' were cultured at 37°C, 90% humidity and 5% CO2 in RPMI + 10% Foetal calf serum (FCS) with added insulin (2.5 jig ml1 '), streptomycin (100 ,pg ml1 ), pyruvate (2 mM), penicillin (100 IU ml-') and glutamine (8 mM). Once the cell line was established and the tumour cells were proliferating, the medium was changed to HITES serum-free defined medium (Carney et al., 1980) to remove contaminating fibroblast cells. WX331 The tumour was cut into small pieces and the tumour cells were teased out. Thereafter, the resulting cell suspension was pelleted by centrifugation (1,000g for 10 min), washed in RPMI and passed over a Ficoll-Hypaque density gradient. The interface cells were washed and checked for viability by vital dye exclusion as before. Aliquots of 105 cells were cultured in HITES serum-free defined medium or HITES + 2+% FCS at 37°C, 90% humidity and 5% CO2. Measurement of clonogenicity The soft agar assay (Courtney et al., 1978) was used without the addition of irradiated feeder cells. Semi-liquid cultures were set up in agar (0.3% v/v). Fresh medium (1 ml) was added weekly. Colonies (> 50 cells) were scored after 28
days. Measurement of doubling times Cells were cultured in 35 mm wells. Duplicate cells were harvested daily and the cells counted. Population doubling times were calculated for cells in exponential phase of growth. Each experiment was repeated three times.
Tumourogenicity studies Cells in exponential growth in culture were harvested, centrifuged (1,000 g for O min), the pellet was washed and resuspended in phosphate buffered saline (PBS) at a density of I07 cells ml-'. 0.25 ml of cell suspension was injected subcutaneously into the flanks of nude mice. All animals were observed weekly until their tumours grew to a mean diameter of 1 cm or for at least 2 months in the absence of tumours. Biochemical assays The biochemical markers L-dopa decarboxylase (DDC) and creatinine kinase (CK) were measured by established methods (Gazdar et al., 1980; Gazdar et al., 1981).
44
F.G. HAY et al.
Immunohistochemistry The immunoperoxidase studies of the cell lines were performed using the Avidin-Biotin complex (ABC) system (Dako, UK). Briefly, cells were placed onto multispot slides (Hendley, (Essex) Ltd, UK) at approximately 2 x 104 cells per spot. Cells were fixed in methanol/acetone (1:1) for 10min then they were incubated at room temperature with 0.5% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity. After washing in TBS (Tris 0.5 M, pH 7.6, diluted in saline 1:10), they were incubated with rabbit serum: TBS (1:10) for 20min followed by the appropriate dilution of mouse monoclonal antibody (mab) for 30 min. Thereafter biotinylated rabbit anti-mouse preparation was applied (1:200) for 30min followed by the ABC complex also for 30 min. All steps were separated by three washes in TBS. The peroxidase was localised by treatment of the slides with freshly prepared 0.1% 3,3'diaminobenzidine and 0.01% hydrogen peroxide in Trisimidazole (pH 7.6) buffer for 10 min. After washing in water these samples were then counterstained with haematoxylin. For rat mabs an indirect immunoperoxidase system was used. The Workshop panel of mabs was used. In addition, the cell lines and the original material used in their derivation, were studied using HMFG2, CAM 5.2, AUA1, SM3 and UJ13A obtained from ICRF, London, EGFR1 and EGFRF4 (Gullick et al., 1986), 123C3 (Schol et al., 1988), and JSB1 (Scheper et al., 1988). Electron microscopy For transmission electron microscopy the tumour cells were pelleted and incubated in 2% glutaraldehyde in PBS. Post fixation was performed in 2% osmium tetroxide in PBS. The material was embedded in Epon and further preparations were according to routine procedures. Results Establishment and morphological characteristics of the cell lines WX330 grew very slowly at first but after the fifth passage became well established and has now reached passage 20. The cell line is routinely maintained in RPMI + 10% FCS, but also grows in HITES serum-free defined medium if
required. The cells grow as large aggregates of tightly packed cells which frequently undergo central necrosis with the formation of hollow sphericules. The doubling time of this cell line is now 57 h with a colony forming efficiency of 14.3% in agar. WX330 is resistant to storage in liquid nitrogen and is tumourogenic in nude mice, giving rise to tumours with an undifferentiated histology. The origin of both the cell line and the xenografted tumour from human SCLC cells has been confirmed by light and electron microscopy. The second cell line, WX331, became established very quickly after being put directly into culture in HITES serumfree defined medium. This cell line has now been in continuous culture for 11 months, grows as loose aggregates of cells, has a doubling time of 31 h, a colony forming efficiency of 33.7% in agar, recovers readily from liquid nitrogen storage and is tumourogenic in nude mice. This cell line has also been confirmed to be of the human SCLC type by light and electron microscopy. Values of L-dopa decarboxylase in WX330 were 2043 + 93.8 uIU mg' protein, creatinine kinase 2.26 ± 0.17 U mg-' protein. CKBB constituted 95% of total CK and CKMM 5%. WX331 was not tested. Both cell lines were immunophenotyped using the complete panel of mabs of the Second International Workshop. Results are shown in Table I. The pleural effusion which was used to derive WX330 stained with the anti-cytokeratin mab CAM 5.2, epithelial mabs AUA1 (cluster 2) HMFG2 and cluster 1 mabs 123C3 and UJ13A. The early passages of WX330 were also reactive with these mabs. At passages 12, 16, 17, 18 and 20, reactivity with CAM 5.2 had been lost and reactivity with EGFRF4 (internal portion of the epidermal growth factor) and with JSBl had emerged. A cell suspension prepared from the solid tumour sample used in the derivation of WX331 was reactive with CAM 5.2, HMFG2, SM3 and 123C3. Passages 1-4 of the WX331 cell line were strongly reactive with the cluster 1 mabs, 123C3 and UJ13A and reactive to a lesser extent with HMFG2, AUA1 and SM3. This cell line did not react with the JSBI or the EGFRF4 mabs. Discussion In this paper, we describe the establishment and characterisation of two new SCLC cell lines. WX330 grows in the manner of a classic SCLC cell line having a growth forma-
Table I Reactivity of WX330 and WX331 to Workshop antibodies
WX330 and WX331. Antibody name (and Workshop number)
WX330 alone. Antibody name (and Workshop number) S-L 11.14(34) SEN 6(60)
2
RNL 1(4), Moc 1(12), Moc 191(21), NCC-LU 246(3 1), NCC LU 243(48), SEN 36(61), NE 150(74), NE 25(77) Moc 31(11), Moc 58(14), PE35(75)
4
SWA1 1(59), SWA21(66), SWA22(67)
Cluster 1
Moc 151(61), Moc 181(20), S-L 4.20(85),
SL 2.21(90) 5
5A 6 7 8 Unclustered
SEN 31(64), SWA20(70) Mov 15(45), NCC LU 152(47)
NCC-CO-450(98) CAM 5.2(3, 56, 28, 71), Moc 171(18), MLuC3(43), NSE H124(38), NCC-RAS-044(57)
A-80(37) 43/9F(7), lA9-D4(8), 6A3-C8(9), Moc 172(9), BS 150(22), BS 153(24), SMI(27), Muc 54(29), Muc 18(30), MBRI(40), MLcC2(42), MLuC4(44), 443A6(46), NCC-LU-279(55), Fib 75(78), Mc 5(79), HMFG1(81), 2A6(83), S-L 3.5(35)
NEW SLCL CELL LINES
tion of tight sperical aggregates, a relatively long doubling time, high levels of the BB-isoenzymes of creatine kinase and dense core granules visible by electron microscopy. This cell line does, however, have a relatively high colony forming efficiency for a classic SCLC cell line. This cell line was reactive with a wide variety of the Second International Workshop panel of mabs and staining of the original tumour sample and later passages of the cell line indicated that the phenotype of this cell line is relatively stable. The reactivity of the cell line with the multiple drug resistance mab, JSBl, may reflect the non-responsiveness to chemotherapy of the patient from whom the cell line was derived. In vitro drug studies should define the relative resistance levels as against in vitro-induced resistance and also the pleiomorphism of the pattern. WX331 has biological characteristics which suggest that it
45
may be of the variant type of SCLC. It grows in loose clusters, has a short doubling time and a very high colonyforming efficiency in agar. Dense core granules were not apparent on electron microscopic examination. The CK and DDC values for this cell line are not yet available. WX331 should be a valuable resource for further elucidating the genetic lesions that link 13q (the retinoblastoma (RB) growth regulatory gene) to SCLC. By comparing changes seen in the human derived tissue with original germ-line DNA and with germ-line DNA from his affected siblings we hope to detect the functional changes at 13q or at other candidate loci which are specific to SCLC as opposed to RB. We thank Dr J. Plumb for performing biochemical assays, Dr M. McIntyre for advice in the interpretation of histological details and Ms J. Kerr for secretarial assistance.
References CARNEY, D.N., BUNN, P.A., GAZDAR, A.F., PAGAN, J.A. & MINNA,
HARBOUR, J.W., SHINN-LIANG LAI, WHANG-PENG, J., GAZDAR,
J.D. (1980). Selective growth of small cell carcinoma of the lung obtained from patient biopsies in serum free hormone supplemented medium. Proc. Natl Acad. Sci. USA, 78, 3185.
A.F., MINNA, J.D. & KAYE, F.J. (1988). Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science, 241, 353.
COURTNEY, V.D., SELBY, P.J., SMITH, I.E., MILLS, J. & PECKHAM,
NAYLOR, S.L., JOHNSON, B.E., MINNA, J.D. & SUKAGUCHI, A.Y.
M.J. (1978). Growth of human tumour cell colonies from biopsies using two soft agar techniques. Br. J. Cancer, 38, 77. GAZDAR, A.F., CARNEY, D.N., RUSSELL, E.K. & 5 others (1980). Establishment of continuous, clonable cultures of small-cell carcinoma of the lung which have amine precursor uptake and decarboxylation cell properties. Cancer Res., 40, 3502.
(1987). Loss of heterozygosity of chromosome 3p markers in small-cell lung cancer. Nature, 329, 451. SCHEPER, R.J., BULTE, J.W.M., BRAKKEE, J.G.P. & 8 others (1988). Monoclonal antibody JSBI detects a highly conserved epitope on the P-glycoprotein associated with multi-drug-resistance. Int. J. Cancer, 42, 389.
GAZDAR, A.F., ZWEIG, M.H., CARNEY, D.N., VAN STEIRTEGHEN,
SCHOL, D.J., MOOI, W.J., VAN DER GUGTEN, A.A., WAGENAAR,
A.C., BAYLIN, S.B. & MINNA, J.D. (1981). Levels of creatine kinase and its BB isoenzyme in lung cancer specimens and cultures. Cancer Res., 41, 2773.
S.J.Sc. & HILGERS, J; (1988). Monoclonal antibody 123C3, identifying small cell carcinoma phenotype in lung tumours recognises mainly, but not exclusively, endocrine and neurone-supporting normal tissues. Int. J. Cancer, 2, 34.
GULLICK, W.J., MARSDEN, J.J., WHITTLE, N., WARD, B., BOBROW, L. & WATERFIELD, M.D. (1986). Expression of epidermal growth
factor receptors on human cervical, ovarian and vulval carcinomas. Cancer Res., 46, 285.
