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The combined effect of interferon and methotrexate on human osteosarcoma and lymphoma cell lines
10 (1980) 83-90 0 Elsevier/North-Holland Scientific
a3 Publishers
Ltd
THE COMBINED EFFECT OF INTERFERON AND METHOTREKATE ON HUMAN OSTEOSARCOMA AND LYMPHOMA CELL LINES
LARS-dKE
BROSTRilM
From the Department Stockholm (Sweden)
of Orthopaedic Surgery and Radiumhemmet,
Karolineka Hospital,
(Received 25 February 1980) (Revised version received 1 April 1980) (Accepted 18 April 1980)
SUMMARY
Two human osteosarcoma cell lines and 1 human lymphoblastoid were tested for the growth inhibitory effect of methotrexate and leukocyte interferon, both separately and in various combinations. agents inhibited the cell lines. No synergistic, inhibitory or blocking on tumor cell growth were detected.
cell line human Both effects
INTRODUCTION
Exogenous interferon and high-dose methotrexate therapy for tumors has been used to an increasing extent in the last few years. Both drugs are employed in adjuvant treatment for human osteosarcoma [ 2,4,5,7,9] . That interferon exerts an inhibitory effect on tumor cell growth in tissue culture is well established [ 1,10,15,21]. However, some cell lines have proved, less sensitive than others [ 13,20 1. Methotrexate is known to exert an anti-tumor effect both in viva and in vitro [ 5,9,18,22]. Methotrexate-resistant tumor development is familiar. The drug has been combined with others, such as adriamycin, both in the treatment of patients and in culture studies [ 5,11,23]. At the Karolinska Hospital a clinical trial in which interferon is being administered as an adjuvant therapy in osteosarcoma has beenin progress since 1971 [2]. When some of the patients have developed pulmonary metastases the interferon has been replaced by high-dose methotrexate treatment, with varying success.
Addrers all correspondence S-104 01 Stockholm,
to: Lars-Ake BrostrSm, Radiumhemmet,
Sweden.
Karolinska Hospital,
84
The purpose of this study was to examine whether there is any interaction between interferon and methotrexate such as that reported for interferon and cortisol [ 81. MATERIALS AND METHODS
Cells
Three human cell lines, sensitive to bothrmethotrexate and interferon were used in this study. Two of them, namely 393 T [16] and T2 56 [17] have been cultivated from osteosarcoma tumors. The third cell line was a lymphoblastoid one, Daudi derived from Burkitt’s lymphoma [ 121. Interferon (LJF)
Human leucocyte interferon suspensions were induced with Sendai virus. Partially purified interferon was obtained by selective precipitation of inert protein from an ethanolic solution with rising pH [6]. The L-IF batch used in this study had a specific activity of 6.4 X IO6 units/mg of protein. The interferon was kindly provided by Dr Kari Cantell, Helsinki, Finland. Methotrexate
Highly purified methotrexate (82.9%) in powder form was kindly provided by Lederle, Cyanamide Nordiska AB, Stockholm, Sweden. Serial dilutions in T&buffer gave nanomolar concentrations. Cell-growth experiments
The osteosarcoma cell lines were grown as mono-layer cultures in Eagle’s minimal essential medium supplemented with 10% of heat-inactivated calf serum, 1.6 mg of sodium bicarbonate/ml, 125 IU of penicillin/ml, and 125 pg of streptomycin/ml. The cells were seeded in 20 mm glass Petri dishes containing 2 ml of medium/dish. Five X lo4 cells/ml were added and dishes were incubated at 37.0%in.a humified atmosphere containing 5% of CO1. Control dishes contained only medium and cells. To the experimental dishes were also added either methotrexate or L-IF or both in a range of concentrations. Twice a week the cultures were split so as to contain 5 X lo4 living cells/ml and at the same time fresh L-IF and methotrexate were added. When splitting up the cells the cultures were rinsed with 2 ml of balanced salt solution (BSS) prior to adding 1 ml of 0.25% trypsin. Cell viability was determined by the trypan blue exclusion test. The cells were counted in a Biirker chamber. The growth inhibition is expressed as the percentage ratio: Counts of living cells in experimental dishes x 100 Counts of living cells in control dishes The lymphoblastoid
cells were grown in suspension in tubes seeded with
85
4 X 10’ cells in 2 ml of RPM1 medium, supplemented with 8% of heatinactivatedcalf serumand 1.6 mg of sodium bicarbonate, 250 unitsof penicillin and 250 pg of streptomycin. These cells were also incubated at 37.O”C in the same CO2 incubator. Twice a week the cultures were maintained by adjustingthe cell concentration to 2 X lo5 livingcells/ml with fresh medium. Fresh methotrexate and L-IF were added at the same time. The living cells were counted in a Biirker chamber, and the percentage calculated. Experimental design The cell lines used were tested for their sensitivityto variousconcentrations of both L-IF and methotrexate,and the concentrations givinga lo-15% inhibition of cell growth after 1 week’s treatmentwere chosen for the interaction study. To establishwhether there is any drug interaction 2 types of experiments were performed. In the first experiment the inhibitory effect of the 2 drugs
I
I
4
7
I
11
J 14
Culture period, days Fig. 1. The Daudi cell line. The effect of interferon (L-IF) and methotrexate (Wtx) on cell growth. The drug effect is exprewed as the percentage ratio of the number of living cells in the cultures containing the drug or drugs, to the number in the control cultures, run in parallel. Interferon: l - - - -e, 0.1 unit/ml; o- - - -0,0.3 unit/ml; X - - - -X 1.0unit/ml.Methotrexate:o---~o,1.9nM;o-----o,3.0n~;x---~x,10.0~l. 0.1 unit L-IF + 1 nM Mtx; o-, 0.3 unit Interferon + methotrexate: l-, , 1.0 unit L-IF + 10 nM Mtx. L-IF + 3 nM Mtx; x -X
,
86
in combination was compared with the separate effects of the 2 drugs. In the tests of the combined effect of the drugs they were mixed in the same dish. The second type of experiment was performed to examine whether pretreatment with 1 drug interfered with effect of the other. The cells were pretreated with either drug at a concentration that would produce an obvious inhibitory effect but not exceeding 50% after 7-11 days. The cells were then divided into 3 groups for further treatment with medium, methotrexate or interferon. Each dish contained 5 X lo4 cells/ml. RESULTS
All 3 cell lines tested were sensitive both to methotrexate and to L-IF, as is evident from the dose response curves presented in Figs. l-3. Solutions of methotrexate of 1,3 and 10 nM strength were used in the experiments and the interferon concentrations ranged from 0.1 to 10 units/ml. The Daudi cell line was more sensitive to interferon than were either of the osteosarcoma lines. At all the concentrations of the 2 drugs used, there was definite inhibition of tumor growth after 1 week of treatment.
z 6 ”
.-c v) =
J Irn
10 -
.E >
z +
0
2c
2
”
5-
)
1
I
4
I
Culture Fig. 2. The osteoaarcoma celI line units/ml; 1 unit/ml; 0----0,3 1nM;o-~-*--o,3nM;x-*-* 1 unit L-IF + nM Mtx, o -0, 10 nM ?vItx.
I
11
1
14 days
period, days T,56 (m legend to Fig. 1). Interferon: o----o, , 10 unite/ml. Methotrexate : l - * - -0, x - ---X -X ,10 nM. Interferon + methotrexate : l, , 10 unite L-IF + 3unitsL-IF t 3nMMtx;X -X l
I
I
I
4
7
11
1 14
Culture period, days
Fig. 3. The osteosarcoma cell line 393T (see legend to Fig 1). Interferon: o----e, 3 units/ml; X----X, 10 units/ml. Methotrexate: l . -. - se, 1 unit/ml; 3----e, 1 n&f; DO-n--.0, 3 nIv¶;x a-•-•x, 10 nM. Interferon + methotrexate: 0’. , 10 units L-IF t 3 unite L-IF t 3 d!f Vtx; X -X 1 unit L-IF t 1 nM Mtx; 0-0, 10 nM Mtx.
For the effect of the 2 drugs combined, dose response curves were drawn for all 3 experiments. A comparison of the combined effect of the 2 drugs with the effect of each drug separately disclosed no definite synergistic or inhibitory effect. In the experiments in which the cells were pretreated with 1 of the 2 drugs and then exposed to the other one, no blocking effect was evident from a comparison with the initial drug and with dishes containing only medium. (Fig. 4). DISCUSSION
If adjuvant treatment is given to the osteosarcoma patient there is a better chance of avoiding a fatal outcome [ 3,7,9,14]. Where interferon and methotrexate have been given separately as adjuvant drugs the results have not been uniformly good. In 30-60% of these patients the tumor disease has ultimately progressed: and pulmonary metastases have developed [ 2,5] . Whether this is due to selection of resistant cells is not known. Studies on animals have shown that tumor cells can develop resistance to both methotrexate and interferon [8,10] .
.c_.c =
m =
u
u
vl WW
lg
mm c .-c ._
> z;
20
201
4
7
11
14
17
21
Culture period, days
Fig. 4. The oeteosarcoma cell line T,66. The effect of pretreatment of either interferon or control or medium only; x ----x , methotrexate. (See legend to Fig. 1). •~. interferon 10 units/ml; X - - * X , methotrexate 10 n%. l
l
The purpose of this study was to find whether there is any interaction between these 2 drugs when they are used together in concentrations which, when used separately, exert a definite though not excessive inhibition of tumor growth. The cell lines used in this in vitro study were all sensitive to both methotrexate and interferon. The drug concentrations were close to those found in the serum of patients receiving these drugs 16,191. No synergistic, inhibitory or blocking effects on tumor growth were detected; the possible clinical implications of this remain to be discovered.
89
ACKNOWLEDGEMENT
I wish to express my indebtedness to Dr. Hans Strander for his guidance and positive criticism and for generously providing laboratory facilities: I also thank Dr. Lambert Skoog and Dr. Stefan Einhom for stimulating discussions and Professor Kari Cantell for providing the interferon. The excellent technical assistance of Mrs Waltraut Szczerba is gratefully acknowledged. Grants in support of this study have been received from the Cancer Society of Stockholm and the Swedish Cancer Society, REFERENCES 1 Adams, A., Strander, H. and Cantell, K. (1976) Sensitivity of the Epstein-Barr virus transformed human lymphoid cell lines to interferon. J. Gen. viral., 2% 207. 2 Adamson, U., Aparlsi, T., Brosttim, L-A ., Cantell, K., Einhorm, S., Hall K., Ingimarsson, S., Nilsonne, Il., Strander, H. and Siirlerberg, G. (1979) Interferon treatment of hyman osteosarcoma. Study week of the Pontificial Academy of Sciences, Vatican city, Oct. 17-21, 1977. “The role of non-specific immunity in the prevention of cancer”. 3 Brostrijm, L-A.., Aparlsi, T., Ingimarsson, S., Lagergren, C., Nilsonne, U., Strander, H. and Siiderberg, G. (1978) Can historical controls be used in current clinical trails in osteosarcoma? Analysis of prognostic factors in a historical and a contemporary group. Int. J. Rad. Oncol. Biol. Phys. In press. 4 Brostrijm, L-A., Aparlsi, T., Ingimarsson, S., Lagergren, C., Nilsonne, U., Strander, H. and Siiderberg, G. (1980) Can historical controls be used in current clinical trials in osteosarcoma? Metastases and survival in a historical and a contemporary group. Int. J. Radiat. Oncol. Biol. Phys. In press. 5 Cancer Treatment Reports (1978) Proceedings of Osteosarcoma Study Group Meeting Febr. 1978. National Cancer Institute (NIH), USA. Cancer Treat. Rep., 62, 187. 6 Cantell, K., Pyh&l&, L. and Strander, H. (1974) Circulating human interferon after intramuscular injection into animals and man. J. Gen. Virol., 2,463. 7 Cartes, E.P., Holland, J.F., Wang, J.J., Sinks, L.F., Blom, J., Senn, H., Bank, A. and Glidewell, 0. (1974) Amputation and adriamycin in primary osteosarcoma. N. Engl. J. Med., 291, 998. 8 Filipic, B., Lllar, M., Schauer, P., Schara, M., Sentjurc, M. and Nemec, M. (1978) Cell receptors for interferon and cortlsol ESR measurements. Interferon Scientific Memoranda, Dec. 3rd. 9 Frei, E. III (1976) Methotrexate revisited. Med. Ped. Oncol., 2,227. 10 Gresser, I. (1977) Antitumor effects of interferon. Cancer. A comprehensive treatise Vol. 5, p. 521. Editor: F. Becker. New York. lf Hill, B., Price, L.A. and Goldie, J.H. (1976) The value of adriamycin in overcoming resistance to methotrexate in tissue culture. Eur. J. Cancer, 12, 641. 12 Klein, E., Klein, G., Nadkami, J.S., Nadkami, J.J., Wigxell, H. and Clifford, P. (1976) Surface IgM kappa specificity on a Burkitt lymphoma cell in vitro and in derived culture lines. Cancer Res., 28, 1300. 13 Kuwata, T., Fuse, A. and Morlnaga, N. (1976) Effect of interferon on cell and virus growth in transformed human cell lines. J. Gen. Vlrol., 33, 7. 14 Lockshin, M.D. and Higgins, I.T.T. (1968) Prognosis in osteogenic sarcoma. Clin. Orthop. Relat. Res., 68, 86. 16 Paucker, K., Cantell, K. and Henle, W. (1962) Quantitative studies on viral interference in suspended L cells. III. Effect of interfering viruses and interferon on the growth rate of cells. Virology, 17,324.
16 Pont&, J. (1975) Neoplastic human glie cells in culture. In: Human tumour cells in vitro, C&apt. 7, p. 175. Editor: J. Fogh. Plenum Press, New York and London. 17 Pontin, J. and Sake&, E. (1967) tie establtid in vitro cell lines from human meeencbymal tumor& Int. J. Cancer, 2,434. 18 Slltrak, F.M. Donrbacb, R.C., Dorick, D.M. and Moccio, D.M. (1976) Tiesue pharmacoklneticr, inhibition of DNA synthesis, and tumor cell kill after high dose metho trexate in murine tumor models. Cancer Rer.. 36,4672. 19 Skoog, L., Nordenskjiild, B., Humla, S. and IUgerstriim, T. (1976) Effects of methotrexata on deoxyrlbonucleomide poolr and nucleic acid eyntbesis in human osteosarcoma cella. Eur. J. Cancer, 12,839. 20 Strander, H., (1977) Interferons: Anti-neoplaetic drugs? Blut, 35,277. 21 Strander, H. and Einhom, 5. (1977) Effect of human leukocyte interferon on the growth of human otsteorarcoma celle in tissue culture. Int. J. Cancer, 19,468. 22 Tattersall, M.H.N. and Harrap, K.R. (1973) Changes in the deoxyribonucleoride tiphoephate pools of mouee 5178Y lymphoma celh following exposure to methotrexate or 5-fluorouracil. Cancer Res., 33, 3086. 23 Tatteraall, M.H.N., Jackson, R.C., Connors, T.A. and Harrap, K.R. (1973) Combination chemotherapy: the interaction of methotrexate and 5-fluorouracil. Eur. J. Cancer, 9, 733.
a3 Publishers
Ltd
THE COMBINED EFFECT OF INTERFERON AND METHOTREKATE ON HUMAN OSTEOSARCOMA AND LYMPHOMA CELL LINES
LARS-dKE
BROSTRilM
From the Department Stockholm (Sweden)
of Orthopaedic Surgery and Radiumhemmet,
Karolineka Hospital,
(Received 25 February 1980) (Revised version received 1 April 1980) (Accepted 18 April 1980)
SUMMARY
Two human osteosarcoma cell lines and 1 human lymphoblastoid were tested for the growth inhibitory effect of methotrexate and leukocyte interferon, both separately and in various combinations. agents inhibited the cell lines. No synergistic, inhibitory or blocking on tumor cell growth were detected.
cell line human Both effects
INTRODUCTION
Exogenous interferon and high-dose methotrexate therapy for tumors has been used to an increasing extent in the last few years. Both drugs are employed in adjuvant treatment for human osteosarcoma [ 2,4,5,7,9] . That interferon exerts an inhibitory effect on tumor cell growth in tissue culture is well established [ 1,10,15,21]. However, some cell lines have proved, less sensitive than others [ 13,20 1. Methotrexate is known to exert an anti-tumor effect both in viva and in vitro [ 5,9,18,22]. Methotrexate-resistant tumor development is familiar. The drug has been combined with others, such as adriamycin, both in the treatment of patients and in culture studies [ 5,11,23]. At the Karolinska Hospital a clinical trial in which interferon is being administered as an adjuvant therapy in osteosarcoma has beenin progress since 1971 [2]. When some of the patients have developed pulmonary metastases the interferon has been replaced by high-dose methotrexate treatment, with varying success.
Addrers all correspondence S-104 01 Stockholm,
to: Lars-Ake BrostrSm, Radiumhemmet,
Sweden.
Karolinska Hospital,
84
The purpose of this study was to examine whether there is any interaction between interferon and methotrexate such as that reported for interferon and cortisol [ 81. MATERIALS AND METHODS
Cells
Three human cell lines, sensitive to bothrmethotrexate and interferon were used in this study. Two of them, namely 393 T [16] and T2 56 [17] have been cultivated from osteosarcoma tumors. The third cell line was a lymphoblastoid one, Daudi derived from Burkitt’s lymphoma [ 121. Interferon (LJF)
Human leucocyte interferon suspensions were induced with Sendai virus. Partially purified interferon was obtained by selective precipitation of inert protein from an ethanolic solution with rising pH [6]. The L-IF batch used in this study had a specific activity of 6.4 X IO6 units/mg of protein. The interferon was kindly provided by Dr Kari Cantell, Helsinki, Finland. Methotrexate
Highly purified methotrexate (82.9%) in powder form was kindly provided by Lederle, Cyanamide Nordiska AB, Stockholm, Sweden. Serial dilutions in T&buffer gave nanomolar concentrations. Cell-growth experiments
The osteosarcoma cell lines were grown as mono-layer cultures in Eagle’s minimal essential medium supplemented with 10% of heat-inactivated calf serum, 1.6 mg of sodium bicarbonate/ml, 125 IU of penicillin/ml, and 125 pg of streptomycin/ml. The cells were seeded in 20 mm glass Petri dishes containing 2 ml of medium/dish. Five X lo4 cells/ml were added and dishes were incubated at 37.0%in.a humified atmosphere containing 5% of CO1. Control dishes contained only medium and cells. To the experimental dishes were also added either methotrexate or L-IF or both in a range of concentrations. Twice a week the cultures were split so as to contain 5 X lo4 living cells/ml and at the same time fresh L-IF and methotrexate were added. When splitting up the cells the cultures were rinsed with 2 ml of balanced salt solution (BSS) prior to adding 1 ml of 0.25% trypsin. Cell viability was determined by the trypan blue exclusion test. The cells were counted in a Biirker chamber. The growth inhibition is expressed as the percentage ratio: Counts of living cells in experimental dishes x 100 Counts of living cells in control dishes The lymphoblastoid
cells were grown in suspension in tubes seeded with
85
4 X 10’ cells in 2 ml of RPM1 medium, supplemented with 8% of heatinactivatedcalf serumand 1.6 mg of sodium bicarbonate, 250 unitsof penicillin and 250 pg of streptomycin. These cells were also incubated at 37.O”C in the same CO2 incubator. Twice a week the cultures were maintained by adjustingthe cell concentration to 2 X lo5 livingcells/ml with fresh medium. Fresh methotrexate and L-IF were added at the same time. The living cells were counted in a Biirker chamber, and the percentage calculated. Experimental design The cell lines used were tested for their sensitivityto variousconcentrations of both L-IF and methotrexate,and the concentrations givinga lo-15% inhibition of cell growth after 1 week’s treatmentwere chosen for the interaction study. To establishwhether there is any drug interaction 2 types of experiments were performed. In the first experiment the inhibitory effect of the 2 drugs
I
I
4
7
I
11
J 14
Culture period, days Fig. 1. The Daudi cell line. The effect of interferon (L-IF) and methotrexate (Wtx) on cell growth. The drug effect is exprewed as the percentage ratio of the number of living cells in the cultures containing the drug or drugs, to the number in the control cultures, run in parallel. Interferon: l - - - -e, 0.1 unit/ml; o- - - -0,0.3 unit/ml; X - - - -X 1.0unit/ml.Methotrexate:o---~o,1.9nM;o-----o,3.0n~;x---~x,10.0~l. 0.1 unit L-IF + 1 nM Mtx; o-, 0.3 unit Interferon + methotrexate: l-, , 1.0 unit L-IF + 10 nM Mtx. L-IF + 3 nM Mtx; x -X
,
86
in combination was compared with the separate effects of the 2 drugs. In the tests of the combined effect of the drugs they were mixed in the same dish. The second type of experiment was performed to examine whether pretreatment with 1 drug interfered with effect of the other. The cells were pretreated with either drug at a concentration that would produce an obvious inhibitory effect but not exceeding 50% after 7-11 days. The cells were then divided into 3 groups for further treatment with medium, methotrexate or interferon. Each dish contained 5 X lo4 cells/ml. RESULTS
All 3 cell lines tested were sensitive both to methotrexate and to L-IF, as is evident from the dose response curves presented in Figs. l-3. Solutions of methotrexate of 1,3 and 10 nM strength were used in the experiments and the interferon concentrations ranged from 0.1 to 10 units/ml. The Daudi cell line was more sensitive to interferon than were either of the osteosarcoma lines. At all the concentrations of the 2 drugs used, there was definite inhibition of tumor growth after 1 week of treatment.
z 6 ”
.-c v) =
J Irn
10 -
.E >
z +
0
2c
2
”
5-
)
1
I
4
I
Culture Fig. 2. The osteoaarcoma celI line units/ml; 1 unit/ml; 0----0,3 1nM;o-~-*--o,3nM;x-*-* 1 unit L-IF + nM Mtx, o -0, 10 nM ?vItx.
I
11
1
14 days
period, days T,56 (m legend to Fig. 1). Interferon: o----o, , 10 unite/ml. Methotrexate : l - * - -0, x - ---X -X ,10 nM. Interferon + methotrexate : l, , 10 unite L-IF + 3unitsL-IF t 3nMMtx;X -X l
I
I
I
4
7
11
1 14
Culture period, days
Fig. 3. The osteosarcoma cell line 393T (see legend to Fig 1). Interferon: o----e, 3 units/ml; X----X, 10 units/ml. Methotrexate: l . -. - se, 1 unit/ml; 3----e, 1 n&f; DO-n--.0, 3 nIv¶;x a-•-•x, 10 nM. Interferon + methotrexate: 0’. , 10 units L-IF t 3 unite L-IF t 3 d!f Vtx; X -X 1 unit L-IF t 1 nM Mtx; 0-0, 10 nM Mtx.
For the effect of the 2 drugs combined, dose response curves were drawn for all 3 experiments. A comparison of the combined effect of the 2 drugs with the effect of each drug separately disclosed no definite synergistic or inhibitory effect. In the experiments in which the cells were pretreated with 1 of the 2 drugs and then exposed to the other one, no blocking effect was evident from a comparison with the initial drug and with dishes containing only medium. (Fig. 4). DISCUSSION
If adjuvant treatment is given to the osteosarcoma patient there is a better chance of avoiding a fatal outcome [ 3,7,9,14]. Where interferon and methotrexate have been given separately as adjuvant drugs the results have not been uniformly good. In 30-60% of these patients the tumor disease has ultimately progressed: and pulmonary metastases have developed [ 2,5] . Whether this is due to selection of resistant cells is not known. Studies on animals have shown that tumor cells can develop resistance to both methotrexate and interferon [8,10] .
.c_.c =
m =
u
u
vl WW
lg
mm c .-c ._
> z;
20
201
4
7
11
14
17
21
Culture period, days
Fig. 4. The oeteosarcoma cell line T,66. The effect of pretreatment of either interferon or control or medium only; x ----x , methotrexate. (See legend to Fig. 1). •~. interferon 10 units/ml; X - - * X , methotrexate 10 n%. l
l
The purpose of this study was to find whether there is any interaction between these 2 drugs when they are used together in concentrations which, when used separately, exert a definite though not excessive inhibition of tumor growth. The cell lines used in this in vitro study were all sensitive to both methotrexate and interferon. The drug concentrations were close to those found in the serum of patients receiving these drugs 16,191. No synergistic, inhibitory or blocking effects on tumor growth were detected; the possible clinical implications of this remain to be discovered.
89
ACKNOWLEDGEMENT
I wish to express my indebtedness to Dr. Hans Strander for his guidance and positive criticism and for generously providing laboratory facilities: I also thank Dr. Lambert Skoog and Dr. Stefan Einhom for stimulating discussions and Professor Kari Cantell for providing the interferon. The excellent technical assistance of Mrs Waltraut Szczerba is gratefully acknowledged. Grants in support of this study have been received from the Cancer Society of Stockholm and the Swedish Cancer Society, REFERENCES 1 Adams, A., Strander, H. and Cantell, K. (1976) Sensitivity of the Epstein-Barr virus transformed human lymphoid cell lines to interferon. J. Gen. viral., 2% 207. 2 Adamson, U., Aparlsi, T., Brosttim, L-A ., Cantell, K., Einhorm, S., Hall K., Ingimarsson, S., Nilsonne, Il., Strander, H. and Siirlerberg, G. (1979) Interferon treatment of hyman osteosarcoma. Study week of the Pontificial Academy of Sciences, Vatican city, Oct. 17-21, 1977. “The role of non-specific immunity in the prevention of cancer”. 3 Brostrijm, L-A.., Aparlsi, T., Ingimarsson, S., Lagergren, C., Nilsonne, U., Strander, H. and Siiderberg, G. (1978) Can historical controls be used in current clinical trails in osteosarcoma? Analysis of prognostic factors in a historical and a contemporary group. Int. J. Rad. Oncol. Biol. Phys. In press. 4 Brostrijm, L-A., Aparlsi, T., Ingimarsson, S., Lagergren, C., Nilsonne, U., Strander, H. and Siiderberg, G. (1980) Can historical controls be used in current clinical trials in osteosarcoma? Metastases and survival in a historical and a contemporary group. Int. J. Radiat. Oncol. Biol. Phys. In press. 5 Cancer Treatment Reports (1978) Proceedings of Osteosarcoma Study Group Meeting Febr. 1978. National Cancer Institute (NIH), USA. Cancer Treat. Rep., 62, 187. 6 Cantell, K., Pyh&l&, L. and Strander, H. (1974) Circulating human interferon after intramuscular injection into animals and man. J. Gen. Virol., 2,463. 7 Cartes, E.P., Holland, J.F., Wang, J.J., Sinks, L.F., Blom, J., Senn, H., Bank, A. and Glidewell, 0. (1974) Amputation and adriamycin in primary osteosarcoma. N. Engl. J. Med., 291, 998. 8 Filipic, B., Lllar, M., Schauer, P., Schara, M., Sentjurc, M. and Nemec, M. (1978) Cell receptors for interferon and cortlsol ESR measurements. Interferon Scientific Memoranda, Dec. 3rd. 9 Frei, E. III (1976) Methotrexate revisited. Med. Ped. Oncol., 2,227. 10 Gresser, I. (1977) Antitumor effects of interferon. Cancer. A comprehensive treatise Vol. 5, p. 521. Editor: F. Becker. New York. lf Hill, B., Price, L.A. and Goldie, J.H. (1976) The value of adriamycin in overcoming resistance to methotrexate in tissue culture. Eur. J. Cancer, 12, 641. 12 Klein, E., Klein, G., Nadkami, J.S., Nadkami, J.J., Wigxell, H. and Clifford, P. (1976) Surface IgM kappa specificity on a Burkitt lymphoma cell in vitro and in derived culture lines. Cancer Res., 28, 1300. 13 Kuwata, T., Fuse, A. and Morlnaga, N. (1976) Effect of interferon on cell and virus growth in transformed human cell lines. J. Gen. Vlrol., 33, 7. 14 Lockshin, M.D. and Higgins, I.T.T. (1968) Prognosis in osteogenic sarcoma. Clin. Orthop. Relat. Res., 68, 86. 16 Paucker, K., Cantell, K. and Henle, W. (1962) Quantitative studies on viral interference in suspended L cells. III. Effect of interfering viruses and interferon on the growth rate of cells. Virology, 17,324.
16 Pont&, J. (1975) Neoplastic human glie cells in culture. In: Human tumour cells in vitro, C&apt. 7, p. 175. Editor: J. Fogh. Plenum Press, New York and London. 17 Pontin, J. and Sake&, E. (1967) tie establtid in vitro cell lines from human meeencbymal tumor& Int. J. Cancer, 2,434. 18 Slltrak, F.M. Donrbacb, R.C., Dorick, D.M. and Moccio, D.M. (1976) Tiesue pharmacoklneticr, inhibition of DNA synthesis, and tumor cell kill after high dose metho trexate in murine tumor models. Cancer Rer.. 36,4672. 19 Skoog, L., Nordenskjiild, B., Humla, S. and IUgerstriim, T. (1976) Effects of methotrexata on deoxyrlbonucleomide poolr and nucleic acid eyntbesis in human osteosarcoma cella. Eur. J. Cancer, 12,839. 20 Strander, H., (1977) Interferons: Anti-neoplaetic drugs? Blut, 35,277. 21 Strander, H. and Einhom, 5. (1977) Effect of human leukocyte interferon on the growth of human otsteorarcoma celle in tissue culture. Int. J. Cancer, 19,468. 22 Tattersall, M.H.N. and Harrap, K.R. (1973) Changes in the deoxyribonucleoride tiphoephate pools of mouee 5178Y lymphoma celh following exposure to methotrexate or 5-fluorouracil. Cancer Res., 33, 3086. 23 Tatteraall, M.H.N., Jackson, R.C., Connors, T.A. and Harrap, K.R. (1973) Combination chemotherapy: the interaction of methotrexate and 5-fluorouracil. Eur. J. Cancer, 9, 733.