c 1991 S. Karger AG. Basel 0030 2414 91 0483 0196 $2.75 0
Oncology 1991;48:196-201
Research on the Differentiation of Human and Murine Neuroblastoma Cells E. Busse, O. Bartsch, B. Kornhaber Zentrum der Kinderheilkunde. Abteilung für Hämatologie und Onkologie. Johann Wolfgang Goethe-Universität. Frankfurt Main. BRD
Key Words. Neuroblastoma • Cyclic nucleotide • Differentiation Abstract. In vitro, we were able to induce a differentiation of human (SK-N-MC, I MR-32, Leo-2) and murine neuroblastoma cells (NA-2, C-1300, NIE-115) with dibutyryl cyclic 3'5'-adenosine monophosphate (dbcAMP), hypothalamic factor (HF). and somatostatin. As morphological criteria of cellular dilTerentiation we used the decrease in cell proliferation and the formation of neurites. Functional parameters were the increase of A cholinesterase activity, cAMP level, and protein content, and the decrease ofcGMP level. After application of dbcAMP and HF, the effects were stronger than after somatostatin. We believe that the action of HF and somatostatin is caused by an increase in cAMP levels. In the in vivo experiments, human and murine neuroblastoma cells (NA-2, C-1300, and Leo-2) were transplanted into nude/nude mice. After HF treatment of 14 mice with NA-2 tumors. 4 of the mice were tumor-free, and mean tumor weight was reduced to one-third of the controls. Of the animals with C-1300 and Leo-2 tumors, half became tumor-free, and mean tumor weight was reduced to one-fourth. The results indicate that the induction of cellular differentiation by factors and hormones may in future become a method of therapy for human neuroblastoma.
Introduction Differentiation of neuroblastoma (NB) cells can be achieved in vitro by treatment with vitamins [1-3], phorbolesters [1-6], hormones, transmitter sub stances, and second messenger cAMP [7-11], cytostat ic drugs, and X-rays [12]. In addition, a hypothalamic growth factor (HF) which has been isolated in mam mals leads in vitro to the dilTerentiation of murine leukemic cells (K-523) and to an increase in intracel lular cAMP levels [13], This study investigates the effects of HF on murine and human NB cells in vitro and in vivo.
were grown as monolayers in Eagle MEM culture medium with 5% FCS in a humidified atmosphere with 5% CO 2 in air. Neurite formation was evaluated by phase-contrast microscopy [14], Protein content was determined as described by Lowry et al. ¡15]. Cyclic AMP and cGMP were prepared from the cells according to Bach [16] and measured with the 1251radioimmunoassay kit from NEN. Acetylcholine esterase (AChF.) was determined according to Ellman et al. [17], For the in vivo experiments, NA-2, C-1300, and Leo-2 NB cells were subjected to 10-12 passages and transferred into nude/nude mice. Treatment began on day 2 after transplantation with in traperitoneal injection of dibutyryl cyclic 3'5'-adcnosine monophos phate (dbcAMP) 50 mg/'kg/day, dbcAMP 10 mg/kg/day. HF 2 x 10 pg protein/kg day, 1IF 2 x I pg protein kg day. or soma tostatin 10 pg kg day. Animals with NA-2 grafts were sacrificed on day 12. and those with C-1300 and Lco-2 tumors on day 16.
Materials and Methods Results In vitro Experiments
The effects of dbcAMP, HF, and somatostatin on human and murine NB cells in vitro are summarized in table 1. The morphological differentiation of the
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HF was prepared as deseribed in Sindell [3]. Somatostatin and acetylcholine were obtained from Sigma, dbcAMP from Boehringer Mannheim, the human NB cell lines SK-N-MC. IMR-32, and Leo-2 from the Deutsches Krebsforschungszentrum. Heidelberg, and the murine NB cell lines NA-2. C-1300. and NIF.-115 from Dr. F. Keller. Institut für Pharmazie der Freien Universität Berlin. Cells
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Cell line
Number of experiments
Compound
Leo-2
10 7 7 7 7
controls controls DBcAMP 0.1 mM somatostatin 10 pg HF 10 pg
C-1300
11 11 7 7 7
controls controls DBcAMP 0.1 m XI somatostatin 10 pg HF 10 pg
NA-2
10 9 6 6 6
controls controls DBcAMP 0.1 mM somatostatin 10 pg HF 10 pg
Number of treatments, days
Cell number per ml x 10'
Protein content mg/10s cells
Ncurile formation %
1.8
0.032 0.039 0.220 0.052 0.230
6 9 72 36 76
1.0 41.0 6.2 20.0 3.9
0.025 0.020 0.200 0.053 0.300
7 10 68 26 86
0
1.0
10 10
89.0 7.0 8.9 4.1
0.010 0.012
8 10 79 40 81
0
1.0
10 10
63.0 5.3
10
11.0
0 10 10 10 10
10 10
0.190 0.070 0.270
Fig. 1. Morphological and functional differentiation of SK-N-MC (a) and NIE-115 NB cells in vitro (b: n = 5). ■ = Controls; O = treatment with dbcAMP (0.1 mXI. 100 pi, 10.000 cells day); □ = treatment with somatostatin (5 pg in 100 pi 10.000 cells day): • = treatment with HF' (5 pg in 100 pi 10.000 cells/day).
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Table 1. Leo-1 and C -1300 were treated for a period of 10 days. NA-2 for 6 days with DBcAMP 10 4.W/day or HF 5 pg protein/day
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b
a
Fig. 2. a Normal morphology of cultured murine N B cells (SK-N-MC) in vitro, b Neurite formation in this cell line after administration of IIF (5 pg in 100 pi 10.000 cells/day). Phase-contrast microscopy, x 200.
///
vivo Experiments
Murine (NA-2, C-1300) and human (Leo-2) NB cell lines were transplanted into nude mice. The ani mals were then treated with dbcAMP. HF, and soma tostatin, as described in the methods section. The
Table 2. Cyclic nucleotide levels in human and murine cultured NB Cell line
Compound
cAMP
cGMP
C -1300
controls dbcAMP 10”4 V/ dbcAMP 10' 5A/ HE 10 pg HF 1 pg
13.6 ±2.9 29.2 ± 3.9* 20.1 ±2.0* 39.0 ± 5.6* 28.7 ±4.3*
5.3 ± 0.9 2.7 ±0.4* 3.2 ±0.5* 2.0 ±0.5* 2.9 ± 0.4*
NA-2
controls dbcAMP 10 4A/ H FI0 pg
7.6 ±0.9 36.0 ± 6.0* 43.0 ± 5.4*
4.9 ±0.7 2.4 ±0.5* 2.4 ±0.3*
N IE-115
controls dbcAMP 10 4 HF 10 pg
20.0 ±4.7 76.0 ±21* 68.0 ± 19*
2.5 ±0.4 1.6 ±0.2* 1.4 ±0.3*
I.co-2
controls dbcAMP 10 4M dbcAMP 10 5A/ HF 10 pg HF 1 pg
4.6 ± 0.3 12.8 ± 2.1* 9.7 ±1.1* 36.7 ± 4.2* 21.5 ± 3.1*
1.8 ± 0.3 0.8 ±0.1* 0.9 ±0.2* 0.5 ±0.1* 0.7 ±0.2*
SK-N-MC
controls dbcArnP 10 4M Il F 10 pg
12.4 ± 1.8 44.0 ±4.7* 58.3 ±9.0*
6.9 ± 0.9 4.9 ±0.7* 4.5 ±0.6*
1MR-32
controls dbcAMP 10 *M HF 10 pg
2.4 ±0.5 9.2 ± 0.9* 10.3 ±0.8*
1.9 ±0.3 0.9 ±0.1* 0.8 ±0.1*
NA-2 cells were treated for 6 days, the other cell lines for 10 days. * p < 0.05 Downloaded by: Dahlgren Memorial Library, GUMC 141.161.91.14 - 8/21/2017 4:36:34 AM
tumors is reflected by the rates of cell replication and neurite formation. Their functional differentiation is described by the intracellular protein content and by the AChE activity. All three substances mentioned above decreased the replication rate in all cell lines. HF had the stron gest effects, and somatostatin the weakest. Examples are shown in figure 1. After 15 days of treatment, dbcAMP and HF had induced neurite formation in approximately 80% of the cells, and somatostatin in 30 40%. as shown in figure 2. The intracellular protein content multiplied with the rate of neurite formation. It increased 10-fold after administration of dbcAMP and HF. After the ap plication of somatostatin no significant changes in protein content resulted when compared to the con trols. In NIF.-115 cells, not only neurite formation and protein content increased, but also AChE levels. In all six cell lines, cAMP levels were significantly higher and cGMP levels were significantly lower than in the controls (table 2). These changes were depen dent on the dose applied.
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Table 3. Effects of HF. dbcAMP. and somatostatin treatment of nude mice with neuroblastoma transplants Cell line
Compound
Total number of mice
Mean tumor weight g
Number of tumor-free mice
controls I1F 10 pg kg HF 1 pg kg dbcAMP 50 mg/kg dbcAMP 10 mg/kg somatostatin 10 pg kg
12 14 10 14 12 10
1.85 ± 0.46 0.59 ± 0.37* 0.92 ± 0.28* 1.02 ±0.31* 1.32 ±0.42* 1.42 ± 36*
0 4 0 0 0 0
controls HF 10 pg kg HF 1 pg/kg dbcAMP 50 mg kg dbcAMP 10 mg/kg somatostatin 10 pg kg
11 15 10 10 6 6
2.00 ± 0.53 0.50 ±0.10* 0.90 ± 0.20* 1.13 ±0.29* 1.86 ± 0.32* 1.43 ± 0.49
0 7 1 0 0 0
controls HF 10 pg/kg HF 1 pg/kg dbcAMP 50 mg kg dbcAMP 10 mg kg
12 10 10 10 9
1.86 ±0.33 0.41 ±0.21* 0.89 ±0.11 * 1.23 ±0.22* 1.46 ±0.21*
0 5 1 0 0
NA-2
C-1300
Leo-2
* p < 0.05.
results are summarized in table 3. Treatment with HF and w'ith high doses of dbcAMP (50 mg/kg) signifi cantly reduced the mean tumor weight, while lower doses of dbcAMP (10 mg/kg) and somatostatin
(1 pg/kg. not shown, and 10 pg/kg) caused no signifi cant changes. HF had the strongest effects: Using a dosage of 10 gg/kg the tumors disappeared in 25 50% of the mice, but with a dosage of 1 gg/kg disappear
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Fig. 3. a Normal morphology of murine NB cell transplant (C-1300) in vivo, b The same cell line after treatment with HF. Only isolated NB cells have survived. They lie embedded in connective tissue and fat.
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Discussion
After the administration of the hypothalamic growth factor HF, dbcAMP, and somatostatin, the cultured NB cells of murine and human origin showed signs of differentiation. This differentiation can be characterized by reduction in the cellular replication rate, the formation of neurites, and increases in the protein content and the AChE activity. These obser vations comply with the findings of other authors [2, 3, 5. 7-9, 18]. In vitro, HF and dbcAMP produced in the cultured cells significant increases in cAMP levels and de creases in cGMP levels. Cytotoxic or cytolytic effects were not detected. Similar results have been reported by Lim et al. [19]. From these findings we may con clude that HF produces its effects by regulative mech anisms which are controlled by second messenger cAMP. In vivo HF had the most pronounced effect on the morphological differentiation of the cells. Some ani mals became tumor-free after HF administration, and there was a positive correlation between the HF doses and the percentage of tumor-free mice. The decreases in mean tumor weight after HF and dbcAMP also were dependent on the level of dosage. The histologic findings between the in vitro and the in vivo experiments vary. In vitro, the NB cells dif ferentiated after administration of dbcAMP and HF. After treatment in vivo, the cells had diminished in numbers, were surrounded by connective tissue and fat, and apparently had lost their invasive growth pattern. The clinical relevance of HF has still to be deter mined. Other differentiation-inducing factors have already been introduced into the treatment of human cancer. We believe that HF or comparable substances may also prove to be of value in the treatment of human NB in the future. Like HF, DbcAMP has dilferentiating effects in vitro, and limits tumor growth in vivo. This substance cannot be used in man, but there are already approved drugs which increase intracellular cAMP levels. Such drugs may also be
effective in the treatment of NB. The third substance studied, somatostatin, activates the adenyl cylase sys tem [20], Although it decreased the mean tumor weight in our experiments, the very high doses needed indicate that it has no place in the treatment of human NB. Acknowledgements We thank the society "Hilfe für krebskranke Kinder" for the financial support during this study, the Krebsforschungszentrum I feidelberg for the gift of the SK-N-MC cell line, and A. Wcith and K. Goebel of the Department of Otolaryngology for taking the photographs.
References 1 Prasad. K.N.; Edwards-Prasad. J.; Ramanuja. S.; Sakamoto. A.: Vitamin E increase the growth inhibitory and differentiating effects of tumor therapeutic agents on neuroblastoma a glioma cells in culture. Proc. Soc. exp. Biol. Med. ¡64: 158 163 (1980). 2 Prasad. K.N.; Sinha. P. K.: In Saunders Cell differentiation and neoplasia, p. 111 (Raven Press. New York 1978). 3 Sindell. N.: Retionic acid induced growth inhibition and morphologica differentiation of human neuroblastoma cells in vitro. J. nail. Cancer Inst. 68: 589-593 (1982). 4 Gartc. S.J.: Differential effects of phorbol ester on the [)adrenergic response of normal and RAS-transformed NIN 3T3 cells. Biochem. biophys. Res. Commun. 133: 702-708 (1985). 5 Liu. A.Y.C.: Chen. K.Y.: Differential effects of tumor pro moter phorbol-12-myristate- 13-acetate on the morphological and biochemical differentiation of N-18 neuroblastoma cells. J. cell. Physiol. 125: 387-393 ( 1985). 6 Spinelli. W.: Sonnenfeld. K.H.: Ishii. D.N.: Effects of phorbol ester tumor promotor and nerve growth factor on neurite out growth in cultured human neuroblastoma cells. Cancer Res. 42: 5067-5073 (1982). 7 Elfman. L.: Lindgrcn. E.; Fredholm. B. B.: Adenosine analogues stimulate cyclic AMP-accumulation in cultured neuroblastoma and glioma cells. Acta pharmac. 55: 297 302 (1984). 8 McKenny, M.: Richclson. E.: Blockade of N IE-115 murine neuroblastoma muscarinic receptor function by agents that af fect the metabolism of arachidonic acid. Biochem. Pharmacol. 35: 2389-2397 (1986). 9 Prasad. K.N.: Kumar. S.: Role of cyclic AMP in differentiation of human neuroblastoma cells in culture. Cancer 36: 1338-1343 (1975). 10 Prasad. K.N.; Kumar. S.: Gilmar. K.; Vernadakis. A.: Cyclic AMP induced differentiation neuroblastoma cells. Biochem. biophys. Res. Commun. 50: 973-977 (1973). 11 Sandquist. D.; Williams. T I L ; Sahu, S.K.; Kataoka. S.: Morpholocical differentiation of murine neuroblastoma clone in monolayer culture induced by dexaméthasone. F.xpl. Cell Res. 113: 375-381 (1978). 12 Prasad. K.N.: X-ray induced morphological differentiation by mouse neuroblastoma cells in vitro. Nature 234:4 7 1 473 ( 1971 ).
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ance of tumors occurred in 10% of the animals. The effects of HF are shown in figure 3: After treatment, only a few isolated cells of the murine NB (C-1300) can be seen. These lie embedded between fat and connective tissue.
Research on the Differentiation of NB
19 Lim. R.; Hicklin. D.J.; Ryken. T.C.; Man, M.; Liu. K.N.; Miller. J.F. Baggcnstoss. A.B.: Suppression of glia growth in vitro and in vivo by glia maturations factor. Cancer Res. 46: 5241 5247 (1986). 20 Montminy. M R.: Scvarino. A.K.: Wagner. J.A.; Mandel. G.: Goodman. R.IL: Identification of a cyclic AMP responsive element within the rat somatostatine gene. Proc. natn. Acad. Sei. USA 83: 6682 6686 (1986).
Dr. E. Busse Zentrum der Kinderheilkunde Abteilung für Hämatologie und Onkologie Johann Wolfgang Goethe-Universität Theodor-Stcrn-Kai 7 D-W-6000 Frankfurt 70 (FRG)
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13 Busse, E.: Wirkung von Glia-Maturations-Faktor(HF) auf Kul turen leukämischer Zellen. Z. Naturf. 42: 49 51 (1987). 14 Busse, E.; Bartsch, O.: Schneider. A.; Kornhuber. B.: Influence of mctoclopramidc and bromocriptine upon the growth of hu man murine neuroblastoma cells. Oncology 47: 199-205 (1990). 15 Lowry, O.H.: Roscbrough. N.F.: Farr. A.L.; Randall. A.J.: Protein measurement with the folinphenol reagent. J. Biol. Chem 193: 265 -267 (1951). 16 Bach. M.A.: Differences in cyclic AMP changes after stimula tion by prostaglandins and isoproterenol in lymphocyte sub populations. J. clin. Invest. 55: 1074-1081 (1975). 17 Ellman. G.L.; Courtney. K.D. Andreas. V.: A new rapid col orimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88-95 (1961). 18 Kindel. Y.: Palfrey. C.; Spector. I.; Barak. Y.; Littaucr. U.Z.: Maturation of neuroblastoma cells in the presence of dimcthylsulfoxyde. Proc. nail. Acad. Sei. USA 73: 462-466 (1976).
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Oncology 1991;48:196-201
Research on the Differentiation of Human and Murine Neuroblastoma Cells E. Busse, O. Bartsch, B. Kornhaber Zentrum der Kinderheilkunde. Abteilung für Hämatologie und Onkologie. Johann Wolfgang Goethe-Universität. Frankfurt Main. BRD
Key Words. Neuroblastoma • Cyclic nucleotide • Differentiation Abstract. In vitro, we were able to induce a differentiation of human (SK-N-MC, I MR-32, Leo-2) and murine neuroblastoma cells (NA-2, C-1300, NIE-115) with dibutyryl cyclic 3'5'-adenosine monophosphate (dbcAMP), hypothalamic factor (HF). and somatostatin. As morphological criteria of cellular dilTerentiation we used the decrease in cell proliferation and the formation of neurites. Functional parameters were the increase of A cholinesterase activity, cAMP level, and protein content, and the decrease ofcGMP level. After application of dbcAMP and HF, the effects were stronger than after somatostatin. We believe that the action of HF and somatostatin is caused by an increase in cAMP levels. In the in vivo experiments, human and murine neuroblastoma cells (NA-2, C-1300, and Leo-2) were transplanted into nude/nude mice. After HF treatment of 14 mice with NA-2 tumors. 4 of the mice were tumor-free, and mean tumor weight was reduced to one-third of the controls. Of the animals with C-1300 and Leo-2 tumors, half became tumor-free, and mean tumor weight was reduced to one-fourth. The results indicate that the induction of cellular differentiation by factors and hormones may in future become a method of therapy for human neuroblastoma.
Introduction Differentiation of neuroblastoma (NB) cells can be achieved in vitro by treatment with vitamins [1-3], phorbolesters [1-6], hormones, transmitter sub stances, and second messenger cAMP [7-11], cytostat ic drugs, and X-rays [12]. In addition, a hypothalamic growth factor (HF) which has been isolated in mam mals leads in vitro to the dilTerentiation of murine leukemic cells (K-523) and to an increase in intracel lular cAMP levels [13], This study investigates the effects of HF on murine and human NB cells in vitro and in vivo.
were grown as monolayers in Eagle MEM culture medium with 5% FCS in a humidified atmosphere with 5% CO 2 in air. Neurite formation was evaluated by phase-contrast microscopy [14], Protein content was determined as described by Lowry et al. ¡15]. Cyclic AMP and cGMP were prepared from the cells according to Bach [16] and measured with the 1251radioimmunoassay kit from NEN. Acetylcholine esterase (AChF.) was determined according to Ellman et al. [17], For the in vivo experiments, NA-2, C-1300, and Leo-2 NB cells were subjected to 10-12 passages and transferred into nude/nude mice. Treatment began on day 2 after transplantation with in traperitoneal injection of dibutyryl cyclic 3'5'-adcnosine monophos phate (dbcAMP) 50 mg/'kg/day, dbcAMP 10 mg/kg/day. HF 2 x 10 pg protein/kg day, 1IF 2 x I pg protein kg day. or soma tostatin 10 pg kg day. Animals with NA-2 grafts were sacrificed on day 12. and those with C-1300 and Lco-2 tumors on day 16.
Materials and Methods Results In vitro Experiments
The effects of dbcAMP, HF, and somatostatin on human and murine NB cells in vitro are summarized in table 1. The morphological differentiation of the
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HF was prepared as deseribed in Sindell [3]. Somatostatin and acetylcholine were obtained from Sigma, dbcAMP from Boehringer Mannheim, the human NB cell lines SK-N-MC. IMR-32, and Leo-2 from the Deutsches Krebsforschungszentrum. Heidelberg, and the murine NB cell lines NA-2. C-1300. and NIF.-115 from Dr. F. Keller. Institut für Pharmazie der Freien Universität Berlin. Cells
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Cell line
Number of experiments
Compound
Leo-2
10 7 7 7 7
controls controls DBcAMP 0.1 mM somatostatin 10 pg HF 10 pg
C-1300
11 11 7 7 7
controls controls DBcAMP 0.1 m XI somatostatin 10 pg HF 10 pg
NA-2
10 9 6 6 6
controls controls DBcAMP 0.1 mM somatostatin 10 pg HF 10 pg
Number of treatments, days
Cell number per ml x 10'
Protein content mg/10s cells
Ncurile formation %
1.8
0.032 0.039 0.220 0.052 0.230
6 9 72 36 76
1.0 41.0 6.2 20.0 3.9
0.025 0.020 0.200 0.053 0.300
7 10 68 26 86
0
1.0
10 10
89.0 7.0 8.9 4.1
0.010 0.012
8 10 79 40 81
0
1.0
10 10
63.0 5.3
10
11.0
0 10 10 10 10
10 10
0.190 0.070 0.270
Fig. 1. Morphological and functional differentiation of SK-N-MC (a) and NIE-115 NB cells in vitro (b: n = 5). ■ = Controls; O = treatment with dbcAMP (0.1 mXI. 100 pi, 10.000 cells day); □ = treatment with somatostatin (5 pg in 100 pi 10.000 cells day): • = treatment with HF' (5 pg in 100 pi 10.000 cells/day).
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Table 1. Leo-1 and C -1300 were treated for a period of 10 days. NA-2 for 6 days with DBcAMP 10 4.W/day or HF 5 pg protein/day
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b
a
Fig. 2. a Normal morphology of cultured murine N B cells (SK-N-MC) in vitro, b Neurite formation in this cell line after administration of IIF (5 pg in 100 pi 10.000 cells/day). Phase-contrast microscopy, x 200.
///
vivo Experiments
Murine (NA-2, C-1300) and human (Leo-2) NB cell lines were transplanted into nude mice. The ani mals were then treated with dbcAMP. HF, and soma tostatin, as described in the methods section. The
Table 2. Cyclic nucleotide levels in human and murine cultured NB Cell line
Compound
cAMP
cGMP
C -1300
controls dbcAMP 10”4 V/ dbcAMP 10' 5A/ HE 10 pg HF 1 pg
13.6 ±2.9 29.2 ± 3.9* 20.1 ±2.0* 39.0 ± 5.6* 28.7 ±4.3*
5.3 ± 0.9 2.7 ±0.4* 3.2 ±0.5* 2.0 ±0.5* 2.9 ± 0.4*
NA-2
controls dbcAMP 10 4A/ H FI0 pg
7.6 ±0.9 36.0 ± 6.0* 43.0 ± 5.4*
4.9 ±0.7 2.4 ±0.5* 2.4 ±0.3*
N IE-115
controls dbcAMP 10 4 HF 10 pg
20.0 ±4.7 76.0 ±21* 68.0 ± 19*
2.5 ±0.4 1.6 ±0.2* 1.4 ±0.3*
I.co-2
controls dbcAMP 10 4M dbcAMP 10 5A/ HF 10 pg HF 1 pg
4.6 ± 0.3 12.8 ± 2.1* 9.7 ±1.1* 36.7 ± 4.2* 21.5 ± 3.1*
1.8 ± 0.3 0.8 ±0.1* 0.9 ±0.2* 0.5 ±0.1* 0.7 ±0.2*
SK-N-MC
controls dbcArnP 10 4M Il F 10 pg
12.4 ± 1.8 44.0 ±4.7* 58.3 ±9.0*
6.9 ± 0.9 4.9 ±0.7* 4.5 ±0.6*
1MR-32
controls dbcAMP 10 *M HF 10 pg
2.4 ±0.5 9.2 ± 0.9* 10.3 ±0.8*
1.9 ±0.3 0.9 ±0.1* 0.8 ±0.1*
NA-2 cells were treated for 6 days, the other cell lines for 10 days. * p < 0.05 Downloaded by: Dahlgren Memorial Library, GUMC 141.161.91.14 - 8/21/2017 4:36:34 AM
tumors is reflected by the rates of cell replication and neurite formation. Their functional differentiation is described by the intracellular protein content and by the AChE activity. All three substances mentioned above decreased the replication rate in all cell lines. HF had the stron gest effects, and somatostatin the weakest. Examples are shown in figure 1. After 15 days of treatment, dbcAMP and HF had induced neurite formation in approximately 80% of the cells, and somatostatin in 30 40%. as shown in figure 2. The intracellular protein content multiplied with the rate of neurite formation. It increased 10-fold after administration of dbcAMP and HF. After the ap plication of somatostatin no significant changes in protein content resulted when compared to the con trols. In NIF.-115 cells, not only neurite formation and protein content increased, but also AChE levels. In all six cell lines, cAMP levels were significantly higher and cGMP levels were significantly lower than in the controls (table 2). These changes were depen dent on the dose applied.
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Table 3. Effects of HF. dbcAMP. and somatostatin treatment of nude mice with neuroblastoma transplants Cell line
Compound
Total number of mice
Mean tumor weight g
Number of tumor-free mice
controls I1F 10 pg kg HF 1 pg kg dbcAMP 50 mg/kg dbcAMP 10 mg/kg somatostatin 10 pg kg
12 14 10 14 12 10
1.85 ± 0.46 0.59 ± 0.37* 0.92 ± 0.28* 1.02 ±0.31* 1.32 ±0.42* 1.42 ± 36*
0 4 0 0 0 0
controls HF 10 pg kg HF 1 pg/kg dbcAMP 50 mg kg dbcAMP 10 mg/kg somatostatin 10 pg kg
11 15 10 10 6 6
2.00 ± 0.53 0.50 ±0.10* 0.90 ± 0.20* 1.13 ±0.29* 1.86 ± 0.32* 1.43 ± 0.49
0 7 1 0 0 0
controls HF 10 pg/kg HF 1 pg/kg dbcAMP 50 mg kg dbcAMP 10 mg kg
12 10 10 10 9
1.86 ±0.33 0.41 ±0.21* 0.89 ±0.11 * 1.23 ±0.22* 1.46 ±0.21*
0 5 1 0 0
NA-2
C-1300
Leo-2
* p < 0.05.
results are summarized in table 3. Treatment with HF and w'ith high doses of dbcAMP (50 mg/kg) signifi cantly reduced the mean tumor weight, while lower doses of dbcAMP (10 mg/kg) and somatostatin
(1 pg/kg. not shown, and 10 pg/kg) caused no signifi cant changes. HF had the strongest effects: Using a dosage of 10 gg/kg the tumors disappeared in 25 50% of the mice, but with a dosage of 1 gg/kg disappear
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Fig. 3. a Normal morphology of murine NB cell transplant (C-1300) in vivo, b The same cell line after treatment with HF. Only isolated NB cells have survived. They lie embedded in connective tissue and fat.
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Discussion
After the administration of the hypothalamic growth factor HF, dbcAMP, and somatostatin, the cultured NB cells of murine and human origin showed signs of differentiation. This differentiation can be characterized by reduction in the cellular replication rate, the formation of neurites, and increases in the protein content and the AChE activity. These obser vations comply with the findings of other authors [2, 3, 5. 7-9, 18]. In vitro, HF and dbcAMP produced in the cultured cells significant increases in cAMP levels and de creases in cGMP levels. Cytotoxic or cytolytic effects were not detected. Similar results have been reported by Lim et al. [19]. From these findings we may con clude that HF produces its effects by regulative mech anisms which are controlled by second messenger cAMP. In vivo HF had the most pronounced effect on the morphological differentiation of the cells. Some ani mals became tumor-free after HF administration, and there was a positive correlation between the HF doses and the percentage of tumor-free mice. The decreases in mean tumor weight after HF and dbcAMP also were dependent on the level of dosage. The histologic findings between the in vitro and the in vivo experiments vary. In vitro, the NB cells dif ferentiated after administration of dbcAMP and HF. After treatment in vivo, the cells had diminished in numbers, were surrounded by connective tissue and fat, and apparently had lost their invasive growth pattern. The clinical relevance of HF has still to be deter mined. Other differentiation-inducing factors have already been introduced into the treatment of human cancer. We believe that HF or comparable substances may also prove to be of value in the treatment of human NB in the future. Like HF, DbcAMP has dilferentiating effects in vitro, and limits tumor growth in vivo. This substance cannot be used in man, but there are already approved drugs which increase intracellular cAMP levels. Such drugs may also be
effective in the treatment of NB. The third substance studied, somatostatin, activates the adenyl cylase sys tem [20], Although it decreased the mean tumor weight in our experiments, the very high doses needed indicate that it has no place in the treatment of human NB. Acknowledgements We thank the society "Hilfe für krebskranke Kinder" for the financial support during this study, the Krebsforschungszentrum I feidelberg for the gift of the SK-N-MC cell line, and A. Wcith and K. Goebel of the Department of Otolaryngology for taking the photographs.
References 1 Prasad. K.N.; Edwards-Prasad. J.; Ramanuja. S.; Sakamoto. A.: Vitamin E increase the growth inhibitory and differentiating effects of tumor therapeutic agents on neuroblastoma a glioma cells in culture. Proc. Soc. exp. Biol. Med. ¡64: 158 163 (1980). 2 Prasad. K.N.; Sinha. P. K.: In Saunders Cell differentiation and neoplasia, p. 111 (Raven Press. New York 1978). 3 Sindell. N.: Retionic acid induced growth inhibition and morphologica differentiation of human neuroblastoma cells in vitro. J. nail. Cancer Inst. 68: 589-593 (1982). 4 Gartc. S.J.: Differential effects of phorbol ester on the [)adrenergic response of normal and RAS-transformed NIN 3T3 cells. Biochem. biophys. Res. Commun. 133: 702-708 (1985). 5 Liu. A.Y.C.: Chen. K.Y.: Differential effects of tumor pro moter phorbol-12-myristate- 13-acetate on the morphological and biochemical differentiation of N-18 neuroblastoma cells. J. cell. Physiol. 125: 387-393 ( 1985). 6 Spinelli. W.: Sonnenfeld. K.H.: Ishii. D.N.: Effects of phorbol ester tumor promotor and nerve growth factor on neurite out growth in cultured human neuroblastoma cells. Cancer Res. 42: 5067-5073 (1982). 7 Elfman. L.: Lindgrcn. E.; Fredholm. B. B.: Adenosine analogues stimulate cyclic AMP-accumulation in cultured neuroblastoma and glioma cells. Acta pharmac. 55: 297 302 (1984). 8 McKenny, M.: Richclson. E.: Blockade of N IE-115 murine neuroblastoma muscarinic receptor function by agents that af fect the metabolism of arachidonic acid. Biochem. Pharmacol. 35: 2389-2397 (1986). 9 Prasad. K.N.: Kumar. S.: Role of cyclic AMP in differentiation of human neuroblastoma cells in culture. Cancer 36: 1338-1343 (1975). 10 Prasad. K.N.; Kumar. S.: Gilmar. K.; Vernadakis. A.: Cyclic AMP induced differentiation neuroblastoma cells. Biochem. biophys. Res. Commun. 50: 973-977 (1973). 11 Sandquist. D.; Williams. T I L ; Sahu, S.K.; Kataoka. S.: Morpholocical differentiation of murine neuroblastoma clone in monolayer culture induced by dexaméthasone. F.xpl. Cell Res. 113: 375-381 (1978). 12 Prasad. K.N.: X-ray induced morphological differentiation by mouse neuroblastoma cells in vitro. Nature 234:4 7 1 473 ( 1971 ).
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ance of tumors occurred in 10% of the animals. The effects of HF are shown in figure 3: After treatment, only a few isolated cells of the murine NB (C-1300) can be seen. These lie embedded between fat and connective tissue.
Research on the Differentiation of NB
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Dr. E. Busse Zentrum der Kinderheilkunde Abteilung für Hämatologie und Onkologie Johann Wolfgang Goethe-Universität Theodor-Stcrn-Kai 7 D-W-6000 Frankfurt 70 (FRG)
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