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A low molecular weight calf pineal peptide with an inhibiting effect on the growth of L1210 and HL60 cells
967
Cell Biology lnternational Reports. VoL 16. No. 10. 1992
A LOW MOLECULAR WEIGHT CALF PINEAL PEPTIDE WITH AN INHIBITING EFFECT ON THE GROWTH OF L1210 AND HL60 CELLS. Petrelli, C.*, Moretti, P., Petrelli, F. and Bramucci, M. Department of Molecular, Cellular and Animal Biology, University of Camerino, 62032 Camerino (MC), Italy. t
Corresponding author
ABSTRACT The peptide isolated by us from calf pineal gland causes a reduction of RNA synthesis in vitro in L1210 and HL60 tumoral cells. This peptide also causes inhibition of cell proliferation; the cell viability is not modified. The effects are dose-dependent and reversible. INTRODUCTION In the literature a physiological relationship between the pineal gland and neoplastic growth has been discussed. In pinealectomized hamsters the growth and metastatic spread of the transplantable melanotic melanoma no 1 (Das Gupta and Terz, 1967) and the number of dimethylbenzanthracene-induced melanomas significantly increased (Aubert et al., 1970). In the same tissue of pinealectomised rats an increase has been observed in DNA synthesis and in mitotic rate (Bindoni, 1971; Quay, 1974). Relationships between administration of mammalian pineal compounds and malignancy have been demonstrated (Lapin, 1976; EI-Domeiri and Das Gupta, 1976; Bindoni et al., 1976; Lapin and Ebels, 1976; Dilman et al. 1979; Bartsch and Bartsch, 1981). From these it can be concluded that the mammalian pineal, that produce melatonin and other still unknown pineal compounds, may influence the manifestation of neoplastic diseases (Carr and Axetrod, 1981). Following Blask (1984) and Blask and Leadem (1987), current knowledge indicates a pineal-mediated neuroendocrine modulation of tumour growth. Investigations are currently in progress to determine structures of various pineal growth-inhibiting iactors other than melatonin ( Bartsch et al., 1987; Noteborn et al., 1988 ). We have isolated from calf pineal gland of low molecular weight a peptide with high inhibiting activity on DNA transcription in vitro . This peptide has molecular weight of about 1200 Dalton . Amino acid composition, in the approximate molar ratio referred to glycine taken as 1, is the following: glycine 1, threonine 1, lysine 1, glutamic acid 2, alanine 6 (Petrelli et al., in press ). This peptide is different from peptides having antimitotic or antiblastic action extracted from pineal glands by other Authors (Noteborn et al. , 1988 ). In this paper we report the results of studies focused on the action on the growth of L1210 and HL60 cells in vitro of the pineal peptide we,isolated. 0309-1651/92/100967-8/$08.00/0
© 1992 Academic Press Ltd
968
Cell Biology lnternational Reports, Vol. 16, No. 10. 1992
MATERIALS AND METHODS
Pineal glands tissue:- Pineal glands were removed from calves (from 5 to 8 months old)immediately after death,cleaned of contaminating material,wiped with filter paper, weighed and stored at -80 ° C until required for experimental use. Extraction and purification of peptide active fraction from pineal gland:- Pineal aqueous extract and the isolation of a peptide fraction with inhibiting activity on DNA transcription were carried.out according to Petrelli et al. (in press). The peptide purification from aqueous extract was performed using five successive purification steps: a) ultrafiltration on SM 121-36 membrane (Mr cutoff 10,000); b, c) gel filtration on Sephadex G25 and gel filtration on Sephadex G10 equilibrated and eluted with bidistilled water; d) thin layer ascendent chromatography on aluminium sheet ceitutose in t-butanol formic acid - water (75:15:15); e) high-performance liquid chromatogr_aphy (HPLC) using a Supercosil LC 318 (particle 5 p.m) reverse phase column (4.6 mm x 25 cm) with precolumn (4.6 mm x 2 cm) equilibrated and eluted for 20 min with solvent A ( 0 . 1 % trifluoro acetic acid in water) followed by a linear gradient (from 0 to 100 % ) solvent B ( 0 . 1 % t.ricloroacetic acid in acetonitrile). Protein and peptide determination:- Protein and peptide determinations were performed following the procedure of Lowry et al. (1951) and Nakai et al.(1 974), respectively. Cell culture:- L1210 mouse leukemia cells and HL60 human promyelocytic leukemia cells were used to test the growth-inhibiting activity of the calf pineal peptide fraction obtained by high performance liquid chromatography (HPLC) that inhibited DNA transcription activity . The L1210 and HL60 cells were grown at 37 ° C in a humidified atmosphere containing 5% CO 2 and 95 % air in RPMI 1640 medium, supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine, penicillin ( 100 unit / ml ), and streptomycin (100 p.g / ml )( Flow Laboratories, Scotland). Cell numbers were monitored by direct counting using a hemocytometer Coulter Counter ZM. Cell viability was assessed by Trypan blue exclusion. I n c u b a t i o n and assay for RNA s y n t h e s i s . Determination of the radioactivity into RNA a c i d - i n s o l u b l e fraction:-The incubation and assay for RNA synthesis was carried out according to the method of Marushige and Bonner (1966). The determination of the radioactivity into RNA acid-insoluble fraction was performed as described by Petrelli et al. (in press). Uptake of labeled uridine:- Uptake of labeled [U-14C]uridine was determined after 2-4 min pulses. Samples were immediately diluted with ice-cold phosphate -buffered solution supplemented with unlabeled uridine 10 times more concentrated than the labeled compound. After two washings, cell aliquots (106 cells) were lysed in 0.5% sodium dodecyl sulfate and total radioactivity was determined.
Cell Biology International Reports, Vol. 16, No. 10, 1992
969
RESULTS AND DISCUSSION. The kinetic data in Table 1 show that the presence in the incubation mixture of the peptide extracted by us from pineal glands causes a pronounced reduction of the transcriptional capacity of DNA tested in vitro by DNA dependent-RNA polymerase reaction. The inhibitor effect on RNA synthesis is dose-dependent. These data confirm those reported in the previous paper (Petrelli et al., in press).
Table 1 Effect' of peptide purified from calf pineal on DNA transcription activity in vitro. Peptide concentration in incubation mixture: 125, 250, 500 n g / ml. Each value is the mean of data from five determinations; mean _+ SD. Percent inhibition
Peptide concentration
125 ng / ml 250 ng / ml 500 ng / ml
5th rain
10th rain
40.00 + 3.08 72.20 +_3.56 94.60 + 4.56
60.20 + 4.44 85.20 + 4.55 100.00 + 0.00
The data in Table 2 show that the calf pineal peptide purified by us causes a dose-dependent inhibition of RNA synthesis in L1210 and HL 60 cell lines in vitro. This effect is more evident in HL60 than in L1210 cells. It is noteworthy that treatment with the peptide does not modify the cell viability.
Table 2 Effect of peptide purified from calf pineal on RNA synthesis in L1210 and HI60 cells. RNA synthesis was measured by incorporation of 3H-Uridine. The peptide was added at 0 time. Two hours of labeling from 4 to 6 hrs of culture. Mean values obtained from four experiments. Percentage inhibition of 3H-Uridine incorporation in treated tumoral cells with respect to untreated specimens. Mean _+SD. Peptide ( n g / m l ) Inhibition ( % ): L1210
HL60
Cell viability
(%)
60
125
9.80 + 1.92
15.20 _+ 4.15
1 5.40 _+ 3.05
+
96
+
250
500
40.20 3.70
68.00 + 2.77
35.40 4.83
65.40 _+ 4.50
98
95
+
80.00 2.62 97
970
Cell Biology Intemational Reports, Vol. 16, No. 10, 1992
Uptake of labeled uridine in the tumoral cells HL60 after 2-4 min pulses was comparable in the presence and in the absence of the peptide, the addition of which did not impair uptake of the label. This excludes the possibility that the decreased incorporation of radioactive uridine into RNA of cells in culture is due to an effect on the transport of uridine through the cell membrane (Table 3).
Table 3 Effect of the peptide on uptake of [U _14C] uridine in HL60 cells: cpm/106 ceils. The HL60 (after 30 min of preincubation) were incubated at a concentration of 5 x 1 0 6 cells per ml in the presence of [U -14C] uridine (23 p.Ci /ml, 453mCi/mmol; Radiochemical Center, Amersham), With occasional shaking under air containing 5% carbon dioxide. The peptide purified (O.1 ml, corresponding to 500 ng) was added per ml of incubation mixture; control was given 0.1 ml of saline. We chose the concentration of the peptide that causes an 100% inhibition of the DNA transcription. 14 C cpm at Sample
2 min
HL60 (control)
10,683 ns + 1,497
HL60 + Peptide
10,750 _+ 1,502
4 rain
13,880 ns + 1,367 13,304 _+ 1,308
Values are expressed as mean + SD of the data obtained from 5 samples. Statistically significant differences between control and HL60 cells treated with peptide (Student's -t,, test): ns not significant differences.
In agreement with data in Table 2, also the proliferation of both cell lineages is affected by peptide effector ( Figures 1, 2 ). The presen(~e of peptide in culture medium causes a dose-dependent inhibition of the cell proliferation. The L1210 cells seem to be more sensitive than HL60 ones to peptide effector. The inhibiting effect may be reversed by washing and reseeding the cells in fresh medium not containing the peptide effector (Figures 3,4). Such data demonstrate that pineal peptide does not modify irreversibly the metabolic activity of the two cell lineages we studied. In conclusion the results reported in this work show that the peptide isolated by us from calf pineal glands, having a molecular weight of about 1200 Dalton, besides having inhibiting activity on DNA transcription in vitro (Petrelli et al., in press), has inhibiting activity on RNA synthesis and on proliferation of L1210 and HL60 tumoral cells. The inhibitor effect is dose-dependent and reversible.
Cell Biology lnternational Reports, Vol. 16, No. 10, 1992 Figure 1 Effect of purified active peptide from calf pineal on the growth of L 1210 cells expressed as number of cells / ml ( x 105). Initial inoculum : 1 x 105 cells / ml. The peptide was added at 0 time.The cells were cultured for O, 48, 72 h without and with peptide: 60, t25, 250, 375, 500 ng /ml. Each time point indicates means obtained from four experiments.
971
4O ..o. Co~uol SOng
125ng •e - 2'50 nO 375 n.g ~ 50On, O
/
f I
24
48
72
TIME (h)
Figure 2 Effect of purified active peptide from calf pineal on the growth of HL 60 cells expressed .as number of cells / ml ( x 105). Initial inoculum: 1 x 105 cells / ml. The peptide was added at 0 time. The cells were cultured for O, 48, 72, 96 h without and with peptide: 60, 125, 250, 375, 500 ng / ml. Each time point indicates means obtained from four experiments.
2°1 -a- control ,sonG 125n o •41- 250rig "+" 375 ,~g
~,
10"
of 0
24
48 "flUE (h)
72
06
972
Cell Biology lnternational Reports, Vol. 16. No. 10, 1992 Figure 3 Effect of the removal of purified active peptide from calf pineal on the growth of L 1210 cells expressed as number of cells / ml ( x 105 ) . initial inoculum: 1 x 105 cells-/ ml. The peptide was added_at 0. time. Thecells were cultured for 0, 24, 48, 72, 96 h without (13) and with (m) peptide (500 ng / ml). Untreated and tre-ated cells were washed and reseeded in flesh medium with (m) and without (13, x) peptide at 48 h. Each time point indicates means obtained from three experiments.
w o
OI 24
0
48
96
72
1 1 ~ (h)
Figure 4 Effect of the removal of purified active peptide from calf pineal on the growth of HL60 cells expressed as number of cells / ml ( x 105 ). Initial inoculum: 1 x 105 cells / ml. The peptide was added at 0 time. The cells were cultured for 0, 24, 48, 72, 96 h without (13) and with ( I ) peptide (500 n g / ml). Untreated and treated cells were washed and reseeded in fresh medium with ( I ) and without (13, x) peptide at 48 h. Each time point indicates means obtained from three experiments.
i
TM
0 0
,
,
,
,
24
48
72
96
TreE (h)
Cell Biology lnternational Reports. VoL 16. No. 10. 1992
973
REFERENCES
Aubert, C., Prade, M. and Bohoun, C.
(1970). Effect de la pin~alectomie sur les tumeurs m~laniques de Hamster dor~ induites par I'administration (per os) d'une seule dose de 9,10-dim~thyl -1,2-benzanthrac~ne. C.R. Acad. Sci. (Paris) 271, 2465-2466. Bartsch, H. and Bartsch, C. (1981). Effect of melatonin on experimental tumors under different photoperiods and times of administration. J. Neural Transm. 52, 269-279. Bartsch, H., Bartsch, C., Noteborn, H.P-.J.M., Flehmig, B., Ebels, I. and Salemink, C.A. (1987). Growth-inhibiting effect of crude pineal extracts on human melanoma cells in vitro is different from that of known synthetic pineal substances. J. Neural Transm. 69, 299-311. Bindoni, M. (1971). Relationship between the pineal gland and the mitotic activity of some tissues. Arch. Sci. Biol. 55, 3-21. Bindoni, M., Jutisz, M. and Ribot, G. (1976). Characterization and partial purification of a substance in the pineal gland which inhibits cell multiplication in vivo. Biochim. Biophys. Acta 437, 577-588. Blask, D. E. (1984). The pineal: an oncostatic gland? In: Reiter, R. J. led) The pineal gland. Raven Press, New York, p.p. 253-284. Blask, D. E. and Leadem, C.A. (1987). Neuroendocrine aspects of neoplastic growth: a review. Neuroendocrinol. Lett. 9 (2), 63-73. Carr, D. B. and Axelrod, L. (1981). The pineal gland, pineal dysfunction and cancer. Rev. Endocrine-related Canco 8, 13-21. Das Gupta, T. K. and Terz, J. (1967). Influence of pineal gland on the growth and spread of melanoma in the hamster. Cancer Res. 27, 1306-1311. Dilman, V. M., Anisimov, V. N. , Ostroumova, M. N., Morosov, V. G., Khavinson, V. Kh. and Azarova, M. A. (1979). Study of anti-tomor effect of polypeptide pineal extract. Oncology 36, 274- 280. EI-Domeiri, A. A. H. and Das Gupta, T. K. (1976). The influence of pineal ablation and administration of melatonin on growth and spread of hamster melanoma.. J. Surg. Oncol. 8, 197-205. Lapin, V. (1976). Pineal gland and malignancy. Osterr. Z. Onkol. 3, 51-59. Lapin, V. and Ebels, I. (1976). Effects of some low molecular weight sheep pineal fractions and melatonin on different tumors in rats and mice. Oncology 33, 110-113. Lowry, O. H., Rosenbrough, M. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. Marushige, K. and Bonner, J. (1966). Template properties of liver chromatin. J. Mol. Biol. 15, 160-174. Nakai, N., Lai, C. Y. and Horecker, B. L. (1974). Use of fluorescamine in the chromatographic analysis of peptides from proteins. Anal. Biochem. 58, 563-570 . Noteborn, H. P. J. M., Bartsch, H., Bartsch, C., Marts, D. R. A., Weusten, J. J. A . M . , Flehmig, B., Ebels, I. and Salemink, C. A. (1988). Partial purification of (a) low molecular weight ovine
974
Cell Biology lnternational Reports, Vol. 16, No. 10, 1992 pineal compound(s) with an inhibiting effect on the growth of human melanoma cells in vitro. J. Neural. Transm. 73, 135-155.
Petrelli,
C.,
Moretti,
P.,
Petrelli,
F.
and
Barra,
D.
(1991).
Purification of a low molecular weight calf pineal peptide controlling DNA transcription in vitro. It. J. Biochem. in press. Quay, W. B. (1974). Temporal mitotic patterns around a brain lesion; cellular and regional asynchronisms and an effect of pinealectomy. Chronobiologia 1, 237-258.
Paper received 22.01.92.
Revised paper accepted 18.07.92.
Cell Biology lnternational Reports. VoL 16. No. 10. 1992
A LOW MOLECULAR WEIGHT CALF PINEAL PEPTIDE WITH AN INHIBITING EFFECT ON THE GROWTH OF L1210 AND HL60 CELLS. Petrelli, C.*, Moretti, P., Petrelli, F. and Bramucci, M. Department of Molecular, Cellular and Animal Biology, University of Camerino, 62032 Camerino (MC), Italy. t
Corresponding author
ABSTRACT The peptide isolated by us from calf pineal gland causes a reduction of RNA synthesis in vitro in L1210 and HL60 tumoral cells. This peptide also causes inhibition of cell proliferation; the cell viability is not modified. The effects are dose-dependent and reversible. INTRODUCTION In the literature a physiological relationship between the pineal gland and neoplastic growth has been discussed. In pinealectomized hamsters the growth and metastatic spread of the transplantable melanotic melanoma no 1 (Das Gupta and Terz, 1967) and the number of dimethylbenzanthracene-induced melanomas significantly increased (Aubert et al., 1970). In the same tissue of pinealectomised rats an increase has been observed in DNA synthesis and in mitotic rate (Bindoni, 1971; Quay, 1974). Relationships between administration of mammalian pineal compounds and malignancy have been demonstrated (Lapin, 1976; EI-Domeiri and Das Gupta, 1976; Bindoni et al., 1976; Lapin and Ebels, 1976; Dilman et al. 1979; Bartsch and Bartsch, 1981). From these it can be concluded that the mammalian pineal, that produce melatonin and other still unknown pineal compounds, may influence the manifestation of neoplastic diseases (Carr and Axetrod, 1981). Following Blask (1984) and Blask and Leadem (1987), current knowledge indicates a pineal-mediated neuroendocrine modulation of tumour growth. Investigations are currently in progress to determine structures of various pineal growth-inhibiting iactors other than melatonin ( Bartsch et al., 1987; Noteborn et al., 1988 ). We have isolated from calf pineal gland of low molecular weight a peptide with high inhibiting activity on DNA transcription in vitro . This peptide has molecular weight of about 1200 Dalton . Amino acid composition, in the approximate molar ratio referred to glycine taken as 1, is the following: glycine 1, threonine 1, lysine 1, glutamic acid 2, alanine 6 (Petrelli et al., in press ). This peptide is different from peptides having antimitotic or antiblastic action extracted from pineal glands by other Authors (Noteborn et al. , 1988 ). In this paper we report the results of studies focused on the action on the growth of L1210 and HL60 cells in vitro of the pineal peptide we,isolated. 0309-1651/92/100967-8/$08.00/0
© 1992 Academic Press Ltd
968
Cell Biology lnternational Reports, Vol. 16, No. 10. 1992
MATERIALS AND METHODS
Pineal glands tissue:- Pineal glands were removed from calves (from 5 to 8 months old)immediately after death,cleaned of contaminating material,wiped with filter paper, weighed and stored at -80 ° C until required for experimental use. Extraction and purification of peptide active fraction from pineal gland:- Pineal aqueous extract and the isolation of a peptide fraction with inhibiting activity on DNA transcription were carried.out according to Petrelli et al. (in press). The peptide purification from aqueous extract was performed using five successive purification steps: a) ultrafiltration on SM 121-36 membrane (Mr cutoff 10,000); b, c) gel filtration on Sephadex G25 and gel filtration on Sephadex G10 equilibrated and eluted with bidistilled water; d) thin layer ascendent chromatography on aluminium sheet ceitutose in t-butanol formic acid - water (75:15:15); e) high-performance liquid chromatogr_aphy (HPLC) using a Supercosil LC 318 (particle 5 p.m) reverse phase column (4.6 mm x 25 cm) with precolumn (4.6 mm x 2 cm) equilibrated and eluted for 20 min with solvent A ( 0 . 1 % trifluoro acetic acid in water) followed by a linear gradient (from 0 to 100 % ) solvent B ( 0 . 1 % t.ricloroacetic acid in acetonitrile). Protein and peptide determination:- Protein and peptide determinations were performed following the procedure of Lowry et al. (1951) and Nakai et al.(1 974), respectively. Cell culture:- L1210 mouse leukemia cells and HL60 human promyelocytic leukemia cells were used to test the growth-inhibiting activity of the calf pineal peptide fraction obtained by high performance liquid chromatography (HPLC) that inhibited DNA transcription activity . The L1210 and HL60 cells were grown at 37 ° C in a humidified atmosphere containing 5% CO 2 and 95 % air in RPMI 1640 medium, supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine, penicillin ( 100 unit / ml ), and streptomycin (100 p.g / ml )( Flow Laboratories, Scotland). Cell numbers were monitored by direct counting using a hemocytometer Coulter Counter ZM. Cell viability was assessed by Trypan blue exclusion. I n c u b a t i o n and assay for RNA s y n t h e s i s . Determination of the radioactivity into RNA a c i d - i n s o l u b l e fraction:-The incubation and assay for RNA synthesis was carried out according to the method of Marushige and Bonner (1966). The determination of the radioactivity into RNA acid-insoluble fraction was performed as described by Petrelli et al. (in press). Uptake of labeled uridine:- Uptake of labeled [U-14C]uridine was determined after 2-4 min pulses. Samples were immediately diluted with ice-cold phosphate -buffered solution supplemented with unlabeled uridine 10 times more concentrated than the labeled compound. After two washings, cell aliquots (106 cells) were lysed in 0.5% sodium dodecyl sulfate and total radioactivity was determined.
Cell Biology International Reports, Vol. 16, No. 10, 1992
969
RESULTS AND DISCUSSION. The kinetic data in Table 1 show that the presence in the incubation mixture of the peptide extracted by us from pineal glands causes a pronounced reduction of the transcriptional capacity of DNA tested in vitro by DNA dependent-RNA polymerase reaction. The inhibitor effect on RNA synthesis is dose-dependent. These data confirm those reported in the previous paper (Petrelli et al., in press).
Table 1 Effect' of peptide purified from calf pineal on DNA transcription activity in vitro. Peptide concentration in incubation mixture: 125, 250, 500 n g / ml. Each value is the mean of data from five determinations; mean _+ SD. Percent inhibition
Peptide concentration
125 ng / ml 250 ng / ml 500 ng / ml
5th rain
10th rain
40.00 + 3.08 72.20 +_3.56 94.60 + 4.56
60.20 + 4.44 85.20 + 4.55 100.00 + 0.00
The data in Table 2 show that the calf pineal peptide purified by us causes a dose-dependent inhibition of RNA synthesis in L1210 and HL 60 cell lines in vitro. This effect is more evident in HL60 than in L1210 cells. It is noteworthy that treatment with the peptide does not modify the cell viability.
Table 2 Effect of peptide purified from calf pineal on RNA synthesis in L1210 and HI60 cells. RNA synthesis was measured by incorporation of 3H-Uridine. The peptide was added at 0 time. Two hours of labeling from 4 to 6 hrs of culture. Mean values obtained from four experiments. Percentage inhibition of 3H-Uridine incorporation in treated tumoral cells with respect to untreated specimens. Mean _+SD. Peptide ( n g / m l ) Inhibition ( % ): L1210
HL60
Cell viability
(%)
60
125
9.80 + 1.92
15.20 _+ 4.15
1 5.40 _+ 3.05
+
96
+
250
500
40.20 3.70
68.00 + 2.77
35.40 4.83
65.40 _+ 4.50
98
95
+
80.00 2.62 97
970
Cell Biology Intemational Reports, Vol. 16, No. 10, 1992
Uptake of labeled uridine in the tumoral cells HL60 after 2-4 min pulses was comparable in the presence and in the absence of the peptide, the addition of which did not impair uptake of the label. This excludes the possibility that the decreased incorporation of radioactive uridine into RNA of cells in culture is due to an effect on the transport of uridine through the cell membrane (Table 3).
Table 3 Effect of the peptide on uptake of [U _14C] uridine in HL60 cells: cpm/106 ceils. The HL60 (after 30 min of preincubation) were incubated at a concentration of 5 x 1 0 6 cells per ml in the presence of [U -14C] uridine (23 p.Ci /ml, 453mCi/mmol; Radiochemical Center, Amersham), With occasional shaking under air containing 5% carbon dioxide. The peptide purified (O.1 ml, corresponding to 500 ng) was added per ml of incubation mixture; control was given 0.1 ml of saline. We chose the concentration of the peptide that causes an 100% inhibition of the DNA transcription. 14 C cpm at Sample
2 min
HL60 (control)
10,683 ns + 1,497
HL60 + Peptide
10,750 _+ 1,502
4 rain
13,880 ns + 1,367 13,304 _+ 1,308
Values are expressed as mean + SD of the data obtained from 5 samples. Statistically significant differences between control and HL60 cells treated with peptide (Student's -t,, test): ns not significant differences.
In agreement with data in Table 2, also the proliferation of both cell lineages is affected by peptide effector ( Figures 1, 2 ). The presen(~e of peptide in culture medium causes a dose-dependent inhibition of the cell proliferation. The L1210 cells seem to be more sensitive than HL60 ones to peptide effector. The inhibiting effect may be reversed by washing and reseeding the cells in fresh medium not containing the peptide effector (Figures 3,4). Such data demonstrate that pineal peptide does not modify irreversibly the metabolic activity of the two cell lineages we studied. In conclusion the results reported in this work show that the peptide isolated by us from calf pineal glands, having a molecular weight of about 1200 Dalton, besides having inhibiting activity on DNA transcription in vitro (Petrelli et al., in press), has inhibiting activity on RNA synthesis and on proliferation of L1210 and HL60 tumoral cells. The inhibitor effect is dose-dependent and reversible.
Cell Biology lnternational Reports, Vol. 16, No. 10, 1992 Figure 1 Effect of purified active peptide from calf pineal on the growth of L 1210 cells expressed as number of cells / ml ( x 105). Initial inoculum : 1 x 105 cells / ml. The peptide was added at 0 time.The cells were cultured for O, 48, 72 h without and with peptide: 60, t25, 250, 375, 500 ng /ml. Each time point indicates means obtained from four experiments.
971
4O ..o. Co~uol SOng
125ng •e - 2'50 nO 375 n.g ~ 50On, O
/
f I
24
48
72
TIME (h)
Figure 2 Effect of purified active peptide from calf pineal on the growth of HL 60 cells expressed .as number of cells / ml ( x 105). Initial inoculum: 1 x 105 cells / ml. The peptide was added at 0 time. The cells were cultured for O, 48, 72, 96 h without and with peptide: 60, 125, 250, 375, 500 ng / ml. Each time point indicates means obtained from four experiments.
2°1 -a- control ,sonG 125n o •41- 250rig "+" 375 ,~g
~,
10"
of 0
24
48 "flUE (h)
72
06
972
Cell Biology lnternational Reports, Vol. 16. No. 10, 1992 Figure 3 Effect of the removal of purified active peptide from calf pineal on the growth of L 1210 cells expressed as number of cells / ml ( x 105 ) . initial inoculum: 1 x 105 cells-/ ml. The peptide was added_at 0. time. Thecells were cultured for 0, 24, 48, 72, 96 h without (13) and with (m) peptide (500 ng / ml). Untreated and tre-ated cells were washed and reseeded in flesh medium with (m) and without (13, x) peptide at 48 h. Each time point indicates means obtained from three experiments.
w o
OI 24
0
48
96
72
1 1 ~ (h)
Figure 4 Effect of the removal of purified active peptide from calf pineal on the growth of HL60 cells expressed as number of cells / ml ( x 105 ). Initial inoculum: 1 x 105 cells / ml. The peptide was added at 0 time. The cells were cultured for 0, 24, 48, 72, 96 h without (13) and with ( I ) peptide (500 n g / ml). Untreated and treated cells were washed and reseeded in fresh medium with ( I ) and without (13, x) peptide at 48 h. Each time point indicates means obtained from three experiments.
i
TM
0 0
,
,
,
,
24
48
72
96
TreE (h)
Cell Biology lnternational Reports. VoL 16. No. 10. 1992
973
REFERENCES
Aubert, C., Prade, M. and Bohoun, C.
(1970). Effect de la pin~alectomie sur les tumeurs m~laniques de Hamster dor~ induites par I'administration (per os) d'une seule dose de 9,10-dim~thyl -1,2-benzanthrac~ne. C.R. Acad. Sci. (Paris) 271, 2465-2466. Bartsch, H. and Bartsch, C. (1981). Effect of melatonin on experimental tumors under different photoperiods and times of administration. J. Neural Transm. 52, 269-279. Bartsch, H., Bartsch, C., Noteborn, H.P-.J.M., Flehmig, B., Ebels, I. and Salemink, C.A. (1987). Growth-inhibiting effect of crude pineal extracts on human melanoma cells in vitro is different from that of known synthetic pineal substances. J. Neural Transm. 69, 299-311. Bindoni, M. (1971). Relationship between the pineal gland and the mitotic activity of some tissues. Arch. Sci. Biol. 55, 3-21. Bindoni, M., Jutisz, M. and Ribot, G. (1976). Characterization and partial purification of a substance in the pineal gland which inhibits cell multiplication in vivo. Biochim. Biophys. Acta 437, 577-588. Blask, D. E. (1984). The pineal: an oncostatic gland? In: Reiter, R. J. led) The pineal gland. Raven Press, New York, p.p. 253-284. Blask, D. E. and Leadem, C.A. (1987). Neuroendocrine aspects of neoplastic growth: a review. Neuroendocrinol. Lett. 9 (2), 63-73. Carr, D. B. and Axelrod, L. (1981). The pineal gland, pineal dysfunction and cancer. Rev. Endocrine-related Canco 8, 13-21. Das Gupta, T. K. and Terz, J. (1967). Influence of pineal gland on the growth and spread of melanoma in the hamster. Cancer Res. 27, 1306-1311. Dilman, V. M., Anisimov, V. N. , Ostroumova, M. N., Morosov, V. G., Khavinson, V. Kh. and Azarova, M. A. (1979). Study of anti-tomor effect of polypeptide pineal extract. Oncology 36, 274- 280. EI-Domeiri, A. A. H. and Das Gupta, T. K. (1976). The influence of pineal ablation and administration of melatonin on growth and spread of hamster melanoma.. J. Surg. Oncol. 8, 197-205. Lapin, V. (1976). Pineal gland and malignancy. Osterr. Z. Onkol. 3, 51-59. Lapin, V. and Ebels, I. (1976). Effects of some low molecular weight sheep pineal fractions and melatonin on different tumors in rats and mice. Oncology 33, 110-113. Lowry, O. H., Rosenbrough, M. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. Marushige, K. and Bonner, J. (1966). Template properties of liver chromatin. J. Mol. Biol. 15, 160-174. Nakai, N., Lai, C. Y. and Horecker, B. L. (1974). Use of fluorescamine in the chromatographic analysis of peptides from proteins. Anal. Biochem. 58, 563-570 . Noteborn, H. P. J. M., Bartsch, H., Bartsch, C., Marts, D. R. A., Weusten, J. J. A . M . , Flehmig, B., Ebels, I. and Salemink, C. A. (1988). Partial purification of (a) low molecular weight ovine
974
Cell Biology lnternational Reports, Vol. 16, No. 10, 1992 pineal compound(s) with an inhibiting effect on the growth of human melanoma cells in vitro. J. Neural. Transm. 73, 135-155.
Petrelli,
C.,
Moretti,
P.,
Petrelli,
F.
and
Barra,
D.
(1991).
Purification of a low molecular weight calf pineal peptide controlling DNA transcription in vitro. It. J. Biochem. in press. Quay, W. B. (1974). Temporal mitotic patterns around a brain lesion; cellular and regional asynchronisms and an effect of pinealectomy. Chronobiologia 1, 237-258.
Paper received 22.01.92.
Revised paper accepted 18.07.92.