- Email: [email protected]
Growth inhibition and induction of phenotypic alterations by l -histidinol in B16 mouse melanoma cells
193
Cancer Letters, 47 (19:39) 193- 197 Elsevier Scientific Publishers lreland Ltd.
Growth inhibition and induction of phenotypic L-histidinol in B16 mouse melanoma cells. J. Nordenberg”,
L. Wassermana,
"Rogoff ResearchInstitute, Qeportment ofMedicine, Tel-Aviv Uniuersity (Israel)
H. Gutmanb,‘,
ofSurgery B,
alterations
by
E.Beery” and A. Novogrodsky”,’
Beilinson Medical Center, Petah-Tikua
49100 and cSackler School
(Received 19 January 1989) (Revision received 25 May 1989) (Accepted 26 May 1989)
Summary L-Histidinol, a histidine analog was recently shown to be an inducer of differentiation in the promyelocytic cell line HL-60. In the present study growth inhibition
we
show
that
L-histidinol
inhibits
the
of B16 melanoma
alterations
is that
cells in vitro. Growth accompanied by phenotypic include
a marked
increase
in
of NADPH cytochrome c reductase and y glutamyl transpeptidase, lipid accumuiation and cell enfargement. These phenotypic alterations are similar to those induced by othe;r chemical inducers of differentiation in melanoma cells. the activities
Keywords: B16 melanoma; L-histidinol; growth-inhibition; phenotypic alterations; differentiation. Introduction L-Histidinolis ,anormal precursor of histindine and is also known as an aminoacyl tRNA synthetase inhibitor. This compound has been shown to induce cell differentiation in the promyelocytic cell line HL-60 [l]. Its effect on these cells was attributed to the depletion of a
Correspondence to: J. Nordenburg.
single amino acid, rather than overall inhibition of protein synthesis [ 1,2]. Recent studies have revealed that L-histidinol improves in vivo treatment efficacy of some conventional antitumor agents in murine tumor models [3--51. In the present study it is shown that L-histidinol markedly inhibits the growth of B16 melanoma cells in vitro and induces phenotypic alterations that might partly be associated with a more differentiated phenotype. Materials and methods L-histidinol was obtained from Sigma Chemical Co. Media and tissue culture reagents were from Biol. Industries, Israel. B16, FlO melanoma cells were cultured in RPM1 1640, supplemented with 10% fetal calf serum as previously described [6]. For growth experiments 3.5 x lo4 cells were plated in 1 ml growth medium in tissue culture plates (3.3 cm). Two hours later, after most cells had attached, L-histidinol was added. The cells were incubated in the presence and absence of L-histidinol for different time intervals. Medium was replaced after 48 h when the incubation lasted more than 72 h. Following incubation cells were detached with EDTA (1 mM), dissolved in phosphate buffered saline, without Ca2+ and Mg2+ and counted in a Coulter counter.
0304.3835/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland Ltd Published and Printed in Ireland
194
The activities of NADPH cytochrome c reductase and y glutamyl transpeptidase were determined spectrophotometrically as previously described [7]. NADPH cytochrome c reductase activity was expressed as nmol acceptor reduced/h per mg DNA. y Glutamyl transpeptidase activity was expressed as pmol product formed/h per mg DNA. DNA was fluorometric method measured by the described by Labarca and Paigen [8]. For the demonstration of lipid droplets, cells were stained by the oil red 0 method [9] following fixation with formol-calcium as previously described [7]. Statistical significance was evaluated by the paired Student’s t-test. Results and discussion B16, F10 melanoma cells were incubated in the absence or presence of various concentrations of i_-histidinol for different timeperiods. The number of untreated cells was found to increase 35-fold during 92 h of incubation (Fig. 1). L-Histidinol is shown to inhibit cell growth in a concentration dependent manner, leading to complete growth arrest at a concentration of 2 mM (Fig. 1). Cells did not detach, following L-histidinol treatment and remained 95% viable as assessed by the Trypan blue exclusion test. These data suggest that the effect of L-histidino1 on B16 melanoma cells is cytostatic. In order to examine whether L-histidinol-treated cells (1 and 2 mM) resume growth following removal of i_-histidinol the following experiment was performed. Untreated and 72 h i_-histidinol (1 and 2 mM) pretreated cells were plated at 4 x lo4 cells/ml in 1 ml culture medium in the absence of i_-histidinol. After 72 h incubation the cells were counted. The number of untreated cells increased approximately lo-fold (4.3 X lo5 f 0.4 X 105). The number of 1 mM t_-histidinol-pretreated cells increased approximately 7.5-fold (3.00 x lo5 2 0.7 x 105) and 2 mM L-histidinolpretreated cells increased only approximately 3-fold (1.15 x lo5 f 0.18 x 105). How-
a20.5to20 42 h
Fig. 1. The anti-proliferative effect of L.-histidinol on B16 melanoma cells. 3.5 x lo4 Cells/ml (that correspond to 4 X lo3 cells/cm2 were incubated in culture medium in the presence and absence of various concentrations of t_-histidinol for 42, 68 and 92 h. Cells were detached and counted as described in Materials and methods. Values are mean /pm/ S.D. of triplicate determinations.
ever, incubation of the 2 mM-pretreated cells for an additional 92 h resulted in an 18-fold increase in their number (7.16 x lo5 f 0.20 X 105). These results show that the removal of L-histidinol from the growth medium leads to reversibility of the anti-proliferative effect. Our previous studies have revealed that known chemical inducers of differentiation, such as sodium butyrate, dimethylsulfoxide, dimethylthiourea and tetramethylurea inhibit the growth of B16 melanoma cells and induce alterations that include phenotypic morphological changes, altered melanin conNADPH tent, increased activities of cytochrome c reductase and y glutamyl transpeptidase and accumulation of lipid droplets
195
Table 1. The effect of histidinol cytochrome c reductase in B16 melanoma Treatment
None Histidinol(1 Histidinol(2
on NADPH cells.
NADPH cytochrome reductase activity (nmol/mg DNA)
mM) mM)
c
9.76 + 0.80 17.31 +. 1.60 24.76 + 3.42
Cells (7 X lO“/mi) were incubated in 10 ml culture medium (g-cm plates) in the absence or presence of t_-histidinol for 72 h. Cell lysates were prepared by repeated freezing and thawing of 6-8 x lo6 cells in 0.5 ml buffer and 25--50 ~1 were used for determination of enzyme activity according to the method described previously [7]. Values are mean + S.E. for 5-6 independent experiments. Histidinol (1 or 2 mM) vs. none, P< 0.01.
[6,7, lo- 131. !Several of these phenotypic alterations seem to reflect differentiated features of melanoma cells. Based on our previous studies we have followed the effect of L-histidinol on the phenotypic expression of the B16 F10 melanoma cells.
The effect of histidinol Table 2. peptidase in B16 melanoma.
on r-glutamyl
trans-
Treatment
y Glutamyl transpeptidase activity (nmol/mg DNA)
None Histidinol (1 mM)
2.36 f 0.23 14.86 + 4.40
Cells (7 x 104) were incubated in 10 ml culture medium (g-cm plates) in the presence and absence of r-histidinol for 72 h. Cells (106), dispersed in 0.1 ml Tris-HCI buffer (0.1 N, pH = 8) were used for enzymatic determination as previously described [7]. Values are mean + S.E. for 5 independent experiments. Histidinol vs. none, P < 0.05.
The results depicted in Table 1 show the effect of L-histidinol on the activity of the enzyme NADPH cytochrome c reductase, an associated with the endoplasmic enzyme reticulum. L-histidinol induces a significant increase in the activity of this enzyme. Increased NADPH cytochrome c reductase activity might reflect a differentiated feature, since normal melanocytic differentiation was described to be associated with development of the endoplasmic reticulum [14]. It has recently been reported that the activity of NADPH cytochrome c reductase is reduced in hepatocellular carcinoma as compared with normal hepatic tissue [ 151. Melanin was not significantly changed by i_-histidinol. It should be noted that its content was differently affected by various chemical inducers of differentiation [6,12]. The activity of the plasma membrane-bound enzyme y glutamyl transpeptidase was also found to be markedly enhanced (Table 2). The relevance of increased activity of glutamy1 transpeptidase to a more differentiated phenotype remains unclear. Induction of its activity has been described in association with carcinogenesis [ 161 as with cell differentiation
1171. Light microscopy reveals that I_-histidinol induces accumulation of lipid droplets. The treated cells also seem to be enlarged (Fig. 2). Lipid accumulation has been suggested as a differentiated characteristic in fibroblasts and breast cancer cells [ 18,191. It should be noted that i_-histidinol was to potentiate the effects of reported chemotherapeutic agents in the B16 melanoma tumor mode1 in vivo [5]. The direct effect of i_-histidinol on B16 F10 cell growth and phenotypic expression might contribute to potentiation of the effects of the chemotherapeutic drugs. L-Histidinol belongs to the chemical group of imidazole compounds. Other imidazole have compounds (imidazotetrazinones) recently been described as broad spectrum antitumor agents in murine tumors and human tumor xenografts [20].
196
Fig. 2. Lipid accumulation induced by L-histidinol in B16 melanoma (a) Untreated, (b) 1 mM histidinol-treated cells. (x ZOO)
cells. Light micrographs
of oil red O-stained
cells
197
References 1
2
3
4
5
6
7
8
9 10
Pilz, R.B., Van den Berghe, G. and Boss, G.R. (1987) Induction of HL-60 differentiation by starvation for a single essential amino acid but not by protein synthesis inhibitors. J. Clin. Invest, 790, 1006-1009. Warrington, R.C., Wratten, N. and Hechtman R. (1977) i_-histidinol inhibits specifically and reversibly protein and ribosomal RNA synthesis in mouse L-cells. J. Biol. Chem., 252,5241-5257. Warrington, R.C., Muzyka, T.G. and Fang W.D. (1984) Histidinol-mediated improvement in sensitivity of 1$-oarabinofuranosylcytosine and 5fluorouracil in L 1210 leukemia-bearing mice. Cancer Res., 44, 2929-2935. Warrington, R.C. (1988) i_-histidinol improves the selectivity and the efficacy of several conventional anti-cancer drugs in murine tumor models. Fed. Am. Sot. Exp. Biol. J., 2,332. Ridgway, R.J. and Stewart, D.P. (1988) L-histidinol improves therape’utic efficacy of some conventional antitumor agents against the advanced B16 melanoma in mice. Fed. Am. Sot. Exp. Biol. J., 2,480. Nordenberg, J., Wasserman, L., Beery, E., Aloni, D., Malik, H., Stenzel K.H. and Novogrodsky, A. (1986) Growth inhibition of murine melanoma by butyric acid and dimethylsulfoxide. Exp. Cell Res., 162, 77-85. Nordenberg, J., Wasserman, L., Peled A., Malik, Z., Stenzel, K.H. and Novogrodsky, A. (1987) Biochemical and ultrastructural alterations accompany the anti-proliferative effect of butyrate on melanoma cells. Br. J. Cancer, 55,493-497. Labarca, C. and Paigen, K. (1980) A simple, rapid and sensitive DNA assay procedure. Anal. Biochem., 102, 344-352. Pearse, A.G.E. (1968) Histochemistry Theoretical and Applied, 3rd edn., p. 697. Churchill, London. Nordenberg, J., Panet, C., Wasserman, L., Malik, Z., Fuchs, A., Stenzel, K.H. andNovogrodsky, A. (1987) The anti-proliferative effect of lithium chloride on melanoma cells and its reversion by myoinositol. Br. J. Cancer, 55. 41-46.
11
12
13
14
15
16
17
18
19
20
Nordenberg, J., Aloni, D., Wasserman, L., Beery, E., Stenzel. K.H. and Novogrodsky, A. (1985) Dimethylthiourea inhibition of melanoma cell growth in vitro and in vivo. J. Natl. Cancer Inst., 75, 891-895. Malik, H., Nordenberg, J., Novogrodsky, A., Fuchs, A. and Malik, Z. (1987) Chemical inducers of differentiation dimethylsulfoxide, butyric acid and dimethylthiourea induce selective ultrastructural patterns in B16 melanoma cells. Biol. Cell 60, 32-40. Nordenberg, J., Fuchs, A., Wasserman, L., Malik. Z. and Novogrodsky, A. (1987) Anti-proliferative effects and phenotypic alterations induced by methyl urea derivatives in B16 mouse melanoma. In: Modern Approaches to Animal Cell Technology. pp. 125-137. Editors: R.E. Spier and J.B. Griffiths. Butterworth Publishers, U.K. Jimbo, K. and Vesugi, T. (1982) New melanogenesis and photobiological processes in activation and proliferation of precursor melanocytes after UV-exposure: Ultrastructural differentiation of precursor melanocytes from Langerhans cells. J. Invest. Dermatol., 78, 108-115. Hamamoto, I., Tanaka, S., Maeba, T., Chikaishi, K. and Ichikawa, Y. (1989) Microsomal cytochrome p-450.linked monooxygenase systems and lipid composition of human hepatocellular carcinoma. Br. J. Cancer., 59, 6- 11. Fiala, S., Fiala, A.E. and Dixon. B. (1972) r-glutamyl transpeptidase chemically induced rat hepatoma and spontaneous mouse hepatomas. J. Natl. Cancer Inst., 48. 1393- 1401. Novogrodsky, A., Tam, S.S.R. and Meister, A. (1976) a lymphoid cell-surface y-Glutamyl transpeptidase, marker. Relationship to blastogenesis, differentiation and neoplasia. Proc. Nat]. Acad. Sci. USA, 73, 2414-2418. Wawra, E. (1986) Long term effect of sodium butyrate on mouse fibroblasts. A model for differentiation. Cell. Mol. Biol., 32, 121-129. Costlow, M.E. (1984) Differentiation-inducing agents decrease cryptic prolactin receptors in culture mammarytumor cells. Exp. Cell. Res., 156, 17-23. Tisdale, M.J. (1987) Antitumor imidazotetrazines - xv. Role of guanine 06.alkylation in the mechanism of cytotoxicity of imidazotetrazinones. Biochem. Pharmacol., 36, 457-462.
Cancer Letters, 47 (19:39) 193- 197 Elsevier Scientific Publishers lreland Ltd.
Growth inhibition and induction of phenotypic L-histidinol in B16 mouse melanoma cells. J. Nordenberg”,
L. Wassermana,
"Rogoff ResearchInstitute, Qeportment ofMedicine, Tel-Aviv Uniuersity (Israel)
H. Gutmanb,‘,
ofSurgery B,
alterations
by
E.Beery” and A. Novogrodsky”,’
Beilinson Medical Center, Petah-Tikua
49100 and cSackler School
(Received 19 January 1989) (Revision received 25 May 1989) (Accepted 26 May 1989)
Summary L-Histidinol, a histidine analog was recently shown to be an inducer of differentiation in the promyelocytic cell line HL-60. In the present study growth inhibition
we
show
that
L-histidinol
inhibits
the
of B16 melanoma
alterations
is that
cells in vitro. Growth accompanied by phenotypic include
a marked
increase
in
of NADPH cytochrome c reductase and y glutamyl transpeptidase, lipid accumuiation and cell enfargement. These phenotypic alterations are similar to those induced by othe;r chemical inducers of differentiation in melanoma cells. the activities
Keywords: B16 melanoma; L-histidinol; growth-inhibition; phenotypic alterations; differentiation. Introduction L-Histidinolis ,anormal precursor of histindine and is also known as an aminoacyl tRNA synthetase inhibitor. This compound has been shown to induce cell differentiation in the promyelocytic cell line HL-60 [l]. Its effect on these cells was attributed to the depletion of a
Correspondence to: J. Nordenburg.
single amino acid, rather than overall inhibition of protein synthesis [ 1,2]. Recent studies have revealed that L-histidinol improves in vivo treatment efficacy of some conventional antitumor agents in murine tumor models [3--51. In the present study it is shown that L-histidinol markedly inhibits the growth of B16 melanoma cells in vitro and induces phenotypic alterations that might partly be associated with a more differentiated phenotype. Materials and methods L-histidinol was obtained from Sigma Chemical Co. Media and tissue culture reagents were from Biol. Industries, Israel. B16, FlO melanoma cells were cultured in RPM1 1640, supplemented with 10% fetal calf serum as previously described [6]. For growth experiments 3.5 x lo4 cells were plated in 1 ml growth medium in tissue culture plates (3.3 cm). Two hours later, after most cells had attached, L-histidinol was added. The cells were incubated in the presence and absence of L-histidinol for different time intervals. Medium was replaced after 48 h when the incubation lasted more than 72 h. Following incubation cells were detached with EDTA (1 mM), dissolved in phosphate buffered saline, without Ca2+ and Mg2+ and counted in a Coulter counter.
0304.3835/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland Ltd Published and Printed in Ireland
194
The activities of NADPH cytochrome c reductase and y glutamyl transpeptidase were determined spectrophotometrically as previously described [7]. NADPH cytochrome c reductase activity was expressed as nmol acceptor reduced/h per mg DNA. y Glutamyl transpeptidase activity was expressed as pmol product formed/h per mg DNA. DNA was fluorometric method measured by the described by Labarca and Paigen [8]. For the demonstration of lipid droplets, cells were stained by the oil red 0 method [9] following fixation with formol-calcium as previously described [7]. Statistical significance was evaluated by the paired Student’s t-test. Results and discussion B16, F10 melanoma cells were incubated in the absence or presence of various concentrations of i_-histidinol for different timeperiods. The number of untreated cells was found to increase 35-fold during 92 h of incubation (Fig. 1). L-Histidinol is shown to inhibit cell growth in a concentration dependent manner, leading to complete growth arrest at a concentration of 2 mM (Fig. 1). Cells did not detach, following L-histidinol treatment and remained 95% viable as assessed by the Trypan blue exclusion test. These data suggest that the effect of L-histidino1 on B16 melanoma cells is cytostatic. In order to examine whether L-histidinol-treated cells (1 and 2 mM) resume growth following removal of i_-histidinol the following experiment was performed. Untreated and 72 h i_-histidinol (1 and 2 mM) pretreated cells were plated at 4 x lo4 cells/ml in 1 ml culture medium in the absence of i_-histidinol. After 72 h incubation the cells were counted. The number of untreated cells increased approximately lo-fold (4.3 X lo5 f 0.4 X 105). The number of 1 mM t_-histidinol-pretreated cells increased approximately 7.5-fold (3.00 x lo5 2 0.7 x 105) and 2 mM L-histidinolpretreated cells increased only approximately 3-fold (1.15 x lo5 f 0.18 x 105). How-
a20.5to20 42 h
Fig. 1. The anti-proliferative effect of L.-histidinol on B16 melanoma cells. 3.5 x lo4 Cells/ml (that correspond to 4 X lo3 cells/cm2 were incubated in culture medium in the presence and absence of various concentrations of t_-histidinol for 42, 68 and 92 h. Cells were detached and counted as described in Materials and methods. Values are mean /pm/ S.D. of triplicate determinations.
ever, incubation of the 2 mM-pretreated cells for an additional 92 h resulted in an 18-fold increase in their number (7.16 x lo5 f 0.20 X 105). These results show that the removal of L-histidinol from the growth medium leads to reversibility of the anti-proliferative effect. Our previous studies have revealed that known chemical inducers of differentiation, such as sodium butyrate, dimethylsulfoxide, dimethylthiourea and tetramethylurea inhibit the growth of B16 melanoma cells and induce alterations that include phenotypic morphological changes, altered melanin conNADPH tent, increased activities of cytochrome c reductase and y glutamyl transpeptidase and accumulation of lipid droplets
195
Table 1. The effect of histidinol cytochrome c reductase in B16 melanoma Treatment
None Histidinol(1 Histidinol(2
on NADPH cells.
NADPH cytochrome reductase activity (nmol/mg DNA)
mM) mM)
c
9.76 + 0.80 17.31 +. 1.60 24.76 + 3.42
Cells (7 X lO“/mi) were incubated in 10 ml culture medium (g-cm plates) in the absence or presence of t_-histidinol for 72 h. Cell lysates were prepared by repeated freezing and thawing of 6-8 x lo6 cells in 0.5 ml buffer and 25--50 ~1 were used for determination of enzyme activity according to the method described previously [7]. Values are mean + S.E. for 5-6 independent experiments. Histidinol (1 or 2 mM) vs. none, P< 0.01.
[6,7, lo- 131. !Several of these phenotypic alterations seem to reflect differentiated features of melanoma cells. Based on our previous studies we have followed the effect of L-histidinol on the phenotypic expression of the B16 F10 melanoma cells.
The effect of histidinol Table 2. peptidase in B16 melanoma.
on r-glutamyl
trans-
Treatment
y Glutamyl transpeptidase activity (nmol/mg DNA)
None Histidinol (1 mM)
2.36 f 0.23 14.86 + 4.40
Cells (7 x 104) were incubated in 10 ml culture medium (g-cm plates) in the presence and absence of r-histidinol for 72 h. Cells (106), dispersed in 0.1 ml Tris-HCI buffer (0.1 N, pH = 8) were used for enzymatic determination as previously described [7]. Values are mean + S.E. for 5 independent experiments. Histidinol vs. none, P < 0.05.
The results depicted in Table 1 show the effect of L-histidinol on the activity of the enzyme NADPH cytochrome c reductase, an associated with the endoplasmic enzyme reticulum. L-histidinol induces a significant increase in the activity of this enzyme. Increased NADPH cytochrome c reductase activity might reflect a differentiated feature, since normal melanocytic differentiation was described to be associated with development of the endoplasmic reticulum [14]. It has recently been reported that the activity of NADPH cytochrome c reductase is reduced in hepatocellular carcinoma as compared with normal hepatic tissue [ 151. Melanin was not significantly changed by i_-histidinol. It should be noted that its content was differently affected by various chemical inducers of differentiation [6,12]. The activity of the plasma membrane-bound enzyme y glutamyl transpeptidase was also found to be markedly enhanced (Table 2). The relevance of increased activity of glutamy1 transpeptidase to a more differentiated phenotype remains unclear. Induction of its activity has been described in association with carcinogenesis [ 161 as with cell differentiation
1171. Light microscopy reveals that I_-histidinol induces accumulation of lipid droplets. The treated cells also seem to be enlarged (Fig. 2). Lipid accumulation has been suggested as a differentiated characteristic in fibroblasts and breast cancer cells [ 18,191. It should be noted that i_-histidinol was to potentiate the effects of reported chemotherapeutic agents in the B16 melanoma tumor mode1 in vivo [5]. The direct effect of i_-histidinol on B16 F10 cell growth and phenotypic expression might contribute to potentiation of the effects of the chemotherapeutic drugs. L-Histidinol belongs to the chemical group of imidazole compounds. Other imidazole have compounds (imidazotetrazinones) recently been described as broad spectrum antitumor agents in murine tumors and human tumor xenografts [20].
196
Fig. 2. Lipid accumulation induced by L-histidinol in B16 melanoma (a) Untreated, (b) 1 mM histidinol-treated cells. (x ZOO)
cells. Light micrographs
of oil red O-stained
cells
197
References 1
2
3
4
5
6
7
8
9 10
Pilz, R.B., Van den Berghe, G. and Boss, G.R. (1987) Induction of HL-60 differentiation by starvation for a single essential amino acid but not by protein synthesis inhibitors. J. Clin. Invest, 790, 1006-1009. Warrington, R.C., Wratten, N. and Hechtman R. (1977) i_-histidinol inhibits specifically and reversibly protein and ribosomal RNA synthesis in mouse L-cells. J. Biol. Chem., 252,5241-5257. Warrington, R.C., Muzyka, T.G. and Fang W.D. (1984) Histidinol-mediated improvement in sensitivity of 1$-oarabinofuranosylcytosine and 5fluorouracil in L 1210 leukemia-bearing mice. Cancer Res., 44, 2929-2935. Warrington, R.C. (1988) i_-histidinol improves the selectivity and the efficacy of several conventional anti-cancer drugs in murine tumor models. Fed. Am. Sot. Exp. Biol. J., 2,332. Ridgway, R.J. and Stewart, D.P. (1988) L-histidinol improves therape’utic efficacy of some conventional antitumor agents against the advanced B16 melanoma in mice. Fed. Am. Sot. Exp. Biol. J., 2,480. Nordenberg, J., Wasserman, L., Beery, E., Aloni, D., Malik, H., Stenzel K.H. and Novogrodsky, A. (1986) Growth inhibition of murine melanoma by butyric acid and dimethylsulfoxide. Exp. Cell Res., 162, 77-85. Nordenberg, J., Wasserman, L., Peled A., Malik, Z., Stenzel, K.H. and Novogrodsky, A. (1987) Biochemical and ultrastructural alterations accompany the anti-proliferative effect of butyrate on melanoma cells. Br. J. Cancer, 55,493-497. Labarca, C. and Paigen, K. (1980) A simple, rapid and sensitive DNA assay procedure. Anal. Biochem., 102, 344-352. Pearse, A.G.E. (1968) Histochemistry Theoretical and Applied, 3rd edn., p. 697. Churchill, London. Nordenberg, J., Panet, C., Wasserman, L., Malik, Z., Fuchs, A., Stenzel, K.H. andNovogrodsky, A. (1987) The anti-proliferative effect of lithium chloride on melanoma cells and its reversion by myoinositol. Br. J. Cancer, 55. 41-46.
11
12
13
14
15
16
17
18
19
20
Nordenberg, J., Aloni, D., Wasserman, L., Beery, E., Stenzel. K.H. and Novogrodsky, A. (1985) Dimethylthiourea inhibition of melanoma cell growth in vitro and in vivo. J. Natl. Cancer Inst., 75, 891-895. Malik, H., Nordenberg, J., Novogrodsky, A., Fuchs, A. and Malik, Z. (1987) Chemical inducers of differentiation dimethylsulfoxide, butyric acid and dimethylthiourea induce selective ultrastructural patterns in B16 melanoma cells. Biol. Cell 60, 32-40. Nordenberg, J., Fuchs, A., Wasserman, L., Malik. Z. and Novogrodsky, A. (1987) Anti-proliferative effects and phenotypic alterations induced by methyl urea derivatives in B16 mouse melanoma. In: Modern Approaches to Animal Cell Technology. pp. 125-137. Editors: R.E. Spier and J.B. Griffiths. Butterworth Publishers, U.K. Jimbo, K. and Vesugi, T. (1982) New melanogenesis and photobiological processes in activation and proliferation of precursor melanocytes after UV-exposure: Ultrastructural differentiation of precursor melanocytes from Langerhans cells. J. Invest. Dermatol., 78, 108-115. Hamamoto, I., Tanaka, S., Maeba, T., Chikaishi, K. and Ichikawa, Y. (1989) Microsomal cytochrome p-450.linked monooxygenase systems and lipid composition of human hepatocellular carcinoma. Br. J. Cancer., 59, 6- 11. Fiala, S., Fiala, A.E. and Dixon. B. (1972) r-glutamyl transpeptidase chemically induced rat hepatoma and spontaneous mouse hepatomas. J. Natl. Cancer Inst., 48. 1393- 1401. Novogrodsky, A., Tam, S.S.R. and Meister, A. (1976) a lymphoid cell-surface y-Glutamyl transpeptidase, marker. Relationship to blastogenesis, differentiation and neoplasia. Proc. Nat]. Acad. Sci. USA, 73, 2414-2418. Wawra, E. (1986) Long term effect of sodium butyrate on mouse fibroblasts. A model for differentiation. Cell. Mol. Biol., 32, 121-129. Costlow, M.E. (1984) Differentiation-inducing agents decrease cryptic prolactin receptors in culture mammarytumor cells. Exp. Cell. Res., 156, 17-23. Tisdale, M.J. (1987) Antitumor imidazotetrazines - xv. Role of guanine 06.alkylation in the mechanism of cytotoxicity of imidazotetrazinones. Biochem. Pharmacol., 36, 457-462.