Increase in Melanin Formation and Promotion of Cytotoxicity in Cultured Melanoma Cells Caused by Phosphorylated Isomers of L-Dopa1

[CANCER RESEARCH 46, 493-497, February 1986]

John M. Pawelek and Marilyn Murray

Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT cells can be induced to undergo autodestruction when exposed A new class of compounds, termed "dopa phosphates," is to tyrosine, dopa, dopachrome, or DHI (5, 8,9). The melanocyte and its malignant counterpart, the malanoma cell, thus function described. The compounds contain phosphate ester linkages at under precarious conditions in that their ability to synthesize positions 3 and/or 4 of the phenylalanine ring. Dopa phosphates melanin can also lead to cellular death. are highly soluble compounds which are stable over a wide range One problem in many of the studies on the regulation of of pH values and are not hydrolyzed by boiling in concentrated pigmentation is that the key metabolite, namely L-dopa, is rela acid. Synthetic yields of greater than 90% can be obtained using tively insoluble (maximum solubility is approximately 5 mu in dopa as starting material. Exposure to alkaline phosphatase aqueous solution at neutral pH) and is converted readily to results in hydrolysis of the phosphate moieties and production melanin in the presence of O2 above pH 7. Thus many studies of dopa. Dopa phosphates do not inhibit dopa oxidase (tyrosin- with cultured pigment cells or with melanomas in animals are ase, EC 1.14.18.1) activity. Dopa oxidase does not catalyze the difficult to carry out or to interpret when L-dopa is a component conversion of dopa phosphates into melanin unless the dopa of the experiment. With this problem in mind, procedures were phosphates are first treated with alkaline phosphatase. Dopa designed for attaching phosphate groups to the oxygens on phosphates, when compared to u-dopa, are stable in the pres positions 3 and 4 of the phenylalanine ring (10). The rationale ence of 02 and are not oxidized by serum proteins. In the was that such compounds would be stable to oxidation and far presence of cultured melanoma cells, dopa phosphates are more soluble than dopa itself. It was reasoned that if melanocytes readily converted into melanin, indicating that the cells are able were able to take up these compounds, the cellular phospha- to produce dopa from dopa phosphates. At high concentrations, tases might catalytically hydrolyze the phosphate groups and in dopa phosphates are cytotoxic toward melanoma cells in culture. the process produce free L-dopa. In this report, we present The cytotoxicity is enhanced at least 3-fold by pretreatment of evidence that phosphorylated isomers of L-dopa (referred to cells with melanotropin and is prevented by phenylthiourea, an collectively as "dopa phosphates") are indeed taken up by mel inhibitor of dopa oxidase activity. These results, combined with anoma cells in culture, that the phosphate moieties are removed studies on the uptake of radioactive forms of dopa phosphates by cellular phosphatases, and that L-dopa is a product of the t32? and 14C),indicate that phosphorylated isomers of dopa are reaction. efficiently taken up by Cloudman melanoma cells and are readily converted by the cells into a melanin precursor, presumably L- dopa. MATERIALS AND METHODS

INTRODUCTION Synthesis and Isolation. Synthesis of dopa phosphates was carried out by modifications of the procedure of Plimmer (11 ) for hydroxyamino acids. L-Dopa (3 g) was dissolved in phosphoric acid (8 ml, 85% w/v) to Pigment biosynthesis in mammals involves the stepwise con which phosphoric acid anhydride (8.5 g) was then added. The mixture version of tyrosine or dopa into a number of compounds which was heated (100Â°C)with occasional swirling for 24-96 h. After heating, polymerize to form the end product, melanin (1) (Fig. 1). The process takes place in subcellular organdÃes known as meiano- the clear brown solution was poured over ice (130 ml) and swirled until the ice was completely melted and the resultant mixture was homoge somes. It is under hormonal control and involves several regu nous. The solution was then applied to a Dowex 50 column (2.5 x 60 latory proteins which are specifically expressed in melanocytes. cm) equilibrated with H2O. Fractions (35 x 20 ml) were collected and The enzyme dopa oxidase (tyrosinase, EC1.14.18.1) catalyzes monitored in a spectrophotometer at a wave length of 280 nm. Phos at least three steps: the conversion of tyrosine to dopa; dopa to phoric acid eluted first from the column, followed by the dopa phos dopa quinone; and DHI2 into rnelanochrome (3). The conversion phates. Elution of the dopa phosphates was preceded by a rise in pH in of dopachrome to DHI-2-carboxylic acid is catalyzed by a factor the fractions. Fractions 20-30 contained the majority of material absorb which is distinct from dopa oxidase (4, 5). This factor is thought ing at 280 nm and these fractions were pooled and applied to a Oowex 1 column (2.5 x 30 cm) equilibrated with H2O. A gradient of HCI (0-0.15 to be an enzyme (6). In amelanotic melanocytes, a factor is N, total volume of 800 ml) was applied to this column, and 25-ml fractions present which blocks pigment production at the level of either DHI-2-carboxylic acid or DHI (5). It is possible that this "blocking were collected. Fractions were monitored spectrophotometrically at 280 factor" is identical to the activity which converts dopachrome to nm, and fractions 16-26, which contained the majority of absorbing material, were pooled, frozen, and lyophilized to dryness. The resulting DHI-2-carboxylic acid (6, 7). The intermediary metabolites in white material (2.8 g) was dissolved by step-wise addition of Tris base melanin synthesis are highly toxic. In culture, pigment-producing (2 M) and adjusted to pH 7.O. High Performance Liquid Chromatography and Nuclear Magnetic Received 4/3/85; revised 9/4/85; accepted 10/28/85. 1Supported by grants from the Skin Cancer Foundation and Plough, Inc. Resonance Analyses. The material synthesized and isolated by the 2The abbreviations used are: DHI, 5,6-dihydroxyindote; HPLC, high performance above procedures was subjected to reverse phase HPLC. Analyses were liquid chromatography; PTU, phenylthiourea; MSH, melanotropin. performed with an Ultrasil ODS column (Altex Instruments, Inc.; 4.6 x

CANCER RESEARCH VOL. 46 FEBRUARY 1986 493

COO"

Leuco Dopachrome Dopachrome Â«cvc/ v UOfXDopachrome Conversion Factor COj

:Â»? Fig. 2. Schematic structures of the two principal products of the reaction described in 'Materials and Methods* for the synthesis of phosphodopas. The 5,6-Dihydroxindole MELANIN structures were deduced from nuclear magnetic resonance measurements of the isomers separated by HPLC (see "Materials and Methods"). A 3-phospho,-4- Tyrosinase hydroxyphenylalanine. B, 3-hydroxyl,-4-phosphophenylalanine.

3.0

3, 4-Dihydroxyphenylalanine Indole-5, 6 -Qui none 23

Melonochrome 2.0 â€¢ AbsorbÂ«Â«* (540nm) 1.5 â€¢ Tyrosine Fig. 1. Stages in the oxidation of tyrosine to melanin, modified from the scheme of Hariey-Mason and Paper (2). Melanin is a mixed heteropolymer and can be tO formed by interactions between many of the intermediates in the pathway, as denoted by the different arrows indicating melanin synthesis. The arrow indicating evolution of C02 denotes the conversion of 5,6-dihydroxyindole-2-carboxylic acid 05 (not shown) to 5,6-dihydroxyindole. The chemical mechanism of conversion of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid to 5,6-dihydroxyindole is as yet an open question3 and may differ somewhat from the denotation in this figure. 0.0 1x10-4 5x10-4 1x10-3 250 mm). The column was packed with octadodecylsilane particles. The Motes/liter Fig. 3. Oxidation of u-dopa and dopa phosphates in Ham's F-10 culture medium mobile phase was methanol (10%) and H3PO4 (0.1%) in water (v/v). The containing 10% horse serum. The compounds were incubated separately in the flow rate was maintained at 1.0 ml/min and the products of the dopa presence of medium at the concentrations indicated. After 24 h at 37Â°Cin a gassed, phosphate synthesis were detected spectrophotometrically at a wave humidified incubator, the extent of oxidation was assessed by measuring spectrc- length of 278 nm. Two peaks of approximately equal size eluted at 5 photometrically the amount of melanin formed. Unincubated, plain culture medium and 5.5 min, respectively. The material in each of these peaks was was used as a blank. dissolved in 100% D2O, and the proton nuclear magnetic resonance phenylalanine, isomerie at positions 3 and 4 of the ring (Fig. 2). spectra at 400 MHz were examined. Tyrosine and phosphotyrosine were Data in this report were obtained with the mixed isomers isolated used as standards. By analyses of coupling constants and chemical from the synthesis as described in "Materials and Methods," I.e., shifts of the aromatic protons, it was determined that the first HPLC peak was 3-phospho-4-hydroxyphenylalanine and that the second peak the isomers were not separated from one another. Results similar was 3-hydroxyl-4-phosphophenylalanine.4 In the studies presented here, to those presented here were also obtained with each of the the two isomers were not separated by HPLC and were therefore referred individual isomers separated by HPLC (see "Materials and Meth to as "mixed isomers" of dopa phosphate. ods").5 Thin Layer Chromatography. Dopa and dopa phosphates were sep Solubility. Dopa phosphates were soluble in aqueous solu arated by thin layer chomatography. Samples were applied to cellulose- tions at neutral pH to concentrations of at least 0.5 M at room coated plastic strips and run in a solvent system consisting of n-butyl temperature. This was based on an empirical determination that alcohol, isopropyl alcohol, formic acid, and H2O (3:1:1:1, v/v). The com a 1 HIM solution had an absorbance of 2.4 at a wave length of pounds were detected by staining with ninhydrin (0.5%, w/v, in acetone) 280 nm in a 1-cm light path. The limits of solubility were not or by UV absorption. The R( values for the dopa phosphates and dopa were 0.25 and 0.42, respectively. This system did not resolve the tested. isomerie forms of the dopa phosphates. Exposure to alkaline phospha- Stability in Serum. Dopa is readily converted to melanin in the tase (0.2 units/ml, bovine intestine, type VII-S; Sigma Chemical Co.) for presence of culture medium. This is due to both spontaneous 1 min prior to spotting on the thin layer chromatogram caused complete oxidation of dopa, which occurs above pH 7 in the presence of conversion of the dopa phosphates to dopa (R( = 0.25) and presumably O2, and by catalytic oxidation, which occurs in the presence of P, (not detected). serum proteins. A comparison was made between the relative Cell Culture. A Cloudman S91 melanoma cell line PS-lwt (12) was stabilities of dopa and dopa phosphates in Ham's F-10 culture used for all of the studies and was grown either in monolayer culture in Ham's F-10 culture medium containing 10% horse serum (13) or as a medium containing 10% horse serum (Fig. 3). The compounds were incubated separately in the presence of medium for 24 h melanoma tumor in DBA/2J mice. at 37Â°Cin a gassed, humidified incubator. After the incubation, the extent of oxidation was assessed by measuring spectropho- RESULTS tometrically the amount of melanin formed. Dopa phosphates Structures. The principle products of the synthesis described were at least 10-fold more stable than dopa. in "Materials and Methods" were monophosphomonohydroxy- Substrate for Dopa Oxidase (Tyrosinase). Comparisons were made between the abilities of dopa and dopa phosphates 3 G. Prota, personal communication. 4J. M. Pawelek, Clark, and Chedekal, unpublished observations. 5J. M. Pawelek and M. Murray, unpublishedobservations.

CANCER RESEARCH VOL 46 FEBRUARY 1986 494

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1986 American Association for Cancer Research. EFFECT OF DOPA PHOSPHATES ON MELANOMA CELLS to act as substrates for dopa oxidase. Dopa oxidase was purified from Cloudman S91 mouse melanoma tumors as described previously (3). Dopa oxidase activity was assessed by measuring spectrophotometrically the production of dopachrome (5) (Fig. 4). Dopa phosphates were not substrates for dopa oxidase 0.8 unless alkaline phosphatase was included in the reaction mixture. AbsorbincÂ» (540nm) rj.6 Alkaline phosphatase catalyzed the hydrolysis of dopa phos phates to free dopa and presumably PÂ¡asassessed by thin layer chromatography (see "Materials and Methods"). Dopa was a substrate for dopa oxidase whether or not alkaline phosphatase was included in the reaction mixture. Alkaline phosphatase did not inhibit the dopa oxidase reaction nor did it have any intrinsic dopa oxidase activity. It should be noted, however, that dopa oxidase activity was consistently enhanced about 20% when alkaline phosphatase was included in the mixture. In another series of experiments, it was tested whether dopa phosphates might inhibit dopa oxidase activity (data not shown). Dopa phosphates did not interfere with the ability of L-dopa to AbsorbwcÂ» be converted to dopachrome by dopa oxidase as assessed (540nm) spectrophotometrically, nor did it inhibit the conversion of tyrosine to dopa by the same enzyme, as assessed by the method of Pomerantz and Chuang (14). Production of Melanin by Cells in Culture. Comparisons were made between the abilities of dopa and dopa phosphates to be converted to melanin by Cloudman S91 melanoma cells growing 1x10-3 in culture (Fig. 5). The experiments were performed in the pres ence and absence of PTU, an inhibitor of dopa oxidase activity. Fig. 5. Melanin accumulation in culture medium in the presence of Cloudman Spectrophotometric measurements were made of melanin re S91 melanoma cells. Cells (4 x 10s) were inoculated into Coming 25-cm2 tissue leased by the cells to the culture medium. When L-dopa was culture flasks in Ham's F-10 culture medium (4 ml) and placed in a gassed humidified incubator at 37Â°C.After 6 h, the medium was removed and fresh media containing added to the culture medium, melanin was formed even in the either no additions, phenylthiourea (10"3 M), L-dopa, dopa phosphates, or various presence of PTU (Fig. 5A), indicating that dopa was oxidized in combinations were added. After 15 h, media were removed and measured spec trophotometrically as absorbance at 540 nm. Unincubated culture medium was used as a blank. Results are averages of media from triplicate flasks and the 0.30 â€¢ variation between triplicates was less than Â±15%. These experiments were re peated 3 times with similar results.

0.25 the medium as shown in Fig. 3. However, in the absence of PTU, there was increased melanin formation indicating that cellular

0.20 â€¢ oxidation of dopa also occurred. When dopa phosphates were added to the culture medium OOPACHROME very little melanin was formed in the presence of PTU (Fig. 50; (00 47Snm) 0.15 â€¢ note difference in scales of absorbance between Fig. 5A and Fig. 5ÃŸ).In the absence of PTU there was increased melanin forma 0.10 â€¢ tion from dopa phosphates and this was assumed to be due to cellular oxidation of dopa following cleavage of the phosphoryl o.os bonds by cellular phosphatase activity. Note that the differences in melanin formation in the presence and absence of PTU, i.e., those attributable to cellular oxidation, were quantitatively similar 0.00 for dopa and dopa phosphates. 30 40 50 time (minutisi Cytotoxicity of Dopa and Dopa Phosphates. It has been well Fig. 4. Abilities of L-dopa and dopa phosphates to act as substrates for dopa established that intermediates in the malanin pathway are poten oxidase. Dopa oxidase was purified from Cloudman S91 melanoma tumors as described previously (3). Activity was assessed by measuring the formation of tially cytotoxic to cells and that cells actively synthesizing pig dopachrome (orange) monitored spectrophotometrically as absorbance at 475 nm. ment run the risk of self-destruction (e.g., see Ref. 5). Experi Each reaction was performed in a plastic cuvette at 25Â°Cin a total volume of 0.6 ml, in 0.04 MTris buffer, pH 7.4. The concentrations of L-dopa and dopa phosphates ments were carried out to determine the relative cytotoxicity of were 1 x 10' ' M. Each reaction contained 100 ng of dopa oxidase. Appropriate dopa and dopa phosphates toward Cloudman cells in culture blanks in which dopa oxidase was omitted showed no absorbance throughout the Table 1). Both compounds were toxic; however, concomitant experiment. Alkaline phosphatase (bovine intestine, type VII-S) was obtained from treatment of the cells with PTU reduced the toxicity of dopa Sigma and was added at a final concentration of 20 units/ml, 15 min before the addition of dopa oxidase. The addition of dopa oxidase was defined as f = 0 for phosphates but not that of dopa. This difference was probably the experiment. Dopa phosphates (â€¢);dopaphosphates plus alkaline phosphatase due to the nonspecific oxidation of dopa which occurs in the (O); L-dopa (â€¢);L-dopaplus alkaline phosphatase (lÃ¬).Curves represent averages of numbers from duplicate cuvettes and the variation between duplicates was less presence of serum proteins and which is not prevented by PTU than Â±10%. The experiments were repeated several times with similar results. (cf. Fig. 5).

CANCER RESEARCH VOL. 46 FEBRUARY 1986 495

Table1 Table2 Toxidty of L-dopaand dopa phosphatestoward Cloudmanmelanomacells in Uptakeof ^P-labeled dopa phosphatesby CloudmanS91 melanomacells in culture: effects of phenylthiourea culture Cells (4 x 10s) were inoculated into Coming 25-cm2 tissue culture flasks in Cellswere inoculatedinto Coming25-cm2tissuecultureflasks at aconcentration Ham's F-10 culture medium.Three h after inoculation, more than 95% of the cells of 10* cells/flask in 4 ml medium.After 6 h, "P-tobeted dopa phosphates(400,000 had attached to the substratum and assumed a flattened morphology typical of cpm, 222 /iCi/nmoi) were added to the culture medium. After 1.5 and 24 h of incubation in a gassed, humidified incubator (37Â°C),the culture medium was Cloudman cells in monolayer culture. Six h after inoculation, the medium was removed and fresh media containing either no additions, phenylthiourea, L-dopa, removed, and the cells were detached from the flasks with EDTA, washed in dopa phosphates,or various combinationswere added. The next day media were calcium-magnesiumfree saline,and precipitatedonto Whatman No. 3MM cellulose removed,cells were detachedwith EDTA(1 HIM)incalcium-magnesiumfree saline, filter discs with 10% trichloroaceticacid by standard techniques(e.g., Ref. 16).The and counted in a Coulter Counter. Results are averages of determinations from discs were counted in a liquid scintillation counter and the results represent triplicate flasks and the variation between triplicates was less than Â±15%.These averagesof triplicate samples.Variation between triplicates was less than Â±15%. experimentswere repeated 3 times with similar results each time. Time of ""P incorporated (cpm/105cells) Additions Celino. % of control incubation(h) None 3.4x10* 100 1.5 51 PTU(10'3M) 3.0 x 105 100 24 7091 u-Dopa(10~3M) 1.9 x 10* 5.6 u-Dopa+ PTU 1.3 x 10* 4.3 Dopa phosphates(10'3 M) 1.5 x 10* 4.4 Dopa phosphates + PTU 2.6 x 105 87

500

400

.MELANOMA TUMORS

Ã‹ 300 O O

200 LIVER

-5 IO'" IO'3 IO" 6X IO [P-DOPA] (MOLES/LITER) EYES -, Fig. 6. Effects of /3-MSHon the toxicity of dopa phosphates toward Cloudman loo BRAIN l T melanomacells. Cellswere inoculatedas described in legendto Table 1 (â€¢),except HEART / * that one set of cells was preexposed to /3-MSH(2 x 10~7M)for 48 h prior to the LUNG J experiment (O). Results are averages of triplicate determinationswith the variation between triplicates being less than Â±15%.The experiment was repeated several times with similar results.

200 400 600 800 In a second series of experiments, the effect of 0-MSH on the I4C-P-DOPA INJECTED INTO MICE (pi) cellular sensitivity to the dopa phosphates was examined (Fig. Fig.7. Incorporation of "C-labeled dopa phosphates into melanoma tumors 6). Pretreatment of cells with Â¿-MSHfor 48 h increased their and other tissues of mice. DL["C]dopa with the label in position 3 of the phenylal- aninering was obtainedfrom ResearchProducts InternationalCorp. and was used sensitivity to the cytotoxic effects of the dopa phosphates. The as starting materialfor the synthesisof dopa phosphatesas describedin "Materials concentration of dopa phosphates for 50% lethality was reduced and Methods.' DBA/2J mice, bearing s.c. palpable Cloudman S91 melanomas approximately 3-fold by pretreatment of cells with MSH. were injected with various amounts of [14C]dopaphosphate s.c. on the opposite Cellular Uptake of Radiolabeled Dopa Phosphates. Dopa side of the body from the tumor. The next day the mice were sacrificed by ether anesthesia, and various tissues were excised, weighed, dissolved by boiling in phosphates were synthesized in a reaction where the phosphate alkali, and counted in a liquid scintillation counter. Results represent averages Â± moieties were labeled with MP. When a small amount of this SE (bars) for triplicate determinations, 750 n\ (the largest amount injected) repre material was added to culture medium, Cloudman melanoma sented approximately20 nmol and 1 nC\of material. Expenseprevented repetition cells showed significant incorporation of radioactivity into acid- of this experiment. precipitable material after 24 h of incubation (Table 2). amount of 14C-labeled dopa phosphates injected (750 u\ or about In another series of studies, dopa phosphates were synthe 20 nmol/100 mg wet tissue) was on the linear portion of the sized in a reaction in which the L-dopa was labeled with 14C.This uptake curve and therefore not saturating for the tumors. It was material was injected into DBA/2J mice bearing palpable mela not possible to determine whether the 14C label entered the nomas. The next day the mice were sacrificed and various tumors as dopa phosphates. tissues were analyzed for radioactivity (Fig. 7). The following points were established, (a) Most of the 14Clabel was excreted DISCUSSION in the urine (not shown); however, there was significant and We have described the synthesis, isolation, biological activity, selective incorporation of 14C into tumors, (b) The greatest and some additional characteristics of a new class of com-

CANCER RESEARCH VOL. 46 FEBRUARY 1986 496

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1986 American Association for Cancer Research. EFFECT OF DOPA PHOSPHATES ON MELANOMA CELLS pounds, "dopa phosphates." The evidence is indirect but to peutic agents for malignant melanoma. The data in Table 2 gether indicates that dopa phosphates are taken up by Cloudman suggest that 32P-labeled dopa phosphates might be used to melanoma cells and converted to L-dopa and PI and that the L- specifically increase 32P incorporation into melanoma cells, a dopa is subsequently utilized as a substrate for melanin biosyn situation that would also have therapeutic potential for malignant thesis. Cytotoxic intermediates are generated during the conver melanoma. sion of L-dopa into melanin. The Pi is incorporated into acid- In addition to the above considerations, it has recently been precipitable material, possibly nucleic acids and proteins. found that dopa phosphates are effective in increasing the mel Evidence that dopa phosphates are taken up by cells with the anin content in the skin of SKH-2 pigmented hairless mice phosphoryl ester linkages intact is as follows, (a) Studies with following topical applications (15). These results raise the pos phenylthiourea (Fig. 5; Table 1) suggested that dopa phosphates sibility that dopa phosphates might be effective in producing were converted to melanin by the cells, but not by the culture natural barriers to solar radiation. Such findings would have far- medium. That is, since dopa phosphates were not substrates reaching implications for the prevention of carcinomas of the skin for dopa oxidase (Fig. 4), cellular phosphatases most probably in humans. catalyzed the hydrolysis of the phosphoryl bonds in order for the production of melanin to occur, (b) Direct measurements of ACKNOWLEDGMENTS incorporation of 32P-labeled dopa phosphates indicated that the 32p label was taken up efficiently and incorporated into acid- We thank Drs. John McLane and Susan Edelstein for their help with the precipitable material. It is most likely that the 3i'P entered into manuscript. cells attached via ester linkages to the phenylalanine ring be REFERENCES cause the concentration of PI in the culture medium (as KH2P04 and Na2HPO4) was about 2 x 1fr3 M or about 4000 times the 1. Pawetek,J. M., and Komer, A. The biosynthesis of melanin in mammals.Am. Sci., 70: 136-145,1982. concentration of the 32P label that was added. Had the "P- 2. RÃ¢per.H.S. The aerobic oxidases. Physiol. Rev., 8: 245-282,1928. labeled dopa phosphates been hydrolyzed in the culture medium, 3. Komer, A., and Pawelek, J. Mammaliantyrosinase catalyzes three reactions the specific activity of the 32P>would have been too low to in the biosynthesis of melanin.Science(Wash.DC),277: 1163-1165.1982. 4. Komer, A., and Pawelek, J. Dopachrome conversion: a possible regulatory observe significant uptake of radioactivity by the cells, (c) Direct point in melanin biosynthesis.J. Invest. Dermatol., 75:192-195,1980. measurements of the incorporation of 14C-labeled dopa phos 5. Pawelek, J., Komer, A., Bergstrom, A., and Bologna, J. New regulators of melanin biosynthesis and the autodestruction of melanoma cells. Nature phates into melanoma tumors indicated specific uptake of the (Lond.),286: 617-619. 1980. material (Fig. 7). However, it was not possible to determine 6. Barber, J. I., Townsend, D., Olds, D. P., and King, R. A. Dopachromeoxido- whether or not the 14Clabel entered the tumors with the phos reductase: a new enzyme in the pigment pathway. J. Invest. Dermatol., 83 145-149,1984. phoryl bonds intact. 7. Pawelek, J. A new enzyme in the pigment pathway? J. Invest. Dermatol., 84: Efficient uptake of dopa phosphates by melanoma cells would 234,1985. 8. Pawelek, J., Wong, Q., Sansone, M., and Morowitz. J. Molecular controls in suggest the possibility of a specific transport system for these mammalianpigmentation.Yale J. Biol. Med., 46: 430-443,1973. 9. Pawelek, J., and Lerner, A. B. 5'.6'-Dihydroxymdole is a melanin precursor compounds, since, in general, phosphorylated compounds do exhibiting potent cytotoxicity. Nature (Lond.),276: 627-628, 1978. not cross plasma membranes. Alternatively, it is possible that 10. Pawelek, J. Diphosphophenylalanine:a new compound exhibiting selective there were cellular phosphatases associated with the plasma toxicity toward melanomacells. J. Invest. Dermatol., 78: 357-358,1982. 11. Plimmer, R. H. A. Esters of phosphoric acid: phosphoryl hydroxyamino acids. membranes which catalyzed the hydrolysis of the dopa phos Biochemistry,35: 461-469,1941. phates before the compounds passed through the membrane 12. Pawelek, J., Sansone, M., Koch, N., Christie, G., Halaban, R., Hendee, J., barrier. Lemer, A., and Varga, J. Melanomacells resistant to inhibition of growth by melanocyte stimulating hormone. Proc. Nati. Acad. Sci. USA, 72: 951-956, The findings presented here, that dopa phosphates can act as 1975. precursors to melanin biosynthesis, raise several possibilities for 13. Pawelek, J. M. Regulationof pigmentation and proliferation in cultured mela- these compounds. For example, increased stability and solubility nocytes. In: Methods for Serum-freeCulture of Neuronaland Lymphoid Cells, pp. 57-66. New York: Alan R. Liss, Inc., 1984. make them attractive substitutes for L-dopa in experiments in 14. Pomerantz, S. H., and Chuang, L. Effects of ÃŸ-MSH,Cortisol,and ACTH on which L-dopa is a component of the reaction. This consideration tyrosinase in the skin of newborn hamsters and mice. Endocrinology,87:302- applies not only to pigment producing systems but also to other 310,1970. 15. Agin, P. P., Sayre, R. M., and Pawelek, J. M. Phosphorylatedmixed isomers systems in which L-dopa is a key metabolite, e.g., in catechol- of L-dopaincreasemelanincontent in skins of SKH-2 pigmentedhairlessmice. amine biosynthesis or the production of dopamine. Also, the Photochem. Photobiol.,47s: 73,1985. 16. Pawelek, J. M. Evidence suggesting that a cyclic AMP-dependent protein cytotoxic effects of dopa phosphates against melanoma cells kinase is a positive regulator of proliferationin CloudmanS91 melanomacells. raise the possibility that they might be effective as chemothera- J. Cell. Phystol.,9fl: 619-625,1979.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1986 American Association for Cancer Research. Increase in Melanin Formation and Promotion of Cytotoxicity in Cultured Melanoma Cells Caused by Phosphorylated Isomers of l-Dopa

John M. Pawelek and Marilyn Murray

Cancer Res 1986;46:493-497.

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