Download Selective Incorporation of i_-3,4

[CANCER
RESEARCH
37. 2123-2125,
July 1977]
Selective Incorporation of i_-3,4-Dihydroxyphenylalanme by
S-91 Cloudman Melanoma in Wfro1
Michael M. Wick and Emil Frei, III
The Sidney Farber Cancer Institute
and Harvard Medical School,
Boston,
Massachusetts
02115
SUMMARY
MATERIALS
The incorporation of precursors of the biopigment mela
nin into melanotic and amelanotic S-91 Cloudman mela
noma, mouse fibroblast L-929, and Chinese hamster ovary
cells was studied. Tyrosine did not selectively accumulate
in pigmented cells compared to that in nonpigmented con
trol cells. Inhibition of protein synthesis with cycloheximide
provided an estimate of the partition of tyrosine between
protein (95%) and pigment biosynthesis (5%).
L-3,4-Dihydroxyphenylalanine, a more proximal precursor
of melanin, was selectively incorporated into pigmented
cells up to 60 times that of control lines. a-Melanocytestimulating hormone and theophylline, agents that en
hance pigmentation, further increased the incorporation of
L-3,4-dihydroxyphenylalanine
into melanocytic cells. A
unique property of melanoma cells thereby has been de
fined that may permit a selective chemotherapeutic and
diagnostic approach.
Materials. L-Tyrosine, L-dopa, and cycloheximide were
from Sigma Chemical Co., St. Louis, Mo. L-[3,5-3H]Tyrosine
(specific activity, 43 Ci/mmole) and L-[3H]dopa (specific
activity, 21 Ci/mmole) were from New England Nuclear,
Boston, Mass. All other chemicals were reagent grade and
were used without further purification.
Cell Culture. S-91A, a melanotic Cloudman melanoma,
was obtained from the American Type Culture Collection
(CCI 53.1), Rockville, Md. S-91B, an amelanotic clone, was
a gift from Dr. Jewel Cobb, Connecticut College, New Lon
don, Conn., and has been fully described (2). Mouse fibro
blast L-929 and CHO have been maintained in our labora
tory for 7 months. Cells were maintained as monolayers
in Falcon plastic flasks in McCoy's Medium 5A supple
INTRODUCTION
Malignant melanoma is comprised of cells that possess a
biochemical pathway for the enzymatic production of the
biopigment melanin (3). According to the accepted scheme
(Chart 1), the enzyme tyrosinase oxidizes tyrosine first to Ldopa2 and then to dopa quinone, which undergoes sponta
neous cyclization and polymerization to melanin. Since ty
rosinase, a copper-containing polyphenol oxidase, is re
stricted to melanocytic cells, a potential basis for a selective
chemotherapeutic or diagnostic approach to neoplasms of
this type may exist. Several previous studies (1, 5, 7) have
attempted to demonstrate selective incorporation of precur
sors in vivo, with conflicting results that possibly reflect the
complex metabolic degradation of the intermediates.
In order to examine the feasibility of utilizing this special
biochemical attribute of melanoma cells, we have investi
gated the incorporation of precursors of melanin into me
lanotic and amelanotic clones of S-91 Cloudman melanoma
and compared them to the nonpigment control cells mouse
fibroblast L-929 and CHO. A highly selective incorporation
of L-dopa into pigmented cells was observed that could be
enhanced further by agents that promoted differentiation
and pigmentation of melanocytic cells in culture.
1 This investigation was supported in part by NIH Grant CA-06516.
2 The abbreviations used are: L-dopa, L-3,4-dihydroxyphenylalanine;
CHO,
Chinese hamster ovary; a-MSH, a-melanocyte-stimulating
hormone.
Received January 14, 1977; accepted April 1, 1977.
AND METHODS
mented with 15% fetal calf serum, 100 units of streptomycin
and 100 ¿igof penicillin per ml in 10% humidified air at
37.5°.
Experimental Cultures. Prior to uptake studies, singlecell suspensions were prepared in medium following trypsinization and were inoculated into 60-mm Falcon plastic
Retri dishes at 5 x 105 cells in 5 ml of medium. Cultures
were maintained for 72 hr before exposure and were in
exponential growth phase.
Incorporation Studies. Medium was removed and cells
were washed once with Hanks' balanced salt solution. For
tyrosine uptake, fresh medium was added containing la
beled tyrosine (2 /¿Ci/ml).L-Dopa uptake was performed in
Hanks' balanced salt solution containing 0.01 mW L-dopa
with labeled L-dopa (2 /nCi/ml). Experiments were per
formed in triplicate with parallel cultures maintained at 4°to
correct for nonspecific binding. Reactions were terminated
by aspirating the medium, by washing 3 times with 0.9%
sodium chloride solution and by adding 10%trichloroacetic
acid for 30 min. After removal of the acid, the precipitate
was washed twice with 0.9% sodium chloride solution, and
1 ml of 1.0 N KOH was added. After standing at 4°for 24 hr,
an aliquot was added to scintillation fluid (Aquasol, New
England Nuclear) and counted in a Beckman LS 335 scintil
lation counter. Quenching was corrected by addition of an
internal toluene standard. A parallel set of cultures was
harvested by trypsinization and counted in a Model Z Coul
ter counter. Trypan blue exclusion indicated greater than
95% viability.
Protein Synthesis Inhibition. Studies of the effect of inhi
bition of protein synthesis with cycloheximide on tyrosine
incorporation were performed as described above except
that 0.01 mM cycloheximide was added at 0 time.
Effect of Differentiation. For a determination of the effect
JULY 1977
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2123
M. M. Wick and E. Frei, III
of a-MSH and theophylline on the uptake of L-dopa, theophylline (1.0 mM) or a-MSH (0.01 HIM)was added 24 hr after
plating, and cultures were then treated as described above.
minimally measurable enzyme (2% of S-91A), also displayed
evidence of L-dopa incorporation.
Effect of a-MSH and Theophylline. After treatment with
a-MSH and theophylline, cells were larger, dendrites had
formed, and a gross increase in melanin pigment was visi
ble. It is evident from Table 2 that a profound increase in LRESULTS
dopa incorporation was effected. S-91A had a 56% increase
Tyrosine Incorporation. The degree of incorporation of L- in uptake with a-MSH and a 500% increase for the combina
tyrosine into the various cell lines (Chart 2) is linear with tion of a-MSH plus theophylline. S-91B, which is normally
time with little variation among cell lines. S-91A was the not pigmented, became heavily melanized and exhibited an
slowest growing line with a doubling time of 30 hr. In an 18-fold increase in L-dopa uptake with combined treatment.
attempt to reveal differences among the cell lines, uptake a-MSH and theophylline had no effect on L-dopa uptake by
CHO and L-929 cells.
was examined in the presence of a specific protein inhibi
tor. Table 1 presents the effect of cycloheximide on tyrosine
incorporation. At 0.01 mM, cycloheximide inhibited 95% of
protein synthesis in S-91B and L-929 cells although only DISCUSSION
90% in S-91A. Since S-91A possesses an alternate pathway
Malignant melanoma is a chemotherapeutically resistant
for tyrosine metabolism, which is unaffected by cyclohexi
mide, an estimate is provided therefore of the partition of
Table 1
tyrosine between protein biosynthesis and alternate path
Inhibition of L-¡3H]tyrosine uptake by cycloheximide
in pigmented
ways.
and nonpigmented
cells
L-dopa Incorporation. Chart 3 depicts the incorporation
dpm/105 cells after 90-min incuba
of L-dopa as a function of time, which is in marked contrast
tion
to tyrosine incorporation. A highly selective incorporation
of L-dopa into S-91A, approximately 60-fold over that into
Cell
typeS-91A
(0.01
mM)382
inhibition90
L-929 and CHO, was observed. S-91B, which does have
±180"
±94
S-91B
6384 ± 64
491 ±40
95
L-929Control3941
5930 ±121Cycloheximide
229 ±11%
96
" Mean ±S.E. of 3 samples; differences were statistically signifi
OOH
cant (p < 0.05).
5000-
PROTEIN
4000
o
3000
"o
^ 2000
5,6,
MHYDROXYINDOLE
1000
Chart 1. Biosynthetic scheme for the conversion of L-tyrosine to melanin
by the enzyme tyrosinase.
30
IOOOO-
60
90
MINUTES
Chart 3. Incorporation of L-[3H]dopa into cell lines S91A (•),
S-91B (•),L929 (O), and CHO (D). Each value represents the mean ±S.E. for 3 samples.
Table 2
Effect of o-MSH and theophylline
on uptake of L-[3H]dopa by
melanoma cells
dpm/105 cells after 90-min exposure
(0.01
HIM) and
theophylline
mM)931
(1.0
Chart 2. Incorporation of L-[3H]tyrosine into cell lines S-91A (•),S-91B
(•),L-929 (O), and CHO (D). Values represent the mean for triplicate sam
ples ±S.E.
2124
(0.01
Cell
typeS-91
HIM)2386
mM)2637
(1.0
±30"
A
±69
±78
1 ±930
S-91 BControl1531
236 ±92a-MSH297 ±91Theophylline
1398 ±28a-MSH
4201 ±421
" Mean ±S.E. of 3 samples.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
VOL. 37
Incorporation
tumor that possesses a biochemical pathway for production
of the pigment melanin. Attempts to exploit this aspect
pharmacologically have focused on the incorporation of
pigment precursors into melanqcytes. The rate of incorpo
ration of L-tyrosine, the initial substrate for the biosynthesis
of melanin, does not serve to distinguish pigmented from
nonpigmented cells. The variation observed is well within
the range that can be explained on the basis of differences
in cellular protein content and growth rate.
A quantitative estimate of the relative importance of the
pigment pathway in tyrosine metabolism may be obtained
by comparing the degree of inhibition of incorporation of
tyrosine by the specific protein synthesis inhibitor, cycloheximide. It is apparent from Table 1 that cycloheximide is
able to inhibit greater than 95% of tyrosine incorporation
into nonpigmented cells but inhibits only 90% in melanotic
cells, which suggests that approximately 5% of label is
entering the pigment pathway under these conditions.
These results are in agreement with those of Schachtschabel (8) who examined partition of tyrosine in the HardingPassey melanoma by means of double labeling techniques.
In contrast, L-dopa, a more proximal precursor of mela
nin, demonstrated a high degree of selectivity for pig
mented cells. Presumably, this selectivity resulted from an
absence of alternate routes of metabolism for L-dopa other
than that of the incorporation into melanin. Agents that
enhance pigmentation and differentiation of melanocytes
further increased incorporation. a-MSH, a peptide hormone
specific for melanocytes, and theophylline, an inhibitor of
phosphodiesterase, have been shown to extensively stimu
late pigment production and tyrosinase activity of mela
noma in vitro (9,10). Each agent also stimulated incorpora
tion of L-dopa and, as would be anticipated from the pre
sumed mechanism of action, were highly synergistic. Inter
estingly, S-91B clone, which is normally nonpigmented,
was melanized to the extent that it exceeded the control
pigmented line in L-dopa uptake.
Selective localization of radiolabeled compounds for tis
sue identification has been a useful technique; a prime
example is adrenal gland imaging with 131l-labeled 19-cholesterol (6). However, this technique is completely depend
ent on a selective biological behavior of the tissue involved.
JULY 1977
of ¿-Dopaby Melanoma
In view of the conflicting evidence in the literature con
cerning L-dopa concentration by melanoma, it was of pri
mary importance to determine whether a melanoma cell
could incorporate L-dopa if metabolic degradation was min
imal. Our results suggest that melanoma cells are capable
of selectively incorporating exogenously administered Ldopa and that possibly with appropriate pharmacological
manipulation, e.g., by controlling rates of infusion and/or
blocking metabolic degradation, a useful localization might
be achieved in vivo.
ACKNOWLEDGMENTS
Dr. Michael M. Wick would especially like to acknowledge Dr. Thomas B.
Fitzpatrick who was responsible for initiating interest in the melanoma prob
lem and first expressed many of the ideas contained herein.
REFERENCES
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2. Cobb, J. P., and McGrath, A. S-91 Mouse Melanoma Sublines following
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1972.
3. Fitzpatrick, T. B. Mammalian Melanin Biosynthesis. Trans. St. John's
Hosp. Dermatol. Soc.,5i. 1-26, 1965.
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2125
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
Selective Incorporation of l-3,4-Dihydroxyphenylalanine by S-91
Cloudman Melanoma in Vitro
Michael M. Wick and Emil Frei III
Cancer Res 1977;37:2123-2125.
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