Methotrexate
Mechanism of Action on DNA Synthesis in Psoriasis
Gerald D. Weinstein, MD; Gary Goldfaden, MD; and Phillip Frost, MD, Miami, Fla
Methotrexate is extremely effective for control
of severe psoriasis; however, its mode of action
is not yet fully understood. Deoxyribonucleic acid
synthesis and its inhibition by methotrexate in nor-
mal and psoriatic epidermis was studied autora-
diographically. Intradermally given methotrexate
inhibits DNA synthesis for 12 to 16 hours in nor-
mal and psoriatic skin. Intramuscularly it causes
complete inhibition in psoriasis but only partial
inhibition in normal epidermis. Psoriatic epider-
mis is more sensitive to the action of metho-
trexate than normal epidermis. This may be due
to increased drug sensitivity of the individual ab-
normal cell, as well as to increased percentage
of psoriatic cells in the methotrexate-susceptible
part of the cell cycle (S period). The local (cu-
taneous) effect of methotrexate to inhibit DNA
synthesis suggests the further investigation of a
topically administered form of methotrexate or its
analogues for the treatment of psoriasis.
IN 1951, the folie acid antagonist, aminop-
terin, was first demonstrated to be effective
for the therapy of psoriasis.1 Since then,
methotrexate has been used extensively for
the treatment of severe, widespread, and
disabling psoriasis.2'3 Although the exact
mode of action of methotrexate is not yet
fully understood, it is thought to affect
DNA synthesis or the S phase of the cell
cycle.4 Methotrexate binds directly to dihy-
drofolic acid reductase preventing the for¬
mation of tetrahydrofolate, thereby limiting
Accepted for publication Feb 11, 1971.
From the Department of Dermatology, University
of Miami (Fla) School of Medicine.
Reprint requests to Department of Dermatology,
University of Miami School of Medicine, 1600 NW
Tenth Ave, Miami, Fla 33136 (Dr. Weinstein).
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the availability of 1-carbon units needed
for the conversion of deoxyuridylic acid
(DUMP) to thymidylic acid (TMP) in the
de novo pathway for DNA production5 (Fig
1).
There is limited information on the mech¬
anism and duration of action of methotrex¬
ate on normal or psoriatic skin in vivo.8'8 In
the present study, psoriatic patients were
treated with intradermally or intramuscular¬
ly administered methotrexate in an attempt
to elucidate its action in both normal and
psoriatic epidermis. This report is concerned
with the effect of methotrexate on DNA
synthesis and the duration of action after its
administration. Previous studies have shown
that, at any moment there are six times as
many psoriatic cells in the S phase (DNA
synthesis) of the proliferative cell cycle as
compared to normal skin (thymidine label¬
ing index 22.7% vs 3.5%).89 Therefore,
there are six times as many psoriatic cells in
a susceptible phase when exposed to a dose
of methotrexate. Above and beyond this in¬
creased number of cells which can be affect¬
ed by methotrexate, this study will provide
evidence that the individual psoriatic cell is
also more sensitive to the action of metho¬
trexate compared to the normal epidermal
cell.10
Methods and Results
DNA Synthesis in Normal and Psoriatic
Skin.—Tritiated deoxyuridine-6-3H (specific
activity =5.59 curies/millimol), 5 microcuries
in 0.1 ml saline, was injected intradermally in
normal skin and psoriatic skin.11 Biopsies of
 Deoxyuridine Thymidine
Thymidine Kinase Thymidine Kinose

Deoxyuridine-5 Thym,dylaie Thymidine-5

Synlhetase Fig 1.—Part of de novo and
Monophosphate *-
Monophosphate —•»- DNA "salvage" pathways involved in
(DUMP) (TMP) DMA synthesis.

N5,NI0-Metriylene Dihydrofolate
Tetrahydrofolale
Totrn— „^^Dihydrofolate
le,ru -*^
reducíase
Hydrofolate
-

Fig 2.—Normal skin after intra¬

dermal injection deoxyuridine'H (5
microcuries/0.1 ml). Heavily la¬
beled cells are seen in basal layer
in same distribution as thymidine-
'H labeled cells.

Fig 3.—Normal skin six hours

after intramuscular injection of
methotrexate and then labeled for
45 minutes with deoxyuridine-3H.
Lightly labeled cells are seen (ar¬
rows) indicating only a partial ca¬
pability for DNA synthesis at this
time.

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 these sites were taken 45 min¬ Table 1.—Effect of Intradermally Administered Methotrexate
utes after the injection and on Deoxyuridine-'H Incorporation Into Epidermal DNA
prepared histologically for au- Test Agent Deoxyuridine-^H
toradiography. Slides coated Used at Injected Intradermally
with photographic emulsion Zero Time _After (hr):_Normal Skin* Psoriasis*
(Kodak NTB-3) were ex¬ Methotrexate
posed for one to four weeks (0.25 tng/0.1 ml) 1
and were developed and
-

stained as previously de¬

scribed (Fig 2).
It has been previously dem¬
onstrated that deoxyuridine- _16 _-I-_-i-
3H is converted to DUMP by _18_-_+_
thymidine kinase.12 A methyl _24_+ + _+ + + +
* DNA synthesis equal to control (no methotrexate inhibition);
group is made available fol¬ + =

complete methotrexate inhibition of DNA synthesis.

lowing the conversion of folie
—
.

acid to tetrahydrofolic acid

Table 2.—Effect of Intramuscularly Administered Methotrexate
utilizing dihydrofolic acid re¬ on Deoxyuridine-'H Incorporation Into Epidermal DNA
ducíase (DHFR) (Fig 1).
This methyl group is then at¬ Test Agent Deoxyuridine-3H
Used at Injected Intradermally
tached to DUMP by the en¬ Normal Skin*
Zero Time After (hr): Psoriasis*
zyme thymidylate synthetase Methotrexate 2 L +
to produce TMP which is in¬
(25 to 50 mg 4
corporated into DNA. There¬ intramuscularly
intramuscularly-¡-
fore, deoxyuridine-3H can be to each
patient)
1 I I
used as a marker of DNA
synthesis in the same way
10 | I
that thymidine-3H has been
12 -I I I I
16 + + + +1 + + + +
used (Fig l).11 Using deoxy- 18 + +
uridine-3H and autoradi-
ography, black silver grains _24_+ _20_+ + + + + + + +
+ + + + + + + + -*-
from tritium emissions ap¬ *
+ DNA synthesis equal to control (no methotrexate inhibition);
= =

peared over the nuclei of cells complete methotrexate inhibition of DNA synthesis; J, partial methotrexate
= —

that were synthesizing DNA inhibition.

at the time of pulse label in-
jection (Fig 2). In order to test further that
deoxyuridine-3H was incorporated into DNA,
control specimens were subjected to DNAase.13
The isotope label was removed upon using
DNAase, demonstrating that the tritium was in
the destroyed nuclear DNA.
Effect of Intradermally Administered Metho-
trexate on DNA Synthesis Pathway in Normal
and Psoriatic Skin.—The incorporation of
deoxyuridine-3H into DNA in normal and pso¬
riatic skin is dependent upon the normal func¬
tioning of dihydrofolic reductase, the major site
of action of methotrexate. Methotrexate (0.25
mg in 0.1 cc saline) was injected intradermally
into both normal and psoriatic skin of seven

Fig 4.—Psoriasis six hours after intramuscular in¬

jection of methotrexate followed by deoxyuridine-3H
intradermally for 45 minutes. This is a high-power
magnification of a dermal papilla surrounded by acan-
thotic psoriatic epidermis. No labeling is present in
epidermal cells while light labeling is seen in the der¬
mal cell population (arrows). This indicates that iso-
topic compound penetrated to this area and that
dermal cells are less sensitive to inhibitory effects of
methotrexate than are psoriatic epidermal cells.

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 0.25
METHOTREXATE
mg/0.1 ml administered intradermally
Fig 5.—Effect of methotrexate inhibition on DNA
synthesis in normal and psoriatic epidermis. Metho¬
trexate was injected intradermally in different concen¬
trations followed by deoxyuridine-:,H at two (solid line)
or eight hours (dashed line). Each point represents
patients. varying from 2 to
After time intervals
24 hours, deoxyuridine-3H injected into the
was
same methotrexate-injected sites. All biopsy
specimens were taken 45 minutes after the
injection of deoxyuridine-3H. No labeled nuclei
were seen in specimens taken from 2 to 12
hours after the methotrexate injection, suggest¬
ing that DNA synthesis was blocked by metho¬
trexate. By 16 hours, labeled nuclei once again
appeared indicating that DNA synthesis was
no longer being inhibited by methotrexate (Ta¬
ble 1). Tritiated thymidine injected after meth¬
otrexate produced labeling patterns similar to
those obtained with premethotrexate control
injections of thymidine-3H or deoxyuridine-3H.
Effect of Intramuscularly Administered
Methotrexate on the DNA Synthesis Pathway.
—Single intramuscular injections of 10 to 50
mg of methotrexate were given to nine patients.
At various times thereafter deoxyuridine-3H
was injected intradermally into areas of normal
and psoriatic skin. Biopsies were performed 45
minutes later, and the autoradiographic results
are shown in Fig 3 and 4.
In psoriatic skin there were, as before, no
labeled cells seen until 16 hours following the
injection of methotrexate (Table 2). However,
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average of severalexperiments. At at dose of 0.0001
mg of methotrexate/0.1 ml, significantly more inhi¬
bition of DNA synthesis is found in psoriasis (open
circles) compared to normal skin (solid circles).
in normal skin specimens taken from 2 to 12
hours after methotrexate administration, there
was partial labeling present as evidenced by
fewer cells labeled and fewer grains per labeled
cell compared to controls (Fig 3). This autora¬
diographic technique may be considered as a
semiquantitative assay to measure the extent of
DNA synthesis and its inhibition.
Dose and Time Response for Intradermally
Administered Methotrexate on Psoriatic and
Normal Skin.—In order to assess the action of
methotrexate as a function of concentration in
the skin, but independent of blood flow to
normal and psoriatic skin, various concentra¬
tions of methotrexate progressively diluted in
saline were injected intradermally into both
normal and psoriatic skin. Deoxyuridine-3H
was then injected into these same sites approxi¬
mately two and eight hours later and biopsied
45 minutes later. Autoradiographs were then
analyzed to determine the approximate number
of grains per cell and labeled cells per section.
By comparison to the control (no methotrex¬
ate) specimens from the same patients consid¬
ered as 100%, the depression of DNA synthesis
(or percent of methotrexate inhibition) was
estimated as the decrease in grain counts and
 number of labeled cells (Fig 5). While this dermis for up to 16 hours. At 16 hours
technique is not as exact as liquid scintillation labeling reappears indicating that DNA syn¬
counting, it permits reasonable approximations thesis is once again taking place and the
on individual specimens of 0%, 25%, 50%, effect of methotrexate has disappeared.
75%, and 100% labeling compared to controls. When methotrexate was administered in¬
When a concentration of 0.0001 mg/0.1 cc of
tramuscularly, inhibition of DNA synthesis
methotrexate was injected, DNA synthesis in
in psoriasis was identical in degree and du¬
psoriatic epidermis was completely inhibited
after two hours, as evidenced by no incorpora¬ ration to the intradermal administration of
tion of deoxyuridine-3H in autoradiographs. methotrexate, whereas, only partial inhibi¬
The same concentration of methotrexate in tion of normal epidermis was found. Metho¬
normal skin caused only partial inhibition of trexate inhibition is gone by 16 hours despite
DNA synthesis. However, eight hours after the the fact that a slight blood level per¬
injection of 0.0001 mg/0.1 cc of methotrexate in sists for at least 12 hours; while following
normal skin, almost no inhibition of DNA syn¬ intradermal administration, the bulk of the
thesis was detected; whereas, in psoriatic skin
there was still 50% inhibition of DNA synthe¬
drug has probably been dissipated within
minutes from the local site. If the primary
sis.
mechanism of action of methotrexate is to
Comment inhibit DNA synthesis as postulated by
others,5 then it is apparent from our data
These experiments were designed to study that methotrexate administered locally pro¬
the mechanism of action of methotrexate in duces an inhibition of DNA synthesis simi¬
psoriatic and normal epidermis in vivo. Nu¬ lar to systemic administration. Therefore,
merous studies in the past have demonstrat¬ methotrexate blocks DNA synthesis in the
ed that methotrexate blocks the pathway for skin by a local action and not by an effect
DNA synthesis by inhibiting folie acid re¬ on some other organ, eg, the liver. If the
ducíase with a consequent decrease in the clinical effectiveness of methotrexate in pso¬
availability of methyl groups needed for sev¬ riasis works through this mechanism, then it
eral biochemical steps, primarily the conver¬ is reasonable to search for a way to use it
sion of DUMP to TMP. The methotrexate topically.
effect on the latter conversion was confirmed Resumption of DNA Synthesis After
in this study by exposing normal and pso¬ Methotrexate.—There are several possible
riatic epidermis to intradermally or intra¬ explanations for the resumption of DNA
muscularly administered methotrexate and synthesis after the methotrexate effect. (1)
then determining whether deoxyuridine-3H A new population of cells enters S phase
was incorporated into epidermal cell nuclei. that produce DHFR for DNA synthesis.14'15
Whereas pretreatment of skin with intrader¬ (2) The original S-phase cells that have
mally administered methotrexate prevented methotrexate-inhibited DHFR produce ad¬
deoxyuridine-3H incorporation, under the ditional enzyme. (3) Methotrexate is re¬
same conditions tritiated thymidine was in¬ leased from its binding to DHFR making
corporated into DNA. This indicates that the enzyme available. (4) The same or dif¬
the block is bypassed using thymidine-3H ferent cells produce a new type of DHFR
and that methotrexate does not block the which is unaffected by methotrexate.
cell's capability of synthesizing DNA except 1. The proliferative cell cycle of the pso¬
by limiting the availability of the nucleic riatic epidermis has been described previous¬
acid precursor, thymidylic acid. ly.9 The S period (DNA synthesis period)
Local or Systemic Effect of Methotrexate of the psoriatic cell is 8.5 hours which is
on DNA Synthesis.—Intradermally admin¬ substantially shorter than the normal epi¬
istered methotrexate, in a relatively large dermal cell or the basal cell carcinoma cell.18
concentration of 0.25 mg/0.1 cc saline, pre¬ At any time, 22.7% of the psoriatic prolif¬
vented labeling with deoxyuridine-3H auto¬ erative cell population is in the S phase, and
radiographs of normal skin and psoriasis it is the S-phase cells which are considered
until 16 hours. This suggests that there was to be primarily affected by methotrexate. If
complete inhibition of DNA synthesis by the methotrexate effect is to inhibit irrever¬
methotrexate in normal and psoriatic epi- sibly further DNA synthesis, then the cells

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 will be unable to divide to produce the rapidly proliferating epidermal cell popula¬
hyperproliferative skin lesion of psoriasis. tions.
The subsequent recurrence of DNA synthe¬
sis at 16 hours in psoriasis may then repre¬ trexate on psoriatic epidermis was found
sent the entrance of a new group of cells into
S phase from the Gx population that was
not significantly affected by the dose of
methotrexate.4'14'15
2. Methotrexate inhibition of dihydrofolic
reducíase is well established; however, the
events controlling the production of addi¬
tional enzyme by the same cell are not well
known. If the methotrexate-exposed S-phase
cells continue to produce dihydrofolic reduc¬
íase, a level of new enzyme should be
reached which will be greater than the avail¬
able methotrexate in the cell needed to in¬
hibit it. At that time this cell, if not lethally
damaged by some other mechanisms, should
be capable of renewing DNA synthesis.
Thus, the continued inhibition of DNA syn¬
thesis by methotrexate may be regarded as a
function of the effective concentration of the
drug in that cell. Methotrexate binds very
tightly to dihydrofolic reductase, and any
excess methotrexate would be in equilibrium
with the tissue fluids and serum. The serum
levels of methotrexate fall rapidly after sys¬
temic administration,17 and, therefore, an
effective tissue level would not be available
for very long.
3. The avidity of methotrexate for dihy¬
drofolic reductase is extremely high, with an
inhibitor constant (Ki) of less than 10 7M.5
We are not aware of any studies in skin that
measure the rate of release of methotrexate
from dihydrofolic reductase, but would an¬
ticipate that this would be very low and not
a likely means of restoring enzyme function.
4. There is no evidence that cells in hu¬
man tissues produce a new or different type
of dihydrofolic reductase that is resistant to
methotrexate. However, Rothenberg18 sug¬
gests that methotrexate may be altered in
leukemic cells affecting its affinity for folie
reductase. In some cell systems increased
amounts of enzymes may appear on a cell in
response to methotrexate, producing a state
of resistance to methotrexate.5
Differential Effect of Methotrexate on
Normal and Psoriatic Epidermis.—Of ex¬
treme importance in this study is the finding
that there is a differential effect of metho¬
trexate on DNA synthesis in normal and
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The selective inhibitory activity of metho¬
first after intramuscular doses when normal
skin had partial inhibition only, while psori¬
asis was completely inhibited. This observa¬
tion was confirmed using intradermal doses
that were titrated to a minimal effective
dose. At the lowest concentration 0.0001
mg/0.1 ml (O.ljug/O.l ml), significant differ¬
ences in the degree of inhibition are found
between normal and psoriatic epidermal
cells. This negates the possibility that in¬
creased blood flow in psoriasis could account
for the greater drug effect after systemic
administration. If anything, greater blood
flow would tend to produce a decreased
inhibition after local injection because of a
rapid disappearance of drug in the psoriatic
skin.
As opposed to the greater activity of
methotrexate on psoriatic cells, a lower level
of inhibitory activity similar to that found
in normal skin also occurred in fibroblasts,
eccrine duct cells, and hair epithelial cells
(Fig 4). One explanation suggested for the
selective inhibition of psoriasis after paren-
teral administration may result from the
increased blood flow to psoriasis. However,
the fibroblasts and epidermal appendage
cells in the dermis juxtaposed to the psoriat¬
ic cells, presumably just as well supplied by
blood, are not as sensitive. Therefore, the
psoriatic cell must have a unique sensitivity
to methotrexate.
The significant differences based on cell
proliferation kinetics between the two cell
cycles is the shorter S phase and the shorter
overall cell cycle of psoriatic cells.9 The
superior effect of methotrexate to inhibit
DNA synthesis preferentially in the psoriat¬
ic cells suggests that the drug's effect on
these cell populations parallels the growth
rate of the cells.
Current concepts of cancer chemothera-
peutic drug action are based to a large
extent on the cellular kinetics of a tumor
population (perhaps analogous to the hyper-
proliferative cell population of psoriasis)
compared to normal cells. Bruce et al4 and
Skipper19 have correlated the selective toxic-
ity of antimetebolites in tumor cells with the
rate of cell reproduction; eg, the more cells
 in DNA synthesis during drug exposure, the compared to normal epidermal cells. (3) A
more sensitive that population would be to rapidly proliferating cell may have a smaller
an S-phase inhibitor like methotrexate. supply of enzyme available and thereby
However, our studies suggest that the pref¬ more easily blocked by a limited amount of
erential effectiveness of methotrexate in pso¬
riasis results from three different factors:
1. At any one time sixfold more psoriatic
cells are in S phase and are, therefore, ex¬
posed to methotrexate (thymidine labeling
index 22.7% vs 3.5%).
2. The duration of DNA synthesis for
each cell population with respect to the
duration of drug exposure will alter the net
effect of that drug. Since S phase of psoria¬
sis is about twice as fast as normal (8.5 vs
16 hours), during a period of drug exposure,
an increasing proportion of susceptible pso¬
riatic cells with enter the S phase and be
exposed to methotrexate. For example, if
methotrexate exposure were prolonged for
16 hours, during that time about 44% of
psoriatic cells would be affected (two groups
of S-phase cells), while in normal skin only
3.5% of the cells or one group of S-phase
cells would be affected.
3. The individual psoriatic cell is bio¬
chemically more sensitive to methotrexate
than the normal epidermal cell.
To our knowledge, this is the first demon¬
stration in humans that an antimetabolite
has a greater effect on the S-phase cells of
the target population (in this case, psoria¬
sis) compared to the S-phase cells of the
normal population. A similar observation
has recently been reported by Hryniuk et
al20 in L5178Y mouse leukemia cells grown
in culture. By comparing the effect of meth¬
otrexate on cells when they were growing at
a logarithmic rate compared to a resting
rate, it was found that the faster the growth
rate, the greater was the inhibition of deox-
yuridine incorporation into DNA produced
by methotrexate.
Several possibilities to explain the selec¬
tive effect in rapidly proliferating cells may
be considered: (1) In addition to the block¬
ade of DUMP conversion to TMP, there
may be a second action occurring in the
rapidly reproducing cells involving the inhi¬
bition of de novo purine biosynthesis, neces¬
sary for both DNA and RNA synthesis.19
(2) There may be an alteration in the trans¬
port of the drug into cells which is greater
for the rapidly reproducing cells in psoriasis
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inhibitor. At this time the reason for the
apparent selective effect on the rapidly pro¬
liferating cell population cannot be defined.
Cutaneous Drug Levels Required for In¬
hibition.—The titrations of intradermal
methotrexate doses indicate the critical min¬
imal levels necessary to obtain inhibition of
DNA synthesis. While inhibition remains
relatively effective using l.O^g/0.1 ml, at the
next dilution (0.1/xg/0.1 ml), inhibitory ac¬
tivity disappears within a few hours. As
might be anticipated, these doses are in the
same range as the peak blood and tissue
levels achieved after parenteral administra¬
tion of methotrexate. Following 50 mg of
methotrexate intravenously, the peak blood
level is approximately 0.15/xg/0.1 ml serum
at one to two hours, but then rapidly falls to
0.02/xg/O.l ml by eight hours.1720 Tissue lev¬
els of methotrexate are reported in the range
of 0.4 to 0.9/xg/gm of skin five hours after
drug administration.21 It would, therefore,
seem that only when the blood level is near
its peak is there an adequate amount of
drug available to a cell for enzyme inhibi¬
tion. Inhibition would then remain from the
peak dose exposure for at least 12 hours, but
could then be renewed presumably by an¬
other dose of drug. This latter possibility
has been applied clinically in the design of
new oral schedule of methotrexate adminis¬
tration.8 Methotrexate is administered oral¬
ly at 12-hour intervals for three doses each
week to provide inhibition of DNA synthe¬
sis for 36 hours, the length of the cell cycle.
Therapeutic Action of Methotrexate.—
While it is possible to demonstrate that
methotrexate inhibits DNA synthesis which
is then followed by a drop in mitotic activ¬
ity,6-22 it is not known how these temporary
effects are translated into resolution of skin
lesions. Mitoses begin to reappear by 24
hours. A few methotrexate-damaged cells
(presumably lethal) are noted within 24
hours, but they do not seem adequate in
number to produce an epidermis of normal
thickness.22'23
It is postulated that a "thymineless state"
is produced followingexposure to metho¬
trexate because of the inability to convert
 DUMP to TMP and this leads to cell
death.24 Unless the previously described
damaged cells are a manifestation of this,
we cannot yet identify the result of the
thymine-deficient cell. In fact, we can dem¬
onstrate that following methotrexate the
number of labeled cells, as well as the grain
counts after tritiated thymidine, increase in
both normal and psoriatic cells. This would
not be anticipated if the cells were dying. It
is possible that the effect of methotrexate on
a cell is delayed and /or cumulative from
several doses since it is rare to see signifi-
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This investigation was supported bv research
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and the National Institutes of Health (AM-14887
pnd AM-11559). The facilities of the General Clini¬
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