[CANCER RESEARCH 42, 2223-2226, June 1982] 0008-5472/82/0042-OOOOS02.00 Dose-related Effects of Psoralen and Ultraviolet Light on the Cell Cycle of Murine Melanoma Cells1
Janos M. Varga,2 Gary Wiesehahn, James C. Bartholomew, and John E. Hearst
Department of Dermatology, Yale University School of Medicine [J. M. V.], New Haven, Connecticut 06510. and Chemistry Department IG. W.. J. E. H.] and Biodynamics Laboratory [J. C. B.¡,Lawrence Berkeley Laboratories, University of California, Berkeley, Berkeley, California 94720
ABSTRACT of tyrosinase is known to occur in the G2 phase of the cell cycle (20). Cloudman (S91) murine melanoma cells were treated with 4'-hydroxymethyltrioxsalen (HMT), a bifunctional psoralen and We have investigated the effects of a bifunctional psoralen (HMT) by flow cytometry of melanoma cells stained with pro exposed to long-wavelength (365 nm) ultraviolet light. DNA pidium iodide, and in parallel experiments we have determined content of the cells stained with propidium iodide was mea the binding of [3H]HMT to DNA. Our studies showed that at sured by flow cytometry, and cell cycle phases were delineated extremely low levels of binding to DNA, HMT caused a G2 from the DNA histograms by using a curve-fitting routine. We block. However, as the number of substitutions increased, cells found that HMT in combination with long-wavelength (365 nm) became arrested in the S phase and then throughout the cell ultraviolet irradiation blocked melanoma cells in different cycle. phases of the cell cycle, depending on the dose of long- wavelength (365 nm) ultraviolet light and the concentration of HMT. The binding of [3H]HMT to DNA was measured parallel MATERIALS AND METHODS
with cell cycle analyses. Treatments with HMT at concentra Cells and Culture Conditions. The PcIA subclone (22) of the murine tions corresponding to about 1 HMT bound per 106 base pairs S91 Cloudman melanoma cell line was cultured in Ham's F-10 medium of DNA led to the accumulation of cells with predominantly G2 containing 12.5% horse serum, 2.5% fetal calf serum, and gentamicin DNA content. At higher concentrations (2 to 3 HMT/106 base (50 jug/ml) (11, 21, 22). Tissue culture materials were obtained from pairs), the cells were blocked in the S and G, phases. In Grand Island Biological Co., Grand Island, N. Y. Viability was deter conclusion, we have shown that extremely sparse substitution mined by the dye exclusion test (14). Cells were counted in a Coulter of HMT to DNA blocks melanoma cells in the G2 phase or other Counter (Coulter Electronics, Inc., Hialeah, Fla). phases of the cell cycle in a dose-dependent manner. Treatment with Psoralens Plus UV-A. The culture medium of cells in the logarithmic phase of growth was replaced with fresh medium containing HMT. After incubation in the dark for 15 min, the cells were INTRODUCTION exposed to UV-A from a Black Light (Spectroline, XX-15; Spectronics Co., Westbury, Long Island, N. Y.). The dose of UV irradiation was Derivatives of psoralen (e.g., trimethylpsoralen, trioxsalen) in measured by ferrioxalate chemical radiometry as described earlier (3). combination with UV irradiation (UV-A,3 365 nm) are used Immediately following irradiation, the medium was removed, the cul therapeutically (PUVA therapy) in proliferative skin disorders tures were rinsed, fresh medium containing no psoralens was added, such as psoriasis (13). The inhibition of cellular proliferation by and the cultures were further incubated at 37°for 24 hr. PUVA treatment has been attributed to the inhibition of DNA Flow Cytometry. The cells were lifted from the substrate by a treatment with 1 mw EDTA in Joklik's medium (Grand Island Biological synthesis (2, 16, 19, 23). On a molecular level, formation of covalently linked mono- and diadducts between psoralens and Co.). They were washed once with a saline:glucose medium (1.5 mM DNA have been demonstrated (4, 10). Na2HPO4:1.1 mM KH2PO4:1.1 mM glucose:0.14 M NaCI:5 mM KCI, pH 7.4) and were fixed for 1 hr in 25% ethanol in saline:glucose medium Another effect of PUVA treatment is the enhancement of containing 15 mM MgCI2. RNase (1 mg/ml, 5 times crystallized; Cal- cutaneous pigmentation (7). We have investigated a murine biochem, La Jolla, Calif.) was added, and the cells were incubated for melanoma cell line as a model to study the mechanism of 1 hr at 37°.The RNase was removed by 2 washes with saline;glucose hormonally induced pigmentation (11 ) and found that treatment medium, and the cells were stained with propidium iodide (50 /jg/ml) of these cells with trioxsalen plus UV-A increased tyrosinase in saline:glucose medium for 15 min at room temperature. The cells activity [the enzyme responsible for melanin synthesis (3)]. In were then washed once with saline:glucose medium, and the cell addition, cell growth was inhibited and cells with high DNA clumps were removed by filtration through a 37-/im mesh Nitex filter content accumulated. A preliminary analysis of the DNA con (Tetko, Inc., Monterey Park, Calif.). The stained cells were passed tent by flow cytometry revealed that cells treated with low through a flow cytometer similar in design to that described by Stein- concentrations of trioxsalen were arrested predominantly in kamp ef al. (18) but without the sorting mode of operation. The stained the G2 phase of the cell cycle (5). The arrest of the cells in the cells traversed the flow chamber at a rate of approximately 500 cells/ sec and were illuminated by an argon ion laser beam tuned to 514 nm. G2 phase could explain the melanogenic effect, since activation The emitted light, collected at an angle of 90°, was passed through a
' This investigation was supported by Grant CA-26081 awarded by the Na series of filters into the photomultiplier. Signals from each cell were processed electronically and displayed as histograms of pulse-ampli tional Cancer Institute and by the Division of Biomedicai and Environmental Research, United States Department of Energy, Contract W-7405-ENG-48. tude frequency distribution on a multichannel pulse-height analyzer. 2 Recipient of Research Career Development Award 5K04 A1 00154 from The data were stored and processed by a Sigma-2 computer (Xerox USPHS, to whom requests for reprints should be addressed. 3 The abbreviations used are: UVA, long-wavelength (365 nm) ultraviolet light; Co., Rochester, N. Y.). Further analysis of the histograms was carried PUVA treatment, treatment with psoralens in combination with long-wavelength out by means of a program based on the approach, according to the (365 nm) ultraviolet light; HMT, 4'-hydroxymethyltrioxsalen; MSH, melanotropin. method of Fried (8). The data were processed by a CDC 6600 computer Received June 15, 1981; accepted March 12, 1982. after the transformation of the DNA histograms to log scale. Gt spread
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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. J. M. Varga et al. functions were extracted from untreated control cell samples with prominent Gìpopulations (1). Measurement of [3H]HMT Binding to DNA. Cells were treated with [3H]HMT followed by UV-A irradiation, or they were processed as "dark controls." The DNA was extracted and banded in a CsCI gradient (10); the syntheses of HMT and [3H]HMT were described earlier (9, 24).
RESULTS Effects of HMT and UV-A Dose on the Cell Cycle. HMT (3 /IM) without UV-A irradiation showed no significant effect on the DNA histograms obtained 24 hr after treatment (Chart i A). By increasing the UV-A dose from 0.1 to 0.3 J/sq cm, the proportion of cells in G2 increased (maximum at 0.3 J/sq cm), and at higher doses the proportion declined. UV-A (0.3 J/sq cm) without HMT had no significant effect on the DNA histo grams. At low (1 to 3 ¡IM)HMT concentration, a predominant G2 population accumulated (Chart 10). At concentrations above 30 ¡IM,HMT had no major effect on the DNA content of cells. UV DOSE (J/sq cm) Cell Cycle Blockage and Growth Inhibition by HMT. The number of viable cells present 24 hr after treatment was deter Chart 2. Comparison of growth inhibition and effects of HMT plus UV-A treatment on the cell cycle. Bottom, plot of values of cell cycle phases obtained mined parallel with measurements of the cell cycle following from the curve-fitting analysis (1). Top curve, degree of growth inhibition, deter treatment with HMT and UV-A (Chart 2). By increasing the dose mined by counting the number of viable cells at 0 time and at the end of the of UV-A at constant (3 /IM) HMT concentration, 100% growth experiment. inhibition was attained by doses above 0.3 J/sq cm. At 0.3 J/sq cm, cells that were leaving G, accumulated in roughly - 100 equal numbers in the S and G2 phases. Following higher UV-A o doses (2 J/sq cm), the growth of cells was stopped at the ^ 80 OD phase in which they were at the time of treatment. Similar £ results were obtained when the HMT concentration was in 60 U) creased at constant (0.3 J/sq cm) UV-A dose. At 1 IXMHMT 5 u, 40 concentration, growth was inhibited by 60%, and the cells were _l blocked primarily in the G2 phase. HMT (3 ^M) caused a nearly o complete inhibition of growth with cells accumulating in equal o 20 numbers in the S and G2 phases at the expense of Gì.At UJ higher HMT levels, the cell cycle phase distribution of the o 230 arrested cells was similar to that of the untreated cells. DAYS
At HMT concentrations that caused G2 and S blocks, the Chart 3. Kinetics of cell cycle phase distribution. The cell cycle was analyzed by flow cytometry. A, untreated control; B, cells treated with 2 ftM HMT plus UV- A irradiation (0.3 J/sq cm). This experiment has been performed 3 times with qualitatively similar results each time. 4O§2X3UJOtt0
effects of PUVA treatment on growth and cell cycle were found to be temporary. Similarly to our observations made by using trioxsalen (3), cells treated with HMT recovered their ability to grow within 2 days after the blockage and become indistin guishable from untreated cells in terms of growth rate and cell —1.0i!yÕO/M!iA; .30\lOO^MA cycle phase (data not shown). 4{E|2 Kinetics of Cell Cycle Changes. The distribution of cell cycle phases was analyzed by flow cytometry at 0, 1, 2, and 3 days following treatment of cells with HMT plus UV-A together
2AtB.CortTotUControl-A;jUV-A*J.'sqcn04HMT=cpuM)'/•¡1^M'L/WQl/l1PJLe-3/ M2.0k30^Miziv. \. first day; subsequently, it declined to 11%. The G2 compart ment showed a steady increase from 9 to 83% during the same DNA CONTENT (RELATIVE) Chart 1. Effects of UV-A irradiation and HMT on the cell cycle. DNA histo period. A reversal of this trend was seen from the second to grams were obtained by flow cytometry. A, first histogram, untreated control; the third day following treatment (Chart 38). second to the seventh histograms, UV-A dose increased from 0 to 3 J/sq cm. The depletion of the Gìcompartment and simultaneous ac The HMT concentration was kept constant at 3 UM B, first histogram, untreated control; second to the seventh histograms, HMT concentration increased from 0 cumulation of cells in the S and G2 phases could have been to 100 UMwhile a constant dose of UV-A irradiation (0.3 J/sq cm) was maintained. caused either by a blockage of cells in S and G... or alterna- 2224 CANCER RESEARCH VOL. 42 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. Effects of Psora/ens on the Melanoma Cell Cycle lively, psoralen plus UV treatment could have stimulated cells tion causes the accumulation of cells in the G2, S, or all phases to enter DNA synthesis. The kinetics of cell cycle phase distri of the cell cycle, depending on both the dose of UV-A irradiation bution showed that while the G, population decreased from 65 and the concentration of HMT. Parallel with cell cycle analysis, to 19% during the first 24 hr (with a simultaneous increase in we have determined the amount of [3H]HMT bound to DNA and the S and G2 populations), cell growth was arrested completely. found that at a low level of HMT binding (corresponding to By Day 2 following treatment, there were only 5% cells left in about one molecule bound per 106 base pairs) cells accumulate the Gìcompartment; the percentage of S phase decreased in the G2 phase. We explain the emergence of G2 block by from 43 to 11,..and 83% of the cells had G2 DNA content. assuming that an extremely sparse substitution of HMT to DNA Parallel with these changes, the number of viable cells did not (corresponding to approximately 1 molecule bound through change significantly. Since these dramatic changes in the cell the length of 200 replicons; Ref. 17) may allow DNA replication cycle occurred while the growth of cells was arrested, accu to proceed but inhibit cells to enter mitosis by preventing the mulation of cells with S and G2 DNA content can be explained separation of DNA strands cross-linked by HMT. The fact that by an inhibition of cell cycle traverse by the treatment with angelicin, a monofunctional angular psoralen, had no similar psoralen plus UV. effect on the cell cycle (data not shown) points to the impor Binding of [3H]HMT to DNA. Cells were exposed to [3H]HMT tance of diadduct formation by HMT. Also, in another system, under conditions identical to those used in the cell cycle Chinese hamster cells were treated with trioxsalen under sim studies. DNA was extracted and fractionated, and the bound ilar conditions as used in our studies, and 11 % of the DNA- [3H]HMT was determined as described in "Materials and Meth bound psoralen was found to be in the form of diadducts (2). ods." "Dark-binding" was measured, and it was found to be in By increasing the number of substitutions, DNA replication is the range of 1.5 to 9.3% of the values obtained following UV- hindered and cells accumulate in the S phase. It is interesting A treatment. The uptake of [3H]HMT into DNA increased linearly to note that a relatively small increase in HMT concentration from about 1 to 5 molecules of [3H]HMT per 106 base pairs as (I.e., 1 to 3 /iM) changes the proportion of cells in G2 relative to the free [3H]HMT concentration increased from 0.7 to 3.5 JUM S from 2:1 to 1:1 (see Chart 2B). The dose dependence of cell (Chart 4). In parallel experiments, the effect of [3H]HMT on the cycle blocks could then explain the dichotomy between earlier cell cycle was investigated and found to be the same as that of data, that showed DNA synthesis inhibition as the major effect cold HMT (data not shown). of psoralens (2,19, 21 ), and our own observation, that showed G2 block when low concentrations of psoralens were used (3, DISCUSSION 5). We hypothesized earlier that enhanced cutaneous pigmen The cytostatic effects of furocoumarins are explained by tation, following the exposure of skin to psoralens plus UV light, their intercalation with DNA in a "dark reaction" (6) followed was due to cell cycle modulation of normal skin melanocytes by the formation of covalently linked mono- and diadducts (3). Since activation of tyrosinase and formation of melanized upon absorption of UV-A (4,10). We have shown that treatment granules take place in the G.. phase (20), blocking the cells in of melanoma cells with HMT in combination with UV-A irradia- the same phase could have had a positive effect on pigmenta tion. Indeed, melanoma cells treated with 0.2 JIM trioxsalen plus UV-A accumulate in G2 (5). However, as we have now K ä shown, whether cells are blocked in the G2, S, or G, phases depends on a narrow margin of the dose of PUVA treatment. l m For example, treatment of cells with 1 ¡MHMT plus 0.3 J/sq cm UV-A blocked cells in G2. However, when the dose of UV irradiation was increased 3-fold, cells were arrested in G, plus S (Chart 2A). From these dose-related effects, we can antici pate that there is an optimal dose of PUVA treatment to en § I- 3 hance pigmentation. Such an UV dose optimum has been found earlier for the stimulation of tyrosinase enzyme (3). The concentration-dependent effects of psoralens may pro vide a tool to modulate the cell cycle to increase the effective ness of cell cycle-dependent anticancer drugs. As an example, melanoma cells of the same strain that were used in these studies can be destroyed selectively by an MSH:daunomycin conjugate that is recognized by MSH receptors present on cells in the G2 phase of the cell cycle (22). However, the cell cycle dependence of MSH receptors permits cells to escape l 2 [SH] HMT ADDED from chemotherapy, thereby reducing the therapeutic effec tiveness of the conjugate. The use of MSH:daunomycin in Chart 4. Binding of [3H]HMT to DNA. In parallel experiments, cells were exposed to | :H]HMT plus UV-A. the DNA was extracted and banded in CsCI combination with PUVA treatment, which blocked melanoma gradient. The concentration of [3H]HMT was measured in a liquid scintillation cells in the G2 phase, has increased the cytotoxic effectiveness counter, and the corresponding DNA concentration was determined by UV of the conjugate (21). absorbance of the solution at 265 nm. The number of [3H]HMT molecules bound per 10" base pairs was calculated, using the specific radioactivity of [3H]HMT (4 In conclusion, we have correlated the effects of HMT on the x 10' cpm//ig) and the extinction coefficient of E = 6600 M~' (phosphorus) for cell cycle of melanoma cells with the amount of radiolabeled DNA. HMT bound to the DNA of the same strain of cells. At a very JUNE 1982 2225 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. J. M. Varga et al. low level of binding, HMT blocks cells in the G2 phase of the DNA photoreaction: controlled production of mono- and di-adducts with nanosecond ultraviolet laser pulses. Science (Wash. D. 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Varga, Gary Wiesehahn, James C. Bartholomew, et al. Cancer Res 1982;42:2223-2226. Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/42/6/2223 E-mail alerts Sign up to receive free email-alerts related to this article or journal. Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected]. Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/42/6/2223. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research.