Effect of Dihydroxyanthraquinoneand Radiationon G2progre@Sion1
Total Page：16
File Type：pdf, Size：1020Kb
[CANCER RESEARCH 40, 42-46, January 1980] 0008-5472/80/0040-0000$02.00 Effect of DihydroxyanthraquinoneandRadiationon G2Progre@sion1 Bruce F. Kimler Radiation Biology Laboratory, Department of Radiation Therapy, University of Kansas Medical Center, Kansas City, Kansas 66 10@3 ABSTRACT MATERIALS AND METHODS The effect of dihydroxyanthraquinone (DHAQ), a potential Cell and Culture Conditions. Chinese hamster ovary cells anticancer chemotherapeutic agent, on the progression of were maintained in exponential groMh as monolayer cultures Chinese hamster ovary cells into mitosis and on the division at 37°in6% CO2. McCoy's modified@Medium5Awas supple delay induced by ionizing radiation was studied using the mented with 10% fetal calf serum and penicillin plus strepto mitotic selection procedure for cell cycle analysis. mycin (KC Biological, Lenexa, Kans.) Under these conditions, Following the addition of DHAQ, the number of mitotic cells the cells displayed a doubling time of pproximately 14 hr with @ selected from an asynchronous population remained unaltered G1 5 hr, S 7 hr, G2 i .5 hr, and 0.7 hr as determined for a refractory period and then decreased. This effect was by autoradiographic studies of sync onous cells. Cells were concentration dependent with transition points between the S/ monitored periodically with a fluoresc nt stain technique (i ) to G2 boundary at iO-4 @.tg/mland the G2/M boundary at :i02 assure the absence of Mycop!asma c ntamination. @g/ml.The duration of the transient division delay was de Mitotic Selection Procedure. The itotic selection proce pendent upon the concentration of drug used and the duration dure for cell cycle analysis (6) was utilized to monitor the ofpulseexposure. perturbatory effects of DHAQ upon elI cycle progression. When cells were treated with pulses of DHAQ in addition to Briefly, ten 75-sq cm tissue culture fI ks containing approxi X-irradiation, there was no change in the location of the radia mately I 0@asynchronous cells in e ponential growth were tion transition point. There was an increase in the duration of shaken for 20 sec every i 0 mm on a horizontal shaker. The division delay compared to that produced by X-ray alone that medium, now containing the detached, ounded-up cells in late was dependent upon the concentration and duration of drug mitosis, was collected on ice and repl ed with i 0 ml of fresh treatment. medium. The cell suspension was c unted using a Coulter The effect of DHAQ is similar to that of other cancer chem counter coupled to a frequency size a lyzer to verity that the otherapeutic agents (Adriamycin, bleomycin, and lucanthone), collected cells were predominately mit tic cells with a volume and the same cautions should therefore be considered when twice that of the smallest (G1) cells. @Themitotic index, as combining DHAQ and radiation for clinical use. determined by fixing and staining with acetoorcein, was cus tomarily greater than 90 to 95%. This chnique resulted in a record of the number of cells that mov into a narrow region INTRODUCTION of mitosis, approximately 4 to 22 mm ior to cytokinesis (6), DHAQ2(NSC 279836) is a newly synthesized compound that i.e., the selection window, every i 0 in (the mitotic rate). has been shown to be effective in preliminary antitumor testing Treatment (9 treatment flasks and 1 co rol flask) was accom (8). At present, the drug is entering Phase I clinical investiga pushed by either irradiating the flask or replacing the control tions. Since the compound is structurally related to a family of medium with medium containing the de ired concentration of intercalating antibiotics that includes Adriamycin, actinomycin drug for the required number of shakes. D, and lucanthone, care must be taken to avoid any significant X-lrradiation. A Norelco300 kVp X-ra unitwas operatedat problem with normal tissue damage. Preliminary data suggest 10 ma with i .2 mm aluminum addition I filtration (half-value that this compound has much less cardiotoxicity and nephro layer, i .0 cm aluminum), producing a se rate of i .2 Gy3/ toxicity than do the conventional antibiotics. It seems likely that mm at 45 cm. The irradiation procedure cessitated the flasks DHAQ will one day be utilized with radiation therapy in a being out of the incubator at room tem rature for 4 mm, but combined modality approach. Therefore, it seems prudent to this did not cause any significant pertur ation of mitotic rate. establish some guidelines by which to estimate the potential DrugTreatment. DHAQwas obtained omthe DrugSynthe for synergistic action of the modalities. The mitotic selection sis and Chemistry Branch, Division of C ncer Treatment, Na procedure for cell cycle analysis (6) was used to investigate tional Cancer Institute. The drug was diss Ived In balanced salt the effects of DHAQ on cell cycle progression, the concentra solution, sterilized by filtration, and stor as a concentrated tion-dependent blockade in the G2 phase, and the interaction solution in the freezer until needed. Pr' r to treatment, the of DHAQ and radiation-induced division delay. It is hoped that concentrated solution was diluted in co plete medium. The these results will assist the oncologist in the design of combined drug solutions were protected from hg t so as to prevent modality therapeutic schedules using radiation therapy and photodegradation. Drug-containing medi at the appropriate DHAQ so as to avoid the severe complications that resulted concentrations was added to the flasks fo the desired number from the early use of actinomycin 0 and Adriamycin in combi of shakes before control medium was onc again used. nation with radiation therapy. RESULTS 1 Supported in part by Mid-America Cancer Center Program, National Cancer The effect of a 60-mm exposure to various concentrations of Institute Grant 5 P30 CAl 5080-04. DHAQ (1 o-@ to i 0°jig/mI) on the selectiofl of mitotic Chinese 2 The abbreviation used Is DHAQ, dihydroxyanthraquinone. Received April 30, 1979; accepted October 4, 1979. 3 One Gy = 100 red. 42 CANCER @ESEARCHVOL.40 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1980 American Association for Cancer Research. @@@@ @8O I DHAQ-Radiation Division Delay hamster ovary cells is shown in Chart i . For comparison, the tion of this experiment. In other experiments, longer treatment effect of X-irradiation (2.0 Gy) on the induction of a progression times produced the same lower plateau region with no recovery blockade (midpoint, 40 mm) and the subsequent period of as long as the drug was present. division delay (206 mm) is also shown. Mitotic cells continued The midpoint of progression blockade as a function of the to enter the selectionwindowat the controlrate for a periodof logarithm of DHAQ concentration is plotted in Chart 2. The 40 to 80 mm after initiation of drug treatment and then declined composite curve from 14 experiments was described by a to a concentration-dependentminimum.The midpointof pro straight line since the midpoint occurred closer to mitosis with gression blockade (the time from initiation of treatment to the increasing drug concentration. Identical midpoints were ob time at which the mitotic rate is 50% of the control; Ref. 5) tamed for pulse exposures (i 0 to 60 mm) as for continuous decreased with increasing concentration of DHAQ. The in treatment, indicating that the response is indeed the result of creasing concentration also resulted in a progressive shorten different points of action in G2 and is not due to different rates ing of the time that cells were selected at the control rate. of drug diffusion, uptake, or intercalation. A plateau was at Concentrations of i 0@ and 10_2 @g/mldid not completely tamed where the midpoint did not decrease further with in reduce the number of cells selected per shake to the constant creasing concentration. This minimum value is coincident with background level of nonmitotic cells and debris over the dura the value obtained for radiation-induced blockade, as has been shown for a number of cancer chemotherapeutic agents (5). @@T_ I I ‘ ‘ The mean value from i 4 experiments for the X-ray transition . 2.0 Gy X-ray EXP K 98 point (36 mm) was used to define T0(see below). It was often DHAQ difficult to obtain data for concentrations above i 0' @ig/mldue to the increasedselectionof nonmitoticcells. However, in no experiment was there a drug transition point less than the X i05 ray transition point, confirming the concept of a minimum transition point. The linear portion of the curve was calculated w by least-squares regression analysis from i O@to i 02 @sg/mlto I fit the equation U, w 0. T= T0+ m log(C0/C) ‘I, .J -J where T is the midpoint value in mm prior to the selection Midpoint window, T0is the minimum transition point attained (36 mm), m 40mm is the slope constant to be derived, C0 is the concentration at 64 mm which T0is obtained (i 02 ,@g/ml;2.2 x i 02 SM), and C is the 75 mm independent variable concentration. The value of i 0 for the 85 mln slope constant is significantly lower than are the values for actinomycin D, Adriamycin, lucanthone, mitomycin C, and bleo V lO@}tg/ml 105 mln -a mycin (a range of 25 to 68) that have been previously reported @ ‘U- iôo 200 300 400 500 (5). This difference cannot be due to the choice of a value of TIMEAFTERINITIATIONOFTREATMENT,MINUTES 102 @sg/mlfor C0 since Co would have to change by several Chart 1. Effect of various concentrations of DHAQ on the selection of mitotic orders of magnitude to appreciably alter the value of the slope cells.