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[CANCER RESEARCH 43, 541-545, February 1983] 0008-5472/83/0043-0000502.00 Cytotoxicity of Alkyl-lysophospholipid Derivatives and Low-AlkyI-Cleavage Enzyme Activities in Rat Brain Tumor Cells Wolfgang E. Berdel, 1 Eva Greiner, 2 Ulrich Fink, Dimitrios Stavrou, Anneliese Reichert, Johann Rastetter, Dennis R. Hoffman, 3 and Fred Snyder 4 Division of Hematology and Oncology, Department of Medicine I, Technical University [W. E. B., E. G., U. F., A. R., J. R.], and Department of Functional Pathology, Ludwig-Maximilians-University [D. S.], 8000 Munich, Federal Republic of Germany, and the Medical and Health Sciences Division, Oak Ridge Associated Universities, Oak Ridge, Tennessee 37830 [D. R. H., F. S.] ABSTRACT Alkyl-lysophospholipids (ALP) and related derivatives inhibited the in v i t r o incorporation of [3H]thymidine into seven different permanent cell lines derived from rat brain tumors. The cytostatic effect of ALP was dependent on dosage and incubation time. Naturally occurring 2-1ysophosphatidylcholine did not exhibit cytostatic effects; under these conditions, the incorporation rates of [3H]thymidine were generally more than 100% of the controls. The trypan blue dye exclusion test, which was used to assess severe cell damage, correlated with the extent that [3H]thymidine incorporation was inhibited by ALP. Preincubation of ALP ( r a c - 1 -octadecyl-lyso-glycero-3-phosphocholine) for more than 8 min with a tetrahydropteridinedependent O-alkyl cleavage enzyme preparation from rat liver microsomes destroyed almost all of the cytotoxic properties of ALP when tested at a concentration that previously inhibited tumor growth by more than 50%. [3H]Thymidine incorporation rates were greater than 1 00% for astrocytoma cells incubated with ALP after exposure to the alkyl cleavage enzyme. Comparison of the microsomal activities of the tetrahydropteridine-dependent alkyl-cleavage enzyme present in astrocytoma 78-FR-G-299 cells and the pleomorphic glioma 78-FRG-21 9 / $ 4 cells to that found in normal skin fibroblasts and rat livers revealed a markedly reduced activity in the neoplastic cell lines. Moreover, those tumor cells that were more resistant to ALP cytotoxicity (pleomorphic glioma, 7 8 - F R - G - 2 1 9 / S 4 ) had a 3-fold higher tetrahydropteridine-dependen t cleavage activity than a more cytotoxic sensitive line (astrocytoma cells, 78-FR-G-299). Our results indicate that the Iow-alkyl-cleavage enzyme activities in these neoplastic cells in comparison to normal cells might be a factor in explaining the relatively high cytotoxicity of ALP in tumor cells. INTRODUCTION ALP 5 and related derivatives (synthetic analogues of naturally Received November 19, 1981; accepted November 4, 1982. 1 Supported by Deutsche Forschungsgemeinschaft Grants Be 822/2-2 and Sta 134/2. To whom requests for reprints should be addressed: Division of Hematology and Oncology, Department of Medicine I, Technical University, Ismaninger Str. 22, 8000 Munich 80, Federal Republic of Germany. 2 In partial fulfillment of the M.D. degreeof the Technical University of Munich. 3 Fellowship supported by Grant CA 09336 awarded by the National Cancer Institute to the University of Tennesseeand the Oak Ridge National Laboratory. 4 Supported by the Office of Energy Research, United States Department of Energy (Contract DE-AC05-760R00033), and the American Cancer Society (Grant BC-70M). 5 The abbreviations used are: ALP, alkyl-lysophospholipids; Pte.H4, tetrahydropteridine; ET-18-H, rac-l-octadecyl-2-hydride-glycerophosphocholine; ET18-OH, rac-l-octadecyl-lyso-glycerophosphocholine; ET-18-OCH3,rac-l-octadecyl-2-methoxy-glycerophosphocholine; ET-12-H, rac-l-dodecyl-2-hydride- occurring 2-LPC) represent a new kind of antitul"nor agent with immune-modulating potential (1 5). It has been shown that ALP inhibits the growth of human leukemic cells (1, 24), hypernephroma cells (6), cells from various urological tumors (7), and different human malignoma cells (4) in vitro. In contrast, human embryonic fibroblasts (7) as well as bone marrow cells (1, 1 4) are less affected by the same doses of ALP that are toxic to tumor cells. In vivo, ALP is also able to prevent or retard tumor growth and to inhibit metastasis in several syngeneic tumor models in mice (3, 5, 1 5, 23). The mode of action for the antineoplastic activity of ALP has been explained as a direct cytotoxic activity on tumor cells and on their ability to generate tumoricidal macrophages (5, 8, 9). In contrast to normal cells, neoplastic tissues are characterized by the lack of a Pte.H4-dependent ether cleavage enzyme activity (1 8). Therefore, ether-linked lipids are not catabolized in tumors as they are in normal cells, and the accumulation of the " l y s o " type of ether phospholipids can interfere with vital pathways of phospholipid metabolism of the cell (1 4). It appears likely that the direct cytotoxic effect of ALP is dependent on the ether-linked alkyl moiety of the glycerophosphocholine lipid and the lower activity of the O-alkyl cleavage enzyme system in tumor cells (1 8). Our study describes the cytostatic and cytotoxic effect of ALP in various rat brain tumor cells and deals with the question of whether 2-LPC, the ester phospholipid analogue, is less cytotoxic than the ether-linked analogue. Furthermore, the influence of pretreatment of the O-alkyl lipid analogues with the Pte. H4-dependent O-alkyl cleavage enzyme on the tumor cytotoxicity of ALP has been tested. Data were also obtained that demonstrate that the cytotoxicity of ALP is inversely related to the microsomal activities of the cleavage enzyme. MATERIALS AND METHODS ALP. ALP (ET-18-H, ET-18-OH, ET-18-OCH3, and ET-12-H) were kindly provided by Dr. H-U. Weltzien (Max-Planck-lnstitute for Immunobiology, Freiburg, Federal Republic of Germany). Details about their chemical structure and synthesis are given elsewhere (2, 4, 15, 25). The ALP and the naturally occurring ester analogue, 2-LPC (ES-16/ 18-OH; Fa. Roth, Karlsruhe, Federal Republic of Germany) were dissolved in RPMI Medium 1640 (GIBCO No. 240; Gibco, Glasgow, Scotland), containing 10% fetal calf serum (GIBCO No. 176), sterilized by micropore filtration (0.22/~m; Millex; Millipore, Molsheim, France), and stored at - 2 0 ~. Cells. Seven permanent cell lines of malignant rat brain tumors were used in this study. The tumors were experimentally induced in 6- glycerophosphocholine; 2-LPC, (ES-16/18-OH), rac-l-hexadecyl/octadecyllyso-2-glycerophosphocholine, i.e., 2-1ysophosphatidyl-choline; RPMI, Roswell Park Memorial Institute. FEBRUARY 1983 Downloaded from cancerres.aacrjournals.org on August 2, 2017. © 1983 American Association for Cancer Research. 541 W. E. B e r d e l e t al. month-old Fischer (F344) rats by N - m e t h y I - N - n i t r o s o u r e a in the drinking water. Details of induction and cultivation of these cells have been published (20, 22); the histological origin of each cell line is listed in Table 1, whereas the code designating each line is explained elsewhere (21). All cell lines have gone through more than 50 passages. In order to prepare single-cell s u s p e n s i o n s from the monolayers, supernatants were removed from the flasks (75 sq cm; Falcon Plastics, Oxnard, Calif.), and the monolayers were overlaid with 3 to 4 ml of a 0.9%-NaCI solution:trypsin:EDTA solution. Cells were incubated at 37 ~ for 5 rain and then washed from the surface with a jet of medium (RPMI Medium 1 6 4 0 supplemented with 10% fetal calf serum, containing 50 units penicillin and 50 #g streptomycin per ml). The cells were gently pipeted into tubes, centrifuged at 2 5 0 x g for 10 rain, and resuspended in medium to a final concentration of 1 x 105 c e l l s / m l . Viability of the cells, based on trypan blue dye exclusion (11 ), was greater than 95%. [ 3 H ] T h y m i d i n e I n c o r p o r a t i o n . The cytostatic effect of the substances tested was based on a decrease of [3H]thymidine incorporation into the tumor cells. A 100-/~1 aliquot of the tumor cell suspension (1 x 105 c e l l s / m l ) was placed into each well of a Microtiter plate (Greiner, Nurtingen, Federal Republic of Germany), and the ALP (100/~1) was then added at various concentrations in triplicate. Controls contained 100 #1 of pure medium instead of the test substance. Plates were incubated at pH 7.2, at 37 ~ in an atmosphere of 5% CO2 and high humidity for 24, 48, 72, or 96 hr. All cultures were pulsed for 24 hr with 0.5/~Ci of [6-3H]thymidine per well (5 C i / m m o l ; Amersham and Buchler, Braunschweig, Federal Republic of Germany). The samples were harvested and radioassayed after freezing and thawing as described previously (7); standard deviations were less than -4-_10%. E n z y m a t i c Studies. Microsomes were isolated from the livers of Fischer (F344) rats (17). The ALP used as substrate was ET-18-OH. The standard assay system (16) was modified as follows. Fifty/~g of the microsomal protein suspended in 0.25 M sucrose were added to the system containing 30, 40, 50, or 60 #g of ET-18-OH, Pte.H4 (1 mM), reduced glutathione (5 raM), and (NH4)2SO4 (5 raM) dissolved in borate (0.1 mM):NaOH buffer at pH 8.8; the final volume was 1 ml. Samples were incubated for 4, 8, 15, 30, 60, or 90 min at 37 ~ in a shaking water bath. The reaction was stopped by placing the samples in an ice bath. O-Alkyl clevage was tested by comparing the cytostatic effect of enzymatically treated ET-18-OH with that of unreacted ET-18OH. An aliquot of 100 #1 of the tumor cell suspension (1 x 10 s c e l l s / ml) was incubated for 24 hr under the conditions mentioned earlier with 100 #1 of the incubation solution in triplicate for each concentration Table 1 Influence of different ALP compounds on [3H]thymidine incorporation into 7 cell lines of rat brain tumors [3H]Thymidine incorporation (% of control) Histology 78-FR-G-299a (astrocytoma) 78-FR-G-284 (mixed glioma) 78-FR-G-344 (mixed glioma) 79-FR-G-41 (astrocytoma) 78-FR-G-219/$1 (pleomorphic glioma) 78-FR-G-219/$2 (pleomorphic glioma) 78-FR-G-219/$4 (pleomorphic ALP Dosage (/~g/ml) Incubation for 1 day Incubation for 2 days ET-18-H 10 20 40 b 20 28 14 ET-18-OH 10 20 38 24 32 17 ET-18-H 10 20 81 36 78 26 ET-18-OCH3 10 20 71 32 30 15 ET-18-H 10 20 130 64 80 72 ET-18-OH 10 20 98 109 90 70 10 20 61 43 69 30 ET-18-OCH3 10 20 55 42 36 17 ET-18-H 10 20 96 65 52 16 ET-18-OCH3 10 20 78 48 18 23 ET-18-H 10 20 75 71 52 53 ET-18-OCH3 10 20 >100 >100 62 60 ET-18-H 10 20 30 102 72 34 41 10 20 30 84 65 22 47 39 22 ET-18-H glioma) ET-18-OCH3 46 19 a For explanation of the code designating the cell lines see Ref 21. b Mean values of triplicate cultures. For culture conditions and standard deviations, see "Materials and Methods." 542 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on August 2, 2017. © 1983 American Association for Cancer Research. Cytotoxic Effect of Ether Lipids in Rat Brain Tumor Cells and incubation time. Control suspensions were incubated with the same concentrations of unreacted ET-18-OH (30, 40, 50, or 60/~g/ ml) dissolved in sucrose-supplemented borate buffer containing Pte. H4, reduced glutathione, and (NH4)2SO4. In order to assess the effect of rat microsomes on tumor cell growth, the control cells were incubated in medium (RPMI Medium 1640 with 10% fetal calf serum) containing supplemented borate buffer and an aliquot of the alkyl cleavage assay system minus ET-18-OH. Cytostasis was measured by [3H]thymidine incorporation; standard deviations were less than _+10%. In another set of experiments, the activity of the Pte.H4-dependent alkyl clevage enzyme was assayed in microsomes as described before (16), except that 1-[9,10-3H]octadecylglycerol (20 #Ci//zmol) served as substrate. Microsomes were prepared from near-confluent cultures of normal human skin fibroblasts (Detroit 551), rat astrocytoma cells (78-FR-G-299), rat pleomorphic glioma cells (78-FR-G-219/S4), and the liver of a male 180-g CDF rat. Microsomal protein (10 to 20/~g) was incubated with the reaction mixture (16) for 10 min. The product, fatty aldehyde, was isolated chromatographically and then analyzed by liquid scintillation spectroscopy (19). Alkyl cleavage of 1-[9,10-3H] octadecylglycerol was linear with rat liver microsomes up to 20 /~g protein and over an incubation time of 20 min. Microsomal protein concentrations were determined according to the procedure of Lowry et al. (13). 150 / ~ p- 5O A Z 0 I + # : + # i : k-- 0 Z RESULTS IJ.J Z Table 1 shows the cytostatic potency of the most effective ALP analogues (i.e., 18-carbon alkyl chains) with 7 established rat brain tumor cell lines. No correlation was found between resistance against ALP and the number of cell passages (data not shown). The cytostatic effect of ALP increased with dose and incubation time (Table 1 ) but varied considerably between different cell lines. The dose-response relationship for the various ALP analogues is shown in Chart 1. Cells of astrocytoma 78-FR-G-299 were incubated with 3 types of ALP and 2-LPC (ES-1 6 / 1 8 - O H ) for 1 , 2 , 3, and 4 days. Naturally occurring 2-LPC was ineffective and in some instances even stimulated the [3H]thymidine incorporation rate above control values (Chart 1 A). The 3 ALP samples revealed a clear dose and time dependence of antitumor action. After 3 days of cultivation of the tumor cells with ALP concentrations of 20 #g/ml, the [3H]thymidine incorporation was less than 1 5% of the controls. However, there were a few exceptions from the dose and time dependence of the ALP-induced cytostasis that lack an explanation (Table 1 ; see 79-FR-G-41 and 7 8 - F R - G - 2 1 9 / S 2 ; also Chart 1). The trypan blue dye exclusion test was performed to make sure that treatment with ALP not only decreased the proliferation of tumor cells but actually caused the death of the cells. Viability expressed as percentage of control correlated well with the corresponding values obtained for the inhibition of [3H]thymidine incorporation (Table 2). Viability of cells treated with ALP also proved to be dependent on incubation time (Chart 2). The requirement of the O-alkyl bond for expression of the antitumor properties of ALP becomes obvious by comparing [3H]thymidine incorporation before and after the incubation of ET-18-OH with a microsomal preparation of the O-alkyl cleavage enzyme, which is a Pte.H4-dependent monooxygenase (Chart 3). Two different sublines of an astrocytoma and 2 different microsomal preparations were tested. The values at an incubation time of 0 min represent the cytostatic effect of unreacted ET-18-OH. Four concentrations of ALP were incu- -II--- 50. I........... "~l--........ u..-f / J ~--"~....... 'lk- ...... 1 ~ D C 2 3 4 1 2 3 4 DAYS OF CULTIVATION Chart 1. [3H]Thymidineincorporation into cells from an astrocytoma line (78FR-G-299) during incubations for 24, 48, 72, or 96 hr with 3 types of ALP or 2LPC (ES-16/18-OH) at different concentrations. See "Materials and Methods" for culture conditionsand standarddeviations. Incubations:A, ES-16/18-OH; B, ET-18-OH; C, ET-18-OCH3; D, ET-18-H. Concentrations(#g/ml): 0, 5; m, 10; A, 20. bated for different times with a suspension of rat liver microsomes containing the Pte. H4-dependent O-alkyl cleavage enzyme as described in "Materials and Methods." Tumor cells were then incubated with the enzymatically treated ET-18-OH. The percentage of metabolized substrate and the kinetics of the enzymatic reaction are reflected by the alteration of the antitumor effect of ALP during the incubation period. At a concentration of 30 # g / m l of ET-18-OH, an incubation of 8 min was sufficient to remove all of the tumor cytotoxicity associated with the ALP based on [3H]thymidine incorporation. Optimal cleavage activity was observed after 30 to 60 min of incubation with a concentration of 40 #g ET-18-OH per ml and an enzyme:substrate relationship of 50/~g protein:40/~g substrate. At a substrate concentration of 60 # g / m l , very little effect on [3H]thymidine incorporation was observed, probably because of an insufficient amount of enzyme for the large amount of substrate incubated. Control samples containing boiled microsomes (data not shown) did not alter the [~H]thymidine incorporation by the cells. Assays of the Pte. H4-dependent alkyl monooxygenase activity in microsomes isolated from cells that are highly sensitive (astrocytoma cells) to ALP cytotoxicity and cells that are more FEBRUARY 1983 Downloaded from cancerres.aacrjournals.org on August 2, 2017. © 1983 American Association for Cancer Research. 543 W. E. B e r d e l e t al. Table 2 Correlation between [~H]thymidine incorporation and viability of rat brain tumor cells as tested with trypan blue dye exclusion after incubation with ALP or 2-LPC (ES-16 /18-OH) 78-FR-G-219/$1 a Substance [3H]Thymidine incorporation c Viability c Control ET-18-H ET-18-OH ET-18-OCH3 ET-12-H ES-16/18-OH 100 73 56 57 71 66 1O0 54 59 34 62 74 78-FR-G-219/$2 a [3H]Thymidine incorporation 1O0 84 75 72 95 94 78-FR-G-344 b Viability [aH]Thymidine incorporation Viability 100 64 73 84 90 95 100 7 24 47 89 93 100 4 20 20 40 90 a After an incubation period of 48 hr with concentrations of 20 #g/ml. b After an incubation period of 24 hr with concentrations of 50/~g/ml. c [3H]Thymidine incorporation and the number of viable cells are expressed as percentage of the control samples, i.e. cells incubated without the test phospholipids. IA 100 /~'-- ~100 ~ g ~ B z o rr o 13.. (3: o (J z- 6 LLI -~-, r'~ u >-1--'I - - ~,_ 50 ~'o (,1 ~ 'k t ...... x I I 1 2 DAYS OF I N C U B A T I O N Chart 2. Correlation between [3H]thymidine incorporation and viability (based on trypan blue dye exclusion) of pleomorphic glioma cells (78-FR-G-219/S1) after 24- or 48-hr incubations with 20-#g/ml concentrations of ALP: 0, ET-18-H; II, ET-18-OCH3. (pleomorphic glioma c e l l s ) to A L P cytotoxicity vealed that the latter had a 3 times higher O-alkyl re- cleavage a c t i v i t y t h a n d i d t h e m o r e s e n s i t i v e t y p e c e l l s ( T a b l e 3). T h e much higher levels of Pte. H4-dependent ties associated with normal these experiments. the human alkyl cleavage activi- c e l l s (1 8 ) w a s a l s o o b s e r v e d Microsomal skin fibroblasts in preparations from rat liver and had alkyl cleavage were 32- and 19-fold (respectively) the astrocytoma cells. 3b 6b 9b '~ - o 4 8 15 30 6o 9o " TINE OF INCUBATION (mini Chart 3. Comparison of the effect of unreacted ET-18-OH (0 min) and enzymatically treated ET-18-OH on [3H]thymidine incorporation into 2 different sublines (A and B) of an astrocytoma. ET-18-OH was incubated with the rat liver microsomal protein and O-alkyl cleavage enzyme reaction mixture for O, 4, 8, 15, 30, 60, or 90 min. See "Materials and Methods" for details. Cells were incubated with 4 different concentrations of reacted and unreacted ET-18-OH. Values are means of triplicate cultures; standard deviations are given in "Materials and Methods." Concentration of ET-18-OH: ~g/ml): V, 3 0 ; . , 40; 0 , 50; A, 60. I resistant ~ TINE OF INCUBATION (rain) :t < :-:--rZ 7 i Table 3 Activity of Pte. H4-dependent alkyl cleavage enzyme in microsomes from normal human skin fibroblasts (Detroit 551), rat astrocytoma cells (78-FR-G-299), rat pleomorphic glioma cells (78-FR-G-219 / $4), and CDF rat liver Specific activity a (pmol//~g protein/min) Microsomal source Rat liver Skin fibroblasts Astrocytoma Pleomorphic glioma 8.85 5.40 0.28 0.88 _+ 0.49 b ___0.91 _+ 0.07 +_ 0.14 a Activity represents cleavage of 1-[9,10-3H]octadecylglycerol as described in "Materials and Methods." b Mean ___S.D. of 2 separate microsomal preparations, each done in duplicate. activities that h i g h e r t h a n t h a t f o u n d in s h o w e d v a r y i n g d e g r e e s o f s e n s i t i v i t y to A L P c y t o t o x i c i t y ( T a b l e 1). A c l e a r d o s e - r e s p o n s e relationship was found (Chart 1), and the cytostatic effect of ALP increased with incubation time DISCUSSION ( T a b l e 1 ; C h a r t 1). O n l y t h e e t h e r - c o n t a i n i n g sessed the cytotoxic Our data indicate that ALP inhibit the incorporation of [3H]thymidine into rat brain tumor cells (10). All cell lines tested 544 activity, whereas logue of the lysophospholipid, substances pos- 2-LPC, the ester ana- was ineffective (Chart 1 A). Results of the trypan blue dye exclusion test correlated well C A N C E R RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on August 2, 2017. © 1983 American Association for Cancer Research. Cytotoxic w i t h r e s u l t s on the i n h i b i t i o n of [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n b y A L P ( C h a r t 2; T a b l e 2). B o t h i n h i b i t i o n of cell p r o l i f e r a t i o n and cell d e a t h o c c u r after A L P t r e a t m e n t of the cells. T h e s e r e s u l t s s u p p o r t p r e v i o u s s t u d i e s , in w h i c h a c y t o t o x i c e f f e c t of A L P w a s o b s e r v e d w i t h h u m a n l e u k e m i c cells (1, 24), h y p e r n e p h r o m a cells (6), and cells from v a r i o u s u r o l o g i c a l t u m o r s (7). A f t e r e n z y m a t i c t r e a t m e n t of ET-1 8 - O H w i t h the m i c r o s o m a l p r e p a r a t i o n c o n t a i n i n g the P t e . H 4 - d e p e n d e n t alkyl c l e a v a g e e n z y m e , the c y t o t o x i c e f f e c t of the p h o s p h o l i p i d w a s a l m o s t c o m p l e t e l y r e m o v e d ( C h a r t 3). T u m o r cells, in c o n t r a s t to normal cells, e x h i b i t m u c h l o w e r levels of alkyl c l e a v a g e a c t i v i t y (Ref. 1 8, T a b l e 3). T h e s e r e s u l t s e x p l a i n w h y n o r m a l cells t h a t c a n e f f e c t i v e l y c l e a v e the e t h e r l i n k a g e are less a f f e c t e d b y A L P (1, 7, 1 4). Also, o n l y the a n a l o g u e s c o n t a i n i n g an O - a l k y l b o n d e x h i b i t c y t o t o x i c a c t i v i t y w i t h the t u m o r cells. T h e e s t e r p h o s p h o l i p i d s , e . g . , 2 - L P C , w e r e i n a c t i v e (Refs. 1 a n d 14; see also C h a r t 1). T h e d i f f e r e n c e in c l e a v a g e e n z y m e a c t i v i t i e s b e t w e e n t u m o r and normal cells and the f a c t t h a t A L P is a d d e d in m e d i u m c o n t a i n i n g s e r u m , w h i c h b i n d s A L P , t e n d s to rule o u t the o c c u r r e n c e of a d i r e c t c y t o l y s i s via a d e t e r g e n t a c t i v i t y of the l y s o p h o s p h o l i p i d s . It is k n o w n t h a t after e x p o s u r e of n e o p l a s t i c cells to A L P s e v e r a l vital p a t h w a y s of p h o s p h o l i p i d m e t a b o l i s m are d i s t u r b e d , w h e r e a s in normal cells t h e s a m e p a t h w a y s are less a f f e c t e d (1 4). T h e s e e a r l i e r i n d i r e c t o b s e r v a t i o n s led to t h e c o n c l u s i o n t h a t the h i g h c y t o t o x i c i t y of A L P in n e o p l a s t i c cells is d u e to the i n a b i l i t y of s u c h cells to d e g r a d e the alkyl e t h e r l i n k a g e (1 4). In the p r e s e n t s t u d y , the d i r e c t d e m o n s t r a t i o n of the i n v o l v e m e n t of the O - a l k y l b o n d a n d t h e c o r r e s p o n d i n g l o w a c t i v i t y of the alkyl c l e a v a g e e n z y m e in n e o p l a s t i c cells ( T a b l e 3) w i t h the a n t i t u m o r a c t i o n of A L P has b e e n d o c u m e n t e d . S u b s t r a t e s p e c i f i c i t i e s in the b i o c l e a v a g e of t h e O - a l k y l b o n d ( 1 2 , 17) m i g h t p r o v i d e an e x p l a n a t i o n for the c o m p a r a b l y h i g h c y t o t o x i c a c t i v i t y of the a n a l o g u e s m o d i f i e d at t h e s n - 2 p o s i t i o n of the m o l e c u l e (1). T h e O - a l k y l m o i e t y ( s n - 1 ) of c o m p o u n d s s u b s t i t u t e d w i t h f u n c t i o n a l g r o u p s at the s n - 2 p o s i t i o n o t h e r t h a n s n 2 - h y d r o x y l are not s u i t a b l e s u b s t r a t e s for the alkyl c l e a v a g e e n z y m e (12, 1 7). S i n c e an o b j e c t i v e t u m o r a n d l e u k e m i a res p o n s e has b e e n o b s e r v e d in p a t i e n t s t r e a t e d w i t h ET-1 8 - O C H 3 in a clinical P h a s e I s t u d y (4), the use of A L P - t y p e p h o s p h o l i p i d s in c a n c e r p a t i e n t s , w i t h d u e c a u t i o n , a p p r o a c h to a n t i c a n c e r t h e r a p y . could 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. provide a new 20. ACKNOWLEDGMENTS 21. The authors thank Dr. Gerd Baumann for his helpful discussion and Dr. Tenching Lee and Veronica Fitzgerald for their important contributions to this investigation. 22. REFERENCES 23. 1. Andreesen, R., Modolell, M., Weltzien, H. U., Eibl, H., Common, H. H., Lohr, G. W., and Munder, P. G. Selective destruction of human leukemic cells by alkyl-lysophospholipids. Cancer Res., 38: 3894-3899, 1978. 2. Arnold, D., Weltzien, H. U., and Westphal, O. Synthesen von Cholinphosphatiden. III. Uber die Synthese von Lysolecithinen und ihren Ather-analoga. Ann. Chem. (Justus Liebigs), 709: 234-239, 1967. 3. Bausert, W. Der Einfluss von synthetischen Lysolecithin-analoga auf das 24. 25. E f f e c t o f E t h e r L i p i d s in R a t B r a i n T u m o r C e l l s Wachstum experimenteller Tumoren der Maus. Ph.D. Dissertation, University of Freiburg, Freiburg, Federal Republic of Germany, 1978. Berdel, W. E., Bausert, W. R. E., Fink, U., Rastetter, J., and Munder P. G. Anti-tumor action of alkyl-lysophospholipids (review). Anticancer Res., 1: 345-351, 1981. Berdel, W. E., Bausert, W. R., Weltzien, H. U., Modolell, M. L., Widmann, K. H., and Munder, P. G. The influence of alkyl-lysophospholipids and lysophospholipid-activated macrophages on the development of metastasis of 3-Lewis lung carcinoma. Eur. J. Cancer, 16:1199-1204, 1980. Berdel, W. E., Fink, U., Egger, B., Reichert, A., Munder, P. G., and Rastetter, J. Alkyl-lysophospholipids inhibit the growth of hypernephroid carcinomas in vitro. J. Cancer Res. Clin. Oncol.,101: 325-330, 1981. Berdel, W. E., Fink, U., Egger, B., Reichert, A., Munder, P. G., and Rastetter, J. Inhibition by alkyl-lysophospholipids of tritiated thymidine uptake in cells of human malignant urologic tumors. J. Natl. Cancer Inst., 66: 813-817, 1981. Berdel, W. E., Fink, U., Egger, B., Reichert, A., Munder, P. G., and Rastetter, J. Growth inhibition of malignant hypernephroma cells by autologous lysophospholipid incubated macrophages obtained by a new method. Anticancer Res., 1: 135-140, 1981. Berdel, W. E., Fink, U., and Munder, P. G. Synthetic lysophospholipids render macrophages cytotoxic. In: Fifth Meeting of the International Society of Haematology, European and African Division, Abstract I., p. 24. Hamburg, Germany: University Press, 1979. Berdel, W. E., Greiner, E., Fink, U., Stavrou, D., Reichert, A., Rastetter, J., and Snyder, F. Cytotoxic effect of alkyl-lysophospholipids (ALP) in rat brain tumor cells. Proc. Am. Assoc. Cancer Res., 23:198, 1982. Hudson, L., and Hay, F. C. Practical Immunology, pp. 29-32. Oxford, England: Blackwell Scientific Publications, 1976. Lee, T-c., Blank, M. L., Fitzgerald, V., and Snyder, F. Substrate specificity in the biocleavage of the O-alkyl bond: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and platelet-activating lipid) and its metabolites. Arch. Biochem. Biophys., 208: 353-357, 1981. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with Folin phenol reagent. J. Biol. Chem., 193: 265-275, 1951. Modolell, M., Andreesen, R., Pahlke, W., Brugger, U., and Munder, P. G. Disturbance of phospholipid metabolism during the selective destruction of tumor cells induced by alkyl-lysophospholipids. Cancer Res., 39: 46814686, 1979. Munder, P. G., Weltzien, H. U., and Modolell, M. Lysolecithin analogs: a new class of immunopotentiators. In: P. A. Miescher (ed.), VII International Symposium on Immunopathology, Bad Shachen, Germany, 1976. pp. 411 424. Basel: Schwabe and Co, 1977. Rock, C. O., Baker, R. C., Fitzgerald, V., and Snyder, F. Stimulation of the microsomal alkylglycerol monooxygenase by catalase. Biochim. Biophys. Acta, 450: 469-473, 1976. Snyder, F., Malone, B., and Piantadosi, C. Tetrahydropteridine-dependent cleavage enzyme for O-alkyl lipids: substrate specificity. Biochim. Biophys. Acta, 316: 259-265, 1973. Soodsma, J. F., Piantadosi, C., and Snyder, F. The biocleavage of alkylglyceryl ethers in Morris hepatomas and other transplantable neoplasms. Cancer Res., 30:309-311, 1970. Soodsma, J. F., Piantadosi, C., and Snyder, F. Partial characterization of alkylglycerol cleavage enzyme system of rat liver. J. Biol. Chem., 247: 3923-3929, 1972. Stavrou, D. Beitrag zur Morphologie und Enzymhistochemie experimenteller Tumoren des Zentralnervensystem der Ratte. I. Morphologische Befunde. Acta Neuropathol. (Berl.), 15: 220-230, 1970. Stavrou, D., Hulten, M., Anzil, A. P., and Bilzer, T. The humoral antibody response of rats immunized with chemically modified syngeneic brain cells and glioma ceils. Int. J. Cancer, 26: 629-637, 1980. Stavrou, D., Osterkamp, U., Schroder, B., Anzil, A. P., and Zanker, K. Selected morphological immunocytochemical and growth characteristics of three experimental rat gliomas and of their cells in vitro. Exp. Cell. Biol., 47: 3-21, 1979. Tarnowski, G. S., Mountain, I. M., Stock, C. C., Munder, P. G., Weltzien, H. U., and Westphal, O. Effect of lysolecithin and analogs on mouse ascites tumors. Cancer Res., 38: 339-344, 1978. Tidwell, T., Guzman, G., and Vogler, W. R. The effects of alkyl-lysophospholipids on leukemic cell lines. I. Differential action on two human leukemic cell lines, HL60 and K562. Blood, 57: 794-797, 1981. Weltzien, H. U., and Westphal, O. Synthese yon Cholinphosphatiden. IV. Omethylierte and O-acetylierte Lysolecithine. Ann. Chem., (Justus Liebigs), 709: 240-243, 1967. FEBRUARY 1983 Downloaded from cancerres.aacrjournals.org on August 2, 2017. © 1983 American Association for Cancer Research. 545 Cytotoxicity of Alkyl-lysophospholipid Derivatives and Low-Alkyl-Cleavage Enzyme Activities in Rat Brain Tumor Cells Wolfgang E. Berdel, Eva Greiner, Ulrich Fink, et al. Cancer Res 1983;43:541-545. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/43/2/541 Sign up to receive free email-alerts related to this article or journal. 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