rTHESlS 3 1293 10607 5314 , -—-\ LIBMRY Michigan State University ‘ #— This is to certify that the dissertation entitled CHARACTERIZATION OF A HUMAN TERATOCARCINOMA CELL ASSAY FOR INHIBITORS OF METABOLIC COOPERATION presented by Terrance J. Kavanagh has been accepted towards fulfillment of the requirements for Doctoral degree in Genetics/ Environmental Toxicology Major professor Qflwa C0 74014.0 / ( Date ”‘4‘“‘1 />j /;f~5/ /T L/ James E. Trosko MS U is an Affirmative Action/Equal Opportunity Institun'on 0-12771 MSU RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from .—_— your record. FINES wil] be charged if book is returned after the date stamped below. 57 a??? J s3 / CHARACTERIZATION OF A HUMAN TERATOCARCINOMA CELL ASSAY FOR INHIBITORS OF METABOLIC COOPERATION By Terrance James Kavanagh A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program and Center for Environmental Toxicology 1985 ‘- / W 7 v {3 .. NJ ACKNOWLEDGEMENTS I wish to express my appreciation to Dr.J. E.Trosko and Dr. C.C. Chang for their guidance. inspiration, and support during my predoctoral training. The consultation and helpful suggestions of Dr. T.B. Friedman and Dr. J.V. Higgins are also gratefully acknowledged. I would also like to express my thanks to Drs. AJLC. Medcalifi NLH. Wade, R. Loch, C. Jone. Z.X. Lin. T.H. Chen. C. F. Aylsworth. C. W. Nelsch. R.K. Jensen. S. Sleight. J.I. Goodman, and S.D..Aust for their freindship, assistance, and advice. The technical assistance of Ms. Beth Rupp is also appreciated. Finally, I wish to thank my parents. my family. and my wife Angelika for their unending patience. love. and understanding. ii TABLE OF CONTENTS Page LIST OF FIGURES . . . . . . . . . . . . . V LIST OF ABBREVIATIONS . . . . . . . . . . . Vi INTRODUCTION . . . . . . . . . . . . . . 1 MATERIALS AND METHODS . . . . . . . . . . . 9 1. Characterization of the Metabolic Cooperation Assay . . . . 9 1.1. Chemicals . . . . . . . . 9 l.2.Cells muiCulture Methods . . . . . . 9 1.3. Chromosome Analysis . . . 10 ‘L4u Selection of 6- TG Resistant Variants . . . 10 1.5. Test oleetabolic Cooperation . . . . . 11 1.6. Test of Metabolic Cooperation with Other Human Cells . . .‘ . 11 1.7. Effect of TPA on Metabolic Cooperation Between HTXTG- l and HTP3- 4 Cells . . . . 12 1.8. Effect of TPA Analogues on Metabolic Cooperation . . . . . . . . . . 1s 2. Effects of the Polybrominated Biphenyls FM, 245-HBB. 345-HBB, and 34-TBB on Metabolic Cooperation between HTXTG—l and HTP3-4 Cells . . 15 2.1. Chemicals . . . . . . 15 2.2. Metabolic Cooperation Assay. 16 3. Effects of TPA on Metabolic Cooperation in Teratocarcinoma Cell Embryoid Bodies . . . . 17 3ft Formation of Embryoid Bodies . . . . . 17 3.2. Measuring Metabolic Cooperation in Composite Embryoid Bodies . . 17 3.3. Effects of TPA on Metabolic Cooperation in Composite Embryoid Bodies . . . . . 18 RESULTS» 22 1. Characterization of the Metabolic Cooperation Assay . . . . . . . . . . . 22 1.1. Plating Efficiency and Chromosome Analysis . 22 iii 1.2. Metabolic Cooperation Between HTP3-4 and 6 HTXTG Variants . 1.3. Test of Metabolic Cooperation Between HTXTG- 1 and Other Human Cells . . 1.4. Effect of TPA on Metabolic Cooperation Between HTXTG- 1 and HTP3- 4 Cells . 1.5. Effect of TPA Analogues on Metabolic Cooperation . . . . . . 2. Effects of FM. 245-HBB. 345—HBB. and 34-TBB on Metabolic Cooperation . . . . . . . 3. Effect of TPA on Metabolic Cooperation in Embryoid Bodies . . . . . . . 3(L Formation of Embryoid Bodies 3.2. Metabolic Cooperation In Composite Embryoid Bodies . 3.3. Effect of TPA on Metabolic Cooperation in Embryoid Bodies . . . . . DISCUSSION . . . . . 1. Usefulness of Human Teratocarcinoma Cells as an Assay for Inhibitors of Metabolic Cooperation 2. Inhibition of Metabolic Cooperation by Selective PBB Compounds . . . 3. Human Teratocarcinoma Cell Embryoid Bodies as a Model for Assessing the Deleterious Effects of Chemicals on Early Human Embryos CONCLUSIONS . LITERATURE CITED iv Page 22 22 28 36 36 41 41 41 45 51 51 56 6O 69 71 LIST OF FIGURES Figure l. 2. 10. 11. 12. 13. 14. 15. 16. Protocol for Metabolic Cooperation Assay Protocol for Embryoid Body Metabolic Cooperation Assay . . . . . . . Karyotype of HTP3-4 Cells Appearance of HTP3-4 Cells in Monolayer Cultures Metabolic Cooperation Between Six X-ray Induced 6-TG Resistant Variants and HTP3-4 Cells . . Metabolic Cooperation Between HTXTG-l Cells and Other Human Cells . . . . . . . . Effect of TPA on Metabolic Cooperation Between HTXTG—l Cells and HTP3-4 . . Effect of TPA on HTXTG-l Cell Colony Morphology . Recovery of HTXTG-l Cells Cocultured with Various Densities of HTP3-4 Cells by TPA . . . . Effect of TPA Analogues on Metabolic Cooperation Effect of Various PBB Compounds on Metabolic Cooperation . . . . . . Appearance of Embryoid Bodies . . . . . . Metabolic Cooperation in Embryoid Bodies Effect of TPA on Metabolic Cooperation in Embryoid Bodies . . . . . . . . Effect of TPA on Compaction of Embryoid Bodies Recovery of HTXTG-l Cells from Embryoid Bodies Treated With TPA and 6-TG at Various Concentrations Page 14 20 24 26 27 29 31 33 35 38 4O 43 44 46 49 SO AHH cAMP DMSO FCS FM GGT 245-HBB 345-HBB HGPRT 3—MC MEZ PB PBB PBS PCB 4 -PDD POD 34-TBB 2.3.7.8-TCDD 6-TG TPA LIST OF ABBREVIATIONS aryl hydrocarbon hydroxylase adenosine 3'.5'-monophosphate dimethylsulfoxide fetal calf serum Firemaster BP—6 gamma-glutamyl transpeptidase 2,2'.4,4'.5.5'-hexabromobiphenyl 3,3'.4.4',5.5'-hexabromobiphenyl hypoxanthine—guanine phosphoribosyltransferase 3-methylcholanthrene mezerein phenobarbital polybrominated biphenyl phosphate buffered saline polychlorinated biphenyl 4 -phorbol-12.13-didecanoate phorbol—12.lB-didecanoate 3.3',4.4'-tetrabromobiphenyl 2,3.7.8-tetrachlorodibenzo-p-dioxin 6—thioguanine lZ-O-tetradecanoylphorbol—13-acetate vi INTRODUCTION Gap junction mediated intercellular communication has been regarded as an important determinant for normal cell growth and differentiation, reproduction, neuroendocrine function. cardiac function. and a whole host of other normal physiological states (Andrew gt git, 1981; Brower and Schultz. 1982; Decker and Freind. 1974; De Mello. 1982, 1984; Gilula. 1980; Gilula gt al.. 1976; Larsen gt a1.. 1981; Lawrence gt git, 1978; Loewenstein. 1979. 1981; Mark _t gig. 1973; Midgeley. 1983) . Modulation of this phenomenon by either natural physiological states or by xenobiotics has been demonstrated in many studies (Hooper. 1982: Larsen. 1983; Pitts, 1980; Trosko and Chang, 1984). Inhibition of this form of intercellular communication has been considered an attributing factor in carcinogenesis (Trosko and Chang. 1981; Trosko gt git, 1983a; Enomoto gt gig, 1984; Potter, 1983), teratogenesis (Trosko gt gig. 1982a; Loch-Caruso gt gig, 1984; Welsch and Stedman. 1984; Warner gt git. 1984). reproductive dysfunction (Malcolm gt gt” 1984; Loch and Trosko. 1985), and other chemically induced disease states (Trosko and Chang, 1981. 1984). Intercellular communication is a process necessary for homeostasis in organisms composed of functionally specialized cells. Gap junctions have probably evolved to provide one 1 communication mechanism by which groups of cells could provide coordinated responses to external stimuli. When this communication is altered through perturbations of gap junctional coupling. the consequences can clearly be deleterious. One such example might be the perturbation of gap junction mediated intercellular communication induced during the process of chemical carcinogenesis. A wide range of experimental evidence indicates that chemically induced cancer is a multistage process (Cairns. 1978; Cairns and Logan. 1984) consisting of a primary genetic event which results in irreversible "initiation" of cells. and the secondary outgrowth of some of these cells. or "promotion". This initiation/promotion model of chemical carcinogenesis has gained support from many 1_ titg_and t_ liELQ studies. Initiation is thought to be a consequence of gene mutation. However. the exact mechanisms underlying promotion are not known. although a role for inhibited intercellular communication has been proposed (Trosko and Chang. 1980). This hypothesis offers the following scenario: Under the influence of a tumor promoter. one might hypothetically envisage the release of an initiated cell from the growth controlling regulation provided by surrounding cells with which it had been "cooperating" metabolically. If this cell could preferentially multiply in response to the promoter. it might ultimately reach the preneoplastic nodule stage. where further genetic damage and neoplastic progression could take place. This model predicts that under 3 some circumstances. the rate limiting step in chemical carcinogenesis may be the promotion phase. since the effective target size for secondary genetic events would be determined by the relative number of preneoplastic cells. Hence. chemicals which promote the isolation and growth of initiated cells by blocking intercellular communication may influence the probability that multiple hits occur in these cells. Since the initial observations that 12-O-tetradecanoyl- phorbol —13-acetate (TPA) (a powerful tumor promoter in the mouse skin initiation/promotion model (Boutwell. 1974; Slaga t git. 1982)) was capable of inhibiting metabolic cooperation between mammalian cells 1 tjt£31(Yotti gt gig, 1979; Murray and Fitzgerald. 1979). TPA and related phorbol esters have been shown to inhibit metabolic cooperation in many different mammalian cell types. including human cells (Davidson gt git. 1984b; Enomoto gt git. 1981a. 1981b. 1984; Fitzgerald and Murray. 1979. 1982; Friedman and Steinberg. 1982; Guy gt 21;: 1981; Kinsella. 1981; Mosser and 80115. 1982; Newbold and Amos. 1981; Trosko gt git. 1981c; Umeda gt git. 1980; Vitkauskas gt gig. 1983; Walder and Lutzelschwab. 1984). These observations have recently been extended to include 1. 1112 experiments in which a dose dependant decrease in the number of gap junctions in the epidermis of TPA treated mice was reported (Kalimi and Sirsat. 1984a. 1984b). The inhibition of metabolic cooperaton by many different classes of tumor promoters besides the phorbol esters. in a variety of cell types has now been well documented (Aylsworth gt al.. 1983; Chen gt gig. 1984; Fitzgerald gt gig. 1983; Hartman and Rosen. 1983; Jone gt git. 1982; Kurata. 1982; Loch—Caruso gt git, 1984; Malcolm and Mills. 1983. 1985; Noda gt 21;: 1981; Rubenstein gt 21:1 1985; Slaga gt git. 1981; Telang gt gig. 1982; Trosko gt gig. 1980. 1981a. 1982b. 1981b; Tsushimoto gt git. 1982a. 1982b. 1983a. 1983b; Umeda gt git. 1980; Walder and Lutzelschwab. 1984; Warren gt 21;: 1982; Williams gt gig, 1981. 1984). Because gap junctional communication is implicated in cellular growth and differentiation. it is clear that disruption of this communication during embyronic development could lead to abnormal histogenesis and morphogenesis. TPA has been shown to adversely affect development in several developmental systems including coelenterates (Shiba. 1981). amphibians (Ellinger. 1982). and rodents (Huber and Brown. 1983; Sawicki and Mystkowska. 1981). The fact that many chemical carcinogens are also teratogenic. and that many of the genetic syndromes which result in congenital anomaly are also associated with a high incidence of tumors. led Trosko gt El; (1982) to propose that inhibited intercellular communication may be an etiologic factor in teratogenesis. Although the numbers of teratogenic compounds tested for their ability to inhibit metabolic cooperation is not as extensive as the number of tumor promoters tested. there is mounting evidence that some classes of teratogens clearly can inhibit metabolic cooperation (Lock gt git. 1984; Welsch and Stedman. 1984). S If indeed there is a relationship between inhibited cell-cell communication and these various chemically induced disease states. the detection of these chemicals by a reliable. easily conducted assay would be desirable. A Chinese hamster V79 cell-based assay for the detection of agents which inhibit metabolic cooperation has been developed for such a porpose (Yotti gt 21;: 1979; Trosko gt gig. 1981b). Although there have been several other types of metabolic cooperation assays described (Davidson gt gig, 1984; Enomoto gt gig. 1984; Fitzgerald and Murray. 1982; Ledbetter and Lubin. 1979; Murray and Fitzgerald. 1979; Vitkauskas gt gl_._1983;). the V79 cell assay seems to be the most widely used. This assay measures the recovery of 6- thioguanine (6-TG) resistant mutant colonies following the cocultivation of 6-TG resistant (HGPRT—) cells and many wild type 6-TG sensitive cells (HGPRT+). in the presence of 6- thioguanine. The number of recovered clones is inversely related to the amount of transfer of a toxic metabolite (phosphoribosylated 6-TG) via gap junctions from HGPRT+ (6—TG sensitive) to HGPRT- (6-TG resistant) cells. If the process of chemically induced inhibition of metabolic cooperation is similar to other cellular responses to toxicants (e.gu. metabolism). the effectiveness of many inhibitors of metabolic cooperation is likely to be species dependant. Therefore. it would be desirable for purposes of human risk assessement. to use human cells in an it tittg assay for inhibitors of metabolic cooperation. It would also be of value to have a human cell line that is capable of differentiation 1 titro. so that chemically induced inhibition of metabolic cooperation might be correlated with chemically induced abberations in development and differentiation. However. most continuously growing human cell lines pose the problem of an unstable aneuploid chromosome complement (which could possibly affect the physiological responsiveness of the cells). or are incapable of differentiation to more specialized cell types. A candidate cell line which seems to possess nearly all of the desirable features for such an assay is the human ovarian teratocarcinoma cell line PA-l. first cultured by Giovanella gt gig (1974) and subsequently characterized for its 1g titro growth and differentiation properties by Zuethen gt El; (1981). These cells are easy to grow in culture. have good colony forming ability. and have a stable pseudodiploid karyotype (46XX. t(15;20)). One of the most salient features of teratocarcinoma cells is their ability to form embryoid bodies which go through many of the same developmental processes as early mammalian embryos (Martin. 1980; Martin and Evans. 1975; Nicolas gt 21;: 1981). Many murine teratocarcinoma lines have been shown to be capable of differentiation. both biochemically and morphologically. to cell types remarkably similar to those in early embryos 1_ _1tg. The pluripotent nature of some of these teratocarcinoma lines is evidenced by their ability to participate in the formation of a wide spectrum of tissues when they are chimaerized with early mouse embryos (Bradley gt gig. 1984; Brinster. 1974; Martin gt al.. 1984; Mintz and Illminsee. 1975; Mintz and Cronmiller. 1981; Papaioannou gt 21;: 1975. 1978; Stewart. 1982; Stewert and Mintz. 1981). Although most human teratocarcinoma cell lines are not pluripotent. some have been shown to undergo limited differentiation. Zuethen gt gig (1981). and Rasilo gt g1; (1981) have shown this to be the case for PA-l cells. These cells will also form embryoid bodies 1 titro. Therefore. in addition to the possible use of PA-l cells in screening for inhibitors of metabolic cooperation. they could also serve as a model for investigating the effects of chemicals on human embryonic development and differentiation. In this dissertation I will describe experiments which were designed to 1) characterize the metabolic cooperation properties of subcloned derivatives of PA-l cells in monolayer cultures; 2) measure the effects of TPA and three related analogues on metabolic cooperation in monolayer cultures of these cells; 3) test the ability of various polybrominated biphenyl compounds to inhibit metabolic cooperation in monolayer cultures of these cells; 4) characterize the metabolic cooperation properties of these cells during embryoid body formation; and 5) establish the effects of TPA on metabolic cooperation in these embryoid bodies. The results of experiments conducted to test the responsiveness of these cells to TPA and three related analogues. are consistant with their tumor promoting potency 12.!112v and with previously reported observations regarding the ability of these compounds to inhibit metabolic cooperation in other mammalian cells. The polybrominated biphenyls Firemaster BP-6 (FM). 2.2'.4.4'.5.5'- hexabromobiphenyl (245-HBB). 3.3'.4.4'.5.5“— hexabromobiphenyl (345-HBBL and 3.3H4“4V—tetrabromobiphenyl (34-TBB) are known liver carcinogens in rodents. In a separate series of studies (Kavanagh gt gig. 1985) it was reported that these compounds failed to induce mutations in mammalian cells. Therefore. it is felt that some other property of these compounds other than mutagenicity. might be responsible for their hepatocarcinogenicity. I report here the findings that. as previously shown in Chinese hamster cells (Trosko gt gig. 1981; Tsushimoto gt gig, 1983). FM and 245-HBB inhibit metabolic cooperation in human teratocarcinoma cells. whereas 34-TBB and 345-HBB are cytotoxic but fail to inhibit metabolic cooperation in these human cells. I also report here that embryoid bodies composed of these subcloned cells undergo limited differentiation similar to the parental PA—l cell line; that cells in these embryoid bodies can metabolically cooperate with each other; and that TPA can both inhibit metabolic cooperation and cause abberant morphologic changes in these embryoid bodies. MATERIALS AND METHODS 1. Characterization gt the Metabolic Cooperation AssayL ‘Ll. Chemicals. 6-TG was obtained from Sigma Chemical Co“. St. Louis. MO. lZ—O-tetradecanoylphorbol-13-acetate (TPA). mezerein (MEZ). phorbol-12.13-didecanoate (P00). and 4a-phorbol-12.13- didecanoate (4a-PDD) were obtained from Consolidated Midland Corporation. Brewster. NY. Colcimid was purchased from Gibco. Grand Island. NY. 1.2. Cgllg_and Culture Methods. The cells used in these studies (designated P3). were a gift from Dr. E. Huberman (Argonne National Laboratory) and were originally derived from PA-l cells (Huberman et a1" 1984). PA-l cells are an outgrowth of an ovarian germ cell tumor taken from a recurrent ascities tumor of a 12 year old girl (Giovanella et al.. 1974). Both PA-1 cells and P3 cells have a pseudodiploid karyotype (46XX. t(15;20)). and grow without the aid of a feeder layer. A subclone of P3. designated HTP3-4. was derived from a single cell colony by the glass cylinder method of Ham and Puck (1962). This clone had high colony forming ability. and tight. uniform colonies. Cells were routinely grown in modified Eagle's minimal essential medium with Earl's salts. with 50% increase in 9 10 vitamins and essential amino acids. except glutamine; 100% increase in non-essential amino acids; 1mM sodium pyruvate. and 52 fetal bovine serum. Penicilin (E.I. Lilly. Indianapolis. IN) and streptomycin (Pfizer. Hoffman Estates. IL) were added to the medium at 100 units/ml and 100 ug/ml. respectively. The cells were incubated at 37 0C in humidified air containing 5% C02' C9115 were checked for the presence of mycoplasma by the Hoechst 33258 method (Chen. 1977). and were free of contamination. Stocks of cells were stored frozen in 10% DMSO in growth medium in liquid nitrogen. and thawed as needed. 1.3. Chromosome Analysis. Karyotyping and chromosome analysis were kindly provided by the Michigan State University Cytogenetics Laboratory. under the direction of Dr. J.V. Higgins. Log-phase cultures of HTP3-4 cells were treated with colcimid (0.5 ug/ml final concentration) for 3 hr. Cells were then trypsinized. treated with hypotonic (0.562) KCl for 10 min. centrifuged. and the cells from the resulting pellet were resuspended in Carnoy's fixative. Cells were refrigerated in fixative overnight. and prepared for giemsa banding by the method of Seabright (1971). 1.4. Selection gt 6-TG Resistant tariants. HTP3-4 cells were trypsinized and irradiated in suspension with a 300 R dose using a Torex—150 X—ray machine. operated at 120 kV. and 5 mA. Cells were maintained in 75 cm2 tissue culture flasks (Corning Glass Works. Corning. NY) in growth medium. and after a 10 day expression time 11 (Huberman et al.. 1984). were replated into 100 mm tissue culture dishes (Corning) with 10 ml of 6-TG containing medium (10 ug/ml) at a cell density of approximately 103 cells per cm2. 96 surviving colonies were screened for colony forming ability and colony morphology and 6 clones were selected from these (designated HTXTG-1 through HTXTG-6) for metabolic cooperation testing. 1.5. Iggt gt Mgtgggltg Coogeration. 200 6-TG resistant cells from each of the 6 HTXTG lines were plated into separate 60 mm plates with either 0. 1. 2. 3. 4. or 5 X 105 HTP3-4 cells. After a 4 hr attachment period. 6-TG was added to the medium (10 ug/ml. final). Since the 6—TG resistant mutants are presumptive HGPRT-. they will only be killed by 6-TG if the lethal metabolite is transferred to them via gap junctions. The selection medium was replaced on days 3 and 7. and after 10 days. colonies were rinsed with 0.9% saline. stained and fixed with 4% crystal violet in 10% ethanol. and the number of surviving colonies was visually scored. 1.6. Test gt Metaboltg Cooperation with Other Human Cellgg A similar protocol was used to evaluate the ability of HTXTG-1 to metabolically cooperate with two other types of human cells. Cells tested were a normal diploid fibroblast strain (MSU-1) and an epithelial line derived from human bladder (HCV-29). which was a gift from Dr. C.Y. Wang. Mich. Cancer Foundation. to Dr. C.C. Chang. Michigan State University. 12 1.7. Effect gt TPA gg Metaboltg Cooperation Between HTXTG—1 and HTP3-4 Cellgg The protocol used to test the effects of TPA (and the other chemicals below) on metabolic cooperation is essentially the same as that used in the V79 cell assay (Yotti et al.. 1979; and Figure 1). Briefly. 100 HTXTG-1 cells and 3 X105 HTP3—4 cells were added to each of ten 60 mm plates in growth medium. After a 4 hr attachment period. TPA was added at various concentrations in a volume of 10 ul. 2 hr after chemical treatment. 6-TG was added to the medium at 10 ug/ml (final concentration). 3 days later. treatment medium was replaced with fresh 6-TG containing medium (10 ug/ml). and the cells were again refed on day 7. On day 10. colonies were rinsed. stained. and scored as above. Cytotoxicity plates containing 100 HTXTG-1 cells were treated in parallel with the metabolic cooperation plates. After 3 days in treatment medium. these plates were similarly given fresh selection medium. and scored as above on day 10. Cells were next tested for their responsiveness to TPA at different HTP3-4 cell densities. 200 HTXTG-1 cells and either 0.25. 1. and 3 X 105 HTP3—4 cells were added per 60 mm plate. and after a 4 hr attachment period TPA was added at various doses. After a 2 hr exposure to TPA. 6-TG was added to the medium at 10 ug/ml. This treatment medium was replaced with selection medium on day 3. and the medium was changed again on day 7. Plates were scored on day 10. Concurrent cytotoxicity plates containing 100 HTXTG-1 cells/plate were treated in parallel in the fashion described above. 13 Figure 1. Protocol for Metabolic Cooperation Assay. 200 HTXTG-l cells are cocultured with 3 X 105 HTP3—4 cells in a 60 mm tissue culture dish. After attachment of cells (4 hr) test chemical is added. 2 hr later 6-TG (10 ug/ml final) is added. After 3 days. treatment media is replaced with fresh selection media. On day 7. media is changed again. and on day 10 surviving colonies are fixed. stained. and scored. (After Trosko, gt_gl;. 1981b.) 14 5335 mmcmcu .. mama 5.. .fi enamfim 33m Em .Smum .XE .. mama a: £3 1:2 SN 25 LEEEEM 32¢ 15 1.8. Effect gt TPA Analogues gg Metaboltg Cooperation. As for the TPA experiment above. 100 HTXTG-1 cells and 3 X105 HTP3-4 cells were cocultivated in 60 mm dishes. The phorbol esters TPA. PDD. 4a-PDD. and the structurally related non-phorbol analogue MEZ. were tested at various concentrations for their ability to inhibit metabolic cooperation. Concurrent cytotoxicity plates containing 100 HTXTG-1 cells were also treated in parallel. Plates were stained and scored as indicated above. 2. Effects gt the Polybrominated Biphenylg FM. 245—HBBL 345- HBB. and 34-TBB gg Metaboltg Cooperation Between HTXTG-1 and HTP3-4 Cellgg 2.1. Chemicals. The PBB congeners used in these studies were purified from Firemaster BP-6 (FM). which was manufactured by Michigan Chemical Corporation (St. Louis. MI) (Moore and Aust. 1978). or from a mixture manufactured by RFR Corporation. (Hope. RI). The former mixture was the source of the 2.2'.4.4'.5.5'—hexabromobiphenyl (245-HBB). while the latter provided the 3.3'.4.4'.5.5'—hexabromobiphenyl (345-HBB) congener. These two compounds and 3.324.45- tetrabromobiphenyl (34-TBB) were purified and kindly provided by Dr. S. Aust. Department of Biochemistry. Michigan State University. Concentrations tested were up to the maximum solubility achievable with a solvent (DMSO) volume of 25 ul in 5 ml of growth medium. 16 2.2. Mgtaboli Cooperation AssayL The assay used to test the various PBB compounds is essentially that as described above for TPA (Material and Methods Section 1.7; and Figure 1). 200 6-TG resistant HTXTG-1 cells were cocultured with 3 X 105 HTP3-4 cells in 60 mm dishes containing 5 ml of growth medium. After a 4 hr attachment period. the test chemicals. or positive (TPA at 0.75 ng/ml final). or solvent (DMSO) control were added to their respective plates (5 replicates per dose) in a volume of 25 ul. After 2 hr of treatment. 6-TG was added to each plate (final concentration 10 ug/ml). and cultures were incubated in this treatment-selection medium for 3 days. At this time. treatment medium was replaced with fresh selection medium (6-TG at 10 ug/ml). and on day 7 this medium was again replaced. On day 10 plates were rinsed with 0.9% saline. and fixed and stained as above. Surviving colonies were scored visually. To measure the cytotoxicity of each treatment. 200 HTXTG-1 cells were plated into 60 mm dishes containing 5 m1 of growth medium. Treatment of these dishes was done in parallel with the metabolic cooperation plates except that the second medium change on day 7 was omitted. On day 10 these plates were rinsed. fixed and stained. and survival of the colonies was recorded. 17 3. Effect gt TPA gg Metaboltg Cooperation 1g Teratocarcinoma tgll Embryoid Bodies. 3.1. Formation gt Embryoid Bodies. Embryoid bodies were formed by plating 2.5 X 106 HTP3-4 cells into each of 24 bacterial grade 100 mm plastic petri dishes (VWR Scientific. San Francisco. CA) containing 10 ml of growth medium supplemented with 15% fetal bovine serum. Cells began to aggregate after 8-10 hr under these conditions. and at 24 hr. they had formed embryoid bodies. 3.2. Measuring Mgtgggltg Cooperation tg Composite Embryoid godies. Composite embryoid bodies were formed in the manner described above. except that 5 X 103 HTXTG-1 (6—TG resistant) cells were added to the petri dishes in addition to the 2.5 X 105 HTP3-4 cells. At 24 hr. 6-TG was added at either 10 or 20 ug/ml (final) to series of separate plates. Controls received saline in an equal volume. At 1. 4. 12. or 24 hr. the medium and embryoid bodies were collected from their dishes with a wide bore pipet and centrifuged at 250 X G for 5 min. The embryoid bodies were washed by gently pipeting the pellet to cause it to dissociate. and resuspending in 10 ml of phosphate buffered saline. Embryoid bodies were again centrifuged. and the pellet was dissociated in 5 ml trypsin (0.01%). and placed into a 37 0C water bath for 10 min. After this trypsin treatment. embryoid bodies could be dissociated into individual cells by gently pipeting them. This cell suspension was then diluted with an equal volume of 18 complete medium (growth medium supplemented with 5% FBS) ‘containing 6-TG (10 ug/ml). and cell density was recorded with a hemocytometer. Cells were further diluted with 6-TG medium so that when 5 ml was plated into tissue culture dishes (8 plates per treatment). it gave a final cell number of 5 X103 per 60 mm dish. Cells on these dishes were given fresh 6-TG containing medium on days 3 and 7 after plating. On day 10 dishes were rinsed. and surviving colonies were fixed and stained. and recorded as above. 3.3. Effect gt IRA gg Metabolic Cooperation tg Composite Embryoid Bodies. The protocol for measuring the effects of chemicals on metabolic cooperation in embryoid bodies is outlined in Figure 2. Composite embryoid bodies containing 5 X 103 HTXTG-1 cells and 2.5 X106 HTP3—4 cells were formed as above. After 24 hrs of embryoid body formation. TPA was added to each of 8 plates in a volume of 20 ul at final concentrations ranging from 0.25 to 10 ng/ml. Ethanol (20 ul) was added to the control plate. 6-TG (20 ug/ml final concentration) was added to plates 2 hr after chemical treatment. Embryoid bodies were incubated in this treatment medium for 24 hr. and then washed and trypsinized as outlined above. Cells were then replated into dishes (10 per treatment) containing 6-TG medium (10 ug/ml). as for the composite embryoid bodies above. Plates were given fresh 6— TG medium on days 3 and 7 after replating. and were rinsed. fixed and stained. and scored on day 10. 19 Figure 2. Protocol for Embryoid Body Metabolic Cooperation Assay. 5 X 105 HTXTG—1 cells and 2.5 X 106 HTP3-4 cells are cocultured in a 100 mm bacterial grade petri dish in growth medium plus 15% FCS. 24 hr later. the test chemical is added. 2 hrs after this. 6-TG is added (20 ug/ml final). Embryoid bodies are trypsined 12 hrs after the beginning of 6-TG treatment. diluted. and cells from this suspension are replated into fresh 60 mm tissue culture dishes containing 6- TG medium (10 ug/ml). After 10 days. with 2 medium changes in between. surviving colonies are scored. 20 GROW STOCK CULTURES TO CONFLUENCE IN "0" MEDIUM + 5% FCS i TRYPSINIZE*AND COUNT PLATE 2.5 x 106 SENSITIVE CELLS + 5.000 RESISTANT CELLS INTO PETRI DISH WITH 10 ML "0" + 15% FCS 24 HR 1 EMBRYOIE BODIES ADD TEST CHEMICAL l 2 HR 0 ADD 6-TG (20 UG/ML) I 24 HR v DISLODGE. WASH WITH PBS TRYPSINIZE AND REPLATE INTO 60 mm TISSUE CULTURE DISH WITH 5 ML "0" +5% FCS I 3 DAYS CHANGEIMEDIUM 4 DAYS i CHANGE MEDIUM 1 3 DAYS i WASH WITH 0.9% SALINE FIX AND STAIN WITH CRYSTAL VIOLET 6 SCORE Figure 2. 21 In order to determine whether the inhibition of metabolic cooperation by TPA could be overcome by a higher dose of 6-TG. an experiment similar to the one above was carried out except that three different doses of 6—TG were employed (10. 20. and 50 ug/ml). In addition. plates designed to measure toxicity of the various TPA and 6-TG treatments were treated either without TPA (solvent/background controls). or with TPA only (6-TG treatment excluded while cells were in the embryoid body state) so as to measure the cytotoxic effects of TPA at the doses tested in the metabolic cooperation plates. RESULTS 1. Characterization gt the Metabolic Cooperation Assay. 1.1. Elating Efficiency and Chromosome Analysis. The subclone HTP3-4 was found to have a plating efficiency of approximately 65%. and karyotype analysis (Figure 3) revealed a chromosome pattern (46XX. t(15;20)) similar to the parental line. PA-l. from which these cells were derived (Zeuthen gt gig. 1981). Furthermore. the colony morphology and appearance of these cells was similar to the parental phenotype (Figure 4). 1.2. Metaboltg Cooperation Between HTP3-4 and g flltlfi Variants. Figure 5 shows that the relative survival of all 6 HTXTG lines was progressively diminished as the number of wild type cells was increased. This indicated that all 6 lines were capable of undergoing metabolic cooperation with the HTP3-4 cells. HTXTG-1 was selected for further testing based on its cloning efficiency. and colony morphology. 1.3. Test f Metaboltg Cooperation Between HTXTG—1 and Other Human Cellgg The ability of HTXTG-1 teratocarcinoma cells to metabolically cooperated with normal human diploid fibroblasts (MSU-1). and a human bladder cell line (HCV-29) 22 23 Figure 3. Karyotype of HTP3-4 Cells. Log—phase cultures were treated with colcemid (0.5 ug/ml) and hypotonic KCl (0.56%), fixed, and giemsa banded by the method of Seabright (1971). The pseudodiploid karyotype shows a translocation involving chromosomes 15 and 20 (46XX, t(15;20)). 24 x NN Taco: 2. or to no. 1O... 1 I vwlm .m chamwm up IEhSLm 25 Figure 4. Appearance of HTP3—4 Cells in Monolayer Cultures. A. Phase contrast photomicrograph of HTP3-4 cells (X 150). Note the epithelioid character of these cells when grown to confluence.lL Photomicrograph of typical single cell derived colcany of HTPB-Aw Note that cel ls tend toiaile up in the middle of a colony (X 60). Figure 4. 27 ’4 SURUIUAL n on [Llal a T'T'r'l'lfififi'l'I'F'I'I'I'T'I'rrr'rrrfi'l'ril B .1. 2 3 4 5 HTP3-4/PLaTE (X 105) Figure 5. Metabolic Cooperation Between Six X-ray Induced 6-TG Resistant Variants and HTP3-4 Cells. 200 cells from each of six cell lines resistant to 6-TG were seperately cocultured with increasing numbers of HTP3-4 cells in 60 mm tissue culture dishes in the presence of 6-TG. Fresh 6-TG medium was replaced on day 3. Plates were stained and colonies scored on day 10. 28 can be seen in Figure 6. As is apparent from the figure. the teratocarcinoma cells communicate quite well with both cell types. 1.4. Effect f TP _g Metaboltg Cooperation Between HTXTG-l and HTP3-4 Cellgg TPA was effective in causing a dose dependant increase in the recovery of HTXTG-1 cells cocultivated with wild type cells at a density of 3 X105 cells per 60 mm plate in the presence of 6-TG (Figure 7). This inhibition appeared to be maximal in the range of 0.75-1.0 ng/ml. Aboveethese doses and at this cell density. TPA began to be cytotoxic (data not shown). The morphology of surviving HTXTG-1 colonies was quite varied. with many colonies appearing whispy and faint. containing flattened cells with many processes. This was in contrast to the tight. epithelial morphology of the few surviving control colonies (Figure 8). This effect on cell morphology was not as pronounced in the TPA treated cytoxicity plates. suggesting that this was an effect related to cell density. TPA was also capable of inhibiting metabolic cooperation at lower cell densities (Figure 9). but the recovery of mutants appeared maximal at between 0.75 and 1.0 ng/ml. under different cell density conditions. The background of HTXTG-1 cells escaping metabolic cooperation in the controls was of course higher with decreasing cell density. and thus the most effective cell density of the 3 densities tested. for detecting inhibition of metabolic cooperation by TPA. was with awild type cell density of 3 X105 cells per 60 mm 29 .11. Q 9‘ SURUIUFIL C0 h) (n G) (9 G) i-1-1-1-1;1-1L1L1L1.1L1r1. g... Q L Q *r'r r I'ITFTITI'I'T'I'I'I'I'Iriff'r'l'rrl'I'l a - '1' 2 3 4 5 1.11m TYPE CELLS/PLATE (x 105) Figure 6. Metabolic C00peration Between HTXTG-1 Cells and Other Human Cells. 200 HTXTG-1 cells were cocultured with increasing numbers of either normal human diploid fibro- blasts (MSU-l In); or with increasing numbers of a human bladder cell line (HCV-29 l ) in the presence of 6-TG. 30 Figure 7. Effect of TPA on Metabolic Cooperation Between HTXTG-l Cells and HTP3-4. 100 HTXTG-1 cells were plated alone (top panel) or cocultured with 3 X 105 HTP3—4 cells (lower panel) in 60 mm plates. TPA was added 4 hr after plating. 6-TG was added 2 hr later. Fresh 6-TG medium was given on day 3 (and 7 for metabolic cooperation plates. lower panel). Colonies were scored on Day 10. 31 _l <1 2- H D O: :3 to X 23-: ‘1 18-: 9 'r*['v'l'irl'r*['r'[rtf[*r'['1r{'IfI'l'lrlr['I'l 130* 1 q 937 m d j 881 Lu '1 o 1 p: 331 m {I ‘1 u I— 56" 3 ‘0 J o H 43‘ o 0" . ml” ... 1 m (z J81 a 1 to 26‘ [.- ' l m 18" B l'l'['fi['rr[*r*[*1']rl'['T"*rrf'rfl'I'lfl'lrrfi B .25 ,5 .75 1.8 TPH (ng/ ml) 32 Figure 8. Effect of TPA on HTXTG-1 Cell Colony Morphology. A. Photomicrograph of colony recovered from metabolic cooperation plate treated with 0.75 ng/ml TPA. 8. Control (background) colonies from metabolic cooperation plate. C. Colony from TPA cytotoxicity plated (0.75 ng/ml). 0. Colony from control cytotoxicity plate. (X 60% 33 Figure 8. 34 Figure 9. Recovery of HTXTG-1 Cells Cocultured with Various Densities of HTP3—4 Cells by TPA. 200 HTXTG—1 cells were plated with 0(a). 0.25011). 1 (i). or 3 X105(-)HTP3-4 cells per 60 mm dish. TPA was added at various concentrations at 4 hr post plating. 6-TG was added 2 hr later. Fresh 6-TG medium was given on days 3 and 7. Colonies were scored on day 9. xRECOUER‘I’ OF 6-T6 RESISTRHT CELLS 198 99 so 73 so 58 4a 39 29 la ’11 SURUIUFIL a r'tfl'rfrfl'I'I'r'I'lrirI'I'l'l'r‘Iflrljr'r'tfrfi] 183 so so 78 ea 59 4a 38 23 la a I'I'I'V'l'I'ITI'I'Urr'I'rrrftflfi'lTI'ITT'I'I'I'I'I .25 .5 .75 .LB 1m (ng/ml) ' Figure 9. 36 dish. giving approximately a 9-fold increase in recovery over the control. 1.5. Effect gt TPA Analogues gg Metabgli Cooperation. Figure 10 displays the effect of TPA. P00. 4a-PDD. and MEZ on metabolic cooperation. PDD was as effective as TPA in inhibiting metabolic cooperation. while MEZ was intermediate in its effect. and 4a-PDD was quited ineffective. However. all four compounds showed relatively little difference in cytotoxicity. 2. Effects gt EM; 245-HBB. 345-HBB. and 34—TBB gg Metaboltg Cooperation. Figures 11 shows the results of both cytotoxicity and metabolic cooperation studies for all four compounds. FM and 245-HBB caused a dose dependant increase in the recovery of the HTXTG—1 cells above background (5% recovery). at relatively non-cytotoxic concentrations. Conversely. 345-HBB was exceedingly toxic at low doses and failed to increase the recovery of 6—TG resistant variants in the metabolic cooperation plates (in fact. the recovery was below background. again indicating toxicity). 34—TBB was moderately toxic. and also failed to increase recovery of the HTXTG-1 cells. TPA (positive control) was effective in recovering 70% of the HTXTG-1 cells plated. 37 Figure 10. Effect of TPA Analogues on Metabolic Cooperation. 100 HTXTG—1 cells were plated alone (top panel) or cocultured with 3 X105 HTP3-4 cells (lower panel) in 60 mm plates. TPA (I). P00 (0). MEZ (a), and 4a-PDD (j). were added at various doses 4 hr after plating. 6-TG was added 2 hr later. Fresh 6-TG medium was given on day 3 (and on day 7. lower panel). and colonies were scored on day 10. ‘ OUER‘I’ OF I m. J 33RE 38 3“ SURUIURL “’1 '3 iTrTl'T'TT‘TI'VT"W”‘l'TTTrl'Trl'T'i'l’t'l'rrl 139 so 813 721 so 5121 // 4a / a. '08 ,/ ELLS a“ RESISTRHT 213 18 6-T6 BITI'I'fif'I"'I'lfir"TTi'i"|'o'l'[r1'i'[fl'I'cfl . 2 5 ,5 .7 5 1.13 TPB (ng/ m1:I Figure 10. 39 Figure 11. Effect of Various PBB Compounds on Metabolic Cooperation. 200 HTXTG-l cells were plated alone (top panel) or cocultured with 3 X105 HTP3-4 cel ls (lower panel) in 60 mm dishes. FN|(.). 245-HBB (o). 345-HBB (I). and 34-TBB (0). were added 4 hr after plating. 6-TG was added 2 hr later. Fresh 6-TG medium was given on day 3 (and day 7. lower panel). and colonies were scored on day 10. xRECOUER‘I’ 0F 6-T6 RESISTANT CELLS 9‘ SURUIURL 40 188 SB 83 7B 88 513 4B 313 26 113 a‘I'Irlrr'rrr'rrl'I'Trr*r'r'l'T'I'r'r'r'i'rrirx'i'l 1891 .V.' I '.'.'.‘ v. V V r f v V ' V ' V ‘ ' ' ' '.‘.V.‘.'-V.V.V.V' . . ' v V " v V v v V v ‘ fl ooooooooooooooooooooooooooooooooooooooooooooooooooooo nnnnnnnn T a a ~ g - .LL.L A L'L‘A A A. 'L ' A‘L. .A. L.‘.A.A.¢A.A‘ . H .AAL.A. .L'L.L L’A. .L. 'A. ' .L .A.L L‘u'L‘L'; U) 6’ fi- (fl 55 ii 1‘ h) (0 G? (S G) /)\o——-0——o 'V'I'I'rfr'r'rriTI*I'I'rrrrrfrfT'rrlrrrrrrfl 18 20 38 43 50 003E (ug/ml) 6) Figure 11. 41 3. Effect gt It_ gg Metaboltg Cooperation tg Embryoid Bodies. 3.1. Formation gt Embryoid Bodies. As previously shown by Zeuthen gt gig (1981). PA-l cells are capable of forming embryoid bodies. Although the chromosome compliment of HTP3-4 cells is stable after many passages 1 yitro (Huberman gt al.. 1984). the ability to undergo limited differentiation in culture might not have been expected to be retained. Therefore. it was necessary to establish that these cells could still form embryoid bodies. In figure 12. the chronological formation of these bodies is shown. Typically. these embryoid bodies begin to form aggregates 2 to 4 hr after plating; these aggregates begin to organize into morula-like clusters by 6-8 hr. and then they undergo a "compaction" like process beginning at about 12 hr after plating. in which the surface cells appear to flatten. creating a relatively smooth surface as opposed to the rounded morphology of the surface cells of aggregates. Subsequent to the compaction stage. the bodies begin to attach. albeit loosely. to the substratum. and by 36 hr. one can observe the appearance of extremely flattened trophoectodermal—like cells anchoring the embryoid bodies to the plate. 3.2. Metabolic Cooperation 1g Composite Embryoid Bodies. It was possible to show metabolic cooperation in embryoid bodies composed of both HTXTG-1 (6-TG resistant) and HTP3-4 (6-TG sensitive) cells (Figure 13). There was a time and dose dependant reduction in the recovery of HTXTG-1 cells 42 Figure 12. Appearance of Embryoid Bodies. Embryoid bodies were formed by plating 2.5 X106 HTP3-4 cells into 100 mm bacterial grade petri dishes containing 10 ml medium supplemented with 15% FCS. A. 4 hr after plating loose aggregates begin to appear. 8. By 8 hr morula-like clusters of cells predominate. C. At 12 hr bodies appear more symmetrical. D. 24 hr after plating. cells appear as compacted masses with smooth. flattened surface cells. E. By 36 hr many of the embryoid bodies have attached with pseudopod like cells anchoring them to the substrate. F. 48 hr after plating. many bodies have begun to compress and flatten against substratum. forming cystic masses. (X 150). 43 Figure 12. 44 1J38 398 138 '78 138 ‘1 .1 :1 7 a 1 1 D. 591 h. \ I2 " \. 03:. 4a; \\ \ I“! 381 \3‘. N :28 \\\\ el'l'rrT'I' lrr'T'I'I'I'T rtrrl'rrr'll'l'lflr 'I'I'I 0 2 4 6 8 10 12 14 16 13 20 22 24 EXPOSURE TIME (hr) Figure 13. Metabolic Cooperation in Embryoid Bodies. s x 103 HTXTG 1 cells and 2. s x 106 HTPS 4 cells were cocultured in bacterial grade petri dishes in 10 ml medium supplemented with 15% FCS. 24 hr after plating, 6-TG was added at either 10 (0), or 20 C.) ug/ml, (I control). At 1, 4, 12, and 24 hr, embryoid bodies were trypsinized and resulting cells were diluted and replated into 60 mm tissue culture dishes containing 6-TG medium (10 ug/ml). Plates were given fresh 6-TG medium on days 3 and 7, and were scored on day 10. 45 in the presence of 6—TG and contacting HTP3-4 cells. At a dose of 20 ug/ml. and an embryoid body incubation time of 12 hrs seemed to give a maximal killing (12% survival relative to control). 3.3. Effect gt TP n Metaboltg Cooperation 1g Embryoid Bodies. TPA was able to block. partially. metabolic cooperation in embryoid bodies (Figure 14). just as was found for these teratocarcinoma cells in monolayer cultures (Results Section 1.4). The TPA induced increase in recovery of HTXTG—l cells from embryoid bodies was not as dramatic as that found in monolayer cultures (two times the background as opposed to a nine fold increase. respectively). but this could be related to the numeric ratio of the two cell types present in the embryoid bodies. or perhaps to the fact that the HTXTG-1 cells had more cell membrane surface contact with the HTP3-4 cells (three dimensional contact) and hence there was a greater degree of gap junctional transfer to block. The concentration of TPA necessary to elicit a maximal effect on, recovery was approximately an order of magnitude higher than that required in monolayer cultures. However. the total cell number per plate in these embrybid body experiments was also roughly ten times that of monolayer culture experiments (approximately 2.5 X 106 for embryoid bodies. and 3 X 105 for monolayer cultures). so that the concentration of TPA per cell was nearly the same for both sets of experiments. It was noted that TPA had an effect on embryoid body morphology. causing a temporary decompaction-like phenomenon. which was 46 1881 U) -4 (D (D G) (9 CD 6) .h E) fiRECOUERY OF m G) 6-T6 RESISTANT CELLS (n G) B l'r'rr"l'ITTrI'IrFTI'I'l'lf 9123455'} *r r' ITI'F'T'I'I sf 5' 19 ll l2 TPA (mg/m1) Figure 14. Effect of TPA on Metabolic Cooperation in Embryoid Bodies. S X 103 HTXTG—1 cells were cocultured with 2.5 X 106 HTP3-4 cells in 100 mm petri dishes. 24 hr after plating, TPA was added to plates at various concentrations. 2 hr later, 6-TC was added. 24 hr later embryoid bodies were trypsinized, and cells were replated into 60 mm tissue culture dishes in 6-TG medium. Plates were given fresh medium on days 3 and 7. Colonies were scored on day 10. 47 apparent at approximately 12 hr post TPA treatment (Figure 15). with the highest concentrations causing reversion to loose aggregates. Resumption of the developmental course of these embryoid bodies seemed to occur at a rate that was inversely correlated with the TPA concentration. for at 24 hr post TPA treatment. the number of embryoid bodies that had overcome the decompaction and showed attachment and cell spreading on the substratum was dependant upon the concentration of TPA present. Figure 16 shows the results of an experiment designed to indicate if increased 6~TG concentration could overcome the ability of TPA to inhibit metabolic cooperation in embryoid bodies. Although TPA caused an increase in the recovery of HTXTG-1 cells in embryoid bodies at 10 and 20 ug/ml 6-TG. it was not very effective in overcoming the killing effect of 50 ug/ml. The effect of the TPA treatment alone on survival of HTXTG—1 cells was minimal. 48 Figure 15. Effect of TPA on Compaction of Embryoid Bodies. Appearance of embryoid bodies at 12 hrs post TPA treatment. A. 1 ng/ml. B. 5 ng/ml. C. 10 ng/ml. 0. Control. 49 Figure 15. 0‘) _l .1 DJ [Lo 01"- 3.2 95¢ ml— 30) 0H 00) mm mix X 1.0 i... I m 4 e 'I'Err'rTT'I'I'T‘I'I'I'T'I'Iri'rfl'I'rfIfT 8 5 18 15 28 TPA (nQ/ml) Figure 16. Recovery of HTXTG-1 Cells from Embryoid Bodies Treated With TPA and 6-TG at Various Concentrations. 5 x 103 HTXTG-l cells were cocultured with 2.5 X 106 HTP3-4 cells in petri dishes. 24 hr later TPA was added at various , concentrations. 2 hr later. 6-TG was added at 10 (I). 20 (I). or 50 03) ug/ml (9 control. TPA only). 24 hr later. embryoid bodies were trypsinized. and cells were replated in 60 mm tissue culture plates. containing 6-TG medium. Fresh 6-TG medium was given on days 3 and 7. Colonies were scored on day 10. DISCUSSION 1. Usefulness gt Human Teratocarcinoma Cellg tg _g Assay for Inhibitors gt Metaboltg Cooperation. The PA—l human teratocarcinoma cell derived lines used in these studies promise to be good candidates for a human cell based assay for inhibitors of metabolic cooperation. These cells. in spite of many passages have retained their originally described pseudodiploid karyotype (46XX. t(15;20)). Their high colony forming ability. tight colony morphology. and reletively short doubling time (approximately 16 hrs. (Huberman gt gig. 1984)). and responsiveness to known tumor promoters. make them an ideal candidate for a human cell based assay for inhibitors of metabolic cooperation. Huberman gt El; (1984). while designing a mutation assay with PA-l derived P3 cells as target cells. demonstrated a cell density dependant effect on recovery of 6-TG resistant mutants. which was the initial indication that these cells were capable of metabolic cooperaton. As shown in these studies (Figures 5 and 6). they are capable of undergoing metabolic cooperation. not only with themselves. but equally as well with at least two other types of human cells. As with the results found in studies of other animal and human cells (cited in the introduction). TPA inhibited metabolic cooperation in a dose dependent manner in these 51 $2 teratocarcinoma cells. In addition P00 and MEZ were highly effective and moderately effective. respectively. in their ability to inhibit metabolic cooperation. 4a-P00 was negative. as its 1 iv and previously demonstrated tg yitro activity would predict. Results similar to these were also found with these and other phorbol ester analogues in previously published reports (Enomoto gt gig, 1981b. 1984; Fitzgerald and Murray. 1982; Guy gt al.. 1981: Mosser and Balls. 1982; Newbold and Amos. 1981; Trosko gt gig. 1981b). Although the mechanism of action of TPA induced reduction in metabolic cooperation is not known. several recent reports offer some clues. Yancey gt gig (1982) have shown the disappearance of gap junction from the surface of V79 cells treated with TPA. Kalimi and Sirsat (1984b) applied TPA and MEZ to the skin of mice and were able to show a dose dependant decrease in the number of gap junctions in the basil and suprabasil layers of interfollicular epidermis. as well as an increase in the intercellular spaces. These effects were apparent for MEZ only at high doses. agreeing in principle with the results obtained here. and the known relative weakness of this substance as stage 1 promoter (Slaga gt gig. 1982). The observation that the intercellular spaces are increased in the basil layer of mouse epidermis by TPA (Kalimi and Sirsat. 1984b; Raick gt gig, 1972). suggests a change in normal celltrlar adhesion mechanisms. Kanno gt g]; (1984) have shown the critical nature of Ca++ dependant adhesion molecules in gap junction 53 mediated intercellular communication. Antibodies raised to these molecules can inhibit metabolic cooperation in mouse teratocarcinoma cells (see Discussion Section 3). At the molecular level. recent advances in the understanding of TPA action have come from receptor binding studies (Ashendal gt gig. 1983; Castagna gt glg. 1982; Kraft and Anderson. 1983; Neidel gt gig. 1983). which show that the Ca++ and phospholipid dependant protein kinase (protein kinase C) binding of TPA is correlated with its biological activity. This kinase is normlly activated by diacylglycerol formed from phospholipase C mediated phosphotidylinositol turnover (Castagna. 1983; Flockhart and Corbin. 1982; Nishizuka. 1984). Many molecular signals such as hormones. growth factors. lectins. etc.. are known to stimulate phosphotidylinositol metabolism via interaction of their respective receptors with phospholipase C (Castagna. 1983; Nishizuka. 1984). This series of reactions is sensitive to the antagonistic effects of cyclic 3.5-adenosine monophosphate (CAMP). Thus. many other molecular signals which increase cAMP. can ultimately depress protein kinase C mediated phosphorylation. while stimulating cAMP dependant protein kinase (protein kinase A) phosphorylation events. TPA. through its tight binding to protein kinase C. bypasses this regulatory feedback control mechanism. and supports continous protein kinase C catalyzed phosporylation. It is known that cAMP can act to increase the junctional coupling between cells (Azarnia gt gig. 1981; Flagg-Newton gt gig. 1981). Enomoto gt El; (1984). have recently been able to 54 demonstrate cAMP antagonism of the TPA induced reduction in gap junction permeability to dye transfer. Thus. it appears that competing phosphorylation reactions catalysed by either of these two kinases may ultimately lead to increased or decreased gap junction mediated intercellular communication. One possible consequence of the ensueing phosphorylation cascade known to occur subsequent to TPA binding. is the ultimate modification of gap junction protein. or proteins associated with its assembly. Consistant with this model is the observation by Atkinson gt gig. (1981) that temperature sensitive Rous sarcoma virus introduced into cells produced a temperature dependant reduction in metabolic cooperation. It is known that the transforming activity of this virus is associated with the pp60$rc protein. a tyrosine specific kinase (Hunter and Sefton. 1980). This oncogene product is known to locate proximal to gap junctions in the membrane. and thus may. as part of its transforming action reduce metabolic cooperation via phosphorylation of gap junction protein or associated protein such as vinculin. Alternatively. Erickson and coworkers have shown that pp60$rc is also capable of phosphorylating membrane phospholipid. and inducing phosphotidylinositol turnover (Sugimoto gt gig. 1984). which would subsequently stimulate protein kinase C. with its consequent phosporylations. Recently. Chang gt glt_(manuscript submitted) have found that pp60$rc transformed NIH-3T3 cells have a reduced metabolic cooperation ability. Incidental to these SS observations is that one of the major breakdown products of phospholipase C catalyzed phosphotidylinositol breakdown is inositol triphospate. This substance has recently been implicated in stimulating the release of intracelluar Ca++ stores (Berridge and Irvine. 1984; Rasmussen and Barrett. 1984). Increases in intracellular Ca++ are known to reduce gap junction permeability (Perecchia. 1978;_Perecchia and Perecchia. 1980; Rose gt 21;: 1977). perhaps through the action of gap junction associated calmodulin (Peracchia and Bernardini. 1984). This may be one of the additional mechanisms by which molecular signals that stimulate phospholipase C (and hence protein kinase C). act to modulate gap junction permeability. Another possible cause of the decreased metabolic cooperation induced by TPA involves still other cell surface changes. TPA is known to modify the nature of glycoconjugates at the cell surface (Srinivas and Colburn. 1982). potentially influencing cell recognition and adhesion mechanisms. and indirectly effecting a block in gap junction assembly or function. Transformation has been described as a dedifferentiation or retrodifferentiaton process (Markert. 1968; Pierce. 1967; Pitot. 1968; Potter. 1978). The fact that these human teratocarcinoma cells are capable of limited differentiation (Zeuthen gt gig. 1980; Rasilo gt gig. 1982). and that compounds reported here which were able to block intercellular communication were also capable of inducing differentiation suggests a link between these two phenomena. 56 at least as far as TPA like compounds are concerned. TPA has been shown to induce differentiation in a number of other systems (reviewed in Yamasaki. 1984). Recently published results by Enomoto and Yamasaki (1984) and Yamasaki gt gig (1985) have shown that i yitro transformation of Balbc-3T3 cells is associated with a reduction in gap junction mediated dye transfer between transformed and contacting normal cells. The results thus give added weight to the concept that selective communication plays a role in dedifferentiation and tumor development. The t_ _tyg potency of the phorbols as tumor promoters and their capacity to affect differentiation are correlated (Yamasaki. 1984). 2. Inhibition gt Metaboltg Cooperation gy elegttvg Egg Compounds. The results reported here indicating the ability of FM and its major congener 245-HBB to inhibit metabolic cooperation at non-cytotoxic concentrations; and the failure to inhibit metabolic cooperation but marked cytotoxicity of 345-HBB and 34-TBB. in human teratocarcinoma cells. corroborate earlier work reported by Trosko gt gig (1981aL and Tsushimoto gt gig (1982a). who used the V79 cell assay to first demonstrate these effects. It was found in the latter series of experiments that a number PBB congeners substituted with a bromine atom at the ggttg position. were relatively non-cytotoxic compared to the similar congeners lacking this substitution. At the same time. the ortho substituted 57 congeners were capable of inhibiting metabolic cooperation in V79 cells. whereas the more coplaner congeners were relatively inactive in this regard. A similar set of findings with various analogous polychlorinated biphenyl (PCB) congeners tested in V79 cells was also reported by Tsushimoto gt gig (1983a). The moderately toxic effects of 34-TBB and its failure to inhibit metabolic cooperation suggests activity similar to 345-HBB. Persuasive arguments have been made which implicate a receptor mediated toxicity for various coplaner congeners of PCBs. PBBs. polychlorinated napthalenes. polychlorinated dibenzofurans. and polychlorinated dibenzodioxins. with 2.3.7.8—tetrachorodibenzo-p-dioxin (2.3.7.8-TCDD) being the species with the highest avidity for this receptor (aryl hydrocarbon hydroxylase (AHH) receptor; TCDD receptor). and causing the most severe effects (for review see Safe. 1984). And although the classic toxicological symptoms (thymic and splenic involution. hepatotoxicity. chloracne. immunotoxicity. wasting syndrome) have been correlated with the AHH receptor binding affinity of the various congeners. the commercial mixture of PBB used in these studies (FM) contains only small amounts of these coplaner congeners (Sundstrom gt gig. 1976). Yet this same mixture is a potent inducer of a mixed type of cytochrome P450 mixed function oxidases (Dent gt al.. 1978; Gibson. 1978; Moore gt al.. 1978). and is quite capable of generating toxicity 1 ‘yivg (see Safe. 1984). indicating that there are enough of these toxic congeners in this mixture. either alone or in 58 combination to elicit these various AHH receptor mediated effects. The non-coplaner congeners. including the principle congener 245—HBB. exhibit very slight toxicity (in terms of the above described effects) as one might predict from their low AHH receptor binding. However. it has been shown by Jensen gt 31; (1982). that both FM and its major congener 245-HBB are very effective liver tumor promoters of the phenobarbital (PB) type in that they greatly enhanced the appearance of gamma-glutamyl transpeptidase positive (GGT+) foci in the Pitot liver tumor promotion model. These effects were noted at concentrations in the diet that were well below the dose necessary to elicit symptoms of hepatotoxicity. Thus. adverse consequences can result from exposure to some polyhalogenated biphenyls which are not contingent upon their binding to the AHH receptor. This is not to say that the non-coplaner congeners are wholly responsible for the hepatocarcinogenic properties of PCB and PBB mixtures. Indeed. Jensen gt gig (1983) have pointed out that the tumor promoting effects of 345-HBB found at doses which caused toxic hepatocellular changes might have been due to the necrotizing effects of this compound. leading to compensatory hyperplasia. and outgrowth of preneoplastic foci from surviving initiated hepatocytes. This outgrowth. in itself. may have been a consequence of indirect inhibition of metabolic cooperation. since it has been shown that gap junctions disappear from the surface of hepatocytes during liver regeneration (Meyer gt gig. 1981; Yee and Revel. 1978). 59 There is good reason to classify most of the polyhalogenated biphenyls in the category of epigenetic carcinogens. as most of the evidence indicates they are non- mutagenic (Kavanagh gt gig. 1985; Schoeny. 1979. 1982; Williams 22.21;: 1984). and many act as promoters in various animal models of hepatocarcinogensis (Maslansky and Williams. 1981; Williams. 1980. 1983). The results reported here add additional evidence that these compounds are associated with nongenotoxic phenomena. by either directly inhibiting metabolic cooperation (FM and 245-HBB) or by indirectly inhibiting metabolic cooperation via hepatocellular necrosis with consequent compensatory hyperplasia (345-HBB). 34-TBB was not as toxic to these cells as was 345-HBB. a result consistant with its t_‘ytyg toxicity (Robertson gt git. 1983). This compound however. is a very effective promoter of GGT+ foci in the Pitot liver tumor promoter model at non—necrogenic doses (S. Sleight. pers. commJ. This suggests that perhaps a metabolite of this compound might be active in inhibiting metabolic cooperation. Since the cells used in this metabolic cooperation assay are not capable of cytochrome p450 mediated metabolic activation (Huberman gt 21;: 1984). it is unlikely that these cells would have detec- ted the effects. if any. of this metabolite. The lack of metabolic capability is a recognized shortcoming of both the V79 cell and human teratocarcinoma cell intercellular commu- nication assays. A series of investigations are currently under way to test various congeners of PBB in a rat liver cell line based assay for inhibitors of metabolic cooperation. 60 3. Human Teratocarcinoma Cell Embryoid Bodies gg g Model for Assessing Gap Junction tg tgtly Human Embryos. Gap junction mediated intercellular communication is an important aspect of early embryonic development which has been investigated in both teratocarcinoma cell embryoid bodies and early embryos. This phenomenon seems to be involved in many critical stages of embryonic development. One such stage is the morula stage in which compaction occurs. Cell coupling via gap junctions immediately precedes compaction in 8 cell mouse embryos (Lo and Gilula. 1979a; Goodall and Johnson. 1982.1984). It is clear form the work of McLachlin gt glg_(1983). that all the gap junction proteins needed for the compaction process are present early on in development. but that assembly occurs just prior to compaction. The compaction process in mouse morulae is dependant upon extracellular divalent cations (notably Ca++)(Ducibella and Anderson. 1979; Hyafil gt gig. 1981). cytoskelatal elements (Surani gt git. 1980). and glycosylation cell surface proteins (Atienza-Samols gt gig. 1981; Surani gt gig. 1981). In an effort to define the role of gap junctions in compaction. Strum gt g_l_t (1984) concluded that generation of compacted morphology was independant of gap junction formation. since gap junctions were present in both compacted and noncompacted mouse embryonal carcinoma cell aggregates. They concluded from this study and from other previous work 61 that gap junctions have a permissive effect on compaction not a directive one. However. in this electron micrograph study. no attempt was made to establish the functionality of these junctions. It is worthwhile to note that there was a marked difference in the average gap junction plaque size in compacted aggregates as compared to noncompacted aggregates (3.5 um2 vs. 0.17 um2. respectively). This 20- fold increase in plaque size suggests at least some difference in functionality between noncompacted and compacted states. Another critical role for gap junctional communication in the developing organism is the establishment of a primordium from within a seemingly uniform conglomeration of cells. The boundaries of morphogenetic fields have been correlated with the extent to which gap junctional communication is compartmentalized among cells within developing tissues. This has been shown to occur in many phylogenetically diverse organisms (Lo and Gilula. 1979b; Weir and Lo. 1984; Kimmel gt gig. 1984; Warner and Lawrence. 1982; de Laat gtglg. 1980; Warner gt _a__l__._. 1984). Guthrie (1984) has pointed out that the unequally distributed degree of dye coupling in early amphibian embryos may be related to positional information which could impact upon determination. Thus. as cells begin to differentiate towards precursor types they may limit their metabolic cooperation with other cell types. distinguishing themselves as a tissue. In support of this notion. are the observations that mouse teratocarcinoma stem cells fail to metabolically cooperate 62 with their differentiated derivatives (Nicolas _t 21;. 1978). or at least seem to limit their communication to a unidirectional communication mode (Lo and Gilula. 1980). Other studies suggest that the capacity for selective intercellular communication may be correlated with the ability of these cells to participate in normal development. The studies of Pierce and coworkers (1984). and Rossant and Papaioannou (1985) have shown that the exertion of growth control over teratocarcinoma cells by blastocysts into which the teratocarcinoma cells were injected. depended on their being in physical contact with either the trophoblast or the inner cell mass. The inner cell mass cells are known to be closely associated. both in terms of their cell surface antigens and their totipotency. with many embryonal carcinoma lines. Gap junctional communication may have been an important factor in this control. When embryonal carcinoma cells are chimaerized with blastocysts and allowed to grow it gtgtg they often times will differentiate and colonize many diverse tissues in the embryo. Their participation in normal development does depend however on the proper ratio of teratocarcinoma to embryonic cells. for when there are too many teratocarcinoma cells involved. there is pronounced abnormal development (Fujii and Martin. 1983). Thus. under these circumstances it may be necessary to have enough normal neighboring cells with which to selectively communicate in order for these embryonal carcinoma cells to differentiate properly. 63 The mechanism by which the selectivity of intercellular communication is achieved may be related to the expression or modification of Ca++ dependant glycosaminoglycans. These cell surface molecules are know to be important in intercellular recognition and adhesion (Edelman. 1976,1983). Specific intercellular recognition seems to be a widespread phenomenon. as it is known that cells from disaggregated tissues tend to reassociate. when reaggregated. with the counterparts they are normally associated with tg‘_tyg (Moscona. 1982). An example germane to the present discussion is that embryonal carcinoma cells selectively adhere to nondifferentiated cells and preferentially aggregate to each other when comingled with various fibroblastic lines (Takeichi gt gig. 1981). This preference was Ca++ dependant. Thus it seems the results obtained with the human cells used in these studies. that demonstrated their ability to communicate with normal human fibroblasts. need further explanation (see below). Low extracellular Ca++ concentration is known to induce decompaction in murine teratocarcinoma cell embryoid bodies (Kartha gt git. 1983). This is presumed to be mediated by Ca++ requiring adhesion molecules on the cell surface. since the calcium ionophore A23187 could not induce compaction in low Ca++ medium. Extracellular Ca++ and Mg++ have been shown to be necessary for the gg ggyg establishment of gap junction mediated intercellular communication in human fibroblasts (Davidson gt git. 1984b). 64 Antibodies or Fab fragments directed towards cell surface glycoproteins have been shown to inhibit or reverse compaction (Kemler gt 21:1 1977; Johnson gt gig. 1979; Ducibella. 1980; Hyafil gt git, 1980). Bird and Kimber (1984) have shown that specific fucose N-acetylglucoseamine moieties. when applied to mouse morulae can similarly inhibit compaction. Prolonged decompaction can result in morphological abnormalities (Johnson gt 21;: 1979). The role of these glycosaminoglycans in both compaction and metabolic cooperation was recently demonstrated by Kanno _t alg (1984) who found that antibodies to Ca++ dependant cell surface embryonic adhesion molecules could inhibit metabolic cooperation. The importance of this interdependancey between cell surface glycoproteins and gap junctional communication in later stages of development was recently demonstrated by Suzuki gt El; (1984). who observed that when lectins bind to cell surface glycoproteins. they interfere with neural induction in anuran embryos. and at the same time inhibit electrical coupling between inducing chorda—mesoderm and reacting ectdoderm cells. Disruption of cell coupling with intracelltllarly injected antibodies to gap junction protein resulted in dire consequences for the development of Xenopus embryos. as well (Warner gt gig. 1984). Similarly. it has been pointed out that chemicals which inhibit gap junction mediated intercellular communicaton are likely to be teratogens (Trosko gt al.. 1982). Indeed many chemicals known to inhibit intercellular 65 communicaton. are also known to be teratogenic. (Loch-Caruso gt glt. 1984; Trosko and Chang. 1984; Welsch and Stedman. 1984). Thus. the findings reported here. that TPA can simultaneously block metabolic cooperation and induce a temporary decompaction in these embryoid bodies. may be attributable to a common mechanism. As was discussed above (Discussion Section 1). the exact mechanism of TPA's ability to inhibit metabolic cooperation is not known. although several hypotheses have been offered. One of these hypotheses is based on the fact that TPA is known to affect the character of plasma membrane glycoconjugates. TPA could be acting through stimulation of protein kinase C with subsequent phosphorylation of enzymes critical to the formation of cell surface glycoproteins (e.g. neuraminidase (Srinivas and Colburn. 1982)). The development of a system which could measure the ability of a chemical to inhibit metabolic cooperation and simultaneously monitor its effects upon early embryonic differentiation would be desirable. It would also be desirable to utilize human cells for this purpose. so as to minimize the effect of species differences. The cells used in these studies seem to be an ideal candidate for such studies. A major drawback to using any carcinogenic cell line. however is the possibility of these cells acquiring genetic abnormalities in culture. Although PA-l derived cells have an exceptionally stable karyotype. there have been some 66 differences noted between early and late passage cells. Late passage PA-l cells are capable of growth in soft agar (Pavlik _t gig. 1983; Tainsky gt gig. 1984). are carcinogenic in nude mice. and seem to have acquired an activated N-Ras oncogene during serial passage in culture. because early passage cells do not exhibit the tumorigenic phenotype and do not have the activated oncogene. The observation that these cells can communicate with normal fibroblasts and with bladder cells. as well as amoung themselves. seems to be at odds with notion that differentiated cells fail to communicate with stem cells. However. in an assessment of the intercellular communication abilities of tumor cells it was noted by Fentiman t _a__l__._ (1979) that although some tumor cells had lost the ability to communicate with their neighbors. some would communicate with cell types that their normal counterparts would not. It may be that these human teratocarcinoma cells are abnormal in the sense that they are "universal" communicators. It would. therefore. be interesting to investigate the communication properties of chemically induced differentiated derivatives of HTP3-4 cells . One feature that is desirable in teratocarcinoma cells is an ability to respond to retinoic acid by differentiating lg ytttgg Even though the subcloned cells used in these studies will respond to certain agents by differentiating in culture (e.g. TPA. PDD). retinoic acid proved ineffectual in 67 inducing PA-l cells to differentiate in work reported by Kikuchi gt_glt_(1984). Although PA—l cells posess qualities which make them suited for monolayer culture studies (good plating efficiency. growth without the aid of a feeder layer. tight colony morphology. and stable karyotype) other human teratocarcinoma cells have differentiation properties that are more like the early embryo. One of these human teratocarcinoma cell lines is the HT—H line. which forms embryoid bodies very similar to preimplantation embryos (Ducibella gt gig. 1982). An encouraging feature of these cells is there development of a blastocoele—like cavity when they form embryoid bodies. and there is evidence from mouse teratocarcinoma cell studies that differentiation capacity is correlated with formation of this cavity (Uno. 1982). However these cells have a modal chromosome number of 96. making it less attractive in terms of mutation induction. Thus. it would have probably been difficult to obtain a 6—TG resistant mutant from this line. in order to conduct the studies described here. In order to answer the question of whether or not the capacity to undergo metabolic cooperation is a prerequisite for going through compaction. I tried to isolate a metabolic cooperation deficient mutant from the HTXTG-1 cell line. The method used was to mutagenize a population of these cells with BrdU/Black light (this increases the likelyhood of hemizygosity (C.C. Chang. pers. comm.)). and subsequently treat the surviving cells with ethylnitrosourea. These 68 mutagenized cells were then cocultivated with HTP3-4 cells at high cell density in the presence of 6-TG. Under these conditions. only HTXTG—1 cells which are not communicating with HTP3-4 cells should survive. The surviving clones were pooled. and put through 3 more rounds of this selection procedure. Finally. 24 candidate clones were tested for their metabolic cooperation ability with HTP3-4 cells. In preliminary screening. none of the clones demonstrated lower metabolic cooperation (higher survival) with HTP3-4 cells than did the original HTXTG-1 line (data not shown). Using a similar protocol. Slack gt El; (1978) reported on the isolation of a metabolic cooperation deficient mutant from mouse embryonal carcinoma cells. No mention was made by the authors as to whether or not these metabolic cooperation deficient variants were capable of forming embryoid bodies and undergoing compaction. CONCLUSIONS The results obtained in these studies agree in general with metabolic cooperation studies that have been performed on other mammalian cells. TPA and its analogues affected metabolic cooperation and differentiation in a manner that was consistant with their previously known activity in yitro and it _lyg. Similarly. the PBB compounds FM and 245-HBB were able to block metabolic cooperation. whereas 345—HBB and 34-TBB were unable to do so but caused. instead. a cytotoxic reaction. This is also consistant with known effects of these compounds in other biological systems. and is consistant with previously determined structure activity relationships. The ability of the PA-l derived cells used in these studies to form embryoid bodies makes them an interesting model system for investigating correlates of early human embryonic development. Metabolic cooperation could be demonstrated in these bodies. as well as adverse effects on compaction by TPA in limited studies. 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