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RESULTS Medification of the Recovery of Induced.Mutations by TPA The primary aim of this report was to test the hypothesis that TPA, the most powerfu1 tumor promoter in the mouse skin system, was exerting its effect through an inhibition of some mode of DNA repair (47,48). If such an hypothesis were true, one of the major biological consequences of the inhibition of DNA repair would be the formation of somatic mutations in those cells previously exposed to the action of an initiator/mutagen. A previous report in the literature (116) had clearly demonstrated that TPA was capable of significantly increasing the recovery of both 6-thioguanine resistant and ouabain resistant V79 cells if it were present during the mutation expression period (that period fellowing ultraviolet light treatment), well after the completion. of the period of DNA repair. Since the repair of ultraviolet light induced DNA damage is thought to be different from the repair of X-ray induced damage (54), the effect of TPA could possibly be different if administered after X-irradiation. TherefOre, an attempt was made to examine the influence of TPA upon the recovery of X-ray induced 6-thioguanine resistant V79 cells. Prior to determining the effect of TPA upon the recovery of speci- fic somatic mutant cells, a number of preliminary experiments were conducted to characterize better the response of V79 cells to TPA. Figure 3 represents a cytotoxicity experiment intended to measure V79 cell survival with increasing doses of TPA. The % survival falls quite rapidly at doses of TPA above 3/4g/ml, consequently the dose of TPA 39 40 % Survival l _ l 1 1 1° 1 2 3 4 TPA (pg/ml.) Figure 3. Survival of V79 cells to increasing doses of TPA. 41 chosen for subsequent mutation experiments was 1 [43/ ml. (approximately 94% survival). If one attempts to determine the effects of TPA on the recovery of X-ray induced mutations it should first be demonstrated that TPA is not in itself mutagenic. Utilizing auxotrophic strains of Salmonella typhimurium with single-base substitution and frame-shift mutations to histidine dependence, Soper and Evans (104) detected no mutagenic or toxic effects on the bacterial text strains with TPA at l fig/ml. In order to rule out fully the possibility that TPA was acting as a mutagen, an experiment examining the effect of TPA upon "urscheduled" DNA synthesis was performed. Most mutagens, including ultraviolet radiation and activated chemical carcinogens (72), interact covalently with DNA; the alteration of which serves as a substrate for excision repair enzymes. Therefore a determination of "urscheduled" DNA syn- thesis (excision repair) can be good evidence of a potential mutagenic event. Figure 4 illustrates the results of the experiment examining the effect of TPA upon urscheduled DNA synthesis. It is clearly evident from Figure 4 that TPA even at doses which are cytotoxic (3 ,qg/ml.) cannot significantly enhance the level of urscheduled DNA synthesis. Even at 3 Ag/ml. the amount of excision repair is not significantly elevated above control levels and is well below a very small dose of UV (0.5 J/mz), included as an internal control. From the preceding discussion it is clear that TPA is not capable of interacting covalently with DNA to the extent that a cell recognizes the interaction as a substrate for DNA excision repair enzymes. Additionally, TPA is clearly not mutagenic itself; bacterial data srpport this claim. 42 .«E. 3 mfioo m9 mo 0.5888 wcgofiom 335:? <5 eoagofimcb 23; Zeus—co: coco we: on n... adds-g a :23... . S”: \ x \\ $52, 083% x :52. 0.23.. O. m. ON oomvuo W/ wdp) Kunmoo ommds 43 However, to explicate the mechanism of tumor promotion by TPA and other promoters, one must still consider the hypothesis proposed by Gaudin e_t_ a_1_. (47,48), i.e. all tutor promoters may be acting, in part, by inhibiting the relatively error free process of INA excision repair. Support for such an hypothesis was provided by Teebor _e_t_ _a_l_. (111) who demonstrated that TPA was very effective at inhibiting the removal of pyrimidine dimers induced by high doses of UV in HeLa cells. However, molecular evidence refuting the contention that TPA inhibits 1 DNA repair was provided by Trosko _e_t a. (119), mo demonstrated a negative effect of TPA on INA repair synthesis occurring after low doses of N-acetoxy-acetylaminofltorene and ultraviolet light in hulan amnion cells. Additionally, TPA had no effect upon post-replication repair synthesis following UV in V79 cells. The work of Trosko gt; _a_l_. was more relevant than the previous reports in that the doses of carcinogens were well within the range of biological survival. Pre- vious reports had either not indicated dosage or had utilized doses of carcinogens which allowed very low levels of cell survival. It would seem quite obvious that tumor promoters can promote only living (surviving) cells and that the biological relevance of the inhibition of INA repair can (mly exist in those cells which are not killed by the action of the mutagen. Consequently, the credibility of the hypothesis that tulor promoters inhibit DNA repair had been called into question. As a further test of the Gaudin hypothesis, Trosko _e_t_ al. (116) examined the effects of TPA Lpon mutageresis (resistance to ouabain and 6-thioguanine) in V79 Chinese hamster cells. The rationale for this series of experiments was as follows: if TPA inhibits excision 44 repair and if excision repair is an error free process, then the conse- quence of inhibiting an error free process wOuld be increased reliance of the cells upon more error4prone types of repair, i.e. post-replicaticur, "gap filling" repair. Error-prone repair while conferring cell sur- vival would.be manifested as an increase in the number of cells har- boring mutant alleles. Therefbre, TPA should.increase the frequency of induced mutations. This is exactly the result Observed by Trosko §£_al,; however, TPA was effective in increasing the recovery of ouabain resistant and 6-thioguanine resistant cells only if it was present after the mutation expression time, fOllowing completion of the DNA.repair period. This result coupled with the observation that TPA did.not sensitize the cells to the killing effect of ultra- violet light led the authors to conclude that the mechanism through which TPA acts is unrelated to the repair of nuclear DNA. .Alternatively, the hypothesis was proposed that TPA was acting epigenetically, i.e. repressing or derepressing mutated and.non-mutated genes. This alter- native hypothesis would be much more compatible with the observation in whole animals that a tumor promoter can be applied up to several months after initiation and still effectively promote tumor cell growth. Consequently, it had become increasingly obvious that the inhi- bition of DNA excision repair was not a viable explanation fer the action of tumor promoters, specifically TPA. However, the Observation that TPA was indeed capable of modulating (increasing) the recovery of specific somatic mutant cells after mutagen treatment was of considerable interest. Lankas §t_al, (64) confirmed this observation utilizing chemical carcinogens as mutagens and TPA as the gene 45 modulator. It was, therefore, apparent that TPA if applied to cells at the time of mutation selection could increase the recovery of specific induced mutants. Consequently, the experiment illustrated in Figure 5 was performed to determine the effects, if any, of TPA Lpon the recovery of X-ray induced 6-thioguanine resistant Chinese hamster cells. The complete protocol of this experimert is presented in Figure 5; including both spontaneous and induced mutation frequencies . Groups A, B, and C were not X-irradiated and, as can be seen, groups B and C, both of which were treated with TPA have a lower spontaneous mutation frequency than the non-treated cells. This reduction of spontaneous mutation frequency by TPA has not beer consistently observed (data not included) and, as such, is given little significance. The cells in groups D-H have all been X-ray treated (700R, nine days prior to addition of selective medium). Group D was a control group treated with absolute ethanol (vehicle for TPA), while Groups E-H were all treated with TPA (1 lag/ml.) during the time periods indicated. Figure 5 clearly indicates that TPA treatment at all time periods with the exception of the period of DNA repair was effective in elevating the induced Initation frequency. TPA treatment only during mutation selection (Grorp G) resulted in the largest increase in mutation freqiency; however Groups F and H each exhibited mutation frequencies which were significantly (p < 1%) elevated above the non-treated cells. The most important point to be made is that TPA was effective in in- creasing the recovery of 6-thioguanine resistant cells only if it was present during the time of mutation selection, i.e. that period of growth in 6-thioguanine containing medium. There was a slight eleva- tion of mutation frequency in Group F in which the TPA was removed 24 46 T.P.A. Treatment after X-lrradiation %:1 hr % 23 hr § 7 gigs 44 23 hr % 1 hr 4 t e growth median —: 1 X-ray 6 thioguaninc A B [IUIUIUIUIHIHIHJ C lllllllI[llllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll D L 11—11—111 P llllllllllllllllllllllllIIlIIIIlllll 0 [IIIIIIIIIIIITIIIIII] [11]] T.P.A. Treatment Survival and recovery of 6 thioguaninc resistant mtants X-ray T.I’.A. Treatment Total No. Survival No. of Nation of cells in (Replating Mutants Frequency/ 24 plates efficiency) 106 survivors 0 A 4.8 x 10° 1.1 358 67 0 B 4.8 x 10° 1.1 192 36 0 c 4.8 x 10° 1.1 177 34 700m 0 4.8 x 10° 0.8 539 133 700m B 4.8 x 10° 1.0 495 103 700R F 4.8 x 106 0.7 $08a 155 7003 c 4.8 x 10° 0.8 645 174 700m 11 4.8 x 10° 0.6 484 166 a mutation frequencies of T.P.A.-troated cells (F,G,ll) were highly significant, p < ll when couparcd to the control cells (D). Figure 5. Protocol and results of TPA modification of X-ray induced mutations to 6-thioguanine resistance in V79 cells. 47 hours prior to the addition of 6-thioguanine. This effect, I feel, represents a lingering effect of TPA treatment upon the cell membrane of those cells treated during the period of mutation expression. The significance of these observations will be discussed.below (see section concerning Metabolic Cooperation). The results discussed above with X-rays closely parallel the experiments previously reported with UV and TPA. TPA is capable of increasing mutation frequencies only if it is administered to muta- genized cell populations after the DNA repair period, preferably during the period of mutation selection. In an attempt to determine if the TPA effect was discernible at increasing doses of X-irradiation the experiment illustrated in Figure 6 was conducted. The protocol for this experiment was identical to that of Group G in Figure 5. TPA was added to the cells after replating, one hour prior to the addition of 6-thioguanine. It is clearly apparent that the ability of TPA to increase the recovery of 6-thioguanine resistant mutant cells is quite pronounced at all three doses of X-rays (300R, 700R, and lOOOR). .At this point, therefore, the observation that TPA.can increase the recovery of X-ray induced.mutant cells appears sound.and reproducible. The interpretation of the above results presented some serious problems. Clearly, TPA was not inhibiting the repair of X-ray damaged DNA. This conclusion was made on the basis of the observation that TPA was effective in terms of increasing mutation frequencies only if it was administered to X-irradiated cells well beyond that period of DNA repair. As an alternative hypothesis to explain the TPA effect, one could evoke the hypothesis previously proposed by Trosko §t_§l, (116), 48 300 200 6T6.R induced mutation lrequency per 106 cells 100 o NO TPA I WITH TPA 0 300 700 1000 X- Ray Dose (R) Figure 6. X-ray induced mutations to 6-thioguanine resistance in V79 cells: dose response. 49 i.e. TPA is capable of modulating gene activities by repressing and derepressing genes. This interpretation when applied to UV-induced mutants (6-thioguanine resistant and ouabain resistant) is quite consistent on the basis of UV inducing point mutations (base substitu- tions). Derepression of such mutated, but non-expressed, genes by TPA could explain the increase in mutant recovery. However, the experi- ments under discussion in this report utilized X-rays as the mutagen . source. There is ample evidence in the literature stating that X-rays induce primarily deletion type mutations and not point mutations in higher organisms (74,106,107). This observation has recently been confirmed by Chang e1; a_1_. (25) who report the failure of both "hard" and "soft" X-rays to produce ouabain resistant mutants in Chinese hamster cells. Ouabain resistance requires a point mutation of the gene coding for the membrane bound enzyme Na+, ICATPase . X-rays are apparently incapable of producing base substitution type mutations which would result in the ouabain resistant phenotype. Therefore, it would seem that the data under consideration do not lend themselves to the interpretation that TPA is modulating gene activity. The locus in question (HG-PRT) is not essential for cellular growth; the deletion of this gene by X-irradiation would result in a perfectly viable 6-thioguanine resistant cell. It is difficult to conceive of a TPA derepression of a deleted gene. If TPA were either derepressing or, equally likely, repressing the HG-PRT locus, one would expect to observe a consistent and significant alteration (decreasing if there is gene derepression and increasing if there is gene repression) of spontaneous mutation frequercies with TPA treatment. Such fluctuations in spontaneous mutation frequencies at the HG-PRT locus in V79 cells 50 are net seen (data not included). Therefore, one is left without a valid hypothesis to explain the increase in the recovery of X-ray induced 6-thioguanine resistant mutants with TPA treatment. TPA Modification of Metabolic Cooperation Inherent problems exist when one attempts to measure mutation frequencies in mammalian cells. One of the most important factors to consider is the effect of cell density upon the recovery of the mutant cells. One type of intercellular communication which must be taken into consideration prior to establishing proper experimental conditions is metabolic cooperation. Metabolic cooperation is a form of cell to cell communication in which the phenotype of enzyme deficient cells can be corrected by wild type cells. The most extenSively studied example of metabolic cooperation is the HG-PRT (EC 2.4.2.8) system first described by Subak-Sharpe and his colleagues (109) . In this system, mutant cells deficient in the HG-PRT enzyme when co-cultivated with wild type cells (HG-PRT+) will incorporate the phosphoribosylated purines of hypoxanthine or guanine into their nuclei. On the basis of radioautographic data it appears as if metabolic cooperation in this system requires cell to cell contact and an enzyme product Or its derivative (inosine monophosphate) is transferred from wild type cells to mutant (HG-PRT-) cells (31). Figure 7 graphically illustrates the process of metabolic cooperation as it occurs with the natural substrates for the HG-PRT enzyme. The HG-PRT system can be adapted for use as a mutation assay simply by supplying the cells with the-purine analogue, 6-thioguanine. 6-Thioguanine can serve as a substrate for the HG-PRT enzyme. Its incorporation as 6-thioguanylic acid into 51 .903 had: 85 8 £8 mS an Emofioeoou 0:088: .N 8%: ALmaéIv #2555. A+._.ma-czv mat. 04:5 .Nxzuxcmzigo 42¢ i u x. mzihzsxofi: z Ozwo azz ux. “.2256 i ux. 3.52583: 52 nuclear DNA.and RNA will result in cell death. Figure 8 illustrates the mechanism.through which 6-thioguanylic acid (6-thioguanosine monophosphate) can be transferred to a mutant cell (HG-PRT’) resulting in its death. As is apparent in Figure 8 the mutant cell, if it were not communicating with a wild type cell, would.be resistant to the cytotoxic effect of 6-thioguanine; however if the cell density (ratio of wild type cells: mutant cells) is above a critical point there will be a considerable amount of cell to cell contact resulting in the communication which will eventually destroy a.mutant cell. Therefore, it is absolutely critical that .a proper protocol be developed which eliminates cell density effects (metabolic cooperation).‘ Only under these conditions is it possible to compare two cell lines or treated versus untreated cells in terms of their mutation frequencies. One experiment which will provide the data to rule out the effects of cell density upon mutation frequency is a reconstruction experi- ment.' As the name implies, the experiment is intended to reconstruct the conditions under which the mutation frequency was originally derived. This is accomplished by seeding a known, small number (usually 100) of preexisting mutant cells (6-thioguanine resistant) with increasing numbers of wild type cells in the same dishes. Figure 9 illustrates the results of a reconstruction.experiment Conducted with and without TPA. It is apparent that the recovery of the 6 TGR mutant cells falls off quite precipitously as the number of wild type cells increases. For example, the recovery of the mutant cells falls to approximately 75 per cent if there are 2 x 105 wild type cells seeded in.the same dishes. Hewever, if TPA is added to the plates immediately fellowing attachment of the two cell lines the recovery of the 6 TGR 53 63538 ounce ”803 590mm 05 um mfioo mg 5 839838 630%qu AFCQuOIV 5.24.242 A+hmauarv wa>._. 04:5 weooom .3 mafia 3.85 2258: 4% a 508.. t «N o _ the Q\ - u — O 8 81198 1919901111 10 asuaseud 9111 U! $1110an .1919 10 1119110993 °/. 00. 58 the mutant cells to nearly 100. It is clear from this experiment that ten minutes exposure of the two cell lines to TPA is sufficient to indme membrane modifications which are sufficiently enduring to reduce intercellular conmnmication and, consequently, increase the recovery of the mutant cells. Table 1 illustrates the results of an experiment intended to determine if the TPA reduction of metabolic cooperation were dose responsive, i.e. did increasing doses of TPA result in increasing percent recoveries of the 6 TGR mutant cells. As is apparent in Table 1 all doses of TPA were maximally effective at increasing the recovery of the mutant cells. Of major significance, however, was the result obtained with phorbol, the parent alcohol of TPA and the phorbol esters. Phorbol which has little or no mouse skin in 3.119. tumor promoting ability at a dose equimolar to the highest concentration of TPA proved to be entirely negative in this system. The implications of this observation are of clear cut importance. If a lmown non-tumor promoter is incapable of modifying intercellular conmunicatim (meta- bolic cooperation) while a strong promoter tests very positively in this _i_n_ y_i_tr_o_ system, then perhaps the system could be useful for the in 11.1519. detection of tumor promoters. This modification of the TPA effect upon metabolic cooperation will be discussed more fully below. Figure 11 demonstrates a definite dose response relationship with TPA. A dose of 0.01 ng/ml. (0.00001 Ag/ml.) has very little effect upon the recovery of the 6 TGR cells while 1 ng/ml. (0.001 Ag/ml.) is of maximun effectiveness. 59 . HWOH .mfinox .. HHNEEZuwfioflspw 23 on wfiwhouom $53 8. v a 05 pa H9550 ~26 c.3963 5383339...“ 0.83 (E. mo 3me H2 « om 4.3135 - 2: m3 x a «ma - .ERISé 2: m2 x 5 «NS - .dfimia 2: m3 x a «ma - .E\m1m.o 2: 1: x a «8 - £35 2: mg on a om - - OOH mOH x a ma - aims: OS - ooH . . OOH - mwwowomw Bfiofi .5: an: 0 mm: o 33m. .5 m Mom 330 mo .02 .Honpofi 98. «E. mo momop wait? mo 8:895 .23 5 mHHoo m8. 0 mo Egouom .H 033. 60 .3833 «wow "3H8 .HB. 0 mo .CESuoH 05 mo :oflmoflflooe $3. .2 95mm :Eos 8:228:00 4n: aw! awe. .owo. 6.30. o Ir IO N 9 81:83 .919 40 Memea °/. IQ .. OO— 61 Phorbol Ester Analogue Modification of Metabolic Cooperation At this point it is clear that lZ-O-tetradecanoyl phorbol-l3- acetate (TPA) , the most powerful mouse skin tumor promoter yet identi- fied, is capable of reducing cormmmication between 6-thioguanine sensitive and 6-thioguanine resistant cells. The effect of TPA, which is manifested as an increase in the recovery of the 6-thioguanine resistant cells, has also been demonstrated to be close respcmsive. However, only phorbol, a known ineffective mouse skin tumor promoter has been shown to be negative in this system. Therefore, it was deemed necessary to validate the system with a series of graded phorbol derivates, synthetically produced, each of which exhibited a degree of in mg tumor promoting activity. Table 2 illustrates the results of the experiment. Excellent correlation was observed between the E 2.119 tumor promoting activity and the _in 2.1.2.9. percent recovery for seven separate phorbol derivates (including phorbol and TPA). In addition, the assay system demonstrated excellent precision, in that the i_n_ Egg assay, rank order of promoters identically paral- leled the i_n_ m tumor promoting rank order. This observation provides considerable credibility for the claim that this system is capable of detecting known whole animal tumor promoters. Timor Promoter Modification of Metabolic Cooperation The series of phorbol derivatives is certainly of major importance in any discussion of tumor promoters; however any system which intends to detect tumor promoters must examine a series of proven whole animal tumor promoters. Consequently, a group of experiments was undertaken in an attempt to refine and further validate the system. 62 mumpoom-mH m.~ u. 2: +1... oompflbéfi 398% 2: m2 x w N.N n ma 1.. Baofiomfieéffi Hofiofi 2: mg on w a; ... 93 I 82,338-32 Hofiofi 2: m2 x w opmuoum-mH m; a. gm + 833:5-2 Hofiofiafime-o-a 2: E x w 3 ... 3m + 88088-2 .2 398% 2: m2 x w m; a. 9:. - Baofiuogeifi Hofiofi- a 2: m2 x w mg n. mi - Hofiofi 2: m2 x w m; u. 58 - 20558 H828 2: 1: x m Spam Emecfim .3932 magenta 83:5 Hofiofi add 0 mm: o w xho>oomm w HQESH o>m> :H .moswofimcw poumo Honhogm mo oucmmopm any :a mpmamfleeo a you mHHou mo .02 mHHoo ace 0 mo th>ouom .N oHan 63 The drugs chosen for examination in the in_vitrg_assay system were chosen on the basis of their ability to represent a specific class of tumor promoter. Just as there are specific and different classes of tumor initiators, i.e. UV, chemical carcinogens, and ionizing radiation; there are various classes of tumor promoters. Among the various classifications of tumor promoters are (l) exogenous chemicals, (2) wounding, (3) cytotoxicity, (4) hepatectomy, and (5) growth stimuli (45,114). The particular in_vitrg_system.under discussion obviously lends itself best for the detection of exogenous chemical promoters. Chemical promoters themselves can be classified into specific types, i.e. skin promoters such as the phorbol ester series and TWeen 80, butylated hydroxytoluene as a representative promoter of lung tumors in mice, and phenobarbital as a representative rat liver tumor promoter. Clearly these categories are only arbitrary and.do not imply that there are specific chemical configurations whiCh determine the class into which any tumor promoter falls; however such categories are useful in that they better characterize an.individual promoter and allow a more meaningfu1 interpretation of the results of tumor promoter administra- tion. Table 3 represents the results of an experiment intended to measure the response of two well documented rodent tumor promoters, phenobarbital and butylated hydroxytoluene , in the i_n_ v_i_t_r2 tunor promoter assay. As is apparent in Table 3 the average percent recovery of 100 6-thioguanine resistant cells when seeded with 7 x 105 6-thioguanine sensitive cells is approximately 41%. TPA, included as an internal control, more than doUbled the control percent recovery and both phenobarbital and butylated hydroxytoluene significantly 64 .umou xzm .8. v a £38 @385 LB: 96%.. 3353 39533:me mm: modem @335 035 23 mo some mo rogue 25989 9:. HS”2 OOH .HauHOmeocmeO .O 4O.x_OO .memsHoprogeAn OmomHApsm .m .N .H .HEAMyx H.O .oomumum-mH-HOOpoga HAoaauoeeeoou-O-NH OH «ma OH.N A H.~O mocmsHOprOAwA; eopmHAusn OOH mOH x A OO.~ w O.AA NHmOHOAmOoemee OOH mOH x A m~.m A O.NO HOmA OOH mOH x A AO.N A O.HO fiHoemepoO HOAocou OOH mOH x A «Show 235% ..... bo>ouom w 9395.5 90.5% m6? 0 mdé o 339 .50 m pom v.28 mo .02 .Hmfifimnocofi dam mom—Soggo»: woumgusn mo oocomoym 23 5 330 m8. 0 mo bgoumm .m 3an 65 (p < 1%) increased the recovery of the thioguanine resistant cells. This result is consistent with published reports in the literature which indicate that phenobarbital is a more effective promoter of rat liver tumors than is butylated hydroxytoluene (86) . Therefore, the _‘11_1_ m assay system identified as positive two well researched chemical promoters and, in addition, was precise enough to rank them in order of effectiveness. Table 4 reports the data testing a weak mouse skin tumor promoter (47), the common laboratory detergent Tween 80. The weakness of this promoter in whole animals is reflected in the _in _v_i__t_r_g assay. TPA more than doubled the average percent recovery of the thioguanine resistant cells while 'Ilveen 80 produced a much smaller, but statisti- cally significant, increase above non-treated cells. Table 5 illustrates the results of an _i_n \_r_i_£r_o test of mezerein. Mezerein is a phorbol-like diterpene which shares with TPA the ability to act synergistically with phytohemagglutinin in the mitogenic stimulation of bovine lymphocytes (61). Mezerein is nearly as proficient as TPA in this system and is an excellent candidate as a mouse skin tunor promoter (studies now in progress, T. Slaga, personal comnunication). The results of the i_n_ yi_tr_g test certainly parallel the E 1119 results; mezerein increases the recovery of 6-thioguanine resistant cells from 43% to 95%. This increment of increase is identical to the response of TPA; therefore on the basis of the results obtained in this i_n_ 11.9.9. assay of tumor promotion one would predict that mezerein would be an excellent mouse skin tumor promoter. Another drug that has yet to be tested in a whole animal tumor promotion system is melittin, a 26 amino acid polypeptide which is 66 .pmop xzm .Ho.o v a .mHHoo woumopu-:o: o>onm commohucH xHquuHchmHm .m .>\> ONOOO .OO :85. .N .HE\wv\ H .mumpoom-mH-Honhocm onomoowmnpop-o-mH mH ouom w H m m oumHmleU m pom mHHou mo .02 .ow coozp mo oocomopm ecu :H mHHou mob 0 mo xuo>ooom .v oHnmh 67 .umou Mzm 65 on méuouum Sod v 3 30.580 96am @3863 .AHHmoHumHumpm ohm modem @385 5.0.832 one «an. .m .HEOX H.O £3351 :A .dfimvx Hod .opmuoom-mH-Hon.HonOH onqmooombouééH 3 <3. .H OO + + OOH OOH x O OO + - OOH OOH x O OOO - + OOH OOH x O OO - - OOH OOH x O AA - - OOH - RWWOWOWOO NEBONQ Hs: Odo. O Ode. O 3qu .5 m .Hom mHHoo mo .02 .5289: mo 858.5 23 5 330 m8. 0 mo bgooom .m 633. 68 the major constituent of bee venom. In a C3H/10T‘1 mouse embryo cell culture system melittin has been shown to act very similarly to TPA, i.e. to inhibit differentiatim of mouse melanoma cells, to enhance anchorage-independent growth of virus-transformed rat enbryo cells, and to induce prostaglandin synthesis (77). Therefore, melittin was chosen to be tested in the _i_n_ _vi_t£g system. The results appear in Table 6. The lowest concentration of melittin effectively doubled the recovery of the thioguanine resistant cells and increasing doses of nelittin resulted in increasing recovery of the thioguanine resistant cells. Therefore, on the basis of the in _vit_r_o_ test results one would predict that melittin would be an effective whole animal tumor promoter. The whole animal studies are currently in progress (T. Slaga, personal commmication). PBB, a compound of considerable interest to those of us living in Michigan, was the next drug to be tested in the i_n_ \_r_i_t_1_‘_o_ system. PBB, a widely distributed fire retardant, is a complex of polybromi- nated biphenyls known as Firemster BP-6 . There have appeared in the literature reports possibly implicating PBB as a tumor promoter (1) . Table 7 indicates the results. PBB at two non-cytotoxic concentra- tions did not statistically significantly increase the average percent recovery of the thioguanine resistant cells. Therefore, on the basis of these results, one would predict that PBB is not a tumor promoter. However, technical problems arising from the handling the drug (possible photosensitivity) necessitate repeat testing. In my judgment the _i_n_ 112E system at this point had been ade- quately verified as an assay capable of detecting known or strongly suspected tunor promoters. The system was further characterized in 69 .pmou VHzm .HohEou 96am mHO.O. v 8 35>on OHHonumHumum .dfiwi H 50339588 :HpuHHoz .m .38 OHzm £838 96%.. HHO.O v 3 @3963 zHHmuHumHumum .HE\O( H.O .coHpmeHHmoqou 532% .v #93 Mzm .HoHEou 269m mHO.O v & 39.963 xHHmuHumHumpw .He\w1 HO.O 50322850 5320: .m .Eo:o> Poop .532on .N .HEOI HOO.O .BSOUOOHHOOSOO HOSSOOOSBOOH OH «E. .H - x mOm + OOH mOH O - x Own + OOH mOH O mom + - OOH OOH x O OO - + OOH mOH x O OH - - OOH mOH x O OO - - OOH - me O mo :HpuHHmz ouom .O 338. m 70 .O-mm OopmmemuHm .menoann woumcHEOOpromsmo ongEou O OH mmm .N .oumaoom-mH-Honhozm oncmoowmupoa-O-NH mH ooom O pouosogm #0559 m.o.e O m.o.e O mpmHm .20 O pom mHHoo mo .02 .mecoann OouquEOOOOHom mo ongEou a mo OUQOOOOm.ogu :O mHHoo OOH O mo Ahm>ouom .O oHOmH 71 this laboratory (117) . Chemical compounds as structurally and func- tionally dissimilar as DDT, anthralin, saccharin, Ween 60, deoxycholic acid, lithocholic acid, and cytochalasin B have all tested positively in the in _v_i_t_r_g system. All of these substances are either known or strongly suspected tunor promoters. Therefore, the validity of the _in 1i_t_rg assay system has been substantially demonstrated. One class of compound not yet discussed is the anti-tumor promoter. These compounds when applied concurrently with known tumor promoters are capable of inhibiting the hyperplastic, proliferative response of the promoter. As a consequence the early onset and increased fre- quency of induced tumors is effectively prevented. Two of the most extensively discussed anti-tumor promoters are the retinoids (synthetic amalogs of Vitamin A) and dibutyryl adenosine 3' ,S'-monophosphate (dibutyryl cyclic AMP). The anti-tumor promoting ability of the retinoids has been thoroughly documented (105). In addition there have been a number of reports verifying the anti- tumorigenic capacity of cyclic adenosine 3' ,S'-monophosphate and its derivatives (26,58,97,130) . In an attempt to determine if the _ip_ \_ri_t_r_g tunor promoter assay were capable of detecting anti-tumor promoters the experiment illus- trated in Table 8 was conducted. Retinoic acid and dibutyryl cyclic AMP were each applied to the cells concurrently with TPA. Neither compomd proved to be effective in eliminating the TPA effect. The recovery of the 6-thioguanine resistant cells in the presence of TPA was essentially unchanged by retinoic acid and dibutyryl cyclic AMP. 72 42.O H .OHOO OHOOHOOO OH OO .O .25 O0.0 .O2ooom .O OHOOH m 73 Characterization of Metabolic Cooperation in Human Diploid Fibroblasts The experiments discussed above were all conducted with V79 Chinese hamster fibroblasts. There is, however, no a priori reasm to suspect that such a system would not function in human diploid fibro- blasts. If such a system were developed it would eliminate the extra- polation one is forced to make from transformed rodent cell line to human cell line. Of course the extrapolation from cell culture results to the i_n_ 3.119. condition still must be made. Metabolic cooperation at the HG-PRT locus exists and is well docu- mented in hunan diploid fibroblasts (31,121) . The form of metabolic cooperation in diploid human fibroblasts is identical to that of V79 cells, i.e. wild type (HG-PRT") cells can transfer phosphoribosylated 6-thioguanine to mutant 6-thioguanine resistant cells (HG-PRT'). The result of this cross-feeding is the suppression and eventual death of the 6-thioguanine resistant cells. In the humn system the 6-thioguanine resistant cells are derived from patients with Lesch-Nyhan syndrome. These diploid fibroblasts are genetically deficient in the HG-PRT enzyme and, consequently, are constitutively resistant to 6-thioguanine. Given these considerations one would predict that lco-cultivation of Lesch-Nyhan cells with increasing numbers of 6-thioguanine sensitive cells would result in decreasing recovery of the mutant cells. Figure 12 confirms this prediction, i.e. 100 Lesch-Nyhan cells plated with 8 x 104 6-thioguanine sensitive cells allows for the recovery of approximately 45% of the mutant cells and there is less than 1% recovery of the Lesch-Nyhan cells when they are grown in the presence of 3.2 x 105 wild type cells. However, as is apparent in Figure 12, TPA does not affect the human fibroblasts in the same manner as it 74 IOO~ IO O-—-O WITHGJT TPA. 0-—-‘ WITH T.P.A. as RECOVERY etc." LESCH-NYHAN CELLS I l l l I l o 02 04 on LG 32 NO. or WILD TYPE (6 T.G.') CELLS PER 9 cu. PLATE 1: I05 0. Figure 12. Reconstruction experiment with and without TPA: human diploid fibroblasts. 75 does the V79 Chinese hamster cells. Quite the contrary, TPA actually reduces the recovery of the 6-thioguanine resistant Lesch-Nyhan cells. At all densities of wild type cells TPA slightly lowers the average percent recovery of the Lesch-Nyhan cells. The experiment was con- ducted twice and.the results were identical each time. Two additional chemicals, phenobarbital and.butylated hydroxy- toluene, were tested in the in y_i£1_'_o_ system using diploid hunan fibro- blasts. Preliminary results (data not included) would indicate that neither of these two strong rodent tumor promoters tested positively in the hunan fibroblast system. However, the single chemical which tested.positively in the human fibroblast system was cytochalasin B. Cytochalasin B is a compound known to disrupt microfilaments and to bind to cell membranes (31). It has also been demonstrated to significantly reduce metabolic cooperation (measured autoradiographically) between BHK (baby hamster kidney) fibrOblasts and Lesch-Nyhan human fibroblasts (31). Table 9 clearly indicates that cytochalasin.l3 effectively increases the recovery of 6-thioguanine resistant diploid human.fibrob1asts when co-cultivated with 6-thioguanine sensitive cells. The increase in the recovery of the Lesch-Nyhan cells is from 5% to almost 37%. 76 OO. u. O.OO O :HOOHOHHBHOU OOO OOH x O.H OO. H NO HHoHafiOO H828 OOO OOH x O.H Hotm whmfiqmum MOHOEOHOH Umpumfimm m.U.B o m.U..H o - b0>o00m O + 821:0 O 8% mHH0u mo .02 .m 5930:0005 mo 00:08.5 05 5 mHH00 §§ZLH0O0H mo r0>000m .O 030g. DISCUSSION The hypothesis which generated the experiments discussed above was that tumor promoters may be acting by inhibiting the repair of DNA. One would predict that one of the major biological consequences of the inhibition of DNA repair would be an increase in the appearance of mutant cells (69). The experiments described above have provided evidence which helps rule out the possibility that TPA, as a represen- tative tumor promoter, is acting by inhibiting DNA repair. TWO lines, of evidence support the contention that the effect of TPA is not related to an influence upon DNA repair. First, TPA.has been demonstrated to have no specific inhibitory effect upon UV-induced unscheduled DNA synthesis in human cells or upon post-replication repair in Chinese hamster cells (119), and secondly, TPA is effective at increasing the recovery of specific somatic mutant cells only if it is present duming the time of mutation selection (116), well beyond.the period of DNA repair. In addition, the work described above has provided quite reasonable information as to the mechanism.through which TPA increases the recovery of 6-thioguanine resistant cells. TPA has been demonstrated to be influencing the ability of two cell lines to conmmicate with one another, i.e. to cooperate metabolically with one another in the 6-thioguanine - HG-PRszutation system. Although it is beyond the scope of this report to demonstrate directly the alteration of intercellular communication by TPA, good circumstantial evidence has been presented which suggests strongly that TPA acts at 77 78 the level of the membrane by altering structure and/or function. The best evidence that TPA Operates in our system at a time when cells are commznicating with one another is the observation that TPA is effective in increasing the recovery of the mutant 6-thioguanine resistant cells only if it is present when wild type and mutant cells are alive and.communicating. TPA had no specific inhibitory or stimulatory effect upon the colony forming ability of the 6-thioguanine resistant cells when they were grown alone. This Observation is consistent with previous mutation studies (64,116) which indicated a TPA effect upon mutation frequencies only if the drug were present during the time of mutation selection, i.e. when wild type and.induced :mutant cells were seeded together in the presence of selective medium. The literature recently'has tended to support the view that the cell membrane may be an important target site for the phorbol ester class of tumor promoters. TPA is a highly lipid soluble molecule (57) which has been demonstrated to cosediment with nuclear membranes in CsCl equilibriun density gradients (63). In addition, TPA has been shown to associate rapidly with a cell membrane-riCh fraction isolated from 3T3 cells previously exposed to the drug (100). Rapid changes in membrane morphology and permeability (99) fOllowing TPA treatment of cells in culture have also been observed. TPA.has also been shown to reduce significantly and reversibly a transfbrmation-sensitive membrane protein (LETS) (18). The putative early changes in cell membrane permeability caused by TPA are reflected.in a stimulation of 8612b+ and.32Rb+ uptake within 5 to 10 minutes of incubation of mouse 3T3 cells (124). .Also implicating the cell membrane as a target site for phorbol ester tumor promoters is the observation that phorbol 79 myristate acetate (TPA) is an effective mitogen for cultured human peripheral blood lymphocytes (41) . And finally there is evidence that TPA is capable of stirmilating certain membrane bomd enzymes (NA+,K+-ATPase and S'-nucleotidase) (98), while at the same time modu- lating membrane protein conformation (129) . Considered collectively these observations offer convincing evidence that the cell membrane is a critical target site for TPA and its related phorbol ester tumor promoters. The fact that TPA is known to interact with cell membranes is of particular importance in any discussion of the TPA perturbation of metabolic c00peration. The type of metabolic cooperation mder discussion is a form of junctional transmission that allows the direct flow of matter between cell interiors (103). These permeable jmctions appear to be membrane protein appositions which are highly infiltrated with aqueous channels. The channels are thought to be contained within the membrane and have been referred to as gap junctions (70). Current thought is that the passage of small molecules through gap jmctions is responsible for tissue homeostasis, perhaps by regulating ionic and metabolic pools (11,67). In addition, gap junctions are thought to be the channels through which the "signals" responsible for the induction of developmental processes, as well as for the control of growth and replication, may be transmitted (29). Therefore, it appears reasonable to suggest that TPA is in some mknown manner altering membrane structure and/ or membrane function in such a way as to significantly reduce the transfer of toxic nucleo- tide(s) from a wild type (6-thi‘oguanine sensitive) cell to a mutant (6-thioguanine resistant) cell. As a consequence of this disruption 80 of membrane integrity, which is postulated to include gap junction protein irregularities, the mutant cells will be rescued and will proliferate in the 6-thioguanine containing medium. This is the most reasonable hypothesis to explain the tumor promoter induced reductim of intercellular communication seen between wild type and mutant Chinese hamster cells. The speculation could be offered that tumor promoters in general may Operate _i_n_ Lilia by disrupting the flow of regulatory substances from normal cells to initiated, preneoplastic cells, eventually allowing initiated cells to express their transformed phenotype. This speculation is consistent with a number of experi- mental and theoretical reports, each of which points to the importance of intercellular communication between normal or wild type cells in physical contact with mutant or transformed cells. Borek and Sachs (19) have demonstrated that non-transformed hamster or rat cells could inhibit the replication of transformed cells when they were cultured together. Sivak and VanDuuren (101) noted that a purified fraction of croton resin enhanced the recovery of virus-transformed cells co- cultivated with high densities of nor-transformed mouse 3T3 fibro- blasts. Bertram (16) and Lloyd et a1. (66) showed that untransformed lOTlg cells can by co-cultivation suppress the expressibn of trans- formed CSH lOT‘Is cells. Recently, Bertram (17) has demonstrated that the mechanism, by which non-transformed lOT": cells cause the inhibition of growth of co-cultivated malignant cells, (seems to involve the modu- lation of cyclic nucleotides in mediating intracellular communication between normal and transformed cells. Three additional observations may be taken into consideration: (1) transformed human cells exhibit reduced contact mediated comrmmication when compared to normal cells 81 (29); (2) malignant mouse melanoma cells do not form tumors when mixed with cells of a non-malignant clone (79); and (3) TPA.has been demon- strated to directly alter the intercellular connections (including the membrane and desmosomes) of mice epidermal cells (62). Clearly these reports demonstrate the ability of normal (non-transformed) cells to suppress the transformed or malignant phenotype of cells with which they are in physical contact. .Additionally, it would appear that tumor promoters, specifically TPA, are capable of disrupting the flow of regulatory information fromunormal to "initiated" cells. The result of such disruption may possibly be proliferation of the initiated cell population until it reaches a critical mass, at which point it could become promoter independent. Recently (9), a.mathe- matical model was postulated to explain just how a single transfbrmed cell could proliferate until a "critical mass" was reached. The theory upon which the model was based depended upon the postulation of a locally reduced mitotic inhibitor (chalone) allowing proliferation of the transformed cell. It is, therefore, unequivocal that the transfbrmed phenotype may remain unexpressed as long as there is an uninterrupted flow of "information" from the surrounding environment of normal cells. In addition, since tumorigenicity has been shown to be a recessive trait (108) in all but virally transfermed cells, it is likely that tumorigenic cells can be converted into normal cells when regulatory sUbstances from normal cells are transferred by metabolic cooperation. The work described in this report provides evidence that one consequence of tumor promotion administration may be a disruption of intercellular communication, eventually leading to proliferation of 82 the transformed cell. Speculatively it may be suggested that TPA might possibly be acting by altering membrane permeability and thereby influencing the flux of calcium ions. Any alteration in the intracellular level of calcium ions would be predicted to have major consequences on the rate at which a cell divides and on the physical integrity of the plasma membrane. Perhaps the effect of TPA upon metabolic cooperation is mediated by calcium.ions, such that TPA.perturbations of membrane permeability are reflected in calcium dependent alterations in the manner in.which cells communicate with one another. As should be Obvious, the exact mechanism of tumor promotion has yet to be reported. The experiments reported above do, however, provide some evidence, albeit circumstantial, that tumor promoters alter the manner in which cells communicate with one another. Whether this alteration of intercellular communication by tumor promoters is operative in_vivg_will have to be determined. .As noted in the Results section above, TPA was completely ineffec- tive in terms of altering metabolic cooperation in a diploid human fibrdblast system. For the fellowing reasons these results were not entirely unexpected. The vast majority of all human cancers are carcinomas (epithelial in origin); therefore observing a negative response to tumor promoters using diploid.human fibrOblasts is not surprising. Attempts are currently underway in our laboratory to characterize a human epithelial cell line with which we could repeat the metabolic cooperation experiments with TPA. Hewever, TPA may not be the most appropriate tumor promoter to test in any human _i_n_ mpg test. There is evidence that TPA is metabolized completely 83 differaltly in four human fibroblast cell lines in comparison to Syrian hamster embryo cells (81,82). The search for a human system to detect tumor promoters must, therefore, continue. SlM/IARY The rationale behind the experiments reported above was that tumor promotion in the sequential two-stage view of carcinogenesis was molecularly different from the initiation step. Evidence at this point in time would favor the view that tumor initiation is probably as a result of a somatic mutation in a gene whose product is essential to the regulation of cell division. However, the exact molecular mechanism of tumor promotion has proven to be much more elusive. In this report, it has been demonstrated that lZ-O-tetradecanoyl phorbol- lS-acetate (TPA), the most powerful mouse skin tumor promoter, is not acting as a mutagen. Quite the contrary, it appears as if the initial target site of TPA, at least in a Chinese hamster fibroblast system, is the cell membrane. If a genetic marker (6-thioguanine resistance) is introduced into V79 Chinese hamster cells such that a phenotypically distinct cell line can be cloned, it is possible to observe the consequences of the phenomenal of intercellular communication between the two cell lines. The conseqLence of the type of intercellular communication occurring between 6-thioguanine resistant and 6- thioguanine sensitive Chinese hamster fibroblasts is a reduction in the recovery of the mutant 6-thioguanine resistant cells. This reduction in recovery can be essentially completely prevented by TPA. Since it has been demonstrated in other laboratories that the type of intercellular communication we are observing requires cell to cell contact and is postulated to occur through membrane bound gap julctions, we have postulated that the initial consequence of TPA is a disruption 84 85 of membrane structure and/or fonction such that communication between the two cells is disturbed- The TPA effect has been demonstrated to be dose responsive and excellent correlation has been Observed between the in;yivg_tumor promoting ability and the in_vitgg_response of a graded series of phorbol ester derivatives. 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