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    Int. J. Dev. Bio, 35: 239 1-249 (1991) 239 The influence of mouse sera, regenerating liver extracts and bacterial products on the abi ities of different cells in vitro NEVEN ZARKOVIC, MAJA OSMAK, DURDICA NOVAK, NELLA LERS and MISLAV JURIN* Department of Experimental Biology and Medicine, Ruder Boskovic Institute, Zagreb, Repubile of Croatia, Yugoslavia ABSTRACT In the complexity of host tumor relations, the rege! ‘tumor is growing, or in some other tissua in the orga tion of the tissue in which the sm, could influence the maturation of tumor cells, Le. tumor reversion. Clinical observations and experiments on plants, lower animals, or animal ‘embryos, performed by several authors, and our results on the influence of regenerating mouse liver (on the abilities of tumor transplanted there or elsewhere in the organism led us to study the ‘growth of different calls or bactoria exposod to tho from these animals. Further, sterile used bacterial medi respectively. Depending on the model, liver extracts - particularly extracts and regenerating liver ~ were shown to inhibit radioactive thymidine incorporation ‘or colls per culture was lower than in otherwise treated experiments, the number of bacter corresponding cultures. Further, used sterile me vitro xxtracts of normal or regenerating liver and/or sora \dded to bacterial or cell cultures, ra from mice with ‘the cells. In these of bacterial cultures stimulated the growth of bacteria but inhibited thymidine incorporation into fibrosarcoma cells in vitro. Whether this means that one or several common regulators ‘open question, The very provocative. in nature appears as an intriguing, but still completely ‘of controlling tumor growth by using such regulatory growth factors seems KEY WORDS: reyenerating tissue extracts, tumor cell growth in vitro, used bacterial media, partial hepatectomy Introduction Although intensively investigated, the regulatory mechanisms af cell growth and development (cell maturation) are still not entirely Understood. This topic is of essential importance, not only for Sciontists working in different fields of biomedical research, but ‘also for 8 practical approach to the therapy of various diseases, especially cancer. The possibility of controlling tumor growth by Inducing malignant cell differentiation sooms very attractive. Nu- merous factors, such as vitamins, xanthines, growth factors, etc, have been successfully used for this purgose in particular exper mental systems (Che Chung and Neweomer, 1977; Santora et a, 1979; Tsuchya, 1982; Bakrenashvile, 1982; Sohingler and Sherman, 1984; Pavelic and Spaventl, 1987), However, the useful ness of these agents In cancer therapy is stil doubtul (Moore, 1977; Sachs, 1978; Caims, 1979; Scnwarz eta, 1982; Nomure, 1983; Pavelic et al, 1990). On tne otherhang, the phenomenon of spontaneous cancer regression of even reversion taward normal cells indicates that there are certain unknown mechanisms in the ‘organism which could control tumor growth (Everson and Cole, 1966). Since the spontaneous regression of cancer cannat be generally explained by successful immune reaction of the organisin Fax 425.407 against tumor ces, the mechanism of this phenomenon remains only speculative (Jurin, 1973; Levison, 1976), Tumor regression ot the reversion of malignant cells can also be obtained in plants and lower vertebrates by exposing the tumor to the influence of regen: crating tissue (Rose and Waligiord, 1948; Braun, 1961; Melns, 1977), Furthermore, murine malignant cells can also be induced to ‘iferentiate into normal cells if injected into an eary stage embryo (McBurney, 187). Aiming to test whether adult animals also possess the capability ofcontroliingtumorgrowtn, we nave analyzed the growth of murine malignant tumors in regenerating liver. During ‘the growth ofregenerating verthe anaplastcityof methyicholantrene Induced fibrosarcoma decreased, as well as the incidence of Ive cells and the intensity of pigmentation in melenoma B46 tissue Abhecations sain his paper CS, al serum; EGF, eptvelial growth factor GS fecal call serum: HPT, hepatoprotein A: HPTB, hepatoprotcin Bl, Ioan serum: t-6,interieukin 6: LE, ler extract NUE. Iner extract fromm normal mouse; NMS, normal mouse serum; PHLE, liver extract from partially hepatectomized mouse; PHMS, serem from partially hepstectomized monse; SLE, liver extract ftom sham-hepatectomined mouse; SHMS, serum fom sham-hepatectomized mouse; Sisline; TGF 8, teanstorming growth fctor = for raprinte: Department of Exparimental Biology end Madicina, Ruder Boskowlnettuta,BijanGkae. 4, 41000 Zegrab, Croatia, Yugosavi, 240. Zarkovie et al. B58 8 8 NMS SHMS PHNS © w. 2. ° ea number per culture (Nx 104) ce ‘60 : an mi cs MLE SHLE PALE cH es Fig. 1. Numbers of L829 cells following cultivation in aif serum (CS), ormalmeuse serum (NMS), sharmhapatectarized reuse serum (SHMS). partly hepatectomzed mouse serum (PHMS), norma| mouse ver extract (NLE), sham-hepatectorzed mouse liver extract ISMLEI, parvally ‘hepatactorized mouse iver extract (PHLE), combinations of CS and iver ‘exacts, enimmediumauzoloments withan adequate volume ofsaine'!, respectively (Zerkovie et a, 1985}. Furthermore, It was also noticed that the growth of melanoma B16 transplanted into the hind leg of 8 ssyngenele mouse was inhibited ifthe host's Iver was regenerating after partial hepatectomy (Zarkovie et a, 1990). Similar, Dut less pronounced tumor growth inhibition was observed in sham hepatectomized tumor bearing animals. Hence, it has been sup posed that tissue regeneration, particularly iver regeneration in ‘mammais, lke tissue regeneration in plants orin lower vertebrates, ‘can modify tumor growth features, probably through certein ‘mechanisms i.e. actors controling tissue regeneration. Such factors ‘could be synthesized by regenerating ussve itself, acting a local regulatory factors and systemic factors regulating cell proliferation In remote tissue, To test this hypothesis we have analyzed the effects of liver extracts and/or sera from nonoperated, sham hepatectomized or partially hepatectomized mice on in vitro growth abilities of diferent cell nes, primary cultures of embryonal and ‘tumor cells, end bacterial cultures, respectively. Results ‘The effects of murine sera and liverextracts on cell growthin vitro L929 cells were cultivated in vitroin MEM supplemented with calf serum (CS) 0 obtain a 10% suspension, or an adequate volume of saline, Further, instead of CS, different murine sera, ie. serum from the normal mouse (NMS), sham-hepatectomized (SHMS). or par tially hepatectomized (PHMS) mouse were used, respectively. Liver ‘extracts (LE} from normal (NWLE}, shan-hepatectomized (SHLE), or partially hepatectomized (PHLE) animals were used alone or in ‘combination with ifferent mouse sera orwith CS. The cell numbers inthese cultures were determined and the results are presented in Fig. 1. In comparison to the cultures where salino was added, a stimulation of cell growth was observed in CS cultures (P<0.05) ‘while suppression wes observed in those with NMS (P<0.05). Further, SHMS ang particularly PHMS stimulated the growth of cells better than NMS (P-0.05), Onthe other hand, alliver extracts used inhibited L929 coll gowth and the inhibition was about the seme if NMLE, SHLE or PHLE were used, respecthely (Fg, 1) Further, LE 20 20 10 Fes, PHMS 30 Cell number per culture (Nx 10‘) an 20. 10. ; nem FCS Sl ONMUE SHLE PHLE Fig. 2. Numbers of HEp-2 calls following cultivation in meciurn with {elalcal serum FCS), normalmouse serum (NIUS) sharrepatectornzed ‘mouse serum (SHAS), parisly hepatactomized mouse serurn (PAMS), fnormal mouse fiver extract INL), sham-hopatectomized mouse var loxttact (SALE, partial hanstectomized mouse lve’ extract (PHLE) oF in ‘medium sucplementedunthan adequate volume of saline (SI, respactvey ‘combined with 10% CS also suppressed L929 cell growth fn vito. The effect was less pronounced if NMLE was used (P<0.08). The growth of HEp-2 cells in the presence of NMS was slighty, but significantly (P<0.05), suppressed in comparison to growth bilities in the presence of fetal calf serum (FOS) as presented in Fig. 2, However, the sera from operated mice, ie. either SHMS or PHS, stimulated HEp2 cell growth in comparison toFCS (P<0.05) Various ver extracts didnot influence HEp:2 cell gowth, i. twas the same as observed for the medium when saline was added. It should be noted that HEp-2 cells growing in FCS supplemented Regenerating tissue extracts and cell growth 241 jn. medium supolement medium were characterized by thelr typical epithelial morphology. Honever, fany murine sera was used instead of FCS the morpho! ‘ogy was completely different, ie. the cells were not solattened and gave the impression of fibroidsike cel’s (Fig. 3). This phenomenon \was not observed if these cells were cultivated in any serumfree medium used. “Murine fetal cell growth was mare stimulated in the presence of, FCS (P-=0.05), than if NMS was used (Fig. 4). Cell numbers in the cultures were even smaller ifthe sera from operated mico (SHMS (or PHMS, respectively} were used (P<0.05).0n the other hand, iver 242. Zarkovie etal. 6 . st eas te Tr mi e Zz 2 21 2 oo 3 Fes ows PHMS € ree & 3 x i af I 1 of sl MLE. SHLEPHLE Fig. 6. Numbers of urine etalcellsfollowing cultivation wi foraicatt ‘tur (FCS), normal muse serum (NUS) shanvhepatectamized mouse serum (SHIMS), partly hepatectomized mouse serum (PHMS), normal ‘mouse liver extract NMLE), shamvnocstectomizea mouse iver ax (SHLE, partially epazectorized mouse ver exact (PHL, orinmadiara supplemented with an equate volume of saline (SI, respectively extracts did not show any influence on fetal cell growth in compar on to the values obtained for serunsfree media (Fig, 4). Primary ell cultures were performed from melanoma 816 tumor rowing in ©57BI/Gozer mouse hind limb, intact liver or regenerat ing liver, respectively. The cultures originating from melanoma transplanted in iver grew much better than those obtained from the ‘tumor transplanted in the ima ifthe media ware supplemented with 110% FCS. (Fg. 8). Similar differences were observed If tumor cells were culated in the presence of NMS or without any sera in the medium (Fig 8). Further, as presented, in vlrogrowth of melanoma cells isolated from the tumor growing in regenerating liver was suppressed in comparison to the cells isolated from melanoma growing in intactiver (Fig. 8). FCS was mare convenientthan any MS ‘or melanoma cell growth in vitro, However, here was no significant difference in the growth of melanoma cells originating trom the ‘tumor ransplantedin te lim ifthe cells were cultured in any of the murine sera used (Fig. 5). NMS stimulated the growth of cells isolated from the tumor transplanted in intact Iver better than the sera from opereted mice, while PHMIS was the best of all murine sera used for the cultnation of melanoma cells from the tumor _growing in regenerating liver (Fig, 5}. The different growth features ‘of melanoma cella, due to the different transplantation procedures, Invivo, could also be observed ifthe colis wore incubated in sorum- free modium. There were no significant differences in cell growth abilities diferent LE were added to the madium or ifthe calls were ‘nebated in medium on\y (Fig. 5) Different LE addedtothe medium ‘comprising 10% FCS innibited equally walthe growth of melanoma cells isolated fram the tumor transplanted in regenerating liver (in comparison to the cultures without LE, i.e. with 10% FCS only) However, NLE even stimulated the growth of calls isolated from 0. TUMOR TRANSPLANTED IN an Hino Leo RECENERATING gy SSS So Sig SI MLE SHLE PALE Cell number per culture (N x10") SHE PALE ——- w% ree —_ Fig.5. Numbers of melanoma 16 calls following cultivation wih fata if serum (FCS). normal mouse serum (NMS|, shar-hepatectomized ‘mouse serum (SHINS), pertaly hepstectomized mouse serum) (PHNMS), formal mouse Iver extract MMLE), shan-hepatectorized lar extract (SHLE) partaliynepatactamizediverextactPHLE) acombination of sera iver extracts,” n mealum supplemented with an adequate volume (fsa (SI, respectively. Melanoma cells were obtained frm the tumar transplanted inthe mouse hind lo, inact Iver, ofthe Iver regenerating aftr partial hepatectomy, respect vely E.coli AB1I57 1 t i 50- - ° SOME SMLE PLE a Proteus 2 aw = § 10 : a | 0 5 Tae sme Pme a Citrobacter wo i ‘00- aa ° = me Fig. 6. Numbers of particular bacterial colonies per plate following plating with the extracts from normal mouse liver (NLE), sham hepatactomzed mouse iver extract (SHLE,, partly hapatectomized ‘mouse iver extract PRLE), on madiurn supplemented with an edequsts volume of sane (5, respectively. ‘melanoma transplanted in intact iver in comparison tothe cultures without LE. Further, there was no difference in the growth abilities of these cells if there was no LE in the culture medium, or i ether SHLE or PHLE were added, respectively (Fg. 5) The influence of liver extracts (LE) on the plating eticlency of bacteria The plating efficiency of E. coli AB 1157, Proteus, or Citrobacter, ‘expoced to particular LE, was determined. The results were com pared te thase obtained withaut LE and are presented in Fig. 6. NLE aid not infuonce the plating efficiency of €. col, SHLE stimulated it and PHLE inhibited it. Further, all LE significantly (P0.05) stimulated the plating eficioney of Proteus and the influence of NLE Regenerating tissue extracts and cell growth 243 \was the most prominent (Fig. 6). In comparison to the control the Plating efficiency of Citrobacter was about the same if LE was ‘added. However, SHLE in comparison to NLE inhibited (P<0.05) the Plating efficiency of Citrobacter (Fi. 6) The modulation of bacterial growth In liquid medium “The medium for Proteus oF Citrobacter cultivation was supple: mented with different concentrations of used media, .e.the media Inwhich bacteria wore growing proviously. As prosontedin Fig. 7 the addition of used medium of Proteus in the concentration of 10% or 50% significantly stimulated the growh of Proteus cultures. Used medium of Citrobacter stimulated Proteus cutture growth only i it was present In a 50% concentration (Fig. 7). This stimulation was Wy A 108 2 ZO 3 . 3 THE ccucctrnatons 0 3 “oruseoweon™ 2 Eu; 8 € 0 1 2 3 4 5 Hours of incubation Fig.7. Number of Proteus strain bacteria in 1 mi of culture following fultivation 1 mineral medium (MA) supplamoted with aliferent oncontraions of used med of Proteus (al or Cirabscter (stain bectena, respectively 244 N. Zarkovie et a. wy 5 = w ° 2 a 5 Hours of incubation Fig.8 Number Citrobacter strainbacterlain 1mlofculturefollowing cultivation in mineral medium WA) supplemented with different Concentrations of used mesia of Proteus (A) or Civobacter (8) stain Dsctora, respectively very prominent during the first hour of cultivation when the number of bacteria significantly decreased in other cultures. However, ‘these used media were not able to modify the growth of Citrobacter (Fig. 8). Further, tne grow of Proteus culture was very pronounced if any ofthe two used media was added in 90% concentrations (Fig 9). If Citrabacter was cultivated under these concitions a pro- ouncedinhibition of culture growth accurred (Fg. 9). Thestimulation of Proteus culture growth, and the inhibition of Citrobacter in the presence of 90% used medium were observed during the fist 10 hours of cultivation. Final analysis of the numbers of bacteria was, performed after 24 hours for Proteus and after 48 hours for Citrobacter cultivation, respectively. As presented in Fig. 9, at Particular times, either Proteus cultures or Citrobacter cultures ‘contained more bacteria than ontrolcuituresif they were cultivated ‘rom the start with the adtltion of 90% of used medium, The Influence of bacterial media, murine sera and iver extracts on ‘murine tumor cell growth in vitto Primary cultures af either melanoma B16 or methyicholanthrene- induced fibrosarcoma were performed from tumors growing in the hind leg of syngeneic mice. The cells were cultivated in Parker's ‘medium supplemented with human AB serum (HS), mouse sera (NMS, SHMS, PHMS),livor extracts (NLE, SHLE, PHLE), or used bacterial media, respectively. The intensity of radioactive thymidine incorporation was determined in these cultures. As prosented in Fig, 10, thymidine incorporation was inhibited if murine sera were Used instead of human serum (P<0.05), Inhibition caused with ‘SHMS was stronger than if other murine sera were used (P=0.05}, Further, melanoma cells incorporates thyridine equally well wth or Without HS or if SHLE was added, but the growin was significantly Inhibited (<0.05) fNLE or PHLE were aided (Fig, 10), Similarly, the ‘addition of either fresh or used bacterial medium did not influence thymidine Incorporation in melanoma cells. Fibrosarcoma cells were also sensitive to mouse serum, i.e. tiymidine incorporation was suppressed in comparison to the cultures with HS. However, HS stimulated incorporation of thymidine in comparison to the cultures without supplements (Fg. 14). NLE did not influence thymidine incorporation, but the extract from operated mice liver did (0.05). Furthermore, used bacterial media, paticulrly those Inwhich Proteus were growing, strongly(P<0.05) inhibited thymidine Incomoration (Fig. 14) Discussion ‘There ate several factors incorporsted in host-tumor relations that ate cetrimental for tumor growth. The site where a tumor ‘appears, of; in the experiments, the site where its transplanted 15 Important for blood formation, possible Immunologieal relations ‘and hormone influences, but certain other conditions in the host tissue (where the tumor is developing) seam to be of further Importance. Our particular interest focuses on the influence of regenerating tissue on tumor characteristics. Liver regenerating after partial hepatectomy significantly changed fibrosarcoma ‘anaplastilty and the intensity of pigmentation in melanoma cells (Zermovie et a, 1985, 1987) indicating that the tumor cells had become less malignant. It iS known that the Iner regeneration Induced by partial hepatectomy is at least in part regulated by humoral factors (Glions, 1956; Weinbren, 1959; Bucher, 1963; Leong eta., 1964; Moolten and Bucher, 1967; Sakal, 1970; Nadal al, 1876; MeMahon and lype, 1980). Although most of these factors, like hormones or growth factors, are known to be patent growth regulators, they are not specific factors, ie, they do not influence liver regeneration only (Bucher and Swaffield, 1975; Bucher et al, 1978: Farer et al, 1979; Barbason et al, 1987), Honever, there are other factors that could be responsible for the systemic control of liver regeneration. Thus hepatocyte growth factor (HGF) was isolated from rat platelets, but its activity was inhibited by adding transfoming growth factor & (TGF 8) in vitro (Hayashi and Carr, 1985; Nakamura et al., 1985, 1986 ab). Honever, whether HGFs the main factor regulatingliver regeneration is stil uncertain. Two other factors of different molecular weight higher than 120,000 or lower than 3,000 D, named hepatoprateins ‘and 8 (HPTA and HPTB), respectively, nave bean described as very Potent growth factors in vitro (Michalopoulos et al., 1984). These factors sumulate proferably tne proliferation of cultured livor coll. Aight stimulation of rat skin fibroblast proliferation in vitro was also noticed (Michalopoulos et a, 1984), \wmether tne mentioned factors could be responsible for the effects of murine sera and liver extracts on cell growth in vitro, as presented in this article, remains uncertain. We have observed 3 The number of bacteria Regenerating tissue extracts and cell growth 245 2 NORMAL MEOIUM 15 USED MEDIUM OF PROTEUS (9 USED MEDIUM OF CITROBACTER o 2 ft és wv 2 @ Hours of incubation Fig. 9. Numbers of Proteus (A) or Citrobacter (8) strin bacteria in 1 ml of culture fllowing cultivation in mineral medium (VA) supelamsnted wih 90% of particular used medium. respecte, significant influence of the sera and liver extracts used, although they were heated at 56°C for 30 min. itis probable that HPTA\s not ‘the main active component of murine sera and liver extracts, while its effect could be significantly decreased after heating the serum ‘at 60°C for 30 min. The presented results indicate that in vitro ‘growth of several cell lines or primary tumor cell cultures could be ‘modified by adding liver extracts and/or sera from normal or from ‘operated mice (with or without partial hepatectomy). Liver extracts Inhipitedthe growth of L929 cells utnat of HEp-2celllines, pimary ‘culture of either mouse fetal cells or melanoma cells. Further, the sera from these mice were generally less convenient in comparison tw other sera used, except for HEp2 cells. t should be mentioned that PHMS wore more convenient than other mice sera for L929 and {or melanoma cells. We cennot explain these specificities, but itis ‘an indication thatthe phenomenan obtained depends on the model ‘chosen. The possibilty that the effects of sera are more pro nounced than thase af liver extracts is not easy to explain. One can ‘speculate that active components are not synthesized only in the Ivor, oF, ifmainly produced there, they are more actve tor coupling to certain sera proteins. Hence, the obtained results seem to be ‘irnar to the effects of the rat serum glycopeptide described by Nadal (1987). The inhibitory effects of serum glycopeptide on hepatocyte proliferation in vive cauld be abolished ay normal adult for baby rat serum, as well as with mouse or human serum, but nat with partially hepatectorized or shamhepatectomized rat serum. It seams that surgeal treatment can modify the activity of both stimulatory and inhibitory serum growth factors, and their interac tions caninfluencecell growth. Theinteraction of serum glycoprotein and acute phase proteins seems very provocative suggesting the possible systemic effects of various disorders (i. inflammation, malignant tumors, trauma) on the growth capacity of liver cells. Recently. it was described that interleukin 18 can significantly inhibit adult rat hepatocyte growth in vitro and induce production of acute phase proteins ivercells (Darlington etal, 1986, Nakamura et al., 1988) It also stimulates the proliferation of fibroblasts, but Inhibits the growth of human melanomacells, L329 mouse fibroblasts ‘and manimary carcinoma cells (Schmidt eta, 1982; Onozeki eta 4985: Gattny and Tsai, 1986), Furthermore, there are numerous data indicating the important role of interleukin (iL6) in the regulation of an organism's response (.0. the acule phase response) to various kinds of ssttessr, including surgery (Seh¢al et ai, 1989). Among other ef focts, IL'6 is known to be a potent multifunctional regulator of {growth and aifferentiation of aifferent cells, including hepatocytes (Sohgal, 1989). Other factors playing an important rolo in homeostasis are vheat-shocke proteins, which could be responsible Tor the biological effects oftissue extracts (Currie ang White, 1984; Jaattela ef al, 2980 ), However, their role In iver regeneration is Uncertain, On the other hand, ubiquitin, which could be included ‘among heat-shock proteins, modifies the metabolism of proteins 246 N. Zarkovic etal, o Hh 30 an 10 Ws NMS SHMS PHMS 20. 20. 10 CPM (Nx10°) Hs S| NLE. SHLE PHLE 50 vof 7 30: 20: Hs MA Fig. 10. Radioactive thymidine incorporation rate in melanoma B 16 cells following cultivation in hurnan AB serum (MS), nore mouse ‘serum NMS), shamnepatectomized mouse serum (SHMS), partly epatectamzedenouse serum (PHMS),rormalmause iverextact NMLE) ‘sham hepatectomized mouse var extract SHLE, cally hepatactomeed ‘mouse iver exact PHLE), madlum supplemented with en adequate volume of sane (SI, mineral medium (MA), used media of Proteus P) or Citrebactor (C stain bactar, raspoctvay ‘and determines growth features of the colls Parag et al,, 1987). However, itis not certain thet ubiquitin could be a factor regulating tissue regeneration, or, further, that It 16 responsible for the “obsorved offects of liver extracts on cell growth in vitro. Our fincings ‘suggest that there are factors that modify lwer regeneration but also influence the growth of other cell types. The activity of such regulatoryerowtn factors could be modified by different pathological ‘oF mayo physiological processes (i.e. issue regeneration), espe- ciallythose reflectedby acute phase response (Carr, 1983; Kushner, 11988), Hence, our results could be analyzed as another argument for this hypotresis, ‘The different acthity of ver extracts acided to the cell cultures incubated in the presence of ea serum or in serum tree conditions, is in agreement with the stated hypothesis. The stimulatory effects of one growth factor in vitro could be significantly modified by the ‘addition of another growth factor, especially a bitunctional growth fector, such as TOFS (Todaro eta, 1980; SowenPope otal, 1983; Roberts et a)., 1985; Spo and Roberts, 1985; Carr et a, 1986; Biyckaer. et al, 4988). twas also shown thatinterferon-ycould inhib the mitogenic activity of different growth factors analyzed on both human and murine foroblasts in vitro (Oleszak, 1988). In contrast, the stimulatory effects of HPTA and HPTB is potentiated in the presence of EGF (Michalopoulos et si, 1984}. Thus, the modifica tion ofthe effects of liver extracts in vitro by the presence of calf ‘serum could be explained as the result of simultaneous effects or the interaction between the growth factors present in murine liver ‘extracts and the growth factors from calf serum. The modification in tumor growth characteristics in vitro, resulting from different ‘vansplantation procedures of metanoma B16, Indicate that tumor characteristics change while growingiinlver tissue. The stimulation of cell growth in vtrowas more pronounced if melanoma cells were ‘wansplanted ito the ntact iver, than ifthey were transplanted into regenerating liver tissue, suggesting that liver regeneration some- how ciminished the capability of melanoma B16 cell growth in vitro. This result could be explained by the increased sensitivity of melanoma cells to the effect of inhibitory factors probably present iinliver tissue, This speculation is mainly based on the observation ‘obtained with tumor cells cultured in medium supplemented with 1105 fetal calf serum and 10% liver extracts. Its also in agreement with tumor growth inhibition observed during Iver regeneration (Zarkovie et al, 1990). Moreover, it could mean thatthe simultane- ‘us presence of liver tissue factors and serum factors is necessary to obtain the controlled proliferation of melanoma cells in vitro. & similar phenomenon of modulation of in vitro growth was obtained by adding liver extracts to bacterial cultures. All liver extracts stimulated Proteus platingefficiency and did notinfluence Citrobacter abilities. NLE was more stimulative than the others for Proteus, Further, used bacterial media, regardless of origin, stimulated the growth of Proteus more ifthe concentration increased, but inhibited Citrabacterin highconcentrations, Supematents of Proteus cultures were able to inhibit the incorporation of radioactive thymidine in fibrosarcoma but notin melanoma cel cultured in vitro. This might indicate that the factors regulating cell mitosis and the increase in coll numbers are present even in bacteria. Whether it means that fone or several common reguiators exist in nature appears as @ provocative, but still complotely open question. However, the data indicating the importance of factors of bacterial origin in influencing the growth of liver tissue in vivo point in this direction (Schulte Herman eta, 1976). The expression of protooncogene ras during Iver rogeneration suggests tat controlled proliferation of iver cells. ‘during regeneration probably results from the autocrine control and the influence of humoral factors (Goyette eta, 1983), Whether the ‘exposure of melanoma cells to the processes controlling liver regeneration induced the programmed cell death (apoptosis) (Kerr fe at, 1972, Bursch e al, 1984; Sarraf and Bowen, 1988) in ‘tumorous tissue, tus changing its Sensitivity to growth reguiatlon factors whentumnorcelis were exposed to liver extracts and fetalcalf serum in vivo, remains unsolved. Wells and Miotto (1986) have : . wo ft : : of [ca ; oa sim , o - a 1 as - zw. oe : : «oy [> i : : : Jn human AB serum (HS), normal mouse serum INMS), sham hepatectomized mouse serum (SHMS, partaly hepelectoriws mouse serum (PHIMS) norm mouse Ivar exractINMLE), shemhepatectamized ‘mouse liver extract (SHLE), partly nepatectomzed mouse lver extract (PHLE), mecium supplemented with an adequate volume of saline (Si). mineral meatum (Ma), used! medka of Proteus Por Civobactor(C stan bactena, respectively shown that neuroblastoma growth was inhibited iftumor eels were Injected into the mouse embryo. The inhibition of tumor formation in an 8.59.5dayold mouse embryo could be obtained only if malignant cells were injected in the somite region. The same effect \was obtained in 13-1 7-layold embryos regaraless of the site of ‘tumor cell implantation. What is more, the condltioned media from Regenerating tissue extracts and cell growth 247 embryonic lim bud tissue culture sioned down the growth of neuroblastoma cells in vitro (Wells and Mlotto, 1986}, Thus, itis possible that embryonic tssue, adult regenerating tissue and ‘Serum contain regulatory growth factors which could be responsible for apoptosis or controled cell proliferation and differentiation. Numerous factors are involved in the contral of regeneration. However, it's not known whether the same or similar factors are fls0 responsible for tumor growth inhibition or for embryonal development. The idea of controling tumor growth by using such regulatory growth factors is quite intriguing. Unfortunately, the nature of these factors and the mechanisms oftheir action are not yet sufficiently known, Materials and Methods Animals Fory- ve CBA/HZgr male mice (Ruse BoskoweInsetue) three months. ol, weighing 20-22 g, were used for the preparation ofthe sera ana ver fxtacts. The animals were maintained in standerd eandisans with unte- Sioted accase to food and water Surgical removal of the medial anterior liver lobe ater Figure (30% patial hepatectomy) was performed under sterile constions according to ‘the metnec of Higgins and Anerson (higgins and Anderson, 1931). Partial hepatectomy was performedon fifteen animals, while the remaining 30rce were either shamshepatectomizod of only anesthetized by Lp. nection of ‘300 re/ig Chloralnydrate solution (Kem, Zagreb). ‘Proparation ofthe sera and her oxtracts ‘Allanimals wore sacfioes 72 haus afer he treatmant. The mice were ‘anesthetized with ether and sacrificed by cutting the blood vesse's of the neck, The obtained blood sampies wore pooied together according tothe experimental groups as: nonoperated, shamhepatectomizea ara partially hhepstectomized mice blood. During the ret four hours the blood was kept atroom temperature anc arenwards centfuged at 70 G for 15 min The sera were collected and used for futher procedures Liver extracts were prepeed by pooling together tre iver tissue from all the animals according te experimental groupe. Liver tissue was rowed fiom sactiiced dono's under sterile conditions and kept in saline solution cooled on ie. Lver tissue was homogenized in saline (Lg of Iver per 45 rmlofsaline) atroom temperature for fue minutes usingamer (Ulta Turax, Jenke und Kunke KG Liver extracts and sora woro futher kept ina warm bath at 86% for 30 rin and ten centhfuged at 15x10° G for 20 min. Supomatants were separated from te pele, luted with str saline in @ 1:20 rato, passed ‘uvough a 0.224 Mlipore titer and razen. The protein cotent in murine ‘sera and liver extracts was determined aecaraing tothe method of Lowry (Lowry ot at, 1953) col eutures Inthese experiments our difference cutures were used. Two cantina ‘us cal lines ~ mouse fioblasts L329 and human layngoal earcinama HEp2 were grown as monolayer cultures in Eagle's Minimum Essential "Meat um (MEM, supplementedwth 20% eal serum (L929 ells or fetal calf serum (HEp2 cel Primary melanoma 616 cel cultures wore obtained eter from tumors Uwansplanted into te hind leg. oF fom those transplanted into Incat or operates ipatial hepatectom) Iver of C5781/Gozer mice, The tuners were ‘taken rom donors (three snimals ineach group), bearmathe ith generation ofdiferertiytransplantedtumors, whieh were saciieedonthe 14" day after {mor col injection. Tumor tissues were pooled together from all donors ‘ccorcing to group, and cutinto smal pleces under sterile cancitans. The Somales were typsinized three times with 1% bovine pancreas tnypsin Solution for 10 min at 37°C. Attar each ‘ypsinization RPMI 1640 medium containing 10% fetal calf serum (FCS) was added, Folowing the last tpsinization the cells were censfuged for 10 min at 250.6, The pallet was 248 N. Zarkovic etal resuscended in RPMI 1640 meclum supplemented with 10% FCS, andthe cells pate. ‘The same procedure a6 forthe preparation ofthe col suspension was usec for C,H murine feat cells, abtaines fom 15dayoldemby0s, sac fee by ether The cells were seeded in von RPMI 1640 medium sup- plemented with 20% FCS. 1529 and HEp2 celle were pated In 0 35 mm Petr dishes at 23108 concentration, while melanoma B46 and feta alls were scodod at 4x10* ‘concentreten, and ineubated overnight, The next day, the samples were washed twice with sterle phosphate buffer and divided according to treatment into diffrent grours. The cultures fom the first group Were inevbate inthe medium containing 10% serum fram nonoperated, sha hepatectozed ar partially nepatectomized animals. The samples fom the Second group receved medium containing 10% of lvor extracts obtained frem nonoperated, sham hepatectom zed oF pataly hepatectom ze0 mice. The tira group of cultures comprised those incubated inthe presence of 410% of various Iher extracts inthe medium supplemented with 10% cat serum (L929 ells} or 10% FCS melanoma B16). Simultaneous, two Controls ware Set, ntlly th oes were cutured in medium under seruny free canctons, end the serum wae thon substituted with an adequate lume of strle saline. The cultures were incubated in 2 humidified ltmosphere containing 5% CO, at 27°C for the folowing fve days, afer ‘hich thecelis were typeinized and counted, Each experimental and contol ‘sOUp of cultures comprsed four equally treated samaies. Melanoma B16 cel, as well as metyicholanthrene induced murine ‘ibrasareams CMCi cells, were also usod forte anaysis ofthe intensityof xanymicita incorporation. Tumor cell suspensions. were prepared 23, already deserted and seeded (10° cells per culture) into meroeytplates in the presonce of Parker's mec um supplementedvtn 20%human AB serum. According 10 the experimental procedure the cells were cultured in the presence of mutine seracrlver extraets orinthe resarce of minoralmecia {ced previously or the neubaton of enterobacteria Beare the murine sera ‘and Iver extracts, a6 wel as media of bacterial cultures, were used in the experiment the protein sortartin tho samples was determined according ‘0 tremethodof Lowry (Lowry etal, 1951),and adjusted by dition with stele saline, The proteln concentration of bacterial media adler extracts was 35 ya/ml and of murine sere 3.5 mg/ml. Aner 4 nour preincubation, *H Fymidine (metny!"H-Thymisne, 25 Ci/mmol, Amersham) clivted ith rmeciurn st 2 1;25 rato was added ang the cells cuted in humidified ‘atmosphere contami 5% CO,, forthe following 24 hours. aftr tra, the Celis were washed aver a iter and he radlanctvy of incorporate “H-TAR ‘was detected in abeta counter (Beckman LS 1000). Each graup of cuturos ‘comptsee four samples. Bacteral cultures “ines different trains of erterobactera were used for these exper: ments: stander laboratory stain of Ecol ABL1S7, and native strains of Proteus and Ctroboeter stain tained fom the Institut of Mierobiology, Zagre). Bacteria wee cultured at 30°C fr 24 hous inliqid Tryon broth itn azration, after which 50 jl samples ofeach stain culture were cited instere saline up to 1:10" ation. The last clution of bacteria suspension wae ata 1-10 ati, ether sallaeor nonoperated, shershepetectomized ‘or parialy hepatectomized mics Iver extracts. Thus, the obtained suspen ‘ions wore poured onto agar ga plates and cultured in normal atmasphere ‘a 30°C for tne foloing 24 hours, when the number of developed colonies inciting te plating ecioncy of bacteria in the presence of various iver ‘extracts or saline was counted, There were thyee samples In each exper ‘mental group dependingon the train ofbacteria and the tye oflivrenract used, ‘acto of the Civabacter and Proteus sain were also cutured in neutral (pH 7) mineral MA mesium containing: {NH,},50,, £.0 , K:HPOg, 10.0 g KH,PO, 45 g, Na crate x5 H,0, 0.5 1, MgS0, 7 H.0, 0.1 gH,0, 1000.0 mi. Thus prenered MA mecium was supplemented with 0.4% glucose ang bacteria were incubated in 50 mi of MA mecium for 24 hours at 30°C with ‘aeration. Theodiained cultures were cenityzed at 40* Gfor 15min atroom temperature, after which tho obtained supematant was kept at 5° inwarm Dat for 30 min, an itera through @@ 0.224 Milnor ite. The protein content of the media was determined according tothe method of Lomty (Lonny ot al, 1951), The used meciar were urthor added at ciferent ‘concentrations tothe tesh Medium inwhich eitherProteuso-Cyobacter Stain bacteria were culture et 30°C with aeration for atleast 24 hours. Bepending onthe concentration of used medias added as a supplement. dition of fesh MA medium with water was adusted to achieve equal concentrations of mineral ingredients for all meth, At diferent time inten afer bectena were seeded in medium, the samples of Bacterial cultures wece tke, diluted with MA medium and cultured on agar plates, to determine the cumbor of bactora inthe samples, eto analyze tre inven of «sed maciae on bacterial growth in Hig medium. Statistics “The resuts were analyzed using the MannWhiney test. 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