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Variation of nucleolar activity and size in liver parenchymal cells during hepatocarcinogenesis
Meetmg Proceedmgs
867
adenosyl-L-methlonme (SAM) decarboxylase and decrease the SAM pool A decrease of the SAM pool is also caused by long-term feeding of phenobarbital (PB), a well-known promoting agent which reduces ODC actw~ty On th~s bas~s, ~t has been hypothesized that PB could reduce polyamme synthesis during hepatocarcmogenesls Th~s effect, as well as the promoting effect, could be regulated by variations m the SAM cellular pool This hypothesis has been tested m rats, m which hver carcinogenesis has been induced by N-nxtrosodxethylamlne (NDEA), using an experimental system m whxch groups of rats were given some or all of the following treatments 2 wk on basal &et with/without a dose of NDEA (200 mg/kg, ip) at the end of wk 1, then 2 wk on &et containing 0 04°0 2-acetylamlnofluorene (2AAF) with a partial hepatectomy (pH) at the mid-point of this period, followed by basal &et w~th/wlthout 0.05% PB until termination. After the end of 2AAF feeding, rats were treated with SAM (six 4-hourly lm injections of 50 mg SAM/kg in buffer) or with buffer alone In the hvers of rats dosed only with 2AAF, with or w~thout PH, no ),-glutamyltransferase-posmve ( T G T + ) loci were found during wk 18 from the start of 2AAF feeding, even when the rats recewed PB On days 21, 35 and 49 after the start of 2AAF feeding, the livers were 14, 18 and 25°0 ),GT+ after NDEA/2AAF/PH treatment and 23, 32 and 38°0 after NDEA/2AAF/PH/PB. SAM treatment led to a sxgmficant decrease m the number and dmmeter of ),GT+ loci in the former group and abohshed the PB induction of 7 G T + focl m the latter In the NDEA..'2AAF/PH rats, there was a 19-40% decrease m the hver content of SAM between days 14 and 49 from the start of 2AAF feeding, but control values were restored by SAM injection In NDEA/2AAF/PH/PB-treated rats, a large decrease in the hepatic SAM pool was restored to 62°0 of the control level by SAM treatment. Some 2 wk after the start of 2AAF feeding, hver ODC actwxty was five times higher m rats subjected to 2AAF.'PH than in control rats or m those treated w~th 2AAF alone Thereafter, enzyme activity decreased progressively In rats subjected to NDEA/'2AAF/PH, ODC actw~ty increased to a peak at day 14, decreasing thereafter, but remaining well above control values 49 days from the start of 2AAF feeding The increase in ODC actw~ty was even greater m the hvers of rats treated w~th NDEA/2AAF/PH/PB SAM treatment markedly inhibited ODC actwmty. However, SAM concentrations between 0 05 and 0 5 mM had no effect on the ODC actlwty of the hver homogenates. The increase in ODC activity during the promotion of hepatocarcmogenesls was coupled with an increase m spermxdme/spermme (SPD/SPE) ratio m the hver of hepatectom~zed rats This effect, however, was observed for more than 14 days after the start of 2AAF feeding only m the rats subjected to NDEA/2AAF/PH + PB Again the SPD/SPE ratio was higher in the hver of PB-treated rats. SAM injection caused a fall m the SPD/SPE ratio to control values m both PB-treated and untreated rats It seems that the liver content of SAM regulates ODC actlvxty and polyamme synthesis. PB-mduced promotion seems to be linked to an increase m polyamme synthesis at least in the first stages of promotion A decrease m liver Ilpotrope content ~s not restricted to promotion by hpotrope-defic~ent d~ets. It appears to be revolved also m PB-supported promotion, probably through the regulation of polyamme biosynthesis 10 LIVER FOCI' OVEREXPRESSION OF AN ADAPTIVE PROGRAMME IN THE LIVER? R Schulte Hermann
Instltut fur Toxlkologle und Pharmakologle der Umversltfit Marburg, Pdgnmsteln 2, 355 Marburg aA Lahn, Federal Repubhc of Germany Liver tumour promoters, such as phenobarbital (PB) and ~-hexachlorocyclohexane (~t-HCH), reduce a programme of adaptive response m normal hver This programme comprises (1) increases m activity of various drug-metabohzlng enzymes, (2) growth by hypertrophy and/or hyperplasla and (3) changes in carbohydrate metabohsm, such as an increase in enzymes of the pentose-phosphate pathway. Many of these responses to PB are expressed m putatwe preneoplast~c foc~ of rat hver, even in the absence of a promoter Treatment with promoters reinforces expression of these adapuve responses in focl. We have therefore developed the hypothesis that focal cells, as a basic regulatory defect, are committed for overexpressxon of the adaptive programme inducible by PB-type promoters This hypothesis would explain why so many inducers of drug-metabohzmg enzymes can apparently promote the development of hver tumours. 1l. VARIATION OF NUCLEOLAR ACTIVITY AND SIZE IN LIVER PARENCHYMAL CELLS D U R I N G HEPATOCARCINOGENESIS M Kirsch-Volders and A Deleener Laboratorlum Antropogenetlka, Vrlje Universlte~t Brussel, Brussels, Belgaum The sliver staining method facdltates the demonstration of nucleolar orgamzing regions (NORs) or sites of ribosomal DNA on metaphase chromosomes and in interphase nuclei Moreover there is a good correlation between the rate of rRNA synthesis and silver staining.
868
Meeting Proceedings
A quantitative cytophotomemc technique was used to show that nucleolar acnv~ty ~s reflected accurately by quantitative analys~s of the mterphase nucleoh but not b~ the number of silver-stained NORs in metaphase plates, and that there is a form of regulation of nucleolar activity m aneuplold mahgnant HeLa-CCL, cells similar to that in PHA-stlmulated human lymphocytes With the same quantitative eshmatlon of silver stalmng, analyses were performed on hepatocyes from Wlstar rats subjected to malignant transformation by sequential treatment with N-nltrosodlethylamme (NDEA) and phenobarNtal. In hepatocytes obtained by perfusmn, the cytophotometrlc analysis of the nucleoh indicated that during the first steps of neoplastic transformation m the liner, both NDEA and phenobarbital modified the nucleolar transcriptional activity in different ways, as wsuahzed by the sllver-stalmng method In liver slices, prepared as serial sections, silver staining was compared with staining v~lth methylgreen pyronine, with the Feulgen reaction and with haematoxyhn eosm The decrease and increase of silver staining correlated with nucleolar area, DNA content and basophfllclty of the parenchymal liver cells at different stages of the carcinogenic process
12 INTERMEDIATE FILAMENT CYTOSKELETON IN HEPATOCYTES AND NEOPLASTIC NODULES OF MOUSE LIVER H Denk Institute of Pathology, Um~erslt.~ of Graz, Austria Cytoskeletons enriched m lntermediate-s~zed filaments were prepared from murme and human hver, as well as from hepatocellular and cholanglocellular carcinomas and grIseofulwnqnduced neoplastic nodules, by high-salt buffer-detergent extraction and were analysed by one- and two-dimensional gel electrophoresls In addition, neoplastic and non-neoplastic hver tissues were analysed by redirect ~mmunofluorescence microscopy using antibodies to major c~tokeratin polypeptmdes Hepatocytes and hepatocellular carcinomas of human origin showed identical cytokeratm polypeptlde composmon, with two major components (molecular weights (Mr) c 52,000 and 46,000) and identical lsoelectnc pH values In mouse material, major polypeptldes (Mr 55,000 and 49,000) were also identical in non-neoplastic and neoplastic hepatocytes, but different proportions of lsoelectrlc variants were observed m two-dlmenmonal gels Cholang~ocellular carcinomas contained cytokeratins d~fferent from those present m hepatocytes, partlcularl.~ m respect of the presence of higher Mr components Immunofluorescence microscopy re~ealed strong cytoplasmic fibrfllar staining by antibodies to a mouse-liver cytokeratln component of M r 49,000 (component D) in neoplastic and non-neoplastic cells, but the arrangement of filaments was less regular in the tumour cells The results show that cytokeratlns represent a fairly conservative cytoskeletal system w~th a high degree of organ specificity and continue to be expressed after neoplastic transformation
13 INITIATION, CLONAL GROWTH AND PROGRESSION OF PRENEOPLASTIC POPULATIONS IN THE LIVER H M Rabes. R Kerler, Ch Schuster, G Rode, M Legner, k Muller and Th Bucher Departments of Pathology and Biochemistry, University of Mamch, Federal Republic of German~ Preneoplastlc loci consisting of hepatocellular sub-populations with an aberrant enzymatic phenotype occur in the livers of animals treated with chemical carcinogens Several groups have used this marker for the analysis of factors involved in initiation and promotion, but very little Is known about the cellular origin of these focl. This question was studied by means of a specific mouse strain (Aarhus) with a mutant expression of the X-chromosomal enzyme phosphoglycerate klnase (PGK) With selective breeding it is possible to raise hybrid females which contain--owing to lionization during early embryonic development--a mosaic of cells expressing either the paternal mutant P G K - I A or the maternal wild type enzyme (PGK-IB) These allozymes can be separated by a mlcroelectrophoretlc fluonmetrlc method (Bucher et al F E B S Lett 1980, !!5, 319L which allows the determination of relative PGK proportions in very small tissue samples Such samples of liver and several other organs always contain both PGK allozymes However, preneoplasttc focx arising after 2-acetylaminofluorene feeding express selectweb either PGK-1A or PGK-IB It ~s consMered hLghl~ probable that preneoplastlc cell populations originate by clonal grov, th from a single lmtlated hepatocyte, the progeny of which show a growth advantage o~er normal hepatocytes, possibly because of internally regulated stimulation Judging from the small number of preneoplastlc loci resulting from a single exposure to a carcinogen, it appears that initiation ~s a fairly rare event in normal adult non-prohferatmg rat hver After exposure to alkylatmg N-nxtroso compounds, this low rate of initiation may be caused by high acttv~t) of DNA-repmr processes in the liver The promutagenlc DNA base modtficanon. O~-methylguanlne. for instance, is rapidly
867
adenosyl-L-methlonme (SAM) decarboxylase and decrease the SAM pool A decrease of the SAM pool is also caused by long-term feeding of phenobarbital (PB), a well-known promoting agent which reduces ODC actw~ty On th~s bas~s, ~t has been hypothesized that PB could reduce polyamme synthesis during hepatocarcmogenesls Th~s effect, as well as the promoting effect, could be regulated by variations m the SAM cellular pool This hypothesis has been tested m rats, m which hver carcinogenesis has been induced by N-nxtrosodxethylamlne (NDEA), using an experimental system m whxch groups of rats were given some or all of the following treatments 2 wk on basal &et with/without a dose of NDEA (200 mg/kg, ip) at the end of wk 1, then 2 wk on &et containing 0 04°0 2-acetylamlnofluorene (2AAF) with a partial hepatectomy (pH) at the mid-point of this period, followed by basal &et w~th/wlthout 0.05% PB until termination. After the end of 2AAF feeding, rats were treated with SAM (six 4-hourly lm injections of 50 mg SAM/kg in buffer) or with buffer alone In the hvers of rats dosed only with 2AAF, with or w~thout PH, no ),-glutamyltransferase-posmve ( T G T + ) loci were found during wk 18 from the start of 2AAF feeding, even when the rats recewed PB On days 21, 35 and 49 after the start of 2AAF feeding, the livers were 14, 18 and 25°0 ),GT+ after NDEA/2AAF/PH treatment and 23, 32 and 38°0 after NDEA/2AAF/PH/PB. SAM treatment led to a sxgmficant decrease m the number and dmmeter of ),GT+ loci in the former group and abohshed the PB induction of 7 G T + focl m the latter In the NDEA..'2AAF/PH rats, there was a 19-40% decrease m the hver content of SAM between days 14 and 49 from the start of 2AAF feeding, but control values were restored by SAM injection In NDEA/2AAF/PH/PB-treated rats, a large decrease in the hepatic SAM pool was restored to 62°0 of the control level by SAM treatment. Some 2 wk after the start of 2AAF feeding, hver ODC actwxty was five times higher m rats subjected to 2AAF.'PH than in control rats or m those treated w~th 2AAF alone Thereafter, enzyme activity decreased progressively In rats subjected to NDEA/'2AAF/PH, ODC actw~ty increased to a peak at day 14, decreasing thereafter, but remaining well above control values 49 days from the start of 2AAF feeding The increase in ODC actw~ty was even greater m the hvers of rats treated w~th NDEA/2AAF/PH/PB SAM treatment markedly inhibited ODC actwmty. However, SAM concentrations between 0 05 and 0 5 mM had no effect on the ODC actlwty of the hver homogenates. The increase in ODC activity during the promotion of hepatocarcmogenesls was coupled with an increase m spermxdme/spermme (SPD/SPE) ratio m the hver of hepatectom~zed rats This effect, however, was observed for more than 14 days after the start of 2AAF feeding only m the rats subjected to NDEA/2AAF/PH + PB Again the SPD/SPE ratio was higher in the hver of PB-treated rats. SAM injection caused a fall m the SPD/SPE ratio to control values m both PB-treated and untreated rats It seems that the liver content of SAM regulates ODC actlvxty and polyamme synthesis. PB-mduced promotion seems to be linked to an increase m polyamme synthesis at least in the first stages of promotion A decrease m liver Ilpotrope content ~s not restricted to promotion by hpotrope-defic~ent d~ets. It appears to be revolved also m PB-supported promotion, probably through the regulation of polyamme biosynthesis 10 LIVER FOCI' OVEREXPRESSION OF AN ADAPTIVE PROGRAMME IN THE LIVER? R Schulte Hermann
Instltut fur Toxlkologle und Pharmakologle der Umversltfit Marburg, Pdgnmsteln 2, 355 Marburg aA Lahn, Federal Repubhc of Germany Liver tumour promoters, such as phenobarbital (PB) and ~-hexachlorocyclohexane (~t-HCH), reduce a programme of adaptive response m normal hver This programme comprises (1) increases m activity of various drug-metabohzlng enzymes, (2) growth by hypertrophy and/or hyperplasla and (3) changes in carbohydrate metabohsm, such as an increase in enzymes of the pentose-phosphate pathway. Many of these responses to PB are expressed m putatwe preneoplast~c foc~ of rat hver, even in the absence of a promoter Treatment with promoters reinforces expression of these adapuve responses in focl. We have therefore developed the hypothesis that focal cells, as a basic regulatory defect, are committed for overexpressxon of the adaptive programme inducible by PB-type promoters This hypothesis would explain why so many inducers of drug-metabohzmg enzymes can apparently promote the development of hver tumours. 1l. VARIATION OF NUCLEOLAR ACTIVITY AND SIZE IN LIVER PARENCHYMAL CELLS D U R I N G HEPATOCARCINOGENESIS M Kirsch-Volders and A Deleener Laboratorlum Antropogenetlka, Vrlje Universlte~t Brussel, Brussels, Belgaum The sliver staining method facdltates the demonstration of nucleolar orgamzing regions (NORs) or sites of ribosomal DNA on metaphase chromosomes and in interphase nuclei Moreover there is a good correlation between the rate of rRNA synthesis and silver staining.
868
Meeting Proceedings
A quantitative cytophotomemc technique was used to show that nucleolar acnv~ty ~s reflected accurately by quantitative analys~s of the mterphase nucleoh but not b~ the number of silver-stained NORs in metaphase plates, and that there is a form of regulation of nucleolar activity m aneuplold mahgnant HeLa-CCL, cells similar to that in PHA-stlmulated human lymphocytes With the same quantitative eshmatlon of silver stalmng, analyses were performed on hepatocyes from Wlstar rats subjected to malignant transformation by sequential treatment with N-nltrosodlethylamme (NDEA) and phenobarNtal. In hepatocytes obtained by perfusmn, the cytophotometrlc analysis of the nucleoh indicated that during the first steps of neoplastic transformation m the liner, both NDEA and phenobarbital modified the nucleolar transcriptional activity in different ways, as wsuahzed by the sllver-stalmng method In liver slices, prepared as serial sections, silver staining was compared with staining v~lth methylgreen pyronine, with the Feulgen reaction and with haematoxyhn eosm The decrease and increase of silver staining correlated with nucleolar area, DNA content and basophfllclty of the parenchymal liver cells at different stages of the carcinogenic process
12 INTERMEDIATE FILAMENT CYTOSKELETON IN HEPATOCYTES AND NEOPLASTIC NODULES OF MOUSE LIVER H Denk Institute of Pathology, Um~erslt.~ of Graz, Austria Cytoskeletons enriched m lntermediate-s~zed filaments were prepared from murme and human hver, as well as from hepatocellular and cholanglocellular carcinomas and grIseofulwnqnduced neoplastic nodules, by high-salt buffer-detergent extraction and were analysed by one- and two-dimensional gel electrophoresls In addition, neoplastic and non-neoplastic hver tissues were analysed by redirect ~mmunofluorescence microscopy using antibodies to major c~tokeratin polypeptmdes Hepatocytes and hepatocellular carcinomas of human origin showed identical cytokeratm polypeptlde composmon, with two major components (molecular weights (Mr) c 52,000 and 46,000) and identical lsoelectnc pH values In mouse material, major polypeptldes (Mr 55,000 and 49,000) were also identical in non-neoplastic and neoplastic hepatocytes, but different proportions of lsoelectrlc variants were observed m two-dlmenmonal gels Cholang~ocellular carcinomas contained cytokeratins d~fferent from those present m hepatocytes, partlcularl.~ m respect of the presence of higher Mr components Immunofluorescence microscopy re~ealed strong cytoplasmic fibrfllar staining by antibodies to a mouse-liver cytokeratln component of M r 49,000 (component D) in neoplastic and non-neoplastic cells, but the arrangement of filaments was less regular in the tumour cells The results show that cytokeratlns represent a fairly conservative cytoskeletal system w~th a high degree of organ specificity and continue to be expressed after neoplastic transformation
13 INITIATION, CLONAL GROWTH AND PROGRESSION OF PRENEOPLASTIC POPULATIONS IN THE LIVER H M Rabes. R Kerler, Ch Schuster, G Rode, M Legner, k Muller and Th Bucher Departments of Pathology and Biochemistry, University of Mamch, Federal Republic of German~ Preneoplastlc loci consisting of hepatocellular sub-populations with an aberrant enzymatic phenotype occur in the livers of animals treated with chemical carcinogens Several groups have used this marker for the analysis of factors involved in initiation and promotion, but very little Is known about the cellular origin of these focl. This question was studied by means of a specific mouse strain (Aarhus) with a mutant expression of the X-chromosomal enzyme phosphoglycerate klnase (PGK) With selective breeding it is possible to raise hybrid females which contain--owing to lionization during early embryonic development--a mosaic of cells expressing either the paternal mutant P G K - I A or the maternal wild type enzyme (PGK-IB) These allozymes can be separated by a mlcroelectrophoretlc fluonmetrlc method (Bucher et al F E B S Lett 1980, !!5, 319L which allows the determination of relative PGK proportions in very small tissue samples Such samples of liver and several other organs always contain both PGK allozymes However, preneoplasttc focx arising after 2-acetylaminofluorene feeding express selectweb either PGK-1A or PGK-IB It ~s consMered hLghl~ probable that preneoplastlc cell populations originate by clonal grov, th from a single lmtlated hepatocyte, the progeny of which show a growth advantage o~er normal hepatocytes, possibly because of internally regulated stimulation Judging from the small number of preneoplastlc loci resulting from a single exposure to a carcinogen, it appears that initiation ~s a fairly rare event in normal adult non-prohferatmg rat hver After exposure to alkylatmg N-nxtroso compounds, this low rate of initiation may be caused by high acttv~t) of DNA-repmr processes in the liver The promutagenlc DNA base modtficanon. O~-methylguanlne. for instance, is rapidly