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Biomedical subjects

H M Rabes

Publications and source records attributed to H M Rabes.

At least 55 records · Page 3Linked to original sources
PubMed1989

Deoxyribonucleoside triphosphate pools in regenerating rat liver: effect of hydroxyurea and exogenous deoxypyrimidines.

Hydroxyurea (HU) causes inhibition of DNA synthesis in regenerating rat liver due to an inhibition of the ribonucleotide reductase. We studied the consequences of a continuous HU infusion for deoxyribonucleoside triphosphate (dNTP) pools in the liver after partial hepatectomy and tried to modify imbalances by application of deoxyribonucleosides in vivo. In normal liver, an intracellular concentration of 0.16, 0.84, 0.33 and 0.27 pmol/micrograms DNA was observed for dATP, dCTP, dGTP and dTTP, respectively. In regenerating liver the dNTP pools show minor changes until 18 h after partial hepatectomy. During and after a continuous HU infusion 14--24 h after partial hepatectomy, the intracellular dNTP pools change considerably. At 19.5 h after partial hepatectomy, 5.5 h after the start of HU infusion, and at 25 h after partial hepatectomy, 1 h after termination of HU infusion, the dTTP pool was more than 10-times, and the dGTP pool about 2-times higher than in controls, while the dATP and dCTP pools remain relatively unchanged. Simultaneous infusion of HU and deoxythymidine (dThd) 14--25 h after partial hepatectomy results in a further increase of the dTTP pool during and after HU infusion. Administration of deoxycytidine (dCyd) leads to a moderate increase of the dCTP pool and a weak decrease of the dTTP pool during HU infusion. The combined application of dCyd and dThd after HU infusion had similar effects on dNTP pools as observed with dThd alone. These results show that intracellular pools of dNTPs in hepatocytes can be altered by exogenous factors in a controlled pattern. This system can be used as a model for studying the implications of induced dNTP pool dysbalances for the initiation of liver carcinogenesis by mutagenic chemicals.

Animals↗
PubMed1989

Chromosomal analysis of a diethylnitrosamine-induced tumorigenic and a nontumorigenic rat liver cell line.

A chromosomal analysis was performed on two cell lines which were derived from the liver of two rats exposed to diethylnitrosamine in vivo. The cells were obtained by collagenase perfusion of the liver at an early stage of development of ATPase-deficient putative preneoplastic populations, and propagated from foci of epithelial cells which started growth in vitro. Cell line CL 38 proved to be tumorigenic after transplantation into nude mice, giving rise to hepatocellular carcinomas and metastases. Cell line CL 44 was nontumorigenic after transplantation into nude mice and was therefore considered preneoplastic. The diploid nontumorigenic line CL 44, with a modal number of 42 chromosomes, showed a deletion of chromosome 1 and a translocation of chromosomes 3 and 14 [t(3q12;14q21)]. The hyperdiploid neoplastic cell line CL 38 has a modal chromosome number of 52 and showed tri- or tetrasomy of chromosomes 3, 7, 9, 11, and 12 and a marker chromosome that might have originated from aberrant chromosome 1. One or two homologues of chromosome 3 showed terminal deletions (q42, q41, or q35). In both cell lines rearrangements of chromosome 11 were observed [rob(11q;?) or +11 or -11 or del(11)(q12)]. Some of these karyotype abnormalities are located on the same chromosome as described for transplantable hepatomas and for other chemically induced tumors of the rat.

Animals↗
PubMed1989

Sequential changes in growth kinetics and cellular phenotype during hepatocarcinogenesis.

Sequential changes in cell proliferation and cellular phenotype during hepatocarcinogenesis induced in rats with N-nitrosomorpholine were investigated by autoradiographic determination of the [3H]thymidine-labelling index in morphologically defined focal lesions and extrafocal hepatic tissue at different times between 4 and 48 weeks after withdrawal of the carcinogen (stop model). The labelling index was found to be significantly increased in all types of preneoplastic and neoplastic hepatic lesions as compared to both the liver tissue of untreated controls and the extrafocal parenchyma of N-nitrosomorpholine-treated rats. However, the extent of the increase in labelling index differed in the phenotypically diverse types of preneoplastic and neoplastic lesions. There was a significant but relatively small increase in the labelling index in clear and acidophilic cell foci. A much stronger elevation of cell proliferation was characteristic of mixed and basophilic cell foci. The development of hepatocellular adenomas and carcinomas from preneoplastic hepatic foci was further characterized by an additional increase in cell proliferation. Each specific cellular phenotype was associated with a rather uniform proliferation rate, which remained elevated at all time points studied, suggesting that the rate of cell proliferation in the phenotypically diverse preneoplastic hepatic foci mainly reflects the intrinsic growth potential of the respective cellular phenotypes. The results support the concept that the predominant sequence of cellular changes in hepatocarcinogenesis induced by the stop model leads from the clear and acidophilic cell foci, storing glycogen in excess, through mixed and basophilic cell foci to hepatocellular adenomas and carcinomas. The fact that the labelling index of the extrafocal liver tissue of N-nitrosomorpholine-treated rats was also significantly higher than that of the normal parenchyma of untreated controls might indicate an involvement of extrafocal hepatocytes, in addition to that of foci of altered hepatocytes, in hepatocarcinogenesis.

Animals↗
PubMed1989

Intralobular distribution of preneoplastic foci in rat liver after a single dose of N-methyl-N-nitrosourea (MNU) following partial hepatectomy.

ATPase-deficient foci in rat liver, considered to be clonal in origin and possible precursors of hepatocellular carcinomas, were induced by injecting male Wistar AF/Han (200-220 g) rats with N-methyl-N-nitrosourea (MNU) (25 mg/kg) 16 or 22 h after partial hepatectomy and feeding for 80 days with a diet containing phenobarbital. The animals were killed after 90 days and the intralobular distribution of the preneoplastic foci was analysed quantitatively. The locations of 48 foci in the 16-h group and 22 foci in the 22-h group were determined from serial sections and blocks of liver from five animals. The mean distance of the foci from the portal vein in the 16-h group (385 microns) was 30% less than the distance of randomly selected points (546 microns), while the mean distance of the foci in the 22-h group (450 microns) was 16% less than the random points (535 microns). The mean diameter of the foci (235 microns) was 17% greater in the 22-h group than in the 16-h group (196 microns). We suggest that the cells in early S phase which are periportally situated 16 h after partial hepatectomy and occupy an intermediary position at 22 h represent the sensitive target population for initiation.

Animals↗
PubMed1988

Preneoplastic rat liver cells in vitro: slow progression without promoters, hormones, or growth factors.

In order to determine early changes in liver cells during carcinogenesis and to compare them with normal or neoplastic hepatocytes, an experimental model was established which allowed enrichment of this population at early stages of carcinogenesis and provided sufficient viable material for biochemical and cytogenetic analysis. This paper describes a method that allows in vitro selection and propagation of hepatocytes after in vivo initiation by alkylating agents, without the use of hormones, growth factors, or promoters which might affect their progression. From 6 different rat livers (5 initiated by continuous diethylnitrosamine feeding, 1 by a single exposure to N-methyl-N-nitrosourea) we established slow-growing lines, each of which had its own typical characteristics of growth behavior, morphology, and chromosome number. One of these lines (CL 38) transformed spontaneously after 8 weeks in primary culture, with an abrupt change to typical tumor cell behavior such as focal growth, anchorage independence, cloning ability in soft agar, and tumorigenicity in nude mice and newborn rats. In none of the other lines (now in culture for 11-15 months) has a similar abrupt change yet been observed, but all of them show a steady, albeit slow progression towards the properties of neoplastic liver cells, together with a reduction in chromosome number.

Adenosine Triphosphatases↗
PubMed1987

Transformation sensitivity in early S-phase and clonogenic potential are target-cell characteristics in liver carcinogenesis by N-methyl-N-nitrosourea.

Changes in the transformation sensitivity of hepatocytes during the cell cycle were in relation to clonogenicity investigated in partially resected rat liver. The hourly rate of the G1-S transit was measured in a control group by means of tritium-labelled thymidine (3H-TdR). At defined periods after partial hepatectomy the animals were injected with a single dose of N-methyl-N-nitrosourea (MNU) (25 mg/kg) and subsequently exposed to phenobarbital (0.05% in the diet) for 80 days. ATPase-deficient cell populations which arise by clonal growth (Rabes et al., 1982) were determined in the liver 90 days after MNU treatment and served as a marker for the initiating action of the carcinogen. The number of foci appeared to be related to the hourly rate of influx of hepatocytes into DNA synthesis at the time of MNU administration. Few foci were observed after MNU exposure in G1 and their frequency increased steeply when MNU treatment coincided with the first peak of cellular G1-S transit. As the rate of influx into S-phase decreased, so did the number of foci, despite the proportion of cells in S-phase still being at a maximum. The possibility of a gradient of clonogenicity existing within the cells of the liver lobule is envisaged. It is suggested that proliferative cells of the peri-portal region, which begin DNA synthesis early after partial hepatectomy, have the greatest ability to form clones of ATPase-deficient putative pre-neoplastic foci after initiation by a carcinogen and might thus represent, if exposed to carcinogen in early S-phase, the effective target cell population for the induction of hepatocellular carcinomas.

Adenosine Triphosphatases↗
PubMed1987

Proliferation of human testicular tumours.

Whole tumour perfusion with radioactive DNA precursors reveals that proliferation of human testicular tumours differs widely depending on type and stage of development. In seminomas a homogeneous distribution of proliferating cells at early stages is followed by a shift of the growth fraction to the periphery at advanced stage. Embryonal carcinomas show a chessboard-like pattern of proliferating fractions with high 3H-thymidine labelling indices and short tpot. In mature teratomas proliferation is low. It increases in immature teratocarcinomas. Lack of proliferation is observed in beta-hCG-positive syncytiotrophoblastic cells of choriocarcinomas, the growth fraction being limited to the cytotrophoblastic compartment.

Adult↗
PubMed1986

DNA adducts and cell cycle.

Cell cycle-dependent differences of transformation sensitivity may be due to alterations in the formation of ultimate electrophilic carcinogens during the cell cycle, preferential primary adduct formation during specific phases of the cell cycle, e.g. binding to single stranded DNA at the replication fork, base-mispairing and mutation of transformation-related genes replicating at critical phases of DNA synthesis, or cell cycle-related differences in the repair of DNA adducts. Some recent data on these subjects are summarized, mainly in context of cell cycle-dependent transformation sensitivity of regenerating rat liver.

Animals↗
PubMed1986

Correlations between ploidy and initiation probability determined by DNA cytophotometry in individual altered hepatic foci.

Single cell DNA cytophotometry of ATPase-deficient putative preneoplastic foci in rat liver, induced by a single dose of N-methyl-N-nitrosourea (25 mg/kg or 50 mg/kg) and subsequent phenobarbital feeding (0.05% in the diet), revealed three different types: the majority of foci consisted of an almost exclusive diploid cell population, others showed a preferential tetraploid pattern and a few large foci contained a mixture of di-, tetra- and octoploid hepatocytes. The results of this selective measurement of individual preneoplastic foci in Feulgen-stained 20-microns-thick cryostat sections indicate that not only diploid hepatocytes are a target for the transforming action of a carcinogen, but also tetraploid cells, and that both initiated cell types are capable of expanding as a homogeneous clone. However, clonal homogeneity appears to be lost when foci enlarge. A few preneoplastic foci, heterogeneous with respect to DNA content and endowed with an increased proliferative potential, may represent cell populations at high risk of further development into malignancy.

Adenosine Triphosphatases↗
PubMed1986

Reduced number of gap junctions in rat hepatocarcinomas detected by monoclonal antibody.

A new rat monoclonal antibody was characterized which recognized the 26K protein in gap junctions of mouse, rat and human liver as shown by immunoblot, indirect immunofluorescence, and immunogold electron microscopy. This monoclonal antibody was used to investigate the abundance of gap junctions in chemically induced rat hepatocarcinomas. In comparison with livers of control animals we found in hepatocarcinomas an average decrease of 71% in the number of gap junctional immunofluorescent spots. A corresponding decrease of the total amount of the 26 K protein was detected by quantitative immunoblot. Changes in the proliferative state as well as in intercellular adhesion of hepatocarcinoma cells in contrast to normal hepatocytes might have contributed to cause this decrease of gap junctions in tumor tissue. Possibly the partial loss of gap junctions provided a selective advantage for those preneoplastic liver cells which developed into rapidly proliferating tumor cells.

Animals↗
PubMed1986

Cell cycle-dependent initiation of adenosine triphosphatase-deficient populations in adult rat liver by a single dose of N-methyl-N-nitrosourea.

Changes in the sensitivity of hepatocytes to initiation during the cell cycle were investigated in partially resected hydroxyurea-synchronized regenerating rat liver. At defined periods of the cell cycle the animals were given injections of a single dose of N-methyl-N-nitrosourea (MNU) (25 mg/kg) and were subsequently exposed to diethylnitrosamine for 30 days (2 mg/kg/day) or to phenobarbital (0.05% in the diet) for 80 days. Adenosine triphosphatase-deficient cell populations in the liver, determined 90 days after MNU treatment, served as a marker for the initiating action of the carcinogen. Few foci were observed when MNU treatment was performed during early G1. Their frequency increased steeply after MNU injection at G1-S boundary and reached a maximum after carcinogen exposure in early S phase, when the number of adenosine triphosphatase-deficient foci was higher by a factor of 5 (after diethylnitrosamine feeding) or 10 (after phenobarbital feeding) than after MNU exposure in early G1 phase. A rapid decline was observed in middle S phase. The frequency of altered foci after MNU in late S phase and during G2-M was in the same range as in early G1. Their size distribution was similar in all groups. The results confirm and extend earlier observations of an increased initiating effect of a carcinogen during liver regeneration. Under in vivo conditions, hepatocytes are, after HU synchronization, at the highest risk of being initiated by a carcinogen when they traverse the early S phase of the cell cycle.

Adenosine Triphosphatases↗
PubMed1985

Analysis of proliferative compartments in human tumors. II. Seminoma.

Growth pattern and cell kinetics of 12 human seminomas were determined by means of vascular organ perfusion after orchiectomy. The arteria testicularis of the tumor-bearing testis was perfused up to 5 hours with dextran-diluted blood under normothermic and normotonic, simulated physiologic conditions. At defined periods, the specimen was exposed to tritiated and/or carbon 14-labeled thymidine. Autoradiograms prepared of whole tumor sections revealed a dependence of growth pattern on the stage of development. A homogeneous distribution of DNA synthesizing seminoma cells was found in small tumor foci. With increasing size, the zone of proliferation shifted to the periphery of the nodule giving rise to nodular subpopulations of high proliferative activity. In nodules of a diameter of more than about 2 cm the growth compartment consists of a highly proliferating invading cell layer at the edge of the tumor and intratumoral patches of proliferating cells near the vascular stroma. The largest part of the tumor remains at this stage in a quiescent state (G0). The mean labeling index of the seminoma cells was 11.6 +/- 1.4%, with the highest values found immediately adjacent to tumor vessels. High mitotic activity in an anaplastic seminoma was coupled with maximum labeling indices up to 41.9%. DNA synthesis time ts was 15.9 +/- 2.0 hours. The potential population doubling time for the proliferating fraction was in the range of 5 days. Lymphocytic infiltration reduced the proliferative activity in some parts, but was without effect in other areas of the seminoma. The seminoma is an example of a malignant human tumor with a rather regular growth pattern: The distribution of the proliferating compartment appears less dependent on cytologic or histologic structure, but more on tumor geometry and size.

Adult↗
PubMed1985

O6-Methylguanine repair of methylated DNA in vitro: cell cycle-dependence of rat liver methyltransferase activity.

O6-Methylguanine DNA transferase activity was investigated in liver proteins obtained at various intervals after partial hepatectomy and/or after hydroxyurea-induced synchronization of the liver cell cycle. Liver proteins were incubated with 3H-methylated calf thymus DNA as previously described by Pegg et al. (1981). The loss of O6-methylguanine was measured by radiochromatography of DNA hydrolysates. The extent of O6-methylguanine repair differed during the cell cycle: the activity increased in late G1, reached a maximum in early S phase and declined in late S phase and G2M. These results indicate that hepatocytes are endowed with an increased DNA repair capacity for this promutagenic lesion during the period of highest transformation sensitivity in the cell cycle. Though increased, however, this repair potential does not, because of its exhaustibility, appear to be sufficient to prevent initiation of transformation after high doses of alkylating carcinogens.

Animals↗
PubMed1985

X-linked allozymes of phosphoglycerate kinase as tools in experimental carcinogenesis in the mouse.

Experimental conditions are discussed that are necessary for a useful application of genetically marked laboratory animals to distinguish between clonal and nonclonal origin of induced tumors: quantitative discrimination of biochemical markers, definition of the "patch" sizes, evaluation of possible admixture of "contaminating" cells and the approach to congenicity of parental animal strains.

Alleles↗
PubMed1985

Cell proliferation and DNA repair in the liver during early stages of chemical carcinogenesis.

X-chromosomal phosphoglycerate kinase mocaicisms in organ samples of female heterozygous mice provided a means to prove, because of a selective expression of one of the two allozymes, the clonal origin of carcinogen-induced preneoplastic hepatocellular populations (see also ref. 61). The occurrence of these clonal preneoplastic foci was used in rats to determine cell cycle dependent differences of transformation sensitivity and DNA repair. The highest transformation rate was found after carcinogen exposure in late G1/early S phase of the cell cycle. Experimental disturbance of DNA precursor pools by continuous thymidine infusion during carcinogen exposure results in an increased formation of preneoplastic clones. This is a further argument in favor of an essential role of base-mispairing during initiation. Cell cycle dependent fluctuations of O6-methylguanine DNA transferase with an increasing enzyme activity in late G1 and a maximum in early S phase indicate that cells possess an increased potential for eliminating promutagenic O6-methylguanine during the most transformation sensitive parts of the cell cycle to prevent base-mispairing during DNA replication or transcription. However, this putatively protective effect is limited because the enzyme is rapidly expended in the reaction and drops again in later stages of the cell cycle.

2-Acetylaminofluorene↗
PubMed1984

O6-methylguanine repair in liver cells in vivo: comparison between G1- and S-phase of the cell cycle.

To compare the formation and persistence of alkylated DNA bases in the G1- and S-phase compartments in liver in vivo, regenerating rat liver was exposed to [14C]dimethylnitrosamine (0.57 mg/kg, IP injection) or N-[methyl14C]-N-nitrosourea (3.3 mg/kg, intraportal injection) during the G1 phase of the cell cycle (12 h after partial hepatectomy), or at 24 h after partial hepatectomy with 30% hepatocytes in DNA synthesis, or at 43 h after partial hepatectomy, 4 h after an hydroxyurea block from 14 to 39 h after operation with 80% hepatocytes in DNA synthesis. At 120 min after dimethylnitrosamine and 90 s, 5, 10, or 60 min after the intraportal pulse of N-methyl-N-nitrosourea the molar fractions of 7-methylguanine (7megua), O6-methylguanine (O6megua), and 3-methyladenine (3mead) and of metabolically labeled guanine were determined from DNA hydrolysates by Sephadex-G10 radiochromatography. After dimethylnitrosamine only minor differences were observed for 7megua formation in the three groups; the 3mead/7megua ratio remained constant irrespective of the number of cells in S phase. In contrast, the O6megua/7megua ratio revealed a loss of O6megua, the extent of which appeared proportional to the fraction of DNA-synthesizing cells in the liver. The rapid loss of O6megua in S-phase cells was confirmed after intraportal administration of N-methyl-N-nitrosourea. During the first 10 min after the methylnitrosourea pulse the O6megua/7megua ratio was constant in G1 cells and dropped from 90 s to 10 min by about 15% in liver containing 30% S-phase cells and by about 40% with 80% cells in DNA synthesis. DNA-synthesizing hepatocytes are apparently endowed with a higher O6megua DNA transferase activity than nonproliferating liver cells. The rapid, though exhaustible elimination of O6megua during S-phase might result in partial protection of DNA-synthesizing cells from base-mispairing and/or from hypomethylation at G-C sites.

Alkylation↗
PubMed1984

Cell cycle kinetics of rat hepatocytes in early putative preneoplastic lesions in hepatocarcinogenesis.

This set of experiments is the first of a series designed to explore facets of cell proliferation of hepatocytes during the carcinogenic process induced in liver by chemical carcinogens. A rat model for hepatocarcinogenesis, the resistant hepatocyte model, was used. A major advantage of this model is the unusual degree of synchrony in the early steps. Carcinogenesis was initiated by the administration of a necrogenic dose of diethylnitrosamine. Resistant hepatocytes so induced were stimulated rapidly to proliferate by partial hepatectomy in the presence of a brief exposure to dietary 2-acetylaminofluorene sufficient to inhibit the proliferation of the majority of hepatocytes, the nonresistant population. Cell cycle parameters were measured in the early carcinogen-altered resistant hepatocyte populations and in regenerating hepatocytes. Growth fraction and doubling time were experimentally determined in the altered hepatocytes. The mean cell cycle length of the resistant cells was 38.6 hr, somewhat longer than that of regenerating hepatocytes, which was 33.6 hr. Most of the increase was due to a prolonged S phase which was 13.6 hr in the altered cell population as compared to 7.0 hr in hepatocytes in regenerating control liver. The hepatocytes in normal regenerating liver had a mean duration of 21.6 hr for G1 as compared to 20.4 hr for the altered hepatocytes and a G2 of 3.4 hr as compared to 3.0 hr for carcinogen-altered hepatocyte. M was assumed to be 1.6 hr in both populations. The growth fraction in the altered cell population was determined to be a minimum of 0.83, and the doubling time was about 45 hr. Thus, the resistant hepatocytes which represent an early putative preneoplastic population show, in general, a prolongation of the cell cycle, mostly due to a prolonged S phase.

2-Acetylaminofluorene↗