DNA alkylation and tumor induction in regenerating rat liver after cell cycle-related continuous N-nitrosodimethylamine infusion.

Synchronized regenerating rat liver after partial hepatectomy was used to study cell cycle-related DNA base alkylation and liver carcinogenesis. A continuous iv infusion of [14C]N-nitrosodimethylamine (DMN) at a dose of 0.5 mg/kg/hour was given to inbred male Wistar Af/Han rats over a period of 8 hours either during the G1 phase, hydroxyurea-synchronized DNA synthesis, or the G2+M-phase of regenerating liver or to untreated rats (G0-phase liver--carcinogen dose, 1.5 mg/kg/hour). Two hours after the end of the infusion, the amount of 7-methylguanine was highest in the G0-phase liver, with a decrease in the G1 phase, the S-phase, and the G2+M-phase. After continuous DMN exposure, the O6-methylguanine:7-methylguanine ratio was lower in the S-phase and G2+M-phase livers than in the G0-phase and G1-phase livers, indicating an increased O6-methylguanine repair during DNA synthesis and the G2+M-phase. Liver tumors in rats treated by continuous DMN infusion either during the G0 phase or the S-phase developed only after carcinogen exposure during DNA synthesis.

Absence of G(s)alpha gene mutations in childhood thyroid tumors after Chernobyl in contrast to sporadic adult thyroid neoplasia.

Heterotrimeric G proteins participate in the signal transduction cascade. Adult thyroid tumors have been shown to harbor specific point mutations in codons 201 and 227 of the G(s)alpha subunit of the adenylate cyclase stimulator. This protein affects the GDP/GTP turnover and finally results in an enhanced activation of G(s) and thus adenylate cyclase. We attempted to find out if G(s)alpha gene mutations were present in thyroid tumors of children from Belarus after the Chernobyl nuclear accident. Paraffin sections of 20 thyroid tumors were used for PCR amplification by oligonucleotide intron primers flanking exons 8 and 9, encompassing codon 201 and 227, respectively. By direct sequencing of the 274-bp amplification product, we did not detect any mutations of the G(s)alpha gene in codon 201 or 227. In contrast to thyroid neoplasia of adults, G(s)alpha gene mutations do not play a role in the development of childhood thyroid tumors after the Chernobyl reactor accident.

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.

A novel type of RET rearrangement (PTC8) in childhood papillary thyroid carcinomas and characterization of the involved gene (RFG8).

As part of ongoing studies on the RET rearrangement frequency in children with papillary thyroid carcinoma (PTC) after their exposure to radioactive iodine after the Chernobyl reactor accident, new methods for the detection of novel types of RET rearrangements are being developed. In this study, an improved reverse transcription-PCR strategy is used successfully to identify a new type of RET rearrangement. This rearrangement is designated PTC8 and the involved RET-fused gene (RFG) as RFG8. The identification of two reciprocal transcripts coding for the RFG8/RET and RET/RFG8 fusions suggests that the PTC8 rearrangement results from a balanced chromosomal translocation. With a view to clarify its role in tumor induction, we compared the fusion products with those of previously described RET rearrangements. We therefore sequenced and characterized the RFG8 cDNA, which showed no significant similarity to any functional protein described as yet. RFG8 is located on chromosome 18q21-22 and is expressed ubiquitously. Bioinformatic analysis predicts with a high probability that the corresponding rfg8 protein is located in the cytoplasm and is involved putatively in intracellular transport processes. Furthermore, we identified coiled-coil structures upstream of the breakpoint with one of the coiled-coils showing dimerization capability. Thus the rfg8/ret fusion protein exhibits structures for oncogenic activation that are similar to those observed in previously described RET fusions.

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.

Amplification, rearrangements, and enhanced expression of c-myc in chemically induced rat liver tumors in vivo and in vitro.

c-myc expression, amplification, and rearrangement were studied in neoplastic hepatic nodules of rats after diethylnitrosamine treatment, in hepatocellular carcinomas induced by N-methyl-N-nitrosourea, and in tumorigenic liver cell lines derived from rat hepatocytes transformed by diethylnitrosamine in vivo. Steady-state levels of c-myc transcripts, as measured by Northern blot hybridization, were slightly increased in neoplastic hepatic nodules and showed high levels in some hepatocellular carcinomas and some tumorigenic liver cell lines. DNA of the samples was analyzed after restriction nuclease treatment by Southern blot hybridization, using different probes specific for the three exons of the c-myc gene. The results suggest rearrangements of the complete gene and/or amplification in some cases. Rearrangements include the 5'-part of the first exon and a stretch upstream c-myc supposed to contain control elements of c-myc expression. Aberrations of this kind are most pronounced in advanced metastasizing hepatocellular carcinomas and highly tumorigenic, metastasizing liver cell lines. Heterogenicity of genomic alterations and c-myc transcript levels were found among different samples of the same hepatocellular carcinoma indicating subclonal diversification.

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.

Detection of a novel type of RET rearrangement (PTC5) in thyroid carcinomas after Chernobyl and analysis of the involved RET-fused gene RFG5.

A novel type of RET rearrangement, PTC5, was detected in papillary thyroid carcinomas of two patients exposed to radioactive fallout after Chernobyl. Reverse transcription-PCR and rapid amplification of 5'-cDNA ends revealed a fusion of the ret tyrosine kinase (TK) domain with a sequence identical to that described previously as ret-II. Ret-II is a transfection artifact in NIH3T3 cells and has not yet been detected in any human tumor. Overlapping sequences found in the expressed sequence tag databases enabled us to sequence the COOH terminus of the ret-fused gene 5 (RFG5). The combined data made it possible to assemble a full-length rfg5 protein sequence. Computer-assisted analysis of this sequence reveals four putative coiled-coil structures, possibly involved in dimerization, but no membrane-binding sequences. Northern blots show a ubiquitous RFG5 expression in various normal tissues, including the thyroid gland. In addition to the RFG5/RET, we also detected the reciprocal RET/RFG5 transcript in both tumor samples, suggesting that the rearrangement is based on a balanced reciprocal translocation. In agreement with other rearranged TKs, it is concluded that the transforming action of the new fusion protein rfg5/ret in thyroid tumors may be due to an activation of the ret TK by constitutive expression and dimerization potential of the 5'-fused rfg5 protein. Ret immunohistochemistry indicates that the fusion protein is expressed in all cells of PTC5 tumors, suggesting that RFG5/RET rearrangement is an early event in thyroid carcinogenesis.

Synchronization of hepatocellular DNA synthesis in regenerating rat liver by continuous infusion of hydroxyurea.

Hydroxyurea (HU) inhibited DNA synthesis in the livers of partially hepatectomized rats. After an i.v. infusion of HU begun in the late G1 phase and continued for periods of up to 30 hr, all hepatocytes scheduled to embark on DNA synthesis in a characteristic intralobular sequence during this time interval accumulated at the G1-S boundary. The effective dose was 1.25 mmoles/kg/hr, preceded by a single injection of 1.69 mmoles/kg. Serum levels of HU rose slightly during continuous infusion and decreased after termination, with a half-life of about 80 min. Liver weight increased during HU infusion. The G1-S blockade was rapidly reversed in the liver after the end of HU infusion. The specific activity of DNA increased to a maximum between 3 and 5 hr. [3H]Thymidine labeling indices reached about 80%. Intralobular distribution of labeled hepatocytes was congruent to the pattern seen in partially hepatectomized rats after a continuous [3H]thymidine infusion of equal duration. The beginning of DNA synthesis in nonparenchymal cells was delayed, as compared with hepatocytes. Vincristine infusion for 12 hr after release from the HU block arrested about 40% of the hepatocytes in mitosis, indicating that a large fraction of cells progressed through the cycle after the prolonged HU block. Partially resected rat liver appeared to be rather resistant to the unfavorable consequences of "unbalanced growth" during the protracted inhibition of DNA synthesis, providing a useful model for synchronization of DNA synthesis in a differentiated resting organ triggered into active growth.

The transcription coactivator HTIF1 and a related protein are fused to the RET receptor tyrosine kinase in childhood papillary thyroid carcinomas.

Children exposed to radioactive iodine as a consequence of the Chernobyl reactor accident have an increased risk of papillary thyroid carcinomas (PTC). The predominant molecular lesions in these tumors are rearrangements of the RET receptor tyrosine kinase (tk). Here we report on two novel types of RET rearrangement, PTC6 and 7, and describe the fusion products and the ret fused gene (rfg) proteins. Like the other rfg proteins identified so far they are ubiquitously expressed, not membrane-bound and contain coiled coil domains required for constitutive activation of the ret tk domain. In the PTC6 rearrangement the ret tk domain is fused to the aminoterminal part of the human transcription intermediary factor htif 1. In the PTC7 rearrangement the ret tk domain is fused to a novel protein that is strongly related to htif1. Like htif1 it contains a RBCC motif (ring finger, B boxes, coiled coil domain) located in the aminoterminal part and a phd finger and a bromodomain in the carboxyterminal part. Htif1 and related proteins are transcription coactivators for nuclear receptors, thus participating in controlling cellular development, differentiation and homeostasis. This is the first report on their involvement in human thyroid carcinogenesis.

High prevalence of RET rearrangement in thyroid tumors of children from Belarus after the Chernobyl reactor accident.

RET rearrangement was studied in papillary thyroid carcinomas (PTC) of children exposed to radioactive fallout in Belarus after the Chernobyl accident. To detect RET rearrangement in small tissue samples from thyroidectomy specimen (12 PTC of children; 2 PTC and 1 follicular carcinoma of adults; non-tumorous thyroid tissue of 4 children and 4 adults as controls), a RT-multiplex PCR was developed using primers suited to amplify fragments in different quantities depending on the presence or absence of RET rearrangements in the tissues. The type of rearrangement was determined by RT-PCR and direct sequencing using primers for ret/PTC1, 2 and 3. Two-thirds of the papillary thyroid carcinomas of the children revealed a RET rearrangement, with ret/PTC3 being more frequent by a factor of 3 than ret/PTC1. ret/PTC2 was not detected. All RET rearrangement-positive tumors had lymph node metastasis while half of the tumors with wild-type cRET had not. More than half of the cases with ret/PTC3 expressed not only the ELE/RET transcript as expected, but also the RET/ELE transcript. Intrachromosomal rearrangement involving RET and the adjacent H4 or ELE gene on chromosome no. 10 is a very frequent event in thyroid cancer of children of the Chernobyl-contaminated zone of Belarus.

A new form of RET rearrangement in thyroid carcinomas of children after the Chernobyl reactor accident.

Intrachromosomal rearrangements involving the RET and the adjacent H4 or ELE1 gene are very frequent events in thyroid cancer of children from Belarus after the Chernobyl reactor accident (Klugbauer et al., 1995). The fusion product between ELE1 and RET (RET/ PTC3) seems to be the prevailing type of rearrangement as shown in a recently published study using a novel RT multiplex PCR approach in combination with the identification of the rearrangement type by RT-PCR and direct sequencing. Now we found a new type of RET rearrangement: By the 5'RACE method we demonstrated in cDNA the fusion of the tyrosine kinase domain of RET with a truncated ELE1 gene shorter than the ELE1 in RET/PTC3. Sequencing of genomic DNA revealed a rearrangement breakpoint at position 41 of a new ELE1 intron (522 bp in length). The new oncogene RET/ delta PTC3 is shortened by one ELE1 exon of 144 bp in length. Structural considerations of the ele1 amino terminal of RET/ delta PTC3 suggest that the transforming activity of the fusion protein is apparently not affected by this truncation. The exon lacking in RET/ delta PTC3 was found to code in the reciprocal transcript RET/ delta ELE1 and increased its size by 144 bp. Obviously the new and possibly additional ELE/RET fusion molecules might even increase the high prevalence of ELE1/RET rearrangements in thyroid carcinomas of children after the Chernobyl reactor accident.

Molecular analysis of new subtypes of ELE/RET rearrangements, their reciprocal transcripts and breakpoints in papillary thyroid carcinomas of children after Chernobyl.

A high prevalence of RET rearrangements is found in papillary thyroid carcinomas (PTC) of children from Belarus after the Chernobyl reactor accident. The ELE/RET rearrangement (PTC3) is prevailing. Aberrant types of ELE/RET rearrangement have been found with a truncated ELE1 gene: As compared with the common form (PTC3r1) one aberrant type is shorter by one 144 bp exon (PTC3r2) (three cases); in the second atypic form (PTC3r3) the ELE1 part is 18 bp shorter than in PTC3r1. In agreement with the observation that the oncogenic RET is generated by a paracentric inversion at chromosome 10, we found not only ELE/RET, but also RET/ELE transcripts in these tumors. Sequencing of the breakpoint regions at the genomic DNA level revealed DNA modifications that might be relevant for illegitimate recombination after DNA doublestrand breaks. The high prevalence of ELE/RET rearrangements and various subtypes appears to be typical for radiation-induced thyroid carcinomas of children after the Chernobyl reactor accident.

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.

Pattern of radiation-induced RET and NTRK1 rearrangements in 191 post-chernobyl papillary thyroid carcinomas: biological, phenotypic, and clinical implications.

Molecular genetic aberrations and the related phenotypes were investigated in 191 papillary thyroid carcinomas (PTCs) from patients exposed at young age to radioiodine released from the Chernobyl reactor. A high prevalence of RET gene rearrangements (62.3%) with a significant predominance of ELE1/RET (PTC3) over H4/RET (PTC1) rearrangements was found in PTCs of the first post-Chernobyl decade. NTRK1 rearrangements were rare (3.3%). In 3.3%, we observed novel types of RET rearrangements: GOLGA5/ RET (PTC5), HTIF/RET (PTC6), RFG7/RET (PTC7), and an as yet undefined RFGX/RET.RET rearrangements, preferentially ELE1/RET, are related to rapid tumor development. At longer intervals after exposure to ionizing radiation, the prevalence of RET rearrangements declines with a shift from ELE1/RET to H4/RET, most significantly in female patients. The prevalence of specific types of rearrangements is independent of age at irradiation. A significantly higher prevalence of ELE1/RET was observed in the most heavily contaminated Oblasts, Gomel and Brest, suggesting a preferential formation of this type of rearrangement after high thyroid doses. RET rearrangement is related to aggressive growth: Rearrangement-positive PTCs were in a more advanced pT category and more frequently in the pN1 category at presentation than rearrangement-negative PTCs. ELE1/RET is related to the solid variant of PTC, H4/RET more frequently to typical papillary structures. The genotype/phenotype evaluation of post-Chernobyl PTCs reveals a characteristic spectrum of gene rearrangements that lead to typical phenotypes with important biological and clinical implications.

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.

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.

Proliferative heterogeneity of human renal cell carcinomas and prevalence of ras gene point mutations.

The variable prevalence and a possible stage-dependent increase of ras gene point mutations in human tumors might correspond to clonal growth advantages of ras-activated cells. Tumor areas with activated ras genes might thus differ in proliferative activity from those lacking ras gene activation. This hypothesis is studied in a series of human renal cell carcinomas that had been used previously for an analysis of proliferative compartments after post-operative vascular [3H]/[14C]thymidine perfusion [Rabes et al. (1979) Cancer 44: 799-813]. The growth fraction of different subcompartments of these tumors was studied by immunohistochemistry with mib1 antibody, recognizing a fixation- and embedding-resistant epitope of Ki-67 protein. Thirty subpopulations of 14 human renal cell carcinomas that exhibited a broad spectrum of proliferative activity were chosen for an analysis of the prevalence of K-ras point mutations in exon 1 by a mutation-enriching primer-mediated restriction-fragment-length-polymorphism analysis and/or direct sequencing of polymerase-chain-reaction-amplified material. The combined autoradiographic and immunohistochemical analysis confirmed the intra- and intertumoral proliferative heterogeneity. Compared to [3H]/[14C]thymidine labeling indices, mib1 labeling indices are higher. The ratio of mib1 to [3H]/[14C]thymidine labeling indices varies from 1.9 to 4.1 for the individual tumor subcompartments. However, neither in K-ras codons 12/13 nor in adjacent codons did we detect any mutations in the various tumor compartments. The results suggest that neither mode of proliferation nor type of differentiation is related to K-ras exon 1 point mutations in human renal cell carcinomas.

Carcinogen-induced liver tumours of Wistar rats: absence of activated ras genes and of N-rasC.

We examined mutational activation of ras genes in rat liver preneoplasias and tumours induced by diethylnitrosamine and N-methyl-N-nitrosourea (NMU). In accordance with previous reports on H- and K-ras genes, no mutations were detected in the investigated hepatic tumours and prestages suggesting that neither mutations at codons 12, 13 and 61 of H- and N-ras nor a mutation in the last intron of the H-ras gene are involved in initiation and progression of rat hepatocellular carcinomas. In the course of this investigation we found two N-ras genes (N-rasA, N-rasB). Surprisingly N-rasC, which is present in the germ line of Fischer rats, is missing in Wistar rats. This suggests different numbers of germline N-ras genes in members of one species. Two out of eight NMU-induced liver tumours exhibited additional N-ras-related sequences of unknown origin.

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.