RESUMEN

Cell cycle checkpoints are barriers to carcinogenesis as they function to maintain genomic integrity. Attenuation or ablation of checkpoint function may enhance tumor formation by permitting outgrowth of unstable cells with damaged DNA. To examine the function of cell cycle checkpoints in rat hepatocarcinogenesis, we analyzed the responses of the G (1), G (2) and mitotic spindle assembly checkpoints in normal rat hepatocytes, hepatic epithelial stem-like cells (WB-F344) and transformed derivatives of both. Normal rat hepatocytes (NRH) displayed a 73% reduction in the fraction of nuclei in early S-phase 6-8 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G (1) checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G (1) checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G (1) checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G (1) checkpoint function. Attenuation of G (1) checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/attenuated induction of p21 (Waf1) by IR, as well as aberrant function of the spindle assembly checkpoint. NRH displayed 93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative measure of G (2) checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G (2) checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G (2) checkpoint function. Abnormalities in the function of cell cycle checkpoints were detected in transformed hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis that aberrant checkpoint function contributes to carcinogenesis.

Asunto(s)

Ciclo Celular , Hepatocitos/citología , Hígado/citología , Animales , Secuencia de Bases , Línea Celular Transformada , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Cartilla de ADN , Células Epiteliales/citología , Células Epiteliales/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Polimorfismo Conformacional Retorcido-Simple , Ratas , Ratas Endogámicas F344 , Huso Acromático , Proteína p53 Supresora de Tumor/genética

RESUMEN

Recent evidence suggests that adult-derived stem cells, like their embryonic counterparts, are pluripotent. These simple, undifferentiated and uncommitted cells are able to respond to signals from their host tissue microenvironment and differentiate, producing progeny that display a phenotype characteristic of the mature cells of that tissue. We used a clonal stem cell line (termed WB-F344) that was derived from an adult male rat liver to investigate the possibility that uncommitted stem cells from a nonmyogenic tissue source would respond to the tissue microenvironment of the heart in vivo and differentiate into cardiac myocytes. Male WB-F344 cells that carry the Escherichia coli beta-galactosidase gene were identified in the left ventricular myocardium of adult female nude mice 6 weeks after transplantation. We confirmed the presence of a rat Y-chromosome-specific repetitive DNA sequence exclusively in the beta-galactosidase-positive myocytes by polymerase chain reaction and fluorescence in situ hybridization. Immunohistochemistry, using a cardiac troponin T-specific monoclonal antibody, and ultrastructural analysis confirmed a cardiac myocyte phenotype of the stem cell-derived myocytes. The beta-galactosidase-positive myocytes ranged from < 20 microm to 110 microm in length. The longer of these cells contained well-organized sarcomeres and myofibrils, and formed intercalated disks and gap junctions with endogenous (host-derived) myocytes, suggesting that WB-F344-derived myocytes participate in the function of the cardiac syncytium. These results demonstrate that adult liver-derived stem cells respond to the tissue microenvironment of the adult heart in vivo and differentiate into mature cardiac myocytes.

Asunto(s)

Trasplante de Células , Hígado/citología , Miocardio/citología , Trasplante de Células Madre , Animales , Diferenciación Celular , Línea Celular , Linaje de la Célula , Femenino , Masculino , Ratones , Ratones Desnudos , Miocardio/ultraestructura , Ratas , Ratas Endogámicas F344 , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

Liver regeneration after two-thirds surgical partial hepatectomy (PH) in rats treated with the pyrrolizidine alkaloid retrorsine is accomplished through the activation, expansion, and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). We have examined expression of the major liver-enriched transcription factors, cytochrome P450 (CYP) enzymes, and other markers of hepatocytic differentiation in SHPCs during the protracted period of liver regeneration after PH in retrorsine-exposed rats. Early-appearing SHPCs (at 3-7 days after PH) express mRNAs for all of the major liver-enriched transcription factors at varying levels compared to fully differentiated hepatocytes. In addition, SHPCs lack (or have significantly reduced) expression of mRNA for hepatocyte markers tyrosine aminotransferase and alpha-1 antitrypsin, but their expression levels of mRNA and/or protein for WT1 and alpha-fetoprotein (AFP) are increased. With the exception of AFP expression, SHPCs resembled fully differentiated hepatocytes by 14 days after PH. Expression of AFP was maintained by most SHPCs through 14 days after PH, gradually declined through 23 days after PH, and was essentially absent from SHPC progeny by 30 days after PH. Furthermore, early appearing SHPCs lack (or have reduced expression) of hepatic CYP proteins known to be induced in rat livers after retrorsine exposure. The resistance of SHPCs to the mitoinhibitory effects of retrorsine may be directly related to a lack of CYP enzymes required to metabolize retrorsine to its toxic derivatives. These results suggest that SHPCs represent a unique parenchymal (less differentiated) progenitor cell population of adult rodent liver that is phenotypically distinct from fully differentiated hepatocytes, biliary epithelial cells, and (ductular) oval cells.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Regeneración Hepática/fisiología , Hígado/citología , Alcaloides de Pirrolicidina/farmacología , Células Madre/citología , Animales , Biomarcadores/análisis , Diferenciación Celular , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Cartilla de ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dipeptidil Peptidasa 4/genética , Hepatectomía , Técnicas para Inmunoenzimas , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/cirugía , Regeneración Hepática/efectos de los fármacos , Masculino , ARN/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tirosina Transaminasa/genética , Tirosina Transaminasa/metabolismo , Proteínas WT1 , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo

RESUMEN

Retrorsine is a member of the pyrrolizidine alkaloid family of compounds whose toxic effects on the liver include a long-lasting inhibition of the proliferative capacity of hepatocytes. Despite the retrorsine-induced blockade of hepatocyte proliferation, retrorsine-exposed rats are able to reconstitute completely their liver mass after surgical partial hepatectomy (PH) via the sustained proliferation of a population of small, incompletely differentiated hepatocyte-like progenitor cells (SHPCs). The extensive proliferation of SHPCs in retrorsine-injured livers is accompanied by the progressive loss of irreversibly injured megalocytes. To study the mechanism by which retrorsine-damaged hepatocytes are removed after PH, we performed TUNEL analysis to establish apoptotic indices for hepatocytes in the livers of retrorsine-exposed and control rats up to 14 days post-PH. Apoptotic indices are highest (approximately 6.0%) in the livers of retrorsine-exposed rats at 1 day post-PH, gradually declining thereafter, yet remaining significantly elevated (approximately 1%) over control rats (<0.1%) at 14 days post-PH (P <.05). After PH, levels of the proapoptotic protein Bax are increased in livers from retrorsine-exposed rats relative to the levels observed in control livers. Similarly, levels of the antiapoptotic protein Bcl-x(L) are significantly decreased (P <.05) compared with controls at t = 0 resulting in an increased (approximately 3.5-fold) Bax/Bcl-x protein ratio that is significantly elevated (P <.05) compared with controls. Finally, increased levels of Bax protein are localized to the mitochondria of retrorsine-exposed rat livers after PH during the same time that cytochrome c is released. These observations combine to suggest that retrorsine-injured hepatocytes are removed after PH via apoptotic pathways dependent on relative levels and localization of Bax and Bcl-x(L) protein.

Asunto(s)

Apoptosis/efectos de los fármacos , Hepatectomía , Hígado/efectos de los fármacos , Proteínas Proto-Oncogénicas/fisiología , Alcaloides de Pirrolicidina/toxicidad , Animales , Western Blotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Hígado/patología , Masculino , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Ratas , Ratas Endogámicas F344 , Proteína X Asociada a bcl-2 , Proteína bcl-X

RESUMEN

Retrorsine is a member of the pyrrolizidine alkaloid (PA) family of naturally occurring compounds found in a large number of plant species worldwide. The cytotoxic, mutagenic, and antimitotic effects of PAs have made them targets for studies designed to determine their potential contributions to carcinogen esis and their usefulness for anticancer therapy. Evidence from the literature suggests that bioactivation of PAs by liver cytochrome P450 (CYP) enzymes is required for their toxicity. However, the specific CYP isozymes that are involved in retrorsine metabolism have not been identified. To address this issue, we administered retrorsine to a cohort of young adult male rats and examined induction or enhanced expression of mRNA and protein for widely studied hepatic CYP isoforms spanning four families together with the essential enzyme CYP reductase. The protein levels of normally expressed CYPs 1A2, 2B1/2, and 2E1 increase significantly in rat liver microsomes from retrorsine-treated rats compared to untreated control rats (P < 0. 05), but protein levels of CYP 4A3, CYP 3A1, and CYP reductase were unchanged after retrorsine treatment. In addition, CYP 1A1 mRNA and protein, which are not detectable in the livers of control rats, were induced after retrorsine exposure. The results of the present study demonstrate enhanced or induced expression of hepatic CYPs 1A1, 1A2, 2E1, and 2B1/2 in response to retrorsine exposure in rats, suggesting that one or more of these enzymes may be involved in retrorsine metabolism.

Asunto(s)

Antineoplásicos Fitogénicos/toxicidad , Sistema Enzimático del Citocromo P-450/biosíntesis , Microsomas Hepáticos/enzimología , Alcaloides de Pirrolicidina/toxicidad , Animales , Western Blotting , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Cartilla de ADN/química , Inducción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Técnicas para Inmunoenzimas , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Microsomas Hepáticos/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Tumor associated gene-1/L amino acid transporter-1 (TA1/LAT-1) was recently identified as a light chain of the CD98 amino acid transporter and cellular activation marker. Our previous studies with primary rat hepatocyte cultures demonstrated that TA1 RNA levels were responsive to media amino acid concentrations, suggesting adaptive regulation. High level TA1 expression associated with transformed cells also suggested a role in tumor progression. The present study examined the relationship of TA1/CD98 expression, adaptive response, and associated amino acid transport to neoplastic transformation using a panel of well characterized rat hepatic cell lines. We found 1) increased expression of TA1 in response to amino acid depletion, specific for arginine but not glutamine; 2) loss of TA1 response to arginine in gamma-glutamyl transpeptidase-positive transformed and tumorigenic cells; 3) no appreciable response of 4F2/CD98 heavy chain to arginine levels; and 4) correlation of system L amino acid transport activity in response to arginine with changes in TA1/LAT-1 mRNA but not total immunoreacting protein. Our results suggest this CD98 light chain may act as an environmental sensor, responding to amino acid availability and that its regulation is complex. We hypothesize that altered TA1 expression is an early event in hepatocarcinogenesis giving neoplastic cells a growth or survival advantage, particularly under conditions of limited amino acid availability.

Asunto(s)

Antígenos CD/química , Antígenos CD/metabolismo , Arginina/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Hígado/metabolismo , Sistemas de Transporte de Aminoácidos , Animales , Transporte Biológico , Northern Blotting , Carcinoma Hepatocelular/metabolismo , Línea Celular , Proteína-1 Reguladora de Fusión , Regulación de la Expresión Génica , Leucina/metabolismo , Masculino , ARN/metabolismo , Ratas , Ratas Endogámicas F344 , Factores de Tiempo , Células Tumorales Cultivadas

RESUMEN

The adult rodent liver contains at least two recognized populations of cells with stem-like properties that contribute to liver repair/regeneration under different pathophysiological circumstances: (i) unipotential committed progenitor cells (differentiated hepatocytes and biliary epithelial cells) and (ii) multipotential nonparenchymal progenitor cells (oval cells). In retrorsine-induced hepatocellular injury the capacity of fully differentiated rat hepatocytes to replicate is severely impaired and massive proliferation of oval cells does not occur. Nevertheless, retrorsine-exposed rats can replace their entire liver mass after 2/3 surgical partial hepatectomy through the emergence and expansion of a population of small hepatocyte-like progenitor cells that expresses phenotypic characteristics of fetal hepatoblasts, oval cells, and fully differentiated hepatocytes, but differ distinctly from each type of cell. The activation, proliferation, and complete regeneration of normal liver structure from small hepatocyte-like progenitor cells have not been recognized in other models of liver injury characterized by impaired hepatocyte replication. We suggest that the selective emergence and expansion of small hepatocyte-like progenitor cells observed in the retrorsine model reflect a novel mechanism of complete liver regeneration in the adult rat. Furthermore, we suggest that these cells may represent a novel progenitor cell population that (i) responds to liver deficit when the replication capacity of differentiated hepatocytes is impaired, (ii) expresses an extensive proliferative capacity, (iii) can give rise to large numbers of progeny hepatocytes, and (iv) can restore tissue mass.

Asunto(s)

Hepatopatías/patología , Hepatopatías/fisiopatología , Regeneración Hepática , Animales , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas , Hepatectomía/métodos , Masculino , Fenotipo , Alcaloides de Pirrolicidina , Ratas , Ratas Endogámicas F344 , Células Madre/fisiología

RESUMEN

We have previously identified and mapped a locus within human chromosome 11p11.2-p12 that suppresses the tumorigenic potential of some rat liver tumor cell lines. In the present study, possible molecular mechanisms of human 11p11.2-p12-mediated liver tumor suppression were investigated by examining gene expression patterns in suppressed and non-suppressed microcell hybrid (MCH) cell lines. The parental rat liver tumor cell lines (GN6TF and GP7TB) express moderate levels of p53 mRNA and protein, overexpress mRNAs for c-H-ras, c-myc, and TGFá, and do not express detectable levels of WT1 mRNA or protein. Suppression of tumorigenicity by human chromosome 11p11.2-p12 was not accompanied by significant alterations in the levels of expression of p53, c-myc, or TGFá. Expression of c-H-ras was decreased significantly in both suppressed and non-suppressed MCH cell lines, suggesting that down-regulation of c-H-ras is not directly responsible for tumor suppression. In contrast, the level of expression of WT1 correlated precisely with tumor suppression in this model system. All suppressed MCH cell lines expressed WT1 mRNA and protein at levels comparable to that of untransformed rat liver epithelial cells (WB-F344), whereas only trace WT1 mRNA and protein were detected in a non-suppressed MCH cell line. PCR analysis demonstrated that two suppressed MCH cell lines do not carry the human WT1 gene, indicating that WT1 expression in these lines originates from the rat locus. Furthermore, RT-PCR analysis showed that each of the four known splice variants of the WT1 mRNA are expressed in these suppressed MCH cell lines, recapitulating the expression pattern observed in the untransformed rat liver epithelial cells. Re-expression of tumorigenicity by suppressed MCH cell lines was accompanied by the coordinate loss of human chromosome 11p11.2-p12 and of WT1 gene expression, suggesting that one or more human 11p11.2-p12 genes are required for sustained expression of WT1 in these cell lines. Together, these results suggest that the molecular mechanism governing human chromosome 11p11.2-p12-mediated liver tumor suppression may involve induction of rat WT1 gene expression under the direct or indirect transcriptional regulation of a genetic locus (or loci) on human 11p11.2-p12.

Asunto(s)

Cromosomas Humanos Par 11 , Proteínas de Unión al ADN/genética , Neoplasias Hepáticas/genética , Factores de Transcripción/genética , Animales , Northern Blotting , Pruebas de Carcinogenicidad , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/biosíntesis , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Células Híbridas , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/prevención & control , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/biosíntesis , Células Tumorales Cultivadas , Proteínas WT1

RESUMEN

The molecular genetic hallmark of mantle cell lymphomas (MCL) is the reciprocal translocation t(11;14)(q13;q32) which juxtaposes the bcl-1 proto-oncogene to one of the joining segments of the immunoglobulin heavy chain gene. This translocation is very common in MCL and occurs in up to 70% of these malignancies. Due to the aggressive nature of MCL, markers identifying tumor progression and clinical outcomes are necessary. In this study we examined whether a corroborative relation exists between p53 mutations, bcl-1 translocation, and the proliferative fraction in MCL. We evaluated the proliferative fraction, p53 gene status, and bcl-1 translocation in 21 patients with confirmed MCL. Controls consisted of normal DNA and 7 B-cell lymphomas. Immunohistochemical detection of Ki-67 was used to assess proliferative activity while molecular techniques were used to detect p53 mutations and the bcl-1 gene translocation. Reactivity to the monoclonal antibody Ki-67 on neoplastic cells ranged from 5% to 40% in typical MCL cases. The bcl-1 gene translocation was detected by PCR in 48% (10/21) of MCLs while no rearrangements were detected by PCR in case control DNA. Screening exons 5-8 of the p53 gene for mutations did not identify a single mutation in any of the MCL cases. No correlation was found between p53 mutations, the presence of a bcl-1 translocation, and the proliferative activity of neoplastic MCL cells. We conclude that these markers may demonstrate independent events which occur during the pathogenesis of MCL.

Asunto(s)

Ciclina D1/genética , Genes p53 , Linfoma no Hodgkin/genética , Mutación , Translocación Genética , Anciano , Anciano de 80 o más Años , Ciclo Celular/genética , División Celular/genética , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 14 , Cartilla de ADN , Femenino , Marcadores Genéticos , Humanos , Antígeno Ki-67/inmunología , Masculino , Reacción en Cadena de la Polimerasa/métodos , Proto-Oncogenes Mas , Análisis de Secuencia de ADN

RESUMEN

We have previously identified and mapped a locus within human chromosome 11p11.2-p12 that suppresses the tumorigenic potential of a rat liver tumor cell line (termed GN6TF) which contains well defined chromosomal aberrations involving rat chromosomes 1, 4, 7, and 10. In the present study, we investigated the potential of this human 11p11.2-p12 liver tumor suppressor locus to suppress the tumorigenic potential of two other rat liver tumor cell lines (GN3TG and GP10TA) following microcell-mediated introduction of human chromosome 11. These tumor cell lines are aneuploid and contain chromosomal abnormalities that are similar to the GN6TF tumor line. The tumorigenic potential and other phenotypic characteristics of GN3TG-11neo and GP10TA-11neo microcell hybrid (MCH) cell lines were variable, and dependent upon the status of the introduced human chromosome 11. MCH cell lines that retained the region of 11p11. 2-p12 delineated by microsatellite markers D11S1385 and D11S903 exhibited suppression of tumorigenicity in vivo (decrease in tumorigenicity and/or elongation of latency), whereas, the tumorigenic potential of one MCH line that lacked markers in this region of human 11p11.2-p12, but retained flanking markers, was not changed from that of the parental tumor cell line. The chromosomal interval between microsatellite markers D11S1385 and D11S903 encompasses the previously localized minimal liver tumor suppressor region, suggesting that a common locus is responsible for tumor suppression among the rat liver tumor cell lines examined. The results of the present study have verified the presence of a liver tumor suppressor locus within human 11p11.2-p12, and have identified a substantial number of microsatellite markers that are closely linked to this tumor suppressor region. These chromosomal markers will facilitate positional cloning of candidate genes from this region, and may prove useful for determining the involvement of this locus in the pathogenesis of human liver cancer.

Asunto(s)

Carcinoma Hepatocelular/terapia , Cromosomas Humanos Par 11 , Genes Supresores de Tumor , Terapia Genética , Neoplasias Hepáticas/terapia , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Diferenciación Celular/genética , División Celular/genética , Mapeo Cromosómico , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratas , Eliminación de Secuencia , Transfección

RESUMEN

Neoplastic cells typically possess numerous genomic lesions, which may include sequence alterations (point mutations, small deletions, and insertions) and/or gross structural abnormalities in one or more chromosomes (large-scale deletions, rearrangements, gene amplifications). Based upon this general observation, it has been suggested that cancer cells are genetically unstable, and that acquisition of genomic instability may represent an early step in the process of carcinogenesis and a general feature of many human tumors. Numerous studies have appeared that characterize the nature and frequency of occurrence of various molecular lesions in human tumors, and significant progress has been made towards the elucidation of the molecular mechanisms that govern genetic stability in normal cells and genetic instability in neoplastic cells. In this review, we examine the evidence that genomic instability plays a significant role in the genesis of various human tumors. Furthermore, we consider the possible molecular pathways to tumorigenesis in humans and how different forms of genetic instability may impact upon these pathways.

Asunto(s)

Transformación Celular Neoplásica/genética , Genoma , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Reparación del ADN , Humanos , Repeticiones de Microsatélite/genética

RESUMEN

Age of host and transplantation-site microenvironment influence the tumorigenic potential of neoplastically transformed liver epithelial cells. Tumorigenic BAG2-GN6TF rat liver epithelial cells consistently form tumors at ectopic sites, but differentially express tumorigenicity or hepatocytic differentiation in the liver depending on host age and route of cell transplantation into the liver. Direct inoculation into host livers concentrates tumor cells locally, resulting in undifferentiated tumors near the transplantation site in both young (3-month-old) and old (18-month-old) rats. Transplantation-site tumors regress within 1 month in the livers of young rats, but grow progressively in old rats. However, inoculation of cells into the spleen distributes transplanted cells individually throughout the liver, resulting in hepatocytic differentiation by tumor cells with concomitant suppression of their tumorigenicity in young rats. When transplanted into livers of old rats by splenic inoculation, or when young hepatic-transplant recipients are allowed to age, hepatocytic progeny of BAG2-GN6TF cells proliferate to form foci, suggesting that the liver microenvironment of old rats incompletely regulates the proliferation and differentiation of tumor cell-derived hepatocytes. Upon removal from the liver, BAG2-GN6TF-derived hepatocytes revert to an undifferentiated, aggressively tumorigenic phenotype. We posit that the spectrum between normal differentiation and malignant potential of these cells reflects the dynamic interaction of the specific transformation-related genotype of the cells and the characteristics of the tissue microenvironment at the transplantation site. Changes in the tissue milieu, such as those that accompany normal aging, may determine the ability of a genetically aberrant cell to produce a tumor.

Asunto(s)

Transformación Celular Neoplásica , Neoplasias Hepáticas Experimentales/patología , Trasplante de Hígado/patología , Envejecimiento , Animales , División Celular , Línea Celular , Células Cultivadas , Hígado/citología , Hígado/patología , Masculino , Fenotipo , Complicaciones Posoperatorias/patología , Ratas , Ratas Endogámicas F344

RESUMEN

Several studies have shown that cultured rat liver epithelial cells transform spontaneously after chronic maintenance in a confluent state in vitro. In the present study, multiple independent lineages of low-passage WB-F344 rat liver epithelial stem-like cells were initiated and subjected in parallel to selection for spontaneous transformation to determine whether spontaneous acquisition of tumorigenicity was the result of events (genetic or epigenetic) that occurred independently and stochastically, or reflected the expression of a pre-existing alteration within the parental WB-F344 cell line. Temporal analysis of the spontaneous acquisition of tumorigenicity by WB-F344 cells demonstrated lineage-specific differences in the time of first expression of the tumorigenic phenotype, frequencies and latencies of tumor formation, and tumor differentiations. Although spontaneously transformed WB-F344 cells produced diverse tumor types (including hepatocellular carcinomas, cholangiocarcinomas, hepatoblastomas, and osteogenic sarcomas), individual lineages yielded tumors with consistent and specific patterns of differentiation. These results provide substantial evidence that the stochastic accumulation of independent transforming events during the selection regimen in vitro were responsible for spontaneous neoplastic transformation of WB-F344 cells. Furthermore, cell lineage commitment to a specific differentiation program was stable with time in culture and with site of transplantation. This is the first report of a cohort of related, but independent, rat liver epithelial cell lines that collectively produce a spectrum of tumor types but individually reproduce a specific tumor type. These cell lines will provide valuable reagents for investigation of the molecular mechanisms involved in the differentiation of hepatic stem-like cells and for examination of potential causal relationships in spontaneously transformed rat liver epithelial cell lines between molecular/cellular alterations and the ability to produce tumors in syngeneic animals.

Asunto(s)

Transformación Celular Neoplásica , Hígado/patología , Animales , Trasplante de Células , Células Cultivadas , Células Clonales/patología , Células Clonales/trasplante , Células Epiteliales/patología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/ultraestructura , Microscopía Electrónica , Trasplante de Neoplasias , Fenotipo , Ratas , Ratas Endogámicas F344

Asunto(s)

Biología Molecular , Neoplasias/diagnóstico , Neoplasias/genética , Southern Blotting , Neoplasias de la Mama/genética , Ciclo Celular/genética , División Celular/genética , Transformación Celular Neoplásica/genética , Reparación del ADN , Enzimas de Restricción del ADN , Amplificación de Genes , Humanos , Neuroblastoma/genética , Análisis de Secuencia de ADN , Translocación Genética

RESUMEN

A model of spontaneous malignant transformation was used to evaluate the molecular changes that take place in WB-F344 rat liver epithelial cells during neoplastic transformation and tumorigenesis. A comparison of wild-type low-passage WB-F344 cells to spontaneously transformed tumor cell lines revealed that the majority of the tumor cell lines have an increased capacity for autonomous proliferation and motility when maintained in serum-free media. In the current study, we show that c-met is expressed at some level in wild-type WB-F344 cells and in all of the spontaneously transformed tumor cell lines, and that 9/16 of the tumor cell lines have acquired hepatocyte growth factor (HGF) expression. In vitro growth of HGF-expressing tumor cell lines is inhibited as much as 68% by the addition of neutralizing antibodies to HGF or antisense HGF oligonucleotides, indicating that the production of HGF by the tumor cells is partially responsible for driving autonomous proliferation in a subset of tumor cell lines. Furthermore, conditioned media collected from HGF-expressing tumor cell lines stimulates DNA synthesis in wild-type WB-F344 cells, and this effect can be abrogated by pre-incubation of the conditioned media with neutralizing antibodies to HGF. Because HGF is a motility-promoting growth factor, all cell lines were evaluated to determine if expression of HGF stimulated motogenesis. All tumor cell lines (regardless of HGF expression) were highly motile in comparison with wild-type WB-F344 cells, with a 3.5-fold to 20-fold greater number of motile cells. The high basal rate of motility characteristic of the tumor cell lines is not a result of the production of HGF, because it is also a property of the cell lines that do not express HGF messenger RNA. Furthermore, tumor cell motility is not inhibited by antisense oligonucleotides or neutralizing antibodies. Establishment of an autocrine HGF/c-met loop in a subset of spontaneously transformed WB-F344 cell lines may influence development and/or expression of the tumorigenic phenotype by driving cellular proliferation.

Asunto(s)

Comunicación Autocrina , Transformación Celular Neoplásica , Factor de Crecimiento de Hepatocito/fisiología , Hígado/patología , Proteínas Proto-Oncogénicas c-met/fisiología , Células Madre/patología , Animales , Anticuerpos/farmacología , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Medios de Cultivo Condicionados , Medio de Cultivo Libre de Suero , ADN/análisis , ADN/biosíntesis , Células Epiteliales/patología , Expresión Génica , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Factor de Crecimiento de Hepatocito/genética , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-met/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas

RESUMEN

The genomic evolution of a cohort of WB-F344 rat liver epithelial cell lineages undergoing spontaneous neoplastic transformation was followed to define the mechanistic relationship between genomic instability and progression to the neoplastic phenotype. Eighteen independent populations of WB-F344 cells (initiated from a single diploid-founding population) were subjected to 12 cycles of selective growth at confluent cell density, and cellular DNA contents were measured after each selection cycle. Flow cytometry demonstrated significant gains in the amount of G1 DNA after selection cycles 3, 6, and 7 in 44% (8 of 18), 89% (16 of 18), and 39% (7 of 18) of the cell populations, respectively. All populations subsequently lost DNA and returned to a diploid or pseudo-diploid DNA content within 1 to 2 selection cycles after the appearance of an increased DNA content. Additionally, appearance and subsequent disappearance of aneuploid or tetraploid subpopulations was observed in 11% (2 of 18) and 83% (15 of 18) of the experimental lineages, respectively. Although perturbations of G1 DNA content were apparent as early as selection cycle 3, at least 8 cycles of selective growth were required for the acquisition of tumorigenicity. While the independent lineages demonstrated significant fluctuations in G1 DNA content between selection cycles 3 and 8, the majority (11 of 13) of the populations contained a diploid or pseudo-diploid DNA content at the time tumorigenicity was expressed. Genomic instability preceded the acquisition of tumorigenic potential in rat liver epithelial cells subjected to selective growth conditions of maintenance at confluence, and may be required for its expression.

Asunto(s)

Transformación Celular Neoplásica/genética , Hígado/citología , Hígado/fisiología , Aneuploidia , Animales , Pruebas de Carcinogenicidad , Ciclo Celular/fisiología , Células Epiteliales/fisiología , Citometría de Flujo , Genoma , Ratas

RESUMEN

We previously demonstrated that a locus (or loci) linked to the D11S436 marker, which is within the approximately 6-Mb cen-p12 region of human chromosome 11, suppresses the tumorigenic potential of some rat liver epithelial tumor microcell hybrid (MCH) cell lines. To more precisely map this putative liver tumor suppressor locus, we examined 25 loci from human chromosome 11 in suppressed MCH cell lines. Detailed analysis of these markers revealed a minimal area of overlap among the suppressed MCH cell lines corresponding to the chromosomal region bounded by (but not including) microsatellite markers D11S1319 and D11S1958E and containing microsatellite markers D11S436, D11S554, and D11S1344. Direct examination of the kang ai 1 (KA/1) prostatic adenocarcinoma metastasis suppressor gene (which is closely linked to D11S1344) produced evidence suggesting that this locus was not responsible for tumor suppression in this model system. In addition, our data strongly suggested that the putative liver tumor suppressor locus was distinct from other known 11p tumor suppressor loci, including the multiple exotoses 2 locus (at 11p11.2-p12), Wilms' tumor 1 locus (at 11p13), and Wilms' tumor 2 locus (at 11p15.5). The results of this study significantly narrowed the chromosomal location of the putative liver tumor suppressor locus to a region of human 11p11.2-p12 that is approximately 950 kb. This advance forms the basis for positional cloning of candidate genes from this region and, in addition, identified a number of chromosomal markers that will be useful for determining the involvement of this locus in the pathogenesis of human liver cancer.

Asunto(s)

Mapeo Cromosómico , Cromosomas Humanos Par 11 , Genes Supresores de Tumor , Neoplasias Hepáticas Experimentales/genética , Animales , Genes del Tumor de Wilms , Humanos , Células Híbridas , Neoplasias Hepáticas Experimentales/patología , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa , Ratas , Células Tumorales Cultivadas

RESUMEN

After intrahepatic transplantation into livers of adult syngeneic German-strain Fischer 344 rats that are deficient for the bile canalicular enzyme dipeptidyl peptidase IV (DPP-IV), cultured WB-F344 rat liver epithelial cells (without exogenous marker genes) integrate into hepatic plates and differentiate into hepatocyte-like cells that are morphologically and functionally indistinguishable from mature hepatocytes. In this model system, the differentiated progeny of transplanted WB-F344 cells are identified among the DPP-IV-negative host hepatocytes by their expression of bile canalicular DPP-IV enzyme activity. DPP-IV-positive hepatocyte-like cells also expressed other markers of hepatocytic differentiation, including albumin, transferrin, and alpha-1-antitrypsin, suggesting that the progeny of transplanted WB-F344 cells express a complete hepatocyte differentiation program. These results complement our previous studies indicating WB-F344 cells can serve as stem-like precursor cells for differentiated hepatocytes and strengthen the suggestion that WB-F344 rat liver epithelial cells represent the cultured counterpart of liver stem-like hepatocyte progenitor cells present in the normal adult rat liver.

Asunto(s)

Trasplante de Células , Dipeptidil Peptidasa 4/metabolismo , Hígado/patología , Células Madre/patología , Animales , Diferenciación Celular/genética , Dipeptidil Peptidasa 4/genética , Epitelio/patología , Inmunohistoquímica , Hígado/enzimología , Ratas , Ratas Endogámicas F344

RESUMEN

Age is the biggest risk factor associated with the development of cancer. Whereas the incidence of neoplastic disease increases dramatically in aging humans and experimental animals, the effects of aging on tumorigenesis are poorly understood. Using a rodent model, we have previously shown that the microenvironment of the hepatic parenchyma regulates hepatic tumor formation from transplanted neoplastic cells in an age-dependent manner. In the current study, we have investigated the mechanistic basis for the age-dependent suppression of tumor formation by transplanted BAG2-GN6TF rat liver epithelial tumor cells. Examination of liver tissue at 7 and 14 days after transplantation of liver tumor cells revealed the presence of injection-site tumors in both young and old animals. With time, these tumors spontaneously regressed from young adult livers, leaving no tumor remnant and without evidence of injury to the parenchyma. In contrast, tumors detected in old animals at early time points after transplantation persisted for the remainder of the life of the host. Reduced cell proliferation and increased apoptotic cell death were detected in hepatic tumors in young rats relative to hepatic tumors in old rats. These observations suggest that the regression of hepatic tumors from young rats was the direct result of an increased ratio of cell death to cell birth, whereas the persistence and expansion of hepatic tumors in old rats was related to increased cell proliferation relative to cell death. Because young adult rats developed persistent (nonregressing) tumors after transplantation of BAG2-GN6TF cells to extrahepatic sites, the consistent regression of BAG2-GN6TF tumors from livers of young rats seemed to be largely a result of interactions between tumor cells and factors specific to the liver microenvironment. These data indicate that the hepatic microenvironment of young rats can negatively regulate the growth of transformed liver epithelial cells, but with increasing age, the ability of the hepatic microenvironment to suppress the growth of neoplastic tissue deteriorates. Age-associated alterations in tissue microenvironments may thus permit the development of tumors late in life.

Asunto(s)

Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas/patología , Hígado/citología , Factores de Edad , Animales , Apoptosis , Comunicación Celular , Diferenciación Celular , División Celular , Supervivencia Celular , Trasplante de Neoplasias , Ratas , Ratas Endogámicas F344

Asunto(s)

Hígado/citología , Hígado/fisiología , Animales , Diferenciación Celular , División Celular , Células Cultivadas , Colágeno , ADN/biosíntesis , Combinación de Medicamentos , Embrión de Mamíferos , Sustancias de Crecimiento/farmacología , Laminina , Hígado/efectos de los fármacos , Proteoglicanos , Roedores , Albúmina Sérica/biosíntesis , Células Madre/citología