Cytokine-dependent activation of small hepatocyte-like progenitor cells in retrorsine-induced rat liver injury.

Complete liver regeneration after partial hepatectomy (PH) in rats exposed to the pyrrolizidine alkaloid retrorsine is accomplished through the activation, expansion, and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). The mechanism(s) governing activation of SHPCs after PH in retrorsine-injured rats has not been investigated. We examined the possibility that SHPCs require cytokine priming prior to becoming growth factor responsive in this model of liver injury and regeneration. Male Fischer 344 rats were treated with retrorsine (30 mg/kg ip) at 6 and 8 weeks of age. Retrorsine-exposed and age-matched control rats were randomized into dexamethasone-treated and no DEX groups. DEX-treated animals were either given a single dose of DEX (2 mg/kg ip) at the time of PH or multiple DEX treatments (2 mg/kg ip each) at 24 and 1 h before PH and 1, 2, and 3 days post-PH. A subset of rats received 10 microg of recombinant IL6 protein, administered intravenously 30 min after PH. Liver tissues were harvested at 7, 14, 21, and 30 days post-PH. Treatment of retrorsine-exposed rats with the cytokine inhibitor dexamethasone (DEX) effectively blocked the emergence of SHPCs resulting in an inhibition of liver regeneration and producing significant short-term mortality. The livers of DEX-treated retrorsine-exposed rats displayed decreased numbers and smaller SHPC clusters compared to retrorsine-exposed rats in the absence of DEX treatment. Administration of recombinant IL6 to DEX-treated retrorsine-exposed rats restored the emergence of SHPCs and SHPC-mediated regenerative response. The livers of DEX-treated retrorsine-exposed rats that received IL6 displayed numbers of expanding SHPC clusters comparable to that of retrorsine-exposed rats in the absence of DEX treatment. These results combine to suggest that SHPC activation after PH in retrorsine-exposed rats is cytokine dependent and may specifically require IL6.

Isolation, short-term culture, and transplantation of small hepatocyte-like progenitor cells from retrorsine-exposed rats.

BACKGROUND: Complete liver regeneration after partial hepatectomy (PH) in rats treated with the pyrrolizidine alkaloid retrorsine can be accomplished through the activation, expansion, and differentiation of a novel population of small hepatocyte-like progenitor cells (SHPCs). These cells have not been isolated in pure form, established in primary culture, or transplanted into syngeneic rats to examine their differentiation potential. METHODS: Primary liver cells enriched for SHPCs were prepared by differential centrifugation of primary liver cell dispersions from retrorsine-exposed rats 6-8 days and 13-15 days after PH. Isolated SHPCs were characterized for cell size, morphology, and expression of cell type-specific markers (including hepatocyte and bile duct-oval cell markers), and established in short-term primary culture. Isolated SHPCs were transplanted into the livers of syngeneic rats to evaluate their ability to engraft and differentiate into mature hepatocytes. RESULTS: SHPCs obtained from retrorsine-exposed rats 6-8 days and 13-15 days after PH were small (10-12 microm in diameter), morphologically resembled hepatocytes, and were predominantly H.4 antigen-positive, alpha-fetoprotein-positive, and OV6-negative. SHPCs did not proliferate in culture and could not be passaged, but short-term cultures were established using protein substrates (collagen or laminin) and defined medium containing epidermal growth factor and nicotinamide. After transplantation into the livers of syngeneic hosts, SHPCs insert into hepatic plates and give rise to differentiated hepatocyte progeny. The SHPC-derived hepatocyte progeny express a differentiated phenotype (albumin-positive, transferrin-positive, alpha-fetoprotein-negative) and are able to proliferate in vivo in response to the growth stimulus provided by PH. CONCLUSIONS: The results demonstrate that enriched SHPC populations can be isolated from retrorsine-exposed rats and established in short-term culture, and they can engraft and differentiate after transplantation into the livers of syngeneic rats.

Molecular analysis of an immature ovarian teratoma with gliomatosis peritonei and recurrence suggests genetic independence of multiple tumors.

Immature ovarian teratoma is a common germ cell tumor of young women. Patients with immature teratoma often exhibit multiple neoplasms, including tumors outside the ovaries, and occasionally a rare benign condition termed gliomatosis peritonei (GP). These multiple neoplasms are generally believed genetically-linked progeny of the ovarian tumor resulting from local recurrence/spread. In this study, we performed a molecular analysis of a single patient clinically diagnosed with immature ovarian teratoma, GP, and recurrent pelvic mucinous teratoma. Microsatellite PCR and amplicon analysis was performed to genetically characterize tissue samples from omental glial implants and multiple peritoneal tumors. PCR-based amplification of microsatellite markers identifies unique genetic differences (allelic variation) between tumors resulting from divergent natural histories among multiple tumor nodules in a single patient. A total of 21 different microsatellite markers were employed, and seven provided informative results (D3S1744, D6S1056, D7S2846, D14S306, D16S764, D18S858, D22S420). These markers demonstrated mutually exclusive genetic differences among the tumors from this patient, establishing the neoplasms as genetically distinct from each other (non-identical), and that no lineage relationship exists among them. This observation suggests that the multiple tumors arising in this patient with immature ovarian teratoma, GP, and recurrent pelvic mucinous neoplasm represent multiple independent tumors rather than true tumor recurrence/spread. The results of this study suggest strongly that patients with recurrent teratoma may be afflicted with a tumor-prone syndrome where one or more peritoneal cell types or populations are predisposed to neoplastic conversion and formation of tumors as a result of an endogenous or exogenous neoplastic stimuli.

Suppression of tumorigenicity of rat liver tumor cells by human chromosome 13: evidence against the involvement of pRb and BRCA2.

Aberrations of chromosome 13, including large-scale deletions and rearrangements, have been implicated in the development of a significant fraction of human hepatocellular carcinomas, suggesting that liver tumor suppressor genes may be located on this chromosome. In this study, we have employed a microcell hybrid-based model system to investigate the presence of liver tumor suppressor loci on human chromosome 13. The parental GN6TF rat liver epithelial tumor cells are highly tumorigenic in vivo and exhibit altered cellular morphology and growth characteristics in vitro. The GN6TF cells form tumors in 100% of syngeneic animals with short latency, are not contact inhibited or anchorage-dependent in cell culture, and do not express mRNAs for rat Rb1 and BRCA2. Microcell-mediated introduction of human chromosome 13 into the rat liver tumor cell line GN6TF resulted in the generation of clonal microcell hybrid (MCH) cell lines that differentially exhibited tumor suppression and/or alteration of other transformation-associated phenotypes in vitro. Two GN6TF-13neo MCH lines exhibited characteristics indicative of suppression by the human chromosome, including a normalized cellular morphology and growth pattern, loss of anchorage-independent growth potential, partial restoration of contact inhibition, reduction in tumorigenic potential in vivo, and dramatic elongation of tumor latency. In contrast, three GN6TF-13neo MCH cell lines were minimally affected by the introduction of the human chromosome and were nearly indistinguishable from the parental GN6TF tumor cells, exhibiting a highly aggressive tumorigenic phenotype in vivo. Both suppressed and non-suppressed GN6TF-13neo MCH cell lines express Rb1 and BRCA2 mRNA in vitro, and tumors derived from the non-suppressed GN6TF-13neo MCH cell lines continue to express Rb1 and BRCA2 mRNA in vitro, and express pRb in vivo. The results suggest that: i) human chromosome 13 contains a liver tumor suppressor locus, ii) expression of Rb1 and/or BRCA2 is insufficient to produce tumor suppression in this rat liver tumor cell line, and iii) that the human chromosome 13 liver tumor suppressor may represent a novel tumor suppressor gene, distinct from Rb1 and BRCA2.

Discovery of a spontaneous genetic mouse model of preeclampsia.

Preeclampsia remains a leading cause of maternal and fetal morbidity and mortality but has an unknown etiology. Women with elevated baseline blood pressure have an increased risk of this disorder. We hypothesized that BPH/5 mice, an inbred mouse strain with mildly elevated blood pressure, would develop a pregnancy-induced hypertensive syndrome. Nonpregnant female BPH/5 and C57BL/6 mice underwent thoracic aortic implantation of telemeters. After 7 days of recovery and 5 days of baseline mean arterial blood pressure (MAP) recording, strain-matched timed matings were carried out. MAP was recorded continuously during pregnancy and for 1 week after birth. In separate mice in metabolic cages, urinary protein was tracked, followed by renal histological analysis. Before pregnancy, the BPH/5 strain had elevated baseline MAP compared with the C57BL/6 strain, but both strains had similar total urinary protein levels and renal histology. MAP remained stable in both groups during the first 2 weeks of pregnancy. However, at the start of the last trimester, MAP began to rise further in the BPH/5 mice; it rose to peak levels just before delivery and returned to prepregnancy levels by 2 days after delivery. This was accompanied by late-gestational proteinuria and progressive glomerulosclerosis. No changes were observed in the C57BL/6 group except for a small decrease in MAP at mid gestation. The BPH/5 group delivered significantly smaller litters despite normal numbers of fetuses early in gestation, and longitudinal ultrasound studies documented fetal demise before the onset of hypertension and renal disease. This is the first report of an animal model that spontaneously develops a syndrome that bears close resemblance to preeclampsia, and it should have an impact on our understanding of the pathophysiology of this disorder.