Oxidative stress-induced phospholipase C-gamma 1 activation enhances cell survival.

Phospholipase C-gamma1 (PLC-gamma1) is rapidly activated in response to growth factor stimulation and plays an important role in regulating cell proliferation and differentiation through the generation of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C (PKC) and increased levels of intracellular calcium, respectively. Given the existing overlap between signaling pathways that are activated in response to oxidant injury and those involved in responding to proliferative stimuli, we investigated the role of PLC-gamma1 during the cellular response to oxidative stress. Treatment of normal mouse embryonic fibroblasts (MEF) with H2O2 resulted in time- and concentration-dependent tyrosine phosphorylation of PLC-gamma1. Phosphorylation could be blocked by pharmacological inhibitors of Src family tyrosine kinases or the epidermal growth factor receptor tyrosine kinase, but not by inhibitors of the platelet-derived growth factor receptor or phosphatidylinositol 3-kinase. To investigate the physiologic relevance of H2O2-induced tyrosine phosphorylation of PLC-gamma1, we compared survival of normal MEF and PLC-gamma1-deficient MEF following exposure to H2O2. Treatment of PLC-gamma1-deficient MEF with H2O2 resulted in rapid cell death, whereas normal MEF were resistant to the stress. Pretreatment of normal MEF with a selective pharmacological inhibitor of PLC-gamma1, or inhibitors of inositol trisphosphate receptors and PKC, increased their sensitivity to H2O2, whereas treatment of PLC-gamma1-deficient MEF with agents capable of directly activating PKC and enhancing calcium mobilization significantly improved their survival. Finally, reconstitution of PLC-gamma1 protein expression in PLC-gamma1-deficient MEF restored cell survival following H2O2 treatment. These findings suggest an important protective function for PLC-gamma1 activation during the cellular response to oxidative stress.

Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state.

gadd153, also known as chop, is a highly stress-inducible gene that is robustly expressed following disruption of homeostasis in the endoplasmic reticulum (ER) (so-called ER stress). Although all reported types of ER stress induce expression of Gadd153, its role in the stress response has remained largely undefined. Several studies have correlated Gadd153 expression with cell death, but a mechanistic link between Gadd153 and apoptosis has never been demonstrated. To address this issue we employed a cell model system in which Gadd153 is constitutively overexpressed, as well as two cell lines in which Gadd153 expression is conditional. In all cell lines, overexpression of Gadd153 sensitized cells to ER stress. Investigation of the mechanisms contributing to this effect revealed that elevated Gadd153 expression results in the down-regulation of Bcl2 expression, depletion of cellular glutathione, and exaggerated production of reactive oxygen species. Restoration of Bcl2 expression in Gadd153-overexpressing cells led to replenishment of glutathione and a reduction in levels of reactive oxygen species, and it protected cells from ER stress-induced cell death. We conclude that Gadd153 sensitizes cells to ER stress through mechanisms that involve down-regulation of Bcl2 and enhanced oxidant injury.

Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival.

The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. Here we investigated the role of PI3-K/Akt during the cellular response to oxidant injury. H(2)O(2) treatment elevated Akt activity in multiple cell types in a time- (5-30 min) and dose (400 microM-2 mm)-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevented H(2)O(2)-induced Akt activation. Akt activation by H(2)O(2) also depended on epidermal growth factor receptor (EGFR) signaling; H(2)O(2) treatment led to EGFR phosphorylation, and inhibition of EGFR activation prevented Akt activation by H(2)O(2). As H(2)O(2) causes apoptosis of HeLa cells, we investigated whether alterations of PI3-K/Akt signaling would affect this response. Wortmannin and LY294002 treatment significantly enhanced H(2)O(2)-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibited cell death. Constitutive expression of v-Akt likewise enhanced survival of H(2)O(2)-treated NIH3T3 cells. These results suggest that H(2)O(2) activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis.

Induction of rat WT1 gene expression correlates with human chromosome 11p11.2-p12-mediated suppression of tumorigenicity in rat liver epithelial tumor cell lines.

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.

Plasticity of the neoplastic phenotype in vivo is regulated by epigenetic factors.

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.

Evaluation of the differentiation potential of WB-F344 rat liver epithelial stem-like cells in vivo. Differentiation to hepatocytes after transplantation into dipeptidylpeptidase-IV-deficient rat liver.

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.

Localization of a putative liver tumor suppressor locus to a 950-kb region of human 11p11.2-p12 using rat liver tumor microcell hybrid cell lines.

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.

Age-dependent induction of hepatic tumor regression by the tissue microenvironment after transplantation of neoplastically transformed rat liver epithelial cells into the liver.

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.

Suppression of the tumorigenic phenotype of a rat liver epithelial tumor cell line by the p11.2-p12 region of human chromosome 11.

Comparative chromosomal mapping studies and investigations of tumor-associated chromosomal abnormalities suggest that the development of hepatic tumors in humans and rats may share a common molecular mechanism that involves inactivation of the same tumor suppressor genes or common genetic loci. We investigated the potential of human chromosomes 2 and 11 to suppress the tumorigenic phenotype of rat liver epithelial tumor cell lines. These tumor cell lines (GN6TF and GP7TB) display elevated saturation densities in culture, efficiently form colonies in soft agar, and produce subcutaneous tumors in 100% of syngeneic rat hosts with short latency periods. Introduction of human chromosome 11 by microcell fusion markedly altered the tumorigenicity and the transformed phenotype of GN6TF cells. In contrast, the tumorigenic potential and phenotype of GP7TB cells was unaffected by the introduction of human chromosome 11, indicating that not all rat liver tumor cell lines can be suppressed by loci carried on this chromosome. Introduction of human chromosome 2 had little or no effect on the tumorigenicity or cellular phenotype of either tumor cell line, suggesting the involvement of chromosome 11-specific loci in the suppression of the GN6TF tumor cell line. The GN6TF-11neo microcell hybrid cell lines displayed significantly reduced saturation densities in monolayer cultures, and their ability to grow in soft agar was completely inhibited. Although GN6TF-11neo cells ultimately formed tumors in 80-100% of syngeneic rat hosts, the latency period for tumor formation was much longer. Molecular characterization of GN6TF-11neo microcell hybrid cell lines indicated that some of the clonal lines had spontaneously lost significant portions of the introduced human chromosome, partially delineating the chromosomal location of the putative tumor suppressor locus to the region between the centromere and 11p12. Molecular examination of microcell hybrid-derived tumor cell lines further defined the minimal portion of human chromosome 11 capable of tumor suppression in this model system to the region 11p11.2-p12.

Age-dependent regulation of the tumorigenic potential of neoplastically transformed rat liver epithelial cells by the liver microenvironment.

Neoplastically transformed rat liver epithelial cell lines (GN6TF and GP7TB), which form tumors with short latency at s.c. or i.p. transplantation sites of syngeneic rats, did not form tumors or were weakly tumorigenic following transplantation into the livers of young adult rats and expressed increasing tumorigenicity in livers of increasingly aged rats. These results suggest that progressive alterations in the hepatic parenchyma with increasing age enabled tumor formation by providing a less suppressive microenvironment for expression of the tumorigenic phenotype. Age is widely recognized as a significant risk factor in the development of neoplasia; this study describes a model for investigation of the influence of age-dependent changes in the hepatic microenvironment on the development of hepatic cancer.