In vitro analysis of ovarian cancer response to cisplatin, carboplatin, and paclitaxel identifies common pathways that are also associated with overall patient survival.

BACKGROUND: Carboplatin and cisplatin, alone or in combination with paclitaxel, have similar efficacies against ovarian cancer (OVCA) yet exhibit different toxicity profiles. We characterised the common and unique cellular pathways that underlie OVCA response to these drugs and analyse whether they have a role in OVCA survival. METHODS: Ovarian cancer cell lines (n=36) were treated with carboplatin, cisplatin, paclitaxel, or carboplatin-paclitaxel (CPTX). For each cell line, IC(50) levels were quantified and pre-treatment gene expression analyses were performed. Genes demonstrating expression/IC(50) correlations (measured by Pearson; P<0.01) were subjected to biological pathway analysis. An independent OVCA clinico-genomic data set (n=142) was evaluated for clinical features associated with represented pathways. RESULTS: Cell line sensitivity to carboplatin, cisplatin, paclitaxel, and CPTX was associated with the expression of 77, 68, 64, and 25 biological pathways (P<0.01), respectively. We found three common pathways when drug combinations were compared. Expression of one pathway ('Transcription/CREB pathway') was associated with OVCA overall survival. CONCLUSION: The identification of the Transcription/CREB pathway (associated with OVCA cell line platinum sensitivity and overall survival) could improve patient stratification for treatment with current therapies and the rational selection of future OVCA therapy agents targeted to these pathways.

Predicted amino acid sequences for 100 JCV strains.

DNA sequence variation between JCV genotypes is confined largely to noncoding intergenic regions and introns. Nevertheless, evidence suggests that the amino acid sequence variations among the 8 genotypes of JCV can influence the potential for neurovirulence of the virus. In the current study, the amino acid sequences for 100 JCV genomes were translated and grouped into genotype families. Subtype consensus sequences were determined and the type-specific amino acid sequence variants were identified.

Synthesis of type II interleukin-1 receptors by human corneal epithelial cells but not by keratocytes.

PURPOSE: The purpose of this study was to determine whether human corneal epithelial cells and keratocytes synthesize both the soluble and membrane forms of the type II IL-1 receptor (IL-1RII). METHODS: Primary cell cultures of human corneal epithelial cells and keratocytes were established from human corneas. RT-PCR was used to analyze cell cultures for expression of IL-1RII mRNA. The capacity of corneal cells to synthesize membrane-bound IL-1RII was determined by immunofluorescence microscopy, whereas ELISA was used to quantitate synthesis of soluble IL-1RII after IL-1alpha and TNF-alpha stimulation. RESULTS: Corneal epithelial cells expressed IL-1RII mRNA. The cells also stained positive for membrane-bound IL-1RII, and media harvested from epithelial cell cultures contained up to 50 pg/ml of soluble IL-1RII. Both IL-1alpha and TNF-alpha significantly enhanced the amounts of soluble IL-1RII released from epithelial cell surfaces. In contrast to epithelial cells, corneal keratocytes did not express IL-1RII mRNA. Membrane-bound IL-1RII was not detected on keratocytes, nor was soluble IL-1RII detected in culture media harvested from these cells. CONCLUSIONS: Human corneal epithelial cells but not corneal keratocytes synthesize both membrane and soluble forms of IL-1RII. Because both forms of IL-1RII can function as IL-1alpha antagonists, the results suggest that human corneal epithelial cells but not corneal keratocytes have evolved the capacity to dampen IL-1alpha responses through the production of IL-1RII.

Human corneal cells and other fibroblasts can stimulate the appearance of herpes simplex virus from quiescently infected PC12 cells.

A two-cell system for the stimulation of herpes simplex virus type 1 (HSV-1) from an in vitro model of long-term (quiescent) infection is described. Rat pheochromocytoma (PC12) cells differentiated with nerve growth factor were infected with HSV-1 strain 17. Little, if any, cytotoxicity was observed, and a quiescent infection was established. The long-term infection was characterized by the absence of all detectable virus in the culture medium and little, if any, detectable early or late viral-gene expression as determined by reverse transcriptase PCR analysis. The presence of HSV-1 DNA was determined by PCR analysis. This showed that approximately 180 viral genomes were present in limiting dilutions where as few as 16 cells were examined. The viral DNA was infectious, since cocultivation with human corneal fibroblasts (HCF) or human corneal epithelial cells (HCE) resulted in recovery of virus from most, if not all, clusters of PC12 cells. Following cocultivation, viral antigens appeared first on PC12 cells and then on neighboring inducing cells, as determined by immunofluorescent staining, demonstrating that de novo viral protein synthesis first occurred in the long-term-infected PC12 cells. Interestingly, the ability to induce HSV varied among the cell lines tested. For example, monkey kidney CV-1 cells and human hepatoblastoma HepG2 cells, but not mouse neuroblastoma cells or undifferentiated PC12 cells, mediated stimulation. This work thus shows that (i) quiescent HSV infections can be maintained in PC12 cells in vitro, (ii) HSV can be induced from cells which do not accumulate significant levels of latency-associated transcripts, and (iii) the activation of HSV gene expression can be induced via neighboring cells. The ability of adjacent cells to stimulate HSV gene expression in neuron-like cells represents a novel area of study. The mechanism(s) whereby HCF, HCE, and HepG2 and CV-1 cells communicate with PC12 cells and stimulate viral replication, as well as how this system compares with other in vitro models of long-term infection, is discussed.

Differential induction of GRO alpha gene expression in human corneal epithelial cells and keratocytes exposed to proinflammatory cytokines.

PURPOSE: GRO alpha is an inducible neutrophil chemotactic factor synthesized in inflamed corneal tissues. In this study, the regulation of GRO alpha synthesis after exposure of human corneal cells to proinflammatory cytokines was investigated. METHODS: Pure cultures of human corneal epithelial cells and keratocytes were exposed to increasing concentrations of either interleukin-1 alpha (IL-1 alpha) or tumor-necrosis factor-alpha (TNF-alpha). At selected times postexposure, the amounts of GRO alpha produced by the cultures were quantitated by enzyme-linked immunosorbent assay. The RNA was harvested from stimulated cultures to monitor GRO alpha mRNA and pre-mRNA levels by the reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Both IL-1 alpha and TNF-alpha induced significant levels of GRO alpha synthesis in keratocytes. However, IL-1 alpha-stimulated cells produced > 10 times more GRO alpha protein and 12 times more GRO alpha mRNA than did TNF-alpha-stimulated keratocytes. In contrast to the log differences in mRNA levels and protein synthesis, there was a less than twofold difference between IL-1 alpha and TNF-alpha in their capacity to induce GRO alpha specific pre-mRNA synthesis. When actinomycin D was added to stimulated keratocytes to inhibit transcription, the half-life of GRO alpha mRNA in TNF-alpha-treated cells was < 1 hour, whereas the half-life of the GRO alpha mRNA synthesized in IL-1 alpha-stimulated cells was > or = 9 hours. In contrast to keratocytes, exposure of corneal epithelial cells to IL-1 alpha and TNF-alpha did not induce significant increases in steady-state levels of GRO alpha pre-mRNA, mRNA, or protein. CONCLUSIONS: The results suggest that keratocytes are the major producers of GRO alpha in the human cornea. The capacity of IL-1 alpha to stimulate the synthesis of significantly higher quantities of GRO alpha in keratocytes compared to TNF-alpha is caused by the fact that GRO alpha-specific transcripts are more than nine times more stable in cells exposed to IL-1 alpha than in cells exposed to TNF-alpha.

Differences in interleukin-6 gene expression between cultured human corneal epithelial cells and keratocytes.

PURPOSE: To determine whether interleukin-6 (IL-6) can be synthesized by human corneal keratocytes and epithelial cells. METHODS: Epithelial cells and keratocytes isolated from the same donor corneas were grown in vitro. After 2 to 3 passages, the cultures were exposed to varying concentrations of recombinant human interleukin-1 (IL-1 alpha) or tumor necrosis factor (TNF-alpha). Culture supernatants subsequently underwent enzyme-linked immunosorbent assays for cytokine content. The levels of cytokine mRNA in cell lysates were monitored by the reverse transcription-polymerase chain reaction. RESULTS: Cultured human keratocytes stimulated with 100 U/ml IL-1 alpha for 18 hours produced more than 160 ng IL-6 per 10(6) cells. Under the same conditions 500 U/ml TNF-alpha induced approximately 5 ng IL-6. IL-6 mRNA, evident by 3 hours after exposure to either cytokine, accumulated and persisted through 18 hours. Exposure of epithelial cells to IL-1 alpha or TNF-alpha induced minimal and transient expression of IL-6 mRNA and < 0.5 ng protein product per 10(6) cells. The poor production of IL-6 did not reflect an inability of epithelial cells to respond to IL-1 alpha and TNF-alpha because both cytokines induced these cells to make copious amounts of IL-8. CONCLUSIONS: These results demonstrate that both IL-1 alpha and TNF-alpha could induce keratocytes to produce nanogram levels of IL-6 but IL-1 alpha was a 30-fold more effective inducer. In contrast, neither cytokine could stimulate epithelial cells to make more than picogram quantities of IL-6. The abundant IL-6 synthesized by keratocytes may promote various activities including specific immune responses in surrounding lymphoid tissues.

Differential regulation of granulocyte-macrophage colony-stimulating factor gene expression in human corneal cells by pro-inflammatory cytokines.

Neutrophils and Langerhans cells participate in inflammatory reactions within the human cornea. Because granulocyte-macrophage (GM)-CSF is a chemotactic and activating factor for these two cell types, we investigated whether this cytokine is produced by human corneal epithelial cells and corneal fibroblasts. Cultures of each cell type were exposed to increasing concentrations of IL-1 alpha or TNF-alpha. Culture supernatants were assayed for GM-CSF by using ELISA and cytokine mRNA levels were monitored by using reverse transcriptase-PCR. IL-1 alpha treatment of both cell types resulted in the appearance of GM-CSF mRNA and the production of > 480 pg protein/10(6) cells. However, TNF-alpha treatment yielded divergent results. Stimulation of epithelial cells with TNF-alpha resulted in the appearance of > 560 GM-CSF mRNA molecules per cell and production of > 1300 pg GM-CSF/10(6) cells. In contrast, stimulation of corneal fibroblasts resulted in < 16 GM-CSF mRNA molecules/cell and < 60 pg GM-CSF/10(6) cells. Binding studies with 125I-labeled TNF-alpha revealed that corneal fibroblasts had as many receptor sites as did corneal epithelial cells. Furthermore, corneal fibroblasts could respond to TNF-alpha-receptor-mediated signal transduction because they produced nanogram amounts of IL-6 after being treated with this cytokine. The results suggest that both cell types synthesize GM-CSF in response to IL-1 alpha, but that only corneal epithelial cells produce significant amounts of GM-CSF after TNF-alpha exposure. Differences in the responses of the two cell types to TNF-alpha may reflect a means of limiting accumulation of neutrophils and Langerhans cells and, thus, minimize corneal damage.

IL-8 gene expression in cultures of human corneal epithelial cells and keratocytes.

PURPOSE: To determine if human corneal keratocytes and epithelial cells synthesize and release IL-8 after stimulation with selected proinflammatory cytokines. METHODS: Human corneal keratocytes and epithelial cells were isolated from human corneal buttons and grown independently in vitro. Epithelial cell cultures stained positive in immunofluorescent tests with antibody specific for keratin (AE1/AE3), whereas keratocyte cultures were unreactive. Both cell types reacted with anti-vimentin antibody. Cultures of the two cell types were treated with various concentrations of human recombinant interleukin-1 alpha or TNF-alpha. Culture supernatants were then assayed at timed intervals by enzyme-linked immunosorbent assay for IL-8 content. Cytokine mRNA levels in cell lysates were monitored by Northern blot analysis. RESULTS: Exposure of corneal keratocytes and epithelial cells to either interleukin-1 alpha or TNF-alpha stimulated IL-8 mRNA synthesis and IL-8 production in a dose-response fashion. It was also found that TNF-alpha stimulated the synthesis of comparable amounts of IL-8 in both cell types. However, when IL-8 synthesis between the two cell types was compared after interleukin-1 alpha stimulation it was found that keratocytes synthesized 33 times more IL-8 than did epithelial cells. CONCLUSIONS: The results establish that pro-inflammatory cytokines can induce IL-8 synthesis in both human corneal epithelial cells and human corneal keratocytes. They also suggest that interleukin-1 alpha may play a more active role in amplifying inflammatory responses in the stroma than in the epithelial layer of the cornea.

Induction of interleukin-8 gene expression is associated with herpes simplex virus infection of human corneal keratocytes but not human corneal epithelial cells.

Interleukin-8 (IL-8) is a proinflammatory cytokine released at sites of tissue damage by various cell types. One important function of IL-8 is to recruit neutrophils into sites of inflammation and to activate their biological activity. Stromal keratitis induced by herpes simplex virus type 1 (HSV-1) is characterized by an initial infiltration of neutrophils. This study was carried out to determine whether cells resident in the cornea synthesize IL-8 after virus infection. Pure cultures of epithelial cells and keratocytes established from human corneas were infected with HSV-1, and the medium overlying the cells was subsequently assayed for IL-8 by an enzyme-linked immunosorbent assay. Cytokine mRNA levels in cell lysates were monitored by Northern (RNA) blot analysis. It was found that virus infection of keratocyte cultures led to the synthesis of IL-8-specific mRNA with more than 30 ng of IL-8 made per 10(6) cells. Neither UV-inactivated virus nor virus-free filtrates collected from HSV-1-infected keratocytes could induce IL-8 protein or mRNA, suggesting that viral gene expression was needed for induction of IL-8 gene expression. Unlike keratocytes, HSV-1-infected epithelial cells failed to synthesize IL-8 protein or mRNA. However, these cells readily produced both molecules following tumor necrosis factor alpha stimulation. HSV-1 had similar titers in both cell types. Thus, the failure to induce IL-8 synthesis was not due to an inability of the virus to replicate in epithelial cells. The capacity of HSV-1-infected corneal keratocytes to synthesize IL-8 suggests that these cells can contribute to the induction of the acute inflammatory response seen in herpes stromal keratitis.

Starvation-inducible loci of Salmonella typhimurium: regulation and roles in starvation-survival.

Four starvation-inducible loci (stiA, stiB, stiC, and stiE) of Salmonella typhimurium have been extensively characterized as to their genetic and physiologic regulation, and their roles in survival during prolonged simultaneous phosphate (P)-, carbon (C)- and nitrogen (N)-starvation (PCN-starvation). Strains of S. typhimurium LT-2, isogenic with the exception of lacking either the stiA, stiB or stiC locus, died off more quickly and survived at much reduced levels compared with their wild-type parent. When certain sti mutations were combined in the same strain, we found that viability of these cultures declined even more rapidly, and starvation-survival was affected to levels over-and-above the additive effects of each individual mutation, indicating an epistatic relationship between these loci. All four sti loci were, directly or indirectly, under negative control by the crp gene product (cAMP receptor protein, CRP). With the exception of stiB, all were similarly regulated by the cya gene product (i.e., cAMP). This suggests that CRP acts alone, or with a signal molecule other than cAMP, to cause repression of the stiB locus. In addition, all four loci are under positive regulation by the relA gene product (i.e., ppGpp) during C- or N-starvation, but not P-starvation. Since not all relA-dependent sti loci are induced during both C- and N-starvation, we propose that two separate ppGpp-dependent pathways function during C-starvation and N-starvation, respectively. Possible models for separate P-, C- and N-starvation-induction pathways are discussed.