Early gene response in lithium chloride induced apoptosis.

Depending on the cellular context, lithium chloride can lead to enhanced proliferation, cell cycle arrest or apoptosis in mammalian cells. Although substantial work has been made to elucidate the downstream events in the case of lithium chloride-induced cellular proliferation, the molecular response to lithium chloride treatment in the apoptotic scenario is largely undefined. We have used quadruplicate human cDNA arrays with 8000 targets to analyze the early gene response in cultures of human T/C28a cells that undergo apoptosis in response to 20 mM lithium chloride treatment. Incubation of cell cultures with 20 mM lithium chloride for five hours caused alterations in the steady-state mRNA levels of a large number of genes. RT-PCR and real-time RT-PCR confirmed the array results for ten of eleven selected targets. In addition to one protein primarily associated with apoptosis, genes identified as differentially expressed based on microarray data mainly encode proteins involved in basic cellular functions such as signaling, cell cycle control and growth, cell-cell interaction, solute transport and transcription control. We present a list of 50 genes that were differentially expressed in response to lithium chloride treatment and which may represent a reference for further studies to define the pathways governing the apoptotic response to lithium chloride.

Mathematical considerations of competitive polymerase chain reaction.

Reverse transcriptase polymerase chain reaction (PCR) is used frequently to monitor gene expression. It is generally regarded as a qualitative technique, although refinements have been made to improve quantification. The object of this study was to develop competitive PCRs to allow reliable quantification of the rat T cell cytokines interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and interleukin-4 (IL-4). Truncated constructs of cDNA for these cytokines were prepared using appropriate pairs of standard and specially constructed primers designed to allow subsequent co-amplification of the purified competitor construct and the target cDNA. A high resolution capillary electrophoresis (CE) system was used for PCR product detection. The performance of the system was compared with a mathematical model that describes and predicts the exponential nature of the PCR reaction. Co-amplification of the competitor and target were achieved. A high level of resolution and accuracy was achieved using CE to detect and quantify the PCR products. The rates of generation of the respective products conformed closely but not exactly to the predictions of the mathematical model. The competitive PCRs estimated initial numbers of target cDNA within 1.1-5.0-fold relative to the amount of starting material as assessed by conventional spectrophotometric absorbance prior to dilution and amplification. A convenient and flexible competitive PCR strategy has been developed with accurate resolution of products and reliable quantification. Assay variability was far less than biological variability likely to be encountered in experiments investigating immunological responses in rats or other animals.