Degradation of cellular mRNA is a general early apoptosis-induced event.

The fate of cellular mRNAs was analyzed in several cell lines of lymphoid origin, after induction of apoptosis by different mechanisms. Cytoplasmic mRNAs are specifically degraded as part of the early apoptotic response. This degradation is not species restricted and is independent of the cell line, the apoptotic stimulus, the intrinsic half-life of the mRNAs, and the transcriptional status of the gene (constitutive or inducible). mRNA degradation precedes DNA fragmentation and correlates with the appearance of phosphatidylserine in the outer cell membrane. In addition, apoptosis-induced mRNA degradation is an active process that can be dissected from other apoptotic hallmarks (degradation of annexin V, DNA, and poly(ADP-ribose) polymerase [PARP]), which suggests that apoptosis-induced mRNA degradation is controlled by a distinct signaling pathway. Furthermore, mRNA degradation also occurs in vivo, specifically during thymocyte apoptosis. Taken together, these data support the notion that degradation of mRNA is a general early apoptotic event that may become a new apoptotic hallmark.

CCL2 inhibits the apoptosis program induced by growth factor deprivation, rescuing functional T cells.

The precise mechanisms involved in the switch between the clonal expansion and contraction phases of a CD8(+) T cell response remain to be fully elucidated. One of the mechanisms implicated in the contraction phase is cytokine deprivation, which triggers apoptosis in these cells. CCR2 chemokine receptor is up-regulated following IL-2 deprivation, and its ligand CCL2 plays an essential role preventing apoptosis induced by IL-2 withdrawal not only in CTLL2 cells, but also in mouse Ag-activated primary CD8(+) T cells because it rescued functional CD8(+) T cells from deprivation induced apoptosis, promoting proliferation in response to subsequent addition of IL-2 or to secondary antigenic challenges. Thus, up-regulation of the CCR2 upon growth factor withdrawal together with the protective effects of CCL2, represent a double-edged survival strategy, protecting cells from apoptosis and enabling them to migrate toward sites where Ag and/or growth factors are available.

Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments.

Using cell lines and primary cells, it has been shown that translation control plays a key role regulating gene expression during physiological and pathological conditions. The relevance of this type of regulation in vivo (tissues, organs) remains to be elucidated, due to the lack of an efficient method for polysome-bound fractionation of solid tissue RNA samples. A simple and efficient method is described, in which tissue samples were pulverized in liquid nitrogen and lysed with NP40-lysis buffer in the presence of the RNAse inhibitors RNAsin and vanadyl-ribonucleoside complex. After cell lysis, the cytoplasmic extract was loaded into sucrose gradients, fractionated, and RNA prepared from each fraction. The obtained RNA was reverse transcribed with a low efficiency, a problem that was overcome by purifying polyA+ RNA. Aiming to use small quantities of solid tissue samples (10-20 mg/sample), polyA+ RNA purification was discarded, and the different components were individually screened for a negative effect on reverse transcription. The polysaccharide heparin, which is present as a nonspecific RNAse inhibitor, inhibits reverse transcriptase activity, and must be removed from RNA samples for an efficient reaction. Heparin was successfully removed by precipitation of the RNA with lithium chloride, as demonstrated by the reversal of the inhibition on RT-PCR reactions. In summary, we present a reliable method allowing us to prepare high-quality polysome-bound mRNA from small quantities of liquid-nitrogen-frozen solid tissue samples from both human and mouse origin, amenable for Northern blotting, RT-PCR reactions, and expression profiling analyses.