An RNA aptamer with high affinity and broad specificity for zinc finger proteins.

A class of RNA aptamers that demonstrates a high affinity for a large variety of C(2)H(2) zinc finger proteins was isolated from a library of random RNA sequences by the zinc finger protein TFIIIA. These aptamers have one or more copies of the consensus sequence GGGUGGG, which is part of a putative hairpin loop in the proposed structure of the most abundant aptamer, RNA1. Binding of zinc finger proteins to RNA1 relies upon zinc-dependent folding of the protein, the affinity of an individual protein for RNA1 being determined by the number of tandem zinc finger motifs. The properties of RNA1 were compared to the properties of two other aptamers from the same selection experiment: RNA21, which binds to some but not all zinc finger proteins tested, and RNA22, which binds only to the 5S rRNA binding zinc finger proteins TFIIIA and p43. The binding of three different zinc finger proteins to RNA1 was compared, and the results indicate that the RNA1-protein interaction occurs by several distinct mechanisms. Mutagenesis of RNA1 confirmed that the GGGUGGG consensus sequence presented in a hairpin conformation is required for high-affinity binding of zinc finger proteins.

An RNA aptamer with high affinity and high specificity for the 5S RNA binding zinc finger proteins TFIIIA and p43.

The Xenopus zinc finger proteins TFIIIA and p43 bind to 5S RNA in immature oocytes to form 7S and 42S ribonucleoprotein storage particles. To probe the similarities and differences in the RNA binding domains of these two proteins, a library of random RNA molecules was enriched using TFIIIA as the bait protein. One of the abundant aptamers isolated, RNA22, bound to both TFIIIA and p43 derived zinc finger peptides with high affinity and specificity even though the predicted secondary structure of the RNA was unrelated to that of 5S RNA. The interactions of TFIIIA and p43 peptides with RNA22 were compared to their interactions with 5S RNA by characterizing the effects of assay conditions, mutations in RNA22, and mutations in the zinc finger proteins. The similarities and differences in the mechanisms by which these two zinc finger proteins interact with 5S RNA compared to RNA22 suggest they share a common platform for RNA binding with enough flexibility to form specific interactions with both RNAs.

Radiation enhances the invasive potential of primary glioblastoma cells via activation of the Rho signaling pathway.

Glioblastoma multiforme (GBM) is among the most treatment-refractory of all human tumors. Radiation is effective at prolonging survival of GBM patients; however, the vast majority of GBM patients demonstrate progression at or near the site of original treatment. We have identified primary GBM cell lines that demonstrate increased invasive potential upon radiation exposure. As this represents a novel mechanism by which radiation-treated GBMs can fail therapy, we further investigated the identity of downstream signaling molecules that enhance the invasive phenotype of irradiated GBMs. Matrigel matrices were used to compare the extent of invasion of irradiated vs. non-irradiated GBM cell lines UN3 and GM2. The in vitro invasive potential of these irradiated cells were characterized in the presence of both pharmacologic and dominant negative inhibitors of extracellular matrix and cell signaling molecules including MMP, uPA, IGFR, EGFR, PI-3K, AKT, and Rho kinase. The effect of radiation on the expression of these signaling molecules was determined with Western blot assays. Ultimately, the in vitro tumor invasion results were confirmed using an in vivo 9L GBM model in rats. Using the primary GBM cell lines UN3 and GM2, we found that radiation enhances the invasive potential of these cells via activation of EGFR and IGFR1. Our findings suggest that activation of Rho signaling via PI-3K is required for radiation-induced invasion, although not required for invasion under physiologic conditions. This report clearly demonstrates that radiation-mediated invasion is fundamentally distinct from invasion under normal cellular physiology and identifies potential therapeutic targets to overcome this phenomenon.

Survivin enhances radiation resistance in primary human glioblastoma cells via caspase-independent mechanisms.

The observed radioresistance of human glioblastoma multiforme (GBM) poses a major challenge, which, if overcome, may lead to significant advances in the management of this patient population. There is accumulating evidence from correlative studies that Survivin expression is associated with increased malignant potential of human gliomas. The purpose of this study was to investigate whether Survivin plays a direct role in mediating radiation resistance in primary human glioma cell lines, and, if so, investigating the underlying mechanisms. Our panel of GBM cell lines included two that were relatively radiation resistant (GM20 and GM21) and two that were more radiation sensitive (GM22 and GM23), which demonstrated differential levels of Survivin expression between the two groups. Through the use of adenoviral vectors containing either dominant-negative (pAd-S(T34A)) or wild-type Suvrivin (pAd-S(WT)), we were able to inactivate or overexpress Survivin, respectively. Our findings suggest that Survivin plays a critical role in mediating radiation resistance in primary GBM cells, in part through suppression of apoptotic cell death via a caspase-independent manner. We have identified novel mechanisms by which Survivin may enhance tumor cell survival upon radiation exposure such as regulation of double-strand DNA break repair and tumor cell metabolism, which were most evident in the radiation-resistant cell lines. These differences in Survivin function both in radiation-resistant vs radiation-sensitive cell lines and in the presence vs absence of radiation exposure warrant further investigation and highlight potentially important mechanisms of radiation resistance in these tumors.