Identification of temsirolimus exposures required to inhibit total protein synthesis.

<p>(<b>A</b>) Cells were pulse labeled with ³⁵S-methionine/cysteine and analyzed by scintillation counting for incorporated radioactivity normalized to vehicle treated control (top panel) or SDS-PAGE (lower panel). Experiments were performed in duplicate or triplicate and mean values shown +/- standard deviation. One-way ANOVA was performed for statistical analysis and asterisk denotes <i>p</i> = <0.05 for treatment condition compared to vehicle treated control. (<b>B</b>) Human rhabdomyosarcoma cells (Rh30) were treated with 2μM or 20μM temsirolimus for 1.5hrs. Cell lysates were then layered onto 15–45% linear sucrose gradients for polysome analysis. Samples were fractionated and UV absorbance was measured in real-time. The location of 40S, 60S ribosomal subunits as well as 80S ribosome monomers and larger molecular weight polysomes are indicated. (<b>C</b>) Human 143B osteosarcoma cells were treated with vehicle, 20nM or 20μM temsirolimus for the indicated times. Protein lysates were analyzed by Western blot for levels of phospho and total protein for eIF2 alpha, eEF2 and 4EBP1. (<b>D</b>) The same as in (A) using human osteosarcoma cells (HOS and 143B) as well as human rhabdomyosarcoma cells (RD) treated with vehicle, 20nM or 20μM rapamycin for 1hr. Three independent experiments were performed for each treatment condition.</p

Decreased solvent accessibility of 28S rRNA in ribosomes isolated from cells treated with temsirolimus.

<p>(<b>A</b>) Polysome profiles (upper panel) and Western blot analysis of mTOR and FKBP12 protein levels (middle panel) in fractions following sucrose density gradient fractionation. Temsirolimus levels were quantified by HPLC-MS-MS analysis from pooled fractions as indicated in the text. <b>(B</b>) Alignment of X-ray crystal structures from <i>D</i>. <i>radiodurans</i> complexed with rapamycin (PDB 1Z58) and <i>S</i>. <i>cerevisiae</i> (PDB 3U5D) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185089#pone.0185089.ref052" target="_blank">52</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185089#pone.0185089.ref054" target="_blank">54</a>]. (<b>C</b>) Secondary structure model for <i>S</i>. <i>cerevisiae</i> large subunit domains II, IV and V, adapted from the Comparative RNA Website [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185089#pone.0185089.ref055" target="_blank">55</a>]. Arrow denotes location of the entrance to the peptide exit tunnel and the location of nucleotides in (B) are labeled. (<b>D</b>) Protection of 28S rRNA from chemical modification in temsirolimus treated samples. Primer extension analysis of human 28S rRNA from ribosomes isolated from temsirolimus treated or vehicle treated human rhabdomyosarcoma cells. Purified ribosomes were treated with DMS (modifies accessible adenosine and cytosines), CMCT (modifies accessible uracil and guanines) or mock treated. RNA was then purified, quantitated and equal amounts were used as the template for fluorescent primer extension analysis. Capillary electrophoresis was used to separate and resolve products under conditions allowing for single nucleotide resolution along with a DNA size standard. Arrows and shaded peaks denote the position of a DMS or CMCT modifiable ribonucleotide which is protected from modification in temsirolimus treated samples. An asterisk (*) denotes a chemical independent stop in some samples. The human nucleotide sequence appears below the electropherogram with <i>D</i>. <i>radiodurans</i> (DR), <i>E</i>. <i>coli</i> (EC), <i>S</i>. <i>cerevisiae</i> (SC) and <i>H</i>. <i>sapiens</i> (HU) numbering to denote the position of interest. Nucleotide positions were adjusted (+1) to account for the fact that DMS and CMCT modification results in a stop one nucleotide before the modified base.</p

Pharmacologically relevant micromolar doses of temsirolimus provide maximal tumor cell growth inhibition.

<p>(<b>A</b>) Human rhabdomyosarcoma (Rh30, RD) and osteosarcoma (143B, HOS-MNNG) cells were treated for 48hrs with the indicated doses of temsirolimus followed by staining with sulforhodamine B. Growth is represented as percentage of vehicle treated control. Each cell line was assayed at least three times and mean values for each data point are shown. (<b>B</b>) Cell cycle analysis of asynchronous cells following treatment with temsirolimus at different doses. Human osteosarcoma cells were treated for the indicated times with vehicle, 20nM, 2μM or 20μM temsirolimus followed by FACS analysis of propidium iodide stained cells to determine the distribution of cells within each phase of the cell cycle. (<b>C</b>) Treatment with FK-506 rescues low dose growth inhibitory effects of temsirolimus, but not high dose effects. Human osteosarcoma cells were treated with the indicated doses of temsirolimus alone or in combination with FK-506. Both drugs bind the intracellular protein FKBP12, but only temsirolimus/FKBP12 complex can bind to mTOR. Treatment was for 48hrs. Cell growth is expressed relative to FK-506 alone and assayed by staining with sulforhodomine B. Each set of conditions were analyzed in three separate experiments and mean values are shown.</p

Rapamycin and temsirolimus induce Xbp-1 splicing and downstream target gene mRNA levels.

<p><b>(A)</b> Human osteosarcoma cells (HOS-MNNG) were treated for the indicated times with 2, 5 or 20μM temsirolimus. RT-PCR was conducted using a primer pair flanking the unconventional splice site of the Xbp-1 mRNA. (<b>B</b>) Quantitative RT-PCR was conducted using primer pairs specific for the Xbp-1 target genes, Grp78 and CHOP. Expression values for each transcript were normalized to 18S and vehicle treated control. Experiments were carried out on three independent biological replicates, assayed in triplicate. One-way ANOVA was performed for statistical analysis and asterisk denotes <i>p</i> = <0.05. (<b>C</b>) 143B human osteosarcoma cells were treated with vehicle or 20μM rapamycin and analyzed for Xbp1 splicing (upper panel) and downstream target gene activation of BiP/Grp78 and CHOP at the indicated time points as in (A and B). (<b>D</b>) Cells were treated for the indicated times with either 20μM temsirolimus or an ATP competitive inhibitor of mTOR. Total RNA was harvested and Xbp-1 splicing was analyzed by RT-PCR as in (A).</p