Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>The short arm of human chromosome 3 is involved in the development of many cancers including lung cancer. Three bona fide lung cancer tumor suppressor genes namely <it>RBSP3 </it>(AP20 region),<it>NPRL2 </it>and <it>RASSF1A </it>(LUCA region) were identified in the 3p21.3 region. We have shown previously that homozygous deletions in AP20 and LUCA sub-regions often occurred in the same tumor (P < 10^-6).</p> <p>Methods</p> <p>We estimated the quantity of <it>RBSP3, NPRL2, RASSF1A, GAPDH, RPN1 </it>mRNA and <it>RBSP3 </it>DNA copy number in 59 primary non-small cell lung cancers, including 41 squamous cell and 18 adenocarcinomas by real-time reverse transcription-polymerase chain reaction based on TaqMan technology and relative quantification.</p> <p>Results</p> <p>We evaluated the relationship between mRNA level and clinicopathologic characteristics in non-small cell lung cancer. A significant expression decrease (≥2) was found for all three genes early in tumor development: in 85% of cases for <it>RBSP3</it>; 73% for <it>NPRL2 </it>and 67% for <it>RASSF1A </it>(P < 0.001), more strongly pronounced in squamous cell than in adenocarcinomas. Strong suppression of both, <it>NPRL2 </it>and <it>RBSP3 </it>was seen in 100% of cases already at Stage I of squamous cell carcinomas. Deregulation of <it>RASSF1A </it>correlated with tumor progression of squamous cell (P = 0.196) and adenocarcinomas (P < 0.05). Most likely, genetic and epigenetic mechanisms might be responsible for transcriptional inactivation of <it>RBSP3 </it>in non-small cell lung cancers as promoter methylation of <it>RBSP3 </it>according to NotI microarrays data was detected in 80% of squamous cell and in 38% of adenocarcinomas. With NotI microarrays we tested how often LUCA (<it>NPRL2, RASSF1A</it>) and AP20 (<it>RBSP3</it>) regions were deleted or methylated in the same tumor sample and found that this occured in 39% of all studied samples (P < 0.05).</p> <p>Conclusion</p> <p>Our data support the hypothesis that these TSG are involved in tumorigenesis of NSCLC. Both genetic and epigenetic mechanisms contribute to down-regulation of these three genes representing two tumor suppressor clusters in 3p21.3. Most importantly expression of <it>RBSP3, NPRL2 </it>and <it>RASSF1A </it>was simultaneously decreased in the same sample of primary NSCLC: in 39% of cases all these three genes showed reduced expression (P < 0.05).</p

Differential Expression of CHL1 Gene during Development of Major Human Cancers

CHL1 gene (also known as CALL) on 3p26.3 encodes a one-pass trans-membrane cell adhesion molecule (CAM). Previously CAMs of this type, including L1, were shown to be involved in cancer growth and metastasis.We used Clontech Cancer Profiling Arrays (19 different types of cancers, 395 samples) to analyze expression of the CHL1 gene. The results were further validated by RT-qPCR for breast, renal and lung cancer. Cancer Profiling Arrays revealed differential expression of the gene: down-regulation/silencing in a majority of primary tumors and up-regulation associated with invasive/metastatic growth. Frequent down-regulation (>40% of cases) was detected in 11 types of cancer (breast, kidney, rectum, colon, thyroid, stomach, skin, small intestine, bladder, vulva and pancreatic cancer) and frequent up-regulation (>40% of cases)--in 5 types (lung, ovary, uterus, liver and trachea) of cancer. Using real-time quantitative PCR (RT-qPCR) we found that CHL1 expression was decreased in 61% of breast, 60% of lung, 87% of clear cell and 89% papillary renal cancer specimens (P<0.03 for all the cases). There was a higher frequency of CHL1 mRNA decrease in lung squamous cell carcinoma compared to adenocarcinoma (81% vs. 38%, P = 0.02) without association with tumor progression.Our results suggested that CHL1 is involved in the development of different human cancers. Initially, during the primary tumor growth CHL1 could act as a putative tumor suppressor and is silenced to facilitate in situ tumor growth for 11 cancer types. We also suggested that re-expression of the gene on the edge of tumor mass might promote local invasive growth and enable further metastatic spread in ovary, colon and breast cancer. Our data also supported the role of CHL1 as a potentially novel specific biomarker in the early pathogenesis of two major histological types of renal cancer

Crystal Structure Analysis Reveals Functional Flexibility in the Selenocysteine-Specific tRNA from Mouse

Selenocysteine tRNAs (tRNA(Sec)) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec)-interacting factors are incompletely understood.We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec). tRNA(Sec) lacking the single-stranded 3'-acceptor end ((ΔGCCA)RNA(Sec)) yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCA)RNA(Sec) resembles the structure of human tRNA(Sec) determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec) used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Sec)in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+)-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec).We provide the most highly resolved structure of a tRNA(Sec) molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec) support its interaction with proteins

Hydration and metal ion binding of mouse tRNA^Sec.

<p>(<b>A</b>) Hydration of the G27•U43 wobble bps in molecules A (carbon – gold) and B (carbon – silver) of mouse tRNA^Sec, showing a full complement of first-shell water molecules (cyan spheres). Hydrogen bonds are indicated by dashed lines. (<b>B</b>) Anomalous difference Fourier map contoured at the 5 σ level (green mesh) calculated with anomalous differences recorded from a Mn²⁺-soaked crystal and phases obtained from molecular replacement with the native structure as a search model. Molecule A – gold; molecule B – silver. There are three common Mn²⁺ sites (1–3) in the two tRNA^Sec molecules. Sites 4 and 5 were found only in chain B. The boxed region is shown in a close-up view in the following panel. (<b>C</b>) Close-up of the boxed region of panel (B). Mn²⁺ ion (site 4; purple sphere) apparently reinforcing the interaction of U9 (AD-linker) with A48•C45 (first bp of the variable arm).</p

Non-denaturing RNA purification.

<p>(<b>A</b>) Elution profile of <i>in vitro</i> transcribed mouse tRNA^Sec from a MonoQ column. Peak 1 – unincorporated rNTPs, T7 RNA polymerase and other proteins; Peak 2 – abortive synthesis transcripts; Peak 3 – desired RNA sample; Peak 4 – aggregates or higher molecular weight nucleic acids. The gradient (buffer B from 30 to 100%) is shown as a dashed line. (<b>B</b>) Denaturing SDS PAGE analysis of peak fractions from Peaks 1–3. T7 RNA polymerase and molecular weight markers (M) were loaded as references. Protein bands were stained with Coomassie. (<b>C</b>) Denaturing urea PAGE analysis of peak fractions eluted from the MonoQ column. S – crude transcription extract. RNA bands were stained with methylene blue. (<b>D</b>) Elution profile of mouse tRNA^Sec from a Superdex 75 10/300 GL column.</p

Anticodon loop conformations in mouse tRNA^Sec.

<p>Stereo stick model of the closed anticodon loop conformation of molecule A (<b>A</b>) and of the open anticodon loop conformation of molecule B (<b>B</b>). The 2′-oxygen of the U34 ribose in molecule A that is methylated in a subset of cellular tRNA^Sec is shown as a thicker stick. Water molecules – cyan spheres. Hydrogen bonds are indicated by dashed lines.</p

tRNA^Sec constructs for crystallization screening.

<p>RNA 1 represents full-length tRNA^Sec. Canonical tRNA numbering was used throughout. Additional nucleotides (labeled with lower case Latin characters) and gaps (missing numbers) compared to the canonical tRNA numbering are indicated only in the scheme of RNA 1. RNAs 2 and 3 were created by site-directed mutagenesis and contain a UUCG (red) or a kissing loop (green) in place of the wt variable loop, respectively. Using the initial, mutated constructs, further DNA templates were generated for <i>in vitro</i> transcription, which allowed synthesis of tRNA^Sec species with deletion of the 3′-GCCA end (RNAs 4, 5 and 6) or with substitution of the 3′-GCCA with a self-complementary 3′-GCGC overhang (RNAs 7, 8 and 9).</p

Analysis of purified RNA.

<p>(<b>A</b>) Native and (<b>B</b>) denaturing PAGE analysis of fractions collected from Superdex 75 10/300 GL gel filtration. S – concentrated RNA sample after anion exchange chromatography. (<b>C</b>) Analysis of mouse tRNA^Sec by multi-angle static light scattering. The optical density at 260 nm (OD₂₆₀; magenta), Rayleigh ratio (Rq; blue) and differential refractive index (RI; yellow) were monitored during analytical gel filtration on a Superdex 200 10/300 GL column. The measurement was done by Wyatt Technology Europe GmbH.</p

Tertiary interactions of the AD-linker and variable arm.

<p>(<b>A</b>) Water-mediated interaction of the AD-linker with the first bp of the variable arm in mouse tRNA^Sec (molecule A – gold; molecule B – silver). Water molecules – cyan spheres. Hydrogen bonds are shown as dashed lines. (<b>B</b>) Close-up of the variable arm (molecule A). Nucleotides of the variable loop are labeled. (<b>C</b>) Superposition of the mouse tRNA^Sec variable loop (carbon – gold) with a stable GAGA tetraloop from the 23S rRNA sarcin/ricin domain (carbon – cyan; PDB ID 483D).</p

Crystal contacts of ^ΔGCCAtRNA^Sec.

<p>Stacking interactions of the terminal G1-C72 bp of the acceptor stem of molecule A with nucleotides G19 and U20 of the the D-loop of a neighboring molecule A. Left – overview. Right – close-up of the crystal packing interaction. In this and the following figures, atoms are color-coded in identical fashion; carbon – gold (or as the respective molecule); nitrogen – blue; oxygen – red; phosphorus – orange. Hydrogen bonds are indicated by dashed lines. The right view is rotated 45° about the horizontal axis as indicated.</p