ErbB2-dependent downregulation of a pro-apoptotic protein Perp is required for oncogenic transformation of breast epithelial cells.

The ability of breast cancer cells to resist anoikis, apoptosis caused by detachment of the non-malignant epithelial cells from the extracellular matrix (ECM), is thought to be critical for breast tumor growth, invasion and metastasis. ErbB2, an oncoprotein that is often overproduced in breast tumors, can block breast cancer cell anoikis via mechanisms that are understood only in part. In an effort to understand them better we found that detachment of the non-malignant human breast epithelial cells from the ECM upregulates a protein Perp in these cells. Perp is a component of the desmosomes, multiprotein complexes involved in cell-to-cell adhesion. Perp can cause apoptosis via unknown mechanisms. We demonstrated that Perp upregulation by cell detachment is driven by detachment-induced loss of epidermal growth factor receptor (EGFR). We also found that Perp knockdown by RNA interference (RNAi) rescues detached cells from death which indicates that Perp contributes to their anoikis. We observed that ErbB2, when overexpressed in detached breast epithelial cells, causes Perp downregulation. Furthermore, ErbB2-directed RNAi or treatment with lapatinib, an ErbB2/EGFR small-molecule inhibitor used for breast cancer therapy, upregulated Perp in ErbB2-positive human breast and ovarian carcinoma cells. We established that ErbB2 downregulates Perp by activating an ErbB2 effector protein kinase Mek that blocks detachment-induced EGFR loss in a manner that requires the presence of a signaling protein Sprouty-2. Finally, we observed that restoration of the wild-type Perp levels in ErbB2-overproducing breast epithelial cells increases their anoikis susceptibility and blocks their clonogenicity in the absence of adhesion to the ECM. In summary, we have identified a novel mechanism of ErbB2-mediated mechanism of anoikis resistance of ErbB2-overproducing breast epithelial cells. This mechanism allows such cells to grow without adhesion to the ECM and is driven by ErbB2-induced activation of Mek, subsequent EGFR upregulation and further EGFR-dependent Perp loss.

Anoikis of colon carcinoma cells triggered by ß-catenin loss can be enhanced by tumor necrosis factor receptor 1 antagonists.

Detachment of non-malignant epithelial cells from the extracellular matrix causes their apoptosis, a phenomenon called anoikis. By contrast, carcinoma cells are anoikis-resistant, and this resistance is thought to be critical for tumor progression. Many oncogenes trigger not only anti- but also pr-apoptotic signals. The proapoptotic events represent an aspect of a phenomenon called oncogenic stress, which acts as a safeguard mechanism blocking tumor initiation. In cells that become malignant, oncogene-induced antiapoptotic signals outbalance the proapoptotic ones. It is now thought that treatments blocking the antiapoptotic events but preserving the proapoptotic signals can be particularly effective in killing tumor cells. Whether or not oncogenes induce any proanoikis signals that can be used for enhancing the efficiency of approaches aimed at triggering anoikis of cancer cells has never been explored. ß-Catenin is a major oncoprotein that is often activated in colorectal cancer and promotes tumor progression via mechanisms that are understood only in part. We found here that ß-catenin triggers both anti- and proanoikis signals in colon cancer cells. We observed that the antianoikis signals prevail and the cells become anoikis-resistant. We further established that one proanoikis signal in these cells is triggered by ß-catenin-induced downregulation of an apoptosis inhibitor tumor necrosis factor receptor 1 (TNFR1) and subsequent reduction of the activity of a transcription factor NF-κB (nuclear factor-κB), a mediator of TNFR1 signaling. We also found that the effect of ß-catenin on TNFR1 requires the presence of transcription factor TCF1, a ß-catenin effector. We demonstrated that ablation of ß-catenin in colon cancer cells triggers their anoikis and that this anoikis is enhanced even further if low TNFR1 or NF-κB activity is artificially preserved in the ß-catenin-deprived cells. Thus, inhibition of TNFR1 or NF-κB activity can be expected to enhance the efficiency of approaches aimed at blocking ß-catenin-driven anoikis resistance of colon carcinoma cells.

Detachment-induced upregulation of XIAP and cIAP2 delays anoikis of intestinal epithelial cells.

Detachment of normal epithelial cells from the extracellular matrix triggers apoptosis, a phenomenon called anoikis. Conversely, carcinoma cells tend to be relatively more anoikis-resistant than their normal counterparts, and this increased resistance represents a critical feature of the malignant phenotype. Mechanisms that control susceptibility and resistance to anoikis are not fully understood. It is now known that detachment of non-malignant epithelial cells triggers both pro- and antiapoptotic signals, and it is the balance between these signals and the duration of detachment that determine further fate of the cells. Detachment-induced antiapoptotic events delay anoikis and if cells reattach relatively soon after detachment they survive. Direct regulators of apoptosis responsible for this delay of anoikis are unknown. We found that detachment of non-malignant intestinal epithelial cells triggers upregulation of inhibitors of apoptosis protein (IAP) family, such as X-chromosome-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis-2 (cIAP2). We demonstrated that this upregulation requires detachment-dependent activation of the transcription factor nuclear factor-kappaB. We further observed that various IAP antagonists accelerate anoikis, indicating that upregulation of the IAPs delays detachment-triggered apoptosis. We conclude that the IAPs are important regulators of the balance between detachment-triggered life and death signals. Perhaps, not by coincidence, these proteins are often upregulated in carcinomas, tumors composed of cells that tend to be anoikis-resistant.

Photoaffinity labeling of 30S-subunit proteins S7 and S11 by 4-thiouridine-substituted tRNA(Phe) situated at the P site of Escherichia coli ribosomes.

4-Thiouridine, a photoreactive analogue of uridine, was randomly incorporated into yeast tRNA(Phe) precursor molecules by transcription with T7 RNA polymerase and the resulting transcripts were converted into mature tRNA(Phe) by treatment with RNase P RNA. The photoreactive tRNA(Phe) was aminoacylated and bound to the P site of Escherichia coli 70S ribosomes in the presence of a poly(U) template. Irradiation of the complexes with light of 300 nm resulted in the covalent crosslinking of nt U20 in the D loop of the tRNA to protein S11 of the 30S ribosomal subunit, whereas nt U33 in the anticodon loop crosslinked to 30S-subunit protein S7. These results allowed us to map the D loop of P site-bound tRNA to the platform of the 30S ribosomal subunit and provided additional information about contacts between protein S7 and the anticodon loop in the cleft between the platform and the subunit head.

Peptidyl transferase and beyond.

The peptidyl transferase center of the Escherichia coli ribosome encompasses a number of 50S-subunit proteins as well as several specific segments of the 23S rRNA. Although our knowledge of the role that both ribosomal proteins and 23S rRNA play in peptide bond formation has steadily increased, the location, organization, and molecular structure of the peptidyl transferase center remain poorly defined. Over the past 10 years, we have developed a variety of photoaffinity reagents and strategies for investigating the topography of tRNA binding sites on the ribosome. In particular, we have used the photoreactive tRNA probes to delineate ribosomal components in proximity to the 3' end of tRNA at the A, P, and E sites. In this article, we describe recent experiments from our laboratory which focus on the identification of segments of the 23S rRNA at or near the peptidyl transferase center and on the functional role of L27, the 50S-subunit protein most frequently labeled from the acceptor end of A- and P-site tRNAs. In addition, we discuss how these results contribute to a better understanding of the structure, organization, and function of the peptidyl transferase center.

Investigation into the relationship between personality traits and OCD.

In the light of recent research suggesting that obsessive-compulsive disorder is more likely to be associated with constellations of personality traits other than obsessive-compulsive personality traits, the present study was undertaken to explore this relationship further and to remove the possibly confounding effects of general pathology, anxiety and depression. By using a non-clinical sample and partialling out the effects of anxiety and depression, it was found, contrary to recent research, that specific obsessional symptoms were only related to the traits of obsessive-compulsive personality disorder.

Recombinant photoreactive tRNA molecules as probes for cross-linking studies.

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.

Mapping the central fold of tRNA2(fMet) in the P site of the Escherichia coli ribosome.

4-Thiouridine (s4U), a photoreactive analog of uridine, was randomly incorporated into tRNA2(fMet) precursor molecules by transcription with T7 RNA polymerase. The s4U-containing transcripts were trimmed at their 5'-ends with RNase P RNA to yield mature tRNA2(fMet). The photoreactive tRNA2(fMet) derivatives were aminoacylated and bound to the P site of 70S ribosomes from Escherichia coli in the presence of a poly(A,G,U) template. Irradiation of the complexes at 300 nm resulted in the covalent cross-linking of tRNA2(fMet) to ribosomal proteins and rRNAs within both the 50S and 30S subunits. The labeled proteins were identified as L1, L27, and S19. 50S-subunit proteins L1 and L27 were attached to nucleotide U17 or U17.1 within the D loop of tRNA2(fMet), whereas 30S-subunit protein S19 was cross-linked to nucleotide U47 in the variable loop. Both of these sites occur in or near the central fold of the tRNA. These results permit us to map the D loop of P site-bound tRNA to the region between the central protuberance and the L1 ridge on the 50S ribosomal subunit, while the variable loop can be placed above the cleft on the head of the 30S subunit.

Identification of the Escherichia coli 30S ribosomal subunit protein neighboring mRNA during initiation of translation.

To identify the proteins of the 30S ribosomal subunit of E coli that neighbor mRNA in the ternary initiation complex (mRNA*30S subunit*tRNA(fMet), we used an affinity cross-linking approach in which photoactivated groups were attached to different positions along the mRNA chain. A series of mini-genes originating from the 5'-end region of the cro gene of lambda bacteriophage were constructed as templates for mini-mRNA synthesis. Two strategies were used to introduce photo-reactive agents into the message. According to the first, two transcripts were isolated from E coli and chemically derivatized at their 5'-ends with a photoinducible diaziril group. One of these messages allowed for localization of the 5'-end of the Shine-Dalgarno sequence while the other one allowed for labeling of the ribosome at the 5'-end side of the initiation AUG codon in the P site. According to the second approach, 5-azidouridine (5N3U) was randomly incorporated into mRNA transcripts during a T7 RNA polymerase catalyzed reaction by using a mixture of 5N3UTP and UTP. A message that had U residues at either -4, -3, -1, +2 and +14, +19, +20 positions was used (A from cro AUG is +1). Whereas cross-links with the 5N3U transcripts were essentially 'zero-length', the 5'-derivatized transcripts were covalently attached to ribosomal components about 14 A from the 5'-end. We found that proteins S1, S7, S5, S3 and S4 compose, or were close to, the ribosomal mRNA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)