S100 calcium-binding proteins such as S100B are elevated in primary malignant melanoma and are used as markers for this and numerous other cancers. Wild-type p53 protein levels are relatively low in these cancer cells (i.e. when compared with cells without S100B) but are elevated when RNA antisense to S100B is introduced. This result implicates S100B in the down-regulation of p53 and is consistent with the large decreases in p53 protein levels observed previously in transient cotransfections of p53 and S100B (Lin, J., Blake, M., Tang Down-regulation of p53 in primary malignant melanoma cells is likely the result of a direct interaction with S100B, which was observed by co-immunoprecipitation experiments. Furthermore, p53 binds regions of the S100B promoter, one of which matches the 20-nucleotide p53-binding consensus DNA sequence perfectly. Therefore, when p53 levels increase, it contributes to its own demise by up-regulating the transcription of S100B as part of a negative feedback loop. This is analogous to what is found for another protein that down-regulates p53, namely hdm2 (human double mutant 2).

The predominantly nuclear heterogenous ribonucleoprotein A18 (hnRNP A18) translocates to the cytosol in response to cellular stress and increases translation by specifically binding to the 3-untranslated region (UTR) of several mRNA transcripts and the eukaryotic initiation factor 4G. Here, we identified a 51-nucleotide motif that is present 11.49 times more often in the 3-UTR of hnRNP A18 mRNA targets than in the UniGene data base. This motif was identified by computational analysis of primary sequences and secondary structures of hnRNP A18 mRNA targets against the unaligned sequences. Band shift analyses indicate that the motif is sufficient to confer binding to hnRNP A18. A search of the entire UniGene data base indicates that the hnRNP A18 motif is also present in the 3-UTR of the ataxia telangiectasia mutated and Rad3-related (ATR) mRNA. Validation of the predicted hnRNP A18 motif is provided by amplification of endogenous ATR transcript on polysomal fractions immunoprecipitated with hnRNP A18. Moreover, overexpression of hnRNP A18 results in increased ATR protein levels and increased phosphorylation of Chk1, a preferred ATR substrate, in response to UV radiation. In addition, our data indicate that inhibition of casein kinase II or GSK3␤ significantly reduced hnRNP A18 cytosolic translocation in response to UV radiation. To our knowledge, this constitutes the first demonstration of a post-transcriptional regulatory mechanism for ATR activity. hnRNP A18 could thus become a new target to trigger ATR activity as back-up stress response mechanisms to functionally compensate for absent or defective responders. ATR2 belongs to a family of protein serine-threonine kinases whose catalytic domains share evolutionary relationship with mammalian and yeast phosphoinositide 3-kinases (1). The ataxia telangiectasia mutated (ATM) protein kinase as well as the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and hSMG-1 also belong to this family (2). Although ATR and DNA-PKcs share substantial sequence homology and substrates specificity with ATM, they cannot compensate for the lack of a functional ATM, at least not redundantly. ATM, ATR, and DNA-PKcs are responsible for initiating the signaling cascades in response to DNA doublestrand breaks (2), whereas hSMG-1 regulates nonsense-mediated mRNA decay (2). DNA-PKcs activity is apparently restricted to phosphorylation of DNA repair proteins, whereas ATM and ATR phosphorylate a larger repertoire of substrates from cell cycle regulators to DNA repair proteins (3). ATR is primarily activated by UV radiation, replication stress, and single-strand DNA gaps but can also respond to double-strand breaks albeit, at much slower kinetics than ATM (3). The recruitment of ATR to stalled replication fork and single-strand DNA breaks has recently been described and involves the coordinate interaction of several proteins including the replication protein A (RPA), the ATR-interacting protein, and the topoisomerase 2-binding protein TopBP1 (4). The delay in ATR activation in ...

The S100B-p53 protein complex was discovered in C8146A malignant melanoma, but the consequences of this interaction required further study. When S100B expression was inhibited in C8146As by siRNA (siRNA S100B ), wt p53 mRNA levels were unchanged, but p53 protein, phosphorylated p53, and p53 gene products (i.e. p21 and PIDD) were increased. siRNA S100B transfections also restored p53-dependent apoptosis in C8146As as judged by poly(ADP-ribose) polymerase cleavage, DNA ladder formation, caspase 3 and 8 activation, and aggregation of the Fas death receptor (؉UV); whereas, siRNA S100B had no effect in SK-MEL-28 cells containing elevated S100B and inactive p53 (p53R145L mutant). siRNA S100B -mediated apoptosis was independent of the mitochondria, because no changes were observed in mitochondrial membrane potential, cytochrome c release, caspase 9 activation, or ratios of pro-and anti-apoptotic proteins (BAX, Bcl-2, and Bcl-X L ). As expected, cells lacking S100B (LOX-IM VI) were not affected by siRNA S100B , and introduction of S100B reduced their UV-induced apoptosis activity by 7-fold, further demonstrating that S100B inhibits apoptosis activities in p53-containing cells. In other wild-type p53 cells (i.e. C8146A, UACC-2571, and UACC-62), S100B was found to contribute to cell survival after UV treatment, and for C8146As, the decrease in survival after siRNA S100B transfection (؉UV) could be reversed by the p53 inhibitor, pifithrin-␣. In summary, reducing S100B expression with siRNA was sufficient to activate p53, its transcriptional activation activities, and p53-dependent apoptosis pathway(s) in melanoma involving the Fas death receptor and perhaps PIDD. Thus, a well known marker for malignant melanoma, S100B, likely contributes to cancer progression by down-regulating the tumor suppressor protein, p53.In addition to regulating numerous genes and pathways involved in cell cycle control (1), the tumor suppressor protein, p53, is an important component for inducing apoptosis (2-4). The p53 protein activates the transcription of pro-apoptotic factors (BAX, Bak, Fas/APO-1, PIDD, etc.) as well as suppresses the transcription of anti-apoptotic genes (Bcl-2, Bcl-X L , etc.) (5, 6). In addition, p53 itself up-regulates apoptosis, without transcription activation, by directly localizing to mitochondria following DNA damage and interacting with anti-apoptotic proteins such as Bcl-X L to free pro-apoptotic proteins like BAX (2, 3, 5, 7). Under stress, the p53 protein can also contribute to apoptosis by facilitating the transport of death receptors such as Fas/APO-1 and/or Killer/DR5 from cytoplasmic stores to the cell surface as required for programmed cell death (5, 8). Although, it is now clear that p53-dependent pathways of apoptosis are numerous and are regulated in a cell-type and signalspecific manner (5).Apoptosis is initiated by a variety of stimuli, including withdrawal of growth factors, activation of specific receptors, such as Fas antigen and TNF receptor, and/or by exposure to UV radiation, ␥ irradiation, DNA-da...

Extremely high-dose-rate irradiation, referred to as FLASH, has been shown to be less damaging to normal tissues than the same dose administrated at conventional dose rates. These results, typically seen at dose rates exceeding 40 Gy/s (or 2,400 Gy/min), have been widely reported in studies utilizing photon or electron radiation as well as in some proton radiation studies. Here, we report the development of a proton irradiation platform in a clinical proton facility and the dosimetry methods developed. The target is placed in the entry plateau region of a proton beam with a specifically designed double-scattering system. The energy after the double-scattering system is 227.5 MeV for protons that pass through only the first scatterer, and 225.5 MeV for those that also pass through the second scatterer. The double-scattering system was optimized to deliver a homogeneous dose distribution to a field size as large as possible while keeping the dose rate .100 Gy/s and not exceeding a cyclotron current of 300 nA. We were able to obtain a collimated pencil beam (1.6 3 1.2 cm 2 ellipse) at a dose rate of ;120 Gy/s. This beam was used for dose-response studies of partial abdominal irradiation of mice. First results indicate a potential tissuesparing effect of FLASH.

Elevated prostaglandin E 2 (PGE 2 ) production is a common feature of human malignancies. This activity has often been attributed to increased metabolic activity of the cyclooxygenase enzymes, although a direct comparison of these 2 parameters i.e., prostaglandin production and cox protein expression, is rarely performed in the same malignant tissue. Using a murine model of metastatic breast cancer, we show that PGE 2 levels are positively correlated with increased tumorigenic and metastatic potential. Because prostaglandin synthesis is a product of 2 isoforms of the cyclooxygenase enzyme, we examined the expression and activity of both isoforms. All tumor cell lines examined, regardless of phenotype, express both cox-1 and cox-2 proteins in vitro. In contrast to the uniform cox-2 expression in vitro, only tumors resulting from the transplantation of metastatic cell lines express cox-2 in vivo. Cox-1 is detected in both metastatic and nonmetastatic tumors. Thus, this is the first evidence that, in the tumor milieu, cox-2 expression can be regulated differently in metastatic vs. nonmetastatic lesions. Examination of PGE 2 synthesis in vitro reveals that nearly complete inhibition of prostaglandin synthesis occurs in the presence of either indomethacin, which inhibits both isoforms, or NS398, which is selective for the cox-2 isoform. Thus, even though cell lines express both isoforms, the majority of the prostaglandin synthesis stems from the activity of the inducible, cox-2 isoform. Likewise, cell growth is inhibited by both indomethacin and NS398 in a dose-dependent manner, albeit at higher drug concentrations than required to ablate PGE 2 synthesis. Despite the inhibition of prostaglandin synthesis, the cox-2 enzyme levels (protein and mRNA) were increased by either indomethacin or NS398. © 2001 Wiley-Liss, Inc. Key words: cyclooxygenase; prostaglandin; breast cancer; metastasisHigh cyclooxygenase activity is a common feature of human epithelial malignancies, 1,2 however, the biological significance of this metabolic activity has never been established. Recent epidemiologic studies have indicated that prolonged use of nonsteroidal antiinflammatory drugs (NSAIDs) is associated with a decreased risk of several malignancies, most notably colorectal cancers. 3 More recently, similar relationships have been observed in lung, breast and other cancers as well. 4 -6 Because NSAIDs have, as a principle action, inhibition of cyclooxygenases, these findings suggest that prostaglandin synthesis contributes to the risk of developing primary malignancies.We sought evidence for a role of prostaglandin synthesis in late tumor progression, i.e., tumor metastasis. Using a murine model of human breast cancer, we showed that higher prostaglandin E 2 (PGE 2 ) levels were observed in malignant mammary tissue in comparison to normal or premalignant gland. Furthermore, the highest PGE 2 levels were observed in the most malignant and metastatic tumors. 7 Two isoforms of cyclooxygenase (cox) are responsible for prostaglandin synthesis....

Neurotrophin receptor-interacting melanoma antigen-encoding gene homolog (NRAGE) is generally recognized as a tumor suppressor as it induces cell apoptosis and suppresses cell metastasis. However, it has recently been reported that NRAGE is overexpressed in lung cancer, melanoma and colon cancer, implicating a complicated role of NRAGE as we have expected. In the study, we aim to elucidate the functional roles and molecular mechanisms of NRAGE in esophageal carcinoma. We found that both NRAGE mRNA and protein were significantly overexpressed in esophageal tumor tissues. Consistently, both in vivo and in vitro analyses demonstrated that knockdown of NRAGE apparently inhibited cell growth, and cell cycle analysis further demonstrated that NRAGE knockdown cells were mainly arrested in G2M cell phase, accompanied with an apparent reduction of S phase. In the process of exploring molecular mechanisms, we found that either knockdown in vitro or knockout in vivo of NRAGE reduced proliferating cell nuclear antigen (PCNA) protein, expression of which could completely rescue the inhibited proliferation in NRAGE defective cells. Furthermore, NRAGE physically interacted with PCNA in esophageal cancer cells through DNA polymerase III subunit, and knockdown of NRAGE facilitated PCNA K48-linked polyubiquitination, leading PCNA to the proteasome-dependent degradation and a ubiquitin-specific protease USP10 was identified to be a key regulator in the process of K48 polyubiquitination in NRAGE-deleted cells. In conclusion, our study highlights a unique role of NRAGE and implies that NRAGE is likely to be an attractive oncotarget in developing novel genetic anticancer therapeutic strategies for esophageal squamous cell carcinomas.

Mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs) turn out to be a promising source of cell‐free therapy. Here, we investigated the biodistribution and effect of nebulized human adipose‐derived MSC‐EVs (haMSC‐EVs) in the preclinical lung injury model and explored the safety of nebulized haMSC‐EVs in healthy volunteers. DiR‐labelled haMSC‐EVs were used to explore the distribution of nebulized haMSC‐EVs in the murine model. Pseudomonas aeruginosa‐induced murine lung injury model was established, and survival rate, as well as WBC counts, histology, IL‐6, TNF‐α and IL‐10 levels in bronchoalveolar lavage fluid (BALF) were measured to explore the optimal therapeutic dose of haMSC‐EVs through the nebulized route. Twenty‐four healthy volunteers were involved and received the haMSC‐EVs once, ranging from 2 × 108 particles to 16 × 108 particles (MEXVT study, NCT04313647). Nebulizing haMSC‐EVs improved survival rate to 80% at 96 h in P. aeruginosa‐induced murine lung injury model by decreasing lung inflammation and histological severity. All volunteers tolerated the haMSC‐EVs nebulization well, and no serious adverse events were observed from starting nebulization to the 7th day after nebulization. These findings suggest that nebulized haMSC‐EVs could be a promising therapeutic strategy, offering preliminary evidence to promote the future clinical applications of nebulized haMSC‐EVs in lung injury diseases.

Despite the ability of most proteins to form amyloid, very little is know about amyloid fibril structures and the factors that govern their stability. Using amyloid fibrils produced from fulllength prion protein (PrP), we describe a reliable approach for determining both site-specific and global conformational stability of the fibrillar form. To measure site-specific stability, we produced six variants of PrP by replacing the residues at positions 88, 98, 127, 144, 196, and 230 with cysteine, labeled the new cysteines with the fluorescent dye acrylodan, and investigated their conformational status within the amyloid form in guanidine hydrochloride-induced denaturation experiments. We found that the fibrils labeled at positions 127, 144, 196, and 230 displayed cooperative unfolding and showed a very high C1 ⁄ 2 value similar to that observed for the global unfolding of the amyloid structure. The unfolding at residue 98 was also cooperative; however, it showed a C1 ⁄ 2 value substantially lower than that of global unfolding, whereas the unfolding of fibrils labeled at residue 88 was non-cooperative. These data illustrate that there are at least two independent cooperative folding domains within the amyloid structure of the full-length PrP. In addition, kinetic experiments revealed only a partial overlap between the region that constituted the fibrillar cross-␤ core and the regions that were involved in nucleation. This result illustrates that separate PrP regions accounted for the nucleation and for the formation of the conformationally most stable fibrillar core.