Abnormal RNA stability in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) share key features, including accumulation of the RNA-binding protein TDP-43. TDP-43 regulates RNA homeostasis, but it remains unclear whether RNA stability is affected in these disorders. We use Bru-seq and BruChase-seq to assess genome-wide RNA stability in ALS patient-derived cells, demonstrating profound destabilization of ribosomal and mitochondrial transcripts. This pattern is recapitulated by TDP-43 overexpression, suggesting a primary role for TDP-43 in RNA destabilization, and in postmortem samples from ALS and FTD patients. Proteomics and functional studies illustrate corresponding reductions in mitochondrial components and compensatory increases in protein synthesis. Collectively, these observations suggest that TDP-43 deposition leads to targeted RNA instability in ALS and FTD, and may ultimately cause cell death by disrupting energy production and protein synthesis pathways.

Two consecutive randomized controlled pertussis booster trials in children initially vaccinated in infancy with an acellular vaccine: The first with a five-component Tdap vaccine to 5-year olds and the second with five- or monocomponent Tdap vaccines at age 14-15 years.

Prior study children from a DTaP efficacy trial were recruited at ages 5 and 15 years to randomized booster trials addressing immunogenicity and reactogenicity; 475 preschool children received mixed or separate injections of a reduced antigen vaccine (Tdap5, Sanofi Pasteur MSD) and an inactivated polio vaccine, and 230 adolescents received the same or another booster vaccine (Tdap1, SSI, Denmark). Pre-vaccination antibody concentrations against pertussis antigens were significantly higher at 15 than 5 years of age, probably due to natural boosting between the studies. Tdap5 induced comparable anti-PT concentrations at both ages, but antibody responses were significantly higher to filamentous haemagglutinin, pertactin and fimbriae 2/3 in adolescents. As expected, a higher amount of PT (Tdap1, 20µg) induced a stronger anti-PT response than a lower amount (Tdap5, 2.5µg). The frequency of adverse events was low and there were no serious adverse reactions. All local reactions had an early onset and a short duration. A large swelling or redness of more than half of the upper arm circumference was reported in 8/475 5-year-olds and in 6/230 15-year-olds. Children vaccinated with Tdap5 reported more moderate pain in adolescence than at preschool age, whereas itching was only reported in preschool children. Sweden introduced DTaP vaccines in 1996 after a 17-year hiatus with no general pertussis vaccination and pertussis was still endemic at the time of the studies. The frequency of adverse events was nevertheless low in both preschool children and adolescents and antibody responses were adequate. These studies document immunogenicity and reactogenicity in a trial cohort consecutively vaccinated with acellular pertussis vaccines from infancy to adolescence. The adolescent study was registered at ClinicalTrials.gov on 26 March 2009 (NCT00870350).

Effects of physiological and synthetic IAP antagonism on c-IAP-dependent signaling.

Cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/2) have central roles in signal transduction mediated by numerous receptors that participate in inflammatory and immune responses. In certain pathways, such as activation of NF-κB, their degradation is a major regulatory event and is physiologically induced by activation of receptors. In addition, a number of synthetic compounds have been developed that also target the c-IAPs and induce their degradation. However, the extent of a synthetic IAP antagonist's ability to mirror the transcriptional program by a physiological signal remains unclear. Here we take a systems approach to compare the transcriptional programs triggered by activation of CD30, a well-characterized receptor previously shown to induce the degradation of the c-IAPs, to SM-164, a synthetic IAP antagonist that specifically triggers c-IAP degradation. Employing a technique that allows the specific analysis of newly transcribed RNA, we have generated comparative transcriptome profiles for CD30 activation and SM-164 treatment. Analysis of these profiles revealed that the genes regulated by each stimulus were not completely shared, indicating novel functions of IAP antagonists and consequences of c-IAP1/2 degradation. The data identified a role for c-IAP1/2 in the regulation of the ribosome and protein synthesis, which was subsequently confirmed by biological assays. These findings expand our knowledge of the roles of c-IAP1/2 in signaling and provide insight into the mechanism of synthetic IAP antagonists, furthering our understanding of their therapeutic potential.

Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1.

Increased levels of enhancer of zeste homolog 2 (EZH2), a critical regulator of cellular memory, are associated with negative estrogen receptor (ER) expression and disease progression in breast cancer. High levels of EZH2 signal the presence of metastasis and poor outcome in breast cancer patients. To test the hypothesis that deregulation of EZH2 contributes to ER-negative breast cancer progression, EZH2 expression was inhibited in ER-negative breast cancer cells MDA-MB-231 and CAL51 using a lentivirus system. EZH2 knockdown decreased proliferation and delayed the G(2)/M cell-cycle transition, although not affecting apoptosis. In vivo, EZH2 downregulation significantly decreased breast xenograft growth and improved survival. EZH2 knockdown upregulated BRCA1 protein. Of note, BRCA1 knockdown was sufficient to rescue the effects of EZH2 downregulation on proliferation, G(2)/M arrest, and on the levels of hyperphosphorylated mitotic Cdc25C and Cyclin B1 proteins, crucial for entry into mitosis. Invasive ER-negative breast carcinomas show significant overexpression of EZH2 and downregulation of BRCA1 proteins. Taken together, we show that EZH2 is important in ER-negative breast cancer growth in vivo and in vitro, and that BRCA1 is required for the proliferative effects of EZH2. Blockade of EZH2 may provide a prime target to prevent and/or halt ER-negative breast cancer progression.

P53 plays a protective role against UV- and cisplatin-induced apoptosis in transcription-coupled repair proficient fibroblasts.

We previously reported that transcription-coupled repair (TCR)-deficient human fibroblasts are extremely sensitive to UV-induced apoptosis and this sensitivity correlated with the induction of the p53 tumour suppressor. However, we have also found that p53 can be protective against UV-induced apoptosis. Thus, prior to this study, it was not clear whether the induction of p53 in TCR-deficient fibroblasts contributed to their death. To address this issue, we have expressed human papillomavirus E6 (HPV-E6) in primary fibroblasts derived from patients affected with xeroderma pigmentosum (complementation groups A, B and C) and Cockayne syndrome (complementation group B). We found that TCR-deficient (XP-A, XP-B and CS-B) fibroblasts were more sensitive than TCR-proficient cells (XP-C and normal) to both UV light and cisplatin treatment and this increase in sensitivity was not p53 dependent. Importantly, HPV-E6 expression increased the sensitivity of TCR-proficient normal and XP-C fibroblasts to UV- and cisplatin-induced apoptosis. This increase in sensitivity correlated with a decrease in the capacity of HPV-E6 expressing cells to recover mRNA synthesis following UV-irradiation. Therefore, we propose that p53 protects against UV- and cisplatin-induced apoptosis in a TCR-dependent manner and that p53 does not contribute strongly to the induction of apoptosis in TCR-deficient fibroblasts.

Induction of ser15 and lys382 modifications of p53 by blockage of transcription elongation.

Blockage of transcription has been shown to induce the tumor suppressor p53 in human cells. We here show that RNA synthesis inhibitors blocking the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II, such as DRB and H7, induced rapid nuclear accumulation of p53 proteins that were not phosphorylated at ser15 or acetylated at lys382. In contrast, agents that inhibit the elongation phase of transcription, such as UV light, camptothecin or actinomycin D, induced the accumulation of nuclear p53 proteins that were modified at both of these sites. Furthermore, using a panel of DNA repair-deficient cells we show that persistent DNA lesions in the transcribed strand of active genes are responsible for the induction of the ser15 and lys382 modifications following UV-irradiation. We conclude that inhibition of transcription is sufficient for the accumulation of p53 in the nucleus regardless of whether the ser15 site of p53 is phosphorylated or not. Importantly, blockage of the elongation phase of transcription triggers a distinct signaling pathway leading to p53 modifications on ser15 and lys382. We propose that the elongating RNA polymerase complex may act as a sensor of DNA damage and as an integrator of cellular stress signals.

Phenylbutyrate attenuates the expression of Bcl-X(L), DNA-PK, caveolin-1, and VEGF in prostate cancer cells.

Phenylbutyrate (PB) is a histone deacetylase inhibitor that has been shown to induce differentiation and apoptosis in various cancer cell lines. Although these effects are most likely due to modulation of gene expression, the specific genes and gene products responsible for the effects of PB are not well characterized. In this study, we used cDNA expression arrays and Western blot to assess the effect that PB has on the expression of various cancer and apoptosis-regulatory gene products. We show that PB attenuates the expression of the apoptosis antagonist Bcl-X(L), the double-strand break repair protein DNA-dependent protein kinase, the prostate progression marker caveolin-1, and the pro-angiogenic vascular endothelial growth factor. Furthermore, PB was found to act in synergy with ionizing radiation to induce apoptosis in prostate cancer cells. Taken together, our results point to the possibility that PB may be an effective anti-prostate cancer agent when used in combination with radiation or chemotherapy and for the inhibition of cancer progression.

The cyclin-dependent kinase inhibitor roscovitine inhibits RNA synthesis and triggers nuclear accumulation of p53 that is unmodified at Ser15 and Lys382.

Roscovitine has been shown to induce the accumulation of the tumor suppressor p53, to arrest cells in the G(1) and G(2)/M phases of the cell cycle, and to induce apoptosis in human cells. Although these cellular effects of roscovitine are thought to be caused directly by its specific inhibition of cyclin-dependent kinases, other mechanisms may contribute as well. In this study, we investigated whether roscovitine interferes with transcription in human cells. We have previously shown that blockage of transcription is a trigger for the induction of p53 and apoptosis in human fibroblasts. Here we show that mRNA synthesis is suppressed significantly by roscovitine in human cells. Furthermore, our results suggest that the mechanism by which roscovitine inhibits RNA synthesis involves the inhibition of the phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Cells treated with roscovitine at doses that affected transcription were found to accumulate p53 in the nucleus; curiously, however, the nuclear accumulation of p53 was not accompanied by modifications at either the Ser15 or Lys382 sites of p53. We conclude that roscovitine is a potent inhibitor of RNA synthesis and that this inhibition may be responsible for the accumulation of nuclear p53.

Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress.

The tumor suppressor p53 is a nucleocytoplasmic shuttling protein that accumulates in the nucleus of cells exposed to various cellular stresses. One important role of nuclear p53 is to mobilize a stress response by transactivating target genes such as the p21(Waf1) gene. In this study, we investigated more closely the localization of p53 in cells following various stresses. Immunocytochemistry of fixed human fibroblasts treated with either UV light, the kinase and transcription inhibitor DRB or the proteasome inhibitor MG132 revealed abundant p53 localized to the nucleus. When cells treated with UV or DRB were permeabilized prior to fixation to allow soluble proteins to diffuse, the nuclear p53 signal was abolished. However, in cells treated with MG132, residual p53 localized to distinct large foci. Furthermore, nucleolin co-localized with p53 to these foci, suggesting that these foci were nucleolar structures. Interestingly, the MDM2 protein was found to co-localize with p53 to nucleolar structures following proteasome inhibition. Our results suggest that the p53 proteins accumulating in the nucleus following UV-irradiation or blockage of transcription are freely soluble and, thus, should be able to roam the nucleus to ensure high occupancy of p53 binding sites. However, inhibition of proteasome activity may be a unique stress in that it leads to the sequestering of p53 proteins to the nucleolus, thereby blunting the p53-mediated transactivation of target genes.

UV light-induced degradation of RNA polymerase II is dependent on the Cockayne's syndrome A and B proteins but not p53 or MLH1.

It has been hypothesized that the degradation of the largest subunit of RNA polymerase II (polIILS) is required for transcription-coupled repair (TCR) of UV light-induced transcription-blocking lesions. In this study we further investigated the mechanism of UV-induced degradation of polIILS using cell lines with specific defects in TCR or in the recovery of RNA synthesis. It was found that the hypophosphorylated IIa form of polIILS rapidly decreased following UV-irradiation in all cell lines tested. Inhibition of proteasome activity resulted in an increase of the hyperphosphorylated IIo form of polIILS in UV-irradiated cells, while inhibition of CTD-kinases resulted in the retention of the IIa form. In UV-irradiated Cockayne's syndrome cells, which are defective in TCR, the levels of the IIo form increased in a similar manner as when proteasome inhibitors were added to UV-irradiated normal cells. In contrast, TCR-deficient HCT116 cells, which lack the mismatch repair protein MLH1, showed proficient degradation of polIILS as did cells with deficiencies in the recovery of RNA synthesis following UV-irradiation due to defective p53. Furthermore, we found that proteasome function was important for the recovery of mRNA synthesis even in TCR-deficient HCT116 cells. Our results suggest that proteasome-mediated degradation of polIILS is preceded by phosphorylation of the C-terminal domain of polIILS and requires the CS-A and CS-B but not MLH1 or p53 proteins. Furthermore, our results suggest that following UV-irradiation, the degradation of polIILS is required for the efficient recovery of mRNA synthesis but not for TCR per se.

The tumor suppressor p53 can both stimulate and inhibit ultraviolet light-induced apoptosis.

We have previously shown that the tumor suppressor p53 can play a protective role against UV-induced apoptosis in human fibroblasts. In the present study, we investigated whether the protective function of p53 expression is established before or after UV irradiation. Using a stable human cell line expressing a murine temperature-sensitive p53 in which p53 function could be tightly and reversibly regulated, we found that functional p53 stimulated the induction of apoptosis when expressed for as little as 4-12 h after UV irradiation and that this induction was not dependent on de novo protein synthesis. In contrast, expression of p53 for 12 h or more before UV irradiation reduced the extent of apoptosis even when functional p53 expression was maintained after irradiation. The protection conferred by p53 required ongoing protein synthesis and correlated with enhanced recovery of mRNA synthesis. Together, these results suggest that p53 induces distinct proapoptotic and antiapoptotic signals and that these opposing activities can be separated both temporally and by their requirement for de novo protein synthesis. These findings may have important implications for the refinement of gene therapy approaches combining p53 with pharmacological agents that target transcription or translation.

Dial 9-1-1 for p53: mechanisms of p53 activation by cellular stress.

The tumor suppressor protein, p53, is part of the cell's emergency team that is called upon following cellular insult. How do cells sense DNA damage and other cellular stresses and what signal transduction pathways are used to alert p53? How is the resulting nuclear accumulation of p53 accomplished and what determines the outcome of p53 induction? Many posttranslational modifications of p53, such as phosphorylation, dephosphorylation, acetylation and ribosylation, have been shown to occur following cellular stress. Some of these modifications may activate the p53 protein, interfere with MDM2 binding and/or dictate cellular localization of p53. This review will focus on recent findings about how the p53 response may be activated following cellular stress.

Role of p53 in cell cycle regulation and apoptosis following exposure to proteasome inhibitors.

In this study, we explored what effect inhibitors of the 26S proteasome have on cell cycle distribution and induction of apoptosis in human skin fibroblasts and colon cancer cells differing in their p53 status. We found that proteasome inhibition resulted in nuclear accumulation of p53. This was surprising because it is thought that the degradation of p53 is mediated by cytoplasmic 26S proteasomes. Nuclear accumulation of p53 was accompanied by the induction of both p21WAF1 mRNA and protein as well as a decrease in cells entering S phase. Interestingly, cells with compromised p53 function showed a marked increase in the proportion of cells in the G2-M phase of the cell cycle and an attenuated induction of apoptosis after proteasome inhibition. Taken together, our results suggest that proteasome inhibition results in nuclear accumulation of p53 and a p53-stimulated induction of both G1 arrest and apoptosis.

The human Cdc14 phosphatases interact with and dephosphorylate the tumor suppressor protein p53.

The yeast Cdc14 phosphatase has been shown to play an important role in cell cycle regulation by dephosphorylating proteins phosphorylated by the cyclin-dependent kinase Cdc28/clb. We recently cloned two human orthologs of the yeast CDC14, termed hCDC14A and -B, the gene products of which share approximately 80% amino acid sequence identity within their N termini and phosphatase domains. Here we report that the hCdc14A and hCdc14B proteins interact with the tumor suppressor protein p53 both in vitro and in vivo. This interaction is dependent on the N termini of the hCdc14 proteins and the C terminus of p53. Furthermore, the hCdc14 phosphatases were found to dephosphorylate p53 specifically at the p34(Cdc2)/clb phosphorylation site (p53-phosphor-Ser(315)). Our findings that hCdc14 is a cyclin-dependent kinase substrate phosphatase suggest that it may play a role in cell cycle control in human cells. Furthermore, the identification of p53 as a substrate for hCdc14 indicates that hCdc14 may regulate the function of p53.

Repair of radiation-induced DNA strand breaks does not occur preferentially in transcriptionally active DNA.

Certain DNA base lesions induced by ionizing radiation or oxidative stress are repaired faster from the transcribed strand of active genes compared to the genome overall. In this study, it was investigated whether radiation-induced DNA strand breaks are preferentially repaired in active genes compared to the genome as a whole in CHO cells. The alkaline unwinding technique coupled to slot-blot hybridization with specific DNA probes was used to study the induction and repair of DNA strand breaks in defined DNA sequences. Results using this technique showed a linear dose response for the formation of radiation-induced DNA strand breaks in the dihydrofolate reductase (DHFR) gene. Furthermore, the half-life of radiation-induced strand breaks was less than 5 min in the DHFR gene, in the ribosomal genes, and in the genome as a whole. These results suggest that the repair of DNA strand breaks is fast and uniform in the genome of mammalian cells.

Potential roles for p53 in nucleotide excision repair.

Ultraviolet (UV) light-induced DNA damage is repaired by the nucleotide excision repair pathway, which can be subdivided into transcription-coupled repair (TCR) and global genome repair (GGR). Treatment of cells with a priming dose of UV light appears to stimulate both GGR and TCR, suggesting that these processes are inducible. GGR appears to be disrupted in p53-deficient fibroblasts, whereas the effect of p53 disruption on TCR remains somewhat controversial. Normal recovery of mRNA synthesis following UV irradiation is thought to depend on TCR. We have found that the recovery of mRNA synthesis following exposure to UV light is severely attenuated in p53-deficient human fibroblasts. Therefore, p53 disruption may lead to a defect in TCR or a post-repair process required for the recovery of mRNA synthesis. Several different functions of p53 have been proposed which could contribute to these cellular processes. We suggest that p53 could participate in GGR and the recovery of mRNA synthesis following UV exposure through the regulation of steady-state levels of one or more p53-regulated gene products important for these processes. Furthermore, we suggest that the role of p53 in the recovery of mRNA synthesis is important for resistance to UV-induced apoptosis.

Recovery of RNA synthesis from the DHFR gene following UV-irradiation precedes the removal of photolesions from the transcribed strand.

It is thought that recovery of RNA synthesis following UV-irradiation is closely related to the removal of UV-induced lesions from the transcribed strand of active genes. To test this hypothesis, nascent RNA synthesis from three different locations within the DHFR gene in CHO cells was assessed following exposure to UV light (254 nm). Using both in vivo RNA labeling as well as the nuclear run-on technique, it was found that RNA synthesis from the middle and the 3'-end of the gene was inhibited within 20 min by approximately 30 and 70%, respectively, while RNA synthesis from the 5'-end of the DHFR gene was enhanced. RNA synthesis from the middle portion of the gene fully recovered within 30-45 min of post-UV incubation, while recovery was slower from the 3'-end of the gene. Compared with previously published data for the kinetics of removal of UV-induced DNA lesions from the 5'-half of the DHFR gene in these cells, it is concluded that RNA synthesis resumed significantly faster in this region than could be accounted for by the removal of photolesions from the transcribed strand. Thus, although RNA synthesis was initially inhibited by UV-induced photolesions, the results suggest that RNA polymerase II was able to bypass these lesions prior to their removal.

Dose-dependent effects of DNA-damaging agents on p53-mediated cell cycle arrest.

We examined the dose-dependent effects of DNA-damaging agents on G1 arrest in isogenic human cell lines differing in their p53 status. As expected, 5 or 20 Gy of ionizing radiation induced a p53-dependent G1 arrest. In contrast, UV light or actinomycin D induced a modest G1 arrest that was p53-dependent only at lower doses. At higher doses, cells were arrested in G1 in a p53-independent manner coinciding with inhibition of RNA synthesis and abolished cyclin E expression. Interestingly, expression of cyclin E was enhanced after exposure to moderate doses of UV light and actinomycin D, and this enhancement was suppressed by wild-type p53. We propose that agents inducing transcription-blocking DNA lesions will at higher doses inhibit the progression of cells into S phase by a p53-independent mechanism involving the attenuation of E2F-mediated transcription of genes, such as cyclin E.