Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory phenotype was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21. These compounds were identified as selective inhibitors of a transcription-regulating kinase CDK8 and its isoform CDK19. Remarkably, p21 was found to bind to CDK8 and stimulate its kinase activity. p21 and CDK8 also cooperate in the formation of internucleolar bodies, where both proteins accumulate. A CDK8 inhibitor suppresses damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts and reverses the increase in tumor engraftment and serum mitogenic activity in mice pretreated with a chemotherapeutic drug. The inhibitor also increases the efficacy of chemotherapy against xenografts formed by tumor cell/fibroblast mixtures. Microarray data analysis revealed striking correlations between CDK8 expression and poor survival in breast and ovarian cancers. CDK8 inhibition offers a promising approach to increasing the efficacy of cancer chemotherapy.transcriptional damage response | senescence | tumor microenvironment | nucleolus | chemical genomics

Paclitaxel (PTX) and other microtubule inhibitors cause mitotic arrest. However, low concentrations of PTX (low PTX) paradoxically cause G1 arrest (without mitotic arrest). Here, we demonstrated that unexpectedly, low PTX did not cause G1 arrest in the first cell cycle and did not prevent cells from passing through S phase and entering mitosis. Mitosis was prolonged but cells still divided, producing either two or three cells (tripolar mitosis), thus explaining a sub G1 peak caused by low PTX. Importantly, sub G1 cells were viable and nonapoptotic. Some cells fused back and then progressed to mitosis, frequently producing three cells again before becoming arrested in the next cell-cycle interphase. Thus, low PTX caused postmitotic arrest in second and even the third cell cycles. By increasing concentration of PTX, tripolar mitosis was transformed to mitotic slippage, thus eliminating a sub G1 peak. Time-lapse microscopy revealed that prolonged mitosis ensured a p53-dependent postmitotic arrest. We conclude that PTX directly affects cells only in mitosis and the duration of mitosis determines cell fate, including p53-dependent G1-like arrest.

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21Waf1 (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.Oncogene ( Keywords: p21; Rb; p27; damage response p53-inducible cyclin-dependent kinase (CDK) inhibitor p21 (CDKN1A) is the key mediator of damage-induced cell-cycle arrest. p21 interacts with different cyclin/CDK complexes and other regulators of transcription and signal transduction, exerting broad effects on cell survival, gene expression and morphology (Roninson, 2002). p21 effects are partially mediated by Rb, which is inactivated in proliferating cells through phosphorylation by CDK2 and CDK4/6, both of which are inhibited by p21. As a result, p21 induction leads to Rb dephosphorylation and activation, with ensuing G1 arrest.Whereas p21 activates Rb by dephosphorylation, several oncoproteins inactivate Rb by degradation via the proteasome. Proteasome-mediated Rb degradation is promoted by Mdm2 (Sdek et al., 2005) and gankyrin (Higashitsuji et al., 2000), E7 of papilloma virus (Boyer et al., 1996) and Tax of HTLV1 (Kehn et al., 2005). Oncoprotein-induced proteasomal degradation of Rb is one of the mechanisms for Rb inactivation in carcinogenesis (Ying and Xiao, 2006), but Rb degradation has not been described in DNA damage response.Changes in Rb phosphorylation are most commonly detected by immunoblotting through changes in the protein's electrophoretic mobility. Examination of numerous Rb immunoblots published by different groups showed that in many (but not all) cases Rb dephosphorylation, which results from drug treatment, cell senescence or ectopic p21 expression, is associated with a reduction in the Rb protein signal. In the present study, we have asked (i) whether a decrease in the Rb signal in response to p21 reflects protein degradation or merely altered immunoreactivity of dephosphorylated Rb, (ii) if p53 plays a p21-independent role in the decrease in Rb, (iii) whether such decrease can be induced by other CDK inhibitors that induce Rb dephosphorylation and (iv) if the decrease...

A major mRNA decay pathway in eukaryotes is initiated by deadenylation followed by decapping of the oligoadenylated mRNAs and subsequent 5 ′ -to-3 ′ exonucleolytic degradation of the capless mRNA. In this pathway, decapping is a rate-limiting step that requires the hetero-octameric Lsm1-7-Pat1 complex to occur at normal rates in vivo. This complex is made up of the seven Sm-like proteins, Lsm1 through Lsm7, and the Pat1 protein. It binds RNA and has a unique binding preference for oligoadenylated RNAs over polyadenylated RNAs. Such binding ability is crucial for its mRNA decay function in vivo. In order to determine the contribution of Pat1 to the function of the Lsm1-7-Pat1 complex, we compared the RNA binding properties of the Lsm1-7 complex purified from pat1Δ cells and purified Pat1 fragments with that of the wild-type Lsm1-7-Pat1 complex. Our studies revealed that both the Lsm1-7 complex and purified Pat1 fragments have very low RNA binding activity and are impaired in the ability to recognize the oligo(A) tail on the RNA. However, reconstitution of the Lsm1-7-Pat1 complex from these components restored these abilities. We also observed that Pat1 directly contacts RNA in the context of the Lsm1-7-Pat1 complex. These studies suggest that the unique RNA binding properties and the mRNA decay function of the Lsm1-7-Pat1 complex involve cooperation of residues from both Pat1 and the Lsm1-7 ring. Finally our studies also revealed that the middle domain of Pat1 is essential for the interaction of Pat1 with the Lsm1-7 complex in vivo.

Lsm proteins are a ubiquitous family of proteins characterized by the Sm-domain. They exist as hexa-or heptameric RNAbinding complexes and carry out RNA-related functions. The Sm-domain is thought to be sufficient for the RNA-binding activity of these proteins. The highly conserved eukaryotic Lsm1 through Lsm7 proteins are part of the cytoplasmic Lsm1-7-Pat1 complex, which is an activator of decapping in the conserved 59-39 mRNA decay pathway. This complex also protects mRNA 39-ends from trimming in vivo. Purified Lsm1-7-Pat1 complex is able to bind RNA in vitro and exhibits a unique binding preference for oligoadenylated RNA (over polyadenylated and unadenylated RNA). Lsm1 is a key subunit that determines the RNA-binding properties of this complex. The normal RNA-binding activity of this complex is crucial for mRNA decay and 39-end protection in vivo and requires the intact Sm-domain of Lsm1. Here, we show that though necessary, the Sm-domain of Lsm1 is not sufficient for the normal RNA-binding ability of the Lsm1-7-Pat1 complex. Deletion of the C-terminal domain (CTD) of Lsm1 (while keeping the Sm-domain intact) impairs mRNA decay in vivo and results in Lsm1-7-Pat1 complexes that are severely impaired in RNA binding in vitro. Interestingly, the mRNA decay and 39-end protection defects of such CTD-truncated lsm1 mutants could be suppressed in trans by overexpression of the CTD polypeptide. Thus, unlike most Sm-like proteins, Lsm1 uniquely requires both its Sm-domain and CTD for its normal RNA-binding function.

(2016) RPTOR, a novel target of miR-155, elicits a fibrotic phenotype of cystic fibrosis lung epithelium by upregulating CTGF, RNA Biology, 13:9, 837-847, DOI: 10.1080/15476286.2016 ABSTRACTCystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the most frequent of which is F508del-CFTR. CF is characterized by excessive secretion of pro-inflammatory mediators into the airway lumen, inducing a highly inflammatory cellular phenotype. This process triggers fibrosis, causing airway destruction and leading to high morbidity and mortality. We previously reported that miR-155 is upregulated in CF lung epithelial cells, but the molecular mechanisms by which miR-155 affects the disease phenotype is not understood. Here we report that RPTOR (regulatory associated protein of mTOR, complex 1) is a novel target of miR-155 in CF lung epithelial cells. The suppression of RPTOR expression and subsequent activation of TGF-b signaling resulted in the induction of fibrosis by elevating connective tissue growth factor (CTGF) abundance in CF lung epithelial cells. Thus, we propose that miR-155 might regulate fibrosis of CF lungs through the increased CTGF expression, highlighting its potential value in CF therapy.

more efficiently than human hepatocytes and results in lower overall transduction as compared to human clinical samples. Thus, while these models can serve as a surrogate to assess the activity of gene therapy constructs against functions of normal human liver, the doses required for optimal activity may be modestly higher than required in the human clinical setting. 146.Regulation of Inflammation in Cystic Fibrosis Lung Epithelial Cells by miR-155

BACKGROUND: Iniparib (BSI-201) has demonstrated promising efficacy and safety in the treatment of patients with metastatic triple-negative breast cancer (TNBC). The addition of iniparib to gemcitabine (G) and carboplatin (C) improved the clinical benefit rate from 34% to 56% (P = 0.01) and the overall response rate from 32% to 52% (P = 0.02) in a phase II clinical study (O'Shaughnessy et al. N Engl J Med. 2011).We investigated the chemosensitizing properties of iniparib with irinotecan (I) or ionizing radiation (IR). METHODS: The chemosensitizing properties of a single dose of iniparib combined with I or IR were evaluated in TNBC MDA-MB-468 breast carcinoma cells. Cell proliferation was evaluated with FACS-based cell cycle analysis, including DNA staining and bromodeoxyuridine incorporation. RESULTS: FACS analysis showed that in TNBC MDA-MB-468 breast carcinoma cells iniparib potentiated cell cycle arrest induced by I, with cells arrested in S and G2/M phases, or potentiated cell cycle arrest induced by IR, with cells arrested mostly in G2/M, at 72 hours after treatment (Table 1). CONCLUSIONS: These data demonstrate the potentiation of the antiproliferative activity of I and IR by iniparib in TNBC MDA-MB-468 breast carcinoma cells. These data support investigation of iniparib in combination with these DNA-damaging agents in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-401. doi:10.1158/1538-7445.AM2011-LB-401