Copper-Mediated Fluoroalkylation of Aryl Bromides and Chlorides Enabled by Directing Groups.

This report describes the reactions between N-heterocyclic carbene copper(I) fluoroalkyl complexes and aryl halides bearing ortho-directing groups. Pyridine, pyrazole, oxazoline, imine, and ester directing groups are shown to dramatically enhance the reactivity of aryl bromides and chlorides with (IPr)CuI-fluoroalkyl complexes (IPr = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene; fluoroalkyl = difluoromethyl and pentafluoroethyl) to afford aryl-fluoroalkyl coupling products. This approach is leveraged to achieve the Cu-catalyzed directed fluoroalkylation of a series of aryl bromide substrates.

C/EBPß deficiency enhances the keratinocyte innate immune response to direct activators of cytosolic pattern recognition receptors.

The skin is the first line of defense to cutaneous microbes and viruses, and epidermal keratinocytes play a critical role in preventing infection by viruses and pathogens through activation of the type I interferon (IFN) response. Using RNAseq analysis, here we report that the conditional deletion of C/EBPß transcription factor in mouse epidermis (CKOß mice) resulted in the upregulation of IFNß and numerous keratinocyte interferon-stimulated genes (ISGs). The expression of cytosolic pattern recognition receptors (cPRRs), that recognize viral RNA and DNA, were significantly increased, and enriched in the RNAseq data set. cPRRs stimulate a type I IFN response that can trigger cell death to eliminate infected cells. To determine if the observed increases in cPRRs had functional consequences, we transfected CKOß primary keratinocytes with the pathogen and viral mimics poly(I:C) (dsRNA) or poly(dA:dT) (synthetic B-DNA) that directly activate PRRs. Transfected CKOß primary keratinocytes displayed an amplified type I IFN response which was accompanied by increased activation of IRF3, enhanced ISG expression, enhanced activation of caspase-8, caspase-3 and increased apoptosis. Our results identify C/EBPß as a critical repressor of the keratinocyte type I IFN response, and demonstrates that the loss of C/EBPß primes keratinocytes to the activation of cytosolic PRRs by pathogen RNA and DNA to induce cell death mediated by caspase-8 and caspase-3.

C/EBPß suppresses keratinocyte autonomous type 1 IFN response and p53 to increase cell survival and susceptibility to UVB-induced skin cancer.

p53 is activated by DNA damage and oncogenic stimuli to regulate senescence, apoptosis and cell-cycle arrest, which are essential to prevent cancer. Here, we utilized UVB radiation, a potent inducer of DNA damage, p53, apoptosis and skin cancer to investigate the mechanism of CCAAT/enhancer binding protein-ß (C/EBPß) in regulating p53-mediated apoptosis in keratinocytes and to test whether the deletion of C/EBPß in epidermis can protect mice from UVB-induced skin cancer. UVB-treatment of C/EBPß skin conditional knockout (CKOß) mice increased p53 protein levels in epidermis and enhanced p53-dependent apoptotic activity 3-fold compared with UVB-treated control mice. UVB increased C/EBPß levels through a p53-dependent pathway and stimulated the formation of a C/EBPß-p53 protein complex; knockdown of C/EBPß increased p53 protein stability in keratinocytes. These results suggest a p53-C/EBPß feedback loop, whereby C/EBPß, a transcriptional target of a p53 pathway, functions as a survival factor by negatively regulating p53 apoptotic activity in response to DNA damage. RNAseq analysis of UVB-treated CKOß epidermis unexpectedly revealed that type 1 interferon (IFN) pathway was the most highly enriched pathway. Numerous pro-apoptotic interferon stimulated genes were upregulated including some known to enhance p53 apoptosis. Our results indicate that p53 and IFN pathways function together in response to DNA damage to result in the activation of extrinsic apoptosis pathways and caspase 8 cleavage. Last, we observed CKOß mice were resistant to UVB-induced skin cancer. Our results suggest that C/EBPß represses apoptosis through keratinocyte autonomous suppression of the type 1 IFN response and p53 to increase cell survival and susceptibility to UVB-induced skin cancer.

C/EBPß deletion in oncogenic Ras skin tumors is a synthetic lethal event.

Therapeutic targeting of specific genetic changes in cancer has proven to be an effective therapy and the concept of synthetic lethality has emerged. CCAAT/enhancer-binding protein-ß (C/EBPß), a basic leucine zipper transcription factor, has important roles in cellular processes including differentiation, inflammation, survival, and energy metabolism. Using a genetically engineered mouse model, we report that the deletion C/EBPß in pre-existing oncogenic Ha-Ras mouse skin tumors in vivo resulted in rapid tumor regression. Regressing tumors exhibited elevated levels of apoptosis and p53 protein/activity, while adjacent C/EBPß-deleted skin did not. These results indicate that the deletion of C/EBPß de-represses p53 in oncogenic Ras tumors but not in normal wild-type Ras keratinocytes, and that C/EBPß is essential for survival of oncogenic Ras tumors. Co-deletion of C/EBPß and p53 in oncogenic Ras tumors showed p53 is required for tumor regression and elevated apoptosis. In tumors, loss of a pathway that confers adaptability to a stress phenotype of cancer/tumorigenesis, such as DNA damage, could result in selective tumor cell killing. Our results show that oncogenic Ras tumors display a significant DNA damage/replicative stress phenotype and these tumors have acquired a dependence on C/EBPß for their survival. RNAseq data analysis of regressing tumors deleted of C/EBPß indicates a novel interface between p53, type-1 interferon response, and death receptor pathways, which function in concert to produce activation of extrinsic apoptosis pathways. In summary, the deletion of C/EBPß in oncogenic Ras skin tumors is a synthetic lethal event, making it a promising target for future potential anticancer therapies.

Role of p53 in the chronic pulmonary immune response to tangled or rod-like multi-walled carbon nanotubes.

The fiber-like shape of multi-walled carbon nanotubes (MWCNTs) is reminiscent of asbestos, suggesting they pose similar health hazards when inhaled, including pulmonary fibrosis and mesothelioma. Mice deficient in the tumor suppressor p53 are susceptible to carcinogenesis. However, the chronic pathologic effect of MWCNTs delivered to the lungs of p53 heterozygous (p53+/-) mice has not been investigated. We hypothesized that p53+/- mice would be susceptible to lung tumor development after exposure to either tangled (t-) or rod-like (r-) MWCNTs. Wild-type (p53+/+) or p53+/- mice were exposed to MWCNTs (1 mg/kg) via oropharyngeal aspiration weekly over four consecutive weeks and evaluated for cellular and pathologic outcomes 11-months post-initial exposure. No lung or pleural tumors were observed in p53+/+ or p53+/- mice exposed to either t- or rMWCNTs. In comparison to tMWCNTs, the rMWCNTs induced the formation of larger granulomas, a greater number of lymphoid aggregates and greater epithelial cell hyperplasia in terminal bronchioles in both p53+/- and p53+/+ mice. A constitutively larger area of CD45R+/CD3+ lymphoid tissue was observed in p53+/- mice compared to p53+/+ mice. Importantly, p53+/- mice had larger granulomas induced by rMWCNTs as compared to p53+/+ mice. These findings indicate that a combination of p53 deficiency and physicochemical characteristics including nanotube geometry are factors in susceptibility to MWCNT-induced lymphoid infiltration and granuloma formation.

C/EBPα regulates CRL4(Cdt2)-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint.

The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4(Cdt2). Cdt2 is the substrate recognition subunit of CRL4(Cdt2) and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4(Cdt2) mediated degradation of p21.

Stress-stimulated mitogen-activated protein kinases control the stability and activity of the Cdt1 DNA replication licensing factor.

DNA replication is tightly coordinated both with cell cycle cues and with responses to extracellular signals to maintain genome stability. We discovered that human Cdt1, an essential origin licensing protein whose activity must be restricted to G(1) phase, is a substrate of the stress-activated mitogen-activated protein (MAP) kinases p38 and c-Jun N-terminal kinase (JNK). These MAP kinases phosphorylate Cdt1 both during unperturbed G(2) phase and during an acute stress response. Phosphorylation renders Cdt1 resistant to ubiquitin-mediated degradation during S phase and after DNA damage by blocking Cdt1 binding to the Cul4 adaptor, Cdt2. Mutations that block normal cell cycle-regulated MAP kinase-mediated phosphorylation interfere with rapid Cdt1 reaccumulation at the end of S phase. Phosphomimetic mutations recapitulate the stabilizing effects of Cdt1 phosphorylation but also reduce the ability of Cdt1 to support origin licensing. Two other CRL4(Cdt2) targets, the cyclin-dependent kinase (CDK) inhibitor p21 and the methyltransferase PR-Set7/Set8, are similarly stabilized by MAP kinase activity. These findings support a model in which MAP kinase activity in G(2) promotes reaccumulation of a low-activity Cdt1 isoform after replication is complete.

C/EBPα expression is downregulated in human nonmelanoma skin cancers and inactivation of C/EBPα confers susceptibility to UVB-induced skin squamous cell carcinomas.

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G(1) checkpoint, and diminished or ablated expression of C/EBPα results in G(1) checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB.

Analysis of re-replication from deregulated origin licensing by DNA fiber spreading.

A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase. Using an adaptation of DNA fiber spreading we report the direct detection of re-replication on single DNA molecules from human chromosomes. Using this method we demonstrate substantial re-replication within 1 h of S phase entry in cells overproducing the replication factor, Cdt1. Moreover, a comparison of the HeLa cancer cell line to untransformed fibroblasts suggests that HeLa cells produce replication signals consistent with low-level re-replication in otherwise unperturbed cell cycles. Re-replication after depletion of the Cdt1 inhibitor, geminin, in an untransformed fibroblast cell line is undetectable by standard assays but readily quantifiable by DNA fiber spreading analysis. Direct evaluation of re-replicated DNA molecules will promote increased understanding of events that promote or perturb genome stability.

Cdt1 and Cdc6 are destabilized by rereplication-induced DNA damage.

The replication factors Cdt1 and Cdc6 are essential for origin licensing, a prerequisite for DNA replication initiation. Mechanisms to ensure that metazoan origins initiate once per cell cycle include degradation of Cdt1 during S phase and inhibition of Cdt1 by the geminin protein. Geminin depletion or overexpression of Cdt1 or Cdc6 in human cells causes rereplication, a form of endogenous DNA damage. Rereplication induced by these manipulations is however uneven and incomplete, suggesting that one or more mechanisms restrain rereplication once it begins. We find that both Cdt1 and Cdc6 are degraded in geminin-depleted cells. We further show that Cdt1 degradation in cells that have rereplicated requires the PCNA binding site of Cdt1 and the Cul4(DDB1) ubiquitin ligase, and Cdt1 can induce its own degradation when overproduced. Cdc6 degradation in geminin-depleted cells requires Huwe1, the ubiquitin ligase that regulates Cdc6 after DNA damage. Moreover, perturbations that specifically disrupt Cdt1 and Cdc6 degradation in response to DNA damage exacerbate rereplication when combined with geminin depletion, and this enhanced rereplication occurs in both human cells and in Drosophila melanogaster cells. We conclude that rereplication-associated DNA damage triggers Cdt1 and Cdc6 ubiquitination and destruction, and propose that this pathway represents an evolutionarily conserved mechanism that minimizes the extent of rereplication.

Cdc6 stability is regulated by the Huwe1 ubiquitin ligase after DNA damage.

The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC(Cdh1)) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC(Cdh1) ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage.