Terminal differentiation and cellular senescence display common properties including irreversible growth arrest. To define the molecular and ultimately the biochemical basis of the complex physiological changes associated with terminal differentiation and senescence, an overlapping-pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes. This approach involved screening of a subtracted cDNA library prepared from human melanoma cells induced to terminally differentiate by treatment with fibroblast IFN and mezerein with mRNA derived from senescent human progeria cells. overlapping-pathway screen ͉ terminal cell differentiation ͉ senescent phenotype ͉ interferon-inducible gene ͉ evolutionary conserved gene P lasticity of the transformed phenotype is suggested by the ability of differentiation-inducing agents to revert the cancerous properties of specific tumors (1-3). This attribute of tumor cells provides the basis for a potentially less toxic form of therapy, ''differentiation therapy.'' In metastatic human melanoma, a combination of IFN-␤ and the protein kinase C activator mezerein (MEZ) produces irreversible growth arrest, a loss of tumorigenic competence, and terminal differentiation (1, 4). To define gene-expression changes associated with induction of terminal differentiation, a subtracted cDNA library enriched for genes associated with terminal differentiation was constructed (5). This construction was accomplished by subtracting control HO-1 human melanoma mRNAs from IFN-␤ ϩ MEZ-treated HO-1 mRNAs, which were temporally collected over a 24-h period (5). This subtracted cDNA library then was screened by random isolation of phage colonies and Northern blotting, high-density cDNA microarray analysis, and reverse Northern screening followed by Northern blotting (5-7). These approaches have identified both unknown and known genes associated with tumor and normal growth control, cell-cycle regulation, IFN response, differentiation, and apoptosis (5-12). Four classes of melanoma differentiation-associated (mda) genes have been identified (5, 10).Terminal cell differentiation and cellular senescence are characterized by changes in cell morphology, lack of responsiveness to mitogenic stimulation, and irreversible growth arrest (1, 4, 13-18).Normal cells cultured in vitro lose their proliferative potential after a finite number of doublings in a process described as cellular senescence (13). Experiments in human diploid fibroblasts and additional cell types document an inverse correlation between replicative senescence and donor age and a direct relationship between replicative senescence and donor-species life span (13,19,20). In agreement with this relationship, cells from patients with premature aging syndromes such as Werner's syndrome and progeria achieve a quiescent state more rapidly than normal human fibroblasts (21). Although senescence is a time-dependent process, terminal differentiation can be induced in a variety of cell types by appropriate treatme...

To investigate double strand break (DSB) repair and signaling in human glioma cells, we stably transfected human U87 (ATM ؉ , p53 ؉ ) glioma cells with a plasmid having a single I-SceI site within an inactive green fluorescent protein (GFP) expression cassette, allowing for the detection of homologous recombination repair (HRR) by GFP expression. HRR and nonhomologous end joining (NHEJ) were also determined by PCR. DSB repair was first detected at 12 h postinfection with an adenovirus expressing I-SceI with repair reaching plateau levels between 24 and 48 h. Within this time frame, NHEJ predominated over HRR in the range of 3-50-fold. To assess the involvement of ATM in DSB repair, we first examined whether ATM was associated with the DSB. Chromatin immunoprecipitation showed that ATM was present at the site of the DSB as early as 18 h postinfection. In cells treated with caffeine, an inhibitor of ATM, HRR was reduced, whereas NHEJ was not. In support of this finding, GFP flow cytometry demonstrated that caffeine reduced HRR by 90% under conditions when ATM kinase activity was inhibited. Dominant-negative ATM expressed from adenovirus inhibited HRR by 45%, also having little to no effect on NHEJ. Furthermore, HRR was inhibited by caffeine in serum-starved cells arrested in G 0 /G 1 , suggesting that ATM is also important for HRR outside of the S and G 2 cell cycle phases. Altogether, these results demonstrate that HRR contributes substantially to DSB repair in human glioma cells, and, importantly, ATM plays a critical role in regulating HRR but not NHEJ throughout the cell cycle.

Background The oncolytic virus, coxsackievirus A21 (CVA21), has shown promise as a single agent in several clinical trials and is now being tested in combination with immune checkpoint blockade. Combination therapies offer the best chance of disease control; however, the design of successful combination strategies requires a deeper understanding of the mechanisms underpinning CVA21 efficacy, in particular, the role of CVA21 anti-tumor immunity. Therefore, this study aimed to examine the ability of CVA21 to induce human anti-tumor immunity, and identify the cellular mechanism responsible. Methods This study utilized peripheral blood mononuclear cells from i) healthy donors, ii) Acute Myeloid Leukemia (AML) patients, and iii) patients taking part in the STORM clinical trial, who received intravenous CVA21; patients receiving intravenous CVA21 were consented separately in accordance with local institutional ethics review and approval. Collectively, these blood samples were used to characterize the development of innate and adaptive anti-tumor immune responses following CVA21 treatment. Results An Initial characterization of peripheral blood mononuclear cells, collected from cancer patients following intravenous infusion of CVA21, confirmed that CVA21 activated immune effector cells in patients. Next, using hematological disease models which were sensitive (Multiple Myeloma; MM) or resistant (AML) to CVA21-direct oncolysis, we demonstrated that CVA21 stimulated potent anti-tumor immune responses, including: 1) cytokine-mediated bystander killing; 2) enhanced natural killer cell-mediated cellular cytotoxicity; and 3) priming of tumor-specific cytotoxic T lymphocytes, with specificity towards known tumor-associated antigens. Importantly, immune-mediated killing of both MM and AML, despite AML cells being resistant to CVA21-direct oncolysis, was observed. Upon further examination of the cellular mechanisms responsible for CVA21-induced anti-tumor immunity we have identified the importance of type I IFN for NK cell activation, and demonstrated that both ICAM-1 and plasmacytoid dendritic cells were key mediators of this response. Conclusion This work supports the development of CVA21 as an immunotherapeutic agent for the treatment of both AML and MM. Additionally, the data presented provides an important insight into the mechanisms of CVA21-mediated immunotherapy to aid the development of clinical biomarkers to predict response and rationalize future drug combinations. Electronic supplementary material The online version of this article (10.1186/s40425-019-0632-y) contains supplementary material, which is available to authorized users.

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We have used a cDNA clone for human phosphoglycerate kinase (PGK) to examine the chromosomal localization of three members of the human PGK gene family. Using somatic cell hybrids segregating portions of several X-autosome translocations as well as a clone panel of hybrids segregating radiation-induced fragments of the human X chromosome, we assign a PGK pseudogene to the region Xq11-Xq13, proximal to the functional X-linked PGK gene located in Xq13. In addition, using a panel of 24 somatic cell hybrids, we assign an autosomal PGK-related DNA sequence to human chromosome 19.

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