Citations

Abstract We previously utilized a whole genome tiling array to identify novel transcripts which had altered expression in response to the genotoxic effects of the tobacco carcinogen NNK. Our goal was to identify novel long stress-induced non-coding transcripts that could play important regulatory roles within cells and be important targets of alteration during cancer formation. Utilizing very stringent criteria, we examined all transcriptionally active regions that had altered expression in response to NNK treatment and identified a group of 12 transcripts, termed long stress-induced noncoding trancripts (LSINCTs). These novel long non-coding transcripts are between 2 and 4 Kb in length and do not contain any open reading frames or have homology to any mRNAs. 11 LSINCTs are intergenically located and one LSINCT is intragenically located within the genome. We found LSINCTs to be more highly expressed in more proliferative normal human tissues and to be frequently over-expressed in a number of different cancers including breast cancer. We studied one of these transcripts in greater detail, LSINCT5. We found LSINCT5 to be a polyadenylated nuclear transcript of exactly 2,647 base pairs. It is highly over-expressed in both breast and ovarian cancers and knocking down its' expression in either a breast or ovarian cancer cell line resulted in decreased cellular proliferation. We further compared gene expression in the breast cancer cell line, MCF7, before and after knocking down LSINCT5 expression and identified a number of genes whose expression changed more than 2-fold in response. Two of the genes identified were the gene for the major paraspeckle protein PSPC1, and another long non-coding transcript NEAT1. Paraspeckles are irregularly shaped compartments found in the nucleus' interchromatin space. Paraspeckles are RNA-protein structures formed by the interaction of NEAT1 and members of the Drosophila Behavior Human Splicing family of proteins including PSPC1. Paraspeckles appear to be critical to the control of gene expression through the nuclear retention of certain species of RNA. It is though that paraspeckles and their components play a role in controlling gene expression during a number of different cellular processes including differentiation, viral infection and stress responses. Using RNA fluorescent in situ hybridization, we found LSINCT5 to co-localize with NEAT1 within the nucleus. Hence, this novel stress-induced transcript may be another important long non-coding transcript that is involved in the normal cellular function of the paraspeckles. Citation Format: David I. Smith, Silva M. Jessica. Long stress-induced non-coding transcripts (LSINCTs) and Cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A1.

Abstract Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.

Abstract Replication stress and DNA damage activate the ATR-Chk1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and Chk1 kinases in ovarian cancer cells using genetic and pharmacologic inhibitors in combination with cisplatin, topotecan, gemcitabine, and the PARP inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNA interference (RNAi)–mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan, and gemcitabine each activated Chk1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 nor the Chk1 inhibitor MK-8776 blocked ATR-mediated Chk1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-Chk1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. In addition, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan, and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and Chk1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents and that Chk1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-Chk1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells. Cancer Res; 73(12); 3683–91. ©2013 AACR.

Background: Early recognition and treatment of social and emotional disorders in children is significant for school preparation. These disorders are frequently underdetected without the use of standardized screening instruments. The purpose of our study is to describe the implementation of the Ages and Stages Questionnaire: Social–Emotional (ASQ:SE) in primary care practice by mail when children are 30 months old. Methods: In this 4-month study, parents of all 30-month-old children who receive primary care at our study site were mailed the ASQ:SE. In children who did not pass screening or received a call from a registered nurse for parental concerns documented on the questionnaire, short-term clinical outcomes were obtained from the electronic medical record. During the last month of the study, the demographics variables of race and insurance type were analyzed for an association with questionnaire completion by mail. Results: Of the 870 families mailed 30-month ASQ:SE screens, 507 (58.3%) were returned by mail. Out of the children with returned screens, 38 (7.5%) of parents were contacted for either elevated scores or concerning comments and 6 (1.2%) were referred to Early Intervention. Parents of children with government insurance returned the ASQ:SE questionnaire 34.2% (13/38) of the time compared with 65.5% (76/116) of those with private insurance ( P < .001). Conclusion: Our results indicate that mental health screening can be effectively managed in primary care practice by a registered nurse using a follow-up protocol. Mailing the ASQ:SE is likely not an effective way to comprehensively screen most primary care populations.