Biomedical subjects

Eugene S Kandel

Publications and source records attributed to Eugene S Kandel.

7 recordsLinked to original sources

PubMed2006

RNA-mediated response to heat shock in mammalian cells.

The heat-shock transcription factor 1 (HSF1) has an important role in the heat-shock response in vertebrates by inducing the expression of heat-shock proteins (HSPs) and other cytoprotective proteins. HSF1 is present in unstressed cells in an inactive monomeric form and becomes activated by heat and other stress stimuli. HSF1 activation involves trimerization and acquisition of a site-specific DNA-binding activity, which is negatively regulated by interaction with certain HSPs. Here we show that HSF1 activation by heat shock is an active process that is mediated by a ribonucleoprotein complex containing translation elongation factor eEF1A and a previously unknown non-coding RNA that we term HSR1 (heat shock RNA-1). HSR1 is constitutively expressed in human and rodent cells and its homologues are functionally interchangeable. Both HSR1 and eEF1A are required for HSF1 activation in vitro; antisense oligonucleotides or short interfering (si)RNA against HSR1 impair the heat-shock response in vivo, rendering cells thermosensitive. The central role of HSR1 during heat shock implies that targeting this RNA could serve as a new therapeutic model for cancer, inflammation and other conditions associated with HSF1 deregulation.

Animals↗

PubMed2006

RNA interference in cancer.

In the recent years, RNA interference (RNAi) has emerged as a major regulatory mechanism in eukaryotic gene expression. The realization that changes in the levels of microRNAs are directly associated with cancer led to the recognition of a new class of tumor suppressors and oncogenes. Moreover, RNAi has been turned into a potent tool for artificially modulating gene expression through the introduction of short interfering RNAs. A plethora of individual inhibitory RNAs as well as several large collections of these reagents have been generated. The systems for stable and regulated expression of these molecules emerged as well. These tools have helped to delineate the roles of various cellular factors in oncogenesis and tumor suppression and laid the foundation for new approaches in gene discovery. Furthermore, successful inhibition of tumor cell growth by RNAi aimed at oncogenes in vitro and in vivo supports the enthusiasm for potential therapeutic applications of this technique. In this article we review the evidence of microRNA involvement in cancer, the use of short interfering RNAs in forward and reverse genetics of this disease, and as well as both the benefits and limitations of experimental RNAi.

Animals↗

PubMed2005

Mutagenesis by reversible promoter insertion to study the activation of NF-kappaB.

Genetic dissection of signaling pathways in mammalian cells involves screening or selecting phenotypic mutants obtained by a variety of techniques. Limitations in current methods include inadequate genome coverage and difficulty in validating the link between mutation and phenotype. We describe an improved method for insertional mutagenesis with retroviral vectors and show that the ability to induce mutations increases greatly if a randomly inserted promoter directs transcription into the host DNA. The mutant phenotype is due to the expression of a hybrid transcript derived from the vector and the insertion site. Because other alleles of the affected gene remain intact, the phenotype is dominant, but is reversible by inactivating the promoter, for example, by site-specific recombination. Importantly, in mutant clones with multiple inserts, limited excision yields progeny with different patterns of inserts remaining. Characterizing these progeny allows the mutant phenotype to be associated with a specific target gene. Relative simplicity and robust target validation make the method suitable for a broad range of applications. We have used this technique to search for proteins that regulate NF-kappaB-dependent signaling in human cells. Two validated targets are the relA gene, which codes for the NF-kappaB p65 subunit, and the NF-kappaB regulator act1. Overexpression of the corresponding proteins, caused by insertion of a promoter into the first intron of each gene, leads to NF-kappaB-dependent secretion of factors that activate NF-kappaB through cell-surface receptors, establishing an autocrine loop.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Constitutive expression of E2F-1 leads to p21-dependent cell cycle arrest in S phase of the cell cycle.

p21 is a potent inhibitor of cyclin-dependent kinases capable of arresting cell cycle progression. p21 is primarily regulated at the transcriptional level by several transcription factors, including p53. Previously, we reported that certain members of the E2F family of transcription factors may activate p21 transcription via a p53-independent mechanism. To further elucidate the consequences of E2F-1-regulated induction of p21, we developed cell lines with a tamoxifen-dependent form of E2F-1. We confirmed direct interaction of E2F-1 with the proximal region of the p21 promoter. Interestingly, elevated E2F-1 activity was sufficient to arrest a substantial subset of cells in S phase and this effect was correlated to and dependent on the induction of p21 protein. Since E2F proteins control genes required for cell cycle progression and are activated by various oncogenic events, we believe that the p21-dependent arrest described in this report represents an additional mechanism that guards against unrestricted cell proliferation.

Animals↗

PubMed2002

Dominant negative form of signal-regulatory protein-alpha (SIRPalpha /SHPS-1) inhibits tumor necrosis factor-mediated apoptosis by activation of NF-kappa B.

Genetic suppressor element (GSE) methodology was applied to identify new genes controlling cell response to tumor necrosis factor (TNF). A retroviral library of randomly fragmented normalized cDNA from mouse fibroblasts was screened for GSEs capable of protecting NIH3T3 cells from TNF-induced apoptosis. The most abundant among isolated GSEs represented a fragment of cDNA encoding the C-terminal cytoplasmic region of the immunoglobulin family inhibitory receptor, SHPS-1 (mouse homologue of human SIRPalpha). Ectopic expression of this fragment (both from human and mouse versions) increased the NF-kappaB-dependent transcription in three cell lines tested; this effect could be reduced by the expression of full-length SIRPalpha, suggesting that the isolated GSE acts through a dominant negative mechanism. GSE-mediated activation of NF-kappaB depended on the presence of serum, was abrogated by wortmannin, and was associated with phosphorylation of PKB/Akt, suggesting that Akt mediates it. These data indicate that SIRPalpha/SHPS-1 is involved in negative regulation of NF-kappaB signaling.

3T3 Cells↗

PubMed2002

Template switching by RNA polymerase II in vivo. Evidence and implications from a retroviral system.

Transfection of retrovirus packaging cells with linear DNA from a retroviral vector missing the 3' long terminal repeat (3' LTR) results in production of infectious virus. Analysis of the newly formed proviruses indicates that restoration of the 3' LTR sequences necessary for reverse transcription and integration occurred due to end-to-end template switching by mammalian RNA polymerase II (RNAP II) in the packaging cells. These observations argue that RNAP II can utilize double-strand breaks and gaps in DNA to generate "recombinant" transcripts in vivo and suggest a mechanism for mutation and recombination of retroviruses.

Animals↗

PubMed2002

Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage.

Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G(2)/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G(2)/M to the G(1) phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G(2) arrest that is alleviated by activated Akt. Furthermore, the transition from the G(2)/M to the G(1) phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G(2)/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G(2)/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.

Animals↗