Biomedical subjects

Nathan R Wall

Publications and source records attributed to Nathan R Wall.

11 recordsLinked to original sources

PubMed2004

Yin Yang 1 is a negative regulator of p53.

Yin Yang 1 (YY1) is a transcription factor that plays an essential role in development. However, the full spectrum of YY1's functions and mechanism of action remains unclear. We find that YY1 ablation results in p53 accumulation due to a reduction of p53 ubiquitination in vivo. Conversely, YY1 overexpression stimulates p53 ubiquitination and degradation. Significantly, recombinant YY1 is sufficient to induce Hdm2-mediated p53 polyubiquitination in vitro, suggesting that this function of YY1 is independent of its transcriptional activity. We identify direct physical interactions of YY1 with Hdm2 and p53 and show that the basis for YY1-regulating p53 ubiquitination is its ability to facilitate Hdm2-p53 interaction. Importantly, the tumor suppressor p14ARF compromises the Hdm2-YY1 interaction, which is important for YY1 regulation of p53. Taken together, these findings identify YY1 as a potential cofactor for Hdm2 in the regulation of p53 homeostasis and suggest a possible role for YY1 in tumorigenesis.

Alleles↗

PubMed2004

Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase.

A major cause of aging is thought to result from the cumulative effects of cell loss over time. In yeast, caloric restriction (CR) delays aging by activating the Sir2 deacetylase. Here we show that expression of mammalian Sir2 (SIRT1) is induced in CR rats as well as in human cells that are treated with serum from these animals. Insulin and insulin-like growth factor 1 (IGF-1) attenuated this response. SIRT1 deacetylates the DNA repair factor Ku70, causing it to sequester the proapoptotic factor Bax away from mitochondria, thereby inhibiting stress-induced apoptotic cell death. Thus, CR could extend life-span by inducing SIRT1 expression and promoting the long-term survival of irreplaceable cells.

Acetylation↗

PubMed2004

Stable expression of small interfering RNA sensitizes TEL-PDGFbetaR to inhibition with imatinib or rapamycin.

Small molecule inhibitors, such as imatinib, are effective therapies for tyrosine kinase fusions BCR-ABL-TEL-PDGFbetaR-mediated human leukemias, but resistance may develop. The unique fusion junctions of these molecules are attractive candidates for molecularly targeted therapeutic intervention using RNA interference (RNAi), which is mediated by small interfering RNA (siRNA). We developed a retroviral system for stable expression of siRNA directed to the unique fusion junction sequence of TEL-PDGFbetaR in transformed hematopoietic cells. Stable expression of the siRNA resulted in approximately 90% inhibition of TEL-PDGFbetaR expression and its downstream effectors, including PI3K and mammalian target of rapamycin (mTOR). Expression of TEL-PDGFbetaR-specific siRNA (TPsiRNA) significantly attenuated the proliferation of TEL-PDGFbetaR-transformed Ba/F3 cells or disease latency and penetrance in mice induced by intravenous injection of these Ba/F3 cells. Although a 90% reduction in TEL-PDGFbetaR expression was insufficient to induce cell death, stable siRNA expression sensitized transformed cells to the PDGFbetaR inhibitor imatinib or to the mTOR inhibitor rapamycin. TPsiRNA also inhibited an imatinib-resistant TEL-PDGFbetaR mutant, and the inhibition was enhanced by siRNA in combination with PKC412, another PDGFbetaR inhibitor. Although siRNA delivery in vivo is a challenging problem, stable expression of siRNA, which targets oncogenic fusion genes, may potentiate the effects of conventional therapy for hematologic malignancies.

Animals↗

PubMed2004

Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis.

Evasion of apoptosis is a hallmark of cancer, but the molecular circuitries of this process are not understood. Here we show that survivin, a member of the inhibitor of apoptosis gene family that is overexpressed in cancer, exists in a novel mitochondrial pool in tumor cells. In response to cell death stimulation, mitochondrial survivin is rapidly discharged in the cytosol, where it prevents caspase activation and inhibits apoptosis. Selective targeting of survivin to mitochondria enhances colony formation in soft agar, accelerates tumor growth in immunocompromised animals, and abolishes tumor cell apoptosis in vivo. Therefore, mitochondrial survivin orchestrates a novel pathway of apoptosis inhibition, which contributes to tumor progression.

Animals↗

PubMed2003

Small RNA: can RNA interference be exploited for therapy?

CONTEXT: RNA interference (RNAi) is the sequence-specific gene-silencing induced by double-stranded RNA (dsRNA), and gives information about gene function quickly, easily, and inexpensively. The use of RNAi for genetic-based therapies is widely studied, especially in viral infections, cancers, and inherited genetic disorders. RNAi has been used to make tissue-specific knockdown mice for studying gene function in a whole animal. Combined with genomics data, RNAi-directed gene-silencing could allow functional determination of any gene expressed in a cell or pathway. The term RNAi came from the discovery that the injection of dsRNAs into Caenorhabditis elegans interferes with the expression of specific genes containing a complementary region to the delivered dsRNA. Although stalled for a time by the non-gene-specific interferon response elicited by dsRNA molecules longer than about 30 nucleotides in mammalian cells, Tom Tuschl's group found that transfection of synthetic 21-nucleotide small-interfering RNA (siRNA) duplexes were highly selective and sequence-specific inhibitors of endogenous genes. STARTING POINT: siRNA expression has been studied with siRNA from plasmid and viral vectors that efficiently deliver siRNAs into both dividing and non-dividing cells, stem cells, zygotes, and their differentiated progeny. A collection of RNA interference vectors that suppress 50 human de-ubiquitinating enzymes allowed Thijn Brummelkamp and colleagues to study this gene family and to identify de-ubiquitinating enzymes in cancer-relevant pathways (Nature 2003; 424: 797-801). These researchers found that the familial cylindromatosis tumour suppressor gene (CYLD), previously of unknown function, could enhance the activation of the transcription factor NF-kappaB, leading to increased resistance to apoptosis. They have now started to investigate the use of CYLD inhibitors in clinical trials. WHERE NEXT: The ability to efficiently and stably produce and deliver sufficient amounts of siRNA to the proper target tissues require refinement before this new technology can be tried clinically. Initial in-vivo studies reported effective transgene suppression in adult mice by chemically synthesised siRNAs. More recently many researchers have used plasmid and viral vectors for transcription of short-hairpin RNAs, both in vitro and in vivo. With these expression systems, gene expression was more stably inhibited than with the transient knockdown recorded with chemically synthesised siRNA. Human trials exploiting these latest findings are likely to soon follow.

Animals↗

PubMed2003

Suppression of survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis.

Survivin is a member of the inhibitor of apoptosis gene family that is expressed in most human cancers and may facilitate evasion from apoptosis and aberrant mitotic progression. Here, exposure of breast carcinoma MCF-7 or cervical carcinoma HeLa cells to anticancer agents, including Adriamycin, Taxol, or UVB resulted in a 4-5-fold increased survivin expression. Changes in survivin levels after anticancer treatment did not involve modulation of survivin mRNA expression and were independent of de novo gene transcription. Conversely, inhibition of survivin phosphorylation on Thr(34) by the cyclin-dependent kinase inhibitor flavopiridol resulted in loss of survivin expression, and nonphosphorylatable survivin Thr(34)-->Ala exhibited accelerated clearance as compared with wild-type survivin. Sequential ablation of survivin phosphorylation on Thr(34) enhanced tumor cell apoptosis induced by anticancer agents independently of p53 and suppressed tumor growth without toxicity in a breast cancer xenograft model in vivo. These data suggest that Thr(34) phosphorylation critically regulates survivin levels in tumor cells and that sequential ablation of p34(cdc2) kinase activity may remove the survivin viability checkpoint and enhance apoptosis in tumor cells.

Antineoplastic Agents↗

PubMed2003

Therapeutic targeting of the survivin pathway in cancer: initiation of mitochondrial apoptosis and suppression of tumor-associated angiogenesis.

PURPOSE: Molecular antagonists of the inhibitor of apoptosis protein survivin have shown promise as novel anticancer strategies for triggering tumor cell apoptosis, dysregulating mitotic progression, and inhibiting tumor growth in preclinical models. However, how survivin couples to the cell death machinery has remained elusive, and the relevant cellular targets of survivin antagonists have not been completely elucidated. EXPERIMENTAL DESIGN: Human umbilical vein and dermal microvascular endothelial cells were infected with replication-deficient adenoviruses encoding survivin (pAd-Survivin), green fluorescent protein (pAd-GFP), or a phosphorylation-defective survivin Thr(34)-->Ala (pAd-T34A) dominant negative mutant. The effect of wild-type or mutant survivin was investigated on capillary network stability, endothelial cell viability, and caspase activation in vitro and on kinetics of tumor growth and development of angiogenesis in a breast cancer xenograft model in vivo. The cell death pathway initiated by survivin targeting was mapped with respect to cytochrome c release, changes in mitochondrial transmembrane potential, and apoptosome requirements using mouse embryonic fibroblasts deficient in Apaf-1 or caspase-9. RESULTS: Adenoviral transduction of endothelial cells with pAd-Survivin inhibited growth factor deprivation- or ceramide-induced apoptosis, reduced caspase-3 and -7 generation, and stabilized three-dimensional capillary networks in vitro. Conversely, expression of pAd-T34A caused apoptosis in umbilical vein and dermal microvascular endothelial cells and resulted in caspase-3 activity. Cell death induced by survivin targeting exhibited the hallmarks of mitochondrial-dependent apoptosis with release of cytochrome c and loss of mitochondrial transmembrane potential and was suppressed in Apaf-1 or caspase-9 knockout mouse embryonic fibroblasts. When injected in human breast cancer xenografts, pAd-T34A inhibited growth of established tumors and triggered tumor cell apoptosis in vivo. This was associated with a approximately 60% reduction in tumor-derived blood vessels by quantitative morphometry of CD31-stained tumor areas, and appearance of endothelial cell apoptosis by internucleosomal DNA fragmentation in vivo. CONCLUSIONS: Survivin functions as a novel upstream regulator of mitochondrial-dependent apoptosis, and molecular targeting of this pathway results in anticancer activity via a dual mechanism of induction of tumor cell apoptosis and suppression of angiogenesis.

Adenoviridae↗

PubMed2002

Induction and regulation of tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand-mediated apoptosis in renal cell carcinoma.

The lack of effective therapy for disseminated renal cell carcinoma (RCC) has stimulated the search for novel treatments including immunotherapeutic strategies. However, poor therapeutic responses and marked toxicity associated with immunological agents has limited their use. The tumor necrosis factor family member tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2 ligand induces apoptosis in a variety of tumor cell types, while having little cytotoxic activity against normal cells. In this study the activation and regulation of TRAIL-induced apoptosis and TRAIL receptor expression in human RCC cell lines and pathologic specimens was examined. TRAIL induced caspase-mediated apoptotic death of RCC cells with variable sensitivities among the cell lines tested. Compared with TRAIL-sensitive RCC cell lines (A-498, ACHN, and 769-P), the TRAIL-resistant RCC cell line (786-O) expressed lesser amounts of the death-inducing TRAIL receptors, and greater amounts of survivin, an inhibitor of apoptosis. Incubation of 786-O with actinomycin D increased the expression of the death-inducing TRAIL receptors and, concomitantly, decreased the intracellular levels of survivin, resulting in TRAIL-induced apoptotic death. The link between survivin and TRAIL regulation was confirmed when an increase in TRAIL resistance was observed after overexpression of survivin in the TRAIL-sensitive, survivin-negative RCC line A-498. These findings, along with our observation that TRAIL receptors are expressed in RCC tumor tissue, suggest that TRAIL may be useful as a therapeutic agent for RCC and that survivin may partially regulate TRAIL-induced cell death.

Adult↗

PubMed2002

Regulation of microtubule stability and mitotic progression by survivin.

Survivin is a member of the inhibitor of apoptosis (IAP) gene family, which has been implicated in both preservation of cell viability and regulation of mitosis in cancer cells. Here, we show that HeLa cells microinjected with a polyclonal antibody to survivin exhibited delayed progression in prometaphase (31.5 +/- 6.9 min) and metaphase (126.8 +/- 73.8 min), as compared with control injected cells (prometaphase, 21.5 +/- 3.3 min; metaphase, 18.9 +/- 4.5 min; P < 0.01). Cells injected with the antibody to survivin displayed short mitotic spindles severely depleted of microtubules and occasionally underwent apoptosis without exiting the mitotic block or thereafter. Forced expression of survivin in HeLa cells profoundly influenced microtubule dynamics with reduction of pole-to-pole distance at metaphase (8.57 +/- 0.21 microm versus 10.58 +/- 0.19 microm; P < 0.0001) and stabilization of microtubules against nocodazole-induced depolymerization in vivo. These data demonstrate that survivin functions at cell division to control microtubule stability and assembly of a normal mitotic spindle. This pathway may facilitate checkpoint evasion and promote resistance to chemotherapy in cancer.

Chromosomal Proteins, Non-Histone↗

PubMed2002

Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function.

Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala(3)-Ile(19) exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence, nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys(57)-Trp(67) identified a cytosolic pool of survivin, which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala(3)-Ile(19), Met(38)-Thr(48), Pro(47)-Phe(58) and Cys(57)-Trp(67) identified both survivin pools within the same mitotic cell. A ratio of approximately 1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34(cdc2), and was phosphorylated by p34(cdc2) on Thr(34), in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34(cdc2) and was not phosphorylated on Thr(34). Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.

Animals↗

PubMed2002

A p34(cdc2) survival checkpoint in cancer.

A checkpoint surveying the entry into mitosis responds to defects in spindle microtubule assembly/stability. This has been used to trigger apoptosis in cancer cells, but how the spindle checkpoint couples to the cell survival machinery has remained elusive. Here, we report that microtubule stabilization engenders a survival pathway that depends on elevated activity of p34(cdc2) kinase and increased expression of the apoptosis inhibitor and mitotic regulator, survivin. Pharmacologic, genetic, or molecular ablation of p34(cdc2) kinase after microtubule stabilization resulted in massive apoptosis independent of p53, suppression of tumor growth, and indefinite survival without toxicity in mice. By ablating this survival checkpoint, inhibitors of p34(cdc2) kinase could safely improve the efficacy of microtubule-stabilizing agents used to treat common cancers.

Adenine↗