David L. Crowe

Conserved function of mSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis

In Drosophila embryos, the loss of sprouty gene function enhances branching of the respiratory system. Three human sprouty homologues (h-Spry1-3) have been cloned recently, but their function is as yet unknown [1]. Here, we show that a murine sprouty gene (mSpry-2), the product of which shares 97% homology with the respective human protein, is expressed in the embryonic murine lung. We used an antisense oligonucleotide strategy to reduce expression of mSpry-2 by 96%, as measured by competitive reverse transcriptase PCR, in E11. 5 murine embryonic lungs cultured for 4 days [2]. Morphologically, the decrease in mSpry-2 expression resulted in a 72% increase in embryonic murine lung branching morphogenesis as well as a significant increase in expression of the lung epithelial marker genes SP-C, SP-B and SP-A. These results support a striking conservation of function between the Drosophila and mammalian sprouty gene families to negatively modulate respiratory organogenesis.

E2F-1 represses transcription of the human telomerase reverse transcriptase gene

The ends of human chromosomes (telomeres) lose up to 200 bp of DNA per cell division. Chromosomal shortening ultimately leads to senescence and death in normal cells. Many human carcinoma lines are immortal in vitro, suggesting that these cells have a mechanism for maintaining the ends of their chromosomes. Telomerase is a ribonucleoprotein complex that synthesizes telomeric DNA onto chromosomes using its RNA component as template. Telomerase activity is found in most tumor cells, but is absent from normal cells. Little is known about how normal human cells repress telomerase (hTERT) gene expression. Mice carrying an E2F-1 null mutation develop a variety of malignant tumors, suggesting that this transcription factor has a tumor suppressor function. To determine mechanisms by which E2F-1 suppresses tumor formation, we examined the role of this transcription factor in regulation of the hTERT promoter in human cells. We identified two putative E2F-1-binding sites proximal to the transcriptional start site of the hTERT promoter. Mutation of these sites produced dramatic increases in promoter activity. Overexpression of E2F-1 but not a mutant E2F-1 repressed hTERT promoter activity in reporter gene assays. This repression was abolished by mutation of the E2F-1-binding sites in the hTERT promoter. Human cancer cell lines stably overexpressing E2F-1 exhibited decreased hTERT mRNA expression and telomerase activity. We conclude that E2F-1 has an atypical function as a transcriptional repressor of the hTERT gene in human cells.

A retinoid X receptor (RXR)-selective retinoid reveals that RXR-α is potentially a therapeutic target in breast cancer cell lines, and that it potentiates antiproliferative and apoptotic responses to peroxisome proliferator-activated receptor ligands

IntroductionCertain lipids have been shown to be ligands for a subgroup of the nuclear hormone receptor superfamily known as the peroxisome proliferator-activated receptors (PPARs). Ligands for these transcription factors have been used in experimental cancer therapies. PPARs heterodimerize and bind DNA with retinoid X receptors (RXRs), which have homology to other members of the nuclear receptor superfamily. Retinoids have been found to be effective in treating many types of cancer. However, many breast cancers become resistant to the chemotherapeutic effects of these drugs. Recently, RXR-selective ligands were discovered that inhibited proliferation of all-trans retinoic acid resistant breast cancer cells in vitro and caused regression of the disease in animal models. There are few published studies on the efficacy of combined therapy using PPAR and RXR ligands for breast cancer prevention or treatment.MethodsWe determined the effects of selective PPAR and RXR ligands on established human breast cancer cell lines in vitro.ResultsPPAR-α and PPAR-γ ligands induced apoptotic and antiproliferative responses in human breast cancer cell lines, respectively, which were associated with specific changes in gene expression. These responses were potentiated by the RXR-selective ligand AGN194204. Interestingly, RXR-α-overexpressing retinoic acid resistant breast cancer cell lines were more sensitive to the effects of the RXR-selective compound.ConclusionRXR-selective retinoids can potentiate the antiproliferative and apoptotic responses of breast cancer cell lines to PPAR ligands.

Hypoxic induction of HIF-1α and VEGF expression in head and neck squamous cell carcinoma lines is mediated by stress activated protein kinases

Solid tumors must establish a blood supply in order to proliferate and grow. Cancer cells secrete soluble factors which can induce proliferation and migration of capillary endothelial cells. Among the most potent of the angiogenic factors is vascular endothelial growth factor (VEGF). Increased VEGF expression by malignant tumors has been associated with high vascularity, increased cancer cell growth, and lymph node metastasis. Reduced oxygen tension has been shown to increase VEGF production by induction of the transcription factor hypoxia inducible factor 1 alpha (HIF-1alpha). The mechanisms by which hypoxic tumor environments induce HIF-1alpha and VEGF expression are largely unknown. Jun N terminal kinase (JNK1) and p38 kinase are activated by a variety of stress stimuli. To determine if hypoxic activation of these stress activated protein kinases regulated HIF-1alpha and VEGF expression, we assayed JNK1 and p38 activity in squamous cell carcinoma (SCC) lines grown under normoxic or hypoxic conditions. Hypoxia rapidly induced both JNK1 and p38 activity in these cells. This activation correlated with induction of HIF-1alpha expression and DNA binding activity which was blocked by the p38 inhibitor SB203580. Hypoxia also increased VEGF production by SCC lines, which was inhibited by treatment with SB203580. Overexpression of JNK1 or p38 was sufficient to induce HIF-1alpha and VEGF expression. These results indicate that induction of SAPKs by hypoxia regulates HIF-1alpha and VEGF expression in head and neck carcinoma cell lines.

EphB4 provides survival advantage to squamous cell carcinoma of the head and neck

The receptor tyrosine kinase EphB4 and its ligand EphrinB2 play critical roles in blood vessel maturation, and are frequently overexpressed in a wide variety of cancers. We studied the aberrant expression and biological role of EphB4 in head and neck squamous cell carcinoma (HNSCC). We tested the effect of EphB4‐specific siRNA and antisense oligonucleotides (AS‐ODN) on cell growth, migration and invasion, and the effect of EphB4 AS‐ODN on tumor growth in vivo. All HNSCC tumor samples express EphB4 and levels of expression correlate directly with higher stage and lymph node metastasis. Six of 7 (86%) HNSCC cell lines express EphB4, which is induced either by EGFR activation or by EPHB4 gene amplification. EphrinB2 was expressed in 65% tumors and 5 of 7 (71%) cell lines. EphB4 provides survival advantage to tumor cells in that EphB4 siRNA and AS‐ODN significantly inhibit tumor cell viability, induce apoptosis, activate caspase‐8, and sensitize cells to TRAIL‐induced cell death. Furthermore, EphB4‐specific AS‐ODN significantly inhibits the growth of HNSCC tumor xenografts in vivo. Expression of EphB4 in HNSCC tumor cells confers survival and invasive properties, and thereby provides a strong rationale for targeting EphB4 as novel therapy for HNSCC.

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

BackgroundIntegrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines.MethodsAdapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays.ResultsIn the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity.ConclusionsWe concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

Rb and E2F-1 regulate telomerase activity in human cancer cells.

The ends of human chromosomes (telomeres) lose up to 200 bp of DNA per cell division. Chromosomal shortening ultimately leads to senescence and death in normal cells. Many human carcinoma lines are immortal in vitro, suggesting that these cells have a mechanism for maintaining the ends of their chromosomes. Telomerase is a ribonucleoprotein complex that synthesizes telomeric DNA onto chromosomes using its RNA component as a template. Recent studies have shown that inactivation of the retinoblastoma gene product pRb and the cyclin dependent kinase inhibitor p16(INK4A) is required for telomerase activity in epithelial cells. We have demonstrated previously that restoration of functional retinoblastoma (Rb) expression is sufficient to downregulate telomerase activity in carcinoma cells. To determine mechanisms by which Rb regulates telomerase expression, we examined the effects of cyclin dependent kinase (cdk) mediated Rb inactivation and the release of E2F-1 on telomerase activity in human carcinoma cells. Overexpression of cdk2 and cdk4 but not a dominant negative cdk2 rescued Rb mediated downregulation of telomerase activity. Overexpression of the cdk regulatory subunit cyclin D1 also rescued telomerase downregulation and p16 expression alone was sufficient to ablate activity. E2F-1 overexpression was sufficient to rescue Rb mediated reduction of telomerase activity, but an E2F-1 mutant defective in DNA and Rb binding activities failed to produce this effect. Tumor tissue from E2F-1 -/- mice was negative for telomerase activity, indicating a key regulatory role for this transcription factor.

Head and neck squamous cell carcinoma lines produce biologically active angiogenic factors

Angiogenesis is the formation of capillaries from pre-existing blood vessels. Angiogenesis occurs in many developmental, physiologic, and pathologic processes including tumor growth. Previous studies have shown that angiogenesis is required for growth and metastasis of solid tumors. Fibroblast growth factors (FGF-1, -2) and vascular endothelial growth factor (VEGF) are extremely potent angiogenesis inducers by stimulating the proliferation and migration of capillary endothelial cells. Expression of these factors is upregulated in many solid tumors and correlates with high vascularity, lymph node metastasis, and poor clinical prognosis. Few studies have examined whether established head and neck squamous cell carcinoma (SCC) lines produce biologically active angiogenic factors. By immunoprecipitation, we detected FGF and VEGF proteins in cellular lysates of SCC lines. We also detected FGF-1 and -2 proteins in serum-free-conditioned medium from these lines. Conditioned medium from SCC lines significantly increased proliferation of human umbilical vein endothelial cells (HUVEC). This increased proliferation was abrogated by pre-incubation of conditioned medium with neutralizing antibodies to FGFs and VEGF. Conditioned medium from SCC lines also significantly stimulated HUVEC invasion across a reconstituted basement membrane. We concluded that head and neck SCC lines secrete biologically active angiogenic factors.

Smad7 is a TGF-β-inducible attenuator of Smad2/3-mediated inhibition of embryonic lung morphogenesis

Smad7 was recently shown to antagonize TGF-beta-induced activation of signal-transducing Smad2 and Smad3 proteins. However, the biological function of Smad7 in the process of lung organogenesis is not known. Since Smad2/3-mediated TGF-beta signaling is known to inhibit embryonic lung branching morphogenesis, we tested the hypothesis that Smad7 regulates early lung development by modulating TGF-beta signal transduction. An antisense oligodeoxynucleotide (ODN) was designed to specifically block endogenous Smad7 gene expression at both transcriptional and translational levels in embryonic mouse lungs in culture. TGF-beta-mediated inhibition of lung branching morphogenesis was significantly potentiated in cultured embryonic lungs in the absence of Smad7 gene expression: abrogation of Smad7 potentiated TGF-beta-mediated inhibition of lung branching morphogenesis from 76 to 52% of the basal level in lungs cultured in the presence of 5 ng/ml TGF-beta1 ligand. Likewise, TGF-beta1 EC(50) (concentration of TGF-beta1 that induced half maximal branching inhibition) was reduced from 5 to 1 ng/ml when Smad7 gene expression was abrogated in lung culture, indicating an enhanced level of TGF-beta signaling in lung tissue with abolished Smad7 gene expression. By immunocytochemistry, Smad7 protein was co-localized with both Smad2 and Smad3 in distal bronchial epithelial cells, supporting the concept that Smad7 inhibits TGF-beta signaling by competing locally with Smad2 and Smad3 for TGF-beta receptor complex binding during lung morphogenesis. Furthermore, antisense Smad7 ODN increased the negative effect of TGF-beta1 on epithelial cell growth in developing lungs in culture. We also demonstrated that Smad7 mRNA levels were rapidly and potently induced upon TGF-beta1 stimulation of lungs in culture, suggesting that Smad7 regulates TGF-beta responses in a negative feedback loop. These studies define a novel function for Smad7 as an intracellular antagonist of TGF-beta-induced, Smad2/3-mediated inhibition of murine embryonic lung growth and branching morphogenesis in culture. The optimization of TGF-beta signaling during early lung development therefore requires a finely-regulated competitive balance between both permissive and inhibitory members of the Smad family.

Dynamic optimization of micro-operations

Inherent within complex instruction set architectures such as /spl times/86 are inefficiencies that do not exist in a simpler ISA. Modern /spl times/86 implementations decode instructions into one or more micro-operations in order to deal with the complexity of the ISA. Since these micro-operations are not visible to the compiler the stream of micro-operations can contain redundancies even in statically optimized /spl times/86 code. Within a processor implementation, however barriers at the ISA level do not apply, and these redundancies can be removed by optimizing the micro-operation stream. In this paper we explore the opportunities to optimize code at the micro-operation granularity. We execute these micro-operation optimizations using the rePLay Framework as a microarchitectural substrate. Using a simple set of seven optimizations, including two that aggressively and speculatively attempt to remove redundant load instructions, we examine the effects of dynamic optimization of micro-operations using a trace-driven simulation environment. Simulation reveals that across a sampling of SPECint 2000 and real /spl times/86 applications, rePLay is able to reduce micro-operation count by 21% and, in particular load micro-operation count by 22%. These reductions correspond to a boost in observed instruction-level parallelism on an 8-wide optimizing rePLay processor by 17% over a non-optimizing configuration.