Dominick Sinicropi

Tumor-cell resistance to death receptor-induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax

The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor–mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive. Bax was dispensable for apical death-receptor signaling events including caspase-8 activation, but crucial for mitochondrial changes and downstream caspase activation. Treatment of colon tumor cells deficient in DNA mismatch repair with the death-receptor ligand apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selected in vitro or in vivo for refractory subclones with Bax frameshift mutations including deletions at a novel site. Chemotherapeutic agents upregulated expression of the Apo2L/TRAIL receptor DR5 and the Bax homolog Bak in Bax−/− cells, and restored Apo2L/TRAIL sensitivity in vitro and in vivo. Thus, Bax mutation in mismatch repair–deficient tumors can cause resistance to death receptor–targeted therapy, but pre-exposure to chemotherapy rescues tumor sensitivity.

Recovery of Cholinergic Phenotype in the Injured Rat Neostriatum: Roles for Endogenous and Exogenous Nerve Growth Factor

Abstract: Polyclonal antibodies against recombinant human nerve growth factor (rhNGF) potently inhibited PC12 neurite outgrowth, blocked high‐affinity 125I‐rhNGF binding but not its receptor, and cross‐reacted with rat, mouse, and human nerve growth factor (NGF) but not with brain‐derived neurotrophic factor, neurotrophin‐3, ciliary neurotrophic factor, insulin‐like growth factor, epidermal growth factor, or activin A. Immunocytochemistry revealed many NGF‐positive neurons in the rat neostriatum. The NGF‐positive neurons disappeared by 3 days after mechanical injury to the neostriatum and were replaced by intensely NGF‐ and glial fibrillary acidic protein‐positive astrocytes. Enzyme‐linked immunosorbent assay measurements revealed that the NGF content of the injured striatum was elevated by eightfold 3 days postinjury and by twofold 2 weeks later. The high‐affinity choline uptake (HACU) into cholinergic nerve terminals was decreased by 23% at 2 and 4 weeks postinjury, yet choline acetyltransferase (ChAT) activity in these neurons was unchanged at 2 weeks and decreased by 14% at 4 weeks. Daily infusion of 1 μg of rhNGF into the injury area did not alter the loss of HACU. However, this treatment elevated ChAT activity by 23–29% above intact neostriatal levels and by 53–65% relative to HACU at both survival times. Thus, lesion‐induced increases in NGF levels within astrocytes are associated with maintenance of striatal ChAT activity at normal levels following cholinergic injury, even with decreases in HACU. Pharmacologic doses of rhNGF can further augment ChAT activity in damaged cholinergic neurons, showing the usefulness of exogenous NGF even when endogenous NGF is elevated in response to injury.

Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method

Background Molecular tests for diagnosis of disease, particularly cancer, are gaining increased acceptance by physicians and their patients for disease prognosis and selection of treatment options. Gene expression profiles and genetic mutations are key parameters used for the molecular characterization of tumors. A variety of methods exist for mutation analysis but the development of assays with high selectivity tends to require a process of trial and error, and few are compatible with real-time PCR. We sought to develop a real-time PCR-based mutation assay methodology that successfully addresses these issues. Methodology/Principal Findings The method we describe is based on the widely used TaqMan® real-time PCR technology, and combines Allele-Specific PCR with a Blocking reagent (ASB-PCR) to suppress amplification of the wildype allele. ASB-PCR can be used for detection of germ line or somatic mutations in either DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor specimens. A set of reagent design rules was developed enabling sensitive and selective detection of single point substitutions, insertions, or deletions against a background of wild-type allele in thousand-fold or greater excess. Conclusions/Significance ASB-PCR is a simple and robust method for assaying single nucleotide mutations and polymorphisms within the widely used TaqMan® protocol for real time RT-PCR. The ASB-PCR design rules consistently produce highly selective mutation assays while obviating the need for redesign and optimization of the assay reagents. The method is compatible with formalin-fixed tissue and simultaneous analysis of gene expression by RT-PCR on the same plate. No proprietary reagents other than those for TaqMan chemistry are required, so the method can be performed in any research laboratory with real-time PCR capability.

Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue

We report a method for Selective Depletion of abundant RNA (SDRNA) species from Human total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue, here demonstrating removal of ribosomal and mitochondrial transcripts from clinical FFPE tissue RNA archived up to 20 years. Importantly, SDRNA removes 98% of targeted RNAs while preserving relative abundance profiles of non-targeted RNAs, enabling routine whole transcriptome analysis of clinically valuable archival tissue specimens by Next-Generation Sequencing.

Whole Transcriptome RNA-Seq Analysis of Breast Cancer Recurrence Risk Using Formalin-Fixed Paraffin-Embedded Tumor Tissue

RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.

Enhanced tumor killing by Apo2L/TRAIL and CPT-11 co-treatment is associated with p21 cleavage and differential regulation of Apo2L/TRAIL ligand and its receptors

Apo2L/TRAIL exhibits enhanced apoptotic activity in tumor xenograft models when used in combination with the topoisomerase 1 inhibitor CPT-11. To investigate the cellular mechanisms involved in this increased tumor-killing activity, a series of in vitro experiments were conducted using the human colon carcinoma cell line (HCT116). Apo2L/TRAIL induced a transient upregulation of DR5 mRNA, while CPT-11 increased both death and decoy receptor expression. Upregulation of decoy receptors by CPT-11 was partially inhibited by co-administration of Apo2L/TRAIL. CPT-11 treatment resulted in accumulation of cells at G2M-phase and correlated with a substantial increase in the protein levels of the cyclin-dependent kinase inhibitor p21. However, cells co-treated with CPT-11 and Apo2L/TRAIL, or pretreated with CPT-11 for up to 24 h followed by 2 h Apo2L/TRAIL, resulted in a caspase-dependent degradation of p21, reversal of G2-M phase arrest with a concomitant increase in apoptosis. The sequential treatment produced the greatest induction of DR5 and DR4, caspase-3-like cleavage/activation and p21 degradation, as well as increased apoptosis. These data indicate that the up-regulation of Apo2L/TRAIL ligand and its death receptors as well as cleavage of p21 protein in the Apo2L/TRAIL plus CPT-11 treatment contributes to the positive cooperation between these agents in enhancing tumor cell apoptosis.

Improved Potency of Hyperactive and Actin-resistant Human DNase I Variants for Treatment of Cystic Fibrosis and Systemic Lupus Erythematosus

The ability of recombinant human DNase I (DNase I) to degrade DNA to lower molecular weight fragments is the basis for its therapeutic use in cystic fibrosis (CF) patients and its potential use as a treatment for systemic lupus erythematosus (SLE). To increase the potency of human DNase I, we have generated and characterized three classes of mutants: (a) hyperactive variants, which have from one to six additional positively charged residues (+1 to +6) and digest DNA much more efficiently relative to wild type, (b) actin-resistant variants, which are no longer inhibited by G-actin, a potent inhibitor of DNase I, and (c) combination variants that are both hyperactive and actin-resistant. For DNA scission in CF sputum where the DNA concentration and length are large, we measured a ∼20-fold increase in potency relative to wild type for the +3 hyperactive variant Q9R/E13R/N74K or the actin-resistant variant A114F; the hyperactive and actin-resistant combination variant was ∼100-fold more potent than wild type DNase I. For digesting lower concentrations of DNA complexed to anti-DNA antibodies in human serum, we found a maximal enhancement of ∼400-fold over wild type for the +2 variant E13R/N74K. The +3 enzymes have ∼4000-fold enhancement for degrading moderate levels of exogenous DNA spiked into human serum, whereas the +6 enzyme has ∼30,000-fold increased activity for digesting the extremely low levels of endogenous DNA found in serum. The actin resistance property of the combination mutants further enhances the degree of potency in human serum. Thus, the human DNase I variants we have engineered for improved biochemical and pharmacodynamic properties have greater therapeutic potential for treatment of both CF and SLE.

Do nerve growth factor-related mechanisms contribute to loss of cutaneous nociception in leprosy?

Abstract While sensory loss in leprosy skin is the consequence of invasion by M. leprae of Schwann cells related to unmyelinated fibres, early loss of cutaneous pain sensation, even in the presence of nerve fibres and inflammation, is a hallmark of leprosy, and requires explanation. In normal skin, nerve growth factor (NGF) is produced by basal keratinocytes, and acts via its high affinity receptor (trk A) on nociceptor nerve fibres to increase their sensitivity, particularly in inflammation. We have therefore studied NGF‐ and trk A‐like immunoreactivity in affected skin and mirror‐site clinically‐unaffected skin from patients with leprosy, and compared these with non‐leprosy, control skin, following quantitative sensory testing at each site. Sensory tests were within normal limits in clinically‐unaffected leprosy skin, but markedly abnormal in affected skin. Sub‐epidermal PGP 9.5‐ and trk A‐ positive nerve fibres were reduced only in affected leprosy skin, with fewer fibres contacting keratinocytes. However, NGF‐immunoreactivity in basal keratinocytes, and intra‐epidermal PGP 9.5‐positive nerve fibres, were reduced in both sites compared to non‐leprosy controls, as were nerve fibres positive for the sensory neurone specific sodium channel SNS/PN3, which is regulated by NGF, and may mediate inflammation‐induced hypersensitivity. Keratinocyte trk A expression (which mediates an autocrine role for NGF) was increased in clinically affected and unaffected skin, suggesting a compensatory mechanism secondary to reduced NGF secretion at both sites. We conclude that decreased NGF‐ and SNS/PN3‐immunoreactivity, and loss of intra‐epidermal innervation, may be found without sensory loss on quantitative testing in clinically‐unaffected skin in leprosy; this appears to be a sub‐clinical change, and may explain the lack of cutaneous pain with inflammation. Sensory loss occurred with reduced sub‐epidermal nerve fibres in affected skin, but these still showed trk A‐staining, suggesting NGF treatment may restore pain sensation.

Distribution and localization of CMP-N-acetylneuraminic acid hydroxylase and N-glycolylneuraminic acid-containing glycoconjugates in porcine lymph node and peripheral blood lymphocytes.

An immunohistochemical analysis was performed on paraplast-embedded sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold labelling was used to investigate the subcellular localization of the hydroxylase. The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b5 is discussed.

Compaction assay: a rapid and simple in vitro method to assess the responsiveness of a biopolymer matrix to enzymatic modification.

A rapid and simple in vitro method is described which measures the extent of unrecoiled solids compression when a complex biopolymer is subjected to a centrifugal force. This method, termed the compaction assay, was used to assess the response of purulent cystic fibrosis (CF) sputum samples to the addition of recombinant human deoxyribonuclease I (rhDNase). Enzyme treatment resulted in a dramatic decrease in DNA size, a redistribution of total DNA content from the pellet to supernatant, a significant decrease in that pellet volume and a decrease in elastic modulus. Sample elasticity, measured by a dynamic cone and plate viscometer, could be related to compaction assay results. These results suggest that the compaction assay may be a useful in vitro method for rapidly assessing the actions of enzymatic disruption of a complex biopolymer, such as that observed for the actions of rhDNase on purulent airway secretions.