Effect of Repeat Dosing of Engineered Oncolytic Herpes Simplex Virus on Preclinical Models of Rhabdomyosarcoma

Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, is the most common sarcoma of childhood. Despite multidrug chemotherapy regimens, surgical intervention, and radiation treatment, outcomes remain poor, especially in advanced disease, and novel therapies are needed for the treatment of these aggressive malignancies. Genetically engineered oncolytic viruses, such as herpes simplex virus-1 (HSV), are currently being explored as treatments for pediatric tumors. M002, an oncolytic HSV, has both copies of the γ₁34.5 gene deleted, enabling replication in tumor cells but thwarting infection of normal, postmitotic cells. We hypothesized that M002 would infect human RMS tumor cells and lead to decreased tumor cell survival in vitro and impede tumor growth in vivo. In the current study, we demonstrated that M002 could infect, replicate in, and decrease cell survival in both embryonal (ERMS) and alveolar rhabdomyosarcoma (ARMS) cells. Additionally, M002 reduced xenograft tumor growth and increased animal survival in both ARMS and ERMS. Most importantly, we showed for the first time that repeated dosing of oncolytic virus coupled with low-dose radiation provided improved tumor response in RMS. These findings provide support for the clinical investigation of oncolytic HSV in pediatric RMS.     

Sequence-Dependent Upstream DNA-RNA Polymerase Interactions in the Open Complex with λPR and λPRM Promoters and Implications for the Mechanism of Promoter Interference

Upstream interactions of Escherichia coli RNA polymerase (RNAP) in an open promoter complex (RPo) formed at the P_R and P_RM promoters of bacteriophage λ have been studied by atomic force microscopy. We demonstrate that the previously described 30-nm DNA compaction observed upon RPo formation at P_R [Rivetti, C., Guthold, M. & Bustamante, C. (1999). Wrapping of DNA around the E. coli RNA polymerase open promoter complex. EMBO J., 18, 4464-4475.] is a consequence of the specific interaction of the RNAP with two AT-rich sequence determinants positioned from − 36 to − 59 and from − 80 to − 100. Likewise, RPos formed at P_RM showed a specific contact between RNAP and the upstream DNA sequence. We further demonstrate that this interaction, which results in DNA wrapping against the polymerase surface, is mediated by the C-terminal domains of α-subunits (carboxy-terminal domain). Substitution of these AT-rich sequences with heterologous DNA reduces DNA wrapping but has only a small effect on the activity of the P_R promoter. We find, however, that the frequency of DNA templates with both P_R and P_RM occupied by an RNAP significantly increases upon loss of DNA wrapping. These results suggest that α carboxy-terminal domain interactions with upstream DNA can also play a role in regulating the expression of closely spaced promoters. Finally, a model for a possible mechanism of promoter interference between P_R and P_RM is proposed.     

ERK‐1 MAP kinase prevents TNF‐induced apoptosis through bad phosphorylation and inhibition of Bax translocation in HeLa Cells

Extracellular signal‐regulated kinase (ERK) 1/2 signaling is involved in tumor cell survival through the regulation of Bcl‐2 family members. To explore this further and to demonstrate the central role of the mitochondria in the ERK1/2 pathway we used the HeLa cellular model where apoptosis was induced by tumor necrosis factor (TNF) and cycloheximide (CHX). We show that HeLa cells overexpressing ERK‐1 displayed resistance to TNF and CHX. HeLa cells overexpressing a kinase‐deficient form of ERK‐1 (K71R) were more sensitive to TNF and CHX. In the ERK‐1 cells, Bad was phosphorylated during TNF + CHX treatment. In the HeLa wt cells and in the K71R clones TNF and CHX decreased Bad phosphorylation. ERK‐1 cells treated with TNF and CHX did not release cytochrome c from the mitochondria. By contrast, HeLa wt and K71R clones released cytochrome c. Bax did not translocate to the mitochondria in ERK‐1 cells treated with TNF + CHX. Conversely, HeLa wt and K71R clones accumulated Bax in the mitochondria. In the HeLa wt cells and in both ERK‐1 transfectants Bid was cleaved and accumulated in the mitochondria. The caspase‐8 inhibitor IETD‐FMK and the mitochondrial membrane permeabilization inhibitor bongkrekic acid (BK), partially prevented cell death by TNF + CHX. Anisomycin, a c‐Jun N‐terminal kinases activator, increased TNF‐killing. The ERK‐1 cells were resistant to TNF and anisomycin, whereas K71R clones resulted more sensitive. Our study demonstrates that in HeLa cells the ERK‐1 kinase prevents TNF + CHX apoptosis by regulating the intrinsic mitochondrial pathway through different mechanisms. Inhibition of the intrinsic pathway is sufficient to almost completely prevent cell death. J. Cell. Biochem. 108: 1166–1174, 2009. © 2009 Wiley‐Liss, Inc.     

Bovine carboxylesterases: Evidence for two CES1 and five families of CES genes on chromosome 18

Predicted bovine carboxylesterase (CES) protein and gene sequences were derived from bovine (Bos taurus) genomic sequence data. Two bovine CES1 genes (CES1.1 and CES1.2) were located on chromosome 18 encoding amino acid sequences that were 81% identical. Two forms of CES1.2 were also observed apparently caused by an indel polymorphism encoded at the C-terminus end. Two CES gene clusters were observed on chromosome 18: CES5–CES1.1–CES1.2 and CES2–CES3–CES6. Bovine CES1, CES2, CES3, CES5 and CES6 shared 39–45% identity with each other, but showed 71–76% identity with each of the five corresponding human CES family members. Phylogeny studies indicated that bovine CES genes originated from five ancestral gene duplication events which predated the eutherian mammalian common ancestor. In addition, a subsequent CES1 gene duplication event is proposed during mammalian evolution prior to the appearance of the Bovidae common ancestor ~ 20 MY ago.     

Phosphorylation of the activation loop tyrosines is required for sustained Syk signaling and growth factor-independent B-cell proliferation

The Syk kinase is regarded as a promising target for the treatment of antigen-driven B-cell malignancies, considering its essential role in propagating antigenic stimuli through the B-cell receptor (BCR). In certain common B-cell malignancies Syk is activated even in the absence of BCR engagement, suggesting a wider role for this kinase in lymphomagenesis. In this paper, we have profiled molecular differences between BCR-induced and constitutive Syk activation in terms of phosphorylation of regulatory tyrosine residues, downstream signaling properties and capacity to sustain B-cell proliferation. Analysis of primary chronic lymphocytic leukemia B-cells and diffuse large B-cell lymphoma cell lines revealed that constitutive and BCR-induced Syk activation differ with respect to the phosphorylation status of the regulatory tyrosines at positions 352 and 525/526, with only the first site being phosphorylated in the case of constitutive and both sites in the case of BCR-induced Syk activation. Syk phosphorylated only on Y352 is capable of downstream signaling, as evidenced by experiments with a phosphomimetic mutant in which the activation loop tyrosines (YY525/526) were replaced with phenylalanines. However, phosphorylation at YY525/526 was shown to significantly increase the enzymatic activity of Syk and to be required for sustained PLCγ2, Akt and ERK signaling as well as B-cell transformation. These data demonstrate that constitutively active Syk and Syk activated by BCR crosslinking represent separate stages of Syk activation with distinct signaling properties and transforming capacities.     

Peptide gomesin triggers cell death through L-type channel calcium influx,MAPK/ERK,PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurria gomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species.     

Age and growth of tarpon,Megalops atlanticus,larvae in the eastern Gulf of Mexico,with notes on relative abundance and probable spawning areas

Synopsis Leptocephali were collected in June 1981 and July 1989 over the continental shelf and slope of the Florida west coast. Tarpon larvae ranged 5.5–24.4 mm standard length (SL) and were the second most abundant leptocephalus species. Sagittae examined with compound microscopes and scanning electron microscopy had increments that were presumed to be formed daily. Increment counts made using the two microscopic techniques were not significantly different. Estimated ages ranged 2–25 days with a growth rate (± standard error) of 0.92 ± 0.04 mm d^–1 The least squares linear regression equation SL = 2.78 + 0.92 (age in days) best described the relationship between estimated age and length. Adult tarpon appear to undergo a substantial spawning migration from inshore areas frequented during spring and summer to offshore spawning grounds. Spawning occurs during May, June, and July, although the spawning season may be of greater duration.     

Water utilization of tropical hardwood hammocks of the Lower Florida Keys

Summary Predawn water potential of representative plant species, together with stable isotope composition of stem water and potential water sources were investigated in four low-elevation tropical hardwood hammocks in the Lower Florida Keys, during a one year period. Hammock species had the lowest water potentials when soil water content was low and/or soil salinity was high, but differences in groundwater salinity had no effect on the water potential. Comparison of D/H ratio of plant stem water with soil and ground water corroborates the conclusion that they are primarily utilizing soil water and not groundwater. Thus, tropical hardwood hammocks are buffered from saline groundwater, and are able to thrive in areas where groundwater salinity is as high as 25. The effect of sea level rise on these forests may depend more on changes in the frequency of tidal inundation of the soil surface than on changes in groundwater salinity.     

Selecting barcoding loci for plants: evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants

There has been considerable debate, but little consensus regarding locus choice for DNA barcoding land plants. This is partly attributable to a shortage of comparable data from all proposed candidate loci on a common set of samples. In this study, we evaluated the seven main candidate plastid regions (rpoC1, rpoB, rbcL, matK, trnH‐psbA, atpF‐atpH, psbK‐psbI) in three divergent groups of land plants [Inga (angiosperm); Araucaria (gymnosperm); Asterella s.l. (liverwort)]. Across these groups, no single locus showed high levels of universality and resolvability. Interspecific sharing of sequences from individual loci was common. However, when multiple loci were combined, fewer barcodes were shared among species. Evaluation of the performance of previously published suggestions of particular multilocus barcode combinations showed broadly equivalent performance. Minor improvements on these were obtained by various new three‐locus combinations involving rpoC1, rbcL, matK and trnH‐psbA, but no single combination clearly outperformed all others. In terms of absolute discriminatory power, promising results occurred in liverworts (e.g. c. 90% species discrimination based on rbcL alone). However, Inga (rapid radiation) and Araucaria (slow rates of substitution) represent challenging groups for DNA barcoding, and their corresponding levels of species discrimination reflect this (upper estimate of species discrimination = 69% in Inga and only 32% in Araucaria; mean = 60% averaging all three groups).