Targeted Suicide Gene Therapy with Retroviral Replicating Vectors for Experimental Canine Cancers.

Cancer in dogs has increased in recent years and is a leading cause of death. We have developed a retroviral replicating vector (RRV) that specifically targets cancer cells for infection and replication. RRV carrying a suicide gene induced synchronized killing of cancer cells when administered with a prodrug after infection. In this study, we evaluated two distinct RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) in canine tumor models both in vitro and in vivo. Despite low infection rates in normal canine cells, both RRVs efficiently infected and replicated within all the canine tumor cells tested. The efficient intratumoral spread of the RRVs after their intratumoral injection was also demonstrated in nude mouse models of subcutaneous canine tumor xenografts. When both RRVs encoded a yeast cytosine deaminase suicide gene, which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil, they caused tumor-cell-specific 5-FC-induced killing of the canine tumor cells in vitro. Furthermore, in the AZACF- and AZACH-cell subcutaneous tumor xenograft models, both RRVs exerted significant antitumor effects. These results suggest that RRV-mediated suicide gene therapy is a novel therapeutic approach to canine cancers.

Therapeutic Efficacy of Prodrug Activator Gene Therapy Using Retroviral Replicating Vectors for Human Ovarian Cancer.

BACKGROUND/AIM: Retroviral replicating vectors (RRV) have exhibited efficient tumor transduction and improved therapeutic benefits in a variety of cancer models. In this study, we validated two RRV created from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which use different cell receptors for virus entry, in human ovarian cancer (OC) cells. MATERIALS AND METHODS: Expression levels of the receptors for AMLV (PiT-2) and GALV (PiT-1) in human OC cell lines (A2780, Caov3, RMG-1, SKOV-3), fibroblasts and HEK293 cells were evaluated using quantitative RT-PCR. In vitro RRV-GFP replication was monitored using flow cytometry, and cytotoxicity quantitated using AlamarBlue assay after 5-fluorocytosine treatment of OC cells transduced with RRV expressing the yeast cytosine deaminase prodrug activator gene. In vivo antitumor effect of RRV-mediated prodrug activator gene therapy was investigated in a SKOV-3 subcutaneous tumor model. RESULTS: Quantitative RT-PCR analysis revealed high expression levels of PiT-2 (AMLV receptor) and PiT-1 (GALV receptor) in the RMG-1 and SKOV3 OC cell lines, compared with their levels in non-malignant cells. In RMG-1 and SKOV3 cells, both RRV showed highly efficient RRV replication and spread leading to over 90% transduction by Days 10-13. Additionally, both RRV that express the yeast cytosine deaminase gene demonstrated effective cell killing of RMG-1 and SKOV-3 cells upon treatment with the prodrug 5-fluorocytosine. Notably, RRV-mediated prodrug activator gene therapy showed significant inhibition of subcutaneous SKOV-3 tumor growth in nude mice. CONCLUSION: RRV-mediated prodrug activator gene therapy may be used for treating PiT-expressing human OC.

Retroviral Replicating Vectors Mediated Prodrug Activator Gene Therapy in a Gastric Cancer Model.

Retroviral replicating vectors (RRVs) selectively replicate and can specifically introduce prodrug-activating genes into tumor cells, whereby subsequent prodrug administration induces the death of the infected tumor cells. We assessed the ability of two distinct RRVs generated from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which infect cells via type-III sodium-dependent phosphate transporters, PiT-2 and PiT-1, respectively, to infect human gastric cancer (GC) cells. A quantitative RT-PCR showed that all tested GC cell lines had higher expression levels of PiT-2 than PiT-1. Accordingly, AMLV, encoding a green fluorescent protein gene, infected and replicated more efficiently than GALV in most GC cell lines, whereas both RRVs had a low infection rate in human fibroblasts. RRV encoding a cytosine deaminase prodrug activator gene, which converts the prodrug 5-flucytosine (5-FC) to the active drug 5-fluorouracil, showed that AMLV promoted superior 5-FC-induced cytotoxicity compared with GALV, which correlated with the viral receptor expression level and viral spread. In MKN-74 subcutaneous xenograft models, AMLV had significant antitumor effects compared with GALV. Furthermore, in the MKN-74 recurrent tumor model in which 5-FC was discontinued, the resumption of 5-FC administration reduced the tumor volume. Thus, RRV-mediated prodrug activator gene therapy might be beneficial for treating human GC.

Influences of amino-terminal modifications on amyloid fibril formation of human serum amyloid A.

Human serum amyloid A (SAA) is a precursor protein involved in AA amyloidosis. The N-terminal region of the SAA molecule is crucial for amyloid fibril formation, and therefore modifications in this region are considered to influence the pathogenesis of AA amyloidosis. In the present study, using the N-terminal peptide corresponding to the putative first helix region of the SAA molecule, we investigated the influences of N-terminal modifications on amyloid fibril formation. Spectroscopic analyses revealed that carbamoylation of the N-terminal amino group delayed the onset of amyloid fibril formation. From transmission electron microscopic observations, the N-terminal carbamoylated aggregate showed remarkably different morphologies from the unmodified control. In contrast, acetylation of the N-terminal amino group or truncation of N-terminal amino acid(s) considerably diminished amyloidogenic properties. Furthermore, we also tested the cell toxicity of each peptide aggregate on cultured cells by two cytotoxic assays. Irrespective of carbamoylation or acetylation, MTT assay revealed that SAA peptides reduced the reductive activity of MTT on cells, whereas no apparent increase in LDH release was observed during an LDH assay. In contrast, N-terminal truncation did not affect either MTT reduction or LDH release. These results suggest that N-terminal modification of SAA molecules can act as a switch to regulate susceptibility to AA amyloidosis.

Stability and Oligomerization of Mutated SMN Protein Determine Clinical Severity of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. Approximately 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) gene deletion, while ~5% carry an intragenic SMN1 mutation. Here, we investigated the stability and oligomerization ability of mutated SMN1 proteins. Plasmids containing wild- and mutant-type SMN1 cDNA were constructed and transfected into HeLa cells. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar abundances of transcripts from the plasmids containing SMN cDNA, but Western blotting showed different expression levels of mutated SMN1 proteins, reflecting the degree of their instability. A mutated SMN1 protein with T274YfsX32 exhibited a much lower expression level than other mutated SMN1 proteins with E134K, Y276H, or Y277C. In immunoprecipitation analysis, the mutated SMN1 protein with T274YfsX32 did not bind to endogenous SMN1 protein in HeLa cells, suggesting that this mutation completely blocks the oligomerization with full-length SMN2 protein in the patient. The patient with T274YfsX32 showed a much more severe phenotype than the other patients with different mutations. In conclusion, the stability and oligomerization ability of mutated SMN1 protein may determine the protein stability and may be associated with the clinical severity of SMA caused by intragenic SMN1 mutation.

In silico and in vitro analyses of the pathological relevance of the R258H mutation of hepatocyte nuclear factor 4α identified in maturity-onset diabetes of the young type 1.

Mutations of the hepatocyte nuclear factor 4α (HNF4α) gene give rise to maturity-onset diabetes of the young type 1. Although many such mutations have been identified in affected individuals, part of these mutations has been characterized with regard to their pathological relevance. We here identified a missense mutation (c.773G>A, p.R258H) of HNF4A in a mother and daughter with early-onset diabetes and impaired insulin secretion. In silico simulation and in vitro luciferase reporter analyses showed that the mutation impairs the stability of self-dimerization and the transactivation activity of HNF4α. Although arginine-258 does not appear to participate directly in dimerization, its mutation alters the electrostatic surface potential of the dimer interface. Our results thus suggest that this mutation impairs the function of HNF4α and thereby contributes to the pathogenesis of maturity-onset diabetes of the young type 1.

Strawberry seed extract and its major component, tiliroside, promote ceramide synthesis in the stratum corneum of human epidermal equivalents.

The activation of peroxisomeproliferator-activated receptor (PPAR) α can stimulate the expression of ceramide-related enzymes, and a major component of strawberry seed extract (SSE) tiliroside enhances the expression of PPARα. We determined whether SSE and tiliroside may stimulate ceramide synthesis in the stratum corneum (SC) of the human epidermal equivalents (HEEs) culture model. Treatment with SSE at 1.0 and 3.0 µg/mL elicited a significant increase in the total ceramide content in the SC, which was accompanied by a significant increase in almost all ceramide species except for ceramide [EOS] and [AP]. Treatment with tiliroside at 0.3 µg/mL slightly accentuated the total ceramide content in the SC together with a significant increase in the ceramide [NS, NDS] content. Messenger RNA analysis demonstrated that SSE at 1 or 3 µg/mL significantly stimulated the gene expression of serine palmitoyltransferase (SPT) 2, ceramide synthase (CerS) 3, glucosylceramide synthase (GCS), and ß-glucocerebrosidase (GBA) but not of SPT1, sphingomyelin synthase (SMS) 1/2 and acid sphingomyelinase (ASM). In contrast, tiliroside elicited significant increases in the gene expression levels of GCS and GBA only at 0.3 and/or 0.1 µg/mL. Western blotting analysis revealed that both SSE and tiliroside enhanced the protein expression levels of GCS and GBA but not of SPT2 at 1 or 3 and 0.1 or 0.3 µg/mL, respectively. These findings suggested that both SSE and tiliroside have a distinct potential to stimulate the level of ceramide [NS, NDS] in the SC by enhancing the expression of GCS and GBA. The higher stimulatory effect with SSE than tiliroside on SC ceramide synthesis correlates with the significant increase observed with SSE but not tiliroside in the gene expression levels of SPT2 and CerS3. Therefore, it is anticipated that SSE is effective in improving skin barrier function and moisture retention in several ceramide-deficit skin conditions, including surfactant-induced roughened skin, xerosis, and atopic dermatitis.

SMA mutations in SMN Tudor and C-terminal domains destabilize the protein.

BACKGROUND AND PURPOSE: Most spinal muscular atrophy (SMA) patients are homozygous for survival of motor neuron 1 gene (SMN1) deletion. However, some SMA patients carry an intragenic SMN1 mutation. Such patients provide a clue to understanding the function of the SMN protein and the role of each domain of the protein. We previously identified mutations in the Tudor domain and C-terminal region of the SMN protein in three Japanese SMA patients. To clarify the effect of these mutations on protein stability, we conducted expression assays of SMN with mutated domains. PATIENTS AND METHODS: Patients A and B carried a mutation in SMN1 exon 3, which encodes a Tudor domain, c.275G>C (p.Trp92Ser). Patient C carried a mutation in SMN1 exon 6, which encodes a YG-box; c.819_820insT (p.Thr274Tyrfs). We constructed plasmid expression vectors containing wild-type and mutant SMN1 cDNAs. After transfection of HeLa cells with the expression plasmids, RNA and protein were isolated and analyzed by reverse-transcription PCR and western blot analysis. RESULTS: The abundance of wild-type and mutant SMN1 transcripts in HeLa cells was almost the same. However, western blot analysis showed lower levels of mutant SMN proteins compared with wild-type SMN. In mutant SMN proteins, it is noteworthy that the level of the p.Thr274Tyrfs mutant was much reduced compared with that of the p.Trp92Ser mutant. CONCLUSIONS: SMN mutations may affect the stability and levels of the protein.

Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is currently incurable. SMA is caused by decreased levels of the survival motor neuron protein (SMN), as a result of loss or mutation of SMN1. Although the SMN1 homolog SMN2 also produces some SMN protein, it does not fully compensate for the loss or dysfunction of SMN1. Salbutamol, a ß2-adrenergic receptor agonist and well-known bronchodilator used in asthma patients, has recently been shown to ameliorate symptoms in SMA patients. However, the precise mechanism of salbutamol action is unclear. We treated SMA fibroblast cells lacking SMN1 and HeLa cells with salbutamol and analyzed SMN2 mRNA and SMN protein levels in SMA fibroblasts, and changes in SMN protein ubiquitination in HeLa cells. Salbutamol increased SMN protein levels in a dose-dependent manner in SMA fibroblast cells lacking SMN1, though no significant changes in SMN2 mRNA levels were observed. Notably, the salbutamol-induced increase in SMN was blocked by a protein kinase A (PKA) inhibitor and deubiquitinase inhibitor, respectively. Co-immunoprecipitation assay using HeLa cells showed that ubiquitinated SMN levels decreased in the presence of salbutamol, suggesting that salbutamol inhibited ubiquitination. The results of this study suggest that salbutamol may increase SMN protein levels in SMA by inhibiting ubiquitin-mediated SMN degradation via activating ß2-adrenergic receptor-PKA pathways.

Trinucleotide insertion in the SMN2 promoter may not be related to the clinical phenotype of SMA.

BACKGROUND: More than 90% of spinal muscular atrophy (SMA) patients show homozygous deletion of SMN1 (survival motor neuron 1). They retain SMN2, a highly homologous gene to SMN1, which may partially compensate for deletion of SMN1. Although the promoter sequences of these two genes are almost identical, a GCC insertion polymorphism has been identified at c.-320_-321 in the SMN1 promoter. We have also found this insertion polymorphism in an SMN2 promoter in an SMA patient (Patient A) who has SMA type 2/3. PURPOSE: The aims of this study were to determine the frequency of the GCC insertion polymorphism in SMA patients, and to evaluate its effect on SMN transcription efficiency. PATIENTS AND METHODS: Fifty-one SMA patients, including Patient A, were involved in this study. SMN2 transcript levels in white blood cells were measured by real-time polymerase chain reaction. Screening of the GCC insertion polymorphism was performed using denaturing high-pressure liquid chromatography. The transcription efficiency of the promoter with the insertion mutation was evaluated using a reporter-gene assay. RESULTS: All SMA patients in this study were homozygous for SMN1 deletion. Patient A retained two copies of SMN2, and showed only a small amount of SMN2 transcript in white blood cells. We detected a GCC insertion polymorphism at c.-320_-321 only in Patient A, and not in 50 other SMA patients. The polymorphism had a slight but significant negative effect on transcription efficiency. DISCUSSION AND CONCLUSION: Patient A was judged to be an exceptional case of SMA, because the GCC insertion polymorphism rarely exists in SMN1-deleted SMA patients. The GCC insertion polymorphism did not enhance the transcriptional efficiency of SMN2. Thus, this GCC insertion polymorphism in the SMN2 promoter may not be associated with the milder phenotype of the patient. Patient A suggests that there are other unknown factors modifying the clinical phenotype of SMA.

Resveratrol enhances splicing of insulin receptor exon 11 in myotonic dystrophy type 1 fibroblasts.

INTRODUCTION: Myotonic dystrophy type 1 (DM1) is characterized by splicing abnormalities caused by CUG expansion of the DMPK gene transcript. Splicing of exon 11 of the insulin receptor (IR) gene is deregulated to suppress exon 11 inclusion into mRNA in DM1. Consequently, the exon 11-deleted IR isoform that is less sensitive to insulin is predominantly produced, leading to glucose intolerance in DM1. Upregulation of exon 11 retaining full-length IR mRNA is a potential way to recover insulin sensitivity in DM1. METHODS: We examined candidate chemicals for their ability to enhance inclusion of exon 11 of the IR gene in cultured cells by reverse transcription-PCR amplification of a fragment extending from exons 10 to 12 of IR mRNA. RESULTS: We revealed that resveratrol (RES) enhanced the percentage of exon 11-containing IR mRNA among the total IR mRNA in HeLa cells. The RES-mediated enhancement of exon 11 inclusion was cell-specific and highest in fibroblasts. We tested RES on four fibroblast samples from three generations of one DM1 family. In each sample, RES treatment significantly upregulated the percentage of exon 11-containing IR mRNA to levels higher than that of the control, irrespective of the length of the sample's CTG repeat expansion. DISCUSSION: A natural compound, RES, was shown for the first time to upregulate the full-length IR mRNA in fibroblasts from DM1 cases. Our results provide the justification of RES as a leading compound to improve glucose tolerance in DM1.