Optimised protocols to generate high titre lentiviral vectors using a novel transfection agent enabling extended HEK293T culture following transient transfection and suspension culture.

HIV-1 based lentiviral viruses are considered powerful and versatile gene therapy vectors to deliver therapeutic genes to patients with hereditary or acquired diseases. These vectors can efficiently transduce a variety of cell types when dividing or non-dividing to provide permanent delivery and long-term gene expression. Demand for scalable manufacturing protocols able to generate enough high titre vector for widespread use of this technology is increasing and considerable efforts to improve vector production cost-effectively, is ongoing. Current methods for LV production mainly use transient transfection of producer cell lines. Cells can be grown at scale, either in 2D relying on culturing producer cells in multi-tray flask cell culture factories or in roller bottles or can be adapted to grow in 3D suspensions in large batch fermenters. This suits rapid production and testing of new vector constructs pre-clinically for their efficacy, particle titre and safety. In this study, we sought to improve lentiviral titre over time by testing two alternative commercially available transfection reagents Fugene® 6 and Genejuice® with the commonly used polycation, polyethyleneimine. Our aim was to identify less cytotoxic transfection reagents that could be used to generate LV particles at high titre past the often used 72 h period when vector is usually collected before producer cell death is caused due to post transfection cytotoxicity. We show that LV could be produced in extended culture using Genejuice® and even by transfected cells in glass flasks in suspension. Because this delivery agent is less toxic to 293 T producer cells, following optimisation of transfection we found that LV can be harvested for more than 10 days at high titre. Using our protocol, titres of 109 TU/ml and 108 TU/ml were routinely reached via traditional monolayer conditions or suspension cultures, respectively. We propose, this simple change in vector production enables large volumes of high titre vector to be produced, cost effectively for non-clinical in vivo and in vitro applications or for more stringent downstream clinical grade vector purification.

Dyskeratosis Congenita Links Telomere Attrition to Age-Related Systemic Energetics.

The underlying mechanisms of plasma metabolite signatures of human aging and age-related diseases are not clear but telomere attrition and dysfunction are central to both. Dyskeratosis congenita (DC) is associated with mutations in the telomerase enzyme complex (TERT, TERC, and DKC1) and progressive telomere attrition. We analyzed the effect of telomere attrition on senescence-associated metabolites in fibroblast-conditioned media and DC patient plasma. Samples were analyzed by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. We showed extracellular citrate was repressed by canonical telomerase function in vitro and associated with DC leukocyte telomere attrition in vivo, leading to the hypothesis that altered citrate metabolism detects telomere dysfunction. However, elevated citrate and senescence factors only weakly distinguished DC patients from controls, whereas elevated levels of other tricarboxylic acid cycle (TCA) metabolites, lactate, and especially pyruvate distinguished them with high significance. The DC plasma signature most resembled that of patients with loss of function pyruvate dehydrogenase complex mutations and that of older subjects but significantly not those of type 2 diabetes, lactic acidosis, or elevated mitochondrial reactive oxygen species. Additionally, our data are consistent with further metabolism of citrate and lactate in the liver and kidneys. Citrate uptake in certain organs modulates age-related disease in mice and our data have similarities with age-related disease signatures in humans. Our results have implications for the role of telomere dysfunction in human aging in addition to its early diagnosis and the monitoring of anti-senescence therapeutics, especially those designed to improve telomere function.

The role of BRCA2 in the fragility of interstitial telomeric sites.

We examined frequencies of radiation-induced chromosomal aberrations, using classical cytological methods, and DNA damage in interphase and metaphase cells, using a combination of FISH, CO-FISH, TIF (telomere dysfunction induced assay) and simultaneous detection of DNA damage and telomeric sequences in metaphase chromosomes, in Chinese hamster cells defective in BRCA2 and control cells. Given that the Chinese hamster genome contains large blocks of interstitial telomeric sites, our results allow us to examine the role of BRCA2 in the potential fragility of these sites, but also whether BRCA2 affects DNA repair within terminal telomeric sequences. BRCA2 defective cells exhibited greater frequencies of DNA damage within interstitial telomeric sites, as well as within terminal telomeric sites, relative to control cells. Therefore, BRCA2 deficiency contributes to the telomere dysfunction phenotype in Chinese hamster cells.

Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells.

Human surfactant protein D (SP-D) belongs to the family of collectins that is composed of a characteristic amino-terminal collagenous region and a carboxy-terminal C-type lectin domain. Being present at the mucosal surfaces, SP-D acts as a potent innate immune molecule and offers protection against non-self and altered self, such as pathogens, allergens, and tumor. Here, we examined the effect of a recombinant fragment of human SP-D (rfhSP-D) on a range of breast cancer lines. Breast cancer has four molecular subtypes characterized by varied expressions of estrogen (ER), progesterone (PR), and epidermal growth factor (EGF) receptors (HER2). The cell viability of HER2-overexpressing (SKBR3) and triple-positive (BT474) breast cancer cell lines [but not of a triple-negative cell line (BT20)] was reduced following rfhSP-D treatment at 24 h. Upregulation of p21/p27 cell cycle inhibitors and p53 phosphorylation (Ser15) in rfhSP-D-treated BT474 and SKBR3 cell lines signified G2/M cell cycle arrest. Cleaved caspases 9 and 3 were detected in rfhSP-D-treated BT474 and SKBR3 cells, suggesting an involvement of the intrinsic apoptosis pathway. However, rfhSP-D-induced apoptosis was nullified in the presence of hyaluronic acid (HA) whose increased level in breast tumor microenvironment is associated with malignant tumor progression and invasion. rfhSP-D bound to solid-phase HA and promoted tumor cell proliferation. rfhSP-D-treated SKBR3 cells in the presence of HA showed decreased transcriptional levels of p53 when compared to cells treated with rfhSP-D only. Thus, HA appears to negate the anti-tumorigenic properties of rfhSP-D against HER2-overexpressing and triple-positive breast cancer cells.

Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression.

Narrowing the search for the critical hTERT repressor sequence(s) has identified three regions on chromosome 3p (3p12-p21.1, 3p21.2 and 3p21.3-p22). However, the precise location and identity of the sequence(s) responsible for hTERT transcriptional repression remains elusive. In order to identify critical hTERT repressor sequences located within human chromosome 3p12-p22, we investigated hTERT transcriptional activity within 21NT microcell hybrid clones containing chromosome 3 fragments. Mapping of chromosome 3 structure in a single hTERT-repressed 21NT-#3fragment hybrid clone, revealed a 490kb region of deletion localised to 3p21.3 and encompassing the histone H3, lysine 36 (H3K36) trimethyltransferase enzyme SETD2; a putative tumour suppressor gene in breast cancer. Three additional genes, BAP1, PARP-3 and PBRM1, were also selected for further investigation based on their location within the 3p21.1-p21.3 region, together with their documented role in the epigenetic regulation of target gene expression or hTERT regulation. All four genes (SETD2, BAP1, PARP-3 and PBRM1) were found to be expressed at low levels in 21NT. Gene copy number variation (CNV) analysis of SETD2, BAP1, PARP-3 and PBRM1 within a panel of nine breast cancer cell lines demonstrated single copy number loss of all candidate genes within five (56%) cell lines (including 21NT cells). Stable, forced overexpression of BAP1, but not PARP2, SETD2 or PBRM1, within 21NT cells was associated with a significant reduction in hTERT expression levels relative to wild-type controls. We propose that at least two sequences exist on human chromosome 3p, that function to regulate hTERT transcription within human breast cancer cells.

A mechanistic evaluation of the Syrian hamster embryo cell transformation assay (pH 6.7) and molecular events leading to senescence bypass in SHE cells.

The implementation of the Syrian hamster embryo cell transformation assay (SHE CTA) into test batteries and its relevance in predicting carcinogenicity has been long debated. Despite prevalidation studies to ensure reproducibility and minimise the subjective nature of the assay's endpoint, an underlying mechanistic and molecular basis supporting morphological transformation (MT) as an indicator of carcinogenesis is still missing. We found that only 20% of benzo(a)pyrene-induced MT clones immortalised suggesting that, alone, the MT phenotype is insufficient for senescence bypass. From a total of 12 B(a)P- immortalised MT lines, inactivating p53 mutations were identified in 30% of clones, and the majority of these were consistent with the potent carcinogen's mode of action. Expression of p16 was commonly silenced or markedly reduced with extensive promoter methylation observed in 45% of MT clones, while Bmi1 was strongly upregulated in 25% of clones. In instances where secondary events to MT appeared necessary for senescence bypass, as evidenced by a transient cellular crisis, clonal growth correlated with monoallelic deletion of the CDKN2A/B locus. The findings further implicate the importance of p16 and p53 pathways in regulating senescence while providing a molecular evaluation of SHE CTA -derived variant MT clones induced by benzo(a)pyrene.

The effect of chemotherapeutic agents on telomere length maintenance in breast cancer cell lines.

Mammalian telomeric DNA consists of tandem repeats of the sequence TTAGGG associated with a specialized set of proteins, known collectively as Shelterin. These telosomal proteins protect the ends of chromosomes against end-to-end fusion and degradation. Short telomeres in breast cancer cells confer telomere dysfunction and this can be related to Shelterin proteins and their level of expression in breast cancer cell lines. This study investigates whether expression of Shelterin and Shelterin-associated proteins are altered, and influence the protection and maintenance of telomeres, in breast cancer cells. 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A (TSA) were used in an attempt to reactivate the expression of silenced genes. Our studies have shown that Shelterin and Shelterin-associated genes were down-regulated in breast cancer cell lines; this may be due to epigenetic modification of DNA as the promoter region of POT1 was found to be partially methylated. Shelterin genes expression was up-regulated upon treatment of 21NT breast cancer cells with 5-aza-CdR and TSA. The telomere length of treated 21NT cells was measured by q-PCR showed an increase in telomere length at different time points. Our studies have shown that down-regulation of Shelterin genes is partially due to methylation in some epithelial breast cancer cell lines. Removal of epigenetic silencing results in up-regulation of Shelterin and Shelterin-associated genes which can then lead to telomere length elongation and stability.

The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis.

Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.

Identification of subpopulations of cells with differing telomere lengths in mouse and human cell lines by flow FISH.

BACKGROUND: Telomeres are specialized nucleoprotein structures at chromosome ends that undergo dynamic changes after each cell cycle. Understanding the mechanisms of telomere dynamics is critically dependent on the ability to accurately measure telomere length in a cell population of interest. Techniques such as Southern blot, which measures average telomere length, and quantitative fluorescence in situ hybridization (Q-FISH), which can estimate telomere length in individual chromosomes, are limited in their capacity to determine the distribution of cells with differing telomere lengths in a given cell population. METHODS: We employed flow-FISH to determine whether mouse and human cell lines exhibit subpopulations of cells with differing telomere lengths. RESULTS: Our analysis showed that at least one of four analyzed mouse cell lines had two subpopulations of cells with differing telomere lengths. Differences in telomere length between subpopulations of cells were significant, and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). We also observed TELEFLUCS in 1 of 19 analyzed human nonalternative lengthening of telomere cell lines and in 1 of 2 analyzed human alternative lengthening of telomere cell lines. The existence of cell subpopulations with differing telomere lengths was confirmed by Q-FISH. CONCLUSION: Our results underscore the importance of flow-FISH in telomere length analysis.

An activity associated with human chromosome 21 permits nuclear colocalization of the adenovirus E1B-55K and E4orf6 proteins and promotes viral late gene expression.

The adenovirus E1B-55K and E4orf6 proteins cooperate during virus infection while performing several tasks that contribute to a productive infection, including the selective nucleocytoplasmic transport of late viral mRNA. Previous studies have shown that the E4orf6 protein retains the E1B-55K protein in the nucleus of human and monkey cells, but not in those of rodents, suggesting that primate-specific cellular factors contribute to the E4orf6-mediated retention of the E1B-55K protein in the nucleus. In an effort to identify these proposed primate-specific cellular factors, the interaction of the E1B-55K and E4orf6 proteins was studied in a panel of stable human-rodent monochromosomal somatic cell hybrids. Analysis of this panel of cell lines has demonstrated the existence of an activity associated with human chromosome 21 that permits the E1B-55K and E4orf6 proteins to colocalize in the nucleus of a rodent cell. Additional hybrid cells bearing portions of human chromosome 21 were used to map this activity to a 10-megabase-pair segment of the chromosome, extending from 21q22.12 to a region near the q terminus. Strikingly, this region also facilitates the expression of adenovirus late genes in a rodent cell background while having little impact on the expression of early viral genes.

Connective tissue growth factor and regulation of the mesangial cell cycle: role in cellular hypertrophy.

Connective tissue growth factor (CTGF) is now considered to be one of the important driver molecules for the pathogenesis of diabetic nephropathy (DN) and possibly many other fibrotic disorders. However, the molecular mechanisms by which CTGF functions remain to be established. In an attempt to define these mechanisms, this study was designed to investigate whether CTGF has any effect on the cell cycle of human mesangial cells (HMC), which are known to undergo hypertrophy in DN. This report provides the first evidence that CTGF is a hypertrophic factor for HMC. CTGF stimulates HMC to actively enter the G(1) phase from G(0), but they do not then progress further through the cell cycle. The molecular mechanisms underlying this G(1) phase arrest appear to be due to the induction of the cyclin-dependent kinase inhibitors (CDKI) p15(INK4), p21(Cip1), and p27(Kip1), which are known to bind and inactivate cyclinD/CDK4/6 and the cyclin E/CDK2 kinase complexes. This could account for the maintenance of pRb protein in a non- or very low-phosphorylated state, preventing cell cycle progression. Using CTGF antisense oligonucleotides, the results also indicate that the previously identified transforming growth factor-beta (TGF-beta)-induced hypertrophy in mesangial cells is CTGF-dependent. Mesangial cell hypertrophy is one of the earliest abnormalities of diabetic nephropathy; therefore, therapeutic strategies targeting CTGF may be beneficial in controlling DN.

Deactivation of the biological activity of paraquat in the soil environment: a review of long-term environmental fate.

During the many years of paraquat usage, wide ranges of investigations of its environmental impact have been conducted. Much of this information has been published, but key, long-term field studies have not previously been presented and assessed. The purpose of this review is to bring together and appraise this information. Due to the nature of paraquat residues in soils, the major part (some 99.99%) of a paraquat application that reaches the soil within the typical Good Agricultural Practice (GAP) is strongly adsorbed to soils of a wide variety of textures. This is in equilibrium with an extremely low concentration in soil solution. However, the paraquat in soil solution is intrinsically biodegradable, being rapidly and completely mineralized by soil microorganisms. The deactivation of the biological activity of paraquat in soils, due to sorption, has been investigated thoroughly and systematically. It is recognized that the determination of total soil residues by severe extraction procedures provides no insight into the amount of paraquat biologically available in soil. Consequently, the key assay developed for this purpose, namely, the strong adsorption capacity-wheat bioassay (SAC-WB) method, has proved to be valuable for determination of the adsorption capacity relevant to paraquat for any particular soil. This method has been validated in the field with a series of long-term (>10 years) trials in different regions of the world. These trials have also shown that, following repeated applications of very high levels of paraquat in the field, residues not only reach a plateau but also subsequently decline. This demonstrates that the known biodegradation of paraquat in soil pore water plays an important role in field dissipation. The biological effects of paraquat in the field have been assessed under unrealistically high treatment regimes. These trials have demonstrated that the continued use of paraquat under GAP conditions will have no detrimental effects on either crops or soil-dwelling flora and fauna. Any such effects can occur only under extreme use conditions (above the SAC-WB), which do not arise in normal agricultural practice.