Conservation of antiviral activity and improved selectivity in PMEO-DAPym upon pyrimidine to triazine scaffold hopping.

Acyclic nucleoside phosphonates incorporating 2,4-diaminotriazine (DAT) as a 5-aza-analog of the 2,4-diamino-pyrimidine (DAPym) nucleobase present in PMEO-DAPyms have been synthesized. The lead PMEO-DAT is as inhibitory against HIV, HBV, MSV and VZV replication as the parent PMEO-DAPym and equally inefficient at markedly affecting replication of HSV-1, HSV-2 and HCMV. A rationale for this similar biological profile is proposed on the basis of structural differences in the active site of the viral DNA polymerases. PMEO-DAT is, however, more selective because, unlike PMEO-DAPym, it does not stimulate secretion of ß-chemokines in cultured PBMC.

Refeeding with a high-protein diet after a 48 h fast causes acute hepatocellular injury in mice.

Elucidating the effects of refeeding a high-protein diet after fasting on disease development is of interest in relation to excessive protein ingestion and irregular eating habits in developed countries. The objective of the present study was to address the hepatic effects of refeeding a high-protein diet after fasting. Mice were fasted for 48 h and then refed with a test diet containing 3, 15, 35, 40, 45 or 50 % casein. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and liver immediate-early gene expression levels were sequentially measured for the first 24 h after initiation of refeeding. Refeeding with a 50 % casein diet after 48 h of fasting led to a rapid (within 2-3 h) and abnormal elevation in serum ALT (P = 0·006) and AST (P = 0·001) activities and a marked increase in liver Finkel-Biskis-Jinkins (FBJ) osteosarcoma oncogene (P = 0·007) and nuclear receptor subfamily 4, group A, member 1 (P = 0·002) mRNA levels. In contrast, refeeding of the 3, 15 or 35 % casein diets produced no substantial increases in serum ALT and AST activities in mice. Refeeding of 40, 45 or 50 % casein increased serum ALT and AST activities in proportion to this dietary casein content. In mice refed the 3, 15 or 35, but not 50 %, casein diets, liver heat shock protein 72 transcript levels greatly increased. We conclude from these data that the consumption of a high-protein diet after fasting causes acute hepatocellular injury in healthy animals, and propose that careful attention should be paid to the use of such diets.

Development of novel, highly potent inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF): increasing cellular potency through optimization of a distal heteroaromatic group.

We describe the design, synthesis, and optimization of a series of new inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF), a kinase whose mutant form (V600E) is implicated in several types of cancer, with a particularly high frequency in melanoma. Our previously described inhibitors with a tripartite A-B-C system (where A is a hinge binding pyrido[4,5-b]imidazolone system, B is an aryl spacer group, and C is a heteroaromatic group) were potent against purified (V600E)BRAF in vitro but were less potent in accompanying cellular assays. Substitution of different aromatic heterocycles for the phenyl based C-ring is evaluated herein as a potential means of improving the cellular potencies of these inhibitors. Substituted pyrazoles, particularly 3-tert-butyl-1-aryl-1H-pyrazoles, increase the cellular potencies without detrimental effects on the potency on isolated (V600E)BRAF. Thus, compounds have been synthesized that inhibit, with low nanomolar concentrations, (V600E)BRAF, its downstream signaling in cells [as measured by the reduction of the phosphorylation of extracellular regulated kinase (ERK)], and the proliferation of mutant BRAF-dependent cells. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo.

Heterogeneity in a mouse model of histiocytosis: transformation of Langerin+ dendritic cells, macrophages, and precursors.

Neoplastic diseases of macrophages (M phi) and dendritic cells (DC), collectively called histiocytoses, are relatively rare. The etiology of most forms of histiocytosis is poorly understood, and the development of animal models is crucial for further research in this field. Previously, an animal model for malignant histiocytosis (MH), involving transformed histiocytic cells, has been generated by infecting mice with malignant histiocytosis sarcoma virus (MHSV). However, increased insight into the heterogeneity of M phi and DC, and the associated reappraisal of human proliferative diseases involving these cells inspired us to re-evaluate the mouse model. We analyzed spleen, bone marrow, and lymph nodes of susceptible mice at various time points after infection. From day 11 onwards, a heterogeneous population of cells, consisting of CD8 alpha(+) Langerin(+) DC, ER-MP58(+) CD11b(+) myeloid precursor cells, CD169(+) metallophilic M phi, and CD71(hi) erythroblasts, was affected by viral transformation. In different mice, these subsets expanded at different rates in different organs, causing a variable disease profile in terminal stages. Cell lines, which were generated from MHSV-transformed tumors, showed a DC-like morphology and phenotype, and appeared to be arrested in different stages of maturation. Upon injection into healthy mice, different preferential homing patterns were observed for the various cell lines, and the cells acquired distinct phenotypes depending on the organ of homing. This indicates that these transformed cells adapt to their microenvironment by switching between precursor, DC/Langerhans cell, and M phi phenotypes. Our results demonstrate that the MHSV model represents a heterogeneous neoplastic disease with characteristics of M phi/DC sarcomas.

The utility of the K6/ODC transgenic mouse as an alternative short term dermal model for carcinogenicity testing of pharmaceuticals.

The use of transgenic rodents may overcome many limitations of traditional cancer studies. Regulatory perspectives continue to evolve as new models are developed and validated. The transgenic mouse, K6/ODC, develops epidermal tumors when exposed to genotoxic carcinogens. In this study, K6/ODC mice were evaluated for model fitness and health robustness in a 36-week study to determine oncogenic risk of residual DNA in vaccines from neoplastic cell substrates. K6/ODC and C57BL/6 mice were treated with T24-H-ras expression plasmid, carrier vector DNA, or saline topically or by subcutaneous injection. One group of K6/ODC mice received 7,12-dimethylbenz-[a]anthracene [DMBA] dermally. Only DMBA-treated mice developed papillomas by six weeks, increasing in incidence to 25 weeks. By week 11, many K6/ODC mice showed severe dehydration and dermal eczema. By week 32, (6/8) surviving K6/ODC mice showed loss of mobility and balance. Microscopic evaluation of tissues revealed dermal/sebaceous gland hyperplasia, follicular dystrophy, splenic atrophy, and amyloid deposition/neutrophilic infiltration within liver, heart, and spleen, in all K6/ODC mice. Pathology was not detected in C57BL/6 mice. Progressive adverse health, decreased survival, and failure to develop papillomas to the H-ras plasmid suggest that K6/ODC mice may be an inappropriate alternative model for detection of oncogenic DNA and pharmaceutical carcinogenicity testing.

Factors affecting the performance of different long terminal repeats in the retroviral vector.

The long terminal repeat (LTR) of retrovirus contains the nucleotide sequences that control gene expression. Although several different LTRs have been used in the context of retroviral vector, the activity of the various LTRs has not yet been systematically compared for their level of gene expression. We evaluated the effect of four different LTRs on gene expression using luciferase, stem cell factor, and enhanced green fluorescence protein as reporter genes. LTRs tested in this study were derived from Moloney murine leukemia virus, myeloproliferative sarcoma virus, murine stem cell virus, and spleen focus-forming virus. It was found that the level of gene expression is affected by not only LTRs but also the transgenes and the cell types in which gene expression occurs. Furthermore, the presence of other nucleotide sequences such as the internal ribosome entry site (IRES)-neo cassette could also significantly affect gene expression. Our results suggested that the LTR should be chosen carefully, more or less on an empirical basis.

High levels of protein expression using different mammalian CMV promoters in several cell lines.

With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.

Dimer linkage structure in retroviruses: models that include both duplex and quadruplex domains.

Genome of all known retroviruses consists of two identical molecules of RNA, which are non-covalently linked. The most stable contact site between two RNA molecules is located near their 5' ends. The molecular interactions in the dimer linkage structure (DLS) in mature virions are currently unknown. Recently we suggested that the dimer linkage structure in human immunodeficiency virus 1 (HIV-1) contains both duplex and quadruplex domains and proposed a model of DLS in HIV-1Mal (Central African virus). In this paper we showed that similar models can be also built for HIV- 1Lai, a representative of the North-American and European viruses. One of the double-stranded domains in the model structures represents either an extended duplex formed by different pathways (through base pair melting and subsequent reannealing or by a recombination mechanism) or kissing loop complex. The quadruplexes contain both G- and mixed tetrads, for example, G.C.G.C or A.U.A.U. Phylogenetic analysis of 350 isolates from NCBI database showed that similar models of DLS are predictable practically for all HIV-1 isolates surveyed. A model of dimer linkage structure in Moloney murine sarcoma virus (MuSV) is also presented. The structure includes a duplex formed by the palindromic sequences and several quadruplexes.

Redirecting human T lymphocytes toward renal cell carcinoma specificity by retroviral transfer of T cell receptor genes.

Adoptive T cell therapy of renal cell carcinoma (RCC) is limited by the difficulty in generating sufficient numbers of RCC-reactive T cells in vitro. To circumvent this problem, we cloned T cell receptor (TCR) alpha and beta chains from a tumor-infiltrating lymphocyte clone specific for an RCC tumor antigen and transferred the TCR into human T cell lines and primary T lymphocytes. Efficient TCR expression in primary T lymphocytes was obtained only with a mouse myeloproliferative sarcoma virus (MPSV)-based retroviral vector, not with a Moloney murine leukemia virus (MLV)-based vector, although both viral supernatants were similar in titer, as shown by analysis of copy number integration in transduced T cells. Reverse transcription-polymerase chain reaction analysis revealed a higher amount of TCR-encoding transcripts when T cells were transduced with the MPSV vector in comparison with the MLV vector, indicating that high TCR expression levels can be achieved by appropriate cis-regulatory vector elements. The biological activity of the transferred TCR was shown by specific lysis of RCC cells ((51)Cr release assay) and by interferon gamma and tumor necrosis factor alpha release (enzyme-linked immunosorbent assay) in an antigen-specific and HLA-A*0201-restricted fashion. Comparison of the redirected T lymphocytes with the original tumor-infiltrating lymphocyte clone revealed similar killing and cytokine secretion capabilities. The functional activity of TCR-redirected T lymphocytes was stable over time. The results demonstrate that use of an optimized retroviral vector yielded a high TCR transduction efficiency and stable and high TCR expression in primary human T lymphocytes and redirected their specificity toward RCC cells.

Principios Básicos y simples de la tecnología transgénica y Knock-Out / Basic and simple concepts on the transgenic and knock-out technologies

El desarrollo de los animales knock-out ha permitido grandes avances en la investigación médica moderna y en la creación de nuevas alternativas terapéuticas para diferentes enfermedades. Hasta la fecha, la habilidad humana para manipular el genoma murino ha tranformado dramáticamente la investigación científica a nivel mundial. Durante la última década, las tecnologías transgénetica convencional y de Knock-out genético se han convertido en herramientas de incalculable valor para la modelacion de desordenes genéticos, para la asignacion de diferentes funciones a diferentes genes, para la evaluación de drogas y tóxinas, y para la resolución de importantes preguntas en la investigación básica y aplicada. Actualmente, el uso de ambos, animales transgénicos y knock-out, se ha convertido en una práctica estándar global en la investigación biomédica

FTIR microscopy detection of cells infected with viruses.

Fourier-transform infrared (FTIR) microscopy is considered a comprehensive and sensitive method for detection of molecular changes in cells. The advantage of FTIR microspectroscopy over conventional FTIR spectroscopy is that it facilitates inspection of restricted regions of a cell culture or a tissue. We have shown that it is possible to apply FTIR microscopy as a sensitive and effective assay for the detection of cells infected with various members of the herpes family of viruses and retroviruses. Detectable and significant spectral differences between normal and infected cells were evident at early stages of the infection. Impressive changes in several spectroscopic parameters were seen in infected compared with uninfected cells. It seems that the change in spectral behavior is specific to the infecting virus, because cells infected with herpesviruses showed different spectral changes compared with cells infected with retroviruses.

Synthesis and antiviral activity of some 5'-N-phthaloyl-3'-azido-2',3'-dideoxythymidine analogues.

A variety of substituted 5'-N-phthaloyl-3'-azido-2',3'-dideoxythymidine derivatives has been evaluated for their activity against HIV-1, HIV-2 and Moloney murine sarcoma virus (MSV) in cell culture. Most of the 3'-azido-2',3'-dideoxythymidine (AZT, zidovudine) derivatives showed antiviral activity in the lower micromolar concentration range and there was a close correlation between their anti-HIV and anti-MSV activity (r = 0.99). The adamantyl phthaloyl derivative was active at submicromolar concentrations. None of the compounds showed marked cytostatic activity. They did not inhibit recombinant HIV-1 reverse transcriptase. All compounds were inactive against HIV in thymidine kinase-deficient cells, pointing to the compounds' requirement to release free AZT to afford antiviral efficacy.

FTIR microspectroscopy of malignant fibroblasts transformed by mouse sarcoma virus.

Fourier transform infrared microspectroscopy (FTIR-MSP), which is based on the characteristic molecular vibrational spectra of cells, was used to investigate spectral differences between normal primary rabbit bone marrow (BM) cells and bone marrow cells transformed (BMT) by murine sarcoma virus (MuSV). Primary cells, rather than cell lines, were used for this research because primary cells are similar to normal tissue cells in most of their characteristics. Our results showed dramatic changes in absorbance between the control cells and MuSV124-transformed cells. Various biological markers, such as the phosphate level and the RNA/DNA obtained, based on the analysis of the FTIR-MSP spectra, also displayed significant differences between the control and transformed cells. Preliminary results suggested that the cluster analysis performed on the FTIR-MSP spectra yielded 100% accuracy in classifying both types of cells.

Detection of ecotropic replication-competent retroviruses: comparison of s(+)/l(-) and marker rescue assays.

Guidelines for testing gene therapy products for ecotropic replication-competent retrovirus (Eco-RCR) have not been delineated as they have for amphotropic viruses. To evaluate biologic assays that can detect these viruses, we compared an S(+)/L(-) assay and a marker rescue assay designed specifically for Eco-RCR detection. Moloney murine leukemia virus (Mo-MuLV) obtained from the American Type Culture Collection was used as the positive control. For marker rescue, NIH 3T3 cells were transduced with a retroviral vector expressing the neomycin phosphotransferase gene (3T3/Neo). Inoculation and passage of test material in 3T3/Neo cells for 3 weeks (amplification) and subsequent testing in the S(+)/L(-) assay or the marker rescue assay increased the level of sensitivity for virus detection greater than 10-fold compared with direct inoculation of D56 S(+)/L(-) cells. When serial dilutions of Mo-MuLV stock were evaluated, six of six cultures had detectable virus by the S(+)/L(-) and marker rescue assays at dilutions of 10(-5) and 10(-6). At the 10(-7) dilution, five of six assays had detectable virus in both assays. The ability to detect virus-infected cells was also evaluated in a modification that substituted cells for supernatant. Fifteen 3T3/Neo cultures inoculated with 10(6) 293 cells containing 100 or 10 Mo-MuLV/3T3 cells were all positive by marker rescue. For dilution with 1 virus-infected cell per 10(6) 293 cells, 10 of 15 cultures were positive. At the 0.1-cell dilution only 2 of 15 cultures were positive. If we hope to detect one infected cell in a test article, the probability of detecting virus if the assay is performed in triplicate is 96.3%. In summary, after 3 weeks of amplification the S(+)/L(-) and marker rescue assays can detect virus with similar sensitivities. We prefer the marker rescue assay because of the more reliable growth features of NIH 3T3 cells compared with the D56 cell line. For laboratories analyzing clinical materials, this report may prove useful in establishing detection assays for Eco-RCR.

Functional analysis of the murine sarcoma virus RNA packaging sequence.

We investigated the features of the Moloney murine sarcoma virus leader sequence necessary for RNA packaging function by using a deletion analysis approach. We found that sequences that extend beyond those characterized genetically in previous reports are important for optimal packaging efficiency. A fragment covering a minimum of four potential stem-loop structures is required for the shortest packaging element compatible with gene transfer. Our results reveal the extent to which each of the segments of the packaging sequence contribute to packaging efficiency.

Protection of hematopoietic cells from O(6)-alkylation damage by O(6)-methylguanine DNA methyltransferase gene transfer: studies with different O(6)-alkylating agents and retroviral backbones.

Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.

v-FBR-fos oncogene fails to rescue mammalian cells from growth arrest but affects the responses of human fibroblasts to heparin.

The effects of v-fos oncogene on the proliferation of mammalian cells were studied using several approaches. Constitutive overexpression of v-FBR-fos in normal human fibroblasts (MRC-5) and of v-FBR-fos in human chondrocytes (HAC21) failed to immortalise them, extend their in vitro lifespan, increase their growth rates or induce cellular transformation. Further, v-FBR-fos did not render MRC-5 growth factor-independent or alter their responsivenness to serum, but it markedly suppressed their heparin-induced proliferation. A conditionally immortalized, temperature-sensitive rat embryo fibroblast cell line (tsa14) which undergoes growth arrest upon inactivation of a thermolabile SV40 large T antigen by a temperature shift producing a phenotype that mimmicks the senescent phenotype, was also used to study the effects of v-FBR-fos on cell proliferation. Whereas a wild-type SV40 large T antigen rescued tsa14 from a temperature-dependent growth arrest, v-FBR-fos failed to do so. Hence, v-FBR-fos was not sufficient to, at least, complement the tsa14 growth defect. There was no change in the expression of c-jun and junB, members of the AP-1 transcriptional complex in MRC-5v-fos cells. These data show that v-FBR-fos is not sufficient to rescue mammalian cells from senescence but it can affect the responses of human fibroblasts to heparin suggesting a role of fos in cell proliferation.

Cellular and viral Fos proteins are degraded by different proteolytic systems.

c-Fos proto-oncoprotein is a short-lived transcription factor degraded by the proteasome in vivo. Its mutated forms expressed by the mouse osteosarcomatogenic retroviruses, FBJ-MSV and FBR-MSV, are stabilized two- and threefold, respectively. To elucidate the mechanisms underlying v-Fos(FBJ) and v-Fos(FBR) protein stabilization, we conducted a genetic analysis in which the half-lives and the sensitivities to various cell-permeable protease inhibitors of a variety of cellular and viral protein mutants were measured. Our data showed that the decreased degradation of v-Fos(FBJ) and v-Fos(FBR) is not simply explained by the deletion of a c-Fos destabilizing C-terminal domain. Rather, it involves a complex balance between opposing destabilizing and stabilizing mutations which are distinct and which include virally-introduced peptide motifs in both cases. The mutations in viral Fos proteins conferred both total insensitivity to proteasomal degradation and sensitivity to another proteolytic system not naturally operating on c-Fos, explaining the limited stabilization of the two proteins. This observation is consistent with the idea that FBR-MSV and FBJ-MSV expression machineries have evolved to ensure controlled protein levels. Importantly, our data illustrate that the degradation of unstable proteins does not necessarily involve the proteasome and provide support to the notion that highly related proteins can be broken down by different proteolytic systems in living cells.