Supplementary Figure 2 from Modulation of drug resistance by artificial transcription factors

Supplementary Figure 1 from Modulation of drug resistance by artificial transcription factors

Supplementary Figure 3 from Modulation of drug resistance by artificial transcription factors

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, β and γ, arise from the human gene. We now show that DMP1α, β and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1β and γ did not activate the ARF promoter, whereas only β resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1β reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1β- and γ-isoforms share domains necessary for the inhibitory function of the β-isoform. That DMP1β may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/β in the nucleus. Cells stably expressing DMP1β, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and β-ratios are tightly regulated in hematopoietic cells and DMP1β antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility (“retrohoming”) by a process that requires reverse transcription of a highly structured, 2–2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3′ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

Abstract The efficiency of chemotherapeutic treatments in cancer patients is often impaired by the acquisition of drug resistance. Cancer cells develop drug resistance through dysregulation of one or more genes or cellular pathways. To isolate efficient regulators of drug resistance in tumor cells, we have adopted a genome-wide scanning approach based on the screening of large libraries of artificial transcription factors (ATFs) made of three and six randomly assembled zinc finger domains. Zinc finger libraries were linked to a VP64 activation domain and delivered into a paclitaxel-sensitive tumor cell line. Following drug treatment, several ATFs were isolated that promoted drug resistance. One of these ATFs, 3ZF-1-VP, promoted paclitaxel resistance in cell lines having mutated or inactivated p53, such as MDA-MB-435 and Kaposi's sarcoma cell lines. 3ZF-1-VP also induced strong resistance to etoposide, vincristine, and cisplatinum. Linkage of a repression domain to the selected ATF resulted in enhanced sensitivity to multiple drugs, particularly vincristine, cisplatinum, and 5-fluorouracil. Small interfering RNA–mediated inhibition of p53 revealed that 3ZF-1-VP activated both p53-dependent and p53-independent mechanisms to promote survival, whereas other ATF required intact p53. Real-time expression analysis and DNA microarrays showed that several ATFs up-regulated targets of p53, such as the cyclin-dependent kinase inhibitor p21WAF1/CIP1, and genes participating in the p14ARF-MDM2-p53 tumor suppressor pathway, such as hDMP1. Thus, ATF can be used to map genes and pathways involved in drug resistance phenotypes and have potential as novel therapeutic agents to inhibit drug resistance. [Mol Cancer Ther 2008;7(3):688–97]

Dominant epitope-specific CD8 (cid:1) T-lymphocyte responses play a central role in controlling viral spread. We explored the basis for the development of this focused immune response in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys through the use of two dominant (p11C and p199RY) and two subdominant (p68A and p56A) epitopes. Using real-time PCR to quantitate T-cell receptor (TCR) variable region beta (V (cid:2) ) family usage, we show that CD8 (cid:1) T-lymphocyte populations specific for dominant epitopes are characterized by a diverse V (cid:2) repertoire, whereas those specific for subdominant epitopes employ a dramatically more focused V (cid:2) repertoire. We also demonstrate that dominant epitope-specific CD8 (Biosource International, Inc., Camarillo, CA). The cDNA was amplified for 30 cycles in a Perkin-Elmer 9600 GeneAmp PCR system under the following conditions: 95°C for 10 s, 57°C for 30 s, and 68°C for 60 s. One microliter of each reaction product was mixed with deionized formamide and a TAMRA-500 size standard and then electrophoresed on a 5% acrylamide gel on a 377 DNA sequencer (Applied Biosystems). Data were analyzed for size and fluorescence intensity by using Genescan software version 3.1 (Applied Biosystems). Further cloning of PCR products into the pGEM-T Easy vector (Promega) for sequencing, in conjunction with the CDR3 length display, allowed for the prediction of CDR3 lengths. These lengths were ex- pressed as predicted numbers of amino acids spanning the portion of the VDJ joining segments.

Dominant epitope-specific CD8(+) T-lymphocyte responses play a central role in controlling viral spread. We explored the basis for the development of this focused immune response in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys through the use of two dominant (p11C and p199RY) and two subdominant (p68A and p56A) epitopes. Using real-time PCR to quantitate T-cell receptor (TCR) variable region beta (V beta) family usage, we show that CD8(+) T-lymphocyte populations specific for dominant epitopes are characterized by a diverse V beta repertoire, whereas those specific for subdominant epitopes employ a dramatically more focused V beta repertoire. We also demonstrate that dominant epitope-specific CD8(+) T lymphocytes employ TCRs with multiple CDR3 lengths, whereas subdominant epitope-specific cells employ TCRs with a more restricted CDR3 length. Thus, the relative dominance of an epitope-specific CD8(+) T-lymphocyte response reflects the clonal diversity of that response. These findings suggest that the limited clonal repertoire of subdominant epitope-specific CD8(+) T-lymphocyte populations may limit the ability of these epitope-specific T-lymphocyte populations to expand and therefore limit the ability of these cell populations to contribute to the control of viral replication.

The TRIM5 alpha(tripartite motif 5 alpha protein) has been linked to the cross-species restriction in human immunodeficiency virus type 1 (HIV-1) infection of non-human cells, but the mechanism by which this occurs remains to be fully elucidated. Here we demonstrate that the capsid (CA) protein of HIV-1 is more rapidly degraded in cells expressing monkey TRIM5 alpha than in cells expressing humanTRIM5 alpha. Other proteins encoded by Gag and Pol are not subject to TRIM5 alpha-mediated accelerated degradation. The accelerated CA degradation by TRIM5 alpha apparently occurs via a nonproteosomal pathway. TRIM5 alpha selectively accelerates degradation of the CA population, which reached the cytosol of restrictive cells, but not the CA population, which ended into the vesicular compartment. Given that cytosolic CA represents "productively" entered cores, whereas vesicular CA represents "nonproductively" entered cores, our findings suggest that TRIM5 alpha interrupts the infectious pathway of HIV-1 by acting on the incoming cytosolic CA. The mode of viral entry does not influence the accelerated degradation of cytosolic CA by TRIM5 alpha. Thus, this study reveals a correlation between TRIM5 alpha-mediated HIV-1 restriction and a selective degradation of cytosolic CA normally associated with productive viral entry.

Abstract Background The protease inhibitor, TL-3, demonstrated broad efficacy in vitro against FIV, HIV and SIV (simian immunodeficiency virus), and exhibited very strong protective effects on early neurologic alterations in the CNS of FIV-PPR infected cats. In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates. Results Twenty cats were infected with uncloned FIV-C and half were treated with TL-3 while the other half were left untreated. Two uninfected cats were used as controls. The general health and the immunological and virological status of the animals was monitored for eight weeks following infection. All infected animals became viremic independent of TL-3 treatment and seven of 20 FIV-C infected animals developed severe immunodepletive disease in conjunction with significantly (p ≤ 0.05) higher viral RNA loads as compared to asymptomatic animals. A marked and progressive increase in CD8+ T lymphocytes in animals surviving acute phase infection was noted, which was not evident in symptomatic animals (p ≤ 0.05). Average viral loads were lower in TL-3 treated animals and of the 6 animals requiring euthanasia, four were from the untreated cohort. At eight weeks post infection, half of the TL-3 treated animals and only one of six untreated animals had viral loads below detection limits. Analysis of protease genes in TL-3 treated animals with higher than average viral loads revealed sequence variations relative to wild type protease. In particular, one mutant, D105G, imparted 5-fold resistance against TL-3 relative to wild type protease. Conclusions The findings indicate that the protease inhibitor, TL-3, when administered orally as a monotherapy, did not prevent viremia in cats infected with high dose FIV-C. However, the modest lowering of viral loads with TL-3 treatment, the greater survival rate in symptomatic animals of the treated cohort, and the lower average viral load in TL-3 treated animals at eight weeks post infection is indicative of a therapeutic effect of the compound on virus infection.

Human immunodeficiency virus type 1 (HIV-1) alters gene expression in infected cells, leading to cellular dysfunction. We uncovered a number of host cell genes that are modulated in both CD4(+) T cell lines and primary CD4(+) T lymphocytes infected with HIV-1, using high-density oligonucleotide probe microarray technology. We focused on one gene in particular, nuclear factor I-B2 (NFI-B2), because of its high level of expression. NFI-B2 is a member of the nuclear factor I family of nuclear proteins, which are known to be involved in viral and cellular transcription. To better understand the role of NFI-B2 during HIV-1 infection, we generated a Jurkat T cell line that constitutively expressed NFI-B2. After infection with HIV-1, these cells produced fewer viruses because of a downregulation of surface CD4 expression. The surface expression of the coreceptor, CXCR4, remained unchanged. Furthermore, levels of CD4 mRNA were reduced in NFI-B2-producing cells, suggesting that expression of NFI-B2 impairs CD4 transcription. Modulation of NFI-B2 by HIV-1 may represent yet another mechanism by which HIV infection reduces cell surface expression of CD4.

A global effort has been undertaken to control human immunodeficiency virus (HIV) though the development of vaccines and pharmacologics. Current FDA approved pharmacological inhibitors target two of the three viral enzymes critical to replication and maturation of infectious viral particles: reverse transcriptase (RT) and protease (Pr). Although combination therapies targeting RT and Pr have significantly reduced AIDS related morbidity and mortality, resistance to individual inhibitors is a growing concern. Currently, there are six protease inhibitors in clinical use. These inhibitors target the active site of protease using peptidomimetic transition state analogs based on natural substrates. However, treatment failures arise as a lack of compliance due to HIV-inhibitor pharmacokinetics, toxicity, and tolerance. This allows reduced HIV-inhibitor pressure, increased viral replication, and the emergence of drug resistant mutations. Continued use of protease inhibitors in the face of incomplete viral suppression may result in HIV-1 escape mutants not only being resistant to the protease inhibitor used, but to all clinically available protease inhibitors. Thus, new broad-based protease inhibitors are needed to control the emerging multi-drug, cross-resistant HIV-1. Moreover, given the emergence of cross-resistant HIV-1, there is a need to target novel protease structural sites to reduce the risk of multi-drug cross-resistance. In this review, we discuss the resistance to protease inhibitors and the rationale for new strategies towards drug design for suppressing protease activity. We focus on the structure and function relationship and the influence that drug resistance mutants exert on the evolution of HIV-1 protease.

CD4 + T-cell depletion is a characteristic of human immunodeficiency virus type 1 (HIV-1) infection. In this study, modulation of mRNA expression of 6800 genes was monitored simultaneously at eight time points in a CD4 + T-cell line (CEM-GFP) during HIV infection. The responses to infection included: (1) >30% decrease at 72 h after infection in overall host-cell production of monitored mRNA synthesis, with the replacement of host-cell mRNA by viral mRNA, (2) suppression of the expression of selected mitochondrial and DNA repair gene transcripts, (3) increased expression of the proapoptotic gene and its gene p53-induced product Bax, and (4) activation of caspases 2, 3, and 9. The intense HIV-1 transcription resulted in the repression of much cellular RNA expression and was associated with the induction of apoptosis of infected cells but not bystander cells. This choreographed host gene response indicated that the subversion of the cell transcriptional machinery for the purpose of HIV-1 replication is akin to genotoxic stress and represents a major factor leading to HIV-induced apoptosis.

HIV-1 induces apoptosis and leads to CD4+ T-lymphocyte depletion in humans. It is still unclear whether HIV-1 kills infected cells directly or indirectly. To elucidate the mechanisms of HIV-1-induced apoptosis, we infected human CD4+ T cells with HIV-1. Enzymatic analysis with fluorometric substrates showed that caspase 2, 3, and 9 were activated in CD4+ T cells with peak levels 48 h after infection. Immunoblotting analysis confirmed the cleavage of pro-caspase 3 and 9, and of specific caspase substrates. Release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria was observed in HIV-infected cells. The cytochrome c and AIF release preceded the reduction of the mitochondrial transmembrane potential and nuclear chromatin condensation. H IV infection led to phosphorylation of p53 at the Ser15 residue, detectable as early as 24 h after infection. The p53 phosphorylation was followed by increased mRNA and protein expression of p21, Bax, HDM2, and p53. Up-regulation of surface FasL expression, accompanied by a down-regulation of Fas-associated proteins (FADD, DAXX, and RIP), was observed 72 h after infection. Our results suggest that HIV activates the p53 pathway, leading to cytochrome c and AIF release with ensuing caspase activation.

TRAIL is a member of the Tumour Necrosis Factor (TNF) Family and a potent inducer of apoptosis in many breast carcinoma cell lines but not in normal human mammary epithelial cells. In vivo administration of soluble TRAIL causes regression of breast cancer xenografts without causing measurable toxicity. Combining it with other traditional anti-cancer therapies enhances the efficacy of TRAIL treatment. The basis for the resistance of normal breast epithelial cells to TRAILinduced apoptosis will be investigated using filter arrays and high-density microarrays. Using a similar approach, the synergy between TRAIL and other cancer therapies will be studied.