Phillip Y.-P. Wong

MicroRNAs identified in highly purified liver-derived mitochondria may play a role in apoptosis.

MicroRNAs (miRNAs) are a class of small ~22 nt noncoding (nc) RNAs that regulate gene expression post-transcriptionally by direct binding to target sites on mRNAs. They comprise more than 1,000 novel species in mammalian cells and exert their function by modulating gene expression through several different mechanisms, including translational inhibition, and/or degradation of target mRNAs. Mitochondria maintain and express their own genome, which is distinct from the nuclear transcriptional and translational apparatus. Thus, they provide a potential site for miRNA mediated post-transcriptional regulation. To determine whether they maintain a unique miRNA population, we examined the miRNA profile from highly purified and RNase treated mitochondria from adult rat liver. Fifteen miRNAs were identified by microarray analysis of which, five were confirmed by TaqMan® 5’nuclease assays using rat specific probes. Functional analysis of the miRNAs indicated that they were not targeted to the mitochondrial genome nor were they complementary to nuclear RNAs encoding mitochondrial proteins. Rather, the mitochondria-associated miRNAs appear to be involved in the expression of genes associated with apoptosis, cell proliferation, and differentiation. Given the central role that mitochondria play in apoptosis, the results suggest that they may serve as reservoirs of select miRNAs that may modulate these processes in a coordinate fashion.

Genomewide microRNA down-regulation as a negative feedback mechanism in the early phases of liver regeneration.

The liver is one of the few organs that have the capacity to regenerate in response to injury. We carried out genomewide microRNA (miRNA) microarray studies during liver regeneration in rats after 70% partial hepatectomy (PH) at early and mid time points to more thoroughly understand their role. At 3, 12, and 18 hours post‐PH ∼40% of the miRNAs tested were up‐regulated. Conversely, at 24 hours post‐PH, ∼70% of miRNAs were down‐regulated. Furthermore, we established that the genomewide down‐regulation of miRNA expression at 24 hours was also correlated with decreased expression of genes, such as Rnasen, Dgcr8, Dicer, Tarbp2, and Prkra, associated with miRNA biogenesis. To determine whether a potential negative feedback loop between miRNAs and their regulatory genes exists, 11 candidate miRNAs predicted to target the above‐mentioned genes were examined and found to be up‐regulated at 3 hours post‐PH. Using reporter and functional assays, we determined that expression of these miRNA‐processing genes could be regulated by a subset of miRNAs and that some miRNAs could target multiple miRNA biogenesis genes simultaneously. We also demonstrated that overexpression of these miRNAs inhibited cell proliferation and modulated cell cycle in both Huh‐7 human hepatoma cells and primary rat hepatocytes. From these observations, we postulated that selective up‐regulation of miRNAs in the early phase after PH was involved in the priming and commitment to liver regeneration, whereas the subsequent genomewide down‐regulation of miRNAs was required for efficient recovery of liver cell mass. Conclusion: Our data suggest that miRNA changes are regulated by negative feedback loops between miRNAs and their regulatory genes that may play an important role in the steady‐state regulation of liver regeneration. (HEPATOLOGY 2011;)

p53 dephosphorylation and p21Cip1/Waf1 translocation correlate with caspase-3 activation in TGF-β1-induced apoptosis of HuH-7 cells

The p53 tumor suppressor gene product plays an important role in the regulation of apoptosis. Transforming growth factor β1 (TGF-β1)-induced apoptosis in hepatic cells is associated with reduced expression of the retinoblastoma protein (pRb) and subsequent E2F-1-activated expression of apoptosis-related genes. In this study, we explored the potential role of p53 in TGF-β1-induced apoptosis. HuH-7 human hepatoma cells were either synchronized in G1, S and G2/M phases, or treated with 1 nM TGF-β1. The results indicated that greater than 90% of the TGF-β1-treated cells were arrested in G1 phase of the cell cycle. This was associated with enhanced p53 dephosphorylation and p21Cip1/Waf1 expression, which coincided with decreased Cdk2, Cdk4, and cyclin E expression, compared with synchronized G1 cells. In addition, p53 dephosphorylation coincided with caspase-3 activation, and translocation of p21Cip1/Waf1 and p27Kip1 into the cytoplasm, all of which were suppressed by caspase inhibition of TGF-β1-induced apoptosis. Finally, phosphatase inhibition and pRb overexpression partially inhibited p53-mediated apoptosis. In conclusion, the results demonstrated that TGF-β1-induced p53 dephosphorylation is associated with caspase-3 activation, and cytosolic translocation of p21Cip1/Waf1 and p27Kip1, resulting in decreased expression of Cdks and cyclins. Further, p53 appears to mediate TGF-β1-induced apoptosis downstream of the pRb/E2F-1 pathway.

Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells.

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells.

Tunneling nanotube formation is stimulated by hypoxia in ovarian cancer cells

In this study, we demonstrated that hypoxic conditions stimulated an increase in tunneling nanotube (TNT) formation in chemoresistant ovarian cancer cells (SKOV3, C200). We found that suppressing the mTOR pathway using either everolimus or metformin led to suppression of TNT formation in vitro, verifying TNTs as a potential target for cancer-directed therapy. Additionally, TNT formation was detected in co-cultures including between platinum-resistant SKOV3 cells, between SKOV3 cells and platinum-chemosensitive A2780 cells, and between SKOV3 cells cultured with benign ovarian epithelial (IOSE) cells; these findings indicate that TNTs are novel conduits for malignant cell interactions and tumor cell interactions with other cells in the microenvironment. When chemoresistant C200 and parent chemosensitive A2780 cells were co-cultured, chemoresistant cells displayed a higher likelihood of TNT formation to each other than to chemosensitive malignant or benign epithelial cells. Hypoxia-induced TNT formation represents a potential mechanism for intercellular communication in ovarian cancer and other forms of invasive refractory cancers.

Polysome trafficking of transcripts and microRNAs in regenerating liver after partial hepatectomy

Liver regeneration after 70% partial hepatectomy (PH) in rats induces >95% of hepatocytes to undergo two rounds of semisynchronous cell replication. Gene expression is controlled primarily by posttranscriptional processing, including changes in mRNA stability. However, the translational activity of a specific mRNA can also be modulated after PH, resulting in significant uncoupling of protein and transcript levels relative to quiescent liver for many genes including c-myc and p53. Although the precise mechanism by which this uncoupling occurs is unknown, the polysomal association of mRNA and microRNA (miRNA) can significantly modulate rate of decay as well as translational activity. Thus we characterized the association of c-myc and p53 mRNAs and miRNAs in free and cytoskeleton- and membrane-bound polysome populations 3, 6, and 24 h after PH. The transcripts for c-myc and p53 were differentially distributed in the three discrete polysome populations, and this was dramatically modulated during liver regeneration. Nascent polysome-associated p53 and c-myc proteins were also differentially expressed in the free and cytoskeleton- and membrane-bound polysomes and significantly uncoupled from transcript levels relative to nonresected liver. At least 85 miRNAs were associated with the three polysome populations, and their abundance and distribution changed significantly during liver regeneration. These data suggest that posttranscriptional control of c-myc and p53 protein expression is associated with the translocation of transcripts between the different polyribosomes. The alteration of expression for the same transcript in different polysome populations may, in part, be due to the action of miRNAs.

Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK–β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–β-globin transgene for gene therapy of sickle cell disease.

β-globin sleeping beauty transposon reduces red blood cell sickling in a patient-derived CD34+-based in vitro model

The ultimate goal of gene therapy for sickle cell anemia (SCA) is an improved phenotype for the patient. In this study, we utilized bone marrow from a sickle cell patient as a model of disease in an in vitro setting for the hyperactive Sleeping Beauty transposon gene therapy system. We demonstrated that mature sickle red blood cells containing hemoglobin-S and sickling in response to metabisulfite can be generated in vitro from SCA bone marrow. These cells showed the characteristic morphology and kinetics of hemoglobin-S polymerization, which we quantified using video microscopy and imaging cytometry. Using video assessment, we showed that delivery of an IHK-βT87Q antisickling globin gene by Sleeping Beauty via nucleofection improves metrics of sickling, decreasing percent sickled from 53.2 ± 2.2% to 43.9 ± 2.0%, increasing the median time to sickling from 8.5 to 9.6 min and decreasing the maximum rate of sickling from 2.3 x 10-3 sickling cells/total cells/sec in controls to 1.26 x 10-3 sickling cells/total cells/sec in the IHK-βT87Q-globin group (p < 0.001). Using imaging cytometry, the percentage of elongated sickled cells decreased from 34.8 ± 4.5% to 29.5 ± 3.0% in control versus treated (p < 0.05). These results support the potential use of Sleeping Beauty as a clinical gene therapy vector and provide a useful tool for studying sickle red blood cells in vitro.

Short, single-stranded oligonucleotides mediate targeted nucleotide conversion using extracts from isolated liver mitochondria

Site-specific single-nucleotide changes in chromosomal DNA of eukaryotic cells have been achieved using chimeric RNA/DNA oligonucleotides (ONs) and short single-stranded (SS) ONs. However, a variety of human diseases originate from single-point mutations in the genome of mitochondrial DNA. We previously demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate targeted single-nucleotide changes using chimeric ONs in vitro. However, different factor(s) and/or mechanism(s) appear to be involved in SS and RNA/DNA ON mediated DNA repair. Because mitochondria are deficient in certain factors involved in nuclear DNA repair pathways, we investigated whether mitochondria possess the enzymatic machinery for SS ON mediated DNA alterations. Using in vitro DNA repair assays based on mutagenized plasmids and a bacterial read-out system, SS ONs were designed to correct the point mutations in the genes encoded by the different plasmids. In this system, protein extracts from purified rat liver mitochondria and nuclei catalyzed similar levels of site-specific nucleotide modifications using SS ONs. Interestingly, extracts isolated from quiescent liver mediated significantly higher conversion rates than those isolated from regenerating liver. The results suggest that mitochondria contain the factors necessary for correction of single-point mutations by SS ONs. In addition, at least some are different than those required for DNA repair by RNA/DNA ONs. Moreover, correction with SS ONs appears to occur one strand at a time suggesting that repair of the DNA substrate involves strand transfer. The ability of unmodified SS ONs to mediate targeted alteration of the mitochondrial genome may provide a new tactic for treatment of certain mitochondrial-based diseases.

Tunneling nanotubes: an alternate route for propagation of the bystander effect following oncolytic viral infection

Tunneling nanotubes (TNTs) are ultrafine, filamentous actin-based cytoplasmic extensions which form spontaneously to connect cells at short and long-range distances. We have previously described long-range intercellular communication via TNTs connecting mesothelioma cells in vitro and demonstrated TNTs in intact tumors from patients with mesothelioma. Here, we investigate the ability of TNTs to mediate a viral thymidine kinase based bystander effect after oncolytic viral infection and administration of the nucleoside analog ganciclovir. Using confocal microscopy we assessed the ability of TNTs to propagate enhanced green fluorescent protein (eGFP), which is encoded by the herpes simplex virus NV1066, from infected to uninfected recipient cells. Using time-lapse imaging, we observed eGFP expressed in infected cells being transferred via TNTs to noninfected cells; additionally, increasing fluorescent activity in recipient cells indicated cell-to-cell transmission of the eGFP-expressing NV1066 virus had also occurred. TNTs mediated cell death as a form of direct cell-to-cell transfer following viral thymidine kinase mediated activation of ganciclovir, inducing a unique long-range form of the bystander effect through transmission of activated ganciclovir to nonvirus-infected cells. Thus, we provide proof-of-principle demonstration of a previously unknown and alternative mechanism for inducing apoptosis in noninfected recipient cells. The conceptual advance of this work is that TNTs can be harnessed for delivery of oncolytic viruses and of viral thymidine kinase activated drugs to amplify the bystander effect between cancer cells over long distances in stroma-rich tumor microenvironments.