Robust Institutional Support and Collaboration Between Summer Training Programs in Cancer and Biomedicine Drive the Pivot to a Virtual Format in Response to the COVID Pandemic.

Summer internships serve important roles in training the next generation of biomedical researchers and healthcare providers through laboratory and clinical experiences that excite trainees about these fields and help them make informed decisions about career paths. The SARS-CoV-2 (COVID) pandemic and associated physical distancing restrictions precluded implementation of traditional in-person summer curricula and led to the cancellation of many internships across the USA. COVID-related disruptions also created opportunities for trainees to engage in remote research, become proficient in online learning platforms, and explore multidisciplinary topics. These skills are highly relevant to trainees as virtual interfaces occupy an increasingly mainstream role in their professional paths. The response to the COVID pandemic required real-time adaptations at all levels for major biomedical institutions including the University of Maryland Baltimore (UMB). Pivoting summer programs to a virtual format as part of this response provided a "teachable moment" to expose trainees to the innovation and resilience that are essential components of the biomedical profession. UMB summer programs, which span diverse biomedical disciplines from cancer research to diabetes, consolidated resources and identified mentors with online research projects to develop a robust virtual curriculum. Herein, data from a cancer-focused internship illustrate the collaborative adaptations to established components and creation of new learning modules in the transition to, and implementation of, online training. Outcomes are presented in the context of the COVID pandemic and significant societal issues that arose in the summer of 2020. The utility of virtual components and their impact on future programs is discussed.

A study of the interferon antiviral mechanism: apoptosis activation by the 2-5A system.

The 2-5A system contributes to the antiviral effect of interferons through the synthesis of 2-5A and its activation of the ribonuclease, RNase L. RNase L degrades viral and cellular RNA after activation by unique, 2'-5' phosphodiester-linked, oligoadenylates [2-5A, (pp)p5' A2'(P5'A2')]n, n >=2. Because both the 2-5A system and apoptosis can serve as viral defense mechanisms and RNA degradation occurs during both processes, we investigated the potential role of RNase L in apoptosis. Overexpression of human RNase L by an inducible promoter in NIH3T3 fibroblasts decreased cell viability and triggered apoptosis. Activation of endogenous RNase L, specifically with 2-5A or with dsRNA, induced apoptosis. Inhibition of RNase L with a dominant negative mutant suppressed poly (I).poly (C)-induced apoptosis in interferon-primed fibroblasts. Moreover, inhibition of RNase L suppressed apoptosis induced by poliovirus. Thus, increased RNase L levels induced apoptosis and inhibition of RNase L activity blocked viral-induced apoptosis. Apoptosis may be one of the antiviral mechanisms regulated by the 2-5A system.

The interferon regulated ubiquitin-like protein, ISG15, in tumorigenesis: friend or foe?

The interferon-stimulated gene, 15 kDa (ISG15) is an interferon regulated gene that is induced as a primary response to diverse microbial and cell stress stimuli, and encodes the founding member of the ubiquitin-like protein family. ISG15 post-translationally modifies proteins via a pathway parallel to, and partially overlapping with, that of ubiquitin. In addition, ISG15 is released from cells to mediate extracellular cytokine-like activities. Although the biological activities of ISG15 have yet to be fully elucidated, it is clear that ISG15 has the capacity to modulate diverse cellular and physiologic functions. Consistent with this view, alterations in the ISG15 pathway have been identified in human tumors and in tumor cell lines. Here we review evidence of a role for ISG15 as an endogenous tumor suppressor that, when dysregulated in malignant cells, can be subverted to promote oncogenesis.

Role of 2-5A-dependent RNase-L in senescence and longevity.

Senescence is a permanent growth arrest that restricts the lifespan of primary cells in culture, and represents an in vitro model for aging. Senescence functions as a tumor suppressor mechanism that can be induced independent of replicative crisis by diverse stress stimuli. RNase-L mediates antiproliferative activities and functions as a tumor suppressor in prostate cancer, therefore, we examined a role for RNase-L in cellular senescence and aging. Ectopic expression of RNase-L induced a senescent morphology, a decrease in DNA synthesis, an increase in senescence-associated beta-galactosidase activity, and accelerated replicative senescence. In contrast, senescence was retarded in RNase-L-null fibroblasts compared with wild-type fibroblasts. Activation of endogenous RNase-L by 2-5A transfection induced distinct senescent and apoptotic responses in parental and Simian virus 40-transformed WI38 fibroblasts, respectively, demonstrating cell type specific differences in the antiproliferative response to RNase-L activation. Replicative senescence is a model for in vivo aging; therefore, genetic disruption of senescence effectors may impact lifespan. RNase-L-/- mice survived 31.7% (P<0.0001) longer than strain-matched RNase-L+/+ mice providing evidence for a physiological role for RNase-L in aging. These findings identify a novel role for RNase-L in senescence that may contribute to its tumor suppressive function and to the enhanced longevity of RNase-L-/- mice.

Thioredoxin reductase mediates cell death effects of the combination of beta interferon and retinoic acid.

Interferons (IFNs) and retinoids are potent biological response modifiers. By using JAK-STAT pathways, IFNs regulate the expression of genes involved in antiviral, antitumor, and immunomodulatory actions. Retinoids exert their cell growth-regulatory effects via nuclear receptors, which also function as transcription factors. Although these ligands act through distinct mechanisms, several studies have shown that the combination of IFNs and retinoids synergistically inhibits cell growth. We have previously reported that IFN-beta-all-trans-retinoic acid (RA) combination is a more potent growth suppressor of human tumor xenografts in vivo than either agent alone. Furthermore, the IFN-RA combination causes cell death in several tumor cell lines in vitro. However, the molecular basis for these growth-suppressive actions is unknown. It has been suggested that certain gene products, which mediate the antiviral actions of IFNs, are also responsible for the antitumor actions of the IFN-RA combination. However, we did not find a correlation between their activities and cell death. Therefore, we have used an antisense knockout approach to directly identify the gene products that mediate cell death and have isolated several genes associated with retinoid-IFN-induced mortality (GRIM). In this investigation, we characterized one of the GRIM cDNAs, GRIM-12. Sequence analysis suggests that the GRIM-12 product is identical to human thioredoxin reductase (TR). TR is posttranscriptionally induced by the IFN-RA combination in human breast carcinoma cells. Overexpression of GRIM-12 causes a small amount of cell death and further enhances the susceptibility of cells to IFN-RA-induced death. Dominant negative inhibitors directed against TR inhibit its cell death-inducing functions. Interference with TR enzymatic activity led to growth promotion in the presence of the IFN-RA combination. Thus, these studies identify a novel function for TR in cell growth regulation.

The role of 2'-5' oligoadenylate-activated ribonuclease L in apoptosis.

Apoptosis of viral infected cells appears to be one defense strategy to limit viral infection. Interferon can also confer viral resistance by the induction of the 2-5A system comprised of 2'-5' oligoadenylate synthetase (OAS), and RNase L. Since rRNA is degraded upon activation of RNase L and during apoptosis and since both of these processes serve antiviral functions, we examined the role RNase L may play in cell death. Inhibition of RNase L activity, by transfection with a dominant negative mutant, blocked staurosporine-induced apoptosis of NIH3T3 cells and SV40-transformed BALB/c cells. In addition, K562 cell lines expressing inactive RNase L were more resistant to apoptosis induced by decreased glutathione levels. Hydrogen peroxide-induced death of NIH3T3 cells did not occur by apoptosis and was not dependent upon active RNAse L. Apoptosis regulatory proteins of the Bcl-2 family did not exhibit altered expression levels in the absence of RNase L activity. RNase L is required for certain pathways of cell death and may help mediate viral-induced apoptosis.

Induction of interferon synthesis and activation of interferon-stimulated genes by liposomal transfection reagents.

Liposome-mediated transfection is a widely used technique for the introduction of exogenous DNA into mammalian cells. We observed a significant induction of endogenous interferon (IFN)-stimulated genes (ISGs) in cells treated with the liposomal reagents, lipofectamine and DOSPER, in the absence of DNA. Liposome treatment induced expression of reporter constructs driven by IFN-responsive promoter elements, demonstrating a generalized effect on ISG expression. The kinetics of ISG induction were markedly delayed in response to liposome as compared with IFN or double-stranded RNA. ISG induction could be transferred to naive cells with conditioned medium from liposome-treated cells, suggesting that a secreted factor was responsible for this activity. A cell line defective in IFN signaling was refractory to liposome-induced ISG expression, indicating a role for IFN in this induction. Indeed, liposome treatment directly induced IFN-beta gene expression and, thus, represents a novel IFN inducer. IFN induction by liposomal reagents and its potential effects on transgene expression should be considered in the choice of transfection reagent. The ability of liposomal gene delivery reagents to induce IFN synthesis in the host may prove useful in gene therapy approaches to viral and neoplastic diseases.

Impact of RNase L overexpression on viral and cellular growth and death.

The biologic actions of interferons (IFNs) are complex and involve multiple biochemical mechanisms, including the 2-5A system, a regulated RNA decay pathway. The 2-5A system is implicated in the antipicornavirus activity of IFN and in the control of apoptosis. To further investigate involvement of the 2-5A system in the control of viral and cellular growth and death, human RNase L cDNA was stably expressed in murine 3T3 cells from a constitutive cytomegalovirus (CMV) promoter. A clonal cell line, 3T3/pLZ, was isolated that overexpressed RNase L by >100-fold compared with levels of the endogenous murine RNase L. Interestingly, human RNase L levels in 3T3/pLZ cells decreased 3-fold as cells entered a confluent, growth arrest state, suggesting autoregulation. Overexpression of human RNase L greatly enhanced both the cell growth inhibitory activity of IFN and the proapoptotic activity of staurosporine. Furthermore, high levels of RNase L suppressed the replication of diverse viruses: encephalomyocarditis virus, vesicular stomatitis virus, human parainfluenza virus-3, and vaccinia virus. Additional reductions in viral growth were obtained by treating 3T3/pLZ cells with IFN (a + beta) before infections. These results directly demonstrate the anticellular and antiviral potential of the 2-5A system.

A sensitive assay for the IFN-regulated 2-5A synthetase enzyme.

2-5A synthetase is the central enzyme of the 2-5A system, an important mediator of interferon action. An assay capable of detecting low, yet biologically important levels of 2-5A synthetase enzyme activity is described. The purification of enzyme reaction products on SepPak C-18 cartridges resulted in a significant reduction in background, when a high specific activity substrate was used to label the 2-5A. Quantitation of labeled 2-5A by chromatography and scintillation counting provided a means of detecting femptomolar amounts of 2-5A. The combination of these procedures accounts for a 3-4 log increase in sensitivity over existing assays. This degree of sensitivity should permit a more accurate determination of the 2-5A synthetase activity in vivo leading to a better understanding of the role of the 2-5A system in virus infection and other cellular processes.

Stage-associated overexpression of the ubiquitin-like protein, ISG15, in bladder cancer.

Bladder cancer is among the most prevalent malignancies, and is characterised by frequent tumour recurrences and localised inflammation, which may promote tissue invasion and metastasis. Microarray analysis was used to compare gene expression in normal bladder urothelium with that in tumours at different stages of progression. The innate immune response gene, interferon-stimulated gene 15 kDa (ISG15, GIP2), was highly expressed at all stages of bladder cancer as compared to normal urothelium. Western blotting revealed a tumour-associated expression of ISG15 protein. ISG15 exhibited a stage-associated expression, with significantly (P<0.05) higher levels of ISG15 protein in muscle-invasive T2-T4 tumours, compared with normal urothelium. Although ISG15 is involved in the primary immune response, ISG15 expression did not correlate with bladder inflammation. However, immunohistochemical staining revealed expression of ISG15 protein in both cancer cells and stromal immune cells. Interestingly, a significant fraction of ISG15 protein was localised to the nuclei of tumour cells, whereas no nuclear ISG15 staining was observed in ISG15-positive stromal cells. Taken together, our findings identify ISG15 as a novel component of bladder cancer-associated gene expression.

Involvement of proteasomes in gene induction by interferon and double-stranded RNA.

Cytokine induced gene expression is mediated through the ligand-dependent activation of the janus kinase (jak)/signal transducer and activator of transcription (STAT) signal transduction pathway. The ubiquitin proteasome pathway functions in the controlled degradation of cellular proteins, and regulates cytokine signal transduction through the degradation of specific signaling components. Interferon (IFN) treatment induces genes that function in ubiquitin conjugation, suggesting a reciprocal regulation of proteasome activity and IFN action; however, a role for the proteasome in IFN-alpha-induced gene expression has not been examined. In this report, we find that proteasome inhibitors markedly reduce the induction of interferon-stimulated-gene 15 (ISG15), ISG43, and STAT1 by IFN-alpha and double-stranded RNA (dsRNA). The reduction in gene expression by proteasome inhibitors was dose-dependent, and was specific for ISGs. Neither STAT1 phosphorylation nor ISGF-3 activation was affected by proteasome inhibition at early times post-IFN treatment. Cycloheximide treatment diminished the effect of proteasome inhibitors on ISG induction, implicating an IFN/dsRNA-induced protein in this activity. These findings demonstrate that a functional proteasome is required for optimal ISG induction, and are consistent with a model in which IFN and dsRNA induce a proteasome-sensitive repressor of ISG expression.

A proliferation-related constraint on endogenous and interferon-induced 2-5A synthetase activity in normal and neoplastic Syrian hamster cells.

2-5A Synthetase is one of the most extensively characterized enzymes induced by interferon (IFN) and is the central enzyme in a pathway that may be involved in the control of cellular proliferation. We examined the activity of this enzyme in normal diploid Syrian hamster cells (FC13) and their neoplastically transformed derivatives (BP6T); the former cell strain possesses regulated proliferative control, while the latter cell line has escaped from this control. A significant threefold increase in 2-5A synthetase activity was observed in density-arrested versus proliferating FC13 cells, whereas endogenous enzyme activity was uniformly low in BP6T cultures. The increase in enzyme activity in FC13 cultures was not accompanied by the production of IFN at a detectable level, but was parallelled by an increase in the intracellular level of 2',5'-oligoadenylate. IFN treatment resulted in a differential induction of enzyme activity depending on the proliferative state of FC13 cells. After IFN treatment, BP6T cells and subconfluent FC13 cells responded similarly with a fivefold increase in enzyme activity, whereas confluent FC13 cells displayed only a 1.4-fold increase. 2-5A Synthetase enzyme activity reflected steady-state mRNA levels in BP6T and subconfluent FC13 cells. In contrast, a noncoordinate regulation of 2-5A synthetase mRNA expression and enzyme activity was detected in confluent FC13 cells, suggesting that posttranscriptional mechanisms may be involved. The different patterns of endogenous and IFN-induced 2-5A synthetase enzyme activity in FC13 and BP6T cells found in this comparative study may represent an alteration fundamental to the loss of proliferative control in transformed cells.

Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action.

2-5A-dependent RNAase, an interferon-induced enzyme that is activated by 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A), is implicated in both the molecular mechanisms of interferon action and the fundamental control of RNA stability in mammalian cells. Here we report the expression cloning and analysis of murine and human 2-5A-dependent RNAases. The 2-5A binding properties and RNAse activities of recombinant and naturally occurring forms of 2-5A-dependent RNAase were identical. Interferon induction of 2-5A-dependent RNAse expression was demonstrated by measuring the mRNA levels in cells treated with interferon and cycloheximide. Analysis of aligned murine and human 2-5A-dependent RNAse sequences revealed several intriguing features, including similarity to RNAase E, which is implicated in the control of mRNA stability in E. coli. Interestingly, a duplicated phosphate-binding loop motif was determined by deletion analysis and site-directed mutagenesis to function in the binding of 2-5A.

RNase L mediates the antiviral effect of interferon through a selective reduction in viral RNA during encephalomyocarditis virus infection.

The 2',5'-oligoadenylate (2-5A) system is an RNA degradation pathway which plays an important role in the antipicornavirus effects of interferon (IFN). RNase L, the terminal component of the 2-5A system, is thought to mediate this antiviral activity through the degradation of viral RNA; however, the capacity of RNase L to selectively target viral RNA has not been carefully examined in intact cells. Therefore, the mechanism of RNase L-mediated antiviral activity was investigated following encephalomyocarditis virus (EMCV) infection of cell lines in which expression of transfected RNase L was induced or endogenous RNase L activity was inhibited. RNase L induction markedly enhanced the anti-EMCV activity of IFN via a reduction in EMCV RNA. Inhibition of endogenous RNase L activity inhibited this reduction in viral RNA. RNase L had no effect on IFN-mediated protection from vesicular stomatitis virus. RNase L induction reduced the rate of EMCV RNA synthesis, suggesting that RNase L may target viral RNAs involved in replication early in the virus life cycle. The RNase L-mediated reduction in viral RNA occurred in the absence of detectable effects on specific cellular mRNAs and without any global alteration in the cellular RNA profile. Extensive rRNA cleavage, indicative of high levels of 2-5A, was not observed in RNase L-induced, EMCV-infected cells; however, transfection of 2-5A into cells resulted in widespread degradation of cellular RNAs. These findings provide the first demonstration of the selective capacity of RNase L in intact cells and link this selective activity to cellular levels of 2-5A.

Role for p53 in gene induction by double-stranded RNA.

Cross talk between p53 and interferon-regulated pathways is implicated in the induction of gene expression by biologic and genotoxic stresses. We demonstrate that the interferon-stimulated gene ISG15 is induced by p53 and that p53 is required for optimal gene induction by double-stranded RNA (dsRNA), but not interferon. Interestingly, virus induces ISG15 in the absence of p53, suggesting that virus and dsRNA employ distinct signaling pathways.

A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon.

2-5A-dependent RNase is the terminal factor in the interferon-regulated 2-5A system thought to function in both the molecular mechanism of interferon action and in the general control of RNA stability. However, direct evidence for specific functions of 2-5A-dependent RNase has been generally lacking. Therefore, we developed a strategy to block the 2-5A system using a truncated form of 2-5A-dependent RNase which retains 2-5A binding activity while lacking RNase activity. When the truncated RNase was stably expressed to high levels in murine cells, it prevented specific rRNA cleavage in response to 2-5A transfection and the cells were unresponsive to the antiviral activity of interferon alpha/beta for encephalomyocarditis virus. Remarkably, cells expressing the truncated RNase were also resistant to the antiproliferative activity of interferon. The truncated RNase is a dominant negative mutant that binds 2-5A and that may interfere with normal protein-protein interactions through nine ankyrin-like repeats.

Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase.

2-5A-dependent RNase (RNase L), a unique endoribonuclease that requires 5'-phosphorylated 2',5'-linked oligoadenylates (2-5A), functions in the molecular mechanism of interferon action. Because this enzyme is present at very low levels in nature, characterization and analysis have been limited. The molecular cloning of human, 2-5A-dependent RNase cDNA has facilitated its expression to high levels in insect cells by infecting with recombinant baculovirus. To determine the properties of the enzyme in the absence of other proteins, the recombinant 2-5A-dependent RNase was purified to homogeneity. The purified enzyme migrated as a monomer upon gel filtration in the absence of activator and showed highly specific, 2-5A-dependent RNase activity. The precise activator requirements were determined by stimulating the purified enzyme with a variety of 2',5'-linked oligonucleotides. The activated enzyme was capable of cleaving poly(rU) and, to a lesser extent, poly(rA), to sets of discrete products ranging from between 4 and 22 nucleotides in length. Reduced rates of 2-5A-dependent RNA cleavage were observed even after removal of ATP and chelation of divalent cations. However, optimal RNA cleavage rates required the presence of either manganese or magnesium and ATP.

RNase-L-dependent destabilization of interferon-induced mRNAs. A role for the 2-5A system in attenuation of the interferon response.

The 2-5A system is an interferon-regulated RNA degradation pathway with antiviral, growth-inhibitory, and pro-apoptotic activities. RNase-L mediates the antiviral activity through the degradation of viral RNAs, and the anticellular effects of the 2-5A system are thought to be similarly mediated through the degradation of cellular transcripts. However, specific RNase-L-regulated cellular RNAs have not been identified. To isolate candidate RNase-L substrates, differential display was used to identify mRNAs that exhibited increased expression in RNase-L-deficient N1E-115 cells as compared with RNase-L-transfected cells. A novel interferon-stimulated gene encoding a 43-kDa ubiquitin-specific protease, designated ISG43, was identified in this screen. ISG43 expression is induced by interferon and negatively regulated by RNase-L. ISG43 induction is a primary response to interferon treatment and requires a functional JAK/STAT signaling pathway. The kinetics of ISG43 induction were identical in wild type and RNase-L knock-out fibroblasts; however, the decline in ISG43 mRNA following interferon treatment was markedly attenuated in RNase-L knock-out fibroblasts. The delayed shut-off kinetics of ISG43 mRNA corresponded to an increase in its half-life in RNase-L-deficient cells. ISG15 mRNA also displayed RNase-L-dependent regulation. These findings identify a novel role for the 2-5A system in the attenuation of the interferon response.