Express��o dos fatores de regula����o miog��nica em cultura de mioblastos submetidos ao TNF-��/IFN-��

A insufici��ncia card��aca (IC) est�� associada a miopatia dos m��sculos esquel��ticos dos membros, com perda da massa muscular, diminui����o na propor����o das fibras do tipo I (contra����o lenta) e aumento na propor����o das fibras do tipo II (contra����o r��pida). �� prov��vel que altera����es na express��o de fatores de transcri����o pertencentes �� fam��lia ���basic helix-loop-helix��� (bHLH), da qual fazem parte a MyoD, Miogenina, Myf5 e o MRF-4, conhecidos como fatores de regula����o miog��nica (MRFs), sejam respons��veis pelas mudan��as nos tipos de fibras. Enquanto que a Miogenina �� expressa em n��veis superiores aos da MyoD em m��sculos lentos, o oposto �� verdadeiro para m��sculos r��pidos. Similarmente, a MyoD est�� associada com a express��o das isoformas de miosina de cadeia pesada r��pidas dos tipos IIX e IIB. Estudos in vitro, demonstraram que o TNF-�� inibe a express��o de MyoD e miogenina diminuindo a atividade de genes m��sculo espec��ficos. A a����o do TNF-�� diminuindo a express��o da MyoD mostra-se mais acentuada quando em associa����o com o IFN-��, no entanto, h�� poucas informa����es na literatura a respeito do papel desta associa����o na express��o dos fatores de regula����o miog��nica, in vitro. Avaliar a express��o dos fatores de regula����o miog��nica, MyoD, miogenina, Myf5, e MRF-4 em cultura de mioblastos C2C12 submetidos ao TNF-��/IFN-��. Nossos resultados mostraram um aumentou na express��o dos gene MyoD, Myf5 e miogenina sob tratamento com IFN-�� quando comparado aos grupos controle e TNF-��/IFN-��. A express��o g��nica do MRF-4 na cultura de c��lulas n��o foi detectada em nenhum dos grupos analisados. O GAPDH foi utilizado para normalizar os valores de express��o dos outros genes analisados. O presente estudo demonstrou que o IFN-�� ex��geno administrado �� culturas de mioblastos... (Resumo completo, clicar acesso eletr��nico abaixo)

Leukocytes from wheezing infants release lower amounts of IL-12 and IFN-gamma compared to non-wheezing infants

Objective This study investigated environmental endotoxin exposure during early life, sensitization to aeroallergens, the production of cytokines by LPS-stimulated leukocytes, and the development of a wheezing phenotype in a prospective cohort of infants with high risk of developing allergic diseases. Materials and Methods Eighty-four infants were followed from birth until 30 months of age. We assessed endotoxin concentration in house dust of their homes during the first 6 months of life. At age 30 months they were clinically evaluated to determine the development of wheezing and other clinical events, were skin prick tested, and had blood samples collected for the evaluation of cytokine release by LPS-stimulated peripheral blood mononuclear cells (PBMC). Results The level of endotoxin exposure during early life was not associated with development of a wheezing phenotype. On the other hand a higher incidence of respiratory infections occurred among recurrent wheezing (RW) infants. PBMC from RW children exposed to higher levels of environmental endotoxin (above 50?EU/mg) released less Interleukin (IL)-12p70 and IFN-? compared to the non-RW group. TNF-a, IL-10, IL-4, IL-5, and IL17 production by LPS-stimulated PBMC from RW and non-RW children was equivalent in both groups of environmental endotoxin exposure. Conclusion In this prospective cohort of infants with high risk of developing allergic diseases we observed that RW and non-RW children were exposed to similar levels of endotoxin early in life. LPS-stimulated PBMC from RW infants exposed to higher levels of endotoxin released significantly less IL-12 and IFN-? compared to non-RW infants. Pediatr Pulmonol. 2012. 47:10541060. (C) 2012 Wiley Periodicals, Inc.

IFN-gamma Plays a Unique Role in Protection against Low Virulent Trypanosoma cruzi Strain

Background: T. cruzi strains have been divided into six discrete typing units (DTUs) according to their genetic background. These groups are designated T. cruzi I to VI. In this context, amastigotes from G strain (T. cruzi I) are highly infective in vitro and show no parasitemia in vivo. Here we aimed to understand why amastigotes from G strain are highly infective in vitro and do not contribute for a patent in vivo infection. Methodology/Principal Findings: Our in vitro studies demonstrated the first evidence that IFN-gamma would be associated to the low virulence of G strain in vivo. After intraperitoneal amastigotes inoculation in wild-type and knockout mice for TNF-alpha, Nod2, Myd88, iNOS, IL-12p40, IL-18, CD4, CD8 and IFN-gamma we found that the latter is crucial for controlling infection by G strain amastigotes. Conclusions/Significance: Our results showed that amastigotes from G strain are highly infective in vitro but did not contribute for a patent infection in vivo due to its susceptibility to IFN-gamma production by host immune cells. These data are useful to understand the mechanisms underlying the contrasting behavior of different T. cruzi groups for in vitro and in vivo infection.

Ectoplacental Cone Induces Resistance to Apoptosis in High Doses of Interferon (IFN)-gamma-Treated Decidual Cells

Problem In this study, we explored the relationship between decidual cells (DC) and interferon (IFN)-gamma, in the presence or absence of ectoplacental cone (EC) using a coculture system. Method of study Decidual cells and EC were isolated from pregnant mice on gestation day 7.5. DCs were cultured for 48 hr and then treated with fresh EC. After characterization, they were treated with IFN-gamma, and cell death was evaluated. Results Interferon-gamma drastically increased decidual apoptosis, which was partially reverted by the addition of EC to the IFN-gamma-treated decidual culture. Moreover, the addition of EC to non-treated DC cultures was also capable of attenuating death rates. Conclusion Resistance to apoptosis may be induced in DC by the EC. This suggests that EC may participate in the inhibition of IFN-gamma-dependent apoptosis and, therefore, play important role for DC survival in a cytokineenriched placental environment.

IFN-��-induced priming maintains long-term strain-transcending immunity against blood-stage Plasmodium chabaudi malaria

The mechanism by which protective immunity to Plasmodium is lost in the absence of continued exposure to this parasite has yet to be fully elucidated. It has been recently shown that IFN-�� produced during human and murine acute malaria primes the immune response to TLR agonists. In this study, we investigated whether IFN-��-induced priming is important to maintain long-term protective immunity against Plasmodium chabaudi AS malaria. On day 60 postinfection, C57BL/6 mice still had chronic parasitemia and efficiently controlled homologous and heterologous (AJ strain) challenge. The spleens of chronic mice showed augmented numbers of effector/effector memory (TEM) CD4(+) cells, which is associated with increased levels of IFN-��-induced priming (i.e., high expression of IFN-inducible genes and TLR hyperresponsiveness). After parasite elimination, IFN-��-induced priming was no longer detected and protective immunity to heterologous challenge was mostly lost with >70% mortality. Spontaneously cured mice had high serum levels of parasite-specific IgG, but effector T/TEM cell numbers, parasite-driven CD4(+) T cell proliferation, and IFN-�� production were similar to noninfected controls. Remarkably, the priming of cured mice with low doses of IFN-�� rescued TLR hyperresponsiveness and the capacity to control heterologous challenge, increasing the TEM cell population and restoring the CD4(+) T cell responses to parasites. Contribution of TLR signaling to the CD4(+) T cell responses in chronic mice was supported by data obtained in mice lacking the MyD88 adaptor. These results indicate that IFN-��-induced priming is required to maintain protective immunity against P. chabaudi and aid in establishing the molecular basis of strain-transcending immunity in human malaria.

Differential inflammasome expression and IL-1�� secretion in monocyte-derived dendritic cells differentiated with IL-4 or IFN-��

NLRP3-inflammasome activation was evaluated in monocyte-derived dendritic cells (DC) obtained through IL-4 (IL4-DC) or IFN-�� (IFN-DC) protocols and pulsed with chemically inactivated HIV-1. Inflammasome' genes expression and IL-1�� secretion were compared in DC isolated from 15 healthy subjects (HC) and 10 HIV-1 infected individuals (HIV+). FINDINGS: Whether HIV was able to increased NLRP3-inflammasome genes expression and IL-1�� secretion in IL4-DC from HC, the induction of inflammasome appeared significantly reduced in IFN-DC from HC, suggesting a different responsive state of IFN-DC compared to IL4-DC. No inflammasome activation was observed in IL4-DC as well as in IFN-DC derived from HIV���+���subjects, confirming previous findings on "unresponsive" state of DC derived from HIV���+���possibly due to chronic inflammatory state of these individuals. CONCLUSIONS: Our results showed that IFN-�� differently modulates inflammasome expression during monocytes-DC in vitro differentiation. These findings could be of interest considering the on-going research about DC manipulation and therapeutic strategies for HIV���+���involving DC-based immune-vaccines.

Untersuchung der TNF-alpha vermittelten IFN-beta Signaltransduktion in malignen und nicht-malignen HPV positiven Zellen

Bestimmte humane Papillomviren sind an der Entstehung von Zervixkarzinomen beteiligt. In dieser Arbeit wird gezeigt, da�� maligne HPV-positive Zellen ihre F��higkeit zur Induktion von endogenem IFN-beta nach TNF-alpha verloren haben. Durch Infektion mit Encephalomyocarditis Virus (EMCV) oder Vesicular Stomatitis Virus (VSV) wurde die Induzierbarkeit des endogenen IFN-beta durch TNF-alpha in nicht-tumorigenen Zellen best��tigt. Alle malignen Zellinien zeigten eine intakte IFN Signaltransduktion, wenn Typ I oder Typ II Interferone exogen supplementiert wurden. Dies zeigt, da�� in tumorigenen Zervixkarzinomzellen die Kommunikation zwischen TNF-alpha und IFN-beta

Die Rolle der IFN-gamma Signal��bertragung und von MyD88 in der Angiotensin-II-induzierten vaskul��ren Dysfunkion, Inflammation und arteriellen Hypertonie

Im Rahmen dieser Arbeit wurde die Rolle von myelomonozyt��ren Zellen, IFN-gamma (Interferon gamma), MyD88 (myeloid differentiation factor 88) und zugrundeliegenden Signalwege in der Angiotensin II (ATII)-induzierten vaskul��ren Inflammation, Dysfunktion und arteriellen Hypertonie untersucht. Wie bereits ver��ffentlichte Vordaten aus meiner Arbeitsgruppe zeigten, sch��tzt die Depletion von Lysozym M (LysM)+ myelomonozyt��ren Zellen (Diphteriatoxin-vermittelt in M��usen, die transgen f��r den humanen Diphtheriatoxin-Rezeptor sind, LysMiDTR M��use) vor der ATII-induzierten vaskul��ren Dysfunktion und arterieller Hypertonie, und kann durch adoptiven Zelltransfer von Wildtyp Monozyten wiederhergestellt werden. In meiner Arbeit konnte ich zeigen, dass die Rekonstitution von Monozyten-depletierten LysMiDTR M��usen mit Wildtyp Monozyten den Ph��notyp der vaskul��ren Dysfunktion wiederherstellen kann, die Rekonstitution mit gp91phox-/y oder Agtr1-/- Monozyten jedoch nicht. Die Hypertonus-mediierenden Effekte dieser infiltrierenden Monozyten scheinen demnach von der intakten ATII und NADPH Oxidase Signal��bertragung in diesen Zellen abh��ngig zu sein. Vermutlich ebenfalls f��r die Aktivierung der Monozyten funktionell wichtig sind IFN-gamma, produziert durch NK-Zellen, und der Transkriptionsfaktor T-bet (T-box expressed in T cells), exprimiert von NK-Zellen und Monozyten. IFN-gamma-/- M��use waren partiell gesch��tzt vor der ATII-induzierten vaskul��ren Dysfunktion und charakterisiert durch reduzierte Level an Superoxid im Gef���� im Vergleich zu ATII-infundierten Wildtyp M��usen. IFN-gamma-/- und T-bet defiziente Tbx21-/- M��use zeichneten sich ferner durch eine reduzierte ATII-mediierte Rekrutierung von NK1.1+ NK-Zellen, als ein Hautproduzent von IFN-gamma, sowie CD11b+GR-1low Interleukin-12 (IL-12) kompetenten Monozyten aus. Durch Depletions- und adoptive Transferexperimente konnte ich in dieser Arbeit NK-Zellen als essentielle Mitstreiter in der vaskul��ren Dysfunktion identifizieren und stellte fest, dass T-bet+LysM+ myelomonozyt��re Zellen f��r die NK-Zellrekrutierung in die Gef����wand und lokale IFN-gamma Produktion ben��tigt werden. Damit wurde erstmals NK-Zellen eine essentielle Rolle in der ATII-induzierten vaskul��ren Dysfunktion zugeschrieben. Au��erdem wurde der T-bet-IFN-gamma Signalweg und die gegenseitige Monozyten-NK-Zellaktivierung als ein potentielles therapeutisches Ziel in kardiovaskul��ren Erkrankungen aufgedeckt. Des Weiteren identifizierte ich in meiner Arbeit MyD88 als ein zentrales Signalmolek��l in der ATII-getriebenen Inflammation und vaskul��ren Gef����sch��digung. MyD88 Defizienz reduzierte den ATII-induzierten Anstieg des systolischen Blutdrucks und die endotheliale und glattmuskul��re vaskul��re Dysfunktion. Zus��tzlich waren die vaskul��re Superoxid-Bildung sowie die Expressionslevel der NADPH Oxidase, der wichtigsten Quelle f��r oxidativem Stress im Gef����, in ATII-infundierten MyD88-/- M��usen im Vergleich zum Wildtyp reduziert. Mit Hilfe von durchflusszytometrischen Analysen deckte ich zudem auf, dass die ATII-induzierte Einwanderung von CD45+ Leukozyten, insbesondere CD11b+Ly6G-Ly6Chigh inflammatorischen Monozyten in MyD88-/- M��usen signifikant abgeschw��cht war. Diese Resultate wurden durch immunhistochemische Untersuchung von Aortengewebe auf CD68+, F4/80+ und Nox2+ Makrophagen/Phagozyten sowie Expressionsanalysen von Inflammationsmarkern untermauert. Analysen der mRNA Expression in Aortengewebe zeigten ferner eine in Wildtyp M��usen nach ATII Infusion tendenziell gesteigerte Expression von inflammatorischen Monozytenmakern sowie eine abnehmende Expression von reparativen Monozytenmarken, w��hrend dieser Shift zu einem proinflammatorsichen Ph��notyp in MyD88-/- blockiert zu sein schien. Dies zeigt eine Rolle von MyD88 in der terminalen Differenzierung von myelomonozyt��ren Zellen an. Um dies weitergehend zu untersuchen und aufzudecken, ob die MyD88 Effekte abh��ngig sind von Zellen der h��matopoetischen Linie oder Gewebszellen, wurden Knochenmarktransferexperimente durchgef��hrt. MyD88 Defizienz in Knochenmark-abstammende Zellen reduzierte die ATII-induzierte vaskul��re Dysfunktion und Infiltration der Gef����wand mit CD45+ Leukozyten und inflammatorischen myelomonozyt��ren Zellen. Die protektiven Effekte der MyD88 Defizienz in der Angiotensin II-induzierten Inflammation konnten nicht auf Signalwege ��ber die Toll-like Rezeptoren TLR2, -7 oder -9 zur��ckgef��hrt werden, wie die Untersuchung der vaskul��ren Reaktivit��t entsprechender Knockout M��use zeigte. Zusammenfassend konnte ich in meiner Arbeit zeigen, dass die Infiltration der Gef����wand mit Nox2+AT1R+T-bet+MyD88+ myelomonozyt��ren Zellen und die Wechselwirkung und gegenseitige Aktivierung dieser Zellen mit IFN-gamma produzierenden NK-Zellen eine zentrale Bedeutung in der Pathogenese der Angiotensin II (ATII)-induzierten vaskul��ren Dysfunktion, Inflammation und arteriellen Hypertonie einnehmen.

Absence of MyD88 results in enhanced TLR3-dependent phosphorylation of IRF3 and increased IFN-(beta) and RANTES production

Toll-like receptors are a group of pattern-recognition receptors that play a crucial role in "danger" recognition and induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral dsRNA, resulting in the induction of the anti-viral molecule, IFN- . Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Previous studies have shown that the TLR adaptor, Mal/TIRAP, an activator of TLR4, inhibits TLR3-mediated IFN- induction through a mechanism involving IRF7. In this study, we sought to investigate whether the TLR adaptor, MyD88, an activator of all TLRs except TLR3, has the ability to modulate TLR3 signaling. Although MyD88 does not significantly affect TLR3 ligand-induced TNF- induction, MyD88 negatively regulates TLR3-, but not TLR4-, mediated IFN- and RANTES production; this process is mechanistically distinct from that employed by Mal/TIRAP. We show that MyD88 inhibits IKK -, but not TBK1-, induced activation of IRF3. In doing so, MyD88 curtails TLR3 ligand-induced IFN- induction. The present study shows that while MyD88 activates all TLRs except TLR3, MyD88 also functions as a negative regulator of TLR3. Thus, MyD88 is essential in restricting TLR3 signaling, thereby protecting the host from unwanted immunopathologies associated with the excessive production of IFN- . Our study offers a new role for MyD88 in restricting TLR3 signaling through a hitherto unknown mechanism whereby MyD88 specifically impairs IKK -mediated induction of IRF3 and concomitant IFN- and RANTES production.

Inflammasome-dependent IFN-�� drives pathogenesis in Streptococcus pneumoniae meningitis

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-��) was strongly induced, and IFN-��(-/-) mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-��. Furthermore, production of IFN-�� was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-�� induction. The influence of IFN-�� gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-��(-/-) mice, both monocyte recruitment and CCL2 production were less in infected IFN-��(-/-) mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-��(-/-) mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-��(-/-) mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-�� contributes via multiple pathways to pathology during S. pneumoniae meningitis.

The physiologic increase in expression of some type I IFN-inducible genes during pregnancy is not associated with improved disease activity in pregnant patients with rheumatoid arthritis

During pregnancy, most patients with rheumatoid arthritis (RA) experience a spontaneous improvement in their condition. Since type I interferons (IFN) have immunomodulatory properties, we investigated whether type I IFN-inducible genes are upregulated in pregnant patients with RA. Peripheral blood mononuclear cells were evaluated using quantitative real-time polymerase chain reaction for type I IFN-inducible genes (IFI 35, IFI44, IFI44L, IFIT3, OAS1, and Siglec1) in patients with RA and healthy women during and after pregnancy as well as in nonpregnant controls. IFN-alpha and IFN-beta levels in sera of patients and healthy donors were analyzed by enzyme linked immunosorbent assay. It was found that healthy women did not show a change of gene expression levels from the second trimester until postpartum, yet some type I IFN-inducible genes were significantly upregulated in pregnant and postpartum women compared with nonpregnant individuals. In patients with RA, a pronounced upregulation of IFI35 and IFI44 at the second trimester and a peak expression of Siglec1 at the third trimester were observed. Pregnancy levels of IFI35 and IFI44 in patients with RA were higher than those of nonpregnant patients with RA. No significant association of gene expression levels with disease activity was found. In the sera of patients and healthy women, IFN-beta was undetectable and IFN-alpha levels remained stable throughout pregnancy and postpartum. Thus, pregnancy can give rise to an increased expression of type I IFN-inducible genes, reflecting an upregulation of the innate immune system. However, an association of type I IFN-inducible genes with pregnancy induced disease amelioration seems unlikely.

Anti-HIV state but not apoptosis depends on IFN signature in CD4+ T cells

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4(+) T cell target population, we examined gene expression in CD4(+) T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-alpha-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-alphaR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-alpha. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4(+) T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4(+) T cells, which induces an anti-HIV state but not apoptosis.

Stimulation-specific contribution of p38 and JNK to IFN-beta gene expression in human macrophages

Induction of interferon-beta (IFN-beta) gene expression is a tightly regulated process, and a plethora of studies identified the signal transduction pathway TANK-binding kinase-1 (TBK-1)/IFN regulatory factor-3 (IRF-3) as essential to the induction of IFN-beta gene expression. Data regarding the role of p38 and JNK are rare, however. We investigated the contribution of these kinases to IFN-beta expression in human macrophages treated with poly(I:C), lipopolysaccharide (LPS), Sendai virus, or vesicular stomatitis virus (VSV). We found that all the stimuli induced IFN-beta mRNA, albeit to a different extent. Whereas LPS and VSV induced the phosphorylation of p38 and JNK, neither poly(I:C) nor Sendai virus led to the detection of phosphospecific signals. When inhibiting p38, a VSV-triggered IFN-beta mRNA response was inhibited, whereas inhibiting JNK suppressed an LPS-triggered response, but only when macrophages were primed with IFN-gamma. Neither poly(I:C)-induced nor Sendai virus-induced IFN-beta mRNA expression was affected when p38 and JNK were inhibited. Collectively, the data show that the contribution of p38 and JNK to the expression of IFN-beta occurs in a stimulation-specific manner in human macrophages.

Type I IFN induction in response to Listeria monocytogenes in human macrophages: evidence for a differential activation of IFN regulatory factor 3 (IRF3)

Listeria monocytogenes is a prototypic bacterium for studying innate and adaptive cellular immunity as well as host defense. Using human monocyte-derived macrophages, we report that an infection with a wild-type strain, but not a listeriolysin O-deficient strain, of the Gram-positive bacterium L. monocytogenes induces expression of IFN-beta and a bioactive type I IFN response. Investigating the activation of signaling pathways in human macrophages after infection revealed that a wild-type strain and a hemolysin-deficient strain of L. monocytogenes activated the NF-kappaB pathway and induced a comparable TNF response. p38 MAPK and activating transcription factor 2 were phosphorylated following infection with either strain, and IFN-beta gene expression induced by wild-type L. monocytogenes was reduced when p38 was inhibited. However, neither IFN regulatory factor (IRF) 3 translocation to the nucleus nor posttranslational modifications and dimerizations were observed after L. monocytogenes infection. In contrast, vesicular stomatitis virus and LPS triggered IRF3 activation and signaling. When IRF3 was knocked down using small interfering RNA, a L. monocytogenes-induced IFN-beta response remained unaffected whereas a vesicular stomatitis virus-triggered response was reduced. Evidence against the possibility that IRF7 acts in place of IRF3 is provided. Thus, we show that wild-type L. monocytogenes induced an IFN-beta response in human macrophages and propose that this response involves p38 MAPK and activating transcription factor 2. Using various stimuli, we show that IRF3 is differentially activated during type I IFN responses in human macrophages.

Dual induction of PKR with E2F-1 and IFN-alpha to enhance gene therapy against hepatocellular carcinoma

Overexpression of the transcription factor E2F-1 induces apoptosis in tumor cells. This apoptotic effect is partly mediated through the induction of the double-stranded RNA-activated protein kinase (PKR). Here, we investigate if agents that upregulate PKR could enhance the apoptotic effect of E2F-1 overexpression in liver tumors. In human hepatocellular carcinoma (HCC) cells (Hep3B, HepG2, Huh7), adenovirus-mediated overexpression of E2F-1 (AdCMV-E2F) transcriptionally increased PKR mRNA. The subsequent increase of total and phosphorylated PKR protein was followed by induction of apoptosis. When AdCMV-E2F was combined with the PKR modifier interferon alpha (IFNalpha), PKR was additionally upregulated and both PKR activation and apoptosis were increased. Subcutaneous xenograft tumors were selectively targeted using an adenoviral vector expressing E2F-1 under the control of the human telomerase reverse transcriptase (hTERT) promoter (AdhTERT-E2F). Weekly systemic administration of AdhTERT-E2F inhibited tumor growth. The tumor suppressive effect of AdhTERT-E2F therapy was further enhanced in combination with IFNalpha.Our results demonstrate that PKR activating agents enhance the anti-tumor effect of E2F-1 overexpression in HCC in-vitro and in-vivo. Hence, modulation of PKR is a potential strategy to increase the efficacy of PKR-dependent anti-tumor therapies.