Bortezomib attenuates HIF-1- but not HIF-2-mediated transcriptional activation

Bortezomib is the first proteasomal inhibitor (PI) to be used therapeutically for treating relapse cases of multiple myeloma and mantle cell lymphoma. A proposed mechanism for its action is that it prevents the proteasomal degradation of proapoptotic proteins, leading to enhanced apoptosis. Although the α subunit of hypoxia‑inducible factor (HIF)‑1 is not degraded with bortezomib treatment, the heterodimeric HIF‑1 fails to transactivate target genes. HIF‑1 and HIF‑2 are related hypoxia‑inducible transcription factors that are important for the survival of hypoxic tumor cells. The majority of reports have focused on the effects of bortezomib on the transcriptional activities of HIF‑1, but not HIF‑2. The present study investigated the effects of bortezomib on HIF‑2 activity in cancer cells with different levels of HIF‑1α and HIF‑2α subunits. HIF‑α subunit levels were detected using specific antibodies, while HIF transcriptional activities were evaluated using immunodetection, reverse transcription‑polymerase chain reaction and luciferase reporter assay. Bortezomib treatment was found to suppress the transcription and expression of CA9, a HIF‑1‑specific target gene; however, it had minimal effects on EPO and GLUT‑1, which are target genes of both HIF‑1 and HIF‑2. These data suggest that bortezomib attenuates the transcriptional activity only of HIF‑1, and not HIF‑2. This novel finding on the lack of an inhibitory effect of bortezomib on HIF‑2 transcriptional activity has implications for the improvement of design and treatment modalities of bortezomib and other PI drugs

Expression of transmembrane carbonic anhydrases, CAIX and CAXII, in human development

Background: Transmembrane CAIX and CAXII are members of the alpha carbonic anhydrase (CA) family. They play a crucial role in differentiation, proliferation, and pH regulation. Expression of CAIX and CAXII proteins in tumor tissues is primarily induced by hypoxia and this is particularly true for CAIX, which is regulated by the transcription factor, hypoxia inducible factor-1 (HIF-1). Their distributions in normal adult human tissues are restricted to highly specialized cells that are not always hypoxic. The human fetus exists in a relatively hypoxic environment. We examined expression of CAIX, CAXII and HIF-1 alpha in the developing human fetus and postnatal tissues to determine whether expression of CAIX and CAXII is exclusively regulated by HIF-1. Results: The co-localization of CAIX and HIF-1 alpha was limited to certain cell types in embryonic and early fetal tissues. Those cells comprised the primitive mesenchyma or involved chondrogenesis and skin development. Transient CAIX expression was limited to immature tissues of mesodermal origin and the skin and ependymal cells. The only tissues that persistently expressed CAIX protein were coelomic epithelium (mesothelium) and its remnants, the epithelium of the stomach and biliary tree, glands and crypt cells of duodenum and small intestine, and the cells located at those sites previously identified as harboring adult stem cells in, for example, the skin and large intestine. In many instances co-localization of CAIX and HIF-1 alpha was not evident. CAXII expression is restricted to cells involved in secretion and water absorption such as parietal cells of the stomach, acinar cells of the salivary glands and pancreas, epithelium of the large intestine, and renal tubules. Co-localization of CAXII with CAIX or HIF-1 alpha was not observed. Conclusion: The study has showed that: 1) HIF-1 alpha and CAIX expression co-localized in many, but not all, of the embryonic and early fetal tissues; 2) There is no evidence of co-localization of CAIX and CAXII; 3) CAIX and CAXII expression is closely related to cell origin and secretory activity involving proton transport, respectively. The intriguing finding of rare CAIX-expressing cells in those sites corresponding to stem cell niches requires further investigation

The oncolytic activity of Newcastle disease virus in clear cell renal carcinoma cells in normoxic and hypoxic conditions: the interplay between von Hippel-Lindau and interferon-β signaling

Viral-mediated oncolysis is a promising cancer therapeutic approach offering an increased efficacy with less toxicity than the current therapies. The complexity of solid tumor microenvironments includes regions of hypoxia. In these regions, the transcription factor, hypoxia inducible factor (HIF), is active and regulates expression of many genes that contribute to aggressive malignancy, radio-, and chemo-resistance. To investigate the oncolytic efficacy of a highly virulent (velogenic) Newcastle disease virus (NDV) in the presence or absence of HIF-2α, renal cell carcinoma (RCC) cell lines with defective or reconstituted wild-type (wt) von Hippel-Lindau (VHL) activity were used. We show that these RCC cells responded to NDV by producing only interferon (IFN)-β, but not IFN-α, and are associated with increased STAT1 phosphorylation. Restoration of wt VHL expression enhanced NDV-induced IFN-β production, leading to prolonged STAT1 phosphorylation and increased cell death. Hypoxia augmented NDV oncolytic activity regardless of the cells' HIF-2α levels. These results highlight the potential of oncolytic NDV as a potent therapeutic agent in the killing of hypoxic cancer cells

Partial protection against enterovirus 71 (EV71) infection in a mouse model immunized with recombinant newcastle disease virus capsids displaying the EV71 VP1 fragment.

Enterovirus 71 (EV71) infection may cause severe neurological complications, particularly in young children. Despite the risks, there are still no commercially available EV71 vaccines. Hence, a candidate vaccine construct, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP1 1-100) protein, was evaluated in a mouse model of EV71 infection. Previously, it was shown that this protein construct provoked a strong immune response in vaccinated adult rabbits. That study, however, did not address the issue of its effectiveness against EV71 infection in young animals. In the present study, EV71 viral challenge in vaccinated newborn mice resulted in more than 40% increase in survival rate. Significantly, half of the surviving mice fully recovered from their paralysis. Histological analysis of all of the surviving mice revealed a complete clearance of EV71 viral antigens from their brains and spinal cords. In hind limb muscles, the amounts of the antigens detected correlated with the degrees of tissue damage and paralysis. Findings from this study provide evidence that immunization with the NPt-VP1 1-100 immunogen in a newborn mouse model confers partial protection against EV71 infection, and also highlights the importance of NPt-VP1 1-100 as a possible candidate vaccine for protection against EV71 infections

Immunization with recombinant enterovirus 71 viral capsid protein 1 fragment stimulated antibody responses in hamsters

Enterovirus 71 (EV71) causes severe neurological diseases resulting in high mortality in young children worldwide. Development of an effective vaccine against EV71 infection is hampered by the lack of appropriate animal models for efficacy testing of candidate vaccines. Previously, we have successfully tested the immunogenicity and protectiveness of a candidate EV71 vaccine, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP1(1-100)) protein, in a mouse model of EV71 infection. A drawback of this system is its limited window of EV71 susceptibility period, 2 weeks after birth, leading to restricted options in the evaluation of optimal dosing regimens. To address this issue, we have assessed the NPt-VP1(1-100) candidate vaccine in a hamster system, which offers a 4-week susceptibility period to EV71 infection. Results obtained showed that the NPt-VP1(1-100) candidate vaccine stimulated excellent humoral immune response in the hamsters. Despite the high level of antibody production, they failed to neutralize EV71 viruses or protect vaccinated hamsters in viral challenge studies. Nevertheless, these findings have contributed towards a better understanding of the NPt-VP1(1-100) recombinant protein as a candidate vaccine in an alternative animal model system

Human renal carcinoma cells respond to Newcastle disease virus infection through activation of the p38 MAPK/NF-κB/IκBα pathway

Purpose: Newcastle disease virus (NDV) is an oncolytic virus that is known to have a higher preference to cancer cells than to normal cells. It has been proposed that this higher preference may be due to defects in the interferon (IFN) responses of cancer cells. The exact mechanism underlying this process, however, remains to be resolved. In the present study, we examined the antiviral response towards NDV infection of clear cell renal cell carcinoma (ccRCC) cells. ccRCC is associated with mutations of the von Hippel-Lindau tumor suppressor gene VHL, whose protein product is important for eliciting cellular responses to changes in oxygen levels. The most common first line treatment strategy of ccRCC includes IFN. Unfortunately, most ccRCC cases are diagnosed at a late stage and often are resistant to IFN-based therapies. Alternative treatment approaches, including virotherapy using oncolytic viruses, are currently being investigated. The present study was designed to investigate the mechanistic pathways underlying the response of ccRCC cells to oncolytic NDV infection. Methods and results: We found that NDV induces activation of NF-κB in ccRCC cells by inducing phosphorylation and subsequent degradation of IκBα. IκBα was found to be phosphorylated as early as 1 hour post-infection and to result in rapid NF-κB nuclear translocation and activation. Importantly, p38 MAPK phosphorylation was found to occur upstream of the NDV-induced NF-κB activation. Restoration of VHL in ccRCC cells did not result in a reduction of this phosphorylation. A similar phenomenon was also observed in several other cancer-derived cell lines. Conclusion: Our data provide evidence for involvement of the p38 MAPK/NF-κB/IκBα pathway in NDV infection and subsequent induction of apoptosis in ccRCC cells

Mitogen-activated protein/extracellular signal-regulated kinase kinase 1act/tubulin interaction is an important determinant of mitotic stability in cultured HT1080 human fibrosarcoma cells.

Activation of the mitogen-activated protein kinase (MAPK) pathway plays a major role in neoplastic cell transformation. Using a proteomics approach, we identified alpha tubulin and beta tubulin as proteins that interact with activated MAP/extracellular signal-regulated kinase kinase 1 (MEK1), a central MAPK regulatory kinase. Confocal analysis revealed spatiotemporal control of MEK1-tubulin colocalization that was most prominent in the mitotic spindle apparatus in variant HT1080 human fibrosarcoma cells. Peptide arrays identified the critical role of positively charged amino acids R108, R113, R160, and K157 on the surface of MEK1 for tubulin interaction. Overexpression of activated MEK1 caused defects in spindle arrangement, chromosome segregation, and ploidy. In contrast, chromosome polyploidy was reduced in the presence of an activated MEK1 mutant (R108A, R113A) that disrupted interactions with tubulin. Our findings indicate the importance of signaling by activated MEK1-tubulin in spindle organization and chromosomal instability

Development and utilization of a somatic cell hybrid mapping panel to assign NotI linking probes to the long arm of human chromosome 6

A somatic cell hybrid mapping panel that defines seven regions of the long arm and one region of the short arm of human chromosome 6 has been developed. Utilizing this panel, 17 NotI boundary clones from a NotI linking library were regionally assigned to the long arm of chromosome 6. The majority of these clones (11) were found to localize within band regions 6q24-q27. The nonuniform distribution of NotI sites may indicate a cluster of HTF islands and likely represents a coincidence of coding sequences in this region of chromosome 6. Cross-hybridization of these linking clones to DNA from other species (zoo blots) provides further evidence for transcribed sequences in 7 of the NotI clones. These NotI clones were also used to identify corresponding NotI fragments using pulsed-field gel electrophoresis, facilitating further physical mapping of this region. Finally, regional assignment of five polymorphic probes to the long arm of chromosome 6 is also presented. These hybrids and probes should facilitate the construction of a physical and genetic linkage map to assist in the identification of disease loci along chromosome 6.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30194/1/0000582.pd