Michael A. McNutt

H5N1 infection of the respiratory tract and beyond: a molecular pathology study

Summary Background Human infection with avian influenza H5N1 is an emerging infectious disease characterised by respiratory symptoms and a high fatality rate. Previous studies have shown that the human infection with avian influenza H5N1 could also target organs apart from the lungs. Methods We studied post-mortem tissues of two adults (one man and one pregnant woman) infected with H5N1 influenza virus, and a fetus carried by the woman. In-situ hybridisation (with sense and antisense probes to haemagglutinin and nucleoprotein) and immunohistochemistry (with monoclonal antibodies to haemagglutinin and nucleoprotein) were done on selected tissues. Reverse-transcriptase (RT) PCR, real-time RT-PCR, strand-specific RT-PCR, and nucleic acid sequence-based amplification (NASBA) detection assays were also undertaken to detect viral RNA in organ tissue samples. Findings We detected viral genomic sequences and antigens in type II epithelial cells of the lungs, ciliated and non-ciliated epithelial cells of the trachea, T cells of the lymph node, neurons of the brain, and Hofbauer cells and cytotrophoblasts of the placenta. Viral genomic sequences (but no viral antigens) were detected in the intestinal mucosa. In the fetus, we found viral sequences and antigens in the lungs, circulating mononuclear cells, and macrophages of the liver. The presence of viral sequences in the organs and the fetus was also confirmed by RT-PCR, strand-specific RT-PCR, real-time RT-PCR, and NASBA. Interpretation In addition to the lungs, H5N1 influenza virus infects the trachea and disseminates to other organs including the brain. The virus could also be transmitted from mother to fetus across the placenta.

SIRT1 is regulated by a PPARγ–SIRT1 negative feedback loop associated with senescence

Human Silent Information Regulator Type 1 (SIRT1) is an NAD+-dependent deacetylase protein which is an intermediary of cellular metabolism in gene silencing and aging. SIRT1 has been extensively investigated and shown to delay senescence; however, less is known about the regulation of SIRT1 during aging. In this study, we show that the peroxisome proliferator-activated receptor-γ (PPARγ), which is a ligand-regulated modular nuclear receptor that governs adipocyte differentiation and inhibits cellular proliferation, inhibits SIRT1 expression at the transcriptional level. Moreover, both PPARγ and SIRT1 can bind the SIRT1 promoter. PPARγ directly interacts with SIRT1 and inhibits SIRT1 activity, forming a negative feedback and self-regulation loop. In addition, our data show that acetylation of PPARγ increased with increasing cell passage number. We propose that PPARγ is subject to regulation by acetylation and deacetylation via p300 and SIRT1 in cellular senescence. These results demonstrate a mutual regulation between PPARγ and SIRT1 and identify a new posttranslational modification that affects cellular senescence.

PTENα, a PTEN Isoform Translated through Alternative Initiation, Regulates Mitochondrial Function and Energy Metabolism

PTEN is one of the most frequently mutated genes in human cancer. It is known that PTEN has a wide range of biological functions beyond tumor suppression. Here, we report that PTENα, an N-terminally extended form of PTEN, functions in mitochondrial metabolism. Translation of PTENα is initiated from a CUG codon upstream of and in-frame with the coding region of canonical PTEN. Eukaryotic translation initiation factor 2A (eIF2A) controls PTENα translation, which requires a CUG-centered palindromic motif. We show that PTENα induces cytochrome c oxidase activity and ATP production in mitochondria. TALEN-mediated somatic deletion of PTENα impairs mitochondrial respiratory chain function. PTENα interacts with canonical PTEN to increase PINK1 protein levels and promote energy production. Our studies demonstrate the importance of eIF2A-mediated alternative translation for generation of protein diversity in eukaryotic systems and provide insights into the mechanism by which the PTEN family is involved in multiple cellular processes.

HDAC inhibitors act with 5-aza-2'-deoxycytidine to inhibit cell proliferation by suppressing removal of incorporated abases in lung cancer cells.

5-aza-2′-deoxycytidine (5-aza-CdR) is used extensively as a demethylating agent and acts in concert with histone deacetylase inhibitors (HDACI) to induce apoptosis or inhibition of cell proliferation in human cancer cells. Whether the action of 5-aza-CdR in this synergistic effect results from demethylation by this agent is not yet clear. In this study we found that inhibition of cell proliferation was not observed when cells with knockdown of DNA methyltransferase 1 (DNMT1), or double knock down of DNMT1-DNMT3A or DNMT1-DNMT3B were treated with HDACI, implying that the demethylating function of 5-aza-CdR may be not involved in this synergistic effect. Further study showed that there was a causal relationship between 5-aza-CdR induced DNA damage and the amount of [3H]-5-aza-CdR incorporated in DNA. However, incorporated [3H]-5-aza-CdR gradually decreased when cells were incubated in [3H]-5-aza-CdR free medium, indicating that 5-aza-CdR, which is an abnormal base, may be excluded by the cell repair system. It was of interest that HDACI significantly postponed the removal of the incorporated [3H]-5-aza-CdR from DNA. Moreover, HDAC inhibitor showed selective synergy with nucleoside analog-induced DNA damage to inhibit cell proliferation, but showed no such effect with other DNA damage stresses such as γ-ray and UV, etoposide or cisplatin. This study demonstrates that HDACI synergistically inhibits cell proliferation with nucleoside analogs by suppressing removal of incorporated harmful nucleotide analogs from DNA.

NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules.

The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus and midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated alpha-tubulin, suggesting that it plays a role in maintaining or enhancing stability of alpha-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated alpha-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.

Distinctly different expression of cytokines and chemokines in the lungs of two H5N1 avian influenza patients

The pathogenesis of human H5N1 influenza remains poorly understood and controversial. ‘Cytokine storm’ has been hypothesized to be the main cause of the severity of this disease. However, the significance of this hypothesis has been called into question by a recent report, which demonstrates that inhibition of the cytokine response did not protect against lethal H5N1 influenza infection in mice. Here we showed discrepant findings in two adult H5N1 autopsies and a fetus obtained at autopsy which also raise doubt about this hypothesis. Antigens of 10 cytokines/chemokines which were found to be significantly elevated in previous H5N1‐infected patients and in vitro experiments, and mRNA of eight of these, were absent from the lungs of a pregnant woman and her fetus. In contrast, antigens of seven cytokines/chemokines and mRNA of six of these were found to be increased in the lungs of a male autopsy. The cells expressing these cytokines and chemokines were identified as type II pneumocytes, bronchial epithelial cells, macrophages and vascular endothelial cells. Levels of cytokines and chemokines in the serum of the male case were also significantly higher than those of infectious (infection other than by H5N1) and non‐infectious controls. In comparison with results from our previous study, it appeared that the male case had increased cytokine/chemokine expression but reduced viral load, while the pregnant female had diminished cytokine/chemokine expression but a significantly increased viral load in the lungs. These disparate findings in these two cases suggest that ‘cytokine storm’ alone could not be a sufficient explanation for the severe lung injury of this newly emerging disease. Copyright

Alpha-fetoprotein: A new member of intracellular signal molecules in regulation of the PI3K/AKT signaling in human hepatoma cell lines

Despite its well‐defined role as a serum growth factor during fetal liver development and hepatic oncogenesis, the biological significance of cytoplasmic alpha‐fetoprotein (AFP) remains incompletely understood. Here, we provide evidence to illustrate that cytoplasmic AFP may function as a regulator in the phosphatidylinositol 3‐kinase (PI3K)/AKT pathway in human hepatocellular carcinoma cells. The results demonstrated colocalization and interaction of AFP and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in the cytoplasm of AFP‐producing Bel 7402 and HepG2 cells, with an interaction distance of 12.6 ± 2.7 Å as determined with the fluorescence resonance energy transfer technique. Knockdown of AFP mRNA or inhibition of AFP expression by all trans‐retinoic acid resulted in enhancement of the PTEN level with a synchronous decrease in phosphorylated AKT. Transfection of the afp gene into HLE cells (originally AFP negative) led to a significant activation of AKT signaling. The inhibition of PI3K signaling by LY 294002 was simultaneously reversed by transfection, accompanied by diminution of all trans‐retinoic acid‐induced upregulation of PTEN and enhancement of cell growth. In conclusion, these results demonstrate that cytoplasmic AFP is involved in regulation of hepatocellular growth and tumorigenesis.

Alpha fetoprotein is a novel protein-binding partner for caspase-3 and blocks the apoptotic signaling pathway in human hepatoma cells

Although there is increasing evidence that alpha fetoprotein (AFP) may function as regulatory factor in the growth of tumor cells, the precise mechanism is still unclear. In the current study, we investigated the role of the cytoplasmic AFP in caspase‐3‐mediated signaling of apoptosis. Our results showed that low doses of TNF‐related apoptosis‐inducing ligand (TRAIL) elevated the activity of caspase‐8, but not caspase‐3. Caspase‐3 colocalized and interacted with AFP in the cytoplasm of Bel 7402 cells, and translocated into nuclei in association with the occurrence of apoptosis while cells were under cotreatment with all‐trans retinoic acid (ATRA) or TRAIL. AFP was able to form complexes with caspase‐3 and block onward transmission of signaling from caspase‐8. Knockdown of AFP increased the sensitivity of Bel 7402 cells to TRAIL, and thereby, triggered caspase‐3 signaling. No intermolecule interaction occurred between AFP and caspase‐8, nor was caspase‐8 activity altered after AFP knockdown, demonstrating the selectivity of AFP in interfering with the apoptotic signaling pathway. The effect of AFP on caspase‐3 was further confirmed by transfection of the AFP gene into HLE cells (AFP negative). We conclude that ATRA or TRAIL resistance in AFP producing hepatoma is at least, in part, attributable to the high level of the cytoplasmic AFP. Therefore, it is possible that the combination of AFP gene silencing together with ATRA/TRAIL cotreatment will benefit the enhancement of the chemotherapeutic efficiency of these agents on tumors.

Overexpression of LAPTM4B-35 promotes growth and metastasis of hepatocellular carcinoma in vitro and in vivo.

LAPTM4B-35, encoded by Lysosomal protein transmembrane 4 beta (LAPTM4B) is over-expressed in more than 71% of hepatocellular carcinomas (HCCs) and associated with prognosis of the patients. But the exact role and molecular mechanism in HCC have not been determined. In this study, we explored the effects and mechanisms of LAPTM4B-35 on tumor growth and metastasis in vitro and in vivo by overexpression and depletion of LAPTM4B in HCC HepG2 and Bel7402 cells. These findings suggest that overexpression of LAPTM4B-35 plays a critical role in the growth and metastasis of HCC, and LAPTM4B-35 may therefore be a therapeutic target for HCC.

Elevated serum alpha fetoprotein levels promote pathological progression of hepatocellular carcinoma

AIM To investigate the biological role of alpha fetoprotein (AFP) and its clinical significance in carcinogenesis of hepatocellular carcinoma (HCC). METHODS Clinical analysis of HCC patients and immunohistochemical examination were conducted to evaluate the relationship between serum AFP level and patient mortality. Confocal microscopy, Western blotting, dimethylthiahzolyl-2,5-diphenyl-tetrazolium bromide, Cell Counting Kit-8 assays and flow cytometry were performed to explore the possible mechanism. RESULTS Among the 160 HCC patients enrolled in this study, 130 patients survived 2 years (81.25%), with a survival rate of 86.8% in AFP < 2 0 μg/L group, 88.9% in AFP 20-250 μg/L group, and 69.6% in AFP > 250 μg/L group, demonstrating a higher mortality rate in HCC patients with higher AFP levels. Surgical treatment was beneficial only in patients with low AFP levels. The mortality rate of HCC patients with high AFP levels who were treated surgically was apparently higher than those treated with conservative management. The results of immunohistochemistry showed that AFP and AFP receptor were merely expressed in tissues of HCC patients with positive serum AFP. Consistently, in vitro analysis showed that AFP and AFPS were expressed in HepG2 but not in HLE cells. AFP showed a capability to promote cell growth, and this was more apparent in HepG2 cells, in which the proliferation was increased by 3.5 folds. Cell cycle analysis showed that the percentage of HepG2 cells in S phase after exposure to AFP was modestly increased. CONCLUSION HCC patients with higher AFP levels show a higher mortality rate, which appears to be attributable to the growth promoting properties of AFP.