Genome-wide analysis of aberrant methylation in human breast cancer cells using methyl-DNA immunoprecipitation combined with high-throughput sequencing

<p>Abstract</p> <p>Background</p> <p>Cancer cells undergo massive alterations to their DNA methylation patterns that result in aberrant gene expression and malignant phenotypes. However, the mechanisms that underlie methylome changes are not well understood nor is the genomic distribution of DNA methylation changes well characterized.</p> <p>Results</p> <p>Here, we performed methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) to obtain whole-genome DNA methylation profiles for eight human breast cancer cell (BCC) lines and for normal human mammary epithelial cells (HMEC). The MeDIP-seq analysis generated non-biased DNA methylation maps by covering almost the entire genome with sufficient depth and resolution. The most prominent feature of the BCC lines compared to HMEC was a massively reduced methylation level particularly in CpG-poor regions. While hypomethylation did not appear to be associated with particular genomic features, hypermethylation preferentially occurred at CpG-rich gene-related regions independently of the distance from transcription start sites. We also investigated methylome alterations during epithelial-to-mesenchymal transition (EMT) in MCF7 cells. EMT induction was associated with specific alterations to the methylation patterns of gene-related CpG-rich regions, although overall methylation levels were not significantly altered. Moreover, approximately 40% of the epithelial cell-specific methylation patterns in gene-related regions were altered to those typical of mesenchymal cells, suggesting a cell-type specific regulation of DNA methylation.</p> <p>Conclusions</p> <p>This study provides the most comprehensive analysis to date of the methylome of human mammary cell lines and has produced novel insights into the mechanisms of methylome alteration during tumorigenesis and the interdependence between DNA methylome alterations and morphological changes.</p

Repeated Pancreatectomy for Recurrent Pancreatic Carcinoma after Pylorus-Preserving Pancreatoduodenectomy: Report of Two Patients

Repeated pancreatectomy for pancreatic carcinoma is extremely rare. We report two such patients who underwent pancreatectomy for carcinoma developing in the pancreatic remnant after pylorus-preserving pancreatoduodenectomy (PpPD) for invasive pancreatic ductal carcinoma. One patient underwent PpPD for invasive pancreatic ductal carcinoma and received adjuvant chemotherapy. Follow-up computed tomography (CT) demonstrated a low-density mass in the remnant pancreas, which was diagnosed as a carcinoma by endoscopic ultrasound-guided fine-needle aspiration cytology 5 years 10 months after PpPD. She underwent curative resection of the remnant pancreas and is alive and well 13 months after the second operation. The other patient underwent PpPD for invasive pancreatic ductal carcinoma. Follow-up CT showed a low-density mass in the remnant pancreas after 2 years 11 months. He received systemic chemotherapy with S-1 for 3 months. The tumor shrank, and the patient underwent curative resection of the remnant pancreas 3 years 1 month after the initial operation. Repeated pancreatectomy may provide a chance of long survival for patients with carcinoma developing in the remnant pancreas after pancreatectomy if the recurrence occurring at long term is limited to the remnant pancreas

Retrovirus Entry by Endocytosis and Cathepsin Proteases

Retroviruses include infectious agents inducing severe diseases in humans and animals. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus-induced diseases and efficient exploitation of retroviral vectors. Entry of enveloped viruses into host cell cytoplasm is achieved by fusion between the viral envelope and host cell membranes at either the cell surface or intracellular vesicles. Many animal retroviruses enter host cells through endosomes and require endosome acidification. Ecotropic murine leukemia virus entry requires cathepsin proteases activated by the endosome acidification. CD4-dependent human immunodeficiency virus (HIV) infection is thought to occur via endosomes, but endosome acidification is not necessary for the entry whereas entry of CD4-independent HIVs, which are thought to be prototypes of CD4-dependent viruses, is low pH dependent. There are several controversial results on the retroviral entry pathways. Because endocytosis and endosome acidification are complicatedly controlled by cellular mechanisms, the retrovirus entry pathways may be different in different cell lines

Enterokinase and IAV Infection

Cleavage and activation of hemagglutinin (HA) by trypsin-like proteases in influenza A virus (IAV) are essential prerequisites for its successful infection and spread. In host cells, some transmembrane serine proteases such as TMPRSS2, TMPRSS4 and HAT, along with plasmin in the bloodstream, have been reported to cleave the HA precursor (HA0) molecule into its active forms, HA1 and HA2. Some trypsinogens can also enhance IAV proliferation in some cell types (e.g., rat cardiomyoblasts). However, the precise activation mechanism for this process is unclear, because the expression level of the physiological activator of the trypsinogens, the TMPRSS15 enterokinase, is expected to be very low in such cells, with the exception of duodenal cells. Here, we show that at least two variant enterokinases are expressed in various human cell lines, including A549 lung-derived cells. The exogenous expression of these enterokinases was able to enhance the proliferation of IAV in 293T human kidney cells, but the proliferation was reduced by knocking down the endogenous enterokinase in A549 cells. The enterokinase was able to enhance HA processing in the cells, which activated trypsinogen in vitro and in the IAV-infected cells also. Therefore, we conclude that enterokinase plays a role in IAV infection and proliferation by activating trypsinogen to process viral HA in human cell lines

Raft localization of CXCR4 is primarily required for X4-tropic human immunodeficiency virus type 1 infection.

Human immunodeficiency virus type 1 (HIV-1) infection is initiated by successive interactions of viral envelope glycoprotein gp120 with two cellular surface proteins, CD4 and chemokine receptor. The two most common chemokine receptors that allow HIV-1 entry are the CCR5 and CXCR4. The CD4 and CCR5 are mainly localized to the particular plasma membrane microdomains, termed raft, which is rich in glycolipids and cholesterol. However, the CXCR4 is localized only partially to the raft region. Although the raft domain is suggested to participate in HIV-1 infection, its role in entry of CXCR4-tropic (X4-tropic) virus is still unclear. Here, we used a combination of CD4-independent infection system and cholesterol-depletion-inducing reagent, methyl-beta-cyclodextrin (MbetaCD), to address the requirement of raft domain in the X4-tropic virus infection. Treatment of CD4-negative, CXCR4-positive human cells with MbetaCD inhibited CD4-independent infection of the X4-tropic strains. This inhibitory effect of the cholesterol depletion was observed even when the CXCR4 was over-expressed on the target cells. Soluble CD4-induced infection was also inhibited by MbetaCD. The MbetaCD had no effect on the levels of cell surface expression of CXCR4. In contrast to these infections, MbetaCD treatment did not inhibit CD4-dependent HIV-1 infection in the wild type CD4-expressing cells. This study and previous reports showing that CD4 mutants localized to non-raft domains function as HIV-1 receptor indicate that CXCR4 clustering in the raft microdomains, rather than CD4, is the key step for the HIV-1 entry

Ezrin, Radixin, and Moesin (ERM) proteins function as pleiotropic regulators of human immunodeficiency virus type 1 infection.

Ezrin, radixin, and moesin (ERM) proteins supply functional linkage between integral membrane proteins and cytoskeleton in mammalian cells to regulate membrane protein dynamisms and cytoskeleton rearrangement. To assess potential role of the ERM proteins in HIV-1 lifecycle, we examined if suppression of ERM function in human cells expressing HIV-1 infection receptors influences HIV-1 envelope (Env)-mediated HIV-1-vector transduction and cell-cell fusion. Expression of an ezrin dominant negative mutant or knockdown of ezrin, radixin, or moesin with siRNA uniformly decreased transduction titers of HIV-1 vectors having X4-tropic Env. In contrast, transduction titers of R5-tropic Env HIV-1 vectors were decreased only by radixin knockdown: ezrin knockdown had no detectable effects and moesin knockdown rather increased transduction titer. Each of the ERM suppressions had no detectable effects on cell surface expression of CD4, CCR5, and CXCR4 or VSV-Env-mediated HIV-1 vector transductions. Finally, the individual knockdown of ERM mRNAs uniformly decreased efficiency of cell-cell fusion mediated by X4- or R5-tropic Env and HIV-1 infection receptors. These results suggest that (i) the ERM proteins function as positive regulators of infection by X4-tropic HIV-1, (ii) moesin additionally functions as a negative regulator of R5-tropic HIV-1 virus infection at the early step(s) after the membrane fusion, and (iii) receptor protein dynamisms are regulated differently in R5- and X4-tropic HIV-1 infections

Development of sensory neuropathy in streptozotocin-induced diabetic mice.

Diabetic polyneuropathy is a major complication of diabetes and the most common cause of peripheral neuropathy. Sensory-dominant neuropathy is the most common type. We previously used streptozotocin (STZ)-induced diabetic ddY mice with sensory neuropathy to evaluate the therapeutic effects of vascular endothelial growth factor and placental growth factor isoforms. In this study, to characterize the development of diabetic sensory neuropathy, electrophysiological, behavioral, and histopathological studies were performed in these diabetic mice. A significant difference in sensory conduction velocity in the tail nerve was observed between healthy and diabetic mice at 1 week after STZ injection. Diabetic mice developed hypoalgesia at 5 weeks after STZ injection. Axon area and myelin thickness of the myelinated fibers were increased in 17-week-old healthy mice compared with those in 8-week-old healthy mice. However, these increases were retarded in 17-week-old diabetic mice. In unmyelinated fibers, axon area was significantly reduced in 17-week-old diabetic mice compared with 8- and 17-week-old healthy mice. These findings suggest that both impaired maturation of myelinated fibers and atrophy of unmyelinated fibers simultaneously occur in the early stage of diabetes in these mice. Our mouse model may be useful for studying the pathogenesis of and therapies for diabetic sensory neuropathy.Diabetic polyneuropathy is a major complication of diabetes and the most common cause of peripheral neuropathy. Sensory-dominant neuropathy is the most common type. We previously used streptozotocin (STZ)-induced diabetic ddY mice with sensory neuropathy to evaluate the therapeutic effects of vascular endothelial growth factor and placental growth factor isoforms. In this study, to characterize the development of diabetic sensory neuropathy, electrophysiological, behavioral, and histopathological studies were performed in these diabetic mice. A significant difference in sensory conduction velocity in the tail nerve was observed between healthy and diabetic mice at 1 week after STZ injection. Diabetic mice developed hypoalgesia at 5 weeks after STZ injection. Axon area and myelin thickness of the myelinated fibers were increased in 17-week-old healthy mice compared with those in 8-week-old healthy mice. However, these increases were retarded in 17-week-old diabetic mice. In unmyelinated fibers, axon area was significantly reduced in 17-week-old diabetic mice compared with 8- and 17-week-old healthy mice. These findings suggest that both impaired maturation of myelinated fibers and atrophy of unmyelinated fibers simultaneously occur in the early stage of diabetes in these mice. Our mouse model may be useful for studying the pathogenesis of and therapies for diabetic sensory neuropathy