[Show abstract][Hide abstract] ABSTRACT: The Atg8 autophagy proteins are essential for autophagosome biogenesis and maturation. The γ-aminobutyric acid receptor-associated protein (GABARAP) Atg8 family is much less understood than the LC3 Atg8 family, and the relationship between the GABARAPs' previously identified roles as modulators of transmembrane protein trafficking and autophagy is not known. Here we report that GABARAPs recruit palmitoylated PI4KIIα, a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) and binds GABARAPs, from the perinuclear Golgi region to autophagosomes to generate PI4P in situ. Depletion of either GABARAP or PI4KIIα, or overexpression of a dominant-negative kinase-dead PI4KIIα mutant, decreases autophagy flux by blocking autophagsome:lysosome fusion, resulting in the accumulation of abnormally large autophagosomes. The autophagosome defects are rescued by overexpressing PI4KIIα or by restoring intracellular PI4P through "PI4P shuttling." Importantly, PI4KIIα's role in autophagy is distinct from that of PI4KIIIβ and is independent of subsequent phosphatidylinositol 4,5 biphosphate (PIP2) generation. Thus, GABARAPs recruit PI4KIIα to autophagosomes, and PI4P generation on autophagosomes is critically important for fusion with lysosomes. Our results establish that PI4KIIα and PI4P are essential effectors of the GABARAP interactome's fusion machinery.
Proceedings of the National Academy of Sciences 06/2015; 112(22):201507263. DOI:10.1073/pnas.1507263112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-B cell immunoglobulins (Igs) are widely expressed in epithelial cancer cells. The past 20 years of research have demonstrated that non-B cell Igs are associated with cancer cell proliferation, the cellular cytoskeleton and cancer stem cells. In this study we explored the transcriptional mechanism of IgM production in non-B cells.
The promoter region of a V-segment of the heavy mu chain gene (VH6-1) was cloned from a colon cancer cell line HT-29. Next, the promoter activities in non-B cells and B-cells were detected using the dual-luciferase reporter assay. Then the transcription factor binding to the promoter regions was evaluated by electrophoretic mobility shift assays (EMSAs) and gel supershift experiments.
Our data showed that the sequence 1200 bp upstream of VH6-1 exhibited promoter activity in both B and non-B cells. No new regulatory elements were identified within the region 1200 bp to 300 bp upstream of VH6-1. In addition, Oct-1 was found to bind to the octamer element of the Ig gene promoter in cancer cells, in contrast to B cells, which utilize the transcriptional factor Oct-2.
The regulatory mechanisms among different cell types controlling the production of IgM heavy chains are worth discussing.
Cancer Cell International 12/2014; 14(1):114. DOI:10.1186/s12935-014-0114-8 · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The restriction of immunoglobulin (Ig) expression to B lymphocytes is well established. However, several reports have confirmed that the Ig gene can be expressed in many non-B cancer cells and/or some normal cells. Our aim is to determine whether the Ig gene promoter can be activated in non-B cancer cells and to identify the regulatory mechanism for Ig gene expression. Our results show that the Ig promoter of VH4-59 was activated in several non-B cancer cell lines. Moreover, two novel positive regulatory elements, an enhancer-like element at -800 to -610 bp and a copromoter-like element at -610 to -300 bp, were identified in two epithelial cancer cell lines, HeLa S3 and HT-29. The octamer element (5'-ATGCAAAT-3') located in the Ig promoter, a crucial element for B-cell-derived Ig gene transcription, was also very important for non-B-cell-derived Ig gene transcription. More importantly, we confirmed that octamer-related protein-1 (Oct-1), but not Oct-2, was a crucial transcriptional factor for Ig gene transcription due to its ability to bind to the octamer element of the Ig promoter in epithelial cancer cells. These results suggested the presence of a distinct regulatory mechanism for Ig gene expression in non-B cancer cells.
[Show abstract][Hide abstract] ABSTRACT: It has been believed that the immunoglobulin (Ig) found abundantly in the colostrum of lactating mammalian is derived from serum or secreted by plasma cells present in the mammary gland. The recent finding of Ig gene rearrangements in breast cancer cells and benign hyperplastic breast epithelial cells suggests that it is likely that hyperplastic mammary gland epithelial cells during lactation can also produce Ig. In this study, we have demonstrated the presence of abundant amounts of Ig heavy and light chain transcripts in sorted cytokeratin 18-positive mammary gland epithelial cells of lactating mice. Interestingly, we found two specific Igkappa variable region sequences (V(CW9)J(kappa1) and V(BV9)J(kappa1)) that were dominantly expressed in different strains of mice. Our data demonstrate that IgG is expressed by mammary gland epithelial cells of lactating mice, and suggest that the IgG found in murine colostrum is at least partially produced by the mammary gland epithelial cells.
Cellular and Molecular Life Sciences CMLS 03/2010; 67(6):985-94. DOI:10.1007/s00018-009-0231-z · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has many essential functions and its homeostasis is highly regulated. We previously found that hypertonic stress increases PIP(2) by selectively activating the beta isoform of the type I phosphatidylinositol phosphate 5-kinase (PIP5Kbeta) through Ser/Thr dephosphorylation and promoting its translocation to the plasma membrane. Here we report that hydrogen peroxide (H(2)O(2)) also induces PIP5Kbeta Ser/Thr dephosphorylation, but it has the opposite effect on PIP(2) homeostasis, PIP5Kbeta function, and the actin cytoskeleton. Brief H(2)O(2) treatments decrease cellular PIP(2) in a PIP5Kbeta-dependent manner. PIP5Kbeta is tyrosine phosphorylated, dissociates from the plasma membrane, and has decreased lipid kinase activity. In contrast, the other two PIP5K isoforms are not inhibited by H(2)O(2). We identified spleen tyrosine kinase (Syk), which is activated by oxidants, as a candidate PIP5Kbeta kinase in this pathway, and mapped the oxidant-sensitive tyrosine phosphorylation site to residue 105. The PIP5KbetaY105E phosphomimetic is catalytically inactive and cytosolic, whereas the Y105F non-phosphorylatable mutant has higher intrinsic lipid kinase activity and is much more membrane associated than wild type PIP5Kbeta. These results suggest that during oxidative stress, as modeled by H(2)O(2) treatment, Syk-dependent tyrosine phosphorylation of PIP5Kbeta is the dominant post-translational modification that is responsible for the decrease in cellular PIP(2).
[Show abstract][Hide abstract] ABSTRACT: It was well accepted that only B-lymphocytes and plasma cells expressed immunoglobulin (Ig) gene. However, our group and others have confirmed that non-B-cells, such as epithelial cancer cells, can also express Ig. The aim of this work is to elucidate the role of non-B-cell-derived Ig by investigating the characteristics of the Ig heavy chain (IgH) gene repertoire in epithelial cancer cells. We cloned and sequenced 89 V(H)DJ(H) (V-D-J recombination of the IgH variable region) transcripts by microdissecting cells from eight different types of epithelial cancers and two cancer cell lines (HT-29 and HeLa S3). The cancer-derived Ig gene repertoire showed specific restricted patterns of V(H)DJ(H) recombination with seven sets of predominant V(H)DJ(H) sequences. Surprisingly, within a set of V(H)DJ(H) recombination, the variable (V) sequences derived from different cancer types had not only identical heavy chain variable (VH), diversity (D), and joining (JH) segments usage, but also identical junctions and mutation targets in the V(H) region. The V(Hgamma)DJ(Hgamma) (but not V(Hmicro)DJ(Hmicro)) in the cancer-derived sequences had a high mutation rate; however, it was shown that the mechanism of hypermutation was different from antigen selection in B-cell-derived V(Hgamma)DJ(Hgamma)sequences. In contrast to V(Hmicro)DJ(Hmicro), the V(Hgamma)DJ(Hgamma) sequences did not appear to originate from classical class switching. These results suggest that cancer-derived Ig genes have a distinct repertoire that may have implications for their role in carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) gamma and alpha isoforms, which synthesize PIP(2), during phagocytosis. PIP5K-gamma-/- bone marrow-derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcgammaR clustering. Delivery of exogenous PIP(2) rescued these defects. PIP5K-gamma knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-gamma by RNA interference inhibits particle attachment. In contrast, PIP5K-alpha knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation. We propose that PIP5K-gamma acts upstream of Rac/Rho and that the differential regulation of PIP5K-gamma and -alpha allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.
The Journal of Cell Biology 02/2009; 184(2):281-96. DOI:10.1083/jcb.200806121 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In our previous work, we have reported the expression of immunoglobulin (Ig) molecules by numerous epithelial cancer cells and hyperplastic epithelial cells. In the present study, we extended our investigation to study the frequencies of expression of IgG and IgA in some types of oral epithelial tumor cells, and analyzed the oral tumor-derived V regions characteristic of Ig gamma chain gene transcripts by immunohistochemistry, in situ hybridization, laser capture microdissection-correlated reverse-transcription polymerase chain reaction, and sequencing. IgG and IgA immunoreactivity was prominent in the cytoplasmic or plasma membrane or secretion of malignant cells, pleomorphic adenoma tumor cells, and some normal glandular epithelial cells or squamous cells adjacent to tumors. More importantly, rearranged Ig gene transcripts were identified in these tumor cells, and in some normal glandular epithelial cells, the V-D-J region sequences revealed that IgG transcripts in 2 tested oral tumors were oligoclonal. These results support that the phenomenon of Ig could also be expressed in oral cavity epithelial tumor cells.
Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 06/2008; 16(3):232-8. DOI:10.1097/PAI.0b013e31814c915a · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Within the past 10 years, several investigators have reported the presence of immunoglobulin G in brain neurons. However, because immunoglobulin molecules were only known to be produced by B-lymphocytes, it was suspected that the neurons were taking immunoglobulin G up from the extracellular fluid. The aim of this study was to determine whether immunoglobulin G was actually being produced by the neurons. By immunohistochemistry and Western blotting analysis, we found that immunoglobulin G was also present in adult mouse brain neurons and isolated neonatal mouse neurons, respectively. More importantly, by in situ hybridization, Northern blotting and single cell reverse transcriptase polymerase chain reaction, the transcripts of rearranged immunoglobulin gamma chain and kappa chain were also found in adult mouse brain neurons. Further, confocal imaging of primary culture neurons showed that immunoglobulin G immunoreactivity was localized in the neuron cytoplasm, axons and dendrites. Immunoglobulin G extracted from the primary culture neurons could also be detected by Western blotting. Furthermore, the results of sulphur-35 or iodine-125 pulse-labeled immunoprecipitation provided additional confirmation that brain neurons could produce immunoglobulin G. Taken together, the results indicated that immunoglobulin G originated from both early generated and adult mouse neurons. Although the bioactivity of neuron-derived immunoglobulin G was not yet clear, we believed that immunoglobulin G might play an important role in neuronal development.
The International Journal of Biochemistry & Cell Biology 02/2008; 40(8):1604-15. DOI:10.1016/j.biocel.2007.12.004 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hyperplasia suppressor gene (HSG), also called human mitofusin 2, is a novel gene that markedly suppresses the cell proliferation of hyperproliferative vascular smooth muscle cells from spontaneously hypertensive rat arteries. This gene encodes a mitochondrial membrane protein that participates in mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network. In this report, we showed that an adenovirus vector encoding human HSG (Ad5-hHSG) had an antitumor activity in a wide range of cancer cell lines. We further focused on the lung cancer cell line A549 and the colon cancer cell line HT-29 and then observed that Ad5-hHSG induced apoptosis both in vitro and in vivo. Confocal laser scanning microscopy and electron microscopy revealed that cells infected with Ad5-hHSG formed dose-dependent perinuclear clusters of fused mitochondria. Adenovirus-mediated hHSG overexpression induced apoptosis, cell cycle arrest, mitochondrial membrane potential (DeltaPsim) reduction and release of cytochrome c, caspase-3 activation, and cleavage of PARP in vitro. Overexpression of hHSG also significantly suppressed the growth of subcutaneous tumors in nude mice both ex vivo and in vivo. In addition, Ad5-hHSG increased the sensitivity of these cell lines to two chemotherapeutic agents, VP16 and CHX, and radiation. These results suggest that Ad5-hHSG may serve as an effective therapeutic drug against tumors.
Molecular Cancer Therapeutics 02/2008; 7(1):222-32. DOI:10.1158/1535-7163.MCT-07-0382 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CKLFSF2 is a member of the chemokine-like factor superfamily (CKLFSF), a novel gene family containing CKLF and CKLFSF1-8. Using a combination of data mining and polymerase chain reactions, we determined the full cDNA sequence and genomic structure of human CKLFSF2, a 4-exon gene encoding 248 amino acids and spanning approximately 8.8 kb on chromosome 16q22.1. Expression profile analyses indicated that CKLFSF2 is expressed in a limited number of tissues. Specifically, immunohistochemistry indicated that CKLFSF2 is highly expressed in testis, mainly in spermatogonia and the seminiferous tubular fluid. Subcellular localization experiments suggested that CKLFSF2 is equally distributed in the cytoplasm, and Western blot analysis revealed that overexpressed CKLFSF2 is secreted into the supernatant of cultured cells. The data therefore strongly suggest that CKLFSF2 is a secreted protein that may be functionally relevant during spermatogenesis.
The International Journal of Biochemistry & Cell Biology 09/2005; 37(8):1633-40. DOI:10.1016/j.biocel.2004.04.028 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chemokine-like factor (CKLF) was isolated from PHA-stimulated U937 cells. It is composed of 152 amino acids and located on chromosome 16q22. Utilizing bioinformatics, based on CKLF cDNA and protein sequences, in combination with experimental validation, we identified a novel gene designated chemokine-like factor super family member 1 (CKLFSF1). CKLFSF1 maps on chromosome 16q22, and the full-length gene comprises of seven exons and six introns. Using RACE-PCR, we identified two potential alternative transcription start sites, 1A and 1B. Northern blot and RT-PCR analysis demonstrated that CKLFSF1 is predominantly expressed in human testis tissue, with only lower levels of expression in many other human tissues. RT-PCR and cDNA sequencing identified 23 alternatively spliced isoforms of CKLFSF1 in the testis tissue, which encode protein variants ranging from 36 to 169 amino acids in length. Immunohistochemistry analysis demonstrated that CKLFSF1 proteins are highly expressed in spermatocyte and in tissue fluid of human testes tissue. In light of these findings, we propose that CKLFSF1 may play an important role in spermatogenesis or testicular development.
The International Journal of Biochemistry & Cell Biology 09/2004; 36(8):1492-501. DOI:10.1016/j.biocel.2003.11.017 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immunoglobulins (Igs) are found thus far only to be produced by differentiated B lymphocytes. By immunohistochemistry analysis, in situ hybridization, and laser capture microdissection-assisted single-cell PCR, we demonstrate that human cancers of epithelial origin, including carcinomas of breast, colon, liver, lung, established epithelial cancer lines, as well as some normal lung tissues, also produce IgG in both cytoplasmic and secreted forms. Furthermore, blockade of tumor-derived IgG by either antisense DNA or antihuman IgG antibody increased programmed cell death and inhibited growth of cancer cells in vitro. More importantly, administration of antihuman IgG antibody also suppressed the growth of an IgG-secreting carcinoma line in immunodeficient nude mice. Our results support a role of tumor-derived IgG as growth factor for epithelial cancers. Prevalent expression of IgG in human carcinomas and its growth-promoting functions may have important implications in growth regulation and targeted therapy of human cancers.
Cancer Research 11/2003; 63(19):6488-95. · 9.33 Impact Factor