Xiaohui Zhu

Expression of immunoglobulin gene with classical V-(D)-J rearrangement in mouse brain neurons.

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.

Immunoglobulin Gene Transcripts Have Distinct VHDJH Recombination Characteristics in Human Epithelial Cancer Cells

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 VHDJH (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 VHDJH recombination with seven sets of predominant VHDJH sequences. Surprisingly, within a set of VHDJH 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 VH region. The VHγDJHγ (but not VHμDJHμ) 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 VHγDJHγsequences. In contrast to VHμDJHμ, the VHγDJHγ 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.

Immunoglobulin mRNA and protein expression in human oral epithelial tumor cells.

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 γ 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.

Immunoglobulin gene locus events in epithelial cells of lactating mouse mammary glands

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 Igκ variable region sequences (VCW9Jκ1 and VBV9Jκ1) 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.

Adenovirus-expressed human hyperplasia suppressor gene induces apoptosis in cancer cells

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 (ΔΨm) 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. [Mol Cancer Ther 2008;7(1):222–32]

Thiazolidinediones, a class of anti-diabetic drugs, inhibit Id2 expression through a PPARγ-independent pathway in human aortic smooth muscle cells

Abstract. Inhibitor of DNA binding (Id2) is a member of the helix-loop-helix family of transcription regulators that is known to play important roles in the proliferation and differentiation of many cell types. Overexpression of Id2 has been reported to result in significant enhancement of vascular smooth muscle cell growth via increased S phase entry. We hypothesized that downregulation of Id2 gene expression by thiazolidinediones (TZDs), a class of anti-diabetic drugs and peroxisome proliferator-activated receptor γ (PPARγ) activators, might contribute to the anti-atherosclerotic and anti-hypertensive effects of the PPARγ. Here we document that TZDs, including troglitazone and ciglitazone, repress Id2 gene expression in a doses- and time-dependent manner. However, GW7845, a high-affinity and non-TZD PPARγ activator, had no inhibitory effect on Id2 gene expression. In addition, PPAR& ggr; antagonist GW9662 did not rescue TZD-induced Id2 repression. Taken together, our data suggest that TZDs repress Id2 expression through a PPARγ-independent pathway.