Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.     

Antisense CD11b integrin inhibits the development of a differentiated monocyte/macrophage phenotype in human leukemia cells

Macrophage-like development of myeloid leukemia cells which can be induced by agents such as phorbol esters (TPA) is accompanied by integrin expression and cell adhesion. Thus, in differentiating myeloid leukemia cells CD11b is predominantly expressed which can associate with CD18 to form the functional heterodimeric integrin Mac-1. To elucidate the role of cell adhesion during macrophage-like differentiation, we transfected human U937 myeloid leukemia cells with a vector containing the CD11b gene in antisense orientation. Expression of the CD11b antisense gene in stably transfected U937 cells (as-CD11b cells) resulted in an attenuated response to TPA. As-CD11b cells demonstrated poor adhesion to solid substrate upon TPA treatment in contrast to U937 control cells. Constitutive expression of c-myc in as-CD11b transfectants was higher than in control cells and failed to be repressed by TPA treatment. Moreover, unlike control cells, antisense transfectants failed to induce expression of early response genes such as c-jun and the redox factor ref-1 upon TPA stimulation. Consequently, the induction of monocytic differentiation markers such as the activity of alpha-naphthyl acetate esterase, the capacity to reduce nitroblue tetrazolium and the expression of the vimentin gene was much lower in antisense transfectants than in control U937 cells. According to the failure to undergo a monocytic differentiation program, TPA treatment of as-CD11b cells resulted in a progressively increasing amount of apoptotic cells whereas the differentiated population of U937 control cells remained alive. Taken together, these data suggest that the integrin-mediated (particularly CD11b-mediated) adhesion of myeloid leukemia cells in the course of induced monocytic differentiation is crucial for cell attachment, development of a monocytic phenotype and subsequent survival.     

A novel tumor metastasis suppressor gene LASS2/TMSG1 interacts with vacuolar ATPase through its homeodomain

LASS2/TMSG1 was a novel tumor metastasis suppressor gene, which was first cloned by our laboratory from non‐metastatic and metastatic cancer cell variants of human prostate carcinoma PC‐3M using mRNA differential display in 1999. LASS2/TMSG1 could interact with the C subunit of vacuolar ATPase (V‐ATPase, ATP6V0C) and regulate V‐ATPase activity. In an attempt to provide molecular mechanism of the interaction between LASS2/TMSG1 and V‐ATPase, we constructed four variant transfectants containing different functional domain of LASS2/TMSG1 and stably transfected the variants to human prostate cancer cell line PC‐3M‐1E8 cell with high metastatic potential. Results showed that there were no obvious differences of V‐ATPase expression among different transfected cells and the control. However, V‐ATPase activity and intracellular pH was significantly higher in the variant transfectants with Homeodomain of LASS2/TMSG1 than that in the control using the pH‐dependent fluorescence probe BECEF/AM. Immunoprecipitation, immunofluorescence and immuno‐electron microscope alone or in combination demonstrated the direct interaction of Homeodomain of LASS2/TMSG1 and ATP6V0C. Loss of Homeodomain markedly enhanced the proliferation ability but weakened the apoptotic effect of LASS2/TMSG1 in PC‐3M‐1E8 cells. These lines of results for the first time contribute to the conclusion that LASS2/TMSG1 could regulate V‐ATPase activity and intracellular pH through the direct interaction of its Homeodomain and the C subunit of V‐ATPase. Their interaction could play important roles in the apoptosis of tumor cells. J. Cell. Biochem. 114: 570–583, 2013. © 2012 Wiley Periodicals, Inc.     

Focal adhesion kinase affects the sensitivity of human hepatocellular carcinoma cell line SMMC-7721 to tumor necrosis factor-alpha/cycloheximide-induced apoptosis by regulating protein kinase B levels.

Most cell lines are resistant to tumor necrosis factor-alpha (TNF-alpha) cytotoxicity and require cotreatment of TNF-alpha with cycloheximide (Chx) to undergo apoptosis. Recently, the serine/threonine protein kinase, protein kinase B has been demonstrated to protect cells from apoptosis induced by TNF-alpha. In this study, we have shown that the human hepatocellular carcinoma cell line, SMMC-7721, was insensitive to TNF-alpha cytotoxicity and underwent apoptosis quickly in the presence of TNF-alpha and Chx. PKB levels decreased during TNF-alpha/Chx-induced apoptosis. No significant change in PKB levels was found in the presence of TNF-alpha or Chx alone. It seemed that the level of PKB closely correlated with apoptosis. The protein level of focal adhesion kinase (FAK) was reduced by 66% by transfecting FAK antisense cDNA recombinant vector into SMMC-7721 cells. We determined the apoptosis-induced effect of TNF-alpha/Chx on the FAK antisense cDNA transfectant cells. The results indicated that the percentage of apoptotic cells was enhanced at lower doses of TNF-alpha (10, 20 or 50 U.mL(-1)) and decreased at a higher dose of TNF-alpha (1000 U.mL(-1)) in the transfected cells as compared to the control. Correspondingly, in the FAK antisense cDNA transfectant cells treated with lower doses of TNF-alpha in presence of 10 microg.mL(-1) Chx, the PKB level was lower, but in the FAK antisense cDNA transfectants treated with higher doses of TNF-alpha in presence of 10 microg.mL(-1) Chx, the PKB level was higher. In response to TNF-alpha alone, FAK antisense cDNA transfectants showed a decrease in the level of PKB. However, in the case of TNF-alpha cotreated with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PtdIns3K), the FAK antisense cDNA transfectants produced significantly less amounts of PKB than the control. It seemed that FAK could stimulate PKB levels through a pathway not involving PtdIns3K. These results suggest that FAK can affect the sensitivity of SMMC-7721 cells to TNF-alpha/Chx-induced apoptosis in a biphasic manner by regulating PKB levels.     

Coupled expression of Ca2+ transport ATPase and a dihydrofolate reductase selectable marker in a mammalian cell system.

Stable expression of a full-length cDNA encoding chicken fast muscle Ca2+ transport ATPase was obtained in a Chinese hamster lung cell line (DC-3F), using a dual-promoter expression vector (pH beta FCaA3) in which the ATPase was cloned downstream of a human beta-actin gene promoter, and a mutant dihydrofolate reductase cDNA (A3/DHFR) was cloned downstream of an SV40 promoter-enhancer. Owing to its essentially normal catalytic activity and modest (20-fold) resistance to the antifolate methotrexate (MTX), the A3/DHFR mutant enzyme served as an efficient dominant selection marker in transfected cell populations challenged with MTX and, within a broad range of drug concentrations, allowed subsequent amplification and overexpression of vector sequences. In stable transfectants, the expressed ATPase was targeted to intracellular membranes, and the microsomal fractions from those cells exhibited high rates of Ca2+ transport. In comparative experiments using transient expression in COS1 cells, the level of ATPase per transfected cell was greater, but less than 5% of the transfected population exhibited ATPase expression. Furthermore, as opposed to the stable lines, the transiently expressing cells could not be propagated. Overall, the yield of ATPase was 12-16 and 4-6 micrograms per milligram of microsomal protein in the stable and the transient expression systems, respectively. The advantages of the stably transfected cell lines therefore lie in the homogeneity of ATPase expression and its distribution in cells and microsomes, in the large yield of microsomes obtained by continuous cell propagation, and in the reproducible functional characteristics of the microsomes. Moreover, the microsomes derived from stably transfected cell lines provide a convenient system for studies of Ca2+ transport and ATPase partial reaction, eliminating the need to conduct repetitive transient transfections to obtain sufficient amounts of enzyme for functional studies.     

Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC-8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC-8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC-8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6-8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.     

端粒酶RNA的反义cDNA对乳腺癌细胞凋亡的影响(英文)

 为了进一步探讨端粒酶RNA(hTR)的反义cDNA对乳腺癌MCF 7细胞凋亡可诱导性的影响 ,构建了能将外源基因整合至细胞基因组的整合型腺病毒载体vAd AAV ,并将hTR全长cDNA反向连接至此载体上 ,获得反义重组腺病毒vAdT AAV .vAd AAV和vAdT AAV分别感染MCF 7细胞后 ,获得两个细胞株MCF 7 vAd AAV和MCF 7 vAdT AAV ,其中MCF 7 vAdT AAV细胞基因组内整合有hTR反义cDNA并能稳定表达 .利用生存曲线、细胞形态学观察、DNA片段分析和流式细胞分析来测定NaBu和无血清DMEM诱导后细胞凋亡的反应性 .通过生存曲线 ,发现NaBu诱导的MCF 7 vAdT AAV细胞比对照组MCF 7和MCF 7 vAd AAV细胞更早出现凋亡 .电镜下 ,NaBu或去血清诱导的MCF 7 vAdT AAV细胞更早出现凋亡形态学指标 .流式细胞分析和DNA片段凝胶电泳实验均显示MCF 7 vAdT AAV细胞对凋亡的抵抗力下降 .研究结果表明 ,端粒酶RNA的反义cDNA使乳腺癌MCF 7细胞的凋亡可诱导性增强     

Regulated expression of vimentin cDNA in cells in the presence and absence of a preexisting vimentin filament network

Human cells were transfected with a mouse vimentin cDNA expression vector containing the hormone response element of mouse mammary tumor virus. The distribution of mouse vimentin after induction with dexamethasone was examined by indirect immunofluorescence with antivimentin antibodies specific for either mouse or human vimentin. In stably transfected HeLa cells, which contain vimentin filaments, addition of dexamethasone resulted in the initial appearance of mouse vimentin in discrete areas, usually perinuclear, that always corresponded to areas of the human filament network with the most intense fluorescence. Within 20 h after addition of dexamethasone, the mouse and human vimentin immunofluorescence patterns were identical. However, in stably transfected MCF-7 cells, which lack vimentin filaments, induction of mouse vimentin synthesis resulted in assembly of vimentin filaments throughout the cytoplasm without any obvious local concentrations. Transient expression experiments with SW-13 cell subclones that either lack or contain endogenous vimentin filaments yielded similar results to those obtained with MCF-7 and HeLa transfectants, respectively. Further experiments with HeLa transfectants were conducted to follow the fate of the mouse protein after synthesis had dropped after withdrawal of dexamethasone. The mouse vimentin-specific fluorescence was initially lost from peripheral areas of the cells while the last detectable mouse vimentin always corresponded to the human filament network with the most intense fluorescence. These studies are consistent with a uniform assembly of vimentin filaments throughout the cytoplasm and suggest that previous observations of polarized or vectorial assembly from a perinuclear area to more peripheral areas in cells may be attributable to the nonuniformly distributed appearance of vimentin filaments in immunofluorescence microscopy.     

Caspase-2/NEDD-2 protease mediates execution of apoptosis in AK-5 tumor cells

AK-5 tumour cells have been shown to undergo apoptosis in vitro and in vivo. The efficient killing of tumour cells by necrosis and apoptosis leads to spontaneous regression of the tumour. To investigate a possible involvement of caspase-2/Nedd-2 protease in AK-5 apoptosis, we introduced Nedd-2 gene in antisense orientation and showed inhibition of tumour cell apoptosis. Similarly introduction of the bcl-2 gene in tumour cells also inhibited the apoptotic programme. NK cells which have previously been shown to be the effector cells also fail to induce apoptosis in Nedd-2 antisense and bcl-2 transfected clones whereas NK mediated cytotoxic activity is not altered in the transfectants. These results suggest participation of Nedd-2 protease in the induction of apoptosis in AK-5 cells leading to tumour regression. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction of apoptosis by HIV-1-infected monocytic cells

We have previously described a soluble 6000-Da peptide produced by an HIV-1-infected human macrophage cell line, clone 43(HIV), which induces apoptosis in T and B cells. We have identified this factor as the novel cDNA clone FL14676485 that encodes for the human hypothetical protein, FLJ21908. The FL14676485 cDNA clone was isolated from a 43(HIV) lambda ZAP Escherichia coli expression library and screened with a panel of rabbit and mouse anti-apoptotic Abs. We transfected the FL14676485 clone into Bosc cells and non-HIV-1-infected 43 cells. Western blot analysis of lysates from the FL14676485-transfected 43 cells and Bosc cells using anti-proapoptotic factor Abs revealed a protein with a molecular mass of 66 kDa corresponding to the size of the full-length gene product of the FL14676485 clone, while Western blot of the supernatant demonstrated a doublet of 46-kDa and 6000-Da peptide that corresponds to our previously described proapoptotic factor. Primary HIV-1(BaL)-infected monocytes also produce the FLJ21908 protein. Supernatants from these transfected cells induced apoptosis in PBMC, CD4(+), and CD8(+) T and B cells similar to the activity of our previously described proapoptotic factor. PCR analysis of 43 cells and 43(HIV) cells revealed a base pair fragment of 420 bp corresponding to the FL14676485 gene product in 43(HIV) cells, but not in 43 cells. The FLJ21908 protein induces apoptosis through activation of caspase-9 and caspase-3. We have further demonstrated that the FLJ21908 protein has apoptotic activity in the SH-SY5Y neuronal cell line and can be detected in brain and lymph tissue from HIV-1-infected patients who have AIDS dementia. The FLJ21908 protein may contribute to the apoptosis and dementia observed in AIDS patients.     

Isolation and characterization of glucocorticoid- and cyclic AMP-induced genes in T lymphocytes.

Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a lambda cDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.     

Involvement of Rho GTPases and their effectors in the secretory process of PC12 cells

Intrathymic maturation of thymocytes is essential for the proper formation of T-cell repertoire. This process involves two major biochemical pathways, one initiated by the recognition of MHC/peptide by the T-cell receptor and the other mediated by glucocorticoids. These hormones seem to affect thymocyte maturation by increasing the threshold of TCR-mediated positive and negative selection, and by inducing apoptosis of nonselected thymocytes. We have previously reported that an SV40-immortalized murine thymic epithelial cell line, namely 2BH4, was able to protect thymocytes from dexamethasone-induced apoptosis. Here we show that this protection is independent of cell-to-cell contact and does not seem to involve a Bcl-2-mediated resistance, since incubation of thymocytes with 2BH4 cells or its supernatant does not interfere with the levels of this antiapoptotic molecule. The protection conferred by 2BH4 cells, or by a primary culture of thymic stromal cells, is specific for the CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes, whereas the broad-spectrum caspase inhibitor z-VAD-fmk blocks apoptosis induced by dexamethasone in all thymocyte subpopulations. Our results suggest that positively selected single-positive thymocytes are still susceptible to glucocorticoid-induced apoptosis but are protected from it through the action of a heat-stable protein(s) released by thymic stromal cells.     

Inhibition of human cancer cell growth by inducible expression of human ribonucleotide reductase antisense cDNA

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis and repair. The enzyme consists of two dissimilar subunits, M1 and M2. It is known that the M2 subunit plays a role in tumorgenicity and metastasis. In this study, we transfected human oropharyngeal KB cancer cells with human RR M1 and M2 antisense cDNA expressed by an inducible vector system. The transfectants were double-selected with hygromycin and G418. The clones, designated KB-M1AS, KB-M2AS and KB-CAT, represented transfectant clones that contained M1 antisense cDNA, M2 antisense cDNA, and a CAT reporter gene, respectively. In a colony-forming assay, colony formation for the KB-M2AS clone decreased approximately 50% when M2 antisense mRNA expression was induced by isopropylthiogalactose (IPTG). However, the KB-M1AS clone revealed no significant inhibition under IPTG induction. RR enzyme activity, as measured by 14CDP reduction assay, revealed a 30% decrease in the IPTG-induced KB-M2AS clone relative to non-IPTG-induced samples at 144 hours. As shown by Northern blot, expression of the M2 antisense mRNA showed peaks at 48 hours and 144 hours after induction by IPTG. M2 antisense mRNA expression induced by IPTG was 33-fold greater than the uninduced control at 144 hours. Western blot analysis showed that the M2 subunit protein level decreased in the KB-M2AS clone beginning at 72 hours after induction and continued to decrease to 50% of the uninduced control at 144 hours, then showed a slight recovery at 168 hours. In conclusion, M2 antisense mRNA expression by an inducible system can effectively decrease RR M2 protein expression, reduce enzyme activity, and inhibit growth. Furthermore, this approach can be employed in future antisense investigations.     

The expression of interleukin-6 by a rat macrophage-derived cell line

A stable rat macrophage-derived cell line (RMSV1) was established by transformation of primary peritoneal exudate cells with the SV40 virus. The RMSV1 cell line was used as a model to study the regulation of the interleukin-6 (IL6) gene expression in rate macrophages with respect to lipopolysaccharides (LPS), interleukin-1 (IL1) and glucocorticoids. The IL6 mRNA level in RMSV1 cell lines was induced 20-fold within 4 h by LPS, whereas IL1 had no effect. The glucocorticoids were able to inhibit completely the induction of the IL6 mRNA synthesis by LPS, indicating the negative regulation of the IL6 gene expression by glucocorticoids.