Gene expression profile of human lymphoid CEM cells sensitive and resistant to glucocorticoid-evoked apoptosis

Three closely related clones of leukemic lymphoid CEM cells were compared for their gene expression responses to the glucocorticoid dexamethasone (Dex). All three contained receptors for Dex, but only two responded by undergoing apoptosis. After a time of exposure to Dex that ended late in the interval preceding onset of apoptosis, gene microarray analyses were carried out. The results indicate that the expression of a limited, distinctive set of genes was altered in the two apoptosis-prone clones, not in the resistant clone. That clone showed altered expression of different sets of genes, suggesting that a molecular switch converted patterns of gene expression between the two phenotypes: apoptosis-prone and apoptosis-resistant. The results are consistent with the hypothesis that altered expression of a distinctive network of genes after glucocorticoid administration ultimately triggers apoptosis of leukemic lymphoid cells. The altered genes identified provide new foci for study of their role in cell death.     

Glucocorticoid effect on oncogene/growth gene expression in human T lymphoblastic leukemic cell line CCRF-CEM. Specific c-myc mRNA suppression by dexamethasone

Glucocorticoids induce growth inhibition and eventually cause cell lysis in certain sensitive leukemic cells. To investigate how glucocorticoids interact with cell growth pathways, we studied the expression of 14 growth-related genes in dexamethasone-treated CEM-C7A cells, a steroid-sensitive clone of the CCRF-CEM cell line, and in several closely related clones. The 14 genes studied were chosen to represent four different levels of mitogenic signal transduction. Detectable mRNA levels were found for 8 of the 14 genes, but among these only c-myc expression was obviously suppressed by dexamethasone. The c-myc mRNA levels declined abruptly during the first 12 h after addition of 1 microM dexamethasone, and maximal suppression occurred by 18 h. This change was not seen in the C7A controls, in the glucocorticoid-resistant, receptor-deficient clone ICR-27, or in the glucocorticoid-resistant, receptor-positive clone C1. H.10, a hybrid clone between C1 and ICR-27, showed restoration of the sensitive phenotype, and in H.10 cells the c-myc mRNA was also suppressed by dexamethasone. Our results suggest that: 1) functional glucocorticoid receptor is required for inducing c-myc suppression. 2) In dexamethasone-resistant cells with functional receptors c-myc is not suppressed. 3) The growth arrest induced by glucocorticoids correlates with, and may be regulated via, suppression of c-myc expression.     

Construction and hormone regulation of a novel retroviral vector

We report the analysis of a self-inactivating retroviral vector, constructed to allow inducible gene expression of inserted sequences from the mouse mammary tumour virus hormonal response element. The cloning strategy has been designed to allow for ease of insertion of the genes of interest. The vector contains the aph gene, allowing geneticin-resistance selection in mammalian cells. We have characterised dexamethasone (Dex)-induced increase in gene expression using the reporter gene encoding chloramphenicol acetyltransferase (CAT) inserted into the retroviral vector. We observe low basal levels of CAT activity in infected cells which is increased up to 50-fold by induction with Dex. The induction of pooled clones is 13.3-fold. Variation in Dex-induced CAT activity is observed in independently infected clones, which is not explained by proviral copy number.     

Hormonal regulation of expression of the endogenous and transfected human growth hormone gene

Expression of the endogenous human GH (hGH) gene in response to glucocorticoids, thyroid hormone, and insulin was studied in cultures of dispersed GH-secreting human pituitary adenomas. Results were compared to those obtained when the hGH gene was transfected into rat pituitary tumor cells (GC). In the human pituitary cells the glucocorticoid dexamethasone [(Dex) 10(-6) M] increased the release of GH and the levels of GH mRNA by 2 to 4-fold (P less than 0.05). T3 (10(-8) M) had no effect on GH mRNA but increased hGH release by 2- to 6-fold (P less than 0.01). Insulin (5 x 10(-9) M) alone had no significant effect on either hGH mRNA or protein, but blunted the effect of Dex. Among 11 of 18 GC cell clones transfected with the hGH gene with detectable hGH mRNA expression, Dex increased hGH mRNA levels in seven and T3 treatment reduced hGH mRNA levels in eight. Conversely, rat GH mRNA levels from the endogenous rat gene were increased by either Dex or T3 in all 18 clones. Insulin alone or in combination with T3 or Dex was found to increase hGH mRNA levels in some cell lines and to decrease hGH mRNA levels in others; these effects were correlated strongly (r = 0.88; P less than 0.001) with the influence of insulin on the endogenous rat GH gene, implying that individual cellular differences can simultaneously affect the insulin responsiveness of both genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microarray Analyses of Glucocorticoid and Vitamin D3 Target Genes in Differentiating Cultured Human Podocytes

Glomerular podocytes are highly differentiated epithelial cells that are key components of the kidney filtration units. Podocyte damage or loss is the hallmark of nephritic diseases characterized by severe proteinuria. Recent studies implicate that hormones including glucocorticoids (ligand for glucocorticoid receptor) and vitamin D3 (ligand for vitamin D receptor) protect or promote repair of podocytes from injury. In order to elucidate the mechanisms underlying hormone-mediated podocyte-protecting activity from injury, we carried out microarray gene expression studies to identify the target genes and corresponding pathways in response to these hormones during podocyte differentiation. We used immortalized human cultured podocytes (HPCs) as a model system and carried out in vitro differentiation assays followed by dexamethasone (Dex) or vitamin D3 (VD3) treatment. Upon the induction of differentiation, multiple functional categories including cell cycle, organelle dynamics, mitochondrion, apoptosis and cytoskeleton organization were among the most significantly affected. Interestingly, while Dex and VD3 are capable of protecting podocytes from injury, they only share limited target genes and affected pathways. Compared to VD3 treatment, Dex had a broader and greater impact on gene expression profiles. In-depth analyses of Dex altered genes indicate that Dex crosstalks with a broad spectrum of signaling pathways, of which inflammatory responses, cell migration, angiogenesis, NF-κB and TGFβ pathways are predominantly altered. Together, our study provides new information and identifies several new avenues for future investigation of hormone signaling in podocytes.     

Dexamethasone induces apoptosis in human T cell clones expressing low levels of Bcl-2

Previous results of ours have demonstrated that the same clonotype can express both a sensitive and a resistant phenotype to Dex-mediated PCD induction depending on its cell cycle phase. In particular, we demonstrated that human T lymphocytes, arrested in the G0/G1 phase of the cell cycle, are susceptible, while proliferating T cells are resistant to Dex-mediated apoptosis. In this paper, we have further characterized the sensitive and resistant phenotypes and investigated whether a different expression of the apoptotic genes Fas, FasL, Bcl-2, Bcl-x and Bax is involved in the regulation of Dex-mediated apoptosis. The results show that the amount of Bcl-2 expression, that changes during cell cycle phases, determines susceptibility or resistance to apoptosis induced by Dex. In fact, undetectable expression of Bcl-2 in sensitive cells favors Dex-mediated apoptosis while high expression of Bcl-2 in proliferating cells counterbalances apoptosis induction. Moreover, the addition of exogenous IL-2, in the presence of Dex, fails to up-regulate Bcl-2 expression and to revert Dex-mediated apoptotic phenomena.     

Evidence for trans regulation of apoptosis in intertypic somatic cell hybrids.

The genetic components required for glucocorticoid induction of apoptosis were studied by using somatic cell hybridization. Intertypic whole-cell hybrids were generated by crossing the glucocorticoid-resistant rat liver cell line Fado-2 with the glucocorticoid-sensitive mouse thymoma cell line BW5147.3. Morphological and biochemical criteria were used to assess sensitivity or resistance to glucocorticoid-induced cell death. Both phenotypes were observed, and all of the hybrids retained a functional glucocorticoid receptor as judged by their abilities to induce the metallothionein gene in response to dexamethasone (Dex). Sensitivity to apoptosis did not correlate with morphological phenotype in that not all suspension cells were sensitive. The effect of glucocorticoids on the expression of apoptosis-linked genes was analyzed in a subset of Dex-sensitive and Dex-resistant hybrids. p53 and c-myc mRNAs were present in parental cells as well as sensitive and resistant hybrid cells, and their levels were not affected by glucocorticoid treatment. bcl-2 expression was restricted to the thymoma cell line and was also not affected by glucocorticoids. We did not detect any bcl-2 mRNA in the hepatoma cell line and the hybrids, suggesting that, as with most tissue-specific genes, bcl-2 is regulated in trans. Furthermore, while the majority of hybrids analyzed retained a full complement of mouse chromosomes, sensitive hybrids were missing some rat chromosomes (preferentially chromosomes 16 and 19), indicating that apoptosis is subject to trans repression. Resistant cells thus appear to repress the activity or synthesis of a nuclear factor that interacts with a glucocorticoid-dependent gene(s) to activate the cell death pathway.     

Differential expression and sequence analysis of the maize glyceraldehyde-3-phosphate dehydrogenase gene family.

Two cDNA clones for maize cytosolic glyceraldehyde-3-phosphate dehydrogenase are described. One is about 97% similar in coding capacity to a previously published clone [Brinkmann et al. (1987). J. Mol. Evol. 26, 320-328], while the other shows only 88% similarity. Evidence points toward the three cDNAs being the products of three genes, to be called Gpc1, Gpc2, and Gpc3. When the least similar clone, corresponding to Gpc3, was used to analyze RNA gel blots, anaerobic treatment for 6 hours induced RNA accumulation in the shoots 15.6-fold, while a 1-hour shift from 28 degrees C to 40 degrees C increased accumulation 5.1-fold. Roots had a higher basal level of expression, leading to a 6.0-fold anaerobic induction, and a 2.4-fold heat stress induction. RNA gel blot analysis using the clone corresponding to Gpc2 showed decreased RNA accumulation within 6 hours of anaerobiosis, while analysis with the previously published clone, corresponding to Gpc1, showed a decrease within 24 hours. Neither Gpc1 nor Gpc2 showed heat stress induction, while some other known anaerobic genes did. Through the use of hybrid selection, in vitro translation, and immune precipitation, the relative expression of the three genes is shown. The role of the observed changes in gene expression is discussed in relation to stress physiology.     

Glucocorticoids in malignant lymphoid cells: Gene regulation and the minimum receptor fragment for lysis

We have examined clones of human malignant lymphoid cells for markers that correlate with glucocorticoid-mediated cell lysis. In glucocorticoid-sensitive clones of CEM, a human T-cell lymphoblastic leukemia line, two genes correlate with glucocorticoid-induced cell lysis. The glucocorticoid receptor (GR) itself is induced by standard glucocortoids in sensitive clones and not in insensitive clones. The phenylpyrazolo-glucocortocoid cortivazol (CVZ) is capable of lysing several clones resistant to high concentrations of standard potent glucocorticoids. When these clones were tested for cortivazol responses, they were not only lysed by cortivazol but also showed induction of GR mRNA. Thus receptor induction appears to correlate with the lysis function of receptor in these cells. To determine what parts of the GR are required for lysis, we have mapped this function by transfecting and expressing GR and GR fragment genes in a GR-deficient CEM clone. Our results indicate that none of the known trans-activation regions of the GR are required. Removal of the steroid binding domain gives a fragment that is fully constitutive. Only one and one-half “Zn fingers” of the DNA binding region are required. We also find in CEM cells rapid suppression of the c-myc protooncogene, proceding growth arrest and cell lysis by glucocorticoids. This occurs only in clones possessing both intact receptors and lysis function. Thus the simple presence of GR alone is not sufficient to guarantee c-myc down-regulation. Introduction into the cells of c-myc driven by a promoter that does not permit suppression by glucocorticoids confers resistance to steroids. Furthermore, suppression of c-myc by antisense oligonucleotides also kills the cells. Therefore, c-myc appears to be a pivotal gene related both to ability of steroid to kill and to cell viability.     

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.     

Human SP-A 3'-UTR variants mediate differential gene expression in basal levels and in response to dexamethasone

Human surfactant protein A (SP-A) is encoded by two genes (SP-A1, SP-A2), and each is identified with several alleles. SP-A is involved in normal lung function, innate immunity, inflammatory processes, and is regulated by glucocorticoids. We investigated the role of 3'-untranslated region (UTR) of 10 SP-A variants on gene expression using transient transfection of 3'-UTR constructs in the human lung adenocarcinoma cell line NCI-H441. We found: 1) both basal mRNA and protein levels of the reporter gene of SP-A 3'-UTR constructs are significantly (P < 0.01) reduced compared with controls (vector pGL3 and surfactant protein B pGL3) and that differences exist among alleles; and 2) after dexamethasone (Dex) treatment (100 nM for 16 h), mRNA was reduced (31-51%). Seven alleles showed a significant decrease (P < 0.05) in mRNA, and three did not. Reporter activity was also decreased, from 17% (1A(1)) to 38% (1A), with six alleles showing a significant decrease. The data indicate that the 3'-UTR of SP-As play a differential role in SP-A basal expression and in response to Dex. Therefore, a careful consideration of individual use of steroid treatment may be considered.