Glutathione-S-transferase pi as a determinant of drug resistance in transfectant cell lines

A series of glutathione S-transferase pi (GST-pi) transfectant cell lines have been constructed in activated c-H-ras-transformed NIH-3T3 cells (pT22-3) by using a pKOneo plasmid and an expression vector containing cDNA for GST-pi with a beta-actin gene promoter. From the wild type pT22-3 cells, two clones were selected and designated RGN1 and RGN2. The degree of overexpression of GST-pi was estimated by Northern and Southern blot analysis to be incrementally higher in RGN2 compared with RGN1. Translation of mRNA was estimated by Western blot analysis using isozyme-specific polyclonal antibodies and confirmed the relative GST-pi levels. Each cell line, including the wild type, expressed alpha and mu class isozymes to the same degree and had similar but negligible expression of the mdr 1 gene. Sensitivity to various anticancer drugs and radiation was estimated by a series of cytotoxicity assays. The data confirmed that GST-pi provided a degree of protection against the toxicity of ethacrynic acid and adriamycin, but sensitivity to alkylating agents such as chlorambucil, melphalan, and cis-platinum was not influenced by GST-pi. Similarly, the response to ionizing radiation was similar for each line. Since the levels of intracellular GSH were also not significantly different, the availability of co-substrate was not a factor in determining response. In creating the GST-pi transfectants, these data establish that while increased isozyme levels can play a role in determining sensitivity to some agents, the protective effect is selective.     

Expression of the pregnancy-specific beta 1-glycoprotein genes in human testis

Northern blot analysis with placental pregnancy-specific beta 1-glycoprotein (SP1) cDNA probe showed the presence of SP1 mRNAs in human testis. Presence of translational products of the mRNAs was demonstrated by Western blot analysis with anti-human SP1 antibodies albeit difference in mobilities between the testis and placental proteins was apparent. Screening of human testis cDNA library with placental SP1 probe yielded 4 groups of positive clones. Two groups were identical to human placental SP1 cDNAs previously reported. The other 2 groups consisted of cDNA of incompletely processed mRNAs. These 2 groups were present in high abundance. Sequence analysis suggested that the cDNAs were products of different genes.     

Differential display of human marrow stromal cells reveals unique mRNA expression patterns in response to dexamethasone

Human bone marrow stromal cells (hBMSC) are pluripotent cells that have the ability to differentiate into bone, cartilage, hematopoietic-supportive stroma, and adipocytes in a process modulated by dexamethasone (DEX). To characterize changes in hBMSC in response to DEX, we carried out differential display experiments using hBMSC cultured for 1 week in the presence or absence of 10(-8) M DEX. When RNA from these cells was used for differential display, numerous cDNA bands were identified that were up-regulated and down-regulated by DEX. The cDNA bands were reamplified by PCR and directly used to screen an hBMSC cDNA library. Seven clones were isolated and characterized by DNA sequencing and found to encode the following genes: transforming growth factor-beta-induced gene product ((beta)ig-h3), calphobindin II, cytosolic thyroid-binding protein, 22-kDA smooth muscle protein (SM22), and the extracellular matrix proteins osteonectin/SPARC, type III collagen, and fibronectin. To confirm that these genes were regulated by DEX, the cells were treated continuously with this hormone for periods ranging from 2 to 30 days, and steady-state mRNA levels were measured by Northern blot analysis. All genes showed some level of regulation by DEX. The most profound regulation by DEX was observed in the (beta)ig-h3 gene, which showed a relative 10-fold decrease in mRNA levels after 6 days of treatment. Interestingly, (beta)ig-h3 expression was not altered by DEX in fibroblasts from other human tissues, including thymus stromal fibroblasts, spleen stromal fibroblasts, and foreskin fibroblasts. In summary, differential display of DEX-treated hBMSC revealed unique patterns of gene expression and has provided new information about phenotypic changes that accompany the differentiation of hBMSC toward osteogenesis. J. Cell. Biochem. 76:231-243, 1999. Published 1999 Wiley-Liss, Inc.     

Characterization of two cDNA clones for pyruvate dehydrogenase E1 beta subunit and its regulation in tricarboxylic acid cycle-deficient fibroblast

Two distinct types of cDNA clones encoding for the pyruvate dehydrogenase (PDH) E1 beta subunit were isolated from a human liver lambda gt11 cDNA library and characterized. These cDNA clones have identical nucleotide sequences for PDH E1 beta protein coding region but differ in their lengths and in the sequences of their 3'-untranslated regions. The smaller cDNA had an unusual polyadenylation signal within its protein coding region. The cDNA-deduced protein of PDH E1 beta subunit revealed a precursor protein of 359 amino acid residues (Mr 39,223) and a mature protein of 329 residues (Mr 35,894), respectively. Both cDNAs shared high amino acid sequence similarity with that isolated from human foreskin (Koike, K.K., Ohta, S., Urata, Y., Kagawa, Y., and Koike, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 41-45) except for three regions of frameshift mutation. These changes led to dramatic alterations in the local net charges and predicted protein conformation. One of the different sequences in the protein coding region of liver cDNA (nucleotide position 452-752) reported here was confirmed by sequencing the region after amplification of cDNA prepared from human skin fibroblasts by the polymerase chain reaction. Southern blot analysis verified simple patterns of hybridization with E1 beta cDNA, indicating that the PDH E1 beta subunit gene is not a member of a multigene family. The mechanisms of differential expression of the PDH E1 alpha and E1 beta subunits were also studied in established fibroblast cell lines obtained from patients with Leigh's syndrome and other forms of congenital lactic acidosis. In Northern blot analyses for PDH E1 alpha and E1 beta subunits, no apparent differences were observed between two Leigh's syndrome and the control fibroblasts studied: one species of PDH E1 alpha mRNA and three species of E1 beta mRNA were observed in all the cell lines examined. However, in one tricarboxylic acid cycle deficient fibroblast cell line, which has one-tenth of the normal enzyme activity, the levels of immunoreactive PDH E1 alpha and E1 beta subunits were markedly decreased as assessed by immunoblot analyses. These data indicated a regulatory mutation caused by either inefficient translation of E1 alpha and E1 beta mRNAs into protein or rapid degradation of both subunits upon translation. In contrast, the PDH E1 alpha and E1 beta subunits in two fibroblast cell lines from Leigh's syndrome patients appeared to be normal as judged by 1) enzyme activity, 2) mRNA Northern blot, 3) genomic DNA Southern blot, and 4) immunoblot analyses indicating that the lactic acidosis seen in these patients did not result from a single defect in either of these E1 alpha and E1 beta subunits of the PDH complex.     

Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.     

IL 1 expression in a clone of human T cells

Three human T cell clones, all of which are T3+, T4+, T8-, and T11+, were examined for IL 1 production. Two clones were found to express readily detectable, membrane-bound IL 1 activity upon stimulation with OKT3 antibody, rIL 2, and PMA. Northern blot analysis of RNA from one of the clones shows that cells can be induced to express the genes for both IL 1 alpha and IL 1 beta. Furthermore, the pattern of expression in response to different stimuli suggests that the genes for IL 1 alpha and IL 1 beta are regulated independently.     

Enhancement of expression of an introduced gene by 5-azacytidine in mammalian cell lines

We transfected a mouse myeloma cell line, P3/NS1-Ag4-1 (NS-1), and a Chinese hamster ovary cell line, CHO-K1 with the β-galactosidase (β-Gal) gene of Escherichia coli, and isolated stable transformants designated as NS-1Z/gpt and CHO-Z/neo, respectively. When NS-1Z/gpt cells were incubated with 5–20 μM 5-azacytidine (5-azaC), the specific and total activity of β-Gal were enhanced 2-to 3-fold and 1.5- to 2-fold, respectively. In CHO-Z/neo cells, similar treatment resulted in a 3- to 5-fold increase in the specific β-Gal activity and about a 2-fold enhancement in total enzyme activity. The growth of both cell lines was inhibited by more than 80% in the presence of 10 μM 5-azaC. It was confirmed in immunotitration experiments that the enhancement of β-Gal activities was due to an increase in the enzyme protein. Northern blot analysis revealed that 5-azaC-treatment resulted in the enhanced expression of β-Gal mRNA. 5-AzaC also enhanced the production of human interleukin 2 (IL2) from CHO cells that were transfected with the IL2 gene. On the other hand, 5-azaC did not significantly affect the expression of an endogenous gene like lactate dehydrogenase or β-actin. These results suggest that 5-azaC is a useful agent for up-regulating the expression of introduced genes.     

Tenascin: cDNA cloning and induction by TGF-beta.

cDNA clones coding for tenascin, an extracellular matrix glycoprotein with a restricted tissue distribution, were isolated from a chicken fibroblast cDNA expression library using a specific tenascin antiserum. Antibodies eluted from the cDNA-encoded fusion proteins reacted exclusively with tenascin. Limited trypsin treatment of purified tenascin resulted in a peptide which confirmed the deduced protein sequences. The largest clone encoding 632 amino acids showed a cysteine-rich region containing 13 consecutive epidermal growth factor-like repeats of unusual uniformity. Northern blot analysis revealed 8- to 9-kb messages. Tenascin is shown to be induced in vitro by fetal calf serum as well as by transforming growth factor beta (TGF-beta). A 4-fold increase in tenascin secretion by chick embryo fibroblasts was seen after TGF-beta treatment. The induction of tenascin protein synthesis was preceded by an increase of tenascin mRNA as determined by Northern blot analysis. The induction of tenascin was compared with fibronectin. The accumulation of the two extracellular matrix proteins in the medium was differentially affected by fetal calf serum and TGF-beta and the increase was in both cases higher for tenascin.     

Enhanced casein kinase II activity in COS-1 cells upon overexpression of either its catalytic or noncatalytic subunit.

Casein kinase II consists of catalytic (alpha) and regulatory (beta) subunits complexed into a heterotetrameric alpha 2 beta 2 structure. Full-length cDNAs encoding the alpha and beta subunits of human casein kinase II were subcloned into an expression vector containing the cytomegalovirus promotor, yielding the expression constructs pCMV-alpha and pCMV-beta. Northern analyses of total cellular RNA prepared from COS-1 fibroblasts 65 h after transfection with pCMV-alpha or pCMV-beta or with both expression constructs showed marked specific increases in corresponding alpha and beta subunit RNAs. Immunoblot analysis utilizing anti-casein kinase II antiserum of cytosolic extracts prepared from COS-1 cells co-transfected with pCMV-alpha and pCMV-beta showed 2- and 4-fold increases in immunoreactive alpha and beta subunit protein, respectively, relative to vector-transfected cells. These same cytosolic fractions exhibited an average 5-fold increase in casein kinase II catalytic activity. COS-1 cells transfected with pCMV-alpha alone exhibited a 3-fold increase in immunoreactive alpha subunit protein and a nearly 2-fold increase in cytosolic casein kinase II catalytic activity. Transfection with the cDNA coding for the noncatalytic beta subunit alone also caused a near doubling of cytosolic casein kinase II catalytic activity. No increase in immunoreactive alpha subunit protein was observed in pCMV-beta-transfected cells, and no increase in immunoreactive beta subunit protein was observed in pCMV-alpha-transfected cells. These results indicate that a portion of the endogenous cellular casein kinase II protein is not fully active and that raising the concentration of the alpha or beta subunit stimulates this latent activity.     

SOCS-1 and SOCS-3 inhibit IFN-alpha-induced expression of the antiviral proteins 2,5-OAS and MxA

Although the use of IFN-alpha in combination with ribavirin has improved the treatment efficacy of chronic hepatitis C virus (HCV) infection, 20-50% of patients still fail to eradicate the virus depending on the HCV genotype. Recently, overexpression of HCV core protein has been shown to inhibit IFN signaling and induce SOCS-3 expression. Aim of this study was to examine the putative role of SOCS proteins in IFN resistance. By Western blot analysis, a 4-fold induction of STAT-1/3 phosphorylation by IFN-alpha was observed in mock-transfected HepG2 clones. In contrast, IFN-induced STAT-1/3 phosphorylation was considerably downregulated by SOCS-1/3 overexpression. In mock-transfected cells, IFN-alpha induced 2',5'-OAS and myxovirus resistance A (MxA) promoter activity 40- to 80-fold and 10- to 35-fold, respectively, and this effect was abrogated in SOCS-1/3 overexpressing cells. As detected by Northern blot technique, IFN-alpha potently induced 2',5'-OAS and MxA mRNA expression in the control clones. Overexpression of SOCS-1 completely abolished both 2',5'-OAS and MxA mRNA expression, whereas SOCS-3 mainly inhibited 2',5'-OAS mRNA expression. Our results demonstrate that SOCS-1 and SOCS-3 proteins inhibit IFN-alpha-induced activation of the Jak-STAT pathway and expression of the antiviral proteins 2',5'-OAS and MxA. These data suggest a potential role of SOCS proteins in IFN resistance during antiviral treatment.     

DNA microarray analysis of region-specific gene expression in the mouse epididymis

Microarray analysis was carried out to identify genes with enriched expression in the initial segment region of the mouse epididymis. A set of approximately 15 000 clones developed at the National Institutes for Aging and consisting of expressed sequence tags (ESTs) derived from pre- and peri-implantation embryos, Embryonic Day 12.5 female gonad/mesonephros, and newborn ovary were hybridized with probes generated against the initial segment (epididymal region 1) and the remainder of the epididymis (epididymal regions 2-5). The median values for the normalized ratios of region 1 to regions 2-5 from three independent experiments were averaged for each gene/EST using Genespring 5.0 software. The majority of clones showed a ratio of 1.0, suggesting they were expressed at similar levels in all epididymal regions. In addition, 123 clones exhibited 2-fold or higher expression in the initial segment, including Cres3, prostein, lipocalin 2, ALEX3, synaptotagmin-like 4, erm, and milk fat globule factor, whereas 216 clones, including elafin-like 1, lactotransferrin, Sin3B, zinc-finger protein 91, and membrane-type frizzled-related protein, showed 2-fold or higher expression in epididymal regions 2-5. Northern blot analyses of 12 clones predicted by microarray analysis to be either enriched in the initial segment (n = 8), enriched in epididymal regions 2-5 (n = 2), or similar in all regions (n = 2) were carried out. All clones exhibited the expected region-specific expression, thus confirming the microarray results. The studies presented here show a global survey of region-specific gene expression in the epididymis, identifying 15287 sequences, the majority of which have not previously been shown to be expressed in this organ.