Regulation of a novel immediate early response gene, IEX-1, in keratinocytes by 1alpha,25-dihydroxyvitamin D3

1alpha,25-Dihydroxyvitamin D3 [1alpha,25(OH)2D3] regulates cellular growth and differentiation. We show that in keratinocytes, 1alpha, 25(OH)2D3 reduces concentrations of the messenger RNA of IEX-1, the product of which blocks Fas- or tumor necrosis factor type alpha-induced apoptosis in various cells. In sub-confluent keratinocyte cultures, the addition of 1alpha,25(OH)2D3, in amounts that induce growth arrest, reduces IEX-1 mRNA concentrations. In confluent cells, 1alpha,25(OH)2D3 initially reduces and then increases IEX-1 mRNA concentrations. IEX-1 protein is localized in the nucleus and perinuclear region of keratinocytes. In sub-confluent cells, 1alpha,25(OH)2D3 translocates IEX-1 protein from the nucleus to the perinuclear region and cytoplasm. Since IEX-1 has recently been shown to regulate cell survival and number, we suggest that IEX-1 may play a role in keratinocyte growth and differentiation and that 1alpha,25(OH)2D3 may reduce keratinocyte growth via a reduction in IEX-1 mRNA and a change in the intracellular distribution of IEX-1 protein.     

Role of p53 in UVB-induced apoptosis in human HaCaT keratinocytes

Apoptosis represents an active form of cell death that is involved in the control of tissue homeostasis and in the deletion of DNA-damaged cells. Because the product of the tumor suppressor gene p53 has been demonstrated to be crucial for the induction of apoptosis in certain cell types, the present study was aimed at elucidating its role in ultraviolet-induced apoptosis in HaCaT keratinocytes. After in vitro ultraviolet B irradiation, p53 protein levels were noted to increase prior to the induction of apoptosis in a time- and concentration-dependent fashion. This increase could not be inhibited by the protein synthesis inhibitor cycloheximide. Because HaCaT keratinocytes are known to bear two p53 point mutations and because it is unclear whether p53 in HaCaT cells is still functional regarding induction of apoptosis, HaCaT cells were stably transfected with wild-type p53 cDNA inserted into the expression vector pCMV-Neo-Bam in sense (pC53-SN3) and anti-sense (pC53-ASN) direction. After selection with geniticin, growing colonies were screened for the presence of the transfected cDNA constructs by polymerase chain reaction. Cell clones bearing the anti-sense product were further analyzed for p53 expression by western blotting. Clones showing reduced p53 protein levels were irradiated with ultraviolet B light, and there was a clear reduction of apoptosis in the pC53-ASN bearing cell clones compared with the parental HaCaT cells. These studies demonstrate that blocking mutated p53 can partially block apoptosis in HaCaT keratinocytes and furthermore can confirm the key role for p53 in ultraviolet-induced apoptosis in human keratinocytes. Moreover, HaCaT keratinocytes and their p53-transfectants provide a convenient model that allows for further detailed analyses of apoptosis-associated biochemical and molecular events in human keratinocytes.     

In vitro expression of the diphtheria toxin A-chain gene under the control of human chorionic gonadotropin gene promoters as a means of directing toxicity to ovarian cancer cell lines

OBJECTIVE: Our goal was to determine whether toxicity of the diphtheria toxin A-chain gene regulated by the human chorionic gonadotropin promoter can be directed to malignant ovarian cell lines. STUDY DESIGN: Plasmids containing diphtheria toxin A-chain gene linked to the regulatory elements of the metalloergothioneine and human chorionic gonadotropin promoters were transfected into the cell lines. Expression of diphtheria toxin A-chain gene was determined by the inhibition of a cotransfected luciferase reporter gene. RESULTS: Cytotoxicity of the diphtheria toxin A-chain gene is shown in a dose-responsive manner. Transfection of a plasmid expressing the diphtheria toxin A-chain gene controlled by a constitutive promoter readily inhibits protein synthesis. Specific inhibition of luciferase protein synthesis occurs in ovarian cancer cells transfected with the diphtheria toxin A-chain gene under the control of the human chorionic gonadotropin promoters when compared with normal ovarian epithelial cells or fibroblasts. CONCLUSIONS: These data demonstrate the preferential expression of the diphtheria toxin A-chain gene, regulated by the human chorionic gonadotropin promoter, to ovarian cancer cell lines. This provides an avenue for targeting such cells for suicide, toxin, or cytokine genes.     

Cloning, expression, and chromosome mapping of human galectin-7

The galectins are a family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. Here we report the cloning and expression of a novel member of this family (galectin-7) that correspond to IEF (isoelectric focusing) 17 (12,700 Da; pI, 7.6) in the human keratinocyte protein data base, and that is strikingly down-regulated in SV40 transformed keratinocytes (K14). The cDNA was cloned from a lambda gt11 cDNA expression library using degenerated oligodeoxyribonucleotides back-translated from an IEF 17 peptide sequence. The protein encoded by the galectin-7 clone comigrated with IEF 17 as determined by two-dimensional (two-dimensional gel electrophoresis) analysis of proteins expressed by transiently transfected COS-1 cells, and bound lactose. Alignment of the amino acid sequences with other members of the family showed that the amino acids central to the beta-galactoside interaction are conserved. Galectin-7 was partially externalized to the medium by keratinocytes although it has no typical secretion signal peptide. Immunoblotting as well as immunofluorescence analysis of human tissues with a specific galectin-7 antibody revealed a narrow distribution of the protein which was found mainly in stratified squamous epithelium. The antigen localized to basal keratinocytes, although it was also found, albeit at lower levels, in the suprabasal layers where it concentrated to areas of cell to cell contact. Both, its cellular localization as well as its striking down-regulation in K14 keratinocytes imply a role in cell-cell and/or cell-matrix interactions necessary for normal growth control. The galectin-7 gene was mapped to chromosome 19.     

Human cytomegalovirus increases modified low density lipoprotein uptake and scavenger receptor mRNA expression in vascular smooth muscle cells

Evidence suggests a possible role for human cytomegalovirus (HCMV) in the development of arteriosclerosis. One of the earliest events in plaque formation is the accumulation of lipid-laden foam cells, derived from macrophages and smooth muscle cells (SMCs). The lipid accumulation that occurs depends upon the uptake of oxidized LDL (Ox-LDL), a process in which the scavenger receptor (SR) has been postulated to play an important role. We therefore examined the effects of HCMV on this process. We demonstrate that HCMV infection of human SMCs increases modified LDL uptake and stimulates class A SR gene (SR-A) mRNA expression. In addition, infection of rat SMCs with HCMV, which causes immediate early gene expression (IE72/IE84), but no early or late HCMV gene products and no cytopathic effects, also increases SMC uptake of Ox-LDL and acetylated LDL, with either effect blocked by an excess of either cold Ox-LDL or acetylated-LDL, and by fucoidin, an SR competitor. Cotransfection of an IE72, but not an IE84, expression plasmid and a plasmid containing a Class A SR promoter/reporter gene construct enhances SR promoter activity. Since increased Ox-LDL uptake is believed to play an important role in arteriosclerosis, these results provide a link between HCMV infection and arteriosclerotic plaque formation.     

Integration of proviral DNA into the PDGF beta-receptor gene in HTLV-I-infected T-cells results in a novel tyrosine kinase product with transforming activity

We have previously shown that noninfected human T-cell lines express the canonical 5.7 kb mRNA coding for the type beta platelet-derived growth factor-receptor (PDGF beta-receptor), whereas HTLV-I-infected T-cell lines express a novel PDGF beta-receptor mRNA of 3.8 kb. In this report, we have extended those studies to molecularly characterize the 3.8 kb PDGF beta-receptor mRNA and show that it has resulted from integration of an apparently undeleted HTLV-I provirus into the PDGF beta-receptor gene in an orientation enabling expression of a truncated PDGF beta-receptor mRNA using the 3' HTLV-I long terminal repeat as a promoter. Further, NIH3T3 cells transfected with a plasmid containing the truncated PDGF beta-receptor ORF plasmid generate colonies in soft agar with more cells per colony than untransfected cells, or cells transfected with the Tax 1 or PDGF-B (c-sis) plasmids. These results indicate that the truncated PDGF beta-receptor protein acquires transforming capability and that HTLV-I-induced truncation of PDGF beta-receptor may correlate with HTLV-I-associated neoplasia of human T-cells.     

Characterization of the human aldose reductase gene promoter

The promoter region of the human aldose reductase gene has been identified upstream of the translation start ATG codon. The promoter contains a TATA box, a CCAAT promoter element, and three Sp1 protein binding consensus sequences upstream of the capsite. A 640-base pair insert spanning +31 to -609 directs expression of the reporter gene chloramphenicol acetyltransferase in an orientation-specific manner in transfected Hep G2 cells. The promoter activity remained constant with deletions from base pairs -609 to -186. The TATA and the CCAAT consensus sequences show significant promoter activity, whereas the three Sp1 binding consensus sequences, individually, have no significant promoter activity. A GA-rich region (-186 to -146) contains two CGGAAA/G motifs, which show promoter activity and interaction with Hep G2 nuclear extract and GA-binding proteins (GABP alpha and GABP beta 1) as shown by mobility shift assays and DNase I footprinting. Similar cis-elements in herpes simplex virus type 1 interact with rat liver GABP and the viral VP16 protein to mediate the induction of immediate early viral genes. A GC-rich region (-87 to -31) is identified by mobility shift assay, and a consensus sequence of an androgen response element is present at -396 to -382. The human aldose reductase promoter, thus, has regulatory response elements that may be important during early development and puberty. These regulatory elements may play a significant role in the development of certain diabetic complications.     

Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector

Recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by inclusion of a dominant selectable marker in the viral vector, cell lines can be derived that stably express recombinant genes. A virus was constructed containing two expression cassettes controlled by constitutive mammalian promoters: the cytomegalovirus immediate early promoter/enhancer directing expression of green fluorescent protein and the simian virus 40 (SV40) early promoter controlling neomycin phosphotransferase II. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. In addition to commonly used transformed cell lines such as HeLa, CHO, Cos-7, and 293, this list includes primary human keratinocytes and bone marrow fibroblasts. In all cases, addition of butyrate or trichostatin A (a selective histone deacetylase inhibitor) to transduced cells markedly enhanced the levels of reporter protein expression observed. When transduced cells are put under selection with the antibiotic G418, cell lines can be obtained at high frequency that stably maintain the expression cassettes of the vector DNA and exhibit stable, high-level expression of the reporter gene. Stably transduced derivatives have been selected from a substantial number of different cell types, suggesting that stable lines can be derived from any cell type that exhibits transient expression.     

Tumor-suppressive activity of the growth arrest-specific gene GAS1 in human tumor cell lines

The GAS1 gene product induces growth arrest through a p53-dependent mechanism. To investigate whether GAS1 is a tumor suppressor gene, we transfected GAS1-negative human tumor cells with GAS1 plasmids and analyzed growth characteristics of stable transfectants. When a constitutively expressing GAS1 plasmid was transfected into A549 cells, no stable colonies expressing GAS1 were isolated. When A549 cells were transfected with a dexamethasone-inducible GAS1 plasmid, expression of GAS1 inhibited growth in vitro, and fewer slow-growing tumors arose in nude mice. GAS1 also inhibited proliferation of an HT1080 subline with wild-type (wt) p53 and normal MDM2. However, when the HT1080 subline HTD114 was transfected with the constitutive GAS1 plasmid, there was no reduction in colony number. GAS1-transfectant clones had unaltered growth in vitro, were morphologically unchanged and showed no difference in their ability to form tumors in nude mice. Although HTD114 cells contain wt p53, levels of MDM2 were elevated by 10-15 fold. The HT1080-6TGc5 subline with mutant p53 and normal MDM2 was also refractory to GAS1. Our results show that GAS1 suppresses the growth and tumorigenicity of human tumor cells and overexpression of MDM2 or p53 mutation inhibits the GAS1-mediated growth-suppressing pathway.     

Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax

To understand the mechanism of p56lck protein downregulation observed in human T cells infected by human T-cell leukemia virus type 1 (HTLV-1), we have investigated the ability of the 3' end of the HTLV-1 genome as well as that of the tax and rex genes to modulate p56lck protein expression and p56lck mRNA synthesis. By using Jurkat T cells stably transfected with constructs that expressed either the 3' end of the HTLV-1 genome (JK C11-pMTEX), the tax gene (JK52-Tax) or the rex gene (JK9-Rex), we found that the expression of p40tax (Tax) was sufficient to modulate p56lck protein expression. Similarly, we found that the expression of the mRNA which encoded p56lck was repressed in Jurkat T cells which expressed Tax. This downregulation was shown to be proportional to the amount of tax mRNA found in the transfected cells, as evidenced by experiments that used cells (JPX-9) stably transfected with a tax gene driven by a cadmium-inducible promoter. Furthermore, cadmium induction of Tax in JPX-9 cells transiently transfected with a construct containing the chloramphenicol acetyltransferase (CAT) gene under control of the lck distal promoter (lck DP-CAT) resulted in the downregulation of CAT gene expression. In contrast, cadmium induction of Tax in JPX-9 cells transiently transfected with a CAT construct driven by a lck DP with a deletion extending from position -259 to -253 (a sequence corresponding to a putative E-Box) did not modulate CAT gene expression, suggesting that the effect of Tax on p56lck is mediated through an E-Box binding protein.     

Targeted integration of adeno-associated virus-derived plasmids in transfected human cells

Adeno-associated virus (AAV) integrates its genomic DNA into a defined region of human chromosome 19 (AAVS1). The specificity of integration is dependent on the presence of the inverted terminal repeats (ITR) and on expression of the rep gene. To develop vectors capable of targeting the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether the rep gene can mediate site-specific integration when cloned outside of an ITR-flanked transgene cassette. HeLa and Huh-7 cells were transfected with a plasmid containing the rep gene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Southern blot analysis of individual clones detected Rep-mediated site-specific integration of the ITR-flanked DNA in 25% and 12% of the HeLa and Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescent in situ hybridization analysis of the transfected HeLa clones. Sequence analysis of the ITR-AAVS1 junction of one of the transfected Huh-7 clones indicated that the insertion of the ITR DNA fragment had occurred at nucleotide 1003. These results have implications for the development of AAV-derived vectors capable of directing the site-specific integration of a gene of interest.     

Expression of CTLA4-Ig by biolistically transfected mouse islets promotes islet allograft survival

BACKGROUND: Localized delivery of immunosuppressive molecules, limited to the graft site, may allow transplantation of tissue in the absence of systemic immunosuppressive agents. We tested whether purified mouse islets that had been engineered to produce human CTLA4-Ig locally at the graft site could survive in allogeneic recipients receiving no systemic immunosuppression. METHODS: CBA (H2(k)) islets were subjected to biolistic (gene gun) transfection with a cDNA encoding human CTLA4-Ig under control of the human cytomegalovirus immediate early promoter. After 40-48 hr of culture, the transfected islets (500 per recipient) were transplanted beneath the renal capsule of alloxan-induced diabetic BALB/c (H2(d)) recipients. RESULTS: Control grafts (n=10) consisting of islets biolistically transfected with the expression plasmid alone (i.e., no gene inserted) survived for 12.8+/-3.6 (mean +/- SD) days. In contrast, islets transfected with CTLA4-Ig (n=12) survived 66.8+/-61.5 days (P=0.01), with 50% demonstrating functional survival until follow-up was concluded at 50 (n=2), 130 (n=2), or 165 (n=2) days. Immunohistochemistry on grafts that survived long term showed well-granulated, insulin-positive islets lying adjacent to, but not infiltrated by, dense aggregates of mononuclear cells. CONCLUSIONS: Transfection of allogeneic mouse islets with human CTLA4-Ig results in prolonged allograft survival. Although on histology mononuclear cells are present in the area of the transfected graft, they do not appear to infiltrate or destroy the islet graft.