Expression Of HA Research Articles

Nuclear Factor of Activated T-Cells (NFAT1): Potential Novel Target in T-Cell Acute Lymphocytic Leukemia (T-ALL)

Abstract 4634 Background.T-cell transcription factor NFAT1 (NFATc2) has been reported to regulate cell cycle regulatory proteins including cyclins, cyclin-dependent kinases (CDKs), and p21. MicroRNA-184 (miR-184) has been noted to have close sequence homology to the 3' UTR of NFAT1 and to regulate NFAT1 mRNA translation in human adult and umbilical cord blood (UCB) CD3+CD4+ T-cells. In this study, we are characterizing the functional role of NFAT1 in T-ALL cells to determine whether its dysregulation may alter normal cell cycle regulation and thereby may contribute to T-ALL leukemogenesis. Materials/Methods.In this study we used MOLT-4 cell line to investigate the role of NFAT1 in T-ALL, since MOLT-4 represents early stage of human T-ALL (CD4+CD8+ double positive) compared with other T-cell lines including Jurkat. We developed two distinct approaches to explore NFAT1 signalling, either by manipulating the expression level of NFAT1 protein by blocking miR-184, or by overexpressing a constitutively active form of NFAT1 (caNFAT1) holding a HA-tag (Addgene plasmid 11792, A. Rao, PhD, Harvard University). caNFAT1 was introduced into gateway system expression vector pEF-DEST51. MOLT-4 cells were maintained in complete RPMI 1640 medium according to ATCC guidelines. Electroporation transfection was performed on Amaxa Nucleofector I using program C-05. 200 nM of anti-miR-184 miRNA inhibitor (Dharmacon) or 2 μ g of plasmid containing caNFAT1 was transfected into 2×10e6 MOLT-4 cells in each sample. Transfection efficiency was tested using pmaxGFP (Lonza). Proteins were extracted at various time points after transfection using RIPA buffer (PIERCE) with protease inhibitor cocktail and tested for the expression of NFAT1 (BD Biosciences), phosphorylated NFAT1 (Santa Cruz Biotechnology), HA (Roche), and beta-actin (Sigma-Aldrich). Cell cycle was measured at various time points by pelleting down 0.5–1 X10e6 cells, followed by ethanol fixation, and incubation in propidium iodide (PI) (10 μ g/ml) staining solution and flow cytometric measurement for PI incorporation. Cell apoptosis was measured by PE-conjugated annexin V staining (BD Biosciences) in transfected MOLT-4 cells including empty plasmid controls. Results/Discussion.The over-expression of caNFAT1 in MOLT-4 led to increased proportion of cells in G2 phase than in controls (10.8% vs. 7.3% on day5, and 11.8% vs. 8.1% on day6). In addition to these cell cycle changes in MOLT-4 treated with caNFAT1 plasmid, higher rates of apoptosis were noted on day 2 (28.3% vs. 15.5%), which notably leveled off from day 3 to day 6 potentially attributed to compensatory cellular responses. Previous work by this group has shown higher expression of miR-184 in MOLT-4 by 56 fold compared with normal human CD4/8 double positive T-cells. Here, miR-184 knockdown was also notable for a larger proportion of MOLT-4 cells in G2 cell cycle phase and higher apoptosis, albeit modest changes compared with that exerted by caNFAT1. Conclusion.In MOLT-4 cell line, this preliminary data suggests that increasing active non-phosphorylated NFAT1 is associated with enhanced proportion of cells in G2 phase as well as enhanced resultant apoptosis. Taken together, regulation of NFAT1 may be exploited as potential T-ALL targeted therapy. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Adenosine A2A Receptor Is Involved in Cell Surface Expression of A2B Receptor

The A2A and A2B adenosine receptors (A2AR and A2BR) are implicated in many physiological processes. However, the mechanisms of their intracellular maturation and trafficking are poorly understood. In comparative studies of A2AR versus A2BR expression in transfected cells, we noticed that the levels of cell surface expression of A2BR were significantly lower than those of A2AR. A large portion of the A2BR was degraded by the proteasome. Studies of cell surface expression of A2BR chimeric molecules in transfectants suggested that A2BR does not have the dominant forward transport signal for export from the endoplasmic reticulum to the cell surface. A2BR surface expression was increased in A2BR chimeras where the A2BR carboxyl terminus (CT) was replaced or fused with the A2AR CT. Co-transfection of A2AR with A2BR enhanced surface expression of A2BR though the F(X)(6)LL motif in the A2AR CT. The requirements of A2AR expression for better A2BR cell surface expression was not only established in transfectants but also confirmed by observations of much lower levels of A2BR-induced intracellular cAMP accumulation in response to A2BR-activating ligand in splenocytes from A2AR(-/-) mice than in wild type mice. The results of mechanistic studies suggested that poor A2BR expression at the cell surface might be accounted for mainly by the lack of a dominant forward transport signal from the endoplasmic reticulum to the plasma membrane; it is likely that A2BR forms a hetero-oligomer complex for better function.

Codon-optimization of the hemagglutinin gene from the novel swine origin H1N1 influenza virus has differential effects on CD4 + T-cell responses and immune effector mechanisms following DNA electroporation in mice

DNA electroporation is a powerful vaccine strategy that could be rapidly adapted to address emerging viruses. We therefore compared cellular and humoral immune responses in mice vaccinated with DNA expression plasmids encoding either the wildtype or a codon-optimized sequence of hemagglutinin from the novel swine origin H1N1 influenza virus. While expression of HA from the wildtype sequence was hardly detectable, the H1N1 hemagglutinin was well expressed from the codon-optimized sequence. Despite poor expression of the wildtype sequence, both plasmids induced similar levels of CD4 + T-cell responses. However, CD8 + T-cell and antibody responses were substantially higher after immunization with the codon-optimized DNA vaccine. Thus, efficient induction of immune effector mechanisms against HA of the novel H1N1 influenza virus requires codon-optimization of the DNA vaccines. Since DNA vaccines and several viral vector vaccines employ the same cellular RNA-Polymerase II dependent expression pathway, the poor expression levels from wildtype HA sequences might also limit the induction of immune effector mechanisms by such viral vector vaccines.

Nuclear Export of Influenza A Virus mRNAs Requires Ongoing RNA Polymerase II Activity

Influenza A virus transcribes its segmented negative sense RNA genome in the nuclei of infected cells in a process long known to require host RNA polymerase II (RNAP-II). RNA polymerase II synthesizes pre-mRNAs whose 5'-cap structures are scavenged by the viral RNA-dependent RNA polymerase during synthesis of viral mRNAs. Drugs that inhibit RNAP-II therefore block viral replication, but not necessarily solely by denying the viral polymerase a source of cap-donor molecules. We show here that 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), a compound that prevents processive transcription by RNAP-II, inhibits expression of the viral HA, M1 and NS1 genes at the post-transcriptional level. Abundant quantities of functionally and structurally intact viral mRNAs are made in the presence of DRB but with the exception of NP and NS2 mRNAs, are not efficiently translated. Taking M1 and NP mRNAs as representatives of DRB-sensitive and insensitive mRNAs, respectively, we found that the block to translation operates at the level of nuclear export. Furthermore, removal of DRB reversed this block unless a variety of chemically and mechanistically distinct RNAP-II inhibitors were added instead. We conclude that influenza A virus replication requires RNAP-II activity not just to provide capped mRNA substrates but also to facilitate nuclear export of selected viral mRNAs.

Preparation of gene-viral therapeutic system CNHK200-hA and its antitumor activity on lung cancer

ObjectiveTo develop a novel adenoviral vector system, which combines the advantages of the antiangiogenic gene therapy and virus therapy, and to investigate its antitumor activity on lung cancer.MethodsA new kind of viral vector CNHK200, in which the E1b55kDa gene was deleted and the whole E1a gene was preserved, was constructed. Human angiostatin gene Kringle 1∼5 (hA) was amplified and inserted into the genome of the replicative virus CNHK200, generating CNHK200-hA. The expression of hA and its effect on lung cancer cell growth in vitro and in vivo were studied.ResultsThe novel vector system CNHK200-hA, just like the replicative virus ONYX-015, replicated in p53-deficient tumor cells but not in normal cells, and thus specifically killed tumor cells. In in vitro experiment, both CNHK200-hA and the non-replicative virus Ad-hA could kill tumor cells but the latter needed 100 times more MOI to achieve the same level of cell killing. In in vivo experiment, the therapeutic effect of CNHK200-hA on human lung cancer A549 xenografts in nude mice was significantly better than that of Ad-hA or that of ONYX-015.ConclusionCNHK200-hA, which carries the angiostatin gene, has the advantages of specific tumor targeting, high expression of transgene in tumor cells and potent antitumor activity.

Antiproliferative heparan sulfate inhibiting hyaluronan and transforming growth factor-β expression in human lung fibroblast cells

The objective of this study was to examine the effects of heparan sulfate (HS) on factors involved in the remodeling of connective tissue observed in patients with fibrotic respiratory disorders such as asthma. A suitable working model is to stimulate human fetal lung fibroblasts in vitro with structurally different forms of HS. Highly sulfated and iduronic acid (IdoUA)-rich HS specifically decreased cell proliferaton, production of jyaluronan (HA), transforming growth factor (TGF)-β1, and TFF-β-induced α-smooth muscle actin but did not affect the overall proteoglycan production in the cells. These repressed factors are suggested to play a critical role in the early stages of remodeling and myofibroblast activation. Low sulfated and IdoUA-poor HS did not display any effects on these factors. Furthermore, analysis of the protein expression pattern by two-dimensional gel electrophoresis revealed a 70% increased expression of annexin II, which has previously been shown to have a high affinity for both heparin and HS. Heat-shock protein 27 and arsenite translocating factor, both involved in actin organization and polymerization, were also increased in the HS-stimulated cells. Thus, the reduced expression of HA and TGF-β1, both important in the development of fibrosis, seems to be mediated by pecific changes in protein expression of the fibroblast. The observed inhibition of cell proliferation, HA, and TGF-β1 allows speculation of highly sulfated HS as a antifibrotic candidate in the early stage of remodeling.

Prevention of brain metastasis formation by local expression of interleukin-4 or hemagglutinin antigen.

Expression of hemagglutinin antigen of influenza virus (HA) by the murine colon carcinoma cell line (CT-26) produces systemic immunization against tumor challenges in the cecum, liver and lungs but not in the brain of BALB/c-mice. Immunization with IL-4 expressing CT-26 cells inhibits lung metastases formation. The purpose of our study was to examine the effects of HA or IL-4 expression on brain metastases formation. Using selective internal carotid artery injections, brain metastases formation of HA or IL-4 expressing CT-26 cells with and without subcutaneous pre-immunization was evaluated in Balb/c mice. Systemic pre-immunization with HA or IL-4 expressing tumor cells cannot protect against brain metastases, while the local, intracerebral expression of HA or IL-4 inhibits the growth of hematogenous brain metastases. Pre-immunization with HA or IL-4 expressing tumor cells did produce systemic immunity against liver and lung metastases but not against brain metastases. Local, intracerebral expression of HA or IL-4 prevents from cerebral metastases formation in an animal model.

Apoptosis and catastrophic cell death in benzo[ a]pyrene-transformed human breast epithelial cells

Apoptosis and mitotic death, bi- and multinucleation, giant cells and micronucleation were investigated in human breast epithelial cell lines transformed by benzo[ a]pyrene (BP) (BP1, BP1-E and BP1-E1 cells) and in BP1 cells transfected with the c-Ha- ras oncogene (BP1-T ras cells). Since BP induces apoptosis and the abnormal expression of ras genes elicits catastrophic mitosis, both cell death phenomena were expected to occur in this system, especially in BP1-T ras cells. Regardless of the cell line considered, single-nucleate cells were found to be eliminated preferentially through apoptosis, while bi- and multinucleate cells were eliminated through catastrophic mitosis. Apoptosis and catastrophic mitosis were observed in all cell lines but were significantly more frequent in BP1-T ras cells. The abnormal expression of Ha- ras in the latter cells may enhance in this system the effects of the BP apoptosis path reported for BP-transformed Hepa 1c1c7 hepatoma cells. Transfection with the ras oncogene also enhanced the mitotic disturbances, which produced multi- and micronucleation and mitotic death, possibly because of the genomic instability promoted by this oncogene in the BP-transformed cell line.

Function of a minus-end-directed kinesin-like motor protein in mammalian cells.

CHO2 is a mammalian minus-end-directed kinesin-like motor protein present in interphase centrosomes/nuclei and mitotic spindle fibers/poles. Expression of HA- or GFP-tagged subfragments in transfected CHO cells revealed the presence of the nuclear localization site at the N-terminal tail. This domain becomes associated with spindle fibers during mitosis, indicating that the tail is capable of interaction with microtubules in vivo. While the central stalk diffusely distributes in the entire cytoplasm of cells, the motor domain co-localizes with microtubules throughout the cell cycle, which is eliminated by mutation of the ATP-binding consensus motif from GKT to AAA. Overexpression of the full-length CHO2 causes mitotic arrest and spindle abnormality. The effect of protein expression was first seen around the polar region where microtubule tended to be bundled together. A higher level of protein expression induces more elongated spindles which eventually become disorganized by loosing the structural integrity between microtubule bundles. Live cell observation demonstrated that GFP-labeled microtubule bundles underwent continuous changes in their relative position to one another through repeated attachment and detachment at one end; this results in the formation of irregular number of microtubule focal points in mitotic arrested cells. Thus the primary action of CHO2 appears to cross-link microtubules and move toward the minus-end direction to maintain association of the microtubule end at the pole. In contrast to the full-length of CHO2, overexpression of neither truncated nor mutant polypeptides resulted in significant effects on mitosis and mitotic spindles, suggesting that the function of CHO2 in mammalian cells may be redundant with other motor molecules during cell division.

Differential splicing of type II procollagen mRNA in canine retina

Canine retina expressed two forms of Type II procollagen mRNA, IIA and IIB, due to alternate splicing of exon 2 at the N‐propeptide region. The N‐propeptide region of Type II procollagen showed a high degree of homology with human, approximately 96% both at the nucleotide and the amino acid levels. Expression of HA and IIB forms of Type II procollagen from early stage of development to adult life suggests the important role of both forms for the development as well as maintenance of the ocular structure.

The Distinctive Features of Influenza Virus Infection of Dendritic Cells

CD8 + cytolytic T lymphocytes (CTLs) are considered to be critical mediators for resistance to influenza virus infection. We have previously demonstrated that dendritic cells are potent antigen presenting cells in the development of anti-influenza CTLs. Here we identify distinctive features of the interaction of influenza virus with dendritic cells. Exposure of dendritic cells to influenza virus at MOIs of 2–4:1 leads to >90% infection, as manifested by the expression of the viral proteins HA and NS1. The infection is non-toxic as viral protein expression is sustained for >2 days with retention of viablility, but little infectious virus is produced. Substantial induction of the anti-viral cytokine IFN-α also occurs. Influenza infection of macrophages also results in viral protein expression in a majority of cells, and synthesis of IFN-α. In contrast to dendritic cells, macrophages display evidence of apoptosis within 10–12 hours, and the majority of cells die within 24–36 hours. During this interval macrophages synthesize >10-fold higher levels of virus than dendritic cells. Infected dendritic cells but not macrophages, can induce substantial CTL responses from purified blood CD8 + T cells in the absence of exogenous cytokines such as IL-2. Low levels of infection (MOIs of 0.02) are sufficient to generate potent CTL responses. Influenza virus expressing non-cleaved HA does not elicit CTLs indicating that virus must access the cytoplasm of dendritic cells to utilize traditional class I processing pathways. These observations indicate that DCs are distinct in their handling of influenza virus and for the induction of anti-viral immunity.

In vitro anti-influenza virus activity of a plant preparation from Geranium sanguineum L.

A polyphenolic complex (PC), isolated from the Bulgarian medicinal plant Geranium sanguineum L., was shown to have selective anti-influenza activity in vitro. Expression of HA on the surface of cells infected with A/chicken/Rostock/34, virus-induced cytopathic effect, infectious virus yield and plaque formation were all reduced at non-toxic concentrations of PC. Synthesis of virus proteins was also selectively inhibited. High concentrations of PC (>200 μg/ml) exhibited a strong virucidal effect. Although the action was directed against an early stage of infection (within 3 h of infection), the process directly affected was not identified. The selectivity of antiviral action was confirmed by the variation in sensitivity of different influenza viruses to PC and the selection of variants with reduced drug sensitivity.

Role of protein kinase C in ras-mediated fos-expression

HC-11 mouse mammary epithelial cells stably transfected with a glucocorticoid-inducible Ha- ras construct encoding a transforming (val 12) p21 Ha- ras were cotransfected with a c- fos-CAT construct containing the human c- fos promoter up to position −711 and the CAT reporter gene. Expression of Ha- ras by dexamethasone leads to a transcriptional activation of the fos-CAT construct which was found to be sensitive to the PKC inhibitor ilmofosine (BM41440) and abrogated by PKC depletion following long-term exposure to TPA. The responsiveness to Ha- ras is retained if only the portion of the fos promoter covering the serum response element (SRE) and the adjacent fos AP-1 (FAP) site are put in front of a CAT gene linked to a thymidine kinase (TK) promoter. Further depletion of the FAP-site does not affect the inducibility by Ha- ras. Transcriptional activation of the SRE-FAP-TK-CAT as well as the SRE-TK-CAT constructs by Ha- ras is sensitive to the PKC-inhibitor ilmofosine (BM41440) and blocked by long-term exposure to TPA. Long-term exposure to TPA depletes cells of PKC α and significantly reduces the PKC ϵ levels. Long-term exposure in bryostatin 1 selectively depletes PKC α. Depletion of PKC α by bryostatin 1 does not reduce the transcriptional activation of the SRE-FAP-TK-CAT-construct by Ha- ras. It is concluded that (i) transforming Ha- ras induces c- fos in HC-11 cells via PKC (presumably ϵ), (ii) the signal is mediated to the serum response element (SRE) of the fos promoter and (iii) the fos AP-1 (FAP) site is not required for this mechanism.

Characterization of influenza virus-induced leukocyte adherence to human umbilical vein endothelial cell monolayers.

The adherence of undifferentiated 51Cr-labeled HL-60 (0.5 x 10(6) HL-60 cells/well) cells was monitored on influenza virus-infected HUVEC monolayers. Whereas only 3.0 +/- 1.6% (n = 36) of HL-60 cells adhered to uninfected HUVEC, adherence was increased to 41.7 +/- 2.2% (n = 6), 79.7 +/- 1.2% (n = 6), 83.9 +/- 0.7% (n = 6), and 84.4 +/- 0.5% (n = 6) on HUVEC infected for 7 h at a MOI of 1, 3, 6, and 9, respectively. In comparison, HL-60 cell adherence increased to 35% when HUVEC monolayers were stimulated with LPS (0.2-20 micrograms) for 4 h. Increased adherence to infected HUVEC occurred at 5 h postinfection, peaked at 7 h, and was maintained at 24 h postinfection. Active virus and metabolically active endothelial cells were required to mediate the virus-induced adherence. E-selectin and ICAM-1 Ag were upregulated 78.3- and 4.1-fold, respectively, by LPS (0.02-20 micrograms, 4 h) whereas virus infection (7 h) only increased these proteins 2.6- and 1.4-fold with a MOI > or = 16. Although the time courses of expression for both adhesion molecules after LPS treatment of virus infection were similar, the difference in the magnitude of upregulation suggests that virus-induced adherence is not a result of upregulation of E-selectin and ICAM-1. In contrast, surface expression of HA is involved in HL-60 cell adherence to virus-infected HUVEC because (1) the time course and magnitude of HA AG expression paralleled the time course and magnitude of HL-60 cell adherence after virus infection of HUVEC; (2) HL-60 cell aggregates were absent on infected HUVEC monolayers in the presence of anti-HA; (3) HL-60 cells competed with RBC for infected endothelial cells stained for cellular HA Ag and (4) anti-HA abolished the virus-induced adherence. Furthermore, it appears that HL-60 cells are binding directly to HA because HL-60 cell adherence to a cell-free surface was increased if virus was prebound and neuraminidase treatment of HL-60 cells prevented the HL-60 cell adherence to influenza virus-infected endothelial monolayers.

HETEROGENEOUS EXPRESSION OF ONCOGENES IN SMALL-CELL LUNG-CANCER XENOGRAFTS

The analysis of oncogene expression may provide insights into the pathogenesis of small cell lung cancer (SCLC) and may help to predict clinical behavior. The expression of 8 oncogenes (c-myc, N-myc, L-myc, Ha-ras, Ki-ras, N-ras, erbB-2, v-sis) was evaluated in small cell lung cancer (SCLC) xenografts of tumor samples, recentlly transplanted, taken from 17 different patients. Eight of these 17 SCLC lines expressed the L-myc oncogene and 2 SCLC lines expressed the c-myc oncogene. One SCLC line (SCLC-63M) simultaneously expressed the L-myc and c-myc oncogenes. All SCLC lines examined had almost similar high RNA levels of the Ki-ras oncogene, while the expression of Ha- and N-ras oncogenes was not always observed. The N-myc and v-sis oncogenes were expressed in only one tumor and at a very weak level, and no transcript of the erbB-2 oncogene was observed in any of our 17 SCLC lines. These results indicate that oncogene expression in SCLC lines is heterogeneous, with the exception of the Ki-ras oncogene which is constantly overexpressed.

Clinical and histopathologic evaluation of the expression of Ha-ras and fes oncogene products in lung cancer.

The expression of Ha-ras and fes oncogenes was investigated with the immunohistochemical method in formalin-fixed, paraffin-embedded tissue specimens of 147 lung carcinomas. Positive immunoperoxidase reactions for Ha-ras p21 were found in 80.5% of the adenocarcinomas, 39.5% of the squamous cell carcinomas, 21.4% of the large cell carcinomas, and 15.4% of the small cell carcinomas; those for fes P85 were found in 51.2% of the adenocarcinomas, 26.3% of the squamous cell carcinomas, 35.7% of the large cell carcinomas, and 15.4% of the small cell carcinomas. Both Ha-ras p21 and fes P85 were expressed most frequently and most strongly in adenocarcinoma. In addition, adenocarcinoma showed significantly higher incidence of concomitant expression of Ha-ras p21 and fes P85 as compared with other histologic types of lung cancer. Thus, the authors suggest that the cooperative effects of Ha-ras and fes oncogenes are especially important in the carcinogenesis of adenocarcinoma. In adenocarcinoma, the incidence and grade of Ha-ras p21 expression increased with the degree of histologic differentiation, suggesting that Ha-ras oncogene might be related to cellular differentiation. Papillary adenocarcinoma showed more frequent Ha-ras p21 expression in comparison with acinar adenocarcinoma. In well- or moderately differentiated adenocarcinoma, the incidence and grade of Ha-ras p21 immunoreactivity in the cases with poor prognosis were significantly higher than in those with good prognosis if other major prognostic factors were equivalent in the two groups. The authors propose that the expression of Ha-ras p21 may be one of the useful prognostic factors in such carcinomas.

Interference of Ha- ras with inositol trisphosphate-mediated Ca 2+-release

Expression of a transforming Ha- ras by dexamethasone in NIH3T3 cells transfected with a glucocorticoid-inducible Ha- ras construct results in a rapid desensitization of the intracellular Ca 2+-mobilizing system to bombesin. This effect precedes the down-modulation of inositol trisphosphate (IP 3) formation by several hours and is, therefore, not explained by an uncoupling of phosphoinositidase C. It is demonstrated that expression of Ha- ras attenuates the Ca 2+-release by IP 3 in permeabilized cells. The IP 3 concentration required for half-maximal Ca 2+-release is doubled in Ha- ras expressing cells. Maximal Ca 2+-release which is obtained with 2,μM IP 3 in control cells requires 10μM IP 3 in cells expressing Ha- ras. The desensitization of the IP 3 receptors coincides with the desensitization of the Ca 2+-mobilizing system to bombesin. The results indicate that the Ha- ras mediated desensitization of the Ca 2+-releasing system to bombesin is — at least in part — caused by a decrease in the affinity of the IP 3 receptor to inositol trisphosphate.

Regulatory elements mediating transcription of the human Ha- ras gene

In order to identify transcriptional regulatory elements controlling the expression of the human Ha- ras gene and to quantitatively assess the role of each element, we made mutations of the transcriptional regulatory region, including 5′ and internal deletions, linker scanning and replacement mutations, and combinations of these mutations all fused to the bacterial chloramphenicol acetyltransferase gene. The promoter activity of each of these mutants was determined by measuring the transient expression of chloramphenicol acetyltransferase activity after transfection into human epithelial HeLa cells. We found that the most important regulatory region consists of two closely linked but functionally independent elements, the non-consensus GC-II element, CGGGCGGGC, centered at position −153 from the major transcription start site cluster and a new element, CCGGAA, centered at position −161 directly upstream from GC-II. In addition, there are two functional regulatory elements which make minor contributions to the full promoter activity; a double CCAAT NF-I binding site at position −88 and an unidentified upstream element between positions −199 and −252. Aside from GC-II, the GC boxes, of which there are a total of six between positions −185 and +85, make little or no contribution to Ha- ras promoter activity when individual mutations are tested in growing HeLa cells. The three potential AP2 sites and a weak single NF-I binding site make no contribution. The basal promoter region extending to position −75 from the major start site cluster has no independent activity in this TATA-less gene.

Oestrogen-induced expression of oncogenes in the immature rat uterus

4 h after a single precocious administration of oestrogen there was a considerable but short-lived surge in the uterine levels of myc-encoded polyadenylated mRNA. This was followed by a further peak 28 h after hormone administration. The expression of ras Ha showed a totally different time course with a build up of hybridizable message that peaked 8 h after oestrogen administration.

Elevated expression of Ha-ras is an early event in two-stage skin carcinogenesis in SENCAR mice

Alterations in the expression of the Ha-ras oncogene were investigated in SENCAR mice epidermis at various stages of initiation and promotion during two-stage skin carcinogenesis in SENCAR mice. Adult SENCAR mice were treated with 200 nmol of the (+) enantiomer of benzo[a]pyrene 7,8-diol 9,10-epoxide-anti (BPDE-anti), a potent initiating agent, followed by repetitive treatments with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Other mice were 'sham'-initiated with the (-) enantiomer of BPDE-anti, which is inactive as a tumor initiator. Polyadenylated RNA was isolated from pre-tumor epidermis and tumors at eight different stages of tumorigenesis and analyzed for changes in Ha-ras expression using Northern blot hybridization. Significantly enhanced levels of Ha-ras RNA were observed in TPA-promoted papillomas as early as 7 weeks after initiation. Only trace amounts of Ha-ras RNA were present in untreated epidermis or epidermis treated with the (+) or (-) enantiomer followed by 2-12 treatments with TPA (pre-papilloma stage). Southern blot hybridization of tumor DNA indicated that the increased expression of the Ha-ras oncogene was not due to gene amplification. We conclude that elevated levels of Ha-ras expression can occur at an early stage of tumor development in mouse epidermis in vivo and may play a role in tumorigenesis.