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Query: UMLS:C0027651 (tumor)
 685,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As an alternative to directing plant or bacterial toxins to surface receptors, we are investigating the possibility of killing tumor cells by the expression of an exogenously introduced toxin gene (i.e., cell suicide). Tissue-specific gene regulatory elements might thus be exploited to achieve selective killing. To assess the feasibility of such an approach, we have transfected human cells (HeLa, B-lymphoblastoid, and 293 cells) with plasmids containing the diphtheria toxin A-chain (DT-A) coding sequence. The presence of the DT-A sequence lowered the level of transient expression of chloramphenicol acetyltransferase from a cotransfected plasmid, pSV2cat. This expression level in B-cells was further diminished by the inclusion of an immunoglobulin enhancer in the DT-A plasmid. In cotransfection experiments with a DT-A plasmid lacking an enhancer, chloramphenicol acetyltransferase expression was much more strongly inhibited in 293 cells (which express adenovirus E1A and E1B products) than in the other cell types; furthermore, the presence of the DT-A sequence eliminated recovery of G418-resistant 293 cell transformants after transfection with a plasmid containing the neo selectable marker. These results suggest that cell-specific regulatory mechanisms can be exploited to achieve selective cell killing by expression of an introduced toxin gene.
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PMID:Regulated expression of a diphtheria toxin A-chain gene transfected into human cells: possible strategy for inducing cancer cell suicide. 346 Jun 97

Phorbol ester tumor promoters affect a broad scope of changes in mammalian cells. This report describes the activation of expression of an introduced chloramphenicol acetyltransferase (CAT) reporter gene by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), in a variety of fibroblast and hematopoietic cell lines. PMA-mediated activation appears to be promoter region specific, yet widespread. Enhanced gene expression is observed for four out of five promoter systems tested, and, in some cases, is dependent on the cellular environment. Further experiments indicate that PMA mediates elevated gene expression by rapidly increasing steady state levels of CAT mRNA. The broad range of promoters affected by PMA may help explain the high potency of this agent in tumor production.
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PMID:Inducible gene expression from multiple promoters by the tumor-promoting agent, PMA. 368 79

The effects of rearrangement and insertion of sequences in the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) were investigated. The alterations were made by recombinant DNA manipulations on a plasmid subclone containing an M-MuLV LTR. Promoter activity of altered LTRs was measured by fusion to the bacterial chloramphenicol acetyltransferase gene, followed by transient expression assay in NIH 3T3 cells. M-MuLV proviral organizations containing the altered LTRs were also generated, and infectious virus was recovered by transfection. Infectivity of the resulting virus was quantified by XC plaque assay, and pathogenicity was determined by inoculating neonatal NIH Swiss mice. Inversion of sequences in the U3 region containing the tandemly repeated enhancer sequences (-150 to -353 base pairs [bp]) reduced promoter activity approximately fivefold in the transient-expression assays. Infectious virus containing the inverted sequences (Mo- M-MuLV) showed a 20-fold reduction in relative infectivity compared with wild-type M-MuLV, but the virus still induced thymus-derived lymphoblastic lymphoma or leukemia in mice, with essentially the same kinetics as for wild-type M-MuLV. We previously derived an M-MuLV which carried inserted enhancer sequences from the F101 strain of polyomavirus (Mo + PyF101 M-MuLV) and showed that this virus is nonleukemogenic. In Mo + PyF101 M-MuLV, the PyF101 sequences were inserted between the M-MuLV promoter and the M-MuLV enhancers (at -150 bp). A new LTR was generated in which the PyF101 sequences were inserted to the 5' side of the M-MuLV enhancers (at -353 bp, PyF101 + Mo M-MuLV). The PyF101 + Mo LTR exhibited promoter activity similar (40 to 50%) to that of wild-type M-MuLV, and infectious PyF101 + Mo M-MuLV had high infectivity on NIH 3T3 cells (50% of wild type). In contrast to the nonleukemogenic Mo + PyF101 M-MuLV, PyF101 + Mo M-MuLV induced leukemia with kinetics similar to that of wild-type M-MuLV. Thus, the position of the PyF101 sequences relative to the M-MuLV LTR affected the biological behavior of the molecular construct. Furthermore, PyF101 + Mo M-MuLV induced a different spectrum of neoplastic disease. In comparison with wild-type M-MuLV, which induces a characteristic thymus-derived lymphoblastic lymphoma with extremely high frequency, PyF101 + Mo M-MuLV was capable of inducing both acute myeloid leukemia or thymus-derived lymphoblastic lymphoma, or both. Tumor DNA from both the PyF101 + Mo- and Mo- M-MuLV-inoculated animals contained recombinant proviruses with LTRs that differed from the initially inoculated virus.
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PMID:Rearrangements and insertions in the Moloney murine leukemia virus long terminal repeat alter biological properties in vivo and in vitro. 374 27

We isolated a mutant strain of Agrobacterium tumefaciens, designated Ros, that has a pleiotropic phenotype which includes elevated levels of expression of certain genes in the virulence (Vir) region of tumor-inducing plasmid pTiC58. This mutant and others were isolated by fusing the promoter of the Vir bak gene to a promoterless gene (cat) that encodes chloramphenicol acetyltransferase and then selecting for increased expression of cat in A. tumefaciens. The ros mutation is chromosomal in nature and is characterized by a more-than-300-fold increase in the level of expression of bak and a 12-fold increase in the level of expression of an adjacent divergent operon containing the hdv genes, which are involved in some aspect of host specificity. The Ros mutant is fully virulent and is typified by its unusual colony morphology; the colonies have rough surfaces, uneven edges, and a pit in the center at 24 degrees C and vary markedly in appearance from one growth temperature to another. The Ros mutant is not able to form colonies at 12 degrees C, a temperature at which the wild-type strain forms colonies in 14 days. The ros mutation occurs spontaneously with a frequency of 5 X 10(-8). Genetic and biochemical evidence indicates that the product of the ros locus is a negative regulator of Ti plasmid genes and is related to undefined chromosomally encoded functions that are involved in the mutant phenotype.
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PMID:Regulation of Ti plasmid virulence genes by a chromosomal locus of Agrobacterium tumefaciens. 405 99

We have studied the temporal regulation of simian virus 40 (SV40) late gene expression by construction and transient expression analysis of plasmids containing the transposon Tn9 chloramphenicol acetyltransferase gene placed downstream from the late control region. The SV40 origin region in the early (but not the late) orientation promotes chloramphenicol acetyltransferase gene expression efficiently in monkey cells lacking large tumor (T) antigen. In monkey cells producing T antigen, the promoter activity of the late control region is induced by approximately 1,000-fold above the basal level. By deletion and point mutagenesis, we define two domains of the late control region required for efficient induction with T antigen. Domain I is the minimal replication origin containing T-antigen binding site II. Domain II consists of the 72-base-pair (bp) repeat and a 19-bp downstream sequence up to nucleotide 270. Domains I and II should act synergistically because the absence of either one or the other decreases induction efficiency by 2 orders of magnitude. Though a complete copy of domain II is optimal, the origin-proximal 22-bp portion of this domain is sufficient. The 21-bp repeat, located between domains I and II, is dispensable for this induction, as are sequences located downstream from nucleotide 270 in the late orientation.
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PMID:The simian virus 40 minimal origin and the 72-base-pair repeat are required simultaneously for efficient induction of late gene expression with large tumor antigen. 609 97

Fragment EcoRI 7 from Ti-plasmid pTi Ach5, a part of the T-DNA in octopine tumors, was cloned in both orientations into pACYC184 and expressed in E. coli minicells. The cells synthesized four proteins from four different coding regions on EcoRI 7. Two of the proteins (Mr 25.000 and 26.000) were expressed with promoters from the Ti-plasmid fragment, while transcription for the two other proteins (Mr 18.000 and 74.000) started with a promoter on pACYC184. The Mr 18.000 protein represented a fusion product between chloramphenicol acetyltransferase (CAT) on pACYC184 and a part of lysopine dehydrogenase (LpDH), the enzyme synthesizing octopine and lysopine in plant tumor cells. The results suggest that E. coli minicells are a valuable system to study the proteins coded for by the T-region of Ti-plasmids.
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PMID:Expression of plant tumor-specific proteins in minicells of Escherichia coli: a fusion protein of lysopine dehydrogenase with chloramphenicol acetyltransferase. 611 27

The simian virus (SV40) 72-base pair (bp) tandem repeated sequences have recently been shown to function as activators or enhancers of early viral transcription. A recombinant viral genome was recently constructed by inserting 72-bp tandem repeats from the Moloney murine sarcoma virus (MSV) in place of the 72-bp repeats of SV40. Although this genome replicates in monkey kidney cells, its rate of large tumor antigen expression and replication is considerably slower than that of wild-type SV40. In mouse cells, however, equivalent levels of large tumor antigen appear to be expressed from both wild-type and recombinant genomes, suggesting a relationship between the level of enhancer activity and the host cell. To confirm this observation, we have applied a sensitive quantitative assay for gene expression based on the conversion of chloramphenicol to its acetylated forms. The gene encoding the enzymatic function chloramphenicol acetyltransferase was inserted into two vectors in which the enhancer sequences from SV40 or MSV were placed adjacent to the early SV40 promoter. The SV40 tandem repeats appear to activate gene expression to significantly higher levels in monkey kidney cells, but the MSV repeats are more active in two lines of mouse cells. These findings suggest that the tandem repeat elements may interact with host-specific molecules and, furthermore, may constitute one of the elements determining the host range of these eukaryotic viruses.
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PMID:Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus. 629 1

Aberrant activation of the androgen receptor (AR) may be one of the mechanisms which contribute to progression of prostatic carcinoma to an androgen-independent stage. We investigated effects of growth factors on stimulation of the AR-mediated gene transcription in human prostatic tumor cell lines. DU-145 cells, which do not contain endogenous AR, were cotransfected with an androgen-inducible chloramphenicol acetyltransferase (CAT) reporter gene and an AR expression vector. The reporter gene (CAT) was driven either by artificial promoters consisting of one or two androgen-responsive elements in front of a TATA box or by the promoter of the prostate-specific antigen (PSA) gene, a naturally occurring androgen-inducible promoter. Insulin-like growth factor-I (IGF-I), at a concentration of 50 ng/ml, stimulated AR-mediated reporter gene transcription to the same extent as the synthetic androgen methyltrienolone. This growth factor was effective irrespective of the nature of the androgen-inducible promoter. Keratinocyte growth factor (KGF) and epidermal growth factor (EGF), at concentrations of 50 ng/ml, activated CAT reporter gene transcription only in experiments in which the artificial promoter with two androgen-responsive elements was used. Insulin-like growth factor-II and basic fibroblast growth factor displayed no effect on AR-mediated gene transcription. None of the growth factors stimulated reporter gene activity in control experiments when added to cells cotransfected with the CAT gene and an empty expression vector. AR activation by IGF-I, KGF, and EGF was completely inhibited by the pure AR antagonist casodex, showing that these effects are AR mediated. Activation of endogenous AR by growth factors was studied in the LNCaP cell line by determination of PSA secretion. IGF-I, at a concentration of 50 ng/ml, increased the PSA level in the supernatant of this cell line 5-fold. Again, the IGF-I effect on PSA secretion was blocked by casodex. Our results provide evidence that IGF-I, KGF, and EGF directly activate the AR in the absence of androgens, which means that the androgen-signaling chain may be activated by growth factors in an androgen-depleted environment. These findings may have implications for endocrine therapy for metastatic prostatic carcinoma.
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PMID:Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor. 752 59

By means of transgenic rats, we have recently shown that the GPEI enhancer of the glutathione transferase P (GST-P) gene, which has two one-base-missmatched AP-1 sites locating palindromically with three-base spacing in between, is sufficient for conferring tumor-specific activation of the gene in vivo. It is noted that there is another consensus AP-1 site near the promoter of this gene. By using seven independent transgenic rats, bearing distinct areas of the GST-P gene that are connected to the chloramphenicol acetyltransferase (CAT) coding sequence, we analyzed CAT expression in various tissues (brain, lung, liver, kidney, spleen) in these transgenic rats. We found that the ECAT gene, which has sufficient of the upstream regulatory region (approx. 2.9 kb) of the gene containing GPEI, is trans-activated in the kidney and lung of transgenic rats in a similar manner to endogenous GST-P. When either the GPEI core sequence or the AP-1 site near the promoter is deleted, CAT expression decreases to almost background level. Substitution of the GPEI core or the AP-1 site near the promoter to this silent construct (5CATGPEIcore) reconstituted CAT expression in the transgenic rats. In these rats, CAT was expressed in the brain and lung rather than in the kidney, showing a somewhat different pattern from the endogenous GST-P. In the brain tissue of the 5CATGPEIcore transgenic rat, CAT was demonstrated in the glia cells, which is consistent with endogenous GST-P expression. These results suggest that a relatively long upstream region (approx. 2.9 kb) is required for tissue-specific expression of the GST-P gene and that GST-P expression in the brain may be regulated differently from its expression in other organs.
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PMID:Tissue-specific activation of tumor marker glutathione transferase P transgenes in transgenic rats. 755 45

Testosterone biosynthesis in Leydig cells is dependent on the action of 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 (P450c17), which is encoded by the Cyp17 gene. Tumor necrosis factor-alpha (TNF alpha), a proinflammatory cytokine, inhibits cAMP-stimulated testosterone production in mouse Leydig cells. The inhibition of testosterone production is parallel to the inhibition of P450c17 messenger RNA and protein levels. To examine the mechanism of TNF alpha-mediated inhibition of steroidogenesis, the effect of TNF alpha on cAMP-stimulated induction of Cyp17 expression was investigated. To determine whether the protein kinase C (PKC) signaling pathway is involved in TNF alpha inhibition of steroidogenesis, the effects of the PKC activator, phorbol 12-myristate 13-acetate (PMA), and the PKC inhibitor, calphostin C, were examined. Treatment of normal mouse Leydig cells in primary culture with 50 microM 8-bromo-cAMP (cAMP) plus 1 ng/ml TNF alpha or 10 nM PMA caused a similar (approximately 90%) decrease in testosterone accumulation and cAMP-stimulated P450c17 messenger RNA levels compared to those after treatment with cAMP alone. To determine whether TNF alpha inhibits the cAMP-induced expression of the Cyp17 gene, plasmids containing two different size fragments of the 5'-flanking region of the Cyp17 gene upstream of the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into MA-10 tumor Leydig cells, and the effect of TNF alpha on cAMP-induced CAT activity was determined. Treatment of cells, transfected with either plasmid, with 500 microM cAMP plus increasing concentrations (0.1, 1.0, and 10 ng/ml) of TNF alpha resulted in a dose-dependent repression of cAMP-stimulated CAT activity. Higher concentrations of TNF alpha (up to 100 ng/ml) did not result in greater inhibition. Treatment of transfected cells with 10 nM PMA resulted in a 51 +/- 6.6% inhibition of cAMP-stimulated CAT activity. Calphostin C (1 microM) completely reversed the inhibitory effect of TNF alpha or PMA. Calphostin C alone had no effect on promoter activity. TNF alpha-stimulated PKC alpha translocation was quantitated by Western blot. After treatment for 3 h, the distribution of immunoreactive PKC alpha in cytosol vs. nucleus was 55%/45%, 60%/40%, and 29%/71% in control, cAMP-treated, and TNF alpha-treated cells, respectively. TNF alpha-stimulated PKC alpha translocation was further demonstrated by indirect immunofluorescence assay. PMA, a known activator of PKC, and TNF alpha had a similar inhibitory effect on P450c17 expression, testosterone production, and Cyp17-CAT activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Tumor necrosis factor-alpha inhibition of 17 alpha-hydroxylase/C17-20 lyase gene (Cyp17) expression. 762 89


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