Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

P53 status may be a determinant of chemosensitivity of tumor cells; however, its involvement in cellular resistance to cisplatin remains uncertain. To investigate the relationships between p53 and the development of resistance to cisplatin, the p53 gene status was studied in ovarian carcinoma cell systems which included two cisplatin-resistant variants (IGROV-1/Pt 0.5 and IGROV-1/Pt 1) selected in vitro after prolonged drug exposure of the cisplatin-sensitive parental IGROV-1 cell line. IGROV-1/Pt 0.5 and IGROV-1/Pt 1 cell lines exhibited a degree of resistance of approximately 6 and 14, respectively, following 96-h exposure to the drug and were cross-resistant to other DNA-damaging agents (ionizing radiation and melphalan). Resistance to cisplatin paralleled a reduced cell susceptibility to cisplatin-induced apoptosis. DNA single-strand conformation polymorphism analysis of exons 5-9 demonstrated the presence of two mutants alleles at exon 8 in the two resistant cell lines, in contrast to the parental IGROV-1 cell line which exhibited the wild-type p53 gene. Direct DNA sequencing revealed that the mutations consist of two nucleotide changes in the DNA-binding domain at codons 270 (T/A) and 282 (C/T). The consecutive levels of p53 protein were lower in IGROV-1 than in IGROV-1/Pt cells. Following exposure to ionizing radiation or cisplatin, accumulation of the p53 protein was markedly enhanced only in the sensitive cells. Concomitantly, the expression of WAF-1 protein was strongly induced in the parental IGROV-1 cells, whereas WAF-1 protein remained undetectable in the IGROV-1/Pt 1 subline after DNA-damaging treatment. Consistent with this finding is the observation that ionizing radiation caused a different pattern of cell cycle perturbation in sensitive and resistant cells. Northern blot analysis demonstrated a marked reduction in bax mRNA levels in IGROV-1/Pt 1 cisplatin-resistant cells. Cotransfection assays with wild-type or mutant p53 expression plasmids and a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase were consistent with the role of p53 in regulation of bax expression in these cells. Taken together, these observations support a role for mutations of the p53 gene in the development of cisplatin resistance in ovarian cancer as a consequence of loss of the ability of p53 to transactivate bax, an apoptosis-inducing gene.
Cancer Res 1996 Feb 01
PMID:Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems. 856 71

To investigate multidrug-resistance gene (MDR1) promoter efficacy and drug inducibility in cells with different multidrug-resistance phenotypes, multidrug-resistant HCT15 and drug-sensitive KM12 human colon carcinoma cell lines were transfected with constructs incorporating the chloramphenicol acetyltransferase (CAT) reporter gene, driven by wild-type and point-mutated MDR1 promoter regions. The basal CAT expression level in HCT15 cells was markedly elevated compared to KM12 cells. CAT induction by vincristine was dose-dependent over a broad concentration range (40-500 ng/ml) in both lines. The induction levels were related to the cells' MDR phenotype, with the multidrug-resistant HCT15 cells showing the greater effect. In both cell types, basal and drug-induced CAT expression were significantly enhanced by the point-mutated promoter regions. The findings support the possible exploitation of the MDR1 promoter for construction of drug-inducible and MDR-cell-targeted expression vectors for use in gene therapy.
J Cancer Res Clin Oncol 1996
PMID:Vincristine induction of mutant and wild-type human multidrug-resistance promoters is cell-type-specific and dose-dependent. 860 50

The ornithine decarboxylase enzyme (ODC) is the key regulator of polyamine synthesis and is a member of the cellular proto-oncogene family. Its expression becomes constitutively activated by carcinogens, viruses, and oncogenes. ODC mRNA has a long 5' untranslated region that could be important in the regulation of enzyme levels by affecting translation. To test this hypothesis, we have determined the role of this region on the constitutive ODC hyperexpression measured in AR4-2J cells, an azaserine-induced, tumor-derived pancreatic acinar cell line. Construction of expression vectors in which ODC 5' leader sequence was placed flanking the chloramphenicol acetyltransferase reporter gene allowed us to identify three AR4-2J specific, different alternatively spliced ODC 5' leaders. The 5' ends of exons 2 and 3 were lengthened by 17 and 13 bases, respectively. Translation performed in a cell-free system as well as in COS7 transient transfection experiments demonstrated that AR4-2J isoforms induce a strong increase in the rate of translation. These results provide evidence that alternative splicing observed in tumoral cells, coupled with translation regulation, relieves the translation repression mediated by the long and structured 5' untranslated region of the ODC proto-oncogene.
Cancer Res 1996 Apr 15
PMID:Relief of ornithine decarboxylase messenger RNA translational repression induced by alternative splicing of its 5' untranslated region. 862 Apr 86

Tumor growth is dependent on new blood vessel formation. Inhibition of vascular endothelial growth factor (VEGF), an endothelial cell mitogen and angiogenic factor secreted by a variety of tumors and tumor cell lines, is sufficient to inhibit primary tumor growth. In the present study, we examined the effect of inhibiting VEGF on tumor cell micrometastasis. A transfectant of A431 (a human epidermoid carcinoma cell line) expressing chloramphenicol acetyltransferase (CAT) was injected s.c. into severe combined immunodeficiency (scid) mice, which were then sacrificed after 6 weeks. The presence of A431 metastases at distant sites was demonstrated by detection of CAT activity in whole-organ lysates. Treatment of animals with VEGF-neutralizing antibodies not only inhibited primary tumor growth but also suppressed metastases, as determined by CAT activity in organ lysates. In experiments to determine the mechanism by which anti-VEGF antibody inhibited metastasis, control animals were sacrificed when their tumors had reached the same size as tumors in VEGF antibody-treated animals. Metastases were uniformly present in these control animals. These findings show that inhibition of VEGF alone is sufficient to prevent tumor growth and dissemination in vivo. The inhibitory effect on metastases appears to be distinct from that on primary tumor growth.
Cancer Res 1996 Feb 15
PMID:Vascular endothelial growth factor promotes tumor dissemination by a mechanism distinct from its effect on primary tumor growth. 863 Oct 34

Humoral hypercalcemia of malignancy (HHM) is caused by the secretion of parathyroid hormone-related protein (PTHrP) by tumor cells, and tumors of squamous histology are the ones most commonly complicated by HHM. To determine why some squamous tumors cause HHM and others do not, we quantitated the levels of PTHrP mRNA expression and PTHrP secretion in a series of eight squamous tumor lines. As anticipated, we found that the level of PTHrP mRNA expression in individual lines correlated with their PTHrP secretion rates. However, PTHrP mRNA levels varied widely in individual lines, and only those tumor lines with the highest levels of PTHrP gene expression were able to cause hypercalcemia in athymic mice. We found that a specific segment of the PTHrP promoter could reproduce the relative pattern of PTHrP gene expression when cloned in front of a chloramphenicol acetyltransferase reporter gene and transiently transfected into these squamous lines. Deletional analysis confirmed that specific sequences within the PTHrP gene promoter appeared to be involved in the transactivation of the gene in tumor lines expressing high levels of PTHrP mRNA. These data suggest that the ability of a given squamous tumor to cause HHM is ultimately a function of its level of PTHrP gene expression, which in turn appears to be a function of the ability of specific transcription factors to transactivate PTHrP gene expression.
Cancer Res 1996 Mar 01
PMID:Transactivation of the PTHrP gene in squamous carcinomas predicts the occurrence of hypercalcemia in athymic mice. 864 Jul 59

Cancer-related mutations of the p53 tumor suppressor gene are clustered in the four so-called 'hot spots', codons 175, 248, 273 and 281/282. By using recombination PCR in vitro mutagenesis, we introduced point mutations into the codon 273 of wild-type (wt) p53 (pC53-SN3) from Arg to His (pC53-273H [273H]), Asp (273D), Pro (273P), Lys (273K), Leu (273L) or Thr (273T), and compared their biological and biochemical activities with wt p53 and cancer-derived 175H, 248W and 273H/309S. Among them, 273H/309S, 273H and 273D as well as wt p53 transactivated the chloramphenicol acetyltransferase (CAT) gene placed downstream of the p53 binding consensus, while none of the other mutants including 273L did. Transcriptions from human c-fos and rat PCNA promoters were suppressed by wt p53 and 273D, while they were enhanced variously by all other mutants in Saos-2 and/or NIH3T3 cells. On the other hand, growth of human squamous carcinoma cell lines measured by the plating efficiency of G418-resistant colonies was enhanced by transfection of 175H, 248W, 273H/309S and 273P, while suppressed by not only wt p53, 273D and 273H but also 273L. Thus, 273H/309S enhanced cell growth in spite of its p53-specific transactivation activity, while 273L suppressed cell growth in spite of its complete loss of the p53-specific transactivation. We concluded that the sequence-specific transactivation of p53 is not always correlated with its growth inhibitory activity.
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PMID:The 273rd codon mutants of p53 show growth modulation activities not correlated with p53-specific transactivation activity. 864 76

In this article we describe an improved method to produce a conjugate of anti-erythrocyte growth factor (EGF) receptor monoclonal antibody with polylysine via thio-ether bonds. The resulting antibody/polylysine conjugate was found to be a much more stable DNA (gene) carrier than the previous conjugate formed via disulfide bonds. We designated the conjugate as an "immunoporter" and the immunoporter/DNA (gene) complex as an "immunogene." The fluorescent microscopic observation showed that the immunoporter as well as immunogene bound specifically to the EGF receptors on the cell surface, and the loaded reporter gene, such as beta-galactosidase (beta-GAL), was detected in the cell nucleus at 2 hours after transfection. The enzyme activity from the beta-GAL gene was detected at 12 hours and increased for 3 to 5 days. Similar kinetics were obtained for another reporter gene, chloramphenicol acetyltransferase. Furthermore, the immunoporter delivered the herpes simplex virus thymidine kinase gene and induced substantial suicide effects on tumor cells when gancyclovir or acyclovir was added. Thus, the immunogene approach was successful in delivering therapeutic genes to EGF receptor overexpressing tumor cells. Further technical refinement may prove useful as a supplementary treatment of patients with squamous cell carcinomas.
Cancer Gene Ther
PMID:Immunogene approach toward cancer therapy using erythrocyte growth factor receptor-mediated gene delivery. 872 10

Long-term expression of a reporter gene has previously been reported in skeletal and cardiac muscles after direct injection of naked plasmid DNA. In this study, we have shown that the direct injection of free plasmid DNA into mouse melanoma BL6 solid tumor can also result in a high level of transfection. THe average amount of chloramphenicol acetyltransferase (CAT) expressed by injecting 30 micrograms plasmid DNA containing a CAT gene into a single BL6 tumor was 1.9 +/- 1.0 ng, which is comparable to that reported in the skeletal muscle. Cationic liposomes, Lipofectamine and DC-chol/DOPE, inhibited gene expression in a dose-dependent manner. Transgene expression by free DNA persisted for at least 10 days. The size of tumor did not seem to affect the gene expression, but proper choice of a diluent solution for DNA was an important factor. Genes driven by the CMV promoter were expressed much more efficiently than genes driven by the SV40 or T7 promoter. Optimal dosage of injected DNA was from 30 to 70 micrograms per tumor. Other mouse melanomas, human melanomas and cervical carcinomas are also able to express directly injected plasmid DNA, but the transfection efficiency is lower than the BL6 tumor. Direct injection of free plasmid DNA is a simple and effective approach and might be a potential method for cancer gene therapy.
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PMID:Direct gene transfer to mouse melanoma by intratumor injection of free DNA. 878 4

Gelatinase B is a regulated matrix metalloproteinase with important role in the remodeling of extracellular matrix and many pathological conditions such as tumor invasion and rheumatoid arthritis, physiological processes including embryonic growth and development, migration of blood leukocytes into tissues and tissue remodeling. Elevated levels of certain MMPs are believed to be associated with various pathological states. We cloned the 5'-flanking 600 bp sequence of human gelatinase B gene by PCR, which controls the expression of the gene by ligating it to the chloramphenicol acetyltransferase gene. Four kinds of cell lines were used to transiently transfect. Deletion analysis revealed that 100 bp (-600 to -500 bp) contributed positively to induction by tumour necrosis factor. The 100 bp contains NF-kappa B site, Ap-1 site, PEA3 and Sp-1 site. The expression of the human gelatinase B gene varied in different cells in the presence of TNF.NF-kappa B factor may play an important role in regulating the gene expression. Comparison of the finding with those for the promoter of gelatinase A, collagenase and stromelysin shows that the determinant for the inducibility of the gelatinase B gene is more complex.
Cancer Lett 1996 Sep 10
PMID:Molecular mechanism of transcriptional activation of human gelatinase B by proximal promoter. 884 71

The ability of glucocorticoids (GCs) to induce death in lymphoid-origin cells is the basis for their frequent use in the therapy of various human hematological malignancies. However, the occurrence of primary or secondary GC resistance limits their clinical usefulness. Prior investigations into the mechanism of GC resistance in established human leukemic cell lines revealed loss-of-function mutations in the GC receptor (GR) gene. In this study, we analyzed the GC-resistant human acute T-cell leukemia line CEM-C1, which has been reported to express biochemically functional GR and, thus, was thought to owe its GC resistance to signal transduction changes distal from the GR. Radioligand binding assays revealed a 2-3-fold lower expression of GR in CEM-C1 than in the GC-sensitive sister cell line CEM-C7H2. Analysis of transcriptional activity using mouse mammary tumor virus-long terminal repeat-controlled chloramphenicol acetyltransferase expression in transient transfection assays confirmed the expression of functional GR in CEM-C1 but at levels lower than those in CEM-C7H2 cells. Upon molecular analyses of the GR gene and its transcripts, we found that CEM-C1 cells were heterozygous for the ligand binding domain L753F point mutation in exon 9, which is also present in GC-sensitive CEM-C7H2. No mutations, however, were found on the second GR allele of CEM-C1. To test the possibility that resistance in CEM-C1 cells might be caused by insufficient expression of GR, we established several cell lines stably transfected with rat GR expression vectors. These cell lines differed in exogenous GR expression as determined by Northern blotting and radioligand binding assays. The GR expression level in individual lines correlated well with their sensitivity to GC-induced apoptosis. Thus, GC resistance of CEM-C1 cells might be due to subthreshold expression of functional GR rather than defects in signal transduction pathways distal from the GR. Since several clinical investigations showed a correlation between reduced GR expression and poor response to GC-containing treatment, the CEM-C1 line may represent a valid model for GC resistance in human acute T-cell leukemia.
Cancer Res 1996 Nov 01
PMID:Resistance to glucocorticoid-induced apoptosis in human T-cell acute lymphoblastic leukemia CEM-C1 cells is due to insufficient glucocorticoid receptor expression. 889 60


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