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D W Kufe

Publications and source records attributed to D W Kufe.

At least 91 records · Page 5Linked to original sources
PubMed1993

Reactive oxygen intermediates target CC(A/T)6GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation.

The cellular response to ionizing radiation includes induction of the early growth response 1 gene (EGR1). The present work has examined the involvement of reactive oxygen intermediates (ROIs) in this response. Exposure of human HL-525 cells, an HL-60 subclone deficient in protein kinase C-mediated signaling, to both ionizing radiation and H2O2 was associated with increases in EGR-1 transcripts. These increases in EGR-1 expression were inhibited by the antioxidant N-acetyl-L-cysteine (NAC). Nuclear run-on assays demonstrate that NAC inhibits the activation of EGR1 transcription by these agents. Previous studies have shown that induction of EGR1 by x-rays is conferred by serum response or CC(A/T)6GG (CArG) elements. The present studies demonstrate similar findings with H2O2 and the finding that activation of the EGR1 promoter region containing CArG elements is abrogated by NAC. Moreover, we show that NAC inhibits the ability of a single CArG box to confer x-ray and H2O2 inducibility when linked to a heterologous promoter. Taken together, these findings indicate that ROIs induce EGR1 transcription by activation of CArG elements.

Actins↗
PubMed1993

Regulation of c-jun gene expression in human T lymphocytes.

The present studies have examined the effects of mitogens on induction of early response gene expression in normal peripheral blood T and Jurkat cells. Pokeweed mitogen (PWM) or anti-CD3 significantly increases c-jun messenger RNA (mRNA) levels in T cells. This transient PWM-related increase in c-jun transcripts is maximal after 15 to 30 minutes of exposure of T cells to PWM. PWM induces c-jun gene expression in a concentration-dependent manner. Moreover, PWM similarly induces expression of other genes coding for leucine zipper transcription factors, ie, jun-B and c-fos. Nuclear run on assays demonstrate that PWM treatment is associated with an increased rate of c-jun gene transcription. Transient expression assays with c-jun promoter fragments linked to the chloramphenicol acetyltransferase gene suggest that the PWM-induced increase in transcription is mediated by the AP-1 transcription factor complex. Moreover, treatment of T cells with actinomycin D to block further transcription before their culture with PWM suggests that the increase in c-jun gene expression by PWM is also regulated at least in part by a posttranscriptional mechanism. Cycloheximide does not alter c-jun mRNA induction by PWM. Finally, given that PWM induces B-cell differentiation in an interleukin-6 (IL-6)-mediated, T-cell-dependent manner, the relationship of c-jun and IL-6 gene expression in PWM-stimulated T cells was examined. The induction of IL-6 mRNA in T cells stimulated by PWM occurs after maximal induction of c-jun mRNA, at a time when the latter is no longer detectable. These findings suggest that PWM induces c-jun gene expression in T cells by a transcriptional and posttranscriptional mechanism and that AP-1 confers PWM inducibility of this gene. Because the IL-6 promoter has several potential transcriptional control elements, one of which is an AP-1-binding site, future experiments will evaluate the role of c-jun in the regulation of PWM-induced IL-6 synthesis by T cells.

CD3 Complex↗
PubMed1993

Radiation signaling mediated by Jun activation following dissociation from a cell type-specific repressor.

The promoter regions of several radiation-inducible genes contain AP-1 cis-acting regulatory elements that are dependent upon protein kinase C signaling. We analyzed nuclear protein from irradiated human tumor cell lines for binding to the AP-1 consensus sequence. The increase in nuclear protein binding following irradiation was specific for the AP-1 sequence and was reduced by antibodies to c-Jun and c-Fos. The AP-1 DNA binding sequence was found to regulate transcription in irradiated cells and mutation of the AP-1 site within the c-jun promoter abolished transcriptional induction by radiation. The gene encoding the chimeric transcription factor Gal4-Jun5-253, which includes the DNA binding region of Gal4 and the transcriptional regulatory region of c-Jun, was cotransfected with the reporter plasmid with Gal4 binding sequences (G5B-CAT). Transfection of RIT-3 and HeLa cells revealed that the regulatory region of Jun was sufficient to activate transcription following irradiation. Conversely, Hep G2 cells, which do not contain the cell type-specific Jun repressor, were not responsive to radiation-induced Jun activation. The c-Jun repressor was found to regulate Jun activation by experiments using the expression vector CMV-jun, which competes for Jun inhibitor and eliminates radiation-induction of Jun. We propose transcription factor dissociation from inhibitor proteins may participate in the initiation of cellular responses to ionizing radiation.

Chloramphenicol O-Acetyltransferase↗
PubMed1993

Recombinant human gamma interferon administered by continuous intravenous infusion in acute myelogenous leukemia and myelodysplastic syndromes.

Nine patients (median age, 58; range: 37-74) with relapsed de novo acute myeloid leukemia (AML) (3), AML after prior myelodysplastic syndrome (MDS) (4), or MDS (2) were treated with 2-20 x 10(6) U/m2/day (1-10 mg/m2/day) of recombinant human interferon gamma (rIFN gamma; Biogen) on a 14-day continuous intravenous infusion schedule. The two patients who received the initial dose of 20 x 10(6) U/m2/day (1.0 mg/m2/day) could only tolerate 6 days of therapy because of severe hepatotoxicity. Two patients who received the revised starting dose of 10 x 10(6) U/m2/day also could not complete a full course of rIFN gamma due to renal failure in one case and pulmonary deterioration in the other. A reversible dose-related rise in SGOT was seen in six patients. All patients developed a severe flu-like syndrome characterized by myalgias and fevers. These toxicities were not associated with detectable serum levels of tumor necrosis factor (TNF). Although blasts from three of five assessable patients displayed increased expression of the Ia (HLA-DR) antigen, there were no hematological responses. Steady-state rIFN gamma plasma levels in patients who tolerated a complete infusion were < 40 U/ml, a concentration below that required to induce differentiation of myeloid leukemic cell lines in vitro. We conclude that continuous infusions of rIFN gamma at doses as low as 2 x 10(6) U/m2/day are poorly tolerated in patients with AML and MDS; the maximum tolerated dose is approximately 2 x 10(6) U/m2/day.

Adult↗
PubMed1993

Effect of Bcl-2 on ionizing radiation and 1-beta-D-arabinofuranosylcytosine-induced internucleosomal DNA fragmentation and cell survival in human myeloid leukemia cells.

1-beta-D-Arabinofuranosylcytosine (ara-C) is an anti-leukemic agent that incorporates into cellular DNA leading to inhibition of DNA synthesis and loss of clonogenic survival. In contrast, ionizing radiation induces DNA damage through the generation of reactive oxygen intermediates. Although little is known of the specific determinants of ara-C and ionizing radiation-induced cytotoxicity, recent work has shown that both are capable of inducing internucleosomal DNA fragmentation in a pattern consistent with programmed cell death (apoptosis). In order to assess the importance of apoptosis in drug and ionizing radiation-induced cytotoxicity in the U-937 myelomonocytic cell line, we created cell lines that constitutively express a transfected bcl-2 gene. Bcl-2 was capable of inhibiting 40-50% of the ara-C and ionizing radiation-induced internucleosomal DNA fragmentation at all tested concentrations. However, cell survival following exposure to these agents was only increased in the bcl-2 transfectants at relatively low doses of ara-C and ionizing radiation. These data demonstrate that although bcl-2 is capable of inhibiting ara-C and ionizing radiation-induced DNA fragmentation in myeloid cells, it increases cell survival only at low doses of these agents. This suggests that apoptosis may be a less important mechanism of cytotoxicity at higher doses of ara-C and ionizing radiation than it is at lower doses.

Cell Survival↗
PubMed1993

Ionizing radiation down-regulates histone H1 gene expression by transcriptional and post-transcriptional mechanisms.

The cellular response to ionizing radiation includes growth arrest and DNA repair. However, little is known about the regulation of gene expression by this agent. The present studies demonstrate that exposure to ionizing radiation is associated with a dose-dependent decrease in histone H1 gene expression. Following treatment with 20 Gy, this effect was transient, detectable at 15-30 min, and maximal at 6 h. Nuclear run-on assays demonstrate that this down-regulation is controlled at least in part by transcriptional mechanisms. We also demonstrate that inhibition of protein synthesis with cycloheximide abrogates the down-regulation of both histone H1 gene transcription and mRNA levels in irradiated cells. The results demonstrate that treatment with ionizing radiation is associated with a decrease in the stability of the histone H1 transcript, and that this effect is reversed by inhibition of protein synthesis. These findings demonstrate that ionizing radiation activates at least two distinct signaling pathways that control histone H1 expression at the transcriptional and post-transcriptional levels.

Dose-Response Relationship, Radiation↗
PubMed1993

The role of cytokines in radiation oncology.

Cytokine induction following ionizing radiation exposure occurs through transcriptional activation in specific proteins studied to date. This process can be blocked by inhibition of radiation-induced signaling pathways, which may potentially modify certain deleterious effects of radiotherapy, e.g., effects related to production of TNF or TGF-beta. bFGF is another radiation-inducible protein that may participate in repair and protection of irradiated endothelial cells following a complex interaction with cellular receptors. The cytokines such as IL-6, TNF, and IL-1 may be useful to protect hematopoietic cells from radiation, while TNF may enhance the killing of tumor cells. Our understanding of cytokine/radiation interactions is only beginning to be understood, but knowledge of the biology of these interactions may prove clinically useful.

Animals↗
PubMed1992

Ionizing radiation activates transcription of the EGR1 gene via CArG elements.

The present studies have examined the effects of ionizing radiation on control of the early growth response 1 (EGR1) gene. Exposure of human HL-525 cells to x-rays was associated with increases in EGR1 mRNA levels. Nuclear run-on assays showed that this effect is related at least in part to activation of EGR1 gene transcription. Sequences responsive to ionizing radiation-induced signals were determined by deletion analysis of the EGR1 promoter. The results demonstrate that x-ray inducibility of the EGR1 gene is conferred by a region containing six serum response or CC(A+T-rich)6GG (CArG) motifs. Further analysis confirmed that the region encompassing the three distal or upstream CArG elements is functional in the x-ray response. Moreover, this region conferred x-ray inducibility to a minimal thymidine kinase gene promoter. Taken together, these results indicate that ionizing radiation induces EGR1 transcription through CArG elements.

Base Sequence↗
PubMed1992

Involvement of reactive oxygen intermediates in the induction of c-jun gene transcription by ionizing radiation.

Previous work has demonstrated that the cellular response to ionizing radiation includes transcriptional activation of the c-jun gene. The signaling events responsible for this response, however, remain unclear. The present studies have examined the effects of ionizing radiation on c-jun expression in a variant of HL-60 cells, designated HL-525, which is deficient in protein kinase C (PKC)-mediated signal transduction. The results demonstrate that these cells express low levels of PKC alpha and PKC beta transcripts and exhibit an attenuated induction of c-jun expression following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, HL-525 cells respond to ionizing radiation with an increase in c-jun mRNA which is more pronounced than that in wild-type HL-60 cells. These cells similarly respond to ionizing radiation with increased expression of the jun-B, jun-D, c-fos, and fos-B genes. Nuclear run-on assays demonstrate that X-ray-induced c-jun expression in HL-525 cells is regulated by increases in the rate of c-jun gene transcription. Moreover, mRNA stability studies in irradiated HL-525 cells demonstrate that the half-life of c-jun transcripts is prolonged compared to that in wild-type cells. Studies with N-acetyl-L-cysteine (NAC), an antioxidant, suggest that X-ray-induced transcriptional activation of the c-jun gene is mediated at least in part through the formation of reactive oxygen intermediates (ROIs). In this context, H2O2 also induced c-jun expression in HL-525 cells, and this effect was inhibited by NAC.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Nucleus↗
PubMed1992

Transcriptional regulation of cytokine expression by diethyldithiocarbamate in human HL-60 promyelocytic leukemia cells.

Diethyldithiocarbamate (DDTC) is an investigational agent used to ameliorate chemotherapy- or radiotherapy-induced myelosuppression. We studied the effects of DDTC on the regulation of hematopoietic cytokine production in human myeloid cells. The results demonstrated that DDTC decreases proliferation of human HL-60 promyelocytic leukemia cells in a concentration-dependent manner. DDTC treatment also increased interleukin-alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor (TNF) expression in these cells. Similar findings were obtained in normal human peripheral blood monocytes. Peak induction of these cytokines occurred 6-12 hr after exposure to DDTC; levels returned to those in control cells by 24-48 hr in HL-60 cells. This effect was specific for IL-1 and TNF in that there was no detectable increase in IL-3, macrophage colony-stimulating factor or granulocyte/macrophage colony-stimulating factor RNA expression. Transcriptional run-on analysis demonstrated that exposure to DDTC increased the rate of TNF gene transcription in HL-60 cells. These data suggest that the myeloprotective effects of DDTC may be mediated, at least in part, by the induction of TNF, IL-1 alpha, and IL-1 beta.

Cytokines↗
PubMed1992

Tumor selective reactivity of a monoclonal antibody prepared against a recombinant peptide derived from the DF3 human breast carcinoma-associated antigen.

The DF3 antigen is a member of a family of high molecular weight glycoproteins aberrantly expressed in malignant mammary epithelium. We have generated a monoclonal antibody (MAb), designated DF3-P, against a recombinant DF3/beta-galactosidase fusion protein. Characterization of this MAb has demonstrated reactivity with immature precursors of DF3 antigen and not with the secreted form. These findings are in contrast to those obtained with MAb DF3, a previously described antibody with predominant reactivity against the mature glycoprotein. The finding that deglycosylation of secreted DF3 antigen with neuraminidase and endo-alpha-N-acetylgalactosaminidase is associated with increased MAb DF3-P reactivity provided additional support for the selectivity of this antibody against the protein core. Epitope mapping studies demonstrate that both the DF3-P and DF3 epitopes are located at a TRPAPGS domain in the 20-amino acid tandem repeat. The results of competition studies with synthetic peptides indicate that the proline in this domain is involved in both epitopes, while the potential glycosylation sites at threonine and serine may contribute to the differential reactivity of MAbs DF3 and DF3-P. Taken together, these findings suggest that both antibodies react with a similar epitope that is modified by the presence of carbohydrate moieties. The results of immunoperoxidase staining studies further demonstrate that while MAb DF3-P reacts with formalin-fixed sections of breast carcinomas, this antibody exhibits little if any reactivity with normal mammary epithelium. Selective expression of the DF3-P epitope in malignant breast cells may be useful in identifying this transformed phenotype.

Animals↗
PubMed1992

Functional expression of the macrophage colony-stimulating factor receptor in human THP-1 monocytic leukemia cells.

Macrophage colony-stimulating factor (M-CSF) is required for the proliferation, differentiation, and activation of monocytes. High-affinity receptors for M-CSF are encoded by the c-fms proto-oncogene. In the present study, we show that c-fms transcripts are detectable in human THP-1 myeloid leukemia cells. Furthermore, radiolabeled 125I-M-CSF is rapidly internalized into THP-1 cells and then degraded intracellularly. The results also show that treatment of THP-1 cells with M-CSF is associated with the activation of protein kinase C (PKC) and the induction of tumor necrosis factor (TNF) gene expression. TNF transcript levels were low to undetectable in uninduced THP-1 cells, reached maximal levels by 1 hour of exposure to M-CSF, and returned to those of control cells by 24 hours. Transcriptional run-on analysis showed that a low level of TNF transcription is detectable in untreated THP-1 cells, and M-CSF treatment increased the rate of TNF transcription. Pretreatment of THP-1 cells with pertussis toxin inhibited the increase in PKC activity but not the induction of TNF transcripts by M-CSF. Moreover, exposure of THP-1 cells to inhibitors of protein kinase activity blocked the increase in TNF messenger RNA. These findings suggest that at least two M-CSF-mediated signaling pathways exist in THP-1 cells and that the induction of TNF may be regulated by a protein kinase-dependent mechanism distinct from PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Activation of the AP-1 transcription factor by arabinofuranosylcytosine in myeloid leukemia cells.

Previous studies have shown that 1-beta-D-arabinofuranosylcytosine (ara-C) induces transcription of the c-jun immediate early response gene in human myeloid leukemia cells. The present work has examined the mechanisms responsible for this effect. Deleted forms of the c-jun promoter were linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into KG-1 cells. The results demonstrate that ara-C-induced c-jun transcription is mediated by an element between positions -74 and -20 upstream to the start site. Electrophoretic mobility shift assays with the fragment f(-74/-20) showed an increase in binding with nuclear proteins from ara-C-treated cells as compared with untreated cells. Competition with an oligonucleotide containing the AP-1 consensus sequence indicated that ara-C stimulates binding of nuclear proteins at the AP-1 site in the c-jun promoter. These findings were confirmed in other gel shift studies with the collagenase enhancer AP-1 consensus sequence and with a DNA fragment containing an altered AP-1 site. The binding of JUN/AP-1 was maximal at 1 hour of ara-C treatment and decreased to baseline levels at 12 hours. The finding that ara-C induces AP-1 binding in the absence of protein synthesis indicated that this agent activates already synthesized JUN/AP-1. To confirm these findings, the AP-1 consensus sequence was introduced 5' to the heterologous SV40 promoter. The results show that AP-1 enhances SV40 promoter activity in ara-C-treated cells. Taken together, these findings indicate that: (1) enhancement of JUN/AP-1 activity in ara-C-treated cells involves a posttranslational modification of JUN/AP-1; and (2) binding of activated JUN/AP-1 to the AP-1 site in the c-jun promoter confers ara-C inducibility of this gene.

Base Sequence↗
PubMed1992

Identification of NF-jun, a novel inducible transcription factor that regulates c-jun gene transcription.

In this study we report the identification of a novel transcription factor, termed Nuclear Factor-jun (NF-jun). This factor contributes to inducible transcription of the c-jun gene in human myeloid leukemia cells. NF-jun was, however, undetectable in nuclear proteins from human monocytes, granulocytes, resting T lymphocytes and lung fibroblasts. NF-jun shares several features with the well characterized NF-kappa B in that binding activity can be generated in cytosolic extracts by treatment with dissociating agents. In addition, binding of NF-jun to its recognition site is enhanced by treatment of cells with 12-O-tetradecanoylphorbol-13-acetate, tumor necrosis factor alpha or the protein synthesis inhibitor cycloheximide (CHX). However, as revealed by competition assays and electrophoretic mobility shift assays, purified NF-kappa B fails to bind to the c-jun fragment which contains the NF-jun site, and this fragment fails to compete with NF-kappa B for binding. UV crosslinking showed that NF-jun contains a 55 and a 125 kDa protein species. These findings demonstrate that the c-jun gene can be regulated by a transcription factor distinct from AP-1. Our findings also indicate that while NF-jun has several features in common with the NF-kappa B binding protein including its subcellular localization and its ability to translocate from the cytoplasm to the nucleus, this factor recognizes a unique DNA sequence. Moreover, the activity of this protein is differentially regulated in various cell types. NF-jun might function as a signal transducing molecule in order to mediate rapid induction of the early response gene c-jun in a cell type- and stimulus-specific manner.

Base Sequence↗
PubMed1992

Gene therapy targeted by ionizing radiation.

Gene therapy used for the treatment of neoplastic diseases is not well localized. Ionizing radiation can be used to activate the transcription of exogenous genes that encode cytotoxic proteins. This may be accomplished through the use of radiation responsive elements distal to the transcription start site of such genes. We discuss this concept and describe a technique which can be used to amplify the signal initiated by localized radiation therapy. These techniques may be used to target gene therapy during the treatment of human neoplasms.

Genetic Therapy↗
PubMed1992

Immunocytochemical determination of the estrogen-regulated proteins Mr 24,000, Mr 52,000 and DF3 breast cancer associated antigen: clinical value in advanced breast cancer and correlation with estrogen receptor.

The Mr 24,000 and Mr 52,000 estrogen-regulated cytosol proteins, and the breast cancer-associated antigen DF3 have been studied in an immunocytochemical assay. Primary tumor specimens from 119 patients with advanced breast cancer who received endocrine therapy have been studied. Monoclonal antibodies were used for the detection of the proteins in formalin-fixed paraffin-embedded blocks. No correlation between Mr 52,000-positive specimens and the presence of estrogen receptor (ER) could be established (p = 0.87, chi-square test) whereas a statistically significant association between Mr 24,000 (p = 0.0002), DF3 antigen (p = 0.044) and ER was demonstrated. No intercorrelation was found between Mr 24,000 and Mr 52,000 or DF3 (p = 0.63, 0.98 and 0.12 respectively). Clinical response was evaluated for immunocytochemical findings, Mr 24,000 (p = 0.37), Mr 52,000 (p = 0.61) and DF3 (p = 0.68) showed no association whereas ER was statistically correlated (p = 0.00005). Neither overall survival nor disease-free survival correlated to Mr 24,000 (p = 0.18 and 0.75 respectively, logrank test), Mr 52,000 (p = 0.095 and 0.38), or DF3 (p = 0.22 and 0.13) staining, whereas ER-positive tumors did (p = 0.00005). Discrimination between ER-positive responders and ER-positive non-responders was not possible using either Mr 52,000, Mr 24,000 or DF3 staining. Based on our findings we conclude that immunocytochemical staining for Mr 52,000, Mr 24,000 or DF3 cannot be used as a marker to predict response to endocrine therapy in patients with advanced or recurrent breast cancer.

Adult↗
PubMed1992

Molecular targets to overcome radioresistance.

Our laboratory has cultured cells from 23 patients with head and neck carcinoma and 13 patients with soft tissue sarcoma. The data suggest that, in some patients, inherent tumor cell radioresistance contributes to radiotherapy failure. This paper examines possible mechanisms that lead to increased cell survival following radiation exposure, and their potential as targets to overcome the radioresistant patient population.

Antineoplastic Agents↗