Biomedical subjects

D W Kufe

Publications and source records attributed to D W Kufe.

At least 73 records · Page 4Linked to original sources

PubMed1995

Activation of serine/threonine protein kinases and early growth response 1 gene expression by tumor necrosis factor in human myeloid leukemia cells.

The early growth response 1 (EGR-1) gene is induced by mitogenic and differentiating signals in diverse cell types. The present studies have examined the effects of TNF-alpha on the induction of EGR-1 expression in human myeloid leukemia cells and the potential cytoplasmic signaling cascades that transduce TNF-induced signals to the nucleus. The results demonstrate that treatment of HL-60 cells with TNF is associated with the transient induction of the EGR-1 gene. The results also demonstrate that TNF treatment is associated with activation of the serine/threonine kinase, pp90rsk, which acts upstream to EGR-1 gene induction. Partial purification of pp90rsk by affinity chromatography demonstrated an increase in S6 peptide phosphorylation in response to TNF treatment. Because TNF activates sphingomyelin hydrolysis, we also studied the effects of sphingomyelinase (SMase) on induction of EGR-1 and pp90rsk. The results demonstrate that SMase also activates pp90rsk and induces EGR-1 gene expression. Previous work has demonstrated that mitogen-activated protein (MAP) kinase activates pp90rsk. The present studies further show that treatment with TNF or SMase is associated with induction of both the pp42/44 MAP and the related Jun kinases. Induction of pp42/44 MAP kinase activity is temporally related to activation of pp90rsk and the EGR-1 gene. These findings support the involvement of an MAP kinase/pp90rsk/EGR-1 cascade in the response of myeloid leukemia cells to TNF.

Amino Acid Sequence↗

PubMed1995

Suppression of Bcl-2 messenger RNA production may mediate apoptosis after ionizing radiation, tumor necrosis factor alpha, and ceramide.

Recent studies have proposed that tumor necrosis factor alpha (TNF-alpha) and ionizing radiation induce apoptosis by activating hydrolysis of sphingomyelin to ceramide. Bcl-2 and a related gene, Bcl-X, inhibit several forms of apoptosis. Herein, we report that internucleosomal DNA fragmentation, characteristic of apoptosis and induced by ionizing radiation, is accompanied by concomitant decreases in Bcl-2 and Bcl-X mRNA levels in HL-60 and U-937 human leukemia cells. Apoptotic DNA fragmentation after exposure to TNF-alpha and C2-ceramide was also associated with down-regulation of Bcl-2 mRNA in HL-60 and U-937 cells, while Bcl-X mRNA production was unaffected. These results suggest that modulation of Bcl-2 gene expression may be a target for ceramide-mediated apoptosis following exposure to ionizing radiation and TNF-alpha. Changes in Bcl-2 expression may be the basis for the interactive killing observed between radiation and TNF-alpha in some human and tumor cells.

Apoptosis↗

PubMed1995

Monocyte chemoattractant protein-1 (MCP-1) gene transduction: an effective tumor vaccine strategy for non-intracranial tumors.

Recently, there has been renewed interest in the concept of tumor vaccines using genetically engineered tumor cells expressing a variety of cytokines to increase their immunogenicity. Human MCP-1 (JE) is a potent chemoattractant and activator of monocytes and T lymphocytes and thus a good candidate gene for a tumor vaccine. We therefore evaluated the efficacy of vaccines consisting of irradiated tumor cells transduced with the murine MCP-1 gene in the syngeneic 9L gliosarcoma brain tumor model. 9L cell lines stably expressing murine MCP-1 (9L-JE) and control cell lines expressing neomycin 3' phosphotransferase (9L-Neo) were generated by infection with a Moloney murine leukemia retroviral vector. Fisher 344 rats were immunized with intradermal injections of 5 x 10(5) or 2 x 10(6) irradiated (5000 cGy) 9L-JE, 9L-Neo, and wild-type 9L (9L-WT) cells. Two weeks later immunized and non-immunized animals were challenged with various doses of intradermal (5 x 10(6)-5 x 10(7) or intracerebral (2 x 10(4)-5 x 10(5) 9L-WT cells. Intradermal tumors grew in all non-immunized animals. No tumors grew in animals immunized with irradiated 9L-JE or 9L-Neo cells and challenged with inocula of fewer than 5 x 10(5) 9L-WT cells. With higher inocula up to 10(7) cells, tumors appeared in all the animals, but subsequently regressed in the immunized animals. Tumors in animals immunized with 9L-JE were always smaller than tumors in the other groups. In addition, only the 9L-JE vaccine protected against tumor inocula of 5 x 10(7) cells. Thus vaccination with MCP-1-expressing cells was able to protect animals against at least a 100-fold larger number of challenge tumor cells than vaccination with control cells. In contrast to studies with intradermal tumors, immunization with 9L-JE and 9L-Neo produced only minimal protection against intracerebral tumors. There was no significant difference between the 9L-JE and 9L-Neo vaccines in intracerebral challenge. This study suggests that tumor vaccines expressing cytokine genes such as MCP-1 can increase the antitumor response. However, the protective effect of these vaccines appears to be largely limited to intradermal tumors rather than intracerebral tumors.

Animals↗

PubMed1995

Cyclocreatine in cancer chemotherapy.

Cyclocreatine, an analog of creatine, is an efficient substrate for creatine kinase, but its phosphorylated form is a poor phosphate donor in comparison with creatine phosphate. Cyclocreatine was not very cytotoxic upon 24 h of exposure of human SW2 small-cell lung cancer cells to concentrations of up to 5 mM. However, combinations of cyclocreatine (0.5 mM, 24 h) with each of four antitumor alkylating agents, cis-diamminedichloroplatinum(II), melphalan, 4-hydroperoxycyclophosphamide, and carmustine, resulted in additive to greater-than-additive cytotoxicity toward exponentially growing SW2 cells. The greatest levels of synergy were seen at higher concentrations of 4-hydroperoxycyclophosphamide and carmustine as determined by isobologram analysis. In vivo cyclocreatine (0.5 or 1 g/kg) was more effective if given i.v. rather than i.p. The longest tumor-growth delays, up to 10 days, were produced by extended regimens of cyclocreatine. Cyclocreatine was an effective addition to therapy with standard anticancer agents including cis-diamminedichloroplatinum(II), cyclophosphamide, Adriamycin, or 5-fluorouracil. No additional toxicity was observed when 10 days of cyclocreatine treatment was given with full standard-dose regimens of each drug. The resultant increases in tumor-growth delay were 1.7- to 2.4-fold as compared with those obtained for each of the drugs alone. These results indicate that cyclocreatine may be an effective single agent and an effective addition to combination chemotherapy regimens.

Adenocarcinoma↗

PubMed1995

Spatial and temporal control of gene therapy using ionizing radiation.

Activation of transcription of the Egr-1 gene by X-rays is regulated by the promoter region of this gene. We linked the radiation-inducible promoter region of the Egr-1 gene to the gene encoding the radiosensitizing and tumoricidal cytokine, tumour necrosis factor-alpha (TNF-alpha) and used a replication-deficient adenovirus to deliver the Egr-TNF construct to human tumours growing in nude mice. Combined treatment with Ad5.Egr-TNF and 5,000 cGy (rad) resulted in increased intratumoral TNF-alpha production and increased tumour control compared with treatment with Ad5.Egr-TNF alone or with radiation alone. The increase in tumour control was achieved without an increase in normal tissue damage when compared to tissue injury from radiation alone. Control of gene transcription by ionizing radiation in vivo represents a novel method of spatial and temporal regulation of gene-based medical treatments.

Animals↗

PubMed1995

Breast cancer selective gene expression and therapy mediated by recombinant adenoviruses containing the DF3/MUC1 promoter.

The high molecular weight mucin-like glycoprotein, DF3 (MUC1), is overexpressed in the majority of human breast cancers. Here we demonstrate that replication defective recombinant adenoviral vectors, containing the DF3 promoter (bp -725 to +31), can be used to express beta-galactosidase (Ad.DF3-betagal) and the herpes simplex virus thymidine kinase (HSV-tk) gene (Ad.Df3-tk) in DF3 positive breast carcinoma cell lines. In vivo experiments using breast tumor implants in nude mice injected with Ad.DF3-betagal demonstrated that expression of the beta-galactosidase gene is limited to DF3-positive breast cancer xenografts. Moreover, in an intraperitoneal breast cancer metastases model, we show that i.p. injection of Ad.DF3-tk followed by GCV treatment results in inhibition of tumor growth. These results demonstrate that utilization of the DF3 promoter in an adenoviral vector can confer selective expression of heterologous genes in breast cancer cells in vitro and in vivo.

Adenoviruses, Human↗

PubMed1994

Expression of the multidrug resistance associated protein and P-glycoprotein in doxorubicin-selected human myeloid leukemia cells.

Drug-resistant sublines of the human U-937 myeloid leukemia cell line were selected in doxorubicin concentrations of 10, 40, and 200 ng/mL (designated U-A10, U-A40, and U-A200, respectively). Northern blot analysis showed overexpression of the multidrug resistance-associated protein (MRP) gene, but not MDR1, in U-A10 cells as compared with parental U-937 cells. Prolonged passage of U-A10 cells in 10 ng/mL of doxorubicin had little effect on MRP RNA levels, but increased MDR1 expression. The U-A40 and U-A200 cells, derived by selection of U-A10 cells, showed high levels of both MRP and MDR1 expression. None of the drug-resistant cell lines showed MRP or MDR1 gene amplification as judged by Southern blot analysis. U-A10 cells exhibited minimal decreased net accumulation of anthracycline, whereas U-A40 and U-A200 cells showed more significantly decreased drug accumulation as compared with U-937 cells. Subcellular anthracycline accumulation in U-937 cells as determined by fluorescence microscopy showed daunorubicin fluorescence predominately in the nucleus. However, the drug-resistant cell lines showed minimal nuclear drug accumulation with marked redistribution of drug into a vesicular compartment. Treatment with sodium azide/2-deoxyglucose, 2,4-dinitrophenol, or monensin, but not verapamil, abolished the vesicular accumulation. These studies in doxorubicin-selected U-937 cells indicate that induction of MRP overexpression occurs before that for the MDR1 gene. In addition, the drug-resistant cells possess an energy-dependent redistribution of anthracyclines into a nonnuclear vesicular compartment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Enhancer sequences of the DF3 gene regulate expression of the herpes simplex virus thymidine kinase gene and confer sensitivity of human breast cancer cells to ganciclovir.

A potentially novel therapeutic strategy for breast cancer treatment involves sensitization of tumor cells to chemotherapy through gene transfer. Clinical application of this approach, however, may be limited by the lack of target cell specificity of currently available gene delivery techniques. Development of vectors with tumor-selective gene expression could overcome this problem. The DF3/MUC1 gene encodes a high molecular weight mucin-like glycoprotein which is overexpressed at the transcriptional level in the majority of human breast cancers. To develop a breast tumor-selective enhancer, we cloned the upstream region of the DF3 gene and have identified a 114-base pair enhancer region that can modulate transcription from heterologous promoters. The present studies demonstrate that the DF3 enhancer sequences can direct selective gene expression in DF3-positive breast carcinoma cells. DF3-positive breast carcinoma cell lines transfected with herpes simplex virus thymidine kinase gene expression cassettes modified by the DF3 enhancer were markedly more sensitive to killing by ganciclovir than were the same cells transfected with the expression cassettes lacking the DF3 enhancer. DF3-negative cell lines transfected with the DF3 enhancer constructs, however, were no more sensitive to ganciclovir than were cells treated with the unmodified expression plasmids. Consistent with an innocent bystander effect, nontransfected human breast carcinoma cells were susceptible in a cell density-dependent manner to ganciclovir-induced cell killing when adjacent to transfected cells. The results also demonstrate that the DF3 enhancer sequences can be effectively incorporated into a retroviral vector to mediate selective gene expression following retroviral infection. These findings suggest that the DF3 promoter/enhancer may be useful for incorporation into vectors designed for gene therapy of breast cancer.

Base Sequence↗

PubMed1994

Identification of upstream signals regulating interleukin-6 gene expression during in vitro treatment of human B cells with pokeweed mitogen.

The present studies have characterized the regulation of interleukin-6 (IL-6) gene expression during pokeweed mitogen (PWM)-driven human B-cell differentiation. PWM induced an early and transient increase in the expression of immediate-early response genes of the jun/fos leucine zipper family (c-jun, jun B, c-fos, and fos-B). The induction of c-jun mRNA by PWM was concentration dependent. Nuclear run-on assays showed that PWM treatment is associated with an increased rate of c-jun gene transcription. The induction of c-jun mRNA precedes the induction of IL-6 gene expression and IL-6 secretion by the B cells. c-Jun antisense, but not sense, oligodeoxynucleotide (ODN) significantly decreases PWM-related B-cell (1) proliferation; (2) IL-6 mRNA induction; (3) IL-6 secretion; and (4) nuclear extract binding to AP-1 in electrophoretic mobility shift assay. In contrast, c-Fos anti-sense ODN did not effect either IL-6 mRNA induction or IL-6 secretion triggered in B cells by PWM. The results further show activation of c-Raf-1 kinase in PWM-treated B cells. Raf-1 acts upstream to mitogen-activated protein (MAP) kinase; therefore, studies were performed to assay for MAP kinase activation in these cells. The results show an increase in phosphorylation of myelin basic protein (MBP) and c-Jun "Y" peptide in PWM-treated B cells. Taken together, these findings suggest that PWM is able to initiate an intracytoplasmic signaling cascade in normal human splenic B cells, which, at least in part, involves serine/threonine protein kinases. These results show transient induction of immediate-early response genes in B cells and support a potential role for the c-jun gene product in regulation of IL-6 transcription and secretion.

B-Lymphocytes↗

PubMed1994

Gene therapy targeted by radiation preferentially radiosensitizes tumor cells.

Transcriptional regulation of the promoter/enhancer region of the Egr-1 gene is activated by ionizing radiation. We linked DNA sequences from the promotor region of Egr-1 to a complementary DNA sequence which encodes human tumor necrosis factor (TNF) alpha, a radiosensitizing cytokine. The Egr-TNF construct was transfected into a human cell line of hematopoietic origin, HL525, which was used in an experimental animal system. HL525 (clone 2) cells containing the Egr-TNF construct which exhibits radiation induction of TNF-alpha were injected into human xenografts of the radioresistant human squamous cell carcinoma cell line SQ-20B. Animals treated with radiation and clone 2 demonstrated an increase in tumor cures compared with animals treated with radiation alone or unirradiated animals given injections of clone 2 alone. No increase in local or systemic toxicity was observed in the combined treatment group. The combination of gene therapy and radiation therapy enhances tumor cures without increasing normal tissue toxicity and is a new paradigm for cancer treatment.

Animals↗

PubMed1994

Role of P-glycoprotein in dolastatin 10 resistance.

Dolastatin 10, a cytotoxic pentapeptide isolated from the mollusk Dolabella auricularia, exhibits potent antitumor activity. The present studies demonstrated that sublines of murine PC4 and human U-937 leukemia cells expressing a multidrug resistance (MDR) phenotype are cross-resistant to this agent. We also demonstrated that such resistance was reversed by verapamil. While these findings suggested the involvement of the P-glycoprotein (P-gp) in dolastatin 10 resistance, we performed similar studies in a CHO cell line transfected with the human mdr1 cDNA. Expression of P-gp in the transfected cells was associated with resistance to dolastatin 10 by a verapamil-sensitive mechanism. The demonstration that photoaffinity labeling of P-gp was decreased in the presence of dolastatin 10 further supports the interaction of this cytotoxic peptide with P-gp. Taken together, these findings suggest that resistance to dolastatin 10 is conferred, at least in part, by P-gp and that this cytotoxic peptide is a novel member of the MDR phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Ketoconazole attenuates radiation-induction of tumor necrosis factor.

PURPOSE: Previous work has demonstrated that inhibitors of phospholipase A2 attenuate ionizing radiation induced arachidonic acid production, protein kinase C activation and prevent subsequent induction of the tumor necrosis factor gene. Because arachidonic acid contributes to radiation-induced tumor necrosis factor expression, we analyzed the effects of agents which alter arachidonate metabolism on the regulation of this gene. METHODS AND MATERIALS: Phospholipase A2 inhibitors quinicrine, bromphenyl bromide, and pentoxyfylline or the inhibitor of lipoxygenase (ketoconazole) or the inhibitor of cyclooxygenase (indomethacin) were added to cell culture 1 h prior to irradiation. RESULTS: Radiation-induced tumor necrosis factor gene expression was attenuated by each of the phospholipase A2 inhibitors (quinicrine, bromphenyl bromide, and pentoxyfylline). Furthermore, ketoconazole attenuated X ray induced tumor necrosis factor gene expression. Conversely, indomethacin enhanced tumor necrosis factor expression following irradiation. CONCLUSION: The finding that radiation-induced tumor necrosis factor gene expression was attenuated by ketoconazole suggests that the lipoxygenase pathway participates in signal transduction preceding tumor necrosis factor induction. Enhancement of tumor necrosis factor expression by indomethacin following irradiation suggests that prostaglandins produced by cyclooxygenase act as negative regulators of tumor necrosis factor expression. Inhibitors of tumor necrosis factor induction ameliorate acute and subacute sequelae of radiotherapy. We propose therefore, that ketoconazole may reduce acute radiation sequelae such as mucositis and esophagitis through a reduction in tumor necrosis factor induction or inhibition of phospholipase A2 in addition to its antifungal activity.

Arachidonic Acid↗

PubMed1994

Membrane-derived second messenger regulates x-ray-mediated tumor necrosis factor alpha gene induction.

Cells adapt to adverse environmental conditions through a wide range of responses that are conserved throughout evolution. Physical agents such as ionizing radiation are known to initiate a stress response that is triggered by the recognition of DNA damage. We have identified a signaling pathway involving the activation of phospholipase A2 and protein kinase C in human cells that confers x-ray induction of the tumor necrosis factor alpha gene. Treatment of human cells with ionizing radiation or H2O2 was associated with the production of arachidonic acid. Inhibition of phospholipase A2 abolished radiation-mediated arachidonate production as well as the subsequent activation of protein kinase C and tumor necrosis factor alpha gene expression. These findings demonstrate that ionizing radiation-mediated gene expression in human cells is regulated in part by extranuclear signal transduction. One practical application of phospholipase A2 inhibitors is to ameliorate the adverse effects of radiotherapy associated with tumor necrosis factor alpha production.

Amino Acid Sequence↗

PubMed1994

Involvement of serum response element in okadaic acid-induced EGR-1 transcription in human T-cells.

The present work has examined the effects of okadaic acid, an inhibitor of serine/threonine protein phosphatase, PP-1 and PP-2A, on the regulation of EGR-1 gene expression in normal peripheral blood T- and Jurkat cells. The results demonstrate that okadaic acid treatment is associated with a transient induction of EGR-1 gene expression which was detectable by 30 min to 1 h and peaked at 3-6 h. EGR-1 mRNA was superinduced in cells treated with both okadaic acid and the protein synthesis inhibitor cycloheximide. The half-life of EGR-1 mRNA was similar in both control and okadaic acid-treated cells. In contrast, treatment with both okadaic acid and cycloheximide prolonged the half-life of EGR-1 transcripts. Nuclear run-on assays demonstrated that induction of EGR-1 gene expression by okadaic acid is controlled at least in part by a transcriptional mechanism. Transient expression assays with EGR-1 promotor fragments linked to the chloramphenicol acetyltransferase gene demonstrate that okadaic acid-induced EGR-1 transcription is conferred by the 5' most distal CArG box, CC (AT)6GG, in the EGR-1 promoter. Moreover, chloramphenicol acetyltransferase activity was induced by okadaic acid when the 5' most distal CArG element was linked to the heterologous herpes simplex virus thymidine kinase promoter, and not induced with a similar heterologous construct containing a mutated CArG sequence. These studies demonstrate that okadaic acid regulates EGR-1 gene expression at the transcriptional level via the CArG element and suggest that PP-1 and PP-2A play a role in T-cell activation.

Base Sequence↗

PubMed1994

All-trans retinoic acid reverses phorbol ester resistance in a human myeloid leukemia cell line.

Treatment of human HL-60 leukemic cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with activation of protein kinase C (PKC) and induction of monocytic differentiation. An HL-60 variant cell line, termed HL-525, derived from long-term exposure to TPA (Homma et al, Proc Natl Acad Sci USA 83: 7316, 1986) is resistant to TPA-induced differentiation and displays decreased PKC beta expression compared with the HL-60 parent line. However, this variant exhibits features of granulocytic differentiation, including nitroblue tetrazolium reduction, when exposed to all-trans retinoic acid (ATRA). Whereas treatment of HL-525 cells with ATRA or TPA alone had no effect on features of monocytic differentiation, these agents in combination resulted in cellular adhesion, nonspecific esterase staining, and induction of the c-fms (monocyte growth factor receptor) gene. In order to measure PKC expression associated with the reversal of TPA resistance by ATRA, we exposed HL-525 cells to ATRA and analyzed PKC-mRNA and protein levels. Exposure of HL-525 cells to ATRA for 3 days resulted in induction of PKC beta transcripts, whereas there was little change in PKC alpha mRNA levels. ATRA treatment was also associated with an increase in PKC activity and an induction of cytosolic PKC beta protein levels. These findings are consistent with the hypothesis that ATRA reverses TPA resistance in HL-525 cells by enhancing the expression of PKC.

Cell Differentiation↗

PubMed1994

The role of intracellular calcium in the cellular response to ionizing radiation.

Calcium is required as a cofactor by primer recognition proteins involved in DNA synthesis and by protein kinase C (PKC), which is activated by ionizing radiation. Because these processes may be involved in radiation-mediated regulation of the progression of cells through the phases of the cell cycle, we studied the effects of the intracellular Ca2+ chelator, acetoxymethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (AM-BAPTA), on PKC activation, expression of c-jun and Gadd45 and distribution of cells in the phases of the cell cycle after irradiation. AM-BAPTA prevented ionizing-radiation-induced activation of PKC and expression of c-jun in cells of human tumor cell lines. Conversely, calcium chelation had no effect on X-ray-induced expression of the Gadd45 gene. To determine whether changes in the intracellular Ca2+ concentration ([Ca2+]i) occurred during irradiation, we measured [Ca2+]i in single cells using fura-2-based microfluorimetry. There was no increase in [Ca2+]i during or after irradiation of cells of the human tumor cell lines RIT-3, SQ-20B or HL-60 or normal human fibroblast strain IMR-90. The percentage of human tumor cells crossing the G1/S-phase border was reduced by pretreatment with AM-BAPTA. These data indicate that calcium is required for ionizing radiation-induced cell cycle regulation and PKC activation, but that increases in [Ca2+]i do not occur in cells of the cell lines irradiated in this study.

Amino Acid Sequence↗

PubMed1993

Localization of tumor-associated glycoprotein DF3 in normal, inflammatory, and neoplastic lesions of the colon.

BACKGROUND: The expression of DF3 was assessed by a monoclonal antibody in normal, inflammatory, and neoplastic conditions in the large bowel. METHODS: Using immunohistochemistry, expression was examined in formalin-fixed paraffin-embedded biopsy and resection samples of 19 normal colonic mucosal specimens, 49 inflammatory lesions, 34 adenomas, and 38 primary colonic adenocarcinomas. In addition, Western blots of normal colonic mucosa and adenocarcinoma were examined. RESULTS: DF3 expression was detected in 84% of the adenocarcinomas with coarse membrane staining, intense positivity of luminal secretions, and focal cytoplasmic and intracytoplasmic vacuole staining. Nine of 32 areas of transitional mucosa revealed reactivity along apical membranes in crypt cells. Five adenomas containing carcinoma revealed DF3 positivity in the malignant areas only, whereas the remaining 29 were negative. Staining was membrane, luminal, and intracytoplasmic. Two examples of active ulcerative colitis revealed focal reactivity along the apical membrane of crypt cells. No other areas of staining were noted, including 12 cases containing dysplasia. Four of 10 other inflammatory lesions also revealed similar membrane reactivity in crypt cells. Normal colonic mucosa was nonreactive. Examples of normal colonic mucosa were negative for DF3 by Western blot analysis, whereas two carcinoma samples that reacted immunohistochemically were positive. CONCLUSIONS: DF3 is not detectable in normal colonic tissues. It is expressed focally and predominantly along the apical membrane of crypt cells in some inflammatory lesions and in the transitional mucosa of primary adenocarcinomas. Most adenocarcinomas of the colon and adenomas with foci of invasive carcinoma demonstrate reactivity in the cytoplasm and luminal secretions.

Adenocarcinoma↗

PubMed1993

Defective translocation of protein kinase C in multidrug-resistant HL-60 cells confers a reversible loss of phorbol ester-induced monocytic differentiation.

Previous studies have demonstrated that human HL-60 myeloid leukemia cells differentiate in response to phorbol esters. This event is associated with induction of the c-jun early response gene and appearance of a monocytic phenotype. The present studies have examined the effects of vincristine-selected, multidrug resistance on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 cell differentiation. The results demonstrate that multidrug-resistant HL-60 cells, designated HL-60/vinc, fail to respond to TPA with an increase in c-jun transcripts or other phenotypic characteristics of monocytic differentiation. By contrast, treatment of HL-60/vinc cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases, induces c-jun transcription, growth arrest, and expression of the c-fms gene. Studies were also performed with an HL-60/vinc revertant (HL-60/vinc/R) line that has regained partial sensitivity to vincristine. The finding that HL-60/vinc/R cells respond to TPA with induction of a monocytic phenotype, but not c-jun expression, suggests that c-jun induction is not obligatory for monocytic differentiation. Other studies further demonstrate that the jun-B and fra-1 genes are induced by TPA in both HL-60/vinc and HL-60/vinc/R cells, whereas c-fos expression is attenuated in the HL-60/vinc line. Since TPA activates protein kinase C (PKC), we examined translocation of PKC from the cytosol to the membrane fraction. Although HL-60 and HL-60/vinc/R cells demonstrated translocation of PKC activity, this subcellular redistribution was undetectable in HL-60/vinc cells. Activity of the mitogen-activated protein kinase family with associated phosphorylation of c-Jun Y-peptide was markedly diminished in TPA-treated HL-60/vinc cells, but not in response to okadaic acid. Taken together, these findings suggest that vincristine resistance confers insensitivity to TPA-induced differentiation and can include defects in PKC-mediated signaling events and induction of jun/fos early response gene expression.

Calcium-Calmodulin-Dependent Protein Kinases↗