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J S Haskill

Publications and source records attributed to J S Haskill.

At least 19 recordsLinked to original sources
PubMed2001

Expression and localization of IL-1beta mRNA is interrelated with cytoskeletal rearrangement in monocytes stimulated by adherence: a light microscopy in situ hybridization study.

Differences in IL-1beta mRNA expression, stability and translation between non-adherent monocytes and those stimulated by adherence suggest that cytokine regulation is coupled to the function and assembly of cytoskeletal structures. In situ hybridization studies were performed to visualize expression and positioning of IL-1beta mRNA in adherently cultivated monocytes. IL-1beta mRNA expression was heterogeneous with high transcript levels found in spread or polarized cells. Transcripts were compartmentalized to the perinuclear region in spread cells, and partially redistributed with polarization. In contrast to mRNA distribution in other motile cell populations, IL-1beta mRNA did not localize to the distal or proximal actin cytoskeleton. Perinuclear confinement of transcripts required intact actin microfilaments. Treatment with cytoskeleton disruption and detergent extraction suggested that most non-translated IL-1beta mRNA was associated with intermediate filaments. In monocytes stimulated by LPS, IL-1beta, but not IL-1Ra transcripts were redistributed and partially associated, yet not bound to actin microfilaments. The present study demonstrates that IL-1beta mRNA expression and localization in adherent monocytes is interrelated with the cytoskeletal rearrangement upon adherence, spreading and polarization.

Actins↗
PubMed2000

Inhibition of the calcium-dependent tyrosine kinase (CADTK) blocks monocyte spreading and motility.

Freshly isolated peripheral blood monocytes lack focal adhesion kinase (p125(FAK)) but activate a second member of this kinase family, calcium-dependent tyrosine kinase (CADTK; also known as Pyk2/CAKbeta/RAFTK/FAK2), upon adhesion or stimulation with chemokines. To study the role of CADTK in monocyte adherence and motility, we performed immunocytochemical localization that showed CADTK at the leading edge and ruffling lamellipodial structures in freshly isolated, adhered human monocytes. We next introduced CADTK/CAKbeta-related non-kinase (CRNK), the C-terminal noncatalytic domain of CADTK, into monocytes by electroporation and showed that it inhibited CADTK autophosphorylation. Introduction of the fusion protein glutathione S-transferase (GST)-CRNK also reduced (i) cell spreading, as reflected in a reduced cell area 30 min after adhesion, (ii) adhesion-induced phosphotyrosine increases and redistribution into lamellipodia, and (iii) adhesion-induced extracellular signal-regulated protein kinase (ERK) activation. In control experiments, introduction of GST or GST-C3 transferase (an inhibitor of RhoA GTPase activity) by electroporation did not affect these parameters. Monocytes adhered in the presence of autologous serum were highly motile even after introduction of GST (83% motile cells). However, only 26% of monocytes with introduced GST-CRNK were motile. In contrast, GST-CRNK-treated monocytes were fully capable of phagocytosis and adhesion-induced cytokine gene induction, suggesting that CADTK is not involved in these cellular activities and that GST-CRNK introduction does not inhibit global monocyte functions. These results suggest that CADTK is crucial for the in vitro monocyte cytoskeletal reorganization necessary for cell motility and is likely to be required in vivo for recruitment to sites of inflammation.

Cell Adhesion↗
PubMed2000

IL-8 reduced tumorigenicity of human ovarian cancer in vivo due to neutrophil infiltration.

Paclitaxel is a frontline therapy for ovarian cancer. Our laboratory has shown that paclitaxel induces IL-8, a member of the C-X-C family of chemokines, in subsets of human ovarian cancer cells. However, the critical issue concerns the biological significance of this chemokine in human ovarian cancer. To study the influence of IL-8 on tumor growth, human ovarian cancer cell lines were transfected with an expression vector for human IL-8 and tested for their ability to form tumors in nude mice. IL-8 expression by the transfected cells did not alter their growth properties in vitro. In contrast, tumor growth in vivo was significantly attenuated in animals receiving IL-8-expressing cells when compared with mice injected with control cells. As additional evidence that IL-8 is a crucial factor in tumor growth, it was noted that ovarian cell lines in which constitutive IL-8 expression is elevated did not form tumors. Injection of neutralizing Ab to IL-8 reverted the phenotype and caused tumor growth in vivo. Examination of tissue from the inoculation site revealed a dramatically elevated cellularity, containing neutrophils and macrophages, in mice receiving IL-8-expressing tumor cells. These results suggest that IL-8 production by human ovarian tumor cells can play a role in reducing the rate of tumor growth; this effect may be mediated by the increased targeting of neutrophil and other mononuclear cells to the tumor injection site. These studies indicate a role for IL-8 in ovarian cancer control and suggest that chemotherapy-induced IL-8 may have a positive role in controlling tumor growth.

Animals↗
PubMed2000

Differential role of tyrosine phosphorylation in adhesion-induced transcription, mRNA stability, and cytoskeletal organization in human monocytes.

Monocyte adhesion resulted in rapid tyrosine phosphorylation and subsequent cytokine mRNA induction. The objective of this study was to determine the role of specific tyrosine phosphorylation events, particularly those involving members of the MAP kinase family, in regulating adhesion-induced cytokine expression. Using nuclear run-on analyses, we demonstrated that on adhesion, monocytes rapidly transcriptionally activated numerous cytokine mRNAs, coincident with the activation of the transcription factors NF-KB and AP-1. Both an inhibitor of tyrosine phosphorylation, genistein, and the cytoplasmic tyrosine phosphatase PTP1B, were unable to prevent adhesion-mediated transcriptional activation. However, both blocked adhesion-induced ERK and JNK but not p38 kinase activation and at the same time decreased the stability of interleukin-1beta (IL-1beta) and IL-8 transcripts. In addition, whereas adhesive events occurred in the presence of genistein and PTP1B, monocyte spreading was markedly inhibited. Our results suggest that the majority of protein phosphorylation events are associated with adhesion-induced cytokine expression through transcript stabilization and cytoskeletal organization. A minority of protein phosphorylation events, not sensitive to genistein or PTP1B exposure, may be instrumental in regulating transcription. Thus the spectrum of protein tyrosine kinases required for transcription appear distinct from those involved in maintaining the stability of some cytokine mRNAs and the integrity of the cytoskeleton to which mRNA destined for translation must be associated.

Adult↗
PubMed1999

Cisplatin inhibits paclitaxel-induced apoptosis in cisplatin-resistant ovarian cancer cell lines: possible explanation for failure of combination therapy.

Combination chemotherapy using paclitaxel with a platinum-based regimen is currently the standard first-line therapy for ovarian cancer after surgical cytoreduction. Whereas cisplatin-paclitaxel combination chemotherapy has shown significant efficacy over previous drug combinations in ovarian cancer, 20-30% of patients fail to respond to this combination. These patients are deemed cisplatin-paclitaxel resistant, although it is unclear whether the tumors are resistant to one or both drugs. Because the options available to ovarian cancer patients for second-line therapy are limited, and knowing that mechanistic differences exist between cisplatin and paclitaxel, we assessed the efficacy of combination drug therapy on cisplatin-resistant (cisplatinR) ovarian cancer cells. We found that paclitaxel induced apoptosis in cisplatinR cells as well as in the cisplatin-sensitive parental cell lines. In cisplatinR C-13 cells, the concomitant addition of cisplatin blocked paclitaxel-induced apoptosis as determined by DNA fragmentation assays, fluorescence microscopy, and flow cytometry. Paclitaxel-induced multimininucleation was also inhibited when the cells were exposed sequentially to paclitaxel and then cisplatin. Cisplatin did not block paclitaxel-induced stabilization of microtubules or prevent paclitaxel-induced loss of Bcl-2 expression in cisplatinR cells. Conversely, paclitaxel did not inhibit p53 protein accumulation by cisplatin. These results suggest that cisplatin blocks paclitaxel-induced apoptosis at a point downstream of Bcl-2 degradation and independent of microtubule stabilization. Our research shows that cisplatin can inhibit the effectiveness of paclitaxel in cispatinR cell lines. Therefore, the establishment of a clinical protocol to evaluate the efficacy of paclitaxel alone versus another second-line regimen in patients with cisplatin-paclitaxel-resistant ovarian cancer is warranted.

Antineoplastic Agents↗
PubMed1998

A calcium-dependent tyrosine kinase splice variant in human monocytes. Activation by a two-stage process involving adherence and a subsequent intracellular signal.

Freshly isolated human monocytes do not express p125(FAK) but upon adherence to substrata activate the highly related calcium-dependent tyrosine kinase (CADTK), also known as Pyk2, CAKbeta, RAFTK, and FAK2. The monocyte CADTK was 5 kDa smaller than protein from epithelial cells; isolation and sequencing of the monocyte CADTK cDNA revealed a predicted 42-amino acid deletion between the two proline-rich domains of the enzyme. The nucleic acid sequence suggests that the deletion is caused by alternative RNA splicing. This species was also found in T and B lymphocytes and appears to be the predominant form of cytoskeletal associated tyrosine kinase in non-neoplastic, circulating, hematopoietic cells. CADTK was not activated when monocytes maintained in suspension were treated with agents that produce an intracellular calcium (thapsigargin) or protein kinase C (phorbol 12-myristate 13-acetate) signal including a chemokine, RANTES, that binds to the HIV co-receptor, CCK5. In contrast, monocyte adherence to tissue culture plastic-stimulated CADTK tyrosine phosphorylation, a process that was enhanced by thapsigargin, phorbol 12-myristate 13-acetate, and RANTES but that was completely blocked by preincubation with cytochalasin D. When compared with plastic, adherence to fibronectin- or collagen-coated surfaces produced only minimal CADTK activation but permitted significant stimulation by added thapsigargin. These data suggest that in a cell type that lacks p125(FAK), CADTK plays an early role in post-adherence signaling. Its activation involves two stages, cytoskeletal engagement, which is permissive, and co-stimulatory signals (calcium or protein kinase C) generated by extensive cell surface engagement, agonists, or inflammatory chemokines.

Alternative Splicing↗
PubMed1998

Effects of paclitaxel on cytokine synthesis by unprimed human monocytes, T lymphocytes, and breast cancer cells.

Paclitaxel or Taxol has attracted a great deal of attention in recent years because of its immense success as a chemotherapeutic agent for numerous types of cancer. It is known that paclitaxel stabilizes microtubules, and this characteristic is the presumed primary mechanism for its antitumor activity. Recently, however, paclitaxel's ability to regulate gene expression, particularly in the murine system, has been reported by several groups. Here, we present research examining paclitaxel's ability to alter expression of the interleukin-1beta (IL-1beta) and IL-8 cytokines in primary human monocytes, T lymphocytes, and four human breast cancer cell lines: MCF-7, ZR-75-1, MDA-MB-468, and MDA-MB-231. This report shows for the first time that treatment with 5-50 microM paclitaxel increases steady-state levels of IL-1beta mRNA in unprimed human monocytes, MCF-7, and ZR-75-1 cells. Monocytes from eight donors in 16 experiments showed increased IL-1beta secretion upon treatment; however, the increase in IL-1beta production by monocytes was predicated on culturing in the absence of fetal bovine serum or in the presence of autologous human serum. In contrast to the IL-1beta results, paclitaxel did not have significant effects on IL-8 expression by monocytes, T lymphocytes, or the breast cancer cells. These data show a specific effect of paclitaxel on cytokine synthesis by both immune cells and cancer cells.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Identification of the structural region of taxol that may be responsible for cytokine gene induction and cytotoxicity in human ovarian cancer cells.

PURPOSE: Interleukin-8 (IL-8) is a pleiotropic chemokine with both chemoattractant and angiogenic properties. In addition to its cytotoxic effects on ovarian cancer cells, taxol can transcriptionally activate genes such as IL-8 that may play a role in tumorigenesis. Utilizing IL-8 as a prototypic marker of tumor-derived modulators of growth, we undertook a systematic study of taxol and 11 structurally modified taxol analogs to identify the region of the taxane skeleton responsible for IL-8 gene induction. METHODS: The human ovarian cancer cell line OVCA-420 was exposed to taxol or taxol analogs. IL-8 gene induction was assessed by Northern blot analysis after 6 h and cytotoxicity after 72 h. RESULTS: Changes in the southern hemisphere (C-1 to C-4) of the taxane skeleton had greater effects on IL-8 induction than changes in the northern hemisphere (C-7 to C-11). Some of the taxol analogs modified at positions C-1 and/or C-2 with increased hydrophobicity induced IL-8 expression more than threefold over that induced by taxol or taxotere and more than 20-fold over control cells. Cells that failed to induce IL-8 gene expression in response to taxol were only marginally responsive to the analogs unless first primed with IL-1beta. Modifications to the northern hemisphere did not alter taxol's effect on IL-8 expression in human cells, but did influence TNFalpha expression in murine macrophage cells, suggesting species and/or gene specificity. We found a direct correlation between IL-8 induction and cytotoxicity, in that analogs that dramatically upregulated IL-8 expression proved to be the most cytotoxic, inhibiting cell survival by > 90%. CONCLUSION: Taken together our results demonstrate that changes in the southern hemisphere of the taxane skeleton influence both the gene induction and cytotoxic potential of taxol in human ovarian cancer cells.

Antineoplastic Agents, Phytogenic↗
PubMed1997

NF-kappa B as a target for anti-inflammatory gene therapy: suppression of inflammatory responses in monocytic and stromal cells by stable gene transfer of I kappa B alpha cDNA.

One of the most challenging issues of anti-inflammatory gene therapy is the complexity of inflammatory pathways. Transcription factor NF-kappa B plays a pivotal role in activation of multiple inflammatory molecules, and therefore represents the logical target for intervention. We evaluated the feasibility of suppressing the inflammatory responses in different cell lines through specific inhibition of NF-kappa B by gene transfer of I kappa B alpha, the naturally occurring intracellular inhibitor of NF-kappa B. The I kappa B alpha overexpressing cells were established using retroviral gene transfer or stable transfection with the wild-type (wt) I kappa B alpha cDNA. In all cell types, overexpression of wt I kappa B alpha resulted in a profound (> 100-fold) increase of the I kappa B alpha message and a moderate (two- to three-fold) increase of the I kappa B alpha protein. The effects of the I kappa B alpha overexpression on the NF-kappa B activation and the inflammatory responses varied significantly in different cell lines. In conditionally immortalized human endometrial stromal cells, overexpression of I kappa B alpha prevented both interleukin-1 (IL-1)-inducible degradation of endogenous I kappa B alpha protein and activation of NF-kappa B. Accordingly, induction of cytokines interleukin-8 (IL-8) and Gro gamma was markedly suppressed. In monocytic THP-1 cells, both lipopolysaccharide (LPS)-inducible degradation of I kappa B alpha and NF-kappa B activation were only partially inhibited by overexpression of exogenous I kappa B alpha cDNA. None the less, the LPS-induced transcription of IL-1 beta and secretion of cytokines interleukin-6 (IL-6) and IL-8 were virtually abolished. In epithelial HT-29 cells, no inflammatory responses were inhibited. These results demonstrate the range of responses in various cell lines to gene transfer of I kappa B alpha and indicate the feasibility of suppression of inflammatory responses in appropriate target cells and their progeny by suppression of NF-kappa B.

Cell Line↗
PubMed1997

Adhesion-dependent regulation of an A+U-rich element-binding activity associated with AUF1.

Monocyte adherence results in the rapid transcriptional activation and mRNA stabilization of numerous mediators of inflammation and tissue repair. While the enhancer and promoter elements associated with transcriptional activation have been studied, mechanisms linking adhesion, mRNA stabilization, and translation are unknown. GROalpha and interleukin-1beta (IL-1beta) mRNAs are highly labile in nonadhered monocytes but stabilize rapidly after adherence. GROalpha and IL-1beta transcripts both contain A+U-rich elements (AREs) in the 3' untranslated region (UTR) which have been directly associated with rapid mRNA turnover. To determine if the GROalpha ARE region was recognized by factors associated with mRNA degradation, we carried out mobility gel shift analyses using a series of RNA probes encompassing the entire GROalpha transcript. Stable complexes were formed only with the proximal 3' UTR which contained the ARE region. The two slower-moving complexes were rapidly depleted following monocyte adherence but not direct integrin engagement. Deadherence reactivated the two largest ARE-binding complexes and destabilized IL-1beta transcripts. Antibody supershift studies demonstrated that both of these ARE RNA-binding complexes contained AUF1. The formation of these complexes and the accelerated mRNA turnover are phosphorylation-dependent events, as both are induced in adherent monocytes by the tyrosine kinase inhibitor genistein and the p38 MAP kinase inhibitor of IL-1beta translation, SK&F 86002. These results demonstrate that cell adhesion and deadhesion rapidly and reversibly modify both cytokine mRNA stability and the RNA-binding complexes associated with AUF1.

Cell Adhesion↗
PubMed1997

Identification of tumor-specific paclitaxel (Taxol)-responsive regulatory elements in the interleukin-8 promoter.

Paclitaxel (Taxol) is a novel chemotherapeutic drug that is effective against breast and ovarian cancers. Although the primary target of paclitaxel is microtubules, its efficacy exceeds that of conventional microtubule-disrupting agents, suggesting that it may have additional cellular effects. Previously, we demonstrated that paclitaxel can induce interleukin-8 (IL-8) gene expression at the transcriptional level in subsets of human ovarian cancer lines. In this as well as the previous report, we present evidence that this ability is not linked to the lipopolysaccharide pathway of IL-8 gene induction. The present study identifies the cis-acting elements and trans-acting factors involved in this induction by transfecting DNA constructs containing the 5'-flanking region of the IL-8 gene linked to the chloramphenicol acetyltransferase reporter gene into paclitaxel-responsive and nonresponsive ovarian cancer cells (responsiveness refers to the IL-8 response). Paclitaxel only activated the IL-8 promoter in responsive cells. The AP-1 and NF-kappaB binding sites in the IL-8 promoter are required for activation by paclitaxel; in contrast, a C/EBP site required for IL-8 promoter activation in other cell types is not involved. Gel shift assays demonstrate that paclitaxel causes a marked increase in protein binding to the NF-kappaB and AP-1 consensus binding sequences in the paclitaxel-responsive ovarian cells, but not the nonresponsive cells. The induction of NF-kappaB and AP-1 binding is reduced by the addition of protein kinase C inhibitors and cyclic AMP effector, respectively. These results demonstrate a molecular mechanism for cell-specific paclitaxel-induced IL-8 gene expression which may have clinical relevance.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Taxol-dependent transcriptional activation of IL-8 expression in a subset of human ovarian cancer.

Taxol is important in the treatment of both primary and drug-resistant ovarian cancer. Although Taxol is known to stabilize microtubules and block cell mitosis, the effectiveness of this drug exceeds that of other antimitotic agents, suggesting it may have an additional mode of action. Stimulated by murine macrophage studies indicating cytokine induction by Taxol, we have investigated proinflammatory cytokine expression in a series of cell lines and recent explants of human ovarian cancer. Taxol induced secretion of interleukin (IL) 8 but not IL-6, IL-1alpha, or IL-1beta in 4 of 10 samples. Induction was dependent on transcriptional activation, and, in contrast to murine macrophage studies, was apparently independent of an active lipopolysaccharide signaling pathway. Confluent cultures secreted as much IL-8 as proliferating cells. Taxol did not induce IL-8 in breast carcinoma, endometrial stromal, or T-lymphocyte or monocyte cultures. We propose that the local expression of this chemokine in vivo may elicit a host response similar in effectiveness to that of cytokine gene therapy. These data are the first to suggest that a chemotherapeutic agent may have a direct effect on transcription of cytokine and/or growth factor genes in ovarian cancer, and that this effect may not be restricted to proliferating tumor cells.

Administration, Topical↗
PubMed1996

Suppression of experimental arthritis by gene transfer of interleukin 1 receptor antagonist cDNA.

Restoration of the impaired balance between pro- and antiinflammatory cytokines should provide effective treatment of rheumatoid arthritis. Gene therapy has been proposed as an approach for delivery of therapeutic proteins to arthritic joints. Here, we examined the efficacy of antiinflammatory gene therapy in bacterial cell wall-induced arthritis in rats. Human secreted interleukin 1 receptor antagonist (sIL-1ra) was expressed in joints of rats with recurrent bacterial cell wall-induced arthritis by using ex vivo gene transfer. To achieve this, primary synoviocytes were transduced in culture with a retroviral vector carrying the sIL-1ra cDNA. Transduced cells were engrafted in ankle joints of animals prior to reactivation of arthritis. Animals in control groups were engrafted with synoviocytes transduced with lacZ and neo marker genes. Cells continued to express transferred genes for at least 9 days after engraftment. We found that gene transfer of sIL-1ra significantly suppressed the severity of recurrence of arthritis, as assessed by measuring joint swelling and by the gross-observation score, and attenuated but did not abolish erosion of cartilage and bone. The effect of intraarticularly expressed sIL-1ra was essentially local, as there was no significant difference in severity of recurrence between unengrafted contralateral joints in control and experimental groups. We estimate that locally expressed sIL-1ra was about four orders of magnitude more therapeutically efficient than systemically administered recombinant sIL-1ra protein. These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis.

Animals↗
PubMed1995

The intracellular IL-1 receptor antagonist alters IL-1-inducible gene expression without blocking exogenous signaling by IL-1 beta.

The epithelium-associated tissue distribution of the intracellular IL-1R antagonist (icIL-1Ra) suggests that it functions as a novel regulatory molecule for IL-1 in somatic tissues. We examined the role of the icIL-1Ra in IL-1 beta-induced responses in human ovarian cancer cells because ovarian surface epithelium expresses transcripts for the icIL-1Ra, and the majority of ovarian cancers arise from these cells. Several human ovarian and cervical cancer cell lines spontaneously express the icIL-1Ra. icIL-1Ra-expressing cells did not have altered growth characteristics or altered short term responses to IL-1 compared with icIL-1Ra-nonexpressing cells. While a 90-min exposure to IL-1 beta resulted in increased steady state cytokine mRNA levels in all cells, icIL-1Ra-positive cells were incapable of maintaining IL-1-beta-induced expression of GRO mRNA. This did not result from decreased transcriptional activity of the GRO gene, but reflected differences in mRNA stability and/or degradation. To determine whether the icIL-1Ra altered mRNA stability, we used a retroviral expression vector to express the icIL-1Ra in an icIL-1Ra-negative cell line. The resulting cells displayed a profile of IL-1 beta-induced genes analogous to that found in cells spontaneously expressing icIL-1Ra. These data show for the first time an intrinsic biologic activity for the icIL-1Ra. The capacity to selectively alter IL-1-induced gene expression suggests that this version of the IL-1Ra is a unique intracellular inhibitor that attenuates IL-1 responses at a point downstream of the initial IL-1/IL-1 receptor interaction.

Female↗
PubMed1995

Transcription-independent turnover of I kappa B alpha during monocyte adherence: implications for a translational component regulating I kappa B alpha/MAD-3 mRNA levels.

We identified I kappa B alpha/MAD-3 as an immediate-early gene in human monocytes that is expressed in response to a variety of signals, including adhesion, lipopolysaccharide, and phorbol myristate acetate. Within 5 min of monocyte adhesion, the level of the I kappa B alpha protein is markedly diminished but is rapidly replaced in a cycloheximide-sensitive manner within 20 min. Accompanying the rapid turnover of the I kappa B alpha protein is simultaneous translocation of NF-kappa B-related transcription factors to nuclei of adhered monocytes. The demonstration that NF-kappa B can regulate I kappa B alpha/MAD-3 gene transcription in other cell types suggested that the rapid increase in steady-state I kappa B alpha/MAD-3 mRNA levels we observed within 30 min of monocyte adherence would result from NF-kappa B-dependent transcriptional stimulation of the I kappa B alpha/MAD-3 gene. Nuclear run-on analyses indicated that, instead, while several immediate-early cytokine genes, such as the interleukin 1 beta (IL-1 beta) gene, were transcriptionally activated during monocyte adhesion, the rate of I kappa B alpha/MAD-3 gene transcription remained constant. The adherence-dependent increase in I kappa B alpha/MAD-3 mRNA levels was also not a consequence of mRNA stabilization events. Interestingly, while increases in both IL-1 beta and I kappa B alpha/MAD-3 mRNA levels were detected in nuclei of adherent monocytes, cytoplasmic levels of IL-1 beta mRNA increased during adherence whereas those of I kappa B alpha/MAD-3 mRNA did not. Taken together, our data suggest that two interactive mechanisms regulate monocytic I kappa B alpha/MAD-3 mRNA levels. We propose that adherent monocytes regulate nuclear processing (or decay) of I kappa B alpha/MAD-3 mRNA, thereby increasing mRNA levels without stimulating I kappa B alpha/MAD-3 gene transcription. Moreover, since inhibition of protein synthesis leads to accumulation of I kappa B alpha/MAD-3 mRNA without stimulating I kappa B alpha/MAD-3 gene transcription, we suggest that low cytoplasmic levels of I kappa B alpha/MAD-3 mRNA are maintained by a translation-dependent degradation mechanism.

Antibodies, Monoclonal↗
PubMed1995

Retrovirus mediated in vivo gene transfer to synovium in bacterial cell wall-induced arthritis in rats.

Gene therapy may provide an effective alternative to conventional approaches for treating rheumatoid arthritis. Direct in vivo gene delivery to synovium has a distinct advantage with respect to clinical use. To date, retroviral vectors are the best studied constructs for gene delivery, and almost all approved gene therapy trials in humans rely on retroviral vectors. However, the applicability of retroviral transduction is limited by requirement for cell division, and attempts to transduce normal synovium in situ using retroviral vectors are reported to fail. The present study was undertaken in order to investigate susceptibility of inflamed synovium to retroviral infection in vivo. Using an experimental model of bacterial cell wall (BCW)-induced arthritis in rats, we attempted two approaches for delivery of retroviral vectors to synovium. In the first approach, recombinant retroviral vectors carrying reporter genes lacZ and neo were directly injected into inflamed rat ankle joints. Alternatively, inflamed joints were inoculated with gamma-irradiated murine retroviral vector-producing packaging cells. We found that about 1% of cells in explants from joints inoculated with packaging cells were lacZ-neo-positive. The lacZ+ neo+ cells in joint explants proliferated in culture and were of rat origin as determined using species-specific polymerase chain reaction (PCR) analysis. There was no evidence of transduction in explants from joints directly injected with retroviral vectors or from contralateral, control joints. These findings show that arthritic joints have a population of cells susceptible to retroviral infection in situ and demonstrate the possibility of using retroviral vectors for direct gene delivery to inflamed synovium.

Animals↗
PubMed1994

Constitutive and stimulated MCP-1, GRO alpha, beta, and gamma expression in human airway epithelium and bronchoalveolar macrophages.

Constitutive expression of mRNAs for GRO alpha, GRO beta, GRO gamma, and MCP-1, belonging to the chemokine family of 8- to 10-kDa cytokines with chemotactic properties for granulocytes and monocytes, has been identified in freshly isolated human nasal and bronchial epithelium, and in bronchoalveolar macrophages (AM). Expression of GRO alpha, GRO gamma, and MCP-1, but not GRO beta, was found in airway epithelial cells. AM expressed all three GRO genes in addition to MCP-1. On reverse transcription, chemokine mRNAs yielded 0.5-30 cDNA molecules/cell, depending on the chemokine and cell type, as determined by a semiquantitative reverse transcriptase-polymerase chain reaction technique. When chemokine mRNA expression in AM and bronchial epithelium from healthy nonatopic individuals was compared, AM expressed more GRO alpha, but similar levels of GRO gamma, MCP-1, and interleukin-8 (IL-8), as in the bronchial epithelial cells. Modulation of chemokine expression by tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml) or endotoxin [lipopolysaccharide (LPS), 100 ng/ml] exposure was studied in primary nasal epithelial cell and alveolar macrophage cultures. In epithelial cells, LPS did not induce chemokine expression but GRO alpha, IL-8, and MCP-1 were upregulated approximately 100-fold by TNF alpha; GRO gamma expression was elevated only 1.5- to 4-fold. In AM cultures, all three GROs were strongly induced by LPS with peak mRNA expression 24 h after stimulation (approximately 50- to 100-fold increase compared with control cultures). MCP-1 mRNA expression, on the other hand, was not increased by LPS in AM. GRO protein was present in supernatants of stimulated epithelial cells and AM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗