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Biomedical subjects

D M Sullivan

Publications and source records attributed to D M Sullivan.

At least 37 records · Page 2Linked to original sources
PubMed1998

Broadened clinical utility of gene gun-mediated, granulocyte-macrophage colony-stimulating factor cDNA-based tumor cell vaccines as demonstrated with a mouse myeloma model.

Effective immunization against the murine B16 melanoma by a nonviral approach in which a gene gun is used to transfer GM-CSF cDNA into tumor cells has been described. We have extended this nonviral approach by using the poorly immunogenic murine myeloma MPC11 model. Vaccination with the transfected, GM-CSF-expressing MPC11 cells induced a potent antitumor cytotoxic T lymphocyte response associated with tumor rejection in the majority of the test mice. Furthermore, nearly 100% (27 of 28) of the tumor-free mice were able to reject a tumor rechallenge. While this approach is clinically attractive because of minimal tissue manipulation/culturing and the absence of infectious agents, a number of tested human primary tumors, including myeloma cells, have failed to produce high levels of GM-CSF after gene gun transfection. To circumvent the low transfection efficiency in certain human tumor cells, we showed that combining irradiated tumor cells to provide tumor antigens together with gene gun-transfected fibroblasts to provide GM-CSF induced effective tumor rejection. We also report that normal human skin fibroblasts transfected by the gene gun produce high levels of human GM-CSF (250 ng/10(6) cells/24 hr). These results suggest that combining irradiated tumor cells with gene gun-transfected fibroblasts results in antitumor immune responses and may allow for a wider application of this approach to cancer immunotherapy.

3T3 Cells↗
PubMed1998

Defining the role of novel high-dose chemotherapy regimens for the treatment of high-risk breast cancer.

We have explored several novel high-dose combinations in an attempt to increase antitumor activity while decreasing treatment-related toxicity. From October 1989 through June 1997, we performed phase I/II dose-escalation trials exploring novel high-dose regimens including ifosfamide/carboplatin/etoposide, mitoxantrone/thiotepa, and paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ)/mitoxantrone/thiotepa. We have also evaluated busulfan/cyclophosphamide and cyclophosphamide/thiotepa/carboplatin in phase II trials. Three hundred ninety-three patients have been treated in these trials and followed for a minimum of 3 months. Event-free survival (including relapses and treatment-related mortality; +/-SE) at 3 years by stage and chemosensitivity is as follows: stage II, four to nine positive nodes (n=16), 52%+/-17%; stage II, greater than nine nodes (n=30), 46%+/-11%; stage III (n=59), 50%+/-8%; inflammatory stage III (n=15), 27%+/-17%; stage IV, anthracycline responsive (n=69), 19%+/-5%; stage IV, anthracycline refractory but responsive to salvage therapy with ifosfamide, carboplatin, and etoposide or paclitaxel (n=53), 12%+/-6%; stage IV, refractory (n=128), 5%+/-2%; and stage IV, not evaluable for response (n=23), 10%+/-8%. Treatment-related mortality was 4% for both phase I and II studies involving stage II breast cancer patients, 5% for stage III breast cancer, 15% for inflammatory breast cancer, and 18% for all stage IV breast cancers, responsive and refractory. We conclude that high-dose therapy for the treatment of high-risk early stage breast cancer or metastatic breast cancer results in durable remissions. Chemosensitivity to induction regimens remains the most important prognostic indicator, although long-term survival has been seen even in patients with highly refractory disease. Further studies are necessary to define optimal high-dose strategies based on stage and chemosensitivity of disease.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1997

Retrovirus-mediated gene transfer of ornithine-delta-aminotransferase into keratinocytes from gyrate atrophy patients.

Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferase (OAT). The strategy of using an autologous keratinocyte graft, modified to express high levels of OAT as an ornithine-catabolizing skin-based enzyme sink, is investigated. Two OAT-containing retroviral vectors were constructed with or without a resistance gene. When packaged in a retroviral vector particle generated with the gibbon ape leukemia (GALV) virus envelope (PG13), these vectors could readily transduce >50% of target keratinocytes. The transduced keratinocytes in culture expressed up to 75-fold more OAT than normal control keratinocytes and these gene-modified cells extracted [14C]ornithine more efficiently than controls. The vector prepared without neo transduced cells more efficiently and led to higher levels of OAT expression than the neo-containing vector. Ornithine catabolism was maintained at high levels when the transduced patient keratinocytes were differentiated in vitro as a multilayered cutaneous organoid.

3T3 Cells↗
PubMed1997

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase II alpha associated with nuclear matrices from wild-type and drug-resistant chinese hamster ovary cell lines.

Topo II alpha is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo II alpha associated with the nuclear matrix in drug-resistant SMR16 and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo II alpha in individual nuclear matrices. There are significant variations in topo II alpha amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix-associated topo II alpha than the resistant cell line matrices. Nuclear matrix-associated topo II alpha from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo II alpha in the same matrix; in fact they were inversely related. In situ whole-mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures.

Animals↗
PubMed1997

Identification and characterization of P2Y2 nucleotide receptors in human retinal pigment epithelial cells.

P2 nucleotide receptor expression in cultured human retinal pigment epithelial (RPE) cells was investigated using the photoaffinity ATP analog BzATP, polymerase chain reaction of reverse-transcribed RNA (RT-PCR) and fura-2 fluorescence measurement of changes in intracellular free calcium concentration ([Ca2+]i). In experiments carried out in RPE cells at passage 10-15, addition of micromolar concentrations of ATP, UTP, and ATPgammaS to RPE cells resulted in a rapid, transient 3.5-fold increase in [Ca2+]i followed by a prolonged elevation that was twofold above the original baseline. Similar results were obtained from cells at passage 2. Characteristics of nucleotide-stimulated calcium mobilization in RPE cells, including partial inhibition by pertussis toxin, suggest that a G protein-coupled receptor mediates this response. Consistent with the expression of a P2Y2 nucleotide receptor subtype in RPE cells, [alpha-32P]BzATP labeled a 53-kDa protein in plasma membranes, and RT-PCR revealed the presence of P2Y2 receptor RNA. Adenosine had no effect on [Ca2+]i in RPE cells, indicating that the A2 subtype of P1 receptor described previously in human RPE is not involved in the response to nucleotides. Together the results indicate that human RPE cells express functional P2Y2 nucleotide receptors.

Adenosine↗
PubMed1997

Interleukin-10 immunoadhesin production by a replication-defective adenovirus.

The present study examined the use of replication-defective adenovirus for in vitro production of an immunoadhesin. A recombinant adenovirus, rendered replication defective by deletion of the E1 gene, was constructed to contain the murine interleukin-10 gene fused in frame with the hinge, CH2, and CH3 domains of the murine immunoglobulin gamma 1 heavy chain constant region gene under the control of the human cytomegalovirus promoter. The resultant recombinant virus, Ad5.hCMV.mIL-10:HFc, was used to transduce several cell types. The expressed protein, mIL-10:HFc, is secreted as a disulfide-bonded homodimer. In vitro, a murine pro-B-cell line expressing transfected recombinant murine interleukin-10 receptor proliferated in response to purified mIL-10:HFc. The results obtained demonstrate the relative ease of production of an immunoadhesin using replication-defective adenovirus.

Adenoviridae↗
PubMed1997

Potentiation of etoposide-induced apoptosis by staurosporine in human tumor cells is associated with events downstream of DNA-protein complex formation.

UNLABELLED: Protein kinase inhibitors have demonstrated potential for use in the therapy of human cancers, in particular leukemia. Staurosporine, a protein kinase inhibitor with broad specificity, enhances the cytotoxic effects of various antitumor agents with different modes of action. The topoisomerase II inhibitor, etoposide, has shown clinical activity against a wide range of tumor types. PURPOSE: The purpose of this study was to assess the effects of staurosporine on etoposide-induced cell death processes in a human tumor of epithelial origin. METHODS: Modulation of etoposide-induced apoptosis by staurosporine in HeLa cells was assessed by cell morphology, extraction of low molecular weight DNA, quantitation of DNA-protein complexes, and measurements of rates of DNA synthesis. The effects on cellular genes implicated in apoptosis were determined by Northern and Western blotting, along with assays of cyclin-dependent kinase activities. RESULTS: Staurosporine exhibited a two- to three-fold potentiation of apoptosis caused by etoposide in HeLa cells when applied concurrently, or immediately following etoposide removal, but did not alter the quantity of DNA-protein complexes produced by etoposide. Etoposide-induced apoptosis, and its potentiation by staurosporine, were associated with reduced c-myc expression, and a moderate increase in p21WAF1/CIP1 mRNA and protein levels. Inhibitors of cyclic AMP-dependent protein kinase and protein kinase C, which exhibit greater specificity than staurosporine, were without effect on apoptosis caused by etoposide, whereas use of the tyrosine phosphatase inhibitor, vanadate, resulted in its abrogation. The potentiation of etoposide-induced apoptosis by staurosprine was associated with a significant increase in cyclin A-dependent kinase activity. In addition, etoposide caused substantial inhibition of DNA synthesis. CONCLUSION: These results indicate that staurosporine potentiates apoptosis through events which occur downstream of DNA damage, and implicate unscheduled activation of cyclin A-dependent kinase during inhibition of DNA synthesis as a possible cause.

Apoptosis↗
PubMed1997

High-dose chemotherapy and autologous peripheral blood stem cell transplantation in patients with multiple myeloma and renal insufficiency.

Six patients with multiple myeloma and chronic renal insufficiency (serum creatinine >3.0 mg/dl), including four on dialysis, received high-dose busulfan and cyclophosphamide (BUCY) followed by autologous peripheral stem cell transplantation. Peripheral blood stem cells were collected after priming with cyclophosphamide, etoposide and G-CSF. Patterns of engraftment and toxicities were not apparently different from those seen in myeloma patients with normal renal function. There was one toxicity-related death, resulting from a massive spontaneous subdural hematoma. One patient died of disease progression 6 months after transplant, while the remaining four patients are alive and free of myeloma progression 6 to 39 months after high-dose therapy. Two of these patients have remained in complete remission for 28 and 39 months. Our experience suggests that high-dose therapy with BUCY and autologous peripheral blood stem cell rescue is feasible in patients with multiple myeloma and renal failure.

Adult↗
PubMed1997

Ornithine-delta-aminotransferase expression and ornithine metabolism in cultured epidermal keratinocytes: toward metabolic sink therapy for gyrate atrophy.

There is now strong evidence that the chorioretinal degeneration associated with ornithine-delta-aminotransferase (OAT) deficiency is a consequence of hyperornithinemia. Therefore development of a metabolic system for clearing ornithine from the circulation is being pursued as a potential treatment. The skin is considered an attractive location for such a metabolic system because autologous cells can be safely and easily utilized. This study was undertaken to determine the ornithine metabolizing capacity of epidermal keratinocytes expressing normal and superphysiologic amounts of OAT. The data show that overexpression of OAT in keratinocytes cultured from a gyrate atrophy patient restores ornithine metabolism and results in a rate of ornithine disappearance from the medium that is significantly higher than the rate of disappearance from the medium bathing normal keratinocytes. In addition, OAT activity determined in soluble protein prepared from sonicates suggests that the capacity to maintain plasma ornithine within the normal range is contained within an accomplishable graft of keratinocytes overexpressing OAT. However, the actual rate of ornithine disappearance from the media was significantly less than predicted from enzyme activity assays. Following ornithine metabolite production by intact cells suggests that ornithine metabolism is limited primarily by clearance of downstream metabolites, as opposed to substrate delivery.

Adenoviridae↗
PubMed1997

Drug resistance to DNA topoisomerase I and II inhibitors in human leukemia, lymphoma, and multiple myeloma.

Several antineoplastic agents used in the treatment of hematologic malignancies exert their cytotoxic effects by inhibiting the activity of nuclear DNA topoisomerase (topo) I or II. Mechanisms of drug resistance to topoisomerase inhibitors have been defined at the molecular level from in vitro studies using model cell lines, and include quantitative and qualitative changes in topo I and II. The possible roles of these mechanisms in clinical drug resistance and clinical outcomes for patients with hematologic malignancies are now under investigation. Available data indicate that the blast content of topo II does not correlate with clinical outcome in acute myeloid leukemia (AML), and this may also be true in acute lymphocytic leukemia (ALL). Chronic lymphocytic leukemia (CLL) cells are resistant to topo II inhibitors because they express low levels of topo II. Further studies using sequential biopsy samples and assays of topoisomerase activity should establish the role that changes in topo I and II activity play in the development of drug resistance in hematologic malignancies.

Antineoplastic Agents↗
PubMed1996

Verapamil suppresses the emergence of P-glycoprotein-mediated multi-drug resistance.

Selection protocols were designed to determine whether non-cytotoxic chemomodifiers can influence the evolution of the drug-resistant phenotype. To this end, the human multiple myeloma cell line RPMI 8226 (8226/S) was selected with either doxorubicin, verapamil or doxorubicin plus verapamil. Using this approach low-level multi-drug-resistant (MDR) cell lines were obtained when 8226/S was selected with doxorubicin only or doxorubicin plus verapamil but not with verapamil only. The MDR phenotypes obtained were mechanistically distinct. In doxorubicin only-selected cells (8226/dox4), drug resistance was mediated by over-expression of the MDR1 gene and its cognate protein P-glycoprotein. In contrast, the drug resistance seen in the doxorubicin plus verapamil-selected cells was mediated through decreases in topoisomerase II protein levels and catalytic activity and not by P-glycoprotein over-expression. Cells selected with verapamil alone did not become resistant to any of the drugs tested. None of the 3 selected cell lines showed any changes in MRP gene expression when compared with 8226/S. Our results indicate that the inclusion of verapamil during drug selection with doxorubicin influences the drug-resistant phenotype by preventing the selection of MDR1/P-glycoprotein-positive cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Preservation of amplitude modulation coding in the presence of background noise by chinchilla auditory-nerve fibers.

Sound envelope temporal fluctuations are important for effective processing of biologically relevant acoustic information including speech, animal vocalizations, sound-source location, and pitch. Amplitude modulation (AM) of sound envelopes can be encoded in quiet with high fidelity by many auditory neurons including those of the auditory nerve (AN) and cochlear nucleus. From both neurophysiological and clinical perspectives, it is critical to understand the effects of background masking noise on the processing of AM. To further this goal, single-unit recordings were made from AN fibers in anesthetized chinchillas. Units were classified according to spontaneous firing rate (SR) and threshold. Best frequency (BF) pure-tone bursts and AM (10-500 Hz) tone bursts were employed as stimuli at several sound levels, both in quiet and in the presence of a continuous wideband noise. It was found that (1) in quiet, low SR AN fibers show the strongest AM coding, followed in order by medium SR and high SR fibers, respectively. (2) AN units of all three classes generally preserve their AM coding even in the presence of loud (0 or +6 dB S/N) background noise and at high sound levels (over 75 dB SPL). (3) This preservation is usually achieved by lowering the average firing rate proportionately to decreases in the synchronous (fundamental frequency) response. (4) For a few AN fibers, the AM coding increases or is reduced in the presence of the background noise. These findings suggest that AN preservation of AM coding in the presence of a continuous masking noise results from shifts in the operating ranges and firing rates of AN fibers resulting from cochlear nonlinearities and adaptive mechanisms.

Animals↗
PubMed1996

A regulatory role in mammalian salivary glands for 5-hydroxytryptamine receptors coupled to increased cyclic AMP production.

Although a functional role for serotonin (5-hydroxytryptamine, 5-HT) has been defined in the salivary glands of some lower species, relatively few data supporting a role for 5-HT in the regulation of mammalian salivary glands have been presented. Our initial results from polymerase chain reaction studies in cells of mammalian submandibular gland origin using consensus sequence primers from G protein-coupled receptors suggested the presence of mRNA for a 5-HT receptor in these cells. Based on this observation, the question of a role for 5-HT in mammalian submandibular gland function was re-addressed, using isolated, perfused rat submandibular glands and dispersed-cell aggregates from this gland. In perfused glands, 5-HT decreased the rate of saliva flow initiated by acetylcholine by about 50% and increased the amount of protein in the saliva two-fold. In dispersed-cell aggregates, 5-HT elicited a concentration-dependent increase in the accumulation of adenosine 3',5' monophosphate (cyclic AMP; EC50 = 660 +/- 110 nM). In addition, functional studies, as well as radioligand binding experiments, indicated that the effects of 5-HT are independent of beta-adrenoceptors. Accumulation of cAMP in gland cells was consistent with a direct action of 5-HT on adenylyl cyclase. Similar cyclic AMP responses to 5-HT were observed in cells isolated from mouse and opossum submandibular glands and rat sublingual and parotid glands. Our findings suggest the presence of a 5-HT receptor in mammalian salivary glands coupled to the stimulation of adenylyl cyclase and, at least in rat submandibular gland, involved in modifying the volume and protein content of saliva.

Animals↗
PubMed1996

Adenovirus-mediated gene transfer of ornithine aminotransferase in cultured human retinal pigment epithelium.

PURPOSE: To evaluate the efficacy of adenovirus mediated transfer of ornithine delta-aminotransferase (OAT) into human retinal pigment epithelial (RPE) cells. METHODS: Adenovirus-mediated gene transfer into primary cultures of human RPE was evaluated by measurement of enzyme activity in whole cell extracts and by Western blot analysis. To assess mitochondrial integrity, succinate dehydrogenase activity was measured in transduced RPE cells. Expression of adenovirus early genes was evaluated using reverse transcription-polymerase chain reaction. RESULTS: OAT activity, which was 65 nmol/mg.hour in untransduced cells, could be increased to levels in excess of 20,000 nmol/mg.hour using an adenovirus vector carrying the OAT cDNA. There was, however, a significant reduction in succinate dehydrogenase activity associated with OAT activity greater than 12,000 nmol/mg.hour. Transduced human RPE displayed an altered morphology that appears to be a response to the vector because similar changes could be induced by an adenovirus vector that does not carry the OAT cDNA. Adenovirus early gene expression was detected in transduced RPE. CONCLUSIONS: This study represents a first step in the development of intraocular gene replacement therapy for the treatment of gyrate atrophy. The authors demonstrate that adenovirus is an efficient vehicle for the delivery of OAT into human RPE and that RPE will tolerate greater than a 150-fold increase in OAT-specific activity. Evidence for disruption of mitochondria when OAT activity exceeds 12,000 nmol/mg.hour and vector-induced toxicity indicate that more controlled transgene expression and refinement of the vector systems is needed.

Adenoviruses, Human↗
PubMed1995

Alterations in the topoisomerase II alpha gene, messenger RNA, and subcellular protein distribution as well as reduced expression of the DNA topoisomerase II beta enzyme in a mitoxantrone-resistant HL-60 human leukemia cell line.

A human HL-60 leukemia cell line selected for resistance to mitoxantrone, HL-60/MX2, displays cross-resistance only to agents whose cytotoxicities result from interaction with the nuclear enzyme DNA topoisomerase II (topo II). The topo II catalytic activity is reduced 2-fold in the drug-resistant cell line in association with the absence of the M(r) 180,000 isoform of topo II and the finding of novel M(r) 160,000 topo II alpha-related immunoreactive protein in these cells by immunoblot. The topo II alpha (M(r) 170,000) protein levels in nuclear extracts from the HL-60/MX2 cells were noted on average to be approximately 40% lower than in comparable HL-60 nuclei. Studies of the subcellular localization of topo II by immunohistochemical and fractional extraction techniques demonstrated that the M(r) 160,000 topo II alpha-related protein is primarily localized in the cytoplasm. Levels of the 6.3-kilobase topo II alpha mRNA were noted to be reduced 2-fold in the HL-60/MX2 cells in association with the finding of a novel 4.8-kilobase topo II alpha-related mRNA transcript that was present in HL-60/MX2 but not HL-60 cells. The absence of topo II beta protein in nuclear and whole cell extracts from the HL-60/MX2 cells was associated with the virtual absence of detectable topo II beta mRNA in those cells by Northern blot analysis. Using a reverse transcription-PCR assay we were able to demonstrate the presence of very low levels of topo II beta mRNA in HL-60/MX2 cells, representing < 1% of that found in the HL-60 cells. In contrast, the nuclear catalytic activity and cellular mRNA levels of the related nuclear enzyme DNA topoisomerase I were nearly identical in the two cell types. Southern blot analysis of DNA extracted from the drug-sensitive and drug-resistant cells revealed a structural alteration in one topo II alpha allele in the HL-60/MX2 cells, but there was no evidence of rearrangement or hypermethylation of the topo II beta locus. These results indicate that the reduced levels of topo II alpha and beta isoenzymes observed in mitoxantrone-resistant HL-60/MX2 cells are related to changes in the levels of their respective mRNA transcripts. The identification of structural changes in one topo II alpha allele in the HL-60/MX2 cell line suggests that the altered allele may serve as the source of the unique 4.8-kilobase topo II alpha-related mRNA transcript and the M(r) 160,000 protein discovered in those cells.

Base Sequence↗
PubMed1994

Phosphorylation of the alpha- and beta-isoforms of DNA topoisomerase II is qualitatively different in interphase and mitosis in Chinese hamster ovary cells.

Qualitative differences between interphase and mitotic topoisomerase II were studied in Chinese hamster ovary cells. Differences in sites of phosphorylation of in vivo 32P-labeled topoisomerase II alpha were observed between mitosis and interphase by one-dimensional phosphopeptide mapping of partial tryptic digests. Two-dimensional phosphopeptide mapping of complete trypsin digests revealed two phosphopeptides unique to interphase and three phosphopeptides unique to mitosis. A reduced electrophoretic mobility on denaturing gels (approximately 190 kDa) was observed for the beta-isoform of topoisomerase II in mitosis relative to interphase. Treatment of lysates with alkaline phosphatase demonstrated that this was due to phosphorylation of mitotic topoisomerase II beta. The existence of interphase- and mitosis-specific sites of phosphorylation of topoisomerase II alpha, along with the electrophoretic mobility shift caused by phosphorylation of topoisomerase II beta in mitosis, demonstrates qualitative differences between interphase and mitosis in the phosphorylation state of both isoforms of topoisomerase II.

Animals↗
PubMed1994

Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy.

The Cl- secretory pathway that is defective in cystic fibrosis (CF) can be bypassed by an alternative pathway for Cl- transport that is activated by extracellular nucleotides. Accordingly, the P2 receptor that mediates this effect is a therapeutic target for improving Cl- secretion in CF patients. In this paper, we report the sequence and functional expression of a cDNA cloned from human airway epithelial (CF/T43) cells that encodes a protein with properties of a P2U nucleotide receptor. With a retrovirus system, the human airway clone was stably expressed in 1321N1 astrocytoma cells, a human cell line unresponsive to extracellular nucleotides. Studies of inositol phosphate accumulation and intracellular Ca2+ mobilization induced by extracellular nucleotides in 1321N1 cells expressing the receptor identified this clone as the target receptor in human airway epithelia. In addition, we independently isolated an identical cDNA from human colonic epithelial (HT-29) cells, indicating that this is the same P2U receptor that has been functionally identified in other human tissues. Expression of the human P2U receptor (HP2U) in 1321N1 cells revealed evidence for autocrine ATP release and stimulation of transduced receptors. Thus, HP2U expression in the 1321N1 cell line will be useful for studying autocrine regulatory mechanisms and in screening of potential therapeutic drugs.

Adenosine Triphosphate↗