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Biomedical subjects

J A Roth

Publications and source records attributed to J A Roth.

At least 253 records · Page 14Linked to original sources
PubMed1994

High-efficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus.

A replication-defective and helper-independent recombinant p53 adenovirus was generated. The virus, Ad5CMV-p53, carries an expression cassette that contains human cytomegalovirus E1 promoter, human wild-type p53 cDNA, and SV40 early polyadenylation signal. Four human non-small-cell lung cancer cell lines representing differences in p53 configuration were used to evaluate the Ad5CMV-p53 virus. In the H358 cell line, which has a homozygous deletion of p53, the p53 gene was transferred with 97% to 100% efficiency, as detected by immunohistochemical analysis, when the cells were infected with Ad5CMV-p53 at a multiplicity of infection of 30 to 50 plaque-forming units/cell. Western blots showed that the p53 protein was expressed at a high level. The protein expression peaked at day 3 after infection and lasted for at least 15 days. Growth of the Ad5CMV-p53 virus-infected H358 cells was inhibited 79%, whereas that of noninfected cells or the cells infected with the control virus was not inhibited. Growth of cell line H322, which has a point mutation in p53, was inhibited 72% by Ad5CMV-p53, while that of cell line H460 containing wild-type p53 was less affected (28% inhibition). Tests in nude mice demonstrated that tumorigenicity of the Ad5CMV-p53-treated H358 cells was greatly inhibited. In a mouse model of orthotopic human lung cancer, the tumorigenic H226Br cells, with a point mutation in p53, were inoculated intratracheally 3 days before the virus treatment. Intratracheal instillation of Ad5CMV-p53 prevented tumor formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed1994

In vivo adenoviral-mediated human p53 tumor suppressor gene transfer and expression in rat liver after resection.

BACKGROUND: The aim of this study was to establish a clinically relevant model for gene transfer to liver with an adenoviral vector encoding wild-type p53 as a first step toward use of this class of gene products in the treatment of primary and metastatic liver tumors. METHODS: Full-size or 50% hepatectomized rat livers were subjected to asanguineous portal perfusion with a replication-defective adenoviral vector encoding wild-type p53 (Ad5p53), whereas control animals received adenoviral vector encoding Escherichia coli beta-galactosidase (beta-gal) (Ad5LacZ) or Ringer's lactate only. Liver biopsy specimens, blood samples, and liver weight were serially obtained. Gene transfer and expression were confirmed by X-Gal staining for gamma-gal, DNA/RNA polymerase chain reaction, (PCR) and Western blots for p53 and beta-gal. Liver integrity was assessed by histologic findings, serum transaminase levels, and synthetic function. RESULTS: The gene transfer rate in whole liver and after hepatectomy ranged from 20% to 40%. DNA PCR showed Ad sequences in livers transduced with Ad5p53 and Ad5LacZ. RNA PCR and Western blot confirmed expression and production of recombinant wild-type p53. Liver regeneration was not affected by p53 gene transduction. Liver histologic findings and synthetic function were not different between transduced and control groups. CONCLUSIONS: Ad5p53 gene transfer to full-size or hepatectomized livers is efficient. Liver regeneration and hepatocyte function are unaffected by overexpression of p53. Adenovirus-mediated tumor-suppressor transduction of the liver is a safe and promising adjuvant in cancer gene therapy.

Adenoviridae↗
PubMed1994

The cell and molecular biology of esophageal carcinoma.

An analysis of the molecular events underlying the development of esophageal cancer may yield new strategies for diagnosis, prevention, and therapy. Oncogenes and tumor suppressor genes have been implicated in the development of esophageal cancer. Tumor suppressor gene mutations are present in Barrett's epithelium, the premalignant lesion for adenocarcinoma. Early genetic changes may predict the development of invasive cancer. Recent data suggest that reversal of single gene abnormalities can reduce the tumorigenic potential of cancer cells.

Adenocarcinoma↗
PubMed1993

Influence of left ventricular function on survival and mode of death after implantable defibrillator therapy (Cleveland Clinic Foundation and Montefiore Medical Center experience).

To determine the influence of left ventricular (LV) function on survival and mode of death in patients with an implantable cardioverter-defibrillator (ICD), sudden death, surgical mortality, total arrhythmia-related death, total cardiac death and total death were retrospectively evaluated in 377 consecutive patients. The outcomes were also compared between patients with an LV ejection fraction > or = 30% (214 patients, group 1) and < 30% (148 patients, group 2). Surgical mortality was 3.9% (1.8% in group 1, 7% in group 2). During the follow-up of 25 +/- 20 months, actuarial survival rates of all patients at 3 years were 96% for sudden deaths, 81% for total cardiac deaths and 74% for total mortality. When the 2 groups were compared, survival rates of groups 1 and 2 at 3 years, respectively, were 99 and 90% for sudden death (p < 0.05), 97 and 84% for sudden death and surgical mortality (p < 0.01), 94 and 80% for the total arrhythmia-related death (p < 0.001), 88 and 68% for total cardiac death (p < 0.0001), and 81 and 62% for total mortality (p < 0.002). In group 2, 73% of total cardiac deaths within 1 year were causally related to the arrhythmia. Thus, in patients with an ICD, sudden death rates were very low. However, total cardiac death and total death rates were relatively higher. The outcomes of patients with an ICD were strongly influenced by the degree of LV dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

Actuarial Analysis↗
PubMed1993

Sequence-specific interaction of a conformational domain of p53 with DNA.

Mutations within a conserved "conformational" domain of the p53 protein have frequently been observed in a wide variety of human cancers. A hybrid protein containing the wild-type conformational domain of p53 fused to protein A bound to calf thymus DNA and a specific p53 DNA-binding motif. Hybrid proteins containing mutations in p53 bound to DNA less efficiently than wild-type hybrid protein. In addition, competition experiments showed that mutated p53 DNA-binding motif failed to interact with p53 hybrid proteins. The DNA-binding activity of wild-type p53 hybrid protein was inhibited by the metal chelator 1,10-phenanthroline. These results demonstrate that DNA-binding activity resides in the conformational domain of p53, providing a structural model for disruption of DNA binding by mutation. Furthermore, metal ions may regulate binding of p53 to DNA by modulating its conformation.

Animals↗
PubMed1993

A codon 248 p53 mutation retains tumor suppressor function as shown by enhancement of tumor growth by antisense p53.

Codon 248 in domain iv of the highly conserved region of the p53 gene is a frequent site of mutations associated with sporadic cancers and the familial cancer syndrome (Li-Fraumeni syndrome). Therefore, a characterization of the functional significance of a codon 248 mutation is of interest. We used antisense RNA methodology to study the role of the wild-type and mutated p53 gene in cell growth and tumorigenesis. We introduced wild-type p53 complementary DNA in sense or antisense orientation under control of a beta-actin promoter into human non-small cell lung cancer cell line H322a which has a codon 248 mutation (G to T) and WTH226b which has wild type p53. The biological properties and p53 expression of stable G418-resistant clones were analyzed. We observed that in both cell lines antisense RNA expression significantly reduced p53 mRNA and protein production; it also caused increases in growth rate in cell cultures and in tumorigenicity in nu/nu mice for both cell types, suggesting that the mechanism by which p53 suppresses cell proliferation and tumorigenesis is not always abrogated by a codon 248 mutation.

Animals↗
PubMed1993

A retroviral wild-type p53 expression vector penetrates human lung cancer spheroids and inhibits growth by inducing apoptosis.

Multicellular tumor spheroids approximate the three-dimensional configuration of primary and metastatic tumors. The effects of retrovirus-mediated transduction of wild-type p53 (wt-p53) were studied on multicellular tumor spheroids of human non-small cell lung cancer cell lines H322a, the p53 gene of which is homozygously mutated at codon 248, and WT226b, which has endogenous wt-p53. The growth of WT226b spheroids was not affected by exogenous wt-p53 transduction; the growth of H322a spheroids, however, was significantly inhibited by the addition of wt-p53 virus stocks. Transduction of cells by the wt-p53 retroviral vector and penetration of multiple cell layers in H322a spheroids was demonstrated by in situ polymerase chain reaction/hybridization with the neomycin-resistant neo probe. Apoptotic changes indicating programmed cell death were observed in H322a spheroids treated with the wt-p53 virus. These results suggest that retroviral vectors can penetrate into multiple cell layers of three-dimensional tumor masses and induce potentially therapeutic effects.

Apoptosis↗
PubMed1993

Prevention of orthotopic human lung cancer growth by intratracheal instillation of a retroviral antisense K-ras construct.

An orthotopic human lung cancer model in nu/nu mice was used to study the effect of an antisense K-ras (AS-K-ras) retroviral construct on tumor growth in vivo. A 2-kilobase genomic AS-K-ras DNA fragment linked to a beta-actin promoter was cloned into the LNSX retroviral vector. The recombinant construct was packaged into GP+envAm12 cells and titers greater than 10(6) colony-forming units/ml were obtained. Irradiated (350 cGy) nu/nu mice were first inoculated intratracheally with 10(5) H460a human large cell lung carcinoma cells which have a codon 61 mutation of the K-ras oncogene. Three days later they received intratracheal instillation of viral supernatant (5 x 10(6) colony-forming units/ml) from either LNSX, LNSX-AS-K-ras, LNSX-sense-K-ras producer cells, or medium daily for 3 days. At autopsy, 30 days after tumor cell inoculation, 90% of the control mice had tumors whereas 87% of mice treated with the LNSX-AS-K-ras viral supernatant were free of tumors. The efficacy of the viral supernatant was dose dependent. Intratracheal administration of retroviral LNSX-AS-K-ras supernatant prevents the growth of human lung cancer cells implanted orthotopically in nu/nu mice.

Animals↗
PubMed1993

Manganese induces spreading and process outgrowth in rat pheochromocytoma (PC12) cells.

Mn2+ has been shown to promote cell-substrate adhesion and cell spreading in many cell culture systems. In this study, we present data demonstrating that Mn2+ not only promotes spreading, but also induces process outgrowth in rat pheochromocytoma (PC12) cells. In the presence of 1.0 mM MnCl2, cell spreading was apparent by 6 hr, and nearly 50% of the exposed cells extended neurite-like processes. These morphological effects of Mn2+ were both time- and dose-dependent. In the presence of cycloheximide, a protein synthesis inhibitor, both Mn(2+)-induced spreading and neurite outgrowth were prevented, indicating that de novo protein synthesis is required for the effects of Mn2+ to take place. Of the other divalent cations tested, Mg2+, Cd2+, Cu2+, Ni2+, and Zn2+ were ineffective, and only Co2+ partially mimicked the effects of Mn2+. Although Mn(2+)-induced cell adhesion and spreading have been extensively studied, this is the first report that this divalent cation can cause neurite outgrowth. The neurite outgrowth-promoting effects of Mn2+ were distinct from those of nerve growth factor in that the response to Mn2+ was considerably more rapid, but apparently lacked the ability to sustain continuous outgrowth and networking of neurites. Mn2+ also induced the levels of GAP-43 and peripherin, two proteins associated with neuronal differentiation of PC-12 cells. In cells grown in serum-free defined medium, Mn2+ was capable of promoting neurite outgrowth when the cells were plated on surfaces pretreated with normal growth medium, vitronectin, or fibronectin, while it failed to cause these morphological changes in cells plated on untreated or poly-D-lysine-coated substrata. Similarly, Mn2+ also promoted neurite outgrowth from rat sympathetic neurons attached to laminin-treated substrate, but had no effect on neurons maintained on substrate with polylysine only. The pentapeptide Gly-Arg-Gly-Asp-Ser nearly completely prevented the morphological effects of Mn2+ on PC12 cells. These findings are consistent with a hypothesis that Mn(2+)-mediated alteration of an RGD-dependent extracellular matrix-integrin interaction is responsible for the neuritogenic effects.

Amino Acid Sequence↗
PubMed1993

Discordant p53 gene mutations in primary head and neck cancers and corresponding second primary cancers of the upper aerodigestive tract.

Patients with primary head and neck malignancies have a 3-7% yearly incidence of second primary cancers. It is thought that these second primary cancers arise independently following exposure to a common carcinogen by a process that has been called field cancerization. Since mutations in the p53 tumor suppressor gene represent a genetic alteration occurring during the evolution of premalignant lesions to malignancies of the upper aerodigestive tract, we analyzed mutations in the p53 gene of patients with cancer of the head and neck who developed second primary tumors of the upper aerodigestive tract epithelium to test the field cancerization hypothesis. DNA was extracted from primary head and neck cancers and second primary cancers of 31 patients. DNA from exons 5-8 of the p53 gene was analyzed by the single strand conformation polymorphism technique to identify the locations of the mutations in different regions of the gene. DNA from 6 patients was also sequenced by the chain termination method to confirm the presence of mutations and determine the base substitutions. Twenty-one of the 31 patients had 1 or more p53 mutations. In all 21 cases the genetic lesions were discordant such that the presence or location of the mutations in the initial primary cancer differed from those of the second and third primary cancers. In each of the five patients with mutations in both primary tumors, the mutations occurred in different regions of the p53 gene. Of the other 16 patients, 8 had a p53 mutation in the first primary but not the subsequent primary cancer and the other 8 had no mutation in the initial primary but did have a mutation in subsequent primary cancers. Sequencing confirmed the single strand conformation polymorphism analysis and showed that 73% of the mutations were transitions. The discordant p53 mutations in second primary cancers arising in patients with primary epithelial cancer of the upper aerodigestive tract suggest that these cancers arise as independent events. These observations provide the first demonstration of a molecular basis for field cancerization effects in cancers of the upper aerodigestive tract.

Adult↗
PubMed1993

Enhancement of the increase in intracellular calcium concentration in stimulated neutrophils by Mycoplasma hyopneumoniae.

Neutrophils isolated from the peripheral blood of pigs free of infection with Mycoplasma hyopneumoniae were loaded with a fluorescent indicator (Fura-2) for detection of cytosolic free calcium concentration. The kinetics of the intracellular calcium flux were examined after incubation with or without a pathogenic or a non-pathogenic strain of M. hyopneumoniae. The basal intracellular calcium concentration was not altered by incubation with M. hyopneumoniae. However, the relative increase in cytoplasmic calcium concentration caused by the addition of opsonized zymosan was significantly (p < 0.05) higher in neutrophils incubated with M. hyopneumoniae as compared to neutrophils not incubated with M. hyopneumoniae. Additionally, after zymosan stimulation, the intracellular calcium concentration was greater in neutrophils incubated with a pathogenic strain of M. hyopneumoniae than in those incubated with a non-pathogenic strain. This suggests that M. hyopneumoniae alters the signal transduction mechanisms in neutrophils and that this alteration may be related to virulence.

Animals↗
PubMed1993

Extended resection of pulmonary metastases: is the risk justified?

Extended resection of pulmonary metastases by pneumonectomy or by pulmonary resection en bloc with chest wall or other thoracic structures (diaphragm, pericardium, superior vena cava) is infrequently performed as survival benefit is presumed low. Between 1981 and 1992, 38 patients underwent extended resection for pulmonary metastases (24 men, 14 women; average age, 48 years) from various primary neoplasms. Thirty-three patients (33/38, 87%) had complete resection. Five-year actuarial survival was 25.4%. Mortality was 5.3% (2/38) and occurred in patients undergoing pneumonectomy (2/19, 10.5%). Nineteen patients underwent pneumonectomy, and 19 patients had other assorted resections: pulmonary resection en bloc with chest wall in 11 and pulmonary resection en bloc with other thoracic structures in 8. Actuarial median survival (median, 27 months) did not differ between patients having pneumonectomy and those having pulmonary resection en bloc with chest wall or other thoracic structures. Initial disease-free interval (median) was no different between those patients undergoing pneumonectomy (32 months) or other type resection (35 months; p = 0.16). Median survival for extended resection as the initial operation for pulmonary metastases was 28 months compared with 14 months for all others (p = 0.095). Pneumonectomy for pulmonary metastases may be performed with operative risk equivalent to pneumonectomy for primary bronchogenic carcinoma. Patients may safely undergo extended resection of pulmonary metastases by pneumonectomy or in continuity with chest wall or other thoracic structures. Despite advanced localized metastatic disease, some patients achieve long-term survival after pneumonectomy and extended resection for pulmonary metastases.

Actuarial Analysis↗
PubMed1993

Effect of recombinant human cytokines on porcine neutrophil function.

The activity of four recombinant human cytokines on porcine neutrophils was evaluated. Porcine neutrophils were treated with varying doses of recombinant human tumour necrosis factor-alpha (rHu-TNF), interferon-gamma (rHu-IFN), interleukin-8 (rHu-lL-8), or granulocyte-macrophage colony-stimulating factor (rHu-GM-CSF). The function of treated neutrophils was compared with that of non-treated controls in the following assays: antibody-independent neutrophil cytotoxicity (AINC), antibody-dependent cell-mediated cytotoxicity (ADCC), iodination, Staphylococcus aureus ingestion, cytochrome C reduction, random migration, and chemotaxis. Treatment with rHu-TNF produced significant (P < 0.05) depression of neutrophil random migration (2.5, 25, and 250 ng ml-1 rHu-TNF) and iodination (250 ng ml-1) and a near significant (P = 0.08) depression in ADCC (250 ng ml-1). Treatment with 25,000 U ml-1 of rHu-IFN caused a significant increase in AINC. At lower doses of rHu-IFN, there was a trend (0.05 < P < or = 0.08) toward depression of AINC (250 U ml-1) and ADCC (25 U ml-1) and enhancement of iodination (250 U ml-1). Treatment with 50 ng ml-1 of rHu-IL-8 caused a near significant increase (P = 0.06) in AINC. There were no significant differences noted when porcine neutrophils were treated with rHu-GM-CSF (2.5-2500 U ml-1). No synergism was noted between rHu-TNF and rHu-IFN.

Animals↗
PubMed1993

In vitro detection of bovine immunodeficiency-like virus using monoclonal antibodies generated to a recombinant gag fusion protein.

An Escherichia coli recombinant fusion protein containing the major core protein of bovine immunodeficiency-like virus (BIV) was used to immunize mice for generation of monoclonal antibodies to BIV p26. Eight hybridomas specific for BIV p26 were identified and two antibodies, designated 104 and 142, were further characterized. Both 104 and 142 antibodies were isotyped as IgG1; they reacted specifically with both BIV p26 and the recombinant fusion protein in Western immunoblot analyses. However, the epitope specificity of the antibodies was different. Immunoperoxidase assays were used to determine if antibodies 104 and/or 142 could detect BIV replication in cell culture. Both antibodies were found to react with BIV-induced syncytia and individual BIV-infected cells. The antibodies were also used successfully in a focal immunoassay for quantitation of BIV-infected cells. These antibodies will provide valuable reagents for detection and quantitation of BIV replication in studies of viral pathogenesis and immunity.

Animals↗