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Biomedical subjects

H R Hubbell

Publications and source records attributed to H R Hubbell.

35 records · Page 2Linked to original sources
PubMed1984

Phosphoproteins altered by antiproliferative doses of human interferon- beta in a human bladder carcinoma cell line.

Phosphoproteins of control and IFN-beta treated human bladder carcinoma cells (RT4) were labelled in vitro with [32P]-ATP and analyzed by polyacrylamide gel electrophoresis. Cells treated with antiproliferative doses of IFN had reduced levels of phosphorylated 60 Kd and 40 Kd proteins. IFN also induced within 24 hours the modification of a low molecular weight phosphoprotein doublet in the 22-24,000 molecular weight range. The ability to phosphorylate high molecular weight proteins by the in vitro procedures was generally depressed by IFN treatment. There was a dramatic shift in the phosphorylation of alkali stable phosphoamino acids associated with proteins in the 43-50,000 molecular weight range in IFN-treated cells. Preliminary studies indicate that at least some of the IFN-induced modifications of cellular phosphoproteins may result from transcriptional control of specific oncogenes.

Adenosine Triphosphate↗
PubMed1984

Independent sensitivity of human tumor cell lines to interferon and double-stranded RNA.

The antiproliferative effect of human interferons (IFNs) and double-stranded RNAs (dsRNAs) was measured in eight human tumor cell lines, five of which were derived from carcinomas of the bladder. Dose-response curves were generated for a 72-hr treatment period. The concentration of interferon or dsRNA necessary to inhibit tumor cell growth 50% compared to untreated cells was generated by linear regression analysis of the dose-response data. In the seven of eight cell lines in which a direct comparison could be made, IFN-beta was a more potent inhibitor than IFN-alpha. Polyriboinosinic acid X polyribocytidylic acid consistently gave an increased antiproliferative response compared to its mismatched analogue, rln X r(C12,U)n. Correlations could not be made between either IFN-alpha or IFN-beta and the dsRNA effect. No correlation was seen between IFN or dsRNA sensitivity and cell type, ability to bind IFN, growth rate, or tumorigenicity in nude mice. The antiproliferative effect of dsRNA was studied in the presence of antibodies against IFN-beta in HT1080 Cl 4, a cell line sensitive to both IFN and dsRNA, and A2182, a cell line relatively resistant to IFN-beta but sensitive to dsRNA. In both cell lines, the anti-IFN-beta antibodies inhibited the antiproliferative effect of the dsRNAs. After treatment with a concentration of dsRNA necessary to inhibit tumor cell growth 50% compared to untreated cells, a concentration of IFN-beta necessary to inhibit tumor cell growth 50% was induced in the HT1080 Cl 4 cells; however, only a low level of IFN-beta was detected in the culture medium of the A2182 cells.

Cell Division↗
PubMed1983

Biological activity of mRNA immobilized on nitrocellulose in NaI.

In 12.2 molal NaI and at 25 degrees C or below, mRNA bound to nitrocellulose while DNA and rRNA did not. Neither the poly(A) tract nor the cap were required for binding. The immobilized RNA could be translated, reverse transcribed, hybridized with radioactive probes, or released for further manipulation. mRNA was efficiently transferred from polyacrylamide to nitrocellulose in NaI. Baking was not required to fix NaI-immobilized mRNA to nitrocellulose. When cells dissolved in 12.2 molal NaI were filtered through nitrocellulose, mRNA became selectively bound (quickblot). The quick-blot system utilizing protease and detergents to prepare cells for NaI solubilization was especially suitable in quantitative, rapid screening of cells for expression of specific genes. Expression of highly repeated DNA sequences was detected in human leukemia cells.

Collodion↗
PubMed1981

Differential expression of the globin genes in human leukemia K562(S) cells induced to differentiate by hemin or butyric acid.

Human leukemia K562(S) cells were induced to differentiate by 50 microM hemin or 1.4 mM butyric acid, and the types of hemoglobins synthesized were compared. In both cases, embryonal hemoglobins [Portland, Gower 1, Hb X, and fetal hemoglobin (Hb-F)] were detected. Butyric acid-treated K562(S) cells contained mostly Hb Gower 1 (zeta 2 epsilon 2) and a hemoglobin with the electrophoretic characteristics of Portland (gamma 2 zeta 2). For hemin-treated K562(S), the most abundant hemoglobin synthesized by Hb X (epsilon 2 gamma 2), and the second most abundant was Bart's (gamma 4). Traces of Gower 1 were observed in nontreated K562(S) cells. The kinetics of hemoglobin induction as a result of the two treatments differed; increased hemoglobin synthesis was detected after only 24 hr of hemin treatment, whereas 4 days were required in butyric acid-treated cells. Both hemin and butyric acid were able to induce their respective patterns of hemoglobin synthesis independent of the presence of serum in the K562(S) growth medium. Analysis of the globin chains in induced K562(S) cells induced to differentiate indicated that, with both inducers, adult alpha- but not beta-globin chains were present. Karyotype analysis of K562(S) cells revealed a nearly triploid chromosome complement with a modal number of 68 chromosomes. Three copies of chromosome 11 and four copies of chromosome 16 (coding for the beta-like and alpha-like globin genes, respectively) were present. A large marked chromosome, involving chromosome 7, and a Philadelphia chromosome were also seen. These data characterize the K562(S) subline and also indicate that hemin and butyric acid differ in their effects on the expression of embryonal globin genes.

Butyrates↗
PubMed1979

S1 nuclease definition of highly repeated DNA sequences in the Guinea pig, Cavia porcellus.

Native DNA of the Guinea pig, Cavia porcellus, purified from liver or tissue culture cells, was heat denatured and reassociated to a Cot value of 0.01 (equivalent Cot value of 7.2 x 10(-2)). The reassociated DNA was isolated by digestion with the single-strand DNA specific enzyme S1 nuclease. Spectrophotometric and radioactivity assays demonstrated that 24% of the total DNA was resistant to S1 nuclease treatment. Zero-time reassociation indicated that approximately 3% of the DNA was inverted repeat sequences. Thus, highly repeated sequences comprised 21% of the total genome. CsCl buoyant density ultracentrifugation indicated that this fraction was composed of both main band and satellite sequences. Although actinomycin D - CsCl density gradients failed to give significant separation of the repetitive sequences, distamycin A - CsCl gradients were able to fractionate the DNA into several overlapping bands. The heterogeneity of the repetitive DNA was further demonstrated by the first derivative plots calculated from their thermal denaturation profiles. This analysis revealed six major thermalytes which indicate that there may be at least six discrete components in the repetitive DNA.

Animals↗
PubMed1979

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.

Animals↗
PubMed1977

Identification of nucleolus organizer regions (NORs) in normal and neoplastic human cells by the silver-staining technique.

Silver nitrate has been used to demonstrate the chromosomal location of ribosomal cistrons in nine tissue-culture lines derived from human tumors of various pathological origins. Control individuals have a particular modal number (range 7--10) of D- and G-group chromosomes stained with silver. In the controls, 96.2% of the D- and G-group chromosomes that have a stalk show silver staining, while no relationship can be seen in acrocentric chromosomes without stalks. The tumor cells, whose modal chromosome numbers range from 42 to 68, possess variable numbers of acrocentrics (11--18). The number of chromosomes stained with silver, however, remained at control levels (range, 6--9). These data indicate that, in humans, silver staining may not identify all NORs that contain structural ribosomal genes.

Cell Line↗
PubMedDate not supplied

Dose-related effects of Ampligen (poly(I).poly(C12U)), a mismatched double-stranded RNA, in a bleomycin-mouse model of pulmonary fibrosis.

The antifibrotic effect of the mismatched double-stranded RNA, Ampligen (poly(I).poly(C12U)), was evaluated in a bleomycin-mouse model of pulmonary fibrosis. Mice received a single intratracheal dose of bleomycin (0.125 U/mouse) or saline (50 microL) at the beginning of the experiment, followed by 5 or 6 intraperitoneal injections of Ampligen (1.0, 5.0, 10.0, 15.0, or 25.0 mg/kg) or saline at regular intervals for 2 weeks. Ampligen did not produce increased mortality or weight loss by itself. However, it produced varying degrees of mortality in combination with bleomycin. Five injections of 10 mg/kg Ampligen or three injections of 25 mg/kg Ampligen plus three injections of 10 mg/kg Ampligen in combination with bleomycin .produced significant reductions in lung collagen accumulation as indicated by lung hydroxyproline content compared to the bleomycin control group. Animals receiving bleomycin plus Ampligen at all dosages had significantly reduced prolyl hydroxylase activity compared to the bleomycin control group. Lipid peroxidation and bronchoalveolar lavage fluid (BALF)-supernatant protein content for the groups receiving bleomycin plus Ampligen were not reduced compared to the bleomycin control group. In the BALF-supernatant, the activity of acid phosphatase, a lysosomal enzyme produced by neutrophils, monocytes, and macrophages, was significantly decreased in the group receiving bleomycin plus 10 mg/kg Ampligen. Also, selected BALF differential immune cell counts were reduced in some of the groups receiving bleomycin plus Ampligen, but not in a consistent or dose-dependent manner. The results of this study indicate that Ampligen can significantly reduce the bleomycin-induced increased collagen accumulation and may be therapeutically useful in the management of lung fibrosis in humans.

Acid Phosphatase↗
PubMedDate not supplied

Heterogeneous nuclear RNA from hairy cell leukemia patients activates 2',5'-oligoadenylate synthetase.

Interferon treatment of cells induces double-stranded RNA (dsRNA)-dependent 2',5' oligoadenylate (2-5A) synthetase, an enzyme which has been implicated in the mechanism of growth arrest in tumour cells. Since interferon (IFN) can inhibit the growth of cells that are not infected with virus, natural non-viral dsRNAs should be present in these cells which can activate 2-5A synthetase. If such nuclear dsRNAs are associated with the mechanism of growth control, cells inherently sensitive to growth inhibition by IFN should contain significant levels of 2-5A synthetase-activating dsRNAs. We measured the ability of size fractionated nuclear dsRNAs isolated from patients with hairy cell leukemia (HCL) to activate purified 2-5A synthetase. Peripheral blood mononuclear cells from HCL patients were utilized because of the inherent sensitivity of these patients to IFN treatment. The heterogeneous nuclear RNA fraction from four out of five HCL patients showed high levels of 2-5A synthetase-activating dsRNAs. The 2-5A formed contained biologically active trimers, tetramers, pentamers and hexamers as demonstrated by HPLC analysis and their ability to activate RNase L. In contrast, the nuclear RNA fraction from three out of four healthy controls were unable to activate 2-5A synthetase. These results indicate that natural, nuclear dsRNAs inherently exist in IFN-sensitive cells and imply that these molecules may play a role in the inhibition of cellular growth.

2',5'-Oligoadenylate Synthetase↗
PubMedDate not supplied

Differential effects of human natural interferon-alpha and mismatched double-stranded RNA against a human renal cell carcinoma xenograft.

The antitumor effects of natural human IFN-alpha and mismatched dsRNA against the human renal cell carcinoma cell line 786-0 were studied both in a clonogenic soft agar assay and in the nude mouse. The 786-0 cells were sensitive in vitro to the antiproliferative effects of IFN-alpha in a dose-response manner, up to 3000 IRU/ml. These cells were also sensitive, in a dose-dependent manner, to mismatched dsRNA in the clonogenic assay. Mismatched dsRNA was effective in inhibiting tumor growth (p less than 0.001) in nude mouse xenografts, with regression of the tumor mass seen in all animals. A significant increase in survival (p less than 0.001) was seen in the mismatched dsRNA treated group. In contrast, IFN-alpha did not inhibit tumor growth in vivo, even though significant titers of IFN-alpha (greater than 3,000 IRU/ml) were found in the serum shortly after treatment. Mismatched dsRNA did not induce the production of human IFNs by the tumor cells in vitro. Assays of mouse IFN induction and their in vitro antigrowth effects indicated that the in vivo antiproliferative effect of mismatched dsRNA was probably not due to potentiation of any direct effects by the induced mouse IFNs. Tumor growth inhibition appeared to occur, at least in part, from the significant augmentation (p less than 0.01) of natural killer cell activity by mismatched dsRNA, as measured in the spleen cells of treated mice. These results suggest that, although both IFN-alpha and mismatched dsRNA can be directly antiproliferative against this tumor, either the IFN-independent antitumor effects of mismatched dsRNA or the mismatched dsRNA-induced augmentation of the host immune response plays a major role in tumor regression. Potentially, both mechanisms may be important in this system.

Animals↗
PubMedDate not supplied

Synergistic inhibition of AZT-resistant HIV by AZT combined with poly(I):poly(C12U), without synergistic toxicity to bone marrow progenitor cell elements.

Mutation of human immunodeficiency virus (HIV) to drug resistance is an obstacle to HIV containment, and may account for the transitory nature of the improvement in CD4 cell counts of patients receiving azidothymidine (AZT). The emergence of AZT-resistant (AZTR) virus might be suppressed if a second therapeutic could be added; however, such a regimen would have to confer not only additional control over HIV replication but also no additional toxicity, especially to bone marrow progenitor cells. In the present study, HIV was isolated from patients receiving AZT alone and was studied for sensitivity to the mismatched double-stranded RNA, poly(I):poly(C12U) (ampligen). In addition, the combination of poly(I):poly(C12U) plus AZT was studied in vitro for toxicity to bone marrow CFU-GM and in patients receiving combined therapy for bone marrow toxicity. HIV isolated from patients receiving AZT alone showed higher resistance to AZT than wildtype virus, but remained sensitive to poly(I):poly(C12U). Poly(I):poly(C12U) and AZT were synergistic in inhibiting all isolates of HIV tested, regardless of their AZTR phenotype. Furthermore, the combination of poly(I):poly(C12U) and AZT showed no toxicity in vitro to bone marrow CFU-GM compared to AZT alone. In 11 HIV infected individuals receiving the combinational regimen, bone marrow function gradually improved. These results indicate that poly(I):poly(C12U) was active against AZTR HIV, synergistic with AZT and did not convey added toxicity.

Antiviral Agents↗
PubMedDate not supplied

RNase L and increased endoribonuclease activities in the mononuclear cells of patients with chronic myelogenous leukemia.

During investigations of the interferon-induced 2',5' oligoadenylate synthetase/RNase L system in malignancy, RNase L activity and an increased endoribonuclease activity were observed in peripheral blood mononuclear cell (PBMC) extracts from patients with chronic myelogenous leukemia. The cleavage of rRNA from intact ribosomes was used as the assay for both RNase L and the increased endoribonuclease activities. Novel rRNA cleavage products (NCP) were generated by extracts of Ficoll-purified mononuclear cells from chronic myelogenous leukemia (CML) patients and in the granulocytic fraction of both patients and healthy controls. Determination of the time course of rRNA degradation demonstrated that the novel cleavage products were rapidly derived from the further endoribonucleolytic degradation of the RNase L derived specific cleavage products. Prolonged incubation of mononuclear cell extracts from healthy controls also yielded the novel rRNA cleavage products. Comparisons of the kinetics of NCP production suggest that the novel endoribonuclease activity can be approximately 240-fold greater in PBMC extracts from CML patients than controls. Analysis of peripheral blood WBC count and differential indicated that the increased RNase activities were associated with the presence of immature granulocytic cells in the peripheral blood (p = 0.001, Fisher's exact test). However, these activities were also found in the mononuclear cells of a CML patient in lymphoid blast crisis. Since CML is a stem cell disease, the novel endoribonuclease activity may be indicative of active disease, rather than a marker for immature granulocytes. Thus, the RNase L and increased endoribonuclease activities may play a functional role in the biology of chronic myelogenous leukemia and may be important in the mechanism of action of interferon therapy in this disease.

Endoribonucleases↗