Howard R. Hubbell

Silver Staining as an Indicator of Active Ribosomal Genes

Silver nitrate has been used as a cytological stain since the late 1800s. A modification of the Bielschowsky technique preferentially stains nucleoli and chromosomal nucleolus organizer regions (NORs). The specificity of staining is related to the method of preparation of the cytological specimens. The silver binds proteins and may be associated with the phosphate groups of certain phosphoproteins. Biochemical analyses of nucleolar proteins indicate that a limited array of specific proteins bind silver. A number of investigations have demonstrated that silver staining is indicative of active ribosomal RNA transcription, although a minor component may be associated with the fibrillar centers of cells in which ribosomal genes are inactive. Silver staining is a simple, reliable cytological method for the demonstration of ribosomal gene activity.

Mismatched double-stranded RNA, ampligen (poly(I) : poly(C12U)), demonstrates antiviral and immunostimulatory activities in HIV disease

Mismatched double-stranded RNA (Ampligen) has broad spectrum antiviral and immunomodulatory activities. These activities generate stabilization or improvement in three important surrogate markers of HIV disease progression. Patients with HIV disease treated with Ampligen do not become positive for p24 antigen, in contrast to patients treated with AZT or placebo. Viral burden can also be decreased in patients receiving Ampligen/AZT therapy. In vitro studies indicate that both AZT sensitive and AZT resistant viruses can be inhibited by Ampligen alone and are synergistically inhibited by Ampligen in combination with AZT. The immunomodulatory effects of Ampligen are manifested as a stabilization of CD4 counts. When Ampligen is combined with AZT, an increase in CD4 count is seen. Furthermore, a return or increase in delayed type hypersensitivity to mumps, Candida, and trichophyton was seen in approximately 70% of patients treated with Ampligen. The activity of Ampligen in HIV disease is due to its multifunctional activity as an antiviral and immune stimulating agent. The antiviral effect directly inhibits HIV-infection and other viruses which have been implicated in HIV disease acceleration and progression. The immunomodulatory activity can stabilize, increase, or restore immune function. This enhanced immune function can also lead to the further inhibition of additional infections associated with disease progression. Thus, Ampligen has multiple mechanisms of action against HIV disease.

Differential antiproliferative actions of 2',5' oligo a trimer core and its cordycepin analogue on human tumor cells

The antiproliferative effect of 2′, 5′A3 core and 2′5′‐3′dA3 (cordycepin trimer) core was measured in 8 human tumor cell lines. Cells were treated in a dose‐response manner for 72 hr and the concentration of drug necessary to inhibit cell growth 50% (Gl50) was determined. A wide range of sensitivities to these drugs was found, even among tumors of the same histological type. The cell lines showed different sensitivities and dose‐response curves to the 2′, 5′A3 and 2′, 5′‐3′dA3 cores. Uptake studies of the 2′,5′A3 and 2′,5′‐3′ dA3 cores, using high‐pressure liquid chromatography , demonstrated that both cores were rapidly degraded in the tissue culture medium and taken up as adenosine or cordycepin, respectively. There was a direct correlation between the uptake of cordycepin and the antiproliferative effect. In contrast, there was no correlation between cell sensitivity and the uptake of the 2′,5′A3 core degradation products. Analysis of intracellular nucleosides and nucleotides indicated that differences in intracellular metabolism of adenosine might explain the different sensitivities of the various cell lines to 2′,5′A3 core. Molar equivalent concentrations of adenosine and cordycepin inhibited cell growth; however, equimolar concentrations of these nucleosides were not effective. In addition, the anti‐proliferative effect of both core compounds and their corresponding nucleosides could be potentiated by the addition of the adenosine deaminase inhibitor, deoxycoformycin . The results indicate that these cores act prodrugs and that the active metabolites are their corresponding nucleosides.

Phosphoproteins altered by antiproliferative doses of human interferon-β 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.

Sensitivities of human glioma cell lines to interferons and double-stranded RNAs individually and in synergistic combinations

SummaryThe antiproliferative effects of human interferons (IFNs) and double-stranded RNAs (dsRNAs) were studied in five human glioma cell lines. Dose response curves were generated over a 72 hour treatment period. The concentration of interferon or double-stranded RNA necessary to produce a 50% antiproliferative response (GI50) was calculated by linear regression analysis. Two cell lines were more sensitive to IFN-β than to IFN-α, one cell line was more sensitive to IFN-α than to IFN-β and two cell lines had approximately equal sensitivities to both interferons. All cell lines showed some sensitivity to either IFN-α or IFN-β. IFN-γ had no antiproliferative effect on any of the cell lines. In addition, only one of the cell lines displayed sensitivity to dsRNA, in which the response to poly(I) · poly(C) was greater than that to a mismatched analogue of poly(I) · poly(C), r(I)n · r(C12,U)n (Ampligen). There was no correlation between the sensitivities to type I IFNs (α and β), type II IFN (γ) or the dsRNAs. The antiproliferative effect of combinations of IFNs, or IFNs and Ampligen, was studied in one of the cell lines. A significant synergistic antitumor effect was seen with all of the IFN/Ampligen combinations (p < 0.02), including IFN-γ/Ampligen, even though these cells were resistant to IFN-γ alone. Synergy was also seen in the IFN-α/IFN-γ (p < 0.02) and IFN-β/IFN-γ (p < 0.05) combinations. The IFN-α/IFN-β combination gave an additive antitumor effect. These results indicate that IFN-α and IFN-β alone or combinations of type I IFNs, type II IFNs and Ampligen can be effective in inhibiting the growth of glioma cells.

Antitumor effects of interleukin-2 and mismatched double-stranded RNA, individually and in combination, against a human malignant melanoma xenograft

SummaryThe antitumor effects of recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA) were assessed in tissue culture and in a nude mouse model. Mismatched dsRNA did not show a direct antiproliferative effect against the human malignant melanoma cell line, BRO, in tissue culture. However, treatment of the BRO cells with up to 1000 units/ml rIL-2 in culture showed a slight increase in growth rate. Combined rIL-2/mismatched dsRNA treatment also demonstrated a similar slight enhancement of growth. Nude mice bearing subcutaneous tumors were treated by intraperitoneal injection of low doses (5000–20 000 units) of rIL-2 and mismatched dsRNA (500 µg). The in vivo tumor growth was significantly inhibited by the combined treatments (P <0.05) and survival was significantly increased (P <0.05). Measurement of cytotoxicity using splenocytes from treated animals showed significant augmentation of lytic activity against natural killer(NK)-sensitive YAC-1 cells in all rIL-2/mismatched dsRNA treatment groups, compared to the individual treatments or controls (P <0.05). Cytotoxicity of the splenocytes against the NK-resistant BRO cells was also augmented in animals treated with mismatched dsRNA and the highest rIL-2 dose utilized here (P <0.01). Renal, liver, and hematological toxicity was evaluated by measurement of blood urea nitrogen, creatinine, serum asparrtate aminotransferase, and a complete blood count with differential. There were no significant differences in these parameters in any of the treatment groups. Similarly, no differences in weight of the animals was seen in any treatment group. These results indicate that the combination of low-dose rIL-2 and mismatched dsRNA can potentiate host-mediated antitumor effects, yielding increased survival, without significant toxicity.

Implications of retroviral and oncogene activity in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a stem cell disease which, on a clinical level, progresses from the release from growth control of normally differentiated cells (a preleukemic state) to an acute leukemia. On a molecular level, the evolution of CML to acute leukemia is a multistep process. We propose that an early step, at the stem cell level, is acquisition of the ability for gene movement, which allows subsequent submicroscopic and chromosomal rearrangements that cause changes in the growth characteristics and regulation of the stem cell. A specific platelet DNA polymerase (PDP - reverse transcriptase) may play a role in gene movement. The characteristic reciprocal translocation of chromosomes #9 and #22, causing the activation of the c-abl oncogene, appears to be responsible for the uncontrolled cellular growth. Yet, other growth factors (e.g., platelet derived growth factor) and activated oncogenes (e.g., c-sis) must be responsible for the stimulation, progression, and variability seen during the course of the disease. Because CML is a progressive disease with clinically definable stages, CML appears to be a model system for the study of the molecular basis of the progression of preleukemia to leukemia specifically, and preneoplasia to aggressive neoplasia in general.

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.

Potentiated lymphokine-activated killer cell activity generated by low-dose interleukin-2 and mismatched double-stranded RNA

SummaryLymphokine-activated killer (LAK) cell activity was measured in human peripheral blood mononuclear cells (PBMC) treated in vitro for 3 days with recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA). Lytic activity was measured utilizing K562 (NK-sensitive) and 786-0 (NK-resistant) target cells. PBMC cultured with rIL-2 (10–1000 BRMP U/ml) alone showed concentration-dependent lytic activity against the 786-0 target cells, while cells cultured in unsupplemented medium or medium supplemented with mismatched dsRNA (200 µg/ml) alone could not lyse the 786-0 targets. The combination of mismatched dsRNA with suboptimal concentrations of rIL-2 (10–30 U/ml) showed enhancement of both natural killer (NK) and LAK cell activities. The uptake of [3H]thymidine by treated effector cells was dependent on time and rIL-2 concentration and was not increased in the cells treated with low-dose rIL-2/mismatched dsRNA, compared to those treated with low-dose rIL-2 or mismatched dsRNA alone. Similarly, changes in the expression of CD3, CD4, CD8, CD57, CD16 and CD25 cell surface antigens were dependent on rIL-2 concentration and not altered by the presence of mismatched dsRNA. These results indicate that mismatched dsRNA can potentiate rIL-2-induced LAK cell activity by increasing the functional activity per cell, rather than by increasing the number of activated cells.