The major allergen from timothy grass pollen, Phlp5b (Phleum pratense), was shown to exhibit ribonuclease activity. It turned out that the C-terminal portion of this molecule was the biologically active domain. Here evidence is presented that the allergen is a single-stranded, sugar-nonspecific nuclease with topoisomerase activity. An isomerase-specific active site was identified, and a non-active mutant was constructed by site directed mutagenesis, and showed no nucleolytic activity. In contrast to the wild type (WT), the mutant did not dimerize. Although the binding capacity of IgE antibodies toward the mutant was reduced as compared to the WT, the allergenic activity was retained. We conclude that the allergen Phlp5b is a single-stranded nuclease with an unusual topoisomerase-like activity. This biological activity is not by itself connected to the allergenicity of the molecule. Whether the enzymatic activity is responsible for the induction of the allergic sensitization and inflammation remains an open question.

Allergens↗

PubMedDate not supplied

Bovine seminal ribonuclease inhibits in vivo growth of human neuroblastoma cells.

Bovine seminal ribonuclease (BS-RNase) is a homologue of RNase A with specific antitumor activities. It is selectively toxic for neuroblastoma (NB) cells in vitro with no significant effects on the viability of normal human cells. We evaluated the antitumoral effects of BS-RNase on human NB xenografts from UKF-NB-3 cells in athymic (nude) mice. The efficacy of direct intraneoplastic, subcutaneous and systemic delivery of BS-RNase was explored. Systemic administration of BS-RNase (12.5 mg/kg/day intraperitoneally, for 20 days in the course of four weeks) suppressed tumor growth but was not able to induce any cures. Subcutaneous injections (12.5 mg/kg/day for 20 days in the course of four weeks) and intratumoral BS-RNase treatment using the same schedule resulted in complete tumor regression. During 30 days following cessation of treatment no tumor regrowth was observed and animals were free of tumors. Toxic effects of BS-RNase (e.g., on bone marrow and inner organs) were not apparent. This data indicates that BS-RNase fulfills important criteria for a candidate antitumor agent specific for NB.

Animals↗

PubMedDate not supplied

Bovine seminal ribonuclease exerts selective cytotoxicity toward neuroblastoma cells both sensitive and resistant to chemotherapeutic drugs.

BACKGROUND: Bovine seminal ribonuclease (BS-RNase) exerts selective cytotoxicity toward different types of tumor cells. In the present study, we tested the effects of BS-RNase on cultured neuroblastoma (NB) cells resistant to chemotherapeutic agents. The selectivity of the antitumoral activity of BS-RNase was evaluated using cultures of CD34+ hematopoietic stem cells. MATERIALS AND METHODS: Human NB cell lines including IMR-32, UKF-NB-2 and UKF-NB-3 were selected for resistance against vincristine, doxorubicin or cisplatin by exposure to increasing concentrations of the respective drug. The cytotoxicity of the drugs to NB cells was evaluated using a clonogenic assay in a methylcellulose medium. Peripheral blood progenitor cells were obtained from adult healthy donors by positive selection using specific anti-CD34+ antibodies. The toxicity of BS-RNase to CD34+ cells was assessed in the direct clonogenic assay using methylcellulose medium or in ex vivo expansion culture supplemented with hematopoietic growth factors. RESULTS: In the clonogenic assay it was shown that BS-RNase completely inhibits growth of both parental NB cells and their sublines resistant to chemotherapeutic drugs at concentrations (up to 50 micrograms/ml) which have no significant influence on the growth of colony-forming units, granulocyte macrophage and erythroid burst-forming units. Moreover, BS-RNase had no effect on the ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors. CONCLUSIONS: BS-RNase is a highly efficient agent against NB cells resistant to chemotherapeutic drugs. The lack of toxicity to hematopoietic progenitor cells suggests that BS-RNase is also likely to have tolerable hematopoietic toxicity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

Selective activity of BS-RNase against anaplastic thyroid cancer.

BACKGROUND: Anaplastic thyroid carcinoma is an aggressive solid tumor that fails to adequately respond to any known chemotherapeutic regimen. The development of effective chemotherapy agents would provide the best chance for long-term survival of patients. MATERIALS AND METHODS: The cytotoxic effects of bovine seminal ribonuclease (BS-RNase) against thyroid carcinoma cell lines with different degrees of differentiation in comparison to non-malignant cells, including human foreskin fibroblasts (HFF) and retinal pigment epithelial cells (RPE), were tested using the MTT dye reduction assay. Induction of apoptosis was demonstrated by annexin V assay and expression of proteins related to apoptosis was investigated by flow cytometry. The antitumoral in vivo effects of BS-RNase were assessed on established xenografts of anaplastic thyroid carcinoma cell line 8505C in nude mice using subcutaneous injections of BS-RNase (12.5 mg/kg once a day, on 20 consecutive days). RESULTS: All the tumor cell lines exhibited marked sensitivity against BS-RNase in comparison to HFF and RPE cells. The greatest growth inhibition was seen in the 8505C line, while IC50 values for papillary (B-CPAP) and poorly-differentiated thyroid carcinoma cells were about 6-fold higher. The cytotoxic action of BS-RNase was associated with induction of apoptosis. Expressions of Fas and Fas-ligand were not influenced by BS-RNase completely, while the down-regulation of Bcl-2 in treated cells was observed. In vivo treatment induced significant tumor regression after the course of 20 consecutive days. No apparent toxic effects of BS-RNase toward non-malignant cells were observed during the in vivo treatment. After cessation of therapy (day 20) tumor volume continued to decrease and the tumor was no longer detectable after 30 days of treatment induction in all animals. CONCLUSION: BS-RNase may have beneficial effects for treatment of aggressive anaplastic thyroid cancer.

Animals↗