Identification of immunoreactive forms of thymosin alpha 1 in serum and supernatants by combining HPLC and RIA.

Thymosin alpha 1 (T alpha 1) is a biologically active peptide, originally isolated from the thymus and currently undergoing clinical trials as an immunomodulator in cancer patients, in individuals with chronic active hepatitis, and as an immunoenhancer of vaccines in immunocompromised individuals. Absorption of rabbit antibody to thymosin alpha 1 with a synthetic C-14 fragment of T alpha 1 results in an antiserum with increased affinity for the amino terminal region of T alpha 1 and the precursor protein prothymosin alpha (ProT alpha). Using HPLC methodologies, the predominant form of immunoreactivity in serum and thymus was T alpha 1 not the precursor. Using this assay we detected a decline in mouse serum T alpha 1 following irradiation but not thymectomy, an observation consistent with the existence of an important radiation sensitive lymphoid source of serum T alpha 1. The secretion of authentic T alpha 1 but not the precursor into culture medium by thymic epithelial cells as well as in mitogen-stimulated peripheral blood lymphocytes was also demonstrated by HPLC/RIA. HPLC analysis by molecular weight sizing columns demonstrated that unlike thymic epithelial cells or peripheral blood lymphocytes, the immunoreactive T alpha 1 (IRT alpha 1) form in the supernatants from tumor cells such as MCF-7 breast carcinoma was of a lower molecular weight than authentic T alpha 1. These studies suggest that the authentic form of T alpha 1 is the major immunoreactive form in normal serum and that it is secreted by the medullary thymic epithelial cells as well as by peripheral blood lymphocytes. An additional immunoreactive form, secreted by tumor cells has also been identified and is the subject of future studies.

Biochemical and immunohistological identification of thymosin alpha-1 in MCF-7 breast cancer cells.

Growth of normal and malignant cells is controlled by the interplay of several hormones and growth factors present in the body. The growth of the human breast cancer cell line MCF-7 is stimulated in vitro by estradiol which has been shown to induce the release of numerous polypeptide growth factors into the culture media. In a search to identify polypeptide growth factors for MCF-7 breast cancer cells we have detected the thymic hormone, thymosin alpha one (TA1) in culture supernatants and cytosol preparations of MCF-7 cells grown in TA1 free media. TA1 was identified by reverse phase-high performance liquid chromatography (RP-HPLC) followed by a specific radioimmunoassay for TA1 (TA1-RIA). Indirect immune fluorescence localized TA1 on, or within, the cytoplasm of MCF-7 cells grown in TA1 free media. The results of this data along with our previously published findings fulfills three of the four criteria needed to establish thymosin alpha-1 as an autocrine growth factor for MCF-7 cells in culture.

Localization of thymosin alpha 1 production to thymus medullary epithelial cells by use of monoclonal antibodies.

The thymus is an endocrine organ which modulates T-cell immunity through the production of protein like peptides such as the thymosins. Thymosin alpha 1 was the first biologically active peptide isolated and sequenced from the partially purified thymic preparation, thymosin fraction 5, and has been extensively studied. Using synthetic Thymosin alpha 1, a heterologous rabbit antiserum has been raised and a radioimmunoassay has been developed. Although thymosin alpha 1 antibodies have been used in several histological studies, their use is limited by potential nonspecific cross-reactivities, unpredictable heterogenicity, variable affinities, and a limited unstandardized supply. In the studies, reported here, eight anti-thymosin alpha 1 monoclonal antibodies (MAbs) were produced by somatic cell fusion between spleen cells from immunized BALB/c mice and P3x64 Ag8.653 myeloma cells. The MAbs were screened for anti-thymosin alpha 1 specificity in a solid phase ELISA and a liquid phase RIA. Only those clones which secreted specific antibody as detected by both procedures were characterized for their heavy chain class and epitope specificity. The anti-thymosin alpha 1 monoclonal antibodies were then used for indirect immune fluorescence studies of perfused rat thymus. Thymosin alpha 1 containing cells were found primarily in the thymic medulla, confirming previous studies using the heterologous antisera. These studies demonstrated the specificity of the anti-thymosin alpha 1 monoclonal antibodies for immunochemical studies of intra- and extra-thymic localization of thymosin alpha 1. They also provide an important reagent for biological studies of the role of thymosin alpha 1, in vitro and in vivo.

Three radioligands compared for determining cytoplasmic estrogen-receptor content of human breast carcinomas.

We compared three radioligands for use in a cytoplasmic estrogen-receptor assay, using pooled cytosol from human breast adenocarcinomas. The estrogen receptor content was determined in vitro by a dextran-coated charcoal method involving a 4-h incubation with and without diethylstilbestrol. Tritiated moxestrol failed to come to equilibrium in 4 h, thereby preventing the use of conventional one-component Scatchard analysis. The use of 125I-labeled estradiol resulted in a higher estimate of estrogen-receptor concentration than that obtained with use of tritiated estradiol. This overestimation was not corrected by Scatchard, double-reciprocal, or Woolf plots, or by two different methods of data analysis: least squared and "robust." An underestimation of the specific activity of iodoestradiol with respect to that of tritiated estradiol and an unrecognized second component of nonreceptor binding could explain this disparity.