thymosin and Multiple-Myeloma

Multiple myeloma (MM) is a malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow (BM). Because of the known involvement of thymosin beta4 (Tbeta4) in metastasis of tumor cells, we examined the expression and role of Tbeta4 in MM disease. In a large patient population, we demonstrated that Tbeta4 expression was significantly lower in myeloma cells compared to normal plasma cells and that patients with a high Tbeta4 expression had a longer event free and overall survival. The decreased Tbeta4 expression was also found in the murine 5TMM model. To study its function, we overexpressed the Tbeta4 gene in 5T33MMvt cells by lentiviral transduction. These cells demonstrated a decreased proliferative capability and an increased sensitivity to apoptosis. Mice injected with Tbeta4-overexpressing cells showed a prolonged survival compared to mice injected with controls. In contrast to its role in solid tumors, we found a decreased expression in myeloma cells compared to their normal counterpart and studies with overexpression of the Tbeta4 gene indicated a tumor suppressive function of Tbeta4 in myeloma development.

Topics: Animals; Apoptosis; Bone Marrow; Humans; Mice; Multiple Myeloma; Thymosin

Investigations of human B-cell malignancies have generally focused on the monoclonal B-cell populations. Until recently there has been little emphasis on the thymus (T) lymphocyte in these disorders. Current studies, however, suggest that quantitative and qualitative disorders of T cells are generally seen both in chronic lymphocytic leukemia and in multiple myeloma. This review will focus on two major concepts. First, it will define the quantitative and functional T-cell abnormalities in B-cell malignancies including evidence suggesting a causal link between the T-cell abnormalities and certain observed disease manifestations in chronic lymphocytic leukemia and multiple myeloma. Secondly, it will review data demonstrating that these T cells may be influenced by in vivo and in vitro manipulations and will outline some of the possible resultant clinical effects.

Topics: B-Lymphocytes; Humans; Immunity, Cellular; Killer Cells, Natural; Leukemia, Lymphoid; Leukocyte Count; Lymphocyte Activation; Multiple Myeloma; Spleen; Splenectomy; T-Lymphocytes; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Thymosin

Studies in a variety of animal and human models indicate that thymosin plays a role in the differentiation of a number of T-cell subpopulations. The hypothesis presented is that a normal immune balance depends heavily upon the presence of thymosin-activated suppressor or regulator T-cells. A major thrust in our present research program is to determine whether or not the various disorders discussed here are causally related to abnormal thymosin production by the thymus gland. We are also assessing in animal models the potential value of thymsin in the treatment of specific autoimmune diseases. This information may yield new insights for the management of autoimmune type disorders such as SLE. Results from clinical trials to date suggest that thymosin will have a role in boosting the immune responses of patients with specific thymic malfunctions and may indeed exert an influence via the production of suppressor or regulator T-cells.

Topics: Animals; Autoimmune Diseases; Cell Differentiation; Clinical Trials as Topic; Hodgkin Disease; Humans; Immune Adherence Reaction; Immunologic Deficiency Syndromes; Leiomyosarcoma; Leukemia, Lymphoid; Lung Neoplasms; Melanoma; Mice; Multiple Myeloma; T-Lymphocytes; Thymosin; Thymus Extracts

Multiple myeloma (MM) is characterized by the accumulation of monoclonal plasma cells in the bone marrow and causes several immune alterations in patients. Thymosin α1 (Tα1) is a thymic peptide that has been associated with immuno-stimulating properties. In addition, this peptide exerts anti-tumor effects in several cancer types. Beneficial effects of Tα1 administration have also been shown on immune reconstitution after hematopoietic stem cell transplantation (HSCT), a current treatment modality in hematological malignancies including MM. In this study, we observed a slight reduction in the proliferation of murine and human MM cell lines in the presence of Tα1 in vitro. However, using two immunocompetent murine MM models (5TGM1 and MOPC315.BM), we did not observe any impact of Tα1 administration on MM development in vivo. Furthermore, no beneficial effects of Tα1 treatment were observed on lymphocyte immune reconstitution after transfusion of human hematopoietic stem cells into immunodeficient mice. In conclusion, despite direct effects of Tα1 on human MM cell line proliferation in vitro, Tα1 did not exert anti-myeloma effects in vivo in the two murine models tested. Moreover, Tα1 failed to improve immune recovery in a xenogeneic HSCT model.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Hematopoietic Stem Cell Transplantation; Humans; Immunomodulation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, SCID; Multiple Myeloma; Peptide Fragments; Thymalfasin; Thymosin; Xenograft Model Antitumor Assays

Thymosin beta4 (Tbeta4) is a polypeptide involved in cellular proliferation, differentiation, and migration, over-expressed in several tumor entities. We evaluated its expression and function in 298 newly diagnosed multiple myeloma patients and the murine 5TMM model. Mean Tbeta4 expression was significantly lower in myeloma cells compared to normal plasma cells (P<0.001). The same observation can be made in the 5TMM-mouse model by qRT-PCR and ELISA. Here, Tbeta4 overexpression by lentiviral transduction of 5T33MMvt-cells led to significantly decreased proliferative and migratory capacities and increased sensitivity to apoptosis-induction. Mice injected with Tbeta4 over-expressing myeloma cells showed a longer survival compared to mice injected with controls (88,9 vs. 65,9 days, P<0.05). In 209 MM patients treated with high-dose therapy and autologous stem cell transplantation, expression of Tbeta4 below the median was associated with a significantly shorter event free survival (37.6 vs. 26.2 months, P<0.05). In conclusion, our results indicate a possible tumor suppressive function of Tbeta4.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Down-Regulation; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Multiple Myeloma; Prognosis; Survival Rate; Thymosin; Tumor Suppressor Proteins

Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32P]orthophosphoric acid, they synthesized [32P]prothymosin alpha. The incorporated radioactivity was resistant to DNase and RNases A, T1, and T2, but could be completely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [32P]prothymosin alpha indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine prothymosin alpha and human [32P]prothymosin alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, and 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the phosphopeptide with Asp-N followed by C18 reversed-phase column chromatography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N-terminal 5-mer derived from the unmodified 14-mer. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only in a nested set of phosphorylated fragments composed of the first three, four, and five amino acids. The results prove that prothymosin alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Furthermore, prothymosin alpha appeared to be stable, with a half-life slightly shorter than the generation time. Although prothymosin alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not directly govern mitosis.

Topics: Alkaline Phosphatase; Amino Acid Sequence; Animals; Cattle; Cell Cycle; Humans; Molecular Sequence Data; Multiple Myeloma; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Phosphoserine; Protein Precursors; Protein Processing, Post-Translational; Thymosin; Thymus Gland; Tumor Cells, Cultured

The function of prothymosin alpha has been investigated by using four different antisense oligodeoxyribonucleotides directed at selected regions of its mRNA. In every case, when synchronized human myeloma cells were released from stationary phase by incubation in fresh medium containing antisense oligomers, cell division was prevented or inhibited; sense oligomers and random antisense oligomers had no effect. A detailed analysis of synchronized cell populations indicated that sense-treated and untreated cells divided approximately 17 hr after growth initiation, whereas cells incubated with antisense oligomer 183, a 16-mer targeted 5 bases downstream of the initiation codon, entered mitosis approximately one cell division late. The failure to divide correlated directly with a deficit in prothymosin alpha and with the continued presence of intact intracellular antisense oligomers over a period of at least 24 hr. Because antisense oligomers had no effect either on the timing of the induction of prothymosin alpha mRNA upon growth stimulation or on mRNA levels seen throughout the cell cycle, we concluded that antisense DNA caused specific hybrid arrest of translation. Our data suggest that prothymosin alpha is required for cell division. However, there is no evidence that prothymosin alpha directly regulates mitosis.

Topics: Base Sequence; Cell Cycle; Cell Division; Cell Line; Humans; Kinetics; Molecular Sequence Data; Multiple Myeloma; Oligonucleotides, Antisense; Protein Precursors; RNA, Messenger; Thymosin; Transcription, Genetic

Human prothymosin alpha, virtually alone among proteins, is recovered from the aqueous phase of phenol-extracted cell lysates prepared from human myeloma cells or COS cells that were transfected with the human prothymosin alpha gene. This observation forms the basis for purification of the protein to homogeneity in two steps--phenol extraction followed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels to remove residual contaminants consisting chiefly of carbohydrate and RNA.

Topics: Cell Line; Electrophoresis, Polyacrylamide Gel; Humans; Multiple Myeloma; Phenols; Protein Precursors; Recombinant Proteins; Thymosin; Transfection