Acute myelofibrosis is often associated with acute megakaryoblastic leukemia (AMKBL). Although the exact mechanism for the progression of myelofibrosis in AMKBL is unclear, certain humoral factors from megakaryoblastic cells, the precursors of platelets, may be involved in the enhancement of collagen synthesis by bone marrow fibroblasts. The present study, therefore, is an investigation of the possible pathogenic role of transforming growth factor-beta (TGF-beta), known to be a very potent collagen-stimulating factor found in platelets in the myelofibrosis of AMKBL. The results obtained were as follows: (1) Conditioned media from peripheral megakaryoblasts taken from an AMKBL patient and from established megakaryoblast cell lines (MEG-01) had much greater stimulatory effects on collagen synthesis in bone marrow fibroblasts than conditioned media from other leukemic cell types. (2) Based on an assessment of soft agar colony formation, there was greater TGF-beta activity in media that had been conditioned from megakaryoblasts than in media from other leukemic cell types. (3) When compared with other leukemic-cell types, megakaryoblasts showed substantially greater expression of TGF-beta mRNA that was hybridized at 2.5 kb with a TGF-beta cDNA probe, and TGF-beta polypeptides were detected at 13 Kd with anti-TGF-beta antibodies. (4) The addition of the anti-TGF-beta antibody inhibited the stimulatory effects of the megakaryoblast conditioned medium on collagen synthesis in bone marrow fibroblasts. These results clearly suggest that megakaryoblasts produce and secrete an active form of TGF-beta and stimulate collagen synthesis in bone marrow fibroblasts in a paracrine manner.

Peripheral mononuclear cells from adult T cell leukemia (ATL) patients were analyzed in comparison with other types of leukemia cells, for the expression of transforming growth factor-beta (TGF-beta) mRNA, for the presence of TGF-beta activity (colony stimulating activity for normal rat kidney fibroblasts [NRK]) in conditioned medium and for their susceptibility to exogenous TGF-beta. Highly elevated TGF-beta mRNA levels were observed in all five ATL cell samples tested; however, in three acute myelogenous leukemia (AML) samples, in one acute lymphatic leukemia (ALL), and one chronic myelogenous leukemia (CML), TGF-beta expression was relatively lower. In normal peripheral mononuclear cells TGF-beta mRNA was weakly detectable. Colony stimulating activity for NRK found in the conditioned medium from ATL cells as well as other leukemia cells correlated well with the levels of TGF-beta mRNA expression. In all three ATL samples tested, stimulation of 3H- thymidine uptake by purified TGF-beta from platelets was apparent. These results suggest that ATL cells are secreting active TGF-beta in a relatively high amount, as compared with other leukemia cells, and may proliferate in response to the factor via an autocrine manner. Furthermore, considering that TGF-beta stimulates bone resorption, we can speculate that the relatively high amount of TGF-beta in ATL cells contributes to the hypercalcemia frequently seen in ATL patients.

The clinical significance of radioreceptor assay for transferrin receptors of human cancerous tissues was evaluated. Fresh surgical specimens from various carcinoma tissues were solubilized with 1% Triton X-100 and the extracts were mixed with '25I-labelled diferric transferrin. The free transferrin and the receptor-bound transferrin were separated by 15% polyethylene glycol precipitation. The % specific transferrin binding to gastric, colonic, lung and mammary carcinoma tissues ranged between 3.9 and 13.9%, whereas those for normal stomach and colon were less than 2%. The concentrations of transferrin receptors in these cancerous tissues ranged between 3.7 and 28.3 pmole/g tissues. It was concluded that the amounts of transferrin receptors were significantly increased in all of the tumor tissue extracts examind and may thereby provide a useful marker for the diagnosis of malignancies. transferrin receptor ; transferrin ; radioreceptor assay ; cell proliferationThe first event in cellular iron uptake is the binding of differric transferrin to its receptors (Aisen and Listowsky 1980). In addition to erythroid cells (Jandl and Katz 1963), transferrin receptors (Tf • R) have been found in established cell lines in vitro (Hamilton et al. 1979;Trowbridge and Omary 1981). Recently, Shindelman et al. (1981) and Habeshow and Lister (1983) have reported the presence of Tf.• R in the tissue of breast cancer and malignant lymphoma, respectively. Furthermore, it has become apparent that the Tf.• R are expressed in much greater amounts on malignant cells than on non-malignant cells, although a quantitative study is lacking. These findings have generated a considerable interest on Tf.• R which appear to be a marker of malignant transformation.In the present paper, we investigated Tf.• R in surgical specimens obtained from a variety of different types of human malignancy. The results of our study indicate that Tf•R may be useful as a marker of cell proliferation.

05-Phosphohydroxylysine was chemically synthesized and techniques were established for its identification by combined use of cation-exchange chromatography, thin-layer electrophoresis at pH 1.9 and 3.5, and thin-layer chromatography. Clean separation of phosphohydroxylysine from the other phospho amino acids, phosphoethanolamine, and phosphocholine was achieved. Conditions were also determined to permit hydrolysis of proteins in 2 M HCO without loss of the phosphono group of phosphohydroxylysine residues. Experiments were then performed showing that 32p was incorporated into the hydroxylysine residues of cell-associated collagens when cultured calf aorta medial smooth muscle cells were incubated with [32P]orthophosphate. In other experiments, the cells incorporated [3Hllysine into hydroxylysine residues of cell-associated collagen and then 32p into phosphohydroxylysine residues. The doubly labeled phosphohydroxylysine subsequently isolated showed nearly 1:1 stoichiometry with respect to incorporation of precursor lysine and phosphorus. Finally, in preliminary experiments done with a cell-free extract of the smooth muscle cells, 32p was transferred from [y-32P]ATP to hydroxylysine residues in several kinds of collagenous substrates. Thus, this work shows that smooth muscle cells have the capacity to phosphorylate hydroxylysine residues in their cell-associated collagens and provides preliminary evidence that a protein kinase is involved.With the discovery of proteins containing phosphotyrosine residues (1, 2), all of the coded hydroxy amino acids inserted into polypeptide chains have been shown to undergo posttranslational phosphorylation by action of protein kinases and ATP. Two other hydroxy amino acids are in themselves formed by post-translational hydroxylation reactions; these are hydroxyproline in collagens and elastins, formed from proline residues, and 5-hydroxylysine in collagens, formed from lysine residues. We have begun studies of the phosphorylation of hydroxylysine and hydroxyproline residues in proteins.In its free form, 05-phosphohydroxylysine was identified tentatively in the extracellular fluids of bovine embryos in the early screening applications of paper chromatography (3). More recently, a system has been found in liver for phosphorylation of free hydroxylysine by a hydroxylysine kinase that utilizes GTP as the phosphono donor (4, 5). That reaction is the first step in the complete degradative metabolism of hydroxylysine liberated by digestion of collagenous proteins as, for instance, shown by our laboratory, by the combined actions of trypsin and carboxypeptidase B (6).For many years, we have been concerned with possible functions for hydroxylysine residues. It is known that such residues, like those of lysine, can undergo oxidation at C-6 to form corresponding aldehyde groups, which can participate in crosslink formation (7). The hydroxyl groups of hydroxylysine residues can undergo glycosylation by action of a transferase and UDP-galactose, and the substituted galactose residue c...

The response of a highly metastatic cell line of methylcholanthrene induced A fibrosarcoma (Meth A) to growth factors from platelets was examined. The highly metastatic cell subline (MH) proliferated more rapidly than its parental counterpart cell subline (ML) in a medium containing platelet lysate. However, when the three major growth factors from platelets, ie, platelet-derived growth factor, epidermal growth factor, and transforming growth factor-beta (PDGF, EGF, TGF-beta), were independently examined for their growth promoting activity, the former 2 growth factors preferentially stimulated the proliferation of ML and the latter growth factor rather suppressed the growth of both cells. On the other hand, the combined effects of these factors were more marked on MH. This combination effect was supported by the evidence that the number of receptors for EGF (which is probably an essential growth factor for the Meth A cell) was increased by pretreatment with PDGF or TGF-beta. Thus, the highly metastatic cells of MH were considered to be the most susceptible to growth factors released from platelets. This conclusion is consistent with the concept that platelets may play an important role in the formation of blood-borne metastasis by releasing growth factors to promote the proliferation of tumor cells, following aggregation with tumor cells.