Purified fibrinogen at concentrations of3-30 nM has been found to stimulate continuous growth ofhuman lymphoid and myeloid cell lines under serum-free conditions. A strong proliferative response resulted from the synergism elicited by the addition of fibrinogen to transferrin-supplemented medium. This effect was observed with the pre-B-cell line Raji, the T lymphomaderived JM, and the monocytic cell line U 937, either at high or low cell densities. With the promyelocytic cell line HL 60, fibrinogen did not shorten the doubling time of cultures seeded at high cell densities (2 x 105 cells per ml). However, at cell densities lower by 2 orders of magnitude and in the same medium, it promoted growth with a doubling time similar to that obtained at high cell concentrations. Fibrinogen also was found to increase the plating efficiency and colony size when human bone marrow cells were cultured in semisolid medium containing serum. In long-term bone marrow liquid cultures without fibrinogen, colony-forming cells were no longer detected after 6 weeks. In those cultured with fibrinogen, -50 granulocyte-macrophage colonies per 105 cells were obtained after 6 weeks, and 10, after 12 weeks. Purified fibrinogen fragment D possessed a stimulating activity similar to that of the intact fibrinogen molecule. This fragment cannot form fibrin, thus eliminating fibrin as a source of the mitogenic effect.The use of serum in tissue culture hinders the study of the regulation of cellular proliferation (1)(2)(3)(4)(5)(6) because the roles and interactions amongst various growth factors are difficult to analyze in nondefined media. Sato and his group (for a review see ref. 7) have contributed to the development of the concept of hormonally defined media and have shown how useful these media can be for analyzing the controls of proliferation and differentiation at the molecular level. Iscove and co-workers (8-10) have applied this concept to the culture ofmurine hemopoietic cells.A minimal medium is composed of defined factors that are all necessary in a particular combination for the growth of a particular cell type; removal ofeach factor individually prevents growth. These media facilitate the study of each growth requirement and its effects. By designing minimal media containing purified plasma components, we have analyzed their possible roles in the control of human hemopoietic cell proliferation (11).Fibrinogen is a major component of plasma, where its concentration is about 3 mg/ml. Blood (19), was provided by R. Gallo (National Cancer Institute, Bethesda, MD). Cell lines were maintained in suspension in RPMI 1640 medium with 2 g of sodium bicarbonate per liter, 1% penicillin/streptomycin and 1% L-glutamine stock solutions, and 10% heat-inactivated fetal calf serum in Coming TM 25 flasks. Defined media were prepared as described (11). Doubling times were estimated only when cultures contained more than 98% viable cells as determined by trypan blue exclusion.Long-Term Human Bone Marrow Culture and ColonyForming Cell Ass...

Acetylcholinesterase (AchE), an essentially specific and early marker of rodent thrombocytic series, in several tissues acts both as a constituent of the cellular membrane and as a secretory enzyme. This study presents the ultrastructural transport and fate of this substance in the megakaryocytes of mouse bone marrow, using Tranum-Jensen and Behnke's adaptation of the indirect thiocholine method. It is shown that megakaryoblasts and megakaryocytes undergo a typical secretory cycle consisting of (1) enzyme synthesis and segregation on the endoplasmic reticulum and nuclear envelope, (2) enzyme concentration in AchE-vesicles and AchE-granules formed from the cisternae of the Golgi apparatus, and (3) discharge in the demarcation membrane system and extracellular space. The AchE-vesicles and granules appear to be hitherto unrecognized megakaryocytic organelles as they do not resemble alpha nor the dense granules, and their mode of formation and fate differ from those of primary lysosomes and peroxidase granules. Released platelets reveal AchE activity in the open canalicular system. The data are compatible with the hypothesis that by controlling acetylcholine concentration in hematopoietic tissues, the secretion of AchE by megakaryocytes can modulate the proliferative activity of megakaryocytes progenitors.

Non-linear dynamics, often chaotic, are now accepted as the normal way in which major physiological functions proceed, allowing adaptation. The length of telomeres, the chromosome endings, is critical in limiting cell lifespan and in controlling subsets of downstream genes. Telomere length dynamics in tumoral cells is the net result of mitotic erosion of telomeric DNA and telomere repair by the enzyme telomerase. We observed telomere length oscillations in the long-lived hepatoma Fao cell line, for forty three 6-day passages in culture. Telomerase activity oscillated with similar frequency and opposite amplitude. We mapped the combined data of telomere erosion and telomerase activity. There was reverse symmetry between consecutive periods of 10 passages. Thus, telomere length and telomerase activity can be considered to be a single complex system with strong reciprocal regulation.

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.