Charles T. Beer

Prolactin stimulation of ornithine decarboxylase and mitogenesis in Nb2 node lymphoma cells: The role of protein kinase C and calcium mobilization

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination with calcium ionophores has been shown to bypass the requisite antigen- or lectin-induced signal for lymphocyte mitogenesis. This suggests that protein kinase C activation and calcium mobilization may be early events required for lymphocyte proliferation. Therefore, the relationship(s) of protein kinase C activation and calcium mobilization to ornithine decarboxylase induction and cellular proliferation were examined in a rat node lymphoma cell line (Nb2) which is dependent upon prolactin (PRL) for mitogenesis. TPA enhanced PRL-stimulated Nb2 node lymphoma cell ornithine decarboxylase induction and [3H]thymidine incorporation. Addition of a calcium ionophore (A23187) to cultures containing TPA plus PRL increased ornithine decarboxylase above PRL alone or PRL plus TPA but inhibited proliferation compared to the PRL plus TPA regimen. Exposure of cells to TPA or TPA plus A23187 increased [3H]thymidine incorporation in a similar manner to that demonstrated for low-dose PRL. However, optimal concentrations were only 20-25% as effective as mitogens as was optimal PRL. Protein kinase C and calmodulin antagonists inhibited PRL-stimulated ornithine decarboxylase induction and proliferation. Ca2+ chelation or cation channel antagonism inhibited both PRL-stimulated responses. The cyclic AMP analogue, 8Br-cAMP, inhibited PRL-stimulated ornithine decarboxylase activity as well as cellular proliferation processes assessed by [3H]thymidine incorporation. Finally, tumor-promoting phorbol esters inhibited 125I-rPRL binding. These data strongly suggest that protein kinase C activation and calcium mobilization are requisite events for PRL-stimulated ornithine decarboxylase induction and cellular proliferation in Nb2 node lymphoma cells. An additional component that is linked to alterations in K+ channeling is also implicated. These data support a role for protein kinase C in PRL-coupled mitogenesis. However, other critical Ca2+ and/or ion-induced events are also required.

Hypothalamic prolactin: characterization by radioimmunoassay and bioassay and response to hypophysectomy and restraint stress.

Prompted by immunohistochemical reports of prolactin-like immunoreactivity in cell bodies within the rat hypothalamus, a study was undertaken to quantitate the immunologic and biologic activity of this material. Hypothalamic concentrations of prolactin-like immunoreactivity averaged 402 +/- 23 pg/mg of protein (n = 30). 97% recovery of rat prolactin standards added to homogenates of hypothalamus insured that neuronal tissue, as prepared for these studies, did not interfere with the radioimmunoassay of rat prolactin. Examination of the elution profile from Sephadex G-75 columns of the prolactin-like immunoreactivity in hypothalamic extracts showed that the majority of hypothalamic prolactin-like substance was of a larger molecular size than pituitary prolactin. While increasing amounts of brain extract progressively displaced more I125 prolactin from antibody-binding sites, the displacement curve produced by adding hypothalamic extract was not parallel to that produced by the addition of increasing amounts of anterior pituitary prolactin standards of rat origin. Hypothalamic extracts from hypophysectomized animals, analyzed for biologic activity in the Nb2 lymphoma cell assay, revealed prolactin-like bioactivity, but the bioactivity/immunoactivity (B/I) ratios for hypothalamic extracts were significantly lower than the B/I ratios for pituitary prolactin (0.71 +/- 0.04 for pituitary, vs. 0.19 +/- 0.06 in the hypothalamus; p less than 0.001). Hypophysectomy, which led to the expected fall in serum prolactin to undetectable levels, and restraint stress, which resulted in a statistically significant 4-fold rise in serum prolactin, caused no change in prolactin concentrations in the hypothalamus, indicating that brain prolactin-like substance is regulated independently of pituitary prolactin and circulating serum prolactin levels.

Biochemical response of lymphoma cells to mitogenic stimulation by prolactin

A previous study showed that cultured Nb 2 node rat lymphoma cells stopped replicating when transferred to medium supplemented with horse serum instead of fetal calf serum; resumption of growth could be induced by the addition of prolactin or other lactogens. The present study shows that in the absence of prolactin cells accumulated early in the G1 phase from which, on stimulation by the hormone, they proceeded through the cell cycle in a synchronized fashion. Ornithine decarboxylase and S-adenosyl methionine decarboxylase activities were closely related to the proliferative status of the cells. In stationary cultures the enzyme activity was barely detectable; following the addition of prolactin, the levels increased over 100-fold and displayed well-defined changes as the cells proceeded through the cell cycle. The results suggest the lymphoma cells are very useful for studying biochemical events resulting from the interaction of a mitogenic polypeptide hormone and its target cell.

Differences between vinblastine and vincristine in distribution in the blood of rats and binding by platelets and malignant cells.

Tritiu m labeled vinblastine ( VLB ) and vincristine ( VCR ) were injected i.p. at a subacutely-toxic dose (0.25mg/kg) into rats (Nb strain). In the .first .few hours the levels of VLB in the blood were substantially higher than those of VCR: subsequently, however, the levels of blood radioactivity in the case of VLB .Jell much more rapidly than those of VCR, reaching values well below the latter in 8-10hr. There was a correlation between the different blood labeling profiles of the alkaloids and their binding by platelets--the carriers of a major proportion of the circulating alkaloids. In vitro, platelets, cells isolated from alkaloid sensitive rat lymphomas and cultured L5178Y cells, all took up VLB much more rapidly than VCR; when restored to alkaloid free medium the cells readily released VLB but, in contrast, retained VCR tenaciously. In binding to cells, VLB and VCR have similar equilibrium association constants and are apparently bound to common receptors. It is suggested the preferential retention of VCR by cells may be an important factor underlying the greater toxicity and oncolytic potency of VCR.