Inducible production of human macrophage growth factor, CSF-1.

A panel of human cell lines was screened for production of colony-stimulating factor-1 (CSF-1) using a specific radioreceptor assay and criterion of macrophage colony growth in mouse bone marrow culture. The pancreatic carcinoma lines MIA PaCa and PANC were found to secrete high levels of CSF-1. In a bone marrow proliferation assay, the activities from these two lines were blocked by a CSF-1 specific neutralizing antiserum, confirming the predominant content of this macrophage growth factor. MIA PaCA cells stopped secreting CSF-1 when transferred to various serum-free media. Serum-free production could be reinitiated by phorbol myristic acetate (PMA). Purified CSF-1 from serum-free MIA PaCa cells stimulated the formation of 14-day colonies from total and nonadherent mononuclear human bone marrow cells. Most of the colonies consisted exclusively of large, dispersed macrophages that were intensely stained for nonspecific esterase. Although similar numbers of human 14-day colonies were stimulated by CSF-1 and other CSFs, more CSF-1 was required for the proliferation of human as compared with murine bone marrow progenitors. Northern analysis of mRNA from induced-MIA PaCa cells, using a human CSF-1 oligonucleotide probe, revealed multiple species of CSF-1 mRNA ranging from 1.5 to 4.5 kilobases (kb). Uninduced, serum-free cultures showed only the largest mRNA species, suggesting that serum removal interfered with CSF-1 mRNA processing related to synthesis and/or secretion of the protein. Regulation of the production of CSF-1 may be an important physiological process in hematopoiesis and macrophage functioning.

Purification of human macrophage colony stimulating factor (CSF-1) from medium conditioned by pancreatic carcinoma cells.

Colony Stimulating Factor-1 has been purified to apparent homogeneity from the serum-free medium conditioned by cultured human pancreatic carcinoma cells which had been induced with phorbol myristate acetate. The purification scheme consisted of sequential steps of batchwise adsorption to calcium phosphate gel, adsorption to lentil lectin-Sepharose, binding to immobilized antibodies, hydrophobic interaction chromatography, and reversed-phase high-performance liquid chromatography. The purified glycoprotein was found to have a subunit molecular weight corresponding to the smallest of four species (approximately 40,000, 33,000, 28,000 and 23,000) which were observed when less purified preparations were examined.

Relationship of octanol/water partition coefficient and molecular weight to cellular permeability and partitioning in s49 lymphoma cells.

We have used modified standard methods and derived new formulae to quantitate cell permeability (P), cell/media partitioning (λ), and intracellular sequestration or binding rate constants (m) for cultured S49 murine lymphoma cells in suspension. Using 15 standard compounds and anticancer drugs, we found quantitative relationships among log P, log PO (octanol/pH 7.4 buffer partition coefficient), and molecular weight (MW) such that logP = -4.5 + 0.56log (PO(MW)(-1/2)). A good correlation among P, λ, and MW was also determined with λ = 0.67 + 5890 gm(1/2) cm(-1) sec (P (MW)(1/2)). These studies show that there is a strong partitioning (λ) dependence to molecular weight and permeability that can be predicted even for known carrier-transported and biotransformable compounds. Furthermore, results of this study show that the slope of the plot of permeability and lipophilicity is not necessarily unity as has been postulated from the results of other studies.

Brain, CSF, and tumor pharmacokinetics of alpha-difluoromethylornithine in rats and dogs.

We have determined the pharmacokinetic parameters for diffusion of alpha-[5-14C]-difluoromethylornithine (DFMO) from blood to brain, blood to cerebrospinal fluid (CSF), 9L rat brain tumor to adjacent brain, and blood to the subcutaneously-implanted 9L tumor in rats, and within the CSF of beagle dogs. DFMO diffusion across the blood-brain and blood-CSF barriers is quite restricted in both rats and dogs, but diffusion across the defective capillary system of both subcutaneous and intracerebral 9L tumors in rats is not. Under steady-state plasma conditions in rats, uptake of DFMO by the intracerebral 9L tumor and diffusion from tumor 5-6 mm into adjacent brain is not restricted; tissue/plasma ratios were approximately 1. Therapeutic efficacy will therefore not be limited by transport of DFMO into tumor or to the extracellular environment of tumor.

In situ drug delivery.

Drug delivery to tumours and the accessibility of tumour cells to drugs remains a problem of foremost importance and is affected by many factors. We will discuss models we have developed for drug delivery from tumour capillaries to tumour cells, and drug delivery from well perfused, well oxygenated tumour regions to less well perfused hypoxic regions in terms of computer simulation of drug delivery for the anticancer agent BCNU and the hypoxic cell radiosensitizer misonidazole.

Hexose transport and phosphorylation by capillaries isolated from rat brain.

Hexose transport and phosphorylation were studied in capillary segments isolated from rat brain. Uptake of 3-O-methyl-D-glucose (3MG) could be inhibited by cytochalasin B, phloretin, and phlorizin, but not by 2,4-dinitrophenol or ouabain. 2-Deoxy-D-glucose (2DG), D-glucose, galactose, and mannose inhibited 3MG uptake, while L-glucose, fructose, and ribose did not. Accelerative exchange diffusion of 3MG was demonstrated. At equilibrium, the intracellular concentration of hexose did not exceed the external concentration, and transport was, therefore, equilibrative rather than accumulative. Transport of 2DG and D-glucose was not rate limiting for metabolism. When incubated in 5 mM D-glucose, the endothelial cells contained a large pool of free glucose. L-Glucose entered capillaries more slowly than other hexoses and served as a marker for simple diffusion of sugars into the cells. Our results suggest that sugar uptake into isolated brain capillaries occurs by a transport system similar to the one responsible for glucose transport across the blood-brain barrier in vivo.

Influence of thyroid hormones on myelin proteins in the developing rat brain.

This communication describes developmental changes in myelin proteins prepared from control, hypothyroid, and hyperthyroid rat brain. In the 10- to 37-day postnatal period studied, total myelin protein was found to double, and this change mainly reflected the increase in proteolipid and basic protein constituents. Thyroid states affect differentially the various myelin proteins. Hypothyroidism decreases the proteolipid and slow-moving basic protein, but has no effect on the fast basic or minor proteins. In hyperthyroidism, an increase was observed in proteolipid as well as both slow- and fast-moving proteins. The protein alterations were correlated to the changes previously found in lipid composition of myelin consequent upon hypo- and hyperthyroidism, and the role of thyroid hormones in brain development.