Establishment and identification of small cell lung cancer cell lines having classic and variant features.

Using a chemically defined medium containing hydrocortisone, insulin, transferrin, 17 beta-estradiol and selenium, with or without serum supplementation (2.5% v/v), continuous cell lines can be established from 72% of all fresh biopsy specimens of small cell lung cancer (SCLC) containing tumor cells. No differences were observed in the rate of establishing cell lines from newly diagnosed untreated patients, or from patients who have relapsed from prior therapy, or from a variety of different organ sites. Biochemical characterization of 50 SCLC cell lines for the expression of L-dopa decarboxylase; bombesin-like immunoreactivity; neuron-specific enolase, and the brain isozyme of creatine kinase, revealed that SCLC cell lines can be subdivided into two distinct classes: classic SCLC cell lines (35 lines), which express elevated levels of all four biomarkers; and variant SCLC cell lines (15 lines) which have undetectable levels of L-dopa-decarboxylase and bombesin-like immunoreactivity, but continue to express neuron-specific enolase and the brain isozyme of creatine kinase. The presence of the latter two markers distinguishes variant lines fron non-SCLC cell lines. In addition, four distinct classes were identified morphologically. The biomedical differences among established SCLC cell lines may account for the differences in response rates to cytotoxic therapy observed in newly diagnosed SCLC patients. A prospective study of biomarker characterization of SCLC tumors will determine if clinical differences exist between classic and variant SCLC tumors.

[3H]nitrobenzylthioinosine binding as a probe for the study of adenosine uptake sites in brain.

The binding of the potent adenosine uptake inhibitor [3H]nitrobenzylthioinosine ([3H]NBI) to brain membrane fractions was investigated. Reversible, saturable, specific, high-affinity binding was demonstrated in both rat and human brain. The KD in both was 0.15 nM with Bmax values of 140-200 fmol/mg protein. Linear Scatchard plots were routinely obtained, indicating a homogeneous population of binding sites in brain. The highest density of binding sites was found in the caudate and hypothalamus in both species. The binding site was heat labile and trypsin sensitive. Binding was also decreased by incubation of the membranes in 0.05% Triton X-100 and by treatment with dithiothreitol and iodoacetamide. Of the numerous salt and metal ions tested, only copper and zinc had significant effects on [3H]NBI binding. The inhibitory potencies of copper and zinc were IC50 = 160 microM and 6 mM, respectively. Subcellular distribution studies revealed a high percentage of the [3H]NBI binding sites on synaptosomes, indicating that these sites were present in the synaptic region. A study of the tissue distribution of the [3H]NBI sites revealed very high densities of binding in erythrocyte, lung, and testis, with much lower binding densities in brain, kidney, liver, muscle, and heart. The binding affinity in the former group was approximately 1.5 nM, whereas that in the latter group was 0.15 nM, suggesting two types of binding sites. The pharmacologic profile of [3H]NBI binding was consistent with its function as the adenosine transport site, distinct from the adenosine receptor, since thiopurines were very potent inhibitors of binding whereas adenosine receptor ligands, such as cyclohexyladenosine and 2-chloroadenosine, were three to four orders of magnitude less potent. [3H]NBI binding in brain should provide a useful probe for the study of adenosine transport in the brain.

The development of immunoreactivity for neuron-specific enolase of preoptic and septal neurons in dissociated cultures.

The immunocytochemical visualization of neuron-specific enolase, which is a marker protein for differentiated neurons, was applied to follow the differentiation of preoptic and septal neurons in dissociated cultures. From 4 to 24 days in vitro, the relative numbers of stained neurons were counted and the staining intensity of individual neurons determined by absorbency measurements using a television-based densitometer. Whereas few stained cells could be observed at 4 DIV, 80% of the neurons were neuron-specific enolase-positive at 13 days in vitro. This value remained constant up to 24 days in vitro. The density of the immunoreaction product increased dramatically from 13 to 17 days in vitro and was still higher at 24 days in vitro. The glial and ependymal cells of the carpet, as well as neuroblasts, remained unstained. Comparison with morphological observations and immunocytochemical demonstration of neuronal peptides made earlier shows that expression of neuron-specific enolase closely parallels neuronal differentiation. These observations indicate that cultures derived from preoptic and septal neurons represent a viable model system for the study of neuronal maturation in vitro.