Treatment of chromaffin cells with nitric oxide (NO) donors (SNP and SNAP) and peroxynitrite produces a time- and dose-dependent necrotic and apoptotic cell death. Necrotic cell death was characterized by both an increase in lactate dehydrogenase and ATP release and changes in nuclei and cell morphology (as seen with fluorescence microscopy analysis with propidium iodide and Hoechst 33342). Apoptotic cell death was characterized by nuclear fragmentation and presence of apoptotic cell bodies, by a decrease in DNA content, and by an increase in DNA fragmentation. Treatment of chromaffin cells with lipopolysaccharide (LPS) or cytokines (interferon-gamma, tumor necrosis factor-alpha) resulted only in apoptotic cell death. Apoptotic effects of NO-inducing compounds were specifically reversed, depending on the stimuli, by the NO scavenger carboxy-PTIO (CPTio) or by the NOS inhibitors L-NMA and thiocitrulline. NO-induced apoptotic death in chromaffin cells was concomitant to a cell cycle arrest in G0G1 phase and a decrease in the number of chromaffin cells in the G2M and S phases of cell cycle. All NO-producing compounds were able to induce activation of caspase 3 and cytochrome c release, and specific inhibitors of caspase 3 and 9, such as Ac-DEVD-CHO (CPP32) and Ac-Z-LEHD-FMK, respectively, prevented NO-induced apoptosis in chromaffin cells. These results suggest that chromaffin cells could be good models for investigating the molecular basis of degeneration in diseases showing death of catecholaminergic neurons, phenomenon in which NO plays an important role.

The Chinese hamster lung fibroblast cell line (CC139) has high anchorage dependence for growth and has retained the high serum dependence of secondary cultures of adult fibroblasts. This cell line is tumorigenic in nude mice; however, the resulting tumor cells have different properties than those of the cell line injected. The tumor-derived cells had strongly reduced or even lost both the high anchorage and the high serum dependence of CC139 cells. This finding suggests that an in vivo selection is necessary for CC139 cells to acquire the malignant phenotype. After mutagenesis, which increases the frequency of CC139 colony formation in agarose up to 8-fold, we selected and analyzed 15 anchorage-independent colonies. No correlation between the colony-forming ability in agarose and serum-growth factor requirement for DNA synthesis was observed. Each of these clones were injected into nude mice and the growth factor dependence of the ensuing tumor cells was compared to that of corresponding injected cells. All of the anchorage-independent colonies with the exception of one (A71), had acquired in vivo a stable phenotype allowing for partial or total escape of growth factor requirement. A71, the only clone which maintained the same growth factor requirement after two passages in vivo (A71 T1 and A71 T2) had already gained, in vitro, the minimal growth factor "relaxation" compatible with in vivo growth. A71 and A71 T1 tumor cells arrested in G0/G1 can reinitiate DNA synthesis in the presence of mouse plasma, low concentrations of serum, or thrombin. The fact that none of the tumors analyzed (more than 20) were found to have retained the high serum dependence of CC139 cells strongly suggests that the partial loss of serum growth factor requirement acquired in vivo is an essential malignant character for bypassing the hormonal growth restraints imposed by the host upon CC139 cells.

Previous work of our group stated that exogenously added and endogenous nitric oxide (NO) generated by cytokines induce apoptosis in chromaffin cells. In this work, we investigate the specific regulation of the NO synthase (NOS) isoforms, inducible NOS (iNOS) and neuronal NOS (nNOS), and their particular participation in cell death induced by interferon gamma (IFNγ). Lipopolysaccharide (LPS) and IFNγ increase iNOS expression, with no effect on nNOS expression. On the other hand, dexamethasone increases basal nNOS expression but decreases LPS + IFNγ‐induced iNOS expression. IFNγ‐induced cell death was abolished by W‐1400, a specific iNOS inhibitor, but only partially by nNOS inhibitors [N‐ω‐propyl‐l‐arginine (N‐PLA), 3‐Bromo‐7‐nitroindazol (7‐NI), l‐methyl thiocitrulline and N‐methyl l‐arginine], indicating the main iNOS participation in chromaffin cell death. IFNγ and LPS induce nuclear factor κB (NFκB) translocation to the nucleus, a process implicated in activation of iNOS expression, as inhibition of NFκB translocation, by SN50, decreased iNOS expression. In addition, IFNγ and LPS induce 847Ser‐nNOS phosphorylation, inhibiting nNOS activity. Both processes, nNOS phosphorylation and iNOS expression induced by LPS + IFNγ, are regulated by Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway, as IFNγ increases 727STAT‐3 phosphorylation and specific inhibitors of JAK/STAT pathway, such as AG490, inhibited both processes. Taken together, these results support the hypothesis of an inactivating phosphorylation of nNOS by IFNγ, via JAK/STAT, in bovine chromaffin cells. Low NO concentrations achieved by this event, would activate NFκB translocation, increasing iNOS expression and generating, this last, high apoptotic NO concentrations.

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The Chinese hamster lung fibroblast line, CCl39, displays the properties characteristic of normal secondary cultures of Chinese hamster fibroblasts including: reversible G0 growth arrest (less than 2% labeled nuclei), anchorage dependence, and high serum-growth factor dependence. Injection of CCl39 cells, or anchorage-independent variants, in nude mice leads to tumor formation; however, as we have previously shown (Pérez-Rodriguez et al., 1981b), the resulting tumor clones no longer possess the high serum dependence of injected CCl39 cells. Hormonal growth restraints imposed by the host create an in vivo selection for diminished, or "relaxed," growth factor requirement. To characterize this growth factor "relaxation" further, we have analyzed the mitogenic response of parental CCl39 cells, anchorage-independent clones, and selected tumoral derivatives, to purified growth factors. Two highly purified growth factors, thrombin and insulin, together fulfill the growth factor requirements of CCl39 cells; thrombin (1 U/ml) stimulates the reinitiation of DNA synthesis in G0-arrested CCl39 cells, and insulin (10 micrograms/ml) maximally potentiates this stimulation to the level obtained with 10% fetal calf serum. First, we found no correlation between loss of anchorage dependence and growth factor relaxation. Second, we found that A71 (anchorage independent), a tumoral variant of CCl39 capable of growth arrest, and tumor-derived cells all display an increased sensitivity to thrombin and a diminished requirement for the potentiating action of insulin. Examination of thrombin binding to CCl39, A51 (nontumoral, anchorage independent), and A71 cells revealed that the increased sensitivity to thrombin of A71 cells is not attributable to an alteration in thrombin cell surface receptor number or affinity for thrombin. Rather, under standard conditions of serum or growth factor removal (30 hr), A71 cells maintain a metabolically elevated growth-arrested state, different from that of their nontumoral counterparts. Consequently, much lower concentrations of growth factors are needed to induce a proliferative response in these tumoral cells.