1. The synthesis and efflux of N-acetyl-l-aspartate from brain mitochondria of rats of different ages has been studied. 2. Brain mitochondrial State 3 (+ADP) respiration rate, using 10mm-glutamate and 2.5mm-malate as substrates, increases during the suckling period and reaches approx. 50% of the adult value at 17 days after birth [adult State 3 respiration rate=160+/-7ng-atoms of O/min per mg of mitochondrial protein(mean+/-s.d.; n=3)]. 3. The influence of 5mm-pyruvate or 10mm-dl-3-hydroxybutyrate on aspartate efflux from brain mitochondira from rats of different ages oxidizing glutamate and malate was studied. In all cases the aspartate efflux in State 3 was greater than in State 4, but, whereas the aspartate efflux in State 3 increased as the animals developed, that of State 4 showed only a small increase. However, the rate of aspartate efflux in the presence of pyruvate or 3-hydroxybutyrate as well as glutamate and malate was approx. 60-65% of that in the presence of glutamate and malate alone. 4. An inverse relationship between aspartate efflux and N-acetylaspartate efflux was observed with adult rat brain mitochondria oxidizing 10mm-glutamate and 2.5mm-malate in the presence of various pyruvate concentrations (0-5mm). 5. N-Acetylaspartate efflux by brain mitochondria of rats of different ages was studied in States 3 and 4, utilizing 5mm-pyruvate or 10mm-dl-3-hydroxybutyrate as acetyl-CoA sources. A similar pattern of increase during development was seen in State 3 for N-acetylaspartate efflux as for aspartate efflux (see point 3 above). Also only very small increases in N-acetylaspartate efflux occurred during development in State 4.6. Rat brain mitochondria in the presence of iso-osmotic N-acetylaspartate showed some swelling which was markedly increased in the presence of malate. 7. It is concluded that N-acetylaspartate may be synthesized and exported from both neonatal and adult rat brain mitochondria. It is proposed that the N-acetylaspartate is transported by the dicarboxylic acid translocase and may be an additional mechanism for mitochondrial/cytosolic carbon transport to that of citrate.

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The polyamines spermidine and spermine and their precursor putrescine are intimately involved in and are required for cell growth and proliferation. This study examines the mechanism by which polyamines modulate cell growth, cell cycle progression, and signal transduction cascades. IEC-6 cells were grown in the presence or absence ofdl-α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the first rate-limiting enzyme for polyamine synthesis. Depletion of polyamines inhibited growth and arrested cells in the G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the level of p53 protein and other cell cycle inhibitors, including p21Waf1/Cip1 and p27Kip1. Induction of cell cycle inhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike many other cell lines. Although polyamine depletion decreased the expression of extracellular signal-regulated kinase (ERK)-2 protein, a sustained increase in ERK-2 isoform activity was observed. The ERK-1 protein level did not change, but ERK-1 activity was increased in polyamine-depleted cells. In addition, polyamine depletion induced the stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) type of mitogen-activated protein kinase (MAPK). Activation of JNK-1 was the earliest event; within 5 h after DFMO treatment, JNK activity was increased by 150%. The above results indicate that polyamine depletion causes cell cycle arrest and upregulates cell cycle inhibitors and suggest that MAPK and JNK may be involved in the regulation of the activity of these molecules.

Non-small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFb secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFb was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFb receptor in HCC4006 cells prevented EMT but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared with cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly developed EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele was lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy. Cancer Res; 75(20); 4372-83. Ó2015 AACR.