Specific transport proteins package classical neurotransmitters into vesicles so that their release can be regulated by neural activity. Previous studies have suggested that a single activity mediates the vesicular transport of monoamines in the adrenal gland, brain, and other tissues such as mast cells and platelets. However, molecular cloning has recently identified two vesicular transporters for monoamines. Although the predicted proteins are closely related in sequence, they show a range of differences in their physiologic and pharmacologic properties. To clarify further the biological significance of the observed functional differences, we have generated anti-peptide antibodies to the C-termini of the two transporters and used them to determine the distribution and localization of the proteins in the rat. We have detected expression of vesicular monoamine transporter 1 (VMAT1) in adrenal chromaffin cells but not in neural cells. Interestingly, some adrenal chromaffin cells also express VMAT2 but the amount of VMAT2 relative to VMAT1 appears much lower than in the bovine adrenal gland. In contrast, sympathetic ganglion cells express only VMAT2, as do enteric neurons and enterochromaffin-like cells of the stomach. Thus, although adrenal chromaffin cells, sympathetic and enteric neurons derive from the neural crest, they express different vesicular amine transporters. In the CNS, dopamine, norepinephrine, epinephrine, 5-HT, and histamine cell groups all express VMAT2. These findings are consistent with the functional characteristics of VMAT1 and VMAT2 and help to explain several classic pharmacological observations. VMAT2-immunoreactivity is generally stronger in cell bodies, proximal dendrites and axonal processes, indicating the potential for monoamine storage at each of these sites. Surprisingly, dopaminergic interneurons in the olfactory bulb show no detectable immunoreactivity for either VMAT1 or VMAT2.

The toxin N-methyl-1,2,3,6-tetrahydropyridine produces a model of neural degeneration very similar to idiopathic Parkinson disease. To understand the cellular mechanisms that modulate susceptibility to its active metabolite N-methyl-4-phenylpyridinium (MPP+), we have transfected a cDNA expression library from the relatively MPP+-resistant rat pheochromocytoma PC12 cells into MPP+-sensitive Chinese hamster ovary (CHO) fibroblasts. Selection of the stable transformants in high concentrations of MPP+ has yielded a clone extremely resistant to the toxin. Reserpine reverses the resistance to MPP+, suggesting that a transport activity protects against this form of toxicity, perhaps by sequestering the toxin within an intracellular compartment. In support of this hypothesis, dopamine loaded into the CHO transformant shows a localized distribution that is distinct from the pattern observed in wild-type cells and is also reversed by reserpine.Systemic administration of N-methyl-1,2,3,6-tetrahydropyridine (MPTP) results in the death of dopaminergic neurons in the substantia nigra (1). Since MPTP reproduces the pattern of neuronal degeneration observed in Parkinson disease (PD), this form of toxicity has been used as a model system to dissect the cellular components responsible for selective neuronal vulnerability in the idiopathic human disease. Toxicity requires conversion of MPTP to the active metabolite N-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase B (2-4). Dopaminergic cells of the substantia nigra then accumulate high levels of MPP+ by uptake through the high-affinity plasma membrane catecholamine transporter (5,6). Inside the cell, MPP+ enters mitochondria (7) and inhibits respiration at the level of complex I, apparently by binding near the site of action of the other mitochondrial toxins rotenone and piericidin (8,9).Studies of patients with PD support the utility of MPTP as a model for idiopathic PD. Both brain tissue and circulating platelets from patients with PD show a selective reduction in the same mitochondrial component affected by MPTP, respiratory complex I (10-12). Deprenyl, which inhibits the enzyme that activates MPTP, monoamine oxidase, also appears to slow the rate of progression in idiopathic PD (13). However, parkinsonism induced by MPTP develops over days to weeks, whereas idiopathic PD develops over years. Explanations for the relatively slow rate of progression in idiopathic PD include chronic, low levels of exposure to an environmental toxin similar to MPTP, oxidative stress related to the cytoplasmic accumulation of dopamine, or the trapping of free radicals by deposited dopamine and lipofuscin that predisposes these cells to mitochondrial injury (14).The MPTP model has suggested multiple pathogenetic factors that may also contribute to idiopathic PD. The amelioration of toxicity with an excitatory amino acid antagonist implies that neural excitation plays a role in the injury produced by MPP+ in vivo (15). In addition, neurotrophic factors such as brain-derived neurotrophic...

Background: Palbociclib (P) is a potent, oral, highly selective, inhibitor of cyclin-dependent kinase (CDK) 4 and 6. In the Phase 3 PALOMA-2 study, P in combination with letrozole (L) demonstrated a significant improvement in progression-free survival (PFS) versus placebo (PBO) plus L. To further explore predictive markers for sensitivity and resistance to CDK4/6-based therapy or endocrine therapy (ET) alone, we performed a large gene expression analysis of baseline tumor tissues using mature PFS data with extended follow-up. Methods: Postmenopausal women (N=666) with no prior systemic therapy for ER+/HER2- ABC were randomized 2:1 to P+L or PBO+L until disease progression, death, unacceptable toxicity, or consent withdrawal. All patients provided tumor tissues to participate in the study. 455 of 666 intent-to-treat (ITT) patients had tumor tissue appropriate for gene expression evaluation (303 [68%] P+L arm and 152 [68%] PBO+L arm). EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics; Tucson, AZ) was used for mRNA profiling, assessing 2534 genes involved in a variety of cancer-related pathways. Using systematic and unsupervised approaches, gene expression levels were interrogated for identification of potential genes and/or pathways associated with a treatment effect of P+L compared with PBO+L. Results: With median follow-up of 38 months, mPFS of P+L vs L + PBO was 27.6 mos vs 14.5 mos (HR=0.563 [p<0.000001] in ITT). PFS in the biomarker-assessed group was similar. Using a supervised approach, expression of genes involved in the Cyclin D-CDK4/6-RB pathway were analyzed. This demonstrated that patients receiving P+L had a consistent benefit similar to the ITT population irrespective of various expression levels of CCND1, CCNE1/2, CDK2/4/6, RB1, and CDKN2A. These results are consistent with findings from previous IHC data (CCND1, RB, p16). Unsupervised analysis revealed that tumors with higher levels of growth factor receptors (eg. FGFR2 FDR 0.032, interaction p=0.056 and ERBB3 FDR 0.221, interaction p=0.043,) were associated with greater sensitivity to P+L vs L alone. Using parallel gene signature-based analyses, tumors with higher expression of a growth factor signature had longer PFS in the P+L arm. In addition, higher CDK4 expression was identified as a resistance marker for PBO+L arm (FDR 0.095, interaction p=0.016). Gene expression-based molecular subtyping demonstrated that both luminal A and B subtypes benefited equally from P+L vs PBO+L. Patients with lower level of tumor PD-1 expression showed more benefit from P+L (FDR 0.099, interaction p=0.021). Conclusion: These results confirm efficacy of palbociclib + letrozole in ER+/HER2- breast cancer, and support the use of ER+ as a biomarker for sensitivity to CDK 4/6 inhibition. Expression levels (whether high or low) of genes in the Cyclin D-CDK4/6-RB pathway did not correlate with benefit from palbociclib + letrozole. These data provide evidence that the interplay between steroid hormone and peptide growth factor signaling in ER+ breast cancer drives dependence on CDK 4/6 and benefit from CDK 4/6 inhibition with palbociclib. These data can be used to guide additional therapeutic opportunities in ER+/HER2- ABC. Sponsor: Pfizer Citation Format: Finn RS, Liu Y, Martin M, Rugo HS, Dieras V, Im S-A, Gelmon K, Harbeck N, Zhu Z, Lu D, Gauthier E, Bartlett CH, Slamon DJ. Comprehensive gene expression biomarker analysis of CDK 4/6 and endocrine pathways from the PALOMA-2 study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-10.