Photoaffinity labeling of P-glycoprotein in multidrug resistant cells with photoactive analogs of colchicine

Two photoactive radiolabeled analogs of colchicine, N-(p-azido[3,5-[3H]benzoyl)aminohexanoyldeacetylcolchicine ([3H]NABC]) and N-(p-azido-[3-125I]salicyl)aminohexanoyldeacetylcolchicine ([125I]NASC) were synthesized and used to identify colchicine-specific acceptor(s) in membrane vesicles from multidrug resistant (MDR) variant DC-3F/VCRd-5L Chinese hamster lung cells. Both [3H]NABC and [125I]NASC specifically photolabeled a prominent 150-180 kDa polypeptide in membrane vesicles from DC-3F/VCRd-5L cells. The photolabeled polypeptide was immunoprecipitated by monoclonal antibody C219 specific for the MDR-related P-glycoprotein (P-gp) indicating the identity of this protein with P-gp. Colchicine at 1000 microM reduced [3H]NABC photolabeling of P-gp by 72%. Furthermore, 100 microM of colchicine, vincristine, vinblastine, doxorubicin and actinomycin D inhibited [125I]NASC photolabeling by 45, 88.8, 91.1, 61.5, and 51% respectively. However, methotrexate did not affect the [125I]NASC photolabeling of P-gp, indicating the multidrug specificity of the P-gp colchicine acceptor for drugs to which these cells are resistant.     

Incidence of novel and potentially archaeal nitrogenase genes in the deep Northeast Pacific Ocean

Archaea have been detected throughout the oceanic water column and are quantitatively important members of picoplankton in the deep ocean. Two common groups, group I Crenarchaeota and group II Euryarchaeota, are consistently detected in warm hydrothermal fluid and are assumed to have been drawn into the subseafloor, mixed with hydrothermal fluid and then expelled. However, because they remain resistant to cultivation, very little is known about their physiology. Here we show that cold deep-seawater from the axial valley of Endeavour Segment on the Juan de Fuca Ridge contains not only groups I and II archaea as expected, but also unique potentially archaeal nitrogenase (nifH) genes, which are required for nitrogen fixation. These nifH genes are phylogenetically distinct and have dissimilar G+C content compared with those of hydrothermal vent archaea, suggesting that they belong to non-thermophilic deep-sea archaea. Furthermore, this sample did not contain mcrA genes, which are present in methanogens, the only known archaeal nitrogen fixers. These nifH genes were not detected in upper water column samples, or in a deep-seawater sample 100 km away from the spreading axis of the Juan de Fuca Ridge. We propose that these unique nifH genes may be localized to archaea that circulate through the nitrogen-poor subseafloor at the mid-ocean ridge as part of their life cycle.     

Extensive natural variation for cellular hydrogen peroxide release is genetically controlled

Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H₂O₂ release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H₂O₂ release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (n = 279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p<5.00×10−8) and 54 suggestive associations (p<1.00×10−5), one of which confirmed the linked region on Hsa15. To replicate our findings, we performed GWAS using 58 HapMap individuals and ∼2.1 million SNPs. We identified 40 genome-wide significant and 302 suggestive SNPs, and confirmed genome signals on Hsa1, Hsa12, and Hsa15. Genetic loci within 900 kb from the known candidate gene p67phox on Hsa1 were identified in GWAS in both cohorts. We did not find replication of SNPs across all cohorts, but replication within the same genomic region. Finally, a highly significant decrease in H₂O₂ release was observed in Down Syndrome (DS) individuals (p<2.88×10−12). Taken together, our results show strong evidence of genetic control of H₂O₂ in LCL of healthy and DS cohorts and suggest that cellular phenotypes, which themselves are also complex, may be used as proxies for dissection of complex disorders.     

Retinoids regulate the formation and degradation of gap junctions in androgen-responsive human prostate cancer cells

The retinoids, the natural or synthetic derivatives of Vitamin A (retinol), are essential for the normal development of prostate and have been shown to modulate prostate cancer progression in vivo as well as to modulate growth of several prostate cancer cell lines. 9-cis-retinoic acid and all-trans-retinoic acid are the two most important metabolites of retinol. Gap junctions, formed of proteins called connexins, are ensembles of intercellular channels that permit the exchange of small growth regulatory molecules between adjoining cells. Gap junctional communication is instrumental in the control of cell growth. We examined the effect of 9-cis-retinoic acid and all-trans retinoic acid on the formation and degradation of gap junctions as well as on junctional communication in an androgen-responsive prostate cancer cell line, LNCaP, which expressed retrovirally introduced connexin32, a connexin expressed by the luminal cells and well-differentiated cells of prostate tumors. Our results showed that 9-cis-retinoic acid and all-trans retinoic acid enhanced the assembly of connexin32 into gap junctions. Our results further showed that 9-cis-retinoic acid and all-trans-retinoic acid prevented androgen-regulated degradation of gap junctions, post-translationally, independent of androgen receptor mediated signaling. Finally, our findings showed that formation of gap junctions sensitized connexin32-expressing LNCaP cells to the growth modifying effects of 9-cis-retinoic acid, all-trans-retinoic acid and androgens. Thus, the effects of retinoids and androgens on growth and the formation and degradation of gap junctions and their function might be related to their ability to modulate prostate growth and cancer.     

Soluble Abeta inhibits specific signal transduction cascades common to the insulin receptor pathway

Numerous studies have now shown that the amyloid beta-protein (Abeta), the principal component of cerebral plaques in Alzheimer disease, rapidly and potently inhibits certain forms of synaptic plasticity. The amyloid (or Abeta) hypothesis proposes that the continuous disruption of normal synaptic physiology by Abeta contributes to the development of Alzheimer disease. However, there is little consensus about how Abeta mediates this inhibition at the molecular level. Using mouse primary hippocampal neurons, we observed that a brief treatment with cell-derived, soluble, human Abeta disrupted the activation of three kinases (Erk/MAPK, CaMKII, and the phosphatidylinositol 3-kinase-activated protein Akt/protein kinase B) that are required for long term potentiation, whereas two other kinases (protein kinase A and protein kinase C) were stimulated normally. An antagonist of the insulin receptor family of tyrosine kinases was found to mimic the pattern of Abeta-mediated kinase inhibition. We then found that soluble Abeta binds to the insulin receptor and interferes with its insulin-induced autophosphorylation. Taken together, these data demonstrate that physiologically relevant levels of naturally secreted Abeta interfere with insulin receptor function in hippocampal neurons and prevent the rapid activation of specific kinases required for long term potentiation.     

Parthenolide sensitizes cells to X-ray-induced cell killing through inhibition of NF-kappaB and split-dose repair

Human cancers have multiple alterations in cell signaling pathways that promote resistance to cytotoxic therapy such as X rays. Parthenolide is a sesquiterpene lactone that has been shown to inhibit several pro-survival cell signaling pathways, induce apoptosis, and enhance chemotherapy-induced cell killing. We investigated whether parthenolide would enhance X-ray-induced cell killing in radiation resistant, NF-kappaB-activated CGL1 cells. Treatment with 5 microM parthenolide for 48 to 72 h inhibited constitutive NF-kappaB binding and cell growth, reduced plating efficiency, and induced apoptosis through stabilization of p53 (TP53), induction of the pro-apoptosis protein BAX, and phosphorylation of BID. Parthenolide also enhanced radiation-induced cell killing, increasing the X-ray sensitivity of CGL1 cells by a dose modification factor of 1.6. Flow cytometry revealed that parthenolide reduced the percentage of X-ray-resistant S-phase cells due to induction of p21 waf1/cip1 (CDKN1A) and the onset of G1/S and G2/M blocks, but depletion of radioresistant S-phase cells does not explain the observed X-ray sensitization. Further studies demonstrated that the enhancement of X-ray-induced cell killing by parthenolide is due to inhibition of split-dose repair.