Low‐volume, high‐throughput sandwich immunoassays for profiling plasma proteins in mice: Identification of early‐stage systemic inflammation in a mouse model of intestinal cancer

Mouse models of human cancers may provide a valuable resource for the discovery of cancer biomarkers. We have developed a practical strategy for profiling specific proteins in mouse plasma using low‐volume sandwich‐immunoassays. We used this method to profile the levels of 14 different cytokines, acute‐phase reactants, and other cancer markers in plasma from mouse models of intestinal tumors and their wild‐type littermates, using as little as 1.5μl of diluted plasma per assay. Many of the proteins were significantly and consistently up‐regulated in the mutant mice. The mutant mice could be distinguished nearly perfectly from the wild‐type mice based on the combined levels of as few as three markers. Many of the proteins were up‐regulated even in the mutant mice with few or no tumors, suggesting the presence of a systemic host response at an early stage of cancer development. These results have implications for the study of host responses in mouse models of cancers and demonstrate the value of a new low‐volume, high‐throughput sandwich‐immunoassay method for sensitively profiling protein levels in cancer.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135703/1/mol2200712216-sup-mmc1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135703/2/mol2200712216.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135703/3/mol2200712216-sup-mmc2.pd

A statistical analysis of spot variation using the two-dimensional polyacrylamide gel electrophoresis

For the development of valid algorithms for matching protein spots between two-dimensional gels, it is essential that one has an understanding of the relative roles of the many sources of variability affecting the location of spots. We first consider the contribution of observers to the measurement variability of spot location, arriving at a simple model for these effects. Next we present an analysis of the variability in spot locations for a sample of gels containing duplicate gels for each sample. Our data indicate that both differences between duplicates and between samples are considerable, and that the size of the effects depends on the region of the gel being considered. In the discussion we examine several matching strategies that match large groups of gels based on algorithms which match two gels at a time.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26270/1/0000355.pd

Comprehensive Proteomic Profiling of Aldehyde Dehydrogenases in Lung Adenocarcinoma Cell Lines

We have explored the potential of proteomic profiling to contribute to the delineation of the range of expression and subcellular localization of aldehyde dehydrogenases (ALDHs) in lung adenocarcinoma. In-depth quantitative proteomics was applied to 40 lung adenocarcinoma cell lines resulting in the identification of the known members of the ALDH family. Substantial heterogeneity in the level and occurrence of ALDHs in total lysates and on the cell surface and in their release into the culture media was observed based on mass spectrometry counts. A distinct pattern of expression of ALDHs was observed in cells exhibiting epithelial features relative to cells exhibiting mesenchymal features. Strikingly elevated levels of ALDH1A1 were observed in two cell lines. We also report on the occurrence of an immune response to ALDH1A1 in lung cancer

Lineage tracing suggests that ovarian endosalpingiosis does not result from escape of oviductal epithelium

Most high‐grade serous carcinomas are thought to arise from Fallopian tube epithelium (FTE), but some likely arise outside of the tube, perhaps from ectopic tubal‐type epithelium known as endosalpingiosis. Importantly, the origin of endosalpingiosis is poorly understood. The proximity of the tubal fimbriae to the ovaries has led to the proposal that disruptions in the ovarian surface that occur during ovulation may allow detached FTE to implant in the ovary and form tubal‐type glands and cysts. An alternative model suggests that cells present in ectopic locations outside the Müllerian tract retain the capacity for multi‐lineage differentiation and can form glands with tubal‐type epithelium. We used double transgenic Ovgp1‐iCreERT2;R26RLSL‐eYFP mice, which express an eYFP reporter protein in OVGP1‐positive tissues following transient tamoxifen (TAM) treatment, to track the fate of oviductal epithelial cells. Cohorts of adult mice were given TAM to activate eYFP expression in oviductal epithelium, and ovaries were examined at time points ranging from 2 days to 12 months post‐TAM. To test whether superovulation might increase acquisition of endosalpingiosis, additional cohorts of TAM‐treated mice underwent up to five cycles of superovulation and ovaries were examined at 1, 6, and 12 months post‐TAM. Ovaries were sectioned in their entirety to identify endosalpingiosis. Immunohistochemical staining for PAX8, tubulin, OVGP1, and eYFP was employed to study endosalpingiosis lesions. Ovarian endosalpingiosis was identified in 14.2% of TAM‐treated adult mice. The endosalpingiotic inclusion glands and cysts were lined by secretory and ciliated cells and expressed PAX8, tubulin, OVGP1, and eYFP. Neither age nor superovulation was associated with a significant increase in endosalpingiosis. Endosalpingiosis was also occasionally present in the ovaries of pre‐pubertal mice. The findings imply that ovarian endosalpingiosis in the mouse does not likely arise as a consequence of detachment and implantation of tubal epithelium and other mechanisms may be relevant. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151844/1/path5308.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151844/2/path5308-sup-0001-FigS1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151844/3/path5308_am.pd

Comparison of seven methods for producing Affymetrix expression scores based on False Discovery Rates in disease profiling data

BACKGROUND: A critical step in processing oligonucleotide microarray data is combining the information in multiple probes to produce a single number that best captures the expression level of a RNA transcript. Several systematic studies comparing multiple methods for array processing have used tightly controlled calibration data sets as the basis for comparison. Here we compare performances for seven processing methods using two data sets originally collected for disease profiling studies. An emphasis is placed on understanding sensitivity for detecting differentially expressed genes in terms of two key statistical determinants: test statistic variability for non-differentially expressed genes, and test statistic size for truly differentially expressed genes. RESULTS: In the two data sets considered here, up to seven-fold variation across the processing methods was found in the number of genes detected at a given false discovery rate (FDR). The best performing methods called up to 90% of the same genes differentially expressed, had less variable test statistics under randomization, and had a greater number of large test statistics in the experimental data. Poor performance of one method was directly tied to a tendency to produce highly variable test statistic values under randomization. Based on an overall measure of performance, two of the seven methods (Dchip and a trimmed mean approach) are superior in the two data sets considered here. Two other methods (MAS5 and GCRMA-EB) are inferior, while results for the other three methods are mixed. CONCLUSIONS: Choice of processing method has a major impact on differential expression analysis of microarray data. Previously reported performance analyses using tightly controlled calibration data sets are not highly consistent with results reported here using data from human tissue samples. Performance of array processing methods in disease profiling and other realistic biological studies should be given greater consideration when comparing Affymetrix processing methods

Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1WT versus Kir2.1Δ314-315, a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases.This work was supported by the National Institutes of Health (NIH) through the National Heart, Lung, and Blood Institute (NHLBI) grant R01HL122352 awarded to J.J., as well as the National Institute of General Medical Sciences (NIGMS) grant R01GM094231 and the National Cancer Institute (NCI) grant U24CA210967 awarded to A.I.N. R.K. is supported by the NCI support grant P30CA046592 awarded to the University of Michigan Rogel Cancer Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.S

Detection of human somatic cell structural gene mutations by two-dimensional electrophoresis

The feasibility of detecting human somatic structural gene mutations by two dimensional electrophoresis has been investigated. A lymphoblastoid cell line was grown as a mass culture in the presence of ethylnitrosourea, after which cells were regrown as single cell clones. A total of 257 polypeptide spots were analyzed in gels derived from 186 clones. Four structural mutations were detected by visual analysis of the gels. Computer analysis of gels corresponding to the mutant clones was also undertaken. At a spot size threshold of 200 spots to be matched using a computer algorithm, all four mutant polypeptides were detected. These results indicate the usefulness of the two-dimensional approach for mutagenesis studies at the protein level.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38517/1/340020103_ftp.pd

The PIAS-like Coactivator Zmiz1 Is a Direct and Selective Cofactor of Notch1 in T Cell Development and Leukemia

SummaryPan-NOTCH inhibitors are poorly tolerated in clinical trials because NOTCH signals are crucial for intestinal homeostasis. These inhibitors might also promote cancer because NOTCH can act as a tumor suppressor. We previously reported that the PIAS-like coactivator ZMIZ1 is frequently co-expressed with activated NOTCH1 in T cell acute lymphoblastic leukemia (T-ALL). Here, we show that similar to Notch1, Zmiz1 was important for T cell development and controlled the expression of certain Notch target genes, such as Myc. However, unlike Notch, Zmiz1 had no major role in intestinal homeostasis or myeloid suppression. Deletion of Zmiz1 impaired the initiation and maintenance of Notch-induced T-ALL. Zmiz1 directly interacted with Notch1 via a tetratricopeptide repeat domain at a special class of Notch-regulatory sites. In contrast to the Notch cofactor Maml, which is nonselective, Zmiz1 was selective. Thus, targeting the NOTCH1-ZMIZ1 interaction might combat leukemic growth while avoiding the intolerable toxicities of NOTCH inhibitors

CLU blocks HDACI-mediated killing of neuroblastoma

Clusterin is a ubiquitously expressed glycoprotein with multiple binding partners including IL-6, Ku70, and Bax. Clusterin blocks apoptosis by binding to activated Bax and sequestering it in the cytoplasm, thereby preventing Bax from entering mitochondria, releasing cytochrome c, and triggering apoptosis. Because increased clusterin expression correlates with aggressive behavior in tumors, clusterin inhibition might be beneficial in cancer treatment. Our recent findings indicated that, in neuroblastoma cells, cytoplasmic Bax also binds to Ku70; when Ku70 is acetylated, Bax is released and can initiate cell death. Therefore, increasing Ku70 acetylation, such as by using histone deacetylase inhibitors, may be therapeutically useful in promoting cell death in neuroblastoma tumors. Since clusterin, Bax, and Ku70 form a complex, it seemed likely that clusterin would mediate its anti-apoptotic effects by inhibiting Ku70 acetylation and blocking Bax release. Our results, however, demonstrate that while clusterin level does indeed determine the sensitivity of neuroblastoma cells to histone deacetylase inhibitor-induced cell death, it does so without affecting histone deacetylase-inhibitor-induced Ku70 acetylation. Our results suggest that in neuroblastoma, clusterin exerts its anti-apoptotic effects downstream of Ku70 acetylation, likely by directly blocking Bax activation

PCNA levels in neuroblastoma are increased in tumors with an amplified N- myc gene and in metastatic stage tumors

N- myc oncogene amplification in neuroblastoma has been found to be significantly associated with advanced stage disease and tumor progression. However, there is a lack of data on tumors, regarding the relationship between N- myc gene amplification and proliferation activity. Proliferating cell nuclear antigen (PCNA) is a proliferation-induced 36 kD nuclear protein that is the auxiliary component of DNA polymerase δ. PCNA levels in tissues have been found to correlate with proliferative activity. We have examined PCNA levels in neuroblastomas in relation to N- myc gene amplification and tumor stage. Statistically, significantly higher levels of PCNA were observed in tumors with an amplified N- myc gene relative to tumors with a single gene copy. The highest levels of PCNA were observed in advanced stage tumors with an amplified N- myc gene. Treatment of neuroblastoma cells in culture with retinoic acid, which induces differentiation, resulted in a substantial decrease in PCNA. Our results suggest that PCNA levels may reflect differences in proliferative activity between neuroblastomas, related to stage of the disease and to N- myc gene copy number.[/p ]Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42581/1/10585_2004_Article_BF00880069.pd