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

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

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

ICF, An Immunodeficiency Syndrome: DNA Methyltransferase 3B Involvement, Chromosome Anomalies, and Gene Dysregulation

The immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) is the only disease known to result from a mutated DNA methyltransferase gene, namely, DNMT3B. Characteristic of this recessive disease are decreases in serum immunoglobulins despite the presence of B cells and, in the juxtacentromeric heterochromatin of chromosomes 1 and 16, chromatin decondensation, distinctive rearrangements, and satellite DNA hypomethylation. Although DNMT3B is involved in specific associations with histone deacetylases, HP1, other DNMTs, chromatin remodelling proteins, condensin, and other nuclear proteins, it is probably the partial loss of catalytic activity that is responsible for the disease. In microarray experiments and real-time RT-PCR assays, we observed significant differences in RNA levels from ICF vs. control lymphoblasts for pro- and anti-apoptotic genes (BCL2L10, CASP1, and PTPN13); nitrous oxide, carbon monoxide, NF-κB, and TNFa signalling pathway genes (PRKCH, GUCY1A3, GUCY1B3, MAPK13; HMOX1, and MAP4K4); and transcription control genes (NR2F2 and SMARCA2). This gene dysregulation could contribute to the immunodeficiency and other symptoms of ICF and might result from the limited losses of DNA methylation although ICF-related promoter hypomethylation was not observed for six of the above examined genes. We propose that hypomethylation of satellite 2at1qh and 16qh might provoke this dysregulation gene expression by trans effects from altered sequestration of transcription factors, changes in nuclear architecture, or expression of noncoding RNAs

High‐grade serous carcinomas arise in the mouse oviduct via defects linked to the human disease

Recent studies have suggested that the most common and lethal type of ‘ovarian’ cancer, i.e. high‐grade serous carcinoma (HGSC), usually arises from epithelium on the fallopian tube fimbriae, and not from the ovarian surface epithelium. We have developed Ovgp1‐iCreERT2 mice in which the Ovgp1 promoter controls expression of tamoxifen‐regulated Cre recombinase in oviductal epithelium – the murine equivalent of human fallopian tube epithelium (FTE). We employed Ovgp1‐iCreERT2 mice to show that FTE‐specific inactivation of several different combinations of tumour suppressor genes that are recurrently mutated in human HGSCs – namely Brca1, Trp53, Rb1, and Nf1 – results in serous tubal intraepithelial carcinomas (STICs) that progress to HGSC or carcinosarcoma, and to widespread metastatic disease in a subset of mice. The cancer phenotype is highly penetrant and more rapid in mice carrying engineered alleles of all four tumour suppressor genes. Brca1, Trp53 and Pten inactivation in the oviduct also results in STICs and HGSCs, and is associated with diffuse epithelial hyperplasia and mucinous metaplasia, which are not observed in mice with intact Pten. Oviductal tumours arise earlier in these mice than in those with Brca1, Trp53, Rb1 and Nf1 inactivation. Tumour initiation and/or progression in mice lacking conditional Pten alleles probably require the acquisition of additional defects, a notion supported by our identification of loss of the wild‐type Rb1 allele in the tumours of mice carrying only one floxed Rb1 allele. Collectively, the models closely recapitulate the heterogeneity and histological, genetic and biological features of human HGSC. These models should prove useful for studying the pathobiology and genetics of HGSC in vivo, and for testing new approaches for prevention, early detection, and treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138263/1/path4927_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138263/2/path4927.pd