Analysis Of Human Gene Expression Research Articles

Differential Gene Expression Signatures and Cellular Signaling Pathways induced by Lamin A/C Transcript Variants in MCF7 Cell Line.

Lamins are the major component of nuclear lamina. Alternative splicing of the 12 exons comprising lamin A/C gene creates five known transcript variants, lamin A, lamin C, lamin AΔ10, lamin AΔ50, and lamin C2. The main objective for this study was to examine the association of critical pathways, networks, molecular and cellular functions regulated by each Lamin A/C transcript variants. Ion AmpliSeq Transcriptome Human Gene Expression analysis was performed on MCF7 cells stably transfected with lamin A/C transcript variants. Lamin A or lamin AΔ50 upregulation was associated with activation of cell death and inactivation of carcinogenesis while both lamin C or lamin AΔ10 upregulation activated carcinogenesis and cell death. Data suggest anti-apoptotic and anti-senescence effects of lamin C and lamin AΔ10 as several functions, including apoptosis and necrosis functions are inactivated following lamin C or lamin AΔ10 upregulation. However, lamin AΔ10 upregulation is associated with a more carcinogenic and aggressive tumor phenotype. Lamin A or lamin AΔ50 upregulation is associated with a predicted activation of increased cell death and inactivation of carcinogenesis. Thus, different signaling pathways, networks, molecular and cellular functions are activated/inactivated by lamin A/C transcript variants resulting in a large number of laminopathies.

Multiplexed Human Gene Expression Analysis Reveals a Central Role of the TLR/mTOR/PPARγ and NFkB Axes in Burn and Inhalation Injury-Induced Changes in Systemic Immunometabolism and Long-Term Patient Outcomes.

Burn patients are subject to significant acute immune and metabolic dysfunction. Concomitant inhalation injury increases mortality by 20%. In order to identify specific immune and metabolic signaling pathways in burn (B), inhalation (I), and combined burn-inhalation (BI) injury, unbiased nanoString multiplex technology was used to investigate gene expression within peripheral blood mononuclear cells (PBMCs) from burn patients, with and without inhalation injury. PBMCs were collected from 36 injured patients and 12 healthy, non-burned controls within 72 h of injury. mRNA was isolated and hybridized with probes for 1342 genes related to general immunology and cellular metabolism. From these specific gene patterns, specific cellular perturbations and signaling pathways were inferred using robust bioinformatic tools. In both B and BI injuries, elements of mTOR, PPARγ, TLR, and NF-kB signaling pathways were significantly altered within PBMC after injury compared to PBMC from the healthy control group. Using linear regression modeling, (1) DEPTOR, LAMTOR5, PPARγ, and RPTOR significantly correlated with patient BMI; (2) RPTOR significantly correlated with patient length of stay, and (3) MRC1 significantly correlated with the eventual risk of patient mortality. Identification of mediators of this immunometabolic response that can act as biomarkers and/or therapeutic targets could ultimately aid the management of burn patients.

Suitable primers for GAPDH reference gene amplification in quantitative RT-PCR analysis of human gene expression

In molecular biology, real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) has been commonly used for the evaluation of gene expression in human cells and tissues. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is commonly used as a reference gene for the analysis of RT-qPCR experiments. However, recent studies have recognized several variants of GAPDH gene transcripts in human tissues. Thus, the application of the GAPDH gene in RT-qPCR amplification was also established with false interpretations of differential gene expression analysis. Thereof, a consistent amplification of reference gene transcript is essentially required from various tissues or cells treated under differential conditions. In this study, we designed a set of oligo primers for consistent amplification of the human GAPDH transcripts as a reference for human gene amplification. The designed primers for the GAPDH gene showed equalized amplification of GAPDH transcripts from human fibroblasts cells for differential gene expression analysis under various experimental conditions.

1088 Prenatal diagnosis of TBC1D32-associated disease by exome sequencing: Further evidence of a new syndrome

Prenatal exome sequencing is increasingly utilized to determine the etiology of congenital anomalies. It also provides an opportunity to identify novel candidate genes critical to human development due to the extreme phenotypes often identified prenatally. We present four new cases associated with variants in TBC1D32, a novel candidate gene for Orofacial Digital (OFD) syndrome, Type IX, not yet cataloged in Online Mendelian Inheritance of Man. Cases were identified through an ongoing prenatal exome sequencing study. Anomalous fetuses with normal karyotype and microarray were enrolled and exome sequencing was completed on fetus-mother-father trios. Confirmatory testing was completed in a clinical laboratory. Novel candidate genes with rare variants suspected to affect protein function were entered into Gene MatcherTM, an online database connecting researchers/clinicians to patients with similar phenotypes. A systematic literature review was completed using PubMed. A consanguineous couple with two fetuses with multiple congenital anomalies and pathogenic variants in TBC1D32 was identified. Gene MatcherTM identified 14 additional cases, two of which are included. Seven cases have been published in the literature. Table 1 summarizes the four new cases harboring recessive TBC1D32 variants. We present four cases associated with variants in TBC1D32 in addition to the seven cases published in the literature, supporting TBC1D32 as a causative gene for OFD, type IX. Proteomic studies have identified TBC1D32 as a primary ciliary protein and animal studies demonstrate its involvement in the development and function of cilia. In human gene expression analyses, TCBC1D32 is identified in the developing hypothalamus and pituitary gland. We present the first prenatal case with absence of the pituitary gland, a finding in four of the published cases with postnatal presentations. This case highlights how extreme prenatal phenotypes can help identify genes critical to human development and the importance of fetal autopsy and deep phenotyping in the context of prenatal exome sequencing.

Atlas of RNA sequencing profiles for normal human tissues

Comprehensive analysis of molecular pathology requires a collection of reference samples representing normal tissues from healthy donors. For the available limited collections of normal tissues from postmortal donors, there is a problem of data incompatibility, as different datasets generated using different experimental platforms often cannot be merged in a single panel. Here, we constructed and deposited the gene expression database of normal human tissues based on uniformly screened original sequencing data. In total, 142 solid tissue samples representing 20 organs were taken from post-mortal human healthy donors of different age killed in road accidents no later than 36 hours after death. Blood samples were taken from 17 healthy volunteers. We then compared them with the 758 transcriptomic profiles taken from the other databases. We found that overall 463 biosamples showed tissue-specific rather than platform- or database-specific clustering and could be aggregated in a single database termed Oncobox Atlas of Normal Tissue Expression (ANTE). Our data will be useful to all those working with the analysis of human gene expression.

High throughput screening at novel striatum orphan GPCRs identified by human transcriptomic profiling

The striatum performs essential brain functions including movement control, regulation of attention and is a critical component of reward systems. Current druggable targets have displayed limited efficacy in treating striatal diseases including addiction, schizophrenia and Parkinson's disease. G protein‐coupled receptors (GPCRs) are essential drug targets with ~35% of FDA approved medicines targeting these receptors. Of the 350 non‐sensory GPCRs, nearly 120 remain orphan GPCRs, whose endogenous ligands and G protein signaling are unknown. A previous challenge with orphan receptors has been the design of reliable drug screens, but a recent advance in the PRESTO‐Tango assay now allows for a measurement of receptor activation for over 300 GPCRs. Here we pursue the objective of identifying new druggable GPCRs, selectively expressed in the human striatum, using human tissue transcriptomics and the PRESTO‐Tango platform. We conducted a comprehensive genome‐wide survey of human tissue‐selective gene expression using 1640 high‐quality RNA‐seq samples from the Genotype Tissue Expression (GTEx) project. To identify receptor function and screen ligands, human clones of receptors were expressed in HEK293 cells and signaling was assessed using the Glo‐sensor cAMP and Tango beta‐arrestin assay. As a ligand discovery proof‐of‐concept, the Tango assay was used to screen the LOPAC library against five striatal orphan GPCRs. Human gene expression analyses identified expression of 18 striatal‐selective GPCRs. 11 of these receptors have known ligands including established therapeutic drug targets (e.g. dopamine D1, D2 receptors). The remaining 7 human striatal GPCRs, namely, GPR6, 52, 55, 88, 101, 139 and 149 were identified as class A orphan receptors. Biochemical and CRISPR/Cas9 knockout studies confirmed GPR6, GPR52 and GPR101 increased cAMP via Gs/olf G proteins, while GPR88 decreased cAMP levels via Gi/o G proteins. Screening the LOPAC library in parallel against five orphans (6, 52, 88, 101, 149) using the Tango assay revealed 60 compounds that selectively induced a 2‐fold activity above or below vehicle treated cells for one of the five receptors. Dose response assays confirmed two compounds with agonist activity for GPR88 (clozapine and carbachol) and one with agonist activity for GPR149 (PDTC). Interestingly, carbachol and clozapine are FDA approved drugs. Clozapine is a classic atypical antipsychotic medication and inverse agonist at serotonin and DA receptors; and carbachol is a cholinergic agonist currently used for the treatment of glaucoma. PDTC agonist activity for GPR149 represents the first described agonist compound for this orphan receptor. This research provides a template for identifying brain region specific targets using human tissue RNAseq transcriptomic datasets. Our findings also suggest the seven identified human orphan GPCRs are potential drug targets amenable to screening, whose pharmacological modulation may be therapeutic for treating striatum‐related neurological diseases.Support or Funding InformationRising Star Award, UT System (JAA); UTMB institutional funding (JAA); T32 DA07287 (DEF)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spectrum of Microbial Sequences and a Bacterial Cell Wall Antigen in Primary Demyelination Brain Specimens Obtained from Living Patients

Multiple sclerosis (MS) is an autoimmune disease characterized by multiple lesions in the brain and spinal cord. We used RNA sequencing to identify microbial sequences and characterize human gene expression patterns in 30 human brain biopsy specimens. RNAs which aligned to known microbial taxa, were significantly enriched in 10 of 12 primary demyelination (MS) brain specimens compared to a group of 15 epilepsy controls, leading to a list of 29 MS microbial candidate genera from 11 different phyla. Most of the candidate MS microbes are anaerobic bacteria. While there were some shared candidates, each of the 10 MS samples with significant microbial RNA enrichment had a distinct set microbial candidates. The fraction of microbial sequencing reads was greater for the MS group (128.8 PPM) compared to the controls (77.4 PPM, p = 0.016). Bacterial peptidoglycan was demonstrated in brain tissue sections from several MS subjects. Human gene expression analysis showed increased expression of inflammation-related pathways in the MS group. This data shows that demyelinating brain lesions are associated with the presence of microbial RNA sequences and bacterial antigen. This suggests that MS is triggered by the presence of a diverse set of microbes within a lesion.

Beta-catenin represses protein kinase D1 gene expression by non-canonical pathway through MYC/MAX transcription complex in prostate cancer.

Down regulation of Protein Kinase D1 (PrKD1), a novel serine threonine kinase, in prostate, gastric, breast and colon cancers in humans leads to disease progression. While the down regulation of PrKD1 by DNA methylation in gastric cancer and by nuclear beta-catenin in colon cancer has been shown, the regulatory mechanisms in other cancers are unknown. Because we had demonstrated that PrKD1 is the only known kinase to phosphorylate threonine 120 (T120) of beta-catenin in prostate cancer resulting in increased nuclear beta-catenin, we explored the role of beta-catenin in gene regulation of PrKD1. An initial CHIP assay identified potential binding sites for beta-catenin in and downstream of PrKD1 promoter and sequencing confirmed recruitment of beta-catenin to a 166 base pairs sequence upstream of exon 2. Co-transfection studies with PrKD1-promoter-reporter suggested that beta-catenin represses PrKD1 promoter. Efforts to identify transcription factors that mediate the co-repressor effects of beta-catenin identified recruitment of both MYC and its obligate heterodimer MAX to the same binding site as beta-catenin on the PrKD1 promoter site. Moreover, treatment with MYC inhibitor rescued the co-repressor effect of beta-catenin on PrKD1 gene expression. Prostate specific knock out of PrKD1 in transgenic mice demonstrated increased nuclear expression of beta-catenin validating the in vitro studies. Functional studies showed that nuclear translocation of beta-catenin as a consequence of PrKD1 down regulation, increases AR transcriptional activity with attendant downstream effects on androgen responsive genes. In silico human gene expression analysis confirmed the down regulation of PrKD1 in metastatic prostate cancer correlated inversely with the expression of MAX, but not MYC, and positively with MXD1, a competing heterodimer of MAX, suggesting that the dimerization of MAX with either MYC or MXD1 regulates PrKD1 gene expression. The study has identified a novel auto-repressive loop that perpetuates PrKD1 down regulation through beta-catenin/MYC/MAX protein complex.

Neuromuscular junction degeneration in muscle wasting.

Denervation is a hallmark of age-related and other types of muscle wasting. This review focuses on recent insights and current viewpoints regarding the mechanisms and clinical relevance of maintaining the neuromuscular junction to counteract muscle wasting resulting from aging or neural disease/damage. Activity-dependent regulation of autophagy, the agrin-muscle specific kinase-Lrp4 signaling axis, and sympathetic modulation are principal mechanisms involved in stabilizing the neuromuscular junction. These findings are derived from several animal models and were largely confirmed by human gene expression analysis as well as insights from rare neuromuscular diseases such as amyotrophic lateral sclerosis and congenital myasthenic syndromes. Based on these insights, agrin-derived fragments are currently being evaluated as biomarkers for age-related muscle wasting. Tuning of autophagy, of the agrin pathway, and of sympathetic input are being studied as clinical treatment of muscle wasting disorders. Basic research has revealed that maintenance of neuromuscular junctions and a few signaling pathways are important in the context of age-dependent and other forms of muscle wasting. These findings have recently started to enter clinical practice, but further research needs to substantiate and refine our knowledge.

Cross-species gene expression analysis identifies a novel set of genes implicated in human insulin sensitivity

Objective:Insulin resistance (IR) is one of the earliest predictors of type 2 diabetes. However, diagnosis of IR is limited. High fat fed mouse models provide key insights into IR. We hypothesized that early features of IR are associated with persistent changes in gene expression (GE) and endeavored to (a) develop novel methods for improving signal:noise in analysis of human GE using mouse models; (b) identify a GE motif that accurately diagnoses IR in humans; and (c) identify novel biology associated with IR in humans.Methods:We integrated human muscle GE data with longitudinal mouse GE data and developed an unbiased three-level cross-species analysis platform (single gene, gene set, and networks) to generate a gene expression motif (GEM) indicative of IR. A logistic regression classification model validated GEM in three independent human data sets (n=115).Results:This GEM of 93 genes substantially improved diagnosis of IR compared with routine clinical measures across multiple independent data sets. Individuals misclassified by GEM possessed other metabolic features raising the possibility that they represent a separate metabolic subclass. The GEM was enriched in pathways previously implicated in insulin action and revealed novel associations between β-catenin and Jak1 and IR. Functional analyses using small molecule inhibitors showed an important role for these proteins in insulin action.Conclusions:This study shows that systems approaches for identifying molecular signatures provides a powerful way to stratify individuals into discrete metabolic groups. Moreover, we speculate that the β-catenin pathway may represent a novel biomarker for IR in humans that warrant future investigation.

Editors' pick: transcriptomes of 1000 genomes.

Next generation sequencing (NGS) technologies facilitate massive human DNA sequence variation data to be produced in a remarkable manner and speed. The functional effects of these variants can now also be analyzed by high-throughput RNA sequencing (RNA-seq) of the transcriptome. Prior to RNA-seq, analysis of human gene expression was performed by expression arrays with more limited coverage. In the recent issue of Nature, Lappalainen et al.[1] have, for the first time, used the full capacity of RNA-seq to create the largest existing catalog of potential causative functional variants in the genome and to characterize transcriptome variation in human populations in a subset of individuals from the 1000 Genomes Project. They reported a deep analysis of high-quality mRNA and miRNA sequences from lymphoblast cell lines from > 450 individuals belonging to five populations (CEPH-CEU Europeans, Finns, British, Toscani Italians, and Yoruba from Nigeria). The authors addressed several questions with their dataset. First, they investigated human transcriptome variation at the population level, which can manifest in overall expression levels or in splicing (in one gene). In a genome-wide perspective, Lappalainen et al. showed that population differences account for about 3% of the total variation, but they also identified 263–4.379 genes with differential expression and/or transcript ratios between population pairs. In the pairwise analysis, they observed something interesting: the African-European population pairs had a higher contribution of genes with differential splicing (75 to 85%) than the European populations had between each other (6 to 40%). Although this phenomenon has not been previously observed in humans, it is in agreement with phylogenetic differences between species being better captured by splicing rather than expression levels. Lappalainen et al. analyzed 644 autosomal miRNAs that could be quantified from > 50% of the individuals. Sixty of those miRNAs (9.3%) had significant cis-expression QTLs (eQTLs) for miRNA expression levels, indicating that genetic effects on miRNA expression are more widespread than had been known. The authors also provide evidence for the existence of feedback loops for mRNA and miRNA genes to have an effect on each other’s expression supporting the idea that miRNAs offer robustness in the expression programs. Altogether, Lappalainen et al. have produced the largest and most diverse catalogue of cis-regulatory variants in a single tissue to date. The authors used their data to analyze the functional properties of the newly found set of regulatory variants and transcriptome effects of protein-truncating loss-of-function variants. This collaborative effort further used the potential of RNA-seq to discover regulatory variants that affect not only expression levels but also splicing. Of the 7.825 genes with expression QTLs (eQTLs), 34% have a second, independent eQTL for any of their exons. The authors concluded that in the transcriptome there exists allelic heterogeneity for regulatory effects on a single gene and independence of exons within the same gene. As an ancillary benefit, the authors demonstrated that RNA-seq data is consistent even when produced in different laboratories, with more detailed analysis in a companion paper in Nature Biotechnology[2]. Namely, in the replicate analyses of RNA-seq data from seven laboratories, there was a smaller amount of variation among the laboratories than seen among the individuals. The study is a great example of integration of genomic sequencing and cellular phenotype data. Obviously, the amount of data produced in this study is enormous, and it can easily be expected that the data will also be used to support other pivotal work. Meanwhile, it will take some time to completely digest the magnitude of data and appreciate the impact it will have on functional genomics.

Abstract SY01-03: Comparative genomics as a tool for the identification of novel Kras synthetic lethal interactions

Abstract Gene expression profiling has been widely used as a tool to characterize genome-wide changes in expression level in various cancers. Expression profiling is generally used to identify specific genes with altered expression or to identify patterns that distinguish cancer subtypes. More recently, several approaches to use expression data to identify interactions between genes or build cancer-specific networks have been described. We have previously demonstrated that cross-species gene expression analysis can be used as a tool to identify oncogene-specific alterations in gene expression. Cross-species analysis is useful for this because it allows for filtering of noise inherent in human gene expression analysis. The identification of an oncogenic Kras-specific gene expression signature was accomplished using this approach. A central focus of our laboratory has been to use this and other oncogene-specific signatures to identify new critical downstream pathways necessary for oncogene function. Using several computational approaches, we identified several upstream transcriptional regulators of our previously identified oncogenic Kras signature. We hypothesized that some of the transcriptional regulators represent specific dependencies that are required for Kras to function as an oncogene. Thus, inhibition of the transcription factors could uncover novel synthetic lethal interactions specific for cells that express oncogenic Kras. Using an shRNA pooled library, we have confirmed one such synthetic lethal interaction betweeen oncogenic Kras and the Wilms-tumor 1 (Wt1), a gene known to function as a tumor suppressor in some contexts. Loss of Wt1 induces senescence in primary cells expressing oncogenic Kras. Human non-small cell lung cancer cells expressing oncogenic Kras also become senescent when Wt1 is knocked-down using shRNA. Thus, synthetic senescence is induced on Wt1 loss in oncogenic Kras expressing cells. A role for Wt1 in Kras-driven oncogenesis was further confirmed using a mouse model for lung cancer in which a mouse carrying a conditional Kras allele was crossed a mouse carrying an allelel that allows for conditional deletion of Wt1. The biological signficance of this intereation was further demonstrated by using a “Wt1 “high” or “Wt1 low” signature to classify human NSCLC gene expression profiles. This allowed us to determine that human tumor samples that express oncogenic Kras and express high levels of Wt1-regulated genes have a worse prognosis compared to samples expressing oncogenic Kras but having low low level of Wt1-regulated genes. In human NSCLC cell lines, loss of Wt1 leads to significant upregulation of the cell cycle regulator p21. Wt1 appears to regulate p21 levels posttranscriptionally, as p21 mRNA levels did not change. Further studies to elucidate the mechanisms leading to p21 upregulation upon Wt1 loss are underway and preliminary results will be discussed. To take these studies further, we began by generating a more refined Kras-specific signature using FACs-sorted tumor cells from the LSL-KrasG12D mouse model crossed to a fluorescent reporter. We have used this mouse-generated Kras signature for cross-species analysis using a much larger NSCLC dataset than was possible in our previously published studies. We then applied the ARACNE algorithm to identify transcription factors that regulate this Kras-signature. Using this approach, we have identified other transcription factors that are potential regulators of oncogenic Kras. The fact that Wt1 once again was identified as a regulator of oncogenic Kras using this novel method suggests the validity of this approach. Preliminary results testing the functional significance of these transcription factors for Kras-driven oncogenesis will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY01-03. doi:10.1158/1538-7445.AM2011-SY01-03

Role of sex and time of blood sampling in SOD1 and SOD2 expression variability

Objectives This study investigates how some of human physiological parameters, in particular sex and daytime, can contribute to the normal variation in blood gene expression. Design and methods The expressions of two genes: SOD1 (Cu/Zn) and SOD2 (Mn) in white blood cells of 19 healthy individuals (10 men and 9 women) were examined by qPCR at 09.00, 12.00, and 15.00 h over three consecutive weeks at 7-day intervals. SOD1 and SOD2 expressions differ between sexes, higher expression being exhibited in males ( P < 0.005). In the male group both genes had lower expression ( P < 0.005) with smaller variation at 09.00 h. In females, both inter- and intra-subject, variability in SOD1 expression was increased at ovulation. Intra-individually, the expressions of genes positively correlated in males, but less in females. Conclusions SOD1 and SOD2 expressions demonstrate how sex and daytime, merit being controlled when human gene expression analyses are evaluated, particularly within the framework of clinical trials or cohort studies.

Detection of allelic variations of human gene expression by polymerase colonies

BackgroundQuantification of variations of human gene expression is complicated by the small differences between different alleles. Recent work has shown that variations do exist in the relative allelic expression levels in certain genes of heterozygous individuals. Herein, we describe the application of an immobilized polymerase chain reaction technique as an alternative approach to measure relative allelic differential expression.ResultsHerein, we report a novel assay, based on immobilized polymerase colonies, that accurately quantifies the relative expression levels of two alleles in a given sample. Mechanistically, this was accomplished by PCR amplifying a gene in a cDNA library in a thin polyacrylamide gel. By immobilizing the PCR, it is ensured that each transcript gives rise to only a single immobilized PCR colony, or "polony". Once polony amplified, the two alleles of the gene were differentially labeled by performing in situ sequencing with fluorescently labeled nucleotides. For these sets of experiments, silent single nucleotide polymorphisms (SNPs) were used to discriminate the two alleles. Finally, a simple count was then performed on the differentially labeled polonies in order to determine the relative expression levels of the two alleles. To validate this technique, the relative expression levels of PKD2 in a family of heterozygous patients bearing the 4208G/A SNP were examined and compared to the literature.ConclusionsWe were able to reproduce the results of allelic variation in gene expression using an accurate technology known as polymerase colonies. Therefore, we have demonstrated the utility of this method in human gene expression analysis.

DNA microarray analysis of human gene expression induced by a non-lethal dose of cadmium.

Cadmium (Cd) is a hazardous heavy metal present in working and living environments. Cd affects many cellular functions, but little is known about the mechanisms of its toxicity and cellular defense against it. Recently, advanced gene expression analysis employing DNA microarrays provided us the means to profile the expression of thousands of genes simultaneously. We describe here a study of Cd-induced gene expression profile. Messenger RNA was prepared from HeLa cells exposed to a non-lethal dose of CdSO4, and analyzed by the use of an array consisting of 7,075 human cDNAs. Many stress response genes including those coding for metallothioneins and heat shock proteins were observed to be induced by Cd. The cellular metabolism inclined toward the synthesis of cysteine and glutathione after Cd exposure. Anti-oxidant genes also appeared to be induced to protect cell components and to quench reactive oxygen species. Ubiquitin pathway was activated as well probably to degrade proteins which might not be renatured. These data suggest that human cells mobilize every genomic resource (induction of some genes and repression of others) to overcome cytotoxicity caused by Cd.

Monoclonal Antibodies to Human Urothelial Cell Lines and Hybrids: Production and Characterization

Eleven independent monoclonal antibodies, the LBS series, were isolated after immunization of mice with RT112 cells, a continuous cell line derived from a transitional cell carcinoma of the human bladder. These antibodies were tested by indirect immunofluorescence on a panel of 28 human cell lines, of which 17 were urothelial carcinoma-derived, 4 of non-urothelial carcinoma origin, 3 fibroblast cell lines, 4 lymphoblastoid lines and 7 murine cell lines. Also tested were 7 somatic cell hybrid clones derived by fusion of human RT112 cells with murine bladder carcinoma MB63T/H cells. None of the LBS antibodies reacted with mesenchyme-derived cells, although all reacted with RT112 cells. On the basis of reactivity with the cell line panel, the antibodies were divided into 3 groups. Group I (LBS-1 and 19) reacted with all human epithelium-derived cell lines. Group II (LBS-2, 8, 15 and 17) reacted only with human urothelium-derived cells, tending to recognise the least anaplastic types. Group III antibodies (LBS-10, 20A, 20B, 21 and 34) were urothelium-specific on the human continuous cell line panel, but additionally reacted with murine urothelial and epithelial cell lines. The 6 human-specific antibodies (Group I and II) were used for preliminary analysis of human gene expression in a series of 7 mouse × human urothelial somatic cell hybrids. Each hybrid reacted with at least 1 LBS antibody, although there were changes in gene expression with time in culture, indicating both loss and unmasking of human genes. These data suggest the LBS-series antibodies recognise different determinants associated with epithelial and urothelial cell differentiation, and thus may be valuable probes in the study of normal differentiation and malignant transformation in human urothelial cells.