Isolation of genes from complex sources of mammalian genomic DNA using exon amplification.

Modifications to exon amplification have been instituted that increase its speed, efficiency and reliability. Exons were isolated from target human or mouse genomic DNA sources ranging from 30 kilobases (kb) to 3 megabases (Mb) in complexity. The efficiency was dependent upon the amount of input DNA, and ranged from isolation of an exon for every 20 kb to an exon for every 80 kb of target genomic DNA. In these studies, several novel genes and a smaller number of genes isolated previously that reside on human chromosome 9 have been identified. These results indicate that exon amplification is presently adaptable to large scale isolation of exons from complex sources of genomic DNA.

The early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein.

Early-onset torsion dystonia is a movement disorder, characterized by twisting muscle contractures, that begins in childhood. Symptoms are believed to result from altered neuronal communication in the basal ganglia. This study identifies the DYT1 gene on human chromosome 9q34 as being responsible for this dominant disease. Almost all cases of early-onset dystonia have a unique 3-bp deletion that appears to have arisen idependently in different ethnic populations. This deletion results in loss of one of a pair of glutamic-acid residues in a conserved region of a novel ATP-binding protein, termed torsinA. This protein has homologues in nematode, rat, mouse and humans, with some resemblance to the family of heat-shock proteins and Clp proteases.

Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family.

Holt-Oram syndrome is a developmental disorder affecting the heart and upper limb, the gene for which was mapped to chromosome 12 two years ago. We have now identified a gene for this disorder (HOS1). The gene (TBX5) is a member of the Brachyury (T) family corresponding to the mouse Tbx5 gene. We have identified six mutations, three in HOS families and three in sporadic HOS cases. Each of the mutations introduces a premature stop codon in the TBX5 gene product. Tissue in situ hybridization studies on human embryos from days 26 to 52 of gestation reveal expression of TBX5 in heart and limb, consistent with a role in human embryonic development.

Role of Rel-related factors in control of c-myc gene transcription in receptor-mediated apoptosis of the murine B cell WEHI 231 line.

Treatment of immature murine B lymphocytes with an antiserum against their surface immunoglobulin (sIg)M results in cell death via apoptosis. The WEHI 231 B cell line (IgM, kappa) has been used extensively as a model for this anti-Ig receptor-mediated apoptosis. Anti-sIg treatment of WEHI 231 cells causes an early, transient increase in the levels of c-myc messenger RNA and gene transcription, followed by a rapid decline below control values. Given the evidence for a role of the c-myc gene in promoting apoptosis, we have characterized the nature and kinetics of changes in the binding of Rel-related factors, which modulate c-myc promoter activity. In exponentially growing WEHI 231 cells, multiple Rel-related binding activities were detectable. The major binding species was identified as p50/c-Rel heterodimers; only minor amounts of nuclear factor kappa B (NF-kappa B) (p50/p65) were detectable. Cotransfection of an inhibitor of NF-kappa B (I kappa B)-alpha expression vector reduced c-myc-promoter/upstream/exon1-CAT reporter construct activity, indicating the role of Rel factor binding in c-myc basal expression in these cells. Treatment with anti-sIg resulted in a rapid transient increase in the rate of c-myc gene transcription and in the binding of Rel factors. At later times, formation of p50 homodimer complexes occurred. In cotransfection analysis, p65 and c-Rel expression potently and modestly transactivated the c-myc promoter, respectively, whereas, overexpression of the p50 subunit caused a significant drop in its activity. The role of activation of Rel-family binding was demonstrated directly upon addition of the antioxidant pyrrolidinedithiocarbamate, which inhibited the anti-sIg-mediated activation of the endogenous c-myc gene. Similarly, induction after anti-sIg treatment of a transfected c-myc promoter was abrogated upon cotransfection of an I kappa B-alpha expression vector. These results implicate the Rel-family in Ig receptor-mediated signals controlling the activation of c-myc gene transcription in WEHI 231 cells, and suggest a role for this family in apoptosis of this line, which is mediated through a c-myc signaling pathway.

Change in lung tumor volume as a biomarker of treatment response: a critical review of the evidence.

SPECIFIC AIM: To review the evidence indicating that volumetric image analysis of computed tomography scans meets specifications for qualification as a biomarker in clinical trials or the management of individual patients with lung cancer. METHODS: Claims of value were broken down into questions about technical feasibility, accuracy, the precision of measurement, sensitivity, the correlations with health outcomes, and the risks of producing misleading information. For each claim, the pertinent literature was reviewed. RESULTS: Technical feasibility has now been shown, but only in limited contexts. Accuracy has been demonstrated, but only for tumors with favorable anatomical features. Measurement error still makes the assessment of change in small nodules precarious in diagnostic settings unless rigorous image acquisition and analysis procedures are followed. Precision is sufficient in some larger masses to make volumetrics a sensitive biomarker. In a few trials, correlations with clinical outcomes have been higher for volumetric-based measures than for unidimensional or bidimensional diameters. Value in clinical practice settings and clinical trials has been suggested, but not proven. CONCLUSION: The weight of the evidence indicates there are circumstances in which volumetric image analysis adds value to clinical trial science and the practice of medicine.

Data sets for the qualification of volumetric CT as a quantitative imaging biomarker in lung cancer.

The drug development industry is faced with increasing costs and decreasing success rates. New ways to understand biology as well as the increasing interest in personalized treatments for smaller patient segments requires new capabilities for the rapid assessment of treatment responses. Deployment of qualified imaging biomarkers lags apparent technology capabilities. The lack of consensus methods and qualification evidence needed for large-scale multi-center trials, as well as the standardization that allows them, are widely acknowledged to be the limiting factors. The current fragmentation in imaging vendor offerings, coupled with the independent activities of individual biopharmaceutical companies and their contract research organizations (CROs), may stand in the way of the greater opportunity were these efforts to be drawn together. A preliminary report, "Volumetric CT: a potential biomarker of response," of the Quantitative Imaging Biomarkers Alliance (QIBA) activity was presented at the Medical Imaging Continuum: Path Forward for Advancing the Uses of Medical Imaging in the Development of New Biopharmaceutical Products meeting of the Extended Pharmaceutical Research and Manufacturers of America (PhRMA) Imaging Group sponsored by the Drug Information Agency (DIA) in October 2008. The clinical context in Lung Cancer and a methodology for approaching the qualification of volumetric CT as a biomarker has since been reported [Acad. Radiol. 17, 100-106, 107-115 (2010)]. This report reviews the effort to collect and utilize publicly available data sets to provide a transparent environment in which to pursue the qualification activities in such a way as to allow independent peer review and verification of results. This article focuses specifically on our role as stewards of image sets for developing new tools.

Two-step stimulation of B lymphocytes to enter DNA synthesis: synergy between anti-immunoglobulin antibody and cytochalasin on expression of c-myc and a G1-specific gene.

Previously we demonstrated that stimulation of resting murine splenic B lymphocytes with goat anti-mouse immunoglobulin antibody (GaMIg) plus cytochalasin D (CD) led to DNA synthesis; GaMIg and CD added simultaneously, or GaMIg added before CD, induced this response (T. L. Rothstein, J. Immunol. 136:813-816, 1986). Cells similarly treated with GaMIg or CD alone did not enter S phase. Here we have measured the effects of this two-signal stimulation on the c-myc, 2F1, and gamma-actin genes. The expression of these growth-related genes is known to change either during the G0-to-G1 transition or in the G1 phase of the cell cycle. For the 2F1 and c-myc genes, neither the GaMIg nor CD stimulus alone led to a prolonged increase in mRNA levels, whereas GaMIg plus CD allowed for continuous elevated expression of these genes. Furthermore, GaMIg pretreatment rendered expression of the c-myc and 2F1 genes susceptible to subsequent action by CD. In contrast, CD alone was sufficient to produce changes in gamma-actin gene expression. Thus there are synergistic effects of competence- and progressionlike factors on the expression of the c-myc and 2F1 genes, and these effects correlate with the progression of B lymphocytes to DNA synthesis.

Isolation, characterization, and expression of the murine Wilms' tumor gene (WT1) during kidney development.

The human Wilms' tumor predisposition gene, WT1, is a Cys-His zinc finger polypeptide which appears to be a transcription factor controlling gene expression during embryonic kidney development. In order to analyze the role of the WT1 gene in nephroblast differentiation, we have isolated the murine homolog of human WT1. An extremely high level of amino acid sequence conservation (greater than 95%) extends throughout all regions of the predicted mouse and human WT1 polypeptides. Two alternative splices within the WT1 transcript have been conserved between mice and humans, suggesting that these have functional significance. Expression of the mouse WT1 mRNA in fetal kidney increases during late gestation, peaks just prior to or shortly after birth, and declines dramatically by 15 days postpartum. Developmental regulation of WT1 expression appears to be selective for the kidney. The restriction of WT1 expression to a limited number of tissues is in contrast to previously described tumor suppressor genes. In addition, the narrow window of time during which WT1 is expressed at high levels in the kidney is consistent with the origin of Wilms' tumor from primitive nephroblasts and the postulated role of this gene as a negative regulator of growth.

Independent regulation of transcription of the two strands of the c-myc gene.

Previously we demonstrated the existence of transcripts from the noncoding strand of a rearranged, truncated c-myc gene in murine plasmacytomas in which this oncogene is translocated to an immunoglobulin constant-region gene element (M. Dean, R. B. Kent, and G. E. Sonenshein, Nature [London] 305:443-446, 1983). Here we report on the transcription of the two strands of a normal, unrearranged c-myc gene. We examined the effects of gene rearrangements, growth state transitions, and differentiation on the relative levels of usage of the two strands. Transcription from intron 1 to exon 3 of the murine c-myc gene was studied in in vitro nuclear runoff assays. The level of transcription of the noncoding strand across this region of a germ line c-myc gene in a murine B-cell lymphoma line was comparable to the level observed in plasmacytomas with translocated c-myc genes. Rapid changes in transcription of the coding strand of the c-myc gene could be seen during growth arrest of WEHI 231 cells and during activation of splenic T lymphocytes. Transcription of the noncoding strand was constitutive during these growth state transitions and during activation of primary cultures of quiescent calf aortic smooth muscle cells as well. In contrast, differentiation of murine erythroleukemia cells was accompanied by an early drop in transcription of the two strands of this gene. The ramifications of these findings with respect to measurements of c-myc gene transcription and to the regulation of this gene are discussed.

Mutational analysis of CDKN2 (MTS1/p16ink4) in human breast carcinomas.

The CDKN2 gene that encodes the cell cycle regulatory protein cyclin-dependent kinase-4 inhibitor (p16) has recently been mapped to chromosome 9p21. Frequent homozygous deletions of this gene have been documented in cell lines derived from different types of tumors, including breast tumors, suggesting that CDKN2 is a tumor suppressor gene involved in a wide variety of human cancers. To determine the frequency of CDKN2 mutations in breast carcinomas, we screened 37 primary tumors and 5 established breast tumor cell lines by single-strand conformation polymorphism analysis. In addition, Southern blot analysis was performed on a set of five primary breast carcinoma samples and five breast tumor cell lines. Two of the five tumor cell lines revealed a homozygous deletion of the CDKN2 gene, but no mutations were observed in any of the primary breast carcinomas. These results suggest that the mutation of the CDKN2 gene may not be a critical genetic change in the formation of primary breast carcinoma.

p16 alterations and deletion mapping of 9p21-p22 in malignant mesothelioma.

To determine whether p16 is altered in human malignant mesothelioma (MM), molecular analysis of multiple 9p loci was performed on 40 cell lines and 23 primary tumors from 42 MM patients. We identified homozygous deletions of p16 in 34 (85%) cell lines and a point mutation in 1 line. Down-regulation of p16 was observed in 4 of the remaining cell lines, 1 of which displayed a DNA rearrangement of p16. Homozygous deletions of p16 were identified in 5 of 23 (22%) primary tumors; no mutations or rearrangements were found in these specimens. Four cell lines displayed a single homozygous deletion proximal to or distal to p16; 4 others had 2 nonoverlapping deletions, one involving p16 and the other involving a region proximal to this locus. These data indicate that alterations of p16 are a common occurrence in MM cell lines and, to a lesser extent, in primary tumors. Furthermore, deletions of 9p21-p22 outside of the p16 locus may reflect the involvement of other putative tumor suppressor genes that could also contribute to the pathogenesis of some MMs.

Isolation of conserved sequences from yeast artificial chromosomes by exon amplification.

Exon amplification is a technique designed to solve a central problem in mammalian molecular genetics--specifically, the isolation of genes from large regions of genomic DNA. This technique allows exons to be isolated from genomic DNA following the selective removal of introns by the eukaryotic splicing mechanism. It is a relatively rapid procedure and can theoretically be applied to test the coding potential of very long stretches of genomic DNA. In this paper we describe the application of exon amplification to a mouse yeast artificial chromosome (YAC) 175 kb in length. A number of potential coding sequences were amplified, and two sequences were shown to be conserved across a wide variety of species representing potential genes.

Isolation of genes from the Batten candidate region using exon amplification. Batten Disease Consortium.

In order to identify genes originating from the Batten disease candidate region, we have used the technique of exon amplification to identify transcribed sequences. This procedure produces trapped exon clones, which can represent single exons or multiple exons spliced together and is an efficient method for obtaining probes for physical mapping and for screening cDNA libraries. The source of DNA for these experiments was a collection of chromosome 16 cosmid contigs isolated by the direct subcloning of region-specific yeast artificial chromosomes (YACs) and hybridization of inter-alu PCR products from these YACs to the flow-sorted Los Alamos chromosome 16 cosmid library. We are now using the resulting exon probes to screen retina and brain cDNA libraries for candidate JNCL genes.

hREC2, a RAD51-like gene, is disrupted by t(12;14) (q15;q24.1) in a uterine leiomyoma.

A balanced translocation between chromosomes 12 and 14 is commonly seen in uterine leiomyoma (UL). We have previously cloned and characterized a 2 Mb segment of human chromosomal subband 14q24.1, and have shown that the t(12;14)(q15;q24.1) breakpoints from several ULs map within this region. Exon trapping of DNA clones spanning one such breakpoint revealed coding sequences from hREC2, a gene that shows significant amino acid sequence identity to the double-strand break repair enzyme RAD51. We report here that this breakpoint is located within a 19 kb intron of the hREC2 gene and that the translocation results in the premature truncation of the major hREC2 transcript. Mapping and sequence analyses show that alternative transcripts of the hREC2 gene, including novel isoforms identified in testis and uterus, are not interrupted by the translocation.

WT1: a novel tumor suppressor gene inactivated in Wilms' tumor.

The development of Wilms' tumor, a pediatric kidney cancer, has been linked to the inactivation of a tumor suppressor gene both by epidemiologic studies and by genetic analyses. Like retinoblastoma, Wilms' tumors can occur bilaterally in individuals with apparent genetic susceptibility to this disease. This led Knudson and Strong to propose in 1972 that two genetic events were rate limiting in tumor development and that predisposed individuals had already inherited one mutation in the germline. The observation of karyotype abnormalities in predisposed children and studies of the molecular genetics of Wilms' tumor specimens enabled the identification of chromosome band 11p13 as one genetic locus inactivated in Wilms' tumor. The recent isolation of the WT1 gene, which is the specific target within that locus, offers new insight into the etiology of Wilms' tumor. This gene has properties distinct from those of other known tumor suppressor genes. WT1 encodes a zinc finger transcription factor that is alternatively spliced and has high sequence homology to the early growth response genes (EGR). Unlike the retinoblastoma (RB1) and p53 genes that are expressed ubiquitously, WT1 is expressed in specific cells of the kidney and only during a short period in development. Thus, disruption of a gene that is active during a critical period in the development of a specific organ can lead to neoplastic growth in that organ. Future studies are aimed at exploring the link between the role of the WT1 gene in normal development and in tumorigenesis of the kidney.

Interaction of an NF-kappa B-like factor with a site upstream of the c-myc promoter.

The c-myc protooncogene has been implicated in control of growth and differentiation of mammalian cells. For instance, growth arrest is often preceded by reduction in c-myc mRNA and gene transcription. To elucidate the mechanisms of control of c-myc gene transcription, we have begun to characterize the interaction of nuclear factors with the 719-base-pair (bp) c-myc regulatory domain, located 1139-421 bp upstream of the P1 start site of the mouse gene. Nuclear extracts from exponentially growing WEHI 231 murine B-lymphoma cells formed multiple complexes in mobility-shift assays. Changes in complex distribution were observed in growth-arrested WEHI 231 cells, and a major site of this interaction mapped to a 21-bp sequence that is similar to the sequences recognized by the NF-kappa B family of proteins. Binding of NF-kappa B-like factors was demonstrated by oligonucleotide competition. Induction of complex formation upon 70Z/3 pre-B- to B-cell differentiation, enhancement of binding by GTP, and detergent-induced release of inhibitor protein suggested that NF-kappa B itself is one member of the family that can bind. Transfection of thymidine kinase-chloramphenicol acetyltransferase constructs containing the 21-bp c-myc sequence into Jurkat cells demonstrated increased chloramphenicol acetyltransferase activity upon phorbol ester and phytohemagglutinin treatment. These results suggest the involvement of NF-kappa B-like factors in the regulation of c-myc transcription.

Transcriptional control of c-myc gene expression during stimulation of murine B lymphocytes.

The binding of ligand to surface IgR results in the initial activation of B cells. As shown by experiments in which B cells are polyclonally stimulated with anti-Ig antibody, this includes an early increase in c-myc gene expression. In our study a correlation between increases in the rate of gene transcription and the level of c-myc mRNA was observed both with the brief increase in c-myc expression that is induced by anti-Ig, as well as with the more intense and prolonged expression of c-myc that follows treatment with anti-Ig plus the cytoskeleton perturbing agent, cytochalasin. Elevation of the rate of initiation was detected with both stimulatory regimens although the combination of anti-Ig plus cytochalasin increased the rate of elongation in addition to the rate of initiation. These results suggest that stimulation of B lymphocytes alters expression of trans-acting factors that regulate transcription of the c-myc gene.