Veterinary and human biobanking practices: enhancing molecular sample integrity.

Animal models have historically informed veterinary and human pathophysiology. Next-generation genomic sequencing and molecular analyses using analytes derived from tissue require integrative approaches to determine macroanalyte integrity as well as morphology for imaging algorithms that can extend translational applications. The field of biospecimen science and biobanking will play critical roles in tissue sample collection and processing to ensure the integrity of macromolecules, aid experimental design, and provide more accurate and reproducible downstream genomic data. Herein, we employ animal experiments to combine protein expression analysis by microscopy with RNA integrity number and quantitative measures of morphologic changes of autolysis. These analyses can be used to predict the effect of preanalytic variables and provide the basis for standardized methods in tissue sample collection and processing. We also discuss the application of digital imaging with quantitative RNA and tissue-based protein measurements to show that genomic methods augment traditional in vivo imaging to support biospecimen science. To make these observations, we have established a time course experiment of murine kidney tissues that predicts conventional measures of RNA integrity by RIN analysis and provides reliable and accurate measures of biospecimen integrity and fitness, in particular for time points less than 3 hours post-tissue resection.

Germline mutations in the ribonuclease L gene in families showing linkage with HPC1.

Although prostate cancer is the most common non-cutaneous malignancy diagnosed in men in the United States, little is known about inherited factors that influence its genetic predisposition. Here we report that germline mutations in the gene encoding 2'-5'-oligoadenylate(2-5A)-dependent RNase L (RNASEL) segregate in prostate cancer families that show linkage to the HPC1 (hereditary prostate cancer 1) region at 1q24-25 (ref. 9). We identified RNASEL by a positional cloning/candidate gene method, and show that a nonsense mutation and a mutation in an initiation codon of RNASEL segregate independently in two HPC1-linked families. Inactive RNASEL alleles are present at a low frequency in the general population. RNASEL regulates cell proliferation and apoptosis through the interferon-regulated 2-5A pathway and has been suggested to be a candidate tumor suppressor gene. We found that microdissected tumors with a germline mutation showed loss of heterozygosity and loss of RNase L protein, and that RNASEL activity was reduced in lymphoblasts from heterozyogous individuals compared with family members who were homozygous with respect to the wildtype allele. Thus, germline mutations in RNASEL may be of diagnostic value, and the 2-5A pathway might provide opportunities for developing therapies for those with prostate cancer.

Aberrant CREB1 activation in prostate cancer disrupts normal prostate luminal cell differentiation.

The molecular mechanisms of luminal cell differentiation are not understood well enough to determine how differentiation goes awry during oncogenesis. Using RNA-Seq analysis, we discovered that CREB1 plays a central role in maintaining new luminal cell survival and that oncogenesis dramatically changes the CREB1-induced transcriptome. CREB1 is active in luminal cells, but not basal cells. We identified ING4 and its E3 ligase, JFK, as CREB1 transcriptional targets in luminal cells. During luminal cell differentiation, transient induction of ING4 expression is followed by a peak in CREB1 activity, while JFK increases concomitantly with CREB1 activation. Transient expression of ING4 is required for luminal cell induction; however, failure to properly down-regulate ING4 leads to luminal cell death. Consequently, blocking CREB1 increased ING4 expression, suppressed JFK, and led to luminal cell death. Thus, CREB1 is responsible for the suppression of ING4 required for luminal cell survival and maintenance. Oncogenic transformation by suppressing PTEN resulted in constitutive activation of CREB1. However, the tumor cells could no longer fully differentiate into luminal cells, failed to express ING4, and displayed a unique CREB1 transcriptome. Blocking CREB1 in tumorigenic cells suppressed tumor growth in vivo, rescued ING4 expression, and restored luminal cell formation, but ultimately induced luminal cell death. IHC of primary prostate tumors demonstrated a strong correlation between loss of ING4 and loss of PTEN. This is the first study to define a molecular mechanism whereby oncogenic loss of PTEN, leading to aberrant CREB1 activation, suppresses ING4 expression causing disruption of luminal cell differentiation.

Gastrointestinal stromal tumors with KIT mutations exhibit a remarkably homogeneous gene expression profile.

Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the digestive tract, are believed to arise from the interstitial cells of Cajal. GISTs are characterized by mutations in the proto-oncogene KIT that lead to constitutive activation of its tyrosine kinase activity. The tyrosine kinase inhibitor STI571, active against the BCR-ABL fusion protein in chronic myeloid leukemia, was recently shown to be highly effective in GISTs. We used 13,826-element cDNA microarrays to define the expression patterns of 13 KIT mutation-positive GISTs and compared them with the expression profiles of a group of spindle cell tumors from locations outside the gastrointestinal tract. Our results showed a remarkably distinct and uniform expression profile for all of the GISTs. In particular, hierarchical clustering of a subset of 113 cDNAs placed all of the GIST samples into one branch, with a Pearson correlation >0.91. This homogeneity suggests that the molecular pathogenesis of a GIST results from expansion of a clone that has acquired an activating mutation in KIT without the extreme genetic instability found in the common epithelial cancers. The results provide insight into the histogenesis of GIST and the clinical behavior of this therapeutically responsive tumor.

Myeloid-specific TGF-ß signaling in bone promotes basic-FGF and breast cancer bone metastasis.

Breast cancer (BCa) bone metastases cause osteolytic bone lesions, which result from the interactions of metastatic BCa cells with osteoclasts and osteoblasts. Osteoclasts differentiate from myeloid lineage cells. To understand the cell-specific role of transforming growth factor beta (TGF-ß) in the myeloid lineage, in BCa bone metastases, MDA-MB-231 BCa cells were intra-tibially or intra-cardially injected into LysM(Cre)/Tgfbr2(floxE2/floxE2) knockout (LysM(Cre)/Tgfbr2 KO) or Tgfbr2(floxE2/floxE2) mice. Metastatic bone lesion development was compared by analysis of both lesion number and area. We found that LysM(Cre)/Tgfbr2 knockout significantly decreased MDA-MB-231 bone lesion development in both the cardiac and tibial injection models. LysM(Cre)/Tgfbr2 knockout inhibited the tumor cell proliferation, angiogenesis and osteoclastogenesis of the metastatic bones. Cytokine array analysis showed that basic fibroblast growth factor (bFGF) was downregulated in MDA-MB-231-injected tibiae from the LysM(Cre)/Tgfbr2 KO group, and intravenous injection of the recombinant bFGF to LysM(Cre)/Tgfbr2 KO mice rescued the inhibited metastatic bone lesion development. The mechanism by which bFGF rescued the bone lesion development was by promotion of tumor cell proliferation through the downstream mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK)-cFos pathway after binding to the FGF receptor 1 (FGFR1). Consistent with animal studies, we found that in human BCa bone metastatic tissues, TGF-ß type II receptor (TßRII) and p-Smad2 were expressed in osteoclasts and tumor cells, and were correlated with the expression of FGFR1. Our studies suggest that myeloid-specific TGF-ß signaling-mediated bFGF in the bone promotes BCa bone metastasis.

Effect of gliquidone on insulin binding to rabbit erythrocytes.

A study was made of the effects of acute or chronic treatment of rabbits with 10 mg gliquidone (Glurenorm)/kg body weight on erythrocyte insulin receptors. The binding of 125I-labelled insulin to the population of whole red blood cells was not affected by either treatment. Because of the heterogeneity of the erythrocytes, subpopulations were selected according to their specific gravity, which is directly related to cell age. The lowest-density fraction showed a 30% greater capacity of the insulin receptors without any change in affinity after chronic therapy with gliquidone, but no effects were observable after acute treatment. The highest-density fraction was not affected by either acute or chronic administration of gliquidone. Therefore, all other things being equal (blood sugar, plasma insulin levels), young erythrocytes are sensitive to sulphonylurea, whereas old ones are not. Irrespective of the compound tested, this observation suggests that receptor sensitivity might be tissue-specific or age-specific or both, and that the stage of maturation of a cell is a determining factor.

Cellular characteristics of immunolabeled luteinizing hormone releasing hormone (LHRH) neurons.

This study utilized the preembedding immunocytochemical technique in order to identify LHRH-containing neurons in rat brain and define their ultrastructural characteristics. LHRH-containing neurons in the vertical limb of the diagonal band of Broca, medial septum, triangular nucleus of the septum and other regions were studied by taking ultrathin serial sections. These neurons had scant cytoplasm surrounding a centrally-located, spheroid, euchromatic nucleus. Neurosecretory granules were evenly distributed throughout the cell, but many tended to lie directly under the plasmalemma. The cytoplasm was organized in such a way that the most extensive portion of the rough endoplasmic reticulum was polar opposite to areas having high concentrations of Golgi complex, lysosome-like bodies, smooth endoplasmic reticulum and ribosomes. The perikarya had no axosomatic synapses but functional interaction via unspecialized appositions to the plasmalemma cannot be discounted. Many of the perikarya bore at least one cilium. Processes from immunonegative cells were occasionally observed to penetrate the cytoplasm of the LHRH perikaryon or its processes. At their points of origin, dendrites were found to be broadened processes containing many elements common to the cytoplasm: ribosomes, smooth endoplasmic reticulum, cristal and lamellar mitochondria, neurotubules, and an occasional alveolate caveola. Infrequently, some of the LHRH axons were partially myelinated. This method of studying serial-sectioned immunocytochemically-identified cells is suggested as a means of describing the cellular and subcellular characteristics of other specific peptide-containing cells.

An inability of subcutaneous vasopressin to affect passive avoidance behavior.

The effects of subcutaneous injections of vasopressin on the passive avoidance behavior of rats were investigated in an extensive study. 200 male Wistar rats were tested in a step-through passive avoidance task. The animals were assigned randomly to 1 of 20 experimental groups consisting of five vasopressin injection and four shock level conditions. Each animal was trained to enter a dark compartment, then subjected to 0.25, 0.10, 0.05 mA or no foot shock for 2 sec. 60 min prior to a retention test administered 24 h after the foot shock, each animal was given a single injection of 0.30, 0.12, 0.06, or 0.03 IU of vasopressin or of saline. Time to reenter the shock compartment was tested 24 and 48 h after the foot shock. Latencies in both retention tests indicated that, although there was a significant effect of shock level on latency scores, there was no effect of vasopressin with any dose level tested. The inability to find an effect of vasopressin in this study is contrary to results of other studies. Several factors, including general reactivity of the animals or the distribution system for vasopressin in the brain, might provide the underlying reason for these dramatic differences.

Presence of immunoreactive luteinizing hormone in the rat forebrain.

This study demonstrates the presence of immunoreactive LH in rat brain by radioimmunoassay and immunocytochemistry. High levels of radioimmunoassayable LH were identified in the hypothalamus, while significant but lesser quantities were found in the amygdala, septal area, preoptic area, thalamus, caudate nucleus, and hippocampus. Correlative immunocytochemistry localized immunopositive fibers in hypothalamic and several extrahypothalamic brain structures. Immunoreactive cell bodies were seen in colchicine-treated rats in the arcuate nucleus of the hypothalamus. Gel chromatography of hypothalamic extracts revealed that immunoassayable LH coeluted with LH standard and rat pituitary extracts. Possible mechanisms related to the origin and functional neuronal LH are discussed.

Immunocytochemical distribution of luteinizing hormone in rat central nervous system.

This paper presents the first detailed localization of luteinizing hormone (LH)-containing cells and fibers in the rat central nervous system. These immunoreactive elements were identified by four LH antisera, two directed against the intact LH molecule and two against LHb. Cell bodies, immunoreactive for LH were found throughout the rostral-caudal extent of the hypothalamic arcuate and ventromedial nuclei, the periarcuate area ventral to the ventromedial nucleus, and the retrochiasmatic area. Immunopositive fibers were traced to numerous structures within the brain including discrete regions of the hypothalamus, septal area, nucleus of the diagonal band, bed nucleus of stria terminalis, amygdala, thalamus, periaqueductal gray, raphe nuclei, brainstem reticular nuclei, locus ceruleus, parabrachial nucleus, dorsal motor nucleus of vagus, and the nucleus of the solitary tract, with a few fibers extending into spinal cord central gray. This pattern of fiber distribution corresponds closely with those described for fibers containing several other anterior pituitary hormones. The extensive projection for LH may provide neuroanatomical substrate mediating reproductive events as it does in the pituitary, or it may serve some modulatory function in brain which is independent of its role in reproduction.

Measurements of scattered light from mirrors and lenses.

The limitations arising from scattered light from lenses and mirrors in a program of daytime astrometry near the sun are discussed. Measurements were made of the angular distribution of the scattered radiance for (1) a coronagraphic lens, (2) a 2.54-cm thick coronagraph quality window, and (3) flat quartz mirrors. Results are also given for the optical elements after they have been exposed in a vacuum for 48h. The results indicate that the accuracy of a daytime astrometry program would be determined by the shot noise of the scattered light produced by the refracting elements and the sky, each producing equal contributions.

Improved procedure for fluorescence in situ hybridization on tissue microarrays.

BACKGROUND: The recently developed tissue microarray (TMA) technology allows the arrangement of up to a thousand tissue specimens on a single microscope slide. This technology enables researchers to perform gene copy number studies on very large series of archival formalin-fixed tissues using fluorescence in situ hybridization (FISH). However, the hybridization properties of individual archival specimens can vary considerably. Therefore a highly optimized protocol is needed to fulfill the task of producing evaluable hybridization signals simultaneously in hundreds of specimens in a TMA. METHODS: The performance of two different FISH protocols, the standard protocol for paraffin embedded tissues and our new optimized protocol, was tested on TMAs using probes for the HER-2 and ZNF217 genes as well as the chromosome 17 centromere. RESULTS: The new protocol resulted in greatly increased signal intensity and an almost 30% increase in the number of tissue samples with evaluable hybridization signals. CONCLUSIONS: Our improved protocol for FISH on TMAs provides standardized hybridization conditions leading to high-quality hybridization signals in the majority of specimens. The increases in the signal intensity and the number of evaluable samples are extremely important for the successful analyses of TMAs by FISH and will allow the utilization of the TMA technology in its full potential.