Lack of correlation of stem cell markers in breast cancer stem cells.

BACKGROUND: Various markers are used to identify the unique sub-population of breast cancer cells with stem cell properties. Whether these markers are expressed in all breast cancers, identify the same population of cells, or equate to therapeutic response is controversial. METHODS: We investigated the expression of multiple cancer stem cell markers in human breast cancer samples and cell lines in vitro and in vivo, comparing across and within samples and relating expression with growth and therapeutic response to doxorubicin, docetaxol and radiotherapy. RESULTS: CD24, CD44, ALDH and SOX2 expression, the ability to form mammospheres and side-population cells are variably present in human cancers and cell lines. Each marker identifies a unique rather than common population of cancer cells. In vivo, cells expressing these markers are not specifically localized to the presumptive stem cell niche at the tumour/stroma interface. Repeated therapy does not consistently enrich cells expressing these markers, although ER-negative cells accumulate. CONCLUSIONS: Commonly employed methods identify different cancer cell sub-populations with no consistent therapeutic implications, rather than a single population of cells. The relationships of breast cancer stem cells to clinical parameters will require identification of specific markers or panels for the individual cancer.

Don't forget the fragment! An unusual case of occult fragment embolisation following penetrating neck injury.

Fragment embolisation following vascular injury is uncommon. The case of a 31 year old soldier, who sustained a penetrating fragment injury to the neck with distal arterial embolisation, is presented and the discussion illustrates both the importance of expedient assessment and management of cervical vascular injuries and of thorough correlation of clinical and radiological findings to avoid missed emboli.

Phytochromes: photosensory perception and signal transduction.

The phytochrome family of photoreceptors monitors the light environment and dictates patterns of gene expression that enable the plant to optimize growth and development in accordance with prevailing conditions. The enduring challenge is to define the biochemical mechanism of phytochrome action and to dissect the signaling circuitry by which the photoreceptor molecules relay sensory information to the genes they regulate. Evidence indicates that individual phytochromes have specialized photosensory functions. The amino-terminal domain of the molecule determines this photosensory specificity, whereas a short segment in the carboxyl-terminal domain is critical for signal transfer to downstream components. Heterotrimeric GTP-binding proteins, calcium-calmodulin, cyclic guanosine 5'-phosphate, and the COP-DET-FUS class of master regulators are implicated as signaling intermediates in phototransduction.

Glucose-dependent insulin release from genetically engineered K cells.

Genetic engineering of non-beta cells to release insulin upon feeding could be a therapeutic modality for patients with diabetes. A tumor-derived K-cell line was induced to produce human insulin by providing the cells with the human insulin gene linked to the 5'-regulatory region of the gene encoding glucose-dependent insulinotropic polypeptide (GIP). Mice expressing this transgene produced human insulin specifically in gut K cells. This insulin protected the mice from developing diabetes and maintained glucose tolerance after destruction of the native insulin-producing beta cells.

HIF-1alpha contributes to tumour-selective killing by the sigma receptor antagonist rimcazole.

We have previously reported tumour-selective killing by the sigma (sigma) receptor ligand rimcazole. We now report that rimcazole elevates hypoxia inducible factor-1alpha (HIF-1alpha) protein levels under normoxic conditions in colorectal (HCT-116) and mammary carcinoma (MDA MB 231) cells but fails to induce HIF-1alpha in normal fibroblasts or mammary epithelial cells. Combining the sigma-1 agonist (+)-pentazocine with rimcazole substantially reduces the accumulation of HIF-1alpha, confirming that the effect is mediated at least partly by antagonism of sigma-1 sites. HIF-1alpha knockdown by RNA interference attenuates rimcazole-induced cell death in both cell types. Thus, the induction of HIF-1alpha by rimcazole contributes to tumour cell killing. In a comparison of HCT-116p53+/+ and HCT-116p53-/- cells, HIF-1alpha levels are consistently higher after rimcazole treatment in HCT-116p53+/+ cells. Furthermore, although rimcazole kills HCT-116p53-/- cells, it has a more potent apoptosis-inducing effect in HCT-116p53+/+ cells. This suggests that the presence of functional p53 protein may enhance death induction by rimcazole in part through greater induction of HIF-1alpha. p53 is not required, however, for the rimcazole-induced engagement of HIF-1alpha in proapoptotic mode as HIF-1alpha knockdown attenuates rimcazole-induced death to comparable extents in p53 mutant and wild-type cell systems. Knowledge of HIF-1alpha involvement may assist the re-profiling of rimcazole and other sigma ligands as cancer therapeutics.

Oat Phytochrome Is Biologically Active in Transgenic Tomatoes.

To determine the functional homology between phytochromes from evolutionarily divergent species, we used the cauliflower mosaic virus 35S promoter to express a monocot (oat) phytochrome cDNA in a dicot plant (tomato). Immunoblot analysis shows that more than 50% of the transgenic tomato plants synthesize the full-length oat phytochrome polypeptide. Moreover, leaves of light-grown transgenic plants contain appreciably less oat phytochrome than leaves from dark-adapted plants, and etiolated R1 transgenic seedlings have higher levels of spectrally active phytochrome than wild-type tomato seedlings in direct proportion to the level of immunochemically detectable oat polypeptide present. These data suggest that the heterologous oat polypeptide carries a functional chromophore, allowing reversible photoconversion between the two forms of the molecule, and that the far-red absorbing form (Pfr) is recognized and selectively degraded by the Pfr-specific degradative machinery in the dicot cell. The overexpression of oat phytochrome has pleiotropic, phenotypic consequences at all major phases of the life cycle. Adult transgenic tomato plants expressing high levels of the oat protein tend to be dwarfed, with dark green foliage and fruits. R1 transgenic seedlings have short hypocotyls with elevated anthocyanin contents. We conclude that a monocot phytochrome can be synthesized and correctly processed to a biologically active form in a dicot cell, and that the transduction pathway components that interact with the photoreceptor are evolutionarily conserved.

Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome.

We constructed Aspergillus nidulans transformation plasmids containing the A. nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11). The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies. Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus. Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered. The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes. The CEN11 sequence had little or no effect on chromosome stability. Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans.

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA, and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting the expression of nonregulated genes. The three genes code for poly(A)+ RNAs that begin to accumulate at different times during conidiation. The brlA- and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.

Profile of phospholipase A2 activity in subcellular fractions and lamellar bodies of developing, neonatal and adult rabbit lung. Correlation with intracellular levels of disaturated phosphatidylcholine.

Phospholipase A2 activity was determined in subcellular fractions and lamellar bodies of fetal, neonatal and adult rabbit lungs. Specific activity in most fractions decreased from the 24th to the 28th day of gestation. All fractions except the mitochondrial and the nuclear fractions exhibited a sharp increase in activity in the newborn lung. Specific activity in the adult lung generally declined in comparison to neonatal values. During gestation total enzyme activity per gram of lung was concentrated in the cytosolic fraction. With the exception of the lamellar body fraction, the total content of phospholipase A2 activity increased dramatically in all fractions from the neonatal lung. The lamellar body fractions displayed both low specific activity and low total enzyme activity during gestation. Specific activity increased dramatically in the neonatal and adult lung but still accounted for only a small fraction of the activity in comparison to the other subcellular fractions. The subcellular content of disaturated phosphatidylcholine (PC) appeared to correlate well with the activity of phospholipase A2 in the neonatal mitochondrial, microsomal and cytosolic fractions. Since decreasing prenatal enzyme levels are associated with increasing disaturated PC content, the alkaline and calcium-dependent phospholipase A2 may not be directly involved in disaturated PC synthesis in the fetus. However, postnatally, the correlation between the pattern of production of disaturated PC and the activity of the phospholipase A2 indicates a role for this enzyme in surfactant-related disaturated PC synthesis.

Functional GIP receptors are present on adipocytes.

In addition to its important role in maintaining glucose homeostasis, it has recently become apparent that glucose-dependent insulinotropic polypeptide (GIP) is also involved in different steps of lipid metabolism. GIP has been shown to stimulate the release of lipoprotein lipase from fat, as well as increase the rate of fat incorporation into adipose tissue. Moreover, GIP has been shown to increase the clearance rate of chylomicrons in the circulation and to inhibit the action of glucagon. Despite evidence for GIP effects on fat tissue, GIP receptors have not been identified in fat cells or tissues. The present study was undertaken to identify GIP receptors in isolated adipocytes, as well as to identify GIP receptors in the established fat cell line, differentiated 3T3-L1. RNAse protection analysis demonstrated the presence of GIP receptor transcripts in rat adipocytes. A polyclonal GIP receptor antiserum directed at the N-terminus of the receptor detected the presence of GIP receptors in both rat fat and differentiated 3T3-L1 cells by Western blot analysis. Moreover, [125I] GIP binding assays revealed both specific and displaceable GIP binding sites in differentiated 3T3-L1 cells (IC50 = 10(-9) M). When undifferentiated 3T3-L1 cells, which appear to express relatively few GIP receptors, were incubated in the presence of GIP, no effect on intracellular cAMP accumulation was detected. In contrast, the inclusion of 10 nM GIP in the incubation medium increased cAMP accumulation in rat fat cells and differentiated 3T3-L1 cells. This increase in cAMP accumulation was abolished with the specific GIP receptor antagonist GIP(7-30)NH2. The results of these studies indicate that GIP receptors are present in fat cells and are up-regulated when 3T3-L1 cells undergo differentiation to become adipocytes. Furthermore, the increase in intracellular cAMP accumulation detected upon ligand binding indicates that these receptors are functional.

Cloning and gene structure of rat phosphatidylcholine transfer protein, Pctp.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic lipid transfer protein that promotes intermembrane transfer of phosphatidylcholines but no other phospholipids. Although its physiological function remains unknown, phosphatidylcholine transfer protein is enriched in liver and evidence from model systems suggests a role in hepatocellular selection and transport of biliary phospholipids. To facilitate in vivo studies, a cDNA encoding rat PC-TP was cloned by library screening and 5'-rapid amplification of cDNA ends. Genomic cloning demonstrated the rat Pctp gene spans 10. 8kb and is comprised of six exons. The putative transcription initiation site was identified 50bp upstream of the translation initiation site. Nucleotide sequence analysis of the 5'-flanking region revealed a CAAT- but no TATA-box. Transient transfection of a series of 5'-deleted Pctp-promoter-firefly luciferase constructs into Reuber H35 rat hepatoma cells, which express Pctp mRNA, and Gunn rat fibroblasts, which do not, suggest that cis-acting elements in a 637bp promoter region contribute to enhanced expression of PC-TP in liver.

Patterns of tachykinin expression and localization in developing feline neostriatum.

The development of tachykinins in the neostriatum was determined qualitatively in order to characterize the ontogeny of an early-forming neostriatal peptidergic system. Tachykinins were detected by immunohistochemistry in fetal, postnatal, and adult cats. Neostriatal cells and neurites expressed tachykinins as early as fetal age 30 and increased in frequency progressively with age. Initial tachykinin expression occurred in neostriatal neurons during their postmitotic migration. In the head of the caudate nucleus, clusters of tachykinin-containing cells and fibers formed between fetal days 35 and 45, when the distribution of labeled neurons changed from a dispersed to an aggregated pattern. Between fetal days 45 and 50, tachykinin-rich neuronal clusters increased in frequency and were distributed throughout the rostral caudate nucleus. In contrast to neurons in clusters, neurons in the complementary neuropil expressed tachykinins largely postnatally. Postnatal morphological maturation of tachykinin-containing neurons paralleled the morphogenesis of medium spiny neostriatal cells. In addition, the caudate nucleus and putamen followed different spatiotemporal gradients of tachykinin expression. These results indicate that tachykinins are expressed in neostriatal neurons during the early ontogeny of the neostriatum and may function as trophic factors before synaptogenesis.

Serum and cerebrospinal fluid soluble Fas levels in clinical subgroups of multiple sclerosis.

Elevated sFas levels have been described in multiple sclerosis (MS) patients with active disease. The aim of this study was to assess the diagnostic potential of serum and cerebrospinal fluid (CSF) sFas measurements in differentiating clinically defined MS patient subgroups. Levels of sFas and sFas indices were determined in patients with stable relapsing-remitting MS (RRMS), active RRMS, primary progressive MS (PPMS), secondary progressive MS (SPMS) and patients with inflammatory (IND) and noninflammatory neurological diseases (NIND). Serum sFas modulation over 32 weeks IFN-beta1a therapy was also investigated. Serum and CSF sFas levels and sFas indices were elevated in MS compared to NIND and IND patients. Within the MS group, serum and CSF sFas levels were highest in PPMS, with active RRMS patients demonstrating the highest sFas indices. This may reflect an ongoing disease process which is occurring acutely (active disease) or incessantly (progressive disease). IFN-beta1a induced a transient increase in circulating sFas following initiation of therapy. Whilst evidence was provided for variable sFas expression in clinical subgroups of MS, there was insufficient definition between the respective groups to advocate sFas measurements as a diagnostic marker of clinical subgroups of MS.

Interferon-beta1a administration results in a transient increase of serum amyloid A protein and C-reactive protein: comparison with other markers of inflammation.

Putative markers of inflammation such as serum beta2-microglobulin and neopterin have been shown to be transiently upregulated following interferon-beta (IFN-beta) administration to multiple sclerosis (MS) patients. However, to date the role of the important inflammatory mediators serum amyloid A protein (SAA) and C-reactive protein (CRP) have not been described. Here we show that SAA but not CRP is elevated in relapsing-remitting MS patients compared to normal healthy individuals, and furthermore that both are transiently upregulated following intramuscular injection with IFN-beta1a (Avonex). This pattern of expression was found to parallel that of beta2-microglobulin and neopterin following injection and was mirrored by a selective activation of peripheral monocytes with respect to upregulation of receptors known to be involved in the inflammatory response (HLA-DR, CD16 and CD86). Injection of saline solution intramuscularly to six healthy control individuals did not produce a similar upregulation of any of the inflammatory markers investigated. Following IFN-beta1a injection, all inflammatory responses were attenuated at week 12 of therapy in comparison to those following the initial injection in a group of follow-up patients. In addition, IFN-beta1a injected on a weekly basis did not produce a sustained modulation of any of the markers investigated in patients treated for 32 weeks.

Changes in insulin-like growth factor-I receptor expression and binding protein secretion associated with tamoxifen resistance and estrogen independence in human breast cancer cells in vitro.

Conditioned medium from a series of human breast cancer cell lines representing the phenotypes of estrogen dependence ZR-75-1, MCF-7), tamoxifen resistance (Z(+)-75-9a1, LY2) and estrogen independence (ZR-PR-Lt) contained four detectable insulin-like growth factor binding proteins (IGFBPs) of MW 24, 34, 44 and 70 kDa. in the tamoxifen resistant lines secretion of the 24 kDA IGFBP was depressed in comparison to the parent lines whilst secretion of the 44 kDa IGFBP was unaffected. Secretion of this species by ZR-PR-LT cells was reduced. Following incubation in medium containing 1% serum both tamoxifen resistant cell lines secreted higher levels of the 44 kDa IGFBP than the respective parent lines. These changes in IGFBP secretion associated with the development of tamoxifen resistance and estrogen independence were accompanied by changes in the expression of receptors for IGF-1.

Functional in vitro assays for the isolation of cell transformation effector and suppressor genes.

Malignant transformation may be viewed as an imbalance between signals inducing cell growth and signals leading to growth inhibition, differentiation, or senescence. A basic understanding of how these counterbalancing forces interact to regulate normal cell growth is the prerequisite to comprehending the mechanisms of tumorigenesis. Identification and characterization of the gene products implicated in these regulatory pathways is the first step toward understanding the disease process. The studies outlined here provide the potential basis for isolating and molecularly characterizing transformation effector and suppressor genes, which must respectively function in the positive and negative regulation of normal cell growth. The general strategy used involves the isolation and molecular characterization of nontransformed variants (revertants) from populations of tumor cells. The selection of revertants is facilitated by the ability to separate normal from transformed cells by fluorescence-activated sorting. The basis for this separation is the differential retention of the fluorescent dye rhodamine 123 in the mitochondria of normal versus transformed cells. Using this approach, we have isolated revertants from a mutagenized population of v-fos-transformed Rat-1 fibroblasts. Characterization of these clones indicated that they had sustained causal mutations in transformation effector genes. The unmutated effector genes are being identified and molecularly cloned by isolating retransformed clones from revertant cell lines that have been transfected with DNA or cDNA from normal primary cells. The same selection protocol has also been used to isolate revertants from tumor cell lines that have been transfected with DNA or cDNA from primary cells. The putative tumor-suppressor genes present in these revertants are currently being analyzed.

Glucose-dependent insulinotropic peptide (GIP) gene expression in the rat salivary gland.

Previous studies have indicated that following nutrient ingestion, GIP is released principally from the upper small intestine. In addition to its presence in the rat small intestine, GIP transcripts have also been localized to the submandibular salivary gland (SSG). The present studies were directed to further characterize expression of the GIP gene in the SSG. Pregnant rats were sacrificed at gestational days 18 and 20, followed by the removal of rat fetuses. The duodenum pancreas, and SSG were then excised from the fetuses, as well as from neonatal pups at ages 1, 3, 7, 10, 14, and 21 days. RNA was extracted and measured by Northern blot analysis using specific rat GIP probes. GIP transcripts were first detected in the duodenum in the 18-day fetus and reached maximum levels at birth. In contrast, GIP mRNA was not observed in the SSG until 10 days postnatally and was not detected at all in either the fetal or neonatal pancreas. In situ hybridization of the SSG using an 35S-labelled antisense GIP RNA probe demonstrated expression of the GIP gene to be limited to ductal cells, with no transcripts present in acini. In separate experiments, rats fasted overnight were given water or 10% glucose. While no changes were detected in water-fed rats following oral glucose ingestion, small, but significant increases in SSG GIP gene expression were detected at 60 and 240 min. The results of these initial studies suggest the possibility of a functional role for GIP in the rat salivary gland by the demonstration of GIP mRNA in the SSG by Northern analysis and in situ hybridization, as well as by an increase in SSG GIP gene expression following a glucose meal.

Atheroembolism from the ascending aorta. An emerging problem in cardiac surgery.

As the ages of patients undergoing cardiac operations have increased, noncardiac causes of death have increased. To identify these causes of death, we analyzed the autopsy findings in 221 patients undergoing myocardial revascularization or valve operations between 1982 and 1989. Mean age was 65.6 +/- 9.5 years and the range was from 32 to 94 years; 130 patients (58.8%) were male. Autopsies were complete in 129 patients (58.4%) and limited to the chest and abdomen in the remainder. Embolic disease was identified in 69 patients (31.2%). Atheroemboli or abnormalities consistent with atheroemboli were identified in 48 patients (21.7%). Fourteen patients had thromboembolism and 7 had disseminated intravascular coagulation. The prevalence of atheroembolic disease increased dramatically from 4.5% in 1982 to 48.3% in 1989 (p = 0.001). Atheroembolic disease was found in the brain in 16.3% of patients, spleen in 10.9%, kidney in 10.4%, and pancreas in 6.8%. Thirty (62.5%) of the 48 patients had multiple atheroembolic sites. Atheroemboli were more common in patients undergoing coronary artery procedures (43/165; 26.1%) than in those undergoing valve procedures (5/56; 8.9%) (p = 0.008). There was a high correlation of atheroemboli with severe atherosclerosis of the ascending aorta. Atheroembolic events occurred in 46 of 123 patients (37.4%) with severe disease of the ascending aorta but in only 2 of 98 patients (2%) without significant ascending aortic disease (p less than 0.0001). Forty-six of 48 patients (95.8%) who had evidence of atheroemboli had severe atherosclerosis of the ascending aorta. There was a direct correlation between age, severe atherosclerosis of the ascending aorta, and atheroemboli. Incremental risk factors for atheroembolic are peripheral vascular disease and severe atherosclerosis of the ascending aorta.

Surgical treatment of isolated left anterior descending coronary stenosis. Comparison of left internal mammary artery and venous autograft at 18 to 20 years of follow-up.

To assess the long-term results of the surgical treatment of isolated left anterior descending coronary artery stenosis and compare surgical strategies for graft selection, we reviewed 100 consecutive patients receiving left internal mammary artery-to-left anterior descending artery grafts and 100 consecutive patients who received a saphenous vein autograft to the left anterior descending artery. All patients underwent operation from 1971 through 1973. The internal mammary artery and saphenous vein graft groups were equivalent with regard to preoperative clinical and angiographic variables, except that patients receiving left internal mammary artery grafts had a higher prevalence of noncritical disease (less than 50% stenosis) in the circumflex and right coronary arteries than did the saphenous vein graft group. Mean follow-up for the internal mammary artery and saphenous vein graft groups was 18.7 years and 20.7 years, respectively. The 18-year outcome was superior for the internal mammary artery group. Cox regression analysis confirmed that patients with left internal mammary artery grafts had superior survival, intervention-free survival, and event-free survival (all p < 0.01). The presence of noncritical disease in other vessels adversely affected intervention-free survival and event-free survival for both groups (all p < 0.03) and decreased survival for the saphenous vein graft group (p = 0.01) but not for the internal mammary artery group (p = 0.24). We conclude that in long-term follow-up of surgically treated isolated left anterior descending artery stenosis (1) the left internal mammary artery consistently yields better overall and intervention-free survival than does the saphenous vein graft, (2) outcome is influenced by the presence of noncritical disease in other vessels at the initial operation, and (3) deployment of the left internal mammary artery in the treatment of isolated left anterior descending artery stenosis yielded 18 years of intervention-free survival of 60.5% and provides a standard for comparison with other forms of therapeutic intervention.

Structure of the rat glucose-dependent insulinotropic polypeptide receptor gene.

The Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of the secretin-vasoactive intestinal polypeptide family of G-protein coupled receptors possessing seven transmembrane domains. We report here the cloning and the exon-intron structure of the rat GIPR gene, along with the identification and characterization of its 5'-flanking region. The coding region of the GIPR gene spans approximately 10.2 kilobases and contains 13 exons. Three additional exons, two encoding either 5' or 3' untranslated sequences and one contained in a novel alternatively spliced mRNA, were identified. The 5'-flanking sequences contained a number of transcription factor binding motifs, including a cAMP response element, an octamer binding site, three SP1 sites and an initiator element. However, neither a CAAT motif nor TATA box were found. Transient transfection assays demonstrated that the 5'-flanking region of the GIPR gene can efficiently promote transcription in RIN38 cells and that deletion of 50 base pairs containing a potential SPI binding sites leads to a 2.4-fold loss of transcriptional activity. In addition, transient transfection experiments comparing the relative promoter activities of 5'-flanking sequences of the GIPR gene in RIN38 and rat-2 cells suggests that distal negative regulatory sequences may control cell-specific expression.