Expansion of CD133(+) colon cancer cultures retaining stem cell properties to enable cancer stem cell target discovery.

BACKGROUND: Despite earlier studies demonstrating in vitro propagation of solid tumour cancer stem cells (CSCs) as non-adherent tumour spheres, it remains controversial as to whether CSCs can be maintained in vitro. Additional validation of the CSC properties of tumour spheres would support their use as CSC models and provide an opportunity to discover additional CSC cell surface markers to aid in CSC detection and potential elimination. METHODS: Primary tumour cells isolated from 13 surgically resected colon tumour specimens were propagated using serum-free CSC-selective conditions. The CSC properties of long-term cultured tumour spheres were established and mass spectrometry-based proteomics performed. RESULTS: Freshly isolated CD133(+) colorectal cancer cells gave rise to long-term tumour sphere (or spheroids) cultures maintaining CD133 expression. These spheroid cells were able to self-renew and differentiate into adherent epithelial lineages and recapitulate the phenotype of the original tumour. Relative to their differentiated progeny, tumour spheroid cells were more resistant to the chemotherapeutic irinotecan. Finally, CD44, CD166, CD29, CEACAM5, cadherin 17, and biglycan were identified by mass spectrometry to be enriched in CD133(+) tumour spheroid cells. CONCLUSION: Our data suggest that ex vivo-expanded colon CSCs isolated from clinical specimens can be maintained in culture enabling the identification of CSC cell surface-associated proteins.

CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers.

CD133/prominin-1 is a pentaspan transmembrane glycoprotein overexpressed in various solid tumours including colorectal and glioblastomas. CD133 was found here to be highly expressed in >or=50% of pancreatic, gastric and intrahepatic cholangiocarcinomas. Quantitative flow cytometric analysis showed that a panel of established hepatocellular, pancreatic and gastric cancer cell lines expressed CD133 at levels higher than normal epithelial cells or bone marrow progenitor cells. A murine anti-human CD133 antibody (AC133) conjugated to a potent cytotoxic drug, monomethyl auristatin F (MMAF), effectively inhibited the growth of Hep3B hepatocellular and KATO III gastric cancer cells in vitro with IC(50) values of 2-7 ng ml(-1). MMAF induced apoptosis in the cancer cells as measured by caspase activation. The anti-CD133-drug conjugate (AC133-vcMMAF) was shown to internalise and colocalised with the lysosomal marker CD107a in the sensitive cell lines. In contrast, in the resistant cell line Su.86.86, the conjugate internalised and colocalised with the caveolae marker, Cav-1. Addition of ammonium chloride, an inhibitor of lysosomal trafficking and processing, suppressed the cytotoxic effect of AC133-vcMMAF in both Hep3B and KATO III. Anti-CD133-drug conjugate treatment resulted in significant delay of Hep3B tumour growth in SCID mice. Anti-CD133 antibody-drug conjugates warrant further evaluation as a therapeutic strategy to eradicate CD133+ tumours.

Interferon-kappa, a novel type I interferon expressed in human keratinocytes.

High throughput cDNA sequencing has led to the identification of interferon-kappa, a novel subclass of type I interferon that displays approximately 30% homology to other family members. Interferon-kappa consists of 207 amino acids, including a 27-amino acid signal peptide and a series of cysteines conserved in type I interferons. The gene encoding interferon-kappa is located on the short arm of chromosome 9 adjacent to the type I interferon gene cluster and is selectively expressed in epidermal keratinocytes. Expression of interferon-kappa is significantly enhanced in keratinocytes upon viral infection, upon exposure to double-stranded RNA, or upon treatment with either interferon-gamma or interferon-beta. Administration of interferon-kappa recombinant protein imparts cellular protection against viral infection in a species-specific manner. Interferon-kappa activates the interferon-stimulated response element signaling pathway and a panel of genes similar to those regulated by other type I interferons including anti-viral mediators and transcriptional regulators. An antibody that neutralizes the type I interferon receptor completely blocks interferon-kappa signaling, demonstrating that interferon-kappa utilizes the same receptor as other type I interferons. Interferon-kappa therefore defines a novel subclass of type I interferon that is expressed in keratinocytes and expands the repertoire of known proteins mediating host defense.

Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis.

In this report we describe the discovery of Eppin (Epididymal protease inhibitor), a gene on human chromosome 20 expressing three mRNAs encoding two isoforms of a cystine-rich protein containing both Kunitz-type and WAP-type four disuffide core protease inhibitor consensus sequences. Analysis of Eppin's genomic sequence from chromosome 20q12-13.2 predicts the existence of all three splice variants of Eppin and that all the exons conform to the AG/GT splicing rule. The presence of single bands on a Southern blot of human genomic DNA suggests that Eppin is a single copy gene. TATA box transcription initiation sites are present for both of the different Eppin 5' UTRs and examination of the promoter region 1800 bp upstream of the start codon revealed a number of putative transcription enhancer binding sites typical of genes expressed in the epididymis or testis. Northern blot and tissue specific PCR data indicate Eppin-1 is expressed only in the testis and epididymis; Eppin-2 is expressed only in the epididymis and Eppin-3 only in the testis. Antiserum prepared against recombinant EPPIN recognizes several strong bands on Western blots of human epididymal extracts from the caput and corpus regions. Immunohistochemistry indicates a strong pattern of expression by the ciliated cells of the efferent ducts and strong staining of ejaculated spermatozoa. Eppin represents the first member of a family of protease inhibitors characterized by dual inhibitor consensus sequences, both WAP-type and Kunitz-type consensus sequences. A second family member is predicted to exist on chromosome 20 approximately 4 kb downstream from Eppin's exon I, which has two WAP-type sequences and one Kumtz-type consensus sequence.

Tumor necrosis factor (TNF) receptor superfamily member TACI is a high affinity receptor for TNF family members APRIL and BLyS.

An expression cloning approach was employed to identify the receptor for B-lymphocyte stimulator (BLyS) and identified the tumor necrosis factor receptor superfamily member TACI as a BLyS-binding protein. Expression of TACI in HEK293T cells confers on the cells the ability to bind BLyS with subnanomolar affinity. Furthermore, a TACI-Fc fusion protein recognizes both the cleaved, soluble form of BLyS as well as the membrane BLyS present on the cell surface of a recombinant cell line. TACI mRNA is found predominantly in B-cells and correlates with BLyS binding in a panel of B-cell lines. We also demonstrate that TACI interacts with nanomolar affinity with the BLyS-related tumor necrosis factor homologue APRIL for which no clear in vivo role has been described. BLyS and APRIL are capable of signaling through TACI to mediate NF-kappaB responses in HEK293 cells. We conclude that TACI is a receptor for BLyS and APRIL and discuss the implications for B-cell biology.

A novel cytokine receptor-ligand pair. Identification, molecular characterization, and in vivo immunomodulatory activity.

As part of a large scale effort to discover novel secreted proteins, a cDNA encoding a novel cytokine was identified. Alignments of the sequence of the new protein, designated IL-17B, suggest it to be a homolog of the recently described T cell-derived cytokine, IL-17. By Northern analysis, EST distribution and real-time quantitative polymerase chain reaction analysis, mRNA was detected in many cell types. A novel type I transmembrane protein, identified in an EST data base by homology to IL-17R, was found to bind specifically IL-17B, as determined by surface plasmon resonance analysis, flow cytometry, and co-immunoprecipitation experiments. Readily detectable transcription of IL-17BR was restricted to human kidney, pancreas, liver, brain, and intestines and only a few of the many cell lines tested. By using a rodent ortholog of IL-17BR as a probe, IL-17BR message was found to be drastically up-regulated during intestinal inflammation elicited by indomethacin treatment in rats. In addition, intraperitoneal injection of IL-17B purified from Chinese hamster ovary cells caused marked neutrophil migration in normal mice, in a specific and dose-dependent manner. Together these results suggest that IL-17B may be a novel proinflammatory cytokine acting on a restricted set of target cell types. They also demonstrate the strength of genomic approaches in the unraveling of novel biological pathways.

HE2beta and HE2gamma, new members of an epididymis-specific family of androgen-regulated proteins in the human.

HE2 is an epididymis-specific sperm-binding secretory protein. We isolated a family of HE2-related complementary DNAs from a human caput/corpus library. The transcripts code for identical 71-amino acid N-termini and different C-termini, and 5'- and 3'-untranslated regions. Compared with the original HE2, HE2beta and HE2gamma proteins have a 25-amino acid deletion near the C-terminus, and HE2gamma isoforms have a second deletion. These frame-shifting deletions result in C-termini differing in length, amino acid sequence, including number of cysteines, and isoelectric point. Identical sequences and deletion start and stop points indicate the HE2 isoforms are derived from alternative splicing of 8 or more exons of a single gene. Northern hybridization revealed that the 0.9-kb messenger RNA (mRNA) is most abundant in human caput; there is much less of it (20%) in corpus and little (<5%) in cauda. In castrated Macaca mulatta, HE2 mRNA decreased to 10% of sham-operated levels. Testosterone replacement maintained HE2 mRNA 3- to 5-fold higher than castrate levels, indicating its androgen dependence. Immunohistochemical staining revealed that the beta1 form is highly expressed in principal cells of the initial segment and caput. It is secreted into the lumen and binds to the sperm surface in the postacrosomal and neck regions. The beta2 form is expressed in principal cells primarily in efferent ducts.

VEGI, a new member of the TNF family activates nuclear factor-kappa B and c-Jun N-terminal kinase and modulates cell growth.

Recently a new member of the human tumor necrosis factor (TNF) family named as VEGI was reported. However, very little is known about the biological activities displayed by this cytokine. In this report, we show that in myeloid cells VEGI activated the transcription factor kappa B (NF-kappa B) as determined by the electrophoretic mobility shift assay, induced degradation of I kappa B alpha, and nuclear translocation of p65 subunit of NF-kappa B. VEGI also activated NF-kappa B-dependent reporter gene expression. In addition, VEGI activated c-Jun N-terminal kinase. When examined for growth modulatory effects, VEGI inhibited the proliferation of breast carcinoma (MCF-7), epithelial (HeLa), and myeloid (U-937 and ML-1a) tumor cells; and activated caspase-3 leading to PARP cleavage. VEGI-induced cytotoxicity was potentiated by inhibitors of protein synthesis. VEGI also induced proliferation of normal human foreskin fibroblast cells. The activity of VEGI could neither be neutralized by antibodies against TNF, nor could it compete with TNF binding, indicating that the activity of VEGI is not due to TNF and it binds to a distinct receptor. These results suggest that VEGI, a new member of the TNF family, has a signaling pathway similar to TNF and is most likely a multifunctional cytokine.

Outcome predictors in pregnant opiate and polydrug users.

A retrospective case note study of 93 women was performed in order to assess the effect of maternal factors on neonatal outcome in a group of women attending a specialist clinic for pregnant drug users. There were no significant differences in outcome for chaotic drug users compared with non-chaotic drug users, or for cocaine users compared with non-cocaine using drug users. Women who reduced their methadone dose during pregnancy delivered babies of significantly higher birth weight than those whose methadone dose remained the same or increased (median 3027 g, range 1780-3629 g vs 2645 g, range 580-3720 g). Women who abused benzodiazepines during pregnancy produced babies of significantly lower birth weight than those women who did not use benzodiazepines (median 2100 g, range 580-3520 g vs 2767 g, range 1530-3720 g). The results of this study give healthcare staff evidence to use in encouraging drug-using women to avoid benzodiazepines during pregnancy and to reduce their methadone dosage. The treatment received from a specialist clinic may mitigate against some of the other recognised effects of drug use during pregnancy.

Effects of keratinocyte growth factor-2 (KGF-2) on wound healing in an ischaemia-impaired rabbit ear model and on scar formation.

Keratinocyte growth factor-2 (KGF-2), also described as fibroblast growth factor-10 (FGF-10), is a member of the fibroblast growth factor family. KGF-2 shares 57 per cent sequence homology to previously reported KGF-1 (FGF-7). In skin, both growth factors are expressed in the dermal compartment. KGF-1 and KGF-2 bind to the same receptor with high affinity, the KGFR isoform of FGFR2, which is exclusively expressed by epithelial cells. This study examines the in vivo function of topically applied KGF-2 on wound healing using an ischaemia-impaired rabbit dermal ulcer model, in young and aged animals. Histological analysis of the wounds showed that KGF-2 significantly promoted re-epithelialization in both young and old animals. Similar results have been observed with KGF-1 in this model. In addition, KGF-2 enhanced granulation tissue formation in both young and old rabbits, a biological effect not found with KGF-1, suggesting a possible indirect mechanism which enhances neo-granulation tissue formation. Immunohistological staining of day 7 wounds with proliferating cell nuclear antigen (PCNA) antibody demonstrated a significant increase of dermal cell proliferation in KGF-2-treated wounds compared with placebo wounds. These results suggest a mesenchymal-epithelial interaction that is mediated by a paracrine feedback loop of KGF-2. Because of the wound healing impairment observed with ageing, the wound healing response to KGF-2 was also studied in ischaemic wounds of aged animals. Administration of KGF-2 led to significant stimulation of epithelial growth and granulation tissue formation. The effects seen in the old animals were delayed compared with the young animals. Lastly, the effect of KGF-2 was examined in a rabbit model of scar formation. Quantification of scar elevation index showed no significant differences in scar formation when KGF-2 was compared with buffer placebo. Compared with other growth factors, including KGF-1 and TGF-beta which have previously been examined in these models, KGF-2 is the most effective and causes no obvious scarring.

BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator.

The tumor necrosis factor (TNF) superfamily of cytokines includes both soluble and membrane-bound proteins that regulate immune responses. A member of the human TNF family, BLyS (B lymphocyte stimulator), was identified that induced B cell proliferation and immunoglobulin secretion. BLyS expression on human monocytes could be up-regulated by interferon-gamma. Soluble BLyS functioned as a potent B cell growth factor in costimulation assays. Administration of soluble recombinant BLyS to mice disrupted splenic B and T cell zones and resulted in elevated serum immunoglobulin concentrations. The B cell tropism of BLyS is consistent with its receptor expression on B-lineage cells. The biological profile of BLyS suggests it is involved in monocyte-driven B cell activation.

TRAIL (Apo-2L) and TRAIL receptors in human placentas: implications for immune privilege.

Mechanisms accounting for protection of the fetal semiallograft from maternal immune cells remain incompletely understood. In other contexts, interactions between TRAIL (TNF-related apoptosis-inducing ligand/Apo-2L) and its receptors kill activated lymphocytes. The purpose of this study was therefore to investigate the potential of the TRAIL/TRAIL-R system to protect the placenta against immune cell attack. Analysis by Northern blotting demonstrated mRNAs encoding TRAIL as well as the four TRAIL receptors (DR4, DR5, DcR1/TRID, DcR2/TRUNDD) in human placentas. Immunohistochemical experiments demonstrated that TRAIL protein is prominent in syncytiotrophoblast, an uninterrupted placental cell layer that is continuously exposed to maternal blood, as well as in macrophage-like placental mesenchymal cells (Hofbauer cells). Studies on cell lines representing trophoblasts (Jar, JEG-3 cells) and macrophages (U937, THP-1 cells) showed that both lineages contained TRAIL mRNA and that steady state levels of transcripts were increased 2- to 11-fold by IFN-gamma. By contrast, cell lineage-specific differences were observed in expression of the TRAIL-R genes. Although all four lines contained mRNA encoding the apoptosis-inducing DR5 receptor, only trophoblast cells contained mRNA encoding the DcR1 decoy receptor and only macrophages contained DcR2 decoy receptor transcripts. DR4 mRNA was present only in THP-1 cells and was the only TRAIL-R transcript increased by IFN-gamma. Cytotoxicity assays revealed that the two trophoblast cell lines were resistant, whereas the two macrophage lines were partially susceptible to killing by rTRAIL. Collectively, the results are consistent with a role for the TRAIL/TRAIL-R system in the establishment of placental immune privilege.

Identification and characterization of a novel member of the fibroblast growth factor family.

A new member of the fibroblast growth factor (FGF) family, FGF-13, has been molecularly cloned as a result of high throughput sequencing of a human ovarian cancer cell library. The open reading frame of the novel human gene (1419 bp) encodes for a protein of 216 a.a. with a molecular weight of 22 kDa. The FGF-13 sequence contains an amino-terminal hydrophobic region of 23 a.a. characteristic of a signal secretion sequence. FGF-13 is most homologous, 70% similarity at the amino acid level, to FGF-8. Northern hybridization analysis demonstrated prominent expression of FGF-13 in human foetal and adult brain, particularly in the cerebellum and cortex. In proliferation studies with BaF3 cells, FGF-13 preferentially activates cell clones expressing either FGF receptor variant, 3-IIIc or 4. The signal transduction pathways of FGF-13 and FGF-2 were compared in rat hippocampal astrocytes. The two FGFs induce an equivalent level of tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) and c-raf activation. However, FGF-13 is more effective than FGF-2 in inducing the phosphorylation of phospholipase C-gamma (PLC-gamma). Treatment of neuronal cultures from rat embryonic cortex with FGF-13 increases the number of glutamic acid decarboxylase immunopositive neurons, the level of high-affinity gamma-aminobutyric acid (GABA) uptake, and choline acetyltransferase enzyme activity. The GABAergic neuronal response to FGF-13 treatment is rapid with a significant increase occurring within 72 h. We have identified a novel member of the FGF family that is expressed in the central nervous system (CNS) and increases the number as well as the level of phenotypic differentiation of cortical neurons in vitro.

Expression and reconstitution of NF-kappaB from insect cells using a baculovirus vector.

NF-kappaB is a pleiotropic transcriptional activator originally identified by its ability to regulate immunoglogulin kappa light chain expression. Purification of this DNA-binding complex demonstrated that NF-kappaB is a heterodimer composed of two subunits, NFKB1 and RelA. Previous studies have shown that truncated versions of these proteins could be expressed and purified from bacterial cells. In the present study, we utilize a baculovirus expression vector system (BEVS) to overexpress each subunit independently to produce homodimers or together to reconstitute functional NF-kappaB. These proteins can be enriched to >70% homogeneity on a kappaB-agarose DNA- affinity column. The purified proteins are active in DNA binding as measured by electrophoretic mobility shift assays. Finally, transcriptional activation of these recombinant proteins can be measured by their ability to activate a kappaB-CAT reporter plasmid in transiently transfected/infected SF-9 cells. Thus, BEVS provides a method for production of full-length, transcriptionally active NF-kappaB proteins.

The development of healthcare services for drug misusers and prostitutes.

With the advent of human immunodeficiency virus (HIV) and the increase of drug misuse in the UK, the Government wishes primary care to play a greater part in treating drug problems in the hope of preventing the spread of HIV. Drug misusers do not avail themselves of traditional services and many are not registered with general practitioners. In response to this Liverpool Health Authority and Family Health Service Authority commenced a new salaried post to provide primary care services to special groups such as injecting drug misusers and prostitutes. Judgemental attitudes towards drug misusers, their high mobility and being a transient population play a part in the reasons why drug misusers find it difficult to access primary healthcare. Drug misusers have high morbidity related to their drug misuse. Many of these conditions, if treated early, can prevent the need for more intensive intervention. Although drug misusers may present with a condition requiring immediate treatment, the opportunity is used to provide other healthcare such as hepatitis B vaccinations, sexually transmitted infection screening, contraception and HIV/hepatitis B testing. The sero prevalence of anti-HBc in injecting drug misusers is 45.5%. Due to their high morbidity and associated costs, the requirements of these groups may conflict with the objectives of budget-holding practices. If general practitioners are unable to respond to their problems, then health care providers and purchasers will have to consider similar schemes in areas which have a higher prevalence of drug misuse in order to provide appropriate healthcare for these vulnerable groups.

Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis.

By searching a database of expressed sequences, we identified a member of the signal transducers and activators of transcription (Stat) family of proteins. Human and murine full-length cDNA clones were obtained and sequenced. The sequence of the human cDNA was identical to the recently published sequence for interleukin-4 (IL-4)-Stat (J. Hou, U. Schindler, W.J. Henzel, T.C. Ho, M. Brasseur, and S. L. McKnight, Science 265:1701-1706, 1994), while the murine Stat6 amino acid and nucleotide sequences were 83 and 84% identical to the human sequences, respectively. Using Stat6-specific antiserum, we demonstrated that Stat6 is rapidly tyrosine phosphorylated following stimulation of appropriate cell lines with IL-4 or IL-3 but is not detectably phosphorylated following stimulation with IL-2, IL-12, or erythropoietin. In contrast, IL-2, IL-3, and erythropoietin induce the tyrosine phosphorylation of Stat5 while IL-12 uniquely induces the tyrosine phosphorylation of Stat4. Inducible tyrosine phosphorylation of Stat6 requires the membrane-distal region of the IL-4 receptor alpha chain. This region of the receptor is not required for cell growth, demonstrating that Stat6 tyrosine phosphorylation does not contribute to mitogenesis.