Thrombopoietin, flt3-ligand and c-kit-ligand modulate HOX gene expression in expanding cord blood CD133(+) cells

Haemopoietic stem/progenitor cell (HSPC) development is regulated by extrinsic and intrinsic stimuli. Extrinsic modulators include growth factors and cell adhesion molecules, whereas intrinsic regulation is achieved with many transcription factor families, of which the HOX gene products are known to be important in haemopoiesis. Umbilical cord blood CD133(+) HSPC proliferation potential was tested in liquid culture with 'TPOFLK' (thrombopoietin, flt-3 ligand and c-kit ligand, promoting HSPC survival and self-renewal), in comparison to 'K36EG' (c-kit-ligand, interleukins-3 and -6, erythropoietin and granulocyte colony-stimulating factor, inducing haemopoietic differentiation). TPOFLK induced a higher CD133(+) HSPC proliferation (up to 60-fold more, at week 8) and maintained a higher frequency of the primitive colony-forming cells than K36EG. Quantitative polymerase chain reaction analysis revealed opposite expression patterns for specific HOX genes in expanding cord blood CD133(+) HSPC. After 8 weeks in liquid culture, TPOFLK increased the expression of HOX B3, B4 and A9 (associated with uncommitted HSPC) and reduced the expression of HOX B8 and A10 (expressed in committed myeloid cells) when compared to K36EG. These results suggest that TPOFLK induces CD133(+) HSPC proliferation, self-renewal and maintenance, up-regulation of HOX B3, B4 and A9 and down-regulation of HOX B8 and A10 gene expression.

Comparative validation of c-kit exon 11 mutation analysis on cytology samples and corresponding surgical resections of gastrointestinal stromal tumours

Objective:

c-Kit+ progenitors generate vascular cells for tissue-engineered grafts through modulation of the Wnt/Klf4 pathway

The development of decellularised scaffolds for small diameter vascular grafts is hampered by their limited patency, due to the lack of luminal cell coverage by endothelial cells (EC) and to the low tone of the vessel due to absence of a contractile smooth muscle cells (SMC). In this study, we identify a population of vascular progenitor c-Kit+/Sca-1- cells available in large numbers and derived from immuno-privileged embryonic stem cells (ESCs). We also define an efficient and controlled differentiation protocol yielding fully to differentiated ECs and SMCs in sufficient numbers to allow the repopulation of a tissue engineered vascular graft. When seeded ex vivo on a decellularised vessel, c-Kit+/Sca-1-derived cells recapitulated the native vessel structure and upon in vivo implantation in the mouse, markedly reduced neointima formation and mortality, restoring functional vascularisation. We showed that Krüppel-like transcription factor 4 (Klf4) regulates the choice of differentiation pathway of these cells through β-catenin activation and was itself regulated by the canonical Wnt pathway activator lithium chloride. Our data show that ESC-derived c-Kit+/Sca-1-cells can be differentiated through a Klf4/β-catenin dependent pathway and are a suitable source of vascular progenitors for the creation of superior tissue-engineered vessels from decellularised scaffolds.

Hepatocyte growth factor (HGF) protects c-met-expressing Burkitt''s lymphoma cell lines from apoptotic death induced by DNA damaging agents

The relative sensitivity of neoplastic cells to DNA damaging agents is a key factor in cancer therapy. In this paper, we show that pretreatment of Burkitt's lymphoma cell lines expressing the c-met protooncogene with hepatocyte growth factor (HGF) protects them from death induced by DNA damaging agents commonly used in tumour therapy. This protection was observed in assays based on morphological assessment of apoptotic cells and DNA fragmentation assays. The protection was dose- and time-dependent — maximal protection requiring pre-incubation with 100 ng/ml HGF for 48 h. Western blotting analysis and flow cytometric studies revealed that HGF inhibited doxorubicin- and etoposide-induced decreases in the levels of the anti-apoptotic proteins Bcl-XL, and to a lesser extent Bcl-2, without inducing changes in the pro-apoptotic Bax protein. Overall, these studies suggest that the accumulation of HGF within the microenvironment of neoplastic cells may contribute to the development of a chemoresistant phenotype.

Kit positive cells in the guinea pig bladder.

PURPOSE: We describe the presence of interstitial cells of Cajal (ICC) throughout the wall of the guinea pig bladder. MATERIALS AND METHODS: Bladders obtained from male guinea pigs were prepared for immunohistochemical investigations using various primary antibodies, including the specific ICC marker c-kit (Gibco BRL, Grand Island, New York). Enzymatically dispersed cells with a branched morphology were identified as ICC using anti-c-kit. They were loaded with fluo-4acetoxymethyl (Molecular Probes, Eugene, Oregon) and studied using confocal laser scanning microscopy. RESULTS: Anti-c-kit labeling demonstrated that ICC were oriented in parallel with the smooth muscle bundles that run diagonally throughout the bladder. Double labeling with anti-smooth muscle myosin (Sigma Chemical Co., St. Louis, Missouri) revealed that ICC were located on the boundary of smooth muscle bundles. When anti-c-kit was used in combination with the general neuronal antibody protein gene product 9.5 (Ultraclone Ltd., Isle of Wight, United Kingdom) or anti-neuronal nitric oxide synthase, it was noted that there was a close association between nerves and ICC. Enzymatic dissociation of cells from tissue pieces yielded a heterogeneous population of cells containing typical spindle-shaped smooth muscle cells and branched cells resembling ICC from other preparations. The latter could be identified immunohistochemically as ICC using anti-c-kit, whereas the majority of spindle-shaped cells were not Kit positive. Branched cells responded to the application of carbachol by firing Ca2+ waves and they were often spontaneously active. CONCLUSIONS: ICC are located on the boundary of smooth muscle bundles in the guinea pig bladder. They fire Ca2+ waves in response to cholinergic stimulation and can be spontaneously active, suggesting that they could act as pacemakers or intermediaries in the transmission of nerve signals to smooth muscle cells.

A quality control program for mutation detection in KIT and PDGFRA in gastrointestinal stromal tumours.

BACKGROUND: Although most gastrointestinal stromal tumours (GIST) carry oncogenic mutations in KIT exons 9, 11, 13 and 17, or in platelet-derived growth factor receptor alpha (PDGFRA) exons 12, 14 and 18, around 10% of GIST are free of these mutations. Genotyping and accurate detection of KIT/PDGFRA mutations in GIST are becoming increasingly useful for clinicians in the management of the disease. METHOD: To evaluate and improve laboratory practice in GIST mutation detection, we developed a mutational screening quality control program. Eleven laboratories were enrolled in this program and 50 DNA samples were analysed, each of them by four different laboratories, giving 200 mutational reports. RESULTS: In total, eight mutations were not detected by at least one laboratory. One false positive result was reported in one sample. Thus, the mean global rate of error with clinical implication based on 200 reports was 4.5%. Concerning specific polymorphisms detection, the rate varied from 0 to 100%, depending on the laboratory. The way mutations were reported was very heterogeneous, and some errors were detected. CONCLUSION: This study demonstrated that such a program was necessary for laboratories to improve the quality of the analysis, because an error rate of 4.5% may have clinical consequences for the patient.

Mechanisms of Disease: specialized interstitial cells of the urinary tract--an assessment of current knowledge.

Scientists interested in the smooth muscles of the urinary tract, and their control, have recently been studying cells in the interstitium of tissues that express the c-kit antigen (Kit(+) cells). These cells have morphologic features that are reminiscent of the well-described pacemaker cells in the gut, the interstitial cells of Cajal (ICC). The spontaneous contractile behavior of muscles in the urinary tract varies widely, and it is clear that urinary tract Kit(+) interstitial cells cannot be playing an identical role to that played by the ICC in the gut. Nevertheless, there is increasing evidence that they do play a role in modulating the contractile behavior of adjacent smooth muscle, and might also be involved in mediating neural control. This review outlines the properties of ICC in the gut, and gives an account of the discovery of cells in the interstitium of the main components of the urinary tract. The physiologic properties of such cells and the functional implications of their presence are discussed, with particular reference to the bladder. In this organ, Kit(+) cells are found under the lamina propria, where they might interact with the urothelium and with sensory nerves, and also between and within the smooth-muscle bundles. Confocal microscopy and calcium imaging are being used to assess the physiology of ICC and their interactions with smooth muscles. Differences in the numbers of ICC are seen in smooth muscle specimens obtained from patients with various pathologies; in particular, bladder overactivity is associated with increased numbers of these cells.

Cholinergic-induced Ca2+ signaling in interstitial cells of Cajal from the guinea pig bladder.

Acetylcholine released from parasympathetic excitatory nerves activates contraction in detrusor smooth muscle. Immunohistochemical labeling of guinea pig detrusor with anti-c-Kit and anti-VAChT demonstrated a close structural relationship between interstitial cells of Cajal (ICC) and cholinergic nerves. The ability of guinea pig bladder detrusor ICC to respond to the acetylcholine analog, carbachol, was investigated in enzymatically dissociated cells, loaded with the Ca(2+) indicator fluo 4AM. ICC fired Ca(2+) transients in response to stimulation by carbachol (1/10 microM). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 microM), an M(3) receptor antagonist, but not by the M(2) receptor antagonist methoctramine (1 microM). The source of Ca(2+) underlying the carbachol transients in isolated ICC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 microM) or Ni(2+) (30-100 microM) to block Ca(2+) channels or the removal of external Ca(2+) reduced the amplitude of the carbachol transients. Application of ryanodine (30 microM) or tetracaine (100 microM) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 microM), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 microM) caused a significant reduction and Xestospongin C (1 microM) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a Ca(2+) indicator showed distinctively different patterns of spontaneous Ca(2+) events in smooth muscle cells and ICC. Both cell types responded to carbachol by an increase in frequency of these events. In conclusion, guinea pig bladder detrusor ICC, both as isolated cells and within whole tissue preparations, respond to cholinergic stimulation by firing Ca(2+) transients. PMID: 18171995 [PubMed - indexed for MEDLINE]

Interstitial Cells in the Urinary Bladder-Localization and Function

Aims: This review summarizes the currently available literature on the localization and proposed functions of a novel group of cells in the urinary bladder known as interstitial cells or interstitial cells of Cajal (ICC).

Methods: On-line searches of "Pubmed" for bladder, c-Kit, ICC, interstitial cell and myofibroblast were performed to identify relevant studies for the review.

Results: The literature contains substantial data that several sub-populations of ICC are present in the wall of the mammalian urinary bladder. These are located in the lamina propria and within the detrusor with distinctive cell shapes and morphological arrangements. Bladder ICC are identified with transmission electron microscopy or by immunohistochemical labeling using antibodies to the Kit receptor which is an established ICC marker. Lamina propria-ICC form a loose network connected via Cx43 gap junctions and are associated with mucosal nerves. Detrusor ICC track the smooth muscle bundles and make frequent contacts with intramural nerves. Both groups of ICC exhibit spontaneous electrical and Ca²⁺-signalling and also respond to application of neurotransmitter substances including ATP and carbachol. There is emerging evidence that the expression of ICC is upregulated in pathophysiological conditions including the overactive bladder.

Conclusions: There is now a convincing body of evidence that specialized ICC are present in the urinary bladder making important associations with other cells that make up the bladder wall and possessing physiological properties consistent with a role of bladder activity modulation. Neurourol. Urodynam. 29: 82–87, 2010. © 2009 Wiley-Liss, Inc.

Comparison of mechanical and electrical activity and interstitial cells of Cajal in urinary bladders from wild-type and W/W-v mice

Background and purpose: W/Wv and wild-type murine bladders were studied to determine whether the W/Wv phenotype, which causes a reduction in, but not abolition of, tyrosine kinase activity, is a useful tool to study the function of bladder interstitial cells of Cajal (ICC).

Experimental approach: Immunohistochemistry, tension recordings and microelectrode recordings of membrane potential were performed on wild-type and mutant bladders.

Key results: Wild-type and W/Wv detrusors contained c-Kit- and vimentin-immunopositive cells in comparable quantities, distribution and morphology. Electrical field stimulation evoked tetrodotoxin-sensitive contractions in wild-type and W/Wv detrusor strips. Atropine reduced wild-type responses by 50% whereas a 25% reduction occurred in W/Wv strips. The atropine-insensitive component was blocked by pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid in both tissue types. Wild-type and W/Wv detrusors had similar resting membrane potentials of -48 mV. Spontaneous electrical activity in both tissue types comprised action potentials and unitary potentials. Action potentials were nifedipine-sensitive whereas unitary potentials were not. Excitatory junction potentials were evoked by single pulses in both tissues. These were reduced by atropine in wild-type tissues but not in W/Wv preparations. The atropine-insensitive component was abolished by pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid in both preparations.

Conclusions and implications: Bladders from W/Wv mice contain c-Kit- and vimentin-immunopositive ICC. There are similarities in the electrical and contractile properties of W/Wv and wild-type detrusors. However, significant differences were found in the pharmacology of the responses to neurogenic stimulation with an apparent up-regulation of the purinergic component. These findings indicate that the W/Wv strain may not be the best model to study ICC function in the bladder.

Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells

Overexpression of Hoxb4 in bone marrow cells promotes expansion of hematopoietic stem cell (HSC) populations in vivo and in vitro, indicating that this homeoprotein can activate the genetic program that determines self-renewal. However, this function cannot be solely attributed to Hoxb4 because Hoxb4(-/-) mice are viable and have an apparently normal HSC number. Quantitative polymerase chain reaction analysis showed that Hoxb4(-/-) c-Kit(+) fetal liver cells expressed moderately higher levels of several Hoxb cluster genes than control cells, raising the possibility that normal HSC activity in Hoxb4(-/-) mice is due to a compensatory up-regulation of other Hoxb genes. In this study, we investigated the competitive repopulation potential of HSCs lacking Hoxb4 alone, or in conjunction with 8 other Hoxb genes. Our results show that Hoxb4(-/-) and Hoxb1-b9(-/-) fetal liver cells retain full competitive repopulation potential and the ability to regenerate all myeloid and lymphoid lineages. Quantitative Hox gene expression profiling in purified c-KIt(+) Hoxb1-bg(-/-) fetal liver cells revealed an interaction between the Hoxa, b, and c clusters with variation in expression levels of Hoxa4, -a11, and -c4. Together, these studies show a complex network of genetic interactions between several Hox genes in primitive hematopoietic cells and demonstrate that HSCs lacking up to 30% of the active Hox genes remain fully competent.

Rational Design of Acridine-Based Ligands with Selectivity for Human Telomeric Quadruplexes

Structure-based modeling methods have been used to design a series of disubstituted triazole-linked acridine compounds with selectivity for human telomeric quadruplex DNAs. A focused library of these compounds was prepared using click chemistry and the selectivity concept was validated against two promoter quadruplexes from the c-kit gene with known molecular structures, as well as with duplex DNA using a FRET-based melting method. Lead compounds were found to have reduced effects on the thermal stability of the c-kit quadruplexes and duplex DNA structures. These effects were further explored with a series of competition experiments, which confirmed that binding to duplex DNA is very low even at high duplex:telomeric quadruplex ratios. Selectivity to the c-kit quadruplexes is more complex, with some evidence of their stabilization at increasing excess over human telomeric quadruplex DNA. Selectivity is a result of the dimensions of the triazole-acridine compounds; and in particular the separation of the two alkyl-amino terminal groups. Both lead compounds also have selective inhibitory effects on the proliferation of cancer cell lines compared to a normal cell line, and one has been shown to inhibit the activity of the telomerase enzyme, which is selectively expressed in tumor cells, where it plays a role in maintaining telomere integrity and cellular immortalization.

Lymphocytic follicles and aggregates are a determinant of mucosal damage and duration of diarrhea

Nonspecific changes (nonspecific chronic inflammation) in patients with chronic diarrhea represent the commonest diagnosis in colorectal biopsy interpretation, but these changes are of little clinical significance.