Single-cell analysis of K562 cells: an imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein.

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

Prophylactic pre-operative bilateral ureteric catheters for major gynaecological surgery.

PURPOSE: The use of prophylactic pre-operative bilateral ureteric catheters for major gynaecological surgery is controversial. The aim of this study was to investigate the frequency of ureteric catheter-associated morbidity in our Unit, where systematic pre-operative ureteric catheterisation is performed. METHODS: We conducted a retrospective casenote review of 337 gynaecology patients undergoing laparotomy at Salford Royal Hospital between January 2007 and September 2010. RESULTS: The mean age was 56.36 (range 17-89). Procedures included TAH BSO (n = 249, 74 %), BSO (n = 17, 5 %), radical hysterectomy (n = 36, 11 %), and other (n = 35, 10 %), for indications of ovarian (n = 189, 56 %), uterine (n = 88, 26 %) or cervical cancer (n = 18, 5.3 %), massive fibroids (n = 27, 8 %), severe endometriosis (n = 6, 1.78 %), or other (n = 9, 2.67 %). Bilateral ureteric catheters were attempted in most patients and successfully placed in 315/337 (93 %) patients. In 22 patients (7 %), either no ureteric catheters or a single ureteric catheter was placed due to pre-existing ureteric anomaly, technical difficulty, or surgeon choice. Bilateral ureteric catheterisation took an average of 5.4 min (SD 2.0, range 3.2-9.2) for an experienced consultant or 8.4 min (SD 3.9, range 6.4-18.6) for an SpR trainee to complete. There were no intra-operative ureteric complications. Post-operative complications included urinary tract infection (5/337 patients, 1.48 %), acute renal failure (2/337, 0.6 %), and uretero-vaginal fistulae (1/337 patients, 0.3 %). CONCLUSIONS: Prophylactic pre-operative ureteric catheters are quick and easy to insert and associated with low complication rates. Routine use before major gynaecological surgery can expedite intra-operative identification of the ureters and may reduce accidental ureteric injury.

Desmosomal adhesiveness is developmentally regulated in the mouse embryo and modulated during trophectoderm migration.

During embryonic development tissues remain malleable to participate in morphogenetic movements but on completion of morphogenesis they must acquire the toughness essential for independent adult life. Desmosomes are cell-cell junctions that maintain tissue integrity especially where resistance to mechanical stress is required. Desmosomes in adult tissues are termed hyper-adhesive because they adhere strongly and are experimentally resistant to extracellular calcium chelation. Wounding results in weakening of desmosomal adhesion to a calcium-dependent state, presumably to facilitate cell migration and wound closure. Since desmosomes appear early in mouse tissue development we hypothesised that initial weak adhesion would be followed by acquisition of hyper-adhesion, the opposite of what happens on wounding. We show that epidermal desmosomes are calcium-dependent until embryonic day 12 (E12) and become hyper-adhesive by E14. Similarly, trophectodermal desmosomes change from calcium-dependence to hyper-adhesiveness as blastocyst development proceeds from E3 to E4.5. In both, development of hyper-adhesion is accompanied by the appearance of a midline between the plasma membranes supporting previous evidence that hyper-adhesiveness depends on the organised arrangement of desmosomal cadherins. By contrast, adherens junctions remain calcium-dependent throughout but tight junctions become calcium-independent as desmosomes mature. Using protein kinase C (PKC) activation and PKCα-/- mice, we provide evidence suggesting that conventional PKC isoforms are involved in developmental progression to hyper-adhesiveness. We demonstrate that modulation of desmosomal adhesion by PKC can regulate migration of trophectoderm. It appears that tissue stabilisation is one of several roles played by desmosomes in animal development.

Calcium-independent desmosomes of keratinocytes are hyper-adhesive.

Desmosomes in tissues are resistant to disruption by chelation of extracellular calcium. It has been suggested that this represents a hyper-adhesive state of these intercellular junctions that is crucial for the maintenance of epidermal integrity. Desmosomes change to a lower affinity, calcium-dependent adhesive state when cells are cultured at low density or when an intact epithelial cell sheet is wounded. Here we demonstrate that cells of the immortalized human keratinocyte line HaCaT acquire calcium-independent desmosomes in confluent culture. An adhesion assay shows that HaCaT cells with calcium-independent desmosomes are more cohesive than cells with calcium-dependent desmosomes. This assay relates directly to desmosomal adhesion because it involves splitting of the desmosomal adhesive material. Moreover, the difference in adhesiveness between calcium-dependent and calcium-independent desmosomes involves no quantitative change in the known protein composition of desmosomes. Instead, switching between the two adhesive states can be achieved by activation or inhibition of protein kinase C (PKC), suggesting a direct effect of PKC signalling on desmosomal adhesion. These results provide direct support for the concept of hyper-adhesiveness in desmosomes.

Assays for the calcium sensitivity of desmosomes.

Epithelial cells in vivo exist as confluent cell sheets, but this confluence is disrupted if the sheets are wounded, if the cells are undergoing morphogenesis, or if they are taking part in invasion and metastasis. Desmosomes are one of the principal types of adhesive junctions in epithelia and are responsible for maintaining tissue integrity. It is likely that modulation of desmosomal adhesion is required to facilitate cell motility in response to alterations in the tissue architecture. Desmosomal adhesion changes from a calcium-dependent state to a calcium-independent state when cells become confluent. Our laboratory has shown that the alpha isoform of protein kinase C is involved in signaling the response of desmosomes to calcium concentration and wounding, in cultured epithelial cells and in mouse epidermis (in vivo).

Targeted deletion of mek5 causes early embryonic death and defects in the extracellular signal-regulated kinase 5/myocyte enhancer factor 2 cell survival pathway.

To elucidate the physiological significance of MEK5 in vivo, we have examined the effect of mek5 gene elimination in mice. Heterozygous mice appear to be healthy and were fertile. However, mek5(-/-) embryos die at approximately embryonic day 10.5 (E10.5). The phenotype of the mek5(-/-) embryos includes abnormal cardiac development as well as a marked decrease in proliferation and an increase in apoptosis in the heart, head, and dorsal regions of the mutant embryos. The absence of MEK5 does not affect cell cycle progression but sensitizes mouse embryonic fibroblasts (MEFs) to the ability of sorbitol to enhance caspase 3 activity. Further studies with mek5(-/-) MEFs indicate that MEK5 is required for mediating extracellular signal-regulated kinase 5 (ERK5) activation and for the regulation of the transcriptional activity of myocyte enhancer factor 2. Overall, this is the first study to rigorously establish the role of MEK5 in vivo as an activator of ERK5 and as an essential regulator of cell survival that is required for normal embryonic development.

Desmosomal cadherins.

New evidence from blocking desmosomal adhesion with anti-adhesion peptides reveals a role for desmosomes in cell positioning in morphogenesis. Desmosomal adhesion is necessary for the stability of adherens junctions in epithelial cell sheets. Knockout and mis-expression of desmosomal cadherins in mice suggests that they may function directly or indirectly in regulating epidermal differentiation. Protein kinase C signalling and tyrosine phosphorylation appear to regulate desmosomal adhesion. There are new insights into the role of desmosomal cadherins in autoimmune, infectious and genetic disease.

Desmosomal adhesion: structural basis, molecular mechanism and regulation (Review).

Desmosomes are adhesive intercellular junctions of epithelia and cardiac muscle. They have an essential function in maintaining the integrity of tissues, which is compromised in human genetic and autoimmune disease that targets desmosomal components. Recent evidence (1) suggests new roles for the function of desmosomal adhesion in tissue morphogenesis, (2) gives new insights into the molecular mechanism of adhesion, (3) indicates that the desmosomal adhesion molecules, desmocollin and desmoglein, may contribute to the regulation of epidermal diffentiation, and (4) shows that the affinity of desmosomal adhesion is regulated by protein kinase C.

Suprabasal desmoglein 3 expression in the epidermis of transgenic mice results in hyperproliferation and abnormal differentiation.

The desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) isoforms of the desmosomal cadherins are expressed in the suprabasal layers of epidermis, whereas Dsg3 and Dsc3 are more strongly expressed basally. This differential expression may have a function in epidermal morphogenesis and/or may regulate the proliferation and differentiation of keratinocytes. To test this hypothesis, we changed the expression pattern by overexpressing human Dsg3 under the control of the keratin 1 (K1) promoter in the suprabasal epidermis of transgenic mice. From around 12 weeks of age, the mice exhibited flaking of the skin accompanied by epidermal pustules and thinning of the hair. Histological analysis of affected areas revealed acanthosis, hypergranulosis, hyperkeratosis, localized parakeratosis, and abnormal hair follicles. This phenotype has some features in common with human ichthyosiform diseases. Electron microscopy revealed a mild epidermal spongiosis. Suprabasally, desmosomes showed incorporation of the exogenous protein by immunogold labeling but were normal in structure. The epidermis was hyperproliferative, and differentiation was abnormal, demonstrated by expression of K14 in the suprabasal layer, restriction of K1, and strong induction of K6 and K16. The changes resembled those found in previous studies in which growth factors, cytokines, and integrins had been overexpressed in epidermis. Thus our data strongly support the view that Dsg3 contributes to the regulation of epidermal differentiation. Our results contrast markedly with those recently obtained by expressing Dsg3 in epidermis under the involucrin promoter. Possible reasons for this difference are considered in this paper.