No abstract

No abstract

Lung cancer is the major human malignancy, accounting for 30% of all cancer-related deaths worldwide. Poor survival of lung cancer patients, together with late diagnosis and resistance to classic chemotherapy, highlights the need for identification of new biomarkers for early detection. Among different cancer biomarkers, small non-coding RNAs called microRNAs (miRNAs) are considered the most promising, owing to their remarkable stability, their cancer-type specificity, and their presence in body fluids. However, results of multiple previous attempts to identify circulating miRNAs specific for lung cancer are inconsistent, likely due to two main reasons: prominent variability in blood miRNA content among individuals and difficulties in distinguishing tumor-relevant miRNAs in the blood from their non-tumor counterparts. To overcome these impediments, we compared circulating miRNA profiles in patients with lung squamous cell carcinoma (SCC) before and after tumor removal, assuming that the levels of all tumor-relevant miRNAs would drop after the surgery. Our results revealed a specific panel of the miRNAs (miR-205, -19a, -19b, -30b, and -20a) whose levels decreased strikingly in the blood of patients after lung SCC surgery. Interestingly, miRNA profiling of plasma fractions of lung SCC patients revealed high levels of these miRNA species in tumor-specific exosomes; additionally, some of these miRNAs were also found to be selectively secreted to the medium by cultivated lung cancer cells. These results strengthen the notion that tumor cells secrete miRNA-containing exosomes into circulation, and that miRNA profiling of the exosomal plasma fraction may reveal powerful cancer biomarkers.

Gap‐junctional intercellular communication (GJIC) in 20 primary human liver tumors with different degrees of malignancy has been studied at the functional and molecular levels. When GJIC capacity was determined by dye‐transfer assay performed directly with freshly removed tumor tissue, significant reduction was found in all samples, regardless of their morphology. In addition, a selective lack of GJIC between tumor and surrounding non‐tumorous cells was observed in some cases, probably due to the physical separation between them resulting from encapsulation of tumors. There was, however, no essential change in the level of expression of the major liver gap‐junction protein, connexin (ex) 32, in liver tumors as measured by Northern and Western blot analyses. Immunohistochemical study revealed aberrant localization of ex 32 in the majority of malignant liver tumors. Instead of cytoplasmic membrane localization at intercellular contacts, ex 32 was detected mainly either intracytoplasmically or in plasma membrane free from contact with other cells. We did not detect any mutation in the coding sequence of the ex 32 gene from any of the human liver tumors we tested. Thus it is likely that the aberrant localization of ex 32 in tumor cells is due to disruption of the mechanisms for establishment of this protein into gap‐junction plaques, rather than to structural abnormality of the ex 32 protein itself. Another member of the connexin family, ex 43, not detectable in non‐tumorigenic hepatocytes, was expressed in several tumors, especially in invasive areas, but was detected in only a few tumor cells and was localized intracytoplasmically, suggesting that ex 43 protein is not involved in GJIC in the tumors. © 1994 Wiley‐Liss, Inc.

Abstract. The carboxyterminal cytoplasmic portions (tails) of desmosomal cadherins of both the desmoglein (Dsg) and desmocollin type are integral components of the desmosomal plaque and are involved in desmosome assembly and the anchorage of intermediate-sized filaments. When additional Dsg tails were introduced by cDNA transfection into cultured human epithelial cells, in the form of chimeras with the aminoterminal membrane insertion domain of rat connexin32 (Co32), the resulting stably transfected cells showed a dominant-negative defect specific for desmosomal junctions: despite the continual presence of all desmosomal proteins, the endogenous desmosomes disappeared and the formation of Co32-Dsg chimeric gap junctions was inhibited. Using cell transfection in combination with immunoprecipitation techniques, we have examined a series of deletion mutants of the Dsgl tail in Co32-Dsg chimeras. We show that upon removal of the last 262 amino acids the truncated Dsg tail still effects the binding of plakoglobin but not of detectable amounts of any catenin and induces the dominant-negative phenotype. However, further truncation or excision of the next 41 amino acids, which correspond to the highly conserved carboxyterminus of the C-domain in other cadherins, abolishes plakoglobin binding and allows desmosomes to reform. Therefore, we conclude that this short segment provides a plakoglobin-binding site and is important for plaque assembly and the specific anchorage of either actin filaments in adherens junctions or IFs in desmosomes. INTERCELLULAR adhering junctions (9) are important structures contributing to stable and positionally ordered cell-cell attachment as well as to the anchorage and specific intracellular arrangement of cytoskeletal filaments and contain calcium-dependent transmembrane cell adhesion molecules, which are members of the cadherin family of proteins. While intercellular contact formation and adhesion is effected by the aminoterminal and N-glycosylated part of the cadherin molecule, the association of cytoskeletal filaments involves the carboxyterminal intracellular portion and is mediated by certain submembranous dense plaques of 10-35 nm thickness. Despite their structural similarities and the fact that all junctions of this group contain a common major plaque protein, plakoglobin, their biochemical composition is markedly different. In epithelial cells, for example, two major types of adhering junctions can be generally distinguished in relation to composition and the specific type of filament attached (4,6,8,16,20,47,52): (a) Actin microfilaments anchor at adherens junctions which occur in diverse sizes, shapes, and positions (e.g., zonula adhaerens, fascia adhaerens, and puncture adhaerens), and contain E-or N-cadherin whose cytoplasmic portion is associated with certain cytoplasmic proteins such as a-and /3-catenin, vinculin, cx-actinin, and radixin, all of which contribute to plaque formation.(b) Intermediate-sized filaments (IFs) 1 are anchored at desmosomes (maculae adhaerentes) of var...

No abstract

No abstract

We have developed a simple method to measure gap-junctional intercellular communication (GJIC), by means of microinjection/dye transfer assay, in liver slices freshly removed from the rat. Using this method and immunostaining of connexin 32 (cx32), the major liver gap junction protein, we studied sequential changes of GJIC during chemical hepatocarcinogenesis in male Fischer-344 rats under a modified Solt-Farber protocol (3 weeks 4 day exposure regimen). Four weeks after commencement of the protocol, there was a substantial decrease in GJIC in the liver parenchyma, which was free from focal lesions. The decrease in GJIC persisted up to at least the 15th week of treatment, while a decrease in the number of immunoreactive cx32 spots was evident only at 4 weeks of post-protocol commencement. Most enzyme-altered (GST-P-positive) focal lesions showed markedly lower GJIC and a significantly lower number of cx32-positive spots than surrounding hepatocytes. Most GST-P-positive foci showed a selective lack of GJIC with surrounding heptocytes. Hepatocellular carcinomas arising 1 year after the carcinogenic regimen had significantly reduced communicational capacity accompanied by a large decrease in cx32 expression. These results suggest that a progressive decrease in homologous as well as heterologous GJIC in preneoplastic lesions occurs during rat hepatocarcinogenesis, and that preneoplastic lesions with the most prominent disorders in GJIC may be more likely to develop into carcinomas.