Relating cell and tissue mechanics: Implications and applications

The Differential Adhesion Hypothesis (DAH) posits that differences in adhesion provide the driving force for morphogenetic processes. A manifestation of differential adhesion is tissue liquidity and a measure for it is tissue surface tension. In terms of this property, DAH correctly predicts global developmental tissue patterns. However, it provides little information on how these patterns arise from the movement and shape changes of cells. We provide strong qualitative and quantitative support for tissue liquidity both in true developmental context and in vitro assays. We follow the movement and characteristic shape changes of individual cells in the course of specific tissue rearrangements leading to liquid-like configurations. Finally, we relate the measurable tissue-liquid properties to molecular entities, whose direct determination under realistic three-dimensional culture conditions is not possible. Our findings confirm the usefulness of tissue liquidity and provide the scientific underpinning for a novel tissue engineering technology. Developmental Dynamics 237:2438-2449, 2008. (c) 2008 Wiley-Liss, Inc.     

Tumour specific promoter region methylation of the human homologue of the Drosophila Roundabout gene DUTT1 (ROBO1) in human cancers

The human homologue of the Drosophila Roundabout gene DUTT1 (Deleted in U Twenty Twenty) or ROBO1 (Locus Link ID 6091), a member of the NCAM family of receptors, was recently cloned from the lung cancer tumour suppressor gene region 2 (LCTSGR2 or U2020 region) at 3p12. DUTT1 maps within a region of overlapping homozygous deletions characterized in both small cell lung cancer lines (SCLC) and in a breast cancer line. In this report we (a) defined the genomic organization of the DUTT1 gene, (b) performed mutation and expression analysis of DUTT1 in lung, breast and kidney cancers, (c) identified tumour specific promoter region methylation of DUTT1 in human cancers. The gene was found to contain 29 exons and spans at least 240 kb of genomic sequence. The 5' region contains a CpG island, and the poly(A)(+) tail has an atypical 5'-GATAAA-3' signal. We analysed DUTT1 for mutations in lung, breast and kidney cancers, no inactivating mutations were detected by PCR-SSCP. However, seven germline missense changes were found and characterized. DUTT1 expression was not detectable in one out of 18 breast tumour lines analysed by RT-PCR. Bisulfite sequencing of the promoter region of DUTT1 gene in the HTB-19 breast tumour cell line (not expressing DUTT1) showed complete hypermethylation of CpG sites within the promoter region of the DUTT1 gene (-244 to +27 relative to the translation start site). The expression of DUTT1 gene was reactivated in HTB-19 after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The same region was also found to be hypermethylated in six out of 32 (19%) primary invasive breast carcinomas and eight out of 44 (18%) primary clear cell renal cell carcinomas (CC-RCC) and in one out of 26 (4%) primary NSCLC tumours. Furthermore 80% of breast and 75% of CC-RCC tumours showing DUTT1 methylation had allelic losses for 3p12 markers hence obeying Knudson's two hit hypothesis. Our findings suggest that DUTT1 warrants further analysis as a candidate for the tumour suppressor gene (TSG) at 3p12, a region defined by hemi and homozygous deletions and functional analysis.     

Successful conversion to rapamycin for calcineurin inhibitor-related neurotoxicity following liver transplantation

INTRODUCTION: Neurotoxicity is a well-recognized side effect of calcineurin inhibitors. Rapamycin is considered to be significantly less neurotoxic than calcineurin inhibitors (CNIs). The aim of this study was to retrospectively analyze a group of post-liver transplant patients who had been converted to rapamycin because of CNI-related neurotoxicity. PATIENTS AND METHODS: Orthotopic liver transplantation (OLT) was performed in 56 consecutive patients between April 1, 2003, and August 15, 2004. Immunosuppression was administered with tacrolimus, mycophenolic acid, and corticosteroids. RESULTS: Seven patients were converted to rapamycin due to new-onset neurotoxicity or exacerbation of previous neurological symptoms secondary to CNI. None of the patients had toxic levels tacrolimus (>15 ng/mL) at the time of symptoms, which persisted despite reduction of CNI dose. The indications for conversion were: (1) peripheral neuropathy; (2) seizure; (3) metabolic encephalopathy; and (4) central pontine myelinolysis. All patients showed improvement or resolution of their neurological symptoms after conversion to rapamycin. Two patients died, the first due to a hypoxic event and the second due to central pontine myelinolysis with limited improvement and a family decision to withdraw care. There were no complications directly attributed to rapamycin. Specifically, there were no thrombotic events, wound complications, or biliary leaks. Three patients had a rejection episode that was successfully treated with pulse corticosteroids and low-dose tacrolimus (levels < 5 ng/mL). CONCLUSIONS: Rapamycin can be safely used in OLT recipients with severe neurological symptoms ascribed to or exacerbated by CNIs. Rapamycin monotherapy may be inadequate to control rejection early after transplantation. Rapamycin can be combined with low doses of CNI to prevent rejection.     

Nicotinic receptors and cognition in Parkinson’s Disease: the importance of neuronal synchrony

Parkinson's Disease (PD) is associated with cognitive deficits. The earliest impairment is evident for executive abilities, visuospatial orientation and memory. Dopamine deficiency is unlikely to be singly responsible for all cognitive changes in PD. Acetyl-choline has an essential role in cognition, thus cholinergic transmission may have an important role in non-dopaminergic cognitive changes. If so, some cognitive defects could possibly be treated with choline-esterase inhibitors. A concern is the potential negative motor effect of cholinergic medication in PD. Surprisingly, these are reported only in a few patients studied. Establishing the relationship between select cognitive deficits and nicotinic neurotransmission may lay the foundation for rational pharmacotherapy of cognitive dysfunction in PD.We summarize anatomical, physiological and pharmacological aspects of nicotinic receptor function. The focus is on those nicotine receptor dependent cognitive dysfunctions which are likely to contribute to motor impairment. Lastly, we discuss hypotheses concerning cholinergic involvement in neuronal synchrony and sensorimotor integration in PD.     

Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression

BACKGROUND: The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers. METHODS: We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures. We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. All statistical tests were two-sided. RESULTS: RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 64% of breast cancer lines, in 30% of primary NSCLCs, and in 49% of primary breast tumors but in none of the nonmalignant lung tissues. RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P =.046). Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity. CONCLUSION: RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.