Modified Cantilever Arrays Improve Sensitivity and Reproducibility of Nanomechanical Sensing in Living Cells

Mechanical signaling involved in molecular interactions lies at the heart of materials science and biological systems, but the mechanisms involved are poorly understood. Here we use nanomechanical sensors and intact human cells to provide unique insights into the signaling pathways of connectivity networks, which deliver the ability to probe cells to produce biologically relevant, quantifiable and reproducible signals. We quantify the mechanical signals from malignant cancer cells, with 10 cells per ml in 1000-fold excess of non-neoplastic human epithelial cells. Moreover, we demonstrate that a direct link between cells and molecules creates a continuous connectivity which acts like a percolating network to propagate mechanical forces over both short and long length-scales. The findings provide mechanistic insights into how cancer cells interact with one another and with their microenvironments, enabling them to invade the surrounding tissues. Further, with this system it is possible to understand how cancer clusters are able to co-ordinate their migration through narrow blood capillaries

T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-α. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein-protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexe

Genetic changes in human testicular germ cell tumours

Most testicular germ cell tumours (TGCTs) have a modal chromosome number in the hypotriploid range and and tumour progression is thought to result from net loss of chromosomes from a near tetraploid carcinoma in situ cell. Around 1.5% of TGCTs occur in a familial form thus suggesting a possible role for tumour suppressor genes in testicular tumorigenesis. To find out whether particular areas of the TGCT genome show marked loss of heterozygosity (LOH) allele loss studies were carried out on tumours from 41 patients using a number of polymorphic ezyme and DNA RFLP markers covering 23 chromosome arms. In addition aneuploid cultures were successfully established from seven cases and the results from these cultures were compared with those from corresponding tumours. LOH at a frequency of >10% was found on the following chromosome arms; 3p (2 losses out of 18 informative individuals), 3q (1/8), 4 (1/10), 5q (6/33), 11p (4/13), 11q (4/24), 12p (1/7), 13q (3/18), 16p (3/24) and 18p (1/5). LOH at a fequency of <10% was found on 1p (2/37), 1q (3/34), 5p (1/30), 7p (1/27), 7q (2/22), 9q (1/19), 14q (2/22), 15 (1/23), 18q (2/25) and 20 (1/26). No losses were found on 6p (six informative individuals), 12q (33) and 17p (23). Additional losses on 1p, 1q, 3p, 9q, 11p, 11q, 14q, 18p and 18q were observed in the tumour cultures. The relatively high frequency of allele loss on 3p, 5q, 11p, 11q, 13q and 16p may indicate the presence of tumour suppressor genes on these chromosome arms which are important in the development of TGCTs. The extent of allele loss as defined by fractional allele loss was not found to be significantly different between the two histologic subtypes seminoma and nonseminoma. An examination of allele intensity in tumour DNA has demonstrated allelic evenness in over 70% of tumours for probes on 1p36, 3q, 4, 5p, 5q, 7q, 11p, 11q, 12q14, 13q and 15q. The only chromosome arm demonstrating an uneven pattern in more than 70% of tumours is 12p. These findings suggest that the changing pattern of genomic organization occuring during tumour progression is non-random. An isochromosome for the short arm of chromosome 12 is a highly specific cytogenetic abnormality found in the majority of TGCTs. The retention of heterozygosity for all informative markers on 12q suggests that formation of the i(12p) markers occurs following polyploidization. Mutations in the p53 gene which is located on 17p are considered to be among the most common genetic alterations in human cancers. The retention of heterozygosity for all informative 17p loci suggests that alterations in the p53 gene are not important for the predisposition and development of TGCTs. Consistent with this finding is the lack of any consistent change in p53 mRNA levels in TGCTs. Inactivation of the retinoblastoma (RB) tumour suppressor gene is associated with the development of several human malignancies. An investigation of RB mRNA revealed decreased levels in all TGCTs analysed but no alterations in transcript size. Rearrangemet of the RB gene was demonstrated in a single TGCT. These findings suggest regulation of transcription and/or transcript half- life or gene mutations may be responsible for the decreased RB mRNA levels in human TGCTs. Analysis of the H-ras oncogene mRNA revealed decreased levels in nearly all tumours examined. The reduced levels did not always coincide with LOH at this locus thus suggesting a decrease in the level of transcription and/or half-life of H-ras mRNA. There was no consistent change in the levels of N-ras and MCC40 mRNA levels

Silencing of androgen-regulated genes using a fusion of AR with the PLZF transcriptional repressor

The androgen receptor (AR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and plays a key role in the development and progression of prostate cancer. Current therapies include the use of antiandrogens aimed at inhibiting the transcriptional activation of AR-regulated genes by AR. Here, we explore a strategy aimed at obtaining silencing of AR-regulated genes, based on the properties of the transcriptional repressor promyelocytic leukamia zinc-finger protein (PLZF). In order to do this, we have made a fusion protein between PLZF and AR, named PLZF-AR, and show that PLZF-AR is able to bring about silencing of genomically encoded AR-regulated genes and inhibit the androgen-regulated growth of LNCaP prostate cancer cells. Together, our results show that this strategy is able to bring about potent repression of AR-regulated responses and, therefore, could be of value in the development of new therapies for prostate cancer