El-Nasir Lalani

Disruption of the developmentally regulated Rev3l gene causes embryonic lethality

The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis [2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals.

Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer

Fluorescence lifetime imaging (FLIM) depends on the fluorescence decay differences between tissues to generate image contrast. In the present study FLIM has been applied to fixed (but unstained) breast cancer tissues to demonstrate the feasibility of this approach for histopathological assessment. As the FLIM method relies on natural autofluorescence, it may be possible to circumvent tissue processing altogether and so FLIM has the potential to be a powerful new method of in vivo tissue imaging via an endoscopic or per‐operative approach in a variety of organs, as well as a research tool for in vivo animal models of disease. Unstained, alcohol‐fixed tissue samples from 13 patients were stimulated by laser pulses at 415 nm. The temporal decay of the autofluorescence was imaged over a period of 2 ns after cessation of the pulse. The decay rate at each image pixel was calculated as the ‘lifetime’ factor τ. A tissue classification scheme was used to define regions in each image. The average lifetimes of different tissue regions were compared. A total of 167 tissue regions were measured. Within individual fields, stroma had a larger τ (slower decay) than epithelium (p < 0.001). Within individual patients (taking the mean τ of a given tissue type across all fields from each patient), there was a statistically significant difference between benign and malignancy‐associated stroma (p < 0.05). Also, benign collagen had a longer τ than benign epithelium (p < 0.05). Multivariate analysis showed a significant difference between benign stroma, malignancy‐associated stroma, blood vessels, and malignant epithelium (p < 0.05). Statistically significant differences between benign and malignancy‐associated stroma were obtained even with small patient numbers, indicating that lifetime‐based instruments can be developed for real‐time diagnostic imaging with microscopic resolution. Copyright

Molecular and cellular biology of prostate cancer

Prostate cancer is an enigmatic disease. Although prostatic-intraepithelial neoplasia appears as early as the third decade and as many as 80% of 80 year old men have epithelial cells in their prostate that fit the morphological criteria for cancer, only about 10% of men will ever have the clinical disease and less than 3% will die from it. There have been no significant proven interventions which have altered the natural history of the disease since hormone down regulation was introduced in the 1940s and new research has been poorly supported. There is however an urgent need to develop new criteria to distinguish those patients with localised disease who will benefit from intervention from those that do not require it or who will have occult extra prostatic metastases. Similarly, there is an urgent need to develop new treatment for those in whom the disease is extra-prostatic and therefore incurable by conventional treatments. This review covers the latest developments in epidemiology, cellular and molecular biology including new areas such as ion channels in the field of prostate cancer.

Intestinal trefoil factor (TFF 3) and pS2 (TFF 1), but not spasmolytic polypeptide (TFF 2) mRNAs are co-expressed in normal, hyperplastic, and neoplastic human breast epithelium

pS2–TFF 1 is expressed in breast cancers and has been investigated as a potential prognostic factor reflecting oestrogen dependence. The relationship to the expression of other trefoil peptides, human spasmolytic polypeptide (hSP–TFF 2) and intestinal trefoil factor (hITF/hPI.B–TFF 3) is documented here. Fifty‐seven breast specimens were selected from surgical pathology archives and included five normal breasts (two lactating), seven benign proliferative lesions, 11 ductal carcinomas in situ (DCIS), three lobular carcinomas in situ (LCIS), 24 invasive ductal carcinomas (IDC), and seven invasive lobular carcinomas (ILC). The comparative distribution of trefoil mRNAs was assessed by in situ hybridization using 35S‐labelled riboprobes and immunohistochemical staining for pS2–TFF 1 and hSP–TFF 2. pS2–TFF 1 and hITF/hPI.B–TFF 3 mRNA were focally present at low signal intensity in normal and benign breast. Both pS2–TFF 1 and hITF/hPI.B–TFF 3 were expressed in all DCIS, LCIS and ILC, and 21/24 IDC. Overall, expression patterns of pS2–TFF 1 and hITF/hPI.B–TFF 3 coincided, but hITF/hPI.B–TFF 3 mRNA was usually found in a greater proportion of cells. Expression of hSP–TFF 2 peptide or mRNA was not detected in any of these cases. MCF 7 breast carcinoma cells also expressed hITF/hPI.B–TFF 3 and pS2–TFF 1 mRNAs but not hSP–TFF 2. hITF/hPI.B–TFF 3 co‐expression with pS2–TFF 1 may act as a prognostic factor, but also raises questions about the regulatory pathway for pS2–TFF 1 hITF/hPI.B–TFF 3. Trefoil factors have effects on cell motility and spreading in vitro, and co‐expression of hITF/hPI.B–TFF 3 with pS2–TFF 1 could be functionally significant if they form a heterodimer or compete for receptor binding. Absence of hSP–TFF 2 expression may be of equal relevance to tumour cell biology.

CD151 Regulates Tumorigenesis by Modulating the Communication between Tumor Cells and Endothelium

The tetraspanin CD151 forms stoichiometric complexes with laminin-binding integrins (e.g., α3β1, α6β1, and α6β4) and regulates their ligand-binding and signaling functions. We have found that high expression of CD151 in breast cancers is associated with decreased overall survival (3.44-fold higher risk of death). Five-year estimated survival rates were 45.8% (95% confidence interval, 16.4-71.4%) for CD151-positive patients and 79.9% (95% confidence interval, 62.2-90.0%) for CD151-negative patients. Furthermore, CD151 was positively associated with axillary lymph node involvement. To study the biological significance of this observation, we investigated the contribution of CD151 in breast cancer tumorigenesis using MDA-MB-231 cells as a model system. Stable down-regulation of this tetraspanin by short-hairpin RNA decreased the tumorigenicity of these cells in mice. Detailed immunohistologic analysis of CD151(+) and CD151(−) xenografts showed differences in tumor vascular pattern. Vascularization observed at the subcutaneous border of the CD151(+) tumors was less pronounced or absent in the CD151(−) xenografts. In vitro experiments have established that depletion of CD151 did not affect the inherent proliferative capacity of breast cancer cells in three-dimensional extracellular matrices, but modified their responses to endothelial cells in coculture experiments. The modulatory activity of CD151 was dependent on its association with both α3β1 and α6β4 integrins. These data point to a new role of CD151 in tumorigenesis, whereby it functions as an important regulator of communication between tumor cells and endothelial cells. These results also identify CD151 as a potentially novel prognostic marker and target for therapy in breast cancer. (Mol Cancer Res 2009;7(6):787–98)

Human MUC1 Mucin: A Potent Glandular Morphogen

Human MUC1 mucin is a high‐molecular‐weight transmembrane glycoprotein, which is apically expressed in the majority of glandular epithelia. During embryonic development, changes in the pattern of MUC1 mucin expression coincide with the onset of glandular differentiation. This mucin is also frequently overexpressed and aberrantly glycosylated in carcinomas. To investigate the potential role of MUC1 mucin in morphogenesis, a full length MUC1u2009cDNA was transfected into murine mammary adenocarcinoma (410.4) and Madin‐Darby canine kidney (MDCK) cells. This generated four clonal cell lines. Western blotting, FACS analysis, and immunohistochemistry confirmed expression of MUC1. All four MUC1‐expressing clones demonstrated altered morphogenesis when cultured in three‐dimensional type I collagen gels. While parental and vector control 410.4 cells formed compact spherical structures, the MUC1‐expressing clones formed complex branching structures. Similarly, while parental and vector control MDCK cells formed small circumscribed colonies with a central lumen, the MUC1‐expressing clones formed elongated tubules. MUC1 expression was also associated with reduced cellular cohesion and enhanced migration on type I collagen‐coated surfaces for all except one of the clones, which expressed only low levels of MUC1 on the cell surface. These results show that MUC1 expression stimulates morphogenetic changes in two distinct epithelial cell lines. Taken together with previous observations on MUC1 expression in embryonic development and carcinomas, this finding suggests that MUC1 may induce changes in tissue architecture in both normal development and cancer. Copyright

MUC 1: a genetic susceptibility to infertility?

In man and some animals regulation of embryo implantation by endometrial expression of the highly polymorphic MUC 1 mucin has been suggested. We assessed the polymorphism of MUC 1 in women known to be fertile and those with infertility due to suspected failure of embryo implantation. The median of the lower allele size in the infertile group was only 2.5 kb compared with 3.4 kb in the fertile group (p=0.0029, difference 0.9, [95% CI 0.1-1.3]). Women with unexplained infertility might have a genetic susceptibility to failure of embryo implantation due to small MUC 1 allele size.

Differentially expressed genes in hormone refractory prostate cancer: association with chromosomal regions involved with genetic aberrations.

Differential gene expression between the androgen sensitive human prostate cancer cell line LNCaP and an insensitive clonal variant, LNCaP-r, was demonstrated by suppression subtractive hybridization. Twenty-one sequences were identified of which 9 are homologous to known genes, 11 are represented by expressed sequence tags (ESTs), and 1 is novel. We present data for 5 of 7 sequences confirmed to be differentially expressed by Northern blot analysis and semiquantitative RT-PCR. Only one gene, fibronectin (FN), was highly overexpressed (>60-fold) in LNCaP-r cells, consistent with previously reported overexpression of FN in prostate cancer. Four sequences were down-regulated in LNCaP-r cells, including an inactive variant of the E2 ubiquitin conjugating enzyme (UEV-1), a novel metalloproteinase-related collagenase (PM5), and a potential tumor suppressor gene (breast basic conserved gene, BBC1). UEV-1 is multifunctional, regulates the cell cycle via cdk1, has homology to MMS2 and likewise functions as a DNA protection protein, and also has homology to TSG101. Aberrant splice variants of TSG101 occur frequently in both breast and prostate cancer, but its mechanism of action is unknown. FN, BBC1, and UEV-1 localize to regions of chromosomal aberration (2q3.4, 16q24.3, and 20q13.2, respectively) associated with advanced prostate cancer and thus may be highly relevant to disease progression.

Differential expression of cell death regulators in response to thapsigargin and adriamycin in Bcl-2 transfected DU145 prostatic cancer cells.

Functional overexpression of Bcl‐2 has been reported to confer an anti‐apoptotic potential in a variety of cell types. The role of Bcl‐2 in epithelial cell‐cycle control and in interactions with other cell‐cycle regulators is not clearly understood. Its expression has been correlated with the hormono‐ and chemo‐resistant phenotype in advanced prostate cancer. The aim of this study was to investigate the mechanisms through which Bcl‐2 mediates increased cytotoxic chemoresistance by assessing alterations in the expression of cell death regulatory molecules. The DU145 human prostatic adenocarcinoma cell line was stably transfected with a Bcl‐2 encoding expression plasmid. Two Bcl‐2 transfectants, DKC9 and DKC11, were expanded for further study. The effects of Bcl‐2 expression on cellular proliferation, cell death (± adriamycin or thapsigargin), and expression of cell‐cycle/death regulators (p53, PCNA, Bax, Bak, Bcl‐XL) were evaluated. Compared with controls, Bcl‐2 transfectants showed no difference in the rate of proliferation, a decrease in p53 (∼two‐fold), an increase in Bax (∼two‐fold) and PCNA (∼three‐fold), and no change in the levels of Bcl‐XL and Bak proteins. DKC9 and DKC11 also exhibited a significantly increased chemoresistance to adriamycin (0.0025–5u2009µM) and thapsigargin (0.0025–5u2009µM) compared with controls. In the presence of thapsigargin or adriamycin, levels of Bcl‐2 and its heterodimeric partner Bax were elevated ∼two‐fold with no change in Bak in Bcl‐2 transfectants in contrast to controls, where Bak was increased (two‐fold). This is the first study to demonstrate that Bcl‐2 transfection modulates the expression of mutant p53, Bax, and PCNA in prostate cancer cells. Moreover, Bcl‐2 overexpression conferred a significant cytotoxic chemoresistance and altered the balance of expression of death promoters (from Bak, a dominant death promoter in controls, to Bax) in response to thapsigargin and adriamycin. Copyright

Topographical expression of class IA and class II phosphoinositide 3-kinase enzymes in normal human tissues is consistent with a role in differentiation

BackgroundGrowth factor, cytokine and chemokine-induced activation of PI3K enzymes constitutes the start of a complex signalling cascade, which ultimately mediates cellular activities such as proliferation, differentiation, chemotaxis, survival, trafficking, and glucose homeostasis. The PI3K enzyme family is divided into 3 classes; class I (subdivided into IA and IB), class II (PI3K-C2α, PI3K-C2β and PI3K-C2γ) and class III PI3K. Expression of these enzymes in human tissue has not been clearly defined.MethodsIn this study, we analysed the immunohistochemical topographical expression profile of class IA (anti-p85 adaptor) and class II PI3K (PI3K-C2α and PI3K-C2β) enzymes in 104 formalin-fixed, paraffin embedded normal adult human (age 33–71 years, median 44 years) tissue specimens including those from the gastrointestinal, genitourinary, hepatobiliary, endocrine, integument and lymphoid systems. Antibody specificity was verified by Western blotting of cell lysates and peptide blocking studies. Immunohistochemistry intensity was scored from undetectable to strong.ResultsPI3K enzymes were expressed in selected cell populations of epithelial or mesenchymal origin. Columnar epithelium and transitional epithelia were reactive but mucous secreting and stratified squamous epithelia were not. Mesenchymal elements (smooth muscle and endothelial cells) and glomerular epithelium were only expressed PI3K-C2α while ganglion cells expressed p85 and PI3K-C2β. All three enzymes were detected in macrophages, which served as an internal positive control. None of the three PI3K isozymes was detected in the stem cell/progenitor compartments or in B lymphocyte aggregates.ConclusionsTaken together, these data suggest that PI3K enzyme distribution is not ubiquitous but expressed selectively in fully differentiated, non-proliferating cells. Identification of the normal in vivo expression pattern of class IA and class II PI3K paves the way for further analyses which will clarify the role played by these enzymes in inflammatory, neoplastic and other human disease conditions.