No description available.

No description available.

No description available.

The endoplasmic reticulum (ER) is the major site for protein synthesis, folding and trafficking, as well as lipid synthesis and Ca2+ storage. To maintain ER integrity, homeostatic response mechanisms to stimuli which perturb ER function, known as the unfolded protein response (UPR) have evolved. Cancer cells can be exposed to extremes of environmental conditions as a direct consequence of their mechanisms of origin, and may have increased cellular proliferation, coupled with poor vascularisation leading to deficiencies in glucose, oxygen and metabolite requirements for efficient cell growth and survival. When ER function is disrupted, the UPR activates pro-survival mechanisms, such as the induction of chaperones and other folding machinery. However, the UPR also activates pro-death stimuli, to remove cells where the stress is to severe or persists for too long. Glucose regulated protein 78 (GRP78) is the major stress regulator and central hub of the UPR, as well as a key chaperone of the ER. GRP78 bind to and inhibits the activation of; protein kinase-like ER kinase (PERK), inositol requiring element 1 (IRE1) and activating transcription factor 6 (ATF6), known as the UPR activators. Solid tumours, such as melanoma and glioblastoma, may have increased expression of GRP78 correlating with disease stage and resistance to chemotherapy. Conversely, increased GRP78 expression in neuroblastoma has been associated with improved prognosis. To test the hypotheses that abrogating GRP78 function increases apoptosis of tumour cells and that GRP78 is a biomarker for the outcome of ER-stress in differing cancer types, neural-crest derived cancers were compared by stoichiometric analysis of GRP78 and the UPR activators, and the downstream components of the UPR, activating transcription factor 4 (ATF4) and X-box binding protein 1 (XBP-1). To determine the importance of GRP78 across cancer types, changes in sensitivity to the ER stress inducers fenretinide or bortezomib with respect to cell death (propidium iodide stained flow cytometry) or inhibition of cell viability (MTS assay) were assessed in response to siRNA mediated knock-down, GRP78 over-expression and GRP78 inhibition. IV There were differences in cellular concentrations of GRP78 between cell lines representing different cancer types; however the expression of UPR activators did not correlate with GRP78 levels. Stoichiometric analysis of the ratio between GRP78 and the UPR activators demonstrated a significant difference between melanoma and glioblastoma, to that of neuroblastoma. Mapping the activation of the UPR by either fenretinide or bortezomib showed cancer-specific and stress-inducer-dependent responses. Importantly, melanoma and glioblastoma demonstrated significantly greater ATF4 induction whereas neuroblastoma showed prolonged XBP-1 splicing. Testing the effect of altering GRP78 expression on sensitivity to ER-stress-induced cell death demonstrated that high expression of GRP78 in melanoma and glioblastoma correlated with resistance and with neuroblastoma GRP78 over-expression enhanced sensitivity. Investigating the effect of inhibiting GRP78 activity on fenretinide- and bortezomibinduced cell death demonstrated enhanced sensitivity in melanoma and glioblastoma, but no significant enhancement in neuroblastoma. Thus, down-regulating GRP78 or its function increased the death of melanoma and glioblastoma cells in response to ER stress; however there are differences in UPR signalling between cancer types, which ultimately results in contrasting prognosis as demonstrated in neuroblastoma. Although all three cancer types responded to ER-stress induced death, interpretation of the relationship between GRP78 and the UPR activators is essential for determining dependence on GRP78. The data suggest that melanoma and glioblastoma demonstrate increased sensitivity when GRP78 is inhibited due to a shift in the dynamic equilibrium of the UPR, promoting activation and downstream cell death. However cancer types expressing higher UPR activator concentrations compared to that of GRP78 do not respond positively to the inhibition of GRP78.

The lymphatic metastasis of tumours to lymph nodes (LN) is a frequent event in many cancers and heralds a poor clinical outcome. Comparison of lymphatic endothelial cells (LECS) from metastasizing murine T-241/VEGF-C fibrosarcomas and normal dermis revealed a tumour-specific LEC profile, characterized by the elevated expression of functionally significant molecules such as endothelial specific adhesion molecule (ESAM) and the TGF-~ coreceptor endoglin (CD105). Moreover, similar induction of ESAM and endoglin by human tumour Iymphatics was seen to dramatically correlate with LN metastasis. To further investigate interactions between tumour cells and Iymphatics, the role of lymphatic endothelial hyaluronan receptor LYVE-1 in LN metastasis was investigated. Using LYVE-1 mAbs and LYVE-1-1- mice, it was shown that a deficiency or perturbation of LYVE-1 expressed by tumour Iymphatics potentiated the lymphatic spread of spontaneously metastasizing T-241/VEGF-C fibrosarcomas and induced de novo metastasis by indolent parental T-241 tumours. Subsequent in vitro experiments using mAbs suggested that LYVE-1 exerts a blocking or 'gatekeeper' function, preventing pro-metastatic tumour cell-LEC interactions. Although largely restricted to the Iymphatics, LYVE-1 is also expressed in murine lung by type I alveolar epithelial cells (AECs). Using a bleomycin model of lung inflammation it was shown that LYVE-1-1- mice are unable to clear infiltrating leukocytes following acute lung injury. Furthermore, in vivo and in vitro experiments using blocking mAb reveal a critical role for LYVE-1 in the transepithelial migration of leukocytes across the alveolar epithelium, an essential step in the resolution of acute lung inflammation. These findings identify a specific role for LYVE-1 in cancer metastasis and the resolution of acute inflammation in the lung.

The LAC and EL4 ascites tumour cells contained both Type A and Type C virions. These virions replicated within the ascites tumour cells. However, neither the tumour cells nor their murine hosts were contaminated with LDV virions. Intraperitoneal transplantation of the LAC and EL4 ascites tumours provoked an oedematous inflammatory response which was accompanied by histological changes within the lymphoid organs of their hosts. Intraperitoneal transplantation of the LAC tumour within MF1 mice induced histopathological changes within the spleens and mediastinal lymph nodes compatible with an immune response. Intraperitoneal transplantation of the EL4 ascites lymphoma within C57BL10 mice culminated in infiltration of EL4 cells into both the spleen and mediastinal lymph nodes. Both the LAC and EL4 ascites tumours induced thymic involution and inflammation within the livers of their murine hosts. Growth of the LAC and EL4 ascites tumours within the peritoneal cavity was accompanied by infiltration of ascites tumour cells into the adipose tissues, pancreas, diaphragm and mesenteries. Whereas successful transplantation of the EL4 ascites lymphoma was achieved in both intraperitoneal and subcutaneous locations, the LAC tumour was only transplanted successfully intraperitoneally but not in subcutaneous locations. Subcutaneous transplantation of EL4 cells within the hind limbs of C57BL10 mice provoked an inflammatory response culminating in angiogenesis and fibroplasia around the transplants. As the growing transplants invaded the surrounding host tissue there was extensive remodelling of the connective tissue matrix followed by degradation of the connective tissue. Host inflammatory cells infiltrated the growing EL4 tumours but ultrastructural studies did not reveal any obvious indications that the host cells exerted an antitumour effect. Instead the macrophages appeared to phagocytose degraded materials within the tumours and therefore behaved as traditional phagocytes. The macrophages within the EL4 tumours had a well developed endoplasmic reticulum and Golgi apparatus, indicative of a secretory status for the cells, and it has been suggested that the macrophages within the EL4 tumours may have a trophic role. Any cytotoxic functions of the macrophages may have been suppressed by tumour derived or host cell derived factors generated within the complex ecosystem of the EL4 tumours. Subcutaneous transplantation of LAC cells within the hind limbs of MF1 mice did not provoke an intense inflammatory response and angiogenesis and fibroplasia was not apparent around the tumours soon after transplantation. It has been speculated that the LAC cells may have suppressed angiogenesis and fibroblast proliferation through production of anti-inflammatory factors. As a consequence of the lack of angiogenesis, the transplanted LAC cells necrosed and only then provoked an inflammatory response from their hosts. The inflammatory response resulted in the formation of scar tissue which gradually replaced the necrosed LAC cells at the site of transplantation. Intraperitoneal transplantation of LAC and EL4 ascites tumour cells provoked an inflammatory response. The inflammatory leucocytes did not appear to impair the viability of the LAC and EL4 cells and although the host inflammatory cells established contacts with the tumour cells they did not appear to sustain any damage and the transplants proliferated to establish ascites tumours. Within the MF1 mice some of the peritoneal macrophages appeared to be damaged and it is suggested that the LAC cells release a factor cytotoxic for macrophages. Within the C57BL10 mice ip injection of the EL4 cells provoked fibrin deposition and the fibrin was phagocytosed by the macrophages. During the course of tumour growth the LAC and EL4 cells bound alpha and gamma globulins to their sufaces and it is believed that these molecules may have afforded the ascites cells 'immunological protection' by masking cell surface antigenic determinants. During the course of the inflammatory response which attended the growth of the ascitic tumours the mesothelial cells covering the mesenteries and adipose tissue underwent a retraction response and exfoliated thus allowing the LAC and EL4 cells to invade these tissues. Whereas ip transplantation of LAC cells into normal MF1 mice resulted in the formation of ascites tumours, ip transplantation of LAC cells into mice which had already injected a subcutaneous transplant of LAC cells, resulted in rejection of the ip transplanted cells. Lymphocytes and macrophages participated in the rejection response and during the course of this event the macrophages became activated.

Metastasis, the process by which cancer spreads in the body, is a complex, multi-step cascade which is poorly understood and is the cause of death of most cancer patients. Aberrant glycosylation is an established characteristic of cancer cells and appears to have a role in metastatic mechanisms. The lectin from Helix pomatia, the Ron (Helix pomatia agglutinin, HPA), recognises aberrant glycans terminating in α-N-acetylgalactosamine (GalNAc). The presence of GalNAc-glycans on cancer cell surfaces, detected by HPA binding, is associated with metastasis and consequent poor survival.

Incidence of neoplasm increases with age. However age-related differences in similar tumours are not well characterised and the biological reasons for the potential differences remain unknown. These are thought to include proliferation, apoptosis, cell survival (telomere maintenance/telomerase expression), chromosomal and genomic instability This increase in expectations of histological reporting has obviously impacted on the workload of pathologists. Consequently there is a move to delegate appropriate parts of this work to suitably trained and qualified senior scientists. This study aims to assess the differences between tumours of the young (<50) compared with those of the old (>70) and use the models derived from the morphological and biological assessments to add to the prognosis of tumours in the mid age group (50 - 70).

MMR-deficiency increases the rate of mutations and often sensitizes cells to DSB-inducing agents (e. g. camptothecin and etoposide) as well as MMC (Jacob et a/., 2001 and Fiumicino et al., 2000). MMR -deficient tumour cell lines are also sensitive to the cytotoxic effects thymidine (Mohindra et al., 2002). This sensitivity is not a direct consequence of MMR -deficiency or alterations of DNA precursor metabolism. Instead, the results described in the present study suggest that MMR -deficient cells are sensitive to thymidine as a result of defects in HRR. The ScNeo recombination reporter substrate was used to determine the integrity of the HRR pathway in several MMR -proficient and -deficient tumour cell lines. Four MMR -deficient tumour cell lines were defective in the production of neo+ recombinants by homology based recombination following the transient expression of a site specific break. Furthermore, all MMR -deficient tumour cell lines tested were sensitive to the cross-linking agent MMC; an effect that is consistent with cells being deficient in HRR (including XRCC2, XRCC3 and BRCAI). To determine the alterations responsible for such HRR defects, genes known to be required for this pathway were screened for mutations in eight tumour cell lines. This revealed a heterozygous frameshift mutation within the RAD51 paralog, XRCC2, (342deIT) in SKUT-1 cells. 342delT was introduced into HRR proficient cells containing the ScNeo substrate. In SW480/SN. 3 transfectants, expression of 342delT conferred sensitivity to thymidine and MMC and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In the MRC5VA/SN. 13 transfectants, expression of 342delT was accompanied by a decreased level of the full-length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of wild-type XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA.

No description available.