Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains

Advances in membrane cell biology are hampered by the relatively high proportion of proteins with no known function. Such proteins are largely or entirely devoid of structurally significant domain annotations. Structural bioinformaticians have developed profile-profile tools such as HHsearch (online version called HHpred), which can detect remote homologies that are missed by tools used to annotate databases. Here we have applied HHsearch to study a single structural fold in a single model organism as proof of principle. In the entire clan of protein domains sharing the pleckstrin homology domain fold in yeast, systematic application of HHsearch accurately identified known PH-like domains. It also predicted 16 new domains in 13 yeast proteins many of which are implicated in intracellular traffic. One of these was Vps13p, where we confirmed the functional importance of the predicted PH-like domain. Even though such predictions require considerable work to be corroborated, they are useful first steps. HHsearch should be applied more widely, particularly across entire proteomes of model organisms, to significantly improve database annotations

Chance mechanisms affecting the burden of metastases

BACKGROUND: The burden of cancer metastases within an individual is commonly used to clinically characterize a tumor's biological behavior. Assessments like these implicitly assume that spurious effects can be discounted. Here the influence of chance on the burden of metastasis is studied to determine whether or not this assumption is valid. METHODS: Monte Carlo simulations were performed to estimate tumor burdens sustained by individuals with cancer, based upon empirically derived and validated models for the number and size distributions of metastases. Factors related to the intrinsic metastatic potential of tumors and their host microenvironments were kept constant, to more clearly demonstrate the contribution from chance. RESULTS: Under otherwise identical conditions, both the simulated numbers and the sizes of metastases were highly variable. Comparable individuals could sustain anywhere from no metastases to scores of metastases, and the sizes of the metastases ranged from microscopic to macroscopic. Despite the marked variability in the number and sizes of the metastases, their respective growth times were rather more narrowly distributed. In such situations multiple occult metastases could develop into fully overt lesions within a comparatively short time period. CONCLUSION: Chance can have a major effect on the burden of metastases. Random variability can be so great as to make individual assessments of tumor biology unreliable, yet constrained enough to lead to the apparently simultaneous appearance of multiple overt metastases

Expression of a protease-resistant insulin-like growth factor-binding protein-4 inhibits tumour growth in a murine model of breast cancer

BACKGROUND: Insulin-like growth factor 1 (IGF1) promotes breast cancer and disease progression. Bioavailability of IGF1 is modulated by IGF-binding proteins (IGFBPs). IGFBP4 inhibits IGF1 activity but cleavage by pregnancy-associated plasma protein-A (PAPP-A) protease releases active IGF1. METHODS: Expression of IGF pathway components and PAPP-A was assessed by western blot or RT-PCR. IGFBP4 (dBP4) resistant to PAPP-A cleavage, but retaining IGF-binding capacity, was used to block IGF activity in vivo. 4T1.2 mouse mammary adenocarcinoma cells transfected with empty vector, vector expressing wild-type IGFBP4 or vector expressing dBP4 were implanted in the mammary fat pad of BALB/c mice and tumour growth was assessed. Tumour angiogenesis and endothelial cell apoptosis were assessed by immunohistochemistry. RESULTS: 4T1.2 cells expressed the IGF1R receptor and IGFBP4. PAPP-A was expressed within mammary tumours but not by 4T1.2 cells. Proliferation and vascular endothelial growth factor (VEGF) production by 4T1.2 cells was increased by IGF1(E3R) (recombinant IGF1 resistant to binding by IGFBPs) but not by wild-type IGF1. IGF1-stimulated microvascular endothelial cell proliferation was blocked by recombinant IGFBP4. 4T1.2 tumours expressing dBP4 grew significantly more slowly than controls or tumours expressing wild-type IGFBP4. Inhibition of tumour growth by dBP4 was accompanied by the increased endothelial cell apoptosis. CONCLUSION: Protease-resistant IGFBP4 blocks IGF activity, tumour growth and angiogenesis

Reliability of MR Enterography Features for Describing Fibrostenosing Crohn Disease

BACKGROUND: Clinical decision making and drug development for fibrostenosing Crohn disease is constrained by a lack of imaging definitions, scoring conventions, and validated end points. PURPOSE: To assess the reliability of MR enterography features to describe Crohn disease strictures and determine correlation with stricture severity

Knockdown of aberrantly expressed nuclear localized decorin attenuates tumour angiogenesis related mediators in oral cancer progression model in vitro

AbstractBackgroundOral cancer accounts for roughly 3% of cancer cases in the world with about 350,000 newly reported cases annually and a 5-year survival rate of only 50%. Majority of oral cancers are squamous cell carcinomas that originate in the oral mucosal epithelial linings. We have previously shown that in human malignant squamous cells carcinoma (SCC-25) as well as in dysplastic oral keratinocytes (DOK), a small leucine-rich multifunctional proteoglycan decorin is aberrantly expressed and localized in the nucleus where it interacts with nuclear epidermal growth factor receptor (EGFR). Post-transcriptional silencing of nuclear decorin significantly reduced IL-8 and IL8-dependent migration and invasion in these dysplastic and malignant oral epithelia. The objective of this study was to further examine the effects of nuclear decorin silencing on angiogenesis and angiogenesis related mediators in this oral cancer progression cell line model.MethodsWe have used multiplex PCR, western blotting, and in vitro endothelial tube formation assay to study angiogenesis and related pathways in nuclear decorin silenced (stable knockdown) DOK and SCC-25 cells.ResultsNuclear decorin knockdown resulted in significant down regulation of IL-8 expression, however IL-10, and TGF-β expression was not affected in either DOK or SCC25 cells as measured by multiplex RT PCR. IL-8 receptor CXCR 1 and 2 expression was slightly lower in nuclear decorin silenced cells indicating a contributing mechanism in previously shown reduced IL-8 mediated migration and invasion phenotype in these cells. IL-8 is known to induce Matrix metalloproteinase 9 (MMP9) which not only plays a role in tumour migration and invasion but also induces angiogenic switch. We found MMP9 to be significantly reduced in nuclear decorin silenced dysplastic and malignant oral epithelia. Other potent angiogenic mediators, VEGF189 and ANG-1 were either significantly reduced or completely abrogated in these cells. Angiogenesis as measured by endothelial tube-like formations of HUVEC cells was reduced by almost 50 percent when HUVECs were incubated in the presence of conditioned medium form nuclear decorin silenced dysplastic and malignant cell lines as compared to respective controls.ConclusionsTogether these results indicate that aberrantly expressed nuclear localized decorin strongly influences angiogenic potential of dysplastic and malignant oral epithelial cells.Peer Reviewe

Molecular biology of breast cancer metastasis: Clinical implications of experimental studies on metastatic inefficiency

Recent technological advances have led to an increasing ability to detect isolated tumour cells and groups of tumour cells in patients' blood, lymph nodes or bone marrow. However, the clinical significance of these cells is unclear. Should they be considered as evidence of metastasis, necessitating aggressive treatment, or are they in some cases unrelated to clinical outcome? Quantitative experimental studies on the basic biology of metastatic inefficiency are providing clues that may help in understanding the significance of these cells. This understanding will be of use in guiding clinical studies to assess the significance of isolated tumour cells and micrometastases in cancer patients

Molecular biology of breast cancer metastasis: Genetic regulation of human breast carcinoma metastasis

The present is an overview of recent data that describes the genetic underpinnings of the suppression of cancer metastasis. Despite the explosion of new information about the genetics of cancer, only six human genes have thus far been shown to suppress metastasis functionally. Not all have been shown to be functional in breast carcinoma. Several additional genes inhibit various steps of the metastatic cascade, but do not necessarily block metastasis when tested using in vivo assays. The implications of this are discussed. Two recently discovered metastasis suppressor genes block proliferation of tumor cells at a secondary site, offering a new target for therapeutic intervention

Cyclo-oxygenase inhibition reduces tumour growth and metastasis in an orthotopic model of breast cancer

The effect of selective and non-selective cyclo-oxygenase inhibition on tumour growth and metastasis in an orthotopic model of breast cancer was investigated. 4T1 mammary adenocarcinoma cells were injected into the mammary fat pad of female BALB/c mice. When tumours reached a mean tumour diameter of 8.4±0.4 mm, mice were randomised into three groups (n=6 per group) and received daily intraperitoneal injections of the selective cyclo-oxygenase-2 inhibitor, SC-236, the non selective cyclo-oxygenase inhibitor, Indomethacin, or drug vehicle. Tumour diameter was recorded on alternate days. From 8 days after initiation of treatment, tumour diameter in animals treated with either SC-236 or indomethacin was significantly reduced relative to controls. Both primary tumour weight and the number of lung metastases were significantly reduced in the SC-236 and indomethacin treated mice. Microvessel density was reduced and tumor cell apoptosis increased in the primary tumour of mice treated with either the selective or non-selective cyclo-oxygenase inhibitor. In vitro, cyclo-oxygenase inhibition decreased vascular endothelial growth factor production and increased apoptosis of tumour cells. Our results suggest that cyclo-oxygenase inhibitors will be of value in the treatment of both primary and metastatic breast cancer

Transfection of interleukin-8 increases angiogenesis and tumorigenesis of human gastric carcinoma cells in nude mice

The growth and spread of tumour cells depends on adequate vasculature. We have previously reported that the expression of interleukin-8 (IL-8) directly correlates with the vascularity of human gastric carcinomas. To provide evidence for a causal role of IL-8 in angiogenesis and tumorigenicity of human gastric cancer, we used the lipofectin method to stably transfect the human TMK-1 gastric carcinoma cells (low endogenous IL-8) with an IL-8 expression vector or control vector. Transfection with IL-8 did not affect the proliferation of cultured cells, yet the culture supernatants of the transfected (but not control) cells stimulated proliferation of human umbilical vein endothelial cells. The IL-8-transfected and control cells were injected into the gastric wall of nude mice. IL-8-transfected cells produced rapidly growing, highly vascular neoplasms as compared to control cells. These results provide direct evidence for the role of IL-8 in the angiogenesis and tumorigenicity of human gastric carcinomas. © 1999 Cancer Research Campaig