IntroductionDesmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens.MethodsWe used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines.ResultsDSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure.ConclusionThese results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.

Integrins play a major role in cell adhesion and migration. Previous work reported that a cleaved form of integrin α6 (α6p) was detected in invasive human prostate cancer tissue, absent in normal prostate tissue and was produced by urokinase-type Plasminogen Activator (uPA) in a plasminindependent manner. Using site-directed mutagenesis we identified amino acid residues R594 and R595, located in the "stalk" region of integrin α6, as essential for cleavage. The cleavage site is located on the extracellular region of the protein between the β-barrel domain and the thigh domain. Prostate cancer cells (PC3N) were stably transfected to over express the cleavable, wild type (PC3N-α6-WT) or the non-cleavable form of integrin α6 (PC3N-α6-RR). The number of cells invading laminin 111 and laminin 332 coated filters by PC3N-α6-WT cells increased by three fold as compared to PC3N-α6-RR cells. Plasminogen Activator Inhibitor-1 (PAI-1) reduced the invasion of PC3N-α6-WT cells by approximately 42% through laminin 332 coated filters and plasmin inhibitor aprotinin had no significant effect. Linear cell migration increased production of integrin α6p in the PC3N-α6-WT cells and not in the PC3N-α6-RR cells and 32% of the PC3N-α6-WT cells migrated on laminin 111 in the linear migration assay as compared to the 5% PC3N-α6-RR cells. These data taken together suggest that the uPA mediated cell surface cleavage of the α6 integrin extracellular domain is involved in tumor cell invasion and migration on laminin.

Of the estimated 565,650 people in the U.S. who will die of cancer in 2008, almost all will have metastasis. Breast, prostate, kidney, thyroid and lung cancers metastasize to the bone. Tumor cells reside within the bone using integrin type cell adhesion receptors and elicit incapacitating bone pain and fractures. In particular, metastatic human prostate tumors express and cleave the integrin A6, a receptor for extracellular matrix components of the bone, i.e., laminin 332 and laminin 511. More than 50% of all prostate cancer patients develop severe bone pain during their remaining lifetime. One major goal is to prevent or delay cancer induced bone pain. We used a novel xenograft mouse model to directly determine if bone pain could be prevented by blocking the known cleavage of the A6 integrin adhesion receptor. Human tumor cells expressing either the wildtype or mutated A6 integrin were placed within the living bone matrix and 21 days later, integrin expression was confirmed by RT-PCR, radiographs were collected and behavioral measurements of spontaneous and evoked pain performed. All animals independent of integrin status had indistinguishable tumor burden and developed bone loss 21 days after surgery. A comparison of animals containing the wild type or mutated integrin revealed that tumor cells expressing the mutated integrin resulted in a dramatic decrease in bone loss, unicortical or bicortical fractures and a decrease in the ability of tumor cells to reach the epiphyseal plate of the bone. Further, tumor cells within the bone expressing the integrin mutation prevented cancer induced spontaneous flinching, tactile allodynia, and movement evoked pain. Preventing A6 integrin cleavage on the prostate tumor cell surface decreased the migration of tumor cells within the bone and the onset and degree of bone pain and fractures. These results suggest that strategies for blocking the cleavage of the adhesion receptors on the tumor cell surface can significantly prevent cancer induced bone pain and slow disease progression within the bone. Since integrin cleavage is mediated by Urokinase-type Plasminogen Activator (uPA), further work is warranted to test the efficacy of uPA inhibitors for prevention or delay of cancer induced bone pain.

The laminin binding integrin α6β1 plays a major role in determining the aggressive phenotype of tumor cells during metastasis. Our previous work has shown that cleavage of the α6β1 integrin to produce the structural variant α6pβ1 on tumor cell surfaces is mediated by the serine protease uPA. Cleavage of α6β1 increases tumor cell motility, invasion, and prostate cancer metastasis, and blockage of uPA inhibits α6pβ1 production. In human tumors uPA and uPAR are expressed in tumor cells and tumor associated macrophages (TAMs). TAMs localize to solid tumors and contribute to increased tumor growth and the metastatic phenotype. In our present study, we utilized a co-culture system of PC-3 prostate tumor cells and macrophages (12-O-tetradecanoylphorbol-13-acetate (TPA) differentiated human leukemia HL-60 cells) to investigate the hypothesis that macrophages stimulate the production of the pro-metastatic variant α6pβ1 on human prostate cancer cells via the uPA/uPAR axis. Our results indicate that adherent macrophages co-cultured with PC-3 cells increased PC-3 uPAR mRNA, uPAR cell-surface protein expression and α6 integrin cleavage. The stimulation does not require macrophage/tumor cell contact since macrophage conditioned medium is sufficient for increased uPAR transcription and α6 cleavage dependent PC-3 cell invasion. The increased cleavage was dependent on uPAR since production was blocked by silencing RNA targeting uPAR. These results indicate that macrophages can stimulate uPA/uPAR production in tumor cells which results in α6 integrin cleavage. These data suggest that tumor associated macrophages promote pro metastatic integrin dependent pericellular proteolysis.

Background:Climate change has emerged as one of the most devastating environmental threats. It is essential to assess the awareness regarding climate change in the general population for framing the mitigation activities.Aim:To assess the awareness regarding climate change in an urban community.Settings and Design:Urban field practice area of a medical college in the Pune city. Observational study.Materials and Methods:The cross-sectional survey was conducted in the urban adult population who had given the written consent. A pre-tested questionnaire was used for a face to face interview. Responses were evaluated.Statistical Analysis Used:Proportions, percentage.Results:Total 733 respondents above 18 years of age were included in the present survey. 672 (91.68%) respondents commented that global climate is changing. 547 (81.40%) respondents opined that human activities are contributing to climate change. 576 (85.71%) respondents commented that climate changing based on their personal experiences. Commonest source of information about climate change was television (59.78%). Poor awareness about UNFCC, Kyoto Protocol and IPCC was found. 549 (74.90%) respondents commented that deforestation contribute most significantly towards climate change. As per 530 (72.31%) respondents water related issues are due to changing climate change. According to 529 (72.17%) respondents, direct physical hazards of extreme climatic events are most important health related impact of climate change. According to 478 (65.21%) respondents, life style changes (63.3%) would be most effective in tackling climate change and for preventing further climate change.Conclusion:The urban general population is aware about changing global climate. Personal efforts are more important in mitigating climate change as per the urban general population. The awareness campaigns regarding mitigation activities are recommended.