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Background: The 3q26.2 chromosomal loci is highly amplified in large set of breast cancers, primarily in aggressive basal tumors are difficult to treat in the clinic. Our amplicon data suggests that 3q26.2 is a large structurally complex amplicon and multiple components in the amplicon contribute to tumor initiation and progression either alone or through cooperative activity. Detailed mapping of the 3q26.2 amplicon by us demonstrated a microRNA, miR-569 is highly amplified as a part of 3q26.2 amplicon in breast cancer patient's samples. The role of microRNAs amplified at 3q26 loci is not well studied and their molecular functions and targets were not well known. Thus our studies provide novel mechanism underlying miR569 oncogenesis. Methods: Following the Institutional Review Board approval, tissues obtained from MD Anderson Cancer Center tumor bank were used to exatract DNA and RNA. Human miR-569 was cloned into pEZX-MR06 lentiviral vector used for the production of amphotropic viruses to infect the target cells. The miRIDIAN microRNA mimics were used to overexpress miRs and the antimiRs were used to knock down the miRNAs. All the transfections were performed using Oligofectamine. Trypsinized cells were grown in 5% matrigel for the 3D morphogenesis of epithelial cells. Results: Our results demonstrated a strong correlation between 3q26.2 amplification and expression of miR569 in patient samples of breast cancer. We subsequently demonstrated that overexpression and knockdown of miR569 in the breast epithelial cells altered their growth, proliferation, and lumen filling in 3-dimensional cultures grown in Matrigel. Importantly ectopic expression of miR569 in breast epithelial cells promoted tumor growth and increased metastatic potential in mouse xenograft models. Seed match based analysis of the microRNA targets, in silico studies and in vitro experiments showed that miR-569 directly target Tumor protein 53-induced nuclear protein1 (TP53INP1) and inhibited the expression of a tumor suppressor gene TP53INP1 expression. Loss of TP53INP1 expression mediated by miR569 altered normal cell growth cycle and subsequently promoted the survival and growth of tumor cells. Our in vitro results showed that knockdown of miR-569 and subsequent increase in TP53INP1 expression enhanced the sensitivity of cancer cell lines to cisplatin. Our immunohistochemical analysis showed that TP53INP1 protein levels were higher in normal tissues compared to cancer tissues. Further, reduced expression of TP53INP1 was observed in invasive cancers as compared to low malignant potential tumors, and decreased TP53INP1 protein levels were associated with worsened outcomes in breast cancer patients. Disscussion: TP53INP1 had previously been identified as a combined target of p53 and p73; however our studies indicate that miR-569 regulates TP53INP1 levels independently of p53 and p73 expression. Based on our preclinical results of antimiR-569 on cell survival, tumor growth and cisplatin sensitivity, inhibiting miR-569 activity or increasing TP53INP1 expression may be valid therapeutic approaches to treat breast cancer. [ABSTRACT FROM AUTHOR]

This article discusses various aspects of pastoralism in the Latin American countries with the largest dryland areas. The topics covered include: social, economic and institutional issues; grasslands and their carrying capacity; production systems and productivity rates; competition for forage resources between domestic livestock and wildlife; and the health status of livestock and wildlife. Most grasslands exhibit some degree of degradation. The percentage of offspring reaching weaning age is low: 47-66% of calves and 40-80% of lambs. Some pastoralists adopt patterns of transhumance. In the main, pastoralists experience a high poverty rate and have poor access to social services. For many pastoralists, wildlife is a source of food and by-products. Argentina, Chile, Mexico and Peru have animal health control agencies, are members of the World Organisation for Animal Health (OIE) and have signed the United Nations Convention to Combat Desertification. Pastoral systems subsist mainly on income unrelated to pastoral farming. The OIE recognises all four countries as free from infection with peste des petits ruminants virus, and from rinderpest and African horse sickness. It is difficult to predict the future of pastoralism in Latin America because the situation differs from country to country. For instance, pastoralism is more important in Peru than in Argentina, where it is a more marginal activity. In the future, lack of promotion and protection policies could lead to a decline in pastoralism or to an adverse environmental impact on drylands.

Primary human keratinocytes with ectopic expression of high-risk human papillomavirus (HPV) E6 and E7 oncoproteins display abnormal centrosome numbers, multipolar mitoses, and aneusomy. However, it has not been explored whether these abnormalities can occur in cells containing HPV episomes where E6 and E7 expression is under viral transcriptional control. Here, we demonstrate that centrosome abnormalities and genomic instability occur in organotypic raft cultures of human keratinocytes with episomal HPV-16 even at low copy numbers. We conclude that HPV-16 DNA, when maintained as an episome, can disturb centrosome homeostasis and subvert genomic integrity of the host cell during early stages of the viral infection.

The transcription factor E2F-1 induces both cell-cycle progression and, in certain settings, apoptosis. E2F-1 uses both p53-dependent and p53-independent pathways to kill cells. The p53-dependent pathway involves the induction by E2F-1 of the human tumour-suppressor protein p14ARF, which neutralizes HDM2 (human homologue of MDM2) and thereby stabilizes the p53 protein. Here we show that E2F-1 induces the transcription of the p53 homologue p73. Disruption of p73 function inhibited E2F-1-induced apoptosis in p53-defective tumour cells and in p53-/- mouse embryo fibroblasts. We conclude that activation of p73 provides a means for E2F-1 to induce death in the absence of p53.

The production of the human papillomavirus type 16 (HPV-16) is intimately tied to the differentiation of the host epithelium that it infects. Infection occurs in the basal layer of the epithelium at a site of wounding, where the virus utilizes the host DNA replication machinery to establish itself as a low-copy-number episome. The productive stage of the HPV-16 life cycle occurs in the postmitotic suprabasal layers of the epithelium, where the virus amplifies its DNA to high copy number, synthesizes the capsid proteins (L1 and L2), encapsidates the HPV-16 genome, and releases virion particles as the upper layer of the epithelium is shed. Papillomaviruses are hypothesized to possess a mechanism to overcome the block in DNA synthesis that occurs in the differentiated epithelial cells, and the HPV-16 E7 oncoprotein has been suggested to play a role in this process. To determine whether E7 plays a role in the HPV-16 life cycle, an E7-deficient HPV-16 genome was created by inserting a translational termination linker (TTL) in the E7 gene of the full HPV-16 genome. This DNA was transfected into an immortalized human foreskin keratinocyte cell line shown previously to support the HPV-16 life cycle, and stable cell lines were obtained that harbored the E7-deficient HPV-16 genome episomally, the state of the genome found in normal infections. By culturing these cells under conditions which promote the differentiation of epithelial cells, we found E7 to be necessary for the productive stage of the HPV-16 life cycle. HPV-16 lacking E7 failed to amplify its DNA and expressed reduced amounts of the capsid protein L1, which is required for virus production. E7 appears to create a favorable environment for HPV-16 DNA synthesis by perturbing the keratinocyte differentiation program and inducing the host DNA replication machinery. These data demonstrate that E7 plays an essential role in the papillomavirus life cycle.

The study of human papillomaviruses (HPVs) in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Additionally, the study of genetic mutations in the HPV genome and its effects on the viral life cycle are difficult using the current method of transfecting molecularly cloned HPV genomes into early-passage human foreskin keratinocytes (HFKs) because of the limited life span of these cells. Unless the HPV genome transfected into the early-passage HFK extends the life span of the cell, analysis of stable transfectants becomes difficult. In this study, we have used BC-1-Ep/SL cells, an immortalized human foreskin keratinocyte cell line, to recreate the HPV-16 life cycle. This cell line exhibits many characteristics of the early-passage HFKs including the ability to stratify and terminally differentiate in an organotypic raft culture system. Because of their similarity to early-passage HFKs, these cells were tested for their ability to support the HPV-16 life cycle. The BC-1-Ep/SL cells could stably maintain two HPV genotypes, HPV-16 and HPV-31b, episomally. Additionally, when the BC-1-Ep/SL cell line was stably transfected with HPV-16 and cultured using the organotypic raft culture system (rafts), it sustained the HPV-16 life cycle. Evidence for the productive stage of the HPV-16 life cycle was provided by: DNA in situ hybridization demonstrating HPV-16 DNA amplification in the suprabasal layers of the rafts, immunohistochemical staining for L1 showing the presence of capsid protein in the suprabasal layers of the rafts, and electron microscopy indicating the presence of virus like particles (VLPs) in nuclei from cells in the differentiated layers of the rafts., (Copyright 1999 Academic Press.)