Despite recent successes in cloning various animal species, the use of somatic cells as the source of donor nuclei has raised many practically relevant questions such as increased abortion rates, high birth weight and perinatal death. These anomalies may be caused by incomplete epigenetic reprogramming of donor DNA. Genome-wide demethylation occurs during early development, 'erasing' gamete-specific methylation patterns inherited from the parents. This process may be a prerequisite for the formation of pluripotent stem cells that are important for the later development. Here, we provide evidence that cloned bovine embryos may have impaired epigenetic reprogramming capabilities. We found highly aberrant methylation patterns in various genomic regions of cloned embryos. Cloned blastocysts closely resembled donor cells in their overall genomic methylation status, which was very different from that of normal blastocysts produced in vitro or in vivo. We found demethylation of the Bov-B long interspersed nuclear element sequence in normal embryos, but not in cloned embryos, in which the donor-type methylation was simply maintained during preimplantation development. There were also significant variations in the degree of methylation among individual cloned blastocysts. Our findings indicate that the developmental anomalies of cloned embryos could be due to incomplete epigenetic reprogramming of donor genomic DNA.

The Akt/protein kinase B (PKB) serine/threonine kinase is well known as an important mediator of many cell survival signaling pathways. Here, we demonstrate for the first time a major role of Akt/PKB in the cell invasion properties of the highly metastatic cell line HT1080. Using confocal microscopic analyses of live samples, we found Akt/PKB to be localized in the leading edge membrane area of migrating HT1080 cells. This localization was dependent on phosphoinositide 3-kinase and required the lipid binding ability of the phosphoinositide binding pleckstrin homology domain of Akt/PKB. We examined the possible function of Akt/PKB in HT1080 invasion. Surprisingly, Akt/PKB potently promoted HT1080 invasion, by increasing cell motility and matrix metalloproteinase-9 (MMP-9) production, in a manner highly dependent on its kinase activity and membrane-translocating ability. The increase in MMP-9 production was mediated by activation of nuclear factor-kappaB transcriptional activity by Akt/PKB. However, Akt/PKB did not affect the cell-cell or cell-matrix adhesion properties of HT1080. Our findings thus establish Akt/PKB as a major factor in the invasive abilities of cancer cells.

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Selenium, an essential biological trace element, has been shown to reduce and prevent the incidence of cancer. Our previous studies have shown that selenite is involved in the chemoprevention of cancer and induction of apoptosis of cancer cells. In this study, we demonstrate that selenite also inhibits the invasion of tumor cells. Cancer cell invasion requires coordinated processes, such as changes in cell-cell and cell-matrix adhesion, degradation of the extracellular matrix, and cell migration. We found that selenite inhibited invasion of HT1080 human fibrosarcoma cells. Adhesion of HT1080 cells to the collagen matrix was also inhibited by treatment with selenite, but cell-cell interaction and cell motility were not affected by selenite. Moreover, selenite reduced expression of matrix metalloproteinase-2 and -9 and urokinase-type plasminogen activator, which are involved in matrix degradation, but increased a tissue inhibitor of metalloproteinase-1. This inhibitory effect of selenite on the protease expressions was mediated by the suppression of transcription factors, NF-B and AP-1. However, selenate showed no remarkable effect on all the steps of cancer cell invasion.Metastasis is the major cause of death among cancer patients. The cancer cells metastasis requires several sequential steps, such as changes in cell-ECM 1 interaction, disconnection of intercellular adhesions, and separation of single cells from solid tumor tissue, degradation of ECM, locomotion of tumor cells into the extracellular matrix, invasion of lymph and blood vessels, immunologic escape in the circulatory system, adhesion to endothelial cells, extravasation from lymph and blood vessels, proliferation of cells, and induction of angiogenesis (1).Attachment of cells to ECM molecules is mediated by the integrin family of extracellular matrix receptors. Integrins are a large family of heterodimeric proteins that transduce a variety of signals from the ECM. Through integrin and matrix interactions, many of the genes, which are critical for cell migration, survival, proliferation, differentiation (2), and ECM degradation, are activated (3, 4). In the majority of metastasizing tumors, cellular interactions with the ECM, which promote adhesion and migration, are thought to be required for primary tumor invasion, migration, and metastasis.The main groups of proteolytic enzymes involved in the tumor invasion are matrix metalloproteinases (MMPs) and serine proteases. The MMPs, a family of zinc-dependent endopeptidases, are involved in tumor invasion, metastasis, and angiogenesis in cancer (5). MMPs are important enzymes for the proteolysis of extracellular matrix proteins such as collagen, proteoglycan, elastin, laminin, and fibronectin (6). MMPs are synthesized as preproenzymes, and most of them are secreted from the cells as proenzymes. Among human MMPs reported previously, MMP-2 (gelatinase A/M r 72,000 type IV collagenase) and MMP-9 (gelatinase B/M r 92,000 type IV collagenase) are thought to be key enzymes for degrading type IV collagen, which ...

A rate-limiting step of tumor cell metastasis is matrix degradation by active matrix metalloproteinases (MMPs). It is known that reactive oxygen species are involved in tumor metastasis. Sustained production of