The miRNA as human cell gene activity regulator after ionizing radiation

A Novel Immunocompetent Rat Model of HCV Infection and Hepatitis

A wealth of evidence attests that the organs of developing embryos, particularly the developing brain, are acutely sensitive to chemical perturbations. However, scientists know very little about how exposures to specific endogenous chemicals actually impact human development or children’s ability to learn. And there are almost no data on how the vast majority of the 84,000 chemicals currently listed in the Toxic Substances Control Act (TSCA) Inventory1—including most of the 201 compounds known to be neurotoxic to adults and the 1,000 chemicals shown to be neurotoxic to animals2—may affect developing infants. It is also unclear whether testing with animals always provides accurate insights into human developmental susceptibility. A new line of research based on human stem cells is providing important insights into how chemicals may affect neonatal development. Stem cells are the master cells capable of producing some or all of the 200-plus different types of cells in the human body. In time, some researchers believe stem cells may enable scientists to amass far more data on how exposure to environmental chemicals affects human development, particularly the development of the brain. Now is a “critical time to be talking about stem cell research in the environmental health context,” says Tracey Woodruff, director of the Program on Reproductive Health and the Environment at the University of California, San Francisco (UCSF) Medical School.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters in various tissues. We recently reported the cloning and expression of human macrophage ACAT cDNA. In the current study, we report the production of specific polyclonal antibodies against ACAT by immunizing rabbits with the recombinant fusion protein composed of glutathione S-transferase and the first 131 amino acids of ACAT protein. Immunoblot analysis showed that the antibodies cross-reacted with a 50-kDa protein band from a variety of human cell lines. These antibodies immunodepleted more than 90% of detergent-solubilized ACAT activities from six different human cell types, demonstrating that the 50-kDa protein is the major ACAT catalytic component in these cells. In multiple human tissues examined, the antibodies recognized protein bands with various molecular weights. These antibodies also cross-reacted with the ACAT protein in Chinese hamster ovary cells. Immunoblot analysis showed that the ACAT protein contents in human fibroblast cells, HepG2 cells, or Chinese hamster ovary cells were not affected by sterol in the medium, demonstrating that the main mechanism for sterol-dependent regulation of ACAT activity in these cells is not change in ACAT protein content. As revealed by indirect immunofluorescent microscopy, the ACAT protein in tissue culture cells was located in the endoplasmic reticulum. This finding, along with earlier studies, suggests that cholesterol concentration in the endoplasmic reticulum may be the major determinant for regulating ACAT activity in the intact cells.

Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells

The tumour suppressor gene, p53, and genes coding for positive signal transduction factors can influence transit through cell-cycle checkpoints and modulate radiosensitivity. Here we examine the effects of RAF1 protein on the rate of exit from a G2/M block induced by γ-irradiation in relation to intrinsic cellular radiosensitivity in human cell lines expressing wild-type p53 (wtp53) protein as compared to mutant p53 (mutp53) protein. Cell lines which expressed mutp53 protein were all relatively radioresistant and exhibited no relationship between RAF1 protein and cellular radiosensitivity. Cell lines expressing wtp53 protein, however, showed a strong relationship between RAF1 protein levels and the radiosensitivity parameter SF2. In addition, when post-irradiation perturbation of G2/M transit was compared using the parameter T50 (time after the peak of G2/M delay at which 50% of the cells had exited from a block induced by 2 Gy of irradiation), RAF1 was related to T50 in wtp53, but not mutp53, cell lines. Cell lines which expressed wtp53 protein and high levels of RAF1 had shorter T50s and were also more radiosensitive. These results suggest a cooperative role for wtp53 and RAF1 protein in determining cellular radiosensitivity in human cells, which involves control of the G2/M checkpoint. © 2000 Cancer Research Campaign

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.

SUMMARY Human leukocyte interferon (HuLeIF) preparations were separated into populations of molecules with different sizes, by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and with different charges, by isoelectric focusing. These populations with different sizes and charges were analysed for their antiviral activity on homologous cells and on heterologous (bovine) cells. The distribution of interferon activity into two broad peaks by SDS-PAGE was similar whether assayed on human or bovine cells. However, within these peaks, the relative ratio of the activity in human cells and bovine cells varied significantly: while most of the size components had similar human/bovine cell activities, the fastest migrating component (apparent mol. wt. ∼ 13500) was more than 100 times more active on bovine cells than on human cells. The peaks of activity in isoelectric focusing were distributed from pH 5.5 to 7.0. There was generally correspondence between human and bovine cell activities, but while the more neutral pH range peaks were consistently slightly more active on human cells than on bovine cells, the more acid range peaks were always slightly more active on bovine cells than on human cells. However, with the most acidic peak, there was more than 100 times greater activity on bovine cells than on human cells. These data show that the heterogeneity of HuLeIFs is greater than merely two size populations, and data confirm that different forms of human leukocyte interferon can vary markedly in biological activity.

Expression of ras has been correlated with increased intrinsic resistance to ionizing radiation. In this study we show that increased EJras expression in human cells is associated with a decrease in the frequency of radiation-induced micronuclei. The experimental system consisted of human osteosarcoma-derived cell lines which quantitatively vary in their EJras expression. There was a dose-dependent relationship between radiation dose and micronuclei formation in all cell lines tested. Human osteosarcoma cells, in which the ras level was undetectable, had the highest frequency of micronuclei production at all radiation doses tested. At 4 Gy the most radioresistant cells exhibited a 41.5 +/- 5% decrease in the production of micronuclei concomitant with high ras expression in comparison with the relatively radiosensitive parental cell line. Cells expressing a low amount of EJras demonstrated a 23 +/- 3% decrease in micronuclei induction compared with parental cells. Treatment of cells with lovastatin, an inhibitor of ras-encoded p21ras post-translational processing via the mevalonate pathway, markedly decreased the yield of micronuclei formation in cells transfected with ras; the drug had no effect on radiation-induced micronuclei formation in parental cells. The use of the in vitro micronuclei assay has provided a convenient way to visualize differences in the genotoxic damage induced by ionizing radiation in cells which express different amount of EJras. The results indicate that elevation of ras expression in human cells can lead to a decrease in the number of radiation-induced micronuclei formed and that this relationship is dependent on membrane association of ras-encoded p21.

Germline development from human pluripotent stem cells toward disease modeling of infertility

Small interfering RNA (siRNA) duplexes induce the specific cleavage of target RNAs in mammalian cells. Their involvement in down-regulation of gene expression is termed RNA interference (RNAi). It is widely believed that RNAi predominates in the cytoplasm. We report here the co-existence of cytoplasmic and nuclear RNAi phenomena in primary human myotonic dystrophy type 1 (DM1) cells by targeting myotonic dystrophy protein kinase (DMPK) mRNAs. Heterozygote DM1 myoblasts from a human DM1 fetus produce a nuclear retained mutant DMPK transcript with large CUG repeats ( approximately 3,200) from one allele of the DMPK gene and a wild type transcript with 18 CUG repeats, thus providing for both a nuclear and cytoplasmic expression profile to be evaluated. We demonstrate here for the first time down-regulation of the endogenous nuclear retained mutant DMPK mRNAs targeted with lentivirus-delivered short hairpin RNAs (shRNAs). This nuclear RNAi(-like) phenomenon was not observed when synthetic siRNAs were delivered by cationic lipids, suggesting either a link between processing of the shRNA and nuclear import or a separate pathway for processing shRNAs in the nuclei. Our observation of simultaneous RNAi on both cytoplasmic and nuclear retained DMPK has important implications for post-transcriptional gene regulation in both compartments of mammalian cells.

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

Researchers have cultured cells that look and act like human primordial germ cells (PGCs)—and they have done it reproducibly, in abundance. Analysis of these cells, derived from both human embryonic stem cells and induced pluripotent cells, reveals some of the factors that drive PGC specification and development [1]. Researchers have previously produced mouse PGCs in a dish, but this transformation has proven more recalcitrant from human stem cell lines, which give rise to PGCs only at low frequency. Jacob Hanna and M. Azim Surani overcame this barrier, starting with a protocol they had previously developed to generate embryonic stem cells or induced pluripotent stem cells. Their protocol yields ‘‘naive’’ pluripotent cells that resemble the blastocyst inner cell mass, in contrast with previous methods, which yielded cells with a slightly more differentiated state, akin to cells of the epithelial postimplantation epiblast. The researchers could handily generate PGC-like cells by applying a molecular cocktail similar to that used to derive PGC-like cells from mouse stem cells. The resulting cells had similar RNA profiles as human germline tumor cells and primordial germ cells derived from aborted human fetuses. The cultured cells also showed signs of epigenetic changes—DNA demethylation—known to occur in PGCs. Further analysis, including gene knockout studies, identified some differences and similarities with mouse PGCs. For instance, the researchers found that the transcriptional regulator SOX17 is a key regulator of PGC-like fate in human cells—a role that it does not seem to serve in mice, where it is better known for specifying endoderm. The researchers identified some of the factors downstream of SOX17, including BLIMP1, which seems to repress somatic genes during specification of PGCs. BLIMP1 is also involved in mouse PGC fate. The findings open the door to the possibility of generating sperm and egg cells in cell culture. But for the more immediate future, this experimental system should be invaluable for answering basic questions about human development at its earliest stages.

The epidermal growth factor receptor (EGFR) is frequently overexpressed in cancer and is an important therapeutic target. Aberrant expression and function of microRNAs have been associated with tumorigenesis. Bioinformatic predictions suggest that the human EGFR mRNA 3'-untranslated region contains three microRNA-7 (miR-7) target sites, which are not conserved across mammals. We found that miR-7 down-regulates EGFR mRNA and protein expression in cancer cell lines (lung, breast, and glioblastoma) via two of the three sites, inducing cell cycle arrest and cell death. Because miR-7 was shown to decrease EGFR mRNA expression, we used microarray analysis to identify additional mRNA targets of miR-7. These included Raf1 and multiple other genes involved in EGFR signaling and tumorigenesis. Furthermore, miR-7 attenuated activation of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2, two critical effectors of EGFR signaling, in different cancer cell lines. These data establish an important role for miR-7 in controlling mRNA expression and indicate that miR-7 has the ability to coordinately regulate EGFR signaling in multiple human cancer cell types.

BIK, a pro-apoptotic BH3-only member of the BCL-2 family, targets the membrane of the endoplasmic reticulum (ER). It is induced in human cells in response to several stress stimuli, including genotoxic stress (radiation, doxorubicin) and overexpression of E1A or p53 but not by ER stress pathways resulting from protein malfolding. BIK initiates an early release of Ca2+ from ER upstream of the activation of effector caspases. Release of the mobile ER Ca2+ stores in baby mouse kidney cells doubly deficient in BAX and BAK, on the other hand, is resistant to BIK but is sensitive to ectopic BAK. Over-expression of p53 stimulates recruitment of BAK to the ER, and both its recruitment and assembly into higher order structures is inhibited by BIK small interfering RNA. Employing small interfering RNA knockdowns, we also demonstrated that release of ER Ca2+ and mitochondrial apoptosis in human epithelial cells requires BIK and that a Ca2+-regulated target, the dynamin-related GTPase DRP1, is involved in p53-induced mitochondrial fission and release of cytochrome c to the cytosol. Endogenous cellular BIK, therefore, regulates a BAX,BAK-dependent ER pathway that contributes to mitochondrial apoptosis.