Cellular & molecular biology research最新文献

Activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappa B), two transcription factors that respond to a wide range of signals, have been shown to be activated by H2O2 in several cell lines. Since H2O2 and related oxidants are implicated in reperfusion injury to the heart, we wished to know if NF-kappa B is present in the myocardium and if cardiac AP-1 and NF-kappa B also respond to oxidants. Rat neonatal cardiac myocytes were exposed to H2O2, and changes in c-fos and c-jun mRNAs, immunoreactive c-Fos and c-Jun proteins (components of AP-1), and immunoreactive p50 subunit of NF-kappa B were determined. Changes in nuclear activities of AP-1 and NF-kappa B were also measured by electrophoretic mobility shift assays. When myocytes were exposed to nonlethal concentrations of H2O2, c-fos and c-jun mRNAs were rapidly induced, reaching peak values at 30-60 min. The levels of c-Fos and c-Jun proteins increased in nuclei as revealed by immunostaining, and DNA binding activity of nuclear AP-1 increased. The presence of p50 subunit of NF-kappa B and its H2O2-induced shift from cytoplasm to nucleus were shown by immunostaining. H2O2-induced myocyte nuclear proteins capable of binding to a DNA probe containing the NF-kappa B element were also demonstrated. The findings suggest that altered expressions of cardiac genes regulated by AP-1 and NF-kappa B may be components of oxidant-induced injury to the heart or a part of the heart's adaptive response to oxidative stress.

Estrogen-mediated accumulation of the avian apolipoprotein (apo) II mRNA is in part due to its stabilization. To identify the biochemical activity responsible for this effect, radiolabeled, capped, and polyadenylated apoII mRNA was incubated in vitro in liver cytosolic extracts from roosters who received either estrogen (estrogen-treated extract) or the vehicle (control extract) parenterally. The mRNA was very stable in estrogen-treated extract but was rapidly degraded in control extract. The RNA was degraded predominantly by endonuclease rather than exonuclease activity. The addition of the estrogen-treated extract to the control extract prevented the degradation of the mRNA in trans. This biochemical activity was heat labile and was also destroyed by proteinase K but not by micrococcal nuclease, indicating that estrogen treatment resulted in the expression of a protein in the liver that stabilized the apoII mRNA by inhibiting its nucleolytic degradation. This mRNA stabilization factor was labile around 60 degrees C, whereas the RNase remained stable up to 80 degrees C. Studies on mRNA protein interaction showed that both control and estrogen-treated extracts contain mRNA-binding (mRNP) proteins that bind apoII mRNA. An increased binding to apoII mRNA by a subset of these proteins was observed with estrogen-treated extract as compared with the control extract. This activity, although it afforded complete protection from nucleolytic degradation to apoII and apo A1 mRNAs, appeared to provide less protection to mRNAs encoding chicken serum albumin and vitellogenin, suggesting differential stabilization of mRNAs. These studies indicate that a cytosolic mRNA-stabilization factor, providing apoII mRNA complete protection from nucleolytic degradation, is expressed in the avian liver upon estrogen treatment. This appears to be the first time that a biochemical activity responsible for hormone-mediated stabilization of mRNAs and estrogen induction of mRNA binding by specific mRNPs have been identified and partially characterized in vitro.

The presence of progenitor or stem cells in the adult liver and their potential roles in oncogenesis are unresolved issues. The study of hepatocyte progenitor cells has been limited by a lack of convenient in vivo systems allowing unequivocal cell localization and demonstration of differentiation into hepatocytes. To develop an in vivo progenitor bioassay, early (E14) fetal Fischer 344 rat hepatoblasts were transplanted into the spleen of syngeneic, weaning rats deficient in dipeptidyl peptidase IV (DPPIV) activity. The donor status of transplanted hepatoblasts was demonstrated by DPPIV expression. Localization of hepatoblasts was facilitated by the use of an ectopic site, as well as weanling recipients, which readily allowed identification of very small numbers of transplanted cells. Fetal rat hepatoblasts were demonstrated to undergo cellular differentiation along the hepatocyte lineage by acquiring glucose-6-phosphatase activity within 5 d of transplantation. A critical review of previous transplantation studies of hepatocyte progenitor cells and the role of the local microenvironment at inducing differentiation indicates that this novel bioassay should facilitate analysis of progenitor cells.

The ovine beta-lactoglobulin gene is expressed abundantly in the mammary gland. This study determines whether the ovine beta-lactoglobulin promoter is sufficient for targeting tissue-specific expression in transgenic mice. To address this, the expression profile of an ovine beta-lactoglobulin promoter driven bacterial chloramphenicol acetyltransferase reporter construct was analysed. Comparison of the expression frequency of this hybrid transgene to that of a genomic beta-lactoglobulin transgene indicates that additional sequences, downstream of the promoter, are required for position-independent expression in transgenic mice. Nevertheless, the hybrid transgene was expressed specifically in the mammary gland. Furthermore, the hybrid transgene was expressed in the appropriate temporal pattern during pregnancy and lactation. Thus, the proximal promoter of the ovine beta-lactoglobulin gene contains sufficient sequence information to target expression to the mammary. This construct constitutes the basis for a compact mammary expression vector.

The tumor suppressor protein p53 is phosphorylated at a C-terminal residue (serine 386 in mouse p53) by the protein kinase CK2. Phosphorylation by CK2 activates the specific DNA binding function of p53 and stimulates its ability to suppress cellular growth. Previous reports have suggested that phosphorylation of p53 at the CK2 site is stimulated in cells expressing the large tumor antigen (T antigen) of simian virus 40 (SV40). To test this idea, we have expressed a C-terminal p53 "mini-protein" which comprises amino acids 154-387 of mouse p53 and therefore lacks the heavily phosphorylated N-terminus. In addition, the serine 309 phosphorylation site (targeted by cyclin-dependent kinases) has been mutated to encode alanine. We have expressed the p53 mini-protein in mammalian cells and shown by phosphopeptide mapping that it is phosphorylated at a single physiological phosphorylation site, serine 386. Using this mini-protein as a cellular target for CK2, we have shown that phosphorylation of p53 by CK2 is not affected by the presence of T antigen. The p53 mini-protein is likely to be a useful tool with which to probe the regulation of p53 phosphorylation by CK2 in response to other factors which influence cell growth.

Activation of expression at the mouse mammary tumor virus (MMTV) promoter is thought to be controlled by nucleosome positioning. On stably integrated MMTV DNA, the long terminal repeat (LTR) region is organized in a phased array of nucleosomes which allegedly occludes transcription factors such as NFI from binding. NFI only binds to the promoter region when the ordered nucleosome structure is apparently disrupted by activated steroid hormone receptors in hormone induced transcription. In certain cell lines, binding sites for the transcription factors NFI and OTF1 are however required for hormone-independent expression of MMTV. We have used stably transfected mouse NIH3T3 and GR cells that exhibit detectable MMTV expression in the absence of hormone for in vivo determination of proteins binding to the MMTV promoter. Here, we present in vivo dimethyl sulfate footprinting data that show that the NFI and OTF binding sites are permanently occupied in vivo in these cells. The contacting guanine residues identified in vivo were demonstrated in in vitro methylation interference assays to correspond to binding by NFI and OTF1. These results demonstrate a novel feature of transcription factor occupancy at the MMTV LTR promoter.

We sought to determine whether the hepatocyte growth factor/scatter factor (HGF/SF)- and keratinocyte growth factor-receptor systems were expressed in normal breast cells, breast carcinoma cell lines, normal breast tissues, and breast cancer tissues. Reverse transcriptase-polymerase chain reaction and hot blotting were used to detect HGF, HGF/SF (met) receptor, KGF, and KGF receptor mRNAs in human mammary epithelial (HME) and stromal (HMS) cells. We also examined breast carcinoma (MDA-MB-157, SCC 38, and SCC 70) and spontaneously immortalized breast epithelial (HMT 3522) cell lines, as well as normal breast and breast carcinoma tissues. PCR products were also confirmed by nucleic acid sequencing. The effects of HGF and KGF, compared to EGF and heparin-binding EGF, on the proliferation of normal human mammary epithelial cells in serum-free defined medium was determined by cell counting. HGF and KGF mRNAs were detected in HMS cells, but not HME cells. KGF receptor mRNA was detected in HME cells, but not HMS cells. HGF/SF receptor mRNA was detected in both HME and HMS cells. mRNAs were also detected in normal breast and breast carcinoma tissues, as well as breast carcinoma and transformed breast epithelial cell lines. Alternative cDNA sequences that are predicted to code for a soluble KGF receptor and a membrane bound, truncated HGF/SF receptor were detected in breast epithelial cells and breast tissues. HGF and KGF maintained viability and stimulated proliferation of HME cells.

Protein kinase CKII is a prevalent serine/threonine protein kinase whose structure is highly conserved among eukaryotic organisms. Its involvement in the eukaryotic cell division cycle has been implicated by genetic experiments in yeast, antisense DNA, and inhibitory antibody experiments in mammalian cells, changes in activity during growth stimulation experiments, and protection of cells from radiation damage to replicating DNA. In addition, the cdc2 protein kinase, which is central to cell division cycle control, serves as a substrate for CKII specifically during the G1 phase of human cells. In this report, extracts of HeLa cells were prepared using neutral, aqueous buffers at low ionic strength. The cells were enriched for specific stages of the cell division cycle by treatment with drugs or by centrifugal elutriation. The results indicate that CKII activity in these extracts is highest during the G1 phase, and there appears to be a reduction in soluble CKII activity during the S phase. These data are consistent with the hypothesis that high CKII is necessary for a normal G1 phase but that progression through the S phase requires inhibition of CKII.