Novel exogenous heme-dependent expression of mammalian cytochrome P450 using baculovirus

Rat P450 IIA1 is a membrane-bound hemoprotein that catalyzes the 7 alpha- and 6 alpha-hydroxylation of testosterone. A recombinant baculovirus, containing the cDNA encoding IIA1, was constructed and used to infect Spodoptera frugiperda cells. Infected cells contained up to 10% IIA1 protein as quantified by Western blot analysis; however, only a small percentage of this protein contained heme and was catalytically active. Addition of hemin to the culture medium during the course of viral infection resulted in a marked sixfold increase in both testosterone hydroxylase activity and heme-containing IIA1 protein. When the exogenously added protoheme was substituted with modified heme molecules containing hydrogen or ethyl groups at the 2 and 4 positions of the protoporphyrin IX, enzymes with only marginal changes in catalytic properties were produced. These data indicate that baculovirus can be used as a system to produce high levels of mammalian cytochrome P450s containing custom modified heme moieties.     

Differences between the endogenous and exogenous DNA sequences of Rous-associated virus-O.

DNA sequences related to the endogenous retrovirus of chickens, Rous-associated virus-O (RAV-O), have been examined using site-specific DNA endonuclease analysis of cellular DNA derived from line 15 and line 100 chickens. Individual embryos from both inbred lines were used as a source of embryonic fibroblasts from which cellular DNA was isolated. Analysis of DNA containing either endogenous RAV-O sequences alone or both endogenous and exogenous RAV-O sequences produced identical patterns of RAV-O-specific DNA fragments after digestion with the endonucleases Eco RI, Hind III, BgI II, Bam HI or Xho I. Similar analysis with endonucleases Hinc II or Hha I, however, produced several RAV-O-specific DNA fragments which were derived from cellular DNA containing both endogenous and exogenous RAV-O sequences but not from cellular DNA containing only endogenous sequences. Although some differences exist between the DNA fragments specific for the endogenous viral sequences of line 15 and line 100 cellular DNA, the DNA fragments specific for the exogenous viral sequences were identical between the two inbred lines. Cleavage of an unintegrated linear RAV-O DNA molecule with Hinc II or Hha I produced DNA fragments identical to those specific for the exogenously acquired RAV-O provirus. This suggests that these characteristic fragments contain no cellular DNA. The potential DNA junction fragments containing both viral and cellular DNA, identified after analysis of DNA that contains both endogenous and exogenous viral sequences, were identical to those observed after analysis of DNA containing only endogenous viral sequences. These results support the following conclusions. First, exogenous proviral sequences are integrated into chicken cell DNA following an interaction between viral and cellular DNA that is specific with respect to the virus and nonspecific with respect to the cell. Second, both the free linear RAV-O DNA intermediate and the newly integrated exogenous provirus contain specific endonuclease sites that are not found in endogenous RAV-O DNA sequences. These results suggest that the formation of the exogenous DNA provirus involves specific alteration of the endogenous viral DNA sequences before reinsertion of the sequences as the exogenous RAV-O DNA provirus. It is possible that newly integrated exogenous RAV-O sequences are characterized by specific differences in the pattern of base methylation and a limited sequence arrangement.     

Genetic exchange between endogenous and exogenous LINE-1 repetitive elements in mouse cells.

The repetitive LINE (L1) elements of the mouse, which are present at about 10(5) copies per genome and share over 80% of sequence homology, were examined for their ability to undergo genetic exchange with exogenous L1 sequences. The exogenous L1 sequences, carried by a shuttle vector, consisted of an internal fragment from L1Md-A2, a previously described member of the L1 family of the mouse. Using an assay that does not require the reconstitution of a selectable marker we found that this vector, in either circular or linear form, acquired DNA sequences from endogenous L1 elements at a frequency of 10(-3) to 10(-4) per rescued vector. Physical analysis of the acquired L1 sequences revealed that distinct endogenous L1 elements acted as donors and that different subfamilies participated. These results demonstrate that L1 elements are readily capable of genetic exchange. Apart from gene conversion events, the acquisition of L1 sequences outside the region of homology suggested that a second mechanism was also involved in the genetic exchange. A model which accounts for this mechanism is presented and its potential implication on the rearrangement of L1 elements is discussed.     

The role of endogenous and exogenous DNA damage and mutagenesis

The field of DNA damage responsiveness in general, and the consequences of endogenous and exogenous base damage in DNA, in particular, has made new and exciting contributions to our increasing understanding of the initiation and progression of neoplasia in humans. This article presents some of the highlights in this area of investigation, with a particular emphasis on DNA repair, the tolerance of DNA damage and its contribution to mutagenesis, and DNA damage checkpoint regulation.     

Both exogenous commensal and endogenous self antigens stimulate T cell proliferation under lymphopenic conditions

Within lymphopenic recipients, naïve T cells undergo proliferation that is induced by homeostatic mechanisms. Earlier studies have demonstrated that commensal antigens play a key role in inducing the proliferation. However, a relative contribution of endogenous self antigens in this process has not been formally investigated. In this study, we utilized a pharmacologic inhibitor that blocks T cell egress from the lymphoid tissues, antibiotics, and germ-free animals to examine the role of commensal and self antigens. The results suggest that T cell proliferation under lymphopenic conditions is a heterogeneous process triggered by both exogenous commensal and endogenous self antigens.     

Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports aging-related processes.

The ongoing DNA damage caused by reactive oxygen species generated during oxidative metabolism is considered a key factor contributing to cell aging as well as preconditioning cells to neoplastic transformation. We postulated before that a significant fraction of constitutive histone H2AX phosphorylation (CHP) and constitutive activation of ATM (CAA) seen in untreated normal and tumor cells occurs in response to such DNA damage. In the present study, we provide further evidence in support of this postulate. The level of ATM activation and H2AX phosphorylation, detected immunocytochemically, has been monitored in WI-38, A549, and TK6 cells treated with H2O2 as well as growing under conditions known or suspected to affect the level of endogenous oxidants. Thirty- to 60-min exposure of cells to 100 or 200 microM H2O2 led to an increase in the level of H2AX phosphorylation and ATM activation, particularly pronounced (nearly fivefold) in S-phase cells. Cell growth for 24-48 h under hypoxic conditions (3% O2) distinctly lowered the level of CHP and CAA while it had minor effect on cell cycle progression. Treatment (4 h) with 0.1 or 0.3 mM 3-bromopyruvate, an inhibitor of glycolysis and mitochondrial oxidative phosphorylation, reduced the level of CHP (up to fourfold) and also decreased the level of CAA. Growth of WI-38 cells in 2% serum concentration for 48 h led to a 25 and 30% reduction in CHP and CHA, respectively, compared with cells growing in 10% serum. The antioxidant vitamin C (2 mM) reduced CHP and CAA by 20-30% after 24 h of treatment, while the COX-2 inhibitor celecoxib (5 microM) had a minor effect on CHP and CAA, though it decreased the level of H2O2-induced H2AX phosphorylation and ATM activation. In contrast, dichloroacetate known to shift metabolism from anaerobic to oxidative glycolysis through its effect on pyruvate dehydrogenase kinase enhanced the level of CHP and CAA. Our present data and earlier observations strongly support the postulate that a large fraction of CHP and CAA occurs in response to DNA damage caused by metabolically generated oxidants. Cytometric analysis of CHP and CAA provides the means to measure the effectiveness of exogenous factors, which either through lowering aerobic metabolism or neutralizing radicals may protect DNA from such damage.     

Enzyme activities in oleaginous yeasts accumulating and utilizing exogenous or endogenous lipids

The activities of ATP: citrate lyase (ACL; EC 4. 1. 3. 8), carnitine acetyltransferase (CAT; EC 2. 3. 1. 7), NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1. 1. 1. 42), isocitrate lyase (ICL; EC 4. 1. 3. 1) and malic enzyme (malate dehydrogenase; EC 1. 1. 1. 40) were measured in four oleaginous yeasts, Candida curvata D, Trichosporon cutaneum and two strains of Rhodosporidium toruloides, grown either to accumulate lipid, or to utilize their own lipid reserves or an exogenous supply of lipid. During lipid utilization, activities of ACL and malic enzyme diminished to low levels; CAT and ICL increased considerably in activity and ICDH activity was slightly increased but catalase (EC 1. 11. 1. 6) diminished in activity in both strains of R. toruloides. In all cases, yeasts utilizing exogenous lipid showed greater changes in enzyme activities than cells utilizing their endogenous reserves. Electron micrographs of Candida curvata D showed proliferation of peroxisomes in starved cells utilizing their own lipid reserve though peroxisomes were more in evidence when the yeast had been grown on exogenous lipid. In Lipomyces starkeyi, which shows only minimal utilization of its stored lipid and furthermore cannot grow on exogenous lipid, only the occasional peroxisome was seen when cells were starved of carbon.     

Phenobarbital and some other liver tumor promoters stimulate DNA synthesis in cultured rat hepatocytes

Hepatocytes isolated from normal adult rats were maintained at sub-confluent density in a defined medium in primary culture. In control cultures with added epidermal growth factor and low concentrations of dexamethasone, the rate of DNA synthesis was initially low but increased after about 30 hours in culture. Addition of the xenobiotic liver tumor promoters phenobarbital, alpha-hexachlorocyclohexane or p,p'-dichlorodiphenyltrichloroethane to cultures after cell attachment caused concentration-dependent stimulation of DNA synthesis measured after 2 days in culture. While dexamethasone (30 nM) alone had little effect on hepatocyte DNA synthesis, the stimulatory effects of the xenobiotics required the permissive presence of the steroid.     

RNA-directed DNA polymerase activity of reticuloendotheliosis virus: characterization of the endogenous and exogenous reactions.

Reticuloendotheliosis virus (REV) contains an endogenously instructed, RNA-directed DNA polymerase activity. Both the endogenous and exogenous DNA polymerase activities exhibited up to 10-fold greater activity at the optimum concentration of manganous ion (0.025 mM for exogenous; 0.25 mM for endogenous) than at any concentration of magnesium ion. Antiserum to the DNA polymerase of an REV group virus (spleen necrosis virus) inhibited both endogenous and exogenous DNA polymerase activity of REV, whereas antiserum to the Rous sarcoma virus (Rous-associated virus-0) [RSV(RAV-0)]DNA polymerase did not. The DNA product of the endogenous reaction is associated with the high-molecular-weight RNA of REV and anneals with REV RNA but not with RNA from Rous sarcoma virus.     

The human estrogen receptor can regulate exogenous but not endogenous vitellogenin gene promoters in a Xenopus cell line.

Transfection of a human estrogen receptor cDNA expression vector (HEO) into cultured Xenopus kidney cells confers estrogen responsiveness to the recipient cells as demonstrated by the hormone dependent expression of co-transfected Xenopus vitellogenin-CAT chimeric genes. The estrogen stimulation of these vit-CAT genes is dependent upon the presence of the vitellogenin estrogen responsive element (ERE) in their 5' flanking region. Thus, functional human estrogen receptor (hER) can be synthesized in heterologous lower vertebrate cells and can act as a trans-acting regulatory factor that is necessary, together with estradiol, for the induction of the vit-CAT constructs in these cells. In addition, vitellogenin minigenes co-transfected with the HEO expression vector also respond to hormonal stimulation. Their induction is not higher than that of the vit-CAT chimeric genes. It suggests that in the Xenopus kidney cell line B 3.2, the structural parts of the vitellogenin minigenes do not play a role in the induction process. Furthermore, no stabilizing effect of estrogen on vitellogenin mRNA is observed in these cells. In contrast to the transfected genes, the endogenous chromosomal vitellogenin genes remain silent, demonstrating that in spite of the presence of the hER and the hormone, the conditions necessary for their activation are not fulfilled.     

Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular clones.

Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.     

Involvement of mouse Rev3 in tolerance of endogenous and exogenous DNA damage

The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta that is implicated in mutagenic translesion synthesis of damaged DNA. To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3. Although some strain-dependent variation was observed, Rev3(-/-) embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities. Rev3(-/-) cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability. Histochemical analysis of Rev3(-/-) embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages. Although increased levels of p53 are detected in Rev3(-/-) embryos, the embryonic phenotype was not rescued by the absence of p53. A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3(-/-) embryos. The inner cell mass of cultured Rev3(-/-) blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene. These combined data are compatible with a model in which, in the absence of polymerase zeta, double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response. Together, these data are consistent with involvement of polymerase zeta in translesion synthesis of endogenously and exogenously induced DNA lesions.     

Transfection by exogenous and endogenous murine retrovirus DNAs.

A low molecular weight (LMW) protein phosphokinase enzyme that binds to actin has been isolated from murine sarcoma virions; this kinase activity is not present in nontransforming murine leukemia viruses. Sephadex G-75 gel filtration and affinity chromatography on actin-Sepharose conjugates allow a significant level of purification of this enzyme. The enzyme associates with microtubular proteins and inhibits the in vitro polymerization of microtubules. This study represents the first isolation of a sarcoma virus-associated protein that possesses the ability to interact directly with two major components of the cytoskeletal system.     

Effect of exogenous DNA on the content and biosynthesis of endogenous DNA in rat bone marrow]

The influence of exogenous DNA on the content of endogenous DNA and the rate of biosynthesis was studied in rat bone marrow. After the injection of highly-polymerized homologous DNA to intact rats the content of rat DNA per 1 gm of the bone marrow decreased within the first 3 days (the most marked fall occurred in 3 days--by 36%), and returned to the normal by the 6th day. The rate of DNA biosynthesis in rat bone marrow increased in 18 hours (doublled in comparison with control), remained elevated within 6 days (by 58%) and approached the normal level from 1 to 3 days after the DNA injection.