Bovine leukemia virus long terminal repeat: a cell type-specific promoter.

The functional activity of the promoter unit contained within the long terminal repeat (LTR) of bovine leukemia virus (BLV) was examined by monitoring transient expression of a heterologous gene placed under its control. Various cell lines were transfected with recombinant plasmids carrying the bacterial chloramphenicol acetyltransferase (CAT) gene coupled to the BLV LTR (pBL-cat). Transient expression of CAT activity directed by the BLV LTR was observed only in the established BLV-producer cell lines derived from fetal lamb kidney (FLK) cells and bat lung cells. The amount of CAT activity transiently expressed in FLK-BLV cells was decreased approximately tenfold by deletion of LTR sequences located within a region 100 to 170 nucleotides upstream of the RNA start site. Surprisingly, removal of the region 50 base pairs downstream of the RNA initiation site to the 3'-end of the LTR reduced the expression of CAT activity by 87 percent. The BLV LTR thus appears to be an unusual promoter unit, functioning in a cell type-specific manner and possessing sequences on both the 5' and 3' sides of the RNA start site that influence gene expression.

dUTP nucleotidohydrolase isoform expression in normal and neoplastic tissues: association with survival and response to 5-fluorouracil in colorectal cancer.

Aberrant dUTP metabolism plays a significant role in the underlying molecular mechanisms of cell killing mediated by inhibitors of thymidylate biosynthesis. dUTP nucleotidohydrolase (dUTPase) is the key regulator of dUTP pools, and significant evidence exists suggesting that the expression of this enzyme may be an important determinant of cytotoxicity mediated by inhibitors of thymidylate synthase (TS). In this study, we have determined the expression patterns of dUTPase in normal and neoplastic tissues and examined the association between dUTPase expression and response to 5-fluorouracil (5-FU)-based chemotherapy and overall survival in colorectal cancer. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections using a monoclonal antibody (MAb), DUT415, that cross-reacts with both nuclear and mitochondrial isoforms of human dUTPase. Nuclear and cytoplasmic staining was observed in both normal and neoplastic tissues. In normal tissues, nuclear dUTPase staining was observed exclusively in replicating cell types. This observation is in agreement with cell culture studies where expression of the nuclear isoform (DUT-N) is proliferation dependent In contrast, cytoplasmic expression of dUTPase does not correlate with proliferation status and was observed in tissues rich in mitochondria. Consistent with this observation, cell culture studies reveal that the mitochondrial isoform (DUT-M) is expressed constitutively, independent of cell cycle status. These data suggest that in normal tissues, nuclear staining with the DUT415 antibody represents the DUT-N isoform, whereas cytoplasmic staining represents the DUT-M isoform. In colon cancer tumor specimens, expression of dUTPase was shown to be highly variable in both amount and intracellular localization. Patterns of dUTPase protein expression observed included exclusive nuclear, exclusive cytoplasmic, and combined nuclear and cytoplasmic staining. Thus, immunohistochemical detection of dUTPase in colon cancers provides distinct intracellular phenotypes of expression that may be of significant prognostic value. To examine the association between dUTPase expression and response to 5-FU-based chemotherapy and overall survival, we initiated a retrospective study including tumor specimens from 20 patients who had received protracted infusion of 5-FU and leucovorin for treatment of metastatic colon cancer. Positive nuclear staining was found in 8 patients, whereas 12 lacked nuclear expression. Of the patients lacking nuclear dUTPase expression, 6 responded to 5-FU-based chemotherapy, 4 had stable disease, and 2 had progressive disease. Of the patients presenting positive nuclear dUTPase expression, 0 responded to chemotherapy, 1 had stable disease, and 7 had progressive disease (P = 0.005). The median survival for patients with tumors lacking nuclear staining was 8.5 months and 6.9 months for patients with tumors demonstrating positive nuclear dUTPase expression (P = 0.09). Time to progression was significantly longer for patients with tumors lacking nuclear staining (P = 0.017). Variable cytoplasmic dUTPase expression was observed in these tumors; however, there was no apparent association with clinical response or survival in this limited study. Nuclear dUTPase staining within these tumors was also associated with TS gene expression (P = 0.06). This study demonstrates that low intratumoral levels of nuclear dUTPase protein expression is associated with response to 5-FU-based chemotherapy, greater time to progression, and greater overall survival in colorectal cancer. Conversely, high levels of nuclear dUTPase protein expression predict for tumor resistance to chemotherapy, shorter time to progression, and shorter overall survival. This report represents the first clinical study implicating dUTPase overexpression as a mechanism of resistance to TS inhibitor-based chemotherapy.

Isolation and characterization of a human cDNA encoding uracil-DNA glycosylase.

DNA repair of genetic information is an essential defense mechanism, which protects cells against mutation and transformation. The biochemistry of human DNA repair is in its beginning stages. Our research has concentrated on the enzymes involved in the removal of atypical bases from DNA. We present information on the identification and characterization of a cDNA isolate encoding uracil-DNA glycosylase. Uracil-DNA glycosylase was purified to homogeneity from HeLa S3 cells and used to generate polyclonal antibodies. These antibodies were in turn used to isolate a uracil-DNA glycosylase specific cDNA from a human T cell (Jurkat) lambda-gt11 library. The identity of this 1.25 kb cDNA was verified using in vitro transcription and translation systems to generate specific uracil-DNA glycosylase activity. Sequence data revealed a 327 amino acid open reading frame, which encodes a protein with a predicted molecular weight of 35351. No significant amino acid homology was found between this human uracil-DNA glycosylase and the glycosylases of yeast, Escherichia coli, herpes simplex virus, or a recently identified 26,000 Da species of human uracil-DNA glycosylase. This apparent lack of homology prompted an investigation of uracil-DNA glycosylase in a variety of eukaryotic species. Western analysis demonstrated the presence of a 36 kDa uracil-DNA glycosylase protein in human fibroblast, human placental and Vero cell extracts. Interestingly, these antibodies did not detect glycosylase protein in Chinese hamster ovary (CHO) or mouse NIH3T3 fibroblast cells. Under conditions of reduced stringency, Southern blot analysis of BamHI digested DNA from human fibroblasts, human placental cells and Vero cells revealed common 12 kb and 3 kb fragments. In contrast, using the same reduced stringency protocol, 6 and 8 kb fragments for CHO and NIH3T3 DNA were seen, respectively, as well as a common 3 kb fragment. Under more stringent wash conditions, the common 3 kb band was absent in all samples analyzed, and no hybridization signal was detected from DNA of hamster or mouse origin. The lack of immunological reactivity between the human uracil-DNA glycosylase and the rodent forms is therefore reflected at the genetic level as well. This distinction in human and CHO hybridization patterns enabled us to localize this human uracil-DNA glycosylase cDNA to chromosome 5 by somatic cell hybridization.

Mode of action of phosphonoformate as an anti-herpes simplex virus agent.

Phosphonoformate inhibited the replication of Herpes simplex virus (HSV) type 1 and type 2 in culture. The concentration required to inhibit the replication of both types of virus by 2 logs at 28 h post-infection was approximately 150 microM. It was more potent than phosphonoacetate against the growth of both virus types. A virus mutant which is resistant to phosphonoacetate was cross-resistant to phosphonoformate. Arsonoacetate, at 300 microM, had no antivirus activity. Phosphonoformate also inhibited HeLa and KB cell growth; at a concentration of about 500 microM, cell growth was inhibited by 50%. The anti-cell growth effects of the drug were completely reversible. The antivirus effect of phosphonoformate was partially reversible, depending on the time and duration of exposure of infected cultures to the drug. To obtain the maximum antivirus effect, phosphonoformate had to be added within the first 3 h post-virus-infection and be continuously present for at least 18 h. Phosphonoformate, added at 0 h post-infection, suppressed the induction of virus-specific DNA polymerase and DNAase activities. dTMP incorporation into DNA was preferentially inhibited in nuclei isolated from infected cells compared to uninfected cells, and the degree of inhibition varied with the ionic strength of the assay. Phosphonoformate was a potent inhibitor of the purified HSV-1 and HSV-2 DNA polymerases, inhibiting DNA polymerase activity by 50% at a concentration of 3 microM and ionic strength of 0.2.

The nature of enzymes involved in uracil-DNA repair: isoform characteristics of proteins responsible for nuclear and mitochondrial genomic integrity.

The absence of uracil from DNA genomes is a consequence of enzyme functions that eliminate intracellular dUTP pools and that purposefully recognize and remove uracil moieties from DNA. These enzymatic functions are dUTP nucleotidohydrolase (dUTPase) and uracil-DNA glycosylase (UDG), respectively. There are distinct nuclear and mitochondrial isoforms of each of these enzymes in human cells. The mitochondrial isoform of dUTPase (DUT-M) begins as a 31 kilodalton precursor protein containing an arginine-rich, amino-terminal presequence required for targeting to the mitochondria. This precursor is processed into a 23 kilodalton protein that resides, in mature form, in the mitochondria. The nuclear isoform of dUTPase (DUT-N) is an 18 kilodalton protein. Both species of dUTPase are nearly identical except for their amino-termini. Analysis of protein expression reveals that DUT-M is constitutive and independent of cell cycle phase or proliferation status of the cell. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication. The common sequence for both nuclear and mitochondrial isoforms includes a cyclin-dependent kinase consensus site. However, only the nuclear form appears to be phosphorylated at this site in vivo. Studies on dUTPase genomic organization reveal that both isoforms are encoded by the same gene. Isoform specific transcripts arise through the use of alternate 5' exons. Uracil-DNA glycosylase (UDG1) is but one of a growing family of enzymes that repairs potentially mutagenic events caused by uracil in DNA. Human cells contain two isoforms of UDG1 which are also nearly identical except for their amino termini. One isoform (UDG1-M), which is constitutively expressed, is targeted to the mitochondria. This form originates as a 35,000 dalton precursor and is N-terminally processed to a mature 29,000 dalton protein as it transits into the mitochondria. The other isoform is targeted to the nucleus and its expression is a function of cellular proliferation status. As with dUTPase, UDG1 isoform specific transcripts arise through the use of alternate 5prie; exons. Both of these enzymatic functions are a unique illustration, in humans, of the use of alternate exons to generate differentially expressed proteins targeted to different organelles. There are questions as to whether the nuclear isoform of UDG (UDG1-N) is also processed (at the N-terminus) to a lower molecular weight form. Polyclonal antisera generated to the unique N-terminal region of this isoform, reveals that UDG1-N exists as a 36,000 dalton protein in human cell nuclei. Since the epitope for this antibody resides in the first 24 amino acids of UDG1-N, it is apparent that the majority of this isoform is not processed and retains its amino terminus. Evidence also indicates that UDG1-N exists as a serine/threonine phosphoprotein and that phosphorylation occurs in the unique N-terminal region. This was initially deduced from the observation that nuclear UDG1-N migrates as multiple bands on SDS-PAGE and as a single band subsequent to phosphatase treatment. Cdc2 kinase is at least one of the enzymes that can phosphorylate UDG1-N. This review will summarize the current information on isoform characteristics of both dUTPase and uracil-DNA glycosylase. It will also focus on evidence for phosphorylation and speculate as to the purpose of these post-translational events.

Intraventricular injection of 6-hydroxydopamine results in an increased number of tyrosine hydroxylase immune-positive cells in the rat cortex.

Previously we have demonstrated that intraventricular injection of 6-hydroxydopamine (6-OHDA) results in increased proliferation and de-differentiation of rat cortical astrocytes into progenitor-like cells 4 days after lesion (Wachter et al., 2010). To find out if these cells express tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine synthesis pathway, we performed immunohistochemistry in the rat cortex following intraventricular injection of 6-OHDA. Four days after injection we demonstrated a strong emergence of TH-positive (TH(+)) somata in the cortices of 6-OHDA-lesioned animals. The number of TH(+) cells in the cortex of 6-OHDA-lesioned animals was 15 times higher than in sham-operated animals, where virtually no TH(+) somata occurred. Combining TH immunohistochemistry with classical Nissl stain yielded complete congruency, and ∼45% of the TH(+) cells co-expressed calretinin, which indicates an interneuron affiliation. There was no co-staining of TH with other interneuron markers or with glial markers such as glial fibrillary acidic protein (GFAP) or the neural stem/progenitor marker Nestin, nor could we find co-localization with the proliferation marker Ki67. However, we found a co-localization of TH with glial progenitor cell markers (Sox2 and S100ß) and with polysialylated-neural cell adhesion molecule (PSA-NCAM), which has been shown to be expressed in immature, but not recently generated cortical neurons. Taken together, this study seems to confirm our previous findings with respect to a 6-OHDA-induced expression of neuronal precursor markers in cells of the rat cortex, although the TH(+) cells found in this study are not identical with the potentially de-differentiated astrocytes described recently (Wachter et al., 2010). The detection of cortical cells expressing the catecholaminergic key enzyme TH might indicate a possible compensatory role of these cells in a dopamine-(DA)-depleted system. Future studies are needed to determine whether the TH(+) cells are capable of DA synthesis to confirm this hypothesis.

Inhibition of phosphorylation of cellular dUTP nucleotidohydrolase as a consequence of herpes simplex virus infection.

During an infection with herpes simplex virus, activity of cellular dUTPase decreases as a function of time, post-infection, while virus-encoded dUTPase activity increases. Prelabeling of cells with 35S-methionine and immunoprecipitation analysis, using monoclonal antibodies, indicates that cellular dUTPase protein levels remain the same (with respect to levels in uninfected cells) throughout the infection period. New synthesis of cellular dUTPase does not occur in infected cells as determined by 35S-methionine labeling during infection. Further characterization of the cellular dUTPase, in uninfected cells, reveals that the protein is post-translationally phosphorylated at serine residues. Pulse labeling of virus-infected cells with 32P-orthophosphate reveals that the phosphorylation rate of the cellular dUTPase protein decreases significantly as a function of time post-infection. In an effort to establish that phosphate turnover was occurring on the cellular dUTPase protein, cells were prelabeled with 32P-orthophosphate and then infected with HSV in the absence of label. Evidence from this experiment indicates that the phosphate moiety is removed from the cellular dUTPase protein during the infection. A series of viable virus mutants was generated by insertional inactivation of the HSV dUTPase gene. These mutants do not express viral dUTPase activity and HSV dUTPase protein is not detected by western blot analysis. However, in contrast to the wild-type situation, these mutant virus retain significant cellular dUTPase activity throughout infection. Interestingly, phosphorylation of cellular dUTPase protein is now readily detectable in each of the mutant virus-infected cells. These studies indicate that cellular dUTPase activity is diminished in wild-type HSV-infected cells by a process of dephosphorylation. It also appears that in mutant HSV, lacking the virus dUTPase, the mechanism of dephosphorylation and thus inactivation of cellular dUTPase is not functional. The end result is that the mutant virus can now rely on the cellular activity for its survival.

Identification of the coding sequence for herpes simplex virus uracil-DNA glycosylase.

We have recently isolated a herpes simplex virus (HSV) type 2 (strain 333)-specific cDNA that encodes uracil-DNA glycosylase. This cDNA lies between 0.065 and 0.08 map units on the HSV genome. Within this region there are five overlapping transcripts which encompass three open reading frames. We have determined that the second open reading frame, UL-2, codes for glycosylase. In vitro transcription of the UL-2 region and subsequent translation yielded uracil-DNA glycosylase activity. Sequence analysis of the UL-2 open reading frame indicated a coding capacity of 295 amino acids. Comparison to the HSV type 1 (strain 17) sequence indicated that there is 74% amino acid homology between the two strains, with most of the conservation occurring in the middle and the 3' end. The 5' end, however, has diverged considerably.

The herpes simplex virus type 2 UL3 open reading frame encodes a nuclear localizing phosphoprotein.

The herpes simplex virus type 2 (HSV-2, strain 333) UL3 open reading frame (ORF) codes for a protein with a predicted molecular weight of 29,681. Comparisons of the UL3, strain 333 ORF show essentially complete amino acid identity with HSV-2 (strain HG52) and a 75% amino acid identity with HSV type 1 (strain 17). To characterize the expression of this gene, a hydrophilic region of the HSV-2 UL3 gene was cloned into a bacterial expression vector. The resulting fusion protein was used to generate antibodies in rabbits. In vitro translation of HSV-2-derived mRNA followed by immunoprecipitation with the rabbit antisera reveals a major 28,000-Da protein as judged by SDS-polyacrylamide gel electrophoresis. This is consistent with the predicted molecular weight of an unmodified UL3 protein. Pulse-labeling of infected cells, with [35S]methionine, followed by immunoprecipitation and electrophoretic analysis reveals three distinct bands of 28,000, 30,500, and 33,000 Da. Labeling infected cells with [32P]orthophosphate shows that the 30,500- and the 33,000-Da species are post-translationally phosphorylated. The 30,500-Da species can be converted to the 28,000-Da species with alkaline phosphatase treatment. Interestingly, the 33,000-Da species is resistant to this treatment. Immunohistochemical analysis of infected cells reveals that the UL3 protein has a perinuclear location early in infection and at later times becomes associated with the nucleus as discrete particles. A mutant of HSV, which has a major deletion of the UL3 coding region does not show any immunohistochemical staining with the UL3 antisera. The wild-type virus-infected cell staining pattern remains the same subsequent to DNAse and RNAse treatment, indicating that the UL3 protein product is not directly associated with nucleic acid.

Cell cycle regulation of a human cyclin-like gene encoding uracil-DNA glycosylase.

The predicted amino acid sequence of a human cDNA encoding uracil-DNA glycosylase activity shows striking similarity to the cyclin protein family. To characterize the expression of this DNA repair enzyme, we have isolated the corresponding genomic clone. This gene is contained within 4.2 kilobases and is composed of only two exons. Sequence analysis of the upstream region shows that it contains two cell cycle box (CCB) regulatory elements which are also found in yeast cyclin genes. Deletional analysis of the promoter reveals the presence of a repressor region located from position -812 to -603. An inverted CCB element (alpha-CCB) and an SP1-like binding site are contained within this region. When uracil-DNA glycosylase mRNA levels are examined in vivo, a 2-3-fold increase is associated with G1 phase in both HeLa S3 and WI38 cells. To examine the role of the 209-base pair repressor region in mediating cell cycle regulation, this fragment was used in gel shift assays with cellular extracts prepared from various stages of the cell cycle. Several specific complexes are formed during S and G2 phases which are not present during M and G1 phases. Two of the complexes are the result of alpha-CCB binding as they can be specifically disrupted by the addition of an oligonucleotide containing the alpha-CCB binding site. Immunoprecipitation studies reveal that uracil-DNA glycosylase protein levels are also elevated during G1 phase. Additionally, we show that the 36-kDa uracil-DNA glycosylase protein is turned over during the course of one cell cycle. These results demonstrate coordinate regulation of uracil-DNA glycosylase at both the transcriptional and the post-transcriptional levels as a function of the cell cycle.

Isolation of a herpes simplex virus cDNA encoding the DNA repair enzyme uracil-DNA glycosylase.

Activity of the DNA repair enzyme uracil-DNA glycosylase has been shown to increase in herpes simplex virus type 2 (HSV-2)-infected cells. When mRNA derived from either HSV-1- or HSV-2-infected HeLa S3 cells was translated in an in vitro translation system, significant uracil-DNA glycosylase activity could be detected in the lysate. This activity was specific for the removal of uracil from DNA. Lysates from in vitro translation of mRNA derived from uninfected HeLa cells did not contain measurable glycosylase activity. A cDNA library was constructed with mRNA derived from HSV-2-infected cells 10 h postinfection. Pooled isolates from this library were used in hybrid-arrest and in vitro translation reactions to isolate a uracil-DNA glycosylase-specific cDNA. In vitro translation of hybrid-selected RNA, by using this cDNA, produced glycosylase activity in the lysate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled products from this translation reaction showed a protein component with a molecular weight of 39,000. This is consistent with the molecular weight determinations of the purified glycosylase enzyme derived from either uninfected or HSV-infected HeLa cells. Northern (RNA blot) analysis of HSV-derived RNA, by using the glycosylase cDNA as a probe, revealed five overlapping transcripts of 3.4, 2.8, 2.4, 1.7, and 1.0 kilobases. Southern analysis indicated that the DNA sequence encoding the HSV-specific uracil-DNA glycosylase was located between 0.065 and 0.08 map units on the prototypic arrangement of the HSV genome.

The human dUTPase gene encodes both nuclear and mitochondrial isoforms. Differential expression of the isoforms and characterization of a cDNA encoding the mitochondrial species.

We have previously identified distinct nuclear and mitochondrial isoforms of dUTPase in human cells, reporting the cDNA sequence of the nuclear isoform (DUT-N). We now report a cDNA corresponding to the mitochondrial isoform (DUT-M). The DUT-M cDNA contains an 252-amino acid open reading frame, encoding a protein with a predicted Mr of 26,704. The amino-terminal region of the protein contains an arginine-rich, 69-residue mitochondrial targeting presequence that is absent in the mature protein. In vitro transcription and translation of the DUT-M cDNA results in the production of a precursor protein with an apparent molecular mass of 31 kDa as judged by SDS-polyacrylamide gel electrophoresis. The DUT-M precursor is enzymatically active and immunoreacts with a dUTPase-specific monoclonal antibody. Mitochondrial import and processing studies demonstrate that the DUT-M precursor is processed into a 23-kDa protein and imported into mitochondria in vitro. Isoelectric focusing experiments demonstrate that the DUT-N has a pI of 6.0, while the processed form of DUT-M has a more basic pI of 8.1, measurements that are in agreement with predicted values. Studies aimed at understanding the expression of these isoforms were performed utilizing quiescent and replicating 34Lu human lung fibroblasts as a model cell culture system. Northern blot analysis, employing an isoform-specific probe, demonstrates that DUT-N and DUT-M are encoded by two distinct mRNA species of 1.1 and 1.4 kilobases, respectively. Western and Northern blot analysis reveal that DUT-M protein and mRNA are expressed in a constitutive fashion, independent of cell cycle phase or proliferation status. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication status. Because DUT-N and DUT-M have identical amino acid and cDNA sequences in their overlapping regions, we set out to determine if they were encoded by the same gene. The 5' region of the gene encoding dUTPase was isolated and characterized by a combination of Southern hybridization and DNA sequencing. These analyses demonstrate that the dUTPase isoforms are encoded by the same gene with isoform-specific transcripts arising through the use of alternative 5' exons. This finding represents the first example in humans of alternative 5' exon usage to generate differentially expressed nuclear and mitochondrial specific protein isoforms.

Specific association of cyclin-like uracil-DNA glycosylase with the proliferating cell nuclear antigen.

We have previously isolated a human gene that encodes a cyclin-like protein with uracil-removing activity (UDG2) (Muller, S.J., and Caradonna, S. 1993. J. Biol. Chem. 268, 1310-1319). The structural and regulatory similarities shared between this uracil-DNA glycosylase and cyclins suggested that it may interact with additional proteins. Using a unique affinity purification protocol (Ugi-Sepharose) and anti-UDG2 antibodies, we have identified a physical interaction between the cyclin-like uracil-DNA glycosylase and PCNA in extracts derived from HeLa cells. Conversely, we show that anti-PCNA immunoprecipitates possess significant uracil-DNA glycosylase activity. This activity is specifically blocked by the addition of uracil-DNA glycosylase inhibitor protein (Ugi) derived from bacteriophage PBS2. To further characterize this association, we performed in vitro mixing experiments using 35S-labeled PCNA and uracil-DNA glycosylase (UDG2) that were generated in a coupled transcription/translation system. We show that UDG2 and PCNA are coprecipitated using anti-PCNA antibodies and anti-UDG2 antibodies as well as Ugi-Sepharose. When PCNA is preincubated with synthetic peptides corresponding to amino acid residues 73-90 of UDG2, the PCNA-UDG2 association is prevented. By contrast, addition of synthetic peptides corresponding to amino acid residues 208-223 has no effect on this interaction. These findings suggest that the UDG2 domain encompassing amino acids 73-90 is directly involved in binding PCNA.

Purification and properties of the deoxyuridine triphosphate nucleotidohydrolase enzyme derived from HeLa S3 cells. Comparison to a distinct dUTP nucleotidohydrolase induced in herpes simplex virus-infected HeLa S3 cells.

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) (EC 3.6.1.23) derived from HeLa S3 cells has been purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of about 16,000 nmol of dUMP hydrolyzed per min/mg of protein. The dUTPase enzyme derived from HeLa S3 cells appears to be composed to two equal molecular mass subunits, each being about 22,500 daltons. Association of these subunits to produce a 45,000-dalton protein is promoted by MgCl2. In the presence of EDTA enzyme activity is abolished and the enzyme dissociates into its monomeric form. MgCl2 will completely reverse the inhibition caused by EDTA and promote subunit association. MnCl2 will also promote association of the dUTPase subunits. However, MnCl2 will not completely reverse inhibition by EDTA. In addition, purified dUTPase, extensively dialyzed to remove contaminating ions, is activated almost 2-fold by the addition of 5 mM MgCl2. In contrast, addition of 5 mM MnCl2 to the dialyzed enzyme preparation will cause more than a 50% decrease in enzyme activity. This data indicates that Mg2+ is the natural prosthetic group for this enzyme. The Km value of dUTP for the purified enzyme is 3 X 10(-6) M in the presence of MgCl2. The turnover number for this enzyme has been calculated to be 550 molecules of dUTP hydrolyzed per min under standard assay conditions. Infection of HeLa S3 cells with herpes simplex type 1 virus induces a distinct species of dUTPase. This new species of dUTPase has an isoelectric point of 8.0, compared to an isoelectric point in the range of 5.7 to 6.5 for the HeLa S3 dUTPase. Molecular weight determinations of this new species of dUTPase indicate that the native enzyme is monomeric with a molecular weight of about 35,000. The virally induced dUTPase is inhibited by EDTA and this inhibition is reversed by MgCl2. Unlike the HeLa S3 dUTPase, the virally induced enzyme does not appear to be composed of subunits.

Uracil DNA-glycosylase. Purification and properties of this enzyme isolated from blast cells of acute myelocytic leukemia patients.

The enzyme uracil DNA-glycosylase has been purified from blast cells of patients with acute myelocytic leukemia. A 1000-fold purification has been achieved and the enzyme appears highly enriched for the uracil glycosylase activity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of the purified enzyme is 30,000. Uracil DNA-glycosylase exhibits activity in the absence of any added metal and the addition of MgCl2, MnCl2, CaCl2, NaCl, or KCl causes inhibition. EDTA as well as EGTA can inhibit enzyme activity. An interesting finding is the biphasic effect of spermine. At a concentration of 25 microM, spermine will cause a 2.5-fold activation of enzyme activity, whereas at concentrations of 100 microM and higher, spermine will inhibit enzyme activity. An Arrhenius plot of glycosylase activity in the presence of 25 microM spermine shows a biphasic curve with the transition temperature being 36 degrees C. Initial velocity studies in the presence of varying concentrations of spermine indicate a change in both the apparent Km and Vmax of the enzyme. Various uracil analogs were tested to establish a structure-activity relationship for this enzyme. It appears from this data that uracil DNA-glycosylase is very specific for uracil moieties. Uracil, acting as a product inhibitor, gives a Ki value of 220 microM.

Induction of uracil-DNA glycosylase and dUTP nucleotidohydrolase activity in herpes simplex virus-infected human cells.

HeLa BU cells infected with either the type 1 or the type 2 forms of herpes simplex virus show an increase in the activities of uracil-DNA glycosylase and dUTP nucleotidohydrolase. Under optimal conditions, uracil-DNA glycosylase activity increases approximately 40-fold in HSV type 2-infected cells. In herpes simplex virus (HSV) type 1-infected cells, uracil-DNA glycosylase activity increases only 6-fold. At a KCl concentration of 100 mM, uracil-DNA glycosylase derived from HSV type 2-infected cells is activated 2-fold, while the glycosylase extracted from mock infected HeLa BU cells is inhibited almost 90% at 100 mM KCl. dUTP nucleotidohydrolase activity increases 4-fold and 3-fold, respectively, in HSV type 1- and HSV type 2-infected HeLa BU cells. Nondenaturing polyacrylamide gel electrophoresis of extracts derived from the type 1- and type 2-infected cells indicates distinct electrophoretic mobilities from the host cell enzyme. dUTP nucleotidohydrolase RF values for the mock infected cells, HSV type 1, and HSV type 2 are 0.5, 0.25, and 0.33, respectively. Serum from rabbits immunized against cells infected with herpes simplex virus type 1 or type 2 specifically neutralizes the dUTPase and uracil-DNA glycosylase activities extracted from herpes simplex virus-infected cells. This serum does not neutralize dUTPase or uracil-DNA glycosylase activity derived from mock infected cells.

DNA glycosylases.

Various DNA glycosylases exist, which initiate the first step in base-excision repair. A summary of the kinetic and physical characteristics of three classes of DNA glycosylases are presented here. The first class discussed, include glycosylases which recognize alkylated DNA. Various data from enzymes derived from both prokaryotic and eukaryotic sources is discussed. The second class deals with a glycosylase that recognizes and initiates the excision of pyrimidine dimers in DNA. To date, this enzyme has only been uncovered from two sources, Micrococcus luteus and the T4 bacteriophage of E. coli. The third class consists of the most studied of the glycosylases, the uracil-DNA glycosylase enzymes. Various characteristics are presented for the uracil-DNA glycosylases derived from various sources. Recent information from our laboratory is presented implicating that herpes simplex virus may mediate a uracil-DNA glycosylase activity in productivity infected cells.

The nuclear isoform of the highly conserved human uracil-DNA glycosylase is an Mr 36,000 phosphoprotein.

We have previously demonstrated that human cells contain multiple forms of uracil-DNA glycosylase (Caradonna, S. J., Ladner, R., Hansbury, M., Kosciuk, M., Lynch, F., and Muller, S. J. (1996) Exp. Cell Res. 222, 345-359). One of these is an Mr 29,000 processed form of the highly conserved uracil-DNA glycosylase (UDG1) located in the mitochondria. The others are located in the nucleus and migrate as a group of at least three distinct bands within the 35,000-37,000 molecular weight range. In this report, we perform a detailed characterization of the Mr 35,000-37,000 purified proteins. To accomplish this, uracil-DNA glycosylases were affinity purified from HeLa cell nuclear extracts. The proteins were separated by SDS-PAGE, and their identities were verified by renaturation and activity assays. The three protein bands were individually digested with cyanogen bromide, and the resulting peptide fragments were analyzed by direct amino acid sequencing. Peptide sequence, derived from each band, was identical and corresponded to a recently identified isoform of UDG1. This isoform (UDG1A) has a unique 44-amino acid N-terminal region and a C-terminal region that is identical to UDG1. To begin to study the signals required for nuclear targeting, the N-terminal regions of UDG1 and UDG1A were isolated and cloned into pEGFP-N2 to generate fusions with a red-shifted variant of green fluorescent protein (GFP). When these constructs were transfected into NIH3T3 cells, UDG1/pEGFP was targeted to the mitochondria, and UDG1A/pEGFP was targeted to the nucleus. Further studies, using deletion mutants, demonstrate that the nuclear localization signal resides within the first 20 amino acids of UDG1A. To investigate the possibility that the heterogeneity observed on SDS-PAGE results from post-translational modification(s), the UDG/pEGFP fusion constructs were transfected into NIH3T3 cells, and the cells were metabolically labeled with [32P]orthophosphate. Results from these experiments show that UDG1A is a phosphoprotein. Subsequent phosphoamino acid analysis revealed that UDG1A is phosphorylated on both serine and threonine residues. As a final characterization, RNase protection assays were performed to examine expression of each of these isoforms. These studies demonstrate that UDG1A is expressed in a wide variety of cell types and that message levels are elevated in transformed cells.

The effects of platelet-derived growth factor and receptor on normal and neoplastic human ovarian surface epithelium.

UNLABELLED: Several growth factors have been shown to stimulate or inhibit the growth of human ovarian surface epithelial (HOSE) cells. Platelet-derived growth factor (PDGF) is likely to be released onto the ovarian surface epithelium during follicular wound repair. We undertook the evaluation of this factor and its receptor in normal and malignant ovarian cells. OBJECTIVES: The goal of this study was to evaluate the response of HOSE cells to PDGF in vitro and identify PDGF receptors on normal and malignant ovarian epithelial cells. In addition, we wanted to examine the prognostic value of the PDGF receptors in clinical specimens. METHODS: Normal HOSE cells were cultured and growth response to PDGF assayed by 3H uptake. PDGF receptor status on HOSE cells, established ovarian carcinoma cell lines, and paraffin tissue was performed by immunohistologic techniques. Data on ovarian cancer patients relapse-free survival (RFS) were abstracted from the Lankenau Hospital Tumor Registry and RFS was plotted using the Kaplan-Meier method. RESULTS: HOSE cells increased 3H uptake in a dose-dependent manner in response to PDGF. HOSE cells stain positively for both alpha and beta receptors, as does the chemotherapy naive cell line A2780. The platinum-resistant CP30 cell line loses PDGF alpha staining. Of 21 ovarian cancer specimens, only 1 retained PDGF alpha receptors while 8 retained PDGF beta receptors. Those patients positive for PDGF receptor beta had a significantly longer relapse-free survival than PDGF beta receptor-negative patients. CONCLUSIONS: PDGF enhances the growth of HOSE cells in vitro and may play a role in ovarian cancer development. Patients whose tumors retain PDGF receptor beta staining positivity have a prolonged relapse-free survival.