Protection against TNF-induced liver parenchymal cell apoptosis during endotoxemia by a novel caspase inhibitor in mice.

Excessive apoptotic cell death is implicated in a growing number of acute and chronic disease states. Caspases are critical for the intracellular signaling pathway leading to apoptosis. The aim of this investigation was to evaluate the efficacy and the mechanism of action of the novel caspase inhibitor CV1013 in a well-characterized model of TNF-induced apoptosis. Administration of 700 mg/kg galactosamine/100 microg/kg endotoxin (Gal/ET) induced hepatocellular apoptosis in C3Heb/FeJ mice as indicated by increased caspase-3 activity (706% above controls) and enhanced DNA fragmentation (3400% above controls) at 6 h. In addition, apoptosis was aggravated by the neutrophil-induced injury at 7 h (ALT activities: 4220 +/- 960 U/L and 48 +/- 4% necrosis). All animals died 8-12 h after Gal/ET treatment from shock and liver failure. A dose of 10 or 1 mg/kg of CV1013 administered three times (3, 4.5, and 5.5 h after Gal/ET) effectively prevented caspase-3 activation and parenchymal cell apoptosis at 6 h as well as the subsequent neutrophil-induced aggravation of the injury at 7 h after Gal/ET treatment. Animals treated with 10 mg/kg CV1013 survived for 24 h without liver injury. CV1013 reduced the processing of caspase-3 and caspase-8. This suggests that CV1013 may have inhibited the small amount of active caspase-8 generated at the receptor level. Because of the multiple amplification loops used to activate the entire caspase cascade, blocking the initial intracellular signal by CV1013 was highly effective in preventing apoptotic cell death. CV1013 has therapeutic potential for disease states with excessive apoptosis.

Triple-strand-forming methylphosphonate oligodeoxynucleotides targeted to mRNA efficiently block protein synthesis.

Antisense oligonucleotides are ordinarily targeted to mRNA by double-stranded (Watson-Crick) base recognition but are seldom targeted by triple-stranded recognition. We report that certain all-purine methylphosphonate oligodeoxyribonucleotides (MPOs) form stable triple-stranded complexes with complementary (all-pyrimidine) RNA targets. Modified chloramphenicol acetyltransferase mRNA targets were prepared with complementary all-pyrimidine inserts (18-20 bp) located immediately 3' of the initiation codon. These modified chloramphenicol acetyltransferase mRNAs were used together with internal control (nontarget) mRNAs in a cell-free translation-arrest assay. Our data show that triple-strand-forming MPOs specifically inhibit protein synthesis in a concentration-dependent manner (> 90% at 1 microM). In addition, these MPOs specifically block reverse transcription in the region of their complementary polypyrimidine target sites.

DNA replication in vitro by recombinant DNA-polymerase-alpha-primase.

DNA-polymerase-alpha--primase complex contains four subunits, p180, p68, p58, and p48, and comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits of DNA-polymerase-alpha--primase from human and mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. We have over-expressed the single polypeptides and co-expressed various subunit complexes using baculovirus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase activity. Purification of co-expressed Mp48 and Mp58 subunits yielded stable DNA primase of high specific activity. Co-expression of all four subunits yielded large quantities of tetrameric DNA-polymerase-alpha--primase. The p180, p58 and p48 polypeptides were also co-expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric and trimeric DNA-polymerase-alpha--primase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA-polymerase-alpha--primase synthesized double-stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.

The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.

DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV40 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and expressed it as a fusion protein in bacteria. The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusion protein promotes formation of a complex containing T antigen and the catalytic subunit (subunit A) of DNA polymerase alpha, suggesting that it serves to tether the two proteins. These physical interactions are functionally significant, since the ability of T antigen to stimulate the activity of the catalytic subunit of DNA polymerase alpha is highly dependent upon the B subunit. We suggest that the interactions mediated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating the subsequent priming and synthesis of DNA chains on the lagging strand template. The protein may play similar roles in cellular DNA replication.

Overproduction and functional analysis of DNA primase subunits from yeast and mouse.

Eukaryotic DNA primases are composed of two distinct subunits of 48-50 and 58-60 kDa. The amino acid sequences derived from the nucleotide sequences of the cloned genes are known only for the yeast and mouse polypeptides, and the extensive homology between the corresponding mouse and yeast subunits suggests conservation of functional domains. We were able to express in Saccharomyces cerevisiae the homologous and mouse primase-encoding genes under the control of both the constitutive ADH1 and the inducible GAL1 strong promoters, thus obtaining strains producing relevant amounts of the different polypeptides. In vivo complementation studies showed that neither one of the wild-type mouse primase-encoding genes was able to rescue the lethal or temperature-sensitive phenotype caused by mutations in the yeast PRI1 or PRI2 genes, indicating that these proteins, even if structurally and functionally very similar, might be involved in critical species-specific interactions during DNA replication.

Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen.

The retinoblastoma susceptibility gene (RB) encodes a phosphoprotein of 110 kd (pp110RB) that forms specific complexes with SV40 T antigen and the transforming proteins of several other DNA tumor viruses. Interaction with RB is thought to contribute to transformation by these viruses as demonstrated by genetic analyses. To help understand the function of these interactions, the regions of RB that are involved in binding to T have been mapped. An in vitro protein synthesis system capable of producing full-length RB protein has been developed to facilitate the mapping study. A 5- to 10-fold increase in translational efficiency in the reticulocyte lysate was obtained when the 5' non-coding region of RB mRNA was replaced with that of beta-globin mRNA or a plant viral RNA, alfalfa mosaic virus (AMV) RNA4. A series of mutated RB polypeptides produced from this system were assayed for T binding. Two non-contiguous regions of the RB protein, amino acid residues 394-571 and 649-773, were found to be necessary for binding to T: mutations in either region abolished T-RB complex formation. These results are consistent with the finding that, in all the cases analyzed so far, mutated RB proteins in human tumor cells also failed to bind to T antigen due to deletions including at least one of the two required regions. Thus the regions of RB defined in vitro as necessary for interaction with T might be physiologically relevant as well, and might play a fundamental role in normal RB protein function.

C-terminal truncation of the retinoblastoma gene product leads to functional inactivation.

Mutational inactivation of the retinoblastoma (RB) gene has been implicated in the genesis of retinoblastoma, osteosarcoma, and other human tumors. Our strategy has been to characterize naturally occurring mutants from tumor cells to pinpoint potential domains of RB protein crucial for tumor suppression. We show here that osteosarcoma cell line Saos-2 contains an abnormal endogenous RB protein of 95 kDa (p95) that is located mainly in the cytoplasm. This protein was identified by antibodies recognizing several different RB epitopes, but not by one directed solely against the C terminus, suggesting C-terminal truncation. This conclusion was supported by analysis of mRNA and genomic DNA, which revealed that a transcriptionally active RB allele had a deletion of exons 21-27. In contrast to normal RB protein, this truncated protein was not phosphorylated and did not bind to the large tumor (T) antigen encoded by simian virus 40. We previously reported that introduction of normal RB protein into Saos-2 cells suppressed their neoplastic phenotype, indicating functional inactivation of their endogenous RB genes. These results provide an initial step to elucidate domains crucial to the cancer-suppression function of RB protein; its C-terminal portion is evidently important for this activity.

Peptide production from proteins separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis.

The development of amino acid sequencers with subnanomolar sensitivities has increased the need for both selective and highly efficient methods for both protein and peptide isolation. In this paper, we describe a simple procedure that utilizes the high resolving capacity of polyacrylamide gel electrophoresis to isolate a single target polypeptide, which can subsequently be subjected to proteolytic digestion and sequencing. Polypeptides are visualized in polyacrylamide gels as dodecyl sulfate/protein complexes, which are passively diffused from gel slices. Free dodecyl sulfate eluted with the protein solution is removed by KCl precipitation, allowing protein digestion with small amounts of trypsin or other proteolytic enzymes. Following enzymatic digestion, the peptide solution is made 6 M guanidine-HCl to remove interfering contaminants and thereby improve resolution of the digest by reverse-phase high-performance liquid chromatography. The peptides generated by this method are suitable for amino acid sequencing with good overall yields, averaging 15-30% on a gas-phase sequenator. The method described is useful for obtaining multiple peptide sequences from a single polypeptide isolated from a complex protein mixture.

Sequence and structural requirements for primase initiation in the SV40 origin of replication.

Primase synthesizes decaribonucleotides for priming of lagging and possibly leading strand synthesis at a replication fork. The sites of initiation by purified mouse primase were shown to be highly specific within the SV40 origin of replication. This study further examines the role of the 27-bp inverted repeat in the origin for initiation. A site is observed on the L-strand template at nucleotide position (np) 22 positioned a similar distance from the 27-bp inverted repeat as sites previously reported on the E-strand. The initiations adjacent to the 27-bp repeat have a higher Km for rATP than other sites. A deletion within the inverted repeat eliminated initiation at sites proximal to the hairpin on both E and L strands but had no effect at more distant sites. A deletion mutant which left the inverted repeat intact but deleted the initiation sites at np 5210-5220 on the E-strand was not active as a template for proximal sites. These results indicate that primase has two modes of recognition, one that requires the SV40 inverted repeat structure and a specific sequence and another that requires sequence alone. Additional regions of the SV40 genome have also been examined and of approximately 2000 nucleotides of single stranded template examined, only one additional site was observed at np 2412 on the E-strand. This indicates that primase initiations are highly specific for the SV40 origin and their potential functional role is discussed.

DNA polymerase alpha activity is not affected by protein kinases or alkaline phosphatase.

Recent studies with crude or partially purified cell extracts have suggested that DNA polymerase alpha activity may be regulated by enzymatic phosphorylation. To further investigate these findings, we have examined the effects of protein kinases and phosphatases on highly purified DNA polymerase alpha from mouse cells. Incubation of DNA polymerase alpha with a variety of protein kinases, including protein kinase C, had no effect on polymerase activity. In addition, treatment of the polymerase with soluble calf intestinal alkaline phosphatase had no effect on DNA polymerase alpha activity, further indicating that phosphorylation does not have a direct role in modulating polymerase activity. In contrast, incubation of DNA polymerase alpha with calf intestinal alkaline phosphatase crosslinked to agarose beads resulted in a time dependent disappearance of polymerase activity. This loss of DNA polymerase activity was dependent on phosphatase activity, as the alkaline phosphatase inhibitors, potassium phosphate or levamisole, prevented the loss of polymerase activity in the presence of the beaded phosphatase. The loss of DNA polymerase alpha activity following beaded phosphatase treatment was not a general phenomena as the large fragment of Escherichia coli DNA polymerase I, T4 DNA polymerase or mouse primase were not affected by similar treatment. The decreased DNA polymerase activity following incubation with phosphatase beads correlated with the binding of the DNA polymerase polypeptides, p185 and p68, to the agarose beads and this binding could not be reversed by either 150 mM potassium chloride or sodium sulfate. The binding of the polymerase to the agarose beads was dependent on the phosphatase activity, as the polymerase could be first treated with soluble calf intestinal phosphatase and subsequently bound to added Sepharose 4B beads. Surprisingly, Sepharose CL4B, a highly desulfated agarose preparation, did not bind the phosphatase-treated polymerase suggesting that sulfated polysaccharides are required for polymerase binding. The physiological correlate of this binding is unknown, but it has been reported that sulfated polysaccharides exist in a variety of intracellular compartments. It would be interesting to speculate that phosphorylation controls the intracellular compartmentalization of DNA polymerase alpha.

Mouse primase p49 subunit molecular cloning indicates conserved and divergent regions.

Primase is a specialized RNA polymerase that synthesizes RNA primers for initiation of DNA synthesis. A full cDNA clone of the p49 subunit of mouse primase, a heterodimeric enzyme, has been isolated using a primase p49-specific polyclonal antibody to screen a lambda gt11 mouse cDNA expression library. The cDNA indicated the subunit is a 417-amino acid polypeptide with a calculated molecular mass of 49,295 daltons. The p49 mRNA is approximately 1500 nucleotides long with a 5'-untranslated region of 74 nucleotides and a 3'-untranslated region of 200 nucleotides. Comparison with a similar sized primase subunit from yeast showed highly conserved amino acid sequences in the N-terminal halves of the polypeptides and included a potential metal-binding domain suggesting the functional importance of this region for DNA binding. In contrast, the 3' portion of the cDNA has rapidly diverged in nucleotide sequence, as primase mRNA can be detected in mouse and rat cells with a 3' probe (including coding and noncoding) but not in RNA from hamster or human cells. A full-length cDNA probe detected mRNA from hamster and human cell lines, indicating a conserved 5' portion and divergent 3' region of the expressed gene. The rapid divergence may be related to the species-specific protein interactions found for the DNA polymerase alpha-primase complex. The mRNA is detected in proliferating but not in quiescent cells consistent with its function in DNA replication.

Isolation of the DNA polymerase alpha core enzyme from mouse cells.

DNA polymerase alpha has been purified from mouse hybridoma cells approximately 30,000-fold using a combination of conventional and high performance liquid chromatography. In contrast to previous characterizations of mammalian DNA polymerase alpha, this enzyme has a single high molecular mass polypeptide (185 kDa) in tight association with a 68-kDa polypeptide and this structure appears to be the core DNA polymerase of the mouse cells. The biochemically purified enzyme, with a specific activity of approximately 200,000 units/mg protein, has an estimated molecular mass by gel filtration chromatography of 240 kDa and sedimentation value of 9 S, consistent with the enzyme being a heterodimer of 185 and 68 kDa. The enzyme is sensitive to both N-ethylmaleimide and aphidicolin and insensitive to ddTTP. Using an activated DNA template, the apparent Km values for the deoxynucleotide triphosphates are approximately 0.5-1 microM. The purified DNA polymerase has neither exonuclease nor primase activities and is the predominant DNA polymerase alpha activity in the mouse cells.

Mouse primase initiation sites in the origin region of simian virus 40.

The sites of initiation of DNA synthesis by purified mouse DNA primase in the origin-of-replication region of simian virus 40 (SV40) were examined. Using as template the separated strands of a cloned fragment of SV40 approximately equal to 300 base pairs (bp) long that includes the origin, we observed specific sites of initiation on the two strands. On the early strand that is the template for early mRNA synthesis, the primary starts are at four positions within 10 nucleotides of each other around nucleotide 5215 and an additional site around nucleotide 5147 that is used at one-sixth the frequency of the major sites. The major start sites on the early strand are within the 65-bp minimal origin of replication and lie between tumor antigen binding sites I and II. On the late strand that is the template for late mRNA synthesis, six major initiation sites were observed, each within the 3' C-C-C-G-C-C 5' sequence in the template that is repeated twice within each of the three 21-bp repeats that lie adjacent to the minimal origin, on its late side. A 6-bp deletion in the 65-bp minimal origin that eliminates its function as an origin reduced the major initiations around nucleotide 5215 on the early strand by 90% but did not affect initiations at the minor start site on the early strand or initiations on the late strand. Mouse DNA primase is able to recognize specific regions on the SV40 DNA. Those on the early strand are within the minimal origin of replication and those on the late strand are within the 21-bp repeat region necessary for maximum replication.

Mechanism for exclusion of 5-fluorouracil from DNA.

5-Fluorodeoxyuridine 5'-triphosphate is hydrolyzed by the enzyme deoxyuridine triphosphate diphosphohydrolase (EC 3.6.1.23). Uracil-DNA glycosylase removes 5-fluorouracil (FUra) from FUra-containing DNA similar to its removal of uracil from uracil-containing DNA. The absence of FUra in DNA following exposure of cells to FUra can be explained by the activities of these two enzymes.