Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy.

BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.

Processivity of the gene 41 DNA helicase at the bacteriophage T4 DNA replication fork.

The gene 41 protein is the DNA helicase associated with the bacteriophage T4 DNA replication fork. This protein is a major component of the primosome, being essential for coordinated leading and lagging strand DNA synthesis. Models suggest that such DNA helicases are loaded only onto DNA at origins of replication, and that they remain with the ensuing replication fork until replication is terminated. To test this idea, we have measured the extent of processivity of the 41 protein in the context of an in vitro DNA replication system composed of eight purified proteins (the gene 43, 44/62, 45, 32, 41, 59, and 61 proteins). After starting DNA replication in the presence of these proteins, we diluted the 41 helicase enough to prevent any association of new helicase molecules and analyzed the replication products. We measured an association half-life of 11 min, revealing that the 41 protein is processive enough to finish replicating the entire 169-kilobase T4 genome at the observed replication rate of approximately 400 nucleotides/s. This processivity of the 41 protein does not require the 59 protein, the protein that catalyzes 41 protein assembly onto 32 protein-covered single-stranded DNA. The stability we measure for the 41 protein as part of the replication fork is greater than estimated for it alone on single-stranded DNA. We suggest that the 41 protein interacts with the polymerase holoenzyme at the fork, both stabilizing the other protein components and being stabilized thereby.

Evidence for an imino intermediate in the T4 endonuclease V reaction.

Reductive methylation and site-directed mutagenesis experiments have implicated the N-terminal alpha-amino group of T4 endonuclease V in the glycosylase and abasic lyase activities of the enzyme. NMR studies have confirmed the involvement of the N-terminal alpha-amino group in the inhibition of enzyme activity by reductive methylation. A mechanism accounting for these results predicts that a (imino) covalent enzyme-substrate intermediate is formed between the protein N-terminal alpha-amino group and C1' of the 5'-deoxyribose of the pyrimidine dimer substrate subsequent to (or concomitantly with) the glycosylase step. Experiments to verify the existence of this intermediate indicated that enzyme inhibition by cyanide was substrate-dependent, a result classically interpreted to imply an imino reaction intermediate. In addition, sodium borohydride reduction of the intermediate formed a stable dead-end enzyme-substrate product. This product was formed whether ultraviolet light-irradiated high molecular weight DNA or duplex oligonucleotides containing a defined thymine-thymine cyclobutane dimer were used as substrate. The duplex oligonucleotide substrates demonstrated a well-defined gel shift. This will facilitate high-resolution footprinting of the enzyme on the DNA substrate.

Site-directed mutagenesis of the NH2 terminus of T4 endonuclease V. The position of the alpha NH2 moiety affects catalytic activity.

Reductive methylation of the alpha NH2 moiety of the DNA repair enzyme T4 endonuclease V has been shown previously to eradicate both the N-glycosylase and apyrimidinic/apurinic lyase activities of the enzyme (Schrock, R. D., III, and Lloyd, R. S. (1991) J. Biol. Chem. 266, 17631-17639). The present study uses the technique of site-directed mutagenesis to investigate the important parameters involved in the cleavage mechanism. The prediction was that the addition of an amino acid in the immediate NH2-terminal region of the protein would alter the proximity of the alpha NH2 moiety of Thr2 to its target, thereby severely compromising the enzyme's catalytic activity. However, substitutions in this region generally should be tolerated. To test this hypothesis, three substitutions of the NH2-terminal amino acid were produced: Ser2 (T2S), Val2 (T2V), and Pro2 (T2P). An addition mutant was also produced by adding a glycine between the first and second amino acids of the protein (Thr2-Gly-Arg3) (+Gly). The T2P and +Gly mutants had negligible pyrimidine dimer-specific N-glycosylase activity as well as negligible pyrimidine dimer-specific nicking activity in vitro. Conversely, the T2S enzyme exhibited wild type levels of activity and the T2V exhibited intermediate levels of activity in vitro. Results from ultraviolet (UV) survival studies of the mutant enzymes indicated that the in vivo activities of these enzymes were directly correlated to the enzymes' ability to cleave at pyrimidine dimers in vitro. These results indicate that a critical parameter for the functionality of endonuclease V is the relative distance between the primary alpha NH2 group in the active site of the enzyme and those elements responsible for DNA binding and pyrimidine dimer recognition.

Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities. Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis.

Endonuclease V, a pyrimidine dimer-specific DNA repair enzyme, was chemically modified by reductive methylation, a technique that specifically methylates primary amino groups. Upon reaction of endonuclease V with [14C]formaldehyde (14CH2O) in the presence of the reducing agent sodium cyanoborohydride (Na-CNBH3), it was discovered that 0.8 methylation/endonuclease V molecule was required to reduce both the glycosylase and the phosphodiester lyase activities by 70-80%. Pyrimidine dimer-specific binding was not eradicated at a level of methylation equivalent to 0.8 CH3/endonuclease V molecule but was eradicated at higher levels of methylation. Endonuclease V that had been modified with an average of 1.6 CH3/molecule was digested with Staphylococcus aureus strain V8 protease and the peptides subsequently separated by reverse-phase high performance liquid chromatography. Radiolabel was found exclusively on the peptide including the amino terminus, as determined by the percent amino acid composition. Neither intact CH3-endonuclease V nor radiolabeled peptides were able to be sequenced by Edman degradation indicating blockage of the amino terminus by methylation. This study shows strong evidence for the unusual involvement of the alpha NH2 moiety in the chemical mechanisms of endonuclease V. A reaction mechanism that incorporates these findings is presented.

Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding.

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function by virtue of its ability to function in the presence of metal-chelating agents. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer.

Changing capital needs means changing capital solutions. Roundtable on capital financing.

For a full day, seven individuals representing varied backgrounds and viewpoints within the healthcare industry discussed the issues confronting capital financing for healthcare institutions--capital requirements and investment, tax reform, capital costs under PPS, and alternative methods of financing. This discussion, sponsored by the Healthcare Financial Management Association and Smith Barney, Harris Upham & Co., Inc., provided a public forum for discussion of upcoming capital financing issues and concerns. The article presented here, taken from that discussion, focuses on the upcoming capital needs of the industry. A "redeployment" of capital in the healthcare industry is going to occur because the industry is moving away from the acute care setting into long-term and ambulatory care. The mainstay of the healthcare industry--acute care--will be broken down into different components, requiring a significant redeployment of capital.