Robert Schlegel

Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells.

Homocysteine is an important and independent risk factor for arteriosclerosis. We showed previously that homocysteine stimulates vascular smooth muscle cell proliferation, a hallmark of arteriosclerosis. We show here that homocysteine and serum increased DNA synthesis synergistically in both human and rat aortic smooth muscle cells (RASMCs). Treatment of quiescent RASMCs with 1 mM homocysteine or 2% calf serum for 36 h increased cyclin A mRNA levels by 8- and 14-fold, respectively, whereas homocysteine plus serum increased cyclin A mRNA levels by 40-fold, indicating a synergistic induction of cyclin A mRNA. Homocysteine did not increase the half-life of cyclin A mRNA (2.9 h), but it did increase the transcriptional rate of the cyclin A gene in nuclear run-on experiments. The positive effect of homocysteine on cyclin A gene transcription was confirmed by our finding that homocysteine increased cyclin A promoter activity and ATF-binding protein levels in RASMCs. Finally, 1 mM homocysteine increased cyclin A protein levels and cyclin A-associated kinase activity by threefold. This homocysteine-induced expression lesions by promoting proliferation of vascular smooth muscle cells.

The human papillomavirus-16 E6 oncoprotein decreases the vigilance of mitotic checkpoints

The E6 and E7 proteins of the high risk human papillomaviruses (HPVs) are consistently expressed in HPV-positive cervical carcinomas. We investigated the ability of HPV-16 E6 and E7 to disrupt mitotic checkpoints in normal diploid human cells. Acute expression of HPV-16 E6, but not HPV-16 E7, decreased the fidelity of multiple checkpoints controlling entry into and exit from mitosis. After irradiation, nearly 50% of cells containing HPV-16 E6 readily entered mitosis as opposed to less than 10% of control cells. Consistent with this, asynchronous populations of cells expressing HPV-16 E6 had increased cdc2-associated histone H1 kinase activity relative to control populations. In addition, HPV-16 E6 increased sensitivity to chemically-induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function relative to control populations. HPV-16 E6 mutants with a reduced ability to target p53 for degradation were unable to abrogate mitotic checkpoints, suggesting a possible mechanism by which HPV-16 E6 disrupts mitotic checkpoints. Expression of a mutant p53 gene yielded an intermediate phenotype relative to HPV-16 E6, generating moderate increases in sensitivity to chemically-induced S-phase PCC and mitotic spindle disruption and a heightened propensity to enter mitosis after irradiation.

Disappearance of cyclin A correlates with permanent withdrawal of cardiomyocytes from the cell cycle in human and rat hearts.

The regulated expression of cyclins controls the cell cycle. Because cardiomyocytes in adult mammals withdraw permanently from the cell cycle and thus cannot regenerate after injury, we examined cyclin expression during development by comparing cyclin A-E mRNA levels in fetal and adult human hearts. Cyclin B mRNA was detectable in adult hearts, although at a level markedly lower than that in fetal hearts. Levels of cyclin C, D1, D2, D3, and E mRNA were essentially identical in the two groups. In contrast, cyclin A mRNA was undetectable in adult hearts whereas cyclin A mRNA and protein were readily detectable in fetal hearts and cardiomyocytes, respectively. We then measured cyclin A mRNA and protein levels in rat hearts at four stages of development (fetal and 2, 14, and 28 d). Cyclin A mRNA and protein levels decreased quickly after birth (to 37% at day 2) and became undetectable within 14 d, an observation consistent with reports that cardiomyocytes stop replicating in rats by the second to third postnatal week. This disappearance of cyclin A gene expression in human and rat hearts at the time cardiomyocytes become terminally differentiated suggests that cyclin A downregulation is important in the permanent withdrawal of cardiomyocytes from the cell cycle.

Disregulation of mitotic checkpoints and regulatory proteins following acute expression of SV40 large T antigen in diploid human cells

SV40 large T antigen (T) inactivates the tumor suppressor proteins p53 and pRb, and can induce cells to enter DNA replication at inappropriate times. We show here that T also compromises three cell cycle checkpoints that regulate the entry into and exit from mitosis. Human diploid fibroblasts infected with a retrovirus expressing T displayed an attenuated radiation-induced mitotic delay, were more susceptible to chemical-induced uncoupling of mitosis from the completion of DNA replication, and were more likely to exit mitosis and rereplicate their DNA when mitotic spindle assembly was inhibited. Consistent with altered mitotic checkpoint control, cells expressing T displayed elevated protein levels and/or associated activities of the mitotic regulatory proteins cyclin A, cyclin B, Cdc25C and p34cdc2. These changes in mitotic control were evident within 5 – 10 population doublings after retroviral infection, indicating a direct effect of T expression. Cells acutely infected with the T-expressing retrovirus suffered numerical and structural chromosome aberrations, including increases in aneuploidy, dicentric chromosomes, chromatid exchanges and chromosome breaks and gaps. These findings indicate that T rapidly disrupts mitotic checkpoints that help maintain genomic stability, and suggest mechanisms by which T induces chromosome aberrations and promotes the immortalization and neoplastic transformation of human cells.

Some special structural features of intracellular bacteriophage T7 concatemers.

Newly synthesized T7 DNA, formed after infection of Escherichia coli with T7 bacteriophage, is found in long linear molecules predominantly 1, 2 and 3 times the length of the mature molecule. Denaturation of the dimers and trimers and sedimentation of the single chains in alkali reveal unit-length single chains. The fractionation of concatemers broken by shearing on benzoylated, naphthoylated DEAE-cellulose columns reveals that all three length classes adhere to this material by virtue of single-chain regions that are spaced at intervals of about 12 μm, the length of one mature molecule. Annealing experiments with purified “middles” and partially digested mature phage DNA, demonstrate that the single-chain regions are located at the genetic terminals (that is, the XYZABC region) of the monomers and dimers. Studies with an exonuclease, and an endonuclease specific for single chains, reveal that “whiskers” (free-ended single chains) and “gaps” of some sort (a single-chain region having no free ends located in the interior of a double helix) are present. Models that accommodate our findings are shown. It seems likely that these structures are intermediates in the formation of new, mature-sized T7 DNA molecules. A test of likely mutants (those unable to break down concatemeric DNA into mature-size lengths) has turned up no obvious abnormalities in these intermediates. Besides elucidating the structure of T7 intermediates, we have developed techniques which should be generally useful in the study of small single-chain regions of any DNA.

Anticarcinogenic potential of DNA-repair modulators

Effects of compounds that inhibit repair of DNA lesions in cells have been reported frequently. The consequences include altered incidence of carcinogenicity in vivo, tumorigenic transformation of cultured cells, mutations, and increased lethality as well as sister-chromatid exchanges and chromosome aberrations. This literature is reviewed here, with major emphasis on methylxanthines (caffeine in particular) and nicotinamide analogs. Existing information is also summarized on a novel potent repair inhibitor, beta-lapachone. Compounds that inhibit both DNA replication and repair are not discussed in detail since they have been reviewed often, but miscellaneous inhibitors of repair are summarized in a table. The relatively small number of experiments performed on the anticarcinogenic effects of methyl-xanthines and nicotinamide analogs gave very conflicting results. Some investigators report decreased carcinogenicity of DNA-damaging agents when caffeine was provided, but others obtained the opposite effect. The three studies with nicotinamide analogs all reported enhanced tumorigenicity of carcinogens. The data are too few to enable firm conclusions to be drawn regarding the possibility of using repair inhibitors to prevent cancer in humans. Variations of experimental conditions, carcinogens, cells, etc. have provided conflicting results. The possibility of cancer prevention is, nevertheless, so important that further investigations with DNA-repair inhibitors, particularly with human cells, seem very well justified.

Hydrodynamic shear breakage of DNA may produce single-chained terminals

Abstract High molecular weight DNA in solution is very shear labile when passed through narrow capillaries. We previously showed that under certain conditions of hydrodynamic shear, single-chained terminals are produced on the DNA fragments. By the use of chromatography on benzoylated-naphthoylated DEAE-cellulose, a single-chain specific exonuclease, and velocity sedimentation we have investigated the physical characteristics of these shear-broken fragments. As the salt concentration is lowered or the temperature of shearing is raised, the number of shear-broken fragments containing single-chained regions is increased. Essentially all single-chained regions are removed by a single-chain specific exonuclease, indicating that these regions are located at the fragment terminals. A variety of conditions of salt and temperature do not affect the limit fragment size produced by a given diameter capillary. For certain applications, the presence of single-chained terminals on DNA fragments in the 2 · 106–30 · 106 molecular weight range may be desired. The methods detailed in this communication provide means of shearing DNA to produce, or to avoid single-chained terminals.

The HPV E7 oncoprotein inhibits tumor necrosis factor α-mediated apoptosis in normal human fibroblasts

Tumor necrosis factor-α (TNF) is a cytokine that induces programmed cell death, apoptosis, in a number of cell types and is employed by cytotoxic T cells to eliminate virus infected cells. Consequently, many viruses have acquired mechanisms to undermine these host cell defense mechanisms and cause resistance to TNF-mediated apoptosis. Here we show that normal human diploid fibroblasts that express the human papillomavirus type 16 E7 oncoprotein have a decreased propensity to undergo apoptosis in response to TNF treatment. The ability of E7 to undermine TNF-mediated apoptosis correlates with cellular transformation. While E7 does not generally subvert signaling by tumor necrosis factor receptor 1, pro-caspase 8 activation is decreased in E7-expressing cells. E7 also provides some protection from apoptosis caused by stimulation of the TNF receptor 1-related cytokine receptor Fas, where induction of apoptosis occurs much slower in this cell type. Hence, E7-expressing normal human fibroblasts exhibit a specific defect that obstructs cytokine-mediated activation of pro-caspase 8 and apoptosis.

Selective isolation of newly synthesized mammalian mRNA after in vivo labeling with 4-thiouridine or 6-thioguanosine

Newly synthesized mRNA from hamster cells was labeled in vivo with the thionucleoside analogs 4-thiouridine (4-TU) and 6-thioguanosine (6-TG). The thio-substituted RNA was selectively recovered by Affi-Gel 501 phenylmercury affinity chromatography. Following a 1-h labeling period, enrichment for newly transcribed RNA after a single round of chromatography ranged between 10- and 15-fold when compared with total RNA. Exposure of CHO UrdA- cells, a uridine auxotrophic line, to 50 microM 4-TU allowed for optimal recovery of newly transcribed RNA. Increasing the concentration of 4-TU to 100 microM or labeling with 6-TG at concentrations of 3 microM or greater resulted in similar recoveries from uridine-prototrophic hamster cell lines. For shorter term labeling, exposure of prototrophic cells to 500 microM 4-TU or 100 microM 6-TG for 15 min allowed newly synthesized RNA to be selectively recovered. As a specific test case, enrichment for histone H3.2 mRNA was analyzed after hamster cells were labeled with 4-TU under conditions in which the gene was highly transcriptionally active. Northern blot analysis and the specific activity of thio-substituted RNA revealed a 15-fold enrichment when compared to total RNA. In vivo labeling of cellular RNA with 4-TU or 6-TG should provide a useful method for studying inducible gene expression and for isolating and cloning specific mRNAs from mammalian cells.

Analysis of DNA bearing single-chained terminals by BNC chromatography

Abstract Native T7 DNA was treated with λ-exonuclease or exonuclease III in order to produce single-chained terminals on predominately duplex molecules. DNA treated in this fashion binds to columns of benzoylated-naphthoylated DEAE-cellulose (BNC) in the presence of 1.0 M NaCl and is elutable by a linear gradient of 0–1.0% caffeine. The concentration of caffeine needed to elute resected molecules is proportional to the length of the single-chained terminals for single chains ranging from 200 to 1300 nucleotides in length. A gradual decrease in the caffeine concentration necessary to elute molecules with terminals longer than 1500 nucleotides is observed. The BNC column therefore provides a simple method for the fractionation of DNA molecules based on the length of exposed single chains. Shear-breakage of resected molecules reveals that DNA possessing two single-chained terminals binds more tightly to BNC than molecules with only one resected terminal. Based on these results, a comparison was made between DNA resected by one exonuclease and the other. Significant differences were obtained which suggest that these methods should be useful for determining the mode of action of various nucleases.