Pharmacokinetics of ibutilide and its enantiomers in dogs

Signal transduction pathways activated by injury play a central role in coordinating the cellular responses that determine whether a cell survives or dies. GADD153 expression increases markedly in response to some types of cellular injury and the product of this gene causes cell cycle arrest. Using induction of GADD153 as a model, we have investigated the activation of the cellular injury response after treatment with taxol and cisplatin (cDDP). Activation of the GADD153 promoter coupled to the luciferase gene and transfected into human ovarian carcinoma 2008 cells correlated well with the increase in endogenous GADD153 mRNA after treatment with taxol but not after treatment with cDDP. Following treatment with cDDP, the increase in endogenous GADD153 mRNA was 10-fold greater than the increase in GADD153 promoter activity. Likewise, at equitoxic levels of exposure (IC80), cDDP produced a 5-fold greater increase in endogenous GADD153 mRNA than taxol. The tyrosine kinase inhibitor tyrophostin B46 had no significant effect on the ability of taxol to activate the GADD153 promoter, but inhibited activation of the GADD153 promoter by cDDP in a concentration-dependent manner. Tyrphostin B46 synergistically enhanced the cytotoxicity of cisplatin; however, the same exposure had no significant effect on the cytotoxicity of taxol. We conclude that (1) taxol and cDDP activate GADD153 promoter activity through different mechanisms; (2) the signal transduction pathway mediating induction by cDDP involves a tyrosine kinase inhibitable by tyrphostin B46; and (3) that inhibition of this signal transduction pathway by tyrphostin synergistically enhances cDDP toxicity.     

Asymmetric contribution to Ig repertoire diversity by V kappa exons: differences in the utilization of V kappa 10 exons

The mouse has approximately 140 germline V kappa genes, and functional V kappa exons are expressed at roughly equivalent levels in the preimmune repertoire. We have examined the expression of individual members of the V kappa 10 family. V kappa 10A and V kappa 10B genes have been utilized in numerous hybridomas and myelomas, while V kappa 10C has not. In this study, we have cloned the V kappa 10C gene and shown that it is structurally functional, has the expected promoter elements and recombination signal sequences, and that it is capable of recombination. V kappa 10C mRNA, however, is present at levels at least 1000-fold lower than V kappa 10A and V kappa 10B in adult spleens. While there are no sequence differences in the octamer or TATA box between V kappa 10C and V kappa 10A, there are three nucleotide changes in the promoter region. These promoters equally drive the expression of a reporter gene in B cells or plasma cells, but the V kappa 10A promoter is able to drive expression in pre-B cell lines significantly better than the V kappa 10C promoter (p < 0.05). V kappa 10C rearrangements can be detected in bone marrow and splenic DNA. Therefore, the lack of V kappa 10C expression may reflect the inability of V kappa 10C-rearranged cells to undergo positive or negative selection. Our results suggest that the available Ab repertoire is shaped not only by the number of structurally functional genes, but also by the ability of assembled genes to be expressed at critical points during B cell maturation.     

Selective compartmental dominance: an explanation for a noninfectious, multifactorial etiology for acquired immune deficiency syndrome (AIDS), and a rationale for ozone therapy and other immune modulating therapies

Gold(III) complexes, isostructural and isoelectronic with platinum(II) complexes, are potentially attractive as anticancer agents. We have synthesized a group of square planar gold(III) complexes, all containing at least two gold-chloride bonds in cis-position, and tested their in vitro cytotoxicity on a panel of established human tumor cell lines. Remarkably, all these compounds showed significant cytotoxic effects. In particular, the complexes containing the salycilaldiminate ligand induced tumor cell growth inhibitory effects comparable to or even greater than cisplatin. All gold(III) complexes substantially retained their antitumor potency against two cisplatin-resistant tumor cell lines (CCRF-CEM/R leukemia and A2780/R ovarian carcinoma); only minimal cross-resistance with cisplatin was observed. When considering the mechanism of action, it is reasonable to assume that the cytotoxicity of these gold(III) complexes derives from DNA binding. Preliminary spectroscopic results are consistent with this hypothesis; indeed, circular dichroism experiments show that both the salycilaldiminate- and the pyridine-containing gold(III) complexes bind calf thymus DNA in vitro and alter reversibly its B-type solution conformation. These results, however, must be treated with caution; solution studies indicate that gold(III) compounds are poorly stable under physiological conditions, possibly implying that, when injected, only a small amount will reach, unchanged, the DNA target. The results of our investigations are discussed in the perspective of future work on the cytotoxic and antitumor properties of gold(III) compounds.     

Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells

Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage.     

Effect of Salmonella assay negative and positive carcinogens on intrachromosomal recombination in S-phase arrested yeast cells

A wide variety of carcinogens including Ames assay (Salmonella) positive as well as Salmonella negative carcinogens induce intrachromosomal recombination (DEL recombination) in Saccharomyces cerevisiae. We have shown previously that the Salmonella positive carcinogens, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS) and 4-Nitroquinoline-N-oxide (4-NQO, and the Salmonella negative carcinogens, safrole, benzene, thiourea, carbon tetrachloride, and urethane, induced DEL recombination in growing, in G1 and in G2 arrested yeast cells. Since we found interesting differences in response between dividing and arrested cells, we wanted to find out whether these differences were due to the difference between cell division versus cell cycle arrest or to any particular cell cycle phase. In the present paper we incubated cells in the presence of hydroxyurea (HU) for cell cycle arrest in S-phase and exposed them to the above carcinogens, and plated them onto selective medium to determine DEL and interchromosomal recombination (ICR) frequencies. It was surprising that carbon tetrachloride had no effect on DEL recombination or ICR in HU treated cells even though it induced DEL recombination in G1 and G2 arrested as well as dividing cells. Further experiments are in agreement with the interpretation that carbon tetrachloride was responsible for prematurely pushing G1 cells into S-phase. The consequence of this may be replication on a damaged template which may be responsible for the action of carbon tetrachloride. EMS, MMS, 4-NQO and urethane were more recombinagenic in HU treated cells than in previous experiments with G2 arrested cells. None of the carcinogens appeared to be activated by S9 for either DEL recombination or ICR induction. Furthermore, we only detect cytochrome P-450 in dividing but not in arrested cells, arguing that possible differences in the ability to metabolize the compounds does not explain the observed differences for DEL recombination induction in the different cell cycle phases. We discuss these data in terms of the mechanism of induced DEL recombination and the possible biological activities of these carcinogens.     

Abrogation of p53 function affects gadd gene responses to DNA base-damaging agents and starvation

The tumor suppressor p53 is required for induction of its downstream effector genes such as GADD45 and CIP1/WAF1 by ionizing radiation (IR). This response is probably mediated through defined p53 binding sites located in the promoter of CIP1/WAF1 and in the third intron of GADD45. In contrast, the gadd gene stress response to base-damaging agents, such as methylmethane sulfonate (MMS) or UV radiation, or medium depletion (starvation) occurs in all mammalian cells examined to date regardless of p53 status for both GADD45 and also GADD153, which is not IR-responsive in many lines with functional p53. These agents strongly induce the p53 protein and raise the possibility that, although p53 is not required for the typical "gadd" response to these agents, p53 may contribute to these non-IR stress responses. This possibility was confirmed by the finding that disruption of p53 function by transfection with dominant-negative vectors expressing HPV E6, mutant p53, or SV40 T Ag reduced the induction of GADD45 and GADD153 as measured by increases in mRNA and protein levels in human lines with wild-type p53. Similarly, induction of these genes by MMS or UV radiation was consistently stronger in the parental mouse embryo fibroblasts compared to cells derived from mice where both p53 alleles had been deleted. Similar qualitative responses were also seen for CIP1/WAF1. In agreement with reduced induction of p53-regulated genes, the G1 checkpoint activated by MMS or UV radiation was markedly abrogated in p53-wt human MCF-7 breast carcinoma cells by E6 expression. Interestingly, induction of reporter constructs driven by the GADD45 or GADD153 promoters was substantially reduced in human cells transfected with mutant p53 or E6 expression vectors or in cells lacking p53 following treatment with MMS, UV radiation, or starvation. Because neither promoter is inducible by IR, and neither contains a strong p53 binding site, these results indicate that p53 has a synergistic or cooperative role in these non-IR stress responses for both GADD45 and GADD153, and that this role is not mediated through identifiable p53-binding sites.     

Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen

GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.     

Effects of quercetin on drug metabolizing enzymes and oxidation of 2',7-dichlorofluorescin in HepG2 cells

1. The effects of quercetin on drug metabolising enzymes and oxygen radicals were studied in human HepG2 cells. 2. Cytotoxicity of quercetin in HepG2 cells was seen at 50 microM and above as evaluated by lactate dehydrogenase (LDH) leakage, neutral red (NR) uptake, and 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. 3. Quercetin inhibited activity of human cytochrome P-450 towards ethoxycoumarin and ethylresorufin at relatively low substrate concentrations (0.1 microM and above). 4. In comparison to induction by the positive control (beta-naphthoflavone; 1.0 microM), quercetin did not significantly induce the metabolism of ethoxycoumarin or glutathione-S-transferase (GST) protein or activity. 5. Response elements for human CYP1A1, GST lambda a, xenobiotic response element (XRE), fos, HSP70, CRE, p53, NF kappa B and DNA damage (GADD) in HepG2 cells were not activated by quercetin. 6. Quercetin exhibited antioxidant activity in HepG2 cells as evidenced by its ability to inhibit the oxidation of the fluorochrome dichlorofluorescin. 7. The results indicate a range of potential beneficial effects of quercetin with respect to the influence on carcinogen-metabolising enzymes, scavenging of reactive oxygen species and a lack of stress response in HepG2 cells.     

Induced p53 expression in lung cancer cell line promotes cell senescence and differentially modifies the cytotoxicity of anti-cancer drugs

The p53-null human lung cancer cell line H1299 was used in order to generate clones with ecdysone-inducible p53 as well as ecdysone-inducible p21waf1. Induced expression of p53 resulted in irreversible cell growth arrest with characteristics of replicative senescence, suggesting that p53 can prevent immortalization by activating a senescence program. The effect of induced p53 and p21waf1 expression on the cytotoxic action of the anti-cancer drugs etoposide and cisplatin was also analysed. Whereas p21waf1 overexpression conferred increased resistance to killing by either drug, p53 overexpression enhanced the cytotoxic effect of cisplatin but protected against etoposide cytotoxicity. These results imply that the impact of p53 on susceptibility to chemotherapy may depend greatly on the particular drug and type of DNA damage. Moreover, these data demonstrate the importance of using isogenic cell lines to address this issue.     

Induction of BCL2 family member MCL1 as an early response to DNA damage

When ML-1 human myeloid leukemia cells are exposed to DNA damaging agents, they exhibit dramatic changes in the expression of a variety of gene products. This includes an increase in p53 (wild-type), a decrease in BCL2, a p53-dependent increase in the BCL2 family member BAX, and increases in Growth Arrest and DNA Damage-inducible (GADD) genes such as GADD45; these changes occur as early events in a sequence that culminates in DNA damage-induced apoptosis. DNA damaging agents have now been tested for effects on expression of another BCL2 family member, MCL1, a gene expressed during ML-1 cell differentiation. Expression of MCL1 was found to increase upon exposure of ML-1 cells to various types of DNA damaging agents, including ionizing radiation, ultraviolet radiation, and alkylating drugs. The increase in MCL1 occurred rapidly and was transient, levels of the MCL1 mRNA being elevated within 4 h and having returned to near baseline within 24 h. An increase in the Mcl1 protein was also seen, with the maximal increase occurring at an intermediate dose of IR (5 Gray) and lesser increases occurring at either lower or higher doses. The increase in expression of MCL1 was further studied using a panel of human cell lines that includes cells containing or not containing alterations in p53 as well as cells sensitive or insensitive to the apoptosis-inducing effects of DNA damage. The DNA damage-induced increase in MCL1 mRNA did not depend upon p53 as it was seen in cells lacking functional p53. However, the increase did depend upon susceptibility to apoptosis as it was not seen in cells insensitive to apoptosis-induction by DNA damaging agents. These findings demonstrate that cytotoxic DNA damage causes an increase in the expression of MCL1 along with increases in GADD45 and BAX and a decrease in BCL2. Furthermore, while the increase in GADD45 is seen both in cells that undergo growth arrest and in cells that undergo apoptosis in response to DNA damage, alterations in the profile of expression of BCL2 family members occur exclusively in cells that undergo the apoptotic response, with some family members increasing through p53-dependent (BAX) and others through p53-independent (MCL1) pathways. Overall, expression MCL1 can increase during the induction of cell death as well as during the induction of differentiation.     

Chromosome location of genes encoding human blood groups

In this study the transactivation potential and DNA binding activities of p53 protein were examined following exposure of A2780 cells, a human ovarian carcinoma cell line, to the DNA damaging agent, cis-diamminedichloroplatinum II (cisplatin). The endogenous murine double minute-2 gene (mdm-2) was used to monitor the ability of p53 to transactivate genes. Northern analysis showed an induction of mdm-2 mRNA upon cisplatin treatment. It was further demonstrated, using an RNase protection assay, that the p53-responsive, mdm-2 promoter (P2) was activated in cisplatin-treated A2780 cells. However, when p53 protein DNA binding activity was analyzed, there was no detectable increase in p53 sequence-specific DNA binding activity during the period of time following DNA damage when mdm-2 mRNA was induced. Instead the increase in p53 protein observed in nuclear, cytoplasmic, and whole cell extracts correlated with a latent conformation of p53 that lacked sequence-specific DNA binding activity. At low doses of cisplatin, these latent pools of p53 increased in parallel with mdm-2 gene activation and were detectable as early as 4 h following cisplatin treatment. In vitro attempts to convert the latent p53 into an active, sequence-specific DNA binding conformation were unsuccessful. Even though cisplatin-induced p53 lacked sequence-specific DNA binding activity, it does possess an increased affinity for cisplatin-damaged duplex DNA molecules. This represents the first identification where cisplatin treatment induces a p53 protein, lacking sequence-specific DNA binding but with an increased affinity for platinated DNA molecules.