Subgaleal hemorrhage in a neonate with factor X deficiency following a non-traumatic cesarean section.

This case report describes a term infant born by a non-traumatic, non-instrumented cesarean section that presented with respiratory failure and severe metabolic acidosis secondary to subgaleal hemorrhage (SGH). Further evaluation revealed a functional factor X deficiency that was initially treated with fresh frozen plasma infusions. This report is significant for the occurrence of a SGH in a non-traumatic delivery and emphasizes the importance of obtaining a coagulopathy evaluation in patients with similar presentations. In addition, this case suggests that the mechanism of injury that causes SGH may occur more frequently than previously thought, but does not become clinically significant in patients without an underlying coagulopathy.

Binding of etoposide to topoisomerase II in the absence of DNA: decreased affinity as a mechanism of drug resistance.

Despite the prevalence of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, interactions between these agents and topoisomerase II are not well understood. Therefore, to further define interactions between anticancer drugs and the type II enzyme, a nitrocellulose filter assay was used to characterize the binding of etoposide to yeast topoisomerase II. Results indicate that etoposide binds to the enzyme in the absence of DNA. The apparent Kd value for the interaction was approximately 5 microM drug. Etoposide also bound to ytop2H1012Y, a mutant yeast type II enzyme that is approximately 3-4-fold resistant to etoposide. However, the apparent Kd value for the drug (approximately 16 microM) was approximately 3 times higher than that determined for wild-type topoisomerase II. Although it has been widely speculated that resistance to topoisomerase II-targeted anticancer agents results from a decreased drug-enzyme binding affinity, these data provide the first direct evidence in support of this hypothesis. Finally, the ability of yeast topoisomerase II to bind etoposide was dependent on the presence of the hydroxyl moiety of Tyr783, suggesting specific interactions between etoposide and the active site residue that is involved in DNA scission.

The response of eukaryotic topoisomerases to DNA damage.

Beyond the known mutagenic properties of DNA lesions, recent evidence indicates that several forms of genomic damage dramatically influence the catalytic activities of DNA topoisomerases. Apurinic sites, apyrimidinic sites, base mismatches, and ultraviolet photoproducts all enhance topoisomerase I-mediated DNA cleavage when they are located in close proximity to the point of scission. Furthermore, when located between the points of scission of a topoisomerase II cleavage site, these same lesions (with the exception of ultraviolet photoproducts) greatly stimulate the cleavage activity of the type II enzyme. Thus, as found for anticancer drugs, lesions have the capacity to convert topoisomerases from essential cellular enzymes to potent DNA toxins. These findings raise exciting new questions regarding the mechanism of anticancer drugs, the physiological functions of topoisomerases, and the processing of DNA damage in the cell.

Topoisomerase II-mediated DNA cleavage and religation in the absence of base pairing. Abasic lesions as a tool to dissect enzyme mechanism.

The interaction of topoisomerase II with its DNA cleavage site is critical to the physiological functions of the enzyme. Despite this importance, the specific enzyme-DNA interactions that drive topoisomerase II-mediated DNA cleavage and religation are poorly understood. Therefore, to dissect interactions between the enzyme and its cleavage site, abasic DNA lesions were incorporated into a bilaterally symmetrical and identical cleavage site. Results indicate that topoisomerase II has unique interactions with each position of the 4-base overhang generated by enzyme-mediated DNA cleavage. Lesions located 2 bases 3' to the point of scission stimulated cleavage the most, whereas those 3 bases from the point of scission stimulated cleavage the least. Moreover, an additive and in some cases synergistic cleavage enhancement was observed in oligonucleotides that contained multiple DNA lesions, with levels reaching >60-fold higher than the wild-type substrate. Finally, topoisomerase II efficiently cleaved and religated a DNA substrate in which apyrimidinic sites were simultaneously incorporated at every position on one strand of the 4-base overhang. Therefore, unlike classical DNA ligases in which base pairing is the driving force behind closure of the DNA break, it appears that for topoisomerase II, the enzyme is responsible for the spatial orientation of the DNA termini for ligation.

Spontaneous DNA lesions poison human topoisomerase IIalpha and stimulate cleavage proximal to leukemic 11q23 chromosomal breakpoints.

Topoisomerase II-targeted drugs, such as etoposide, "poison" this enzyme and kill cells by increasing levels of covalent topoisomerase II-cleaved DNA complexes. In spite of the success of this drug in the treatment of human cancers, a significant proportion of patients treated with etoposide eventually develop secondary leukemias that are characterized by translocations at chromosome band 11q23. Since similar translocations are associated with primary leukemias in previously untreated infants, we questioned whether they could also be triggered by the actions of "endogenous topoisomerase II poisons". Recent studies, which demonstrated that several forms of spontaneous DNA damage stimulate cleavage mediated by Drosophila topoisomerase II, suggest that DNA lesions may act as these endogenous poisons. Therefore, to determine whether the ability to recognize spontaneous DNA damage has been conserved from this lower eukaryote to mammalian species, the effects of apurinic sites, apyrimidinic sites, and deaminated cytosine residues on human topoisomerase IIalpha were assessed. All three lesions were potent poisons of the human enzyme and stimulated cleavage when located within the four-base overhang generated by enzyme-mediated DNA scission. Furthermore, these lesions increased levels of cleavage at five sites proximal to 11q23 translocation breakpoints and did so with an efficacy that was comparable to or greater than that of therapeutic concentrations of etoposide. Although the physiological relevance of these findings has yet to be established, they suggest a potential role for endogenous topoisomerase II poisons in the initiation of leukemic chromosomal breakpoints.

Spontaneous DNA damage stimulates topoisomerase II-mediated DNA cleavage.

Apurinic sites are position-specific poisons of topoisomerase II and stimulate DNA scission approximately 10-18-fold when they are located within the 4-base overhang generated by enzyme-mediated cleavage (Kingma, P. S., and Osheroff, N. (1997) J. Biol. Chem. 272, 1148-1155). To determine whether other major forms of spontaneous DNA damage also act as topoisomerase II poisons, the effects of position-specific apyrimidinic sites and deaminated cytosines (i.e. uracil:guanine mismatches) on the type II enzyme were determined. Both of these lesions stimulated topoisomerase II-mediated DNA scission with the same positional specificity as apurinic sites but were less efficacious. Moreover, apurinic sites dominated the effects of apyrimidinic sites in substrates that contained multiple lesions. The differential ability of spontaneous lesions to enhance DNA cleavage did not correlate with either a decreased stability of the double helix or the size of the gap formed by base loss. Rather, it appears to be due (at least in part) to increased rates of religation for substrates containing apyrimidinic sites or deaminated cytosines. These results suggest that several forms of spontaneous DNA damage are capable of acting as endogenous poisons of topoisomerase II.

Apurinic sites are position-specific topoisomerase II poisons.

Many anticancer drugs "poison" topoisomerase II by enhancing its double-stranded DNA cleavage activity. To determine whether DNA lesions act as endogenous topoisomerase II poisons, we characterized the effects of position-specific apurinic sites on enzyme activity. Lesions located within the 4-base overhang generated by enzyme-mediated DNA scission stimulated cleavage approximately 10-18-fold without altering the specificity of topoisomerase II. DNA breaks were double-stranded in nature, protein-linked, and readily reversible. In contrast, apurinic sites located immediately outside the cleavage overhang were inhibitory. Thus, apurinic sites, which are the most commonly formed lesion in DNA, are position-specific topoisomerase II poisons. A model is proposed that encompasses the actions of endogenous and exogenous topoisomerase II poisons and provides a pre-existing pathway for the cellular actions of topoisomerase II-targeted anticancer drugs.

Topoisomerase II.etoposide interactions direct the formation of drug-induced enzyme-DNA cleavage complexes.

Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. Cmax) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. Km) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme.drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide. topoisomerase II (rather than etoposide.DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with Cmax values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.

Abasic sites stimulate double-stranded DNA cleavage mediated by topoisomerase II. DNA lesions as endogenous topoisomerase II poisons.

Several clinically relevant anticancer drugs induce genomic mutations and cell death by increasing topoisomerase II-mediated DNA breakage. To determine whether endogenous DNA damage also affects this cleavage event, the effects of abasic sites (the most commonly formed spontaneous DNA lesion) on topoisomerase II activity were investigated. The presence of 3 abasic sites/plasmid stimulated enzyme-mediated DNA breakage > 6-fold, primarily by enhancing the forward rate of cleavage. This corresponds to a potency that is > 2000-fold higher than that of the anticancer drug, etoposide. These findings suggest that abasic sites represent endogenous topoisomerase II poisons and imply that anticancer drugs mimic the cleavage-enhancing actions of naturally occurring DNA lesions.