Replication-dependent and -independent camptothecin cytotoxicity of seven human colon tumor cell lines.

Anticancer inhibitors of topoisomerase I (TOP1, EC 5.99.1.2) cause the reversible stabilization of the TOP1-DNA covalent complex (cleavable complex). The cleavable complex can be converted into a double-strand break, the presumed cytotoxic lesion, by active replication forks. Cytotoxicity independent of DNA replication has also been demonstrated, and suggested to have possible clinical significance. To assess the importance of the replication-independent mechanism of camptothecin (CPT) cytotoxicity we have analyzed replication-dependent and replication-independent cytotoxicity following a brief CPT treatment (40 min) of seven human colon tumor cell lines. The cell lines were exposed to CPT in the presence or absence of aphidicolin, an inhibitor of DNA polymerases alpha, delta or epsilon. The seven cell lines responded similarly to CPT: treatments of less than 0.5 microM caused cytotoxicity only when DNA replication was ongoing, as evidenced by a plateau in the cytotoxicity curve corresponding to the S-phase fraction and the prevention of this cytotoxicity by aphidicolin cotreatment; at higher CPT doses, the cytotoxicity exceeded the S-phase fraction and was not prevented by aphidicolin. The CPT sensitivity among the cell lines, measured as the concentration required to inhibit cell growth by 25%, was between 0.17 and 0.43 microM without aphidicolin and 2-10 microM with aphidicolin cotreatment; with aphidicolin in cotreatment, 20-fold greater CPT concentrations were required, on average among the cell lines, to achieve cytotoxicity equivalent to CPT treatment alone. The potential of the lower dose and longer duration treatments of camptothecins used in the clinical setting to produce cytotoxicity independent of DNA replication is discussed.

The CHO XRCC1 mutant, EM9, deficient in DNA ligase III activity, exhibits hypersensitivity to camptothecin independent of DNA replication.

We have analyzed the X-ray-sensitive CHO mutant cell line EM9 for sensitivity to the topoisomerase I inhibitor comptothecin. These cells exhibit defective repair of single strand DNA breaks. Recently, EM9 were complemented the DNA ligase III interactive protein, XRCC1. Defective XRCC1 apparently accounts for the low DNA ligase III activity that may explain the single-strand break repair deficiency of EM9 cells. Here, we demonstrate cytotoxic hypersensitivity of EM9 cells following a brief camptothecin treatment. Both the S-phase and non-S-phase populations of EM9 exhibited camptothecin sensitivity relative to the parent cell line AA8. In AA8 cells, only the 55% of the population corresponding to the S-phase subpopulation were sensitive to camptothecin, while the remainder of the population were totally resistant to doses as high as 10 microM. The role of DNA replication in the camptothecin sensitivity was studied using the DNA polymerase inhibitor aphidicolin in co-treatment with camptothecin. Aphidicolin treatment fully protected AA8 cells from camptothecin cytotoxicity. In EM9 cells, aphidicolin protected the S-phase fraction to some degree but all the cells remained sensitive to camptothecin cytotoxicity. These results suggest that EM9 cells are sensitized to camptothecin by a mechanism that is independent of DNA replication and may be a consequence of the XRCC1 mutation or the associated deficiency in DNA ligase III activity. Mechanistic models for the replication-independent cytotoxicity of camptothecin in EM9 cells are discussed.

Ubiquitin-dependent destruction of topoisomerase I is stimulated by the antitumor drug camptothecin.

Topoisomerase I (TOP1) relaxes superhelical DNA through a breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site as a transient intermediate. The antitumor drug camptothecin (CPT) and its analogs inhibit the rejoining step of the breakage/rejoining reaction, which traps the enzyme in covalent linkage with DNA (the cleavable complex). Little is known about the fate of cellular TOP1 trapped in the cleavable complex. We have analyzed TOP1 in mammalian cell lines treated with CPT. When CPT-treated cells were lysed with either SDS or alkali and analyzed by Western blotting, greater than 90% of the TOP1 was linked to DNA. Nuclease treatment of the cell lysate to remove the covalently linked DNA from TOP1 revealed a distinct ladder of higher molecular weight bands having properties indicative of multi-ubiquitin (Ub) conjugates of TOP1. Approximately 5-10% of TOP1 was present as these conjugates within minutes of CPT treatment. Consistent with ubiquitination, TOP1 was not modified in ts85 cells at the restrictive temperature for its thermolabile ubiquitin-activating enzyme (E1). Because conjugation with ubiquitin can mark proteins for destruction by the 26S proteasome, we analyzed TOP1 protein levels during prolonged CPT treatment. TOP1 protein levels were reduced to about 25% during CPT treatments of 2-4 h resulting from increased destruction, with the half-life dropping from 10-16 h down to 1-2 h. The destruction of TOP1, like the formation of Ub-TOP1 conjugates, was not observed in ts85 cells at the restrictive temperature. The destruction of TOP1 was also prevented in cells treated with MG-132 and lactacystin, specific inhibitors of the 26S proteasome. Finally, the multi-Ub conjugates of TOP1 were observed whether or not aphidicolin was included in cotreatment with CPT, indicating that replication fork activity was not involved in making TOP1 a substrate for ubiquitination. These results demonstrate that independent of DNA replication, the TOP1 cleavable complex is ubiquitinated and destroyed in cells treated with antitumor drugs that block the religation step of the TOP1 reaction.

Cell cycle-specific and transcription-related phosphorylation of mammalian topoisomerase I.

Eukaryotic DNA topoisomerase I has been recently shown to be associated with the transcriptional machinery and has also been implicated to function in DNA replication and perhaps other DNA transactions. We have identified several differentially phosphorylated forms of mammalian topoisomerase I as electrophoretic variants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These differently phosphorylated forms cleave chromosomal DNA in cells and also relax supercoiled DNA with about equal activity, suggesting that they primarily function other than to activate catalysis. One of the phosphorylated forms is specifically present during mitosis. Upon transition from mitosis into G1 phase, two forms differing in phosphorylation state appear and persist throughout the remainder of interphase. When cells are incubated in a pellet, one of the interphase phosphorylated forms disappears coincidentally with an increase in the abundance of the other; if the cell pellet is disrupted and the cells are reincubated in suspension, the forms rapidly shift back to their original abundance levels. Finally, a shift in relative abundance of the differently phosphorylated interphase forms is observed when transcription is inhibited. These results suggest that dynamic phosphorylation and dephosphorylation regulate topoisomerase I during RNA transcription and cell cycle progression.

Management of giant retinal tears using perfluorodecalin as a postoperative short-term vitreoretinal tamponade: a long-term follow-up study.

To avoid postoperative "compartmentalization" of the vitreous cavity, which may accelerate the recurrence of proliferative vitreoretinopathy (PVR), and to provide a tamponading effect lasting long enough to allow the formation of a firm chorioretinal adhesion by retinopexy, we managed 11 eyes with giant retinal tears and grade-B PVR with lensectomy, vitrectomy, 5-day internal tamponade with perfluorodecalin (PFD), and postoperative supine positioning until the PFD was removed. Baseline characteristics included myopia (10 eyes; range, 5.00 to 15.00 diopters) and perforating trauma (one eye). All patients underwent PFD/fluid exchange 5 days after surgery. Anatomic attachment of the retina was achieved with two operations (the second one being the removal of the PFD) in 9 (82%) of the 11 eyes (median follow up, 18 months). In eight eyes (73%), there was no evidence of reproliferation; in one (successfully reattached after PFD/fluid exchange), a macular pucker developed. The intraocular PFD used as an internal tamponade appeared to be well tolerated for up to 5 days, as judged by static threshold perimetry in the two patients tested, and by the functional outcomes (64% of the reattached eyes had a final visual acuity of 20/40 or better).

Determinants of cellular sensitivity to topoisomerase-targeting antitumor drugs.

It is now clear that topoisomerase activity level is an important determinant of sensitivity to topo drugs. The regulation of topoisomerases is no doubt complex and multifaceted and is probably accomplished through redundancy at many control levels. The mechanism(s) of altered topo I expression in certain tumor types is unknown, but may be related to the central importance of topoisomerases in proliferating cell functions (transcription, replication, etc.), and the aberrant and chronic activation of these functions as a result of specific tumorigenic alterations. Small differences in sensitivity to chemotherapy can have a dramatic effect on cure rates, and therefore subtle cell type-specific differences may be important determinants of drug sensitivity. Whether abnormal topoisomerase quantity and specific activity are associated with resistance or sensitivity to topoisomerase-targeted chemotherapy in the clinic is now being studied. Determinants downstream of cleavable complex formation that affect the sensitivity of tumor versus normal cells to topo drugs in particular and DNA-damaging agents in general are little known. The goal of enhancing selective tumor cell killing relative to the normal cells that are dose limiting may be achieved either by overcoming tumor cell resistance or by protecting normal cells. Both of these strategies will become more feasible as specific molecular differences between tumor and normal cells are being rapidly identified and new combination therapies that take advantage of these differences are being designed and tested.

Perfluorocarbon liquids as postoperative short-term vitreous substitutes in complicated retinal detachment.

To avoid postoperative "compartmentalization" of the vitreous cavity, which can potentially accelerate the recurrence of proliferative vitreoretinopathy (PVR), 32 eyes of 32 selected patients with complicated retinal detachment were managed with lensectomy, vitrectomy, 5-day internal tamponade with perfluorocarbon liquids (PFCL) and postoperative supine positioning until PFCL removal. Intraoperatively, 19 eyes had grade C3 or greater PVR; 10 eyes exhibited ocular trauma and 6 displayed giant tears. All but 5 patients (PFCL/fluid exchange) underwent PFCL/silicone oil exchange 5 days after surgery. Anatomic attachment of the retina was achieved with one operation in 25 (78%) of the 32 eyes with a median follow-up of 8 months (mean 8.4 months). Of the 20 eyes that underwent silicone oil removal, none redetached. Nineteen eyes (61%) showed no or only mild reproliferation; a macular pucker developed in 50% of the 20 eyes successfully reattached after PFCL/silicone oil exchange and in none of the 5 eyes successfully reattached after PFCL/fluid exchange. Intraocular tolerance of PFCL for up to 5 days of internal tamponade appeared to be good as judged by static threshold perimetry in the two patients tested and by our functional outcomes, with 88% of the reattached eyes showing a final visual acuity of 5/200 or better.

The involvement of active DNA synthesis in camptothecin-induced G2 arrest: altered regulation of p34cdc2/cyclin B.

Cell cycle arrest in G2 phase is a common response to a variety of DNA-damaging agents. The coupling between DNA damage and G2 arrest was studied in synchronized HeLa cells using camptothecin, a highly specific inhibitor of topoisomerase I that damages DNA through the formation of reversible topoisomerase I-DNA cleavable complexes. Brief camptothecin treatment of early S-phase HeLa cells caused arrest at G2 phase and abolished the activation of p34cdc2 protein kinase. Both tyrosine dephosphorylation of p34cdc2 and cyclin B accumulation were altered. These cell cycle-dependent changes were not observed when DNA replication was inhibited by aphidicolin during the brief camptothecin treatment. Our results suggest that to produce G2 arrest, active DNA synthesis is required at the time of camptothecin treatment, as was previously shown for camptothecin-induced cytotoxicity. Furthermore, our results suggest that the interaction of the replication fork with DNA damage may ultimately trigger altered regulation of p34cdc2/cyclin B, leading to cell cycle arrest at the G2 phase.

Combined silicone and fluorosilicone oil tamponade (double filling) in the management of complicated retinal detachment.

We evaluated the use of simultaneous double filling with polydimethylsiloxane (PDMS) and fluorosilicone (FSiO) in the repair of complicated retinal detachment in 12 selected cases. Initial retinal reattachment was achieved in all cases. Proliferative vitreoretinopathy (PVR) recurred in 10 patients (83%), 6 of which showed partial retinal detachment. Inferior and superior postoperative residual free spaces were abolished by this procedure, but a new residual fluid space was created, lying horizontally between the bubbles and expanding in a triangular shape nasally to the optic disk and temporally to the macula. Overall, 9 of 10 eyes with postoperative PVR had proliferation involving these areas. These findings support the concept that 'compartmentalization' is of major importance in determining postoperative cell proliferation.

The effect of simultaneous internal tamponade on fluid compartmentalization and its relationship to cell proliferation.

To determine whether the residual free spaces within the vitreous chamber that result after vitreoretinal surgery and internal tamponade may be avoided, and to verify whether such compartmentalization is of real importance in the recurrence of postoperative proliferative vitreoretinopathy (PVR), the use of simultaneous double filling with polydimethylsiloxane (PDMS) and fluorosilicone (FSiO) in the repair of complicated retinal detachment is evaluated in 12 selected cases. Initial retinal reattachment was achieved in all cases. PVR recurred in 10 patients (83%), 6 of whom showed partial retinal detachment. Inferior and superior postoperative residual free spaces were abolished by this procedure, but a new residual fluid space was created, lying horizontally between the bubbles and expanding in a triangular shape nasal to the optic disc and temporal to the macula. Overall, 9 of 10 eyes with PVR after surgery had proliferation involving these areas. These findings support the concept that compartmentalization is of major importance in determining postoperative cell proliferation.

A new device for pupillary dilatation in vitreous surgery.

A completely new surgical technique to obtain pupillary dilatation is presented. It permits achievement of a mydriasis sufficient to observe the vitreous cavity in both phakic and aphakic eyes during vitreous surgery. Miotic immobile pupils not dilatable pharmacologically are enlarged by means of a silastic ring with a C-shaped groove on its outer part. This is introduced into the eye through a limbic opening. When in place, the external sulcus of the ring hosts the pupillary rim. Twelve patients (three phakic and nine aphakic) were treated. In two patients operated upon previously with silicone oil tamponade, some difficulties were encountered during the insertion of the device owing to the lubricating effect of the oil. After the removal of the ring, all the pupils returned to a round shape, with no apparent damage to the iris structure.

Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase poisons.

The primary cytotoxic mechanism of camptothecin has been proposed to involve an interaction between the replication machinery and the camptothecin-mediated topoisomerase I-DNA cleavable complex (Y. H. Hsiang, M.G. Lihou, and L.F. Liu, Cancer Res., 49:5077-5082, 1989). In the present study, we show that killing of V79 cells by the topoisomerase II poisons 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide may involve ongoing RNA synthesis in addition to ongoing DNA synthesis. V79 cells synchronized by mitotic shake-off were treated with topoisomerase poisons in the presence of inhibitors of nucleic acid synthesis. S-Phase V79 cells were more sensitive to the topoisomerase I poison camptothecin and the topoisomerase II poison m-AMSA than G1-phase cells. The greater sensitivity of S-phase cells to killing by m-AMSA and camptothecin was abolished during cotreatment, but not posttreatment, with aphidicolin, suggesting that ongoing DNA synthesis in involved in cell killing by both topoisomerase I and II poisons. Cotreatment with transcription inhibitors, such as 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole or cordycepin, partially protected cells from the cytotoxic effects of m-AMSA but had no effect on camptothecin-mediated cytotoxicity. These results suggest that ongoing RNA transcription may be involved in cell killing by topoisomerase II poisons but not topoisomerase I poisons. Cotreatment with camptothecin reduced m-AMSA-mediated cytotoxicity in G1-phase V79 cells, suggesting a possible antagonism between topoisomerase I and II poisons. This antagonistic effect between topoisomerase I and II poisons could be explained by the strong inhibitory effect of camptothecin on RNA transcription.

Use of molecular cloning methods to map the distribution of epitopes on topoisomerase I (Scl-70) recognized by sera of scleroderma patients.

We report the initial molecular characterization of the autoimmune response against DNA topoisomerase I (topo I; Scl-70). Sera from 36 patients with scleroderma and 4 healthy control subjects were studied using 6 subcloned portions of topo I. Twenty-three sera recognized at least 2 independent epitopes on the molecule. Therefore, anti-topo I, like other non-organ-specific autoantibodies characterized to date, is polyclonal and multifocal. The cloned protein should prove suitable for sensitive early detection of anti-topo I in the clinical setting.

A model for tumor cell killing by topoisomerase poisons.

Our model proposes that topoisomerase-targeting antitumor drugs form reversible drug-enzyme-DNA complexes that collide with the DNA and RNA synthesis machineries. On collision, the complexes lose their reversibility, and generate lethal double-strand DNA breaks. Further investigations of topoisomerase action will allow this model to be refined, and may ultimately lead to the development of more effective anticancer drugs.

Topoisomerase-targeting antitumor drugs.

Much has been learned about the unusual type of DNA damage produced by the topoisomerases. The mechanism by which these lesions trigger cell death, however, remains unclear, but it appears that DNA metabolic machinery transforms reversible single-strand cleavable complexes to overt strand breaks which may be an initial event in the cytotoxic pathway. For the topoisomerase I poisons, they produce breaks at replication forks that appear to be the equivalent of a break in duplex DNA. Indicating that this may be an important cytotoxic lesion is the hypersensitivity to camptothecin of the yeast mutant rad52, which is deficient in double-strand-break-repair. The topoisomerase poisons preferentially kill proliferating cells. In the case of the topoisomerase I poison camptothecin, dramatic S-phase-specific cytotoxicity can explain its preferential action on proliferating cells. For the topoisomerase II poisons, high levels of the enzyme in proliferating cells, and very low levels in quiescent cells appear to explain the resistance of quiescent cells to the drug's cytotoxic effects. Thus, the topoisomerase poisons convert essential enzymes into intracellular, proliferating-cell toxins. The identification of both topoisomerase I and II as the specific targets of cancer chemotherapeutic drugs now provides a rational basis for the development of topoisomerase I poisons for possible clinical use. Knowledge of the molecular mechanisms of cell killing may lead to the identification of new therapies for treating cancer. The topoisomerase poisons appear to be a good tool for studying cell killing mechanisms as they produce highly specific and reversible lesions.

Heritable hypersensitivity to induced mutagenesis in the progeny of cell populations exposed to UVC (254 nm).

The ability of mutagens to transform benign papillomas to malignancy in the mouse skin model of multistage carcinogenesis [Hennings et al. Nature 303, 67-68 (1983)] suggests that multiple events may underlie carcinogenic progression, and that mutagenic exposures separated by time can act synergistically. Such synergism may result from initial mutagenic exposure which induces heritable sensitivity to subsequent mutagenic exposures. For example, progeny of X-irradiated V79 cells are hypersensitive to subsequent mutation induced by psoralen plus long-wave ultraviolet light, PUVA [Frank and Williams, Science 216, 307-308 (1982)]. In the present studies 100 to 200 surviving clones of short-wave ultraviolet light (UVC) irradiated V79 cells were assayed for mutation at two loci. Cultures derived from these cells were found to be hypermutable at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus following exposure to PUVA, but showed mutant frequencies similar to control cells following UVC challenge at the HGPRT and ATPase loci.