Comparison of in vitro activity of epipodophyllotoxins with other chemotherapeutic agents in human medulloblastomas.

Surgical specimens from 15 medulloblastoma patients were used to establish early passage cultures. In vitro sensitivity to a battery of cytotoxic agents, including some in current medulloblastoma treatment protocols, was measured. Drug sensitivity was assessed at clinically relevant drug concentrations using the 3H-thymidine uptake method. Tumours were predicted to be sensitive if greater than 37% were killed by exposure to drugs at clinically achievable levels. A poor response to vincristine (Vcr), cis-platin (CDDP), hydroxyurea (HU) or diaziquone (AZQ) (no responders), and cytosine arabinoside (AraC) (1/12), was seen. Nine of ten tumours tested were sensitive to mafosfamide (Mfs); seven out of 12 were sensitive to carmustine (BCNU), 12 of 13 to teniposide (VM-26) and seven of 13 to etoposide (VP16-213). VM-26 was the best of the agents tested with most tumours responding to very low concentrations of drug, suggesting that the role of epipodophyllotoxins in treatment of brain tumours be further investigated. Despite the marked sensitivity of the medulloblastomas to the epipodophyllotoxins, three early passage cultures were much more resistant to these drugs than the average for the group. The basis of this resistance was investigated. Deficient cellular uptake of drug was excluded as a cause of resistance. One resistant early passage culture displayed low cellular activity of topoisomerase II and decreased levels of drug induced enzyme-DNA strand break activity. This was not the case for the other resistant early passage cultures: the basis of resistance in these cells does not appear to be due to any previously reported mechanism.

Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin.

Camptothecin, which induces an unusual type of DNA damage by trapping cellular topoisomerase I on chromosomal DNA in the form of drug-enzyme-DNA cleavable complexes, inhibits DNA synthesis and specifically kills S-phase cells. Cotreatment of L1210 cells with aphidicolin, which is an inhibitor of replicative DNA polymerases, completely abolished camptothecin cytotoxicity, suggesting the involvement of DNA replication in camptothecin cytotoxicity. In order to study the role of DNA replication in drug action, a cell-free SV40 DNA replication system was used in the present study. Camptothecin inhibited SV40 DNA replication in this cell-free system only in the presence of topoisomerase I. Addition of excess purified calf thymus DNA topoisomerase I to this extract system in the presence of camptothecin resulted in severe inhibition of SV40 DNA replication and the accumulation of linearized replication products, which contained covalently bound DNA topoisomerase I. We propose that the collision between moving replication forks and camptothecin-stabilized topoisomerase I-DNA cleavable complexes results in fork arrest and possibly fork breakage, which are lethal to proliferating cells.

Sensitivity of human neuroblastoma to activated dacarbazine: relationships between cell survival, methyltransferase activity and activation of adenovirus-5.

Early passage cultures of neuroblastoma cells were tested for (i) cellular sensitivity to the methylating agent 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC); (ii) ability to reactivate MTIC-damaged adenovirus (Mer+ phenotype); and (iii) methyltransferase activity. Seven of eight lines were resistant to MTIC. One line had an intermediate level of cellular resistance to MTIC, when compared with Mer+ and Mer- control lines. Methyltransferase activity of the neuroblastomas was intermediate between Mer+ and Mer- control. Unlike other methylation-resistant cell types, the neuroblastomas showed an initial decline in the MTIC dose-response profile for cell survival followed by a plateau at higher doses. In the virus reactivation assay (HCR), the slope (D0) of the virus survival curve at high MTIC doses for cells from three of 10 patients was similar to that of Mer- controls. The D0 for the remaining seven was also much less than for Mer+ controls. However, due to shoulders on the survival curves, all of the neuroblastomas could be classified as Mer+ at low levels of MTIC damage. Overall, the neuroblastoma cells appeared to form a new, though heterogeneous, methylation-resistant group, with cell survival not paralleled by methyltransferase activity or virus reactivation at high methylation levels.

Lack of cross-resistance between cytosine arabinoside and a new halogenated nucleoside analogue, 2-bromo-2'-deoxyadenosine in human acute myeloid leukaemia cells.

2-Bromo-2'-deoxyadenosine (BdA) is one of a group of recently synthesised halogenated deoxyadenosine analogues that are relatively resistant to inactivation by adenosine deaminase (ADA). Its activity has been studied in human acute myeloid leukemia (AML) in vitro. In these studies BdA behaved as a cycle-active, phase-active agent that blocked cells at the G1-S transition. It did not exhibit significant cross-resistance with cytosine arabinoside (Ara-C) in either clinical AML samples (from patients who exhibited Ara-C resistance in vivo) or in HL60 in which Ara-C resistance had been induced in vitro. Deoxycytidine kinase levels were not reduced in resistant lines. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an adenosine deaminase (ADA) inhibitor, with BdA produced a simple additive response without the dramatic synergism reported when it is used with deoxyadenosine. This is consistent with the idea that BdA is a poor substrate for ADA. This group of compounds warrants further investigation to determine their suitability for clinical use, especially in situations where Ara-C resistance is likely to be a problem.

Prediction of remission induction in childhood acute myeloid leukemia.

Prediction of clinical response to induction treatment was studied in a group of 16 children and adolescents with acute myeloid leukemia (AML) by a colony inhibition assay. Samples from ten of 16 patients formed colonies in agar and cells were available from nine of these ten for further study. The system correctly predicted the response of eight of the nine patients (89%) with a confidence level of greater than 80% in all cases and thus produced useful results in half of the series of 16 children. The incorrect prediction occurred in a child whose leukemia had a very low plating efficiency and who relapsed soon after achieving remission. While this methodology requires further development and assessment, it shows great promise in aiding in the selection of appropriate chemotherapy for individual patients with AML.

Adriamycin and cytosine arabinoside contribute equally to prediction of response in acute myelocytic leukemia with improved confidence level.

Samples from 15 patients with acute myeloid leukemia (AML) were studied in a colony-inhibition assay of drug sensitivity. In vitro sensitivity to both Adriamycin (Adr) (1-h incubation) and cytosine arabinoside (AraC) (24-h and continuous incubation) were determined, as were the tritiated thymidine suicide indices (SI) during the 24-h incubation with AraC. The sensitivity of the proportion of cells entering S-phase during the 24-h incubation was also evaluated. Using discriminant analysis a function was derived to separate the patients into two groups, those who failed remission induction therapy, or nonresponders (group 1), and those who entered complete remission (group 2). The only variables that contributed to prediction were the Adr (1 h) and Ara-C (24 h) results. Overall, 87% (13/15) of the patients were correctly grouped using this function, with a confidence level for nine of these 13 patients of greater than 80%. Adr and AraC results contributed equally to the prediction.

Quantitation of chemosensitivity in acute myelocytic leukaemia.

A system for the prediction of clinical response in acute myelocytic leukaemia (AML), based on inhibition of growth of colony forming cells (CFC) was studied. If the product of initial drug concentration and time of exposure (C X T) was constant, the response to adriamycin (Adr) was constant, at T values less than 48 h. No constancy of response to the phase-specific agents cytosine arabinoside (Ara-C) and 6-thioguanine (6TG) was demonstrated with constant C X T (T value range 0.25-48 h). Hence in the predictive test, a 1 h incubation with Adr was employed whilst a continuous exposure to Ara-C and 6TG, with these drugs incorporated in the agar medium, was used. The in vitro sensitivity to Adr, Ara-C and 6TG of 19 AML patients and the predictive value of several parameters of sensitivity were evaluated. 6TG sensitivity was not useful for prediction of remission. Adr sensitivity in vitro made a greater contribution to prediction of remission than did Ara-C sensitivity. Seventy-nine percent of patients were correctly classified if Adr data alone were considered. A multivariate function including Adr and Ara-C results was obtained which resulted in 84% of patients correctly classified as sensitive or resistant to the agents received in remission-induction therapy.