No abstract

Tallimustine or N-deformyl-N-[4-N-N,N-bis(2-chloroethylamino)benzoyl], a distamycin-A derivative (FCE 24517), is a novel anti-cancer agent which alkylates N3 adenine in the minor groove of DNA. The cell-cycle phase perturbations induced by the drug were investigated and compared with those caused by melphalan (L-PAM) in SW626 human ovarian-cancer cells. By coupling bromodeoxyuridine (BUdR) immunoreaction with biparametric flow-cytometric (FCM) analysis, we investigated the cell-cycle phase perturbation induced by tallimustine or L-PAM, considering separately the cells which, during the 1-hr treatment, were in the S phase or in G1-G2/M phases of the cell cycle. L-PAM delayed the S-phase progression of cells exposed to the drug when they were in S phase, with a consequent accumulation of cells as soon as they reached the G2 phase. In contrast, the S-phase cells treated with tallimustine were not perturbed during the DNA-synthesis phase progression, and were blocked in G2 only after they had passed through the G1/S transition of a new cell cycle. In cells which were in G1 or G2/M phases during drug treatment, tallimustine and L-PAM caused similar accumulation in G2. The differences in the cell-cycle perturbation caused by tallimustine and L-PAM may well be related to the different DNA damage the 2 drugs produced. These findings emphasize the different properties of DNA-minor-groove alkylating agents and conventional ones.

Summary Inhibitors of ornithine decarboxylase (ODC), such as a-difluoromethylornithine (DFMO), may influence the cytotoxicity of anti-tumour agents that interact with DNA. Intracellular levels of putrescine and spermidine were markedly reduced by ODC inhibitors while the level of spermine, which is the main polyamine in nuclei, was unchanged. By combining a novel inhibitor of ODC, such as (2R, 5R)-6-heptyne-2,5-diamine (MDL 72.175, MAP), with an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), such as 5'-{[(Z)-4-aminobut-2-enyl]methylamino}-5'-deoxyadenosine (MDL 73.81 1, AbeAdo), spermine was selectively depleted in a human ovarian cancer cell line OVCAR-3 (i.e. spermine became almost undetectable whereas the levels of spermidine and putrescine were not affected). The . The addition of spermine before drug treatment restored the sensitivity to the DNA topoisomerase 11 inhibitors, thus indicating that the reduced effect was related to the intracellular spermine level. The reason for the reduction in cytotoxicity is unclear, but it does not appear to be related to a cell cycle effect or to a decrease in the intracellular level of DNA topoisomerase 11. Drugs that modify polyamine biosynthesis are under early clinical development as potential new anti-tumour agents. These findings illustrate the need for caution in combining such drugs with DNA topoisomerase 11 inhibitors.

Intracellular glutathione (GSH) content was measured by flow cytometry using monochlorobimane (mBCl) and by the enzymatic assay in a set of 6 sublines of murine L1210 leukemia cells made resistant to DNA-interacting agents having distinct mechanisms of action: L-phenylalanine mustard (L-PAM), 1,3-bis(2-chloroethyl)-I-nitrosourea (BCNU), cisplatin (DDP), N-deformyl-N-(4-N,N-bis(2-chloroethylamino) benzoyl) distamycin A (FCE 24517), doxorubicin (DX) and 3'-deamino-3' (2-methoxy-4-morpholinyl)-doxorubicin (FCE 23762). A significant correlation was demonstrated between the mean intracellular mBCl fluorescence values measured by flow cytometry and levels of GSH measured by the classical enzymatic assay, despite the possible influence of glutathione-S-transferases and of other thiols on the mBCl fluorescence. Although less specific, the flow cytometric method is more informative than the enzymatic assay, allowing detection of fluorescence distributions, which we proved to be characteristic of each subline. In order to assess a procedure enabling a quantitative analysis to be made of intercellular GSH heterogeneity, we propose the use of appropriate thresholds and parameters of the mBCl flow cytometric distribution. By use of this analysis procedure, distinct types of alterations, with respect to the heterogeneity distribution of the parental L1210 cell line, have been evidenced in resistant cells. A uniform increase in mBCl fluorescence was observed among cells of the sublines resistant to L-PAM and FCE-24517. The mean mBCl fluorescence increase in sublines resistant to DX and DDP was due to a higher number of cells with fairly high mBCl fluorescence, but still within the range spanned by the parental cell line. A less heterogeneous mBCl fluorescence distribution was found in the L1210 subline resistant to FCE 23762, which was, however, similar to a cloned sensitive line. Though GSH was linked to the principal cause of drug resistance only in the L-PAM-resistant cell line, alterations in heterogeneity, as detected by mBCl fluorescence distributions, were found in 5 out of 6 resistant lines.

Flow cytometric cyclin expression/DNA content analysis, now commonly used, provides useful information on the mechanisms regulating cell cycle progression. However, this biparametric analysis does not make a clear‐cut distinction between G1 and S‐early or between S‐late and G2M phase cells. This paper proposes a new three‐parameter flow cytometric method with which to determine cyclin B1 levels in single cells in different cell cycle phases by coupling bromodeoxyuridine (BrdUrd) immunodetection and DNA content. DNA denaturation by HCl did not alter the level of cyclin B1. Differences in cyclin B1 expression were observed in seven human cancer cell lines of different origin. The percentage of cyclin B1‐positive cells and the cyclin B1 content per cell indicated different patterns. In some cases cyclin B1 accumulation preceded the G2M checkpoint, at which its content usually started to rise. Using available easily reproducible techniques, this flow cytometric approach gives details of intracellular variability in cyclin expression.  Cytometry 31:53–59, 1998. © 1998 Wiley‐Liss, Inc.