Molecular failure of apoptosis: inappropriate cell survival and mutagenesis?

Since cell death by apoptosis is achieved through complex interactions between numerous molecular components, cells may fail to die when stimulated because of molecular abnormalities in the apoptosis pathway or in its control mechanisms. Such inappropriate cell survival is well established when apoptosis is suppressed by elevated expression of bcl-2, at least for some cell types. Many cells undergo apoptosis at moderate levels of DNA damage and suppression of such apoptosis might be expected to increase the rate of mutation because of the persistence of cells with damaged DNA. We and others have now confirmed this prediction in bcl-2 transfected cells. Suppression of the apoptosis pathway can only lead to inappropriate cell survival if it relates to events before the cell becomes committed to die. We have analyzed this question for agents that inhibit the caspases, the site-specific proteases which form the biochemical core of the process of apoptosis. We have shown that inhibition of certain caspases does lead to the survival of Jurkat human T-cells induced to undergo Fas-mediated apoptosis.

Polymeric drug-carriers containing doxorubicin and melanocyte-stimulating hormone: in vitro and in vivo evaluation against murine melanoma.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing doxorubicin (DOX, approximately 8% by weight) bound via the lysosomally degradable spacer Gly-Phe-Leu-Gly and, in certain cases, also melanocyte-stimulating hormone (MSH, 5-10% by weight) were synthesized with the aim of developing a drug conjugate for site-specific delivery to malignant melanoma. Polymer-bound MSH, like free MSH, was able to stimulate tyrosinase activity in B16F10 cells in vitro, confirming the ability of conjugated hormone to interact with the MSH receptor. Similarly, a 125I-labelled conjugate containing MSH was captured by B16F10 cells in vitro more rapidly than a similar polymer without the targeting moiety. HPMA copolymers containing DOX bound via the lysosomally degradable Gly-Phe-Leu-Gly linkage were cytotoxic to a mouse melanoma cell line (M3 S91) in vitro, the MSH-containing conjugate being more active than that without (although the difference in the ID50 was not significant). When administered intraperitoneally or intravenously to C57BL/6J mice bearing intraperitoneal B16F10 tumours, HPMA copolymers containing DOX linked via this biodegradable spacer (with or without MSH) significantly increased animal survival, the maximum ratio of the mean survival of the test group (T) to that of the untreated control (C) T/C observed (approximately 200) over the dose range 5-20 mg DOX/kg being similar to that seen for free DOX. In contrast, neither polymer conjugates containing DOX bound via a non-degradable linkage (Gly-Gly) nor free MSH showed antitumour activity. In mice bearing established subcutaneous B16F10 tumours, biodegradable polymer-bound DOX conjugates given intraperitoneally were more effective than free DOX (which was virtually inactive in this system); conjugates containing MSH were significantly more effective than those without, the maximum T/C being approximately 148 and 324 respectively. Preliminary pharmacokinetic experiments showed evidence of selective MSH targeting of polymer conjugates to subcutaneous B16F10.

Effect of galactose on interaction of N-(2-hydroxypropyl)methacrylamide copolymers with hepatoma cells in culture: preliminary application to an anticancer agent, daunomycin.

A series of copolymers were prepared containing 1,2:3,4-di-O-isopropylidene-6-O-methacryloyl-alpha-D-galactopyranose (0 to 99 mol %), methacryoyltyrosinamide and N-(2-hydroxypropyl)methacrylamide (99 to 0 mol %). The effect of galactose content on interaction with hepatoma cells in vitro was studied. Increased galactose content caused increased accumulation of N-(2-hydroxypropyl)methacrylamide copolymers by two human hepatoma cell lines (Hep G2 and SAH), but accumulation by rat and mouse hepatoma (HTC and NCTC) was not galactose dependent. Accumulation of N-(2-hydroxypropyl)methacrylamide copolymers by Hep G2 was shown to be an active process, being inhibited by low temperature and by the metabolic inhibitor 2,4-dinitrophenol. Addition of N-acetylgalactosamine and polymer-galactose to the incubation medium resulted in a concentration-dependent inhibition of accumulation of galactose-containing polymers. Addition of fucose or galactose was without effect at the concentrations used. Polymers bearing galactosamine or fucosylamine residues and, in addition, daunomycin were evaluated for cytotoxicity against Hep G2 and SAH. N-(2-Hydroxypropyl)methacrylamide copolymer-bound daunomycin produced a dose-dependent inhibition of DNA synthesis (measured by incorporation of [3H]thymidine), and the galactose-containing polymer showed greatest inhibition.

Selective cytotoxicity of hydroquinone for melanocyte-derived cells is mediated by tyrosinase activity but independent of melanin content.

In previous studies we have shown melanotic melanomas to be exquisitely more sensitive to hydroquinone (HQ) inhibition than non-melanotic cell lines in vitro. Indeed, incorporation of [H3] Urd and [H3] Thd have been shown to be respectively 80 and 35 times more sensitive to HQ inhibition. The difference between the cell lines studied was their derivation, marked by their different melanin contents. The presence of melanin was proposed as a possible explanation of the differences. However, comparative experiments reported here demonstrate that amelanotic melanoma cell lines are equally susceptible to HQ inhibition. Thus, the action of HQ is apparently independent of the melanin content of the cell. Significantly, the tyrosinase levels in the melanomas and the amelanomas were found to be comparable and markedly different from that in the non-melanoma control cell lines. Thus, the results reported here support the hypothesis put forward by other workers that hydroquinone melanotoxicity is independent of cellular melanin content but requires the presence of active tyrosinase.