Phase transitions in the growth of C3H 10T1/2 cells.

In systems used to express transformation using focus formation as the end point, nontransformed cells generally express a down-regulation of cell growth and division made evident by the formation of a monolayer of cells that completely covers the growth surface. In C3H 10T1/2 cells, down-regulation is thought to be progressively effected principally by cell-to-cell communication via gap junctions. Starting with a sparse population in asynchronous growth--e.g. containing cells in all phases of the growth cycle--as the area density increases, cells are progressively lost from the distribution in the order M phase, G2 phase, S phase and G1 phase, leading to the accumulation of viable cells out of cycle in so-called G0 phase. We have measured the progressive phase transitions as a function of inoculum size and time. The influence of a promoter and an antipromoter was also examined as well as the expression of the cyclin/cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1 as the cells grew into confluence. Using cells synchronized in mitosis, we found that with increasing cell density the expression of p27 increased and concomitantly p21 decreased.

Amsacrine-induced lesions in DNA and their modulation by novobiocin and 2,4-dinitrophenol.

The cancer chemotherapeutic agent amsacrine, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA), is thought to effect cytotoxicity by inhibiting the ATP-dependent enzyme topoisomerase II in the act of its duplex strand-passing action. Upon protein denaturation, the arrested "cleavable complex" that results gives rise to double- and single-strand breaks (dsbs and ssbs) and DNA-protein cross-links (dpcs). Simultaneous cotreatments with 2,4-dinitrophenol (DNP) or novobiocin (novo) abrogates mAMSA cytotoxicity in Chinese hamster cells (H. Utsumi et al., Cancer Res., 50:2577-2581, 1990). Pulsed-field gel electrophoresis was used to estimate dsbs, velocity sedimentation in alkaline sucrose gradients for ssbs, and alkaline elution without protease digestion for dpcs. Although cotreatment with DNP or novo modulated somewhat the yield of DNA lesions due to mAMSA, quantitatively these changes did not correlate at all with, and therefore could not account for, the reduced lethality that resulted from cotreatments. For example, DNA cotreatment markedly increased the yields of dsbs, ssbs, and dpcs, even though cell killing was appreciably reduced. Furthermore, neither DNP nor novo cotreatment affected the rate, or the completeness of, the repair of mAMSA-induced DNA damage, and neither cotreatment lowered total cellular ATP. Hence, the arresting of the cleavable complex by mAMSA, made evident by lesions in DNA, did not correlate with cytotoxicity. However, cotreatment with either DNP or novo resulted in an enhanced recovery of the mAMSA-induced inhibition of replicative DNA synthesis. Because DNP and novo (transiently) slow down DNA synthesis, it is proposed that these compounds abrogate mAMSA killing of S phase cells by reducing the disorganization of the processing of replicated DNA by topoisomerase II.

Abrogation by novobiocin of cytotoxicity due to the topoisomerase II inhibitor amsacrine in Chinese hamster cells.

Using cultured V79 Chinese hamster cells, we found that novobiocin (or 2,4-dinitrophenol) can abrogate, almost completely, the cytotoxicity due to the topoisomerase II inhibitor amsacrine (mAMSA). V79 cells were sensitive to mAMSA killing at all stages in the cell cycle but mainly in S phase followed by late G1 phase; however, novo rescued cells of all ages. The properties of two kinds of radiation-sensitive Chinese hamster cells were also examined, i.e., the line of V79 cells that can be rescued by caffeine, designated S-10 (H. Utsumi and M.M. Elkind, Radiat. Res., 96: 348-358, 1983); and Chinese hamster ovary cells (P.A. Jeggo and L.M. Kemp, Mutat. Res., 112: 313-327, 1983) which are also sensitive to other DNA-damaging agents. As is the case for exposure to radiation, after mAMSA treatment caffeine rescued V79/S-10 cells. Although Jeggo's Chinese hamster ovary cells were more responsive to mAMSA, novo still abrogated mAMSA toxicity in the mutant cells as well as in the parental Chinese hamster ovary cells 2,4-Dinitrophenol acted similarly to novo with respect to mAMSA killing, but neither compound reduced the ATP content of V79 cells. We propose that one reason for the rescue from mAMSA killing of at least S-phase cells by novo or 2,4-dinitrophenol is their ability transiently to inhibit replicative DNA synthesis.