C.Stephen Downes

The nuclear membrane prevents replication of human G2 nuclei but not G1 nuclei in Xenopus egg extract

We have used synchronized HeLa cells to investigate the role of the nuclear membrane in preventing rereplication in a single cell cycle. Nuclei were prepared with intact nuclear membranes using streptolysin-O or digitonin and assayed for replication in Xenopus egg extracts. Intact G1 nuclei replicate semiconservatively, but intact G2 nuclei do not replicate in egg extract. However, permeabilizing the nuclear membranes of G2 nuclei by treatment with NP-40 allows them all to replicate in egg extract under cell cycle control, suggesting that integrity of the nuclear membrane is required to distinguish G2 from G1 human nuclei and to prevent rereplication within a single cell cycle. The results are discussed in terms of the previously proposed licensing factor model.

Action of caffeine on DNA replication after ultraviolet irradiation in Indian muntjac cells: No connection with action on cell cycle delay

Indian muntjac fibroblasts of the SV40-transformed line SVM are hypersensitivity to UV, and after UV irradiation have defective post-replication recovery and a high level of sister chromatid exchanges and chromosome aberrations. The lethal and clastogenic effects of UV on SVM have elsewhere been shown to be aggravated by caffeine, which overcomes the block to cycle traverse imposed by DNA damage; however, in DM cells, an Indian muntjac line of normal UV sensitivity, caffeine has no effect on cycle traverse, but nevertheless enhances UV killing and sister chromatid exchanges. In this paper, the effects of caffeine on irradiated DM cells are shown to be due to its inhibition of post-replication recovery, with subsequent formation of DNA double-strand breaks at the strand gaps thus produced. By contrast, in SVM cells the limited capacity for post-replication recovery is relatively insensitive to caffeine after UV fluences which permit significant cell survival; however, caffeine still strongly induces DNA double-strand breaks and chromosome aberrations, apparently by an alternative mechanism. The SVM and DM cell lines therefore exemplify separate actions of caffeine on mammalian cells, deficient in the caffeine effects on post-replication recovery and cell cycle progression, respectively.

Abnormal sister-chromatid exchange induction by 3-aminobenzamide in an SV40-transformed Indianamuntaj cell:line: Relationships with DNA maturation and DNA-strand breakage

In SVM cells, an SV40-transformed line of Indian muntjac fibroblasts, levels of sister-chromatid exchanges are known to be abnormally high after UV-irradiation or alkylation. The SVM line is also known to have a defect in the processing of DNA-strand breaks. Sister-chromatid exchange in other cells is known to be stimulated by the poly(ADP-ribose) transferase inhibitor, 3-aminobenzamide, which also retards DNA-break sealing. Sister-chromatid exchanges in SVM cells are found to be hypersensitive to 3-aminobenzamide, or to nicotinamide deprivation which similarly inhibits poly(ADP-ribosyl)ation; DNA-strand breaks are likewise induced by 3-aminobenzamide. Bromodeoxyuridine, needed to detect sister-chromatid exchanges, is more toxic to SVM cells and itself induces sister-chromatid exchanges to a greater extent than it does in normal muntjac cells. However, in contrast to the situation reported for other cell types prone to sister-chromatid exchange (the Chinese hamster ovary mutant EM9 and human Blooms Syndrome cells), SVM cells do not show an abnormal delay in DNA maturation when, under the influence of bromodeoxyuridine and 3-aminobenzamide, they show a high level of sister-chromatid exchange. The mechanism by which BrdU exerts its effects can largely be explained in terms of familiar effects on deoxyribonucleotide pools and DNA integrity. 3-Aminobenzamide, however, induces sister-chromatid exchanges in SVM cells by another mechanism.