RNA synthesis block by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) triggers p53-dependent apoptosis in human colon carcinoma cells.

Most modern chemo- and radiotherapy treatments of human cancers use the DNA damage pathway, which induces a p53 response leading to either G1 arrest or apoptosis. However, such treatments can induce mutations and translocations leading to secondary malignancies or recurrent disease, which often have a poor prognosis because of resistance to therapy. Here we report that 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of CDK7 TFIIH-associated kinase, CKI and CKII kinases, blocking RNA polymerase II in the early elongation stage, triggers p53-dependent apoptosis in human colon adenocarcinoma cells in a transcription independent manner. The fact that DRB kills tumour-derived cells without employment of DNA damage gives rise to the possibility of the development of a new alternative chemotherapeutic treatment of tumours expressing wild type p53, with a decreased risk of therapy-related, secondary malignancies.

Schedule-dependent cytotoxicity of SN-38 in p53 wild-type and mutant colon adenocarcinoma cell lines.

In this study the effects of SN-38 on colon adenocarcinoma cell lines expressing wild-type p53 (LS174T) or mutant non-functional p53 (HT29) have been investigated. On exposure to SN-38, HT29 cells rapidly progressed through G1 and S and arrested in G2/M. Release and concomitant increase in apoptosis after 48 h was concentration- and time-dependent (P < 0.001), being more rapid at higher concentrations, but reaching plateau at 10 ng ml(-1) with prolonged exposure. LS174T cells showed only a small increase in apoptosis, and only at high concentrations (50-100 ng ml(-1)). The main effect of SN-38 in LS174T cells was prolonged cell cycle arrest, which was independent of concentration. Arrest occurred in all phases of the cell cycle, with the distribution depending on concentration (P < 0.001) and not duration (P > 0.05). With increasing concentration, LS174T cells arrested in G2/M, S and G1. Cell cycle arrest was coincident with increased p53 expression in each phase of the cell cycle. Expression in G1 increased with time and concentration (P < 0.001, P = 0.01 respectively)whereas in S and G2/M p53 expression increased only with time (P< 0.001). Dose-dependent p53-associated G1 arrest, in the absence of DNA synthesis indicates an additional cytotoxic mechanism for SN-38, which requires higher concentrations than the S phase mechanism, and detection of which seems to involve p53. For incubations with the same ED (exposure x duration), apoptosis in HT29 cells was significantly higher for prolonged exposure to lower concentrations, whereas in LS174T cells there was a trend towards increased apoptosis with shorter exposures to higher concentrations, indicating a schedule effect of SN-38. Although expression of wild-type p53 leads to a more rapid induction of apoptosis, SN-38 cytotoxicity was generally greater in cells with mutant p53, as wild-type cells escaped apoptosis by p53 associated prolonged cell cycle arrest. Thus, pulsed schedules with higher doses may be more effective in cells expressing wild-type p53, whereas continued exposure with protracted schedules may be more active in cells expressing mutant p53.

An efficient system for active bovine pancreatic ribonuclease expression in Escherichia coli.

Bovine pancreatic ribonuclease (RNase A) is a member of a homologous group of extensively studied proteins. It is a small, basic protein, containing 124 amino acid residues and four stabilizing disulfide bridges. Ribonuclease A catalyzes the hydrolysis of the phosphodiester bonds in ribonucleic acids. Since this degradation of RNA interferes with normal cell functions, the signal peptide of alkaline phosphatase (phoA, Escherichia coli) was cloned onto the gene coding for RNase A, directing the protein to the periplasm. Several expression systems have been evaluated which use T7, trc, or PR promoters to transcribe the RNase A gene. Also, variation in host strains was tested to optimize the protein yield. It was found that the PR system gave better expression than the two other systems. E. coli strain BL21 was shown to be the strain in which export to the periplasm was most effective and recombinant RNase A could be isolated from the periplasmic fraction of these cells. The system provides a stable yield of active recombinant bovine pancreatic RNase of about 45-50 mg/liter of cell culture.