A novel indolocarbazole, ICP-1, abrogates DNA damage-induced cell cycle arrest and enhances cytotoxicity: similarities and differences to the cell cycle checkpoint abrogator UCN-01.

DNA damaging agents such as cisplatin arrest cell cycle progression at either G1, S, or G2 phase, although the G1 arrest is only seen in cells expressing the wild-type p53 tumor suppressor protein. We have reported that 7-hydroxystaurosporine (UCN-01) overcomes S and G2 phase arrest and enhances the cytotoxicity of cisplatin. Abrogation of arrest appears to be selective for cells defective in p53 and therefore provides a potential, tumor-targeted therapy. Unfortunately, UCN-01 binds avidly to human plasma proteins, limiting access to the tumor. A screen of related indolocarbazoles identified analogues with both beneficial and undesirable properties. This led to a synthetic program to develop a novel analogue rationally designed to overcome the obstacles observed with the other analogues. We report the synthesis and analysis of a novel analogue, ICP-1. This analogue abrogated S and G2 phase arrest and enhanced cytotoxicity induced by cisplatin only in p53 defective cells. ICP-1 also abrogated arrest and enhanced cell killing induced by the topoisomerase I inhibitor SN38. Analysis of proteins that regulate cell cycle arrest suggest both drugs inhibit checkpoint kinases Chk1 and/or Chk2. In contrast to UCN-01, checkpoint abrogation by ICP-1 was only slightly inhibited by human plasma. UCN-01 and ICP-1 differed significantly in other regards. UCN-01 potently enhanced the activity of 1-beta-D-arabinofuranosylcytosine in both p53 wild-type and mutant cells, whereas ICP-1 was inactive in this combination. This property of UCN-01 was independent of its ability to inhibit protein kinase C because more specific inhibitors of protein kinase C failed to enhance cell killing induced by 1-beta-D-arabinofuranosylcytosine. High concentrations of UCN-01 also inhibit C-TAK1 that results in S phase-arrested cells directly entering mitosis, but this property was not observed with ICP-1. Hence, ICP-1 appears to be a more selective inhibitor of the S and G2 cell cycle checkpoint than previously studied analogues and is worthy of study in preclinical tumor models.

Abrogation of the S phase DNA damage checkpoint results in S phase progression or premature mitosis depending on the concentration of 7-hydroxystaurosporine and the kinetics of Cdc25C activation.

DNA damage causes cell cycle arrest in G(1), S, or G(2) to prevent replication on damaged DNA or to prevent aberrant mitosis. The G(1) arrest requires the p53 tumor suppressor, yet the topoisomerase I inhibitor SN38 induces p53 after the G(1) checkpoint such that the cells only arrest in S or G(2). Hence, SN38 facilitates comparison of p53 wild-type and mutant cells with regard to the efficacy of drugs such as 7-hydroxystaurosporine (UCN-01) that abrogate S and G(2) arrest. UCN-01 abrogated S and G(2) arrest in the p53 mutant breast tumor cell line MDA-MB-231 but not in the p53 wild-type breast line, MCF10a. This resistance to UCN-01 in the p53 wild-type cells correlated with suppression of cyclins A and B. In the p53 mutant cells, low concentrations of UCN-01 caused S phase cells to progress to G(2) before undergoing mitosis and death, whereas high concentrations caused rapid premature mitosis and death of S phase cells. UCN-01 inhibits Chk1/2, which should activate the mitosis-inducing phosphatase Cdc25C, yet this phosphatase remained inactive during S phase progression induced by low concentrations of UCN-01, probably because Cdc25C is also inhibited by the constitutive kinase, C-TAK1. High concentrations of UCN-01 caused rapid activation of Cdc25C, which is attributed to inhibition of C-TAK1, as well as Chk1/2. Hence, UCN-01 has multiple effects depending on concentration and cell phenotype that must be considered when investigating mechanisms of checkpoint regulation.