David E. MacCallum

Seliciclib (CYC202, R-Roscovitine) induces cell death in multiple myeloma cells by inhibition of RNA polymerase II-dependent transcription and down-regulation of Mcl-1

Seliciclib (CYC202, R-roscovitine) is a cyclin-dependent kinase (CDK) inhibitor that competes for the ATP binding site on the kinase. It has greatest activity against CDK2/cyclin E, CDK7/cyclin H, and CDK9/cyclin T. Seliciclib induces apoptosis from all phases of the cell cycle in tumor cell lines, reduces tumor growth in xenografts in nude mice and is currently in phase II clinical trials. This study investigated the mechanism of cell death in multiple myeloma cells treated with seliciclib. In myeloma cells treated in vitro, seliciclib induced rapid dephosphorylation of the carboxyl-terminal domain of the large subunit of RNA polymerase II. Phosphorylation at these sites is crucial for RNA polymerase II-dependent transcription. Inhibition of transcription would be predicted to exert its greatest effect on gene products where both mRNA and protein have short half-lives, resulting in rapid decline of the protein levels. One such gene product is the antiapoptotic factor Mcl-1, crucial for the survival of a range of cell types including multiple myeloma. As hypothesized, following the inhibition of RNA polymerase II phosphorylation, seliciclib caused rapid Mcl-1 down-regulation, which preceded the induction of apoptosis. The importance of Mcl-1 was confirmed by short interfering RNA, demonstrating that reducing Mcl-1 levels alone was sufficient to induce apoptosis. These results suggest that seliciclib causes myeloma cell death by disrupting the balance between cell survival and apoptosis through the inhibition of transcription and down-regulation of Mcl-1. This study provides the scientific rationale for the clinical development of seliciclib for the treatment of multiple myeloma.

The location of pKi67 in the outer dense fibrillary compartment of the nucleolus points to a role in ribosome biogenesis during the cell division cycle

Although widely used as a marker of cell proliferation, the biochemical properties and function of the Ki67 antigen remain poorly understood. Recent data indicate that it can interact with RNA, DNA, and a number of cellular proteins including elements of the ubiquitin proteolytic pathway and a novel kinase. The evidence for its expression only in cycling cells is extensive and it is not regulated by stress, apoptosis or DNA damage. It was reasoned that a detailed characterization of the localization of pKi67 and analysis of its spatial relationship to other nucleolar proteins may provide insights into its function. Using high‐resolution laser scanning confocal microscopy with double and triple labelling, pKi67 expression in MCF7 cells has been defined in relation to the distribution of nucleolin, fibrillarin, p130 (human Nopp 140 homologue), p120 (Nol 1), RH‐II/Gu helicase, and topoisomerase II β. All of these molecules are perichromosomal during mitosis and all but fibrillarin and p130 show extra‐nucleolar distribution in early G1. The majority of p120 (Nol 1) and RH‐II/Gu helicase co‐localize in the diffuse fibrillar centre (DFC) of nucleoli, while there is only partial overlap with nucleolin and fibrillarin. There is no co‐localization between p130 and pKi67. These data refine current understanding of the distribution of pKi67 and its physical relationship with functional domains of the nucleolus and place pKi67 in a zone of the DFC associated with late rRNA processing. Taken together with recent biochemical data, these observations allow the proposal of a model of pKi67 function in which it acts as an ‘efficiency factor’ in ribosome biogenesis during the heavy metabolic demands placed on a cell during the cell division cycle. Copyright

Expression of the 'dead box' RNA helicase p68 is developmentally and growth regulated and correlates with organ differentiation/maturation in the fetus

The human DEAD box protein p68 is an established RNA‐dependent ATPase and RNA helicase. p68 has been highly conserved in evolution and appears to be essential for normal growth, suggesting that this protein plays an important role in the cell. Although the biochemical activities of p68 are fairly well characterized, little is known about its biological function. This report shows that p68 is detectable in quiescent cell lines, but its expression is induced by serum, suggesting that this protein may play a role in cell growth. It is also shown that both p68 mRNA and protein are differentially expressed in adult tissues; in this case, however, the levels do not always correlate with proliferation status, suggesting that the regulation of expression in the animal may be different from that in cell lines. Finally, it is shown that p68 expression is developmentally regulated and appears to correlate with organ differentiation/maturation. These findings suggest that p68 expression may not simply reflect proliferation/differentiation status and that it appears to be regulated in a more complex way.

The biochemical characterization of the DNA binding activity of pKi67

Ki67 is only expressed in the nucleus of cycling cells. While it is employed as an operational marker of proliferation, little is known of the biochemical properties of this large protein. Using an immunoaffinity strategy for purification of pKi67, this study has shown that it can form higher‐order complexes and can bind to DNA cellulose in vitro. No other co‐purifying proteins could be identified, strongly suggesting that the DNA binding activity is an inherent property of pKi67. Using an electromobility shift assay, the affinity of pKi67 was shown using a range of different forms of DNA as competitors. Single‐stranded DNA was the poorest competitor, followed by double‐stranded DNA, with supercoiled DNA being the best competitor. In addition, it was found that purified pKi67 has a preference for AT‐rich DNA. The DNA binding domain is mapped to the C‐terminal domain of pKi67, and recombinant protein from the terminal 321 residues of pKi67 can bind DNA in vitro. GFP constructs from this domain were used to map regions that could target nucleolar localization and allow DNA binding. Finally, it was found that over‐expression of the C‐terminal 321 residues in cells induced chromatin disruption and apoptosis. These data provide strong evidence that pKi67 has a novel DNA binding activity within the C‐terminal domain and that this protein can influence chromatin structure. Copyright

The Trp53 Pathway Is Induced In Vivo by Low Doses of Gamma Radiation

Abstract MacCallum, D. E., Hall, P. A. and Wright, E. G. The Trp53 Pathway is Induced In Vivo by Low Doses of Gamma Radiation. Radiat. Res. 156, 324–327 (2001). The induction of the Trp53 response after very low doses (0.01–1 Gy) of ionizing radiation was studied in the adult mouse using immunochemical and immunohistochemical methods. We found a detectable response at 0.01 Gy and an increased induction of Trp53 with increasing dose in both radiation-resistant and radiation-sensitive tissues. These results suggest that there is no lower threshold for induction. This response was heterogeneous, since cells that received the same dose had different staining intensities, suggesting that the induction of Trp53 is not based simply on dose-dependent responses to DNA damage. These data also demonstrate the exquisite sensitivity of the Trp53 pathway and show that this response is controlled by cell- and tissue-specific factors that have yet to be defined.

Abstract 3502: Understanding the pathways involved in the repair of CNDAC induced DNA damage

CNDAC is the active metabolite of sapacitabine, which is currently being evaluated in Phase II clinical trials for acute myeloid leukaemia, myelodysplastic syndrome and non small cell lung cancer. CNDAC (2′-C-Cyano-2′-deoxy-β-D-arabino-pentafuranosylcytosine) was designed as a novel cytosine analogue with a unique mechanism of action. The presence of the cyano-group within the ribose moiety of the molecule causes the formation of single-stranded DNA strand breaks, following incorporation of CNDAC into an extending DNA chain. These breaks are difficult to repair and are processed into double-strand DNA breaks that activate the dsDNA damage checkpoint. As a consequence of this unique mechanism of action, CNDAC arrests cells in the G2/M phase of the cell cycle in contrast to other nucleoside agents such as cytarabine and gemcitabine which cause an S-phase arrest. As such CNDAC may have unique therapeutic applications as a single agent in certain tumour types as well as in combination with other agents compared with standard nucleoside analogues. In order to identify options for maximising that activity of CNDAC, two approaches were taken to evaluate the repair mechanisms involved for CNDAC induced DNA damage. First a small scale siRNA screen was used to identify genes that were synthetically lethal with CNDAC. Ten targets were initially selected; prioritising genes known to be involved in DNA repair and including BRCA, ATM, CHK and ERCC1. The most dramatic increase in CNDAC sensitivity was seen when BRCA2 was targeted by siRNA, indicating that the homologous recombination DNA repair pathway is involved in the repair of CNDAC induced DNA damage. The second approach involved a cytotoxicity screen evaluating synergy with commercially available agents that either target DNA repair or induce DNA damage themselves. The most promising combinations were then followed up with flow cytometry analysis to examine the induction of cell death. Using this approach the best synergy was detected between CNDAC and either the ATM inhibitor, KU55933 or the CHK inhibitor PF477736. Again these data indicate that the homologous recombination pathway is involved in the repair of CNDAC induced DNA damage. These data suggest that it would be feasible to explore the clinical use of sapacitabine in diseases where the homologous recombination DNA repair pathway is compromised, such as BRCA deficient tumours. Data from these ongoing studies will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3502.