The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5^KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5^KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5^KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5^KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5^KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels.     

The roles of poly(ADP-ribose)-metabolizing enzymes in alkylation-induced cell death

Poly(ADP-ribose) (PAR) has been identified as a DNA damage-inducible cell death signal upstream of apoptosis-inducing factor (AIF). PAR causes the translocation of AIF from mitochondria to the nucleus and triggers cell death. In living cells, PAR molecules are subject to dynamic changes pending on internal and external stress factors. Using RNA interference (RNAi), we determined the roles of poly(ADP-ribose) polymerases-1 and -2 (PARP-1, PARP-2) and poly(ADP-ribose) glycohydrolase (PARG), the key enzymes configuring PAR molecules, in cell death induced by an alkylating agent. We found that PARP-1, but not PARP-2 and PARG, contributed to alkylation-induced cell death. Likewise, AIF translocation was only affected by PARP-1. PARP-1 seems to play a major role configuring PAR as a death signal involving AIF translocation regardless of the death pathway involved. Received 7 November 2007; received after revision 19 December 2007; accepted 21 December 2007 O. Cohausz, C. Blenn: These two authors contributed equally to this work.     

Influence of agents that act on DNA and RNA synthesis on the activity of poly(ADP-Rib) polymerase.

The activity of poly(ADP-Rib) polymerase is enhanced in the presence of spermine and spermidine. Among the adenosine-like antibiotics tested, only formycin B and showdomycin cause an inhibition of the enzyme, which is competitive to NAD. The activity of poly(ADP-Rib) polymerase is not reduced by rifamycin, alpha-amanitin and 2-phenylethanol.     

Targeting O 6-methylguanine-DNA methyltransferase with specific inhibitors as a strategy in cancer therapy

O ⁶-methylguanine-DNA methyltransferase (MGMT) repairs the cancer chemotherapy-relevant DNA adducts, O ⁶-methylguanine and O ⁶-chloroethylguanine, induced by methylating and chloroethylating anticancer drugs, respectively. These adducts are cytotoxic, and given the overwhelming evidence that MGMT is a key factor in resistance, strategies for inactivating MGMT have been pursued. A number of drugs have been shown to inactivate MGMT in cells, human tumour models and cancer patients, and O ⁶-benzylguanine and O ⁶-[4-bromothenyl]guanine have been used in clinical trials. While these agents show no side effects per se, they also inactivate MGMT in normal tissues and hence exacerbate the toxic side effects of the alkylating drugs, requiring dose reduction. This might explain why, in any of the reported trials, the outcome has not been improved by their inclusion. It is, however, anticipated that, with the availability of tumour targeting strategies and hematopoetic stem cell protection, MGMT inactivators hold promise for enhancing the effectiveness of alkylating agent chemotherapy.     

Targeting NADPH oxidases for the treatment of cancer and inflammation

NADPH oxidases are a family of oxidases that utilize molecular oxygen to generate hydrogen peroxide and superoxide, thus indicating physiological functions of these highly reactive and short-lived species. The regulation of these NADPH oxidases (nox) enzymes is complex, with many members of this family exhibiting complexity in terms of subunit composition, cellular location, and tissue-specific expression. While the complexity of the nox family (Nox1-5, Duox1, 2) is daunting, the complexity also allows for targeting of NADPH oxidases in disease states. In this review, we discuss which inflammatory and malignant disorders can be targeted by nox inhibitors, as well as clinical experience in the use of such inhibitors.     

Effect of a brain-specific protein (S-100 protein) on the nucleolar RNA polymerase activity in isolated brain nuclei

Riassunto Una proteina specifica del sistema nervoso, chiamata S-100, stimola la RNA polimerase nucleolare in nuclei isolati da encefalo immaturo di pollo (11 giorni di incubazione). La proteina non ha alcum effetto sulla RNA polimerase nucleoplasmica del medesimo sistema sperimentale. La stimolazione della S-100 sulla RNA polimerase nucleolare è -amanitina resistente ed è parzialmente antagonizzata dalla actinomicina. Ulteriori ricerche potranno chiarire il ruolo della proteina S-100 sulla espressione genetica del sistema nervoso.     

Understanding microtubule dynamics for improved cancer therapy

Microtubules (MTs), key components of the cytoskeleton, are dynamic polymers of tubulin that form a well-organized network of polarized tube filaments. MT dynamics are highly regulated both spacially and temporally by several MT-related proteins, themselves regulated by several kinases and phosphatases via signaling cascades, and also by coordinated interactions with actin cytoskeleton and adhesion sites. Regulation of MT dynamics is crucial for mitosis, cell migration, cell signaling and trafficking. MT-targeted drugs (MTDs), which constitute a major anticancer drug family with antimitotic and antiangiogenic properties, inhibit tumor progression mainly by altering MT dynamics in both cancer and endothelial cells. Identification of proteins regulating the MT network will lead to a better understanding of tumor progression regulators and will be helpful in improving cancer therapy. Received 22 July 2005; received after revision 8 September 2005; accepted 12 September 2005     

Telomeres and telomerase as targets for cancer therapy

Telomeres are protective structures located at the ends of all eukaryotic chromosomes. Telomere shortening upon cell division restricts the proliferative capacity of most normal human cells due to the lack of telomerase, an enzyme synthesizing telomeric DNA de novo. Since most tumor cells are reliant on the activity of telomerase to maintain the stability of predominantly short individual telomeres, inhibition of this enzyme presents an attractive approach for a mechanism-based anticancer therapy. Here, we review advances and obstacles in targeting telomerase and telomeres and discuss potential applications of such approaches for the clinic. Received 9 November 2006; received after revision 8 December 2006; accepted 17 January 2007