We have evaluated the efficacy of the multinuclear platinum chemotherapeutics BBR3464, BBR3571, and BBR3610 against glioma cells in culture and animal models and investigated their mechanism of action at the cellular level. In a clonogenic assay, BBR3610, the most potent compound, had an IC90 dose (achieving 90% colony formation inhibition) that was 250 times lower than that of cisplatin for both LNZ308 and LN443 glioma cells. In subcutaneous xenografts of U87MG glioma cells, BBR3610 approximately doubled the time it took for a tumor to reach a predetermined size and significantly extended survival when these cells were implanted intracranially. Analysis of apoptosis and cell cycle distribution showed that BBR compounds induced G2/M arrest in the absence of cell death, while cisplatin predominantly induced apoptosis. Interestingly, the BBR compounds and cisplatin both induced extracellular signal-regulated kinase 1/2 phosphorylation, and inhibition of this pathway at the level of MEK antagonized the induction of G2/M arrest or apoptosis, respectively. Analysis of Chk1 and Chk2 status did not show any differential effects of the drugs, and it is thus unlikely to underlie the difference in response. Similarly, the drugs did not differentially modulate survivin levels, and knockdown of survivin did not convert the response to BBR3610 to apoptosis. Together, these findings support continued development of BBR3610 for clinical use against glioma and provide a framework for future investigation of mechanism of action.

Successful clinical development of cancer treatments is aided by the development of molecular markers that allow the identification of patients likely to respond. In the case of broadly cytotoxic drugs, such as the multinuclear series of platinum chemotherapeutic agents that we are evaluating for the treatment of glioma, one route to marker identification is proteomic profiling. We are using the two-dimensional chromatography system, the ProteomeLab PF2D, to compare proteomic profiles of glioma cells in culture before and after drug treatment. The existing software tools allowed the rapid identification of peaks increased by treatment of a given drug as compared with control untreated cells. To compare across these pairs, we developed new software, called the MetaComparison Tool (MCT). The MCT uses the chromatographic characteristics of peaks as identifiers, an approach that was validated by mass spectrometry of two independent isolations of a peak, from cells that were treated with two different platinum compounds. The MCT made it possible to rapidly query whether a given peak responded to more than one treatment and so allowed the identification of peaks that were specific to a given drug. As a result, this analysis greatly reduced the list of peaks whose isolation and downstream analysis by mass spectrometry is warranted, accelerating the search for protein markers of response. Molecular & Cellular Proteomics 5:35-42, 2006.The successful clinical deployment of anticancer drugs relies on being able to identify patients whose tumors are likely to respond. In the case of drugs developed against tumorspecific molecules, detection of the relevant target is the natural approach. For more broadly cytotoxic therapies, including platinum chemotherapeutic agents, such a direct approach is not possible, and so markers of likely response must be identified by molecular comparisons. In this work we present an approach to comparing the response of glioma cells to novel platinum drugs at the protein level as a first step toward this goal. We show that proteome displays generated by two-dimensional liquid chromatography can be compared using new computer software, the MetaComparison Tool (MCT), 1 and that this approach can rapidly lead to the identification of peaks worth further analysis as proteins whose expression is responsive to drug treatment.The need for new clinical agents for the treatment of glioma has led us to investigate the potential of a newer class of platinum compounds, which are multinuclear, and whose clinical profile and mechanism of action are quite distinct from the established platinum compounds. The first member of this class, the trinuclear platinum BBR3464, is effective against cells with an inherent or acquired resistance to cisplatin, including glioma cells (1, 2), suggesting that there are important differences in their mode of action. BBR compounds, exemplified by BBR3464, show increased rates of cellular uptake and more rapid formation of DNA adducts than cisplatin and a different pattern of ad...

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Lack of functional pRb results in attenuated recovery of mRNA synthesis and increased apoptosis following UV radiation in human breast cancer cells. We have previously demonstrated that a human breast cancer cell line, MDA-MB-468, which lacks the retinoblastoma protein (pRb), is particularly sensitive to low doses of ultraviolet (UV) radiation. These cells are 15 ± 20-fold more sensitive to UV radiation than cells with wild-type pRb. In order to understand the mechanisms of the high apoptotic response of MDA-MB-468 cells to UV radiation, we examined the e ects of UV on these cells with regards to both membrane-mediated events and DNA damage. We found that MDA-MB-468 cells were resistant to all ligandinduced death receptor signaling. In addition, although UV activated caspase 8 in MDA-MB-468 cells, a peptide inhibitor of caspase 8 failed to inhibit UV-induced apoptosis. We then tested the possibility that nuclear events mediated the enhanced sensitivity to UV-induced apoptosis in these cells. Unlike UV-resistant cells, MDA-MB-468 cells were unable to recover mRNA synthesis after 5 J/m 2 UVC. We also found that the pRb-null DU-145 cells similarly had attenuated recovery of mRNA synthesis after UV radiation. In UV-resistant cells with wild-type pRb, the inactivation of pRb with HPV-16 E7 resulted in signi®cant inhibition in their ability to recover mRNA synthesis and increased levels of apoptosis following UV radiation. Furthermore, pRb-null cells were de®cient in repair of UV radiation-induced DNA damage. These data suggest that the sensitivity of MDA-MB-468 cells to UV radiation is due to defects in repair of DNA damage and recovery of mRNA synthesis rather than to membrane death receptor pathways. Inactivation of pRb may contribute to an increased sensitivity to UV radiation by attenuating repair of DNA lesions and recovery of mRNA synthesis following UV radiation.