Ernst, Russell J. (2011) The biological activity of rhodium metalloinsertors. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:05272011-113632785
Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. The loss of MMR carries dire consequences, including increased mutation rates, carcinogenesis, and resistance to a variety of clinical anti-cancer agents, such as cisplatin and DNA alkylators. Rhodium metalloinsertors previously developed in our laboratory bind to DNA mismatches with high affinity and specificity, and represent a promising strategy to target mismatches in cells. Thus, uncorrected mismatches can be exploited to provide a basis of discrimination between MMR-deficient, cancerous cells and MMR-proficient, healthy cells. Here we describe the application of rhodium metalloinsertors to inhibit cellular proliferation selectively in MMR-deficient cells compared to those that are MMR-proficient. The colorectal carcinoma cell lines HCT116N and HCT116O serve as an isogenic model system for MMR deficiency. We show that the Δ-isomer of an octahedral rhodium complex containing a bulky chelate ligand for insertion into a DNA mismatch is active both in targeting base mismatches in vitro and in inhibiting DNA synthesis selectively in the HCT116O cell line. A family of derivative complexes with varying ancillary ligands has also been synthesized, and both DNA mismatch binding affinities and anti-proliferative activities against the HCT116 cell lines have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatched sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M-1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. In particular, rhodium metalloinsertors bearing dipyridylamine ancillary ligands are shown to exhibit accelerated cellular uptake. This increased uptake allows us to observe additional cellular responses to these agents, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that also occur preferentially in the HCT116O cell line. Finally, these cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents, and are consistent with the idea that repair proteins are activated in response to DNA mismatch binding.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||Rhodium metallinsertor, DNA mismatch repair, anti-cancer|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Major Option:||Biochemistry and Molecular Biophysics|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||16 May 2011|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Russell Ernst|
|Deposited On:||31 May 2011 23:06|
|Last Modified:||18 Jan 2013 00:48|
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Restricted to Caltech community only until 1 June 2013.
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