RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines

Narendrula, Rashmi; Mispel-Beyer, Kyle; Guo, Baoqing; Parissenti, Amadeo M.; Pritzker, Laura B.; Pritzker, Ken; Masilamani, Twinkle; Wang, Xiaohui; Lannér, Carita

In this study, we have documented that the ability of specific chemotherapy agents to induce cell death/ growth arrest in a clonogenic assay is also reflected in their ability to induce RNA disruption in tumor cells in vitro (Fig. 5) . Our data thus suggests that RNA disruption by chemotherapy agents accurately reflects cellular drug sensitivity and drug resistance (as measured using the highly sensitive clonogenic assay). This reinforces the usefulness of the RNA disruption assay (RDA) as a technique for quantifying response to cytotoxic agents such as chemotherapy drugs. Moreover, the RNA disruption assay appears to be equivalent in sensitivity to the clonogenic assay in evaluating response to treatment, is much less labor intensive, is amenable to high throughput approaches, and requires less time to obtain assay results. It may thus represent a valuable tool for drug discovery. The observations of our current study (See figure on previous page.) Fig. 7 Recovery and proliferation of A2780 cells following docetaxel treatment. In order to assess whether the A2780 cells treated with docetaxel (DXL) that show RNA degradation are able to recover and proliferate, cells were treated with 0, 0.005 or 0.2 μM docetaxel for 24, 48 and 72 h. Following the treatment end point, cells were collected and replated in fresh drug-free medium, and their proliferation was assessed following recovery for 24, 48, 72 and 96 h of replating. a Recovery of cells after 24 h treatment. b Recovery after 48 h drug treatment. c Recovery following 72 h drug treatment also provide in vitro confirmation of the association of RNA disruption with response to chemotherapy in patients with locally advanced breast cancer [18, 20] . Northern blots of total RNA isolated from A2780 cells treated with docetaxel were used to identify the approximate origins of the RNA disruption bands. As described above, the 28S-5 probe hybridized to the fulllength 28S rRNA and to two 28S rRNA fragmentsone migrating faster than the full-length 18S rRNA (1,870 nt) and one migrating between the intact 28S (5,025 nt) and 18S rRNA bands (Fig. 4a) . Their sizes were determined to be 1,630 nt and 3,012 nt respectively (although these calculated sizes should be interpreted as approximate values). Since both fragments were detected by the most 3' probe (28S-5), they must have been derived from the 3'-end of the 28S rRNA, with the smaller fragment derived from the larger (Fig. 4b) . The size of 3,012 nt for the larger fragment suggests that there must be a cleavage site within the full-length 28S rRNA sequence approximately 2,013 nt from the 5' end of the molecule (5,025 minus 3,012 nt). The smaller fragment (1,630 nt) indicates another cleavage site located 3,395 nt from the 5' end of the molecule (5,025 minus 1,630 nt; see Fig. 4b ). This interpretation suggests that another 28S rRNA fragment of about 2,013 nt is produced upon docetaxel treatment (2,013 nucleotides from the 5'-end of the molecule), which was not detected by any of the other probes (28S CD1, 28S VR2, and 28S-1). The reason for this is not clear. It is possible that this 2,013 nt fragment, when generated, is not stable and/or is subjected immediately to RNase digestion. In support of this idea, Mroczek and Kufel report that some rRNA fragments are susceptible to digestion by the exosome while other fragments are not [16] . The cleavage site at 3,395 nt from the 5' end of the 28S rRNA is located in variable region (expansion segment) 8,