Over the last decade, there has been increasing interest in mechanisms that underlie genomic instability which is implicated in tumor progression, development of drug resistance and possibly even oncogenesis. We have observed that dysregulated homologous recombination (HR) is an important contributor to genomic instability. In this study, we aim to broaden our understanding of HR by exploring alternate pathways that may affect genomic instability such as nuclease activity.

Methods

We have developed an assay to measure total cellular nuclease activity and compare activity levels across a spectrum of plasma cell dyscrasias. Total nuclease activity in cell lines and patient samples was assessed using a novel plasmid degradation assay. Briefly, multiple myeloma (MM) cell lines and primary MM patient cells were lysed at equal cell concentration. The lysates were mixed with plasmid DNA in triplicate/quadruplicate and incubated at 37°C for fixed time points ranging from 0 to 30 minutes. Following forced cessation of enzymatic activity, samples were fractionated on agarose gel and resultant image analyzed for band intensity by Kodak 1D Image analysis software. The ratio of degraded plasmid over time was analyzed via nonlinear regression to calculate a half-life and k-constant.

Results

Non-linear regression analysis of data derived from assays involving 6 MM cell lines (RPMI, OPM1, H292, U266, INA6 & MM1S) with three pellets each of equal size taken from non-confluent cell culture with > 85% viability showed statistically significant reproducibility of best fit curve with an average R-squared of 0.93. Similar regression analysis could not be performed on data derived from normal PBMC pellets because they showed minimal degradation with linear regression slope of (-0.62). Lastly, non-linear regression analysis on 22 patient samples with a plasma cell dyscrasia diagnosis, revealed a broader range of activity by half-life. Further differentiation of plasma cell dyscrasia patients into 2 groups revealed a significantly higher half-life in MGUS & smouldering (9499.8 minutes) versus newly diagnosed and refractory or relapsed multiple myeloma patients (9.4,19.3 minutes respectively).

Conclusions

In conclusion, we show that nuclease activity, implicated in elevated recombination and genomic instability, can be measured in a statistically reproducible fashion by our in vitro assay and may be used to differentiate plasma cell dyscrasias across a clinical spectrum. Further analysis of nuclease activity in clinically annotated samples for correlation with clinical features, genomic data and survival outcomes is ongoing.

Disclosures:

Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Topics:
nuclease, plasma cell disorder, gel, multiple myeloma, carcinogenesis, cell culture techniques, lysate, monoclonal gammopathy of undetermined significance, sepharose, signs and symptoms

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2013 by The American Society of Hematology
2013
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