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Dear IMF:

I am writing to give you an update on the project titled "DNA Double Strand Break Repair By Non-Homologous End Joining (NHEJ) In Multiple Myeloma," which had been awarded the 2001 IMF Junior Research Grant for the year 2000 in memory of Sharon Newman.

In February of 2001, I took a position at Loyola University?s Stricht School of Medicine as an Assistant Professor in the division of Hematology and Oncology, section of Bone Marrow Transplantation. The project was submitted to and approved by the Cancer Center protocol review committee and then submitted to the Loyola University?s Institutional Review Board, which gave the final approval.

I have been extremely fortunate in getting excellent mentorship from Dr. Andrew Vaughan, who is an expert in the field of apoptosis and DNA repair by non-homologous end joining. Dr. Vaughan has a specific interest in the mechanisms of translocation formation, concentrating on elements of the apoptotic and NHEJ pathways. Under his guidance, we have made some novel observations in multiple myeloma cell lines which we are eager to carry through and study in primary myeloma patient samples. Hopefully, this will give us new insights into the management of multiple myeloma.


  1. Study the expression of NHEJ proteins in MM cell lines.
  2. Evaluate NHEJ function in MM cell lines ex vivo.
  3. Determine the relationship between karyotypic instability and NHEJ activity in myeloma cell lines.
  4. Study NHEJ protein expression and function in primary MM samples.

Our preliminary studies conducted on multiple myeloma cell lines had indicated that two cell lines, RPMI 8226 and ARK, had reduced levels of XRCC4 expression as determined by Western blotting using XRCC4 MoAb. In addition a truncated, ~34kD transcript was seen in both these cell lines. This data suggested that XRCC4 expression is abnormal in some MM cell lines. This may result from transcriptional abnormalities in XRCC4 expression or as a result of a truncating XRCC4 mutation that may render the molecule nonfunctional.


Following up on these XRCC4 abnormalities, we conducted functional DNA double strand break repair assays to confirm their significance. These DNA repair assays were performed using total cellular protein extracts. Briefly an LM-PCR strategy was employed to evaluate the efficiency of ligation of a double stranded oligonucleotide linker to a linearized vector. This was followed by PCR, using one primer complimentary to the linker and one primer complimentary to the vector. Samples with successful ligation demonstrated a bright PCR band.

For this procedure, cells were collected and cellular extracts were prepared by a modification of the protocol described by Baumann et al (PNAS, 1998; 95(24):14066). Extracts were snap-frozen in liquid nitrogen and stored at ?80°C. Protein concentration was determined and equal protein was used for each assay. Substrate vector was prepared by digesting pCR-Script Amp with Srf I. The enzyme was then inactivated and linearized vector purified using a QiaQuick PCR Purification kit. A double-stranded linker was consructed, with a linker 25 bp oligo (5?-gcggtgacccgggagatctgaattc-3?) and linker 11 bp oligo (5?-gaattc agatc-3?). Repair reaction was carried out using linearized pCR-Script, double-stranded linker, ATP, and protein extract. Control samples (to rule out repair activity by other mechanisms, e.g. homologous recombination etc.) were pre-treated with wortmannin to inhibit DNA-PKcs, and linearized pCR-Script was added subsequent to pretreatment. Other controls included DNA ligase as well as DNA ?PKcs incompetent and wild type cell lines (MO59K and MO59J). Circular vector as well as water controls were used as well.

Ligation Mediated-PCR amplification of the ligation reaction product was performed using repair reaction products, linker 25 oligo and rep1.1 oligo (5?-ggagcccccgatttagagcttgacg-3?). The products were size-fractionated on a 2.0% agarose gel and visualized by ethidium bromide staining.


Using this technique 3 cell lines have been analyzed: RPMI-8226, U266, and OPM-2. Whereas a clear band is seen when protein extracts from U266 and OPM-2 are used in LM-PCR, a very faint band, if any, is seen in the reaction using extracts from RPMI-8226. When controls with wortmannin were performed inhibiting DNA-PKcs kinase activity, DNA ligation was abrogated in all 3 myeloma cell lines as well as in the controls, indicating that the repair activity was mediated by NHEJ. These observations have been confirmed in repeated experiments. This finding is indicative of impaired capability of DNA double strand break repair by non-homologous end joining in cells from the multiple myeloma cell line RPMI-8226. Indeed this finding corroborates the earlier finding of reduced expression and possible truncation of XRCC4 in this cell line.

Having identified a functional deficit in DNA double strand break repair by NHEJ in this cell line, further work is planned to evaluate this in other myeloma cell lines. For this purpose, we are trying to obtain myeloma cell lines ARK, ARP1, HS Sultan. Further work is being conducted to:

  1. Identify the truncating mutation in XRCC4. This will be done using RT-PCR using primers spanning short distances on the mRNA molecule, to isolate the truncated version (if any). Subsequently, sequencing of the transcripts may be performed to identify possible mutations. If a truncating mutation is identified, the readily applicable RT-PCR assay will be applied to patient samples to detect XRCC4 abnormalities in primary myeloma samples.
  2. Assess the fidelity of DNA double strand break repair using a reporter assay utilizing a linearized plasmid with a double strand break introduced in the beta-galactosidase insert of this construct. Colored colonies will give a semi-quantitative estimate of repair of the plasmid and give an evaluation of the fidelity of the repair reaction.


Once further data has been generated consolidating the novel findings described here, further funding will be sought from other extramural sources to expand this study and evaluate the role of DNA double strand break repair in the pathogenesis of multiple myeloma. Since this assay is readily applicable to clinical samples of purified plasma cells, it may give invaluable insight into the origin of complex karyotypic abnormalities in malignant plasma cells.

With sincere regards,

Amir A. Toor, M.D.
Assistant Professor of Medicine
Division of Hematology/Oncology
Cardinal Bernardin Cancer Center
Loyola University Medical Center
Maywood, Illinois