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UBC Theses and Dissertations

Ex vivo bone marrow purging using BPD-mediated photodynamic therapy Yip, Stephen


Photodynamic therapy (PDT) using the second generation photosensitiser benzoporphyrin derivative monoacid ring- A (BPD, Verteporfin®) offers an attractive alternative to purging of contaminating neoplastic cells during autologous haematopoietic stem cell transplantation. Enhancement of PDT cytotoxicity was attempted using two independent approaches: combination treatment with doxorubicin (Dox) and selective protection of normal haematopoietic cells using the tetrapeptide N-AcSDKP. The murine leukaemic cell line L1210 was 45 x more susceptible to the sequenced combination regimen of 1 h 2.5 μM Dox incubation followed by PDT mediated by 5.0 ng/ml BPD and 15 J/cm² red light (Dox -> PDT) than normal murine haematopoietic cells. The significant enhancement in cytotoxicity was dependent on the concentration of BPD used as well as on the sequence of treatment. Specifically, it was observed with 5.0 ng/ml but not with 2.5 ng/ml BPD and only when Dox was used before PDT. The reverse sequence of PDT -> Dox and simultaneous Dox/PDT treatment were not associated with enhanced killing. Interestingly, L1210 cells were much more susceptible to the combination therapy than normal DBA /2 haematopoietic progenitor cells which offered interesting therapeutic implications. BPD uptake and cellular GSH content, appeared not to be responsible for the potentiation of L1210 killing in the Dox-> PDT sequence. Next, the potential of selective stem cell protection in BPD - mediated PDT was investigated. Preincubation of DBA /2 bone marrow cells with 100 nM N-AcSDKP for 1.5 h significantly protected resultant colony formation from PDT by a factor of 1.5- 2 over cells that were incubated with control peptides or with tissue culture medium. Interestingly, L1210 cells were not protected by N-AcSDKP and the control peptides. However, N-AcSDKP mediated photoprotection did not appear to extend to earlier murine haematopoietic cells and stem cells as demonstrated by the long- term bone marrow culture (LTBMC ) assay. The same findings of differential photoprotection were also demonstrated in human haematopoietic cells. The mechanism of protection appeared to be mediated by inhibition of progression to S phase of the cell cycle since depletion of cycling DBA /2 bone marrow cells with 50 μM cytosine arabinoside (ara- C) resulted in cells more tolerant to subsequent PDT cytotoxicity. The above two approaches enhanced selective BPD- mediated PDT cytotoxicity and therefore maybe of merit in the clinical use of PDT in purging.

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