|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1996 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1995 : ¥1,200,000 (Direct Cost : ¥1,200,000)
As an alternative treatment with allogeneic bone marrow transplantation, the possibility of autologous stem cell transplantation for chronic myelogenous leukemia (CML) was studied. The most critical problem in this study is how to purify normal stem cells from autologous marrow or blood cells. Although normal stern cells are present in these patients in chronic phase, absolute amount of these cells are very small and undetectable by regular chromosome analysis.
In the first two reports, we investigated the effect of long-term marrow culture (LTMC) to eliminate leukemic clones. In the first report, we followed the previous report that appearance of Philadelphia chromosome (Ph) negative clones was observed after LTMC of CML patients. We found that addition of interferon-alpha (IFNalpha) in LTMC preferentially decrease Ph cells. In the second study, we investigated the frequencies of leukemic clones in suspending- and adherent- fraction of LTMC by reverse transcriptional-polymerase chain r
eaction (RT-PCR) for bcr-abl, CML specific transcript, and chromosome analysis of a single progenitor derived colony. Addition of IFNalpha in LTMC of CML bone marrow accelerated the elimination of leukemic clones preferentially in the adherent fraction of the culture.
In the third presentation, we investigated the effect of hyperthermic treatment on clonogenic growth of Ph^+ leukemia cell lines, K562 and KU812. Hyperthermic treatment suppressed the clonogenic activities of both cell lines. Addition of AZT,IFNalpha, TNF,and quercetin enhanced the effect of hyperthermic clonogenic inhibition. The optimal in vitro hyperthermia purging technique is heating at 42ﾟC for 1 hour with combined IFN-alpha (100 U/ml) and AZT (0.5 muM) or quercetin (50 muM/ml), depending on the sensitivity of the CML cells to be eliminated. Effect of hyperthermia purging was associated with cellular apoptosis shown by DNA ladder formation in agarose electrophoresis.
For further clinical application of these technique, ex vivo expansion of human hematopoietic stem cells is necessary because only a small number of normal stem cells are available after culture. The last section in the second paper is showing the results of preliminary experiment in this field. Our data represented a possibility of expansion of human hematopoietic progenitors in vitro. Less