Selective Expression of a Number of ABC Transporter Genes in Normal CD34⁺CD38⁻ Versus CD34⁺CD38⁻ Cells, with a Reverse Pattern in a Subgroup of AML Patients.

Several ABC transporters involved in drug transport have been identified in hematopoietic stem cells, including ABCB1, ABCC1 and ABCG2. The ABC transporters play a role in chemotherapy resistant AML, although the relevant information is mostly obtained from the total AML cell population instead of the leukemic stem cells characterized by the CD34⁺CD38⁻ phenotype. In this study we investigated which ABC transporters are selectively expressed in normal CD34⁺CD38⁻ hematopoietic stem cells versus CD34⁺CD38⁺ cells, and to what extent lineage-restricted modulation is aberrantly regulated in AML stem cells. We first investigated murine microarray expression data of 29 ABC transporter genes in lin⁻sca-1⁺c-kit⁺ cells (available on www.webqtl.org). Based on these data 7 of the 29 ABC transporters were selected with a high expression profile (abcg1, abcb2, abca2, abcd1, abcc3, abcc5, and abcg2). Based on data published at www.sciencemag.org/cgi/content/full/1073823/DC1, concerning the lineage restricted expression of genes in lin⁻AA4.1⁺ + c-kit⁺sca-1⁺ murine stem cells, 6 additional stem cell-related ABC transporters (abcb1, abcb11, abcc1b, abcd4, abce1 and abcf2) were selected. The mRNA expression of the 13 ABC transporters was analyzed in the CD34⁺CD38⁻ versus CD34⁺CD38⁺ fraction of human normal bone marrow cells (n=10) by quantitative RT-PCR. Five ABC transporter genes were not detectable in the human CD34⁺CD38⁻ and CD34⁺CD38⁺cells (ABCA2, ABCB11, ABCC3, ABCD1 and ABCF2). Three ABC transporters were expressed equally in both fractions (ABCC5, ABCE1 and ABCG2). However, five ABC transporters were differentially expressed, with a higher expression in the CD34⁺CD38⁻ cells, (ABCB1, ratio of CD34⁺CD38⁺/CD34⁺CD38⁻ expression of 0.22, p<0.001; ABCG1, 0.27, p<0.001; ABCC1, 0.52, p<0.001; ABCD4, 0.60, p<0.001; and ABCB2, 0.71, p<0.02).

Additionally these five ABC transporters were studied in sorted AML subpopulations (n=7). In the sorted AML cells (CD34⁺CD38⁻ versus CD34⁺CD38⁺) a more heterogeneous expression pattern was observed as compared to normal CD34⁺CD38⁻ cells. In general, the expression levels of ABCB1 and ABCC1 in the AML subpopulations were lower than in normal CD34⁺CD38⁻ cells, ABCB2 expression was higher in the AML fractions and ABCG1 and ABCD4 were expressed similar in AML and normal CD34⁺CD38⁻ cells. Downregulation of the ABC transporters in the leukemic CD34⁺CD38⁺ cells was observed in 50%–60% of the samples, the reverse pattern was observed for the remaining cases, independent of FAB classification. Since ABCG1 plays a prominent role in cholesterol transport and was strongly downregulated in normal CD34⁺CD38⁺ cells (ratio 0.27, p<0.001), the mRNA expression of a number of additional cholesterol synthesis genes was investigated. PPARβ, LXRα and HMCGCoA reductase appeared to be downregulated in the CD34⁺CD38⁺ cells (ratios of 0.59, p=0.002, 0.32, p<0.001 and 0.59, p= 0.002 respectively). In conclusion, these results indicate that cholesterol synthesis and transport might play an important role in hematopoietic stem cells. Furthermore, a number of ABC transporter genes appeared to be predominantly expressed in hematopoietic stem cells, and are downregulated upon maturation, whereas the reverse pattern is observed in about 40% of the AML patients suggesting that these more committed leukemic cells might have gained some properties of the leukemic stem cells.