Evidence for keratinocyte stem cells in vitro: Long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes

Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in β1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1–27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo, β-galactosidase (β-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3–33.3% of the colonies thus formed were β-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were β-gal negative also lacked the lacZ transgene. These results show that cells with long-term growth properties in vitro do indeed persist in vivo and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.

The t(8;21) chromosomal translocation in acute myelogenous leukemia modifies intranuclear targeting of the AML1/CBFα2 transcription factor

Targeting of gene regulatory factors to specific intranuclear sites may be critical for the accurate control of gene expression. The acute myelogenous leukemia 8;21 (AML1/ETO) fusion protein is encoded by a rearranged gene created by the ETO chromosomal translocation. This protein lacks the nuclear matrix-targeting signal that directs the AML1 protein to appropriate gene regulatory sites within the nucleus. Here we report that substitution of the chromosome 8-derived ETO protein for the multifunctional C terminus of AML1 precludes targeting of the factor to AML1 subnuclear domains. Instead, the AML1/ETO fusion protein is redirected by the ETO component to alternate nuclear matrix-associated foci. Our results link the ETO chromosomal translocation in AML with modifications in the intranuclear trafficking of the key hematopoietic regulatory factor, AML1. We conclude that misrouting of gene regulatory factors as a consequence of chromosomal translocations is an important characteristic of acute leukemias.

ETO, fusion partner in t(8;21) acute myeloid leukemia, represses transcription by interaction with the human N-CoR/mSin3/HDAC1 complex

The t(8;21) translocation between two genes known as AML1 and ETO is seen in approximately 12–15% of all acute myeloid leukemia (AML) and is the second-most-frequently observed nonrandom genetic alteration associated with AML. AML1 up-regulates a number of target genes critical to normal hematopoiesis, whereas the AML1/ETO fusion interferes with this trans-activation. We discovered that the fusion partner ETO binds to the human homolog of the murine nuclear receptor corepressor (N-CoR). The interaction is mediated by two unusual zinc finger motifs present at the carboxyl terminus of ETO. Human N-CoR (HuN-CoR), which we cloned and sequenced in its entirety, encodes a 2,440-amino acid polypeptide and has a central domain that binds ETO. N-CoR, mammalian Sin3 (mSin3A and B), and histone deacetylase 1 (HDAC1) form a complex that alters chromatin structure and mediates transcriptional repression by nuclear receptors and by a number of oncoregulatory proteins. We found that ETO, through its interaction with the N-CoR/mSin3/HDAC1 complex, is also a potent repressor of transcription. This observation provides a mechanism for how the AML1/ETO fusion may inhibit expression of AML1-responsive target genes and disturb normal hematopoiesis.

Synergistic up-regulation of the myeloid-specific promoter for the macrophage colony-stimulating factor receptor by AML1 and the t(8;21) fusion protein may contribute to leukemogenesis.

AML1 is involved in the (8;21) translocation, associated with acute myelogenous leukemia (AML)-type M2, which results in the production of the AML1-ETO fusion protein: the amino-terminal 177 amino acids of AML1 and the carboxyl-terminal 575 amino acids of ETO. The mechanism by which AML1-ETO accomplishes leukemic transformation is unknown; however, AML1-ETO interferes with AML1 transactivation of such AML1 targets as the T-cell receptor beta enhancer and the granulocyte-macrophage colony-stimulating factor promoter. Herein, we explored the effect of AML1-ETO on regulation of a myeloid-specific AML1 target, the macrophage colony-stimulating factor (M-CSF) receptor promoter. We found that AML1-ETO and AML1 work synergistically to transactivate the M-CSF receptor promoter, thus exhibiting a different activity than previously described. Truncation mutants within the ETO portion of AML1-ETO revealed the region of ETO necessary for the cooperativity between AML1 and AML1-ETO lies between amino acids 347 and 540. Endogenous M-CSF receptor expression was examined in Kasumi-1 cells, derived from a patient with AML-M2 t(8;21) and the promonocytic cell line U937. Kasumi-1 cells exhibited a significantly higher level of M-CSF receptor expression than U937 cells. Bone marrow from patients with AML-M2 t(8;21) also exhibited a higher level of expression of M-CSF receptor compared with normal controls. The upregulation of M-CSF receptor expression by AML1-ETO may contribute to the development of a leukemic state in these patients.