14. Enhancement of Early Retrotransposon (ETn) RNA in Myeloid Leukemia Cells from C3H/He Mice

Hiroshi Ishihara and Izumi Tanaka

Keywords: retrotransposon, gene expression, early-transposon, VL30, intracisternal A-particle, DNA-protein binding

Acute myeloid leukemia (AML) cells from C3H/He mice induced by radiation have an active retrovirus-like retrotransposon, the intracisternal A-particle (IAP) element. Examples of an accumulation of IAP RNA and genomic aberration by the IAP-mediated retrotransposition in AML cells were reported previously. Since the function of the retrotransposon is inhibited in normal and most tumor cells, the AML cells may lack mechanisms to repress the retrotransposons. To confirm this possibility, we analyzed the transcription of other mouse retrotransposons, the early retrotransposon (ETn) and virus-like 30S (VL30) particle elements in the AML cells.

All the AML lines from different mice commonly overexpressed the ETn RNA (Fig. 15 (a) ). The ETn is known as an inactive transposon since the expression is limited to only early embryonic cells. As with other tumor cells, only faint levels of ETn RNA were detected in the cells from other tumors including hepatoma and lymphoma from C3H mouse. By structural analyses of ETn cDNA clones, it was revealed that the ETn RNA molecules overexpressed in AML cells had complete forms except for a larger variation in the polyadenylation sites at the 3'-end of the molecule.

To determine the nucleotide sequence that contributes to the transcription of ETn, electrophoretic mobility-shift assay using the long-teminal repeat sequence of ETn and the nuclear extract of the AML cells was performed. The target sequence against the nuclear protein was the C-rich nucleotide sequence positioned 30 nucleotides upstream from the transcriptinal start site (Fig. 15 (b) ). The C-rich sequence did not have any similarity with previously reported target motifs for enhancers.

Since ETn does not have any open-reading frame, a biological effect due to the overexpression can be ignored in AML cells. However, this finding shows that the cells of all the leukemia lines are activated by a common mechanism that simultaneously transcribes ETn. The activation of this mechanism is probably necessary for the leukemic transformation. Thus the ETn LTR function is useful to study the leukemogenetic process in myloid cells.

Publications:
Tanaka, I. and Ishihara, H. Virology, 280, 107-114, 2001.

Fig.15. Overexpression of ETn in AML cells and binding of ETn LTR sequence to nuclear extracts. (a). RNAs from normal liver cell from 2 different mice, 4 lines of primary hepatic tumor cells, 6 lines of primary AML cells, 4 lines of AML strain cells and 4 lines of primary thymyc lymphoma cell were used in northern analysis probing ETn. (b). Mixtures of the end-labeled double-stranded oligonucleotides (block-1 to 11, shown in numbered boxes on both sides of the arrow) corresponding to ETn LTR (arrow) and nuclear extract from spleen (S) or leukemia lines (L1 or L2) were used for the EMSA assay until free DNAs (F) were overrun from the gel. Areas of gels where bands are retarded are shown as (B). Nucleotide sequence that can bind to the nuclear protein (corresponding to block-7 to 9) is indicated at the bottom.