The present differential gene display analysis identified a number of genes with increased expression in relapsed AML. The increase of each mRNA varied between patients, in keeping with our recent study [
13] which revealed different response of signaling pathways coupled to translational control in a similar panel of patient AML cells. Also array-based AML gene expression profiling studies [
9,
22‐
24] underscore the heterogeneity of AML and the need for personalized therapy [
25,
26].
A strikingly high proportion of the genes with increased expression in relapsed AML (Fig.
1) were related to protein translation. One of them, termed ARAP, has sequence similarities to ribosomal protein P0 and has not previously been cloned. The elucidation of ARAP function and the role proteins involved in protein translation will be the subject of future studies.
Importantly, we found increased mRNA levels in relapsed AML for the general transcriptional co-activator lens epithelium derived growth factor (LEDGF)/p75 and its splice product p52 [
11]. The LEDGF/p75 mRNA was the most consistently upregulated in our series of relapsed AML. AML cells transfected with LEDGF/p75 or p52b were significantly more resistant to apoptosis induction
in vitro by either daunorubicin or the presumed physiological apoptogen cAMP (Fig.
5). Detailed studies in HEK293 cells showed that overexpression of LEDGF/p75 protected against daunorubicin-induced death to a similar extent as bcl-2 (Figs.
3,
4), suggesting that increased LEDGF/p75 might enhance chemotherapy resistance also in non-AML cells. This finding is consistent with LEDGF/p75 protecting cells against stress such as serum deprivation [
14]. LEDGF/p75 and p52 share the same 325 N-terminal residues, in which is found a PWWP domain (residues 1–100) interacting with stress response elements, a nuclear localization signal (residues 146 – 156), and an AT-hook motif (residues 179–198) [
14]. Although p52 only has 8 unique C-terminal amino acids, it can interact selectively with the splicing factor ASF/SF2 [
12]. The unique C-terminal 205 residues of LEDGF/p75 harbor a hepatoma derived growth factor homology domain that binds to chromatin even in the absence of a functional NLS [
27]. The pro-survival function of LEDGF/p75 is believed to depend on the 44 C-terminal residues since LEDGF/p75 (1–486) failed to protect hepatoma cells against serum deprivation [
21]. We found that p52b, which lacks the C-terminal residues of LEDGF/p75 (Fig.
2) did not induce apoptosis but our data suggest that it has some protective activity against daunorubicin-induced death (Fig.
4). This suggests that the C-terminal 205 residues of LEDGF/p75 were dispensable for protection against cell death. In fact, enforced expression of LEDGF/p75 (317–530) not only failed to protect against daunorubicin but induced significant death by itself (Fig.
4). The finding that p52 is not pro-apoptotic helps understand why patient leukemic cells with cytogenetic abnormality, t(9;11), involving the NUP98 gene and encoding NUP98-LEDGF/p75/p52 fusion proteins, have a high expression of both the LEDGF/p75 and p52 fusion products [
28‐
30].
LEDGF/p75 and p52 were also present in NB4 AML cells, in which we detected four additional novel p52 variants, three of which had deleted exon 6. One variant (p52bΔE6) lacked only exon 6, while the others (p52Δ1, p52bΔ1) also lacked exon 7 and parts of exons 5 and 8 (Fig.
2). Exon 6 codes for residues important both for the NLS and the AT-hook of LEDGF/p75, and its absence will therefore presumably abolish or alter the nuclear localization of p52. All three p52 splice variants lacking exon 6 enhanced rather than protected against daunorubicin- induced death (Fig.
4). This means that the LEDGF/p75 gene can create both pro- and anti-apoptotic proteins. We found that full-length LEDGF/p75 protected against all three pro-apoptotic splice variants of p52 as well as against the pro-apoptotic p75 (317–530) fragment of LEDGF/p75 (Fig.
4). This suggests that increased expression of full-length LEDGF/p75 may protect against pro-apoptotic p52 splice variants as well as against truncated LEDGF/p75 variants produced by caspase-dependent processing of LEDGF/p75 [
21]. The high expression of LEDGF/p75 in relapsed AML may therefore protect against apoptosis both by stimulating the transcription of other survival genes and by partially blocking the effect of pro-apoptotic shorter versions of LEDGF/p75 and p52.