A Novel MDMX Transcript Expressed in a Variety of Transformed Cell Lines Encodes a Truncated Protein with Potent p53 Repressive Activity

Abstract

The MDMX gene product is related to the MDM2 oncoprotein, both of which interact with the p53 tumor suppressor. We have identified a novel transcript of the MDMX gene that is expressed in a variety of cell lines, and in particular, in growing and transformed cells. This transcript is identical to the published sequence yet it has a short internal deletion of 68 base pairs. This deletion produces a shift in the reading frame after codon 114, resulting in the inclusion of a stop codon at amino acid residue 127 (full-length MDMX is 489 residues). This truncated MDMX protein is termed MDMX-S ("short form"), represents only the p53-binding domain, and appears to bind p53 better than full-length MDMX. The MDMX-S protein can be detected in cell extracts and when overexpressed is much more effective than MDMX at inhibiting p53-mediated transcriptional activation and induction of apoptosis. Since MDMX-S lacks the central and carboxyl-terminal regions contained within full-length MDMX, it is likely to play a key role in the regulation of cell proliferation and apoptosis in a way distinct from MDMX.The p53 tumor suppressor protein plays an important role in regulating movement through a number of cell cycle checkpoints (1-3, 38). The activity of p53 is in turn modulated through the action of the MDM2 protein which is amplified in a variety of tumors (4 -6). MDM2 can associate with p53 (7) and directly inhibit p53's ability to activate transcription of target genes, such as p21. MDM2 performs this function in a number of ways. First, the amino-terminal domain MDM2 binds tightly to the transcriptional transactivation domain of p53 (4 -6, 8, 9). Through this interaction, MDM2 blocks the ability of p53 to activate transcription of specific target genes by repressing the formation of the preinitiation complex mediated through the TFIIE and TATA-binding protein subunits of RNA polymerase II (10, 11). The second blocking activity of MDM2 is that it targets p53 for proteolytic degradation by the ubiquitin-proteasome pathway (12)(13)(14)40). Third, MDM2 also functions to shuttle p53 out of the nucleus and into the cytoplasm, via a nuclear export signal located within the MDM2 protein (33).While MDM2 was first identified as a protein that inhibits the action of p53 it has recently been shown to affect other components of the G 1 /S transition. MDM2 was found to associate with both the E2F1 transcription factor (15) and the retinoblastoma tumor suppressor (pRb) 1 (16). The interaction with pRb was found to be repressive, in that the association of MDM2 with the carboxyl-terminal domain of pRb inhibited the growth regulatory function of pRb (16). The interaction with E2F1, over the amino terminus of MDM2 and the carboxyl terminus of E2F1, was stimulatory in the sense that MDM2 enhanced the ability of E2F1 to activate target gene expression (15). Thus MDM2 appears to regulate at least three of the important players in the G 1 /S transition: p53, pRb, and E2F1. It has therefore been concluded that MDM2 functions in the fashion of a domi...

Significance of HDMX‐S (or MDM4) mRNA splice variant overexpression and HDMX gene amplification on primary soft tissue sarcoma prognosis

“…The calculated weight of the HDMX-S protein is 15 kDa, and the difference compared to the apparent weight observed after Western analysis suggests that HDMX-S undergoes posttranslational modification. 12 In our analysis, HDMX-S protein could be detected in cell lysates isolated from the samples that also showed elevated HDMX-S/FL-HDMX mRNA ratios. The band intensities have been analyzed by means of densitometry and were normalized against the actin expression levels within the same sample.…”

“…However, this association was not observed in anaplastic gliomas and low-grade gliomas. 8 Studies of Rallapalli et al 12 and de Graaf et al 13 revealed that the truncated proteins encoded by both MDMX-S and HDMX-S are more efficient at suppressing p53-mediated transactivation and induction of apoptosis than FL-MDMX or FL-HDMX, respectively. It is therefore conceivable that the upregulation of HDMX-S might contribute to the neoplastic transformation of cells by inactivating wt-p53.…”

“…Regarding the correlation of HDMX-S overexpression and p53 mutational status, no data can be found in the literature. Riemenschneider et al 8 report that the amount of HDMX-S is higher in relation to FL-HDMX in glioblastomas than in gliomas, and Rallapalli et al 12 describe that HDMX-S is upregulated in transformed cells and downregulated in low passage cells. However, no information is given on the p53 mutational status of the tumors or cells described.…”

“…The truncated proteins are much more effective than full-length MDMX in the inhibition of p53-mediated transcriptional activation and induction of apoptosis. 12,13 To date, no information is available regarding the clinical significance of HDMX alterations in STS. Therefore, we were interested in evaluating gene amplification and mRNA expression of HDMX in STS.…”

“…11 Furthermore, several splice variants of the MDMX/HDMX mRNA have been detected both in murine and human tumor cell lines. 12,13 For example, the MDMX-S/HDMX-S variant (also referred to as HDMX-E) is characterized by the loss of exon 6, which produces a shift of the reading frame, resulting in a premature stop codon and the incorporation of 13 and 26 unique amino acids in MDMX-S and HDMX-S, respectively (Fig. 1).…”