The MDM2-a splice variant of MDM2 alters transformation in vitro and the tumor spectrum in both Arf- and p53-null models of tumorigenesis.

MDM2-A is a common splice variant of murine double minute 2 (MDM2) that is frequently detected in many tumor types. Our previous work has characterized MDM2-A as an activator of p53, and therefore, in a wild-type p53 background, this splice variant would be predicted to confer p53-dependent tumor protection. To test this hypothesis, we used Mdm2-a transgenic mice to assess transformation and tumorigenesis in tumor susceptible murine models. A MDM2-A-dependent decrease in transformation was observed in Arf-null mouse embryonic fibroblasts (MEF) or when wild-type MEFs were exposed to the carcinogen ethylnitrosourea. However, this reduced transformation did not confer tumor protection in vivo; Mdm2-a/Arf-null mice and ethylnitrosourea-treated MDM2-expressing mice developed similar tumor types with equivalent latency compared with their respective controls. Interestingly, when p53 was deleted, MDM2-A expression enhanced transformation of p53-null MEFs and altered tumor spectrum in vivo. In addition, p53-heterozygous mice that expressed MDM2-A developed aggressive mammary tumors that were not observed in p53-heterozygous controls. In conclusion, we found that although MDM2-A expression enhances p53 activity and decreases transformation in vitro, it cannot confer tumor protection. In contrast, MDM2-A seems to exhibit a novel transforming potential in cells where p53 function is compromised. These data show that MDM2 splice variants, such as MDM2-A, may provide protection against transformation of normal tissues having intact p53. However, when such splice variants are expressed in tumors that have defects in the p53 pathway, these isoforms may contribute to tumor progression, which could explain why their expression is often associated with aggressive tumor types.

MDM2-A, a common Mdm2 splice variant, causes perinatal lethality, reduced longevity and enhanced senescence.

MDM2 is the predominant negative regulator of p53 that functions to maintain the appropriate level of expression and activity of this central tumor suppressor. Mdm2-a is a commonly identified splice variant of Mdm2; however, its physiological function is unclear. To gain insight into the activity of MDM2-A and its potential impact on p53, an Mdm2-a transgenic mouse model was generated. Mdm2-a transgenic mice displayed a homozygous-lethal phenotype that could be rescued by a reduction in p53 expression, demonstrating a dependence upon p53. Mdm2-a hemizygous mice exhibited reduced longevity, and enhanced senescence was observed in their salivary glands. In addition, the transgenic mice lacked typical, accelerated aging phenotypes. Growth of transgenic mouse embryonic fibroblasts (MEFs) was inhibited relative to wild-type MEFs, and MDM2-A was shown to bind to full-length MDM2 in an interaction that could increase p53 activity via reduced MDM2 inhibition. Evidence of p53 activation was shown in the Mdm2-a transgenic MEFs, including p53-dependent growth inhibition and elevated expression of the p53 target protein p21. In addition, MDM2-A increased senescence in a p21-independent manner. In conclusion, unexpected roles for MDM2-A in longevity and senescence were identified in a transgenic mouse model, suggesting that Mdm2 splice variants might be determinants of these phenotypes in vivo.

MDM2 displays differential activities dependent upon the activation status of NFkappaB.

MDM2 is an oncoprotein best characterized for its role in the inactivation and degradation of the p53 tumor suppressor. However, MDM2 has many other binding partners and its p53-independent role in the regulation of cell growth and survival appears to be extremely complex. This report describes the expression of MDM2 in two rhabdomyosarcoma cell lines, both expressing a mutant p53 gene. Expression of MDM2 in Rh30 cells enhanced cell growth whereas expression of MDM2 in RD cells suppressed their growth and enhanced the rate of spontaneous apoptosis. The mechanism for these opposite phenotypes was demonstrated to be due to differential effects on the NFkappaB pathway. Previously MDM2 has been shown to activate NFkappaB through activation of transcription of the p65RelA subunit. In Rh30 cells MDM2 acted similarly to previously described, thereby promoting growth of Rh30 cells. In untreated RD cells p65RelA was constitutively overexpressed resulting in activation of the NFkappaB pathway. Expression of MDM2 in RD cells transcriptionally repressed p65RelA and suppressed NFkappaB activity, resulting in a reduced growth rate and enhanced apoptosis. The MDM2-sensitive region of the p65 promoter was localized to a 225 bp fragment to which MDM2 protein was shown to bind. The observation that MDM2 induces apoptosis under certain circumstances may help to explain the apparently surprising clinical studies that have shown that MDM2 expression in tumors is often associated with a favorable prognosis.

Wild-type breast cancer resistance protein (BCRP/ABCG2) is a methotrexate polyglutamate transporter.

The existence of an ATP-dependent methotrexate (MTX) efflux mechanism has long been postulated; however, until recently, the molecular components were largely unknown. We have previously demonstrated a role for the ATP-binding cassette transporter breast cancer resistance protein (BCRP) in MTX resistance (Volk et al., Cancer Res., 62: 5035-5040, 2002). Resistance to this antifolate directly correlated with BCRP expression, and was reversible by the BCRP inhibitors fumitremorgin C and GF120918. Here, we provide evidence for BCRP as a MTX-transporter using an in vitro membrane vesicle system. Inside-out membrane vesicles were generated from both drug-selected and stably transfected cell lines expressing either wild-type (Arg482) or mutant (Gly482) variants of BCRP. In the presence of the wild-type variant of BCRP, transport of MTX into vesicles was ATP-dependent, osmotically sensitive, and inhibited by fumitremorgin C. In contrast, no transport was observed in vesicles containing the mutant form of BCRP. Wild-type BCRP appeared to have low affinity, but high capacity, for the transport of MTX, with an estimated K(m) of 680 micro M and a V(max) of 2400 pmol/mg/min. MTX accumulation was greatly decreased by mitoxantrone, a known BCRP substrate, suggesting competition for transport. Furthermore, and in contrast to the multidrug resistance-associated proteins, BCRP also transported significant amounts of polyglutamylated MTX. Although transport gradually decreased as the polyglutamate chain length increased, both MTX-Glu(2) and MTX-Glu(3) were substrates for BCRP. Together, these data demonstrate that BCRP is a MTX and MTX-polyglutamate transporter and reveal a possible mechanism by which it confers resistance.

Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance.

Previously, we have reported that a multidrug-resistant, mitoxantrone (MX)-selected cell line, MCF7/MX, is highly cross-resistant to the antifolate methotrexate (MTX), because of enhanced ATP-dependent drug efflux (E. L. Volk et al., Cancer Res., 60: 3514-3521, 2000). These cells overexpress the breast cancer resistance protein (BCRP), and resistance to MTX as well as to MX was reversible by the BCRP inhibitor, GF120918. These data indicated that BCRP causes the multidrug-resistance phenotype. To further examine the role of this transporter in MTX resistance, and in particular the role of amino acid 482, we analyzed a number of BCRP-overexpressing cell lines. MTX resistance correlated with BCRP expression in all of the cell lines expressing the wild-type transporter, which contains an Arg at position 482. In contrast, little or no cross-resistance was found in the MCF7/AdVp1000 and S1-M1-3.2 and S1-M1-80 cell lines, which contain acquired mutations at this position, R482T and R482G, respectively. Concomitantly, the greatest reduction in MTX accumulation was observed in the MCF7/MX cells (BCRP(Arg)) as compared with cells expressing the Thr and Gly BCRP variants. Furthermore, the reduction in drug accumulation was sensitive to BCRP inhibition by GF120918. In conclusion, we have demonstrated a novel role for BCRP as a mediator of MTX resistance and have provided further evidence for the importance of amino acid 482 in substrate specificity.