Drug resistance in human cancer is associated with overexpression of the multidrug resistance (MDR1) gene, which confers cross-resistance to hydrophobic natural product cytotoxic drugs. Expression of the MDR1 gene can occur de novo in human cancers in the absence of drug treatment. The promoter of the human MDR1 gene was shown to be a target for the c-Ha-Ras-1 oncogene and the p53 tumor suppressor gene products, both of which are associated with tumor progression. The stimulatory effect of c-Ha-Ras-1 was not specific for the MDR1 promoter alone, whereas a mutant p53 specifically stimulated the MDR1 promoter and wild-type p53 exerted specific repression. These results imply that the MDR1 gene could be activated during tumor progression associated with mutations in Ras and p53.

Abstract(À)-Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, has been shown to inhibit the growth of many cancer cell lines and to suppress the phosphorylation of epidermal growth factor receptor (EGFR). We observed similar effects of EGCG in esophageal squamous cell carcinoma KYSE 150 cells and epidermoid squamous cell carcinoma A431 cells. Pretreatment of KYSE 150 cells with EGCG (20 Mmol/L) for 0.5 to 24 hours in HAM's F12 and RPMI 1640 mixed medium at 37°C, before the addition of EGF, resulted in a decreased level of phosphorylated EGFR (by 32-85%). Prolonged treatment with EGCG (8 or 24 hours) also decreased EGFR protein level (both by 80%). EGCG treatment for 24 hours also caused decreased signals of HER-2/neu in esophageal adenocarcinoma OE19 cells. These effects of EGCG were prevented or diminished by the addition of superoxide dismutase (SOD, 5 units/mL), or SOD plus catalase (30 units/mL), to the cell culture medium. A similar phenomenon on inactivation of EGFR was observed in A431 cells as well. Under culture conditions for KYSE 150 cells, EGCG was unstable, with a half-life of f30 minutes; EGCG dimers and other oxidative products were formed. The presence of SOD in the culture medium stabilized EGCG and increased its half-life to longer than 24 hours and some EGCG epimerized to (+)-gallocatechin-3-gallate. A mechanism of superoxide radical-mediated dimerization of EGCG and H 2 O 2 formation is proposed. The stabilization of EGCG by SOD in the culture medium potentiated the activity of EGCG in inhibiting KYSE 150 cell growth. The results suggest that in cell culture conditions, the auto-oxidation of EGCG leads to EGFR inactivation, but the inhibition of cell growth is due to other mechanisms. It remains to be determined whether the presently observed auto-oxidation of EGCG occurs in vivo. In future studies of EGCG and other polyphenolic compounds in cell culture, SOD may be added to stabilize EGCG and to avoid possible artifacts. (Cancer Res 2005; 65(17): 8049-56)

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor a (RARa) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RARa, which is able to form a heterodimer with PML. It was hypothesized that PML-RARa is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RARa, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RARoa resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RARot resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RARao, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.Acute promyelocytic leukemia (APL) represents a clonal proliferation and expansion of the hematologic precursors at the promyelocyte stage of myeloid differentiation. A nonrandom chromosomal translocation, t(15;17), can be found in over 95% of patients with APL (3, 42), suggesting that this translocation plays a critical role in leukemogenesis. In recent attempts to explore this role further, genes involved in this translocation have been cloned and characterized (4,7,17,37,48). The t(15;17) breakpoint occurs within the second intron of the retinoic acid (RA) receptor ox (RARox) gene and within two major sites of the PML (or MYL) gene (1,8,10,28,47 (VDR) to its target sequence and prevent vitamin D3-dependent activation of VDR-responsive genes. Furthermore, it was shown that transfection of PML-RARot into U937 human myeloid leukemia cells inhibits its ability to respond to vitamin D3 and transforming growth factor 13 and so induce differentiation. These results strongly suggest that the fusion protein encoded from the breakpoint of the t(15;17) translocation plays a critical role in APL pathogenesis. Recently, Doucas et al. (19) reported that PML-RARot, instead of being an inhibitor, is an RA-dependent activator of the transcription factor AP-1. However, since, as Kastner et al. (38)

Genome rearrangements are important in evolution, cancer, and other diseases. Precise mapping of the rearrangements is essential for identification of the involved genes, and many techniques have been developed for this purpose. We show here that end-sequence profiling (ESP) is particularly well suited to this purpose. ESP is accomplished by constructing a bacterial artificial chromosome (BAC) library from a test genome, measuring BAC end sequences, and mapping end-sequence pairs onto the normal genome sequence. Plots of BAC end-sequences density identify copy number abnormalities at high resolution. BACs spanning structural aberrations have end pairs that map abnormally far apart on the normal genome sequence. These pairs can then be sequenced to determine the involved genes and breakpoint sequences. ESP analysis of the breast cancer cell line MCF-7 demonstrated its utility for analysis of complex genomes. End sequencing of Ϸ8,000 clones (0.37-fold haploid genome clonal coverage) produced a comprehensive genome copy number map of the MCF-7 genome at better than 300-kb resolution and identified 381 genome breakpoints, a subset of which was verified by fluorescence in situ hybridization mapping and sequencing.

No abstract

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The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein with growth-and transformationsuppressing ability. Having previously shown it to be a transcriptional repressor of the epidermal growth factor receptor (EGFR) gene promoter, we have now shown that PML's repression of EGFR transcription is caused by inhibition of EGFR's Sp1-dependent activity. On functional analysis, the repressive effect of PML was mapped to a 150-bp element (the sequences between ؊150 and ؊16, relative to the ATG initiation site) of the promoter. Transient transfection assays with Sp1-negative Drosophila melanogaster SL2 cells showed that the transcription of this region was regulated by Sp1 and that the Sp1-dependent activity of the promoter was suppressed by PML in a dose-dependent manner. Coimmunoprecipitation and mammalian two-hybrid assays demonstrated that PML and Sp1 were associated in vivo. In vitro binding by means of the glutathione S-transferase (GST) pull-down assay, using the full-length and truncated GST-Sp1 proteins and in vitrotranslated PML, showed that PML and Sp1 directly interacted and that the C-terminal (DNA-binding) region of Sp1 and the coiled-coil (dimerization) domain of PML were essential for this interaction. Analysis of the effects of PML on Sp1 DNA binding by electrophoretic mobility shift assay (EMSA) showed that PML could specifically disrupt the binding of Sp1 to DNA. Furthermore, cotransfection of PML specifically repressed Sp1, but not the E2F1-mediated activity of the dihydrofolate reductase promoter. Together, these data suggest that the association of PML and Sp1 represents a novel mechanism for negative regulation of EGFR and other Sp1 target promoters.The promyelocytic leukemia gene, PML, was first identified at the breakpoint of the t(15;17) translocation in acute promyelocytic leukemia (APL) (10,14,19,35,37). PML encodes a nuclear phosphoprotein that functions as a transcriptional regulator (9, 50, 58) and belongs to the RING family of proteins, which share a cysteine-rich motif at the N terminus. This motif is divided into a RING finger (C 3 C 4 zinc binding) motif and two B-box (B1 and B2) motifs (18). This region is followed by a predicted ␣-helical coiled-coil (dimerization) domain, which allows PML to homodimerize and form heterodimer complexes with the APL fusion protein PMLRAR␣ and the promyelocytic leukemia zinc finger (PLZF) protein (37,40). PML localizes to distinct domains in the nucleus called PML nuclear bodies, or PML oncogenic domains (PODs) (16,60). In addition to PML, there are several other POD-associated factors, including SP100, the ubiquitin-like protein PIC1, and the interferon-stimulated 20-kDa gene product called ISG20 (3,6,20). PODs are frequently targeted and/or reorganized by viral proteins, such as the herpes simplex virus type 1 (HSV-1) gene product Vmw110 (17), the adenoviral proteins E1A and E4-ORF3 (8), the Epstein-Barr virus-encoded nuclear antigen EBNA-5 (53), and the human cytomegalovirus major immediate-early proteins IE1 and IE2 (1).PMLRAR␣, which r...

There have been speculations that the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA) may have other functions. A recent study has shown that the catalytic (C) subunit of PKA may be regulated in a cAMP- and R subunit-independent manner. However, evidence linking a function to the R subunit apart from inhibiting the C subunit has been elusive. In this report, interaction cloning experiments showed that the RIalpha subunit association with the cytochrome c oxidase subunit Vb (CoxVb) is cAMP-sensitive. Interaction was detected with a GST-RIalpha fusion protein as well as by coimmunoprecipitation. Transient treatment with cAMP-elevating agents inhibited cytochrome c oxidase in Chinese hamster ovary (CHO) cells with a concomitant decrease in cytochrome c levels in the mitochondria and an increase in its release into the cytosol. Furthermore, mutant cells harboring a defective RIalpha show increased cytochrome c oxidase activity and also constitutively lower levels of cytochrome c in comparison to either the wild-type cells or the C subunit mutant. These results suggest a novel mechanism of cAMP signaling through the interaction of RIalpha with CoxVb thereby regulating cytochrome c oxidase activity as well as the cytochrome c levels.