BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore.

Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Deltapsim) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.

Nix and Nip3 form a subfamily of pro-apoptotic mitochondrial proteins.

We have identified Nix, a homolog of the E1B 19K/Bcl-2 binding and pro-apoptotic protein Nip3. Human and murine Nix have a 56 and 53% amino acid identity to human and murine Nip3, respectively. The carboxyl terminus of Nix, including a transmembrane domain, is highly homologous to Nip3 but it bears a longer and distinct asparagine/proline-rich N terminus. Human Nip3 maps to chromosome 14q11.2-q12, whereas Nix/BNip3L was found on 8q21. Nix encodes a 23. 8-kDa protein but it is expressed as a 48-kDa protein, suggesting that it homodimerizes similarly to Nip3. Following transfection, Nix protein undergoes progressive proteolysis to an 11-kDa C-terminal fragment, which is blocked by the proteasome inhibitor lactacystin. Nix colocalizes with the mitochondrial matrix protein HSP60, and removal of the putative transmembrane domain (TM) results in general cytoplasmic and nuclear expression. When transiently expressed, Nix and Nip3 but not TM deletion mutants rapidly activate apoptosis. Nix can overcome the suppressers Bcl-2 and Bcl-XL, although high levels of Bcl-XL expression will inhibit apoptosis. We propose that Nix and Nip3 form a new subfamily of pro-apoptotic mitochondrial proteins.

The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis.

Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3(163) mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

Relationship of c-myc amplification to progression of breast cancer from in situ to invasive tumor and lymph node metastasis.

BACKGROUND: Amplification of the c-myc gene (also known as MYC) occurs in up to 20%-30% of breast cancers and has been associated with poor prognosis. PURPOSE: The purpose of this study was to define the relationship between c-myc amplification and breast cancer progression in order to better understand the biological significance of c-myc amplification. METHODS: We identified invasive tumors with grossly detectable c-myc amplification by using Southern blot analysis to examine frozen tissue from 135 breast carcinomas and polymerase chain reaction (PCR) analysis to examine archival paraffin-embedded tissue from an additional 19 invasive tumors. These 19 tumors were selected on the basis of histologically identifiable in situ and invasive components within the primary tumor and associated lymph node metastases. Amplification of c-myc in these areas was then assessed by quantitative PCR assay. RESULTS: We detected gross c-myc amplification in 10 of the tumors examined--eight of the 135 frozen tissue specimens and two of the 19 archival specimens. We selected five of these 10 invasive tumors for further regional analysis. In all four cases where an in situ component was present, amplification of c-myc was present in both the in situ and the invasive components. However, c-myc amplification was present in the corresponding nodal metastases in only two of the four cases where this could be examined. CONCLUSION: These results suggest that c-myc amplification can occur at an early stage in tumor progression and that amplification does not always persist in the nodal metastasis.

c-myc oncogene expression in estrogen-dependent and -independent breast cancer.

We demonstrate that c-myc gene expression is essential for growth of breast cancer cells. It also plays an important role in the progression of human breast cancer. c-myc gene amplification may be important for cancer cell invasion, but perhaps not essential for nodal metastasis. We also provide compelling evidence that the c-myc oncogene is an estrogen target gene in hormone-responsive breast cancer. Hormonal progression of breast cancer could be brought about by the enhanced expression of the c-myc gene, with gene amplification and enhanced c-myc mRNA stability being two major mechanisms involved.

Mechanism of estrogen activation of c-myc oncogene expression.

The estrogen receptor complex is a known trans-acting factor that regulates transcription of specific genes through an interaction with a specific estrogen-responsive cis-acting element (ERE). In previous studies we have shown that in estrogen-responsive human breast cancer cells estrogen rapidly activates c-myc expression. This activated expression occurs through enhanced transcription and does not require the synthesis of new protein intermediates; therefore, an ERE is present in the human c-myc gene regulatory region. To localize the ERE, constructs containing varying lengths of the c-myc 5'-flanking region ranging from -2327 to +25 (relative to the P1 promoter) placed adjacent to the chloramphenicol acetyl transferase reporter gene (CAT) were prepared. They were used in transient transfection studies in MCF-7 and HeLa cells co-transfected with an estrogen receptor expression vector. These studies reveal that all constructs containing the P2 promoter region exhibited estrogen-regulated CAT expression and that a 116-bp region upstream and encompassing the P2 TATA box is necessary for this activity. Analysis of this 116-bp region failed to identify a cis-acting element with sequences resembling the consensus ERE; however, co-transfection studies with mutant estrogen receptor expression vectors showed that the DNA-binding domain of the receptor is essential for estrogen-regulated CAT gene expression. We have also observed that anti-estrogen receptor complexes can weakly trans-activate from this 116-bp region but fail to do so from the ERE-containing ApoVLDLII-CAT construct. To explain these results we propose a new mechanism of estrogen trans-activation in the c-myc gene promoter.

Transcriptional regulation of c-myc oncogene expression by estrogen in hormone-responsive human breast cancer cells.

Enhanced c-myc oncogene expression associated with peptide mitogen-stimulated cell growth is primarily a result of a post-transcriptional event involving increased mRNA stability. We have recently shown that estradiol stimulates c-myc expression in estrogen receptor-positive human breast cancer cells. In this report, we show that in estrogen-responsive MCF-7 cells, estradiol stimulated the c-myc gene exclusively at the transcriptional level, increasing c-myc mRNA transcription more than 10-fold within 20 min, while having no effect on the c-myc mRNA half-life of 18 min. In addition, pretreatment of the cells with cycloheximide did not prevent induction of the c-myc oncogene, indicating a primary effect of estrogen. Furthermore, the elevated level of c-myc mRNA in estrogen-independent MDA-MB-231 cells was due primarily to a more stable c-myc mRNA with a half-life of 49 min, in the absence of enhanced transcription. These results indicate that different mechanisms of regulation of c-myc oncogene expression exist in hormone-dependent and -independent human breast cancer cells.

Epidermal growth factor gene expression in human breast cancer cells: regulation of expression by progestins.

Epidermal growth factor (EGF) is thought to be important in normal mammary development. The presence of EGF receptors in breast cancer cells suggests that it may also have a role in regulating growth of tumors of the human breast. Using a complementary DNA probe for the human EGF precursor we have examined expression of this gene in a series of human breast cancer cells in long term culture. The T-47D cell line demonstrated the highest level of EGF mRNA. EGF expression was not detectable in the MCF-7, BT 20, or HBL 100 cell lines. Surprisingly, in both T-47D and ZR 75 cells, pretreatment with progestins which exert antiproliferative effects under the conditions used increased EGF mRNA levels approximately 6-fold above untreated controls. This effect, demonstrable with as little as 0.1 nM of medroxyprogesterone acetate, was apparent as early as 12 h after addition of progestin and was reversed with the antiprogestin RU 486. Dexamethasone, estradiol, and dihydrotestosterone had no effect on EGF expression in T-47D cells. There was no evidence that the increased levels of EGF mRNA were due to gene amplification. Immunoprecipitation of biosynthetically labeled T-47D conditioned medium with antibodies to human EGF and EGF-precursor revealed the presence of both Mr 40,000 and 18,000 products. Fully processed Mr 6,000 EGF was not detectable in either conditioned medium or cell lysate. These data provide unequivocal evidence for the expression of the EGF gene in some human breast cell lines.

Stimulation of c-myc oncogene expression associated with estrogen-induced proliferation of human breast cancer cells.

Regulation of c-myc expression is known to be sensitive to a variety of mitogenic stimuli in various cell types. Since estrogen is a well documented mitogen of estrogen-responsive human breast cancer (HBC) cells, we studied the influence of estradiol and its antagonist tamoxifen on the expression of c-myc in HBC cell lines. Using Northern hybridization analysis, we monitored the accumulation of c-myc mRNA in a number of HBC cell lines. The cell lines studied included the estrogen-responsive, estrogen receptor positive (ER+) MCF-7, T-47D, the nonresponsive, estrogen receptor negative (ER-) MDA-MB-231, BT-20, and a nontumorous breast cell line, HBL-100. The effects of endogenous estrogen were minimized by culturing the cells in medium containing 10% (v/v) charcoal-treated fetal bovine serum and tamoxifen (10(-6) M) for 48 h prior to estradiol (10(-7) M) treatment. In the ER+ cell lines the addition of estradiol resulted in a noticeable increase in c-myc expression after 15 min with a maximal (greater than 10-fold) induction in 1-2 h. In the ER- cell lines the level of c-myc mRNA was high and was unaffected by estrogen or tamoxifen; in the ER- cancer cell lines, neither amplification nor rearrangement of the c-myc gene was observed. In contrast, the expression of another oncogene, c-H-ras, remained constant in both ER+ and ER- cell lines and was insensitive to estrogen and antiestrogen. These results suggest that regulation of c-myc expression may be an important step in estrogen-induced proliferation of HBC cells.