HEXIM1 is a critical determinant of the response to tamoxifen.

Tamoxifen resistance is a major problem in the treatment of estrogen receptor (ER)-positive patients. We have previously reported that hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) inhibits ERα activity by competing with ERα for binding to cyclin T1, a subunit of positive transcription elongation b (P-TEFb). This results in the inhibition of the phosphorylation of RNA polymerase II (RNAPII) at serine 2 and the inhibition of transcription elongation of ERα target genes. As HEXIM1 can inhibit ER activity, we examined whether it has a critical role in the inhibitory effects of tamoxifen on ER. We observed that tamoxifen-induced HEXIM1 recruitment to the promoter region of ER target genes and decreased the recruitment of cyclin T1 and serine 2 phosphorylated RNAPII to the coding regions of these genes. Conversely, in cells wherein HEXIM1 expression has been downregulated we observed attenuation of the inhibitory effects of tamoxifen on estrogen-induced cyclin T1 recruitment to coding regions of ER target genes. As a consequence, downregulation of HEXIM1 resulted in the attenuation of the repressive effects of tamoxifen on estrogen-induced gene expression and proliferation. Conferring clinical relevance to our studies is our analysis of human breast cancer tissue samples that indicated association of lower expression of HEXIM1 with tumor recurrence in patients who received tamoxifen. Our studies provide a better understanding of the mechanistic basis for the inhibitory effect of tamoxifen on ER activity and may suggest new therapeutic targets for the treatment of tamoxifen-resistant breast cancer.

Presumptive exercise-associated peracute thoracolumbar disc extrusion in 48 dogs.

Forty-eight dogs were diagnosed with presumptive exercise-associated peracute thoracolumbar disc extrusion. The median age was seven years (range two to 11 years), and median bodyweight was 23 kg (range 10 to 41 kg). The duration of signs before presentation ranged from 0.5 to four days. Twenty-nine dogs were non-ambulatory, of which 17 were incontinent and two had lost pain perception. Pelvic limbs were hyporeflexic or areflexic in 11 dogs. Intervertebral disc narrowing was evident on radiographs in 44 dogs. Myelography demonstrated a small, extradural space-occupying lesion dorsal to an intervertebral disc between T11-12 and L3-4 with adjacent spinal cord swelling. Forty-six dogs were treated non-surgically, one was euthanased and one was managed by hemilaminectomy (and subsequently euthanased). Follow-up information was available for 46 dogs 1.5 to 55 months after injury (median 22 months) showing that pelvic limb function had improved in all cases and all non-ambulatory dogs had regained the ability to walk. Six dogs remained faecally incontinent, and one dog remained urinarily and faecally incontinent.

Laryngeal collapse in seven brachycephalic puppies.

OBJECTIVES: To document the histories, clinical findings, and management of seven puppies with laryngeal collapse occurring secondarily to brachycephalic airway syndrome. METHODS: Seven brachycephalic puppies aged between 4.5 and six months underwent surgery for management of brachycephalic airway syndrome following presentation for exercise intolerance and increased respiratory noise and effort. RESULTS: Stenotic nares of varying severity and an elongated soft palate were common to all dogs. All dogs had tracheal hypoplasia and this was severe in four dogs. Laryngeal collapse was present in all dogs. Two dogs had stage I, four dogs stage II, and one dog stage III laryngeal collapse. The dog with stage III laryngeal collapse and one dog with stage II laryngeal collapse died. There was no apparent association between the changes evident on thoracic radiographs or the degree of tracheal hypoplasia and postoperative outcome. CLINICAL SIGNIFICANCE: The development of severe secondary laryngeal changes in dogs aged six months or less supports the suggestion that immature brachycephalic dogs should undergo assessment and, if indicated, surgery as soon as any clinical signs of BAS are apparent.

Intramural tracheal haematoma causing acute respiratory obstruction in a dog.

Respiratory obstruction resulting from a discrete haematoma within the dorsal tracheal membrane was seen in an 11-year-old neutered female greyhound that had been involved in a fight two days earlier. There was no history or evidence of rodenticide toxicity or other coagulopathy, and it is suggested that the tracheal haematoma resulted from trauma. A right third intercostal thoracotomy was performed and this allowed resection of the haematoma from within the dorsal membrane of the cranial thoracic trachea, relieving the obstruction with no subsequent signs of dyspnoea. Intramural haematoma should be considered as a rare differential diagnosis for dogs presenting with acute respiratory obstruction.

beta-Lapachone-induced apoptosis in human prostate cancer cells: involvement of NQO1/xip3.

beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells.

Calcium is a key signaling molecule in beta-lapachone-mediated cell death.

beta-Lapachone (beta-Lap) triggers apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. Downstream signaling pathway(s) that initiate apoptosis following treatment with beta-Lap have not been elucidated. Since calpain activation was suspected in beta-Lap-mediated apoptosis, we examined alterations in Ca(2+) homeostasis using NQO1-expressing MCF-7 cells. beta-Lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca(2+), from endoplasmic reticulum Ca(2+) stores, comparable to thapsigargin exposures. 1,2-Bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, an intracellular Ca(2+) chelator, blocked early increases in Ca(2+) levels and inhibited beta-Lap-mediated mitochondrial membrane depolarization, intracellular ATP depletion, specific and unique substrate proteolysis, and apoptosis. The extracellular Ca(2+) chelator, EGTA, inhibited later apoptotic end points (observed >8 h, e.g. substrate proteolysis and DNA fragmentation), suggesting that later execution events were triggered by Ca(2+) influxes from the extracellular milieu. Collectively, these data suggest a critical, but not sole, role for Ca(2+) in the NQO1-dependent cell death pathway initiated by beta-Lap. Use of beta-Lap to trigger an apparently novel, calpain-like-mediated apoptotic cell death could be useful for breast and prostate cancer therapy.

Nuclear clusterin/XIP8, an x-ray-induced Ku70-binding protein that signals cell death.

Clusterin [CLU, a.k.a. TRPM-2, SGP-2, or ionizing radiation (IR)-induced protein-8 (XIP8)] was implicated in apoptosis, tissue injury, and aging. Its function remains elusive. We reisolated CLU/XIP8 by yeast two-hybrid analyses using as bait the DNA double-strand break repair protein Ku70. We show that a delayed (2-3 days), low-dose (0.02-10 Gy) IR-inducible nuclear CLU/XIP8 protein coimmunoprecipitated and colocalized (by confocal microscopy) in vivo with Ku70/Ku80, a DNA damage sensor and key double-strand break repair protein, in human MCF-7:WS8 breast cancer cells. Overexpression of nuclear CLU/XIP8 or its minimal Ku70 binding domain (120 aa of CLU/XIP8 C terminus) in nonirradiated MCF-7:WS8 cells dramatically reduced cell growth and colony-forming ability concomitant with increased G(1) cell cycle checkpoint arrest and increased cell death. Enhanced expression and accumulation of nuclear CLU/XIP8-Ku70/Ku80 complexes appears to be an important cell death signal after IR exposure.

Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during beta-lapachone-mediated apoptosis.

beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.

NAD(P)H:Quinone oxidoreductase activity is the principal determinant of beta-lapachone cytotoxicity.

beta-Lapachone activates a novel apoptotic response in a number of cell lines. We demonstrate that the enzyme NAD(P)H:quinone oxidoreductase (NQO1) substantially enhances the toxicity of beta-lapachone. NQO1 expression directly correlated with sensitivity to a 4-h pulse of beta-lapachone in a panel of breast cancer cell lines, and the NQO1 inhibitor, dicoumarol, significantly protected NQO1-expressing cells from all aspects of beta-lapachone toxicity. Stable transfection of the NQO1-deficient cell line, MDA-MB-468, with an NQO1 expression plasmid increased apoptotic responses and lethality after beta-lapachone exposure. Dicoumarol blocked both the apoptotic responses and lethality. Biochemical studies suggest that reduction of beta-lapachone by NQO1 leads to a futile cycling between the quinone and hydroquinone forms, with a concomitant loss of reduced NAD(P)H. In addition, the activation of a cysteine protease, which has characteristics consistent with the neutral calcium-dependent protease, calpain, is observed after beta-lapachone treatment. This is the first definitive elucidation of an intracellular target for beta-lapachone in tumor cells. NQO1 could be exploited for gene therapy, radiotherapy, and/or chemopreventive interventions, since the enzyme is elevated in a number of tumor types (i.e. breast and lung) and during neoplastic transformation.

Cellular and molecular responses to topoisomerase I poisons. Exploiting synergy for improved radiotherapy.

The efficacy of topoisomerase (Topo) I-active drugs may be improved by better understanding the molecular and cellular responses of tumor compared to normal cells after genotoxic insults. Ionizing radiation (IR) + Topo I-active drugs (e.g., Topotecan) caused synergistic cell killing in various human cancer cells, even in cells from highly radioresistant tumors. Topo I poisons had to be added either during or immediately after IR. Synergy was caused by DNA lesion modification mechanisms as well as by concomitant stimulation of two pathways of cell death: necrosis (IR) + apoptosis (Topo I poisons). Cumulative data favor a mechanism of synergistic cell killing caused by altered DNA lesion modification and enhanced apoptosis. However, alterations in cell cycle regulation may also play a role in the synergy between these two agents in certain human cancers. We recently showed that NF-kappa B, a known anti-apoptotic factor, was activated in various cancer cells after poisoning Topo I using clinically active drugs. NF-kappa B activation was dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. Cytoplasmic signaling leading to NF-kappa B activation after Topo I poisons was diminished in cytoplasts (lacking nuclei) and in CEM/C2 cells that expressed a mutant Topo I protein that did not interact with Topo I-active drugs. NF-kappa B activation was intensified in S-phase and blocked by aphidicolin, suggesting that activation was a result of double-strand break formation due to Topo I poisoning and DNA replication. Dominant-negative I kappa B expression augmented Topo I poison-mediated apoptosis. Elucidation of molecular signal transduction pathways after Topo I drug-IR combinations may lead to improved radiotherapy by blocking anti-apoptotic NF-kappa B responses. Recent data also indicate that synergy caused by IR + Topo I poisons is different from radiosensitization by beta-lapachone (beta-lap), a "reported" Topo I and II-alpha poison in vitro. In fact, beta-lap does not kill cells by poisoning either Topo I or II-alpha in vivo. Instead, the compound is "activated" by an IR (damage)-inducible enzyme, NAD(P)H:quinone oxidoreductase (NQO1), a gene cloned as x-ray-inducible transcript #3, xip3. Unlike the lesion modification pathway induced by IR + Topo I drugs, beta-lap kills cells via NQO1 futile cycle metabolism. Downstream apoptosis caused by beta-lap appears to be noncaspase-mediated, involving calpain or a calpain-like protease. Thus, although Topo I poisons or beta-lap in combination with IR both synergistically kill cancer cells, the mechanisms are very different.

Modulation of tumor cell proliferation and apoptosis by polyamine depletion in cells of head and neck squamous cell carcinomas.

These studies were carried out to examine the capacity of alpha-difluoromethylornithine (DFMO) to modulate cell proliferation and apoptosis in cells of squamous cell carcinomas (SCCs) of the head and neck. Exposure of cells to DFMO (5 mM for 48 h) depleted intracellular putrescine and spermidine levels (greater than 5-fold) and inhibited proliferation of the cells without manifestation of cytotoxicity as measured by a clonogenic assay. Exposure of the cells to DFMO did not influence the survival response after exposure to single-dose radiation between 0 and 10 Gy. Treatment of polyamine-depleted cells with 200 nM staurosporine amplified apoptosis 65% (1.65-fold) over that in controls, as determined by flow cytometry. The increased apoptosis after DFMO treatment was effectively inhibited by the addition of 1 mM putrescine or spermidine. Cleavage of poly(ADP-ribose) polymerase (PARP) illustrated that the staurosporine treatment induced apoptosis in the cells within 6 h. Analysis of PARP cleavage indicated that treatment with DFMO accelerated the kinetics of progression of apoptosis but did not influence the sensitivity of cells to 10 nM-1 microM staurosporine. These data suggest an involvement of endogenous polyamines in modulation of proliferation kinetics and apoptosis in human SCCs and suggest opportunities to explore new therapeutic strategies in head and neck cancer patients to be treated with radiation therapy.

Delayed apoptotic responses associated with radiation-induced neoplastic transformation of human hybrid cells.

HeLa X human skin fibroblast hybrid cells have been developed into a model for radiation-induced neoplastic transformation of human cells. Previous studies indicate that the appearance of neoplastically transformed foci in this system is delayed for several population doublings after irradiation and appears to involve the loss of putative tumor suppressor loci on fibroblast chromosomes 11 and 14. We now show that after treatment with 7 Gy of X-rays, transformed foci initiation correlates with delayed apoptosis initiated in the progeny of the irradiated cells after 10-12 cell divisions and with reduced plating efficiency (delayed death). The cells develop classic apoptotic morphology, positive terminal deoxynucleotidyl transferase-mediated nick end labeling and phosphatidylserine (annexin V) staining, and cleavage of poly(ADP-ribose) polymerase. In addition, a delayed induction of the p53 protein and the proapoptotic Bax protein is evident over a week after radiation exposure. We propose that a delayed build-up of mitosis-dependent genomic DNA damage or a loss of genetic material over time (10-12 cell divisions postirradiation) has two relevant outcomes: (a) cell death due to the delayed induction of a p53-dependent apoptosis; and (b) neoplastic transformation of a minor subset of survivors that has lost fibroblast chromosomes 11 and 14 (tumor suppressor loci for this system) and has either evaded apoptosis or not acquired enough genetic damage to induce apoptosis. It is postulated that both phenomena result from X-ray-induced, translesion-mediated genomic instability.

Niemann-Pick human lymphoblasts are resistant to phthalocyanine 4-photodynamic therapy-induced apoptosis.

Stress-induced activation of sphingomyelinase (SMase) leading to generation of ceramide, a lipid mediator, has been associated with apoptosis in several malignant and nonmalignant cell lines. Photodynamic therapy (PDT), with the phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N(CH3)2], is an oxidative stress associated with increased ceramide generation and subsequent induction of apoptosis in various cell types. We assessed the role of SMase in photocytotoxicity. Normal human lymphoblasts accumulated ceramide and underwent apoptosis after Pc 4-PDT. In contrast, Niemann-Pick disease (NPD) lymphoblasts, which are deficient in acid sphingomyelinase (ASMase) activity, failed to respond to Pc 4-PDT with ceramide accumulation and apoptosis, suggesting that ASMase may be a Pc 4-PDT target. NPD lymphoblasts were exposed to exogenous bacterial sphingomyelinase (bSMase) to test whether these defects are reversible. Treatment of NPD cells with bSMase itself led to elevated ceramide formation, which did not translate into induction of apoptosis. However, a combination of Pc 4-PDT + bSMase induced a significant apoptotic response. Thus, the combined treatment of Pc 4-PDT + bSMase, rather than bSMase alone, was required to restore apoptosis in NPD cells. These data support the hypothesis that SMase is a proapoptotic factor determining responsiveness of cells to Pc 4-PDT.

Bcl-2 protects against beta-lapachone-mediated caspase 3 activation and apoptosis in human myeloid leukemia (HL-60) cells.

We previously demonstrated that beta-lapachone (beta-lap) killed cancer cells solely by apoptosis. Beta-Lap induced apoptosis in HL-60 cells in a dose-dependent manner as measured by flow cytometry and DNA ladder formation. Cell cycle changes, such as accumulations in S and G2-phases, were not observed. Apoptosis was accompanied by activation of caspase 3 and concomitant cleavage of poly(ADP-ribose) polymerase (PARP) to an 89 kDa polypeptide. PARP cleavage was blocked by zDEVD-fmk or zVAD-fmk, caspase-specific cleavage site inhibitors. Retrovirally introduced bcl-2 prevented beta-lap-mediated caspase 3 activation and PARP cleavage and increased the viability of Bcl-2-expressing HL-60 cells compared to cells with vector alone. Various beta-lap-related analogs (e.g., dunnione and naphthoquinone derivatives) induced equivalent apoptosis in HL-60 cells, but no compound was more effective than beta-lap. These data provide further evidence that the primary mode of cell killing by beta-lap is by the initiation and execution of apoptosis in human cancer cells.

Induction of apoptosis in MCF-7:WS8 breast cancer cells by beta-lapachone.

Beta-lapachone (beta-lap) affects a number of enzymes in vitro, including type I topoisomerase (Topo I); however, its exact intracellular target(s) and mechanism of cell killing remain unknown. We compared the cytotoxic responses of MCF-7:WS8 (MCF-7) human breast cancer cells after 4-h pulses of beta-lap or camptothecin (CPT), a known Topo I poison. A direct correlation between loss of survival and apoptosis was seen after beta-lap treatment (LD50 = 2.5 microM). A concentration-dependent, transient sub-2 N preapoptotic cell population was observed at 4-8 h. Estrogen deprivation-induced synchronization and bromodeoxyuridine-labeling studies revealed an apoptotic exit point near the G1-S border. Apoptosis activated by beta-lap was closely correlated with cleavage of lamin B but not with increases in p53/p21 or decreases in bcl-2. Loss of hyperphosphorylated forms of the retinoblastoma protein was observed within 5 h, but cyclins A, B1, and E levels were unaltered for up to 72 h after 5 microM beta-lap. Topo I and Topo IIalpha levels decreased at > 24 h. Logarithmic-phase MCF-7 cells were not affected by < or = 1 microM beta-lap. In contrast, dramatic and irreversible G2-M arrest with no apoptosis was observed in MCF-7 cells treated with 1 microM CPT, monitored for 6-10 days posttreatment. MCF-7 cells treated with supralethal doses of CPT (5 microM) resulted in only approximately 20% apoptosis. No correlation between apoptosis and loss of survival was observed. MCF-7 cells exposed to > 5 microM CPT arrested at key cell cycle checkpoints (i.e., G1, S, and G2-M), with little or no movement for 6 days. Ten-fold increases in p53/p21 and 2-5-fold decreases in bcl-2, Topo I, Topo IIalpha, and cyclins A and B1, with no change in cyclin E, were observed. Temporal decreases in bcl-2 and cleavage of lamin B corresponded to the minimal apoptotic response observed. Beta-lap activated apoptosis without inducing p53/p21 or cell cycle arrest responses and killed MCF-7 cells solely by apoptosis. In contrast, concentration-dependent increases in nuclear p53/p21 and various cell cycle checkpoint arrests were seen in MCF-7 cells after CPT. Despite dramatic p53/p21 protein induction responses, CPT-treated MCF-7 cells showed low levels of apoptosis, possibly due to protective cell cycle checkpoints or the lack of specific CPT-activated apoptotic pathways in MCF-7 cells.

Cloning and characterization of a 77-kDa oestrogen receptor isolated from a human breast cancer cell line.

We have cloned and characterized a 77-kDa oestrogen receptor (ER) from an oestrogen-independent subclone of the MCF-7 human breast cancer cell line. This receptor contains an in-frame, tandem duplication of exons 6 and 7, located in the steroid-binding domain of the ER. This mutation has abrogated ligand binding, but not DNA binding, in this mutant ER. We previously described the partial structure of a unique oestrogen receptor (ER) that is expressed in an oestrogen-independent MCF-7:2A subclone of the breast cancer cell line MCF-7 (Pink JJ, Wu SQ, Wolf DM, Bilimoria MM, Jordan VC 1996a, Nucleic Acids Res 24 962-969). Sequence analyses determined the molecular weight of this 80-kDa ER to be 77 kDa, and hereafter this protein will be designated as ER77. Examination of the entire coding sequence of the ER77 mRNA indicates that it contains a tandem duplication of exons 6 and 7. Using a coupled transcription/translation system, a 77-kDa ER, which corresponds to the protein observed in the MCF-7:2A cells, was expressed. The ER77 protein does not bind the ligands [3H] oestradiol or [3H]tamoxifen aziridine. In DNA binding gel shift assays, the in vitro synthesized ER77 binds to a consensus vitellogenin A2 oestrogen-response element. In transient transfection experiments, the mutant ER, alone or in combination with the wild-type ER, does not induce expression of an oestrogen-responsive luciferase reporter construct. In fact, expression of the ER77 in the ER-positive T47D:A18 cell line inhibits E2-induced luciferase expression. Overexpression of wild-type ER in T47D:A18 cells leads to elevated constitutive expression of the luciferase reporter, which was inhibited by co-transfection with ER77. These data suggest that the ER77 can interfere with normal ER activity and does not act as a constitutive activator of oestrogen-independent growth in MCF-7:2A cells. Consequently, the constitutive growth observed in MCF-7:2A cells is probably the result of other ER-mediated pathways.

Irreversible loss of the oestrogen receptor in T47D breast cancer cells following prolonged oestrogen deprivation.

The development of antioestrogen resistance is a major clinical obstacle encountered in the treatment of breast cancer. By long-term growth in oestrogen-free medium, we have derived an oestrogen-independent, anti-oestrogen resistant cell line from the oestrogen receptor (ER)-positive, oestrogen-dependent T47D human breast cancer cell line. This cell line grows maximally in oestrogen-free medium and is resistant to all tested antioestrogens. This cell line does not express any measurable amounts of ER mRNA or protein and, in short-term studies, these cells show no response to either oestrogens or antioestrogens. However, return of these cells to oestrogen-containing medium for more than 8 weeks resulted in the re-expression of ER mRNA and protein. Subsequent limiting dilution subcloning of the T47D:C4 line revealed two phenotypically distinct clones, one which did not express measurable ER after long-term growth in oestrogen-containing medium and one which expressed ER mRNA and protein after a number of weeks in oestrogen-containing medium. In the absence of oestrogen, both types of cells are ER-negative as determined by Northern and Western blotting and lack of any oestrogen-dependent responses. The clone which re-expresses the ER (T47D:C4:5W) now responds to E2 with a 50% increase in growth and a 30-fold induction of an ER-responsive luciferase reporter construct. Long-term growth of the stably ER-negative clone (T47D:C4:2W) causes no measurable oestrogen-mediated responses, as assessed by ER expression, growth stimulation or luciferase induction. Interestingly, ER mRNA can be detected in both cell types by using reverse transcriptase-polymerase chain reaction (RT-PCR). This suggests that the ER mRNA present in the T47D:C4:2W clone is either inefficiently translated or is present at such a low level as to be functionally irrelevant. These novel clonal cell lines will prove to be invaluable in the study of the regulation of ER expression and regulatory pathways leading to oestrogen-independent growth.

Models of estrogen receptor regulation by estrogens and antiestrogens in breast cancer cell lines.

The expression and stability of the estrogen receptor (ER) is the result of a complex process that is modulated by estrogens and antiestrogens. Regulation of the steady-state ER mRNA and protein levels in breast cancer cells appears to be the result of either of two distinct regulatory mechanisms. Estrogen exposure causes a rapid down-regulation of the steady-state level of ER mRNA and protein in model I regulation, as exemplified by the MCF-7:WS8 cell line. Conversely, in model II regulation, as observed in the T47D:A18 cell line, estrogen exposure causes an increase in the steady-state ER mRNA level and a maintenance of the ER protein level. In both these cell lines, the nonsteroidal antiestrogen 4-hydroxytamoxifen has little effect on the mRNA level but causes a net accumulation of the ER protein over time. In contrast, the pure antiestrogen ICI 182,780 causes a dramatic reduction of the ER protein in both the MCF-7:WS8 and T47D:A18 cell lines. This loss has little effect upon the ER mRNA level in the MCF-7:WS8 cells but leads to a decline in the ER mRNA in the T47D:Al8 cells. The estrogen-independent MCF-7:2A cell line, which has adapted to growth in estrogen free media, expresses two forms of the ER, a wild-type Mr66,000 ER and a mutant Mr77,000 ER (ER77). ER77 is the product of a genomic rearrangement resulting in a tandem duplication of exons 6 and 7 (J. J. Pink et al, Nucleic Acids Res., 24:962-969,1996). This exon duplication has abolished ligand binding by this protein. Here we demonstrate that the loss of ligand binding has eliminated the effects of 4-OHT and ICI 182,780 on the steady-state ER77 protein level. However, in the MCF-7:2A cells, antiestrogens affect the wild-type ER protein in the same manner as observed in the MCF-7:WS8 and T47D:A18 cells. Estrogen regulates the ER mRNA and wild-type ER and ER77 proteins in the MCF-7:2A cells in the same manner as observed in the MCF-7:WS8 cells. Interestingly, treatment of the MCF-7:2A cells with ICI 182,780 causes a slight increase in ER mRNA, which is reflected in a net increase in the ER77 protein but a dramatic decrease in the wild-type ER. The models presented here describe the response of two human breast cancer cell lines in short-term studies. These distinct regulation pathways are predictive of the response of these cell lines to long-term estrogen deprivation. This study illustrates two alternative regulation pathways that are present in ER-positive, estrogen-dependent breast cancer cells. This variable response highlights the diversity of responses potentially present in the heterogeneous cell populations of clinically observed breast cancer.

A novel 80 kDa human estrogen receptor containing a duplication of exons 6 and 7.

Alterations in the amino acid sequence of the estrogen receptor (ER) have been shown to have dramatic effects on its function. Recently, mutant ERs have been isolated from both clinical samples and established breast cancer cell lines, primarily through the use of the polymerase chain reaction (PCR). All previously reported mutations have given rise to either alterations or truncations of the ER protein. We determined the structure of a novel 80 kDa ER which is expressed in an estrogen independent subclone of the MCF-7 human breast cancer cell line (MCF-7:2A). This 80 kDa ER was initially detected by Western blot analysis using a variety of ER specific antibodies. PCR mapping and partial PCR mediated subcloning of the ER cDNA were used to demonstrate that this protein was an ER containing an in-frame duplication of exons 6 and 7. This type of duplication has not been previously described for any members of the steroid receptor superfamily. Karyotype analysis coupled with fluorescence in situ hybridization (FISH) demonstrated that MCF-7:2A cells contained 4-5 copies of the ER gene in contrast to 2 copies in MCF-7:WS8 cells. The ER gene was localized by FISH analyses in both the MCF-7:WS8 and MCF-7:2A cells on chromosome 6, which is the source of the ER in normal human cells. The relative expression level of 2:1 is consistent with DNA gene dosage analysis. Genomic PCR was then used to demonstrate that the 80 kDa ER mRNA was not derived from the trans-splicing of two ER mRNAs but was the result of a genomic rearrangement in which exons 6 and 7 were duplicated in an in-frame fashion. This variant ER may prove to be useful in elucidating the mechanism of estrogen action in breast cancer cells.

An estrogen-independent MCF-7 breast cancer cell line which contains a novel 80-kilodalton estrogen receptor-related protein.

Long-term growth of estrogen-responsive human breast cancer cell lines in estrogen-free media leads inevitably to the development of estrogen-independent growth. We have identified and characterized a unique subclone of the MCF-7 human breast cancer cell line, named MCF-7:2A, which grows maximally in the absence of endogenous estrogens but whose growth is inhibited by the antiestrogens 4-hydroxytamoxifen and ICI 164,384. The MCF-7:2A cells express high levels of estrogen receptor (ER; 477 fmol/mg protein), which can be reduced by growth in 10 nM 17 beta-estradiol (201 fmol/mg protein). Basal progesterone receptor synthesis is very low in the 2A cells (< 1 fmol/mg protein) but can be dramatically increased by 10 nM 17 beta-estradiol (384 fmol/mg protein). Clearly, the pathways that control growth and estrogen-regulated genes such as the progesterone receptor are now dissociated in these cells. MCF-7:2A cells also possess two unique characteristics. First, the MCF-7:2A cells constitutively activate an ER-responsive luciferase reporter construct in the absence of any estrogens, and this activation can be blocked by either 4-hydroxytamoxifen or ICI 164,384. This constitutive activity is not observed in the parental MCF-7 cells. Second, they express an 80-kDa protein that cross-reacts with three distinct antibodies to the ER. The MCF-7:2A cells were subjected to an additional round of limiting dilution subcloning, and 10 independent clones were all shown to express both the 66- and 80-kDa ERs as observed in the MCF-7:2A line. This confirms that both ERs are being expressed in each cell and are not the result of a mixed population of cells. While numerous ER variants have been reported previously, no ER has until now been described that is larger than the wild-type 66-kDa ER. The MCF-7:2A cells provide a unique model to use in the study of ER action and the development of estrogen-independent growth in human breast cancer cells.