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Breast cancer treatment

This paper summarizes three papers on breast cancer research. One paper looks at the tumor suppressor gene, maspin and how it is deactivated in breast cancer. One looks at how uptake of cholesterol from HDL promotes cell growth, including the growth of breast cancer cells, and the third looks at a new method of inducing apoptosis in cells through CD47-mediated Gi protein-dependent protein kinase A.

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Abstract biochemical studies aimed at determining the factors causing theproliferation and the restriction of growth of breast cancer cells aboundin the literature In this paper three such studies are analyzed Onelooks at the tumor suppressor gene maspin and how it may be recruited tohelp fight cell proliferation A second looks at the role of high-densitylipoprotein in breast cancer cell proliferation and how it can becounteracted and the third looks at a novel way of killing breast cancercells through a CD mediated Gi

et al. Activation mechanisms of maspin, the tumor suppressor gene, areimportant in controlling development and proliferation of breast cancer;preventing cholesterol uptake from HDL by tumor cells is a mechanism forcontrolling their growth, and CD47-mediated killing of tumor cells is amethod of destroying breast cancer cells in situ. In theseexperiments, only wortmannin was able to block the ability of EGF toprotect against CD47-mediated cell death, suggesting P13K activation isneeded for the EGF survival response.Discussion Khalkhali-Ellis et al (2 4) showed that the nonsteroidal-selectiveER modulator TAM, which is used extensively to treat breast cancer, is ableto induce the tumor suppressor gene maspin in MCF-7 cells, which suggests amechanism of action for this chemotherapeutic drug. This suggests the mitogenic effects of HDLmay involve the activation of AP-1. Raising cellular cAMP reduced apoptosis in breast cancer cells,suggesting CD47-mediated killing is mediated by decreased intracellularlevels of cAMP. The supernatant were removed and the protein contentdetermined. That thiseffect was mediated through the HRE in the maspin promoter is shown by thefact that mutation of this site led to complete abolishment of the hormoneresponse. Transected MCF-7 cells were washed once in serum-freemedium/ .5% BSA. All three of these areasneed further research to clarify the mechanisms now known to exist. Cao et al assessed the functions of CLA-1 and HDL on human breastcancer cells using a mutant CLA-1 (mCLA) from which the COOH-terminalsegment had been removed to see if it played a role in breast cancergrowth. A membrane fraction and a cytosolic fraction were prepared andrun on 1 % SDS-PAGE for cytochrome c analysis and detection of signals wasby chemiluminescence (Manna and Frazier, 2 4).Statistical Analysis: Statistical analysis of the data was performed usingthe unpaired t test (Manna and Frazier, 2 4 ).Results Khalkhali-Ellis et al (2 4) showed that 17$-estradiol, progesteroneand dihydrotestosterone had minimal effects on maspin promoter-derivedluciferase activity in either cell line tested. A second looks at the role of high-densitylipoprotein in breast cancer cell proliferation and how it can becounteracted, and the third looks at a novel way of killing breast cancercells through a CD47 mediated Gi protein-dependent protein kinase Apathway.Biochemical Approaches in Breast Cancer Treatment Breast cancer is the most common cancer found in women and in Westernsocieties it is the leading cause of early death in women (Cao et al 2 4). Regulating the tumor suppressor gene maspin in breast cancer cells. An expression vector encoding adominant-negative mutant of Akt(Akt-DN) was used.HDL Cell Association and Selective Cholesterol Ester (CT) Uptake: Human HDLwas isolated from fresh plasma in EDTA by preparative ultracentrifugation(Cao et al, 2 4). This was also documented by Western blotanalysis and intracellular staining. Equal amounts of cellular protein and experimental treatmentswere subjected to SDS-PAGE and Western blot analysis using antibodyspecific for maspin, and visualized by chemiluminescence.Hormone Regulation of Maspin Promoter: The effect of hormones on promoteractivity was determined by Khalkhali-Ellis et al (2 4) using the maspinpromoter luciferase reporter assay. Immunohistochemistry revealed the presence of maspin inmyopeithelial and epithelial duct cells, with reduced levels in primarytumors. MCF-7 lines were plated at 6x1 5 cells/well in 6-welldishes in hormone-depleted media. Other non-ER mechanisms are also active,including signaling proteins, proto-oncogenes, and the transformation ofgrowth factor $ in TAM-mediated apoptosis. Assessing the actions of a dominant negative mutant of Akt(Akt-DN) on AP-1 promoter activity in MCF-7 cells cotransfected with pAP-LUC plus the Akt-DN or empty vector showed a 2.4-fold increase inluciferase activity after HDL stimulation. 4NIK, 1F7 and platelet-derived TSP1 all induced apoptosis in breast cancer cells, but 4NGG didnot. Fltuamide had someinductive effect on MCF-7 cells, but not HMEC1331 cells. FITC-labeledantihuman Bcl-2, phycoerythrin-labeled anti-active caspase-3, purifiedantihuman caspase-9, mouse antihuman poly(ADP ribose) polymerase antibody,FITC-labeled annexin V apoptosis detection kit, epidermal growth factor,etoposide, doxorubicin, purified mAb (SC12 to human epidermal growthfactor and antihuman cytochrome c antibodies SC-7159 and SC-13561,forskolin, 8-bromo-cAMP, H-89 dihydrochloride, IBMX, wortmannin, PD98 59,AG1478, PD153 35, pertussis toxin (PTX), PKA-inhibitor peptide,staurosporine, antihuman CD95 antibody CH-11, antihuman caspase 3 antibody,antihuman "$ antibody, and cAMP ELISA kit, were all purchased commercially. It is anonsteroidal-selective ER modulator which reduces the risk of both invasiveand non-invasive breast cancer in women. In the work of Manna and Frazier (2 4), both natural and antibodyligands of CD47 caused classic, apoptotic death of four different breastcancer cell lines to varying degree and over different lengths of time.The cell death was unusual in that it did not involve active capsases, DNAladdering, release of cytochrome c from mitochondrial membranes, or loss ofBcl-2. Results were expressed as the increased induction orsuppression of reporter plasmid after normalization against an internalcontrol plasmid.Immunohistochemical Analysis of Tissue Specimens: Tissues were sectioned at4 :M, deparaffinized, and an antigen-recovery protocol was used (Khalkhali-Ellis, 2 4). Abstract biochemical studies aimed at determining the factors causing theproliferation and the restriction of growth of breast cancer cells aboundin the literature. {3H] uptake into fCLA-1 transected clone cells increasedsignificantly in a dose-dependent fashion, bur exposure of mCLA-1 cells toHDL had no effect on {3H] uptake (Figure 5 - use figure 3 from Cao et al).The effect of pharmacological inhibitors of HDL-mediated [3H]thymidineuptake was tested using phosphatidylinositol 3'kinase [P13K], a mitogen-activated extracellular signal-regulated kinase, a JNK, or a p38 mitogen-activated protein kinase. In this paper, three such studies are analyzed. Previousstudies have suggested a role for P13K and Akt in breast cancer and it hasbeen shown that activation of Akt predicts a worse outocme for patientstreated with TAM and/or goserelin. The mechanism of action of TAM is primarily competitive binding tothe ER, reducing its ability to bind 17$-estradiol and other estrogens(KhalKhali-Ellis, 2 4). The researchers speculate that TAM may preventmetastatic spread of breast cancer by inducing the suppressor gene. HDL-mediated [3H] uptake was only sensitive towortmannin, an inhibitor of P13K.Actions of CLA-1 in the Presence of HDL Affect AP-1 Activity: The additionof HDL to cells stimulated the DNA binding activity of AP-1 in the fCLA-1containing cells but not the mCLA-1 cells (Cao et al, 2 4) (Figure 6 - usefigure 4 from Cao et al). Cancer Research, 64, 151-1521.Khalkhali-Ellis, Z., Christian, A.L., Kirschmann, D.A., Edwards, E.M., Rezaic-Thompson, M., Yasef, M.A. Expression of mCLA-1 inhibited growthof MCF-7 cells and promoted apoptosis in transfected cells. The percentage of apoptoticcells was measured by the propidium iodide method. All assays were corrected for $-galactosidase activity and the total protein in each reaction wasidentical. Cholesterolfrom HDL is taken up into cells by a scavenger receptor of the B class (SR-B1)/human homolog of SR-B1, CD36, and LIMPH analogous-1 (CLA-1 and thispromotes cell growth. TAM has been shown to reduce theoccurrence of ER+ invasive cancers by 69 percent, and reduce the occurrenceof non-invasive cancers by 5 percent. The HDL effect is known to be more prominent in ER+ thanin ER- cell. 1 was consideredsignificant.mAbs and Chemicals: Anti-CD47 mAbs, 1F7(IgHG1), 2D3(IgG1) and B6H12(IgG1)have been described previously (Manna and Frazier, 2 4). After 18 hours, hormonesupplemented media was added using 1 -8M 17$-estradiol, progesterone, anddihydrotestosterone, or TAM or flutamide at 1 -7 M final concentration, thelatter based on tissue concentrations found in therapy. cDNA coding for full length CLA-1 (fCLA-1) ormCLA-1 fused to FLAG were obtained by PCR using CLA-1 expression vector.The PCR product was then digested with HindIII/Xba1 and subcloned intopcDNA3 mammalian expression vector and the sequence of the expressionvectors verified by dideoxy sequencing. PTX did inhibit 4NIK-induced apoptosis in a dose-dependent manner. (2 4). mCLA inhibited MCF-7 cell growth and promoted apoptosis oftransected cells. One problem is thatcontinued therapy with TAM leads to resistance, and when this happens, thedrug must be discontinued and alternative therapeutic agents must be used.Khalkhali-Ellis et al hypothesized that the antitumor action of TAM may bemediated in part by its ability to induce the tumor suppressor gene maspin. Luciferase activity was increased 2.5-fold in fCLA-1cells after HDL treatment but HDL did not change the DNA binding or AP-1 ortranscription of the receptor gene in cells expressing mCLA-1. Cultures were transected with full-length, truncated, or HRE-mutated maspin promoter reporter plasmids withcytomegalovirus-$-galactosidase as control. PCR and maspin promoter-specificprimers were used to generate the reporter constructs with amplificationsspanning the maspin promoter region corresponding to 416 to +87, containingthe hormone-response element (HRE), proximal Ets and AP1 site, truncatedpromoter, and -956 to +87 (Figure 1). EFG has an antiapoptotic effect on cancer cells, using theERK/mitogen activated protein kinase and Akt pathways. ReferencesCao, W.M., Murao, K., Imachi, H., Yu, X., Abe, H., Yamauchi, A. The cell death was also unusual in requiring a PTX-sensitive Gprotein, probably a Gi form. Cholesterolis an essential component of rapidly dividing cells, which makes itextremely important for the proliferation of cancer cells. The same effect wasseen with the nonsteroidal antiandrogen flutamide. There is considerable variation in the chemotherapeutic response ofbreast cancers which may be attributed to variations or defects inprogrammed cell death pathways, according to Manna and Frazier (2 4),since a variety of growth factors oppose cell death. (2 4). 17$-estradiol exerted a slight inhibitoryeffect on maspin levels in HMED1331 cells but had minimal effect on MCF-7ells. Antibody to maspin and peroxidase visualization followed,and samples were reviewed by a board-certified pathologist, blinded to theclinical outcome of the patients until scoring was complete. This suggests that inhibiting HDL signaling may be auseful therapeutic direction for study for controlling breast cancerproliferation. Evidence has beenbuilding for a role of thrombospondins (TSPs) 1 and 2 as antitumor factorsfor breast cancer. Cao et al (2 4) showed that CLA-1 mRNA was present in large amountsin breast cancer cells but in very low amount if at all in adjacentnoncancerous tissues (Figure 3 - use Fig 1 from Cao et al). Values for cell-associated HDL, apolipoprotein,(expressed as HDL CE), endocytosed and degraded HDL apolipoprotein, totalcell-associated HDL CE and selective uptake of HDL CE were determined.Western Blot Analysis: Cytoplasmic proteins were lysed inradioimmunoprecipitation buffer and separated on 7.5% SDS-PAGE thentransferred to a polyvinylidene difluoride membrane (Cao et al, 2 4). CD47 apoptosis also did not induce cytochrome crelease from mitochondria as shown by Western blot analysis. Preparation of 4NIK peptide (KRFYVVMWKK) from the COOH-terminal domain ofTSP1, the mutant 4NGG peptide (KRFYGGMWKK) and TSP1 were describedelsewhere.Breast Cancer Cell Lines: MCF-7 breast adenocarcinoma, pleural effusion, AU-565 breast adenocarcinoma, HRL-1 breast epithelial cells with transformedmorphology) and MDA-MB-231 adenocarcinoma cells were obtained commercially,and the cell culture medium used was RPMI 164 supplemented with 1 % fetalbovine serum, 1 :g/ml streptomycin and 1 :g/ml ciprofloxin (Manna andFrazier, 2 4).Apoptosis Induction and Detection: 1x1 5 breast cancer cells in 24-wellplates were treated with 1 :g/ml 1F7, 4N1K, or 4NGG 91 :M) etoposide ordoxorubicin (1 :g/ml) for 24 h (Manna and Frazier, 2 4). The function of mCLA-1was tested by measuring the kinetics of CE uptake using doubly labeled HDL,which showed that mCLA-1 could bind HDL and uptake CE, meaning the receptorwas fully active. TSP1directly promotes endothelial apoptosis and inhibits matrixmetalloproteinases, causing a decrease in proteolytic release andactivation of vascular endothelial growth factor from the matrix.Expression of TSP receptor CD47 leads to increased apoptosis in umbilicalvein endothelial cells, and CD47 seems to function in other aspects ofcellular regulation. Theresearchers propose CD47 as a potential therapeutic target in treatingbreast cancer along with TSP1 and TSP2.Conclusion There are many new and exciting discoveries being made in the fieldof biochemistry in approaches to treating breast cancer. All reactions were incubated at room temperature for 2 minutes, then separated on a native 6% polyacrylamide gel. CLA-1 is primarily found in the liver and steroidogenic tissues suchas mammary gland cells (Cao et al, 2 4). TAM has no effect on ER- invasivecancers. Onelooks at the tumor suppressor gene, maspin. These promoter regions werecloned into pGL3 basic vector and DNA sequences of the constructs confirmedby DNA sequencing. Although DNA fragmentation is common in cell death, neither 1F7 nor4NIK caused DNA fragmentation but treatment with etoposide or doxorubicindid produce fragmentation of DNA. HDL is also thought to have apoptotic effects onsome cell types. (2 4). Tamoxifen(TAM) is a commonly used chemotherapeutic agent in breast cancer. Cells were lysedusing 1.1 ml .1 N NaOH, and the trichloracetic acid-soluble and insoluble125I radioactivity in the lysate, and organic solvent-extractable [3H]radioactivity determined. Thecells were incubated for 1.5 hours at 37C, the medium removed, the cellswashed three times with PBS/ .1% BSA and once with PBS. A mutant high-density lipoprotein receptor inhibits proliferation of breast cancer cells. In this study, treatment of breastcancer cells with P13K inhibitor wortmannin prevented the EGF-mediatedprotection against CD47-mediated cell death. TAM also induced maspin in myoepithelial cells, suggesting it mayexert similar effects on two different types of breast cancers (Khalkhali-Ellis et al, 2 4). The tumorsuppressor gene maspin is known to be present in high concentrations innormal mammary epithelial cells, which also require estrogen for continuedgrowth. Some cells were incubated for 8 h, fixed with 2%paraformaldehyde, and permeabilized with n-octyl-n-glucopyranoside (7mg/mol) before staining with phycoerythrin-conjugated antihuman caspase-3antibody.Flow Cytometric Determination of Apoptosis and Cell Death: Apoptosis wasdetermined by monitoring changes in cell size and the externalization ofphopshatidyl serine by flow cytometry after exposure to FITC-labeledannexin-V, with a minimum of 1 , cells were analyzed in each case withduplicate determinations (Manna and Frazier, 2 4).Determination of Mitochondrial Transmembrane Potential ()Rm): Loss ofmitochondrial membrane potential was measured using flow cytometry usingcells stained with 4 :M CMX-ROS in serum free medium for 45 mins at roomtemperature, washed and analyzed by fluorescence activated cell sorting(Manna and Frazier, 2 4).Quantification of Bcl-2: Cells were treated with 1F7, 4NIK, 4NGG oretoposide, washed in buffered saline, and resuspended in buffer, fixed in2% paraformaldehyde, treated with n-octyl-"glucopyranoside (7mg/mol) for 5min for permeabilization at room temperature, washed in PBS, stained withFITC-labeled antihuman BcL-2 antibody for 45 min and analyzed by flowcytometry, with isotope-matched antibody as control (Manna and Frazier,2 4).Measurement of Cyclic AMP: 1x1 5 cells (MDA-MB-231 and AU-565)) retreatedwith or without PTC (1 ng/ml) were plated in complete medium, incubated24 h, treated with either 4NIK or 4NGG (1 :M each) for 24 h, the cellsharvested and cAMP extracted and determined by cAMP enzyme immunoassays(Manna and Frazier, 2 4).DNA Fragmentation Analysis: 1x1 5 cells were treated with medium alone orindividually with 1F7 (1 :g/ml), 4NIK (1 :M), 4NGG (1 :M), etoposide,or doxorubicin for 4 h. The results of Cao et al showed thatP13K/Akt signaling pathway was involved in HDL-CLA-1-mediated breast cancergrowth. 125Idilactitol tyramine-[3-H]cholesterol oleyl ether HDL3particles were added (1 :g protein/ml) in serum-free medium .5%/BSA. Cells in someexperiments were retreated with a saturating concentration of pan caspaseinhibitor, ZVAD-FMK, 1 :M, for 2 h before exposure to 1F7, 4NIK oretoposide. A synthetic DNA duplex spanning AP-1, nuclear factor "$, andnuclear factor for interleukin-615' used in these studies wasradiolabelled. Site-directed mutagenesis was usedto produce a mutated promoter luciferase reporter construct 416 to +87 withthe HRE region AGATCAGA replaced by CCCCCCCC. Immunohistochemically, 1F7 gave the most intense staining, but all threemAbs used in the study stained breast cancer cells. These data indicate CD47-inducedapoptosis of breast cancer cells is caspase-independent (Figure 9 - use fig5 from Manna and Frazier). The percentage ofapoptotic cells containing subdiploid DNA was determined as the apoptoticcell rate.Statistical Analysis: Cao et al (2 4) used one-way ANOVA and the Student'st test to compare statistical significance, and p< . Previously, TAM hadbeen thought to have its primary action through competition for ER bindingsites. Cao et al focused their attention on Akt as a target becauseof reports in the literature showing the importance of this kinase on theeffects of HDL. A protective role for EGF has been indicated in breast cancer cellapoptosis and the apoptosis of other tumor cells and data from thisexperiment add another pathway for apoptosis of breast cancer cells opposedby EGF (Manna and Frazier, 2 4). TAM showed aninductive effect up to 1 -fold on promoter-driven luciferase activity inboth cells lines which was concentration and time dependent. The researhers stress the importance of furtherresearch in this area because current treatment with TAM may not be insufficient doses to induce maspin to effective levels. When mammarycarcinoma cell line MDA-MB-435 is transected with maspin cDNA, the growthand metastatic ability of these cells in severely inhibited in nude mice.Treatment of human breast cancer cells and prostate cancer cells withrecombinant maspin has been shown to reduce the motility of these cells. CD47-mediated cell death does not causecleavage of nuclear protein poly (ADP-ribose) polymerase, which is adownstream substrate for activated caspases. Using a mutant CLA-1, mCLA-1, which retainedits extracellular domain, facilitating binding of the receptor to HDL, butlacking the intracellular domain that is thought to control HDL signaling,Cao et al created a stably transected MCF-7 cell line which couldproliferate (Figure 4 - use Fig 2 from Cao et al). The increase in AP-1 activity was specificbecause other transcription factors in the same extracts were not affectedby HDL treatment. Thiswas supported by an inverse relationship found between intracellular maspinlevels and the metastatic ability of breast cancer cells. Western blot analysis of cell lysates showed that both TAM andflutamide induced maspin expression (KhalKhali-Ellis et al, 2 4) (Figure 2- from Khalkhali-Ellis et al). HDL-stimulated AP-1 activity was only sensitive to inhibition of P13K bywortmannin. Gi activation by CD47 caused a sharp decreasein intracellular cAMP levels, and restoring these levels inhibited CD47-mediated cell death. Genomic DNA was prepared from the cells .Roughly 5 :g DNA sample was electrophoretically separated on 2% agarose gelcontaining ethidium bromide ( .5 :g/ml) (Manna and Frazier, 2 4).Western Blot Analysis: 1x1 6 cells (MDA-MB231 or AU-565) were treated witheither medium alone, 4NIK, 4NGG, 1F7 or etoposide for 8 h, the cellsharvested, resuspended in solubilization buffer and run on 1 % SDS-PAGE inNuPAGE MOPS SDS running buffer and transferred to nitrocellulose membranesimmunoblotted with primary antibody and peroxidase-conjugated secondaryantibody. Because a large number of women who may be at risk forbreast cancer receive hormone therapy, there is a pressing need to discoverthe role played by estrogen in promoting breast cancer. In spite of high surface expression of FAS/CD95, breast cancer cellsresisted apoptosis induced by cross-linking FAS with mAB CH-11. TSP1, the first identifiedligand of CD47, has been implicated in the proliferation of human breastcarcinoma and has been reported in invasive lobular breast cancer. This sparing effect could be blocked entirely usingPKA inhibitor H89 and the highly specific PKA inhibitor peptide, whichsuggests one or more downstream substrates of PKA blocks CD47 activation ofGi. Cao et al (2 4) showed that mCLA-1, a mutant HDL receptor, inhibitsthe growth of ER+ MCF-7 breast cancer cells, and suppressed HDL activationof AP-1 transcription factor. Cells were then harvested without trypsinization and analyzedfor apoptosis. Tissue from patients receiving TAM therapy exhibited intensestaining for maspin in all cases. The gel wasdried and radioactive signals detected using a Bioimaging Analyzer.Detection of Apoptotic Cells: Phosphatidylserine externalization byapoptotic cells in the early stage was visualized using annexin-V-FITCstain and flow cytometry (Cao et al, 2 4). Most human breast cancers require estrogen for their continued growth, andthese cancers have been shown to respond to antiestrogen therapy alone orwhen it is used in combination with other forms of chemotherapy (Khalkhali-Ellis et al, 2 4). Maspinexpression was determined semiquantitatively by staining intensity.Tissue Samples for HDL/CLA Experiments: Coa et al (2 4) obtained tissuesfrom patients with breast cancer confirmed by histopathological findings.RNA Isolation and Reverse Transcription-PCR Analysis: Cao et al (2 4) usedPCR analysis of the reverse-transcribed RNA to determine CLA-1 expression.The 93 -bp fragment was identified using previously published CLA-1sequence.Immunohistochemical Localization: CLA-1 expression vectors were created toexpress chimeric CLA-1 mutants fused to FLAG in full-length CLA-1 (aminoacids 1-5 9) or mCLA-1 (amino acids 1-464) fused to FLAG in its NH2terminus (Cao et al, 2 4). Its ability to arrest thegrowth of breast cancer cells and to induce apoptosis are considered thereasons for TAM's efficiency as a clinical drug. Caspaseactivation is critically important for the classical apoptotic pathway, andcaspases 3,6 and 7 are thought to be activated after the mitchondrialmembrane is disrupted and releases cytochrome c (Manna and Frazier, 2 4).These researchers found that pretreatment of MDA-MB-231 or AU-565 withbroad spectrum caspase inhibitors did not prevent phosphatidylserineexposure induced by 4NIK and 1F7. Cancer Research, 64, 1 26-1 36. et al. and how it may be recruited tohelp fight cell proliferation. They also point outthat in maspin positive breast tumors, the suppressor activity may bedysfunctional and so higher doses of TAM may be of no use in these cases.For this reason, more research is needed to determine the roles of otherhormone-responsive genes such as p53 and Her-2/neu. Clinical Cancer Research, 1 , 449-454.Manna, P.P., & Frazier, W.A. They tested the ability of TAM to re-express maspin in maspin-deficientbreast cancer cells, normal mammary epithelial cell lines, and breastcancer cell lines. Western blot analysis of cell lysases confirmed the induction ofmaspin by these drugs. The primary effect of TSPs is binding to CD36, a TSPreceptor expressed on microvascular endothelial cells which seems toregulate the proliferation and motility of endothelial cells. The results of Manna and Frazier (2 4) did not show a reduction inBcl2 by CD47 induction. (2 4). CD47 mediates killing of breast tumor cells via Gi-dependent inhibition of protein kinase A. Other studies have shown thatbreast cancer cells acquire cholesterol from HDL in a CLA-!-mediatedprocess. When estrogen binds to the estrogen receptor (ER) on mammaryepithelial cells or breast cancer cells, a conformational change occurswhich allows the ER to attract a coactivator and induce gene expressionwhich is regulated by estrogen (Khalkhali-Ellis et al, 2 4). It is importantto delineate the mechanisms involved in both the proliferative phase andthe apoptotic phase so that treatment can be directed at the appropriatetargets. Apoptosis was inducedwith anti-Fas monoclonal antibodies and the propidium iodide fluorescenceof individual nuclei was measured by flow cytometry. However, maspin is down-regulated in primary breast cancer cells,and is entirely absent in aggressive mammary carcinoma lines. The loss of mitochondrial membrane potential by PTX treatment of the cellssuggests it acts downstream of a heterodimeric G protein of the Gi family,which may inhibit adenylate cyclase activity and lower intracellular levelsof cAMP. The medium was replaced with DMEM pluslipoprotein deficiency serum with or without 1 -5 :g/ml HDL, the cellsincubated for 24 h at 37C, the cells were pulsed with 1 :Ci/well[3H]thymidine (specific activity = 6.7 Ci/mmol), buretted onto glass fiberfilters using an automated cell harvester, and counted in a Packard liquidscintillation counter (Cao et al, 2 4).Transfection of MCF-7 Cells and Luciferase Reporter Gene Assay:The AP-1 reporter gene and purified reporter plasmid were transected intoMCF-7 cells at 6 % confluence using a conventional cationic lappaceumtransfection method (Cao et al, 2 4). Twenty :l aliquots were used for the luciferase assay which wasperformed according to manufacturer's instructions.Electrophoretic Mobility Shift Analysis: MCF-7 cells were treated with orwithout 5 :g/ml HDL for 12 h and nuclear extracts were prepared (Cao etal, 2 4). High-density lipoprotein (HDL) is known to stimulate growth in breastcancer cells and many other cells types, and women with high levels of HDLare at an increased risk for breast cancer (Cao et al, 2 4). PTX toxin inthe highest concentrations used did not increase annexin-V binding, and didnot affect etoposide-induced apoptosis (Figure 1 - use Manna and Frazier,fig 7). Expression of Akt-DN inhibitedthe actions of HDL induction of pAP-LUC activity, supporting the idea ofthe P13K/Akt pathway being required for HDL induction of AP-1 promoteractivity in MCF-7 cells. Manna and Frazier (2 4) have reported previouslythat some monoclonal antibodies (mAbs) to CD47 and natural CD47 ligandTSP1, and a CD47 agonist peptide, 4NIK, derived from TSP1 induce a novelform of apoptosis in transformed and activated normal T cells, chroniclymphocytic leukemia cells, erythroleukemia cells and primary arterialsmooth muscle cells.Experimental Procedures (Materials and Methods)Tissue specimens: KhalKhali-Ellis et al (2 4) used archival tissue,formalin-fixed paraffin-embedded specimens supplied by the Department ofPathology at the University of Iowa from patients with: invasive ducalcarcinoma [ER+, progesterone receptor PR+] after 5 years of TAM therapy,ductal carcinoma in situ (ER-, PR+] after 4 years of TAM therapy, and twospecimens of grade 3 invasive ductal carcinoma (ER-, PR-] after 3 and 4years of TAM treatment. HDL had a demonstrated antiapoptotic effect onMCF-7 cells, but this was not found with mCLA-expressing MCF-7 cells. This wasdemonstrated by Western blot analysis, reverse transcription OCR andimmunohistochemical staining. Cells were thenharvested, lysed in buffer, and luciferase activity of the supernatantsdetermined. Themembrane was incubated with anti-CLA-1 antibody or anti-cyclophilin-Aantibody and the binding was visualized by chemiluminescence detection.Proliferation Assay: 1 4 MCF-7 cells were cultured with 2 :l DMEM with1 % lipoprotein deficiency serum. Three primary tumor specimens were from patientswho had not received any adjuvant hormone therapy.Cell Culture and Hormone Treatments: Control and treated cells were lysedusing buffer A containing 1 mM HEPES pH7.9, 1 mM NaCl, 1 mM DTT, 1 percent glycerol, 15 mM MgCl2, .2 mM EDTA, .1 percent NP4 , proteaseinhibitor mixture, and 1 mM phenylmethanesulfonyl fluoride] and subjectiedto three freeze-thaw cycles and centrifuged at 45 g for 1 mins Khalkhali-Ellis et al, 2 4). FACS analysis showed CD47 present in breast cancer cells (Figures 7and 8 - use figs 1 and 2 from Manna and Frazier) (Manna and Frazier, 2 4).

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