Mammary Carcinoma Cell Line MCF-7 Research Articles

Roles of Cytochrome P450 1A1 and 1B1 in Metabolic Activation of Dibenzo[a, l]Pyrene by Microsomes from the Human Mammary Carcinoma Cell Line MCF-7

Incubation of DB[a, l]P with microsomes from TCDD-treated MCF-7 cells produced mainly (-) anti DB[a, l]P-11,12-diol 13,14-epoxide-dAdo adducts. Addition of antibodies against CYP1A1 and CYP1B1 inhibited the formation of DNA adducts up to 88% and 51%, respectively. The level of P450 1B1 protein was dramatically elevated, but P450 1A1 protein is not detectable by blottings in MCF-7 cells treated with 5 μM or 8 μM DB[a, l]P. MCF-7 cells treated with TCDD or B[a]P contained elevated P450 1A1 and P450 1B1. The current results demonstrate that both P450 1A1 and P450 1B1 are involved in metabolic activation of DB[a, l]P in MCF-7 cells treated with TCDD and suggest that P450 1B1 may be the major DB[a, l]P activating enzyme in MCF-7 cells treated with DB[a, l]P.

Effects of the dietary phytoestrogen biochanin A on cell growth in the mammary carcinoma cell line MCF-7

Studies of the dietary phytoestrogen biochanin A on cell proliferation of the cultured estrogen responsive cells human breast carcinoma MCF-7 showed that biochanin A exhibits biphasic regulation on MCF-7 cells. At concentrations of less than 10 μg/mL, cells respond to biochanin A by increasing cell growth and de novo DNA synthesis. The addition of biochanin A at concentrations of greater than 30 μg/mL significantly inhibited cell growth and DNA synthesis in a dose-dependent fashion, resulting in an IC 50 value of 40 μg/mL. The reversibility of these inhibitory effects by biochanin A appears also to be concentration dependent. Cells previously treated with high concentrations (>60 μg/mL) of biochanin A did not regain normal growth after treatment ceased. Biochanin A was cytostatic at low concentrations (<40 μg/mL) and cytotoxic at higher concentrations. Upon exposure to 100 μg/mL of biochanin A, cell morphology was severely altered, cell volume decreased, and condensation of cell components was clearly noticeable. In addition, biochanin A damaged cell membranes by increasing membrane permeability. These results suggest possible molecular and cellular mechanisms of the action of dietary phytoestrogens on estrogen target cells.

The level of DNA modification by (+)-syn-(11S,12R,13S,14R)- and (-)-anti-(11R,12S,13S,14R)-dihydrodiol epoxides of dibenzo[a,l]pyrene determined the effect on the proteins p53 and p21WAF1 in the human mammary carcinoma cell line MCF-7.

The polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P), the most carcinogenic PAH tested in rodent bioassays, exerts its pathobiological activity via metabolic formation of electrophilically reactive DNA-binding fjord region (+)-syn-(11S,12R,13S,14R)- or (-)-anti-(11R,12S,13S,14R)-DB[a,l]P-dihydrodiol epoxides (DB[a,l]-PDEs). DB[a,l]P is metabolized to these DB[a,l]PDEs which bind to DNA in human mammary carcinoma MCF-7 cells. The molecular response of MCF-7 cells to DNA damage caused by DB[a,l]PDEs was investigated by analyzing effects on the expression of the tumor suppressor protein p53 and one of its target gene products, the cyclin-dependent kinase inhibitor p21WAF1. Treatment of MCF-7 cells with (+)-syn- and (-)-anti-DB[a,l]PDE at a concentration range of 0.001-0.1 microM resulted in DB[a,l]PDE-DNA adduct levels between 2 and 30, and 3 and 80 pmol/mg DNA, respectively, 8 h after exposure. (-)-anti-DB[a,l]PDE exhibited a higher binding efficiency that correlated with a significantly stronger p53 response at low concentrations of the dihydrodiol epoxides. The level of p53 increased by 6-8 h after treatment. The p21WAF1 protein amount exceeded control levels by 12 h and remained elevated for 96 h. At a dose of 0.01 microM (+)-syn-DB[a,l]PDE, an increase in p21WAF1 was observed in the absence of a detectable change in p53 levels. The results indicate that the increase in p53 induced by DB[a,l]PDEs in MCF-7 cells requires an adduct level of approximately 15 pmot/mg DNA and suggest that the level of adducts rather than the specific structure of the DB[a,l]PDE-DNA adduct formed triggers the p53 response. The PAH-DNA adduct level formed may determine whether p53 and p21VAF1 pathways respond, resulting in cell-cycle arrest, or fail to respond and increase the risk of mutation induction by these DNA lesions.

Graves' disease as a model for anti-Gal involvement in autoimmune diseases.

Anti-Gal, which is the most abundant antibody in humans appeared relatively recently in the evolution of Old World monkeys and apes, i.e., approximately 20 million years ago, subsequent to the suppression of α-gal epitope expression (Galili et al., 1988; see the chapter of Galili on the “Evolution of α1,3galactosyltransferase and of the α-gal epitope” in this book). It is well established that humans do not produce α-gal epitopes because they lack the glycosylation enzyme α1,3galacto-syltransferase (α1,3GT). This enzyme synthesizes α-gal epitopes on glycolipids and glycoproteins in nonprimate mammals and New World monkeys (Galili et al., 1987a; 1988; Spiro and Bhyroo 1984; Thall et al., 1991). Nevertheless, several studies have described the in vitro interaction of anti-Gal with various human cells. These included binding of the antibody to: placental cells (Christiane et al., 1992), the mammary carcinoma cell line MCF7 (Galili 1989; Petriniyak et al., 1991), freshly obtained mammary carcinoma cells (Castronovo et al., 1989), human normal senescent red cells (Galili et al., 1986), and some pathologic red cells including red cells from patients with β-thalassemia (Galili et al., 1983; 1984) and sickle cell anemia (Galili et al., 1986). Other studies demonstrated marked increase in antiGal activity in patients with autoimmune diseases such as Graves’ Disease (Etienne-Decerf et al., 1987) and scleroderma (Gabrielli et al., 1991). Taken together, all these observations suggest that despite the evolutionary suppression of α-gal epitope expression in ancestral Old World primates, anti-Gal may interact with human cells under certain circumstances, which are poorly understood at present.

Influence of 5-Lipoxygenase on in vitro Growth of Human Mammary Carcinoma Cell Line MCF-7

The aim of this study was to investigate the direct effect of 5-lipoxygenase (5-LO) on the growth of human mammary cancer cells MCF-7 in vitro. Cell growth was measured according to the level of ³H-thymidine incorporation. 5-LO was shown to inhibit ³H-thymidine incorporation. The inhibitory effect was 19, 42 and 78% when administered at concentrations of 0.1, 0.2 or 0.5 U/ml, respectively. Its effect was time- and dose-dependent and was statistically significant at concentrations of 0.2 and 0.5 U/ml. We have also shown that the specific 5-LO inhibitor MK-886 (1 µM) lifts the inhibitory effect of 5-LO (0.2 U/ml). Moreover, when treated with an activator of 5-lipoxygenase calcium ionophore A23187 (10 µM) MCF-7 cells synthesize LTB4. The results of this study are evidence of the role of 5-lipoxygenase in the regulation of human mammary cancer cells growth in vitro.

Influence of leukotrienes on in vitro growth of human mammary carcinoma cell line MCF-7

Objectives: The aim of this work was to study the action of leukotrienes on the growth of human mammary cancer cells MCF-7. Study design: The growth of the cells was measured by incorporation of 3H-thymidine. The action of leukotriene (LT)B4, LTD4, LTC4, LTE4 or arachidonate (AA) was tested in human mammary cancer cells MCF-7 in vitro. Results: LTB4 or LTD4 but not LTC4 or LTE4 reduced significant incorporation of 3H-thymidine in MCF-7 cells up to 52% or 56% respectively, when administered in concentrations 0.1–1000 pM. Agents in concentrations of 0.01 pM or 10 000 pM did not effect 3H-thymidine incorporation. We have shown, that MCF-7 cells synthesise LTB4 when treated with calcium ionophor A23187 (10 μM). Leukotriene-antagonist LY171883 (10 μM) lifts inhibitory effects of LTB4 or LTD4. Arachidonic acid (10 μM) inhibits 3H-thymidine incorporation up to 72%. 5-lipoxygenase inhibitor MK-886 (100 nM) lifts the inhibitory effect of arachidonate. Conclusions: LTB4 or LTD4 inhibits MCF-7 breast cancer cell growth. LT-receptors mediate the growth-inhibitory effect of LTB4 or LTD4.

Stereoselective activation of dibenzo[a,l]pyrene and its trans-11,12-dihydrodiol to fjord region 11,12-diol 13,14-epoxides in a human mammary carcinoma MCF-7 cell-mediated V79 cell mutation assay.

Dibenzo[a,l]pyrene (DB[a,l]P) represents the most potent carcinogenic polycyclic aromatic hydrocarbon (PAH) yet discovered. Like other PAHs, DB[a,l]P requires metabolic activation to exert its mutagenic and/or carcinogenic activity. In the human mammary carcinoma cell line MCF-7, DB[a,l]P is stereoselectively metabolized to the (-)-anti- and (+)-syn-DB[a,l]P-11,12-diol 13,14-epoxides (DB[a,l]PDE) which both bind extensively to deoxyadenosine residues in DNA. To further characterize the underlying mechanism of its strong carcinogenicity, the relationship between DNA binding and mutagenicity of DB[a,l]P was determined. Racemic DB[a,l]P-11,12-dihydrodiol and the two individual (+)- and (-)-enantiomers, the metabolic precursors of the stereoisomeric fjord region dihydrodiol epoxides, were also investigated. Induction of mutations at the HPRT locus was measured in a MCF-7 cell-mediated Chinese hamster V79 cell mutation assay. The parent hydrocarbon, (+/-)-DB[a,l]P-11,12-dihydrodiol, and (-)-DB[a,l]P-11,12-dihydrodiol were highly mutagenic under the assay conditions. In contrast, (+)-DB[a,l]P-(11S,12S)-dihydrodiol was not mutagenic using MCF-7 cells as the metabolic activating system. Analysis of DNA adducts in the same experiments revealed that MCF-7 cells treated with (-)-DB[a,l]P-11,12-dihydrodiol formed exclusively (-)-anti-DB[a,l]-PDE adducts whereas cells treated with (+)-DB[a,l]P-11,12-dihydrodiol did not contain detectable levels of DNA adducts. These results suggest that specific cytochrome P450 enzymes may have high stereoselectivity for activation of the two DB[a,l]P-11,12-dihydrodiol enantiomers, and this may play an important role in the metabolic activation of the strong carcinogen DB[a,l]P in human cells.

Human and rodent expression pattern of a fusion gene isolated from an MCF7 cDNA library

A cDNA library established from the human mammary carcinoma cell line MCF7 was used to screen for transcripts related to or representing the gene coding for the estrogen receptor-related protein ERD5. Among the two populations of cDNAs isolated, one group (3 clones) represented heat shock protein transcripts (HSP27), the other (2 clones) appeared identical in their insert size and restriction enzyme banding pattern. Detailed analysis of one of them revealed its nature as a fusion sequence composed of two genes. At the 5'-end it consists of the last 157 bp of the HSP28 gene. At its 3'-part, it is identical to the 3'-portion of thr nuclear factor IV gene. The genuine expression of this fusion gene was demonstrated by Northern blots and RT-PCR. It appears to be ubiquitously expressed in a variety of normal tissues and malignant cells in man, mouse and rat.

Influence of ethanol on in vitro growth of human mammary carcinoma cell line MCF-7.

We have shown that ethanol in concentrations of 0.0001%-10% significantly enhances 3H-thymidine uptake in cultured human mammary carcinoma cell line MCF-7. Methanol only enhances 3H-thymidine uptake in a concentration of 0.01%.

Benzo[c]phenanthrene is activated to DNA-binding diol epoxides in the human mammary carcinoma cell line MCF-7 but only limited activation occurs in mouse skin.

Benzo[c]phenanthrene (B[c]Ph) is an environmental contaminant with low carcinogenic activity in rodent bioassays. B[c]Ph-3,4-diol-1,2-epoxides (B[c]PhDE), however, are among the most tumorigenic diol epoxides known. To determine whether human cells are capable of activating B[c]Ph to DNA-binding metabolites, cultures of the human mammary cell line, MCF-7, were exposed to 10 microM B[c]Ph for 48, 72 and 96 h or to 1 microM (+/-)-B[c]Ph-3,4-dihydrodiol for 48 h. The B[c]Ph-DNA adducts were analyzed by 33P-postlabeling and reverse-phase HPLC. The major B[c]Ph-DNA adducts were formed by the trans-addition of (4R,3S)-dihydroxy-(2S,1R)-epoxy-1,2,3,4-tetrahydro-B[c]Ph to deoxyadenosine [(-)-B[c]PhDE-2dAt] and by the cis- and trans-addition of (4S,3R)-dihydroxy-(2S,1R)-epoxy-1,2,3,4-tetrahydro-B[c]Ph to deoxyadenosine [(+)-B[c]PhDE-1dAc and (+)-B[c]PhDE-1dAt]. Smaller amounts of the trans-addition of (-)-B[c]PhDE-2 were bound to deoxyguanosine. To determine whether B[c]Ph can be metabolically activated to diol epoxides in mouse epidermis, female SENCAR mice were treated topically with 2 micromol B[c]Ph for 24, 48 or 72 h or with 0.4 micromol (+/-)-B[c]Ph-3,4-dihydrodiol for 24 or 48 h. In B[c]Ph-treated mice, only small amounts of three B[c]PhDE-DNA adducts were detected [(-)-B[c]PhDE-2dAt, (+)-B[c]PhDE-1dAt and (+)-B[c]PhDE-1dAc] at 24, 48 and 72 h. In contrast, mice treated topically with 0.4 micromol (+/-)-B[c]Ph-3,4-dihydrodiol formed B[c]PhDE-DNA adducts at levels 6-fold greater than those observed with B[c]Ph at 48 h. The higher formation of B[c]PhDE-DNA adducts by (+/-)-B[c]Ph-3,4-dihydrodiol correlates with the greater potency of (+/-)-B[c]Ph-3,4-dihydrodiol than of B[c]Ph as a tumor initiator in mouse skin. The low extent of formation of B[c]PhDE from B[c]Ph in mouse epidermis may explain the low tumorigenicity of B[c]Ph in this tissue. These results indicate activation of B[c]Ph in mouse skin and tumorigenesis results in that tissue may not adequately assess the potential capability of cells from humans to activate B[c]Ph to ultimate carcinogenic metabolites.

Stereoselective activation of dibenzo[a,l]pyrene to (-)-anti (11R,12S,13S,14R)- and (+)-syn(11S,12R,13S,14R)-11,12-diol-13,14-epoxides which bind extensively to deoxyadenosine residues of DNA in the human mammary carcinoma cell line MCF-7.

Dibenzo[a,l]pyrene (DB[a,l]P) is an environmental contaminant and a very potent carcinogen. DB[a,l]P exceeds the carcinogenic potency of both benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in rodent bioassays. Previous studies demonstrated that DB[a,l]P is metabolized to DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE) in the human mammary carcinoma cell line MCF-7. In the present study the major DNA adducts formed in DB[a,l]P-treated MCF-7 cells have been identified through the use of 33P-postlabeling. TLC and HPLC. DB[a,l]P is metabolically activated in MCF-7 cells to form large amounts of three major DNA adducts and smaller amounts of three other adducts. The three major DNA adducts are with deoxyadenosine: two are formed by reaction of (+)-syn-DB[a,l]PDE (11S,12R,13S,14R), the third by reaction of (-)-anti-DB[a,l]PDE (11R,12S,13S,14R). The results demonstrate that DB[a,l] is stereoselectively metabolized in MCF-7 cells to form one enantiomer of each diol epoxide diastereomer; (+)-syn-DB[a,l]PDE and (-)-anti-DB[a,l]PDE. The high extent of binding of these diol epoxides to deoxyadenosine in DNA of MCF-7 cells may help to explain the very high carcinogenic potency of DB[a,l]P and suggests that DB[a,l]P could also pose a carcinogenic threat to humans.

Exposure of mammalian cell cultures to benzo[a] and light results in oxidative DNA damage as measured by 8-hydroxydeoxyguanosine formation

Carcinogenic polycyclic aromatic hydrocarbons induce DNA damage through direct covalent interactions with nucleotides of the DNA in cells in which they are activated to 'ultimate carcinogenic metabolites'. To determine whether they also induce oxidative damage to DNA under the same circumstances, early passage Syrian hamster embryo and human mammary carcinoma cell line MCF-7 cultures were treated for 24 h with 0-5 micrograms/ml benzo[a]pyrene (BaP) or for 1 h with 0-100 microM methylene blue (as a positive control for oxidative damage). The cells were then exposed to fluorescent light for 1 or 4 h or retained in darkness. After cell harvest, DNA isolation and enzymatic digestion of the DNA to deoxyribonucleosides, the amounts of 8-hydroxy-2'deoxyguanosine (8-OH-dGuo) and unmodified deoxyguanosine present were determined by reverse-phase HPLC with electrochemical and UV detection respectively. Cultures treated with methylene blue for 1 h followed by light exposure for 1 h contained 5-fold (10 microM) and 8- to 28-fold (100 microM) higher 8-OH-dGuo levels than cells treated with methylene blue not exposed to light or untreated cells with methylene blue not exposed to light or untreated cells exposed to light. There was no significant change in 8-OH-dGuo levels in cultures treated with 1-5 micrograms/ml BaP for 24 h in the absence of light. However, both the human and hamster cell cultures treated with BaP and then exposed to fluorescent light for 4 h contained 3-fold (1 micrograms/ml) and 8- to 10-fold (5 micrograms/ml) higher 8-OH-dGuo levels than those not exposed to light or not treated with BaP. These results indicate that BaP treatment does not cause 8-OH-dGuo formation in DNA of cells maintained in darkness. Exposure of BaP-treated cells to fluorescent light causes formation of significant amounts of oxidative DNA damage as measured by 8-OH-dGuo formation. These findings suggest that oxidative damage of DNA could be involved in tumor induction by BaP in tissues, such as skin, in which exposure to BaP can occur in the presence of light.

Involvement of Jun and Fos proteins in regulating transcriptional activation of the human pi class glutathione S-transferase gene in multidrug-resistant MCF7 breast cancer cells.

Elevated levels of the human pi class glutathione S-transferase (GSTP1-1) have been implicated in the development of antineoplastic drug resistance. Using GSTP1 promoter deletion constructs we have shown that enhanced GSTP1 transcription (up to 18-fold) is the predominant mechanism responsible for increased GSTP1-1 levels in a multidrug resistant derivative (VCREMS) of the human mammary carcinoma cell line MCF7. Furthermore, disruption of a putative AP-1 response element within the GSTP1 promoter (nucleotides -69 to -63) abrogated GSTP1 transcription in both cell lines. In addition, band shift assays demonstrated binding of a VCREMS nuclear complex to the promoter region C1 (-73 to -54) which could be competed for by a DNA fragment containing a known AP-1 binding site from the human collagenase promoter. However, no such competition was observed for the major MCF7 C1 complex. The role of a Fos-Jun-like complex in regulating GSTP1 transcription in VCREMS cells was further emphasized by the introduction of point mutations within the C1 region which were known to inhibit AP-1 binding and the interaction of antisera raised against human c-Jun and c-Fos with the major C1 complex in VCREMS cells. These studies therefore highlight cell-specific differences in the binding pattern of Jun and Fos proteins to the GSTP1 promoter which are likely to play an important role in regulating transcriptional activation of the GSTP1 gene in drug-resistant breast cancer cells.

Idiotypic Replica of an Anti‐Human Tumour‐Associated Antigen Monoclonal Antibody DNA Sequence Comparison between Ab1 and Ab3

2G-3 is an anti-anti-idiotypic MoAb (Ab3) obtained upon immunization with a monoclonal Ab2 (A3B10), which behaves as the 'internal image' of CaMBr1, a saccharide epitope defined by MoAb MBr1 (Ab1). CaMBr1 is expressed on glycoconjugates of the human mammary carcinoma cell line MCF-7 and on normal and neoplastic mammary gland epithelial cells. Ab1 and Ab3, although exhibiting, in many respects, superimposable paratopic and idiotopic specificities, show a non-identical fine immunoreactivity, since 2G-3 has a preferential reactivity with the saccharidic epitope mounted on glycoproteins, while MBr1 reacts with both glycoproteins and glycolipids. V-region sequence analysis has shown that: (i) the VK genes employed belong to different families (VK1 and VK10); (ii) the JK2 segment is shared by the two L chains (thus a high degree of homology is observed between VK CDR3s); (iii) the VH genes employed derive from the same family VHIIB/J558 (but show CDR homology only in CDR2); (iv) different JH region genes are employed. These data, together with the comparison of deduced secondary structure parameters, give further evidence for the possible production of similar combining sites using different VH and VL germ-line genes.

The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells

Okadaic acid, a specific and potent inhibitor of protein phosphatases 2A and 1, was tested for its effect on the morphology of a number of cell types: freshly isolated rat hepatocytes in suspension or in primary culture, the human mammary carcinoma cell line MCF-7, the human neuroblastoma cell line SK-N-SH, rat pituitary adenoma GH3 cells, and rat promyelocytic IPC-81 cells. All the cell types reacted within a few hours to okadaic acid in the concentration range 0.1 to 1 μ M with profound morphological alterations. Among the changes noted were: condensation of chromatin, shedding of cell contents via surface bleb formation, redistribution and compacting of cytoplasmic organelles, formation of cytoplasmic vacuoles, and hyperconvolution of the nuclear membrane. In some cells nuclear fragmentation was noted. In addition, cells growing as monolayers rounded up and detached from the substratum. The treated cells had no swollen mitochondria and retained the ability to exclude trypan blue until the final stage of dissolution, supporting the hypothesis that the changes were apoptotic rather than necrotic. In hepatocytes the action of okadaic acid was mimicked by another phosphatase inhibitor, microcystin, and was accompanied by shrinkage of the cell volume, as judged by Coulter counter analysis. The action of phosphatase inhibitor was not abolished by protein synthesis inhibitors, Ca 2+-depleted medium, or phorbol ester. Although hepatocyte DNA replication was very sensitive to inhibition by okadaic acid, DNA fragmentation was less pronounced in response to okadaic acid than other agents inducing the morphological appearance of apoptosis.

Effects of the antiestrogen toremifene on growth of the human mammary carcinoma cell line MCF-7

The effects of toremifene, a new antiestrogenic drug, were investigated in vitro on the exponentially growing human mammary carcinoma cell line MCF-7. The drug effects were monitored by serial cell counts and DNA flow cytometry. The inhibitory effect of toremifene on MCF-7 became greater as the drug concentration was increased from 1 microM to 10 microM. At 5 microM toremifene induced a large decrease in the relative percentages of S- and G2/M-phase cells, and an increase in the amount of cell debris, indicating increased cell death. After withdrawal of the drug the mammary cancer cells resumed logarithmic growth similar to that of control cells. The effects caused by toremifene were similar to those caused by tamoxifen both in quality and quantity.

Cytoskeleton in human mammary carcinoma cells forming three-dimensional cellular structures within collagen gels.

Human mammary carcinoma cell line MCF-7 cells grown on type I collagen gels floating in a medium occasionally invaginated into the gels as a cell mass and formed cylindrical or domed structures within it. The 0.05% Triton-insoluble cytoskeleton of such cellular structures sedimented as a white flocculent layer at the boundary between 60 and 70% sucrose layers by ultracentrifugation, and consisted of 4 basal components: 54-kD (beta-tubulin), 45-kD, 42-kD (actin), and 39-kD polypeptides. By contrast, the isolated cytoskeleton of MCF-7 cells grown as monolayers on plastic substratum formed a finer cytoskeletal network with a smaller buoyant density and consisted of two distinct polypeptides with apparent molecular sizes of 80-kD and 65-kD in addition to the 4 basal components found in the morphologically developing cells. The present results indicate that the cytoskeleton of MCF-7 cells forming the three-dimensional cellular structures within collagen gels is lacking in these two polypeptides, and that it has a coarser cytoskeletal network with a greater buoyant density than that of the monolayered cells on plastic.

Idiotypic replica of an anti-human tumor-associated antigen monoclonal antibody. Analysis of monoclonal Ab1 and Ab3 fine specificity.

CaMBr1 is a tissue-specific and tumor-associated saccharidic epitope, defined by mAb MBr1 (Ab1), expressed on glycoconjugates of the human mammary carcinoma cell line MCF-7 and of normal and neoplastic mammary epithelial cells. An anti-anti-idiotypic monoclonal Ab3, 2G-3, identifying a human breast tumor associated antigen, was raised by using as immunogen a mouse anti-idiotypic monoclonal Ab2, A3B10, which behaves as the internal image of CaMBr1. mAb 2G-3, as well as MBr1, defines a saccharidic epitope on glycoconjugates extracted from MCF-7 cells and shows MBr1-like reactivity on normal and neoplastic-tissues. Experimental evidence, however, suggests that the fine immunoreactivity of the two antibodies is not identical, because MBr1 has a preferential reactivity with glycolipids and 2G-3 with glycoproteins. We suggest that a possible biologic explanation for our findings could reside in the nature of the immunogens used to raise the two mAb (glycolipid vs protein "internal image").

Generation of monoclonal antibodies reactive with nuclear proteins of human primary breast tumors.

Nuclear proteins were extracted from purified nuclei of human primary breast tumors (BrT) and bladder tumors and of human normal breast, kidney and lymphocytes by enzymatic treatment. SDS-Polyacrylamide gel electrophoresis of nuclear proteins from breast tumors showed different bands in the molecular weight zones from 25 to 220 kDa which were absent or present only as traces in normal breast tissue. Murine monoclonal antibodies (MAbs) have been produced using nuclear extracts of human primary breast tumors as immunogens. Approximately 2,000 hybridomas were generated from 5 hybridizations. According to their reactivity to BrT nuclear extracts and mammary carcinoma cell line MCF-7, seven hybridomas were selected and cloned. They were further characterized with histological immunoperoxidase assays of formaldehyde-fixed BrT paraffin tissue sections. MAb 6A3 particularly gave strong nuclear staining with all BrT specimens while MAb 1D8 showed both nuclear and cytoplasmic staining with only some of them. Specimens from mammoplasty did not react with these MAbs. Immunoblotting of BrT nuclear extracts as developed with MAbs 6A3 and 1D8 revealed major protein bands with molecular weight of 120 and 130 kDa. The potential use of these MAb-defined BrT-related nuclear proteins as markers for human breast cancer was suggested.

Anti-human tumor antibodies induced in mice and rabbits by "internal image" anti-idiotypic monoclonal immunoglobulins.

MBr1 is a murine monoclonal antibody, defining a saccharidic epitope [CaMBr1] of a human tissue-specific, tumor-associated globoside, present on the mammary carcinoma cell line MCF-7. The same epitope is shared by glycoproteins present on normal and neoplastic mammary epithelial cells, and by mucins from some ovarian cyst fluids. We have used MBr1 as the monoclonal antitumor antibody in an idiotypic sequence of immunizations in order to obtain and characterize "internal images" of the original epitope to be used as substitutes of the nominal antigen in serologic immunoassays. Two monoclonal anti-idiotypic antibodies (beta-1 and beta-2), which reacted with paratope-related idiotopes on MBr1, were obtained. The analysis of the antigenic and immunogenic properties of these molecules by both "antigen" and "antibody" competition assays provided evidence that both beta-1 and beta-2 bear "internal images" of the MBr1-defined epitope. Moreover, when injected in mice and rabbits both beta-1 and beta-2 induced anti anti-idiotypic antibodies, which mimicked MBr1 in binding MCF-7 as well as normal and neoplastic mammary gland epithelial cells. These data are discussed in terms of their possible application to the production of tumor-associated antigen substitutes and their use in serologic immunoassays.