Malignant Mammary Tissues Research Articles

Monoclonal antibody directed against colon cancer mucin has high specificity for malignancy

Exposure of core carbohydrate structures such as NeuAc alpha2-6GalNAc-Ser/Thr (sialosyl-Tn) in mucus glycoproteins is often associated with malignant transformation in a number of different tissues. Reagents that specifically identify such structures would be useful in the diagnosis of cancer. Monoclonal antibody JT10e has been produced against mucins from xenografts of LS174T colon cancer cells but also reacts with mucins of pancreatic cancer xenografts. The following data suggest that JT10e reacts with sialosyl-Tn: (I) reactivity with bovine and ovine submaxillary mucins; (2) reactivity sensitive to neuraminidase or mild acid; (3) inhibition of reactivity by NeuAc and NeuAc alpha2-6 lactose; and (4) lack of reactivity with other glycoproteins with related carbohydrate structures but no sialosyl-Tn. JT10e is distinguishable from 2 other antibodies which react with sialosyl-Tn, B72.3 and TKH2 because JT10e: (a) did not react with normal gastric tissue while B72.3 and TKH2 did; (b) was only partially inhibited by TKH2 and not at all by B72.3; (c) did not react with Tn antigen while B72.3 did; and (d) bound more strongly to pancreatic cancer mucins than did either B72.3 or TKH2. JT10e reacted with a high percentage of malignant colonic, pancreatic, gastric and mammary tissues but not with the corresponding normal tissues. A high percentage of patients with colonic, pancreatic, gastric, mammary and lung cancers had elevated blood levels of JT10e antigen. A number of colonic cancer patients had elevated JT10e antigen levels without corresponding elevations in CA19-9 levels. These results suggest that JT10e antibody could be used in conjunction with other mucin markers to improve the identification of malignancy in colon, and to study the structure of oligosaccharides in cancer mucins.

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract Collagen-binding proteins ( CBPs) of rat mammary tumors are identical to Ca 2+-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I. In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.

The high-affinity folate binding protein in normal and malignant mammary gland tissue.

Human milk contains a soluble 25 kDa folate binding protein (FBP) as well as a membrane-derived FBP with a hydrophobic glycosyl-phosphatidylinositol tail; the enzyme phosphatidylinositol specific phospholipase C reduced the apparent size of the latter FBP from 100 to 25 kDa1. The two FBPs from human milk had identical N-terminal amino acid sequence for 39 cycles and shared antigenic determinants2,3. A high correlation between the concentrations of FBP and folate in milk suggested the secretion of a folate mammary FBP complex into milk4. In the present study we have characterized and compared FBP in the normal mammary gland and mammary tumors.

Expression of Two Antigens Defined by Monoclonal Antibodies in Normal, Benign and Malignant Human Mammary Tissues

We examined the reactivity of two monoclonal antibodies (MAbs), MBrI and MBr8, on sections from normal breast tissues, benign lesions, primary infiltrating carcinomas and distant breast cancer metastases and compared the expression of the recognized antigens (CaMBr1 and CaMBr8 respectively) in these different normal and pathologic conditions. The expression of both antigens was found to significantly decrease in the neoplastic tissues, in comparison to normal breast tissue (34% vs 61% for MBr1 and 52% vs 86% for MBr8; p less than 0.001 for both). Atypical epithelial hyperplasia was found to be reactive on the same level (31%) of the tumors when tested with MBr1, whereas its reactivity was similar to that of normal tissues (78%) when MBr8 was used. Nonatypical epithelial hyperplasia showed the same reactivity with both MAbs (positive staining on about 50% of the cells). 65% of the "in situ" ductal carcinoma cases was MBr1-positive, whereas 50% was MBr8-positive; these values, when compared with the reactivity on normal glands, were significantly lower for MBr8, but not for MBr1. When testing sections from premenopausal women who had biopsies performed on different days during their menstrual cycle a correlation was found between the expression of CaMBr1 in normal breast epithelial cells and the stimulatory phase of the cycle. These findings suggest that both antigens are typical of normal breast and their expression could possibly decrease in pathologic tissues.

Analysis of the tumour-associated antigen TAG-12 by monoclonal antibody 7A9 in normal, benign and malignant mammary tissues

A monoclonal antibody 7A9 was raised against the tumour-associated glycoprotein TAG-12 purified from T47-D breast carcinoma cells. In immunoblots from cytosol of T47-D cells and from sera of breast cancer patients, antibody 7A9 detects the high molecular weight mucin-like TAG-12 antigen. A series of paraffin sections of normal, benign and malignant mammary tissues have been studied with monoclonal antibody 7A9 and the immunoalkaline phosphatase method. In resting gland, proliferating gland and fibroadenoma ducts, reactivity of 7A9 was mainly restricted to luminal membranes of epithelial cells and secretions. 77/79 primary breast carcinomas including ductal, lobular and various other carcinoma types showed cytoplasmic and/or membrane-associated staining with 7A9 in most tumour cells. Metastases (31/31) from different sites were also positive. Strong immunoreactivity with single tumour cells was noted in cytological preparations from freshly resected breast cancer tissue. Thus, monoclonal antibody 7A9 seems to be very useful for the targeting of breast carcinoma cells.

A novel leupeptin-sensitive serine endopeptidase present in normal and malignant rat mammary tissues

N-Methyl-N-nitrosourea (MNU)-induced rat mammary adenocarcinomas contain high levels of a novel leupeptin-sensitive serine endopeptidase. Its properties apparently differ from those of other similar endopeptidases reported to be present in various normal and malignant mammalian tissues. The same leupeptinsensitive serine endopeptidase was also detected in normal rat mammary tissues, but at levels approximately 20 times lower than those in MNU-induced mammary tumors. This enzyme, which is a trypsin-like serine endopeptidase, preferentially hydrolyzes various synthetic endopeptidase substrates at the carboxyl side of an arginyl residue. It has an apparent Mr of approximately 160,000 and a Stokes radius of 49 A, as determined by gel filtration. Its isoelectric points range from 4.5 to 4.8, and it has a pH optimum of approximately 7.0. The enzyme is stable from pH 4.0 to 7.0, but is extremely unstable above pH 7.0. Besides leupeptin, its activity is inhibited by antipain, aprotinin, N alpha-p-tosyl-L-lysine chloromethyl ketone and phenylmethylsulfonyl fluoride, but is not inhibited by soybean trypsin inhibitor. Many other potential inhibitors or activators such as 2-mercaptoethanol, p-hydroxymercuribenzoic acid and EDTA have no effect on its activity. The enzyme is adsorbed to p-aminobenzamidine agarose affinity beads at pH 6.5 and elutes at pH 4.0.

Tenascin distribution in the normal human breast is altered during the menstrual cycle and in carcinoma

Abstract Tenascin is a novel extracellular matrix glycoprotein which appears to have a major role in tissue development [6]. Previous studies have stated that tenascin is absent from the normal human [22], rat [6] and mouse [18] breast, its distribution being restricted to embryonic and malignant mammary tissues [22]. No previous studies have investigated tenascin distribution as a function of the normal menstrual cycle. Therefore this study addresses the cyclical appearance of tenascin in the normal breast and associated changes in distribution in preinvasive cancer (carcinoma-in-situ) and invasive infiltrating ductal carcinoma. Tenascin is present in the normal human adult mammary gland, principally in the basement membrane, sub-basement-membrane zone and delimiting layer of fibroblasts around the ductules. Both the distribution and quantity of tenascin change during the menstrual cycle. In carcinoma-in-situ (preinvasive cancer) tenascin is present in the attenuated basement membrane/sub-basement-membrane zone around the expanded ductules and in small amounts in the stroma. In infiltrating ductal carcinoma, tenascin is absent from the remnants of the basement membrane and sub-basement-membrane zone but greatly increased in the adjacent intralobular and interlobular stroma. Therefore, if tenascin is used as a basement membrane/sub-basement-membrane marker for distinguishing carcinoma-in-situ from invasive ductal carcinoma, the time of the menstrual cycle is of importance in interpreting the biopsy appearance. This study suggests that the optimal time for biopsy is between weeks 3 and 4 of the cycle, to avoid confusion between the normal low levels of tenascin (due to hormonal status) and those due to microinvasive disease. That tenascin expression in the normal breast is cyclically regulated suggests that this molecule may act as a mediator in the hormonal control of mammary epithelial cell behaviour.

Endogenous oestrogens and androgens in normal and malignant endometrial and mammary tissues

Because of a well-established mechanism of action, tissue concentrations of steroid hormones are thought to be more closely related than blood levels to the biological effects exerted by these hormones.The results of studies on oestrogen and androgen concentrations in malignant and normal breast tissues are presented. Normal fatty and epithelial breast tissues and malignant tumour samples which had been obtained from pre- and postmenopausal women of two countries (Poland and The Netherlands) differing in the incidence of this malignancy were studied. In both countries highly comparable oestradiol concentrations in the breast were found. The median hormone levels in tumour tissue of 0.65 pmol/g tissue did not change with age. They were significantly higher than in normal epithelial (0.48 and 0.25 pmol/g in pre- and postmenopausal women) and fatty tissues (0.54 and 0.19 pmol/g respectively). Particularly in postmenopausal women, hormone levels in tumour tissue were much higher than plasma concentrations, which are comparable in both populations. Oestrone levels decreased with age in normal and malignant breast tissues. In both countries median levels in normal and fatty tissues of premenopausal women were similar (1.10 pmol/g tissue) but higher than those in postmenopausal patients (0.45 pmol/g tissue. Significantly lower levels were found in the malignant tissue samples of Polish premenopausal women (0.70 pmol/g) than in Dutch women (1.05 pmol/g); similarly, after menopause the tissue concentrations were higher in Dutch (0.55 pmol/g) than in Polish (0.31 pmol/g) patients. Thus lower oestrone tissue levels were observed in tumours from the country with the lower incidence for breast cancer.In a comparable study of uterine tissues, obtained from pre- and postmenopausal women, higher oestradiol concentrations than in the breast were found, whereas estrone levels were very similar. The levels in the uterus did not correlate with those in the plasma; no relation with histology was observed.The results of androgen measurements in breast tissues were in agreement with the concept that, particularly, androstenedione and testosterone could play a role as substrates for local aromatization. Lower concentrations were observed in the tumours than in the normal and fatty tissues.More extensive investigations will be needed to clarify the role of local formation (aromatization, hydrolysis by sulphatase) of oestrogens in tissues and of the interconversion of less active (oestrone) to more active (oestradiol) oestrogens.

Micromethod for the determination of free and total prolactin receptors: measurement of receptor levels in normal and malignant mammary and prostate tissues.

A sensitive micromethod for the determination of free and total prolactin receptors in normal or malignant tissues has been developed. Positive and negative quality controls are incorporated in the procedure. Either whole tissue or the pellet fraction remaining from tissue that had undergone processing for estrogen receptors can be used. Crude microsomal and plasma membrane fractions obtained by homogenization and differential centrifugation are incubated with labeled prolactin in the presence or absence of increasing amounts of unlabeled hormone. The labeled ligand is prepared by a stoichiometric iodination procedure in which one atom of iodine-125 is incorporated into one molecule of the hormone, resulting in an intact labeled prolactin with a high specific activity of 170-186 muCi/micrograms (1 Ci = 37 GBq). Human prolactin labeled by this procedure has much greater specific binding capacity to various rat tissues than does iodinated rat prolactin. This technique permits an accurate measurement of prolactin receptors in as little as 50 micrograms of membrane protein. Highest levels of free and total prolactin receptors were found in the liver of 60-day-old female rats that served as a positive control. Liver of immature 21-day-old male rats, devoid of prolactin receptors, was used as a negative control. The amount of detectable free receptors was dependent on the level of circulating plasma prolactin. In 3-day postpartum lactating rats with high prolactin levels in plasma, all prolactin receptors in the mammary glands were found to be occupied, and no free receptors could be detected. When these receptors were desaturated from the endogenous prolactin by exposure to 3 M MgCl2, one class of receptors in a high quantity (1.75 nmol/mg of protein) and with a moderate affinity (Kd = 6.41 X 10(-9) M) was detected. A similar type of receptor was found in the mammary glands of rats at midpregnancy and of cycling adult female rats. In malignant rat mammary tissue, however, fewer receptors (27 pmol/mg of protein) but with a very high affinity (Kd = 6.8 X 10(-14) M) were detected. Normal ventral and dorsolateral rat prostate contained two classes of prolactin receptors (Kd = 3.46 X 10(-10) M and 1.93 X 10(-8) M). In the cancerous rat prostate, however, only one of these two classes of receptors was detected, and the number was smaller.

The 52-kDa estrogen-induced protein secreted by MCF 7 cells is a lysosomal acidic protease

An estrogen-induced 52-kDa glycoprotein secreted by human breast cancer cells and able to autostimulate the growth of MCF 7 cells has been purified, using monoclonal antibodies, and characterized. The protein contains mannose 6-phosphate signals on its N-linked high-mannose chains, suggesting that it is a lysosomal enzyme. Both the secreted 52-kDa protein and its processed cellular forms (52-, 48- and 34-kDa) were identified as carboxyl proteinases having an optimal activity at pH 3.5 and being specifically inhibited by pepstatin. This protease is characterized by its inducibility by estrogens and its high concentration in proliferative benign and malignant mammary tissue, when detected by immunohistochemistry. The estrogen-induced secretion of this protease may help to understand how estrogens stimulate mammary tumor growth and/or invasion.

Immunohistochemical detection of ras oncogene p21 product in benign and malignant mammary tissue in man.

The monoclonal antibody RAP-5 generated against a synthetic peptide corresponding to amino acid positions 10-17 of the ras p21 protein was used in an immunohistochemical study of the expression of ras in normal, benign, and malignant breast epithelium in man. The staining intensity and intracellular distribution of RAP-5 was similar in the three epithelial populations and extended to other tissue elements including myoepithelial cells, smooth muscle, myelin, capillary endothelium, and stromal fibroblasts, as well as sebaceous glands and sweat glands overlying the breast. These results suggest that RAP-5 recognises a normal cellular component, the expression of which is not more enhanced in hyperplastic or neoplastic conditions. The detection of mutant forms of p21 exclusively expressed in malignant tumours requires that alternative reagents be developed.

Characterization of lysosomal acid phosphatase from normal and malignant mammary tissue.

Acid phosphatase (EC 3.1.3.2) isoenzyme 3 was purified from normal and malignant human mammary tissue and its properties in each were compared. The relative molecular mass of each was 53 000, as measured by sodium dodecyl sulfate gel electrophoresis. Several phosphomonoesters are good substrates for the isoenzymes, whereas organic and inorganic pyrophosphates and phosphoryl choline are hydrolyzed very slowly or not detectably. The optimum pH for interaction of these isoenzymes with p-nitrophenyl phosphate as substrate ranges from 3.5 to 4.5. L-(+)-Tartrate is a very strong inhibitor, Ki = 0.028 +/- 0.04 mmol/L (mean +/- SE), as are mercuric and fluoride ions in low concentrations. We conclude that type 3 isoenzymes obtained from normal and malignant tissue are very similar, though the malignant tissue appears to have a greater proportion of this type than does normal tissue.

Suppressive effect of inflammation and other forms of stress on the binding of prolactin by rat liver.

We have found that in female rats a variety of stressful stimuli, including sc inflammation, skin incision, endotoxin injection, and cold exposure, cause a significant decrease (30-86%) in the capacity of the hepatic cell membranes to specifically bind [125I]ovine PRL. Stress-induced decrease in food intake was not a factor in these studies, as nourishment was given only by tube feeding. Neither sc inflammation nor cold exposure affected hepatic binding of [125I]insulin. Further, the induction of inflammation in lactating rats and rats bearing 7,12-dimethylbenz[a]anthracene-induced mammary carcinomas did not affect the binding of PRL by the lactating or malignant mammary tissue. The suppressive effect of inflammation on hepatic binding of PRL was demonstrable in adrenalectomized-ovariectomized rats, in hypophysectomized rats receiving hormone replacement, and in adrenalectomized rats that had undergone partial chemical sympathectomy. We conclude that sc inflammation, as well as other forms of stress, decreases hepatic binding of PRL, but does not affect hepatic binding of insulin or mammary binding of PRL. The decrease in hepatic PRL binding is not mediated by a hormone secreted by the adrenals, ovaries, or pituitary, or by catecholamines, but could be mediated by another plasma factor or by peripheral dopaminergic neurons. Stress-induced decrease in hepatic PRL binding, or a related decrease in the binding of other polypeptide hormones, could play a role in the physiological response to stress.

Ultrasonic Doppler study of the hormonal response of blood flow associated with normal and malignant mammary tissue: J.C. Bamber, M. Sambrook, H. Minasian and C.R. Hill, Institute of Cancer Research: Royal Marsden Hospital, Sutton, Surrey, U.K.

Ultrasonic Doppler study of the hormonal response of blood flow associated with normal and malignant mammary tissue: J.C. Bamber, M. Sambrook, H. Minasian and C.R. Hill, Institute of Cancer Research: Royal Marsden Hospital, Sutton, Surrey, U.K.

Magnesium in tumoral tissues, in the muscle and serum of subjects suffering from neoplasia

Mg 2+ content is significantly increased in malignant neoplastic mammary tissue compared with normal mammary tissue and benign neoplastic tissue of the breast. Significant variations of the ion were not found in the skeletal muscle tissue (rectus abdominis and pectoral muscle) of subjects suffering from malignant neoplasia with diffused metastasis. It seems that the variation of Mg 2+ content in malignant neoplastic mammary tissue is a local factor linked with the biological anomalies of the neoplastic cell.

Survival of Mouse Mammary Gland Transplants of Normal, Hyperplastic, and Tumor Tissues Exposed to X-Rays<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

Mouse mammary tissues, including ducts, prelactating lobules, hyperplastic outgrowth lines, and tumors, were exposed to varying doses of X-rays and then transplanted to fat pads of nonirradiated BALB/c mice for study. Estimates of the dose of radiation that would allow survival of 50% of the transplants (SD50) were made with the use of probit analysis. Nearly all duct and lobule transplants survived doses of X-rays from 0 to 800 rad. The survival rate declined rapidly following doses above 800 rad, and the calculated SD50 was 1,020 and 1,260 rad for mammary ducts and lobules, respectively. The three hyperplastic outgrowth lines tested gave very different results. Hyperplastic line Z5C1 transplants had better than 90% survival at doses up to 1,200 rad and an SD50 between 1,200 and 1,600 rad. Hyperplastic line Z5D transplants had an SD50 of between 800 and 1,200 rad. Hyperplastic line D1 transplants had a better than 90% survival following doses of 0-600 rad and an SD50 between 600 and 800 rad. The survival of tumor transplants was 100% following doses of X-rays up to 1,200 rad; the SD50 was in excess of 1,600 rad. The mouse mammary transplantation system can be used to study the direct effect of X-rays on normal, premalignant, and malignant mammary tissues and provides a basis for the study of the radiobiology of mammary tissues.

A preliminary assessment of an ultrasonic Doppler method for the study of blood flow in human breast cancer.

A 10 MHz, continuous wave ultrasonic Doppler system was used to study the blood flow associated with normal and malignant mammary tissue in patients with breast cancer. Some patients were receiving endocrine therapy and were examined repeatedly over a period of months. each of 6 characteristics extracted from the time varying maximum Doppler-shift frequency were averaged over signals obtained from a number of sites in the vicinity of the tumour, and from corresponding sites in the normal breast. A method was devised to allow location of previously examined vessels for subsequent examinations. The preliminary results of analysing approx. 400 recordings of Doppler signals obtained from 16 patients (6 of whom received endocrine therapy) are presented. The most informative of the 6 characteristics were the maximum systolic frequency (A) and the "mean" frequency (M) (= A + B/2 where B is the maximum frequency during end diastole). The average values of A and M obtained from the tumourous breast were always greater than those obtained from the normal breast in the same patient. A and M were roughly proportional to tumour volume, with extrapolated values at zero volume only slightly greater than the corresponding mean values for normal breast tissue. On average, changes in the values of A and M obtained from tumour sites during endocrine therapy appeared to occur in association with, and possibly slightly in advance of, changes in the tumour volume.

Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and guinea pig antikeratin antisera.

Few markers are available to identify the three types of mammary epithelial cells--ductal, alveolar, and myoepithelial--especially in pathological conditions and in cell cultures. We have used antisera to human keratins in immunofluorescence to facilitate the identification of the three mouse mammary epithelial cell types. In frozen tissue sections and primary cell cultures, a rabbit antikeratin antiserum specifically stained cytoplasmic filaments in all three types of epithelial cells. A guinea pig antiserum against the same keratin preparation, however, reacted preferentially with filaments in myoepithelial cells and readily detected this cell type in normal, dysplastic, and malignant mammary tissues and cell cultures. Neither antisera reacted with fibroblasts or any other mesenchymal cells. The combined use of the two antikeratin antisera thereby permits rapid surveys of tissue sections and cultures for the localization of not only all epithelial cells but also the subpopulation of myoepithelial cells. Moreover, when mammary cultures established from late-pregnant or lactating mice were stained simultaneously with guinea pig antikeratin and rabbit anticasein antisera, three populations of epithelial cells were mutually exclusive: those stained by anticasein antiserum, those stained by guinea pig antikeratin antiserum, and those stained by neither, consistent with properties of alveolar, myoepithelial, and ductal cells, respectively. These antisera thus offer a tool for studying different epithelial cell types during mammary development, tumorigenesis, and malignant progression.

Changes in casein sequence abundance in DNA of mouse mammary tumors

The casein gene content of mouse mammary tumor virus-induced mammary tumors was found to be elevated, apparently in proportion to the increase in MTV proviral copy number. Conversely, mammotropic hormone-induced mammary tumor DNA was deficient in casein genes in 2 of 3 tumors. Globin gene abundance, however, in both normal and malignant mammary tissues was unchanged, regardless of changes in casein gene content. These results suggest that perturbations have occurred in a common region of DNA from mammary tumors with 2 different etiologies.

Observations on the binding of testosterone to malignant mammary tumours and other tissues in vitro.

Observations on the binding of testosterone to malignant mammary tumours and other tissues in vitro.