Glycoprotein patterns in normal and malignant cervical tissue.

Glycoproteins from normal and malignant human cervix were studied using an organ culture system and compared by gel electrophoresis and autoradiography. Five glycoproteins of 178 kDa, 95 kDa, 93 kDa, 82 kDa and 38 kDa and 1 glycolipid (46 kDa) were detected more frequently in squamous carcinomas. Certain glycoproteins were shown to be oncofoetal and some had affinity for Concanavalin A (Con A). The 82 kDa glycoprotein was present in 16/17 squamous carcinomas but in only 1/13 normal cervices. This band represented a glycoprotein containing glucosamine, mannose, small quantities of methionine and no fucose. These preliminary results suggest that these glycoproteins and in particular the 82-kDa glycoprotein are worthy of further investigation and characterisation.     

Immunohistochemical demonstration of metallothionein in normal human breast tissue and benign and malignant breast lesions

Metallothioneins (MTs) are a set of low molecular weight proteins with a high binding affinity to metal ions. MT over-expression has been recently demonstrated in invasive ductal carcinoma of the breast with poor clinical prognosis. In the present study, MTs have been immunohistochemically investigated in normal human breast tissue and a variety of benign, pre-invasive, and malignant breast lesions. In normal breast tissue, MTs were present in myoepithelial cells whereas the vast majority of luminal cells were MT negative. In lesions without increased cancer risk (adenosis and scleradenosis), MT was only immunolocalized in myoepithelial cells. In papillomas, MT was also found exclusively in myoepithelial cells. In most cases of epitheliosis, both the luminal and myoepithelial cells expressed MT. Atypicallobular hyperplasia, lobular carcinomain situ, and 13/15 invasive lobular carcinomas showed no MT over-expression. The two invasive lobular carcinomas with MT over-expression were classified as pleomorphic lobular carcinomas with apocrine differentiation. In contrast to lobular cancerization, 12/24 ductalin situ carcinomas and 9/20 invasive ductal carcinomas showed MT over-expression.In situ components found within invasive ductal carcinomas usually reflected the MT status of their invasive counterpart. It is concluded from our immunohistochemical results that breast carcinoma cases with MT overexpression arise from lesions which also show MT overexpression. Thus MT expression in carcinomas may be regarded as a genuine feature of the tumour cells and seems not to be related to endogenous or exogenous factors known to induce MT synthesis.     

Blood group MN antigens and precursors in normal and malignant human breast glandular tissue.

Blood group M and N active substances were demonstrated in human mammary gland tissue in both benign and malignant lesions. The precursor T (Thomsen-Friedenreich) antigen occurred only in cancerous tissue, where it was found regularly in the 15 gross cancers tested. The precursor Tn antigen also was found regularly in cancerous breast tissue; Both antigens were reactive in breasts with in situ carcinoma. The T antigen was not demonstrable in the 6 benign mammary glands studied; similarly, the Tn antigen was unmasked by sialidase treatment of healthy breast tissues. Anti-T antibody, present in all human sera, was severely depressed in many breast cancer patients compared to controls.     

Specific polypeptide differences in normal versus malignant human breast tissues by two-dimensional electrophoresis

Summary Postmitochondrial and cytosolic polypeptides were extracted from human breast tumors and non-malignant breast tissue and analyzed using high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Approximately 800–1000 postmitochondrial and 600–800 silver stained cytosolic polypeptides were detected over the pH range of 4.8 to 7.5 and molecular weight range of 18–120 kDa. The 2D-PAGE patterns of polypeptides from normal and malignant tissue were very similar, although both qualitative and quantitative polypeptide differences were noted. Six cytosolic polypeptides (pI/molecular weight × 10^–3) 5.20/80 kDa, 5.75/43, 6.25/40, 5.43/35, 5.45/34.5, 5.50/34 and 6.15/24 were expressed only in malignant tissues. One constitutive polypeptide, 7.25/52, was not detected in any of the malignant tissue samples. Quantitatively, marked differences in spot density were noted in polypeptides localized mainly in the molecular weight ranges of 22–40 kDa and pI ranges of 5.65–7.00. A general increase in polypeptide expression was noted in malignant tissues as compared to normal. Twenty-two polypeptides were significantly and consistently increased in tumor samples while only one polypeptide was decreased. One polypeptide, p24 (6.15/24) was expressed in greatest concentrations in tumors which also expressed the greatest estrogen receptor content. Expression of p24 was markedly reduced in normal tissue and malignant tissues expressing low levels of estrogen and progesterone receptors.     

Antigenic structure of normal and malignant breast tissue

The study was concerned with 13 antigens identified with the aid of a set of immune sera obtained from rabbits immunized with a nuclear, a membrane and a soluble fractions of normal and malignant mammary tissues as well as with the membranes of fat globules of breast milk. The antigens (1 nuclear, 4 soluble and 8 membraneous) were of different specificity: four--occurring in many organs, two--specific for milk only, three--organ-specific antigens which, however, persist in malignant tissues, and one--present in malignant tissue and absent from normal mammary and 25 other tissues tested. Such antigens as lactoferrin and alpha-lactalbumin were identified by immuno-enzymatic methods in the sera of a third of patients with tumors of different localizations.     

Immunological analysis of 17 beta-hydroxysteroid dehydrogenase in benign and malignant human breast tissue.

The expression of 17 beta-hydroxysteroid dehydrogenase (17-HSD) enzyme protein was studied in benign and malignant human breast tissue using the time-resolved immunofluorometric assay (IFMA), immunoblotting and immunohistochemistry. The presence and distribution of estrogen and progestin receptors was also analyzed immunohistochemically. Cytosolic 17-HSD concentrations in malignant breast specimens were highly variable (less than or equal to 0.2-311 ng/mg protein). As was previously found for the placental enzyme, the molecular weight of the 17-HSD expressed in malignant breast tissue was 35 kDa, estimated following polyacrylamide gel electrophoresis and immunoblotting. The cellular distribution of 17-HSD was further studied by immunohistochemistry. Immunostaining for 17-HSD was observed in 71% of the benign breast lesions (fibroadenomas and cases of mastopathia chronica) and in 47% of the cancer specimens (intra-ductal carcinomas, invasive ductal carcinomas). In benign lesions, the staining was exclusively localized in the cytoplasm of epithelial cells, with no immunoreactivity in the stromal cells. The staining in the cancer specimens was also detected only in the cytoplasm of malignant epithelial cells. A strong or moderate expression of 17-HSD was related to the presence of PR in the specimen (chi 2 = 4.657, p = 0.031). However, the expression of PR was not a prerequisite for expression of 17-HSD in all the cancer specimens. Our data suggest that, in addition to the reported regulation of 17-HSD by progestins, other factors are also involved in this process in breast tissue.     

Endogenous concentration and subcellular distribution of androgens in normal and malignant human breast tissue

The endogenous concentrations and the subcellular distribution of dehydroepiandrosterone (DHEA) and 5-androstene-3 beta,17 beta-diol (ADIOL) were measured in malignant and nonmalignant human breast tissue from both pre- and postmenopausal women. DHEA 3-sulfate was measured only in the cytosol. A greater tissue-plasma gradient of DHEA was present with large variations. The highest concentration of DHEA and ADIOL occurred in the nuclear fraction (average, 2.9 and 1.6 times the concentration in cytosol). With respect to DHEA, this finding is remarkable because no specific binding protein in human breast tissue has been reported. The concentration of DHEA 3-sulfate was significantly higher in the cytosol of nonmalignant than in malignant breast tissues. No significant differences in tissue concentrations of DHEA and ADIOL were found in malignant and nonmalignant breast tissue. The concentration of estrogens was measured in the cytosol and the nuclear fraction of the same tissues, as reported in a previous paper. We found a significantly higher estradiol concentration in malignant tissue as compared to nonmalignant tissue. When the ratio of ADIOL to estradiol was calculated from the combined data, a significant difference was found only in the cytosol of premenopausal cancer patients versus normal women. No difference was seen in the postmenopausal women. No difference in the ADIOL:estradiol ratio was found between normal and malignant breast tissue of patients of the same menopausal status.     

Endogenous concentration and subcellular distribution of estrogens in normal and malignant human breast tissue

The endogenous concentrations and subcellular distribution of estrone and estradiol were measured in malignant and nonmalignant human breast tissue from pre- and postmenopausal women. The most striking finding was the significantly higher concentration of estradiol per g of tissue in the malignant tissues than in the nonmalignant tissues. The tissue concentrations of estradiol in pre- and postmenopausal women were similar despite the large differences in the peripheral plasma levels. No correlation was found between the estradiol receptor content and endogenous concentration and subcellular distribution of estradiol. No difference in the estrone tissue concentration was found between malignant and nonmalignant tissues. In comparison with human uterine tissues, which we have reported previously, human breast tissue "handles" estrogenic hormones differently from human uterine tissue. At equal concentrations of the estradiol receptor, concentrations and subcellular distribution of the estrogens are different in both tissues. It is concluded that the mechanism of action of estradiol via its receptor, a mechanism mainly based on studies in animal uterine tissue, applies only qualitatively to human breast cancer tissue.     

Factor X-activating procoagulant in normal and malignant breast tissue

Factor X-activating activity (FXAA) was determined by a chromogenic assay in normal and malignant breast tissue. FXAA was found in all tissue (n = 38) irrespective of pathology, and the activity of normal tissue was similar to that of tumours. FXAA correlated with tissue hemoglobin in normal breast (p less than 0.02) but not in tumours. FXAA was markedly reduced by aluminium hydroxide, barium citrate, anti-human factor VII, DFP, PMSF and phospholipase C, but was unaffected by iodoacetamide and mercuric chloride. It is concluded that FXAA is a serine protease with the properties of a tissue factor-factor VII complex. FXAA occurs in normal and malignant breast tissue, although the 'normal' activity may be an artefact of the homogenization process.     

An ultrastructural morphometric analysis of normal human mammary tissue and human breast cancer

An ultrastructural morphometric study of normal and tumourous adult human mammary tissue is presented. The data show a characteristic numerical pattern for the different neoplastic stages of breast tissues. By applying a special data-comparison program a standardized diagnostic and prognostic system appears possible through the morphometric analysis of ultrathin epon sections of surgically obtained material.     

Effect of prolactin on lactalbumin production by normal and malignant human breast tissue in organ culture

To determine whether lactalbumin production by normal and neoplastic human mammary tissue is under the same control, the effect of prolactin treatment was studied in organ culture. Of 9 premenopausal normal breast samples, 6 produced lactalbumin in culture, and all 6 responded to prolactin treatment over 4 days. One biopsy of pregnant breast tested also responded to prolactin treatment, producing 200 times more lactalbumin in culture than did normal breast. Two of 4 normal postmenopausal biopsies produced lactalbumin, and one increased synthesis and release after prolonged exposure to prolactin. Of 10 scirrhous carcinomas, 6 produced lactalbumin, but none responded to prolactin treatment. In 2 premenopausal patients, normal breast tissue responded to different concentrations of prolactin, which were without effect on malignant tissue from the same breast. In summary, lactalbumin production in the samples that we have studied can be stimulated in normal but not in malignant breast tissue. This may indicate an absence or deficiency of prolactin receptors in malignant tissue.     

Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue

The Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), is a critical enzyme in the regulation of cell growth. In this report, we demonstrate elevated expression of PKC activity in surgical specimens of eight of nine spontaneous human breast tumors, as compared with the expression of PKC activity in normal breast tissue obtained from the same patients. The mean PKC specific activity in histologically normal breast tissue was 166 +/- 63 pmol 32P/min/mg, whereas the mean PKC specific activity in the breast tumors was 460 +/- 182 pmol 32P/min/mg (P = 0.0003; Student's t test). The low interpatient variability among the PKC levels observed in the histologically normal breast tissue specimens and the significant elevation of PKC levels observed in the tumors indicate that elevated expression of PKC activity in breast tissue is a potential marker for malignant disease in the breast.     

Comparison of procoagulant activities in extracts of normal and malignant human tissue.

Cancer procoagulant A (CPA) was originally described in extracts of tumor tissue, but whether this represented a quantitative and/or a qualitative difference from procoagulant activity in normal tissue extracts was not clear. Procoagulant activity was quantitated in extracts of 12 matched normal and malignant human tissue samples from the large intestine, breast, lung, and kidney. The specific activity of procoagulants in the tumor extracts was not greater than that in the extracts of normal tissue. Two enzymatic characteristics of CPA that distinguish it from tissue thromboplastin are its inhibition by diisopropylfluorophosphate (DFP) and its lack of dependence on factor VII. These specific tests were used to evaluate qualitative differences between procoagulants from normal and malignant intestinal tissues. In the paired normal and malignant tissue extracts, all tumor samples were inhibited by DFP and were active in factor VII-depleted bovine plasma (F7D-BP). In contrast, the extracts of normal tissue were insensitive to DFP and, except for one extract, were inactive in F7D-BP. Four of 9 other tumor extracts (44%) were positive for both of these tests for CPA, whereas the other 5 extracts were positive for only one of the two tests. The results suggest that extracts of normal and malignant tissues contained similar levels of procoagulant. However, malignant tissue contained a procoagulant enzymatically different from normal tissue thromboplastin. Furthermore, most of the malignant tissue extracts seemed to contain little or no thromboplastin.     

Clonal growth of normal and malignant human breast epithelia

We have developed an efficient method for the growth of colonies from single breast epithelial cells derived from nonmalignant [6], primary [7], and metastatic [1] surgical specimens. The immediate sources of the cells were primary and secondary mass cultures. An enriched culture medium and fibroblast feeder layers were used. A linear relationship between the number of cells cultured and the number of colonies obtained was found. Efficiency of colony formation was higher for those derived from nonmalignant specimens (mean 24%) than those derived from malignant specimens (mean 11%). Fibroblast feeder layers increased the efficiency of colony formation and also supported extended growth of colonies.     

Elevated expression of the myc gene in human benign and malignant breast lesions compared to normal tissue

Expression of the c-myc gene in human breast lesions and in adjacent normal tissue was studied by immunohistochemical analysis. The previously described monoclonal antibody Myc1-9E10 (1) which recognizes the p62 c-myc protein was used in paraffin tissue sections. A total of 101 cases of breast disease examined included 38 simple and complex cystic disease, 18 simple and hyperplastic fibroadenomas, 36 ductal and lobular carcinomas and 9 in situ carcinomas. Whereas the adjacent normal tissue was slightly positive, 25 out of 38 cystic disease, 7 out of 18 fibroadenoma, 36 out of 36 carcinoma and 9 out of 9 in situ carcinoma specimens showed moderate to high levels of p62 c-myc expression as indicated by staining intensity. These results suggest that the c-myc protein may play a role in breast neoplasia.     

Endogenous sugar-binding proteins in human breast tissue and benign and malignant breast lesions

Binding of carbohydrate moieties was detected in tissue sections of human breast by employing two types of labeled ligands: neoglycoproteins (chemically glycosylated, histochemically inert carrier protein) and desialylated naturally occurring glycoproteins. Paraffin-embedded, formalin-fixed sections from 40 benign and malignant breast lesions were examined for the presence and distribution of endogenous sugar receptors, employing a panel of 13 biotinylated neoglycoproteins, representing carbohydrates commonly found in naturally occurring glycoconjugates, and four biotinylated glycoproteins. Benign and malignant breast lesions revealed staining with mannosylated carrier neoglycoprotein in comparison to normal breast. A mixed pattern of staining localization and intensity was seen for different types of malignancy with this neoglycoprotein. Similarly, receptors for lactose and N-acetylglucosamine could only be detected within the cytoplasm for certain types of malignancy. Their nuclear localization, however, could also be seen in normal breast specimens. The extent of staining with different glycoproteins, containing different types of galactoside-terminal sugar chains, also appeared to differ between various types of breast cancer. The detection of endogenous sugar receptors by neoglycoproteins is proposed to contribute to an understanding of malignancy-associated alterations in the structure of their potential physiological ligands, the glycoconjugates. Changes in the structure and abundance of such glycoconjugates have commonly been detected with the use of plant lectins in histopathologic studies.