[Quantitative detection of telomerase activity and its association with clinicopathological characteristics in breast cancer].

BACKGROUND & OBJECTIVE: Telomerase is a ribonucleoprotein enzyme, which plays an important role in cell immortalization and carcinogenesis. Recent studies on the association of telomerase activity with prognostic factors of breast cancer were controversial due to different methods. This study was to establish a feasible assay of quantitative detection of telomerase activity based on silver staining, and investigate possible association between telomerase activity and clinicopathological prognostic factors in primary breast cancer. METHODS: Highly sensitive silver-staining telomeric repeat amplification protocol assay (SS-TRAP) was used to quantify telomerase activity in 52 frozen human breast cancer samples and their adjacent breast tissues, 32 benign lesions, and 14 normal mammary gland lesions. The association between telomerase activity and clinicopathological data was analyzed. RESULTS: Telomerase activity was detected in 47 of the 52 breast cancer samples (90.38%), and in 10 of the 32 benign lesions, whereas no activity was detected in 37 of 52 adjacent nonmalignant breast tissues, and all 14 normal mammary gland tissues. The telomerase activity levels were 36.91+/-15.35, 8.27+/-4.37, 14.10+/-5.28, 0 (unit: TPG) in breast cancer, adjacent tissue of cancer, benign lesion, and normal tissue, respectively. The difference of telomerase activity was significant between breast cancer and the other 3 groups by using ANVOA (all P< 0.01). A strong correlation was found between telomerase activity and pathological category, and differentiation degree by logistic regression analysis, i.e. with ongoing tumor progression, telomerase activity appeared to increase in primary breast cancer (P=0.003, and P=0.004). No correlation was seen between telomerase activity and disease course, age, and menopause status of patients (all P >0.05). Telomerase activity level of invasive non-special cancer was higher than that of invasive special cancer(P< 0.05). Telomerase activity level of moderately/poorly differentiated carcer was higher than that of highly differentiated cancer (P< 0.05), while no obvious difference was found between moderately and poorly differentiated cancer (P >0.05). CONCLUSIONS: The activation of telomerase might occur early in breast cancer,and plays a critical role in carcinogenesis and tumor development. Telomerase may serve as a specific marker of early diagnosis and prognosis in mammary gland neoplasm.

Alterations in the expression of the DNA repair/redox enzyme APE/ref-1 in epithelial ovarian cancers.

The DNA base excision repair pathway is responsible for the repair of alkylation and oxidative DNA damage. A crucial step in the base excision repair pathway involves the cleavage of an apurinic/apyrimidinic (AP) site in DNA by an AP endonuclease (APE). The major AP endonuclease in mammalian cells is APE/ref-1, a multifunctional enzyme that acts not only as an AP endonuclease but as a redox-modifying factor for a variety of transcription factors. The purpose of this study was to determine the expression of APE/redox factor-1 (ref-1) in ovarian tissues, particularly ovarian cancers. Formalin-fixed, paraffin-embedded specimens of ovarian tissues (normal, various benign conditions, and epithelial cancers) were studied using both polyclonal and monoclonal antibodies to APE/ref-1. The relationship between APE/ref-1 protein levels and DNA repair activity was studied in ovarian Hey and Hey-C2 cell lines using Western blot and a specific AP-site oligonucleotide cleavage assay. Hey and Hey-C2 cells were fractionated, and the nuclear and cytoplasmic extracts were quantitated for protein levels and assessed for APE/ref-1 with Western blot. Normal ovarian tissues consistently demonstrated strong nuclear staining of the surface epithelium, epithelial inclusions, corpora lutea and albicantia, and stroma. Cytoplasmic staining was absent. A similar pattern was seen for benign conditions including endometriosis. Low malignant potential ovarian cancers stained in a pattern similar to normal ovarian and nonneoplastic tissues; however, two specimens also had areas of cytoplasmic staining. Epithelial ovarian cancers were remarkably different from all other ovarian tissues studied. Both nuclear and cytoplasmic staining of the malignant epithelium were seen and ranged from strong to weak, often with considerable staining heterogeneity within the same tumor. The AP-site oligonucleotide cleavage assay indicated that APE/ref-1 protein levels correlate well with DNA repair activity. The increased levels of APE/ref-1 in the Hey-C2 cells was mainly attributable to increased cytoplasmic enzyme. APE/ref-1 immunoreactivity is altered in malignant ovarian tumors. Further studies will determine whether the altered expression and subcellular location reflect changes in redox regulatory functions.

MT1-MMP and MMP-2 mRNA expression in human ovarian tumors: possible implications for the role of desmoplastic fibroblasts.

Expression of activated MMP-2 (72 kDa type IV collagenase) is highly associated with the malignant phenotype in adenocarcinomas, but predominant expression of the mRNA appears to be in stromal cells. MT1-MMP (membrane type 1-matrix metalloproteinase) is implicated in tumor-epithelial cell surface activation of latent pro-MMP-2, indicating a mechanism for tumor-stromal interaction in invasion. We determined the relative mRNA distribution of these MMPs in human ovarian tumors with a view to analyzing potential variations in the epithelial-mesenchymal interactions dictating ovarian tumor cell spread. In situ hybridization using 35S-labeled riboprobes was used to analyze 33 human ovarian tumors and mouse xenografts of human ovarian (DOV 13, SKOV3) and breast (MCF 7) tumor cell lines known to express MT1-MMP and MMP-2. MMP-2 mRNA was expressed in 31 of 33 and MT1-MMP mRNA was expressed in 29 of 33 tumor cases. MMP-2 mRNA was predominantly expressed in desmoplastic fibroblasts and in the subepithelial stroma. MT1-MMP mRNA showed some colocalization with MMP-2 in stromal cells. Neoplastic epithelial cell labeling for MT1-MMP mRNA was present in borderline and malignant tumors but not in benign tumors, and was invariably less than stromal labeling. Xenografts of DOV 13, SKOV 3, and MCF 7 cells showed some stromal localization of MMP-2 mRNA and weak labeling of DOV 13 cells. There was variable labeling for MT1-MMP mRNA in the neoplastic cells only. The colocalization of MT1-MMP and MMP-2 mRNAs in ovarian carcinoma stroma supports the view that MT1-MMP is closely associated with MMP-2 expression and function. It suggests that either additional mechanisms are involved in regulating MMP-2 activation at the tumor cell surface, or more intriguingly, that desmoplastic fibroblasts may be the primary mediators of extracellular matrix remodeling with respect to this system.

Messenger RNA for urokinase plasminogen activator is expressed in myofibroblasts adjacent to cancer cells in human breast cancer.

Urokinase plasminogen activator (uPA) is a serine proteinase involved in degradation of the extracellular matrix during cancer invasion. uPA is up-regulated in breast cancer, and high levels of uPA in tumor extracts are strongly associated with poor prognosis. Like several other matrix proteinases, uPA is in some types of cancer, including breast cancer, expressed by stromal cells. The present study was undertaken to determine the identity of the uPA-expressing stromal cells in breast cancer tissue. By in situ hybridization, a positive signal for uPA mRNA was in 26 of 28 ductal and four of five lobular carcinomas demonstrated in stromal cells adjacent to nests of cancer cells, whereas only one ductal carcinoma showed a positive reaction in the epithelial component itself. The positive stromal cells were found in both the peripheral and central parts of the tumors. Stromal cells surrounding carcinoma in situ lesions were uPA mRNA positive in a few cases, and no signal was observed in the neighboring nonmalignant tissue. Cell identification was done by immunostaining with Ab to markers for the following cell types: myoepithelial cells, myofibroblasts, smooth muscle cells, macrophages, endothelial cells, and epithelial cells. The only one of these cell types that had a distribution similar to the uPA mRNA-expressing cells was myofibroblasts, recognized as extravascular alpha-smooth muscle actin-positive and cytokeratin-negative cells. On adjacent sections, colocalization was found of cells positive for uPA mRNA and cells positive for alpha-smooth muscle actin and negative for cytokeratin. We concluded that the uPA mRNA-expressing cells are myofibroblasts. The myofibroblasts have previously been found to be abundant in breast cancer tissue. They primarily originate by differentiation of fibroblasts, probably induced by cytokines released from the cancer cells. The present findings suggest that the myofibroblasts, through production of uPA, play an active role in breast cancer invasion.

Relative activities of thymidylate synthetase and thymidine kinase in human mammary tumours.

Thymidylate synthetase (TS) and thymidine kinase (TK) are known to catalyse the methylation of dUMP for the de novo synthesis of dTMP and the phosphorylation of thymidine for the salvage synthesis of dTMP in the pyrimidine pathway, respectively. High activities of TS and TK have been observed in rapidly proliferating tissues. In the present study, both enzyme activities were measured in 8 specimens of normal mammary tissues, 12 fibroadenomas and 10 adenocarcinomas in the human breast. The average activities of TS in fibroadenomas and adenocarcinomas were approximately 5- and 7-fold that in normal mammary tissues, respectively. The average activities of TK in fibroadenomas and adenocarcinomas were 4- and 14-fold that in normal mammary tissues, respectively. Thus, DNA synthesis in benign and malignant mammary tumors may be relatively predominant in de novo and salvage pathways, respectively.

Dipeptidyl peptidase IV expression identifies a functional sub-population of breast fibroblasts.

The immunocytochemical distribution of the cell-surface enzyme dipeptidyl peptidase IV (DPP IV) has been studied in the human breast at the light and ultrastructural level. The presence of the enzyme was demonstrated on the cell membranes of interlobular fibroblasts, whilst intralobular fibroblasts were DPP-IV-negative. A fluorograph, after immunoprecipitation of 35S-methionine-labelled proteins of fibroblasts from primary breast cultures with an anti-serum to DPP IV, demonstrated a band at 135 kDa consistent with the presence of the enzyme. The clear delineation of 2 functionally distinct subpopulations of breast fibroblasts was maintained in benign fibro-adenomas and cystosarcoma phyllodes, both tumour types having growth characteristics of intralobular stroma. This observation has important implications for both normal breast biology and for breast carcinogenesis.

Amine oxidase activities in rat breast cancer induced experimentally with 7,12-dimethylbenz(alpha)anthracene.

The activities and distribution of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) in solid breast tumour induced in the rat by treatment with 7,12-dimethylbenz(alpha)anthracene (DMBA) were studied. The mammary tumours were classified according to anatomopathological criteria into: the benign fibroadenoma (FAD) and the malignant adenocarcinoma (ADC) and infiltrant adenocarcinoma (I-ADC). The proportions of total MAO (15%) and SSAO activities (85%) did not change with malignancy. However, an increasing degree of malignancy was associated with an increase in MAO-A activity and a decrease in MAO-B and SSAO activities. Kinetic constants were calculated for SSAO and for each MAO form separately, using specific substrates. The Km values did not change significantly with the degree of malignancy, but Vmax values for MAO-A increased whereas Vmax for SSAO and MAO-B diminished with malignancy. The dependence of SSAO activity on protein concentration indicated the presence of endogenous reversible inhibitory material in extracts from the more malign tumours. This inhibitor was associated with the microsomal fraction and was not removed by dialysis. It was also present in detergent-solubilized extracts, suggesting that the phenomenon might be due to an association of the enzyme itself producing an inactive species.

Quantitative cytochemistry of glucose-6-phosphate dehydrogenase in benign and malignant breast tumours.

Glucose-6-phosphate dehydrogenase (G6PD) activity was quantified cytochemically in mammary epithelial cells within frozen tissue sections from 38 patients with breast cancer and 44 with benign breast disease. G6PD activities were measured under atmospheres of both N2 and O2. The mean (S.E.) G6PD value 2.5 (0.23) IE U/min measured in N2 in mammary epithelial cells from the group of malignancies was significantly greater than that of 1.6 (0.37) IE U/min in the benign group (P less than 0.001), but there was considerable overlap between individual values. G6PD measured in O2 was detectable in 84% of malignancies compared to only 14% of benign biopsies and the group mean of 1.3 (0.18) IE U/min in the former was significantly greater than that of 0.35 (0.20) IE U/min in the latter (P less than 0.001). Significant correlations between G6PD activities measured in N2 and O2 were observed in both groups. The techniques present a sensitive method of identifying increases in G6PD activity in mammary epithelial cells and provide an assay that in a majority of cases permits the separation of malignant from benign tissues.

Immunohistochemical evaluation of reverse transcriptase in breast carcinoma with polyclonal antibodies raised in rabbit.

In this study, the presence of reverse transcriptase in breast tumours was examined with immunoperoxidase staining using antibodies raised in rabbit against reverse transcriptase of Moloney murine leukemia virus and against reverse transcriptase of avian myeloblastosis virus. The specificity of such antibodies was investigated with ELISA and Western blotting techniques. Five cases of infiltrating ductal carcinomas were found positive with the immune serum anti-reverse transcriptase of Moloney murine leukemia virus on 28 studied infiltrating ductal carcinomas, 2 infiltrating lobular carcinomas and 2 fibroadenomas.

[Aromatase activity in lymphocytes of cancer patients and healthy subjects]. / Aktivnost' aromatazy v limfotsitakh onkologicheskikh bol'nykh i zdorovykh liudei.

Conversion of androgens (aromatization) to estrogens in lymphocytes of cancer patients and healthy subjects in vitro was discovered. Aromatase activity in lymphocytes was comparable to that of fat and breast tumor tissues. It was completely suppressed in one out of two breast cancer patients receiving orimeten (aminoglutethimide). The activity in large bowel cancer was lower than in adenoma patients; however, the two groups were not balanced for age.

Milk-specific RNase as a marker of differentiation of rat mammary tumors.

Various rat mammary tumors were analyzed for the presence of a milk-specific Ca2+-stimulated RNase (Ca2+-RNase). When crude extracts of some differentiated tumors--adenocarcinomas of MT/W9, MT/W9a, R3230AC, DMBA-1, DMBA-8, and DMBA-14 and 3MN squamous cell carcinoma--were assayed for RNase activity under various ionic conditions, it was always highest in the presence of Ca2+/EDTA than under any other ionic condition. The opposite was true in invasive MT/W449a and 13762 adenocarcinomas, poorly differentiated SMT/2A carcinomas, MAMF2/TC fibrosarcoma, and MT/A fibroadenoma. Sephacryl S-200 chromatography separation of tumor extracts confirmed the presence of Ca2+-RNase in those differentiated tumors and absence of the enzyme from other tumors. Expressing the activity as a ratio of Ca2+/EDTA to either Mg2+/EDTA or EDTA alone to more clearly represent the relative level of Ca2+-RNase activity further illustrates the distinct differences between tumor classes. Thus Ca2+-RNase is a sensitive marker for use in the characterization of rat tumors with respect to differentiated mammary functions.

Folate conjugase activity in the plasma and tumors of breast-cancer patients.

The activity of folate conjugase (pteroylpolyglutamate hydrolase, EC 3.4.22.12) was measured in plasma from normal subjects and patients with breast cancer using pteroylglutamyl-gamma-glutamyl-gamma-(U14C) glutamate as the substrate. Conjugase assays also were performed on samples of breast-tumor tissue, normal breast, and fibroadenoma. When assayed at pH 4.5 and 7.4, mean plasma conjugase activity was significantly (p less than 0.05) elevated in a group of patients with anatomically proven metastatic disease (n = 21) when compared with control subjects (n = 12) and a group of patients (n = 13) with no clinical evidence of disease after mastectomies. Mean plasma conjugase activity assayed at pH 4.5 also was significantly higher in the metastatic disease group when compared with breast cancer patients before mastectomy (n = 9) and fibroadenoma patients before biopsy (n = 3). The specific activity of tissue conjugase assayed at pH 4.5 was significantly higher (p less than or equal to 0.05) in infiltrating ductal carcinoma than in normal adjacent breast tissue according to the Wilcoxon test for paired samples (n = 10).

Cytologic characteristics and origin of naked nuclei in breast aspirate smears.

To elucidate the origin of "naked nuclei" in breast aspiration smears, 17 cases of fibroadenoma were studied by light and electron microscopy. The ATPase reaction was also studied at both levels. The aspirates contained two types of naked nuclei: denuded degenerated nuclei and oval to spindle-shaped nuclei with very scanty cytoplasm. The cytoplasm of the latter was rich in free ribosomes and rough-surfaced endoplasmic reticulum but was devoid of cytoplasmic filaments and dense bodies. These cells were negative for ATPase activity. Stromal cells, not myoepithelial cells, characteristically demonstrated such cellular features in the aspirates and tissue sections studied. We conclude that most naked nuclei are derived from stromal cells.

Semiquantitative cytochemical estimation of glucose-6-phosphate dehydrogenase activity in benign diseases and carcinoma of the breast.

The level of glucose-6-phosphate dehydrogenase (G6PDH) activity was semiquantitatively evaluated in fresh imprints of infiltrative ductal carcinoma, fibrocystic disease and fibroadenoma of the breast. A significantly higher level of G6PDH activity was found in the carcinomas. The results suggest that the estimation of G6PDH activity could be a valuable method for evaluating the cells in benign and malignant breast lesions. It is possible that the intensification of G6PDH activity in carcinomas is a sign of the shift of the carbohydrate metabolism from an aerobic path or that the activity of the pentose shunt is higher because of the increased need for nucleic acid precursors in tissues with faster growth rates.

Altered monocyte function in patients with benign breast disease.

Monocyte migration, lysozyme production and phagocytosis was studied in 34 patients with fibroadenosis, 28 patients with fibroadenoma and 48 healthy female controls. In patients with fibroadenosis and fibroadenoma, monocyte migration and phagocytic activity were significantly reduced when compared to controls (P less than 0.001). Conversely, lysozyme production by monocytes from patients with benign breast disease was significantly higher than in controls (P less than 0.001). In 20 patients with benign breast disease, there was no significant difference in monocyte function before and 3 months after operation. The observed impairment of monocyte function in fibroadenosis and fibroadenoma would not appear to be the result of abnormal blood biochemistry or due to a direct serum inhibitor, but is probably related to an intrinsic cellular defect. Further studies are required to evaluate the significance of impaired monocyte function in the pathophysiology of benign breast disease.

Detection of the plasmin system in human mammary pathology using immunofluorescence.

The presence and localization of the plasmin system components urokinase (UPA), tissue type plasminogen activator (TPA), plasminogen (PG), a neoantigen expressed by the plasmin-alpha 2-antiplasmin complex, and plasmin inhibitors alpha 2-antiplasmin (AP) and alpha 2-macroglobulin (MG) have been tested by immunofluorescence on sections of 11 benign and 40 malignant lesions of the breast in an attempt to apply a morphological approach to the problem of tumor invasion in vivo. In benign lesions, TPA was seen in secretions of mammary glands and MG was seen in edematous zones. In one involuting lactating adenoma, UPA, TPA, PG, PAP, and AP were associated with glandular cells. UPA was detected in 11 carcinomas, TPA in 22, PG in 31, PAP in 12, AP in 23, and MG in all 40. All these components were essentially present in invasive territories, with a cellular labeling for UPA and TPA and a fluorescent staining frequently at the periphery of tumoral foci for PG and PAP. AP was more closely associated with cancer cells than MG, which was present in the stroma. Intraductal proliferations were rarely positive and there was no correlation between the localization of PG and the distribution of a basement membrane glycoprotein laminin. These data argue strongly for the involvement of the plasmin system in the infiltrating process of the stroma. This system seems to play a limited role in the breakdown of basement membrane in breast carcinomas in vivo.

gamma-Glutamyltranspeptidase activity in human breast lesions: an unfavourable prognostic sign.

The activity of gamma-glutamyltranspeptidase (gamma GT) (EC 2.3.2.2) was examined by histoenzymatic labelling on frozen sections derived from normal breast tissue, benign lesions and carcinomas. In biopsies from normal tissue and benign lesions, labelling was very intense in lumina and in the apical pole of the cells lining the lumina whilst in the cytoplasm it was slightly positive. In 34 out of 70 carcinomas, gamma GT activity was either undetectable or slightly positive while in the remaining 36 there was intense activity. Statistical examination of the results revealed no obvious correlation of gamma GT activity with histological grade of the tumour, progesterone receptor content or classification of patients by pre- or postmenopausal status. A good correlation between gamma GT activity and the following unfavourable prognostic signs: lymph node metastases and absence of oestradiol receptors. Patients with gamma GT-negative tumours may have a more favourable prognosis than those with gamma GT-positive tumours.

Increased activities of thymidine kinase isozymes in human mammary tumours.

Thymidine kinase, the enzyme in the pyrimidine salvage pathway, and its isozymes were examined in 10 specimens of normal mammary gland, 10 fibroadenomas and 11 adenocarcinomas in human breasts. The average thymidine kinase activities in fibroadenomas and adenocarcinomas were about 3 and 8 times that in normal mammary gland. The mammary thymidine kinase isozymes were separated into two types by diethylaminoethyl (DEAE) cellulose column chromatography. The activity of the thymidine kinase isozyme eluted with 0.1 M sodium chloride in buffer was twofold higher in fibroadenomas and fourfold higher in adenocarcinomas than that in normal tissue. In adenocarcinomas, but not fibroadenomas, the activity of the other isozyme eluted with buffer alone was increased to 22-fold that in normal tissues. As the activity of the latter isozyme was not affected by deoxycytidine triphosphate, it may be involved closely in DNA replication.

Demonstration of human breast carcinoma cells in cryosections and primary monolayer cultures of surgical biopsies by neotetrazolium reductase cytochemistry.

The present study describes a cytochemical approach to demonstrate human breast carcinoma cells in cryosections and in primary monolayer cultures from surgical biopsies. The material consisted of biopsies from 52 carcinomas and 29 benign lesions. Cryosections and cultures were incubated to demonstrate NADPH-neotetrazolium reductase in an atmosphere of 99.5% oxygen. Incubation time and section thickness were adjusted to accomplish the same level of reaction in cells of cryosections and corresponding cultures. Positive reaction was thus confined to epithelial elements and to the wall of some smaller blood vessels. More than one-half of the carcinoma cells showed moderate to strong reaction in cryosections from 29 of 52 carcinomas whereas no reaction was seen in ductules of normal appearance adjacent to these carcinoma cells. Positive reaction was seen in epithelial cell islets in primary cultures of 16 of the 40 carcinomas cultured. In cryosections from fibroadenomas and fibrocystic disease specimens only apocrine metaplasia consistently showed positive reactions compared with less than 10% of other ductular profiles in a given cryosection. This pattern of reaction was reflected in the derived primary cultures in which positive reaction was found in epithelial cell islets in only one of 19 biopsies cultured. The presence of human milk fat globule membrane antigen was used to demonstrate the epithelial nature of the cell islets seen in cultures of biopsies from both benign lesions and carcinomas. NADPH-neotetrazolium reductase positive islets from carcinoma biopsies were frequently aneuploid whereas most negative islets from carcinoma biopsies were diploid as were all islets from benign tissues.

Multiple forms of plasminogen activator in human breast tumors.

Total plasminogen activator (PA) activity, tissue-type PA (t-PA) activity, urokinase-like PA activity, and immunoreactive t-PA were measured in benign breast tumors (fibroadenomas), primary breast carcinomas, axillary node metastases, and chest wall recurrences. Total PA activity did not differ significantly in the different types of tumors. However, benign tumors contained predominantly t-PA activity. Urokinase-like PA activity was significantly higher in the malignant tumors compared with the benign group. Both t-PA activity and immunoreactive t-PA were significantly lower in chest wall recurrences compared with primary carcinomas. The ratio of t-PA to urokinase activity was significantly decreased between stages 1 and 3 in the primary tumors. Also, immunoreactive t-PA levels were significantly lower in stages 2 and 3 compared with stage 1. No correlation was found between PA (either total or its different forms) and tumor grade, histological type, or the presence or absence of axillary node metastases.